Effect of Heavy Metals On Economical Important Plant Dahlia (Coccinea Cav.)

Effect of Heavy Metals on Economical Important Plant
Dahlia (Coccinea cav.)
Research Supervisor:
Dr. Humayun Ajaz (Associate Professor)
Submitted By
Bilal Ehsan
2016-M. Phil-App-Chem-02
Department of Chemistry
University of Engineering and Technology Lahore,

Pakistan.
Effect of Heavy Metals on Economical Important Plant
Dahlia (Coccinea cav.)
This research thesis is submitted to the Department of Chemistry, University of Engineering
& Technology, Lahore for the partial fulfilment of requirement for partial degree of
Master of Philosophy
In
Chemistry
Approved on: _____________
Internal Examiner ______________________

(supervisor)
External Examiner ______________________
Chairperson
(Chemistry Department) ______________________
Dean
(Faculty of Natural Sciences, Humanities & Islamic Studies)
DEPARTMENT OF CHEMISTRY
UNIVERSITY OF ENGINEERING AND TECHNOLOGY
LAHORE - PAKISTAN
DEPARTMENT OF CHEMISTRY,
UNIVERSITY OF ENGINEERING AND TECHNOLOGY
LAHORE – PAKISTAN
From for the release of Thesis for Examination
The Thesis Titled “Effect of Heavy Metals on Economical Important Plant Dahlia (Coccinea
cav.)” is submitted for partial fulfilment of the requirement for the Masters of Philosophy in
Chemistry.
___________________ Date: ________________
Signature of Candidate
I approve that the above thesis be submitted for examination,
___________________ Dated: _____________________
Signature of Supervisor
(Internal Examiner)
This form must be submitted with the thesis to the Chairperson,
Department of Chemistry, University of Engineering and Technology Lahore – Pakistan.

Dedicated to my Loving Parents
And Wish ‘O’ Lord
Have mercy on them (parents) both as they did care
for me when I was little
(Al- Quran 17:24)

Acknowledgement
Verily, all praises are due to ALLAH ALMIGHTY, the most compassionate and the most
merciful who created us, flourished us with insight and cognition to distinguish right from
wrong and provoked us to reach for the mysteries of life and universe. Further He send His
Messenger Hazrat Muhammad (P.B.U.H), the most perfect and exalted who is forever a source
of guidance and knowledge for humanity.
With the grace of ALLAH and HAZRAT Muhammad (P.B.U.H) I have been able to
complete my research work.
It is a matter of great privilege and honor for me to express my deep gratitude and
thanks to my respected, learned, gracious and loving supervisor Dr. Humayun Ajaz for his help
invaluable guidance, precious suggestions and sympathetic attitude and keen observation
which enabled me to complete my work successfully. This ardors job was impossible without
the able guidance, skilled advice and pain taking efforts of my supervisor.
I forward my special thanks to Prof. Dr. Rubina Gilani, Chairman, Department of

Chemistry, for providing me the best possible research facilities during my research.
I cannot remain without expressing my heartiest gratitude to my parents, who made a

great devotion to my studies, always prayed for my betterment and success. Without his
assistance, support and inspiration it would have been just a dream to complete this ambitious
work
Table of Contents
1 INTRODUCTION ............................................................................................................ 16
1.1 Heavy Metal Stress.................................................................................................... 16
1.2 Metal Uptake ............................................................................................................. 17
1.3 Plant’s resistance to heavy metal absorption............................................................. 17
1.4 Choice of plant .......................................................................................................... 18
1.5 Effects of lead on plants ............................................................................................ 19
1.6 Effects of Cadmium on plants ................................................................................... 19
1.7 Effects of Mercury on plants ..................................................................................... 20
1.8 Effects of Zinc on plants ........................................................................................... 21
2 LITERATURE SURVEY ................................................................................................ 23
3 INSTRUMENTATION .................................................................................................... 28
3.1 Atomic Absorption Spectroscopy ............................................................................. 28
3.1.1 Introduction ........................................................................................................ 28
3.1.2 Uses .................................................................................................................... 29
3.1.2.1 Clinical Analysis......................................................................................... 29
3.1.2.2 Pharmaceutical Analysis............................................................................. 29
3.1.2.3 Environmental Analysis ............................................................................. 29
3.1.2.4 Rocks Analysis ........................................................................................... 29
3.1.2.5 Industrial Analysis ...................................................................................... 29
3.1.3 Working ............................................................................................................. 30
3.1.4 Components of AAS .......................................................................................... 31
3.1.4.1 Radiation source ......................................................................................... 31
3.1.4.1.1 Line source .............................................................................................. 31
3.1.4.1.2 Hollow cathode lamp ............................................................................... 32
3.1.4.1.3 Construction............................................................................................. 32
3.1.4.1.4 Sputtering................................................................................................. 32
3.1.4.1.5 Excitation ................................................................................................. 32
3.1.4.1.6 Emission .................................................................................................. 33
3.1.4.1.7 Electrodes discharge lamp ....................................................................... 33
3.1.4.1.8 Continuum line sources ........................................................................... 34
3.1.4.2 Atomizers.................................................................................................... 34
3.1.4.2.1 Flame Atomizer ....................................................................................... 34
3.1.4.2.2 Electro thermal Atomizer ....................................................................... 36
3.1.4.3 Sample Introduction System ....................................................................... 36
3.1.4.3.1 Continuous introduction system .............................................................. 36
3.1.4.3.2 Discontinuous introduction system ......................................................... 36
3.1.4.4 Monochromator .......................................................................................... 36
3.1.4.5 Detector ...................................................................................................... 37
3.1.4.5.1 Sensitivity and Specificity ....................................................................... 38
3.1.4.5.2 Back ground Absorption .......................................................................... 38
3.1.5 Sample Preparation ............................................................................................ 38
3.1.6 Instrumental Setup ............................................................................................. 38
3.2 Cold Vapor Technique .............................................................................................. 39
3.2.1 Instrumentation .................................................................................................. 39
3.2.1.1 Hollow Cathode Lamp................................................................................ 39
3.2.1.2 Atomizer ..................................................................................................... 39
4 MATERIALS & METHODS ........................................................................................... 42
4.1 Plantation................................................................................................................... 42
4.2 Sampling.................................................................................................................... 43
4.3 Sample collection ...................................................................................................... 45
4.4 Sample treatment ....................................................................................................... 45
4.4.1 Electric oven ...................................................................................................... 45
4.4.2 Grinding of sample ............................................................................................ 46

4.4.3 Weighing of sample ........................................................................................... 46
4.4.4 Digestion of grinded sample .............................................................................. 46
4.4.4.1 Method 1 ..................................................................................................... 46
4.4.4.2 Method 2 ..................................................................................................... 46
4.4.4.3 Method 3 ..................................................................................................... 46
4.4.5 Heating of sample .............................................................................................. 47
4.4.6 Dilution of digested sample ............................................................................... 47
4.4.7 Filtration ............................................................................................................. 48
4.4.8 Sample stored ..................................................................................................... 48
4.5 Standard Solution ...................................................................................................... 48
4.5.1 Standard Solution for Lead (Pb) ........................................................................ 48
4.5.1.1 1000 mg/L (Pb) Solution ............................................................................ 49
4.5.1.2 Diluted Solution.......................................................................................... 49
4.5.1.2.1 1000 mg/L → 500 mg/L .......................................................................... 49
4.5.1.2.2 500 mg/L → 100 mg/L ............................................................................ 49
4.5.1.2.3 100 mg/L → 10 mg/L .............................................................................. 49
4.5.1.2.4 10 mg/L → 1 mg/L .................................................................................. 50
4.5.2 Standard Solution for Cadmium (Cd) ................................................................ 50
4.5.2.1 1000 mg/L (Cd) Solution ............................................................................ 50
4.5.3 Standard Solution for Mercury (Hg) .................................................................. 50
4.5.3.1 1000 mg/L (Hg) Solution ........................................................................... 51
4.5.4 Standard Solution for Zinc (Zn) ......................................................................... 51
4.5.4.1 1000 mg/L (Zn) Solution ............................................................................ 51
4.6 Atomic Absorption Spectrophotometer .................................................................... 52
4.7 Analysis ..................................................................................................................... 52
4.7.1 Atomic Absorption Spectrophotometer ............................................................. 52
4.7.2 Cold vapor Atomic Absorption Spectrophotometer .......................................... 52

4.8 Transfer factor ........................................................................................................... 53
4.9 Tolerance Index ......................................................................................................... 53
5 RESULTS ......................................................................................................................... 55
5.1 Effect of Lead (Pb) .................................................................................................... 55
5.1.1 Absorbance of Lead in different parts of plant .................................................. 55
5.2 Statistical Analysis Lead ........................................................................................... 60
5.2.1 Student T test for Lead ....................................................................................... 60
5.2.1.1 Hₒ or Null hypothesis .................................................................................. 60
5.2.1.2 H1 hypothesis .............................................................................................. 61
5.2.1.3 Method for calculation of Student T test: ................................................... 61
5.2.1.3.1 Example: .................................................................................................. 61
5.2.1.3.2 Calculation of mean: ................................................................................ 62
5.2.1.3.3 Calculation of Standard Deviation: ......................................................... 62
5.2.1.3.4 Calculation of True value: ....................................................................... 62
5.2.1.3.5 Calculation of Student T test: .................................................................. 63
5.2.2 Q test rejection of a result for Lead ................................................................... 64
5.2.2.1 Method for calculation of Q test ................................................................. 65
5.3 Effect of Lead on physical parameters ...................................................................... 66
5.4 Effect of Cadmium (Cd) ............................................................................................ 68
5.4.1 Absorbance of Cadmium in different parts of plant .......................................... 68
5.5 Statistical Analysis Cadmium ................................................................................... 73
5.5.1 Student T test for cadmium ................................................................................ 73
5.5.2 Q test rejection of a result .................................................................................. 74
5.6 Effect of Cadmium on physical parameters .............................................................. 75
5.7 Effect of Mercury (Hg) ............................................................................................. 77
5.7.1 Absorbance of Mercury in different parts of plant ............................................ 77
5.8 Statistical Analysis Mercury ..................................................................................... 82

5.8.1 Student T test for Mercury ................................................................................. 82
5.9 Effect of Mercury on physical parameters ................................................................ 84
5.10 Effect of Zinc (Zn) .................................................................................................... 86
5.10.1 Absorbance of Micronutrient Zn in different parts of plant .............................. 86
5.11 Statistical Analysis Zinc ............................................................................................ 91
5.11.1 Student T test for Zinc ....................................................................................... 91
5.12 Effect of Zinc on physical parameters....................................................................... 93
6 DISCUSSION................................................................................................................... 95
6.1 Heavy Metals (Pb, Cd & Hg) .................................................................................... 95
6.2 Micronutrients Zinc and Zn toxicity ......................................................................... 95
7 CONCLUSION ................................................................................................................ 95
8 References ........................................................................................................................ 96
List of Figures
Figure 1: First Atomic Absorption Spectrophotometer. .......................................................... 28
Figure 2: Varian Flame AAS model AA240. .......................................................................... 29
Figure 3: Lambert-Beer law. .................................................................................................... 30
Figure 4: Schematic diagram of AAS. ..................................................................................... 31
Figure 5: Hollow Cathode Lamp. ............................................................................................ 32
Figure 6: Sputtering and Emission processes in Hollow Cathode lamp. ................................. 33
Figure 7: Electrodeless discharge lamp. .................................................................................. 33
Figure 8: High resolution continuum AAS with double monochromator. .............................. 34
Figure 9: Flame Atomization. .................................................................................................. 35
Figure 10: Graphite Tube. ........................................................................................................ 36
Figure 11: Monochromator. ..................................................................................................... 37
Figure 12: Emission process in Detector. ................................................................................ 38
Figure 13: Schematic diagram for Cold Vapor Mercury Technique. ...................................... 40
Figure 14: Seedling of Dahlia Plant. ........................................................................................ 42
Figure 15: seedling in individual pot. ...................................................................................... 43
Figure 16: Dahlia Flowering Plant. .......................................................................................... 44
Figure 17: Collected samples (Tubers, Stems and Leaves) ..................................................... 45
Figure 18: Grinded Sample ...................................................................................................... 46
Figure 19: Digested Sample. .................................................................................................... 47
Figure 20: Filtration of digested acid. ...................................................................................... 48
Figure 21: Effect of Pb on Dahlia Plant. .................................................................................. 56
Figure 22: Absorbance comparison 50 mg/L Pb. .................................................................... 57
Figure 23:Absorbance comparison 0 mg/L Pb. ....................................................................... 57
Figure 24: Absorbance comparison 200 mg/L Pb. .................................................................. 57
Figure 25: Absorbance comparison 100 mg/L Pb. .................................................................. 57
Figure 26: Comparative relation between absorption of Pb in tubers, Stems, leaves & flowers.
.................................................................................................................................................. 59
Figure 27: Blooming Period comparison (Pb). ........................................................................ 66
Figure 28: Days to Flowers comparison (Pb). ......................................................................... 66
Figure 29: Height of Plant (Pb). ............................................................................................... 66
Figure 30: Size of Flower comparison (Pb). ............................................................................ 66
Figure 31: Effect of Cd on Dahlia Plant. ................................................................................. 69
Figure 32: Absorbance comparison 50 mg/L Cd. .................................................................... 70
Figure 33: Absorbance comparison 0 mg/L Cd. ...................................................................... 70
Figure 34: Absorbance comparison 200 mg/L Cd. .................................................................. 70
Figure 35: Absorbance comparison 100 mg/L Cd. .................................................................. 70
Figure 36: Comparative relation between absorption of Cd in tubers, Stems, leaves & flowers.
.................................................................................................................................................. 72
Figure 37: Blooming Period comparison (Cd)......................................................................... 75
Figure 38: Days to Flowers comparison (Cd). ......................................................................... 75
Figure 39: Size of Flower comparison (Cd). ........................................................................... 75
Figure 40: Height of Plant (Cd). .............................................................................................. 75
Figure 41: Effect of Hg on Dahlia Plant. ................................................................................. 78
Figure 42: Absorbance comparison 0 mg/L Hg. ...................................................................... 79
Figure 43: Absorbance comparison 50 mg/L Hg. .................................................................... 79
Figure 44: Absorbance comparison200 mg/L Hg. ................................................................... 79
Figure 45: Absorbance comparison100 mg/L Hg. ................................................................... 79
Figure 46: Comparative relation between absorption of Hg in tubers, Stems, leaves &
flowers...................................................................................................................................... 81
Figure 47: Blooming Period comparison (Hg). ....................................................................... 84
Figure 48: Days to Flowers comparison (Hg).......................................................................... 84
Figure 49: Height of Plant (Hg). .............................................................................................. 84
Figure 50: Size of Flower comparison (Hg). ........................................................................... 84
Figure 51: Effect of Zn on Dahlia Plant................................................................................... 87
Figure 52: Absorbance comparison 100 mg/L Zn. .................................................................. 88
Figure 53: Absorbance comparison 0 mg/L Zn. ...................................................................... 88
Figure 56: Comparative relation between absorption of Zn in tubers, Stems, leaves & flowers.
.................................................................................................................................................. 90
Figure 57: Blooming Period comparison (Zn). ........................................................................ 93
Figure 58: Days to Flowers comparison (Zn). ......................................................................... 93
Figure 59: Height of Plant (Zn). .............................................................................................. 93
Figure 60: Size of Flower comparison (Zn). ............................................................................ 93
List of Tables
Table 1: Different flames with their maximum temperature. ............................................................................... 35
Table 2: Absorbance of Lead (Pb) by different parts of Dahlia plant against different concentration. ................ 55
Table 3:Student T test statistical analysis of different parts of Dahlia plant against different concentration of Pb.
.............................................................................................................................................................................. 60
Table 4: Q test Rejection test of suspected values of different parts of plant of various concentration of Pb. ..... 64
Table 5: Different physical parameters measurement against different concentration of Lead. .......................... 66
Table 6: Absorbance of Cadmium (Cd) by different parts of Dahlia plant against different concentration. ........ 68
Table 7: Student T test statistical analysis of different parts of Dahlia plant against different concentration of Cd.
.............................................................................................................................................................................. 73
Table 8: Q test Rejection test of suspected values of different parts of plant of various concentration of Cd. ..... 74
Table 9: Different physical parameters measurement against different concentration of Cadmium (Cd). ........... 75
Table 10: Absorbance of Mercury (Hg) by different parts of Dahlia plant against different concentration. ........ 77
Table 11: Student T test statistical analysis of different parts of Dahlia plant against different concentration of Hg.
.............................................................................................................................................................................. 82
Table 12: Q test Rejection test of suspected values of different parts of plant of various concentration of Hg. .. 83
Table 13: Different physical parameters measurement against different concentration of Mercury (Hg). ........... 84
Table 14: Absorbance of Zinc (Zn) by different parts of Dahlia plant against different concentration. ............... 86
Table 15: Student T test statistical analysis of different parts of Dahlia plant against different conc. of Zn. ....... 91
Table 16: Q test Rejection test of suspected values of different parts of plant of various concentration of Zn. ... 92
Table 17: Different physical parameters measurement against different concentration of Zinc (Zn)................... 93
Abstract
Dahlia (Coccinea cav.) sprung in soil contaminated with Lead, Cadmium, Mercury and Zinc.
Different metals Pb, Cd, Hg and Zn have different tolerance index. Zn as a micronutrient has
high tolerance index. Lead is one of the toxic metal among Pb, Cd, Hg transported to all parts
of body. Mercury and its movement to higher parts is permanently blocked and trace amount
of cadmium is accumulated to higher parts. The physiological changes by toxic effect of Pb,
Cd, Hg and Zn discerned by visible symptoms which due to deficiency of essential nutrients.
Plant’s height (93.5 cm), Days to flower (151.28 days) and blooming period (39.88 days) in
unpolluted soil was negatively affected and dwindled with increased concentration of each
metal. Zn is a micronutrient: concentration higher than 100 ppm evinced to be toxic. The
minimum and highest absorption of Pb, Cd, Hg and Zn was 0.088-4.575 mg/L, 0.1-2.945 mg/L,
0.012-6.508 mg/L and 4.583-18.786 mg/L. The Dahlia plant was severely influenced by
mercury and descending toxicity order is Pb ˂ Cd ˂ Hg.
Key words: tolerance index, visible symptoms, essential nutrients, Plant’s height, Days to
flower, blooming period, toxicity order.
CHAPTER 1
INTRODUCTION
INTRODUCTION
1 INTRODUCTION
The sources, reactions, transport and effect of chemical species in the soil, water and air and
human’s activity effect are studied in branch of chemistry called environmental chemistry [1].
The heavy metal contamination of soil and water raise a serious threat for environment and
human health and need productive and inexpensive technological solution [2]. The required
concentration of heavy metals exceeds and concentrate due to human activities, absorb in plant,
enter in animal through diet and inhalation and become cellular component of organisms [3].
The intake of heavy metals at chronic low-level become deleterious effects on humans being
and animals because no method discover yet for completely elimination of heavy metals. Pb,
Hg, Cd and Cu are considered as cumulative poisons metals [4]. The agriculture soil
contamination of metals either atmospheric deposition or disposal sewage sludge cause
leaching of metals into top soil and ground water. The source of metals in aquatic ecosystems
in dissolved and solid waste come from domestic, industrial and agriculture run off. Many
industries like tannery, steel, automotive, textile, electroplating, battery producing, mining and
cable manufacturing set free heavy metals like Cu, Cr, Ni, Cd and Pb in waste water [5] The
heavy metals or metalloids are potentially toxic in environment context. The resources for
urban agriculture irrigation are industrial and municipal wastewaters effluent disposal problem
[6]. The sewage effluents have usually low concentration of heavy metals but prolonged usage
of these heavy metals contaminated sewage effluents increase the concentration of metals in
soil [7]. Trace amount of heavy metals are less health hazard but amount exceeded from the
amount recommended value become health hazard for plants [8].
1.1 Heavy Metal Stress

The toxic heavy metal contamination due to industrial effluents and heavy metals in wastes in
sufficient amount cause toxicity to plants [9]. The heavy metal concentration exceeded from
optimum level greatly affect the plant both as directly and indirectly. The direct toxic effect
due to exceeded concentration of heavy metals are oxidative damage [10] [11]. Indirect heavy
metal toxic effect is due to substitution of necessary nutrients at cation exchange sites of roots
in plants [12]. The growth and activities of soil microorganism are negatively affected the
growth of plant. Beneficial soil microorganism and nutrients reduction due to increased
concentration of heavy metals consequently decrease decomposed organic matter and decline
soil fertility. The interference of heavy metals with activities of soil microorganisms hamper
the enzyme activities. The effect of heavy metals both directly and indirectly cause overall
reduction in growth which may lead to death of plants [13]. The heavy metal effect differs on
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INTRODUCTION
the growth and development of plant. Pb. Cd, Hg and As in trace amount which don’t play
significant role in plants have adverse effect for plants in the growth medium. The magnificent
reduction in height of rice plant was noticed with reduction in tiller and panicle formation rice
plant was grown in 1mg/kg Hg contamination soil [14]. The Cd concentration below 5 mg/L
declined the Stems and roots growth in wheat plants [15]. Plant growing in contaminated soil
caused reduction in growth because of reduction in activities of photosynthetic, plant mineral
nutrients and some enzyme activities stopped [16].
1.2 Metal Uptake

The natural and common components found in soil are metals including toxic metals. For
proper growth and development plants need metals (macronutrients and micronutrients) in
correct proportions. The bioavailability of metals depends on chemical form of the element in
the soil solution and soil physiochemical properties e.g., cation exchange capacity, pH and
organic matter contents. In acidic environment, the solubility and bioavailability of a metal
cation increases in contaminated soil [17]. Metals (macronutrients and micronutrients) are
translocated from roots to upper parts through adsorption, absorption, precipitation,
complexation phenomenon. Nature of soil, plant’s genotype, root system, climatic conditions
and seasonal cycles are the factors determine the bioavailability of metals in soil solution.
Cation exchange capacity of clay particles in soil are different for different metal, pH of soil,
oxidation states of metals, ion exchange capacity and carbon contents of soil control
bioavailability of metals in soil solution. The metal ion concentration in ionized state depend
on pH of soil and total contents of metals [18]. The dependent of metals uptake is on soil
chemistry. The absorption of metals is reduced by increased interaction or sorption to soil
particles. The higher capacity of ion exchange capacity, the higher sorption of metals to soil
(availability of metals to plants decline) determine the bioavailability of metals ions to plants.
In acidic conditions, metals from exchange site desorb (available to plants as free ions) due to
compaction of H+ at exchange site. The transportation of metals from soil to groundwater result
in metal accumulation in soil and growth of plants are inhibited [19].
1.3 Plant’s resistance to heavy metal absorption

The high concentration of metals in the soil, plants response in two ways. Avoidance is the first
mechanism, the absorption of metals by roots and transfer of metals in organs are prevented by
plants. Such plants are known as non-accumulator. Metals accumulation by other plants and
such plants can have maximum potential to absorb heavy metals by roots from the soil and
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INTRODUCTION
transferred and stored metals are called hyperaccumulator [20]. The resistance to absorb heavy
metals by some plant species are due to stabilize them in the internal tissue but metal contents
in plants may be health injurious for human beings and animals [21].
1.4 Choice of plant

In this study, Dahlia (Coccinea cav.) which is economically important and ornamental plant to
assess optimum level of toxicity and their effects on growth and development. The Pb, Cd, Hg
and Zn metal accumulation in plants due to contamination of soil by these metals having toxic
effects on plants, animals and many living organisms as heavy metals become part food chain
and have many health issues like kidney dysfunction, malfunction of liver, heart attack, lung
cancer, nose cancer, larynx cancer and respiratory failure. The aim of research is to assess the
tolerance index of Pb, Cd, Hg and Zn and better understanding of heavy metals and toxicity in
Dahlia (Coccinea cav.) to maintain ecological harmony of our planet. The second perspective
of research paper is to find defense response of Dahlia. Dahlia has octoploid chromosome. The
interaction of heavy metals and plants has two aspects. One is negative effect of heavy metals
on plants cause overall growth retardation and second is, some plants have their own defense
response against heavy metals and detoxify heavy metal contaminant [22].
The Dahlia (Coccinea cav.) is medicinal & non-medicinal plant. The tubers of Dahlia
contain inulin. Inulin performed prebiotic function. The prebiotic is component of healthy food
that is not digested by digesting enzymes and believed to play an important role in stimulating
the activity and beneficial bacteria in the digestive tract. Inulin is used as a food ingredient and
has a high forbearance level of food consumption and proved to be safe and has no side effect
[23]. The absorption of calcium and magnesium is increased by using inulin. The flower of
Dahlia is used in salads, cream, cheese and dip as a flavor. The florets of Dahlia are also used
in some recipes include a dahlia dip, sundried tomato & dahlia bread. The tubers of Dahlia
(Coccinea cav.) have antibiotic characteristics and are used as CNS depressants. The Aztecs
used floret and tubers of dahlia for skin treatment like rashes, infected grazes, cracks in skin.
The roots contain antibiotic compounds plentiful in the skin of tubers. The florets crush, mash
up, soothe insect bites or stings. The florets of Dahlia are used in coffee as a flavor. The
polyacetylene with ene-diynediene chromophore in trace amount were found in tubers and
aerial parts of plant. The polyacetylene is considered an important group of nutraceuticals in
vegetable foods an active target for the development of healthier food products [24].
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INTRODUCTION
1.5 Effects of lead on plants

Roots of plants accumulated lead (Pb) because plants have ability to absorb lead from the soil.
The foliage of plant takes up lead and possibly to transport lead to other parts of the plant.
Addition of calcium and phosphorus proved to reduce the absorption of lead from soil by
plants. Lead is distributed omnipresent and is toxic element in the soil. The morphology,
growth and photosynthetic processes were greatly affected by absorption of lead. Seed
inhibition of Spartiana alterniflora, Pinus helipensis were due to absorption of lead [25].
Inhibition of seed germination may be due to interference of lead with essential enzymes.
Mukherji and Maitra [26] noticed the reduction of protease and amylase by 50% in rice
endosperm with the application of 60 μM lead acetate. The inhibition of seedling was also
noticed in soya bean, rice [27] maize [28] barley, tomato and legumes. The root and stem
elongation and leaf expansion in Allium species barley [29] and Raphanus satives was also
inhibited due to absorption of lead. The concentration of lead, ionic composition and pH of the
medium are the factors determined the inhibition of root elongation [30]. The inhibition of root
growth was also seen in Sesamum indicum due to high concentration of lead [31]. The
morphological changes in plants with the application of lead was evinced in many plant species
for example irregular radial thickening in pea roots, lignification of cortical parenchyma and
cell walls of the endodermis [32]. The proliferation effects on the repair process of vascular
plants was induced by lead [33]. The concentration of lead at rate of 100-200 mg/L
administrated to potted sugar beet plants caused reduction in growth and chlorosis [34]. The
oxidative stress by increasing production of ROS in plants was due to high level of lead in soil
[35]. The reduced biomass, suppressed growth, reduction in germination, decrease in plant
protein contents was seen in maize (Zea mays) [36]. Reduced plant height, reduction in number
of leaves and area of leaf in Portia tree (Thespesia populnea) [37] and enzyme activity
inhibition which deteriote CO2 fixation cycle in Oat (Avena sativa) were seen due to
application of lead in soil [38].
1.6 Effects of Cadmium on plants

Cadmium permissible limit in soil of agriculture is 100mg/kg of soil [39]. The visible
symptoms like chlorosis, browning of root tips, growth inhibition and finally death was due to
plant sown in soil containing high level of cadmium concentration [40] [41] [42]. Fe (ΙΙΙ)
reductase caused inhibition in roots by application of cadmium and led to deficiency of Fe (ΙΙ)
and critically affected photosynthesis processes. In general, the uptake and transport of
essential elements Ca, Mg, P and K and water by plants was affected by cadmium [43]. The
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INTRODUCTION
inhibition of nitrate reductase activity in the Stems caused the reduction in absorption of nitrate
ions and in the transportation of nitrate from roots to Stems declined [44]. In plant, Silene
cucubalus significant nitrate reductase activity inhibition was seen [45] . The high
concentration of cadmium in soil caused decrease in nitrogen fixation and primary ammonia
assimilation process in nodules of soyabean plant [46]. Plasma membrane permeability was
affected by metal toxicity particularly cadmium application causing reduction in water content
and disturbing water balance [47]. The soil treated with cadmium caused to reduce the ATPase
activity in plasma membrane fraction of wheat and sunflower roots [48]. The peroxidation of
lipid in membranes of plants due to cadmium treatment severed alteration in functionality of
membranes and chlorophyll biosynthesis inhibition and reduction in enzymes activity disturbed
in chloroplast metabolism and CO2 fixation respectively [49]. The high cadmium concentration
in soil of wheat (Tritium sp.) declined seed germination, decrease in uptake of plant’s nutrients
and reduction in length of Stems and roots was observed [50] [51] Stems growth reduction due
to Cd absorption by garlic (Alium sativum) [52] and root growth inhibition by Maize (Zea
mays) [53] was seen.
1.7 Effects of Mercury on plants

Mercury is not essential but contamination of soil with heavy metal due to usage of fertilizers,
sludges, limes and manures. Several variables e.g., soil pH, cation exchange capacity, soil
creation and plant’s species determine the uptake of mercury from soil and the relationship
between the amount of Hg in soil and uptake of Hg from soil is not linear. The contamination
of Hg in entire food chain has been resulted high concentration of Hg arable lands. Mercury
exist in different forms e.g., HgS, Hg+2, Hg0 in agriculture field but mostly soil is contaminated
with Hg+2 [54]. The absorption of Hg onto sulphides, clay particles, organic matters, Hg remain
in solid phase but mercury as Hg+2in soil of higher and aquatic plants [55] [56]. Phytotoxic,
physiological disorders and visible injuries in plants was due to high level of accumulation of
Hg [57]. Hg+2interfere and bind to protein channel of membrane in plants caused closure of
leaf stomata and water flow obstructed [58]. Induction of mitochondrial activity and oxidative
stress by increased production of ROS was due to high level of Hg+2served disruption of lipids
of bio membranes and cellular metabolism in plants [59]. Decreased in plant height, reduction
in tiller and panicle formation and bioaccumulation of Hg+2 in Stems and roots seedling because
of high concentration of Hg+2was observed in rice (Oryza sativa) [60] [61]. Reduced plant’s
height, germination, flowering reduction, fruit weight loss and chlorosis in tomato
(Lycopersican) plant was noticed.
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INTRODUCTION
1.8 Effects of Zinc on plants

Plants produce chlorophyll with the help of micronutrient zinc. Deficiency of Zn in soil caused
leaf to discolor and plant growth stunted [62]. Leaf discoloration e.g., chlorosis, turn of the
tissue of veins to yellow was caused by deficiency of Zn usually lower surface of leaf near the
base turn yellow and then move to upper side of leaves and leaf may turn into brown or purple
and eventually die. Several metabolic processes of plants affected by micronutrient Zinc [63].
Phytotoxicity of Zn and Cd can be seen by declined growth and development, metabolism and
oxidative stress in different species e.g., Phaseolus vulgarus [64] and Brassica juncea [65].
Catalytic efficiency of enzyme altered due to excessive of Cd and Zn in soil in Phaseolus
vulgarus [66] and pea plants [67]. Contamination of soil with Zn frequently exceed to that is
required as nutrient for healthy growth of plants caused phytotoxicity in plants. The range 150-
300 mg/kg Zn concentration in polluted soil was estimated [68]. Plant’s metabolic function
was spoiled and retarded growth in Senescence because of high concentration of Zn. The
growth of root and Stem limited due to zinc toxicity [69]. Chlorosis in younger leaves due to
Zn toxicity can be extended to older leaves if prolong exposure to high concentration of Zn
[70]. Chlorosis in younger leaves may be due to deficiency of Fe because hydrated Zn+2 and
Fe have same radii [71]. Mn and Cu deficiency in Stems was due to Zn toxicity. Deficiency of
Zn and Cu micronutrients because of hindered transport of these micronutrients from roots to
Stems, ascribed on the fact that Mn and Fe in plants sprung in Zn rich media have high
concentration of Fe and Mn in roots and Stems. The appearance of purple and red color in
leaves because of deficiency of P was due to Zn toxicity [72]. Reduction in germination,
chlorophyll (Cyamopsis tetragonoloba) and amino acids because excess of Zn in soil. Reduced
plant’s growth, chlorophyll, photosynthesis in pea (Pisum sativum), nutrient contents and
photosynthetic energy conversion in rye grass (Lolium perenne).
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LITERATURE SURVEY
LITERATURE SURVEY
2 LITERATURE SURVEY
2.1. Phytoaccumulation to identify uptake of cadmium (Cd) and Lead (Pb)
Rashid; Faisal, M; Azeem, M; Beenish, Aniqa (2014) used Lactuca sativa as model plant for
phytoaccumulation to identify uptake of cadmium (Cd) and Lead (Pb) and their subsequent
accumulation in edible tissue of plant. Increase in bioavailability increase root to Stem uptake
which effects negatively plant biomass. Cadmium transportation from root to Stem was high
which show its strong affinity forming complex with enzyme while Pb accumulation in roots
was higher than Stems. Post-harvest availability of metals after three hours spiking showed
persistence of metals and slow degradation. That metal absorbed on soil surfaces was more
bioavailable for plant uptake. Cd bioavailability and absorption was higher than other metals.
These trends helped in better understanding of limiting growth effect of metals on plant while
changes in plant physiology and cationic balance in soil needs analysis [73].
2.2. Heavy Metal Contamination of Pinus pinaster, P. sytvestris Quercus robur,

and Q. rotundifolium
Ingrid, Peter; Giuseppe, B; Vojtech, Joao (2016) Dirner heavy metal contamination of Pinus
pinaster, P. sytvestris Quercus robur, and Q. rotundifolium was studied in four abandoned
historic Cu deposits from Italy. The highest Cu and Mn contents in anthropogenic soil were
described in Libiola and Caporciano whereas the highest Pb, Zn, As, and Sb contents in Sao
Dominngos. The anthropogenic soil in L’ubietova was shown the highest Co contents. The
area of Sao Domingos was the most acidified. Bioavailability of the heavy metals was generally
independent of pH values. The high Ca and Mg contents in soil can block the transport of heavy
metals to the plant tissues. The bio concentration factor values of all plants taxa in all deposits
indicate predominant strategy of excluders. Only Ag showed excellence bio concentration
ability. In L’ubieyova, Pius sylvestris have a strategy as an accumulator of Pb (2.43 mg/L) and
Zn (2.49 mg/L), Pinuspinastero of Mn (4.97 mg/L), Cd (1.85 mg/L) and Co (5.52 mg/L). the
predominantly low translocation factor values indicated that in most cases the heavy metals
were accumulated in roots, only in a few rare cases migrated to Stems [74].
2.3. Effect of Cadmium, Chromium, Copper, Nickel and Zinc on Alfalfa plant
(Medicago sativa)
C. Aydinalp and S. Marinova (2009) studied the effects of cadmium, chromium, copper nickel
and zinc on Alfalfa plant (Medicago sativa). The solution of different strengths 0 ppm, 5 ppm
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10 ppm and 40 ppm were applied. the seed germination and plant growth were greatly affected
by cadmium and chromium at 10 ppm and by copper and nickel at 20 ppm [75].
2.4. Stress of Heavy Metals Cd, Ni and Pb on cassava in a greenhouse
Ano, A. O, Eke-Okoro, O. N. and Egesi, C. N. (2013) studied the stress of heavy metals Cd,
Ni and Pb on cassava in a greenhouse. The solutions of different strengths (0, 100, 200, and
300 ppm) were used. Heavy metals Cd, Ni and Pb reduced cassava sprouting at four weeks
after planting (WAP) the toxicity level was of following order Ni > Cd > Pb [76].
2.5. Plant Growth Inhibition and Accumulation of Cu, Pb, and Zn by Brassica
juncea L. Czem
Nguyen Xuan (2014) experiment was done to study plant growth inhibition and accumulation
of Cu, Pb, and Zn by Brassica juncea L. Czem. The solutions of different Strength were applied
in pot experiment. The plant growth and yield of Brassica Juncea was decreased significantly
in case of Pb followed by Cu and Zn. At 100 ppm Pb declined yield by 51%, Cu reduced yield
by 29% and effect of Zn at 500 ppm reduced yield by 38% [77].
2.6. Toxic Effect of Heavy Metals in Maize (Zea mays)
Abdul Ghani (2013) studied the toxic effect of heavy metals in Maize (Zea mays) the different
metals were found to have different toxic effect in maize. The phytotoxic effect of Cd was more
severe than Chromium (Cr). Soil contaminated by heavy metals caused to substantial losses of
dry matter and seed yield in maize plant [78].
2.7. Effect of Zinc toxicity on Plant Growth and Metabolism

Gyana Rout, Premananda Das (2003) studied the effect of Zinc toxicity on Plant Growth and
Metabolism. Zinc is micronutrient for plant but caused zinc toxicity at higher concentration of
zinc. Zinc toxicity was noticed in this research with inhibition of growth and decreased in
biomass production. The toxic effect of zinc might be possible due to interaction of toxic ions
i.e., Cd, Cu, Pb with Ca, Mg, Fe, and P. zinc toxicity was certainly due to competition for
uptake [79]
2.8. Tolerance level of Pollutants Elements (Co, Ni, Cd, Cr and Pb)
Rajeev Gopal and Neena Khurana (2011) studied the tolerance level of pollutants elements
(Co, Ni, Cd, Cr and Pb). The experiment was performed to study visible foliar symptoms, tissue
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concentration and some biochemical parameters in sunflower plants. The sunflower plants
were grown at 0.25 mM for each metal. The results showed that Cd had more severe effects
like visual toxicity and maximum oxidative damage was seen by accumulation of
ThioBarbituric Acid Reactive substances (TBARS) and reduced antioxidant capacity than the
sunflower plants exposed to the Co, Cd, Cr, and Pb [80].
2.9. Sunflower Plant to Remediate of Lead and Cadmium contaminated in the

Soil
Sewalem, Naseer; Elfeky, Soad; El-Shintinawy, Fatima (2014) performed an experiment on

sunflower plant to remediate of Lead and Cadmium contaminated in the soil. The soil was
treated with (0, 5, 10, 20, 40, and 80 mM CdCl₂ and Pb(NO₃) ₂. After one week, the seedling
was removed and washed with tap water. The fresh roots and Stems were investigated to study
the effects of both heavy metals. The results were shown that total absorbed amount of Cd
(88.84%) accumulated in roots and total absorbed amount of Pb (71.39%) translocated to
Stems. Finally concluded that sunflower plant remediated Cd from the soil through Phyto-
stabilization and remediated Pb through phytoextraction. The trace amount was also
accumulated in the seeds suggested that sunflower plants were saved to remediate the Cd and
Pb from the contaminated soil [81].
2.10. Heavy Metals Effect on Plant
Asati, Ambika; Pichhode, Mohnish; Nikhil, Kumar (2016) studied heavy metals effect on plant.
The metals like cadmium, copper, lead, chromium, manganese, iron, mercury became a part of
soil due to geologic and anthropogenic activities. The results showed that the soil contaminated
by heavy metals, the growth and photosynthetic pigments of plants affected by these metals
and to be removed by hyperaccumulator plant through phytoremediation [82].
2.11. Effects of Cd, Ni, Cr, Zn, Mn, Co and Cu Heavy Metals on Vegetable
Farzana Bashir (2009) studied the effects of Cd, Ni, Cr, Zn, Mn, Co and Cu heavy metals on
vegetable sample that was collected from six vegetable fields near Lahore. The soil was also
collected to see the irrigation of land with the untreated sewage effluents. One controlled
experiment was also performed in site of fresh canal water irrigation for comparative study.
Following the extraction procedure, the extract was digested and analyzed by Atomic
Absorption Spectrophotometer. The results showed that all the metals were found at its peak
(450%) in the edible part in controlled as well as waste water irrigated [83].
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2.12. Effect of Heavy Metals from Contaminated Soil on Growth in

Pelargonium hortorum
Orrono, DI and Lavado, RS (2009) studied the effect of heavy metals from contaminated soil
on growth in pelargonium hortorum. Plants were grown on Cd, Cr, Cu, Pb, Ni, Zn enriched
soils. The biomass reduction was significantly noticed in pelargonium in effect of these
enriched heavy metal soil. The heavy metal concentration was high in roots than Stems. The
rates were different pattern of uptake of these heavy metals in roots and Stem [84].
2.13. Vegetables Growing in Agriculture Field Near Peshawar and Hayatabad

irrigated with mixed industrial effluents
Noor-ul Amin and Tauseef Ahmed (2016) performed an experiment on vegetables growing in
agriculture field near Peshawar and Hayatabad irrigated with mixed industrial effluents to study
accumulation and bio-concentration of seven heavy metals quantitatively measured by Atomic
Absorption Spectroscopy. The concentration of Co, Cu, Fe, Pb, Cr, Mn, Zn and Ni were found
0.009-0.085 mg/L, 0.044-0.504 mg/L, 0.243-7.737 mg/L, 0.496-0.474 mg/L, 0.005-0.033 mg/L,
0.019-2.019 mg/L, 0.045-0.703 mg/L and 0.017-0.108 mg/L respectively in the edible parts of
different vegetables that showed a health concern. Iron (Fe) was seen to be found in highest
percentage (P<0.05) as compared to other metals and concluded that cultivation of vegetable crops
in the area polluted with industrial effluents is not recommended [85].
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CHAPTER 3
INSTRUMENTATION
INSTRUMENTATION
3 INSTRUMENTATION
3.1 Atomic Absorption Spectroscopy
Atomic Absorption Spectroscopy is a common quantitative analytical technique that is used to
measures the concentration of elements in a sample. Atomic absorption spectroscopy is a very
simple, reliable and sensitive technique that can measure down to parts per billion of a gram
(µgdm-3) in a sample. It is mostly used to detect metal, sand, metalloids. It can analyze
approximately 62 elements in a sample.
3.1.1 Introduction
The modern first Atomic Absorption Spectrophotometer was invented in 1954 by CSIRO team
or Australian scientists Alan Walsh.
Figure 1: First Atomic Absorption Spectrophotometer.
In AAS, the sample is first atomized and for this purpose flame is mostly used but
sometimes graphite furnace can also be used.
Atomization of liquid into gas takes place in following steps.
• Desolation: In which liquid sample is evaporated and dried sample left

behind.
• Vaporization: The solid that left behind is vaporized and converted into gas.
• Volatilization: The vaporized samples are broken down into individual atoms.
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3.1.2 Uses
Atomic Absorption Spectroscopy is used in wide areas of chemistry for different purposes.
3.1.2.1 Clinical Analysis

AAS is used in analysis of body fluids such as bold serum, urine, saliva etc.
3.1.2.2 Pharmaceutical Analysis

AAS is used in measurement and detection of quantities of elements and enzymes in drugs.
3.1.2.3 Environmental Analysis

AAS is used for environmental monitoring, for detection of concentration of different metals
and other elements in water petrol and fruits.
3.1.2.4 Rocks Analysis

Level of metals in rocks can be determined by using AAS. For example, we can estimate
concentrate of Gold in rock.
3.1.2.5 Industrial Analysis

AAS is used for checking of raw materials used in manufacturing processes. It is used to detect
level of the major elements present and concentration of toxic elements.
Figure 2: Varian Flame AAS model AA240.
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3.1.3 Working
Different elements present in samples absorb of different radiations of characteristics wave
lengths from source of light. After absorption of radiations atoms show transitions by excitation
of valence electrons from low energy to high energy orbitals. There excited atoms return to
ground state by emitting same amount of energy. The amount of energy is specific for
transition. The sample is atomized and converted into free gaseous atoms. The excited beam of
atoms can pass through vaporized sample. Some radiations are absorbed by the sample.
Amount absorbed radiations depends upon number of vaporized atoms. In AAS, Beer-
Lambert’s law is followed.
According to this law there is a linear relationship between absorbance and

concentration of the sample and path length of the cell.
A= €lc
• A = Absorbance
• € = Molar absorptivity (or absorption coefficient)
• l = Path length
• c = Concentration
There is a direct relationship between transmittance and analytic concentration while path
length remains constant. We can find unknown analyte concentration by drawing calibration
cure between absorbance a concentration.
Figure 3: Lambert-Beer law.
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3.1.4 Components of AAS

Atomic Absorption Spectroscopy needs three basic components for proper functioning.
1. Radiation source
2. Atomizer
3. Flame
4. Sample introduction system
5. Monochromator
6. Detector
Figure 4: Schematic diagram of AAS.
3.1.4.1 Radiation source

There are two types of radiation sources.
• Line source
• Continuum light sources
3.1.4.1.1 Line source

These are classical sources and proposed by Alan Walsh first time. In this source, high
resolution accompanied by narrow line of emission spectrum of analyte than absorption
spectrum line.
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3.1.4.1.2 Hollow cathode lamp

Hollow cathode lamp is light source which emit light of high intensity and narrow line wave
length of element to be determined.
3.1.4.1.3 Construction
Hollow cathode lamp consists of a glass cylinder which is usually filled with an insert gas of
very low pressure such as Argon or Neon. It consists of two electrodes cathode and anode.
Figure 5: Hollow Cathode Lamp.
Anode is made of tungsten while cathode is made of that metal to be determined. Specific lamp
is used to measure an element. The emission line of the lamp corresponds with the absorption
wave length of the analyte.
3.1.4.1.4 Sputtering
Inert gas is ionized by applying potential difference between electrodes. Positively charged gas
ions strike with oppositely charged cathode and release metal atoms.
3.1.4.1.5 Excitation
Sputtered metal atoms are excited due to bombardment with the ionized gas.
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3.1.4.1.6 Emission
When metal atoms come back to ground state radiations of specific wavelength are emitted.
Hollow cathode lamp has life time shelf life. Lamp life is deceased by increasing current.
Increase in current cans also self-absorption broadening.
Figure 6: Sputtering and Emission processes in Hollow Cathode lamp.
3.1.4.1.7 Electrodes discharge lamp

To overcome the problem of reduction in life of the lamp and low intensity electrodeless
discharge lamps are made. Electrodeless discharge lamps are commonly available for Sb, As,
Bi, Cd, Pb, Hg, K, Rb, Sn, Te. It consists of a bulb made of quartz and filled it with an inert
gas containing salt of metal to be analyzed. It has advantage of low detection limit.
Figure 7: Electrodeless discharge lamp.
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3.1.4.1.8 Continuum line sources

In continuum lights sources deuterium lamps are used to reduce back ground noise. In this
radiation source only high-resolution Monochromators required for measurements. That’s a
great advantage. Along this advantage this technique also has a disadvantage of need of a
separate lamp for each element during analysis. Continuum light source emit electromagnetic
radiation in the wavelength rage from about 250 to 700 nm.
Figure 8: High resolution continuum AAS with double

monochromator.
3.1.4.2 Atomizers
There are two types of atomizers which are commonly used now a day.
• Flame atomizers
• Electro-thermal atomizers
3.1.4.2.1 Flame Atomizer

Flame Atomic Absorption Spectroscopy is a quantitative method for analysis of elements
present in a compound. This is the oldest method and commonly used flames are of acetylene
and nitrous dioxide.
Liquid samples are aspirated into fine droplets called aerosols and then enter the flame.
In which a light beam from fire can pass through the sample and after absorption decrease in
intensity is calculated. Atomization depends upon temperature of the flame. The temperature
and characteristics of different flame sources are:
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Table 1: Different flames with their maximum temperature.
Different Flame Source Max. Flame Speed (cm/s) Max. Temperature (ͦC)
Air-propane 82 1925
Oxygen acetylene 1130 3060
Air-coal gas 55 4840
Oxygen cyanogens 140 4640
Air-acetylene 160 2300
Figure 9: Flame Atomization.
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3.1.4.2.2 Electro thermal Atomizer

Electro-thermal atomizers were firstly used by Boris V. L’vov. In these atomizers graphite
tubes are used. Dimensions of the graphite tube used in these sources 20-25mm in length and
5-6mm inner diameter. The advantage of tube is they offer very low ohmic resistance and
allowed high current to pass through them.
3.1.4.3 Sample Introduction System

The purpose of the sample introduction system is to introduce sample in the AAS instrument
with high efficiency and reproducibility. Sample must be in liquid state. There are two types
of sample introduction systems.
3.1.4.3.1 Continuous introduction system

Fixed amount of sample is added at once to system which is then atomized.
3.1.4.3.2 Discontinuous introduction system

In discontinuous system sample is added in small batches after regular intervals.
After introduction sample is then aspirated and rate of aspiration is directly proportional
to pressure of gas and inversely proportional to viscosity of sample.
Figure 10: Graphite Tube.
3.1.4.4 Monochromator
Monochromator is a device which separate out as specific wavelength from interfering
wavelengths. It is used in many devices. The basic purpose of its use is to isolate the absorbed
radiation from the background radiations. In Atomic Absorption Spectrophotometer, main
function of Monochromator is separate single resonance peak from the others spectrum line. It
only chooses a single wavelength and allowed it to pass through analyte.
Monochromator consists of following components.
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• Entrance slit: It selects the specific beam of light.

• Diffraction grating: It disperse white light into its components.
• Exit slit: It allows single wavelength to enter.
The dispersion of light controlled by collimator. Diffraction grating disperses the light in
to its seven components. Entrance slit focused single selected beam. At exit slit selected beam
of light can fall on analyte solution.
Figure 11: Monochromator.
Optical system can also be used in monochromator. It is complicated system. In this system
combination of mirrors is used to select a wavelength. The advantage of this system is this
increase the path length inside the system unit.
3.1.4.5 Detector
Detector is a device which converts the light into electrical signals. In AAS, photomultiplier
tube detector is used. Choice of detector depends on sensitivity and selectivity. An ideal
detector has high sensitivity and offers low noise and fast response times.
Principle of Photomultiplier detector is the emission of large number of electrons on

exposure to radiations. Photomultiplier detector consists of cathode and several dynodes
arranged in series. When a beam of light strikes to cathode, the electrons are emitted. These
emitted electrons are accelerated to 90 V potential difference and then strikes to series of
dynodes and large number of electrons emitted by secondary emission process. These light
signals simplified and collected at anode where measurement takes place.
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Figure 12: Emission process in Detector.
These are very sensitive to UV-VIS radiations. Photomultipliers tubes are extremely
sensitive to sunlight. Cathode surface can be permanently damaged.
3.1.4.5.1 Sensitivity and Specificity

Sensitivity of an instrument is defined as ability of instrument to detect any change in
concentration in the sample.
Detection limit of instrument is the minimum concentration of analyte that can be

detected by instrument. AAS has very low detection limits. The detection limit in mg/L for Cu,
Cr, Cd, Pb, and mercury are 0.02 mg/L, 0.075 mg/L, 0.01 mg/L, 0.02 mg/L respectively.
3.1.4.5.2 Back ground Absorption

Sometime molecules other than the molecules to be determined may scatter the radiations of
light or they may absorb some quantity of radiations. These are the molecules which cannot be
separated from analytic molecules. They can cause back ground absorption and may affect their
salts. To avoid this problem two lights sources are used. One is hollow cathode lamp and second
is deuterium lamp.
3.1.5 Sample Preparation

Initial state of sample is not important. Firstly, sample weighed and a dilute it with suitable
solvent. Liquid samples such as blood or urine analyzed after dilution. Reference solution was
made for calibration of sample reference and sample should be in liquid state.
3.1.6 Instrumental Setup

Following are the steps of analysis by using AAS:
• Connect the instrument to power supply, computer device and ignition flame.
• Firstly, check the pressure of Acetylene cylinder. It should not be lower than 400psi.
• Open the new file, named it according to the metal to be analyzed.
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• The instrument loaded auto profiles setting for selected element and synchronize line
source and flame.
• Introduce the standards of metal of interest (1 mg/L, 5 mg/L, 15 mg/L) and check the
straight line following the Beer-Lambert Law.
• Introduce the sample.
• After detection, note the readings from read out device and calculate the absorption
according to requirement
• Perform analysis thrice for more accurate measurements.
3.2 Cold Vapor Technique

This procedure is strictly confined for the determination of mercury (Hg). This element is stable
and has no appreciable vapor pressure at room temperature. Its gaseous atoms exist without
need for any special treatment. In the method for determine mercury compounds the procedure
consists of reduction of Hg+2 compounds with SnCl2 to form elemental Hg.
Hg+2 + Sn+2 ͇ Hg0 + Sn+4
3.2.1 Instrumentation
The basic components used for cod vapor technique are:
• Hollow Cathode Lamp

• Atomizer
• Monochromator
• Photocell
• Recorder/Readout device
3.2.1.1 Hollow Cathode Lamp

Hollow cathode lamp is specific and called mercury hollow cathode lamp because cathode is
made up of Hg which generates radiations of specific wavelength.
3.2.1.2 Atomizer
The atomizer consists of a long path cell tube which is 12 inches with an entrance and exit slit.
This tube is connected to the flask which is called ERLENMYER flask. A passage for nitrogen
gas is present in this flask and the reagent is employed through the side tube. The pressure of
nitrogen takes it to the long path cell.
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2ml Hg solution is inserted through the side tube of flask along with SnCl2. Hg+2 ions
is reduced to Hg0. The mercury atoms converted into vapors along with nitrogen and go to the
long path cell. It’s so efficient that it can estimate Hg in ppb as well. The Hg vapors in long
path cell is absorbed by beam which is refracted by monochromator and is recorded as on
recorder at 253.7 nm
Figure 13: Schematic diagram for Cold Vapor Mercury

Technique.
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CHAPTER 4
MATERIALS & METHODS
MATERIALS & METHODS
4 MATERIALS & METHODS

The materials used in this experiment were:
4.1 Plantation
The experiment was started with purchasing of Dahlia’s seed. The seed was purchased from
Pride seed (Seed supplier) M.M. Alam Road, Gulberg Lahore. The soil decomposed organic
matter and coconut peat were bought from Al-Haq Nursey, Kalma chowk Ferozpur road
Lahore. The seedling was initiated in 120 compartments pot tray. The soil and decomposed
was mixed with 3:1 proportion respectively. The soil and decomposed organic matter was
homogeneity mingled in a bath. The pots were filled with 3/4th part of each pot, leaving 1/4th
part left. The dahlia’s seed was spread uniformly and each pot of tray comprised 2 or 3 seed.
The coconut peat (light in weight) was spread over each pot to hold the seed at its place. The
water was showered over the tray 1 meter away from the tray. The tray was covered with
polythene bag. The polythene bag was removed at 11’o clock day time for direct sunlight. The
water was showered for one week and covered again with bag for full night.
Figure 14: Seedling of Dahlia Plant.
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4.2 Sampling
The seedling was emerged from the soil within 7 days. The seedling was transferred to 13
individual’s pots. The individual’s pot was 1.5m long, 0.5m wide from upside and 0.2m wide
from lower side. The pot was filled with soil and dead organic matter mixed with 3:1 proportion
respectively. The individual pot was arranged and properly labelled. The pots were covered
with polythene bag to prevent dahlia plant from frost in winter. The bag was permanently
removed at mid of January (average temperature 20 ͦC). 250ml solution of different strengths
Figure 15: seedling in individual pot.
(50 mg/L, 100 mg/L and 200 mg/L) of Cd, Pb, Hg and 100 mg/L, 300 mg/L and 500 mg/L of
Zn were applied to individual pot. The solution was applied after two weeks for six months.
One controlled experiment was also conducted
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Figure 16: Dahlia Flowering Plant.
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MATERIALS & METHODS
4.3 Sample collection

The leaves, tubers, Stems and flowers from individual’s pots were cut at March 10, 2017. The
Figure 17: Collected samples (Tubers, Stems and Leaves)
leaves, Stems, tubers and flowers were first washed with dil. HCl and then distilled H2O. The
tubers, Stems, leaves and flowers were placed for sun dry for one day, properly labelled and
weighed and placed for 3 weeks in sun for maximum loss water contents and weighed again.
4.4 Sample treatment

Each collected sample after sun dry was treated in the following steps:
4.4.1 Electric oven
The temperature of electric oven was settled at 60 ͦC. The samples were heated uniformly for
36 hours.
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4.4.2 Grinding of sample
Figure 18: Grinded Sample
The samples after heating were grinded gently until each sample was homogenized in
powdered form and ready for analysis.
4.4.3 Weighing of sample
Before weighing of sample, each sample was again heated in electric oven at 60 ͦC for 6 hours
to ensure maximum water loss. The 300 mg of each sample was weighed. Three replicates of
each same sample weighed. Before weighing samples were homogeneity mixed.
4.4.4 Digestion of grinded sample
The weighing sample (300 mg) was digested for metal extraction. Three different acid
digestion methods are:
4.4.4.1 Method 1
Nitric acid and perchloric acid (HNO3-HClO3) are mixed with proportion 2:1 of nitric acid and
perchloric acid respectively
4.4.4.2 Method 2
The only nitric acid is another method for acid digestion.
4.4.4.3 Method 3
Aqua Regia a mixture of nitric acid and hydrochloric acid mingled with proportion 1:3 of HNO3
and HCl respectively.
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Five sample were analyzed by three different acid digestion methods. The most efficient
method that produce good results was Aqua Regia [86]. Freshly prepared 25 ml Aqua Regia
(65% HCl and 37% HNO3) was added in each 300mg sample.
Figure 19: Digested Sample.
4.4.5 Heating of sample
Each same sample mixture was heated over water bath at 95 ͦC for 5-6 hours until contents of
sample mixture were dissolved. After 1 hour standby, the wall of each beaker was rinsed with
10 ml dis. H2O to avoid sample loss.
4.4.6 Dilution of digested sample
To each digested sample, 65 ml dis. H2O was added to make total volume of 100 ml.
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4.4.7 Filtration
To last part of digestion process, each diluted sample was filtered with Whatman filter paper
with
retention of 2-6 micro meter particle size.
Figure 20: Filtration of digested acid.
4.4.8 Sample stored

ͦ
The filtrate collected and stored in 250 ml bottles, lid, properly labelled and stored at 4 C if
necessary.
4.5 Standard Solution

4.5.1 Standard Solution for Lead (Pb)
Salt Lead (ΙΙ) Nitrate, Pb(NO3)2
Molecular weight 331.21 g mol-1
Atomic weight (Pb) 207.2 g
Minimum Assay: 96 %
Maximum limits of impurities:
Chloride 0.05 %
Copper 0.001 %
Iron 0.01 %
48 | P a g e
MATERIALS & METHODS
Cadmium 0.005 %
4.5.1.1 1000 mg/L (Pb) Solution
Molecular Weight of Lead Nitrate
=
Atomic Weight of Lead
331.21
= 1.60 g mol-1
207.2
1.6 g Pb(NO3)2 is dissolved in sufficient amount of distilled water so that total volume of
solution will be one liter to get 1000 mg/L Pb solution or 0.4 g Pb(NO3)2 is dissolved in
sufficient amount of distilled water so that total volume of solution will be 250 mL to get 1000
mg/L Pb solution.
4.5.1.2 Diluted Solution
Standard solutions of different strengths are prepared as:
4.5.1.2.1 1000 mg/L → 500 mg/L
The dilution formula is:
C1V1 = C2V2
1000 V1 = 500 ⨯ 250
V1 = 125 mL
Pipette out 125 mL 1000 mg/L Pb solution in 250 mL measuring flasks and dilute with distilled
water up to the mark so that total volume of solution will be 250 mL. This will be 500 mg/L
Pb solution.
4.5.1.2.2 500 mg/L → 100 mg/L
C1V1 = C2V2
500 V1 = 100 ⨯ 250
V1 = 50 mL
water up to the mark so that total volume of solution will be 250 mL. This will be 100 mg/L
Pb solution.
4.5.1.2.3 100 mg/L → 10 mg/L
C1V1 = C2V2
100 V1 = 10 ⨯ 250
V1 = 25 mL
water up to the mark so that total volume of solution will be 250 mL. This will be 10 mg/L Pb
solution.
49 | P a g e
MATERIALS & METHODS
4.5.1.2.4 10 mg/L → 1 mg/L

C1V1 = C2V2
10 V1 = 1 ⨯ 250
V1 = 25 mL
water up to the mark so that total volume of solution will be 250 mL. This will be 1 mg/L Pb
solution.
4.5.2 Standard Solution for Cadmium (Cd)
Salt Cadmium (ΙΙ) Chloride, CdCl2
Atomic weight (Cd) 112.41 g
Minimum Assay: 95 %
Chloride 0.3 %
Lead 0.002 %
Iron 0.01 %
Arsenic 0.002 %
4.5.2.1 1000 mg/L (Cd) Solution
Molecular Weight of Cadmium Chloride
=
Atomic Weight of Cadmium
183.32
= 1.63 g mol-1
112.41
1.63 g CdCl2 is dissolved in sufficient amount of distilled water so that total volume of solution
will be one liter to get 1000 mg/L Cd solution or 0.41 g CdCl2 is dissolved in sufficient amount
of distilled water so that total volume of solution will be 250 mL to get 1000 mg/L Cd solution.
4.5.3 Standard Solution for Mercury (Hg)
Salt Mercury (ΙΙ) Nitrate, Hg(NO3)2
Atomic weight (Hg) 200.59 g
Minimum Assay: 94 %
Chloride 0.02 %
Lead 0.01 %
50 | P a g e
MATERIALS & METHODS
Iron 0.03 %
Arsenic 0.005 %
4.5.3.1 1000 mg/L (Hg) Solution
Molecular Weight of Mercury Nitrate
=
Atomic Weight of Mercury
342.62
= 1.71 g mol-1
200.59
1.71 g Hg(NO3)2 is dissolved in sufficient amount of distilled water so that total volume of
solution will be one liter to get 1000 mg/L Hg solution or 0.43 g Hg(NO3)2 is dissolved in
mg/L Hg solution.
4.5.4 Standard Solution for Zinc (Zn)
Salt Zinc (ΙΙ) Sulphate monohydrate, ZnSO4.H2O
Atomic weight (Hg) 65.38 g
Minimum Assay: 97 %
Chloride 0.02 %
Iron 0.05 %
Arsenic 0.001 %
4.5.4.1 1000 mg/L (Zn) Solution
Molecular Weight of Zinc Sulphate monohydrate
=
Atomic Weight of Zinc
179.45
= 2.24 g mol-1
65.38
2.24 g ZnSO4.H2O is dissolved in sufficient amount of distilled water so that total volume of
solution will be one liter to get 1000 mg/L Zn solution or 0.69 g ZnSO4.H2O is dissolved in
mg/L Zn solution.
51 | P a g e
MATERIALS & METHODS
4.6 Atomic Absorption Spectrophotometer

The analysis was run in Varian Flame AAS model AA240 at Chemistry Department UET
Lahore. Switch on exhaust, computer, air compressor, power of AAS. The worksheet was
opened in computer. The file was generated and named. Flame was set (select the element for
analysis) in Add method. In Edit method, the following setting was done:
Type/mode → Manual
Measurement → Integration
Smoothing 3.00 point
Read delay 1 second
Standard 1 second
Sample run → one time
The position of lamp was confirmed in lamp setting. The standard values were set (1
mg/L, 5 mg/L, 10 mg/L and 15 mg/L) for calibration curve. The optimization was done as ask
the confirmation of metal, lamp position, Rescale, instrumental zero, optimize lamp and signal.
The flame was initiated by pressing power button of AAS. The acetylene flame pressure should
be 5-10psi. The nebulizer was cleaned by dipping in distilled H2O until blue color appear in
the flame. Standard solutions was run for calibration curve when all setting was done as
standard 1, 2 and 3. The calibration curve obtained. Each labelled sample was run and
absorbance against each sample was recorded in file. The file was saved and kept record for
further analysis.
After analysis, the instrument was shut down. Turn off lamp, switch off flame, air
compressor and computer. Close the valve of acetylene cylinder.
4.7 Analysis
4.7.1 Atomic Absorption Spectrophotometer
The analysis of each sample (Pb, Cd, Zn) was run in Varian flame atomic absorption
spectrophotometer.
4.7.2 Cold vapor Atomic Absorption Spectrophotometer

Cold vapor AAS was used for the analysis of Hg sample
52 | P a g e
MATERIALS & METHODS
4.8 Transfer factor

The transfer factor of metals from contaminated soil to different parts of dahlia plant was
calculated as:
Stem (metal) concentration (mg/L)
Tranfer Factor =
Tuber (metal)concentration (mg/L)
4.9 Tolerance Index

The evaluation of tolerance index in the metal contaminated environment, Wilkins method was
used.
Plant growth in metal containing environment
Tolerance Index = ×100
Plant growth in metal free environment
53 | P a g e
CHAPTER 5
RESULTS & DISCUSSION
5 RESULTS
5.1 Effect of Lead (Pb)
5.1.1 Absorbance of Lead in different parts of plant
Table 2: Absorbance of Lead (Pb) by different parts of Dahlia plant against different concentration.
Concentration (mg/L) 0 50 100 200
Mean ± SD 0.075±0.004 0.013±0.002
Mean ± SD 0.433±0.002 0.435±0.003 0.234±0.002 0.005±0.002
Mean ± SD 1.775±0.002 1.32±0.004 0.655±0.004 0.017±0.003
Mean ± SD 2.387±0.005 1.366±0.005 0.715±0.004 0.107±0.003

Replicates
Replicates
Replicates
Replicates
(mg/L)
(mg/L)
(mg/L)
(mg/L)
Plant's part
0.07 0.43 1.77 2.38

Tubers 0.07 0.43 1.78 2.39
0.08 0.44 1.78 2.39
Absorbance
0.01 0.43 1.32 1.36

Stems 0.01 0.44 1.32 1.37
0.02 0.44 1.32 1.37
BDL 0.23 0.65 0.71
BDL
Leaves BDL 0.23 0.65 0.72

BDL 0.24 0.66 0.72
BDL 0 0.01 0.1
BDL
Flowers BDL 0.01 0.02 0.11

BDL 0.01 0.02 0.11
55 | P a g e
Absorption of Pb was estimated in all parts of plant body (Pb having high tolerance index).
The estimated absorbed amount in 200 mg/L Pb in tubers, Stems, leaves and flowers were
2.387 mg/L, 1.366 mg/L, 0.715 mg/L and 0.107 mg/L respectively. Total absorbed
concentration in 0 mg/L Pb, 50 mg/L Pb, 100 mg/L Pb and 200 mg/L Pb by dahlia plant were
0.088 mg/L, 1.107 mg/L, 3.767 mg/L and 4.575 mg/L respectively. The plant’s height although
was affected by increased concentration of Pb but not too much as in case of Cd and Hg. Similar
result was found in research paper (Mostafa Lamhamdi, Ouiam EI Galiou, Ahmed Bakrim)
[87] studied the effect of different concentration of Pb on spinach and wheat. The transportation
of Pb from roots to leaves was increased with increased concentration of Pb. Lead accumulation
in dahlia plant also caused deficiency of P, K, Mn and Cl. Tip burn, some chlorotic spots,
interveinal yellowish spots and edges burn were discerned.
Figure 21: Effect of Pb on Dahlia Plant.
56 | P a g e
0.075 . 0.4350.433
0.08 0.45
0.07 0.4
0.06 0.35
Absorbance
Absorbance
0.3 0.234
0.05
0.25
0.04
0.2
0.03
0.15
0.02 0.013
0.1
0.01 0 0 0.05 0.005
0 0
1 1
Concentration (mg/L) Concentration (mg/L)
Flowers Leaves Shoots Tubers Flowers Leaves Shoots Tubers
Figure 23:Absorbance comparison 0 mg/L Pb. Figure 22: Absorbance comparison 50 mg/L Pb.
2.387
2.5
2 1.775
2
Absorbance
1.32 1.366
1.5 1.5
Absorbance
1 0.655 1 0.715
0.5 0.5 0.107

0.017
0 0
1 1
Figure 25: Absorbance comparison 100 mg/L Pb. Figure 24: Absorbance comparison 200 mg/L Pb.
57 | P a g e
The maximum absorption of heavy metal Lead (Pb) was seen in tubers except 50 mg/L Pb
where the maximum absorption was observed in Stems although very close to absorption of
Lead in tubers.
In controlled experiment 0 mg/L Pb, the absorption of Lead (Pb) in tubers and Stems
were 0.075 and 0.013 respectively but below detection level to concentration of standard
solution in leaves and flowers. The absorption of Pb was increased in 50 mg/L Pb and 100
mg/L Pb. The accumulation of Lead (Pb) in tubers, Stems, leaves and flowers in 50 mg/L Pb
were 0.433 mg/L, 0.435 mg/L, 0.234 mg/L and 0.005 mg/L respectively. Similar estimated
concentration of Lead (Pb) in 100 mg/L Pb in tubers, Stems, leaves and flowers were 1.775
mg/L, 1.32 mg/L, 0.655 mg/L and 0.017 mg/L respectively. In 200 mg/L Pb, the maximum
absorption of Lead in tubers, Stems, leaves and flowers were 2.387 mg/L, 1.366 mg/L, 0.715
mg/L and 0.107 mg/L respectively.
In shortly, Lead was transported to all parts of plant body (high tolerance index) but
absorption was linearly increased with increased concentration of Lead.
58 | P a g e
2.5
Absorbance
1.5
0 ppm
50 ppm
1
100 ppm
200 ppm
0.5
0
FLOWERS LEAVES SHOOTS TUBERS
Plants's parts
Figure 26: Comparative relation between absorption of Pb in tubers, Stems, leaves & flowers.
The maximum absorption in all four strengths was observed in tubers and minimum absorption
was in flowers. Descending order of Pb accumulated in different parts are: Flowers ˂ Leaves
˂ Stems ˂ Tubers except 50 mg/L Pb.
In conclusion, lead (Pb) accumulation was incredibly increased with increased

concentration of Lead. In case of flower no lead accumulation in 0 mg/L Pb, 50 mg/L Pb but
in 100mg/L Pb and 200mg/L Pb trace amount of lead were absorbed by flowers. Total Pb
contents accumulated in all parts of plant in 0 mg/L Pb, 50 mg/L Pb, 100 mg/L Pb and 200
mg/L Pb were 0.088 mg/L, 1.107 mg/L, 3.767 mg/L and 4.575 mg/L respectively.
59 | P a g e
5.2 Statistical Analysis Lead

5.2.1 Student T test for Lead
Table 3:Student T test statistical analysis of different parts of Dahlia plant against different concentration of Pb.
Tabulated value
Accepted value
Concentration
Hₒ rejection
H1 rejection
Mean ± SD
Plant parts
(mg/L)
(mg/L)
(t ˂ tv)
(t ˃ tv)
t test
(tv)
(μ)
Tubers 0.075±0.004 0.010 4.348 4.303 ˃4.303
Stems 0.013±0.002 0.005 4.303 4.303 ˂4.303
0
Leaves BDL
Flowers BDL
Tubers 0.433±0.002 0.005 4.479 4.303 ˃4.303
Stems 0.435±0.003 0.007 3.795 4.303 ˂4.303
50
Leaves 0.234±0.002 0.004 3.286 4.303 ˂4.303
Flowers 0.005±0.002 0.005 4.303 4.303 ˂4.303
Tubers 1.775±0.002 0.004 3.286 4.303 ˂4.303
Stems 1.32±0.004 0.009 3.879 4.303 ˂4.303
100
Leaves 0.655±0.004 0.009 3.879 4.303 ˂4.303
Flowers 0.017±0.003 0.008 4.382 4.303 ˃4.303
Tubers 2.387±0.005 0.013 4.332 4.303 ˃4.303
Stems 1.366±0.005 0.013 4.332 4.303 ˃4.303
200
Leaves 0.715±0.004 0.009 4.073 4.303 ˂4.303
Flowers 0.107±0.003 0.008 4.382 4.303 ˃4.303
Two methods, one is accepted method (calculated by confidence limit calculation) and other is
test method. A series of replicate measurements on a single sample using two methods can be
done if accepted value is not available
5.2.1.1 Hₒ or Null hypothesis

If t ˃ tv then the estimated heavy metal absorption by plant, the error in the experiment is not
occurred by chance or happened determinantal error. The determinate error may be
instrumental AAS e.g., confirmation of metal, lamp position, lamp obstruction, Rescale,
instrumental zero, optimize lamp and signal. The determinate error may quite possible due to
inaccurate sample preparation, sample grinding(homogenization), sample weighing and error
in acid digestion process.
60 | P a g e
5.2.1.2 H1 hypothesis
If t ˂ tv then the error in the experiment is occurred by chance. Error is not determinantal. The
indeterminate error may be present but not large enough to reject H1 hypothesis or test
performed.
If t ˃ tv H1 hypothesis may or may not be rejected but the value viewed as suspected
value. The rejection of suspected value can be tested by Q test. If Q ˂ Q95 even t ˃ tv (suspected
value) then the test is not rejected, error in the experiment may be occurred indeterminate.
5.2.1.3 Method for calculation of Student T test:

Student T test can be calculated by formula
√𝑁
±t = (x̅ − μ )
𝑠
Here
̅
X 𝑖𝑠 𝑚𝑒𝑎𝑛
μ is accepted/ true/listed value
s is standard deviation
Accepted value can be drawn from the formula:
𝑡𝑠
μ = x̅ ±
√𝑁
Here
t is statistical factor
N is degree of freedom or Number of measurement
‘t’ can be drawn by number of degree of freedom (N-1) and desired confidence limit
required.
5.2.1.3.1 Example:
For example, replicate measurements for 0 mg/L tubers were:
61 | P a g e
Replicate measurements
0.071
0.074
0.079
5.2.1.3.2 Calculation of mean:
∑ Xi
x̅ =
N
0.224
x̅ = = 0.075
3
5.2.1.3.3 Calculation of Standard Deviation:
∑(𝑋𝑖 − x̅)2
s=√
𝑁−1
0.00003
s=√
2
s = ± 0.004
5.2.1.3.4 Calculation of True value:

𝑡𝑠
μ = x̅ ±
√𝑁
‘t’ is statistical factor and can be drawn from tabulated table and depend on number of degree
of freedom and confidence limit or probability required e.g., 95% probability.
For three replicate measurements= N-1 = 3-1 = 2. ‘t’ statistical factor 95% confidence
limit and N-1 = 2, the ‘t’ value from the tabulated table is 4.303.
4.303 ⨯0.004
μ = 0.075 ±
√3
0.017
μ = 0.075 ±
1.732
μ = 0.075 ± 0.01
62 | P a g e
The range 0.065 to 0.085 and 95% confidence that true value lies within this range.
5.2.1.3.5 Calculation of Student T test:
√𝑁
±t = (x̅ − μ )
𝑠
√3
±t = (0.075 − 0.065)
0.004
1.732
±t = (0.01)
0.004
±t = 0.01 ⨯ 433
±t = +4.348
Since t ˃ tv or ‘t’ value exceeded the tabulated value tv, either it is rejected or not? Suspected
value can have tested by Q test (rejection of a result).
In 50 mg/L Pb tubers, the t value calculated by replicate measurements was 4.479

greater than tabulated value 4.303. Similarly, 0 mg/L Pb tubers and 100 mg/L Pb flowers t
values were 4.348 and 4.382 respectively & 200 mg/L Pb tubers, Stems and flowers t values
were 4.332, 4.332 and 4.382 respectively. All these t values are greater than tabulated value
(tv) at confidence limit 95%. The 95% probability the error may be due to determinantal error
and is not occurred by chance.
63 | P a g e
5.2.2 Q test rejection of a result for Lead

Table 4: Q test Rejection test of suspected values of different parts of plant of various concentration of Pb.
Rejection Quotient at
Ranges of replicates
Difference between
Difference between
largest to nearest
largest value and
probability 95%
neighbor value
Concentration
smallest value
Hₒ rejection
Plant parts
(mg/L)
Q˂Q95
(t ˃ tv)
(mg/L)
(Q95)
0 Tubers ˃4.303 0.071 0.074 0.079 0.005 0.008 0.97 0.63
50 Tubers ˃4.303 0.431 0.432 0.435 0.003 0.004 0.97 0.75
100 Flowers ˃4.303 0.014 0.016 0.02 0.004 0.006 0.97 0.67
Tubers ˃4.303 2.382 2.388 2.392 0.004 0.01 0.97 0.40
200 Stems ˃4.303 1.361 1.367 1.371 0.004 0.01 0.97 0.40
Flowers ˃4.303 0.104 0.106 0.11 0.004 0.006 0.97 0.67
The suspected results can be depicted as accidental error. The suspected value increases the
standard deviation and variance which alter mean. Q test is one of all the test, range within
significant observation fall is established if the range is too small then the good data can be
rejected but if the range is too large the retention of erroneous measurements is maximum and
suspected results can be rejected if Q value is equal to or greater than tabulated value.
Q test is the ratio between the difference between the suspected number and its nearest
neighbor (a) number to the difference between largest number and highest number.
𝑎
Q=
𝑤
64 | P a g e
5.2.2.1 Method for calculation of Q test

Replicate measurements
0.071
0.074
0.079
Difference between largest value and nearest neighbor value = a = 0.005
Difference between largest value and smallest value= w = 0.008
𝑎
Q=
𝑤
0.005
Q=
0.008
Q = 0.63
For number of degree of freedom (N-1) = 3-1 = 2 tabulated value at 95% probability
(Q95) is 0.97.
So Q95 = 0.97
And Q ˂ Q95
Results obtained in the estimation of lead in 0 mg/L Pb tuber is not rejected because Q
value is not exceeded to the Q95 and 95% probability or confidence that result values are within
range and are not found outlier of this range.
0 mg/L Pb tubers and 50 mg/L Pb tubers, the suspected values were lie within the range
in the Q test (Q ˂ Q95). Q values were 0.63 and 0.75 respectively. Similarly, 100 mg/L Pb
flowers, Q value was 0.67 and 200 mg/L Pb tubers, Stems and flowers Q values were 0.40,
0.40 and 0.67 respectively. The suspected values are less than the tabulated value (Q ˂ Q95)
consequently 95% probability that suspected values are not outlier of the range so results are
not rejected.
65 | P a g e
5.3 Effect of Lead on physical parameters

Table 5: Different physical parameters measurement against different concentration of Lead.
Effect of Pb on Physical parameters

Parameters 0 mg/L 50 mg/L 100 mg/L 200 mg/L
Days to Flower 151.28 99.61 96.24 84.39
Blooming Period (Days) 39.88 36.79 24.09 20.68
Flower Size (square/cm) 68.82 63.36 59.88 58.5
Plant Height (cm) 93.5 90 80.3 61.4
39.88
151.28 36.79
160 40
140 35
Blooming Period
120 30
Days to Flower
99.61 96.24 24.09

100 84.39 25 20.68
(Days)
80 20
60 15
40 10
20 5
0 0
0 50 100 200 0 50 100 200
Figure 28: Days to Flowers comparison (Pb). Figure 27: Blooming Period comparison (Pb).
100 93.5 90
70 68.82
80.3
68 80
66 61.4
Plant's Height
63.36
Flower's Size
64 60
(sq./cm)
(cm)
62 59.88
60 58.5 40
58
56 20
54
52 0
0 50 100 200 0 50 100 200
Figure 30: Size of Flower comparison (Pb). Figure 29: Height of Plant (Pb).
66 | P a g e
The effect of different concentration of lead (Pb) on different parameters like Days to flower,
Blooming period, Flower size and plant height.
In 0 mg/L Pb, the Dahlia plant was taken 151.28 days to flower, 38.88 days gap between
first and last flower opening. The size of flower was ideal 68.82 square/cm and plant height
was 93.5 cm but in each strength of 50 mg/L Pb, 100mg/L Pb and 200 mg/L Pb lesser number
of days was taken to days to flower are 99.61, 96.24 and 84.39 respectively. The flower
bloomed earlier than normal time. The size of flower and plant’s height was declined with
increased concentration of lead. The plant’s height in 50 mg/L Pb, 100 mg/L Pb and 200 mg/L
Pb were 90cm, 80.3cm and 61.4cm respectively. Similar flower’s size measured in 50 mg/L
Pb, 100 mg/L Pb and 200 mg/L Pb were 63.36 square/cm, 59.88 square/cm and 58.5 square/cm
respectively
In the end, the increased concentration of lead greatly affected the flower size, plant
height and lesser number of days were taken for flowering and blooming.
67 | P a g e
5.4 Effect of Cadmium (Cd)

5.4.1 Absorbance of Cadmium in different parts of plant
Table 6: Absorbance of Cadmium (Cd) by different parts of Dahlia plant against different concentration.
Mean ± SD 0.015±0.004 0.085±0.004
Mean ± SD 0.203±0.004 0.13±0.004 0.118±0.005
Mean ± SD 0.418±0.003 0.453±0.004 0.21±0.006
Mean ± SD 1.855±0.005 0.809±0.006 0.276±0.004 0.015±0.004

Replicates
Replicates
Replicates
Replicates
(mg/L)
(mg/L)
(mg/L)
(mg/L)
Plant's part
0.01 0.2 0.41 1.85

Tubers 0.02 0.2 0.42 1.86
0.02 0.21 0.42 1.86
Absorbance
0.08 0.13 0.45 0.8

Stems 0.09 0.13 0.45 0.81
0.09 0.14 0.46 0.81
BDL 0.11 0.2 0.27
BDL
Leaves BDL 0.12 0.21 0.28

BDL 0.12 0.22 0.28
BDL BDL BDL 0
BDL
BDL
BDL
Flowers BDL BDL BDL 0.01

BDL BDL BDL 0.01
68 | P a g e
Cadmium and Mercury was proved to be toxic for dahlia plant. Absorption of Cd was estimated
showed that the transportation of Cd from tubers to aerial part decreased. The maximum
concentration accumulated in tubers, Stems, leaves and flowers were 1.855 mg/L, 0.809 mg/L,
0.276 mg/L and 0.015 mg/L respectively in 200 mg/L Cd. The total estimated Cd of 0 mg/L
Cd, 50 mg/L Cd, 100 mg/L Cd and 200 mg/L Cd were 0.1 mg/L, 0.451 mg/L, 1.081 mg/L and
2.945 mg/L respectively. The same finding was found in research paper (Hassan Nazarian,
Delara Amouzgar, Hossein Sedghianzadeh) [88]. Cd transportation from roots to stems in
Baasil (Ocimum basilicum L.) declined and plant tolerance index was significantly reduced.
The tolerance index was observed through the analysis of transport factor. The trace amount
of cadmium was absorbed by plant negatively affected plant’s height. Plant’s height was
declined with increased concentration of heavy metal Cd. The results are collaborated with
(Leonel Hernandez-Bautista, Libia Iris Trejo-Tellez, Fernando Carlos Gomez-Merino) [89]
who were discovered the similar results 30 mg/L Cd concentration caused significant
physiological changes in sweet pepper (Capsicum annum L.). Absorption of Cd caused
deficiency of Mn. N, P, K, and Cu manifested by some spot of chlorosis, slightly yellowish
spot in young leaves, edges and tip burns and brownish interveinal spot.
Figure 31: Effect of Cd on Dahlia Plant.
69 | P a g e
0.085
0.09 0.25
0.203
0.08
0.07 0.2
Absorbance
Absorbance
0.06 0.13
0.15 0.118
0.05
0.04
0.1
0.03
0.015
0.02 0.05
0.01 0 0 0
0 0
1 1
Figure 33: Absorbance comparison 0 mg/L Cd. Figure 32: Absorbance comparison 50 mg/L Cd.
0.5 0.453 1.855

0.418 2
0.4
1.5
Absorbance
Absorbance
0.3
0.21
1 0.809
0.2
0.1 0.5 0.276

0
0.005
0
0
1
1
Concentration (mg/L)
Figure 35: Absorbance comparison 100 mg/L Cd. Figure 34: Absorbance comparison 200 mg/L Cd.
70 | P a g e
The maximum cadmium absorption was discerned in tubers except 100 mg/L Cd where
maximum accumulation was in Stems (0.453 mg/L) but very close to the absorption of
cadmium in tubers (0.418 mg/L).
In 0 mg/L Cd, the concentration of cadmium in flowers and leaves was below detection
level to the concentration of standard solution. The absorption of cadmium in Stems and tubers
were 0.085 mg/L and 0.015 mg/L respectively. The absorption of cadmium in 100 mg/L Cd
strength in tubers, Stems and leaves were 0.418 mg/L, 0.453 mg/L and 0.21 mg/L respectively
but below detection level in flowers. Cadmium accumulation in 50 mg/L Cd in tubers, Stems
and leaves were 0.203 mg/L, 0.13 mg/L and 0.118 mg/L respectively and measured
concentration in flowers was below detection level. In 200 mg/L Cd, the concentration of
cadmium accumulated in tubers, Stems, leaves and flowers were 1.855 mg/L, 0.809 mg/L,
0.276 mg/L and 0.005 mg/L respectively.
In shortly, trace amount of cadmium was transported to all parts of plant body but
absorption was linearly increased with increased concentration of cadmium.
71 | P a g e
2
1.8
1.6
1.4
Absorbance
1.2
0 ppm
1
50 ppm
0.8
100 ppm
0.6
0.4 200 ppm
0.2
0
FLOWERS LEAVES SHOOTS TUBERS
Plant's parts
Figure 36: Comparative relation between absorption of Cd in tubers, Stems, leaves & flowers.
The comparative relationship between cadmium absorption in plant’s parts and different
strengths of cadmium applied.
In all four different strengths of cadmium, the maximum absorption of cadmium was
seen in tubers and minimum absorption (Cd) was met in flowers. The descending order of
accumulated cadmium is: Flowers ˂ Leaves ˂ Stems ˂ Tubers except in 100 mg/L Cd, the
descending order is: Flowers ˂ Leaves ˂ Tubers ˂ Stems. Total amount of cadmium
accumulated in plants of 0 mg/L Cd, 50 mg/L Cd, 100 mg/L Cd and 200 mg/L Cd were 0.1
mg/L, 0.451 mg/L, 1.081 mg/L and 2.945 mg/L respectively.
In conclusion, no absorption of cadmium was estimated in flowers in strength of 0 mg/L
Cd, 50 mg/L Cd, 100 mg/L Cd and in 200 mg/L Cd cadmium, the absorption of Cd in flowers
was 0.005 mg/L.
72 | P a g e
5.5 Statistical Analysis Cadmium

5.5.1 Student T test for cadmium
Table 7: Student T test statistical analysis of different parts of Dahlia plant against different concentration of Cd.
Tabulated value
Accepted value
Concentration
Hₒ rejection
H1 rejection
Mean ± SD
Plant parts
(mg/L)
(mg/L)
(t ˂ tv)
(t ˃ tv)
t test
(tv)
(μ)
Tubers 0.015±0.004 0.009 3.778 4.303 ˂4.303
Stems 0.085±0.004 0.010 4.479 4.303 ˃4.303
0
Leaves BDL
Flowers BDL
Tubers 0.203±0.004 0.010 4.348 4.303 ˃4.303
Stems 0.13±0.006 0.014 3.993 4.303 ˂4.303
50
Leaves 0.118±0.005 0.012 4.303 4.303 ˂4.303
Flowers BDL
Tubers 0.418±0.003 0.008 4.611 4.303 ˃4.303
Stems 0.453±0.004 0.010 4.479 4.303 ˃4.303
100
Leaves 0.21±0.006 0.015 4.382 4.303 ˃4.303
Flowers BDL
Tubers 1.855±0.005 0.011 3.881 4.303 ˂4.303
Stems 0.809±0.006 0.016 4.611 4.303 ˃4.303
200
Leaves 0.276±0.004 0.010 4.479 4.303 ˃4.303
Flowers 0.005±0.004 0.010 4.348 4.303 ˃4.303
t test calculated in 0 mg/L Cd Stems and 50 mg/L tubers were 4.479 and 4.348. Similarly, 100
mg/L Cd tubers, Stems and leaves, calculated t values were 4.611, 4.479 and 4.382
respectively, greater than tabulated value tv. 200 mg/L Cd Stems, leaves and flowers, calculated
t values were 4.611, 4.479 and 4.348 respectively. The suspected values are not too much
significantly different from tabulated value (4.303).
The suspected values in each case can be tested by Q test. If Q test tested that the
suspected values are within the range and each value is not lied outside of the range then result
cannot be rejected. Erroneous in measurements may be indeterminate rather than determinantal
error.
73 | P a g e
5.5.2 Q test rejection of a result

Table 8: Q test Rejection test of suspected values of different parts of plant of various concentration of Cd.
Difference between
Difference between
largest to nearest
largest value and
probability 95 %
neighbor value
Concentration
smallest value
Hₒ rejection
Plant parts
(mg/L)
(mg/L)
Q˂Q95
(t ˃ tv)
(Q95)
0 Stems ˃4.303 0.08 0.086 0.088 0.002 0.008 0.97 0.25
50 Tubers ˃4.303 0.199 0.202 0.207 0.005 0.008 0.97 0.62
Tubers ˃4.303 0.414 0.419 0.42 0.001 0.006 0.97 0.17
100 Stems ˃4.303 0.448 0.454 0.456 0.002 0.008 0.97 0.25
Leaves ˃4.303 0.203 0.211 0.215 0.004 0.012 0.97 0.33
Stems ˃4.303 0.802 0.812 0.814 0.002 0.012 0.97 0.17
200 Leaves ˃4.303 0.271 0.277 0.279 0.002 0.008 0.97 0.25
Flowers ˃4.303 0.001 0.006 0.009 0.003 0.008 0.97 0.38
0 mg/L Cd Stems & 50 mg/L Cd tubers, calculated Q values were 0.25 & 0.62 respectively (Q
˂ Q95). 100 mg/L Cd tubers, Stems and leaves calculated Q values were 0.17, 0.25 and 0.33
respectively. Similarly, 200 mg/L Cd Stems, leaves & flowers calculated Q values were 0.17,
0.25 and 0.38 respectively less than tabulated value Q95. 95% assured that suspected values
are not rejected and error in the exxperiment may be due to indeterminate.
74 | P a g e
5.6 Effect of Cadmium on physical parameters

Table 9: Different physical parameters measurement against different concentration of Cadmium (Cd).
Effect of Cd on Physical parameters

Days to Flower 151.28 76.27 71.48 62.11

Plant Height (cm) 93.5 81.5 70.4 46.1
39.88
151.28
160 40
140 35
120 30
Blooming Period
Days to Flowers
23.58
100 25
76.27 71.48
(Days)
18.66
80 62.11 20 15.75
60 15
40 10
20 5
0 0
0 50 100 200 0 50 100 200
Figure 38: Days to Flowers comparison (Cd). Figure 37: Blooming Period comparison (Cd).
70 68.82
100 93.5
68 81.5
66 80 70.4
Flower's Size
Plant's Height
64
(sq./cm)
60
60.85 46.1
(cm)
62
59.28 59.21
60 40
58
20
56
54 0
0 50 100 200 0 50 100 200
Figure 39: Size of Flower comparison (Cd). Figure 40: Height of Plant (Cd).
75 | P a g e
The different strength effect of cadmium on parameters like plant’s height, flower size, days to
flower and blooming period.
In 0 mg/L Cd, 151.28 days to flower were taken by Dahlia plant. The blooming period
in terms of days gap between first and last flower opening was 38.88 days. The height of plant
and flower’s size were 93.5 cm and 68.82 sq./cm respectively.
The less number of days to flower 76.27, 71.48 and 62.11 days and blooming 23.58,
18.66 and 15.75 were noticed in 50 mg/L, 100 mg/L and 200 mg/L strengths of cadmium
respectively. The increased concentration of cadmium negatively affected the plant’s height as
plant height of plant was declined with increased concentration of cadmium. The plant’s height
measured in 50 mg/L Cd, 100 mg/L Cd and 200 mg/L Cd were 81.5cm, 70.4cm and 46.1cm
respectively. The size of flowers was almost constant and days gap between first and last flower
opening were also declined in 50 mg/L Cd, 100 mg/L Cd and 200 mg/L Cd.
76 | P a g e
5.7 Effect of Mercury (Hg)

5.7.1 Absorbance of Mercury in different parts of plant
Table 10: Absorbance of Mercury (Hg) by different parts of Dahlia plant against different concentration.
Mean ± SD 0.012±0.003
Mean ± SD 1.019±0.007 1.741±0.005 0.091±0.005
Mean ± SD
Mean ± SD 4.396±0.004 2.025±0.005 0.087±0.006

Replicates
Replicates
Replicates
Replicates
(mg/L)
(mg/L)
(mg/L)
(mg/L)
Plant's part
0.009 1.013 3.616 4.39
3.62±0.004
Tubers 0.013 1.019 3.62 4.4
0.015 1.026 3.624 4.4
Absorbance
1.687±0.005
BDL 1.736 1.683 2.02
BDL
Stems BDL 1.742 1.687 2.03

BDL 1.746 1.692 2.03
BDL 0.087 BDL 0.08
BDL
BDL
Leaves BDL 0.09 BDL 0.09
BDL 0.096 BDL 0.09
BDL BDL BDL BDL
BDL
BDL
BDL
BDL
Flowers BDL BDL BDL BDL
BDL BDL BDL BDL
77 | P a g e
Mercury was found to be poisoned for dahlia plant. The Hg was more accumulated in tubers
and its transportation to higher part of plant permanently blocked (Hg have less tolerance index
than Pb and Cd in Dahlia plant). The highest concentration was absorbed by dahlia plant in
tubers, Stems and leaves in 200 mg/L Hg were 4.396 mg/L, 2.025 mg/L, 0.087 mg/L
respectively. The maximum amount accumulated in each 0 mg/L Hg, 50 mg/L Hg, 100 mg/L
Hg and 200 mg/L Hg were 0.012 mg/L, 2.851 mg/L, 5.307 mg/L and 6.508 mg/L respectively.
The plant’s height was greatly affected by increased concentration of Hg. All leaves were
damaged in 200 mg/L Hg discerned the deficiency of micronutrients N, K and P and
micronutrients Mn, Mo and Cl. The major symptoms were tip burn, edges burn, interveinal
chlorosis, uniformly yellowish leaves, curling of leaves and few necrotic spots.
Figure 41: Effect of Hg on Dahlia Plant.
78 | P a g e
0.012 1.741
0.012 1.8
1.6
0.01
1.4
1.019
Absorbance
Absorbance
0.008 1.2
1
0.006
0.8
0.004 0.6
0.4
0.002 0.091
0 0 0 0.2 0
0 0
1 1
Figure 42: Absorbance comparison 0 mg/L Hg. Figure 43: Absorbance comparison 50 mg/L Hg.
4.396
4 3.62 4.5
3.5 4
3 3.5
Absorbance
Absorbance
3
2.5
1.687 2.5 2.025
2
2
1.5 1.5
1 1
0.5 0 0 0.5 0 0.087
0 0
1 1
Figure 45: Absorbance comparison100 mg/L Hg. Figure 44: Absorbance comparison200 mg/L Hg.
79 | P a g e
Mercury was accumulated in tubers and Stems more than accumulated in leaves and flowers.
Four strengths 0 mg/L Hg, 50 mg/L Hg, 100 mg/L Hg and 200 mg/L Hg were applied and
absorption of mercury in different parts of plant was estimated. The Hg absorption was
maximum in tubers except in 50 mg/L Hg, the accumulation of Hg was maximum in Stems
(1.741 mg/L) as compared to tubers (1.019 mg/L).
In 0 mg/L Hg, 0.012 mg/L was engrossed in tubers but the absorption of mercury in
Stems, leaves and flowers were below detection level. In 50 mg/L Hg, the measured
concentration in tubers, Stems and leaves were 1.019 mg/L, 1.741 mg/L and 0.091 mg/L
respectively and Hg accumulation in flowers was below detection level. In 100 mg/L Hg, the
concentration of Hg measured in tubers, Stems were 3.62 mg/L and 1.687 mg/L respectively
but Hg absorption in leaves and flowers was below detection level. In 200 mg/L Hg, the
concentration measured in tubers, Stems and leaves were 4.396 mg/L, 2.025 mg/L and 0.087
mg/L respectively but accumulation of Hg in flowers was below detection level. Total contents
of mercury (Hg) accumulated in dahlia plants of strengths 0 mg/L Hg, 50 mg/L Hg, 100 mg/L
Hg and 200 mg/L Hg were 0.012 mg/L, 2.851 mg/L, 5.307 mg/L and 6.508 mg/L respectively.
In shortly, the Hg absorption was increased with increased concentration of Hg.

Mercury proved to be poisoned and trace amount of mercury severely affected the dahlia plant.
80 | P a g e
4.5
4
3.5
3
Absorbance
2.5 0 ppm
2 50 ppm
1.5 100 ppm
1 200 ppm
0.5
0
Flowers Leaves Shoots Tubers
Plant's parts
Figure 46: Comparative relation between absorption of Hg in tubers, Stems, leaves & flowers.
Comparative relationship between absorption of mercury in tubers, leaves, Stems, flowers and
different concentration of mercury can been seen.
It can be inferred that the maximum accumulation of mercury was quantified in tubers
and minimum absorption was estimated in flowers. The descending order of Hg absorption is:
Flowers ˂ Leaves ˂ Stems ˂ Tubers except 50 mg/L Hg where descending order is: Flowers ˂
Leaves ˂ Tubers ˂Stems.
In conclusion, the estimated concentration of mercury in tubers and Stems were more
than the concentration of mercury absorbed in leaves and flowers.
81 | P a g e
5.8 Statistical Analysis Mercury

5.8.1 Student T test for Mercury
Table 11: Student T test statistical analysis of different parts of Dahlia plant against different concentration of Hg.
Tabulated value
Accepted value
Concentration
Hₒ rejection
H1 rejection
Plant parts
Mean ± SD
(mg/L)
(mg/L)
(t ˂ tv)
(t ˃ tv)
t test
(tv)
(μ)
Tubers 0.012±0.003 0.008 4.382 4.303 ˃4.303
Stems BDL
0
Leaves BDL
Flowers BDL
Tubers 1.019±0.007 0.016 4.000 4.303 ˂4.303
Stems 1.741±0.005 0.013 4.332 4.303 ˃4.303
50
Leaves 0.091±0.005 0.011 3.944 4.303 ˂4.303
Flowers BDL
Tubers 3.62±0.004 0.010 4.303 4.303 ˂4.303
Stems 1.687±0.005 0.011 3.881 4.303 ˂4.303
100
Leaves BDL
Flowers BDL
Tubers 4.396±0.004 0.009 3.778 4.303 ˂4.303
Stems 2.025±0.005 0.011 3.881 4.303 ˂4.303
200
Leaves 0.087±0.006 0.015 4.303 4.303 ˂4.303
Flowers BDL _
Two cases were noted when t values exceeded from tabulated value tv (4.303). In 0 mg/L Hg
tubers, t value was calculated 4.382 and 50 mg/L Hg Stems, t value was calculated 4.332.
The suspected values are not significant different from tabulated value (4.303).
Suspected values can be checked by Q test either the suspected values are rejected or not. If
suspected value lies within the range in Q test then results cannot be rejected and error in results
may be occurred accidently rather than determinantal error.
82 | P a g e

Table 12: Q test Rejection test of suspected values of different parts of plant of various concentration of Hg.
Difference between
Difference between
largest to nearest
largest value and
probability 95 %
neighbor value
Concentration
smallest value
Hₒ rejection
Plant parts
(mg/L)
(mg/L)
Q˂Q95
(t ˃ tv)
(Q95)
0 Tubers ˃4.303 0.009 0.013 0.015 0.002 0.006 0.97 0.33
50 Stems ˃4.303 1.736 1.742 1.746 0.004 0.01 0.97 0.40
The suspected values in t test 0 mg/L Hg tubers and 50 mg/L Hg Stems were checked by Q
test. Q value calculated in each case was less than tabulated value (Q ˂ Q95). 95% probability
replicate measurements are not outlier of the range and 95% assured that erroneous
measurements may be occurred accidently rather than determinate error in the experiment. So,
results are not rejected.
83 | P a g e
5.9 Effect of Mercury on physical parameters

Table 13: Different physical parameters measurement against different concentration of Mercury (Hg).
Effect of Hg on Physical parameters

Days to Flower 151.28 115.13 105.64 0
Blooming Period (Days) 39.88 27.58 21.39 0
Flower Size (square/cm) 68.82 61.11 58.68 0
Plant Height (cm) 93.5 47.6 38.8 28.5
39.88
151.28
160 40
140 35
115.13 27.58
120 105.64 30
Blooming Period
Days to Flower
100 25 21.39
80 (Days) 20
60 15
40 10
20 0 5 0
0 0
0 50 100 200 0 50 100 200
Figure 48: Days to Flowers comparison (Hg). Figure 47: Blooming Period comparison (Hg).
68.82
70 100 93.5
61.11 58.68
90
60
80
50 70
Flower;s Size
Plant's Height
(sq./cm)
40 60
47.6
(cm)
50 38.8
30
40 28.5
20 30
10 20
0 10
0 0
0 50 100 200 0 50 100 200
Figure 50: Size of Flower comparison (Hg). Figure 49: Height of Plant (Hg).
84 | P a g e
The relationship between different concentration of mercury and physical parameters like days
to flower, blooming period, plant height and flower size can be met.
The plant height in 0 mg/L Hg, 50 mg/L Hg, 100 mg/L Hg and 200 mg/L Hg were
93.5cm, 47.6cm, 38.8cm and 28.5cm respectively. The number of days to flower blooming in
50 mg/L Hg and 100 mg/L Hg were reduced to 115.13 days and 105.64 days respectively.
Similarly, the days gap for flower blooming in 50 mg/L Hg and 100 mg/L Hg were decreased
to 27.58 days and 21.39 days respectively. No flower was bloomed in 200 mg/L Hg.
In conclusion, the plant height was greatly affected by increased mercury concentration.
Days to flower and Blooming period were noticed to be reduced in 50 mg/L Hg and 100 mg/L
Hg.
85 | P a g e
5.10 Effect of Zinc (Zn)

5.10.1 Absorbance of Micronutrient Zn in different parts of plant
Table 14: Absorbance of Zinc (Zn) by different parts of Dahlia plant against different concentration.
Mean ± SD 1.773±0.005 1.036±0.004 0.968±0.045 0.786±0.006
Mean ± SD 2.459±0.005 2.036±0.005 1.514±0.006 0.977±0.006
Mean ± SD 5.791±0.005 3.466±0.004 2.346±0.004 1.814±0.003
Mean ± SD 7.673±0.004 5.658±0.005 3.448±0.003 2.007±0.005

Replicates
Replicates
Replicates
Replicates
(mg/L)
(mg/L)
(mg/L)
(mg/L)
Plant's part
1.769 2.45 5.79 7.67

Tubers 1.773 2.46 5.79 7.67
1.778 2.46 5.79 7.68
Absorbance
1.032 2.03 3.46 5.65

Stems 1.037 2.04 3.47 5.66
1.04 2.04 3.47 5.66
0.991 1.51 2.34 3.45
Leaves 0.997 1.51 2.35 3.45
0.916 1.52 2.35 3.45
0.792 0.97 1.81 2
Flowers 0.781 0.98 1.81 2.01
0.786 0.98 1.82 2.01
86 | P a g e
Zinc is micronutrient for dahlia plant. Zinc was estimated to all plant’s part (Zn has high
tolerance index). The maximum Zn amount was absorbed by dahlia plant (500 mg/L Zn) in
tubers, Stems, leaves and flowers were 7.673 mg/L, 5.658 mg/L, 3.448 mg/L and 2.007 mg/L
respectively. Total estimated concentration in 0 mg/L Zn, 100 mg/L Zn, 300 mg/L Zn and 500
mg/L Zn were 4.583 mg/L, 6.986 mg/L, 13.417 mg/L and 18.786 mg/L respectively. Zinc at
higher applied concentration than 100 mg/L Zn was proved to be toxic, the more Zn
concentration accumulated in dahlia plant. The plant’s height was affected by increased
concentration of zinc. Deficiency of nutrients K, N, Mn, Mo, Cu and visual symptoms
uniformly chlorosis, tip burn, young leaves chlorosis, curling of young leaves were seen. The
descending order for metal accumulation in plant’s parts is: Flowers ˂ Leaves ˂ Stems ˂
Tubers.
Figure 51: Effect of Zn on Dahlia Plant.
87 | P a g e
2.459
1.773
2.5
1.8 2.036
1.6 2
1.4 1.514
Absorbance
0.9681.036
Absorbance
1.2 1.5
1 0.786 0.977
0.8 1
0.6
0.4 0.5
0.2
0
0
1
1

Figure 52: Absorbance comparison 100 mg/L Zn.

Figure 53: Absorbance comparison 0 mg/L Zn.
5.791 7.673
6 8
7
5 5.658
6
3.466
Absorbance
4
5
3.448
3 2.346 4
Absorbance
1.814
3 2.007
2
2
1
1
0 0
1 1
Figure 55: Absorbance comparison 300 mg/L Zn. Figure 54: Absorbance comparison 500 mg/L Zn.
88 | P a g e
It can be inferred that the maximum absorption of micronutrient zinc was in descending order
Flowers ˂ Leaves ˂ Stems ˂ Tubers in 0 mg/L, 100 mg/L, 300 mg/L and 500 mg/L Zn.
In 0 mg/L Zn, the estimated absorption of Zn in tubers, Stems, leaves and flowers were
1.773 mg/L, 1.036 mg/L, 0.968 mg/L and 0.786 mg/L respectively. In 100 mg/L Zn, the zinc
concentration accumulated in tubers, leaves, Stems and flowers were 2.459 mg/L, 2.036 mg/L,
1.514 mg/L and 0.977 mg/L respectively. In 300 mg/L Zn, the absorption of Zn was 5.791
mg/L, 3.466 mg/L, 2.346 mg/L and 1.814 mg/L in tubers, Stems, leaves and flowers
respectively. In 500 mg/L Zn, the accumulation of zinc in tubers, Stems, leaves and flowers
were 7.673 mg/L, 5.658 mg/L, 3.448 mg/L and 2.007 mg/L respectively. Total Zn contents
deposited in all parts of plant in each strength 0 mg/L Zn, 100 mg/L Zn, 300 mg/L Zn and 500
mg/L Zn were 4.563 mg/L, 6.986 mg/L, 13.417 mg/L and 18.786 mg/L respectively.
In conclusion, Zn is transported to all parts of body and absorption of zinc in tubers,

Stems, leaves and flowers were increased with increased concentration of zinc.
89 | P a g e
6
Absorbance
5
0 ppm
4 50 ppm
3 100 ppm
2 200 ppm
0
Figure 56: Comparative relation between absorption of Zn in tubers, Stems, leaves & flowers.
The comparative relationship between absorption of micronutrient Zn and four strengths 0

mg/L Zn, 100 mg/L Zn, 300 mg/L Zn and 500 mg/L Zn can be convened.
The maximum absorption of zinc in each strength of Zn was in tubers and minimum
was in flowers. This absorption was linearly increased with increased concentration of zinc.
Total Zn contents deposited in all parts of plant in each strength 0 mg/L Zn, 100 mg/L Zn, 300
mg/L Zn and 500 mg/L Zn were 4.563 mg/L, 6.986 mg/L, 13.417 mg/L and 18.786 mg/L
respectively.
In shortly, Zn as a micronutrient has high tolerance index and concentration higher than
100 mg/L evinced to be toxic.
90 | P a g e
5.11 Statistical Analysis Zinc

5.11.1 Student T test for Zinc
Table 15: Student T test statistical analysis of different parts of Dahlia plant against different conc. of Zn.
Tabulated value
Accepted value
Concentration
Hₒ rejection
H1 rejection
Mean ± SD
Plant parts
(t ˂ tv)
(mg/L)
(mg/L)
(t ˃ tv)
t test
(tv)
(μ)
Tubers 1.773±0.005 0.011 4.303 4.303 ˂4.303
Stems 1.036±0.004 0.010 4.348 4.303 ˃4.303
0
Leaves 0.968±0.045 0.112 4.316 4.303 ˃4.303
Flowers 0.786±0.006 0.014 3.950 4.303 ˂4.303
Tubers 2.459±0.005 0.011 3.881 4.303 ˂4.303
Stems 2.036±0.005 0.013 4.333 4.303 ˃4.303
100
Leaves 1.514±0.006 0.014 3.950 4.303 ˂4.303
Flowers 0.977±0.006 0.014 3.950 4.303 ˂4.303
Tubers 5.791±0.005 0.012 4.245 4.303 ˂4.303
Stems 3.466±0.004 0.010 4.479 4.303 ˃4.303
300
Leaves 2.346±0.004 0.009 3.778 4.303 ˂4.303
Flowers 1.814±0.003 0.007 4.303 4.303 ˂4.303
Tubers 7.673±0.004 0.009 3.778 4.303 ˂4.303
Stems 5.658±0.005 0.011 3.882 4.303 ˂4.303
500
Leaves 3.448±0.003 0.007 4.302 4.303 ˂4.303
Flowers 2.007±0.005 0.012 4.303 4.303 ˂4.303
Four cases were reported when t ˃ tv e.g., 0 mg/L Zn Stems and leaves, calculated t values were
4.348 and 4.316 respectively. 100 mg/L Zn Stems, calculated t value was 4.333 and 300 mg/L
Zn Stems, calculated t value was 4.479. Both values are exceeded than tabulated value tv
(4.303). The suspected values can be tested by Q test to see suspected values are lie within the
range at confidence limit 95% (Q95) then suspected values are not rejected and erroneous
measurements may quite possible accidently rather than determinantal error.
91 | P a g e

Table 16: Q test Rejection test of suspected values of different parts of plant of various concentration of Zn.
Difference between
Difference between
largest to nearest
largest value and
probability 95 %
neighbor value
smallest value
Concentration
Hₒ rejection
Plant parts
(mg/L)
Q˂Q95
(mg/L)
(t ˃ tv)
(Q95)
Stems ˃4.303 1.032 1.037 1.04 0.003 0.008 0.97 0.38
0
Leaves ˃4.303 0.916 0.991 0.997 0.006 0.081 0.97 0.07
100 Stems ˃4.303 2.031 2.035 2.041 0.006 0.01 0.97 0.60
300 Stems ˃4.303 3.461 3.467 3.469 0.002 0.008 0.97 0.25
0 mg/L Zn Stems and leaves, calculated Q values were 0.38 and 0.07 respectively (Q ˂ Q95).
Similarly, 100 mg/L Zn Stems and 300 mg/L Zn Stems, calculated Q values were 0.6 and 0.25
respectively. 95% confidence that error in the experiments are not due to determinate and quite
possible error may be occurred by chance or indeterminate error.
92 | P a g e
5.12 Effect of Zinc on physical parameters

Table 17: Different physical parameters measurement against different concentration of Zinc (Zn).
Effect of Zn on Physical parameters

Days to Flower 151.28 167.41 142.65 110.21
Plant Height (cm) 93.5 73.8 61.3 60.7
180 167.41 56.77

60
151.28
160 142.65
50
140 39.88 41.58
Days to Flowers
Blooming Period
110.21 38.2
120 40
(Days)
100
30
80
60 20
40
10
20
0 0
0 100 300 500 0 100 300 500
Figure 58: Days to Flowers comparison (Zn). Figure 57: Blooming Period comparison (Zn).
68.82
69 100 93.5
68
67 80 73.8
66 61.3 60.7
Plant's Height
Flower's Size
65 60
sq./cm)
64
(cm)
62.88
62.18 62.38
63
40
62
61
60 20
59
58 0
0 100 300 500 0 100 300 500
Figure 60: Size of Flower comparison (Zn). Figure 59: Height of Plant (Zn).
93 | P a g e
The relative relationship between effect of different concentration of zinc and physical
parameters on Days to flower, flower size, blooming period and plant height.
Increased concentration of Zn has significantly affected on plant’s height. In 100 mg/L

Zn, 300 mg/L Zn and 500 mg/L Zn concentration, the height of plant was reduced to 73.8cm,
61.3cm and 60.7cm respectively as compared to 93.5cm in 0 mg/L Zn. The days to flowers
110.21, 142.65 and 167.41 were noticed by the Dahlia plants of 500 mg/L Zn, 300mg/L Zn and
100 mg/L Zn to bloom and less number days were taken in higher concentration of Zn for first
flower to emerge as compared to 0 mg/L Zn (151.28 days) except in 100 mg/L where the first
flower to bloom took 167.41 days. The exceptional case was also seen in 100 mg/L Zn and 300
mg/L Zn, 56.77 and 41.58 days gap between first and last flower opening in 100 mg/L Zn and
300 mg/L Zn respectively as compared to 39.88 days in 0 mg/L Zn. In 500 mg/L Zn, the
blooming period was almost same (38.2 days). The flower’s size was also of almost same size
62.88 square/cm, 62.38 square/cm and 62.18 square/cm in 300 mg/L Zn, 500 mg/L Zn and 100
mg/L Zn respectively.
In conclusion, the higher concentration of Zn significantly affected the plant’s height

and less number of days were taken by Dahlia plant to first flower to bloom than 0 mg/L Zn
except 100 mg/L Zn.
94 | P a g e
6 DISCUSSION
6.1 Heavy Metals (Pb, Cd & Hg)
Dahlia plants response differently to heavy metals. Dahlia plants have eight set of
chromosomes and are octoploid while most of the plants are diploid (having two set of
chromosomes). The alleles in a chromosome can be transposed from one position to another.
In conclusion dahlia plants resist to absorb cadmium and trace amount of Cd badly
affected the plant’s height. Mercury accumulation in tubers and Stems and blockage to higher
parts of plant was observed. The height of plant and others parameter like flower’s size, quality
of flowers, days to flower and blooming period proved that Hg is poisoned for dahlia plant.
The days to flower, blooming period, flower’s size and plant’s height were affected by
increased concentration of Lead, Cadmium and Mercury especially plant’s height. The uptake
of micro and macro-nutrients was partially blocked and deficiency of essential nutrients were
signified by various symptoms/diseases in dahlia plant.
Plant’s height, metal accumulation, uptake of essential nutrients, visual

symptoms/diseases, days to flower, blooming period and flower’s size are key factor to decide
the toxicity order. The descending order of toxicity is: Pb ˂ Cd ˂ Hg and the accumulation of
heavy metals in different parts are: Flowers ˂ Leaves ˂ Stems ˂ Tubers exceptional in some
cases are Flowers ˂ Leaves ˂ Tubers ˂ Stems.
6.2 Micronutrients Zinc and Zn toxicity

Zinc is micronutrient for plants. At concentration higher than 100 mg/L Zn, the more Zn
concentration accumulated in dahlia plant. The plant’s height was affected by increased
concentration of zinc. Deficiency of nutrients K, N, Mn, Mo, Cu and visual symptoms
uniformly chlorosis, tip burn, young leaves chlorosis, curling of young leaves. The descending
order for metal accumulation in plant’s parts are Flowers ˂ Leaves ˂ Stems ˂ Tubers.
7 CONCLUSION
The soil contaminated with heavy metals are badly affected the dahlia plant and heavy metals
become part of food chain as inulin extracted from tubers are used by human beings as artificial
sweetener. Similarly, others medicinal usage of this plant are influenced. Dahlia plant should
be sprung in uncontaminated soil for healthy growth.
95 | P a g e
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Effect of Heavy Metals on Economical Important Plant

Dahlia (Coccinea cav.)

Dr. Humayun Ajaz (Associate Professor)

University of Engineering and Technology Lahore,

Approved on: _____________

Internal Examiner ______________________

External Examiner ______________________

UNIVERSITY OF ENGINEERING AND TECHNOLOGY

UNIVERSITY OF ENGINEERING AND TECHNOLOGY

From for the release of Thesis for Examination

___________________ Date: ________________

I approve that the above thesis be submitted for examination,

___________________ Dated: _____________________

This form must be submitted with the thesis to the Chairperson,

Department of Chemistry, University of Engineering and Technology Lahore – Pakistan.

Have mercy on them (parents) both as they did care

for me when I was little

(Al- Quran 17:24)

I forward my special thanks to Prof. Dr. Rubina Gilani, Chairman, Department of

I cannot remain without expressing my heartiest gratitude to my parents, who made a

1.1 Heavy Metal Stress.................................................................................................... 16

1.2 Metal Uptake ............................................................................................................. 17

1.3 Plant’s resistance to heavy metal absorption............................................................. 17

1.4 Choice of plant .......................................................................................................... 18

1.5 Effects of lead on plants ............................................................................................ 19

1.6 Effects of Cadmium on plants ................................................................................... 19

1.7 Effects of Mercury on plants ..................................................................................... 20

1.8 Effects of Zinc on plants ........................................................................................... 21

2 LITERATURE SURVEY ................................................................................................ 23

3.1 Atomic Absorption Spectroscopy ............................................................................. 28

3.1.1 Introduction ........................................................................................................ 28

3.1.2 Uses .................................................................................................................... 29

3.1.2.1 Clinical Analysis......................................................................................... 29

3.1.2.2 Pharmaceutical Analysis............................................................................. 29

3.1.2.3 Environmental Analysis ............................................................................. 29

3.1.2.4 Rocks Analysis ........................................................................................... 29

3.1.2.5 Industrial Analysis ...................................................................................... 29

3.1.3 Working ............................................................................................................. 30

3.1.4 Components of AAS .......................................................................................... 31

3.1.4.1 Radiation source ......................................................................................... 31

3.1.4.1.1 Line source .............................................................................................. 31

3.1.4.1.2 Hollow cathode lamp ............................................................................... 32

3.1.4.1.6 Emission .................................................................................................. 33

3.1.4.1.7 Electrodes discharge lamp ....................................................................... 33

3.1.4.1.8 Continuum line sources ........................................................................... 34

3.1.4.2.1 Flame Atomizer ....................................................................................... 34

3.1.4.2.2 Electro thermal Atomizer ....................................................................... 36

3.1.4.3 Sample Introduction System ....................................................................... 36

3.1.4.3.1 Continuous introduction system .............................................................. 36

3.1.4.3.2 Discontinuous introduction system ......................................................... 36

3.1.4.4 Monochromator .......................................................................................... 36

3.1.4.5 Detector ...................................................................................................... 37

3.1.4.5.1 Sensitivity and Specificity ....................................................................... 38

3.1.4.5.2 Back ground Absorption .......................................................................... 38

3.1.5 Sample Preparation ............................................................................................ 38

3.1.6 Instrumental Setup ............................................................................................. 38

3.2 Cold Vapor Technique .............................................................................................. 39

3.2.1 Instrumentation .................................................................................................. 39

3.2.1.1 Hollow Cathode Lamp................................................................................ 39

3.2.1.2 Atomizer ..................................................................................................... 39

4 MATERIALS & METHODS ........................................................................................... 42

4.3 Sample collection ...................................................................................................... 45

4.4 Sample treatment ....................................................................................................... 45

___ Date:

_ Dated: ___