PHYTOREMEDIATION MEASURES FOR HEAVY METAL CONTAMINATION OF LOKTAK , ,

THESIS SUBMITTED TO UNIVERSITY IN PARTIAL FULFILMENT FOR THE AWARD OF THE DEGREE OF DOCTOR OF PHILOSOPHY IN ENVIRONMENTAL SCIENCE

By MAYANGLAMBAM MUNI SINGH (Ph.D. Registration No - MZU/Ph.D./570 of 13.05.2013)

DEPARTMENT OF ENVIRONMENTAL SCIENCE

SCHOOL OF EARTH SCIENCES & NATURAL RESOURCES

MANAGEMENT, MIZORAM UNIVERSITY

AIZAWL – 796004

2016

I

DECLARATION

I, Shri Mayanglambam Muni Singh, hereby declare that the subject matter of this thesis entitled “PHYTOREMEDIATION MEASURES FOR HEAVY METAL

CONTAMINATION OF LOKTAK LAKE, MANIPUR, INDIA,” is the record of work done by me, that the content of the thesis did not form basis for the award of any previous degree or to anybody else, and that I have not submitted the thesis in any other University/

Institute for any other degree.

This is being submitted to the Mizoram University for the degree of Doctor of

Philosophy in the Department of Environmental Science.

(Dr. Prabhat Kumar Rai) Head Supervisor Department of Environmental Science Department of Environmental Science Mizoram University Mizoram University

(Mayanglambam Muni Singh)

Date:

Place:

II

DEPARTMENT OF ENVIRONMENTAL SCIENCE MIZORAM UNIVERSITY, TANHRIL, AIZAWL 796004 (A Central University Established by Parliament Act No. 8 of 2000)

Dr. Prabhat Kumar Rai Contact No.: +919862315981 Assistant Professor E-mail: [email protected]

CERTIFICATE

This is to certify that the Thesis entitled “PHYTOREMEDIATION MEASURES FOR

HEAVY METAL CONTAMINATION OF LOKTAK LAKE, MANIPUR, INDIA” submitted by Mayanglambam Muni Singh for the award of degree of Doctor of Philosophy of the Mizoram University, Aizawl, embodies the record of original investigation carried out by him under my supervision. He has been duly registered and the thesis presented is worthy of being considered for the award of the Ph.D. Degree. The work has not been submitted for any degree of any other University.

(Dr. Prabhat Kumar Rai) Head Supervisor Department of Environmental Science Department of Environmental Science Mizoram University Mizoram University

Date: Place: Aizawl

III

ACKNOWLEDGEMENTS

I express my feelings of gratitude to the Almighty God, for successful completion of this piece of research work.

I am extremely grateful and deeply indebted to my supervisor Dr. Prabhat Kumar Rai, Department of Environmental Science, School of Earth Sciences and Natural Resources Management, Mizoram University, Aizawl, for his valuable guidance, consistent and stimulating advice, constant encouragement and untiring help throughout the research work.

I am also thankful to Prof. Lalnuntluanga (Head, Department of Environmental Science), Prof B.P. Mishra (Department of Environmental Science) and Prof. H Lalramnghinglova (Department of Environmental Science) who has been a constant source of inspiration to me and also for providing valuable inputs with pleasure, as and when required. I also thank all the faculty members and non-teaching staff of the Department of Environmental Science, for their constant support and necessary helps during the tenure of this work. I also thank Department of Life Science, and Department of Food Nutrition and Safety, Government of Manipur for extending the laboratory Facilities.

Thanks are also due to my all friends and research scholars who provided me friendly atmosphere and helpful attitude at each step of the study in various ways.

Words fail to express my humble gratitude and profound regards to my loving parents and family members for their affection, encouragement, cooperation and blessing during the course of this work which have always been a source of inspiration for me.

I also wish to acknowledge the UGC, New Delhi for providing financial support in form of the Rajiv Gandhi National fellowship for SC.

(Mayanglambam Muni Singh)

Date:

Place: Aizawl

IV

CONTENTS

Page No.

TITLE PAGE I

DECLARATION II

CERTIFICATE III

ACKNOWLEDGEMENTS IV

CONTENTS V

LIST OF FIGURES VI-VII

LIST OF TABLES VIII-IX

LIST OF MAPS AND PHOTO PLATES X

1-16 CHAPTER 1 INTRODUCTION

17-44 CHAPTER 2 REVIEW OF LITERATURE

45-61 CHAPTER 3 MATERIAL AND METHODS

62-98 CHAPTER 4 RESULTS

99-118 CHAPTER 5 DISCUSSION

CHAPTER 6 SUMMARY, CONCLUSION AND 119-127 RECOMMENDATIONS

REFERENCES 128-163

164-170 APPENDIX

V

LIST OF FIGURES Page No.

Figure 4.1. Seasonal variations of Temperature of water from different 63 study sites.

Figure 4.2. Seasonal variations of pH of water from different study sites. 64

Figure 4.3. Seasonal variations of Transperancy of water from different 65 study sites.

Figure 4.4. Seasonal variations of Total Solids of water from different 66 study sites.

Figure 4.5. Seasonal variations of Dissolve Oxygen of water from 68 different study sites.

Figure 4.6. Seasonal variations of Biological Oxygen Demand of water 69 from different study sites.

Figure 4.7. Seasonal variations of Acidity of water from different study 70 sites.

Figure 4.8. Seasonal variations of Alkalinity of water from different 71 study sites.

Figure 4.9. Seasonal variations of Chloride of water from different study 73 sites. Figure 4.10. Seasonal variations of Total Hardness of water from 74 different study sites.

Figure 4.11. Seasonal variations of Turbidity of water from different 75 study sites. Figure 4.12. Seasonal variations of Nitrate of water from different study 76 sites.

Figure 4.13. Seasonal variations of Phosphate of water from different 77 study sites.

Figure 4.14. Seasonal variations of Fe concentrations (in mgL-1) of water 89 from different study sites.

Figure 4.15. Variations of Fe concentration (in mgkg-1) of plants from 84 different study sites.

Figure 4.16. Percentage (%) removal of Fe by selected plant species of 95 different concentrations in 4 days.

VI

Figure 4.17. Percentage (%) removal of Fe by selected plant species of 96 different concentrations in 8 days.

Figure 4.18. Percentage (%) removal of Fe by selected plant species of 97 different concentrations in 12 days.

VII

LIST OF TABLES Page No.

Table 4.1. Temperature (in ºC) with standard deviation of water from 63 different study sites.

Table 4.2. pH with standard deviation of water from different study sites. 64

Table 4.3. Transperancy (in m) with standard deviation of water from 65 different study sites.

Table 4.4. Total Solids (in mgL-1) content with standard deviation of water 67 from different study sites. Table 4.5. Dissolve Oxygen (in mgL-1) content with standard deviation of 68 water from different study sites.

Table 4.6. Biological Oxygen Demand (in mgL-1) content with standard 69 deviation of water from different study sites.

Table 4.7. Acidity (in mgL-1) content with standard deviation of water 70 from different study sites.

Table 4.8. Alkalinity (in mgL-1) content with standard deviation of water 72 from different study sites.

Table 4.9. Chloride (in mgL-1) content with standard deviation of water 73 from different study sites.

Table 4.10. Total Hardness (in mgL-1) content with standard deviation of 74 water from different study sites.

Table 4.11. Turbidity (in NTU) content with standard deviation of water 75 from different study sites.

Table 4.12. Nitrate (in mgL-1) content with standard deviation of water from 76 different study sites.

Table 4.13. Phosphate (in mgL-1) content with standard deviation of water 78 from different study sites.

Table 4.14. Fe concentrations (in mgL-1) of water from different study sites. 79

Table 4.15. Hg concentrations (in mgL-1) of water from different study 80 sites.

Table 4.16. Cd concentrations (in mgL-1) of water from different study 80 sites.

VIII

Table 4.17. As concentrations (in mgL-1) of water from different study sites. 81

Table 4.18. Pb concentrations (in mgL-1) of water from different study sites. 82

Table 4.19. Cr concentrations (in mgL-1) of water from different study sites. 82

Table 4.20. Zn concentrations (in mgL-1) of water from different study sites. 83

Table 4.21. Phytosociological attributes of macrophyte species. 84

Table 4.22. Showing Sorenson’s Similarity Index between different sites. 86

Table 4.23. Family-wise distribution of macrophyte species. 88

Table 4.24. Fe concentrations (in mgkg-1) of plants from different study 90 sites.

Table 4.25. Hg concentrations (in mgkg-1) of plants from different study 90 sites.

Table 4.26. Cd concentrations (in mgkg-1) of plants from different study 91 sites.

Table 4.27. As concentrations (in mgkg-1) of plants from different study 92 sites.

Table 4.28. Pb concentrations (in mgkg-1) of plants from different study 92 sites.

Table 4.29. Cr concentrations (in mgkg-1) of plants from different study 93 sites. Table 4.30. Zn concentrations (in mgkg-1) of plants from different study 94 sites.

Table 4.31. Percentage (%) removal of Fe by selected plant species of 95 different concentrations in 4 days.

Table 4.32. Percentage (%) removal of Fe by selected plant species of 97 different concentrations in 8 days.

Table 4.33. Percentage (%) removal of Fe by selected plant species of 98 different concentrations in 12 days.

IX

LIST OF MAPS AND PHOTO PLATES Page No.

Map. 3.1 Map showing the Loktak lake at Bishenpur District, 48 Manipur.

Map. 3.2 Map showing structure of the Loktak Lake and study sites. 49

Photo plate 3.1 Photo showing study site Site I. 50

Photo plate 3.2 Photo showing study site Site II. 50

Photo plate 3.3 Photo showing study site Site III. 51

Photo plate 3.4 Photo showing study site Site IV. 51

X

Chapter 1 INTRODUCTION

Water

Water is a substance which is composed of hydrogen and oxygen and exists in solid, liquid and gaseous state. It is colourless, tasteless, and odourless liquid at room temperature.

Water exhibits very complex chemical and physical properties that are incompletely understood although its formula seems simple. Its melting point is 0° C (32° F), and its boiling point is 100° C. Compare with the analogous compounds, such as hydrogen sulphide and ammonia, it is much higher than the expected melting and boiling point. In its solid form (ice) water is less dense than when it is liquid, another unusual property.

Water is an integral component of environment, which is responsible for various life processes and hence persistence of life on earth. Water is the major component of human body and if it is extracted from the body, one dies immediately. Water is essential for completion of life cycle in animals and plants because it is needed at one or other stages of life.

They are extremely essential for survival of all living organisms. The important property of water is its ability to dissolve many other substances therefore water is considered as a universal solvent. The dissolve substances have some significant role in many mechanisms of living organisms. Life is also believed to have originated in the world’s oceans, which are complicated solution of water which many substances are dissolved into it. Living organisms use aqueous solutions- e.g. blood and digestive juices- as mediums for carrying out biological processes.

Water is the essence of life on earth and totally dominates the chemical composition of all organisms. The Utility of water in biota is the fulcrum of biochemical metabolism which rests on its unique physical and chemical properties. The characteristics

1 | P a g e of water regulate Lake metabolism. The unique thermal density properties, high specific heat, and liquid-solid characteristics of water allow the formation of a stratified environment that controls the chemical and biotic properties of to a mark degree.

Water provides a tempered milieu in which extreme fluctuations in water availability and temperature are ameliorated relative to conditions facing aerial life. Coupled with a relatively high degree of viscosity, these characteristics have enabled the biota to develop a large number of adaptations that improve sustained productivity (Wetzel, 1983).

Water Distribution

Life is believed to have originated in the world’s ocean, a complicated solution of water with many substances dissolved into it. Of total water reserves, the ocean contains 97%, icecaps and permanent glaciers 2.1% and fresh water is only 0.9% which found in atmospheric water vapour, rivers, lakes, ponds, ground water and soil moisture (Dugan,

1972).

As the earth’s population grows and the demand for fresh water increases water purification and recycling becomes increasingly important. Interestingly the purity requirements of water for industrial use often exceed those for human consumption.

Though, present in larger amount, oceanic water is of little direct use than freshwaters. The freshwater resources have played an important role in the development and evolution of civilizations. This is because the freshwater resources find their use in various human endeavors like drinking, washing, transportation and . Therefore, proper conservation or management is prerequisite so that this small percentage of usable water can be sustained for various purposes which are directly linked with the human survival.

Lakes and surface water reservoirs are the planet’s most important freshwater resources and provide innumerable benefits. They are a source of water for domestic use,

2 | P a g e irrigation and renewable energy in the form of hydro power and are essential resources for industry. Lakes provide ecosystems for fish, thereby functioning as a source of essential protein, and for significant elements of the world’s biological diversity. They have important social and economic benefits as a result of and recreation, and are culturally and aesthetically important for people throughout the world.

Physico-chemical Properties

Water has several unique physical and chemical properties that have influenced life as it has evolved. Indeed the very concept of the earth biosphere is dependent on the special physicochemical properties of water. These characteristics have significantly influenced the structure of inland aquatic ecosystems.

It is very essential and important to check the water before it is used for various purposes i.e. domestic, agricultural and industrial purpose. Water must be tested with different physico-chemical parameters. Selection of parameters for testing of water is solely dependent upon for what purpose we going to use that water and what extent we need its quality and purity. Water may contain different types of floating, dissolved, suspended and microbiological as well as bacteriological impurities. Some physical test should be performed for testing of its physical appearance such as temperature, colour, odour, pH, turbidity, transparency, Total Solid etc, while chemical tests should be perform for its biological oxygen demand, dissolved oxygen, acidity, alkalinity, chloride, hardness, nitrate and phosphate. For obtaining more and more potable/pure water, it should be tested for its trace metal, heavy metal contents and organic i.e. pesticide residue. It is obvious that drinking water should pass these entire tests and it should contain required amount of mineral level. Only in the developed countries all these criteria’s are strictly monitored.

Due to very low concentration of heavy metal and organic pesticide impurities present in water it need highly sophisticated analytical instruments and well trained manpower.

3 | P a g e

Following different physico-chemical parameters are tested regularly for monitoring quality of water.

Temperature affects a number of physical, chemical, and biological processes in natural aquatic systems. The temperature regime of a water source is a function of seasonal/diurnal ambient air temperatures and the morphometry and setting of the water source. Biologically, one of the most important effects of temperature is the decrease in oxygen solubility as the temperature increases. As a result, the increase in temperature can also increase the oxygen demand of biological organisms such as aquatic plants and fish.

In addition, chemical compounds tend to become more soluble as a response to higher temperatures. Temperature is controlled primarily by climatic conditions, but human activity can also influence temperature. Thermal or chemical pollution could adversely alter the distribution and species composition of aqua tic communities.

At prevailing global temperatures most inland water is in liquid form. As liquid water has a special thermal features that minimize temperature fluctuations. First among these features is its high specific heat i.e., a relatively large amount of heat is required to raise the temperature of water. The quantity of heat required to convert water from solid to a liquid state (latent heat of fusion) is high. This capacity to absorb heat has several important consequences for the biosphere, including the ability of inland water to moderate seasonal and diurnal temperature differences both within aquatic ecosystems and to lesser extent beyond them. Most of the heat input to inland waters is in the form of solar energy.

The amount of heat that actually reaches inland waters at any time depends on several factors, including time of the day, season, latitude, altitude and amount of cloud cover. A significant amount of solar radiation that reaches the water column surface is lost through reflection and backscattering. The remaining fractions enter the water column where its energy rapidly diminishes with depth as it is absorbed and converted either to heat by

4 | P a g e physical processes or to chemical energy by the biological processes of photosynthesis. In large lakes most of the energy required by the biota is derived from this biological conversion. In other sorts of inland waters however a large proportions of the required energy by biological communities may come from emergent and nearby terrestrial vegetation. In any event, the amount and nature of solar energy entering inland waters is a principal determinant of the structure and function of the ecosystem.

One of the most significant properties of water is its function as a solvent. Water is considered as universal solvent. In this regard, it has an unrivalled capacity to dissolve in solution exceptionally wide range of substances, including electrolytes (salt, which dissociate into ions in aqueous solution), colloids (particulate matter small enough to remain suspended in solution), and non-electrolytes (substances such as glucose that retain their molecular structure and do not dissociate into ions). A great variety of combinations of dissolved substances can occur in inland waters. The major inorganic solutes are the cations sodium, potassium, calcium, and magnesium and the anions chloride, sulphate, and bicarbonate/carbonate. When the concentration of all these ions (i.e. the salinity, or salt content) is less than 3 grams per litre (i.e. 3 grams per kilogram, or 3 parts per thousand), inland waters are conventionally regarded as fresh. In addition to these major ions, all inland waters contain smaller quantities of other ions, of which phosphate and nitrate essential plant nutrients are particularly significant and certain dissolved gases, especially oxygen, carbon dioxide and nitrogen whose solubilities are inversely correlated with temperature, altitude, and hydrogen ion concentration (pH) are of biological significance.

Physicochemical phenomena affect every body of inland water, creating unique relationships among and within the biotic and abiotic components of the ecosystem. Of particular interest are the pathways or biogeochemical cycles that are travelled by the chemical elements essential to life nitrogen, phosphate, carbon, and variety of

5 | P a g e micronutrients such as iron, sulphur and silica. The degree to which output of a particular element balances input within a given aquatic ecosystem varies according to the type of inland water involved. However all essential elements follow pathways in inland waters that’s numerous, complex, well-defined and often interdependent on the other biogeochemical cycles. In fact, a defining characteristic of all inland aquatic ecosystems, including the simplest temporary bodies of highly saline water, is the occurrence of well- defined biogeochemical cycles (Wetzel, 1983).

Some of the most salient general physiochemical features of inland waters having been indicated its importance to emphasize that these features are expressed differently in various types of inland waters.

Inland waters represent parts of the biosphere within which marked biological diversity, complex biogeochemical pathways, and energetic processes occur. Although from a geographic perspective inland waters represent only a small fraction of the biosphere, when appreciated from an ecological viewpoint, they are seen to be major contributors to biospheric diversity, structure and function. This regulation of the entire physical and chemical dynamics of lakes and the resultant metabolism is governed to a great extent by differences in density. Density increases with increase in concentrations of dissolved salts in an approximately linear fashion.

Water is a vital natural resource, which is essential for multiple purposes. It is an essential constituent of all animal and vegetable matters. It is also an essential ingredient of animal and plant life. Its uses may include drinking and other domestic uses, industrial cooling, power generation, agriculture, transportation and waste disposal. At the present state of national development, agricultural productivity in India, heavily dependent on rainfall and occurring, droughts in various parts of our country during the last decade, have given a series of jolts to the growth of our economy. Growing population, accelerating

6 | P a g e pace of industrialization and intensification of agriculture and also urbanization exert heavy pressure on our vast but limited water resources.

Water Pollution

The Water (Prevention and Control of Pollution) Act, 1974 defines water pollution as

“such contamination of water and such alteration of the physical, chemical or biological properties of water or such discharge of any or trade effluent or any other liquid, gaseous or solid substance into water (whether directly or indirectly) as may or is likely to, create a nuisance or render such water harmful or injurious to the public health or safety, or to domestic, commercial, industrial, agricultural or other legitimate uses, or to the life and health of animals or plants or of aquatic organisms”.

With the increase in the age of the earth, clean water is becoming more precious as water being polluted by several man made activities, e.g. rapid population growth, alarming speed of industrialization and , urbanizations, increasing living standards and wide spheres of other human activities. Ground water, surface water, rivers, sea, lakes, ponds etc are finding more and more difficult to escape from pollution. The term water pollution refers to anything causing change in the diversity of aquatic life. The presence of too much of undesirable foreign substance in water is responsible for water pollution. Water pollution is one of the most serious problems faced by man today. Since water is the vital concern for mankind and essential for man, animal and aquatic. It is the universal enabling chemical which is capable of dissolving or carrying in suspension of a variety of a toxic materials from mainly heavy flux of sewage, industrial effluents, domestic and agricultural waste. That is why it is of special interest to study the water pollution.

Sources of Water Pollution

Sources of contamination of water pollution are as follows.

7 | P a g e i) Sewage and Domestic Wastes. ii) Industrial Effluents iii) Agricultural Discharges iv) Pesticides and Fertilizers v) Soap and Detergents vi) Thermal Pollution etc. i) Sewage and Domestic Wastes

About 75% of water pollution is caused by sewage and domestic wastes. If the domestic waste and sewage are not properly handled after they are produced and are directly discharged into water bodies, the water gets polluted. Domestic sewage contains decomposable organic matter which exerts on oxygen demand and sewage contains oxidisable and fermentable matter which causes depletion of Dissolved Oxygen (DO) level in the water bodies. ii) Industrial Effluents

Industrial effluents are discharged into water bodies containing toxic chemicals, phenols, aldehyaes, ketones, cyanides, metallic wastes, plasticizers, toxic acids, oil and grease, dyes, suspended solids, radioactive wastes etc. The principal types of industries which contribute to water pollution are chemical and pharmaceutical industries, steel plants, coal, soap and detergents, paper and pulp, distilleries, tanneries, foods processing plants etc.

These effluents when discharged through sewage system poison the biological purification mechanism of sewage treatment and pose several pollution problems. iii) Agricultural Discharge

Plant nutrients, pesticides, insecticides, herbicides and fertilizers plants and animal debries are reported to cause heavy pollution to water sources. Now a day fertilizers containing phosphates and nitrates are added to soil, some of these are washed off through rain fall,

8 | P a g e irrigation and drainage into water bodies thereby severely disturbing aquatic system.

Organic wastes increase the Biological Oxygen Demands (BOD) of the receiving water body. Some pesticides which are non-biodegradable, when sprayed remain in the soil for longer time and then carried in water bodies during rainfall. iv) Soap and Detergents

Soap formed the oleic acid and fatty acid, when contact with water. Thus acidity of water increases which perturb the aquatic life. Detergents used as cleaning agents containing several pollutants which severely affect the water bodies. They contain surface activity agents and contribute to phosphates of sodium, silicates, sulphates and several other salt builders in water. Waste water contaminated with detergents carries a huge cap of foam, which is anesthetic for all purposes. Since detergents are composed of complex shosphates, they increase the concentration of phosphates in water making it poisonous and causing eutrophication problems. v) Thermal Pollution

The discharge of pollutants with unutilized heat from nuclear and thermal power adversely affects the aquatic environment. Apart from the electric power plant, various other industries with cooling system, contribute thermal loading of water bodies. These pollutants increase the temperature of water and decrease the D.O value thus making condition unsuitable for aquatic life.

Loktak Lake: An Important Ramsar

Wetlands are defined as ‘lands transitional between terrestrial and aquatic eco-systems where the water table is usually at or near the surface or the land is covered by shallow water (Mitsch and Gosselink, 1986). Nowadays, are fast declining and rapidly deteriorating ecosystems in the world. People around the world will have to make concerted effort for the abatement towards degradation of the lakes. Fresh water lakes are

9 | P a g e of much importance to mankind but they occupy a relatively small portion of the earth’s surface as compared to the marine and terrestrial habitats (Santra, 2001). The Loktak Lake, the largest fresh water lake in is rich in biodiversity and it is considered to be the ‘lifelines for the people of Manipur’ due to its importance in their socio-economic and cultural life (Tombi and Shyamananda, 1994) and has been recognised as a Wetland of

International Importance (Ramsar site no. 463, declared on 16th June, 1993). The

‘phoomdi’ (a Manipuri word meaning floating mats of soil and vegetation) a heterogeneous mass of soil, vegetation and organic matter in different stages of decay, which has a unique ecosystem and it is only found in this particular lake.

Distribution

India has 2167 recorded natural wetlands, covering an area of 1.5 million hectares. Further, there are 65,254 artificial wetlands, spreading over an area of 0.25 million hactares

(Kumar, 1999). Indian wetlands cover the whole range of the ecosystem types found including natural wetlands of the high altitude Himalayan lakes, followed by wetlands of flood plains of the major river systems, saline wetlands and wetlands of the arid and semi- arid regions, coastal wetlands such as lagoons and estuaries; mangrove swamps; coral reefs and marine wetlands, and so on (Prasad et al., 2002). The biodiversity rich northeast India accounts for three Ramsar sites in the country i.e. the Deepor Beel in , Rudra Sagar in and the Loktak Lake in Manipur, being the major wetland-areas in the region.

Values

The Loktak Lake performs numerous valuable functions. It recycle the nutrients, purify water, recharge ground water, provides drinking water, fish, fodder, fuel, wildlife habitat, control rate of runoff in urban area, and act as a recreation centre in the state. The interaction of man with wetlands during the last few decades has been of concern largely due to the rapid population growth accompanied by intensified industrial, commercial and

10 | P a g e residential development further leading to pollution of wetlands by domestic, industrial sewage, and agricultural run-offs as fertilizers, insecticides and feedlot wastes (Prasad et al., 2002). The fact that wetland values are overlooked has resulted in threat to the source of these benefits. Wetlands are often described as “kidneys of the landscape” (Mitsch and

Gosselink, 1986). Hydrologic conditions can directly modify or change chemical and physical properties such as nutrient availability, degree of substrate anoxia, soil salinity, sediment properties and pH. These modifications of the physiochemical environment, in turn, have a direct impact on the biotic response in the wetland (Gosselink and Turner,

1978).

Diversity and resources

The lake supports varied types of habitat due to which the lake is blessed with rich diversity flora and fauna. The lake sustained the lives of the wildlife (flora and fauna) and people who live on the phoomdis.

Animal resources

The Lake is the natural habitat of the most endangered ungulate species, the brow antlered deer (Cervus eldi eldi) locally known as (Kosygin and Dhamendra, 2009). The

Keibul Lamjao National Park, which is approximately 40.5 km2 is the largest among the phoomdis in the lake and is home to the Sangai. 425 species of animals (249 vertebrates and 176 invertebrates) have been spotted in the lake. Rare animals like Indian python, samber and barking deer, Muntiacus muntjak, rhesus monkey, , hog deer, otter, wild boar, fox, jungle cat, golden cat, sambar, etc are found in the lake. 116 species of , including 21 species of waterfowl have been spotted in the lake. Varied species of water fowl and migratory birds could be spotted during November to March. It is also an

Important area as it’s being a potential breeding site for some waterfowl and is a staging site for migratory birds especially from Siberia. The prominent bird species found

11 | P a g e are east Himalayan pied kingfisher, , black kite, lesser skylark, northern hill myna, lesser eastern jungle crow, yellow headed wagtail, spotbill duck, Indian white breasted water hen, among others. Among the vertebrates fishes including Channa striatus and Channa punctatus are found in the park. Fish yield from the lake is reported to be

1500 tonnes every year. Amphibian and reptiles of the park include keel black tortoise, viper, krait, Asian rat snake and python were also found (Anonymous, 2014a; Anonymous,

2014b). Due to anthropogenic activities, agricultural expansion and draining of polluted water to the lake from different sources, the faunal growth and population became consistently decreasing year after year. It specially affects the Sangai (Indeginous deer found only in Loktak Lake) keeping under the near extinct status.

Plant Resources

Ecological studies of the Loktak Lake reported that altogether 86 macrophytic plant species are found distributed in the lake in different seasons of the year. Eichhorinia crassipes, Euryale ferox, Nelumbo nucifera, Nymphea pubescence, Nymphoides indicum,

Trapa natans were the most common species. 13 macrophyte species i.e Ceratophyllum demersum, Eichhornia crassipes, Euryale ferox, Hydrilla verticillata, Nymphoides cristatum, Pistia stratiotes, Potamogeton crispus, Salvinia cucullata, Salvinia natans,

Trapa Natans, Urticularia exoleta, Urticularia flexuosa, and Vallisnaria spiralis, are found distributed in all months of the year (Devi and Sharma, 2002). Important vegetation of the phoomdis includes Eicchornia crassipes, Phragmites karka, Oryza sativa, Zizania latifolia, Cynodon spp., Limnophila spp., Sagittaria spp., Saccharum latifolium, Erianthus pucerus, Erianthus ravennae, Lersia hexandra, Carex spp., etc. are the most dominant species and the floating plants includes Nelumbo nucifera, Trapa natans, Euryale ferox,

Nymphaea alba, Nymphaea nouchali, Nymphaea stellata and Nymphoides indica (Sanjit,

2005).

12 | P a g e

Species of economic importance like Trapa natans, Euryale ferox, Nelumbo nucifera, Nympheae spp. have been found much decreased in the growth and production over the year.

The Depleting

The last few decades act as a very critical time in the degradation of Loktak Lake, due to the agricultural activities expansion. This is due to the activities of the people inhabiting around the lake and also with some river which are drained into the Loktak Lake. The river which drain directly in the lake i.e. Potsangbam, Awang khujairok, Thongjarok,

Merakhong, Nambol and Nambul, are mostly contained heavy load of agricultural chemicals as well as domestic waste from different sources of the city into the lake water and may contribute significantly to water quality deterioration of the lake and if it is not taken into consideration then it may also results in eutrophication of the lake (LDA.

2011). As a result, there is an enormous increase in the unwanted plants production leading to the increase of “Phoomdi”. The lake is also gradually silting and the pollution of its water is increasing day by day due to those activities which lead to shrinkage of the lake

(Roy, 1992). Direct discharge of urban waste from Imphal city into the Loktak Lake through Nambul River has become one of the main causes for polluting the Lake. Some expected heavy metal contamination might be there in the lake due to the excessive discharge of waste from the Imphal city in the river especially Nambul river which after drain into the lake directly as heavy metals are known to have adverse or detrimental effect on the environment and human health by their carcinogenic effect. Some elements were detected in the lake water such as Mg, Al, P, S, Cl, K, Ca, Mn, Fe, Cu, Zn, Se, Br, Rb, and

Sr in which some of them were detected with high concentration leading to the fact that there is some complex relationship between environmental concentrations and bioaccumulation (Singh et al., 2014). Internally the Loktak Lake is getting weakened and

13 | P a g e externally disturbed with dismal future in all manifestation. The water quality of the

Loktak lake, in general, falls within class C to E as per the CPCB's designated best use criteria and the lake water is not fit for direct drinking without treatment but can be used for irrigation purposes (Rai and Raleng, 2011).

Phytoremediation

The use of plants for the removal of pollutants from the environment is termed as phytoremedition (Gardea Torresdey, 2005). Phytoremediation refers to the natural ability of certain plants called hyperaccumulators to bioaccumulate, degrade, or render harmless contaminants in soils, water, or air (Anonymous, 2014c). Phytoremediation is an emerging technology that uses various plants to degrade, extract, contain, or immobilize contaminants from soil and water. This technology has been receiving attention lately as an innovative, cost-effective alternative to the more established treatment methods used at hazardous waste sites. The huge cost burden has opened a path to the marketplace for an innovative technology. The removal of heavy metals using living organisms has recently been attracting a lot of public attention and research and development spending. The expansion of this research work will promote the eco-friendly and cost-effective technology as phytoremediation. Phytoremediation promotes the use of plants for environmental cleanup.

Role of Phytoremediation

The use of phytoremediation as an abatement of water pollution was studied by many researchers. Many macrophytic plants were used to study by researchers to accumulate metals to levels greatly in excess of these in their environment. Macrophytes such as

Eichhornia crassipes, Azolla pinnata, Hydrilla verticillata, Lemna minor, Ipomoea aquatica, Marsilea quadrifolia, Vallisneria spiralis, etc. were use for the abatement of the pollution. Ceratophyllum demersum, Echinochloa pyramidalis, Eichhornia crassipes,

14 | P a g e

Myriophyllum spicatum, Phragmites australis, and Typha domingensis can survive in extreme conditions and can tolerate very high concentrations of heavy metals which make them an excellent choice for phytoremediation and Biomonitoring programs (Fawzy et al.,

2012). Aquatic plants absorb elements through roots and/or shoots (Pip and Stepaniuk,

1992.). In aquatic systems, where pollutant inputs are discontinuous and pollutants are quickly diluted, analyses of plant tissues provide time-integrated information about the quality of the system (Baldantoni et al., 2005). Exhaustive monitoring and assessment of heavy metal pollution and phytoremediation experiments through diverse macrophytes were performed in Singrauli Industrial Region, India (Rai, 2008a, 2008b, 2008c; Rai and

Tripathi, 2009), and subsequently human health implications were assessed indirectly (Rai,

2008b). The use of aquatic macrophytes, such as Azolla pinnata with hyper accumulating ability is known to be an environmentally friendly option to restore polluted aquatic resources (Sood et al., 2012). Once the pollutant are released to the water bodies, only the plants are the only hope which can cleaned up the pollutants by absorbing and metabolising them from the water bodies. Therefore, the plant role in the water pollution abatement is very much important in the present era of rapid industrialization and urbanization.

Phytoremediation technique using the plant species found in the lake will benefit in the prevention against further pollution of the water including heavy metal pollution and it will increase the proper knowledge of utilising the particular plant species which is very much important for the wetland as well as for biodiversity conservation of this lake.

15 | P a g e

There are scanty researches which studied physico-chemical characteristics, heavy metal, phytosociology as well as phytoremediation studies in totality. Henceforth, present study aimed:

 To assess the heavy metal pollutants of the Loktak lake.

 To perform the phytosociological studies of macrophytes.

 To perform heavy metal phytoremediation investigations with selected

macrophytes of the Loktak lake.

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Chapter 2 REVIEW OF LITERATURE

Fresh water resources are extremely relevant for the sustainable development of people present in current Anthropocene era. Wetland being a major fresh water source, the need of its conservation from the contamination is increasingly important. It is the most productive ecosystems on the Earth (Ghermandi et al., 2008). Wetland is defined as land which is transitional between terrestrial and aquatic systems where the table is usually at or near the surface, or the land is periodically covered with shallow water, and which under normal circumstance supports vegetation typically adapted to life in water saturated soil

(Oberholster et al., 2014). Wetlands are categorized into Marine (coastal wetlands), estuarine (including deltas, tidal , and mangrove swamps), lacustarine (lakes), riverine (rivers and ), and palustarine (‘marshy’- marshes, swamps and bogs) based on their hydrological, ecological and geological characteristics (Cowardin et al., 1979;

Bassi et al., 2014).

In current scenario, water in most Ramsar wetlands has been heavily degraded, mainly due to agricultural runoff of pesticides and fertilizers, and industrial and municipal wastewater discharges, all of which cause widespread eutrophication (Prasad et al., 2002;

Liu and Diamond, 2005). Heavy metal pollution in aquatic ecosystems has become most important point for discussion in recent years. Sources of heavy metals are coal mining and its allied industries e.g. thermal power plants and also chemical industries e.g. chlor-alkali plants in developing countries like India. Effluents of these industries pose serious threats to water quality and aquatic biodiversity of rivers, lakes and reservoirs (Rai, 2011, 2012).

The problem of heavy metal pollution is emerging as a matter of concern at local, regional and also at global scales. Heavy metal pollution in aquatic ecosystem pose serious threat to

17 | P a g e aquatic biodiversity and drinking of contaminated water pose severe health hazards in humans. Therefore, focus of attention has been shifted from mere monitoring of environmental conditions to the development of alternative means to solve the environmental problems at local and global levels. The economic aspects and side effects of conventional treatment technologies in aquatic ecosystems paved way to phytoremediation technology. In phytoremediation plants are used to ameliorate the environment from various hazardous pollutants. It is cost-effective and eco-friendly technology for environmental clean-up (Rai, 2011).

Importance of wetlands

Wetlands possess significant high ecosystem service value (Costanza et al. 1997; Zhang et al. 2013). Wetlands have long been providing , irrigation and domestic water supply, recreational and tourisms (Jain et al., 2007). Wetlands are the most productive ecosystems on the Earth (Ghermandi et al., 2008). Wetlands are noted for contributing ground water recharge, support a rich diversity of aquatic flora and fauna (Bassi et al.,

2014), improving water quality, abating flood waters, supporting biodiversity, and storing carbon (Moreno-Mateos et al., 2012; Doherty et al., 2014). Wetlands which include peat- lands, mangroves, mires, marshes, and swamps help in carbon cycle (Bassi et al., 2014).

Wetlands sediments stores carbon in long-term while wetlands biomass of plants, animals, bacteria, fungi and dissolved components in surface and ground water stores in short term

(Wylynko, 1999). Natural wetlands have been used for centuries as a sink for waste, being capable of assimilation large amounts of environmental pollutants (Sheoran and Sheoran,

2006). Wetlands act as a low cost measure to reduce point and non-point pollution

(Bystrom et al., 2000). Wetlands also act as the ‘Kidney of Nature’ which have the capability of trapping and/or efficiently modify broad spectrum of contaminants (Mander

18 | P a g e and Mitsch, 2009). These ecosystems have special characteristics which make them particularly suitable for wastewater treatment: they are semiaquatic systems which normally contain large quantities of water; they have oxic and partly anoxic soils where the biodegradation of organic matter takes place; and they support a highly productive, tall emergent vegetation capable of taking up large amount of nutrients which enhance growth

(Verhoeven and Meuleman, 1999; Arroyo et al., 2013). They support wildlife which may include many rare, threatened and endangered species (EPW, 2004) and perform various ecological functions.

Wetlands are one of the fast deteriorating ecosystems due to urbanization which led to the contribution of increasing pollution and contamination. The introduction of relatively recent anthropogenic originated toxic substances including heavy metals and their massive relocation to different environmental compartments, especially water, has resulted in severe pressure on the self-cleansing capacity of aquatic ecosystems (Rai,

2009a). The heavy metal has serious deleterious effect on the living organisms especially on human. Attaining sustainability and following green approach in pollution remediation phytotechnologies is the need of the hour (Rai, 2012).

Macrophytes as green bio-resource

Macrophytes play a major role in the structural and functional aspects of aquatic ecosystems by altering water movement regimes, providing shelter to fish and aquatic invertebrates, serving as a food source, and altering water quality by regulating oxygen balance, nutrient cycles, and accumulating heavy metals (Sood et al., 2012). Macrophytes have natural abilities to take up, accumulate or degrade organic and inorganic substances

(Lasat, 2000; McIntyre, 2003), heavy metals through the process of bioaccumulation

(Tiwari et al., 2007). On the other hand macrophytes can bioaccumulate, biomagnificate or

19 | P a g e biotransfer certain metals to concentrations high enough to bring about harmful effects

(Opuene et al., 2008). Some macrophyte species are adapted to grow in the areas of higher metal concentrations (Chatterjee et al., 2011), some of which are having indispensable property of metal tolerance (Singh et al., 2003; Chatterjee et al., 2004; Bertrand and

Poirier, 2005). Various species show different behaviour regarding their ability to accumulate elements in roots, stems and/or leaves (Kumar et al., 2008). Therefore, it is necessary and useful to identify the plants organ that absorbs the greatest amount of trace elements (St-Cyr and Campbell, 1994; Baldantoni et al., 2004).

In wetland aquatic ecosystems where pollutants are discontinuous and pollutants are quickly diluted, analyses of plant components provide time-integrated information about the quality of the ecosystem (Baldantoni et al., 2005). Conventional technologies such as electrolysis, reverse osmosis, ion exchange, etc, were used for cleaning heavy metal (Rai, 2009a). Use of such macrophyte species in remediation of water contaminated with heavy metals is a promising cost-effective alternative to the more established treatment methods (Chatterjee et al., 2011).

Heavy metals

Heavy metals are metals of the d block having a specific gravity greater than 5 g/cm3

(Nies, 1999; Rai, 2012). Increasing industrialization and urbanization has given an increasing problem of heavy metals which are listed as priority pollutants by the US-

Environmental Protection Agency. There are over 70,000 chemicals in use in the world, and over 4,000,000 on the American Chemical Society’s computer list of chemicals

(Cairns et al., 1988; Rai, 2009a; Rai, 2012). As hazards lead, mercury, arsenic, and cadmium are ranked first, second, third, and sixth hazards respectively on the list of the US

Agency for Toxic Substances and Disease Registry (ATSDR), which lists all hazards

20 | P a g e present in toxic waste sites according to their prevalence and the severity of their toxicity

(Rai, 2009a).

Pollution of the biosphere with toxic metals has accelerated dramatically since the beginning of the Industrial Revolution (Nriagu, 1979). The problem of heavy metal pollution is emerging as a matter of concern at local, regional and also at global scales. In

Japan, 2252 peoples were affected and 1043 died due to Minamata disease over the past few decades, caused by elevated mercury pollution from a chemical plant (Kudo and

Miyahara, 1991; Rai, 2011, 2012). Recently, in developing countries major problem have arisen such as ground water contamination with As in Bangladesh (Alam et al., 2003; Rai,

2009a, 2012) and heavy metals contamination for example Cd, Pb, Cu and Zn in drinking water sources in Bolivia, Hong Kong and Berlin (Ho et al., 2003; Zietz et al., 2003; Miller et al., 2004). Heavy metals in surface water systems can be from natural or anthropogenic sources. Currently, anthropogenic inputs of metals exceed natural inputs. High levels of

Cd, Cu, Pb, and Fe can act as ecological toxins in aquatic and terrestrial ecosystems

(Balsberg-Pahlsson, 1989; Guilizzoni, 1991; Rai, 2012). Excess metal levels in surface water may pose a health risk to humans and to the environment.

Sources of Heavy metals

Heavy metals in essential and non-essential forms are naturally persistent in the environment (Alhashemi et al., 2011). The inorganic pollutants present in the aquatic bodies originate from natural and anthropogenic sources (Mdegela et al., 2009), but the occurrence of heavy metals in the environment is mainly due to anthropogenic sources

(Zhipeng et al., 2009). The point source discharges of municipal sewage, industrial facility effluents, and non-point-source discharges from domestic waste, fisheries, and agriculture contaminate the aquatic bodies with the toxic heavy metals (Yuwono et al., 2007).

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Agrochemicals including fertilizer and plant nutrients also lead to the increases in the concentrations of heavy metals in the water and soil (Rattan et al., 2005). These increased contamination of the aquatic bodies leading to serious threat in public health from the consumption of fish from these sources (Bickham et al., 2000; Mayon et al., 2006; Fatima et al., 2014). The toxicity of metals is highly influenced geochemical factors that influence metal bioavailability (Fairbrother et al., 2007). The sediments of the wetland effectively sequester hydrophobic chemical pollutants which are readily available from various pollutant discharges (Harikumar et al., 2009). The discharges of specific local sources from dye formulators and paint (Cd, Cr, Cu, Hg, Pb, Se and Zn), metal based industries

(Cd, Cr and Zn from electroplating), discharges from smelters (Cu, Ni and Pb), petroleum refineries (As and Pb), and effluent from chemical factories may leads to metal accumulation in the sediments (Al-Masri et al., 2002; Harikumar et al., 2009).

Anthropogenic sources of heavy metals

Agriculture seems to be the most considerable source of pollution due to runoff from fertilized land (Milovanovic, 2007; Rai and Tripathi, 2008; Rai, 2009a, 2011, 2012).

Industrial processing and solid waste dumps are considered to be the main anthropogenic sources of metal pollution. As a result of mining and metal working in ancient times, a close link has been demonstrated between metal pollution and human history (Nriagu,

1996; Rai, 2011, 2012). The primary sources of metal pollution are the burning of fossil fuels, mining and smelting of metalliferous ores, municipal wastes, fertilizers, pesticides and sewage (Rai, 2007a,2007b, 2008a, 2008b, 2008c, 2009a, 2009b, 2010a, 2010b, 2010c,

2010d; Rai and Tripathi, 2009; Rai et al., 2010). Heavy metal contamination and acid mine drainage (AMD) are very important concerns where waste materials containing metal-rich sulphides from mining activity have been stored or abandoned (Concas et al., 2006. Rai,

22 | P a g e

2009a). Further sources are coal mining (Finkelman and Gross, 1999) and its allied industries e.g. thermal power plants and also chemical industries e.g. chlor-alkali plants are major sources of heavy metals in industrial belts in developing countries like India

(Sharma, 2003; Rai et al., 2007). Effluents of these industries pose serious threats to water quality and aquatic biodiversity of rivers, lakes and reservoirs.

Major sources of mercury include gold and silver mine, the coal industry, untreated discarded batteries, industrial waste disposal (Pilon-Smits and Pilon, 2000; Malar et al.,

2014). Element like Cd is used in a number of industries, such as electronic components, electroplating, metal mechanic processing, and mining (Haddam et al., 2011). It can also be found in plastic toys and food containers (Kumar and Pastore, 2007; Liu et al., 2014).

Anthropogenic sources of environmental manganese include sewage sludge, mining and mineral processing, and combustion of fossil fuels (WHO, 2004). The release of these toxic metals in biologically available forms by human activity may damage or alter both natural and man-made ecosystems (Chatterjee et al., 2011).

Heavy metals as pollutants to wetland and their environment

Heavy metal is becoming one of the major threats and important issue for wetland environment. Many heavy metals which are potential toxic materials and environmental pollutants have drawn a great attention in the world (Liu et al., 2009; Zhang et al., 2012;

Montuori et al., 2013, Zhang et al., 2014). The aquatic ecosystem contaminated from heavy metals has been an urgent problem worldwide (Alhashemi et al., 2011). This heavy metals includes Aluminium (Al), Arsenic (As), Cadmium (Cd), Copper (Cu), Cobalt (Co),

Chromium (Cr), Mercury (Hg), Iron (Fe), Lead (Pb), Magnesium (Mg), Manganese (Mn),

Nickel (Ni), Strontium (Sr), Vanadium (V), Zinc (Zn), etc. These heavy metals are persistent toxic substances and are leading cause of aquatic contamination (Gurcu et al.,

23 | P a g e

2010; Fatima et al., 2014). Heavy metal contamination can cause toxicity in biological systems through bioaccumulation and also can cause hazardous effect in the wetlands environment and the associated living organisms through biomagnification (increase of toxic accumulation in organisms through various food chain systems). Its toxicity has been of great concern since it is very important to the health of people and ecology (Feng et al.,

2008). Occurrence of toxic heavy metals in lake, reservoir, and river water affects the lives of local people that depend upon these water sources for their daily requirements (Rai et al., 2002).

Wetlands have long been recognized as an important heavy metal sink due to the process of chemical, physical and biological involving adsorption, precipitation, sedimentation, setting, and induced biogeochemical changes by plant and bacteria

(Chandra et al., 2013; Jiao et al., 2014; Xin et al., 2014). The aquatic bodies are being degraded by human activities and discharge of environmental pollutants such as persistent organic pollutants (Dsikowitzky et al., 2011) and heavy metals (Noegrohati, 2005). The persistent heavy metal tissue bioaccumulation, due to their redox cycling and their ability to deteriorate plant tissue sulfahydryl groups, leads to oxidative stress, which can be seen through genotoxic parameters such as micronucleus text (MNT) and comet assay (Ali et al., 2009). Excessive concentration of heavy metals in soil solution such as Co, Cu, Fe,

Mn, Mo, Ni, and Zn limits the plant enzyme/protein functions, metabolism, plant growth and development (Mengel at al., 2001; Hänsch and Mendel, 2009). Many microorganisms have genetic variability that allows them to circumvent the toxic effect caused by the heavy metals but the more developed organisms usually succumb to the toxic effects

(Valdman et al., 2001).

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Health impact of heavy metal pollution

Toxic metal contamination of surface and ground waters poses a major environmental and health problem which is still in need of an effective and affordable technological solution

(Rai, 2010a, 2010b, 2010c, 2010d; Rai et al., 2010). Heavy metal exposure of the population may cause neurobehavioral disorders, such as fatigue, insomnia, decreased concentration, depression, irritability, sensory and motor symptoms (Hanninen and

Lindstrom, 1979). Exposure to heavy metals has been linked to developmental retardation, various types of cancers, kidney damage, autoimmunity and even death in some instances of exposure to very high concentrations (Glover- Kerkvilet, 1995). More specifically, methyl-mercury intake through fish and aquatic foods can have a considerable effect on human health. At higher levels, mercury can damage vital organs such as lungs and kidneys. Methyl-mercury may cross the placental barriers and cause foetal brain damage

(Sharma, 2003; Rai, 2009a). Accumulation of Cd in human bodies (principally in kidney and liver) can cause renal dysfunction and bone disease such as Itai-Itai in Japan

(Nordberg, 1996). High concentrations of cadmium have been reported in sewage, irrigation water and vegetables grown in the Gangetic plain of eastern and western regions of India have resulted in carcinoma of gallbladder and production of stones with concentration of cadmium, chromium and lead concentrations being significantly higher in carcinoma of gallbladder than in gallstones (Shukla et al., 1998; Rai and Tripathi, 2008a). Lead poisoning in children causes neurological damage leading to reduced intelligence, loss of short term memory, learning disabilities and coordination problems. The effects of arsenic include cardiovascular problems, skin cancer and other skin effects, peripheral neuropathy and kidney damage (WHO, 1997).

Numerous human health problems such as developmental, reproductive, cardiovascular, gastrointestinal, dermal, immunological, hepatic, haematological, renal,

25 | P a g e neurological, respiratory, genotoxic, mutagenic and carcinogenic effects (such as liver cancer) (Lin et al., 2013; Singh and Prasad, 2015) were cause by heavy metals. Toxic heavy metals such as Pb, Cr and As can cause a serious problems to animal and human such as irritation of sensory organs, respiratory troubles and arsenic poisoning (Yu et al.,

2014). Drinking water containing As and Cd can cause cancer (Steinemann, 2000),

Allergies, hyperpigmentation (Wongsasuluk, 2014) and may also seriously damage the kidney, liver, digestive system, nervous system of human (Wcislo et al., 2002: Li et al.,

2008), Kidneys, lungs, brain and bones (PCD, 2000). Chronic exposure to low doses of cancer-causing heavy metals may induce many types of cancer (Singh and Prasad, 2015).

Cd is carcinogenic heavy metal (Lauwerys, 1979) and can reside inside the body for half- life of 38 years (Berman, 1980). Cd can infiltrate into organisms through respiratory tract and skin and then damage tissues and organs owing to its interactions with biological macromolecules (Nursita et al., 2009). Pb is also effects health such as cardiovascular, nervous system blood and bone diseases (Jarup, 2003).

Another reason for toxic heavy metals causing concern is that they may be transferred and accumulated in the bodies of animals or human beings through food chain, which will probably cause DNA damage and carcinogenic effects due to their mutagenic ability (Knasmuller et al., 1998; Rai, 2008b, 2009a). For example, some species of Cd, Cr, and Cu have been associated with health effects, ranging from dermatitis to various types of cancer (Das et al., 1997; McLaughlin, 1999). In addition, some metals occur in the environment as radioactive isotopes (e.g. 238U, 137Cs, 239Pu, 90Sr), which can greatly increase the health risk (Pilon-Smits and Pilon, 2002).

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Heavy metal and the source management

The chemicals management methodology with reference to heavy metals should be:

• Compatible

• Cost effective

• Flexible enough to handle fluctuations in the quality and quantity of effluent feed;

• Reliable by operating continuously;

• Robust enough to minimize supervision and maintenance;

• Selective enough to remove only the contaminant metals under consideration; and

• Simple enough to minimize automation and the need for skilled operators (Eccles, 1999;

Rai, 2012).

Alternative green sustainable technology-phytoremediation

In 1990’s, there were considerable interest in developing sustainable, cost effective technologies for remediation of heavy metal-contaminated soil and water (Lasat, 2000).

The removal of heavy metals using living organisms has recently been attracting a lot of public attention and R&D (research and development) spending (Rai, 2009a). The use of plants for remediation of metals offers an attractive alternative, because it is solar driven and can be carried out in situ, minimizing cost and human exposure (Salt et al., 1998).

Eccles (1999) through cost benefit analysis proved that biological processes for heavy metals removal are cheaper, when compared to the conventional technologies. The term

‘phytoremediation’ which consist of ‘phyto’ in Greek means plant and ‘remedium’ in

Latin means correct evil (Erakhrumen and Agbontalor, 2007), was coined in the year 1991

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(Sachdeva and Sharma, 2012). Phytoremediation is defined as the use of plants and their associated microbes to extract, sequester, and/or detoxify various kinds of environmental pollutions from water, sediments, soils and air (Memom and Schröder, 2009). It is the plant based green technology that received increasing attention after the discovery of hyperaccumulating plants which are able to accumulate, translocate, and concentrate high amount of hazardous elements in the harvestable part (Rahman and Hasegawa, 2011).

Phytoremediation is a relatively new approach to the cost-effective treatment of wastewater, and soils contaminated by organic xenobiotics, heavy metals, and radionuclides (Gardea-Torresdey, 2003). While there are numerous descriptions of the term, phytoremediation can be summed up with one clear definition: the use of plants for the removal of pollutants from the environment (Gardea-Torresdey, 2003).

Phytoremediation mechanisms in macrophytes

Phytoremediation mechanism consists of several processes such as phytoextraction, rhizofiltration, phytostabilization, phytovolatilization and phytotransformation or phytodegradation. Each of the process have different role in the accumulation and remediation of the heavy metals.

(1) Phytoextraction: Phytoextraction is the use of plants to remove the metals form the

aquatic bodies by accumulating in its tissues of different parts particularly to the

harvestable roots and the shoots (Kumar et al., 1995) and burned to obtain the

metal (Erakhrumen and Agbontalor, 2007). It is based on the hyperaccumulation

mechanisms. Some hyperaccumulator plants absorb unusually large amounts of

metals compared to other plants and the ambient metals concentration

(Padmavathiamma and Li, 2007). The process phytoextraction involves

accumulation, heavy metal liquefaction transformed them into relatively stable

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metal fractions (oxidizable and residual fractions) leading to decreased

bioavailability and eco-toxicity of heavy metals (Yuan et al., 2011). Two basic

important strategies of phytoextraction have developed after several approaches

(Salt et al., 1995, 1997). Phytoextraction comprised of

a. Chelate assisted phytoextraction or induced phytoextration, in which artificial

chelates are added to increase the mobility and uptake of metal contaminant.

b. Continuous phytoextraction in this the removal of metal depends on the natural

ability of the plant to remediate; only the number of plant growth repetitions

are controlled.

(2) Rhizofiltration: Rhizofiltration is the absorption of metals by macrophyte roots

from the aquatic bodies, precipitate and concentrate in their biomass (Erdei et al.,

2005; Dushenkhov et al., 1995). Wetlands plants perform rhizofiltration process to

accumulate and concentrate metals. The process involves raising plants

hydroponically and transplanting them into metal polluted waters where plants

absorb and concentrate the metals in their roots and shoots (Dushenkhov et al.,

1995; Flathhman and Lanza, 1998; Salt et al., 1995). The process involves

chemisorption, complexation, ion exchange, micro precipitate, hydroxide

condensation onto the biosurface, and surface adsorption (Gardea-Torresdey et al.,

2004). The efficiency of these process can be increased by using plants with

heightened ability to absorp and translocate metals (Zhu et al., 1999). Macrophytes

such as Eichhornia crassipes, Lemna minor, Azolla pinnata etc. are being used for

rhizofiltration for the treatment of contaminated wetland (Rai, 2009a).

Rhizofiltration will be a particularly cost-competitive technology in the treatment

of surface or ground water containing relatively low concentration of toxic metals

(Salt et al., 1995).

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(3) Phytostabilization: Phytostabilization is the immobilization of metals through the

use of tolerant macrophytes (Salt et al., 1995; Rai, 2009a) by the absorption and

acuumulation in its tissues, adsorption in its roots, or precipitation within the root

space stopping their transfer in soil, alongwith their movement by erosion and

deflation (Erdei et al., 2005). It is not intended to remove metal contaminant from a

site but rather to stabilize them by accumulation in root zones, reduces the mobility

of contaminants and prevents migration to groundwater or air and also reducing the

risk to human health and the environment (Padmavathiamma and Li, 2007). For the

process of phytostabilization appropriate selection of macrophyte is required.

Characteristics of macrophytes include; tolerance to high levels of the

contaminant(s) of concern; high production of root biomass able to immobilize

these contaminants through uptake, precipitation, or reduction; and retention of

applicable contaminants in roots, as opposed to transfer to shoots, to avoid special

handling and disposal of shoots (Padmavathiamma and Li, 2007).

(4) Photovolatilization: Phytovolatilization is the transpiration of absorbed

contaminant by the plants in modified form to the atmosphere (Erdei et al., 2005)

from their foliage (Lone et al., 2008). Plants can volatilize certain metals such as

highly toxic mercury (Hg2+) and methyl mercury by reducing them using the

enzyme, mercuric reductase, to less harmful Hg (Yadav et al., 2010). The

mechanisms undergo in the aerial parts of the macrophyte such as leave

(Mukhopadhyay and Maiti, 2010).

(5) Phytodegradation: Phytodegradation is elimination or degradation of metals by the

macrophytes by its enzyme or enzyme co-factors (Susarla et al., 2002) and its

associated microbes (Garbisu and Alkorta, 2001). The enzymes such as

dehalogenases, oxygenases and reductases degrade the compounds that contain

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metals inside the plant bodies (Black, 1995). It is also known as

phytotranformation (Mani and Kumar, 2014). Degradation may also occur outside

the plant, due to the release of compounds that cause the transformation

(Mukhopadhyay and Maiti, 2010). With help of the associated microbes in root

zone (rhizosphere) of the plant, rhizodegradation also occurs.

The effectiveness of phytoremediation requires well-planned strategies for the decontamination process. Not all plants can develop in contaminated environments, the first step for the use of phytoremediation is to identify species, which, besides being suitable to local conditions, are tolerant to contaminants and the second step is to evaluate the capacity of the plant to promote decontamination (Marques et al., 2011; Preussler et al., 2014). The plant used for the phytoremediation should have some characteristic features (Sharma et al., 2014). They includes,

a. Fast growing

b. High metal tolerance

c. Resistant to disease, pest etc.

d. Having dense root and shoot system (Couselo et al., 2012)

e. Unattractive to animals so that there should be minimum transfer of metals to

higher trophic levels of terrestrial food chain (Bruce et al., 2003)

f. Should be easy to cultivate and harvest.

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Utility of macrophyte bioresource in phytoremediation

Plants are the most tolerant to pollution, which makes them very useful for new emerging environmental biotechnology – phytoremediation (Gawronski et al., 2011). Many wetlands plants species are successfully used for phytoremediation of heavy metal contaminated water bodies. These wetland aquatic plants and its associated microbes were utilized to absorbed and degrade the metals to prevent from further contamination of the water bodies.

The adequate restoration of these environments requires cooperation, integration and assimilation of such biotechnological advances along with traditional and ethical wisdom to unravel the mystery of nature in the emerging field of bioremediation/phytoremediation

(Mani and Kumar, 2013). Also advances in molecular studies with macrophytes is the need of the hour as it can enhance the efficiency of phytoremediation (Ali et al., 2013).

The metal uptake and distribution within the plants is affected by some factors

(Susarla et al., 2002). They includes,

 Physical and chemical properties of the compound (e.g., water solubility, vapour

pressure, molecular weight, and octanol-water partition coefficient, KOW)

 Environmental characteristics (e.g., temperature, pH, organic matter, and soil

moisture content) and

 Plant Characteristics (e.g., type of root system and type of enzymes).

Macrophytes are beneficial to lake because they provide food and shelter for fish and aquatic invertebrates (Rai, 2008b, 2009a, 2011, 2012). They also produce oxygen, which helps in overall lake functioning, and provide food for some fish and other wildlife.

Macrophytes are considered as important components of the aquatic ecosystem, not only as a food source for aquatic invertebrates, but they also act as an efficient accumulator of heavy metals (Rai, 2008b, 2011, 2012). They are unchangeable biological filters and play an important role in the maintenance of the aquatic ecosystem. Aquatic macrophytes are 32 | P a g e taxonomically closely related to terrestrial plants, but are aquatic phanerogams, which live in a completely different environment. Their characteristics in accumulating metals make them interesting research objects for testing and modelling ecological theories on evolution and plant succession, as well as on nutrient and metal cycling (Fostner and Whittman,

1979; Rai, 2011, 2012). Therefore, it is very important to understand the functions of macrophytes in aquatic ecosystems. The use of aquatic vascular plants for heavy metal phytoremediation is very much emphasized these days for treatment of industrial effluents before discharge into the aquatic ecosystems. Since only aquatic plants can flourish in aquatic environments, naturally requiring simple mineral nutrients and sunlight, they can be conveniently tested for their phytoremediation potential. Aquatic macrophytes e.g. water hyacinth (Eichhornia crassipes), water velvet (Azolla pinnata) and duckweeds

(Lemna minor, Spirodela polyrhiza) are prevalent in lakes, rivers and streams all over the globe. A huge amount of money is invested to remove them from polluted aquatic bodies to conserve the aesthetic character and maintain them as suitable for the eco-tourism industry (Rai, 2009a, 2011, 2012). Wastewater treatment wetlands harbouring rich growth of macrophytes can be both a cost-efficient and effective means to improve water quality before effluents are discharged into major rivers (Hammer, 1992; Kadlec and Knight,

1996; Verhoeven and Mueleman, 1999; Mitsch and Gosselink, 2000; Nzengya and

Wishitemi, 2001; Shutes, 2001; Stone et al., 2004; Nahlik et al., 2006; Rai, 2011, 2012).

Other economic benefits, such as vegetation for animal feed (e.g., floating aquatic plants) and habitat for harvestable fish (e.g. Tilapia), make wetlands an attractive option for meeting water quality standards through nutrient reduction for farmers and small industry (Greenway and Simpson, 1996; Denny, 1997; Rai, 2011, 2012). Free-floating macrophytes provide shading of the water column, thereby providing a cooler habitat for fish and macro invertebrates in what otherwise would be a warm water tropical

33 | P a g e environment. Despite floating aquatic plants providing cooler water temperatures and abundant food sources that help create optimal habitat structure for fish and invertebrates, it has been reported that wetlands with floating aquatics support larger mosquito larvae populations than do open water areas due to reduced dissolved oxygen concentrations

(Greenway et al., 2003; Rai, 2011, 2012).

Water hyacinth (Eichhornia crassipes) is one of the most commonly used plants in constructed wetlands because of its fast growth rate and large uptake of nutrients and contaminants (Rai, 2007a, 2011, 2012). It has been studied for its tendency to bio- accumulate and biomagnify the heavy metal contaminants present in water bodies (Tiwari et al., 2007). It accumulates metals and as the recycling process is run by photosynthetic activity and biomass growth, sustainable process and cost efficient (Garbisu et al., 2002;

Lu et al., 2004; Bertrand and Poirier, 2005). It has the capacity to accumulate metals such as Cd, Cu, Pb and Zn in its root tissues (Nor, 1990). However, it has been reported that the growth of water hyacinth poses problem in functioning of constructed wetlands due to its exotic invasive nature and rapid decomposition in comparison to other plants (Khan et al.,

2000; Rai, 2011, 2012). Macrophytes including Eichhornia crassipes, Pistia stratiotes and

Spirodela polyrrhiza remove Cr from wastewater (Mishra and Tripathi, 2008). Duckweeds

(family Lemnaceae) appear to be the better alternative and have been recommended for industrial effluent treatment as they are (i) more tolerant to cold condition than water hyacinth, (ii) more easily harvested than algae, and (iii) capable of rapid growth (Sharma and Gaur, 1995; Rai, 2011, 2012). Biomass of aquatic macrophytes e.g. Azolla may be used for recycling of municipal wastewater for irrigation by filtering the heavy metals and other pollutants which assist in water resource conservation (Rai, 2007b, 2011, 2012). At the same time the waste biomass of macrophytes produced after the treatment can be used for biogas production in an effort towards achieving sustainability in the energy sector.

34 | P a g e

Azolla pinnata, endemic to India, after treatment with HCl or HNO3 to remove heavy metals absorbed may be of tremendous biofertilizer value due to its association of a cyanobacterium, Anabena azollae (Rai, 2007b, 2011, 2012).

Typha species also act as nutrient pumps, absorbing large amount of nutrients from the sediment and accumulating them in the above ground tissue (Sharma, 2007). Typha augustifolia show higher metal levels in roots than above ground tissues (Wu et al., 2014).

In constructed wetlands Typha domingensis may be a potent macrophytes in the phytoremediation of extremely deleterious mercury (Gomes et al., 2014). Typha domingensis is use in constructed wetlands for enhancement of water quality in water treatment systems (El-Sheikh et al., 2010; Hegazy et al., 2011). It can assess a significant concentration of Cu, Cd, Mg and ash during its growing season (Eid et al, 2012). Cattail

(Typha latifolia) and common reed (Phragmites australis) have been used successfully for the phytoremediation of Pb/Zn mine tailings under water logged conditions (Ye et al.,

2004). Polyculture constructed wetland with Typha latifolia and Phragmites australis may be used to treat mine effluent particularly Boron (Turker et al., 2013). Estuarine sediments colonized by Phragmites australis and Juncus maritimus were spiked with Cd in the absence and presence of an autochthonous microbial consortium resistant to the metal

(Nunes da Silva et al., 2014). They also do not have a significant signs of toxicity in the increase Cd uptake (Nunes da Silva et al., 2014). Certain metals like Zn, Fe, Pb, Cu, Ni,

Cd and Cr were reported to accumulate in higher concentrations inside root followed by leaf and stems of Phalaris arundinacea (reed canary grass), thus making it potent bioresource for trace metals removal from bottom sediments (Polechonska and Klink,

2014). Both roots and shoots of Eleocharis acicularis effectively phytoremediated Indium,

Ag, Pb, Cu, Cd and Zn (Ha et al., 2011).

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Rhizofiltration process involved in accumulation through macrophytes

Macrophytes mostly follow the mechanism of rhizofiltration. Rhizofiltration is the phytoremediative technique designed for the removal of metals in the aquatic environment using macrophytes (Rai, 2009a, 2011, 2012). Therefore, roots are thought to be important for element uptake in free-floating macrophytes (Sharma and Gaur, 1995; Rai, 2011,

2012). Although, aquatic macrophytes have been found to accumulate metals in their shoots also (Greger, 1999; Fritioff et al., 2005), but whether these metals originate from direct uptake from the water or from root-to-shoot translocation is in most cases an open question.

Root exudates and changes in rhizosphere pH also may cause metals to precipitate on root surfaces. As they become saturated with the metal contaminants, roots or whole plants are harvested for disposal (Zhu et al., 1999; Rai, 2011, 2012). Rhizofiltration offers a cost advantage in water treatment because of the ability of plants to remove up to 60% of their dry weight as toxic metals, thus markedly reducing the generation and disposal cost of the hazardous residue. Hence, rhizofiltration will be a particularly cost-competitive technology in the treatment of surface or ground water containing relatively low concentration of toxic metals (Salt et al., 1995; Rai, 2011, 2012).

Most research works have shown that plants for phytoremediation should accumulate metals only in the roots (Dushenkov et al., 1995; Salt et al., 1995; Rai, 2011,

2012). Dushenkov and Kapulnik (2000) explained that the translocation of metals to shoot would decrease the efficiency of rhizofiltration by increasing the amount of contaminated plant residue needing disposal. In contrast, Zhu et al. (1999) suggested that the efficiency of the process can be increased by using plants which have a heightened ability to absorb and translocate metals within the plant. Dushenkov and Kapulnik (2000) described the characteristics of the ideal plants for rhizofiltration. Plants should be able to accumulate 36 | P a g e and tolerate significant amounts of target metals in conjunction with easy handling, low maintenance cost, and a minimum of secondary waste disposal. It is also desirable that plants produce significant amounts of root biomass or root surface area (Rai, 2011, 2012).

Various scientists and researchers perform phytoremediation using many macrophytes for the removal of heavy metals from the aquatic bodies. Most of the workers prefer Eichhornia crassipes as a common plant for the removal of Pb, Cu, Zn, Hg, Cd, Cr and Mn (Tiwari et al., 2007; Kumar et al., 2008; Rai 2009; Rai et al., 2010; Chatterjee et al., 2011; Fawzy et al., 2012; Padmapriya and Murugesan, 2012; Sasidharan et al., 2013;

Mishra et al., 2013). Wetlands plants of more than 60 species were used for phytoremediation studies by various workers. Some of the common macrophytes includes,

Ipomoea aquatic (Kumar et al., 2008), Typha sp (Kumar et al., 2008)., Echinochloa sp.

(Kumar et al., 2008), Hydrilla verticillata (Kumar et al., 2008; Rai, 2009b; Begam and

HariKrishna, 2010), Nelumbo nucifera (Kumar et al., 2008), Vallisneria spiralis (Kumar et al., 2008; Rai and Tripathi, 2009,Rai, 2009b), Aponogeton natans (Rai, 2009b), Cyperus rotundus (Rai, 2009b), Ipomoea aquatic (Rai, 2009b), Marsilea quadrifolia (Rai, 2009b),

Potamogeton pecitnatus (Rai, 2009b), Lemna sp. (Rai et al., 2010), Spirodela polyrhiza,

Azolla pinnata (Rai et al., 2010), Polygonum sp.( Bako and Daudu, 2007; Rai, 2009b),

Ludwigia sp.( Bako and Daudu, 2007), Elodea Canadensis (Begam and HariKrishna,

2010), Salvinia sp. (Begam and HariKrishna, 2010), Wolffia arrhiza (Chatterjee et al.,

2011), Pistia stratiotes (Chatterjee et al., 2011), Trapa bispinosa (Chatterjee et al., 2011),

Cynodon dactylon (Chatterjee et al., 2011), Scirpus sp. (Chatterjee et al., 2011), Colocasia esculenta (Chatterjee et al., 2011), Sagittaria montevidensis (Chatterjee et al., 2011),

Schoenoplectus californicus (Boudet et al., 2011), Ricciocarpus natans (Boudet et al.,

2011), Bidens tripartitus (Branković et al., 2011), Lycopus europaeus (Branković et al.,

2011), Myriophyllum sp. (Fawzy et al., 2012), Phragmites sp. (Fawzy et al., 2012), 37 | P a g e

Spirodela polyrrhiza (Loveson et al., 2013), Juncus effuses (Ladislas et al., 2013), Carex riparia (Ladislas et al., 2013), Jussiaea repens (Mishra et al., 2013), Phragmites australis

(Philippe et al., 2015; Kumari and Tripathi, 2015), Ipomoea aquatic (Mazumdar et al.,

2015), Salvinia minima (Ponce et al., 2015), etc.

Ramsar Sites in India and phytoremediation work

India is having a varying topography and climatic regimes, supports diverse and unique wetland habitats (Prasad et al., 2002). Till date, 201,503 wetlands of India were identified and mapped on 1:50,000 scale (SAC, 2011). Overall India has about 757.06 thousand wetlands of 15.3 m ha (nearly 4.7% of the total geographical area) which include open water, aquatic vegetation (submerged, floating and emergent) and surrounding hydric soils

(Bassi et al., 2014). These wetlands provide numerous products and services to humanity with their unique ecological features (Prasad et al., 2002).

Ramsar sites as per defined as most of the natural bodies such as rivers, lakes, coastal lagoons, mangroves, peat land, coral reefs, etc. constitute the wetland ecosystem. In India there are 26 Ramsar sites or wetlands of International importance (Ramsar secretariat, 2013). Phytoremediation work in India has been done by several researchers and scientists. In Ramsar sites, wetlands of Kashmir

Himalaya were studied to assess the heavy metals such as Al, Mn, Ba, Zn, Cu, Pb, Mo, Co,

Cr, Cd and Ni sequestration capability of the macrophyte species Phragmites australis

(Ahmad, et al., 2014). East Calcutta Wetlands of Kolkata were also studied on the waste metal such as Ca, Cr, Mn, Fe, Cu, Zn and Pb remediation using floriculture of the plant species such as Helianthus annuus, Tagetes patula and Celocia cristata (Chatterjee, 2011).

In the North East India, the three Ramsar sites, Deepor Beel of Assam, Loktak lake of

Manipur and of Tripura has not been studied on phytoremediation. The

38 | P a g e current study will be focusing on the phytoremediation work using the local macrophytes in the Loktak lake of Manipur, which is the largest freshwater lake in the eastern India.

Constructed wetlands and use of phytoremediation

Constructed wetlands are the artificial wetlands planed and developed for the treatment process of contaminated water. They include some biotic and abiotic component found in the natural wetlands such as macrophytes, soil, microorganisms, living organisms etc. EPA

(1993) state that the constructed wetland system are designed to mimic natural wetlands utilising macrophytes, soils and associated microorganisms to remove contaminants from wastewater effluents. The removal of pollutants in these systems relies on a combination of physical, chemical and biological processes that naturally occur in wetlands and are associated with vegetation, sediment and their microbial communities (Farooqi et al.,

2008). They study of large scale constructed wetland can successfully achieve ecosystem functions as replacement for natural wetland and fasten the restoration process, although the restoration effectiveness of ecosystem structures in terms of living biomass and water using energy–value accounting is still inconclusive (Zhang et al., 2013). Phytoremediation and its use in constructed wetlands is one of the effective cheaper technologies comparing to those advanced technology. This promotes sustainable use macrophytes in the treatment.

Creation of the constructed wetlands can be low cost with low technology. The technology is also applicable to large amount of contaminated water. This help in minimising the contamination of heavy metals. Constructed wetland demonstrated to remove significant percentage of Cd, Cu, Pb and Zn from the effluent of road runoff over a period of six years

(Gill et al., 2014). This system is applicable for all the contaminated aquatic bodies which include river, pond, lakes, estuaries etc.

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Nowadays, Constructed wetlands are engineered for effective phytoremediation processes using selective macrophytes. Engineered constructed wetlands or engineered wetlands are special, advanced, semi-passive kinds of constructed wetlands in which operating conditions are more actively monitored, manipulated and controlled in such a manner as to allow contaminant removals to be optimized (Anonymous, 2010). They are aesthetically pleasing, solar driven, passive technique useful for cleaning up wastes including metals, pesticides, crude oil, polyaromatic hydrocarbons, landfill leaches (Zhang et al., 2010), metals and organic contaminants from mine waste, agricultural runoff, industrial effluent (Williams, 2002) and has become increasingly recognized pathway to advance the treatment capacity of wetland systems (Zhang et al, 2010).

Constructed wetlands and natural wetlands

Constructed wetlands are man-made wetlands ecosystem similar to natural wetlands. The most attractive advantage of the natural wetlands is low energy requirement, straightforward operation and maintenance works (García et al., 2001). After observations of the importance of natural wetlands, the constructed wetlands are prepared to meet the need of the human in controlled way. They are designed to perform extra functions than natural wetlands by choosing different selective materials. Most of the constructed wetlands systems were used to clean-up the contaminated water through the process of phytoremediation choosing proportionate wetlands plant. It is created from a non-wetland ecosystem or a former terrestrial environment, mainly for the purpose of contaminant or pollutant removal from wastewater (Hammer, 1994). Compared with natural wetlands, more visits from tourists and lesser financial investment coming in as feedback into the wetland would reduce system environment loading and promote self-support ability, ultimately generating sustainability (Zhang et al., 2013)

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Microbial association and phytoremediation

Most of the plants are always associated with microorganisms which cause them diseases.

They are called endophytes. Endophytes include endophytic bacteria, endophytic fungi and endophytic ectinomycetes (Raghukumar, 2008). All the endophytes do not cause diseases.

Some of them help the plants indirectly. Their activity inside the plants helps and increases in their metabolism forming beneficial association between them. Different mechanisms have been developed by bacteria for avoiding toxicity which include the following (Pavel et al., 2013; Ullah et al., 2014):

a. Active efflux pumps

b. Intra-extracellular sequestration

c. Exclusion through permeable barriers

d. Reduction through enzymes

e. Reduction of cellular sensitivity

Endophytes help plants to enhance growth through phytohormone production, supply nitrogen after nitrogen fixation process, resistance to environmental stresses (heat, cold, drought, salt), producing important medicinal, agricultural, industrial compounds and enhancing phytoremediation after improving uptake of contaminants and degradation of several toxins (Khan and Doty, 2011). They are resistant to heavy metal and capable of degrading the contaminant.

The heavy metal-resistant endophytes belonged to a wide range of taxa; in bacteria these include Arthrobacter, Bacillus, Clostridium, Curtobacterium, Enterobacter,

Leifsonia, Microbacterium, Paenibacillus, Pseudomonas, Xanthomonadaceae,

Staphylococcus, Stenotrophomona, and Sanguibacte, and in fungi Microphaeropsis,

Mucor, Phoma, Alternaria, Peyronellaea, Steganosporium, and Aspergillus (Li et al.,

2012).

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The endophytes are densely colonised inside the plant roots, decreases from stem to the leaves (Porteous-Moore et al., 2006). In comparision with other rhizosphere microorganisms, endophyes interact more closely with their host plants and could more efficiently improve phytoremediation (Zhang et al., 2011). Among the endophytic genera,

Burkholderiaceae, Enterobacteriaceae and Pseudomonaceae are the most common cultivable species found (Khan and Doty, 2011).

Advantages and disadvantages/limitations of phytoremediation

Phytoremdiation technology and its application in the wetland for contamination treatment is a productive method which may have many advantages and disadvantages. Some of the advantages are given below.

 It is adopted by many researchers and scientists because of its low cost, effective

method, easily applicable to wide range of water contaminant, cost free plant for the

method, safe and eco-friendly technology.

 It is low cost option for environmental media, which is suited to large sites which have

relatively low contamination (Ginneken et al., 2007).

 Phytoremediation can be performed aesthetically with clean environment.

 It is also eco-friendly technology and can use only freely available macrophytes for

the process sustainably.

 The biomass production from the technology can also be used in different forms. The

accumulated metals can be obtained from the biomass after burning into ash.

 Phytoremediation can be performed for the treatment of effluents and wastewater of

agriculture, industrial municipal, stormwater etc.

There are also some disadvantages/limitations in phytoremediation.

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 It is a time consuming process and totally dependable to the plant deployed for the

treatment.

 Plant growth takes time and the growth is due to the availability of the proper medium.

 During the process of phytoremediation, plants may be death due to many factors such

as contamination extremity, lake of nutrients, lake of photosynthesis, etc. Due to high

concentration of contaminant, the toxicity level increases which leads to the death of

the plants.

 Appropriate plants selection is required for effective phytoremediation process. The

matured plant growth rate is slow and attends withering processes. Plant with high

growth rate and resistant to environmental stress are more suitable for

phytoremediation. Each plant has the capacity of removing different heavy

metals/contaminants according to their metabolic activities.

 The timely removal of these plants from the site is also required.

 Climatic condition is also one of the important factors supporting the

phytoremediation process.

Future prospects of phytoremediation: Genetic engineering

The application of genetic engineering to modify plants for metal uptake, transport and sequestration may open up new avenues for enhancing efficiency of phytoremediation

(Eapen and D’Souza, 2005). It is a promising way towards the improvment of the phytoremediation efficiency therby enhancing metal tolerance and accumulation properties of plants (Moffat, 1999). Trangenic plants, which detoxify/accumulate metals like As, Cd,

Hg, Pb, Se etc. have been developed (Eapen and D’Souza, 2005). In recent years, several key steps have been identified at the molecular level, enabling us to initiate transgenic approaches to engineer the transition metal content of plants (Clemens et al., 2002).

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Complex interactions of transport and chelating activities control the rates of metal uptale and storage (Rai, 2009a). Metal chelator, metal transporter, metallothionein (MT) such as glutathione (GSH), and phytochelatins (PCs) (Rai, 2009a) genes have been incorporated to plants for improved metal uptake and sequestration (Eapen and D’Souza, 2005). PCs are the family of peptides with general structure (γ-Glu-Cys) n-Gly, where n equals 2-11

(Cobett, 2000; Rauser, 1990), which are rapidly synthesized in response to toxic levels of heavy metals (Cobbett, 1999; Zenk, 1996; Rai, 2009a). PCs are enzymatically synthesized from GSH by phytochelatin synthase (PCS, EC 2.3.2.15) (Cobbett, 2000, Rai, 2009a) and bind heavy metals such as Ag, Ar, Cd, or Cu (Maitani et al., 1996; Schmoger et al., 2000;

Rai, 2009a). PCS genes were cloned from Arabidopsis thaliana, Caenorabditi elegans,

Schizosaccharomyces pombe and Triticum aestivum (Clemens et al., 1999, 2002; Ha et al.,

1999; Vatamaniuk et al., 1999; Rai, 2009a).

Plants such as Populus angustifolia, Nicotiana tabacum and Silene cucubalis have been genetically engineered to overexpress glutamylcysteine synthetase which enhanced heavy metal accumulation as compared with similar wild plants (Fulekar et al., 2009).

Typha latifolia were also used for incorporate standardized Agrobacterium-mediated model transformation system to achieve long term objective of introducing candidate gene for phytoremediation (Nandakumar et al., 2005; Rai, 2009a).

The use of phytoremediation technology using wetland macrophyte bio-resources

should be encouraged in order to replace costly, ineffective and conventional

technologies. It is quite suited for a developing country like India where there is huge

water pollution especially in the wetlands. The use of the invasive plant species like

Eichhornia crassipes for this technology is also an important concept for utilizing freely

available plant resources. The remediation of hazardous heavy metals and other toxic

44 | P a g e contaminants from the wetlands through wetland bio-resources will benefit the associated living being from a toxic free healthy environment. Concerted multidisciplinary approach integrating wetland ecology, pollution science, environmental engineering, genetic engineering, sustainability issue with macrophyte bio-resource may assist in green phyto- technological innovation.

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Chapter 3 MATERIAL AND METHODS

3.1. Description of Study site

Manipur is one of the eight states of Northeast India. The state is bound by in the north, by Mizoram in the south, by Assam in the west, and by the borders of the country Burma in the east as well as in the south. The state capital of Manipur is Imphal.

The state lies at latitude of 23°83’N – 25°68’N and longitude of 93°03’E – 94°78’E. The total area covered by the state is 22,347 km². The capital lies in an oval-shaped valley of approximately 700 square miles (2,000 km2) surrounded by blue mountains and is at an elevation of 790 meters above the sea level. The slope of the valley is from north to south.

The presence of the mountain ranges not only prevents the cold winds from the north from reaching the valley but also acts as a barrier to the cyclonic storms originating from the Bay of Bengal. The southwest monsoon chiefly determines the weather and rainfall throughout the state. The state has tropical to temperate climate depending upon elevation.

Rainfall varies from 1000 mms to 3500 mms and average rainfall is 1500 mms.

Temperature ranges from sub-zero to 36 degrees Celsius. Winter season is from November to February. Then is the Pre-Monsoon season in the months of March and April. The monsoon season is from the month of May to September, and the the post-monsoon season in the month of October and November.

In the heart of Manipur, there lies the Loktak Lake (Map. 3.1), which is rich in biodiversity and considered to be the lifeline of Manipur valley and has been recognised as a Wetland of International Importance (Ramsar site no. 463, declared on 23th March, 1990) which was added in the Montreux Record on the 16th June, 1993. The Loktak lake lies between the latitude of 24°25’– 24°42’N and longitude of 93°46’–93°55’E, located at

Bishnupur district of Manipur and is the largest natural lake in eastern India and its size is

45 | P a g e approximately 26,600 hactares. The lake also supports varied types of habitat due to which the lake is blessed with rich diversity flora and fauna. The ‘phoomdi’ (a Manipuri word meaning floating mats of soil and vegetation) a heterogeneous mass of soil, vegetation and organic matter in different stages of decay, which has a unique ecosystem and it is only found in this particular lake (Ningombam and Bordoloi, 2008). The Keibul Lamjao

National Park, which is approximately 40.5 km2 is the largest among the phoomdis in the

Loktak lake, which is home to the Sangai (Cervus eldi eldi), the Manipuri brow-antlered deer, which is on the brink of extinction. The lake gives sustained the lives of macrophytes, the wildlife and people who live on the phoomdis.

3.2. Selection of sampling sites

Preliminary surveys of the lake was done before selection of the sampling sites for detailed investigation in relation to physico-chemical and phytosociological studies of the water. A total of four sampling sites were selected (in triplicates) for analysis of various physico- chemical characteristics of the water. The study sites (Map 3.2) were selected on the basis of the source pollution and disturbance causing the lake polluted. The Sites are as follows.

1. Site I (Loktak Nambul vicinity):

Site I is the contact point of Loktak Lake and the river, Nambul, which pass

through the Imphal municipal area and then drained in the lake. This river carries

waste of the Imphal municipal area.

2. Site II (Loktak Nambol vicinity):

Site II is the contact point of the Loktak Lake and the river, Nambol, which pass

through the Bishenpur municipal area and then drained in the lake. This river

carries waste of the Bishenpur municipal area.

3. Site III (Loktak Yangoi vicinity):

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Site III is the contact point of the Loktak Lake and the river, Yangoi, which is also

a main source of pollution which drained in the lake.

4. Site IV (Loktak proper):

Site IV is the Joining point of the Loktak Lake and the Canal of National Hydro

Power Corporation Limited (NHPC) of Loktak Lake.

Water sampling were performed in all three seasons i.e. Rainy season (July to October),

Winter season (November to February), and Summer season (March to June) from August,

2013 to July, 2015.

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Map 3.1. Map showing the Loktak lake at Bishenpur District, Manipur.

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Map 3.2. Map showing structure of the Loktak Lake and study sites.

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Photo Plate 3.1. Photo showing study site Site I.

Photo Plate 3.2. Photo showing study site Site II.

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Photo Plate 3.3. Photo showing study site Site III.

Photo Plate 3.4. Photo showing study site Site IV.

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3.3. Analytical Methods

3.3.1. Collection of Samples

Water

Samplings were done in the morning between 6:30 to 9:30 AM and the samples were immediately transported to the laboratory and analysed. Wide mouth bottles were used to collect samples for the analysis. Tag/Labels for each batch and samples were given for easy identification. Thirteen water quality parameters i.e. Temperature, pH, Transperancy,

Total Solids (TS), Dissolved Oxygen (DO), Biological Oxygen Demands (BOD), Acidity,

Alkalinity, Chloride, Hardness, Turbidity, Nitrogen-Nitrate and Phosphate along with the heavy metals analyses were studied. The temperature was measured by digital thermometer and is expressed in degree celsius, pH value was determined by Hanna digital pH-meter, Transparency is measured by using Secchi disc, TS is measured by using filtration and evaporation method, DO and BOD by Winkler Titrimetric method,

Alkalinity and Acidity by using potentiometric titration method, Chloride content was measured by using Mohr’s argentiometric method, Total Hardness by using EDTA titration method, Turbidity by digital turbidity meter, Nitrate content by phenoldisulphonic acid method and Phosphate content by using stannous chloride method.

Macrophytes

Macrophytes sampling were also done in the morning between 6:30 to 9:30 AM and the samples were immediately transported to the laboratory and analysed. Transperant polybags were use for macrophyte samplings. The fresh macrophytes were kept inside the polybags with water in order to prevent drying of the fresh macrophytes samples. Fresh

Macrophyte samples were collected from all sampling sites in different seasons of the year, depending on their growth stages and thoroughly washed with tap water to eliminate remains of lake sediments and were placed in corresponding lake water receiving 8 hours

52 | P a g e of fluorescent light per day. The fresh macrophytes samples were weight and kept in the oven for drying and temperature were maintained at 80ºC for 24 hours. The dried macrophyte samples were again weight and crushed it into powder. The powdered macrophytes samples were then digested using di-acid method.

3.3.2. Analysis

Water

Samples will be analysed immediately within 1-6 hours of collection for physical analysis like Temperature, Turbidity, Transparency, Total Solids, Total Suspended Solids, Total

Dissolved Solids and analysis for chemical parameters like pH, Total Hardness, Acidity,

Alkalinity, Chloride, Nitrate, Phosphate, and Dissolved Oxygen (DO) will be done within

24 hours except for Biological Oxygen Demand (BOD), which takes 5 day for incubation.

The methods for the analysis of various physico-chemical characteristics are as follows.

i. Temperature (Celsius thermometer)

Temperature of water was measured by using a small centigrade thermometer with

the precision of 0.1° C or by means of Digital thermometer. ii. Turbidity (Nephalometer)

Turbidity will be measured either by its effect on the transmission of light which is

termed as Turbidity or by its effect on the scattering of light which is termed as

Nephalometry. Nephalometer will be used for measuring turbidity.

iii. Transparency (Secchi disc)

Transparency will be measured by using Secchi disc. It can be calculated by using

the formula,

Secci Disc Transparency, SDT (cm) = A+B/2

Where,

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A = Depth at which Secchi disc disappears (cm)

B = Depth at which Secchi disc reappears (cm) iv. Total Solids (TS)

Total Solids (TS) will be measured by using filtration and evaporation method.

Total Solids can be calculated by using the formula,

TS (g/l) = W1–W2

Where,

W1 = Final weight of the crucible

W2 = Initial weight of the crucible

V = Volume of water sample evaporated (ml) v. Dissolved Oxygen

Dissolved Oxygen (DO) will be measured by using Winkler’s modified azide

method.

It can be calculated by using the formula,

vi. Biological Oxygen Demand (Winkler’s modified azide method)

Biological Oxygen Demand (BOD) can be measured by using Winkler’s modified

azide method. Determination of DO is required for the analysis of BOD.

The Dissolved Oxygen content can be calculated by using the formula,

Biological Oxygen Demand can be calculated by using the formula

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Biological Oxygen Demand, BOD = DO1 – DO2

Where,

DO1 = Dissolved Oxygen taken before incubation of the sample.

DO2 = Dissolved Oxygen taken after incubation of the sample. vii. pH (digital electronic pH meter)

The pH value of the natural water is an important index of acidity and alkalinity.

pH can be measured by using the Digital pH meter. viii. Hardness (EDTA titration method)

The Hardness will be measured using EDTA titration method. Hardness can be

calculated by using the following formulae, CaCO3

i. Total hardness

Where,

C = Vol, of EDTA required by sample.

D = mg CaCO3 equivalent to 1.0 ml EDTA titrant

ii. Calcium hardness

Where,

Aʹ = Volume of EDTA used by sample

D = mg CaCO3 equivalent to 1.0 ml EDTA titrant

iii. Magnesium hardness

Magnesium hardness as CaCO3 = Total hardness as CaCO3 – Ca hardness as

CaCO3

ix. Acidity (potentiometric titration method)

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Acidity will be measured by using potentiometric titration method.

Acidity can be calculated by using the formula,

where,

A = Acidity due to mineral

B = Acidity due to CO2

x. Alkalinity (potentiometric titration method)

Alkalinity will be measured by using potentiometric titration method.

Alkalinity can be calculated by using the formula

where,

A = Alkalinity due to Phenolphthalein

B = Alkalinity due to Methyl Orange

xi. Chloride (Mohr’s argentometric method)

Chloride content will be measured by using Mohr’s argentiometric method.

It can be calculated by using the formula,

xii. Nitrate (The phenol-di-sulphonic acid method)

The phenol-di-sulphonic acid method was used for the estimation of nitrate N. The

steam dried water samples were dissolved in 2 ml phenol-di-sulphonic acid made

the alkaline medium, by adding 10 ml ammonium hydroxide. The development of

yellow colour indicated the presence of nitrate-N. The colour intensity was

proportional to the amount of nitrate –N. The optical density was measured with

56 | P a g e

the help of colorimeter at 410nm. The final calculation was made with known

standard graph.

Reading of standard x 1000 Nitrogen-N = mg L-1 ml sample

C6H3OH (HSO3)2 + HNO3 → C6H2OH9HSO3)2NO2 + H2O

(Nitrophenol sulphonic acid) xiii. Phosphate (Stannous Chloride Method)

Stannous chloride method was used for the determination of phosphate

concentration in water sample. 4ml ammonium molybdate solution and 10 drops of

stannous chloride solution were added to the 50ml sample water and measured to

100 ml with the distilled water. The development of blue colour indicates the

presence of phosphate-P. The colour intensity was proportional to the amount of

phosphorus present and was measured in terms of optical density with the help of

colorimeter at 680 nm. The final calculation was made with the help of standard

graph, prepared from known concentration of phosphate in solution.

H3PO4 + 12H2MoO4 → H3P (MoO10)4 + 12H2O

(Blue)

57 | P a g e

3.4. Heavy metals

Water samples were acidified with Sulphuric Acid and kept for analysis. Macrophyte samples were digested using Di-acid methods for macrophytes digestion.

Di-Acid Method for macrophyte sample digestion: 0.5 to 1.0 g of dried and processed macrophyte samples were weighed in a 150 ml conical flask. 10 ml of conc. HNO3 were added and place a funnel on the flasks and kept for about 6-8 overnight at a covered place for pre-digestion. After pre-digestion when the solid sample is no more visible, 10 ml of conc. HNO3 and 3 ml of HClO4 were added. The flasks is then kept in a hot plate and heat at about 100ºC for first one hour and then raise the temperature to about 200ºC. The digestion is continued until the contents become colourless and only white dense fumes appear. The acid content is reduced to about 2-3 ml without letting it dried up by continuing heating at the same temperature. The flasks were then removed from the hot plate, cool and about 30 ml of distilled water is added. The content is then filtered through

Whatman filter paper no. 42 into a 100 ml volumetric flask. The volume is then make up to

100 ml. Digested samples was analysed with Atomic Absorption Spectrophotometer

(AAS) and Microwave Induced Plasma Atomic Emission Spectrophotometer (MP-AES).

For the above water quality parameters the methods as described in “Standard

Methods for the Examination of water and waste water” prescribed by the “American

Public Health Association (APHA, 2005)’’ has been adopted for analysis of water quality parameters.

3.5. Phytosociological Analysis

Vegetation analysis was carried out by following the standard methods as outlined in

Misra (1968), Kershaw (1973) and Mueller-Dombois and Ellenberg (1974). Harvest

58 | P a g e methods were adopted for phytosociological analysis pertaining to the macrophytes and quadrats (1mx1m) were used. Macrophytic diversity has been calculated using the following indices.

a) Simpson Index of Dominance, Cd

Where,

pi = proportion of individual in the ith species.

As the Simpson’s index values decreases, diversity decreases. Simpson index is therefore usually expressed as “1–Cd” (Simpson, 1949).

b) Sorenson’s similarity index,

Where:

S1= number of species in community 1.

S2= number of species in community 2.

c = number of species common to both communities (Sorensen, 1948).

59 | P a g e

c) Shannon-Wiener diversity index, H'

Where,

H' = the Shannon-Wiener diversity index

Pi = the proportion of individuals in the ith species i.e. ni/N (Shannon and Weaver,

1949).

3.6. Phytoremediation experiments using macrophytes

Phytoremediation experiments with selected 4 macrophytes were conducted for contamination analysis. Macrophytes were Eichhornia crassipes, Lemna minor, Pistia stratiotes and Salvinia cucullata. Macrophytes were selected according to the adequate availabilities at all the sites and their active role in phytoremediation as discussed in review of literature.

Phytoremediation of Iron (Fe)

As all the metals except Fe were well below the permissible limit prescribed by WHO (see

Appendix VI), I planned microcosm investigation on phytoremediation of Fe only in view of current context of metal pollution in Loktak Lake.

Macrophytes i.e. Eichhornia crassipes, Lemna minor, Pistia stratiotes and Salvinia cucullata were kept separately in a 39 constructed 5 litres capacity aquarium for the experiment. 50 g of the fresh weight of each macrophytes sample were used in 1 litre of each Fe aqueous solutions. FeSO4 were taken as Fe concentration for the experiment. After

60 | P a g e the calculation, FeSO4 were measured keeping in mind that the Fe content in the FeSO4 should be maintained at 1mgL-1, 3mgL-1 and 5mgL-1 respectively in all aquarium in triplicate. For each set of experiment 1 litre of 1mgL-1, 3mgL-1 and 5mgL-1 of solutions of

Fe metals were poured within the aquaria. Further, each of these solutions was treated with respective macrophyte for 4, 8 and 12 days. Each of the set of experiments was repeated thrice with a control for all the concentration where the solution was not treated with plant.

The water samples were collected and analysed after the experiment. The average data of three replications was taken into consideration.

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Chapter 4 RESULTS

4.1. Water Quality Analysis

The analysis for water quality parameters was conducted for two years i.e., from August,

2013 to July, 2015, on seasonal basis considering mean value for each water quality parameter. Results of the various water quality parameters of four sites with standard deviation of with seasonal variation are given in Table 4.1, Table 4.2, Table 4.3, Table 4.4,

Table 4.5, Table 4.6, Table 4.7, Table 4.8, Table 4.9, Table 4.10, Table 4.11, Table 4.12 and Table 4.13.

4.1.1. Temperature

The highest value of temperature was measured 28.6°C at Site II during rainy season

(2013) and the lowest was measured 16.8°C at Site II during winter season (2013) as shown in Figure 4.1. Rainy seasons normally posses higher temperature on the other hand the winter seasons posses the lower temperature. All the water quality parameters relating to physico-chemical characteristics from different seasons were group together site wise and correlated to find the correlation coefficient between them. (Appendix I-IV)

62 | P a g e

35 Temparature

30

25 20 Site I 15 Site II 10

Temperature Temperature inºC Site III 5 Site IV 0 Rainy Winter Summer Rainy Winter Summer 2013-2014 2014-2015 Seasons

Figure 4.1. Seasonal variations of Temperature of water from different study sites.

Table 4.1. Temperature (in ºC) with standard deviation of water from different study sites.

Year 2013-2014 Year 2014-2015

Study Seasons Seasons

Sites Rainy Winter Summer Rainy Winter Summer

Site I 26.5±0.17 17.2±0.26 24.1±0.98 26.7±0.26 18.5±0.26 24.1±0.26

Site II 28.6±0.4 16.8±0.98 26.3±1.79 28.33±0.04 17.36±0.37 24.63±0.61

Site III 26.9±0.2 18.3±0.28 24±0.56 27.26±0.15 18.23±0.15 24.1±0.6

Site IV 27.7±0.49 20±0.64 26.2±0.35 27.63±0.75 18.66±0.25 26.23±0.11

4.1.2. pH

The highest value of pH was measured 7.4 at Site I and Site IV during summer season

(2015) and the lowest of 6.3 at Site IV during rainy season (2013) as shown in Figure 4.2.

The Site I and Site IV values i.e. 6.4 and 6.3 are lower than the permissible limit value set by WHO and ICMR. The summer season has the highest pH value whereas the rainy season has the lowest pH. All the water quality parameters relating to physico-chemical

63 | P a g e characteristics from different seasons were group together site wise and correlated to find the correlation coefficient between them. (Appendix I-IV)

7.6 pH 7.4 7.2 7

6.8

6.6 Site I pH 6.4 Site II 6.2 6 Site III 5.8 Site IV 5.6 Rainy Winter Summer Rainy Winter Summer 2013-2014 2014-2015 Seasons

Figure 4.2. Seasonal variations of pH of water from different study sites.

Table 4.2. pH with standard deviation of water from different study sites.

Year 2013-2014 Year 2014-2015

Study Seasons Seasons

Sites Rainy Winter Summer Rainy Winter Summer

Site I 6.4±0.05 7±0.05 7.4±0.05 6.66±0.15 7.23±0.30 7.3±0.01

Site II 6.6±0.2 6.5±0.17 7.03±0.05 6.76±0.15 6.66±0.23 6.93±0.05

Site III 6.5±0.15 7.2±0.05 7.2±0.05 7±0.17 7.16±0.15 7.23±0.05

Site IV 6.3±0.2 6.8±0.05 7.4±0.05 7.23±0.15 6.83±0.15 7.46±0.15

4.1.3. Transperency

The highest value of Transperancy was measureed 1.7 m at Site IV during winter season

(2013) as well as rainy (2013) and the lowest of 0.74 m at Site III during summer season

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(2014) as shown in Figure 4.3. The winter season has the highest Transperancy and the summer season has the lowest Transperancy of the water. All the water quality parameters relating to physico-chemical characteristics from different seasons were group together site wise and correlated to find the correlation coefficient between them. (Appendix I-IV)

1.8 Transperancy 1.6

1.4 1.2 1 Site I 0.8 0.6 Site II

Transperancym in 0.4 Site III 0.2 Site IV 0 Rainy Winter Summer Rainy Winter Summer 2013-2014 2014-2015 Seasons

Figure 4.3. Seasonal variations of Transperancy of water from different study sites.

Table 4.3. Transperancy (in m) with standard deviation of water from different study sites. Year 2013-2014 Year 2014-2015

Study Seasons Seasons

Sites Rainy Winter Summer Rainy Winter Summer

Site I 1±0.05 1±0.02 0.97±0.05 0.85±0.06 1.04±0.12 0.99±0.03

Site II 1.23±0.05 1.2±0.01 0.94±0.26 1.04±0.04 1.27±0.01 0.93±0.09

Site III 1.04±0.03 1.04±0.01 0.74±0.015 0.91±0.005 1.02±0.005 0.82±0.02

Site IV 1.7±0.03 1.7±0.01 1.01±0.06 1.03±0.01 1.66±0.01 1.06±0.03

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4.1.4. Total Solids (TS)

The highest value of Total Solids (TS) was measured 46.6 mgL-1 at Site III during rainy season (2014) and the lowest of 23.3 mgL-1 at Site III during winter season (2013) and Site

I and Site III during summer season of both the years (2014 and 2015) as shown in Figure

4.4. Normally due to agitation of water with the rains, the rainy season has the highest TS and the winter season has the lowest TS. All the water quality parameters relating to physico-chemical characteristics from different seasons were group together site wise and correlated to find the correlation coefficient between them. (Appendix I-IV)

50 Total Solids

45

1

- 40 35 30 Site I 25 Site II 20 15 Site III

Total Solids Solids Total inmgL 10 Site IV 5 0 Rainy Winter Summer Rainy Winter Summer 2013-2014 2014-2015 Seasons

Figure 4.4. Seasonal variations of Total Solids of water from different study sites.

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Table 4.4. Total Solids (in mgL-1) content with standard deviation of water from different study sites. Year 2013-2014 Year 2014-2015

Study Seasons Seasons

Sites Rainy Winter Summer Rainy Winter Summer

Site I 40±20 26.6±5.77 23.3±5.77 43.33±20.817 23.33±5.77 26.66±5.77

Site II 36.7±5.77 33.3±5.77 33.3±5.77 40±10 36.66±5.77 30±10

Site III 43.3±20.81 23.3±5.77 23.3±5.77 46.66±11.54 26.66±5.77 23.33±5.77

Site IV 36.6±5.77 33.3±5.77 30±10 33.33±5.77 30±10 30±10

4.1.5. Dissolved Oxygen (DO)

The highest Dissolved Oxygen (DO) was measured 9.6 mgL-1 at Site IV during winter season (2013) and the lowest of 6.9 mgL-1 at Site III during summer season (2014) as shown in Figure 4.5. Winter season has the highest DO and summer season has the lowest

DO. All the water quality parameters relating to physico-chemical characteristics from different seasons were group together site wise and correlated to find the correlation coefficient between them. (Appendix I-IV)

67 | P a g e

12 Dissoved Oxygen

10

1 - 8

6 Site I Site II DO in in mgL DO 4 Site III 2 Site IV 0 Rainy Winter Summer Rainy Winter Summer 2013-2014 2014-2015 Seasons

Figure 4.5. Seasonal variations of Dissolve Oxygen of water from different study sites.

Table 4.5. Dissolve Oxygen (in mgL-1) content with standard deviation of water from different study sites. Year 2013-2014 Year 2014-2015

Seasons Seasons Study

Sites Rainy Winter Summer Rainy Winter Summer

Site I 8±0.36 8.7±0.37 7.3±0.15 7.96±0.58 8.9±0.62 7.66±0.32

Site II 7.7±0.35 8±0.5 7.1±0.15 7.66±0.20 8.26±0.40 7.3±0.1

Site III 7.8±0.32 8.6±0.87 6.9±0.26 7.8±0.05 8.46±0.20 7.06±0.15

Site IV 7.5±0.46 9.6±0.26 7.6±0.26 7.83±0.41 8.7±0.2 7.7±0.17

4.1.6. Biological Oxygen Demand (BOD)

The highest value Biological Oxygen Demand (BOD) was measured 2.1 mgL-1 at Site I during winter season (2014) and the lowest of 0.5 mgL-1 at Site III during winter season

(2013) as shown in Figure 4.6. Winter season has the highest BOD and the winter season and rainy season has the lowest BOD. All the water quality parameters relating to physico-

68 | P a g e chemical characteristics from different seasons were group together site wise and correlated to find the correlation coefficient between them. (Appendix I-IV)

2.5 Biological Oxygen Demand

2

1 - 1.5 Site I

1 Site II BOD in mgL in BOD 0.5 Site III Site IV 0 Rainy Winter Summer Rainy Winter Summer 2013-2014 2014-2015 Seasons

Figure 4.6. Seasonal variations of Biological Oxygen Demand of water from different

study sites.

Table 4.6. Biological Oxygen Demand (in mgL-1) content with standard deviation of water from different study sites. Year 2013-2014 Year 2014-2015

Seasons Seasons Study

Sites Rainy Winter Summer Rainy Winter Summer

Site I 2±0.51 1.7±0.61 1.4±0.15 1.7±0.69 2.1±0.81 1.66±0.41

Site II 1.8±0.25 1.4±0.26 1.1±0.3 1.8±0.30 1.76±0.20 1.3±0.1

Site III 1.6±0.55 0.5±0.45 1.3±0.34 1.76±0.23 1.86±0.11 1.46±0.15

Site IV 1.7±0.51 1.9±1.15 1.4±0.45 1.96±0.47 1.9±0.01 1.76±0.41

4.1.7. Acidity

The highest value of Acidity was measured 32.6 mgL-1 at Site III during rainy season

(2013) and the lowest of 9.3 mgL-1 at Site IV during winter season (2013) as shown in

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Figure 4.7. Rainy season has the highest Acidity and winter season has the lowest. All the water quality parameters relating to physico-chemical characteristics from different seasons were group together site wise and correlated to find the correlation coefficient between them. (Appendix I-IV)

Acidity 35

30

1 - 25 20 Site I 15 Site II

10 Site III Acidity inmgL Acidity 5 Site IV 0 Rainy Winter Summer Rainy Winter Summer 2013-2014 2014-2015 Seasons

Figure 4.7. Seasonal variations of Acidity of water from different study sites.

Table 4.7. Acidity (in mgL-1) content with standard deviation of water from different study sites. Year 2013-2014 Year 2014-2015

Study Seasons Seasons

Sites Rainy Winter Summer Rainy Winter Summer

Site I 27±7.81 15.3±1.15 27±4 28.33±8.73 19±2 27.66±3.05

Site II 23.3±4.16 14±3 24.6±4.04 24±1 13.33±1.52 24±2.64

Site III 32.6±7.57 14.3±0.57 29.6±2.08 32.33±8.14 17±1 25.33±2.08

Site IV 26±3.6 9.3±1.15 23±2 26.33±1.52 13.33±1.52 22.33±1.52

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4.1.8. Alkalinity

The Highest value of alkalinity was measured 73.3 mgL-1 at Site IV during winter season

(2013) and 21.3 mgL-1 at Site III of rainy season (2013) as shown in Figure 4.8. Winter season has the Alkalinity and rainy season has lowest. All the water quality parameters relating to physico-chemical characteristics from different seasons were group together site wise and correlated to find the correlation coefficient between them. (Appendix I-IV)

80 Alkalinity

70

1 - 60 50 40 Site I 30 Site II

Alkalinity in mgL Alkalinityin 20 Site III 10 Site IV 0 Rainy Winter Summer Rainy Winter Summer 2013-2014 2014-2015 Seasons

Figure 4.8. Seasonal variations of Alkalinity of water from different study sites.

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Table 4.8. Alkalinity (in mgL-1) content with standard deviation of water from different study sites. Year 2013-2014 Year 2014-2015

Study Seasons Seasons

Sites Rainy Winter Summer Rainy Winter Summer

Site I 24.6±1.15 58.6±5.03 49±6.24 23.33±1.15 50.33±4.72 51±4.35

Site II 26.3±3.05 48.6±4.16 47±2 27±6 49±3.60 45.33±2.88

Site III 21.3±1.15 56±3.46 50±3.6 22±4.35 53.66±2.08 46±4.58

Site IV 23±1 73.3±1.15 64.6±6.02 21.66±1.15 69±4.58 67.66±5.85

4.1.9. Chloride

The highest value of Chloride was measured 45.6 mgL-1 at Site III during winter season

(2014) and the lowest of 20.3 mgL-1 at Site II during winter season (2014) as shown in

Figure 4.9. Winter has the highest Chloride as well as lowest. All the water quality parameters relating to physico-chemical characteristics from different seasons were group together site wise and correlated to find the correlation coefficient between them.

(Appendix I-IV)

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50 Chloride

45

1 40 - 35 30 25 Site I 20 Site II 15 Chloride in mgL Chloridein 10 Site III 5 0 Site IV Rainy Winter Summer Rainy Winter Summer 2013-2014 2014-2015 Seasons

Figure 4.9. Seasonal variations of Chloride of water from different study sites.

Table 4.9. Chloride (in mgL-1) content with standard deviation of water from different study sites. Year 2013-2014 Year 2014-2015

Study Seasons Seasons

Sites Rainy Winter Summer Rainy Winter Summer

Site I 23.9±3.46 39.3±1.15 31.6±2.08 23.65±2.88 38.99±2.64 30.99±4.58

Site II 22.9±2.64 20.9±2 23.6±1.51 23.65±2.08 20.32±2.08 26.32±3.05

Site III 24.6±2.3 42.3±1.52 37.3±3.78 26.99±2 45.65±1.52 37.32±1.15

Site IV 26.6±1.15 21.3±2.3 23.3±1.52 27.99±1.73 22.32±3.78 25.65±1.52

4.1.10. Total Hardness

The highest value of Total Hardness was measured 68 mgL-1 at Site III during winter season (2013) and the lowest of 18.7 mgL-1 at Site III during rainy season (2013) as shown in Figure 4.10. Winter season has the highest Total Hardness lowest in rainy season. All the water quality parameters relating to physico-chemical characteristics from different seasons were group together site wise and correlated to find the correlation coefficient between them. (Appendix I-IV)

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80 Total Hardness 70 60 50 40 Site I 30 Site II 20 Site III 10 Site IV 0 Rainy Winter Summer Rainy Winter Summer 2013-2014 2014-2015 Seasons

Figure 4.10. Seasonal variations of Total Hardness of water from different study sites.

Table 4.10. Total Hardness (in mgL-1) content with standard deviation of water from different study sites. Year 2013-2014 Year 2014-2015

Study Seasons Seasons

Sites Rainy Winter Summer Rainy Winter Summer

Site I 25.3±1.15 62±1.73 55.6±5.68 28.33±4.04 54±4.35 56±3.60

Site II 26.6±1.15 28.3±6.42 33.6±7.5 28.66±4.04 28±9.64 37±3

Site III 18.7±3.05 68±4 51±14.93 22.66±2.88 63.33±4.50 45.66±14.04

Site IV 23.3±3.05 29±1 27±1 25.66±3.05 29.66±3.78 29±7

4.1.11. Turbidity

The Highest value of Turbidity was measured 15.8 NTU at Site IV during summer season

(2014) and the lowest of 0.4 NTU at Site III during winter season (2013) and Site IV during winter season (2013 and 2014) as shown in Figure 4.11. Summer season has the highest Turbity and winter season has the lowest. Some value in summer and rainy seasons

74 | P a g e are higher than the permissible limits set by WHO. All the water quality parameters relating to physico-chemical characteristics from different seasons were group together site wise and correlated to find the correlation coefficient between them. (Appendix I-IV)

18 Turbidity 16

14 12 10 Site I 8 6 Site II Turbidity in NTU Turbidityin 4 Site III 2 Site IV 0 Rainy Winter Summer Rainy Winter Summer 2013-2014 2014-2015 Seasons

Figure 4.11. Seasonal variations of Turbidity of water from different study sites.

Table 4.11. Turbidity (in NTU) content with standard deviation of water from different study sites.. Year 2013-2014 Year 2014-2015

Study Seasons Seasons

Sites Rainy Winter Summer Rainy Winter Summer

Site I 4.8±2.16 0.5±0.05 5.9±1.68 4.13±1.13 0.53±0.11 6.36±0.37

Site II 6.3±0.87 0.5±0 12.5±1.11 5.36±0.97 0.5±0.1 9.16±0.58

Site III 2.8±2.06 0.4±0.05 8±0.4 4.3±0.34 0.53±0.05 8.1±0.26

Site IV 3.3±1.49 0.4±0 15.8±1.05 3.26±0.66 0.43±0.05 15.5±1.75

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4.1.12. Nitrate

The highest value of Nitrate was found 0.62 mgL-1 at Site II during summer season (2014) and the lowest of 0.21 mgL-1 at Site IV during rainy season (2013) as shown in Figure

4.12. Summer season has the highest Nitrate and winter season has the lowest. All the water quality parameters relating to physico-chemical characteristics from different seasons were group together site wise and correlated to find the correlation coefficient between them. (Appendix I-IV)

0.7 Nitrate

0.6

1 - 0.5 0.4 Site I 0.3 Site II

Nitrate inmgL Nitrate 0.2 Site III 0.1 Site IV 0 Rainy Winter Summer Rainy Winter Summer 2013-2014 2014-2015 Seasons

Figure 4.12. Seasonal variations of Nitrate of water from different study sites.

Table 4.12. Nitrate (in mgL-1) content with standard deviation of water from different study sites. Year 2013-2014 Year 2014-2015

Study Seasons Seasons

Sites Rainy Winter Summer Rainy Winter Summer

Site I 0.26±0.1 0.39±0.05 0.54±0.1 0.28±0.05 0.42±0.04 0.54±0.032

Site II 0.37±0.05 0.43±0.02 0.62±0.05 0.37±0.02 0.46±0.02 0.61±0.03

Site III 0.3±0.04 0.4±0.04 0.41±0.026 0.32±0.04 0.41±0.02 0.4±0.04

Site IV 0.21±0.02 0.23±0.005 0.33±0.11 0.24±0.01 0.24±0.03 0.37±0.10

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4.1.13. Phosphate

The highest value of Phosphate was measured 0.3 mgL-1 at Site II during summer season

(2015) and the lowest of 0.06 mgL-1 at Site IV during winter season (2013) as shown in

Figure 4.13. Summer season has the highest Phosphate and winter season has the lowest.

Phosphate is higher than the permissible limit of USPH. All the water quality parameters relating to physico-chemical characteristics from different seasons were group together site wise and correlated to find the correlation coefficient between them. (Appendix I-IV)

0.35 Phosphate

0.3

1 - 0.25 0.2 Site I 0.15 Site II 0.1

PhosphateinmgL Site III 0.05 Site IV 0 Rainy Winter Summer Rainy Winter Summer 2013-2014 2014-2015 Seasons

Figure 4.13. Seasonal variations of Phosphate of water from different study sites.

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Table 4.13. Phosphate (in mgL-1) content with standard deviation of water from different study sites. Year 2013-2014 Year 2014-2015

Study Seasons Seasons

Sites Rainy Winter Summer Rainy Winter Summer

Site I 0.08±0.03 0.17±0.04 0.19±0.15 0.12±0.02 0.13±0.04 0.29±0.02

Site II 0.16±0.04 0.19±0.03 0.28±0.075 0.17±0.04 0.20±0.07 0.3±0.04

Site III 0.08±0.04 0.2±0.04 0.17±0.03 0.11±0.04 0.14±0.03 0.18±0.14

Site IV 0.07±0.02 0.06±0.02 0.13±0.04 0.09±0.02 0.1±0.01 0.14±0.04

4.2. Heavy Metals Analysis

The analysis for heavy metals for the water samples collected from different Sites was conducted for two years i.e., from August, 2013 to July, 2015, on seasonal basis as well for the macrophytes collected from the different Sites but not seasonally. Seven heavy metals i.e. Iron (Fe), Mercury (Hg), Cadmium (Cd), Arsenic (As), Lead (Pb), Chromium (Cr), and

Zinc (Zn) were analysed for the water samples as well as for the macrophytes. The macrophytes collected from different Sites are Eichhornia crassipes, Lemna minor, Pistia stratiotes, and Salvinia cucullata. Results of the heavy metals concentrations of four sites for water and plants with seasonal variations are given in Table 4.14, Table 4.15, Table

4.16, Table 4.17, Table 4.18, Table 4.19, 4.20, 4.21, 4.22, 4.23, 4.24, 4.25, 4.26, and 4.27.

4.2.1. Water

Water samples were collected in all the seasons for all the four Sites. The results are as follows.

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Iron (Fe)

The highest value of Fe concentration was measured 0.17 mgL-1 at Site IV during winter season (2013) and the lowest of 0.01 mgL-1 at Site I during rainy season (2014) and summer season (2015) as shown in Figure 4.14. Winter season has the highest Fe concentration and the rainy season has the lowest.

0.18 Fe Concentrations

0.16

1 - 0.14 0.12 0.1 Site I 0.08 Site II 0.06 Site III

0.04 Fe Concentrations Fe in mgL 0.02 Site IV 0 Rainy Winter Summer Rainy Winter Summer 2013-2014 2014-2015 Seasons

Figure 4.14. Seasonal variations of Fe concentrations (in mgL-1) of water from

different study sites.

Table 4.14. Fe concentrations (in mgL-1) of water from different study sites.

Study 2013-2014 2014-2015 Sites Rainy Winter Summer Rainy Winter Summer

Site I 0.02 0.07 0.03 0.01 0.08 0.01

Site II 0.08 0.13 0.1 0.07 0.15 0.09

Site III 0.04 0.1 0.06 0.06 0.12 0.03

Site IV 0.08 0.17 0.06 0.05 0.15 0.07

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Mercury (Hg)

The Hg were present in a minute negligible amount of <0.001 mgL-1 for all the samples collected from different Sites as shown in Table 4.15. In this study, all the values of Hg concentrations are found far below the limit and not harmful to use any purposes.

Table 4.15. Hg concentrations (in mgL-1) of water from different study sites. Study 2013-2014 2014-2015 Sites Rainy Winter Summer Rainy Winter Summer

Site I <0.001 <0.001 <0.001 <0.001 <0.001 <0.001

Site II <0.001 <0.001 <0.001 <0.001 <0.001 <0.001

Site III <0.001 <0.001 <0.001 <0.001 <0.001 <0.001

Site IV <0.001 <0.001 <0.001 <0.001 <0.001 <0.001

Cadmium (Cd)

The Cd were present in a minute negligible amount of <0.001 mgL-1 for all the samples collected from different Sites as shown in Table 4.16. In this study, all the values of Cd concentrations are found far below the limit and not harmful to use any purposes.

Table 4.16. Cd concentrations (in mgL-1) of water from different study sites. Study 2013-2014 2014-2015 Sites Rainy Winter Summer Rainy Winter Summer

Site I <0.001 <0.001 <0.001 <0.001 <0.001 <0.001

Site II <0.001 <0.001 <0.001 <0.001 <0.001 <0.001

Site III <0.001 <0.001 <0.001 <0.001 <0.001 <0.001

Site IV <0.001 <0.001 <0.001 <0.001 <0.001 <0.001

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Arsenic (As)

The As were present in a minute negligible amount of <0.001 mgL-1 for all the samples collected from different Sites as shown in Table 4.17. In this study, all the values of As concentrations are found far below the limit and not harmful to use any purposes.

Table 4.17. As concentrations (in mgL-1) of water from different study sites. Study 2013-2014 2014-2015 Sites Rainy Winter Summer Rainy Winter Summer

Site I <0.001 <0.001 <0.001 <0.001 <0.001 <0.001

Site II <0.001 <0.001 <0.001 <0.001 <0.001 <0.001

Site III <0.001 <0.001 <0.001 <0.001 <0.001 <0.001

Site IV <0.001 <0.001 <0.001 <0.001 <0.001 <0.001

Lead (Pb)

The Pb were present in a minute negligible amount of <0.001 mgL-1 for all the samples collected from different Sites as shown in Table 4.18. In this study, all the values of Pb concentrations are found far below the limit and not harmful to use any purposes. Pb is a cumulative poison and its effects on human health include gastrointestinal disorder, liver and kidney damage, abnormalities and infertility. Pb poisoning is due to permanent cumulative effects and not due to occasional exposure to small doses. However, in extreme case of Pb poisoning death may result (Trivedy and Goel, 1986).

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Table 4.18. Pb concentrations (in mgL-1) of water from different study sites. Study 2013-2014 2014-2015 Sites Rainy Winter Summer Rainy Winter Summer

Site I <0.001 <0.001 <0.001 <0.001 <0.001 <0.001

Site II <0.001 <0.001 <0.001 <0.001 <0.001 <0.001

Site III <0.001 <0.001 <0.001 <0.001 <0.001 <0.001

Site IV <0.001 <0.001 <0.001 <0.001 <0.001 <0.001

Chromium (Cr)

The Cr were present in a minute negligible amount of <0.001 mgL-1 for all the samples collected from different Sites as shown in Table 4.19. In this study, all the values of Cr concentrations are found far below the limit and not harmful to use any purposes.

Table 4.19. Cr concentrations (in mgL-1) of water from different study sites. Study 2013-2014 2014-2015 Sites Rainy Winter Summer Rainy Winter Summer

Site I <0.001 <0.001 <0.001 <0.001 <0.001 <0.001

Site II <0.001 <0.001 <0.001 <0.001 <0.001 <0.001

Site III <0.001 <0.001 <0.001 <0.001 <0.001 <0.001

Site IV <0.001 <0.001 <0.001 <0.001 <0.001 <0.001

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Zinc (Zn)

The Zn were present in a minute negligible amount of <0.02 mgL-1 for all the samples collected from different Sites as shown in Table 4.20. In this study, all the values of Zn concentrations are found far below the limit and not harmful to use any purposes.

Table 4.20. Zn concentrations (in mgL-1) of water from different study sites. Study 2013-2014 2014-2015 Sites Rainy Winter Summer Rainy Winter Summer

Site I <0.02 <0.02 <0.02 <0.02 <0.02 <0.02

Site II <0.02 <0.02 <0.02 <0.02 <0.02 <0.02

Site III <0.02 <0.02 <0.02 <0.02 <0.02 <0.02

Site IV <0.02 <0.02 <0.02 <0.02 <0.02 <0.02

4.3. Phytosociological Analysis

4.3.1. Macrophytes Species composition

From the phytosociological studies of the different sites, altogether a total of 24 wetland macrophytes species belonging to 23 genera and 17 families were recorded. Of this, 10 species belonging to 8 genera and 8 families, 13 species belonging to 12 genera and 11 families, 12 species belonging to 11 genera and 9 families and 21 species belonging to 20 genera and 15 families were reported from Site I, Site II, Site III and Site IV respectively.

The macrophyte species namely Alternanthera philoxeroides Griseb., Arthraxon lanceolatus (Roxb) Hochst., Azolla pinnata Lam., Ceratophyllum demersum Linn.,

Cyrtococcum accrescens Stafp., Eichhornia crassipes Linn., Enhydra fluctuans Lour.,

Euryale ferox Salisb., Hydrilla verticillata (Linn) Royle., Ipomoea aquatica Forsk., Lemna minor Linn., Nymphaea nouchali Burm. f., Nymphoides cristatum O. Kuntz, Oenanthe

83 | P a g e javanica (Bl) D.C., Pistia stratiotes Linn., Polygonum glabrum Willd., Potamogeton crispus Linn., Rumex nepalensis Spreng., Salvinia cucullata Roxb., Salvinia natans

Hoffm., Spirodela polyrhiza (Linn) Schleid., Trapa natans Linn., Vallisnaria spiralis

Linn., and Zizania latifolia (Griseb) stapf.. were recorded.

Alternanthera philoxeroides Griseb., Azolla pinnata Lam., Eichhornia crassipes

Linn., Lemna minor Linn., Pistia stratiotes Linn., Salvinia cucullata Roxb., Salvinia natans Hoffm., and Spirodela polyrhiza (Linn) Schleid. were the plants with higher density al all the sites.

Table 4.28. shows phytosociological attributes of macrophyte species in the study.

Shannon-Wiener diversity index for macrophyte species was maximum at the Site IV i.e.

2.37 and minimum in the Site I i.e. 1.31. A reverse trend in the results was observed in case of the Simpson index of dominance. The Simpson index of dominance were maximum at Site III i.e. 0.42 and minimum at Site IV i.e. 0.12. Table 4.28. shows the presents dominance and diversity. As the Simpson’s index of dominance values decreases,

Shannon-Wiener diversity increased which is quite appropriate.

Table 4.21. Phytosociological attributes of macrophyte species.

Parameter Site I Site II Site III Site IV

Number of Family 8 11 9 15

Number of Genera 8 12 11 20

Number of Species 10 13 12 21

Simpson Index of Dominance 0.37 0.27 0.42 0.12

Shannon-Wiener Diversity Index 1.31 1.68 1.37 2.37

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4.3.2. Similarity index (Sorenson’s Similarity Index)

Between Site I and Site II, the Sorenson’s Similarity was found to be 0.87, between Site I and Site III, it was found 0.73, between Site I and Site IV, it was 0.65, between Site II and

Site III, it was 0.72, between Site II and Site IV, it was 0.71, and between Site III and Site

IV, it was found to be 0.61 as shown in Table 4.29.

From the phytosociological studies of the different sites, the macrophyte species common/similar to all the sites were Alternanthera philoxeroides Griseb., Azolla pinnata

Lam., Eichhornia crassipes Linn., Lemna minor Linn., Pistia stratiotes Linn., Salvinia cucullata Roxb., Salvinia natans Hoffm., and Spirodela polyrhiza (Linn) Schleid.

The uncommon/disimilar species were Arthraxon lanceolatus (Roxb) Hochst.,

Ceratophyllum demersum Linn., Cyrtococcum accrescens Stafp., Enhydra fluctuans Lour.,

Euryale ferox Salisb., Hydrilla verticillata (Linn) Royle., Ipomoea aquatica Forsk.,

Nymphaea nouchali Burm. f., Nymphoides cristatum O. Kuntz., Oenanthe javanica (Bl)

D.C., Polygonum glabrum Willd., Potamogeton crispus Linn., Rumex nepalensis Spreng.,

Trapa natans Linn., Vallisnaria spiralis Linn., and Zizania latifolia (Griseb) stapf., were recorded.

Majority of the species common to all sites belonging to families Amaranthaceae,

Araceae, Azollaceae, Lemnaceae, Pontederiaceae, and Salviniaceae.

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Table 4.22. Showing Sorenson’s Similarity Index between different sites.

Site I Site II Site III Site IV

Site I

Site II 0.87

Site III 0.73 0.72

Site IV 0.65 0.71 0.61

4.3.3. Dominance of families and Diversity

A total number of 17 families were recorded from all the study sites. Of these, 15 families were reported from Site IV followed by 11 families from Site II, 9 families from Site III and 8 families from Site I. Table 4.30. shows family-wise distribution of macrophyte species.

At Site I, Lemnaceae and Salvinaceae were the dominant family with 2 species each followed by Amaranthaceae with 1 species, Araceae with 1 species, Azollaceae with

1 species, Ceratophyllaceae with 1 species, Hydrocharitaceae with 1 species and

Pontederiaceae with 1 species (Table 4.28).

Similarly, at Site II, Lemnaceae and Salvinaceae were the dominant family with 2 species each followed by Amaranthaceae with 1 species, Araceae with 1 species,

Asteraceae with 1 species, Azollaceae with 1 species, Ceratophyllaceae with 1 species,

Hydrocharitaceae with 1 species, Menyanthaceae with 1 species, Poaceae with 1 species, and Pontederiaceae with 1 species (Table 4.28).

Similarly, at Site III, Lemnaceae, Salvinaceae with one more family than Site II i.e.

Polygonaceae were the dominant families with 2 species each followed by Amaranthaceae

86 | P a g e with 1 species, Araceae with 1 species, Azollaceae with 1 species, Convolvulaceae with 1 species, Poaceae with 1 species and Pontederiaceae with 1 species (Table 4.28).

Similarly, at Site IV, Poaceae was dominant with 3 species followed by

Lemnaceae, Hydrocharitaceae, Nymphaeaceae, and Salvinaceae family with 2 species each followed by Amaranthaceae with 1 species, Apiaceae with 1 species, Araceae with 1 species, Azollaceae with 1 species, Ceratophyllaceae with 1 species, Convolvulaceae with

1 species, Menyanthaceae with 1 species, Pontederiaceae with 1 species,

Potamogetonaceae with 1 species, and Trapaceae with 1 species (Table 4.28).

Amaranthaceae, Araceae, Azollaceae, Lemnaceae Pontederiaceae, and

Salviniaceae were the families common to all the study sites i.e. Site I, Site II, Site III and

Site IV. Apiaceae and Trapaceae are restricted to higher diversity site, i.e. Site IV whereas

Asteraceae and Polygonaceae are restricted to low diversity sites i.e. Site II and Site III respectively.

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Table 4.23. Family-wise distribution of macrophyte species.

Sl. No. Family Site I Site II Site III Site IV

1 Amaranthaceae 1 1 1 1

2 Apiaceae - - - 1

3 Araceae 1 1 1 1

4 Asteraceae - 1 - -

5 Azollaceae 1 1 1 1

6 Ceratophyllaceae 1 1 - 1

7 Convolvulaceae - - 1 1

8 Hydrocharitaceae 1 1 - 2

9 Lemnaceae 2 2 2 2

10 Menyanthaceae - 1 - 1

11 Nymphaeaceae - - - 2

12 Poaceae - 1 1 3

13 Polygonaceae - - 2 -

14 Pontederiaceae 1 1 1 1

15 Potamogetonaceae - - - -

16 Salviniaceae 2 2 2 2

17 Trapaceae - - - 1

(-) Absent

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4.4. Metal analysis in biomass of selected macrophytes

The four macrophytes species samples were collected from all the four Sites in winter season (2014-15). Therefore, only these macrophytes were investigated for metal concentration inside biomass. The results of heavy metal concentration in the collected macrophyte species are as follows.

Iron (Fe)

The highest Fe concentration was measured 28.29 mgkg-1 in Pistia stratiotes at Site II which is higher than the permissible limit set by WHO and the lowest of 1.68 mgkg-1 in

Salvinia cucullata at Site I as shown in Figure 4.15. Salvinia cucullata has the lowest Fe as compared to the other plant species i.e. Eichhornia crassipes, Lemna minor and Pistia stratiotes. The result of Fe concentration of the plants species from different sites wise were grouped together with Fe concentration of the water from different sites wise and correlated to find the correlation coefficient between them. (Appendix VII)

30.00 Fe Concentrations

1 - 25.00

20.00 Eichhornia crassipes 15.00 Lemna minor 10.00 Pistia stratiotes Salvinia cucullata

Fe Concentrations Fe in mgkg 5.00

0.00 Site I Site II Site III Site IV Sites

Figure 4.15. Variations of Fe concentrations (in mgkg-1) of plants from different study

sites.

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Table 4.24. Fe concentrations (in mgkg-1) of plants from different study sites. Name of the plants Site I Site II Site III Site IV

Eichhornia crassipes 0.72 9.77 9.07 0.53

Lemna minor 12.52 12.74 9.92 9.41

Pistia stratiotes 13.01 28.29 12.68 1.09

Salvinia cucullata 1.68 3.14 2.72 2.25

Mercury (Hg)

The highest Hg concentration was measured <0.0069 mgkg-1 in Salvinia cucullata at Site I and the lowest of <0.0017 mgkg-1 in Eichhornia crassipes at Site I as shown in Table 4.22.

Eichhornia crassipes has the lowest Hg as compare to the other macrophytes species i.e.

Lemna minor, Pistia stratiotes and Salvinia cucullata.

Table 4.25. Variations of Hgs concentration (in mgkg-1) of plants from different study sites.

Name of plants Site I Site II Site III Site IV

Eichhornia crassipes <0.00173 <0.00328 <0.00249 <0.00178

Lemna minor <0.00318 <0.00309 <0.00228 <0.00318

Pistia stratiotes <0.00356 <0.00417 <0.00247 <0.00389

Salvinia cucullata <0.00698 <0.00314 <0.00368 <0.00234

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Cadmium (Cd)

Similar with Hg concentration, the highest Cd concentration was measured <0.0069 mgkg-1 in Salvinia cucullata at Site I and the lowest of 0.0017 mgkg-1 in Eichhornia crassipes at Site I as shown in Table 4.23. Eichhornia crassipes has the lowest Cd as compare to the other macrophytes species i.e. Lemna minor, Pistia stratiotes and Salvinia cucullata.

Table 4.26. Cd concentrations (in mgkg-1) of plants from different study sites. Name of plants Site I Site II Site III Site IV

Eichhornia crassipes <0.00173 <0.00328 <0.00249 <0.00178

Lemna minor <0.00318 <0.00309 <0.00228 <0.00318

Pistia stratiotes <0.00356 <0.00417 <0.00247 <0.00389

Salvinia cucullata <0.00698 <0.00314 <0.00368 <0.00234

Arsenic (As)

Similar with Hg and Cd concentrations, the highest As concentration was measured

<0.0069 mgkg-1 in Salvinia cucullata at Site I and the lowest of 0.0017 mgkg-1 in

Eichhornia crassipes at Site I as shown in Table 4.24. Eichhornia crassipes has the lowest

As as compare to the other macrophytes species i.e. Lemna minor, Pistia stratiotes and

Salvinia cucullata.

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Table 4.27. As concentrations (in mgkg-1) of plants from different study sites. Name of plants Site I Site II Site III Site IV

Eichhornia crassipes <0.00173 <0.00328 <0.00249 <0.00178

Lemna minor <0.00318 <0.00309 <0.00228 <0.00318

Pistia stratiotes <0.00356 <0.00417 <0.00247 <0.00389

Salvinia cucullata <0.00698 <0.00314 <0.00368 <0.00234

Lead (Pb)

Similar with Hg, Cd and As concentrations, the highest Pb concentration was measured

<0.0069 mgkg-1 in Salvinia cucullata at Site I and the lowest of 0.0017 mgkg-1 in

Eichhornia crassipes at Site I as shown in Table 4.25. Eichhornia crassipes has the lowest

Pb as compare to the other macrophytes species i.e. Lemna minor, Pistia stratiotes and

Salvinia cucullata.

Table 4.28. Pb concentrations (in mgkg-1) of plants from different study sites. Name of plants Site I Site II Site III Site IV

Eichhornia crassipes <0.00173 <0.00328 <0.00249 <0.00178

Lemna minor <0.00318 <0.00309 <0.00228 <0.00318

Pistia stratiotes <0.00356 <0.00417 <0.00247 <0.00389

Salvinia cucullata <0.00698 <0.00314 <0.00368 <0.00234

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Chromium (Cr)

Similar with Hg, Cd, As, and Pb concentrations, the highest Cr concentration was measured <0.0069 mgkg-1 in Salvinia cucullata at Site I and the lowest of 0.0017 mgkg-1 in Eichhornia crassipes at Site I as shown in Table 4.26. Eichhornia crassipes has the lowest Cr as compare to the other macrophytes species i.e. Lemna minor, Pistia stratiotes and Salvinia cucullata.

Table 4.29. Cr concentrations (in mgkg-1) of plants from different study sites.

Name of plants Site I Site II Site III Site IV

Eichhornia crassipes <0.00173 <0.00328 <0.00249 <0.00178

Lemna minor <0.00318 <0.00309 <0.00228 <0.00318

Pistia stratiotes <0.00356 <0.00417 <0.00247 <0.00389

Salvinia cucullata <0.00698 <0.00314 <0.00368 <0.00234

Zinc (Zn)

The highest Zn concentration was measured <0.13 mgkg-1 in Salvinia cucullata at Site I and the lowest of 0.03 mgkg-1 in Eichhornia crassipes at Site I as shown in Table 4.27.

Eichhornia crassipes has the lowest Cr as compare to the other macrophytes species i.e.

Lemna minor, Pistia stratiotes and Salvinia cucullata.

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Table 4.30. Zn concentrations (in mgkg-1) of plants from different study sites. Name of plants Site I Site II Site III Site IV

Eichhornia crassipes <0.0345 <0.0656 <0.04983 <0.03552

Lemna minor <0.06355 <0.06184 <0.0456 <0.06355

Pistia stratiotes <0.07128 <0.08344 <0.04943 <0.07788

Salvinia cucullata <0.13966 <0.06279 <0.07356 <0.04688

4.5. Phytoremediation

Phytoremediation experiments were conducted using selected macrophytes species which were available at all the sites i.e. Site I, Site II, Site III, and Site IV as per from the study of the phytosociology of the sites. Macrophytes which were used in phytoremediation experiment are Eichhornia crassipes, Lemna minor, Pistia stratiotes and Salvinia cucullata. As per the result of heavy metal analysis of water from the different sites, we conducted the experiment with Fe concentration as it was the only metal found in the highest amount. i.e. above permissible limit The results were given in Table 4.31, Table

4.32 and 4.33.

Fe removal in water under varying ranges of metal concentration i.e. 1 mgL-1,

3mgL-1 and 5 mgL-1 was maximum in Eicchornia crassipes treated water followed by

Pistia stratiotes treated water, Lemna minor treated water and Salvinia cucullata treated water during interval of 4, 8 and 18 days. Student’s t test revealed that decrease in Fe concentrations was significant (at p<0.01) when compared to control.

In 4 days experiment, Eicchornia crassipes treated water showed maximum of 68% removal from 1mgL-1, 40.3% removal from 3mgL-1, 45.4% removal from 5mgL-1 and

Lemna minor treated water showed minimum of 25% removal from 1mgL-1, 21% removal

94 | P a g e from 3mgL-1, Salvinia cucullata showed 26% removal from 5mgL-1 as shown in Figure

4.16.

80 Fe removal

70 60 50 40 1mgL-1 30 3mgL-1 20 5mgL-1

10 Percentage (%) Removal (%) Percentage Removal Fe of 0 Eichhornia Lemna minor Pistia stratiotes Salvinia cucullata crassipes Plants

Figure 4.16. Graph showing percentage (%) removal of Fe by selected plant species of

different concentrations in 4 days.

Table 4.31. Percentage (%) removal of Fe by selected plant species of different

concentrations in 4 days.

Percentage (%) removal of Fe

No. of Concentration Eichhornia Lemna Pistia Salvinia

Days (mgL-1) crassipes minor stratiotes cucullata

1 68 25 27 39

4 3 40.3 21 36 30

5 45.4 34.4 36.8 26

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In 8 days experiment, Eicchornia crassipes treated water showed maximum of 81% removal from 1mgL-1, 55% removal from 3mgL-1, 76.6% removal from 5mgL-1 and Lemna minor treated water showed minimum of 71% removal from 1mgL-1, Salvinia cucullata showed 40.3% removal from 3mgL-1, Pistia stratiotes showed 45.2% removal from 5mgL-1 as shown in Figure 5.17.

90 Fe removal 80 70 60 50 40 1mgL-1 30 3mgL-1 20 5mgL-1

Percentage (%) Removalof (%) Percentage Removalof Fe 10 0 Eichhornia Lemna minor Pistia stratiotes Salvinia cucullata crassipes

Plants

Figure 4.17. Graph showing percentage (%) removal of Fe by selected plant species of

different concentrations in 8 days.

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Table 4.32 Percentage (%) removal of Fe by selected plant species of different concentrations in 8 days.

Percentage (%) removal of Fe No. of Concentration Eichhornia Lemna Pistia Salvinia Days (mgL-1) crassipes minor stratiotes cucullata 1 81 71 78 76 8 3 55 59.3 61.3 40.3 5 76.6 48 45.2 58

In 12 days experiment, Eicchornia crassipes treated water showed maximum of

89% removal from 1mgL-1, 81.3% removal from 3mgL-1, 73.2% removal from 5mgL-1 and

Salvinia cucullata treated water showed minimum of 81% removal from 1mgL-1, 59.3% removal from 3mgL-1, 61.4% removal from 5mgL-1 as shown in Figure 4.18.

Fe removal 100

90 80 70 60 1mgL-1 50 3mgL-1 40 30 5mgL-1 20

Percentage (%) Removalof (%) Percentage Removalof Fe 10 0 Eichhornia crassipes Lemna minor Pistia stratiotes Salvinia cucullata Plants

Figure 4.18. Graph showing percentage (%) removal of Fe by selected plant species of

different concentrations in 12 days.

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Table 4.33. Percentage (%) removal of Fe by selected plant species of different

concentrations in 12 days.

Percentage (%) removal of Fe No. of Concentration Eichhornia Lemna Pistia Salvinia Days (mgL-1) crassipes minor stratiotes cucullata 1 89 87 87 81 12 3 81.3 62.3 75.7 59.3 5 73.2 69.6 63.4 61.4

Although, I confined my quest for phytoremediation potential of 4 macrophytes pertaining to Fe only in context of present status of Loktak Lake pollution, future microcosm studies will also investigate the phytoremediation potential of other observed macrophytes for different ranage of heavy metals.

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Chapter 5 DISCUSSION

5.1. Water Quality

The result of the present study reveals that the lake is under severe stress mainly due to human interventions like the polluted draining rivers in the lake which originate from the municipal areas, weed infestation, pollution, encroachment, overexploitation of resources and siltation thereby causing flooding of the agricultural fields and villages, decrease in flora and fauna production and loss of biodiversity.

5.1.1. Temperature

The temperature of water is a function of seasonal ambient air temperatures. The temperature is one of the important factors in aquatic environment since it regulates physicochemical as well as biological activities (Kumar et al., 1996). Water in the

Temperature range of 7ºC to 11ºC has a pleasant taste and is refreshing. At higher

Temperature, with less dissolved gases, the water becomes tasteless and even does not quench the thirst (Trivedy and Goel, 1986).

There was an increase in temperature with degree of disturbance with some exception. The discharge of pollutants from surroundings field area through run-off along with the rivers which drain in the lake may be attributed to high temperature during rainy season, as energy is released during decomposition of waste present in water. The decomposition of wetland macrophytes of lake may also a factor in the increase of water temperature. The Temperature of water samples, which may not be more important for normal uses, but in the polluted water, it has profound effect on the dissolved oxygen, biological oxygen demand and aquatic life (Hasan and Hoesien, 2008). Similar trend were

99 | P a g e found in Khan et al. (2012), Singh et al. (2010), Umavati and Logankumar (2010) and

Zafar and Sultana (2008).

A positive and significant correlation was obtained for temperature with Acidity and Turbidity in all the study sites. On the contrary, a negative and significant correlation was obtained between Transperancy, and Alkalinity in all the study sites. (Appendix I-IV)

5.1.2. pH

In other words pH value of water is a measure of Acidity or Alkalinity of water and is very important indicator of its quality. It influences the growth of plants and soil organism, therefore, it affects to a great extent, the suitability of water for irrigation. The pH value of water is controlled by the amount of Bicarbonates, Carbonates and Dissolved Carbon dioxide.

There was a decrease in pH in the Rainy season of all the sites. It is also below the permissible limit set by various agencies. In the rainy season, the water should be diluted with rain water and the pH should be increasing but on the contrary the result shows the decrease pH value. This may be due to late raining condition in the year along with the early sampling. It may also be due to the sampling point where the water is being polluted with polluting material nearby.

The decrease pH may be due varying several factors such as interaction with suspended matter, polluting material from the runoff and polluted river which drain in the lakes, decays and other chemicals which were applied in the nearby field for agriculture and other purposes. Significant changes in pH occur due to discharge of agricultural and domestic waste (Khan et al., 2012). The mineral present in the water also affect the pH.

Most of the chemical and biochemical reactions are influenced by the pH of waters.

Toxicity in water increases with increase in acidic content in water (Singh et al., 1989).

100 | P a g e

Similar trend of results was also recorded by Fakayode (2005), Singh (1995),

Sivasubramani (1999) and Unni et al., (1992).

A positive and significant correlation was obtained for pH with Hardness. On the contrary, a negative correlation was obtained between TS in all the study sites. (Appendix

I-IV)

5.1.3. Transperancy

Transperancy of water is one of the important water quality parameter. It indicates the clarity of water depending upon the presence of material in the aquatic bodies. Transparent waters allow more light penetration which has far reaching effects on all aquatic organisms, including their development, distribution and behaviour, etc. (Kaushik and

Saksena, 1999). Tranperant water is consider as a good water where it provide light penetration in the inside of water where the plants growing inside will

Tranperancy varies with the number of minute impurities present in the water bodies. Higher transparency occurred, during winter and summer due to absence of rain, runoff and flood water as well as gradual settling of suspended particles (Khan and

Chowdhury, 1994).

The high value of transperancy in winter is due to the better settlement of solid particle in the water bed which helps the light to penetrate inside while it is low in rainy season due to presence of abundance organisms including macrophytes on the surface of the water and solid material in the water. Kosygin and Dhamendra (2009) noted the transparency values of this lake ranges from 0.51 m to 2.98 m.

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A positive and significant correlation was obtained for Transperancy with

Alkalinity and Chloride in all sites except for Site II. On the contrary, a negative and significant correlation was obtained between Acidity at all the study sites. (Appendix I-IV)

5.1.4. Total Solids (TS)

Total Solids (TS) are the substances present in the water bodies dissolved or non- dissolved. They disturbed the penetration of light in the water bodies. Higher the TS, lower will be the survival of the aquatic organisms. TS are determined as the residue left after evaporation of the unfiltered sample. Higher value of TS makes the water more turbid due to presence of and organic matter.

The higher TS in the rainy season is due to the varying several factors such as suspended matter, polluting material from the runoff and polluted river which drain in the lakes, decays and other organic material. The muddy agitated flowing mixed rain water from the river which afterwards drains in the lake mixed with the lake water affecting the clarity of water increasing the TS.

A positive and significant correlation was obtained for TS with BOD in Site III. On the contrary, a negative and significant correlation was obtained between Hardness, Nitrate and Phosphate at all the study sites. (Appendix I-IV)

5.1.5. Dissolved Oxygen (DO)

Dissolved Oxygen (DO) is one of the most important parameters of water quality which reflects the various processes of physical and biological in water. All living organisms in the water bodies are dependent upon DO in one form or the other to maintain the metabolic processes that produce energy for growth and reproduction. Low DO

102 | P a g e concentrations (< 3 mg/L) in fresh water aquatics systems indicate high pollution level of the waters and cause negative effects on life in this system (Yayıntas et al., 2007).

DO content was low in summer because of enhanced utilization by micro- organisms in the high decomposition of organic matter which indicates a high pollution load in the water. The fluctuations in Oxygen content depend on factors such as

Temperature, decompositional activities, photosynthesis and the level of aeration. The reoxygenation of waters during monsoon might be occurring due to circulation and mixing by inflow after monsoon rains. In summer the D.O depletion was due to high Temperature.

Also low DO may be due to luxuriant growth of algae and aquatic macrophytes resulting to higher photosynthetic rate as a result of increased temperature (Nybakken, 1997).

Oxygen value is an indicative of pollution in water and depicts an inverse relationship with water temperature. Similar trend of dissolved oxygen in fresh water lakes were also observed by Khan et al. (2012), and Pandey et al. (1999).

A positive and significant correlation was obtained for DO with Acidity. On the contrary, a negative and significant correlation was obtained between Turbidity at all the study sites. (Appendix I-IV)

5.1.6. Biological Oxygen Demand (BOD)

Biological Oxygen Demand (BOD) is the most important parameter of water quality. BOD refers to the quantity of Oxygen required by bacteria and other microorganisms in the biochemical degradation and transformation of organic matter under aerobic conditions

(Manivaskasam, 1986). The BOD can be used as a measure of the amount of organic materials present in an aquatic solution which support the growth of microorganisms. The

BOD determines the strength of pollution in natural waters. The test is applied for fresh

103 | P a g e water sources (rivers, lakes), wastewater (domestic, industrial), marine water (estuaries, coastal water).

The high BOD of the Site I is due to the polluted Nambul River and also from the domestic waste from the local areas including the residence in the lake itself. It also may be due to high concentration of organic waste discharged into water leading to high rate of decomposition and resulting into more consumption of oxygen by the microorganisms.

The enormous growth of aquatic macrophytes may leads to high BOD of the site. Hacioglu and Dulger (2009), Naik (2005) and Lalchingpuii et al., (2012) reported the similar trends.

A positive and significant correlation was obtained for BOD with chloride in Site I.

On the contrary, a negative and significant correlation was obtained between phosphate.

(Appendix I-IV)

5.1.7. Acidity

Acidity of the water is its capacity to neutralize a strong base and is mostly due to the presence of strong mineral acids, weak acids (Carbonic, Acetic Acids) and the salts of strong acids and weak bases (e.g. Ferrous Sulphate, Aluminium Sulphate etc.) These salts on hydrolysis produce strong acid and metal hydroxides which are sparingly soluble thus producing the Acidity. Determination of Acidity is significant as it causes corrosion and influences the chemical and biochemical reactions (Kulkarni and Shrivastava, 2000)

The high acidity in rainy season may be due to the presence of high organic load received of runoff, river and the decomposed macrophyte material in the water. Acidic water is less buffered and less productive because sufficient amount of bicarbonates are not dissolved to give CO2 for a high rate of photosynthesis. Lowering of pH in water is result of decomposition of organic matters and finally release of CO2 (Warren, 1971).

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Acidity of water refers to the amount of acids bases present. The adverse effects of most of the acid appear below pH 5.0. There have been no particular limit for acidity and can be expressed in terms of CaCO3. Highly acidic water must be avoided and could be dangerous. Acidity has been not desirable in municipal water system because it tends to increase corrosion.

A positive and significant correlation was obtained for Acidity with Temperature,

DO and Turbidity in all the sites except for Site IV. On the contrary, a negative and significant correlation was obtained between phosphate. (Appendix I-IV)

5.1.8. Alkalinity

The acid neutralizing capacity of water is known as alkalinity. The constituents of

Alkalinity in natural system mainly include Carbonate, Bicarbonate and Hydroxide. These constituents result from dissolution of mineral substances in the soil and atmosphere

(Mittal and Verma, 1997).

Presence of Carbonate and Bicarbonate may be the reason of high Alkalinity in winter seasons. It may originate from microbial decomposition of organic matters. Higher values of Alkalinity in winter season months may be due to liberation of carbon dioxide during decomposition which reacts with water to form bicarbonate which is limited to low volume of water. Water with low alkalinity having a pH range of 6.3 to 7.3 are low in production and support phytoplankton which have low acid and low alkaline adaptation.

A positive and significant correlation was obtained for Alkalinity with Nitrate and

Phosphate at all the sites except for Site IV. On the contrary, a negative and significant correlation was obtained with temperature. (Appendix I-IV)

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5.1.9. Chloride

Chloride is one of the important water parameters and it is found in nature in the form of salts of sodium, potassium and calcium. Chloride is one of the most stable components in water, being unaffected by most physico-chemical and/or biological processes. The major sources of chloride include natural mineral deposits, sea water intrusion, agricultural and . Chlorides occur naturally in all types of waters. One of the most important sources of Chlorides in the waters is the discharge of municipal and agricultural waste.

Man and other animals excrete very high quantities of Chlorides together with nitrogenous compounds

The presence of high Chlorides in the winter season is due to the mixing of discharge of municipal and domestic waste from the polluted river which then drains in the lake water, agricultural waste and surface runoff. Chlorides in water are the indicators of large amount of non-point source pollution by pesticides, grease, oil, metals and other toxic materials (Khare and Jadhav, 2008). Higher values are hazardous to human consumption and create health problems (Kataria and Iqbal, 1995). The chloride in water is under permissible value set by different agencies. A similar trend of results was observed by Saxena et al., (1966), Singh et al. (2010) and Zafar and Sultana (2008).

A positive and significant correlation was obtained for Chloride with Nitrate and

Phosphate at all the sites except for Site I and Site IV. On the contrary, a negative and significant correlation was obtained between Hardness at Site IV. (Appendix I-IV)

5.1.10. Total Hardness

Hardness is the property of water which prevents the leather formation with soap and increases the boiling point of water. Water hardness is a traditional measure of the capacity of water to precipitate soap. Calcium and Magnesium are the principal cations causing

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Hardness. However other cations such as Strontium, Iron and Manganese also contribute to the Hardness. The anions responsible for Hardness are mainly Bicarbonate, Carbonate,

Sulphate, Chloride, Nitrate, Silicates etc (Taylor, 1949).

The high amount of hardness indicates high concentration of calcium and magnesium in the water bodies. In fresh water, the principal hardness causing ions are calcium and magnesium which precipitate soap. Other polyvalent cations also may precipitate soap, but often are in complex form, frequently with organic constituents, and their role in water hardness may be minimal and difficult to define. Similar trend were found by Jana (1973), Kumar (2000) and Palharya et al., (1993).

A positive and significant correlation was obtained for Hardness with pH in all the sites. On the contrary, a negative and significant correlation was obtained between TS.

(Appendix I-IV)

5.1.11. Turbidity

Turbidity is an expression of optical property of a water sample containing insoluble substances which cause light to be scattered rather than transmitted in straight lines. In most of the waters Turbidity is due to colloidal and extremely fine dispersions. Suspended matter such as clay, slit, finely divided organic and inorganic matter, plankton and other microscopic organisms also contribute to Turbidity (Manivaskam, 1986).

Turbidity can be measured by its effect on the scattering light, which is termed as

Nephelometry. Turbidimeter can be used for sample with moderate turbidity and nephelometer for sample with low turbidity. Higher the intensity of scattered lights, higher the turbidity value.

The higher turbidity values were obtained during summer seasons. This could be attributed to raining in the summer season where it discharges pollution load into the lake

107 | P a g e through the rivers which drains and also surface runoff from the adjoining agricultural fields and fish farms that makes the water more turbid.

High turbidity is due to silt, clay and other suspended particles which contribute to the turbidity values during summer seasons and rainy seasons whereas settlement of silt, clay in winter results low turbidity. Decomposition is also high in summer due high microbial activities and the production of organic microbial waste.

A positive and significant correlation was obtained for Turbidity with Temperature, at all the sites. On the contrary, a negative and significant correlation was obtained between DO. (Appendix I-IV)

5.1.12. Nitrate

Nitrate is the stable form of oxidized nitrogen but can be reduced by microbial action to nitrite. In the process of nitrification, nitrogen changes to nitrate ion. Therefore, all sources of nitrogen (including organic nitrogen, ammonia and fertilizers) should be considered as potential source of nitrates. The maximum limit of nitrate in drinking water is 45 mgL-1 for human and 100 mgL-1 for livestock (Hasan, 2008).

High nitrate concentration in Summer season may be due to the higher decomposition rate of the macrophyte and other material in the water. Agricultural chemical fertilizer from the adjoining areas may also help in increasing the nitrate of the water.

High concentration of nitrate in drinking water is toxic (Umavathi et al, 2007). The concentration of Nitrate ion is very important in public water supplies because it causes methemoglobinemia in children (Naidu et al. 1988). The presence of Nitrate indicates, the organic pollution of water and not only causes cyanosis among infants, when present in

108 | P a g e considerable quantity but also has been reported to course gastric cancer, when present in high quantity (Olaniya and Nawlakhe, 1978).

A positive and significant correlation was obtained for Nitrate with pH in all the sites. On the contrary, a negative and significant correlation was obtained between TS.

(Appendix I-IV)

5.1.13. Phosphate

Phosphate is usually derived from leaching of phosphorus rich bedrock and additionally from human wastes, synthetic detergents, industrial and agriculture waste. It is an important micro- nutrient for macrophytes and plays an important role in macrophyte’s growth including phytoplankton. Phosphate helps in eutrophication.

The high phosphate in summer may be due to high decomposition of the macrophytes thus leading to liberation of phosphate in the water. The agricultural waste and agricultural chemical fertilizer with the phosphate content used in the nearby field may also affect the water. Banerjee and Gupta (2010) and Umavathi and Longankumar (2010) reported the similar trends. Consumption of drinking water containing high phosphate may cause osteoporosis and kidney damage.

A positive and significant correlation was obtained for Acidity with Temperature,

DO and Turbidity in all the sites except for Site IV. On the contrary, a negative and significant correlation was obtained between Acidity, BOD and TS. (Appendix I-IV)

5.2. Heavy metals Analysis

Of all the heavy metals tested i.e. Iron (Fe), Mercury (Hg), Cadmium (Cd), Arsenic (As),

Lead (Pb), Chromium (Cr), and Zinc (Zn), Fe is the only metal found in high amount in water as well as in the plant samples. Others metals were found in negligible amount or in other words well below the permissible limit prescribed by scientific agencies.

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5.2.1. Water

Fe was high in water samples due to the pollution caused by draining river and the domestic waste from the surroundings. Fe in excess of 0.3 mg/L causes staining of clothes and utensils. The water is also not suitable for processing food, beverages, ice, dyeing, bleaching etc. The limit on Fe in water is based on aesthetic and taste consideration rather than its physiological effects (Trivedy and Goel, 1986). Kakati and Bhattacharya (1990) studied the water quality of various surface water sources of greater and found that Fe content ranges from 0.112 to 12.8 mg/L. High doses of Fe are known to cause hemorrhagic necrosis, sloughing of mucosa areas in the stomach, tissue damage to a variety of organs by catalyzing the conversion of H2O2 to free radical ions that attack cell membranes, proteins and break the DNA double strands and cause oncogene activation

(Gurzau et al., 2003).

5.3. Phytosociology

The physico-chemical nature of environment and biological peculiarities of the macrophytes themselves play a significant role in the pattern of distribution of macrophytes. Alternanthera philoxeroides Griseb., Azolla pinnata Lam., Eichhornia crassipes Linn., Lemna minor Linn., Pistia stratiotes Linn., Salvinia cucullata Roxb.,

Salvinia natans Hoffm., and Spirodela polyrhiza (Linn) Schleid. were the plants with higher density in all the sites. This shows the resisting capacity and tolerance of these macrophytes with the variation in the degree of pollution and disturbance.

Many scientist and researcher globally are now using the various species diversity index of any given water body to find out the degree of pollution of that water body because the level of pollution is always equal to the loss of species biodiversity (Staub et al., 1970).

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It is believed that pollution reduced biotic diversity (Patrick et al., 1963; Mishra,

2006; Rai, 2008b). There was successive decrease in the number of species with increase in the degree of disturbance and pollution pollution and disturbance cause by anthropogenic activities. Due to these reasons, Site I, Site II and Site III shows decreased diversity. Decreased diversity has been used as an indicator of water quality deterioration.

The Site IV shows more diversity than the remaining Site I, Site II and Site III.

Considerably much higher diversity at Site IV was probably due to higher nutrient availability and dissolved oxygen contents. Communities with a high diversity use the available energy efficiently and have high stability. In other words, they can resist the adversities of changed environment in a better way. Bechtel and Copeland (1970) have shown an increasing trend of diversity with dilution from the effluent discharge points.

Schafer (1973), have suggested that polluted systems display a reduction in diversity indices. Decrease in diversity indices at polluted regions may be also due to cumulative effects of the various effluents. Staub et al. (1970) proposed a scale of pollution in terms of species diversity i.e. 3.0-4.5 clear; 2.0-3.0 light pollution; 1.0-2.0 moderate pollution and

0.0-1.0 heavy pollution.

The Sorenson’s Similarity Index was calculated between the different. Similarity index may successfully be used for two purposes, i.e. whether two or more stands belong to the same community or to determine the extent of resemblance between different communities (Mishra, 2006).

5.4. Plants

Iron (Fe) is one of the vital elements for humans and for other forms of life. Past studies have documented the Fe phytoremediation ability of the obnoxious free-floating macrophytes from nutrient-rich wastewaters (Tripathi and Upadhyay, 2003; Sooknah and

Wilkie, 2004; Jayaweera et al., 2008). Pistia stratiotes accumulate high amount of Fe

111 | P a g e concentration from the lake in site II. The accumulation of heavy metals like Fe, Zn, Cu,

Cr, and Cd did not cause any toxic effect on the Pistia stratiotes which qualifies the plant to be used for the phyto-remediation of waste water for heavy metals on large-scale

(Mishra and Tripathi, 2008). Pistia stratiotes has been considered a promising plant for the remediation of contaminated waters (Maine et al., 2001). Odjegba and Fasidi (2004) revealed the accumulation capacity and resistance of Pistia stratiotes against trace elements and concluded that it can tolerate heavy metals and growth responses of the plant varied inversely with the increase in metal concentration.

5.5. Phytoremdiation

Eichhornia crassipes is the top accumulators of Fe in the phytoremediation experiment at all the concentrations i.e. 1 mg, 3 mg, and 5 mg for 4 days, 8 days and 12 days. Jayaweera et al. (2008) also reported very high accumulation of Fe in Eichhornia crassipes biomass under varying nutrient conditions and showed the highest phytoremediation efficiency of

47% during optimum growth at the 6th week with a highest accumulation of 6707 Fe mg/kg dry weight. Furthermore, previous studies showed that hyacinth roots form plaques of Fe(OH)3 by diffusing photosynthetically produced O2 to the rhizosphere to avoid the

2+ 2+ formation of H2S and to counteract Fe and Mn toxicity at lower Dissolved Oxygen levels (Vesk and Allaway, 1997; Vesk et al., 1999).

Eichhornia crassipes and Pistia stratiotes has been mostly studied for its tendency to bio-accumulate and biomagnify the heavy metal contaminants present in water bodies.

These plants were studies to evaluate the efficiency to remove heavy metals from the effluents of a Steel Foundry located in Hayatabad, Pakistan (Aurangzeb et al. 2014). The potential application Eichhornia crassipes in the removal of heavy metals from water was discovered in the early 1980s (Govindaswamy et al., 2015). It accumulates metals and as

112 | P a g e the recycling process is run by photosynthetic activity and biomass growth, sustainable process and cost efficient (Garbisu et al., 2002; Lu et al., 2004; Bertrand and Poirier,

2005). Due to its exotic invasive nature and rapid decomposition in comparison to other plants, it has been reported that the growth of water hyacinth poses problem in functioning in the aquatic ecosystem e.g. constructed wetlands (Khan et al., 2000; Rai, 2011, 2012) however, it is one of the most suitable plant for phytoremediation used by various researchers and scientists. Eichhornia crassipes colonized natural wetland systems could serve as “nature’s kidneys” for proper effluent treatment to preserve the earth’s precious water resources from the pollutions (Malik, 2007). The application of Eichhornia crassipes as a cleaning agent for phytoremediation is very useful in a way to preserve the aquatic bodies from many variant pollutants specially heavy metals because of its easily availability, low cost, effectiveness and eco-friendly methods.

Eichhornia crassipes is used in various polluted sites such as wetlands, rivers basins, ponds, ditches, sewages, industrial effluents, landfills, etc. for remediation purposes. They is adopted by many researchers and scientists for phytoremediation because of its easily availability, its effectiveness, easily applicable to wide range of water contaminant, cost free plant for the method, safe and eco-friendly technology. Eichhornia crassipes as a common plant for the removal of Fe, Pb, Cu, Zn, Hg, Cd, Cr and Mn

(Tiwari et al., 2007; Kumar et al., 2008; Rai 2009a; Rai et al., 2010; Chatterjee et al.,

2011; Fawzy et al., 2012; Padmapriya and Murugesan, 2012; Sasidharan et al., 2013;

Mishra et al., 2013). A study to assess the growth of Eichhornia crassipes and its ability to accumulate Cu from polluted water with high Cu concentrations and mixture of other contaminants under short-term exposure, in order to use this plants for remediation of highly contaminated sites results in high accumulation and translocate much lesser in leaves than in roots (Melignami et al., 2015). Eichhornia crassipes were also exposed to

113 | P a g e varying concentration of Hg as seedlings under hydroponic system to investigate accumulated mercury level, anti-oxidant defence mechanisms, growth patterns changes, and damaging of DNA from the exposure effect. (Malar et al., 2015). It is also an As hyperaccumulator which can uptake As, have the tolerance, and accumulate in their biomass (Tiwari et al., 2014). Eichhornia crassipes reduce levels of heavy metals in acid- mine water with little sign of toxicity (Falbo and Weaks, 1990).

Eichhornia crassipes in the removal of nutrients from the aquatic bodies is very effective (Roger and Davis, 1972; Wolverton and McDonald, 1979; Trivedy and

Pattanshetty, 2002; Jayaweera and Kasturiarachchi, 2004; Cristina C et al., 2009). Many researchers have studied Eichhornia crassipes with waste water containing high nutrients

(Gamage and Yapa, 2001; Jayaweera and Kasturiarachchii, 2004; Tripathy and Upadhyay,

2003). Domestic sewage purification potential were studied using this plant many parameters which includes Biochemical Oxygen Demands (BOD), Chemical Oxygen

Demand (COD), Faecal coliform count, nitrate and phosphate concentrations, pH value, heavy metals, turbidity, odor and colour (Alade and Ojoawo, 2009). Moreover, Eichhornia crassipes has the capacity to remove nutrients and heavy metals from leachate from landfill which minimize pollution to an acceptable level (Akinbile and Yusoff, 2012).

Significant results were obtained in the physiological response of Eichhornia crassipes to the combined exposure of excess nutrients and Hg (Cristina C et al., 2009). A study also reveals that Eichhornia crassipes grown in nutrient-poor conditions are ideal to remove Fe from wastewater with a hydraulic retention time of approximately 6 weeks (Jayaweera et al., 2008). The plant is also effective for purifying wastewater from an intensive duck farm during its growing season and also the harvested plant had an excellent performance as duck feed (Jianbo et al., 2008). Also the plant can survived extremely eutrophic water with anaerobically digested flushed dairy manure wastewater (Sookah and Wilkie, 2004).

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P. stratiotes has also been extensively used for phytoremediation (Quian et al.,

1999; Skinner et al., 2007). It was also used in lab experiments for the removal of heavy metals (Miretzky et al., 2010). Pistia stratiotes was helpful in the removal of some heavy metals from the industrial effluent and was found to be the best phytoremedient for Pb, Cu as it was successful in removing 70.7% and 66.5% of these metals (Lone et al., 2008).

Similar findings were also reported by Lu (2011) while working on lettuce that was a hyper accumulator of Fe, Pb and Cu. The same kind of findings were also reported by

Mokhtar et al. (2011) and Mishra and Tripath (2008).

Phytochemicals composition

Eichhornia crassipes being a fast growing plant is used for rapid removal of various kinds of pollution in water resulting in positive outcomes. The plant was evaluated for its possible potential of heavy metal accumulations which results in the discovery of high cellulose content and its functional groups including amino (-NH2), carboxyl (-COO-), hydroxyl (-OH-), sulfahydryl (-SH) showing high tolerance and affinity towards heavy metals adsorption (Patel, 2012). Eichhornia crassipes contains many phytochemical such as amino acids including glutamic acid theonine, leucine, lysine, methionine, tryptophan, tyrosine, and valine; flavonoids including apigenin, azaeleatin, chrysoeriol, gossypetin, kaempferol, luteolin, oientin and tricin. (Nyananyo et al., 2007).

P. stratiotes plant extracts consist of various alkaloids, glycosides, flavonoids and phytosterols. Leaf and stem extract consist of 92.9% H2O, 1.4% protein, 0.3% fats, 2.6% carbohydrates, 0.9% crude fiber and 1.9% minerals (mostly potassium and phosphorous).

Leaves are rich in vitamins A & C, stigma-sterol, stigma-steryl, stigma-sterate and palmitic acids are found in abundance (Khan et al. 2014) 2-di-cgl-cosy-flavones of vicenin and lucenin type, anthocyanin cyaniding-3-glucoside, luteolin-7-glucoside and mono-C-glcosyl

115 | P a g e flavones–vitexin and orientin have also been isolated from the plant (Khare, 2005).

Stratioside II (a new C13 norterpene glucoside) is the major component of this plant.

Leaves are rich in proteins, essential amino acids, stigmatane, sito-sterol acyl glycosides and minerals (Ghani, 2003). Vicenin an anticancer agent (Nagaprashantha et al., 2011) and cyanidin-3-glucoside (an anthocyanin) is present (Rastogi & Mehrotra, 1993).

Biofuel production

The biogas produce from the organic matter of plants serves as a cheap mode of replaceable biofuel from the present petroleum fuels. Bhattacharya and Kumar (2010) listed attributes of an ideal biofuel crops. They are:

1. The plant should be naturally grown vegetation, preferably perenials.

2. The plant should content high cellulose with low lignin per unit volume of

dry matter.

3. The plant should be easily degradable.

4. The plant should not compete with arable crop plants for space, light and

nutrient.

5. The plant should resists pest, insects and disease.

6. The plant should not be prone to genetic pollution by cross breeding with

cultivable food crops.

Eichhornia crassipes is abundantly available, perennial, non crop, biodegradabale and high cellulose content which fulfils all the criteria for bio-energy production (Patel, 2012). The cellulose and hemicellulose content in the plant are more easily converted to fermentable

116 | P a g e sugar which results in enormous amount of utilizable biomass for the biomass industry

(Bhattacharya and Kumar, 2010). Eichhornia crassipes mixed with animal waste yields better biogas (Kumar, 2005) and the obtained sludged mixed feed with nitrogen, phosphorous and potassium content can be utilized as good manure (Malik, 2007).

Microbes take a great role in decomposing the organic matter of plants. Patel et al (1992) work in stimulation of microbial activity to increase the biogas production using different biological and chemical additives. Also the plants biomass fermentation with the help of methanogens in a reactors turn out to be positive producing good biofuel (Chanakya et al.,

1993).

Other Utilities

Eichhornia crassipes and Pistia stratiotes is a kind of multi-utility plant that can take into different roles from different perspective utility. It can be used to make traditional basket and weaving purposes (Jafari, 2010). Moreover it can be crafted into coasters, placemeats, mats, shoes, sandals, bags, wallets, vases etc. from its dried petioles (Patel, 2012). Pulp material extracted from Eichhornia crassipes plant has the potential for producing greaseproof paper (Goswami and Saikia, 1994). The plant can also be used for oil sorption in a wide range of temperatures, and sorbed oils can be recovered (Yang et al., 2014).

Eicchornia crassipes and Pistia stratiotes being an infesting plant, it’s used as beneficial utility plants has been restricted by many. However, taking its positive advantages of this plants, many have also developed the technology and used in accumulation and absorption of the heavy metals and other nutrients under phytoremediation from the aquatic bodies, biofuel and biogas production through fermentation and decomposition, and many more utilities which are more beneficial. As the plant is infested throughout the world in every corner and every side, utilization of the

117 | P a g e plant should be done from every angle seeing the positive attributes. It will also help in controlling the infestation of the plant and help in acquiring the positive attributes of the

Eichhornia crassipes and Pistia stratiotes in order to aware and make used the most of it in eco-friendly and sustainable ways.

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Chapter 6 SUMMARY, CONCLUSIONS AND RECOMMENDATIONS

The Loktak Lake is the largest fresh water lake in northeast India is rich in biodiversity and it is considered as ‘lifelines for the people of Manipur’ for its importance in their socio-economic and cultural life. The lake is also supports varied types of habitat due to which the lake is blessed with rich diversity flora and fauna for the reason it has been included under Ramsar convention. The lake sustained the lives of the wildlife (flora and fauna) and people who live on the unique vegetation, phoomdis. However, the last few decades acted as a very critical time in the degradation of this lake the due to the agricultural expansion in the adjoining areas, heavy load of domestic wastes from different sources of the Imphal city into the lake through the inflowing rivers, and anthropogenic activities that contribute significantly to water quality deterioration and may results in eutrophication of the lake.

The present study performed various experiments along with phytoremediation technique using the plant species found in the lake which will benefit in the prevention against further pollution of the water including heavy metal pollution and it will increase the proper knowledge of utilising the particular plant species which is very much important for the wetland as well as for biodiversity conservation of this lake.

There are scanty researches which studied physico-chemical characteristics, heavy metal, phytosociology as well as phytoremediation studies in totality. Henceforth, present study aimed:

 To assess the heavy metal pollutants of the Loktak lake.

 To perform the phytosociological studies of macrophytes.

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 To perform heavy metal phytoremediation investigations with selected

macrophytes of the Loktak lake.

Surveys of the lake were done before selection of the sampling sites. A total of four sampling sites were selected (in triplicates) for analysis of various physico-chemical characteristics of the water. The study sites were selected on the basis of the source pollution and disturbance causing the lake polluted. The Sites are as follows.

5. Site I (Loktak Nambul vicinity):

Site I is the contact point between Loktak Lake and the river, Nambul, which pass

through the Imphal municipal area and then drained in the lake. This river carries

waste of the Imphal municipal area.

6. Site II (Loktak Nambol vicinity):

Site II is the contact point between the Loktak Lake and the river, Nambol, which

pass through the Bishenpur municipal area and then drained in the lake. This river

carries waste of the Bishenpur municipal area.

7. Site III (Loktak Yangoi vicinity):

Site III is the contact point between the Loktak Lake and the river, Yangoi, which

is also a main source of pollution which drained in the lake.

8. Site IV (Loktak proper):

Site IV is the Joining point between the Loktak Lake and the Canal of National

Hydro Power Corporation Limited (NHPC) of Loktak Lake.

Water sampling were performed in all three seasons i.e. Rainy season, Winter season, and

Summer season from August, 2013 to July, 2015. Detailed investigation in relation to physico-chemical, heavy metal, phytosociological studies and phytoremediation. The results of the investigation can be summarised as follows.

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A. Water Quality analysis

1. The highest value of temperature was measured 28.6°C at Site II during rainy

season (2013) and the lowest was measured 16.8°C at Site II during winter season

(2013).

2. The highest value of pH was measured 7.4 at Site I and Site IV during summer

season (2015) and the lowest of 6.3 at Site IV during rainy season (2013).

3. The highest value of Transperancy was measureed 1.7 m at Site IV during winter

season (2013) as well as rainy (2013) and the lowest of 0.74 m at Site III during

summer season (2014).

4. The highest value of Total Solids (TS) was measured 46.3 mgL-1 at Site III during

rainy season (2014) and the lowest of 23.3 mgL-1 at Site III during winter season

(2013) and Site I and Site III during summer season of both the years (2014 and

2015).

5. The highest Dissolved Oxygen (DO) was measured 9.6 mgL-1 at Site IV during

winter season (2013) and the lowest of 6.9 mgL-1 at Site III during summer season

(2014).

6. The highest value Biological Oxygen Demand (BOD) was measured 2.1 mgL-1 at

Site I during winter season (2014) and the lowest of 0.5 mgL-1 at Site III during

winter season (2013).

7. The highest value of Acidity was measured 32.6 mgL-1 at Site III during rainy

season (2013) and the lowest of 9.3 mgL-1 at Site IV during winter season (2013).

8. The Highest value of alkalinity was measured 73.3 mgL-1 at Site IV during winter

season (2013-14) and 21.3 mgL-1 at Site III of rainy season (2013-2014).

9. The highest value of Chloride was measured 45.6 mgL-1 at Site III during winter

season (2014) and the lowest of 20.3 mgL-1 at Site II during winter season (2014).

121 | P a g e

10. The highest value of Total Hardness was measured 68 mgL-1 at Site III during

winter season (2013) and the lowest of 18.7 mgL-1 at Site III during rainy season

(2013).

11. The Highest value of Turbidity was measured 15.8 NTU at Site IV during summer

season (2014) and the lowest of 0.4 NTU at Site III during winter season (2013) and

Site IV during winter season (2013 and 2014).

12. The highest value of Nitrate was found 0.62 mgL-1 at Site II during summer season

(2014) and the lowest of 0.21 mgL-1 at Site IV during rainy season (2013).

13. The highest value of Phosphate was measured 0.3 mgL-1 at Site II during summer

season (2015) and the lowest of 0.06 mgL-1 at Site IV during winter season (2013).

B. Heavy Metals Analysis

Water

1. The highest value of Fe concentration was measured 0.17 mgL-1 at Site IV during

winter season (2013) and the lowest of 0.01 mgL-1 at Site I during rainy season

(2014) and summer season (2015).

2. The Hg were present in a minute negligible amount of <0.001 mgL-1 for all the

samples collected from different Sites.

3. The Cd were present in a minute negligible amount of <0.001 mgL-1 for all the

samples collected from different Sites.

4. The As were present in a minute negligible amount of <0.001 mgL-1 for all the

samples collected from different Sites.

5. The Pb were present in a minute negligible amount of <0.001 mgL-1 for all the

samples collected from different Sites.

122 | P a g e

6. The Cr were present in a minute negligible amount of <0.001 mgL-1 for all the

samples collected from different Sites.

7. The Zn were present in a minute negligible amount of <0.02 mgL-1 for all the

samples collected from different Sites.

C. Phytosociology

1. From the phytosociological studies of the different sites, altogether a total of 24

wetland macrophytes species belonging to 23 genera and 17 families of

angiosperms were recorded. Of this, 10 species belonging to 8 genera and 8

families, 13 species belonging to 12 genera and 11 families, 12 species belonging

to 11 genera and 9 families and 21 species belonging to 20 genera and 15 families

were recorded from Site I, Site II, Site III and Site IV respectively.

2. The macrophyte species namely Alternanthera philoxeroides Griseb., Arthraxon

lanceolatus (Roxb) Hochst., Azolla pinnata Lam., Ceratophyllum demersum Linn.,

Cyrtococcum accrescens Stafp., Eichhornia crassipes Linn., Enhydra fluctuans

Lour., Euryale ferox Salisb., Hydrilla verticillata (Linn) Royle., Ipomoea aquatica

Forsk., Lemna minor Linn., Nymphaea nouchali Burm. f., Nymphoides cristatum

O. Kuntz, Oenanthe javanica (Bl) D.C., Pistia stratiotes Linn., Polygonum

glabrum Willd., Potamogeton crispus Linn., Rumex nepalensis Spreng., Salvinia

cucullata Roxb., Salvinia natans Hoffm., Spirodela polyrhiza (Linn) Schleid.,

Trapa natans Linn., Vallisnaria spiralis Linn., and Zizania latifolia (Griseb) stapf..

were recorded.

3. Shannon-Wiener diversity index for macrophyte species was maximum at Site IV

i.e. 2.37 and minimum at Site I i.e. 1.31.

4. The Simpson index of dominance was maximum at Site III i.e. 0.42 and minimum

at Site IV i.e. 0.12.

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5. Between Site I and Site II, the Sorenson’s Similarity was found to be 0.87, between

Site I and Site III, it was found 0.73, between Site I and Site IV, it was 0.65,

between Site II and Site III, it was 0.72, between Site II and Site IV, it was 0.71,

and between Site III and Site IV, it was found to be 0.61.

6. A total number of 17 families were recorded from all the study sites. Of these, 15

families were recorded from Site IV followed by 11 families from Site II, 9

families from Site III and 8 families from Site I.

D. Metal analysis in biomass of selected macrophytes

1. The highest Fe concentration was measured 28.29 mgkg-1 in Pistia stratiotes at Site

II and the lowest of 1.68 mgkg-1 in Salvinia cucullata at Site I.

2. The highest Hg concentration was measured <0.0069 mgkg-1 in Salvinia cucullata

at Site I and the lowest of <0.0017 mgkg-1 in Eichhornia crassipes at Site I.

3. Similar with Hg concentration, the highest Cd concentration was measured

<0.0069 mgkg-1 in Salvinia cucullata at Site I and the lowest of 0.0017 mgkg-1 in

Eichhornia crassipes at Site I.

4. Similar with Hg and Cd concentration, the highest As concentration was measured

<0.0069 mgkg-1 in Salvinia cucullata at Site I and the lowest of 0.0017 mgkg-1 in

Eichhornia crassipes at Site I.

5. Similar with Hg, Cd and As concentration, the highest Pb concentration was

measured <0.0069 mgkg-1 in Salvinia cucullata at Site I and the lowest of 0.0017

mgkg-1 in Eichhornia crassipes at Site I.

6. Similar with Hg, Cd, As, and Pb concentration, the highest Cr concentration was

measured <0.0069 mgkg-1 in Salvinia cucullata at Site I and the lowest of 0.0017

mgkg-1 in Eichhornia crassipes at Site I.

124 | P a g e

7. The highest Zn concentration was measured <0.13 mgkg-1in Salvinia cucullata at

Site I and the lowest of 0.03 mgkg-1 in Eichhornia crassipes at Site I.

E. Phytoremediation

1. Fe concentration in water under varying ranges of metal concentration i.e. 1 mgL-

1, 3mgL-1 and 5 mgL-1 was maximum in Eicchornia crassipes treated water

followed by Pistia stratiotes treated water, Lemna minor treated water and Salvinia

cucullata treated water during interval of 4, 8 and 18 days.

2. In 4 days experiment, Eicchornia crassipes treated water showed maximum of

68% removal from 1 mgL-1, 40.3% removal from 3 mgL-1, 45.4% removal from 5

mgL-1 and Lemna minor treated water showed minimum of 25% removal from 1

mgL-1, 21% removal from 3mg/L, Salvinia cucullata showed 26% removal from

5mg/L.

3. In 8 days experiment, Eicchornia crassipes treated water showed maximum of

81% removal from 1 mgL-1, 55% removal from 3 mgL-1, 76.6% removal from 5

mgL-1 and Lemna minor treated water showed minimum of 71% removal from 1

mgL-1, Salvinia cucullata showed 40.3% removal from 3 mgL-1, Pistia stratiotes

showed 45.2% removal from 5 mgL-1.

4. In 12 days experiment, Eicchornia crassipes treated water showed maximum of

89% removal from 1 mgL-1, 81.3% removal from 3 mgL-1, 73.2% removal from 5

mgL-1 and Salvinia cucullata treated water showed minimum of 81% removal from

1 mgL-1, 59.3% removal from 3 mgL-1, 61.4% removal from 5 mgL-1.

From the findings of the present study, it can be concluded that the water of the

Loktak lake is polluted in some of the parameter performed along with phytoremediation technique using the plant species found in the lake. The result of the findings may benefit in the prevention against further pollution of the water including heavy metal pollution and

125 | P a g e

it will increase the proper knowledge of utilising the particular plant species which is very much important for the wetland as well as for biodiversity conservation of this lake.

Moreover, proper utilisation of the important macrophyte, Eicchornia crassipes and Pistia stratiotes in the simple technology, using in accumulation and absorption of the heavy metals and other nutrients under phytoremediation from the aquatic bodies, biofuel and biogas production through fermentation and decomposition and many more utilities which are more beneficial were also discussed. Thus, the study proved that the proper utilization of phytoremediation technique using macrophytes may mitigate the heavy metal and other contamination in a eco-friendly and sustainable ways.

Recommendations

From the findings of the present study, following recommendations were made for proper mitigation of pollution and management of Loktak lake:

 Eicchornia crassipes, Pistia stratiotes, Lemna minor and Salvinia cucullata may be

used in phytoremediation of Fe at microcosm level.

 Although, the water of Loktak Lake is contaminated, most of the pollutants were

under permissible limit, therefore precautions and serious attempt should be taken

so that it may not turn into serious water/metal pollution.

 Physico-chemical and heavy metal parameters may be regularly monitored to

check the current condition of the water.

 After seeing the importance, pollution management approach is a must to conserve

a Ramsar Lake like Loktak Lake.

 The Government must take nessesary steps for cleaning the lake, physically as well

as internally to prevent from further contamination.

126 | P a g e

Future perspective

Although, I confined my quest for phytoremediation potential of 4 macrophytes pertaining to Fe only in context of present status of Loktak Lake pollution, future microcosm studies will also investigate the phytoremediation potential of other observed macrophytes for different ranage of heavy metals.

127 | P a g e

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Appendix I. Correlation between physico-chemical parameters of Site I.

Site I Total Temperature pH Transperancy TS DO BOD Acidity Alkalinity Chloride Hardness Turbidity Nitrate phosphate Temperature 1.00

pH -0.45 1.00

Transperancy -0.61 0.35 1.00

TS 0.69 -0.91 -0.68 1.00

DO -0.75 -0.13 0.40 -0.13 1.00

BOD -0.21 -0.44 0.42 0.20 0.72 1.00

Acidity 0.96 -0.20 -0.54 0.49 -0.84 -0.31 1.00

Alkalinity -0.81 0.82 0.60 -0.93 0.25 -0.24 -0.65 1.00

Chloride -0.97 0.65 0.65 -0.84 0.60 0.09 -0.87 0.91 1.00

Total -0.76 0.87 0.53 -0.94 0.18 -0.33 -0.58 0.99 0.88 1.00 Hardness Turbidity 0.83 0.03 -0.33 0.22 -0.96 -0.52 0.92 -0.35 -0.69 -0.29 1.00

Nitrate -0.27 0.95 0.35 -0.85 -0.36 -0.54 -0.01 0.76 0.49 0.81 0.28 1.00

Phosphate -0.12 0.73 0.13 -0.56 -0.38 -0.56 0.11 0.62 0.28 0.67 0.39 0.85 1.00

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Appendix II. Correlation between physico-chemical parameters of Site II.

Site II Temperature pH Transperancy TS DO BOD Acidity Alkalinity Chloride Total Turbidity Nitrate phosphate Hardness Temperature 1.00

pH 0.47 1.00

Transperancy -0.49 -0.89 1.00

TS 0.25 -0.37 0.44 1.00

DO -0.68 -0.85 0.90 0.43 1.00

BOD 0.09 0.09 0.67 0.81 0.66 1.00

Acidity 0.95 0.68 -0.74 -0.03 -0.87 -0.21 1.00

Alkalinity -0.77 0.17 -0.08 -0.67 0.08 -0.64 -0.54 1.00

Chloride 0.67 0.71 -0.84 -0.41 -0.83 -0.37 0.85 -0.23 1.00

Total 0.16 0.83 -0.87 -0.76 -0.75 -0.77 0.47 0.42 0.78 1.00 Hardness Turbidity 0.72 0.88 -0.83 -0.35 -0.97 -0.58 0.88 -0.11 0.78 0.69 1.00

Nitrate -0.03 0.81 -0.71 -0.80 -0.66 -0.87 0.27 0.66 0.50 0.91 0.65 1.00

Phosphate 0.02 0.82 -0.77 -0.81 -0.69 -0.84 0.33 0.59 0.61 0.96 0.66 0.99 1

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Appendix III. Correlation between physico-chemical parameters of Site III.

Site III Temperature pH Transperancy TS DO BOD Acidity Alkalinity Chloride Total Turbidity Nitrate phosphate Hardness Temperature 1.00

pH -0.58 1.00

Transperancy -0.36 -0.46 1.00

TS 0.71 -0.77 0.30 1.00

DO -0.62 -0.08 0.90 0.09 1.00

BOD 0.44 -0.31 -0.10 0.50 -0.22 1.00

Acidity 0.98 -0.57 -0.42 0.67 -0.66 0.50 1.00

Alkalinity -0.87 0.80 -0.13 -0.95 0.16 -0.49 -0.82 1.00

Chloride -0.93 0.78 0.02 -0.86 0.32 -0.33 -0.88 0.96 1.00

Total -0.94 0.77 0.04 -0.88 0.35 -0.52 -0.90 0.98 0.98 1.00 Hardness Turbidity 0.56 0.22 -0.94 -0.16 -0.98 0.16 0.58 -0.09 -0.24 -0.27 1.00

Nitrate -0.75 0.88 -0.34 -0.96 -0.04 -0.35 -0.69 0.97 0.93 0.91 0.12 1.00

Phosphate -0.63 0.87 -0.33 -0.89 -0.05 -0.70 -0.66 0.87 0.76 0.17 0.17 0.87 1.00

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Appendix IV. Correlation between physico-chemical parameters of Site IV.

Site IV Temperature pH Transperancy TS DO BOD Acidity Alkalinity Chloride Total Turbidity Nitrate phosphate Hardness Temperature 1.00

pH 0.21 1.00

Transperancy -0.60 -0.89 1.00

TS 0.33 -0.76 0.47 1.00

DO -0.88 -0.23 0.59 -0.06 1.00

BOD -0.39 -0.24 0.35 0.22 0.53 1.00

Acidity 0.96 0.18 -0.59 0.26 -0.95 -0.36 1.00

Alkalinity -0.69 0.34 0.08 -0.73 0.55 -0.14 -0.74 1.00

Chloride 0.85 0.07 -0.45 0.41 -0.75 0.09 0.89 -0.85 1.00

Total -0.75 0.43 0.01 -0.78 0.62 0.23 -0.75 0.90 -0.70 1.00 Hardness Turbidity 0.51 0.72 -0.76 -0.53 -0.61 -0.74 0.45 0.25 0.15 0.07 1.00

Nitrate 0.27 0.83 -0.73 -0.73 -0.39 -0.51 0.22 0.47 0.01 0.38 0.94 1.00

Phosphate 0.27 0.81 -0.75 -0.79 -0.53 -0.50 0.33 0.35 0.10 0.32 0.93 0.93 1.00

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Appendix V: Permissible limits of physico-chemical parameters of water by different scientific agencies.

Parameter Standards

USPH ISI WHO ICMR

Temperature (◦C) - 40 - -

pH (nano mole Lˉ¹) 6 - 8.5 6 - 9 6.5 - 8.5 7 - 8.5

TS (mgLˉ¹) - - 500 500 - 1500

Turbidity (NTU) - - 5 -

Total Hardness 500 - - 300

( mgL ˉ¹CaCOз)

DO (mgLˉ¹) >4 >5 - -

BOD (mgLˉ¹) - <3 - -

Chloride 250 600 200 250

(mgL ˉ¹ CaCOз)

Total Alkalinity - 200 - -

(mgL ˉ¹CaCOз)

Nitrate (mgLˉ¹) 10 50 - 20

Phosphate (mgLˉ¹) 0.1 - - -

(-) Not found

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Appendix VI: Permissible limits of heavy metals of water and biomass by different scientific agencies.

Parameter Standards

USPH ISI WHO ICMR

Fe (in water ) - - 1 mgL-1 -

(in biomass) - - 20 mgkg-1 -

Zn (in water ) - - 5mgL-1 -

(in biomass) - - 50 mgkg-1 -

Pb (in water ) - - 0.05mgL-1 -

(in biomass) - - 2 mgkg-1 -

Cd (in water ) - - 0.01 mgL-1 -

(in biomass) - - 0.02 mgkg-1 -

Cr (in water ) - - 0.1 mgL-1 -

(in biomass) - - 1.3 mgkg-1 -

Hg (in water ) - - 0.001 mgL-1 -

(in biomass) - - - -

As (in water ) - - 0.05 mgL-1 -

(in biomass) - - - -

(-) Not found

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Appendix VII: Correlation between Fe concentrations of plants and water samples from different sites 2013-2014 2014-2015

Name of plants Rainy Winter Summer Rainy Winter Summer

Eichhornia crassipes 0.21 -0.07 0.73 0.77 0.36 0.34

Lemna minor -0.21 -0.52 0.15 -0.26 -0.35 -0.01

Pistia stratiotes 0.09 -0.30 0.63 0.35 0.08 0.31

Salvinia cucullata 0.62 0.37 0.94 0.96 0.73 0.71

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LIST OF PUBLICATIONS

1. A Microcosm Investigation of Fe (Iron) Removal Using Macrophytes of Ramsar

Lake: A Phytoremediation Approach. International Journal of phytoremediation

(Impact Factor: 2.04)

2. Wetland Plants: Green Bio-resource for heavy metals phytoremediation. Lambert

Academic Publishing. (Book)

3. Eichhornia crassipes as a potential phytoremediation agent and an important

bioresource for Asia Pacific region. Environmental Skeptics and Critics.

4. Seasonal Monitoring of Water quality of a Ramsar site in an Indo-Burma Hotspot

region of Manipur, India. International Research Journal of Environmental

Sciences.

5. Wetland Resources of Loktak Lake in Bishenpur District of Manipur, India: A

Review. Science and Technology Journal (Mizoram University)

6. Wetland Resources of Northeast India: A Case Study of the Loktak Lake, Manipur,

India. Seminar Proceeding (Department of Geography & RM, MZU)

7. A study of depletion water quality and wildlife resources of the Loktak Lake,

Manipur, India. Seminar Proceeding (Department of Environment Science,

MZU)

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