EFFECTS OF COPPER AND LEAD ON THE PHYSIOLOGY OF SALVINIA MOLESTA, PISTIA STRATIOTES AND LEMNA TRISULCA AND THEIR PHYTOREMEDIATION POTENTIALS

TABLE OF CONTENTS

Cover Page --------------------------------------------------------------------------------------------- i

Fly Leaf ------------------------------------------------------------------------------------------------- ii

Title Page ----------------------------------------------------------------------------------------------- iii

Declaration --------------------------------------------------------------------------------------------- iv

Certification -------------------------------------------------------------------------------------------- v

Dedication ---------------------------------------------------------------------------------------------- vi

Acknowledgements ----------------------------------------------------------------------------------- vii

Abstract ------------------------------------------------------------------------------------------------- ix

Table of Contents ------------------------------------------------------------------------------------- xi

List of Tables ------------------------------------------------------------------------------------------ xvi

List of Figures ----------------------------------------------------------------------------------------- xvii

List of Appendices------------------------------------------------------------------------------------- xix

List of Acronyms and Symbols---------------------------------------------------------------------- xxi

CHAPTER ONE

1.0 INTRODUCTION

1.1         General Introduction ---------------------------------------------------------------------- 1

1.2         Background Information------------------------------------------------------------------ 5

1.2.1     Lemna trisulca-------------------------------------------------------------------------------- 5

1.2.2     Pistia stratiotes------------------------------------------------------------------------------- 8

1.2.3     Salvinia molesta------------------------------------------------------------------------------- 9

1.2.4    Anti-oxidant Enzymes----------------------------------------------------------------------- 11

4.3 Morphological Observations/Visual Symptoms by Salvinia molesta,

Pistia stratiotes andLemna trisulca-----------------------------------------------------------                  95

4.4 Metal Accumulation/Concentration in the Aquatic Macrophytes Species----------- 99

4.5 Comparison of Copper and Lead Bioaccumulation by Salvinia molesta,

Pistia stratiotes andLemna trisulca-----------------------------------------------------------                  115

4.6 Principal Component Analysis (PCA) ------------------------------------------------------ 119

CHAPTER FIVE

5.0      DISCUSSION

5.1 Chlorophyll Content (Photosynthetic Pigment)               ----------------------------------- 121

5.1.1 Chlorophyll a content--------------------------------------------------------------------------- 121

5.1.2 Chlorophyll b content -------------------------------------------------------------------------- 123

5.2 Anti-oxidant enzyme activity---------------------------------------------------------------              125

5.2.1 Catalase (CAT) Activity----------------------------------------------------------------------- 126

5.2.2 Peroxidase (POD) Activities------------------------------------------------------------------ 127

5.3         Effect of Copper and Lead on the Visual Symptoms---------------------------------128

5.4         Metals Accumulation in the Aquatic Macrophytes----------------------------------- 130

5.5         Potential of Macrophytes for Phytoremediation Studies---------------------------- 134

CHAPTER SIX

6.0     SUMMARY, CONCLUSION AND RECOMMENDATION

6.1    Summary --------------------------------------------------------------------------------------- 135

6.2   Conclusion -------------------------------------------------------------------------------------- 137

6.3   Recommendation ------------------------------------------------------------------------------ 138

References -------------------------------------------------------------------------------------------- 139

Appendices -------------------------------------------------------------------------------------------  161

List of Tables

Table                                                                                                   Page

Table 4.1: Overall Chlorophyll a content of the 3 macrophytes at different Copper

and Leadconcentrations ---------------------------------------------------------------- 72

Table 4.2: Comparison of Chlorophyll a content among the 3 macrophytes ----------------- 73

Table 4.3: Overall Chlorophyll b contentof the 3 macrophytes at different Copper

and Leadconcentrations ---------------------------------------------------------------- 79

Table 4.4: Comparison of Chlorophyll b content among the 3 macrophytes ----------------- 80

Table 4.5Overall Catalase activities for the various treatments in relation to days---------- 86

Table 4.6: Comparison in catalase activitiesbetween the 3 macrophyte species ------------- 87

Table 4.7Overall Peroxidase activity for the different treatments in relation to days-------  93

Table 4.8: Comparison of peroxidase activities among macrophyte species ----------------- 94

Table 4.9. Symptoms of Chlorosis/Necrosis in Salvinia molestaafter copper and

lead treatment --------------------------------------------------------------------------- 96

Table 4.10.Symptoms of Chlorosis/Necrosis in Pistia stratiotesafter copper and

lead treatment-----------------------------------------------------------------------------------97

Table 4.11.Symptoms of Chlorosis/Necrosis in Lemna trisulcaafter copper and

lead treatment-----------------------------------------------------------------------------------98

Table 4.12. Comparison of Copper accumulation in Salvinia molesta in

different days----------------------------------------------------------------------------109

Table 4.13. Comparison of Lead accumulation in Salvinia molesta in different days------- 110

Table 4.14. Comparison of Copper accumulation in Pistia stratiotes in different days------ 111

Table 4.15. Comparison of Lead accumulation in Pistia stratiotes in different days-------- 112

Table 4.16. Comparison of Copper accumulation in Lemna trisulca in different days------ 113

Table 4.17. Comparison of Lead accumulation in Lemna trisulca in different days--------- 114

Table 4. 18: Comparison of the accumulation of copper and lead in the 3 macrophytes ---- 118

List of Figures

Figure                                                                                                                                 Page

Fig. 4.1: Chlorophyll a content in Salvinia molesta at different Copper and Lead

concentrations------------------------------------------------------------------------------------68

Fig. 4.2: Chlorophyll a contents in Pistia stratiotes at different Copper and Lead

concentration-------------------------------------------------------------------------------------69

Fig. 4.3: Chlorophyll a contents in Lemna trisulcaat different Copper and Lead

concentrations-----------------------------------------------------------------------------------71

Fig. 4.4: Chlorophyll b contents in Salvinia molesta at different Copper and Lead

concentration------------------------------------------------------------------------------------75

Fig. 4.5: Chlorophyll b contents in Pistia stratiotes at different Copper and Lead

concentrations-----------------------------------------------------------------------------------76

Fig. 4.6: Chlorophyll b contents in Lemna trisulca at different Copper and Lead

concentrations-----------------------------------------------------------------------------------77

Fig. 4.7: Catalase activity of Salvinia molesta as a function of different Copper

and Lead concentrations------------------------------------------------------------------------82

Fig.4.8: Catalase activity of Pistia stratiotes as a function of different Copper

and Leadconcentrations------------------------------------------------------------------------83

Fig. 4.9: Catalase activity of Lemna trisulca as a function of different Copper

and Leadconcentrations------------------------------------------------------------------------85

Fig. 4.10: Peroxidase activity of Salvinia molesta at different Copper and Lead

concentrations----------------------------------------------------------------------------------89

Fig. 4.11: Peroxidase activity of Pistia stratiotes at different Copper and Lead

concentrations----------------------------------------------------------------------------------90

Fig. 4.12: Peroxidase activity of Lemna trisulca at different Copper and Lead

concentrations-----------------------------------------------------------------------------------91

Fig. 4.13: Copper concentrationsin Salvinia molesta in 18 days period invitro-----------------100

Fig. 4.14: Lead concentrationsin Salvinia molesta in 18 daysperiodin vitro--------------------101

Fig. 4.15: Copper concentrationsin Pistia stratiotes in 18 daysperiod in vitro------------------103

Fig. 4.16: Lead concentrationsin Pistia stratiotesin 18 days period in vitro---------------------  104

Fig. 4.17: Copper concentrationsin Lemna trisulca in 18 days period invitro-------------------106

Fig. 4.18: Lead concentrationsin Lemna triculcain 18 days period invitro---------------------      107

Fig.4.19: Comparison of Copper Bioaccumulation by Salvinia molesta,

Pistia stratiotesand Lemna trisulca--------------------------------------------------------           116

Fig. 4.20:Comparison of lead Bioaccumulation by Salvinia molesta,

Pistia stratiotesand Lemna trisulca-------------------------------------------------------117

Fig. 4.21: Principal Components Analysis (PCA) showing significant

correlation between physiological responses and metal uptake/

absorption by themacrophytes---------------------------------------------------------------120

List of Appendices

Appendix                                                                                                                                                         Page

Appendix I:        Chlorophyll acontentin Salvinia molesta at different

Cu and Pb concentrations---------------------------------------------------------- 161

Appendix II:        Chlorophyll acontentin Pistia stratiotes at different

Cu and Pb concentrations-------------------------------------------------------- 162

Appendix III:      Chlorophyll acontentin Lemna trisulcaat different

Cu and Pb concentrations-------------------------------------------------------- 163

Appendix IV:      Chlorophyll b content in Salvinia molesta at different

Cu and Pb concentrations--------------------------------------------------------- 164

Appendix V:        Chlorophyll bcontentin Pistia stratiotes atdifferent

Cu and Pb concentrations--------------------------------------------------------- 165

Appendix VI:      Chlorophyll b contentin Lemna trisulca at different

Cu and Pb concentrations--------------------------------------------------------- 166

Appendix VII:   Catalase activity of Salvinia molesta as a function of different

Cu and Pb concentrations-------------------------------------------------------- 167

Appendix VIII: Catalase activity of Pistia stratiotesas a function of different

Cu and Pb concentrations------------------------------------------------------      168

Appendix IX:      Catalase activity ofLemna trisulcaas a function of different

Cu and Pb concentrations-------------------------------------------------------169

Appendix X:        Peroxidase activity of Salvinia molesta at different

Cu and Pb concentrations--------------------------------------------------------170

Appendix XI:  Peroxidase activity of Pistia stratiotes at different

Cu and Pb concentrations--------------------------------------------------------171

Appendix XII: Peroxidase activity ofLemna trisulca at different

Cu and Pb concentrations -------------------------------------------------------- 172

Appendix XIII: Copper concentrations in Salvinia molestain 18 days period in vitro-------173

Appendix XIV: Lead concentrations in Salvinia molestain 18 days period in vitro ---------174

Appendix XV: Copper concentrations in Pistia stratiotes in 18 days period in vitro---------175

Appendix XVI: Lead concentrations in Pistia stratiotesin 18 days period in vitro    ---------176

Appendix XVII: Copper concentrations in Lemna trisulca in 18 days period in vitro-------177

Appendix XVIII: Lead concentrations in Lemna triculcain 18 days period in vitro ---------178

Appendix XIX: Comparison of Copper Bioaccumulation by Salvinia molesta,

Pistia stratiotes and Lemna trisulca-------------------------------------------- 179

Appendix XX: Comparison of lead Bioaccumulation by Salvinia molesta,

Pistia stratiotes and Lemna trisulca-------------------------------------------- 180

Appendix XXI: Picture of Lemnatrisulca L. -----------------------------------------------------  181

Appendix XXII: Picture of Pistia stratiotes L. ---------------------------------------------------- 182

Appendix XXIII: Picture of Salvinia molesta D. Mitch------------------------------------------ 183

List of Abbreviations, Acronyms, Glossary and Symbols

Abbreviations

mgl-1       milligramme per litre      

hrs        hours

kg          kilogramme

mg        milligramme

ml          millilitre

mm        millimetre

mg/kg

Acronyms

ANOVA       Analysis of Variance

EPA            Environmental Protection Agency

ppm           parts per million 

ROS           Reactive Oxygen Species

rpm      rotation per minute 

UNDP                                                                                 

United Nations Development Project  

WHO     World Health Organisation

Glossary

CO2                                  Carbon IV oxide

ceteris paribus       all things being equal

cum                              together/along with

et al                               and others

Symbols

+                             plus

-                                           minus

CHAPTER ONE

1.0         INTRODUCTION

1.1         General Introduction

A pollutant is any substance in the environment, which causes objectionable effects, impairing the welfare of the environment, reducing the quality of life and may eventually cause death. Such a substance has to be present in the environment beyond a set or tolerance limit, which could be either a desirable or acceptable limit. Environment is defined as the totality of circumstances surrounding an organism or group of organisms especially, the combination of external physical conditions that affect and influence the growth, development and survival of organisms (FarlexIncorporated, 2005). It consists of the flora, fauna and the abiotic components, and includes the aquatic, terrestrial and atmospheric habitats. The environment is considered in terms of the most tangible aspects like air, water and food, and the less tangible, though no less important, the communities we live in.

Comprising over 70% of the Earth‟s surface, water is undoubtedly the most precious natural resource that exists on our planet (Terry, 1996). Population growth, urbanization and industrialization have led to rapid degradation of the environment and publichealth due to improper sewage disposal, especially in developing countries. Conventional solutions are inappropriateand expensive because the infrastructures and skilled labour are lacking.

The development of the intensive agriculture in Nigeria between 1960 and 1990 totally neglected the aspect connected with the negative impact of the chemical compounds toxic on the air, water and soil. As one of the consequences of heavy metal pollution in soil, water and air, plants are contaminated by heavy metals. Contamination of the aquatic environment by the heavy metals has become a serious concern in the developing world(Chandra et al., 1997). Heavy metals unlike organic pollutants are the persistent in nature, therefore, tends to accumulate in the different components of the environment (Chandra et al., 1997). Sources of metals in the environment are widespread and data on typical concentrations in the various media and environmental settings exits worldwide (Mwamburi, 2015).These metals are released from a variety of sources such as mining, urban sewage, smelters, tanneries, textile industry and chemical industry.

Water pollution is the contamination of water bodies (e.g. lakes, rivers, oceans, aquifers and groundwater). Water pollution occurs when pollutants are discharged directly or indirectly into water bodies without adequate treatment to remove harmful compounds. Aquatic environments are increasingly affected by human activity because of urban, industrial, mineraland agricultural waste. The use of the ocean as a dumpingground for wastes could lead to high levels of pollution in the aquatic environment (Bramha et al.,2014; Bodin et al., 2013). Water pollution affects plants and organisms living in these bodies of water. In almost all cases, the effect is damaging not only to individual species and populations, but also to the natural biological communities.

Water pollution is a major global problem which requires ongoing evaluation and revision of water resource policy at all levels (international down to individual aquifers as well). It has been suggested that it is the leading worldwide cause of deaths and diseases and that it accounts for the deaths of more than 14,000 people daily(Pink, 2006; West, 2006).

The specific contaminants leading to pollution in water include a wide spectrum of chemicals and pathogens. While many of the chemicals and substances that are regulated may be naturally occurring  (calcium,  sodium,  iron,  manganese,  etc.)  the  concentration  is  often  the  key  in determining  what  is  a  natural  component  of  water,  and  what  is  a  contaminant.  High xxiv

concentrations of naturally occurring substances can have negative impacts on aquatic flora and fauna. Oxygen-depleting substances may be natural materials, such as plant matter (e.g. leaves and grass) as well as man-made chemicals. Other natural and anthropogenic substances such as may cause turbidity (cloudiness) which blocks light and disrupts plant growth, and clogs the gills of some fish species (EPA, 2005).

Heavy metal is the term used for a group of elements that have particular weight characteristics. They are on the "heavier" end of the periodic table of elements. Heavy metals are natural components of the Earth‟s crust. They cannot be degraded or destroyed. The most dangerous heavy metals are Lead, Cadmium, Copper, Chromium, Selenium and Mercury. Some heavy metals – such as Cobalt, Copper, Iron, Manganese, Molybdenum, Vanadium, Strontium, and Zinc – are essential to health in trace amounts. Others are non-essential and can be harmful to health in excessive amounts. These include Cadmium, Antimony, Chromium, Mercury, Lead, and Arsenic – these last three being the most common in cases of heavy metal toxicity.

The term “heavy metals” refers to any metallic element that has a relatively high density and is toxic or poisonous even at low concentration (Huton and Symon, 1986). “Heavy metals” is a general collective term, which applies to the group of metals and metalloids with atomic density greater than 4 g/cm³ , or 5 times or more, greater than water. That is,a specific gravity of greater than 4.0-5.0. The actinides may or may not be included.(Huton and Symon, 1986; Battarbee et al., 1988; Nriagu and Pacyna 1988; Nriagu, 1989; Garbarino et al., 1995).Most recently, the term "heavy metal" has been used as a general term for those metals and semimetals with potential human or environmental toxicity (Chehregani et al., 2005).

All metals, both essential (Cu, Zn, Mg) and toxic (Cd, Pb, Cr, Hg) can cause toxic effects to plants and animals if found in high concentrations in the organisms (when the concentrations xxv

exceeds the standard by WHO and EPA).Heavy metals are dangerous because they tend to bio accumulate. Bioaccumulation means an increase in the concentration of a chemical in a biological organism over time, compared to the chemical‟s concentration in the environment. Compounds accumulate in living things any time they are taken up and stored faster than they are broken down (metabolized) or excreted (http://www.tip2000.com/health/waterpollution.asp). Heavy metal can enter a water supply by industrial and consumer waste, or even from acidic rain breaking down rocks and releasing heavy metals into streams, lakes, rivers and groundwater. Heavy metals present in large water bodies can lead to pollution of the aquatic system, thereby causing several diseases and leading to termination of life of aquatic organisms. It can also in return make fish unsafe for consumption(Xue et al., 2005).

All heavy metals at high concentration have strong toxic effect and are regarded as environmental pollutants (Nedelkoska and Doran,2000; Chehregani et al., 2005). Acute heavy metal intake may damage central nervous function, the cardiovascular and the gastrointestinal (GI) systems, lungs, kidneys, liver, endocrine glands and bones (Jang and Hoffman, 2011; Adal and Wiener, 2013). Chronic heavy metal exposure has been implicated in several degenerative diseases of these same systems and may increase the risk of some cancers (Galaniset al., 2009; Wuet al, 2012).

The presence of heavy metals in aquatic ecosystems, causes severe impacts on the biological components of these environments i.e. heavy metals are highly toxic to the aquatic plants and animals as well as they do not vanish easily from the environment. As a result, serious disorders in human health have been observed as a result of biomagnification processes and the toxic effects within the food chain (Xue et al., 2005; Ljung and Vahter 2007).There are two aspects on the interaction of plants with heavy metals: (i) heavy metals show negative effects on plants, and (ii)   plants have their own resistance mechanisms against toxic effects and for detoxifying heavy metal pollution (Cheng, 2003).

There is no doubt that excessive levels of pollution are causing a lot of damage to human and animal health, plants including tropical rainforests, as well as the wider environment. All types of pollution-air, water and soil pollution have an impact on the living environment (Seth et al., 2007).

1.2         Background Information on Lemna trisulca, Pistia stratiotes and Salvinia molesta

1.2.1     Lemna trisulca L.:

Lemna species commonly known as duckweed belong to the genus Lemna and family Lemnaceae. Duckweeds are among the world‟s smallest flowering plants. Individual lesser duckweed plants are tiny, round, bright green disks, each with a single root. They are found scattered among emergent plants or massed together in floating mats (Appendix XXI). Star duckweed is much less commonly observed. Individual non-flowering plants are longer and narrower than lesser duckweed, commonly floating in masses beneath the water surface. Flowering plants more closely resemble lesser duckweed (Rahman and Hasegawa, 2011).

Lemma species have no true leaves but have a leaf-like body called a thallus, which is flat on the top and slightly rounded on the bottom. Lesser duckweeds are nearly circular to oval, 2-5 mm in diameter; occur as single plants or up-to-five plants may be connected. Star duckweeds are two types: non-flowering plants are elongate or spatula-shaped (6-10 mm long), tapered to a stalked base, connected in branched chains of 8- 30 plants, and submerged beneath the water surface.

Flowering plants are more oval-shaped with a separate margin and a shorter stalk at the base.

They float on the water surface (Mkandawire and Dudel, 2005).

Duckweeds have no stem; flowers are tiny and rarely seen and arise from a pouch in the thallus. Fruit is inconspicuous, usually 1 seeded. Root of lesser duckweed is single short rootlet (unbranched root) that hangs from the underside of each plant. Star duckweeds are often rootless (Landolt, 1986; Rahman and Hasegawa, 2011).

Types:

Duckweed comes in many varieties. Only plants belonging to the genus Lemna are duckweeds, but a number of similar plants often end up lumped under this name. Watermeal or Wolffia species are a close relative, but lack roots. They are the smallest kind of flowering plant at about

½  mm long. Landoltia and Spirodela species are also similar to duckweed, but have two or more roots (Reid and Stanley, 2003)

Propagation:

Duckweed has a fairly simple life cycle that enables it to spread quickly. New plants bud from pockets on either side of the parent plant and eventually break apart. Overwinters as winterbuds on the lake bottom, but rarely reproduces from seeds (Rahman et al., 2007). Distributed by wind and on the bodies of birds, and aquatic animals. A single lesser duckweed plant can reproduce itself about every 3 days under ideal conditions in nutrient-rich waters. It can be hard to determine the point in the life cycle of a specific duckweed specimen. Botanists primarily use the length of the root to decide the age of a piece of duckweed. Longer roots belong to older plants (Reid and Stanley, 2003; Rahman et al., 2007).

Cloning:

Duckweed is very short lived. Lemna minor for instance, survives only five to six weeks. However, the plant almost always appears as a dense mat, since it reproduces primarily asexually. The mature duckweed produces a small outgrowth from a bud on one end, which then breaks off becoming small new duckweed. The new plant produces a root and eventually is indistinguishable from the parent. Immature plants may remain attached to the parent until maturity (Wang et al., 2002).

Importance of plant:

Food for fish and waterfowl and habitat for aquatic invertebrates. Because of its high nutritive value, duckweeds have been used for cattle and pig feed in Africa, India, and Southeast Asia. Sometimes used to remove nutrients from sewage effluent (Wang et al., 2002; Korner et al., 2003).

Distribution: Throughout much of the temperate and subtropical regions of the world.

Habitat: Still and slow moving waters in many freshwater habitats. Often found along the shoreline after water levels have dropped (Wang et al., 2002).

1.2.2     Pistiastratiotes L.:

Pistia stratiotes commonly known as water lettuce or shell-flower belongs to the family Araceae.

Description:

Water lettuce is a floating water plant with 15 cm rosettes of ribbed, Ruffles Potato Chip-like leaves (Appendix XXII). The rosettes are connected by stolons that break easily. The leaves are fleshy-thick, pale green and velvety-hairy, which causes water to bead and keeps them from getting wet. The feathery roots are white, purple and black, and quite showy, hanging down a foot or so below the floating rosettes (Ramey, 2001). Water lettuce frequently forms solid mats on the water's surface and can become a serious pest (Skinner et al., 2007).

Location

Water lettuce thrives in still waters in swamps, ponds, lakes, and sluggish rivers in the tropics and subtropics in both the Old and New Worlds. In the United States it is restricted to Peninsular Florida where it probably was introduced (Ramey, 2001).

Culture:

Light: Water lettuce needs full sunlight or slightly filtered sunlight.

Moisture: Water lettuce typically floats on the surface, but can withstand periods of drawdown as long as the mud does not dry out completely.

Propagation: Waterlettuce propagates vegetatively by growing stolons (stem like shoots) which xxx

produce new rosettes. Seeds are produced in the tropics and these are said to be easy to germinate (Zimmels et al., 2006).

Usage:

Waterlettuce is difficult to maintain in artificial conditions. It can be grown in tropical or heated aquaria with a glass cover and full sunlight, or in a greenhouse pool. The air must be at least 75ºF (24ºC) degrees and permanently humid above the water (Skinner et al., 2007). Waterlettuce can be grown in a temperate water garden, but must be lifted before frost and overwintered on damp sand or peat at no colder than 50 ºF (10ºC) (Odjegba and Fasidi, 2004; Skinner et al., 2007).

1.2.3     Salvinia molesta D. Mitch:

Common name giant Salvinia, familySalviniaceae is a non-native, extremely invasiveaquatic fern that has infested the southern UnitedStates. Native to southeastern Brazil, the weedwas introduced for use in aquariums and gardenponds (Jacono, 2002). Giant Salvinia may also have been broughtin as packing with fresh, iced fish. It has beensold under many common names, including water velvet, Salvinia, African pyle, aquarium watermoss, kariba weed, water fern, and koi kandy. Since its escape, giant Salviniahas become a serious problem in rivers, streams, lakes, dams and rice fields (US Army Corps of Engineers, 2002).

Giant Salviniais a free-floating aquatic fern with small, oblong, ½-inch to 1-inch long spongy green leaves along the stem (Appendix XXIII). Young plants are smaller and the leaves lie flat on the water surface. Stems branch in an irregular fashion. The leaves occur in whorls of three: two floating and one submerged. The plant has no flowers, and the submersed leaves are thread-like and look like roots. As the leaves mature, they become thick and curl at the edges in response to crowding. As infestations grow in size, leaves become more vertical, forming upright xxxi

chains that form mats of floating plants. The leaf surfaces of giant Salviniahave rows of hairs that, when magnified, look much like egg beaters in shape. The hairs give the leaves a velvety appearance and repel water. This characteristic is useful in identifying the species (Oliver, 1993).

The root like submerged fronds of giant Salviniaoften support chains of egg-shaped spore-bearingstructures. Any spores produced, however, appearto be genetically defective, as they do not produceviable plants.Giant Salvinia grows best in quiet waters of lakesand ponds, oxbows, ditches, slow-flowing streamsand rivers, marshes, and rice fields. Its growth isfavored in water with a high nutrient content, suchas eutrophic waters or waters polluted by wastes (Land Protection, 2001).

Salviniaspecies reproduces as fragments break offexisting plants as they mature. New plants alsodevelop as dormant buds break away from theoriginal plant. As each node has up to five lateralbuds, the weed has high potential for growth. Asdormant buds, giant Salvinia will survive periodsof stress from both low temperature anddewatering.Giant Salviniahas small, spongy, vertical leaves.The third leaf forms a root like structure, but theweed is free-floating.Activities that fragment the weed, includingboating, fishing, or intentional harvesting, add toits spread. Giant Salvinia is often introduced tonew areas as people empty aquariums or infestedponds into waterways or as infested boats andtrailers are moved to new waters. Spread bywaterfowl may also occur (WAPMS, 2002).

1.2.4    Anti-oxidant Enzymes:

Plants  possess  several  anti-oxidation  defence  systems  and  enzymes  like  Catalase  (CAT), Superoxide dismutase (SOD), Glutathione  Peroxidase (GPX), Ascorbate Peroxidase  (APX), xxxii

Glutathione Reductase (GR) and Glutathione-S-Transferase (GST) to scavenge toxic reactive oxygen species and to protect themselves from the oxidant stress (Seth et al., 2007). Diverse array of pollutants stimulate a variety of toxicity mechanisms such as oxidative damage to membrane lipids, DNA, Proteins and changes to anti-oxidant enzymes (Valavanidis et al., 2006). Elevated activities of anti-oxidant enzymes may help to alleviate the oxidative damage caused by ROS (Qian et al., 2007). Glutathione –s –Transferase is an enzyme associated with atrazine detoxification in plants by detoxifying ROS through Ascorbate-glutathione cycle (Yadav, 2010). SOD converts superoxide radical into hydrogen peroxide and molecular oxygen, while the Catalase and Peroxidase convert hydrogen peroxide into water. In these two ways, two toxic species, superoxide radical and hydrogen peroxide are converted to the harmless product water (Weydert and Cullen, 2010). Various methods to identify oxidized amino acids in blood proteins as biomarkers of free radical damage, especially for metal-catalyzed oxidations, have been developed recently (Valavanidis et al.,2006).

According to Weydert and Cullen (2010), there are three SOD enzymes that are highly compartmentalized, Manganese-containing Superoxide dismutase (MnSOD) is localized in the mitochondria, Copper and Zinc-containing Superoxide dismutase (CuZnSOD) is located in the cytoplasm, nucleus and extracellular SOD (ECSOD). Catalase is found in peroxisomes and cytoplasm. Biological systems have developed during their evolution adequate enzymatic and non-enzymatic anti-oxidant mechanisms to protect their cellular components from oxidative damage (Valavanidiset al., 2006).

The individual toxicity of heavy metals has been assessed, but they can interact and potentially increase their toxicity on plants and microalgae. Correspondingly, the combined effect of metals could be described qualitatively as antagonistic, non-reaction or synergistic. Generally plants are sensitive to pollutants; these pollutants may affect species composition of the plant community (Walsh, 1999). The toxicity of heavy metal (cadmium) in conjunction with atrazine herbicide was inhibited up to 100ppm of metal concentration in bean plant (Azmat et al., 2006). Mathad et al. (2006) confirmed that Helichrysum stuhmannii and Scenedesmus quadricauda exposed to the various concentration of the bi- metallic combinations of chromium- iron, revealed no marked morphological variations at the lower metal concentration but, exhibited distinct abnormalities such as chlorosis, enlargement or reduction of cells, fragmentation and shrinkage of protoplasm or chloroplast, loss of cellular contents and cell lyses at the higher metal concentrations.

1.3         Statement of Research Problem

Environmental pollution by organic compounds and metals became extensive as mining and industrial activities increased in the 19^th century and have intensified since then (Torres et al., 2008). These days, environmental problems are multiple and complex, especially those arising from the identification and assessment of the toxicity of chemical substances in the aquatic ecosystem (Ma et al., 2006). Both natural and anthropogenic factors are considered as a major environmental concernfor aquatic ecosystems. They can impact on the aquatic environmentby producing polluting components which may enter into thehuman food chain and result in health problems (Kerambrunet al., 2012). Metallic contamination in aquatic environmentshas received huge concern due to its toxicity (Diop et al., 2014).

Adverse effects of heavy metals such as oxidative stress, inhibition of Hill reaction of photosynthesis, reduction of electron transport through photosystem II leading to photoinhibition, reduce utilization of carbon dioxide, decrease in growth or biomass production, alteration of ultra-structures of the cellular organelles and change in community structure on non-target plants are of particular concern because of the annual, wide spread and increasingly worldwide use of these chemicals (Van der Brink and Ter Braak, 1999). Extensive investments in sewage plants during the last two decades have greatly reduced the organic loading of receiving water bodies in a number of countries. However, an equivalent improvement in water quality has not been achieved since there are many small contributors which still have no cleaning of their wastewater discharge; and since leakage of nitrogen from the agricultural land, as a consequence of the increased use of fertilizers has greatly increased over the last thirty years (Peterson et al., 1987).

There is little information about the joint effect of heavy metals on aquatic organisms. As a food source, microalgae may facilitate the uptake of contaminants into higher organisms, increasing the possibility of toxicity (Okayet al., 2000). Therefore, stress effect of both metals on plants can cascade into the food chain. The stresses are numerous and often plant or location specific. They include increased UV-B radiation, water, high salinity, metal toxicity, herbicides, fungicides, air pollutants, light, temperature, topography and hypoxia that is restricted oxygen supply in waterlogged and compacted soil (Ali and Alqurainy, 2000). They further emphasize that stress depend on tissue and/or species, on membrane properties, on endogenous anti-oxidant content and on the ability to induce the response in the anti-oxidant system.

Low concentrations of trace metal for long periods of time can lead to metabolism modification, elimination of the plant and algae species that are unable to adapt themselves to the new conditions which will eventually affect the biodiversity of the environment (Tripath and Gaur, 2006). Cadmium enters plant cells either by means of active transport or by endocytosis through chelating proteins and affects various physiological and biochemical processes of the plant. The toxicity primarily results from their binding to the sulphydryl group in proteins or disrupting protein structure or displacing essential elements (Arunakumara and Xuecheng, 2007). Heavy metal irons could interrupt routine metabolic processes by competing for the Protein binding sites, active enzymes and various biological reactive groups, causing poor or no growth (Arunakumara and Xuecheng, 2007).

These pollutants stimulate a variety of toxicity mechanisms, such as oxidative damage to membrane lipids, DNA, Proteins, Carbohydrates and changes to anti-oxidant enzymes. Oxygen free radicals are essential in the physiological control of cell function in biological systems and are continuously produced in living cells but, during these metabolic processes, a small proportion (2–3%) of free radicals may escape from the protective shield of anti-oxidant mechanisms, causing oxidative damage to cellular components.

Heavy metal ions can cause plasma membrane depolarization and acidification of the cytoplasm. In fact, membrane injury is one important effect of heavy metal ions that may lead to the disruption of cellular homeostasis (Cardozo et al., 2002).However, study on the biochemical composition changes and anti-oxidant enzymes responses will bring in the effect of metals on plants (Tripathi and Gaur, 2006). Metals may displace or substitute for essential trace metals and interfere with proper functioning of enzymes, associated cofactors and disorganizes chloroplast causing the damage of photosynthetic pigments. Metals trigger changes in the transcript levels of numerous genes encoding proteins and induce the synthesis of several proteins, including metallothionein in plants (Torres et al., 2008).Heavy metals can interfere in the photosynthetic activity by increased photoinhibition from excess of light (Heckathorn et al., 2004).

The disposal of large amounts of sewage and the intensified exploitation of agricultural land involving increased amounts of fertilizers, has resulted in pronounced eutrophication of receiving waters. The effects of eutrophication i.e. lower species diversity and decreasing self–purification capacity have been greatly magnified by destruction of the natural physical heterogeneity of the ecosystem. Streams have been viewed simply as conduits and have been deepened and straightened and their vegetation has been removed to augment the drainage of agricultural land. Natural wetland and marshes have been drained and turned into agricultural land(Peterson et al., 1987).

The causes of water pollution may be due to direct and indirect contaminant sources. The former are effluents outputs from refineries, factories and waste treatment plants. Fluids of differing qualities are emitted to the urban water supplies. In the United States and some other countries, these methods are controlled. However, pollutants can still be found in the water bodies. The latter are the water supply from soils/groundwater systems that have fertilizers, pesticides and industrial wastes. Also those through the atmosphere like bakeries, factories emission and automobile discharge. Contaminants can also be divided into inorganic, organic, acid/base and radioactive.

Municipal, industrial and agricultural sources are the different categories of the causes of water pollution. Municipal causes are related to waste water from homes and commercial establishments. The main aim of handling municipal waste water was to decrease the harmful bacteria, oxygen requiring materials, mixed inorganic compounds and suspended solids. Industrial causes vary as per the biochemical demand, suspended solids, inorganic and organic substances. Agricultural sources include commercial livestock and poultry farming. These lead to organic and inorganic pollutants in surface water and groundwater (http//www.buzzle.com/articles/causes-of-water-pollution.html).

Pollutants have a tremendous impact on the biodiversity and productivity of aquatic communities (Relyea, 2005). Adverse effects of pollutants on non-target organisms of aquatic ecosystems are of special concern. These pollutants cause rapid changes in the communities of macrophytes, phytoplankton and other photosynthetic organisms. A common mechanism of metal toxicity in aquatic plants and algae is inhibition of biological processes such as photosynthesis and mitochondrial electron transport (Babu et al., 2005). This leads to inevitable changes in plant cell physiology, growth and biomass yield (Kuster et al., 2007).

Recently, the value of the biological diversity and complexity which prevail in natural ecosystems have been recognized, and attempts have been made to restore streams, rivers and wetlands in order to regain their heterogeneity, and thereby their self–purification capacity and buffering effects.

1.4         Justification

The problem of environmental pollution is now a worldwide phenomenon that needs to be urgently looked into. In line with the foregoing, the Federal Government of Nigeria recently set up a body-Federal Environmental Protection Agency (FEPA) to look into this problem and proffer solutions with respect to the monitoring and control of pollution sources in the country.

In the early eighties, the local municipalities in Nigeria were met with increasing demands for the removal of nitrogen and phosphorus as well as the organic content of wastewater prior to disposal. The traditional solution for small contributors is to collect the sewage from several small villages in one central – treatment facility. Such a solution is however rather expensive, and therefore the municipalities were, and still are searching for more effective solutions.

The legacy of rapid urbanization, industrialization, fertilizer and pesticide use has resulted in major problems in both terrestrial and aquatic environments. In response, conventional remediation systems based on high physical and chemical engineering approaches have been developed and applied to avert or restore polluted sites (Singh and Laban, 2003; Pilon-Smits 2005). Much as these conventional remediation systems are efficient, they are sparsely adopted because of some economical and technical limitations. Generally, the cost of establishment and running deter their use and meeting the demand particularly in countries with a weak economy.

As one of the consequences of heavy metal pollution in soil, water and air in developing countries (Guo,1994;Liao,1993; Suet al., 1994; Wu et al., 1998), plants were also seen to be polluted by heavy metals (Duet al.,1999;Wu et al., 1998; Yin et al.,1999;Zhang et al.,1996), which consequently threatens the health of animals and human beings via the food chain (Wang and Dei, 2001). It is urgently necessary to clean and remediate heavy metals from areas, where crops, vegetables, fruits and pasturages have been grown, in order to protect the health of animals and human beings.

Different methodologies are used for the removal of the different heavy metals viz. electrodialysis, reverse osmosis and adsorption. All of these methodologies are quite costly and energy intensive, none of them could claim to treat all the heavy metals in economically feasible manner (Singh et al., 1996). Economies of developing countries have other investment priority therefore cannot afford the high price involved in the removal of heavy metals from waste water.

Contrary to this phytoremediation which is removal of metals through plants offers an ecofriendly and cost effective methodology for the treatment of heavy metals from waste water.

Plants play an important role in solar energy transport to bio-energy and can clean the environment in an environmentally friendly manner; they would also play an important role in heavy metal remediation (Skinner et al., 2007). To understand the effects of heavy metals on plants and resistance mechanisms would be helpful for using plants to clean and remediate heavy metal pollution. Phytoremediation is potentially least harmful method because it uses naturally occurring organisms and preserves the environment in a more natural state (Maineet al., 2004; Skinneret al., 2007). Therefore, Knowledge of the ability of Salviniamolesta, Pistia stratiotesand Lemnatrisulcato absorb heavy metals would help in pollution control by protecting the environment as well as reducing health hazard.

Having an insight on the factors responsible for differential toxicity and physiological response of plants upon exposure to these metals will improve our ability to predict the impact of aquatic contaminants on freshwater ecosystem. The combined effect of Copper and Lead on the physiological responses of aquatic plants is a subject of intensive investigation, as most studies focus on the individual toxicity of heavy metals on plants. Investigation on the toxicity of heavy metals at environmentally relevant concentrations to fresh water plants will help to provide a scientific basis to assess the ecological risk of the pollutant groups in aquatic ecosystem accurately (He et al., 2012).

Understanding of the regulatory mechanisms of metals tolerance, and the components involved in the mechanism will be helpful in metal removing processes from aquatic ecosystem (Li et al., 2006). The potential of oxygen free radicals and other reactive oxygen species (ROS) to damage tissues and cellular components called oxidative stress, in biological systems has become a topic of significant interest for environmental toxicology studies (Valavanidiset al., 2006). The balance between environmental pollutants and Antioxidant defences (enzymatic and Non-enzymatic) in biological systems can be used to assess toxic effects under stressful environmental conditions, especially oxidative damage induced by different classes of chemical pollutants. Therefore, the role of these Antioxidant systems and their sensitivity can be of great importance in environmental toxicology studies (Valavanidis et al., 2006).

Aquatic ecosystems, especially their biological assemblages, continue to be degraded globally. Anthropogenic enrichment of aquatic ecosystems by heavy metal is an ever increasing phenomenon and, for larger bodies of water, the principal requirement for ecological restoration is the management of degraded water chemistry (Herve et al., 2005). Rivers contribute largely to the biodiversity of macrophytes and macro invertebrates.

Recently, it is evident that durability restoration and long term contamination control in conventional remediation is questionable because in the long run, the pollution problem is only suspended or transferring from one site to another. In view of this, there has been growing interest in the search for alternative remediation technology that is effective, durable and cost-effective. One such technology is phytoremediation, the use of plants and associated microbes for environmental cleanup (Singh and Laban, 2003; Pilon-Smits, 2005).

On an international basis, considerable attention is at present being directed towards the capacity of aquatic macrophytes (Swamp and water plants) to control pollution and to treat municipal and industrial waste water as indicated by the great number of participants at recent international meetings (Athie and Cerri; 1987; Reddy and smith 1987; Hammer, 1989). The interest is partly coupled to the public demand for increasing stringent water quality standards, and partly to the need to develop low–cost decentralized constructions capable of serving small to medium sized-communities. Macrophytes–based wastewater treatment systems have several potential advantages such as low operating costs, low energy requirements, they can often be established at the site where the waste water is produced and they are more flexible and less susceptible to stock loading compared to conventional treatment systems (Brix, 1987).

The utilization of wetland areas as natural filters for the abatement of pollutants transported by water in rivers or lakes is considered to be an effective clean up option to ameliorate the quality of surface waters. Indeed, wetlands have been extensively utilized in the last decades to clean pollutant water almost all over the world (Gopal, 2003).

The vegetation covering the wetland areas plays an important role in sequestering large quantitative of nutrients (Cronk and Fennessy, 2001) and metals (Mays and Edwards, 2001; Baldantoni et al., 2004) from the environment by storing them in the roots and/or shoots. Wetland plants have high remediation potential for macronutrients because of their general fast growth and high biomass production. Some Western Africa estuarine habitats have been seriouslydegraded for the last 30 years by climate changes causing severedroughts with reduction of freshwater flow, combined with theincrease in domestic and industrial effluents (Bouvy et al., 2008).

Bioaccumulation of heavy metals in aquatic ecosystems is gaining tremendous significance globally. Several of the submerged, emergent and free floating aquatic macrophytes are known to accumulate and bioconcentrate heavy metals. Aquatic macrophytes take up metals from the water, producing an internal concentration several fold greater than their surroundings. Many of the aquatic macrophytes are found to be scavengers of heavy metals from water and wetlands (Gopal, 2003).

Shallow, eutrophic, aquatic ecosystems stocked with macrophytes are among the most productive in the world. (Schierup, 1978). Aquatic macrophytes are biological filters that carry out purification of water bodies by accumulating dissolved metals and toxins in their tissues (Begum, 2009). The capacity of such systems to decompose organic matter and assimilate nutrients has long been recognized, and it is well known that streams, lakes, coastal areas, and wetlands contain a considerable self – purification capacity. During the growing season the plants absorb and incorporate the nutrients into their own structures.

The potential of aquatic plants forbio-monitoring of polluted water has increasingly been recognized (Lewis and Wang 1997, Mohan and Hosetti, 1999). Algae have been reported as equally or more sensitive than animals (Lewis, 1992) and have been widely used in toxicity tests for regulatory purposes (ISO 1987; Weber et al.., 1987). However, algae may not necessarily be an indication of overall aquatic plant sensitivity to pollution. In fact, some studies have shown a higher sensitivity of macrophytes as compared to algae and animals (Thomas et al., 1986; Roshon et al., 1999).

Algae and aquatic plants play a key role in aquatic ecosystems because they are at the base of food webs. Also, they are a food resource and provide oxygen and shelter for many aquatic organisms. They also contribute to the stabilization of sediments, thus resulting in their accumulation in sediments (Gabas et al., 1991). In aquatic systems, where pollutant inputs are discontinuous and pollutants are quickly diluted, analyses of plants provide a time integrated information about the quality of the system. Phytoremediation has several advantages and the most significant one is study of sub-lethal levels of bio accumulated contaminants within the tissues or components of organisms, which indicate the net amount of pollutants integrated over a period of time. Bio-monitoring of pollutants using some plants as accumulator species, helps to accumulate relatively large amounts of certain pollutants, even from much diluted solutions without obvious noxious effects (Begum, 2009).

Phytoremediation which is the use of plants to remove pollutants from the environment, is a growing field of research in environmental studies because of the advantages of its environmental friendliness, cost effectiveness and the possibility of harvesting the plants for the extraction of the absorbed contaminants such as metals that cannot be easily biodegraded for recycling among others (Maine et al., 2001, 2004; Malik 2007). Phytoremediation work best when the contaminants discharged into the environment are within the reach of the plant roots. Most aquatic plants possess the qualities that favour their potential use in water and wastewater phytoremediation. There are limited data on phytoremediation of contaminated water bodies in Nigeria as against remediation of soil that is common.

Development of aquatic plants-based wastewater treatment systems is now recognized as suitable alternative to cost-effectively and safely treat sewage (Reddy and Smith, 1987; Cooper and Findlater, 1990). The scientific basis and the technical feasibility of this eco-technology are well established (Wolverton, 1987; Tchobanoglous, 1987) and abundant literature exists on the potentials of several aquatic plants to clean water especially in North America and Europe (Kadlek, 1987; Brix and Shierup, 1989; Brix, 1991). The tropical regions offer several advantages for the development of such technology.Aquatic plants are abundant and the suitable climate means that processes are optimum and operational all year round.

The impact of tropical aquatic ecosystems with high concentrations of metals can be reduced through phytoremediation. Some plants have shown great potential for their use in projects involving the sequestering and mitigation of contaminated aquatic effluents (Hasan et al. 2007; Alvarado et al., 2008). Butterfly fern, water lettuce and duckweed are among the few popular organisms that can be used as tools to evaluate the presence of heavy metals (Claudiaet al., 2008). Enzymes such as catalase and peroxidase participate in protective mechanisms against damage caused by different chemicals (Santandrea et al., 2000). The toxic effect of multiple chemicals plays a vital role in ecotoxicology because chemical mixtures could have a greater negative impact than the individual constituents (Hernando et al., 2003).Several researchers have dealt with toxicity of individual pollutant, but few reports are available on the toxicity of pollutants in combination (Kumar and Han, 2011).

Environmental concerns on heavy metals have been of interest in the world especially in the developing countries including Nigeria. Several studies have described investigations relating to the effects and accumulation of metals in terrestrial and aquatic plants (Fecht-Christoffers et al., 2003; Fernando et al., 2006; Yang et al., 2008; Pollard et al., 2009) outside Nigeria. In Nigeria, some studies have reported the impact of heavy metals in the terrestrial environment using fungi, bryophytes, lichens and plants and very few studies on heavy metal concentrations inaquatic environment. To the best of my knowledge however, no work has been reported in Nigeria on the use of macrophytes for phytoremediation purposes in the aquatic environment and in vitro. Therefore, it became necessary to carry out the phytoremediation potential of copper and lead bybutterfly fern, water lettuce and duckweed;thereby making this research work the first in this part of the world.

1.5         Aim

The aim of the study is:

To evaluate the effects of the copper and lead on the physiology and the potential of Salvinia molesta, Pistia stratiotes and Lemna trisulca as phytoremediation plants.

1.6         Objectives of the Study

The specific objectives of the study are:-

1.      To determine the effects of Cu and Pb on the photosynthetic pigments (chlorophyll a and chlorophyll b) of Salvinia molesta, Pistia stratiotes and Lemna trisulca.

2.      To determine the effects of Cu and Pb on the activity of the antioxidant enzymes (antioxidant responses of catalase and peroxidase) of the three aquatic macrophytes.

3.      To determine the effects of Cu and Pb on the morphology(visual signs/symptoms of chlorosis) of the three aquatic macrophytes.

4.      To determine the uptake and bioaccumulation (metal sequestration) of the pollutants in the different macrophytes tissues in proportion to exposure concentration over time.

5.      To determine which of the species has a potential for phytoremediation in relation to the absorption rate of the heavy metals.

1.7         Research Hypotheses

The hypotheses tested by the research results are:-

1.      There is no significant difference in the effect of the metals on the photosynthetic pigment (chlorophyll a and chlorophyll b) of the different aquatic macrophytes