topic title: hydrilla verticillata, a submerged aquatic plant, as phytoremediation agent of lead (ii)nitrate (pb(no3)2)

THAI NGUYEN UNIVERSITY UNIVERSITY OF AGRICULTURE AND FORESTRY NGUYEN THI THAO TOPIC TITLE: HYDRILLA VERTICILLATA, A SUBMERGED AQUATIC PLANT, AS PHYTOREMEDIATION AGENT OF LEAD II-NIT

THAI NGUYEN UNIVERSITY

UNIVERSITY OF AGRICULTURE AND FORESTRY

NGUYEN THI THAO

TOPIC TITLE: HYDRILLA VERTICILLATA, A SUBMERGED AQUATIC

PLANT, AS PHYTOREMEDIATION AGENT OF LEAD (II)-NITRATE

(PB(NO 3 ) 2 )

BACHELOR THESIS

Study Mode : Full-time

Thai Nguyen, 2015

DOCUMENTATION PAGE WITH ABSTRACT

Thai Nguyen University of Agriculture and Forestry

Degree Program : Bachelor of Environmental Science and Management

Student name : Nguyen Thi Thao

Student ID : DTN 1153110148

Thesis Title : HYDRILLA VERTICILLATA, A SUBMERGED AQUATIC

PLANT, AS PHYTOREMEDIATION AGENT OF LEAD NITRATE (PB(NO3)2)

(II)-Supervisor (s): Dr Arinafril

Dr Ho Ngoc Son Abstract: The experiment of this study were started from March 2015 and finished in June 2015 at Integrated Research Laboratory of Sriwijaya University Palembang in Indonesia, with 4 applied treatments (B1: Pb(NO3)2 0 ppm(control), B2: Pb(NO3)2 5 ppm, B3: Pb(NO3)2 10 ppm and B4: Pb(NO3)2 15 ppm) and 3 replications for each treatment through 5 times of observing in 20 days Given results were totally evidenced

strong capacity of hydrilla verticillata in absorbing and accumulating heavy metals,

especially lead (Pb) which is known as a dangerous toxicity for living organisms This

ability of hydrilla verticillata pulled it up to become an effective instrument for

phytoremediation technique in solving environmental issues

In the research phytoremediation was presented by the use of a submerged aquatic

plant, hydrilla verticillata as a tool of removing Lead (II)-nitrate (Pb(NO3)2) from water The revealed results after experiment of 4 treatments, 3 replications in 5 times of

observing shown that hydrilla verticillata is an useful plant for treating Pb contaminant

in water by taking up and storing the metal in its body After applying Pb(NO3)2 with 4

different treatments, Pb was directly affected hydrilla verticillata morphology based

on higher level of contaminant with the changes in 5 morphological states in order of normal; leaves became yellow; leaves became faintly yellow; partial decolourization, complete decolourization and plant died through 20 days of experiment

Hydrilla verticillata also demonstrated a large percentage of average Pb absorbed

ability is 92.8 % and high ratio of Pb content concentrating in the plant compared to Pb

content in water is 1.88 The study also gave predictions of Hydrilla verticillata

potential in improving water quality, with Pb concentration in water was forecasted to

be removed all from water in treatment B1, B2 and B3 Hence, this ability of Hydrilla

verticillata would be widely known and used for water assessing, monitoring and

cleaning operations in the future

Keywords: Phytoremediation, Hydrilla verticillata, Lead (II)-nitrate

(Pb(NO3)2), absorbed capacity, water heavy metal pollution Number of Pages: 51

Date of Submission: 30/09/2015

Supervisor (Sign)

ACKNOWLEDGEMENT

First and foremost I offer my sincerest gratitude to my supervisor, Dr.-phil

Arinafril of Sriwijaya University, Indralaya, Indonesia, who has supported and

instructed me throughout my research time and also my thesis completion with all of

his patience, devotions, enthusiasms and empathy If I have not had his helps I would

have never finished this study

Also, I would like to show my great appreciation to Dr Ho Ngoc Son, for

supervising, guiding, counseling and advising me in writing and completing the study

Besides my two supervisors, I want to send my sincere thanks to the Integrated

Research Laboratory of Sriwijaya University Palembang and the Research laboratory

in the Department of Chemistry, Faculty of Science, Sriwijaya University Indralaya in

Indonesia with their approvals, supports and helps in using place and equipment in the

research time this study is completed successfully

From the bottom of my heart, I am grateful to Fadila Mutmainnah, S.Si, for her

guidance, support and encouragement during the time of accomplishing this research

in Indonesia

My special thanks go to Ha, also, Chi, Linh, Marisa, Eka and all my

classmates, friends who helped me in my 3 months of internship time

Finally, I would like to express my heartfelt gratitude to my parents and

teachers for having, nurturing and teaching me from the very first steps of my life until

now, and their dedications will be my indications for future life

Thai Nguyen, 30 th September 2015

Student

Nguyen Thi Thao

TABLE OF CONTENTS

List of figures 1

List of tables 2

PART I INTRODUCTION 4

1.1 Research rationale 4

1.2 Research’s objectives 6

1.3 Research questions and hypotheses 6

1.4 Limitations 7

1.5 Definitions 7

PART II LITERATURE REVIEW 9

2.1 Water environment 9

2.1.1 The importance of water 9

2.1.2 Water resources 9

2.2 Heavy metal-polluted water 10

2.2.1 Source of heavy metal contaminants in water 10

2.2.2 Consequences of heavy metal contaminants in water 11

2.3 Lead Compound- Lead (II) Nitrate 11

2.4 Phytoremediation 12

2.5 Hydrilla verticillata 13

PART III METHODS 16

3.1 Sampling site and time 16

3.2 Materials and equipment 18

3.3 Methods 19

3.3.1 Applying treatments 19

3.3.2 Laboratory method of analyzing Pb content in Hydrilla verticillata 20

3.3.3 Laboratory method of analyzing Pb content in water 21

3.4 Data analysis methods 21

3.4.1 Variant analysis 21

3.4.2 Lead (Pb) Reduction rate 21

3.4.3 Time Series analysis: Linear regression 22

3.4.4 Bio-Accumulation Factor 22

PART IV RESULTS 23

4.1 Pb concentration in Hydrilla verticillata 23

4.2 Pb concentration in water 25

4.3 Result of Data analysis 27

4.3.1 Variant analysis 27

4.3.2 Time series analysis: Linear regression 32

4.3.3 Lead (Pb) reduction rate 37

4.3.4 Bio-Accumulation Factor 37

4.5 Hydrilla verticillata morphological changes 38

PART V DISCUSSION AND CONCLUSION 39

5.1 Discussion 39

5.2 Conclusions 43

REFERENCES 45

List of figures

Figure 3.1: Jakabaring lake in Palembang, Indonesia 3’0158.5’S and 104’7965.4’N 16

Figure 3.2: Integrated Research Laboratory of Sriwijaya University, Palembang, Indonesia which is located in 2’9910.5’S and 104’7295.5’N 17

Figure 4.1: Pb concentration in Hydrilla verticillata 24

Figure 4.2: Pb concentration in water 27

Figure 4.3: Forecasting of Pb concentration in water in treatment B3 33

Figure 4.4: Forecasting of Pb concentration in water in treatment B4 33

Figure 4.5: Forecasting of Pb concentration in Hydrilla verticillata in treatment B1 34

Figure 4.6: Forecasting of Pb concentration in Hydrilla verticillata in treatment B2 35

Figure 4.7: Forecasting of Pb concentration in Hydrilla verticillata in treatment B3 35

Figure 4.8: Forecasting of Pb concentration in Hydrilla verticillata in treatment B4 36

List of tables

Table 3.1: Experimental design 19 Table 4.1: Pb concentration in Hydrilla verticillata (ppm) 23 Table 4.2: Pb concentration in water (ppm) 25 Table 4 3: Significantly different effects of treatments on water’s Pb concentration in the 5th day 28 Table 4 4: Significantly different effects of treatments on water’s Pb concentration in the 10th day 28 Table 4 5: Significantly different effects of treatments on water’s Pb concentration in the 15th day 29 Table 4 6: Significantly different effects of treatments on water’s Pb concentration in the 20th day 29 Table 4 7: Significantly different effects of treatments on Pb concentration in Hydrilla verticillata in the 0th day 30 Table 4 8: Significantly different effects of treatments on Pb concentration in Hydrilla verticillata in the 5th day 30 Table 4 9: Significantly different effects of treatments on Pb concentration in Hydrilla verticillata in the 10th day 31 Table 4 10: Significantly different effects of treatments on Pb concentration in

Hydrilla verticillata in the 15th day 31 Table 4 11: Significantly different effects of treatments on Pb concentration in

Hydrilla verticillata in the 20th day 32 Table 4 12: Lead (Pb) reduction rate 37

Table 4 13: Bio-Accmulation Factor (BAF) ratio of Lead (Pb) from water to Hydrilla verticillata 37 Table 4 14: Hydrilla verticillata’s morphological changes observed in different

treatments through time 38

PART I INTRODUCTION

1.1 Research rationale

Playing vital role as a source of life, water have been being an indispensable resource to humankind and also other organisms However, water are now getting in a strong alarmed level of pollution, which can directly impact to the whole planet, Balkis (2012) stated “Water pollution 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 In addition to the acute problems of water pollution in developing countries, industrialized countries continue to struggle with pollution problems as well” Especially, current time, water is seriously polluted by heavy metals in the nearby industrial or populated urban areas, these heavy metals can widely distribute in water and directly or indirectly affect human and living aquatic organisms

due to human activities ( Mohiuddin, et al., 2010)

Lead (II) nitrate (Pb(NO3)2 ) is a toxic compound, and ingestion may cause acute lead poisoning, and also for all soluble lead compounds (USCG, 1999) Besides, when dissolve in water, Lead (Pb) from Lead (II) nitrate (Pb(NO3)2, one of the harmful chemical elements, can be huge influences for human health such as behavioral problems, high blood pressure, kidney damage, memory and learning difficulties, reduced IQ, … (Ernhart, 2006; Spivey, 2007) In term of aquatic systems, lead (Pb) has last and long effects on plants and animals by spreading and existing for long time

of about 100 to 1,000 years in water (UNEP, 2010)

There are many projects and researches had done in treating metal water pollution, but almost all of those projects required high techniques and also high cost for the steps and treatment processes Furthermore, with the variety in kind of metal

contaminants, water pollution could not be treated in some sensitive areas and by former ways (Hogan, 2014)

Phytoremediation, a green technique of treating heavy metal contaminants in water, uses plants to remove pollutants from the environment In order to remove pollutants from water, plants are used to accumulate the heavy metals in contaminated water, this way of dealing with water pollution shows itself as a friendly and cost-

effective method for resolving environmental issues (Salt, et al., 1995) With the

ability of absorbing variety kind of metals, and also the real effectiveness for economics and ecosystems, phytoremediation has been becoming a new favor to use in removing environmental pollutants (Paz-alberto & Sigua, 2012)

socio-There are many absorbents have been used for removing Lead from water; water hyacinth (Okunowo & Ogunkanm, 2010), Orange Barks (Azouaou, 2013) or

dried Lemna perpusilla Torr (Tang, et al., 2013) However, unlike those absorbents, with the very good advantage of living under water, hydrilla verticillata- a submerged

aquatic has its whole body inside water and can grow up to reach the surface which would has higher potential for environmental science in applying of phytoremediation

as Denny & Wilkins (1987) detected that in the purposes of removing contaminants from water “whole plant plays important roles the submergence and simplicity of shoot structure aquatic plants tend to have better potential in heavy metal uptake than other different structure plants” Hence, to effectively solve the problems of heavy

metal pollutants in water, this study with the topic is “Hydrilla verticillata, a

submerged aquatic plant, as phytoremediation agent of Lead (II)-Nitrate (Pb(NO3)2)”

was carefully conducted and completed

1.2 Research’s objectives

1 To understand how the submerged aquatic plant - Hydrilla verticillata works as a

tool of phytoremediation and how it got effect from Lead (II)-nitrate (Pb(NO3)2) polluted water

2 To assess the ability of Hydrilla verticillata in removing Lead (Pb) from water

3 To examine the effectiveness and amount in absorbing and accumulating Lead (Pb)

by Hydrilla verticillata

1.3 Research questions and hypotheses

This research is able to answer and explain these questions and hypothesis as follow:

Research questions:

1 What is Phytoremediation? How could Lead (II)-nitrate (Pb(NO3)2)-polluted water

effect Hydrilla verticillata and be treated by Phytoremediation with Hydrilla

verticillata?

2 How is the absorbed capacity of Lead (Pb) of Hydrilla verticillata?

3 Whether Lead in water could completely be absorbed and accumulated by Hydrilla

1.4 Limitations

Hydrilla verticillata from the sampling site which had Pb content inside may effect on

the experiment results Besides, throughout the transported and selected, acclimated processes, the plants health may be effected which can reduce its absorbed and accumulated ability

1.5 Definitions

Phytoremediation: The application of using plants to remove pollutants from

environment (Peuke and Rennenberg, 2005)

Lead (II)-nitrate (Pb(NO3)2: the water which is polluted by Lead (II)- nitrate

(Pb(NO3)2)- a danger poison, strong oxidizer Contact with other material may cause fire, may be fatal if swallowed or inhaled, causes irritation to skin, eyes and Respiratory tract, neurotoxin, central nervous system, kidneys, blood and reproductive system (MSDS, 2006)

Hydrilla verticillata: Submersed, usually rooted, aquatic perennial herb with slender

ascending stems to 9 m (30 ft) long, heavily branched Fleshy axillary buds (turions) often formed at leaf axils, to 5 cm (2 in) long, with 3 sepals and 3 petals, each about 4

mm (0.3) long, whitish or translucent, floating at water surface, thrive well in waste water (Langeland, et al., 2008)

Absorbability: The state or quality of being absorbable to take, suck up, assimilate or

be removed, as to take up substances into or across tissues, or the skin in to object's body1

1

Absorption (n.d.) Dictionary.com Unabridged Retrieved June 05, 2015, from Dictionary.com website: http://dictionary.reference.com/browse/absorption

Accumulate: to gather or collect, often in gradual degrees; heap up to gather into a

heap, mass, cover, etc… form a steadily increasing quantity2

PART II LITERATURE REVIEW

2.1 Water environment

2.1.1 The importance of water

Water is an essential source for life on the planet, and every living thing needs water

as a factor for growing, developing and surviving, it is now covering more than thirds of the Earth’s surface, with 97% is water in ocean, fresh water only accounts for 3%, in which 68 % is storied in glaciers and 30% is ground water, only 0.3% is surface waters (lakes and rivers) (Shakhashiri, 2014; Gleick, 1996)

two-Water not only is a motive for the growth of economic, but also is a main element for the stability of ecosystems, and a really important integral influence for human life and

health (Min, et al., 2015)

Thus, along with the development of global industrialization and modernization, the issues of environmental pollution, which cause lacking water and water contamination, have been becoming the intense concerns for the whole world society (Revenga & Mock, 2000)

2.1.2 Water resources

Functioning as a source of life, water becomes a definitely valuable resource and it is distributed on the earth in two locations - ground and surface which is respectively called ground water and surface water These two water sources are the living tools for all living things and especially are the great contributions for the developments of

community and the environment (Peter, et al., 2002; James, 2006)

Water exists in the environment in 3 phases of transferred cycle – solid-liquid- gas, in those 3 phases, liquid form of water is occupied for 30% of the earth’s fresh water and mostly is from ground water, which can supply for all human, animals and plants

activities, solid water is occurred in the earth’s poles and vapor form is usually appears

in atmosphere by condensing in the clouds and then falling down as rain drops (Hubbart & Medalye, 2013)

When water is polluted, as the two types of water resources surface waters and groundwater are affected Pollution can occur in two different ways: point-source pollution: pollution comes from a single location such as a waste discharge pipe of a company or an oil spill from a tanker, or someone pouring their car oil to a springs, and nonpoint-source pollution: water pollution happens not from one single source but from many different sources (Woodford, 2015)

2.2 Heavy metal-polluted water

The water which is contaminated by heavy metals such as lead, iron, chromium, copper, selenium, mercury, manganese, etc… is defined as heavy metal-polluted waters, these heavy metals can poison water environment when they are released by nature by natural processes or mostly from human activities (Wyk, 2012)

With high ability of to be accumulated in almost all environments, heavy metals were known as the most dangerous contaminants for human health and ecosystem balance (Thirulogachandar, 2014)

2.2.1 Source of heavy metal contaminants in water

Heavy metals occur in all around the earth, they will become toxic substances for environments and every living organism by natural processes or human activities, and the sources of them in life are divided into 3 principle sources: industry, supplier, and laboratory (Gleick, 2006)

However, the main sources of heavy metal contaminants in water is from industrial activities like mineral mining, electroplating industry, lead-acid battery manufacturing

industry,… which can directly put out huge amounts of heavy metals into water environments by running, leaching or spreading through ground or surface water system (Wyk, 2012)

In the green electric energy industry like geothermal industry, although, all workshops such as hydrothermal systems, binary power systems and vapor systems were designed

in order of producing electricity from hydraulic power of nature, the steps of processing water with high heats and pressures of these workshop is contaminating water while they are running (Sabadell & Axtmann, 1975)

2.2.2 Consequences of heavy metal contaminants in water

The impacts of heavy metal pollution on living organisms are very serious and difficult to solve Being released out to environment, heavy metals are easily dissolved

in water and expanded through areas which can cause significant effects to environment, human health and the origin of life (Heibutzki, 2015)

In all over the world, there is around 1.1 billion people do not have any knowledge about safe and clean drinking water, 6000 children die per day, 2.2 million people die

a year due to using the polluted and unsafe water, and this is also the cause 80% world diseases (Kumar, 2003)

Heavy metals are distributed in water, air and soil, they can be accumulated in a living body by interacting which leads to so many body problems such as the damage of: the peripheral nervous system; heart and cardiovascular system; lungs and respiratory system; skin, muscles and hair; stomach and intestine (Gagnon, 2015)

2.3 Lead Compound- Lead (II) Nitrate

One of the Lead compound, Lead (II) nitrate Pb(NO3)2 has the molecular mass of 331.2, the solubility of 50 gram in 100 gram water, white color or colorless crystals,

melting point at 290°C, evaporation point at 20°C, easy to burn and disperse, by ingesting, inhaling, or contacting this lead compound can be absorbed and accumulated inside body causing hazards for human life (USCG, 1999)

Lead (II) nitrate is considered as a strong toxic substance which can poison any organ when they contact with harmful amount For example, there is a study which had done

an experiment on the effect of Lead (II) nitrate on DNA damage and oxidative stress in diabetic and non-diabetic rat stated that “Lead (II) nitrate caused toxic effects on blood cells” (Kalender, 2015)

Not only Lead (II) nitrate, but also other Lead compounds have been becoming worrisome matters for society by being in the trend of increasing their pollution through ecosystem’s food chain, in which one Lead contaminated living organisms in can be eaten by non-contaminated living organisms, and it is accelerating the lead pollution to a higher rate (Roncero, 1990)

2.4 Phytoremediation

Phytoremediation uses plants as a tool for removing pollutants from the environment

to eliminate or minimize toxic effects for the planet, with the plant advantages of absorbing or concentrating contaminated elements from the environment plant is taking high potentials of becoming the effective materials for phytoremediation technique (Raskin, Smith, & Salt, 1997)

With various processes in phytoremediation: Phytoextraction: plants take the harmful elements the soil are required for their growth by the roots and reduce pollution, phytostabilization reduces element mobility by accumulating them near the roots, phytotransformation and phytostimulation: the degrading of chemicals into less toxic elements, either through plant only or initiating other microorganisms, this method can

be successful applied in the industrial waste dumped areas and Lead is one of the recommended toxic heavy metal to be treat (Chhabra, 2014)

In soil and water environments, when plants interact with contaminants especially heavy metal substances they will immediately be attracted to these compounds and plants will show their ability in having different mechanisms to uptake, transport, accumulation and detoxification heavy metals (Revathi, 2013)

Phytoremediation has ability to reduce diverse types of contaminants in environment, which was proved by many successful experiments of mitigating metals, pesticides, solvents, explosives, and crude oil (Rhodes, 2013)

Some projects and research have done in removing heavy metal contaminants such as Laed (Pb) from water environments Phytoremediation by using water hyacinth to degrade Pb in water have raised a great benifit of this plant and also for other studies (Okunowo and Ogunkanm, 2010)

It also showed quite good results when Orange Barks were used to treat Pb in the environment (Azouaou, 2013) It's difficult to find Orange Barks in so many areas Using dried Lemna perpusilla Torr to remove Pb and improve water quality had done Tang, et al., 2013) However, this is not a material that can be found easy or not the popular one

2.5 Hydrilla verticillata

Hydrilla verticillata (also called water thyme, Florida elodea or waterweed) is a rooted

submerged aquatic plant which can grow with the depth of 6.1 m in water, in the hydro-soil or at nodes the roots are brown, long, whitish, or slender Its leaves are green and small with the long of around 5-20 mm, wide is normally 2 mm, whorls of

3 - 10 leaves in one stem, stems will form dense mats near the surface and around 20%

of the plant's total biomass is the top 10 cm of the densely surface mats occur in bunches, and usually live in rivers, lakes, ponds and springs, can be found in almost all continents (Langeland, 2008)

Hydrilla verticillata can stand and grow well in vary kinds of environment, can live in

low light areas (1% of light), low or high nutrient areas and even in the high chemical range and polluted water, thus according to Langeland (1996), it covered in a high percentage of 43% of the public lakes with about 40,000 hectares water

Similar with some other aquatic plants, Hydrilla verticillata has known as an upstart

source for phytoremediation – a high effective, green and cheap method of tackling the environmental issues by using the plant ability of gathering contaminants like heavy metals to remove them from environments (Tiwari, 2006)

As other plants when absorb heavy metals from environment into its body the internal concentration of that metal is higher than that of surrounding environment (Albers & Camardese, 1993)

Higher internal concentration of heavy mentals in plant can degrad plant absorbed capacity and its growth causing lower absorbed ability by higher applied metals (Singh, 2013)

These reviews of the professional literatures, which are relevant to this study, play important roles in understanding the reason why water envronments need to be treated, which ways gave good resuls and which ways will have high potential of using them

The literature reviews have raised background information about Hydrilla verticillata

as an useful and effective phytoremediation agent of Lead (II) - nitrate (Pb(NO3)2) And also, they are the scientific and theoretical basis for this research in assessing,

examining and investigating Hydrilla verticillata abilities to eliminate Lead (Pb) from

water, absorb and store Lead (Pb) by its body and leaves

PART III METHODS

3.1 Sampling site and time

This study about Phytoremediation of Lead (II)-Nitrate (Pb(NO3)2) from water with a

submerged aquatic plant, Hydrilla verticillata were started from March 2015 and

finished in June 2015 On 09th March 2015, harvesting Hydrilla verticillata for the experiment, the total of 3 plastic containers of Hydrilla verticillata was taken from

submerged waters around Ski Air Lake Jakabaring Palembang, Indonesia Then these

3 plastic containers were moved to integrated research for the Integrated Research Laboratory of Sriwijaya University Palembang, Indonesia in the same day and acclimated for 3 days

Figure 3.1: Jakabaring lake in Palembang, Indonesia 3’0158.5’S and 104’7965.4’N

Figure 3.2: Integrated Research Laboratory of Sriwijaya University, Palembang, Indonesia which is located in 2’9910.5’S and 104’7295.5’N

The experiment was conducted by applying Lead (II) nitrate (Pb(NO3)2) on 13th March

2015 then observed and sampled in 5 times:

 The first time was taken on 13th March 2015 for day 0th

 The second time was taken on 18th March 2015 for day 5th

 The third time was taken on 23th March 2015 for day 10th

 The fourth time was taken on 28th March 2015 for day 15th

 The fifth time was taken on 02sd April 2015 for day 20th

Five times of destructing Hydrilla verticillata for identifying Lead (Pb) concentration

inside the plant, on:

 14th March 2015 for day 0th

 19th March 2015 for day 5th

 24th March 2015 for day 10th

 29th March 2015 for day 15th

 03th April 2015 for day 20th

And 4 times of identifying Lead (Pb) concentration in water (inside 12 plastic

containers and plant (Hydrilla verticillata) in the research laboratory in the

Department of Chemistry, Faculty of Science, University of Sriwijaya, Indralaya, Indonesia, on:

 18th March 2015 for day 0th

 24th March 2015 for day 5th

 27th March 2015 for day 10th

 07th April 2015 for day 15th and for day 20th

3.2 Materials and equipment

 Hot plate, incubator

 Analytical balance, mortar, rod stirrer, pipette

 Glass funnels, glass conical flasks, bunchner flask, measuring cup, vacuum filtered set of tools, glass sample bottles

3.3 Methods

3.3.1 Applying treatments

After moving to Integrated Research Laboratory of Sriwijaya University Palembang,

Indonesia, 3 plastic containers of Hydrilla verticillata was selected with selecting

standard are fresh, green and healthy plants, and washed by tab water from the laboratory Then take 12 samples (12 plastic containers: 4 treatments and 3

replications) of Hydrilla verticillata (which are already selected and washed), 300

gram for each sample, then put 12 of 300 gram samples into 12 plastic containers (diameter: 90cm, height: 30cm), which already contained 4 treatments of Lead (Pb) 0 ppm (control), 5 ppm (mg / l), 10 ppm (mg / l) and 15 ppm (mg / l), 3 replications for each treatment and mixed with 20 liter of clean tab water Lead solution from stock is Pb(NO3)2 1000 ppm These 12 plastic containers were labeled as: Hydrilla verticillata

(A1), treatment (B1 (control), B2, B3 and B4), and replication (1, 2 and 3) (table 3.1) Finished the processes of applying different treatments, 12 plastic containers were kept

in static condition and observed in five times on the day 0th, 5th, 10th, 15th and 20th Experiment was summarized in table 3.1:

Table 3.1: Experimental design

3.3.2 Laboratory method of analyzing Pb content in Hydrilla verticillata

In order to analyze the content of Lead (Pb) in Hydrilla verticillata, AAS- Atomic

absorption spectrometry was used as an analytical technique which can identify and measure the concentrations of metal elements

The procedures of analyzing Pb content in Hydrilla verticillata:

1 Took 50 grams of Hydrilla verticillata from each of 12 plastic containers and

dried them in the 80o C incubator in 24 hours

2 Took them out and grinded until finely ground

3 Weighed as much as ± 3.00 g of each sample to be tested and then put each of them into a conical flask and add 50 ml of distilled water and then stirred with a rod stirrer

4 Added 5 ml of Nitric acid (HNO3 65%) in each conical flask sample, stirred until mixed, covered with a glass funnels and then heated them with hot plates which were set at a temperature range of 105ºC - 120ºC

5 12 solutions were heated until the volume of only 10 ml, then removed and cooled them

6 Filtered the cooled solutions by using vacuum filtered set of tools, bunchner flask and Whattman filter papers, poured each of obtained samples into a 50ml labeled sample bottle and added distilled water to get exactly 50ml of solution And then the samples were measured by using AAS

These procedures to analyze Pb concentration in Hydrilla verticillata were

implemented according to SNI 06-6992.3-2004 reference of using AAS - Atomic absorption spectrometry test Lead (Pb) with acid destruction, and in 5 times of the 0th,

5th, 10th, 15th and 20th day

3.3.3 Laboratory method of analyzing Pb content in water

The processes of measuring Pb content in water by using AAS - Atomic absorption spectrometry were based on the Indonesia approved standard, SNI 6989.8.2009 of using AAS- Atomic absorption spectrometry to test Lead (Pb) in water The 50 ml of water samples from each container was taken to be tested and filtered using Whattman filter paper Then added HNO3 until the pH of the water became 2 After that, use AAS to analyze Pb content in water These processes were carried out in 5 times: 0th,

5th, 10th, 15th and 20th day

3.4 Data analysis methods

3.4.1 Variant analysis

To identify significant differences in effects of treatments on Pb concentrations in

water and Hydrilla verticillata, all the results of Pb concentration of 4 treatments and 3

replications in the plant and water were analyzed by Variant analysis, if real different effects appear, the processes will be continued with the Duncans Multiple Range Test

(DNMRT) at 5% level by using ANOVA one-way analysis in statistics software 8.0 of

statistical significance α = 0.05 was assumed Whenever analysis of variance showed statistically significant effect (p < α = 0.05), Duncan test was performed to determine the significant differences more accurately

3.4.2 Lead (Pb) Reduction rate

The Mean reduction rate of Pb concentration in water was calculated based on the initial and final mean Pb concentration in water of each treatment using the formula of: (R is Pb reduction rate; Ci: initial Pb concentration applied in

water; Cf: final mean Pb concentration in water) (Dalun, 2008)

3.4.3 Time Series analysis: Linear regression

In order to predict the changing trend of Pb concentration in water and also Hydrilla

verticillata in upcoming time if the experiment still continues, the processes of Linear

regression and also the trend line were done in excel software by using data analysis tool, and the forecasted results were gotten by calculating which is based on the formula: Y= a + bX

Note: Y is dependent variable (Pb concentration (ppm)), X is independent variable (Observation time (day)), a is the intercept (of the regression line with Y-axis), and b

is the slope of the regression line (the predicted change in Y per unit of change in X, when b is positive: value of y increases as the value of x increases (positive correlation) and b is negative: value of y decreases as x increases (negative correlation))

3.4.4 Bio-Accumulation Factor

By using the formula of calculating Bio-Accumulation Factor (BAF):

(Where CB is the chemical concentration in the organism and CWD is the

freely dissolved chemical concentration in the water), the ratio of concentrating Pb in

Hydrilla verticillata in 5 observation times of B1, B2, B3 and B4 can be determined

PART IV RESULTS

After finishing all of experiment procedures, with 4 applied treatments of Pb 3 replications for each treatment, 5 times of observing, sampling, the results of Pb

concentration in Hydrilla verticillata and in water will be presented as followed

sections

4.1 Pb concentration in Hydrilla verticillata

The results of Pb concentration in Hydrilla verticillata after analyzing by AAS-

Atomic absorption spectrometry (analytical technique that measures the concentrations

of elements) for 5 sampling times (0th, 5th, 10th, 15th and 20th day) are presented in table 4.1

Table 4.1: Pb concentration in Hydrilla verticillata (ppm)

Table 4.1 shows the Pb concentration in Hydrilla verticillata, in the first sampling time

- 0th day, Hydrilla verticillata in B1 (control) already had Pb content inside their body

with Pb concentration is 0.05 ppm With higher treatments of B2, B3 and B4, the plant had Pb concentration of 0.46 ppm, 0.55ppm and 0.72 ppm respectively

The data in the 5th day in Table 4.1 illustrated that Pb concentration in Hydrilla

verticillata had the significant increases went up to 3.65 ppm in B2, 5.74 ppm in B3,

10.83 ppm in B4 and had no change