Use of coconut shell as natural adsorbent to treat wastewater containing hazardous insecticide compound and its toxicity test on nile tilapia

THAI NGUYEN UNIVERSITY UNIVERSITY OF AGRICULTURE AND FORESTRY AMANA AMALIA USE OF COCONUT SHELL AS NATURAL ADSORBENT TO TREAT WASTEWATER CONTAINING HAZARDOUS INSECTICIDE COMPOUND AND I

THAI NGUYEN UNIVERSITY

UNIVERSITY OF AGRICULTURE AND FORESTRY

AMANA AMALIA USE OF COCONUT SHELL AS NATURAL ADSORBENT TO TREAT WASTEWATER CONTAINING HAZARDOUS INSECTICIDE COMPOUND

AND ITS TOXICITY TEST ON NILE TILAPIA

BACHELOR THESIS

Study Mode : Full-Time

Major : Environmental Science and Management

Faculty : Advanced Education Program

Batch : 2014 - 2018

Thai Nguyen, September 2018

DOCUMENTATION PAGE WITH ABSTRACT

Thai Nguyen University of Agriculture and Forestry

Wastewater Containing Hazardous Insecticide Compound and Its Toxicity Test on Nile Tilapia

Prof Tran Van Dien

Abstract:

To investigate the effects of environmental contaminants, this study explored the

adsorption capacity of Coconut (Cocos nucifera L.) shell towards cypermethrin in

aqueous solutions The purpose of the present study is to evaluate the ability of coconut shell, to alleviate cypermethrin from wastewater and to assess the

histopathological alterations in the gills of Nile Tilapia (Oreochromis niloticus) which

were kept in histopathological alterations would contribute an important role in assessing the harmful effects of cypermethrin Histopathological response of fish exposed to pollutants has been used as (a) sensitive biomarkers Histopathological examination on fish gill indicates that excessive levels of cypermetrhin can damage the tissues of the fish gills The alterations detected were as Telengeactacia, Fusion of

Secondary Lamellae, Epithelial Proliferation of Secondary Lamellae, Congestion and Curling Bend

Cypermethrin, Wastewater, Natural Adsorbent

Supervisor’s Signature

ACKNOWLEDGEMENT

From the deepest feeling in my heart, I would like to say Thank You for all the people who always support me and thank you to Allah SWT who has given his grace so that I could finish this thesis

First, I would like to send my sincere gratitude to Dr.-phil Dipl.-Ing.agr Arinafril of Sriwijaya University, Indralaya, Indonesia as my first supervisor, Prof Tran Van Dien of Thai Nguyen University, Vietnam as my second supervisor and Krisna Murti, MD., M Biotech Stud., Ph.D in the Department of Anatomical Pathology, Faculty of Medicine, Sriwijaya University who assisted and helped me patiently in histopathological examination and also during my thesis writing

I also would like to thank all parties who have supported and assisted in the preparation of this thesis especially to:

1 Prof Dr Ir H Anis Saggaf, MSCE as the Rector of Sriwijaya University, Palembang, Indonesia

2 Prof Dr Ir Andy Mulyana, M.Sc as the Dean of Faculty of Aquaculture, Sriwijaya University, Palembang, Indonesia

3 Prof Dade Jubaedah, S.Pi., M.Si from Faculty of Aquaculture, Sriwijaya University

as a companion lecturer who has kindly accompanied me and guided me when I implemented this research

4 Prof Mochamad Syaifudin, S.Pi., M.Si from Faculty of Aquaculture, Swirijaya University, Palembang, Indonesia

5 Prof Dr Mohammad Amin, S.Pi., M.Si Faculty of Aquaculture, Sriwijaya University, Palembang, Indonesia

6 Mrs Nurhayani as lab analyst from Laboratory of Aquaculture, Sriwijaya University who helped, assisted me and gave some advises during my research

7 Mrs Ana and Mrs Naomi as lab analyst from Laboratory of Fisheries Product of Technology, Sriwijaya University

8 Other staffs and friends from Aquaculture Sriwijaya University who helped and

assisted me during my research

In addition, I would like to express my deepest thanks to my friends who always give me their love, support and advises

Finally, special thanks to my parents and my family for their support throughout

my study

Sincerely,

Amana Amalia

TABLE OF CONTENTS

LIST OF FIGURES vii

LIST OF TABLES viii

PART I INTRODUCTION 1

1.1 Background and Rationale 1

1.2 Objectives 4

1.3 Research Questions and Hypotheses 4

1.3.1 Research Questions 4

1.3.2 Hypotheses 5

1.4 Limitations 5

PART II LITERATURE REVIEW 6

2.1 Cypermethrin Compound 6

2.2 Toxic Effect of Cypermethrin on Organisms 7

2.2.1 Toxicity 7

2.2.2 Histopathological Effects 10

2.3 Test Species – Oreochromis niloticus 12

2.3.1 Scientific classification 12

2.4 Coconut Shell 12

2.5 Activated Carbon 14

2.6 Single Drum Carbonization 15

2.7 Activation of Activated Carbon 16

PART III MATERIALS AND METHODS 21

3.1 Place and Time 21

3.2 Equipments and Materials 21

3.3 Methods 22

3.3.1 Fish Preparation 22

3.3.2 Toxicity Testing 22

3.3.3 Adsorbent Preparation 23

3.3.4 Ash Content Analysis 24

3.3.5 Water Content Analysis 24

3.3.6 Adsorbent Experiment Using Fish as Bio indicator 25

3.3.7 Histopathological Examination 26

PART IV RESULTS AND DISCUSSIONS 29

4.1 Results 29

4.1.1 Preliminary Test LC50 Determination 29

4.1.2 Ash Content Analysis 30

4.1.3 Water Content Analysis 31

4.1.4 Adsorbent Experiment using Fish as Bio Indicator 31

4.1.5 Histopathological Observation of Gills 37

4.2 Discussions 44

PART V CONCLUSION 48

REFERENCES 49

APPENDICES 53

LIST OF FIGURES

Figure 1 Coconut Shell 13 Figure 2 The Condition of Aquarium after 24 hours of Adsorbent Additional Process 32 Figure 3 Normal histological gills structure 37 Figure 4 Histopathological alterations of gills structure of Nile Tilapia treated by using 3.8 ml/L of cypermethrin concentration without adsorbent treatment 38 Figure 5 Histopathological alterations of gills structure of Nile Tilapia treated by using 3.1 ml/L of cypermethrin concentration with adsorbent treatment 39 Figure 6 Histopathological alterations of gills structure of Nile Tilapia treated by using 3.4ml/L of cypermethrin concentration with adsorbent treatment 40 Figure 7 Histopathological alterations of gills structure of Nile Tilapia treated by using 3.8ml/L of cypermethrin concentration with adsorbent treatments 41 Figure 8 Histopathological alterations of gills structure of Nile Tilapia treated by using 4.2 ml/L of cypermethrin concentration with adsorbent treatment 42 Figure 9 Histopathological alterations of gills structure of Nile Tilapia treated by using 4.6ml/L of cypermethrin concentration with adsorbent treatment 43

LIST OF TABLES

Table 1 Physical and Chemical Properties of Cypermethrin 6

Table 2 Toxicity Classification of Cypermethrin 8

Table 3 Characterization of Activated Carbon (SII 0258-88) 15

Table 4 Equipments for Toxicity and Adsorbent Test 21

Table 5 Materials for Toxicity and Adsorbent Test 22

Table 6 Effect of Cypermethrin Concentration on Nile Tilapia in Preliminary Test 29

Table 7 Effect of Cypermethrin on Nile Tilapia in Five Repetitions using Adsorbent Treatments 32

Table 8 Observation of Cypermethrin Effect on Nile Tilapia 35

PART I INTRODUCTION

1.1 Background and Rationale

Along with the extensive increasing of coconut industrial making the waste production is higher This will certainly cause some negative impact to the environment

if it is not managed and utilized wisely Environmental damage caused by technological advancement using substances is very dangerous to living things Furthermore, it will disturb the ecological balance Basically, most of environmental damages are an impact

of human activities, especially those that occurred to be water pollution The contamination of water pollution tends to disrupt and destroy the fragile ecology through indiscriminate discharge of industrial and municipal waste into the sea The contamination affects not only the surrounding area but also indirectly for human health Most of industrial waste dumped into the water leads to extinction of living things The composition of industrial waste cannot be characterized readily by a typical range of values because its makeup is depended on the type of manufacturing process involved (Kurniati, 2008) Sewage, industrial waste and agricultural chemicals, such as fertilizers and pesticides, are the main causes of water pollution Among those wastes, insecticides are the most widely used in agricultural activity, one of them is cypermetrhin (Sarikaya, 2009)

Cypermethrin is a class of pyrethroid insecticides Pyrethroid is a group of protection products that are widely used as insecticides in agricultural settings, gardens and industrial areas Furthermore, they are used to treat ectoparastic diseases (e.g., lice)

plant-in sheep, cats, dogs, and other animals They are structurally similar to pyrethrplant-ins, a class of compounds that are found in chrysanthemum plants, where they work as natural

insecticides In this past 25 years, pyrethroids have become one of the most dominant class of insecticides in agriculture because of their low toxicity on mammals and reduced application rates Because of these properties, organophosphates and carbamates are increasingly being replaced by pyrethroids in arable crops and forestry throughout

Europe and North America (Hartnik, Sverdrup and Jensen, 2008)

In the recent years, water pollution caused by insecticide compound has been a serious problem in developing country that runs farming system as their major opportunity to raise money Generally, pyrethroid insecticides are used extensively in agriculture and have resulted as the cause of increasing level of toxic chemicals in an aquatic environment Cypermethrin, one of insecticides from pyrethroid group, is known as the highest toxic synthetic insecticide which can kill insects by strong excitement in their nervous system It is often transported to surface waters via runoff and erosion as well as the drift from aerial sprays Consequently, it could be dangerous

to benthic and epi-benthic species, as well as fish and other organisms feeding on

benthos (Richterova et al., 2015) Due to the high toxicity to aquatic organisms, the

usage of cypermethrin is restricted for crops and wide area applications (Sarikaya, 2009) Histopathological response of fish exposed to pollutants has been used as the sensitive biomarkers

One of the efforts to mitigate cypermethrin insecticide residues is to use activated charcoal whose ingredients can come from agricultural waste, such as coconut shells The relative increasing of technological development of coconut in Indonesia makes the production of coconut waste higher One of the wastes produced by the coconut is the shell Dried coconut shell contains 33.61% cellulose, 36.51% lignin, 29.27% pentosans

and 0.61% ash (Shelke et al., 2014) The contents make the coconut shell are possibly

used as raw material for making activated carbon or activated charcoal According to

Ratnoji and Singh (2014), coconut shells have high carbon content and hardness which

make coconut shells an excellent raw material source to produce activated carbon which are very effective to adsorb the waste The use of activated charcoal in rice fields able

to increase the number of bacteria and nitrogen fixation bacteria (Azotobacter) in the

soil, especially around the roots of food crops Activated charcoal from coconut shell

increases the microbial population of Citrobacter sp, Enterobacter sp, and Azotobacter

sp in rice cultivation which is where some of these bacteria include pesticide degrading

bacteria and nitrogen fixers The use of activated charcoal in the cultivation of agricultural crops can reduce insecticide residues in soil, water, and agricultural products The direct benefit of using activated charcoal from coconut shells on agricultural land is that it can help to reduce surface water pollution in rivers that can disrupt the life of aquatic organisms and human health, reduce insecticides and heavy metals residues in agricultural products, reduce agricultural waste and provide added

value from agricultural waste (Harsanti et al., 2013)

Fish have been considered as good bio-accumulators of organic and inorganic toxicants Fish are primary sources of protein They comprise large components of most aquatic environment functioning as bio-indicator of heavy metal contamination in the

aquatic ecosystem Oreochromis niloticus is one of the most favoured common fish

species among aqua-culturists It has a comparative advantage, such as ability to tolerate

a wide range of environmental conditions, fast growth, successful reproductive strategies, and ability to feed at different trophic levels, which make it becomes an important commodity of aquaculture These same traits allow them to be an extremely

successful invasive species in subtropical and temperate environments (Grammer et al.,

2012) On the other hand, people are also affected by eating fish especially those areas where main food is fish Terrestrial and aquatic food chains are suitable for accumulating numerous environmental pollutants up to levels that could be toxic to both anthropogenic and aquatic organisms’ health

Fish as one of the biotas of water that can be used as an indicator of the level of water pollution Histopathological analysis of fish can be used as a biomarker to monitor marine environment through observations of fish health The observations can be done

on the organs which functions are important in the metabolism So, it can be used as an

early diagnosis of illness in fish (Zulfahmi, Affandi and Lumban Batu, 2015) One of

the freshwater fishes which sensitive to water pollution is Nile Tilapia

In addition, to face the problem related to the impact of water pollution caused by hazardous waste content such as insecticides This study explored the effective role of coconut shell as activated carbon in reducing the impact of cypermethrin to the water through fish which could be seen by histopathological analysis

1 How does cypermethrin affect the gills of Nile Tilapia?

2 How does cypermethrin affect the gills of Nile Tilapia after adsorbent is given?

1.3.2 Hypotheses

Hypotheses Question 1:

H0 (Null Hypotheses): Exposure to cypermethrin contamination with the treatment

of adsorbent will not result in changes in gills histology of Oreochromis niloticus

HA (Alternative Hypotheses): Exposure to cypermethrin contamination with the

treatment of adsorbent will result in changes in gills histology of Oreochromis niloticus

Hypotheses Question 2:

H0 (Null Hypotheses): Exposure to cypermethrin contamination with the

treatment of adsorbent will not result in changes in gills histology of Oreochromis niloticus after adsorbent is given

HA (Alternative Hypotheses): Exposure to cypermethrin contamination with the

treatment of adsorbent will result in changes in gills histology of Oreochromis niloticus

after adsorbent is given

1.4 Limitations

- There is a lack of experimental methodology using coconut shell as an

adsorbent to adsorb cypermethrin insecticide

- An uncertain fish conditions affects the time of the toxicity testing and

adsorbent experiment

- As well, the lack of experimental results on the histopathological effects on the gills tissue of Nile Tilapia which caused by cypermethrin in literature

PART II LITERATURE REVIEW

2.1 Cypermethrin Compound

In Table 1 shows the physical and chemical properties of cypermethrin insecticide

Table 1 Physical and Chemical Properties of Cypermethrin

Chemical Name

Cyano(3-phenoxyphenyl)methyl dichloro ethenyl)-2,2-

Source: USDA, 2001; Tomlin, 1997

2.2 Toxic Effect of Cypermethrin on Organisms

2.2.1 Toxicity

The toxicity classification of cypermethrin via the designated routes of exposure shows

in Table 2

Table 2 Toxicity Classification of Cypermethrin

Inhalation The respiratory irritation is one of the

major effects of inhalation exposures by directly spraying and getting involved in the packing of the cypermethrin will have symptoms such as hypersensitivity, neumonistic, pleuritic chest pain, non-productive cough and shortness of breath

cypermethrin insecticide effects on human will have symptoms such as nausea, diarrheal, vomiting and epigastric pain or even death

skin by washing with shampoo which contains cypermethrin and skin irritation such as burning, numbness and itching which farmers used it as based plant

Source: Javed et al., 2015 Cyano(3-phenoxyphenyl)methyl3–(2,2–dichloroethenyl)–(2,2

dimethylcyclopropanecarboxylate) which has known as cypermethrin is an active

substance one of type II synthetic pyrethroid insecticide similar to natural pyrethrin isolated from chrysanthemum flowers, which has high insecticidal activity (ATSDR, 2003) Cypermethrin is an active pyrethroid, which intensively controls a wide range of pests in agriculture and animal breeding Cypermethrin is classified as a schedule 6 poison in the standard for the Uniform Scheduling of Drugs and Poisons Most technical grades of cypermethrin contain more than 90% of the active material Cypermethrin has

a very low vapour pressure and solubility in water, but it is highly soluble in a wide range of organic solvents Analytical methods are available for the determination of cypermethrin in commercially available preparations In addition, methods for the determination of residues of cypermethrin in foods and in the environment are well established Cypermethrin is relatively safe to mammals and birds, but is extremely toxic

to fish and aquatic organisms, and should not be applied on or near water, or when drift

is heavily used to replace the more toxic organophosphates Its main use is against foliage pests and certain surface soil pests, such as cutworms, but because of its rapid breakdown in soil, it is not recommended for use against soil-borne pests below the surface

Cypermethrin used to control many pests including lepidopterous pests of cotton, fruit, and vegetable crops and is available as an emulsify-able concentrate or wet-able

powder As a result of its numerous applications, the potential of cypermethrin discharge into aquatic environment has increased When cypermethrin get into the water it will disrupt into the environment of aquatic biota such as fish Fish can show reaction to the physical changes of water as well as pollutant compounds dissolved in accordance with the limits of concentration certain This is one of the reasons why fish can be utilized as

a biota in biological tests (Authman et al., 2015)

2.2.2 Histopathological Effects

There are several studies on histopathological effects of cypermethrin on organisms Histopathological alterations give a reliable, efficient measurable index of

low-level toxic stress to a wide range of environmental contaminants

Ojutiku et al (2014) conducted a research on the toxicity and histopathological

effects of cypermethrin on juveniles of Clarias gariepinus In this experiment the fish

were exposed to six acute concentrations (0.025mg/l, 0.050mg/l, 0.075mg/l, 0.100mg/l, 0.125mg/l and 0.000mg/l) for 96 hours The 96 hours LC50 of the toxicant to the test fish was 0.060mg/L The most common gill changes at all doses of cypermethrin in solution were destruction of gill lamella, epithelial hyperplasia and epithelial hypertrophy Fish gills treated with the lowest concentration of cypermethrin showed gill necrosis, congestion, cartilage and interstitial haemorrhages while in the highest concentration of cypermethrin the treated gills showed a severe gill necrosis with extensive vacuolation within the epithelia of the gills, infiltration, cartilage and interstitial haemorrhages were also found

Rahayu, Zulfatin and Nuriliani (2001) conducted a research about histopathological test of gill tissue, liver and intestine towards Nile Tilapia using λ-cyhalothrin as pollutant The variations concentration of λ-cyhalothrin were 3, 6, 9 and

12 μg/L using 5 treatments and 3 repetitions where each of aquariums filled with 10 Nile tilapia and 10L of water As the results obtained, there were found some damages include the gill tissue, liver and intestine of Nile Tilapia Gills damages were in the form

of hyperplasia, necrosis, edema, secondary lamella fusion, removal of lamella epithelial cells, and primary lamella dilatation Liver damages include hemorrhage, intravascular hemolysis on blood vessels, piknosis, and leukocytes infiltration The intestine damages were fatty degeneration, edema, leukocyte infiltration, hemorhage, cloudy swelling, vacuolisation, necrosis, and arthropy The gills, liver, and small intestine damages of Nile Tilapia range from slight to severe It could be concluded that λ-cyhalothrin caused histopathological effects on the gills, liver, and small intestine of Nile Tilapia Then, continue with study of the effects of cadmium (Cd) on the structures of the gills of catfish

(Clarias batrachus) If the fish were exposed to 0 (control), 1, 2, and 4 ppm of Cd

during 14 days, showed that Cd affected the structure of the gills of catfish, such as edema, hyperplasia, and fusion gill lamella Damage could be happened as the concentration of cadmium is increasing in media The gills of control animals showed the lowest damage (13.9%), whereas the level of gill damage of fish exposed to 1, 2, and 4 ppm were 45.7; 71.6; and 82.1% respectively (Hayati, Ummah and Winarni, 2016)

Histopathological changes due to some chlorinated hydrocarbon pesticides in the

tissues to Cyprinus carpio performed by using the concentration of Aldrin, Dieldrin,

BHC and DDT The concentrations of pollutants were varied from 0.005, 0.002, 1.000 and 0.002 mg L-1 respectively The test fish were kept in the test solution of known concentration for a period extending over to 20 and 30 days After every 24 hours fresh test solution was introduced The experiments were run in replicates for all pesticides

Histopathological changes were observed in vital organs such as damages in gills showed swelling and thickening in their filaments, liver showed abnormal fatty degeneration and kidney tissue showed large vacuolated cells were seen in the glomeruli

Binomial name : Oreochromis niloticus (Linnaeus, 1758)

Oreochromis niloticus is one of the mostly favored common fish species among

aquaculturists It has a comparative advantage which makes it becomes an important commodity of aquaculture because of its ability to tolerate a wide range of environmental conditions, fast growth, successful reproductive strategies, and ability to feed at different trophic levels These same traits allow them to be an extremely

successful invasive species in subtropical and temperate environments (Grammer et al.,

2012)

2.4 Coconut Shell

Coconut plantation industry is one of the most popular sectors in society in Indonesia even the world The processing of coconut itself has many processes which

each of process will certainly produce waste One of the wastes which usually produce from the coconut is coconut shell

Figure 1 Coconut Shell

Coconut shells are one of the sludge-by products of coconut processing industry, which currently creates problems for the environment This is because the waste is produced

in large quantities and difficult to degrade or decompose naturally in the environment Dried coconut shell contains 33.61% cellulose, 36.51% lignin, 29.27% pentosans and

0.61% ash (Shelke et al., 2014) With the content of coconut shells which are very

possible to be used as raw material for making activated carbon or activated charcoal Coconut shells have high carbon content and hardness which make coconut shells as an excellent raw material source to produce activated carbons which are very effective to adsorb the waste Activated coconut shell charcoal on the ground cabbage cropping can reduce chlorpyrifos insecticide residues in water to about 50%, lindan and aldrin residual levels on mustard plants which are given activated charcoal from the shell are relatively high (Ratnoji and Singh, 2014)

2.5 Activated Carbon

Activated carbon is a form of carbon species that is processed and prepared to have high porosity and very large surface area available for adsorption Coconut shell is suitable for preparing micro porous activated carbon due to its excellent natural structure and low ash content Activated carbon can be activated either chemically or physically (Hanum, 2009) The advantages of coconut shell carbons including high density and high purity They are virtually dust-free, since they are harder and more resistant to attrition Activated carbon is used in gas purification, gold purification, metal extraction, water purification, medicine, sewage treatment, air filters in gas masks and respirators,

filters in compressed air and many other applications (Sangotayo et al., 2017)

There are some characteristics to determine whether the activated carbon is good

or not according to SII 0258-88 as can be seen in Table 3

Table 3 Characterization of Activated Carbon (SII 0258-88)

3 The missing part on the heating of

6 Adsorption of Methylene Blue Ml/gram Min 120

Source: Scientific Communication and Information Center, 1997

2.6 Single Drum Carbonization

Carbonization is a pyrolysis process or incomplete combustion (without oxygen) to the base material used to obtain charcoal with carbonization temperature depending on the base material The most important and most influential stage of charcoal quality is the process of burning and turning out fires At the combustion in the way that people usually do, the combustion process is thorough and constantly uncontrolled so that the burned shell first becomes charcoal, will continue to burn following the unburned shell

As a result, many shells become ash and others have not been burned so that the low yield of charcoal is 22.5% (Lindayanti, 2006)

In combustion with a controlled air supply system, the combustion process is controlled by regulating the air supply into the combustion tube In the burned shell portion of the charcoal, the air supply hole is closed and the opening in the top row is opened so that the combustion process only takes place in the portion where the air supply hole is open And so on until the air hole on the top row Thus, in the charcoal of combustion results not found ash and very little shell that does not become charcoal so that the yield of charcoal produced higher, i.e 31.58%

2.7 Activation of Activated Carbon

There are some methods for activation of activated carbon, which by chemically, physically or physic-chemical activation Each of the methods has their own advantages and disadvantages so we can adjust to the needs

1 Chemical Activation

This activation is the process of breaking the carbon chain from organic compounds

by the use of chemicals The activators used are chemicals such as: alkali metal hydroxides, carbonate salts, chlorides, sulfates, phosphates of alkaline earth metals and

in particular ZnCl2, inorganic acids such as H2SO4 and H3PO4 Chemical solutions that can be used in carbon activation include ZnCl2, H2SO4, KOH or CaCl2 Disadvantages

of using mineral ingredients as an activator lie in the washing process of these mineral materials are sometimes difficult to remove whereas the advantage of using mineral materials as activators is the relatively short activation time, the more activated carbon produced The use of different activator substances will produce different pores Factors affecting pore formation are the concentration of the activator and its temperature (Hadi, 2011)

to ash and oxidation by further heating can be reduced (Gumelar, 2015)

3 Physical-Chemical Activation

Physics-Chemical Accumulation is a combination of physical and chemical activation process This activation can produce excellent activated carbon characteristics when compared to physical and chemical activation methods But there is also a weakness if using laboratory equipment which is difficult enough in can especially for rural society Substances that have been carbonized and become charcoal then in the activation of physics that can be pyrolysis using CO2 or nitrogen vapor with a certain time, after that the result of charcoal then chemically activated by soaking the charcoal

in the chemical solution we have set (Hanum, 2009)

The quality of activated charcoal is influenced by the type of raw material, hard raw materials have a high specific gravity so that it will produce high adsorption compared

to raw materials that are light and have low specific gravity According to Meisrilestari (2013) the quality of activated charcoal is influenced by:

1 Water Content (Inherent Moisture)

Inherent water content is the amount of water contained in activated charcoal after carbonated raw materials through the carbonization and activation stages of the chemical, both chemically bound and due to the influence of external conditions such as climate, grain size and screening process This determination aims to determine the hygroscopic properties of activated carbon

a Volatile Matter

Volatile Matter is a value that shows the percentage of the amount of flying substances contained in charcoal H2, CO2, CH4, and vapors that condense like CO2 and

H2O

d Adsorption

The most important active charcoal property is absorption In this case, there are several factors that affect adsorption absorption, namely:

i Absorption Properties

The amount of uptake that can be adsorbed by activated charcoal, but its ability

to adsorb is different from each compound Adsorption will increase according to increasing absorption molecules from the same structure, as in a homologous series

i Temperature

The factors that influence the temperature of the adsorption process are the viscosity and thermal stability of the absorption compound For volatile adsorption compounds, it is carried out at room temperature or if possible, at lower temperatures

ii pH (acidity degree)

For organic acids, the adsorption will increase when pH is lowered, namely by the addition of mineral acids This is due to the ability of mineral acids to reduce the

ionization of organic acids Conversely, if the pH of the organic acid is increased by adding alkali, the adsorption will decrease as a result of the formation of salt.

PART III MATERIALS AND METHODS

3.1 Place and Time

This experiment started from May 2017 until September 2017 Adsorbent adsorption experimental and toxicity testing were conducted at Aquaculture Laboratory, Faculty of Agriculture, Sriwijaya University, Inderalaya Campus Histophatological

examination was conducted at Barokah Laboratory km 3.5 Palembang

3.2 Equipments and Materials

Table 4 Equipments for Toxicity and Adsorbent Test Toxicity Testing Bioadsorbent

Testing

Histopathological Testing

• Magnetic funnels

• Gloves

Apparatus used in laboratory:

Table 5 Materials for Toxicity and Adsorbent Test Toxicity

Testing

Bioadsorbent Testing

Histopathological Testing

3.3 Methods

3.3.1 Fish Preparation

Black Nile Tilapia fish obtained from Balai Benih Ikan Indralaya, Ogan Ilir Fish were selected based on the size (6-8cm) Selected fish were the fish that swim actively, not sick, and no defects They were acclimated to laboratory conditions for 7 days prior

to adapt selected fish to the experiments (Edy, 2001)

3.3.2 Toxicity Testing

3.3.2.1 Preliminary Test LC 50 Determination

A total of 40 fish was chosen randomly for the experiment The test fish were divided into 4 chambers, each containing of 10L of water with 10 fish, 3 chambers were exposed to the test chemical and one control group was not exposed to the test chemical The concentrations used for toxicity estimation were 0, 2ml/L, 4ml/L and 6ml/L which were considered as test solutions During toxicity experiment fish were not fed LC50

values estimated using by probit analysis (SPSS)

3.3.3 Adsorbent Preparation

3.3.3.1 Carbonization

The carbonization procedure of coconut shells using single drum with controlled air supply (Hadi, 2011) is as follows:

1 Coconut shells were dried in the sun till dry and cleaned from dirt

2 A design bans with controlled air supply system were prepared

3 The holes on the controlled air were left opened, the coconut husk is used as an angler

at the first combustion

4 Insert coconut shells up to ¼ drum parts, the holes of controlled air which is located

in the centre and top of the drum were closed

5 When the fire was perfectly alive added the coconut shells slowly until the drum full

6 Drum closed and let the chimney opened

7 Observed the burned shells at the bottom through the controlled air, if it has become the embers then closed the holes after that the centre control hole was opened and so

on

8 Chimneys and control holes were slapped with clay so no air enters the drum

9 Fire will be extinguished by itself ± 1.5 hours after closure due to the absence of air

10 Open the drum cover and the temperature in the drum decreased then take out the formed charcoal

3.3.3.2 Activated Charcoal Procedure

The procedure for activating charcoal chemically (Gumelar, 2015) is as follows:

1 The charcoal smoothed with a crusher and sieved with a mesh size of 50

2 Immersed the charcoal powder in CaCl2 solution for 18 hours

3 Rinsed with distilled water to pH 7