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 i DOCUMENTATION PAGE WITH ABSTRACT Thai Nguyen University of Agriculture and Forestry Degree Program Bachelor of Environmental Science and Management Student Name Amana Amalia Student ID DTN1454290075 Thesis Title Use of Coconut Shell as Natural Adsorbent to Treat Wastewater Containing Hazardous Insecticide Compound and Its Toxicity Test on Nile Tilapia Supervisor (s) Dr.-phil Dipl.-Ing.agr Arinafril 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 i Secondary Lamellae, Epithelial Proliferation of Secondary Lamellae, Congestion and Curling Bend Keywords Coconut Shell, Histopathology, Oreochromis niloticus, Cypermethrin, Wastewater, Natural Adsorbent Number of Pages 55 Date of Submission 26/09/2018 Supervisor’s Signature ii 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: Prof Dr Ir H Anis Saggaf, MSCE as the Rector of Sriwijaya University, Palembang, Indonesia Prof Dr Ir Andy Mulyana, M.Sc as the Dean of Faculty of Aquaculture, Sriwijaya University, Palembang, Indonesia 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 Prof Mochamad Syaifudin, S.Pi., M.Si from Faculty of Aquaculture, Swirijaya University, Palembang, Indonesia iii Prof Dr Mohammad Amin, S.Pi., M.Si Faculty of Aquaculture, Sriwijaya University, Palembang, Indonesia Mrs Nurhayani as lab analyst from Laboratory of Aquaculture, Sriwijaya University who helped, assisted me and gave some advises during my research Mrs Ana and Mrs Naomi as lab analyst from Laboratory of Fisheries Product of Technology, Sriwijaya University 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 iv TABLE OF CONTENTS LIST OF FIGURES vii LIST OF TABLES viii PART I INTRODUCTION 1.1 Background and Rationale 1.2 Objectives 1.3 Research Questions and Hypotheses 1.3.1 Research Questions 1.3.2 Hypotheses 1.4 Limitations PART II LITERATURE REVIEW 2.1 Cypermethrin Compound 2.2 Toxic Effect of Cypermethrin on Organisms 2.2.1 Toxicity 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 v 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 vi LIST OF FIGURES Figure Coconut Shell 13 Figure The Condition of Aquarium after 24 hours of Adsorbent Additional Process 32 Figure Normal histological gills structure 37 Figure Histopathological alterations of gills structure of Nile Tilapia treated by using 3.8 ml/L of cypermethrin concentration without adsorbent treatment 38 Figure Histopathological alterations of gills structure of Nile Tilapia treated by using 3.1 ml/L of cypermethrin concentration with adsorbent treatment 39 Figure Histopathological alterations of gills structure of Nile Tilapia treated by using 3.4ml/L of cypermethrin concentration with adsorbent treatment 40 Figure Histopathological alterations of gills structure of Nile Tilapia treated by using 3.8ml/L of cypermethrin concentration with adsorbent treatments 41 Figure Histopathological alterations of gills structure of Nile Tilapia treated by using 4.2 ml/L of cypermethrin concentration with adsorbent treatment 42 Figure Histopathological alterations of gills structure of Nile Tilapia treated by using 4.6ml/L of cypermethrin concentration with adsorbent treatment 43 vii LIST OF TABLES Table Physical and Chemical Properties of Cypermethrin Table Toxicity Classification of Cypermethrin Table Characterization of Activated Carbon (SII 0258-88) 15 Table Equipments for Toxicity and Adsorbent Test 21 Table Materials for Toxicity and Adsorbent Test 22 Table Effect of Cypermethrin Concentration on Nile Tilapia in Preliminary Test 29 Table Effect of Cypermethrin on Nile Tilapia in Five Repetitions using Adsorbent Treatments 32 Table Observation of Cypermethrin Effect on Nile Tilapia 35 viii 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 plantprotection 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) in sheep, cats, dogs, and other animals They are structurally similar to pyrethrins, a class of compounds that are found in chrysanthemum plants, where they work as natural A F B EP F F CB C Figure Histopathological alterations of gills structure of Nile Tilapia treated by using 3.8ml/L of cypermethrin concentration with adsorbent treatments The alterations were as followed A) fusion of secondary lamellae (F), B) epithelial proliferation of secondary lamellae (EP), C) curling bend (CB) Stained with H&E Magnification 200x As the features (fig 7) shown, in 3.8 ml/L of cypermethrin concentration with adsorbent treatment showing several damages comprise of A showing fusion of secondary lamellae occurred due to epithelial hyperplasia of the gill caused by cells in the secondary lamella multiplying which makes the secondary lamella become larger or become wider than its proper size In B showing epithelial proliferation of secondary lamellae occurred due to thickened of epidermis cell and secondary lamella combined 41 with one another itself while in C showing curling bend due to shrinkage of secondary lamellae EP A B C Figure Histopathological alterations of gills structure of Nile Tilapia treated by using 4.2 ml/L of cypermethrin concentration with adsorbent treatment The alterations were as followed A) epithelial proliferation of secondary lamellae (EP), B) congestion (C) Stained with H&E magnification 400x As the features (fig 8) shown, in this 4.2 ml/L of cypermethrin concentration with adsorbent treatment in A Nile Tilapia showing epithelial proliferation of secondary lamellae occurred due to thickened of epidermis cell and secondary lamella combined with one another itself and In B Nile Tilapia showing congestion occurred due to the dilation in blood vessel 42 A B TS C C EP Figure Histopathological alterations of gills structure of Nile Tilapia treated by using 4.6ml/L of cypermethrin concentration with adsorbent treatment The alterations were as followed A) congestion (C), B) telengeactacia (TS), C) epithelial proliferation of secondary lamellae (EP) Stained with H&E Magnification 400x Increase the concentration will also increase the degree of damage As the features (fig 9) shown, 4.6 ml/L of cypermethrin concentration with adsorbent treatment A showing congestion occurred due to the dilation in blood vessel while in B showing telengeactacia where there is a dilation in distal portion in secondary lamellae, and in C 43 there is an epithelial proliferation of secondary lamellae occurred due to thickened of epidermis cell and secondary lamella combined with one another itself 4.2 Discussions The use of insecticide has been rampant among farmers, although most of these insecticides are dangerous such as the example of one of the pyrethroid insecticide type, cypermethrin Improper use of insecticides has a negative impact on the environment Its accumulation in soil, water and agricultural products that exceed the maximum level of residual limit will reduce the quality of the environment and human health Moreover, decreasing in the quality of the environment in water could possibly have a direct impact on the ecosystem in it, for example fish As known if fish gill is an important organ for breathing, osmoregulation and also it is directly related to the external environment (Zulfahmi, Affandi and Lumban Batu, 2015) There are many ways scientists to reduce residues from insecticides, one of them is by using activated carbon from coconut shells In this study, activated carbon derived from coconut shells is a type of activated carbon that has good adsorption which is appropriate with the quality standards according to SII No 02258-88, where it contains 3.62% of water content and 8.7% of ash content (Appendix 1) Alterations occur in fish from the preliminary test without using adsorbent and test with adsorbent treatments proved that the fish using adsorbent treatments lasted longer The exposure of cypermethrin concentrations towards fish were more liable to die quickly, the total of fish mortality time was about four and a half hours compared to fish exposed to cypermethrin concentration with adsorbent lasted for approximately up to 11 hours 44 The alterations in motoric behaviour of Nile Tilapia with the exposure concentration of cypermethrin of 3.8ml/l without adsorbent treatment occur to swim irregularly, fish begin to falter, fish begin to weaken and swim in the bottom 3.1 ml/l with adsorbent treatment, changes occur when fish started to swim irregularly, fish begin to adapt and swim in the bottom At a concentration of 3.4 ml/l with adsorbent treatment the behaviour changes that occur to swim irregularly, restlessness and fish emit dirt and slow movements At a concentration of 3.8 ml/l with the adsorbent treatment the fish swim in the bottom, the movement of the fish slows down; gasping for breath due to difficulty in breathing, fish swim sideways, and fish emit dirt At a concentration of 4.2 ml/l with adsorbent treatment, fish swim in the bottom, hyperactive movement, weak and faint At a concentration of 4.6 ml /l with adsorbent treatment, there was a change in motor behaviour of tilapia, such as swim in the bottom, hyperactive fish movement, limp fish, and unconscious fish At each concentrations of motoric behaviour changes in tilapia experience differences from each concentration Clinically, animals that are contaminated with the insecticide will show symptoms of stress that are characterized by the behaviour of fish that are starting to be less stable and tend to be at the bottom This is appropriate with Thiery et al (2009), fish exposed to insecticidal toxins can be known by hyperactive movements, erratic tides, fish float with stomach above surface, flutter, faint, and body colour looks darker, appetite decreases and dies Behavioural changes that have been observed in this study are appropriate with Aliza (2014), fish that experience a lack of oxygen will accelerate movement in their body accompanied by taking air above the water Low oxygen in water can have an impact on the consumption of oxygen in fish 45 The use of pesticides can be a potential source of pollutants for resources and the aquatic environment The active ingredients of pesticides at high concentrations that enter the waters can directly kill fish and other aquatic biotas At low concentration even though it does not directly cause death in fish but it can accumulate in the body due to water, sediment and other aquatic biota which can be as a pesticide residue media and accumulator Fish as an aquatic organism, are very sensitive of pollution and against the use of pesticides Insecticides can pollute fish caused by the entry of active ingredients of pesticides into the water adsorbed by the body, and can be carried along with food Gills are organs that work by the mechanism of surface diffusion of oxygenrepelling gases and carbon dioxide in blood and water Oxygen dissolved in water is adsorbed into fish gill capillaries and will be fixed by haemoglobin which will then be distributed throughout the body Whereas carbon dioxide is removed from the cell and the tissue released into the water around the gill organs (Ojutiku et al., 2014) It is clearly can be seen from histopathological result of gill in fig & fig found that gills of Nile Tilapia showing fusion of secondary lamellae Fusion of secondary lamellae could affect the respiration system in fish gills, it is suspected occur because of the injury of secondary lamellae to force an organ to remove a lot of mucus to cover the wound so that there is an attachment occurs between secondary lamellae with each other In addition, fusion occurs because of lamellae experience swelling or hyperplasia so that the respiratory process is disrupted This resulted in the size of the cavity (lumen capillaries) narrowed and the cells in the middle of the secondary lamellae shifted to the other of secondary lamellae which resulted as an attachment (Rizki, S.M and Damanik, 2015) 46 Congestion occurs in the treatment of fig 9, and suspected occur by the entry of toxic substances (insecticides) into the gills This is appropriate with the research from Lujić, Marinović and Miljanović (2013), congestion will result in damming of blood due to disturbances in circulation which can lead to a lack of oxygen and nutrients The occurrence of congestion is preceded by the occurrence of cell swelling Cell swelling is an increase in cell size due to accumulation of water in the cell Epithelial proliferation of secondary lamellae in some treatments (fig 6, fig 7, fig & fig 9) excessive epithelial proliferation of secondary lamellae is suspected cause cell division (especially in cells capable of splitting rapidly) becomes uncontrolled resulting in hyperplasia This proliferation occurs in each treatment exposed to cypermethrin insecticides This is appropriate with the research of Aliza (2014) that cell proliferation occurs in changes in gill fish which causes cell division (especially in cells that are able to divide rapidly) into uncontrolled Rupture or broken of secondary lamellae in gills (fig & 4) is a condition where it loses its cell pillar (epithelial) which serves to maintain stability of secondary lamellae Damage to the structure of the gill tissue can cause the fish to have difficulty in breathing and cause the contents in the blood are decreasing which make haemoglobin difficult and turns out bind the oxygen So that the fish will experience a lack of oxygen and will experience hypoxia this is due to the crushing of the secondary lamellae in the oedema (Ojutiku et al., 2014) Cypermethrin insecticides that have been accumulated in the body of fish through the respiratory organs (gills) cause damage to the gills which could disrupt the respiratory process This can be seen from the test animals that make a hovering motion on the surface of the water before experiencing death 47 PART V CONCLUSION Based on the results of the research it can be concluded if cypermethrin may pose a potential risk to fish As histopathology examination shown, exposure of cypermethrin concentration leads to many deaths of fish where the alterations and damages of gills in 3.8ml/L cypermethrin concentration without adsorbent treatment have more severe damages compared to some concentrations with adsorbent treatment, only few damages were found Also, it has been proved if fish could adapt and survive last longer against the exposure of cypermethrin concentration using adsorbent treatment Throughout the examination, the following damages found were telengeactacia, rupture of secondary lamellae, congestion, curling bend and epithelial proliferation of secondary lamellae In addition, results from adsorbent experiment show that coconut shells as natural adsorbents are able to adsorb insecticide contamination and reduce insecticide content in the water which can reduce the risk of death of the fish or may prolong the life of the fish Also, using coconut shells as natural adsorbent can be used as one effective way to treat polluted water considering the materials used are easily to be found 48 REFERENCES Ahmedna, M., Marshall, W E and Rao, R M (2000) Production of Granular Activated Carbons from Select Agricultural by-Products and Evaluation of Their Physical, Chemical and Adsorption Properties Biosource Technology (71) : 113-123 Arkeblom, N (2004) Agricultural Pesticide Toxicity to Aquatic Organisms- A Literature Review Department of Environmental Assessment Swedish University of 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Turkish Journal of Fisheries and Aquatic Sciences 85–89 Satyanarayan, S., Satyanarayan J P K.A and Verma, S 2012 Histopathological Changes due to Some Chlorinated Hydrocarbon Pesticides in The Tissues to Cyprinus carpio IOSR Journal of Pharmacy (6) : 60–66 51 Shelke, A.S., K R Ninghot, P P Kunjekar and S P Gaikwad 2014 Coconut Shell as Partial Replacement for Coarse Aggregate: Review International Journal of Civil Engineering Research (5): 211-214 Thiery, J P., Acloque, H., Huang, R Y J and Nietto, M A 2009 EphitelialMesenchymal Transitions in Development and Disease (139): 871-890 Zulfahmi, I., Affandi, R and Lumban Batu, D T F 2015 Perubahan Struktur Histologis Insang dan Hati Ikan Nila (Oreochromis niloticus Linnaeus 1758) yang Terpapar Merkuri IV (1) : 25–31 52 APPENDICES Appendix Result of Probit Analysis (SPSS) Confidence Limits Probabilit y a PROBIT 95% Confidence Limits for concentration Estimate Lower Bound Upper Bound 0.01 -.858 0.02 -.309 0.03 039 0.04 301 0.05 514 0.06 695 0.07 854 0.08 997 0.09 1.126 0.1 1.245 0.15 1.739 0.2 2.131 0.25 2.467 0.3 2.769 0.35 3.049 0.4 3.315 0.45 3.572 0.5 3.825 0.55 4.078 0.6 4.335 0.65 4.600 0.7 4.880 0.75 5.182 0.8 5.519 0.85 5.911 0.9 6.404 0.91 6.524 0.92 6.653 0.93 6.795 0.94 6.954 0.95 7.136 0.96 7.349 0.97 7.610 0.98 7.959 0.99 8.507 a A heterogeneity factor is used 53 Appendix Result of Water Content and Ash Content Content Water Content Ash Content Finale Mass Mass of Mass of Cup+Sample Cup+Sample Cup (g) Sample (g) Before (g) After (g) 5.24 7.24 7.17 1.93 3.62 34.64 36.64 36.48 1.84 8.7 of Sample Percentage (%) (g) 54 Appendix The Activity During Research Pyrolisis Process of Coconut Shell Grind Process of Coconut Shell Observation of Gill Tissues of Nile Sample Gill Tissue of Nile Tilapia Tilapia 55 ... ID DTN1454290075 Thesis Title Use of Coconut Shell as Natural Adsorbent to Treat Wastewater Containing Hazardous Insecticide Compound and Its Toxicity Test on Nile Tilapia Supervisor (s) Dr.-phil... 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. .. 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