THAI NGUYEN UNIVERSITY UNIVERSITY OF AGRICULTURE AND FORESTRY DUONG THI HONG NGOC TOXICOLOGICAL EFFECT AND HISTOPATHOLOGICAL CHANGES IN GILLS AND LIVER OF TILAPIA EXPOSED TO NICLOSAMIDE BACHELOR THESIS Study Mode : Full-Time Major : Environmental Science and Management Faculty : International Training and Development Center Batch : 2012 – 2016 Thai Nguyen, 20/06/2016 DOCUMENTATION PAGE WITH ABSTRACT Thai Nguyen University Of Agriculture And Forestry Degree Program: Bachelor of Environmental Science and Management Student name: Duong Thi Hong Ngoc Student ID: DTN 1253110106 TOXICOLOGICAL EFFECT AND HISTOPATHOLOGICAL CHANGES IN GILLS AND Thesis Title: LIVER OF TILAPIA (OREOCHROMIS NILOTICUS) EXPOSED TO NICLOSAMIDE Dr Arinafril Naalim Supervisor (s): Krisna Murti, MD., M Biotech Stud., Ph.D Dr Duong Van Thao Abstract: Niclosamide commonly used as a pesticide and is able to contaminate the aquatic ecosystem as a toxic pollutant from agricultural and domestic washouts The aim of this study was to investigate the toxic effect of niclosamide on gills and liver tissues of the tilapia fish Oreochromis Niloticus exposed to sublethal concentration of 0.035 ppm and 0.05 ppm This experiment was performed in period of three months from March 2016 to May 2016 at the Laboratory of Aquaculture and Pesticide Toxicology of the Integrated Research Laboratory of Sriwijaya University Graduate School and the Department of Anatomic Pathology, Faculty of Medicine, University of Sriwijaya/Dr Mohammad Hoesin General Hospital Palembang The most common changes at all doses of niclosamide in solution were destruction of gill lamellas While, hepatic lesions were characterized by blood congestion in central vein Histological comparison of tissues biopsy indicated that most damage occurred in the gills rather than in the liver The severity of damages on gills and liver of the fish is proportional to the concentration of the pesticides Key-words: Oreochromis Niloticus, Tilapia, Niclosamide, Toxicity, Gills, Liver, Histopathology Number of pages: 40 Date of submission: 20/09/2016 Supervisor’s signature ACKNOWLEDGEMENT I would like to express my deepest appreciation to all those who provided me the opportunity to complete this research Foremost, I would like to express my sincere gratitude and deep regards to my supervisor: Dr.-phil Arinafril of Sriwijaya University, Indralaya, Indonesia, who guided me wholeheartedly when I implemented this research My special thanks go to Krisna Murti, Md., M Biotech Stud., Ph.D., second supervisor - who offered me a warm welcome, assisted me with the histopathological detection in this dissertation; she was very patient with my knowledge gaps and gave me the opportunity to use the research facilities in her department - Department of Anatomic Pathology, Faculty of Medicine, University of Sriwijaya/Dr Mohammad Hoesin General Hospital Palembang Besides my supervisors, I would like to thank Dr Duong Van Thao, Adviser, for his supervision, encouragement, advice, and guidance in writing this thesis In addition, formal thanks should be offered to the Rector of Sriwijaya University, Prof Dr Badia Perizade, MBA., for granting my internship acceptance I also want to express my thanks to the Dean of Faculty of Medicine in Sriwijaya University, Dr dr Mohammad Zulkarnain, M Med.Sc, PKK., and Director of Dr Mohammad Hoesin General Hospital Palembang, Dr Mohammad Syahril, Sp.P., MPH., who gave the permission to use all required equipment and the necessary materials to conduct my research in Laboratory of Department of Anatomic Pathology, Faculty of Medicine, University of Sriwijaya/Dr Mohammad Hoesin General Hospital Palembang I gratefully acknowledge Ms Mirna Fitrani, Mbak Ana Nyayu and Mr Mohammad Zainuri, Laboratory of Aquaculture, Sriwijaya University for helping and providing me necessary equipment as well as knowledge for fish anatomy and how to dissected the fish I wish to thank the technicians of Department of Anatomic Pathology, Faculty of Medicine, University of Sriwijaya/Dr Mohammad Hoesin General Hospital Palembang for their help in tissue preparation My sincere thanks also go to all my classmates – k44 AEP for helping me finish the study Special thanks to Ate Jelly, Van, Dung, Phong, Keraia, Ye, Indonesian friends and all the people who helped me when I stayed in Palembang – Indonesia Finally, I would like to thank my family, for their love and supporting me throughout my life Palembang, April 2016 Student Duong Thi Hong Ngoc TABLE OF CONTENTS LIST OF FIGURES LIST OF TABLES PART I INTRODUCTION 1.1 Background and rationale 1.2 Research’s Objectives 1.3 Research questions and hypotheses 1.4 Limitations PART II LITERATURE REVIEW 2.1 Niclosamide 2.2 Toxic effects of Niclosamide on organisms 2.2.1 Toxicity 2.2.2 Histological Effects 12 2.3 Test Species 14 PART III.MATERIALS AND METHODS 15 3.1 Time and Place 15 3.2 Materials 15 3.3 Equipment 16 3.4 Methods 17 3.4.1 Toxicity test 17 3.4.2 Histopathological examination 17 PART IV.RESULTS 21 4.1 Behavioral changes 21 4.2 Histopathological changes 21 4.2.1 Gills 21 4.2.2 Liver 23 PART V DISCUSSION AND CONCLUSION 26 5.1 Discussion 26 5.2 Conclusion 28 BIBLIOGRAPHY .29 LIST OF FIGURES Figure 3.2: Stock solution of Niclosamide - bayluscide 15 Figure 4.2.1: Histopathological changes observed in the gills 22 Figure 4.2.2a: Histopathological alteration of liver in central vein after treated fish with niclosamide 24 Figure 4.2.2b Histopathological alteration of liver in Portal Vein after treated fish with niclosamide 25 LIST OF TABLES Table 2.1: Physical and Chemical Properties of Niclosamide Table 2.2.1: Toxicology profile of the niclosamide 10 Table 3.3: Listed of Equipment used for this study 16 PART I 1.1 INTRODUCTION Background and rationale Molluscicides are toxic to non-target animals and cause environmental pollution (Wang R & Chen C., 2003; Huang S.S & Zhu H.G., et al., 2014) Some plant molluscicides low toxicity or (and) high for the fish or other animals is not its goal (Wei F.H & Xu X.J., et al., 2002) In the reports of recent years, some moluscicides supposedly are less harmful and effectiveness, for example niclosamide, a potential molluscicide derived from the plant Solanum xanthocarpum, a tropical plant species (Dai J.R & Wang W., et al, 2008) To reduce the harmful effect of plant molluscicides, niclosamide - a new developing molluscicide have been developed lately with desirable and natural molluscicide content Hence, niclosamide becomes more popular due to its nature origin, moreover it is showed less toxic to environment (Rapado L.N., de Sá Pinheiro A., et al., 2013) Since 1960s, niclosamide has been recommending by the WHO (World Health Organization) for usage as a molluscicide and it is still molluscicide of choice up to now (WHO, 1992) It is commercially available as a 50% wettable powder and its content is widely known as niclosamide ethanolamine salt (WPN) (Yang G.J & Li W., et al., 2010) However, WPN is expensive and highly toxic to fish and other aquatic animal, therefore, some economically poor areas of crabs or fish farming are not used it (Fang Y.M & Huang Y.X, 2007; Zhu M.D & Hong L.D., et al., 2005) Niclosamide is a relatively selective molluscicide because it is mainly used to fight the freshwater snails - intermediate hosts of schistosomiasis and fascioliasis It is highly toxic to aquatic vertebrates but less effective for the mammals e.g fish, amphibians and crustaceans (Oliveira-Filho and Paumgartten, 2000), this agent also has a slight effect on aquatic plants and zooplankton (WHO, 2007) Molluscicide niclosamide or Bayluscide has been reported to be effective with all development stages of snails and schistosomiasis (Tchounwou, et al., 1991 & Lowe, et al., 2005) Moreover, niclosamide is sometimes used as a lampricide (Nettles, et al., 2001; WHO, 2003) However, in conditions of certain water quality, dose of Bayluscide that used to kill sea lampreys can also kill rainbow trout (Nettles, et al., 2001) Niclosamide has been used with TFM (a lampricide) to supplement this product and increase its efficacy as a lampricide It kills a wide variety of snails, cestodes and cercariae by affecting the respiration and the carbohydrate metabolism (Nettles, et al., 2001) Worldwide, however, niclosamide is used primarily as a molluscicide (WHO, 2003) and is recommended by WHO for control of schistosome-bearing snails’ (WHO, 2003), because of its high toxicity to aquatic snails (e.g Helisoma trivolvis and Biomphalaria havanensis) (Tchounwou, et al., 1991) The present study is aimed to investigate the effects of niclosamide on the gills and liver of the tilapia - Oreochromis Niloticus Histopathological changes in the organs that are directly exposed to the pesticide such as the gills were taken as a parameter to assess the impact or toxic effect of niclosamide In Eosin – dipped The next step is dipped them on water for times Next, the slides were dipped times in 70%, 80% alcohol for time each and times in alcohol 96% or until excess eosin is removed Afterward, they were washed in running tap water for minutes and dipped in xylene times Finally, the slides after clearing with xylene were mounted Mounting After clearing with xylene, the slides were mounted in Dibutyl Phthalate Xylene (DPX - Adhesives used for fixing the sections on the slides) medium The tissues were then mounted with clean thin glass slides (cover slides) to protect section Light Microscopy Upon examination the slides under microscope, we initially analyze the morphology of gills and liver by observing organs of the control, followed by organs of the treated fish The alteration on the treated organs were compared to the controls 20 PART IV RESULTS 4.1 Behavioral changes Upon treating with lower concentrations of Niclosamide, at the beginning, fishes showed disturbance of swimming movements, rapid opercular movements and surfacing behavior indicative of avoidance response Several abnormal behavior of fishes before they died such as incessant jumping and gulping of air, restlessness, loss of equilibrium, increase opercula activities, surface to bottom movement, sudden quick movement, resting at the bottom, and then all of them died after four minutes Exposure to higher concentrations of niclosamide, fish behaviors mostly similar with the one exposed to lower concentrations But the fish movement became faster and they tried to swallow air by jump out Treating with higher concentrations of niclosamide caused the death of all fishes in only three minutes The rapid mortality, may be due to an acute toxic effect 4.2 Histopathological changes 4.2.1 Gills The normal structures of gills of Oreochromis Niloticus in the control fishes consist of primary lamellae projecting on the lateral sides of the primary and secondary lamellae (respiratory lamellae) The degree of alteration was varied upon the different concentrations of given niclosamide and these alterations can be seen in figure 4.2.1-A 21 MC SL Hr EC In PL A B Dis Des In Cb C D Figure 4.2.1: Histopathological changes observed in the gills (A) Normal gill tissues in control fish Gills structure of the control fish showing primary lamellae (PL), secondary lamellae (SL), epithelial cell (EC), and mucous cell (MC) (B) Gills tissues upon treatment with 0.035 mg/L of Niclosamide showing inflammation (In) and haemorrhage (Hr); (C) and (D) showing the alteration that observed after treatment with 0.05 mg/L of Niclosamide consist of desquamation (Des), inflammation (In), disintegration (Dis) and curling bend (Cb) (All pictutes above were stained with H&E, 40x magnification) The initial alterations in the gills were manifested in groups exposed to 0.035 mg/l, the higher concentration of niclosamide (0.05 mg/l) the more pronounce of alterations The fish gills treated with 0.035 mg/l of niclosamide showed inflammation (In) in both primary and secondary lamellae Haemorrhage 22 (Hr) in primary lamellae was recorded (figure 4.2.1-B) While in fishes exposed to 0.05 mg/l, this was more evident in higher concentration of the pesticide that showed disintegration (Dis) on the tip of primary lamellae with slightly curling bend (Cb) (figure 4.2.1-C) and desquamation (Des) in secondary lamellae, inflammation (In) in primary lamellae (figure 4.2.1-D) The gills exhibit a marked alteration in their epithelia 4.2.2 Liver The microscopic observations of histological sections of liver from control fishes in the experiment showed the typical structure of hepatic tissues The hepatic cells appeared as a continuous mass and distinctly shaped as round or polygonal, containing clear spherical nucleus They were located among sinusoids forming cord like structure known as hepatic cell cords A large number of blood sinusoids were found in the hepatic mass of these cords (figure 4.2.2a-A) Pancreatic tissue was present in association with venous vessels (Figure 4.2.2b-A) Examination of livers sections after exposed to 0.035 mg/l niclosamide showed blood congestion (figure 4.2.2a-B and 4.2.2b-B) The histopathological changes found in the liver with higher concentration of niclosamide (0.05 mg/l) of the examined fishes included blood congestion in blood vessel (figures 4.2.2aC and 4.2.2b-C) with patchy degeneration located around the pancreatic cells due to tissues damage These changes were not found in tissues of fishes exposed to lower concentrations (0.035 mg/l) of niclosamide (Figure 4.2.2b-C) 23 S H CV A Bc Bc B C Figure 4.2.2a: Histopathological alteration of liver in central vein after fishes treated with niclosamide (A) The tissues of liver in control fishes with normal hepatocytes (H), radiating away from central vein (CV), sinusoids (S) (100 x magnification) The changes in the livers treated with 0.035 mg/L and 0.05 mg/L of Niclosamide in (B) (400 x magnification) and (C) showing blood congestion (Bc) (200 x magnification) (All the above pictures were stained with H&E) 24 P A P P Bc Bc B Pd C Figure 4.2.2b Histopathological alteration of livers in Portal Vein after fishes treated with niclosamide (A) Normal livers in Portal Vein of Nile tilapia showing pancreas (P) (B) The changes of fishes liver treated with 0.035 mg/L Niclosamide showing blood congestion (Bc) After exposed with 0.05 mg/L of Niclosamide, Blood congestion (Bc) and patchy degeneration (pd) found in the livers tissue (C) (All the above pictures were stained with H&E, 200 x magnification) 25 PART V DISCUSSION AND CONCLUSION 5.1 Discussion The histological changes on fish was a noteworthy and promising field to understand the extent to which alteration in the structural organization were occurre in the organs of fish due to environmental pollution Fish was sensitive to environmental impacts, such as pollution, acid-base, and ionic change The histological lesions in the fish gills found in the present study might arise also because of the stress that the fish were subjected to within a short period of time Depending on the toxic potential of a particular toxin accumulates in the tissues to determine the extent of damage Hence, when exposed to water contaminated various organs in the fishes can be adversely affected and may lead to toxic effects on organs like the livers and gills (Parvathi A & Sivakumari P., et al., 2011) Different kind of toxicity effects were noticed in various fishes exposed to different pollutants (Olojo E & Olurin K., et al., 2005) Fish gills were multifunctional organs involved in ion transport, gas exchange, acid-base regulation and waste excretion (Wong, 2000; Thophon, et al., 2003; Zayed & Mohamed, 2004) The gills were particularly sensitive to changes in the water, which were depended on quality of the water Hence, gills were considered as the primary target of contaminants (Fernandes & Mazon, 2003; Camargo & Martinez, 2007) The gills of fishes exposed to niclosamide showed the loss of their architecture with inflammation of primary lamellae and desquamation of secondary lamellae at different concentrations of niclosamide 26 (Figure 4.2.1-B and 4.2.1D) Upon their position, gills were in direct contact with pollutants, thus, any kind of damage to the gills tissues of fishes might lead to disorder because of gas exchange process as well as the decrease of ion regulation efficiency via these organs (Fernandes & Mazon, 2003) Histopathology of gills was the important bio-indicator for monitoring pollution (Reethamma, 2014) The treated gills of fishes showed the damage to varying extent depending on the exposure concentrations As the exposure concentrations increased the degree of damage also increased Liver as the main organ of metabolism came into contact with xenobiotics absorbed from the aquatic environment and liver lesions were often associated with exposure to aquatic pollutants (Fernandes C & Fontainhas-Fernandes A., at al., 2008) One of the most important functions of liver was to clean pollutants from the blood coming from the intestine, so it was considered as an indicator of aquatic environmental pollution It was most likely because fishes dead so fast within 3-4 minutes, hence, we cannot find many changes in the livers The livers of the exposed fishes compared to the control showed varying degree of degeneration of cells, which included blood congestion and patchy degeneration elements around the pancreatic cells (figure 4.2.2b-C) and these alterations were dose-dependent Severe congestion in sinusoids and small blood vessels made the blood flow from the hepatic portal vein and hepatic artery into the central vein rather difficult Observe abnormal livers reflect the impacts of pollutants Besides this, oxygen deficiency was the most common cause of the cellular degeneration in the liver (Olojo, et al., 2005; 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Hypothesis): Exposure to niclosamide will result in changes in gills histology of Oreochromis Niloticus Hypothesis 2: HO: Exposure to niclosamide will not result in changes in liver histology of Oreochromis... 4.2.2b Histopathological alteration of livers in Portal Vein after fishes treated with niclosamide (A) Normal livers in Portal Vein of Nile tilapia showing pancreas (P) (B) The changes of fishes liver. .. degrees of histopathological alterations of different organs of fish Therefore, morphologic studies including histopathological analysis could be used as bio-monitoring tools or indicators of health