(Luận văn) toxicological effect and histopathological changes in gills and liver of tilapia (oreochromis niloticus) exposed to niclosamide

THAI NGUYEN UNIVERSITY UNIVERSITY OF AGRICULTURE AND FORESTRY lu DUONG THI HONG NGOC an va n TOXICOLOGICAL EFFECT AND HISTOPATHOLOGICAL CHANGES IN GILLS AND LIVER OF TILAPIA EXPOSED TO NICLOSAMIDE p ie gh tn to d oa nl w BACHELOR THESIS : Environmental Science and Management nf va Major an lu Study Mode : Full-Time : International Training and Development Center Batch : 2012 – 2016 oi lm ul Faculty z at nh z m co l gm @ an Lu n va Thai Nguyen, 20/06/2016 ac th si 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 lu an Thesis Title: LIVER OF TILAPIA (OREOCHROMIS NILOTICUS) va n EXPOSED TO NICLOSAMIDE tn to Dr Arinafril Naalim gh Krisna Murti, MD., M Biotech Stud., Ph.D p ie Supervisor (s): Dr Duong Van Thao nl w d oa Abstract: Niclosamide commonly used as a pesticide and is able to contaminate the an lu aquatic ecosystem as a toxic pollutant from agricultural and domestic washouts nf va The aim of this study was to investigate the toxic effect of niclosamide on gills and oi lm ul 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 z at nh period of three months from March 2016 to May 2016 at the Laboratory of z Aquaculture and Pesticide Toxicology of the Integrated Research Laboratory of @ l gm Sriwijaya University Graduate School and the Department of Anatomic Pathology, m co Faculty of Medicine, University of Sriwijaya/Dr Mohammad Hoesin General Hospital Palembang The most common changes at all doses of niclosamide in an Lu solution were destruction of gill lamellas While, hepatic lesions were characterized n va ac th si 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 lu an Date of submission: 20/09/2016 n va ie gh tn to Supervisor’s p signature d oa nl w oi lm ul nf va an lu z at nh z m co l gm @ an Lu n va ac th si 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 lu an My special thanks go to Krisna Murti, Md., M Biotech Stud., Ph.D., va second supervisor - who offered me a warm welcome, assisted me with the n gh tn to 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 p ie w department - Department of Anatomic Pathology, Faculty of Medicine, University d oa nl of Sriwijaya/Dr Mohammad Hoesin General Hospital Palembang an lu Besides my supervisors, I would like to thank Dr Duong Van Thao, nf va Adviser, for his supervision, encouragement, advice, and guidance in writing this oi lm ul thesis z at nh In addition, formal thanks should be offered to the Rector of Sriwijaya University, Prof Dr Badia Perizade, MBA., for granting my internship z gm @ acceptance m co l 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 an Lu Director of Dr Mohammad Hoesin General Hospital Palembang, Dr Mohammad n va Syahril, Sp.P., MPH., who gave the permission to use all required equipment and ac th si 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 lu how to dissected the fish an va I wish to thank the technicians of Department of Anatomic Pathology, n gh tn to Faculty of Medicine, University of Sriwijaya/Dr Mohammad Hoesin General p ie Hospital Palembang for their help in tissue preparation My sincere thanks also go to all my classmates – k44 AEP for helping me nl w d oa finish the study lu va an Special thanks to Ate Jelly, Van, Dung, Phong, Keraia, Ye, Indonesian oi lm ul nf 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 z at nh throughout my life z Palembang, April 2016 gm @ Student m co l an Lu Duong Thi Hong Ngoc n va ac th si 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 lu an Niclosamide 2.2 Toxic effects of Niclosamide on organisms n va 2.1 Toxicity 2.2.2 Histological Effects 12 2.3 Test Species 14 ie gh tn to 2.2.1 p PART III.MATERIALS AND METHODS 15 3.2 Materials 15 oa nl w Time and Place 15 3.1 Equipment 16 3.4 Methods 17 d 3.3 an lu Toxicity test 17 3.4.2 Histopathological examination 17 oi lm ul nf va 3.4.1 PART IV.RESULTS 21 Behavioral changes 21 4.2 Histopathological changes 21 z at nh 4.1 Gills 21 4.2.2 Liver 23 z 4.2.1 gm @ PART V DISCUSSION AND CONCLUSION 26 l Discussion 26 5.2 Conclusion 28 m co 5.1 an Lu BIBLIOGRAPHY .29 n va ac th si 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 lu an n va p ie gh tn to d oa nl w oi lm ul nf va an lu z at nh z m co l gm @ an Lu n va ac th si 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 lu an n va p ie gh tn to d oa nl w oi lm ul nf va an lu z at nh z m co l gm @ an Lu n va ac th si 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, lu an some moluscicides supposedly are less harmful and effectiveness, for example va n niclosamide, a potential molluscicide derived from the plant Solanum p ie gh tn to xanthocarpum, a tropical plant species (Dai J.R & Wang W., et al, 2008) To reduce the harmful effect of plant molluscicides, niclosamide - a new oa nl w developing molluscicide have been developed lately with desirable and natural molluscicide content Hence, niclosamide becomes more popular due to its d lu va an nature origin, moreover it is showed less toxic to environment (Rapado L.N., de Sá Pinheiro A., et al., 2013) oi lm ul nf Since 1960s, niclosamide has been recommending by the WHO (World z at nh 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 z gm @ powder and its content is widely known as niclosamide ethanolamine salt m co l (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 an Lu areas of crabs or fish farming are not used it (Fang Y.M & Huang Y.X, 2007; n va Zhu M.D & Hong L.D., et al., 2005) ac th si 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 lu an (Tchounwou, et al., 1991 & Lowe, et al., 2005) n va 2001; WHO, 2003) However, in conditions of certain water quality, dose of ie gh tn to Moreover, niclosamide is sometimes used as a lampricide (Nettles, et al., p Bayluscide that used to kill sea lampreys can also kill rainbow trout (Nettles, et oa nl w 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 d lu va an snails, cestodes and cercariae by affecting the respiration and the carbohydrate ul nf metabolism (Nettles, et al., 2001) oi lm Worldwide, however, niclosamide is used primarily as a molluscicide z at nh (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 z gm @ trivolvis and Biomphalaria havanensis) (Tchounwou, et al., 1991) The present study is aimed to investigate the effects of niclosamide on the l m co gills and liver of the tilapia - Oreochromis Niloticus Histopathological changes an Lu 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 n va ac th si 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 lu an After clearing with xylene, the slides were mounted in Dibutyl Phthalate va Xylene (DPX - Adhesives used for fixing the sections on the slides) medium n gh tn to The tissues were then mounted with clean thin glass slides (cover slides) to p ie protect section Light Microscopy oa nl w Upon examination the slides under microscope, we initially analyze the d lu va an morphology of gills and liver by observing organs of the control, followed by oi lm the controls ul nf organs of the treated fish The alteration on the treated organs were compared to z at nh z m co l gm @ an Lu n va ac th 20 si 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 lu an gulping of air, restlessness, loss of equilibrium, increase opercula activities, va n surface to bottom movement, sudden quick movement, resting at the bottom, and p ie gh tn to then all of them died after four minutes Exposure to higher concentrations of niclosamide, fish behaviors mostly oa nl w 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 d lu va an concentrations of niclosamide caused the death of all fishes in only three minutes The rapid mortality, may be due to an acute toxic effect oi lm ul nf 4.2 Histopathological changes z at nh 4.2.1 Gills z gm @ The normal structures of gills of Oreochromis Niloticus in the control fishes consist of primary lamellae projecting on the lateral sides of the primary l m co and secondary lamellae (respiratory lamellae) The degree of alteration was alterations can be seen in figure 4.2.1-A an Lu varied upon the different concentrations of given niclosamide and these n va ac th 21 si MC SL Hr EC In PL lu A B an n va to ie gh tn Dis p Des w In nl D d oa C Cb lu va an 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 nf oi lm ul 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 z at nh 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 z (Des), inflammation (In), disintegration (Dis) and curling bend (Cb) (All pictutes @ above were stained with H&E, 40x magnification) gm The initial alterations in the gills were manifested in groups exposed to l m co 0.035 mg/l, the higher concentration of niclosamide (0.05 mg/l) the more an Lu pronounce of alterations The fish gills treated with 0.035 mg/l of niclosamide showed inflammation (In) in both primary and secondary lamellae Haemorrhage n va ac th 22 si (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 lu 4.2.2 Liver an n va The microscopic observations of histological sections of liver from The hepatic cells appeared as a continuous mass and distinctly shaped as round ie gh tn to control fishes in the experiment showed the typical structure of hepatic tissues p or polygonal, containing clear spherical nucleus They were located among oa nl w 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 d lu oi lm ul nf (Figure 4.2.2b-A) va an 4.2.2a-A) Pancreatic tissue was present in association with venous vessels Examination of livers sections after exposed to 0.035 mg/l niclosamide z at nh 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) z @ of the examined fishes included blood congestion in blood vessel (figures 4.2.2a- gm m co l C 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 an Lu exposed to lower concentrations (0.035 mg/l) of niclosamide (Figure 4.2.2b-C) n va ac th 23 si S H CV lu A an va n Bc tn to p ie gh Bc oa nl w C d B an lu nf va 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 ul oi lm 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 z at nh 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) z m co l gm @ an Lu n va ac th 24 si P A lu an n va P tn to P p ie gh Bc Bc C d oa B nl w Pd lu an Figure 4.2.2b Histopathological alteration of livers in Portal Vein after fishes nf va treated with niclosamide (A) Normal livers in Portal Vein of Nile tilapia oi lm ul 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 z at nh 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 z magnification) m co l gm @ an Lu n va ac th 25 si 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 lu histological lesions in the fish gills found in the present study might arise also an n va because of the stress that the fish were subjected to within a short period of time to gh tn Depending on the toxic potential of a particular toxin accumulates in the p ie tissues to determine the extent of damage Hence, when exposed to water w contaminated various organs in the fishes can be adversely affected and may d oa nl 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 an lu ul nf va exposed to different pollutants (Olojo E & Olurin K., et al., 2005) oi lm Fish gills were multifunctional organs involved in ion transport, gas exchange, acid-base regulation and waste excretion (Wong, 2000; Thophon, et z at nh al., 2003; Zayed & Mohamed, 2004) The gills were particularly sensitive to z changes in the water, which were depended on quality of the water Hence, gills gm @ were considered as the primary target of contaminants (Fernandes & Mazon, l m co 2003; Camargo & Martinez, 2007) The gills of fishes exposed to niclosamide showed the loss of their architecture with inflammation of primary lamellae and an Lu desquamation of secondary lamellae at different concentrations of niclosamide n va ac th 26 si (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 lu an concentrations increased the degree of damage also increased n va absorbed from the aquatic environment and liver lesions were often associated ie gh tn to Liver as the main organ of metabolism came into contact with xenobiotics p with exposure to aquatic pollutants (Fernandes C & Fontainhas-Fernandes A., at oa nl w 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 d lu va an aquatic environmental pollution It was most likely because fishes dead so fast ul nf within 3-4 minutes, hence, we cannot find many changes in the livers The livers oi lm of the exposed fishes compared to the control showed varying degree of z at nh degeneration of cells, which included blood congestion and patchy degeneration elements around the pancreatic cells (figure 4.2.2b-C) and these alterations were z gm @ 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 l m co vein rather difficult Observe abnormal livers reflect the impacts of pollutants an Lu Besides this, oxygen deficiency was the most common cause of the cellular degeneration in the liver (Olojo, et al., 2005; Camargo & Martinez, 2007; n va ac th 27 si Atamanalp, et al., 2008; Mohamed & Velisek, 2009; and Reethamma, 2014) have also reported similar observations upon pesticides contamination in various fish 5.2 Conclusion The histopathological alterations found in the gills and livers of O Niloticus were most likely caused by the niclosamide exposure Based on these lu results, O Niloticus cannot tolerate the levels of niclosamide used in this an n va experiment Therefore, histological analyses of gills and livers showed gh tn to significantly pathological changes at lethal levels p ie Histological comparison of tissues indicated that most damage occurred w in the gills rather than in the livers This might be because short contact (4- oa nl minutes) was not enough time for niclosamide to penetrate into fishes livers The d severity of damages to gills and livers of the fishes were proportional to the an lu concentration of the pesticides oi lm ul nf va z at nh z m co l gm @ an Lu n va ac th 28 si BIBLIOGRAPHY Atamanalp, M , Sisman, T., Geyikoglu, F., and Topal, A (2008) The histopathological effects of copper sulphate on rainbow trout liver (Oncorhynchus mykiss) Journal of Fish , Aquat Sci., (In press) Bancroft, J D., and Gamble, M (2002) Theory and practice of histological techniques (5th ed.) 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