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

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 NICLOSA

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

Thesis Title:

TOXICOLOGICAL EFFECT AND HISTOPATHOLOGICAL CHANGES IN GILLS AND LIVER OF TILAPIA (OREOCHROMIS NILOTICUS) EXPOSED TO NICLOSAMIDE

Supervisor (s):

Dr Arinafril Naalim 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 1

LIST OF TABLES 2

PART I INTRODUCTION 3

1.1 Background and rationale 3

1.2 Research’s Objectives 5

1.3 Research questions and hypotheses 5

1.4 Limitations 6

PART II LITERATURE REVIEW 7

2.1 Niclosamide 7

2.2 Toxic effects of Niclosamide on organisms 9

2.2.1 Toxicity 9

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 7 Table 2.2.1: Toxicology profile of the niclosamide 10 Table 3.3: Listed of Equipment used for this study 16

PART I INTRODUCTION

1.1 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

addition to this, histopathological studies of the liver were performed to understand the effect of absorbed niclosamide on the internal soft tissue

Furthermore, the lethal concentrations of most of the pesticides cause various 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 condition in scope of toxicity studies since they provide early signs of disease (Meyers & Hendricks., 1985) Due to being exposed to pollutants, major structural damages may occur in their target organs, histological structure may change and physiological stress may occur This stress may be caused by some alterations in the metabolic functions The changes in the functions are initiated by the changes in the tissue and

cellular level (Van der Oost, et al., 2003)

1.2 Research’s Objectives

The objectives of this study are to evaluate the toxicological effects of niclosamide on Tilapia as well as to investigate the histopathological alterations

on its gills and liver under different concentrations of niclosamide

1.3 Research questions and hypotheses

Research questions:

− How does niclosamide affect the gills of Tilapia?

− How does niclosamide affect the liver of Tilapia?

Hypotheses:

Hypothesis 1:

HO (Null Hypothesis): Exposure to niclosamide will not result in changes

in gills histology of Oreochromis Niloticus

HA (Alternative Hypothesis): Exposure to niclosamide will result in

changes in gills histology of Oreochromis Niloticus

PART II LITERATURE REVIEW

2.1 Niclosamide

Table 2.1: Physical and Chemical Properties of Niclosamide

Chemical Name

Molecular Weight 327.119 g/mol

Specific Gravity No data available

Physical State Yellowish to grey-greenish powder

CIPAC code numbers 599

In 1964s, the U.S Department of Agriculture (USDA) has recognized niclosamide as a pesticide and it has 5 products niclosamide, which registered in the Environmental Protection Agency as a 70% WP for sea lamprey control, two Special Local Needs labels with the 70% WP, one 3.2% granular formulation, and one 5% granular formulation The registrant has requested voluntary cancellation of the 5% granular product (Environmental Protection Agency, 2004)

Although niclosamide is used to remove oxidative phosphorylation, but

we still do not fully understand its mode of operation In vitro studies demonstrated that niclosamide inhibited rat liver mitochondrial synthesis of ATP (WHO, 2003) Bayluscide inhibits succinate oxidation and causes oxaloacetate

accumulation (Ishak, et al., 1972)

Niclosamide is relatively selective; primarily used against aquatic snails, but it is also used as an anti-parasitic drug in human medicine and veterinary medicine Although it may be toxic to aquatic vertebrates (e.g fishes and amphibians) and crustaceans, but it has slight effect on mammals However, niclosamide is non-persistent in the aquatic environment, it has a very low toxicity to aquatic plants and plankton (WHO, 2003)

It is relatively harmless to humans It is also uses as an antihelminthic in humans, livestock and pets (WHO, 2003) In addition, the side effects of it are frequent gastrointestinal discomfort It has been also reported that the workers

experienced skin irritation after applying niclosamide, but this is thought to be caused rather by formulation ingredients other than niclosamide (WHO, 2003)

2.2 Toxic effects of Niclosamide on organisms

2.2.1 Toxicity

Pesticides, including molluscicides, must be applied in accordance with the full product label as registered by the U.S Environmental Protection Agency (USEPA) U.S Environmental Protection Agency (2004) recommends that:

“Niclosamide has acute oral LD50 values of >1000 mg/kg (Toxicity Category III) The acute dermal toxicity is minimal, as indicated by a LD50 > 2000 mg/kg (Toxicity Category III) (EPA, 2004) It produces slight skin irritation (Toxicity Category IV) and caused eye irritation (unclassified Toxicity Category based on short time interval of eye examination) It was a moderate skin sensitizer The acute inhalation data are not available Niclosamide showed no evidence of causing developmental toxicity, mutagenicity or carcinogenicity (EPA, 2004).” The toxicological and ecotoxicological data included in the summary below was derived from niclosamide having impurity (table 2.2.1)

Table 2.2.1: Toxicology profile of the niclosamide

conditions or guideline adopted

LD50 > 5000 mg/kg bw (Flucke, 1978)

rats, male and

female

Dermal not stated LD50 = >4000 mg/kg bw

only determined for EC

250, not TC, Kröthlinger,

1997 rabbits, male

and female

Dermal not stated LD50 >2000 mg/kg bw

only determined for WP

70, not TC, Nelson & Bauman, 1969

rats, male and

female

Inhalation dust, 1 h

exposure

LC50 = >20.000 mg/m3 , Crawford et al, 1970 Rabbits skin

irritation

not stated irritating, especially at

high doses or with repeated application, Kimmerle, 1971, Lorke & Lischka, 1965, Crawford

& Roney, 1971 Rabbits eye irritation not stated strongly irritating to eyes,

locally corrosive to cornea, Crawford & Roney, 1971, Nelson, 1969, Kimmerle,

1971

guinea pigs skin

sensitization

Buehler patch test not sensitizing, result

obtained from EC 250, not for TC, Stropp, 1997 guinea pigs skin

Niclosamide can be fatal to sea lamprey larvae (LC100 = 0:06 to 0:15 mg / L) Meanwhile it is more toxic to free-swimming sea lamprey juvenile (12-h LC50 = 0.0625 mg / L) and the larvae burrowed sea lampreys (12-h LC50 =

0110 mg / L) (Nettles, et al., 2001) The 24-h LC50 for teleost fish species

(juveniles) ranges from 0.052 to 0.143 mg/L, with salmonids tending to be more susceptible than other species (Nettles, et al., 2001) With the concentration of 0.5 mg / L of niclosamide (48h exposure) is toxic to all species of fish, but not to the zooplankton and aquatic plants, which are need higher concentrations (WHO, 2003) Overall, the soft-bodied invertebrates such as worms, leeches, snails are vulnerable to poisoning of niclosamide (24-h LC50 = 0.03–0.4 mg/L) than hard-bodied invertebrate, such as insect larvae or crustaceans (24-h LC50 =

0.8– > 50.0 mg/L) (Nettles, et al., 2001)

Niclosamide is relatively non-toxic to birds and bees if applied as recommended (WHO, 2003) Concentration range tested the impact of the acute toxicity of low toxicity to highly toxic of niclosamide to aquatic animals is 0.034

to > 50 mg / L The most tolerant species tested were crayfish, dragonflies, snipeflies, and dobsonflies The most sensitive species were turbellarians, snails, and aquatic earthworms and appeared to affect organisms inhabiting sediments With the potential to absorb sediments of niclosamide, the use of formulations specifically designed to slowly release the chemical at the water-sediment interface, and the acute toxicity of niclosamide to aquatic invertebrates is necessary (EPA, 2004)

2.2.2 Histological Effects

A large number of agricultural runoff flowing into the aquatic environment, including pesticides, herbicides, fungicides, fertilizer residues, heavy metals, etc In addition, the aquatic environment must also receive a large variety of industrial waste, domestic waste human and oil compounds (El-Sayed, 2006) Eutrophication, environmental degradation, imbalance of biodiversity and bioaccumulation are impacts likely due to these organic compounds and inorganic caused Therefore, wild and farmed fishes, including tilapia, are predicted to be affected by these pollutions These effects depend on the concentration of pollutants, exposure duration, species, size and environmental conditions However, little pay attention to the impact of environmental pollution on tilapia farming, primarily interested in wild tilapia Despite the fact that hundreds of chemical compounds might be hazardous to these fish, even at

low concentrations, only a few compounds have been considered (El-Sayed, 2006)

One of these organic, phenolic compound, seriously contaminates to aquatic environment These chemical is very toxic to aquatic animals and can cause serious damage to the economic and ecological (El-Sayed, 2006) For

example, Hart, et al., (1998) discovered that Nile tilapia subjected to the

carcinogenic polycyclic aromatic hydrocarbon (PAH) 7, dimethylbenzanthracene (DMBA) suffered from reduced spleen, pronephros and total white blood cell counts This fish shows decreased expression of swimming activities and feeding, but increased mortality and skin pigmentation In another research, Mehrim (2001) have assessed the acceptable maximum level of phonelic to Nile tilapia fingerlings He noticed that the acceptable maximum level was 30ppm, and if more than that, fish exhibited a respiratory manifestation and hyper-irritability, followed by lethargy, increased mucus secretion, skin darkness, fin erosion, gill and liver congestion and distension of the gall bladder

12-There is a phenolic compound widely used as a molluscicide, it is Bayluscide - one of the chloronitrophenol derivatives (niclosamide ethanolamine salt) (5,2-dichloro-4-nitro-salicylic-anilide) It is often used to eradicate the intermediate host snails of schistosomiasis (bilharzia) and fascioliasis It is very toxic to snails and fish (El-Sayed, 2006) Acute toxicity of Nile tilapia after exposed with Bayluscide included erratic and nervous swimming, continuous opening of mouth and gill cover, haemorrhage under the scales and at the base of

the fins, degenerative and necrotic changes in the liver, kidney, spleen, heart and gills (Marzouk and baker, 1991) Abdel-Fattah M El-Sayed discovered that 0.3 mg/l was lethal to Nile tilapia, while 0.1 mg/l was sublethal Fish exposed to 0.15 mg/l (50% of the median lethal concentration (LC50) suffered from nervous and respiratory manifestations, corneal opacity, decrease in red and white blood cell counts, haemoglobin concentration and phagocytic activity, and a high accumulation of Bayluscide in the gills, liver and muscles (El-Sayed, 2006)

Binomial name: Oreochromis Niloticus (Smith, 1840)

The Mozambique tilapia, Oreochromis Niloticus, is an African tilapia cichlid fish, used as food and thereby introduced in aquaculture for commercial products and may be (erroneously) called "Java tilapia" in trade, which was

chosen for the present study (Nagl, S & Tichy, H., el at., 2001)