Toxicological impact and histopathological response of tilapia after lead (II) nitrate (pb (n03)2) contamination

The present study was conducted to assess the histopathological alterations in the gills, heart, dorsal muscles and liver of tilapia Oreochromis niloticus which were kept in aqueous sol

THAI NGUYEN UNIVERSITY

UNIVERSITY OF AGRICULTURE AND FORESTRY

MENDOZA, JIMLEA NADEZHDA A

TOXICOLOGICAL IMPACT AND HISTOPATHOLOGICAL RESPONSE OF TILAPIA AFTER LEAD (II)-NITRATE (Pb (NO 3 ) 2 ) CONTAMINATION

BACHELOR THESIS

Study Mode: Full-time Major : Environmental Science and Management Faculty : International Training and Development Center Batch : 2012-2016

Thai Nguyen, September 2016

DOCUMENTATION PAGE WITH ABSTRACT

Thai Nguyen University of Agriculture and Forestry

Degree Program : Bachelor of Environmental Science and Management

Student name : Mendoza, Jimlea Nadezhda A

RESPONSE OF TILAPIA AFTER LEAD (II)-NITRATE (Pb(NO3)2) CONTAMINATION

Supervisor (s): Krisna MURTI, MD., M Biotech Stud., Ph.D

Duong Van Thao, Ph.D

Abstract: To investigate the effects of environmental contaminants, histopathological response of fish exposed to pollutants have been used as sensitive biomarkers The present study was conducted to assess the histopathological alterations in the gills,

heart, dorsal muscles and liver of tilapia Oreochromis niloticus which were kept in

aqueous solution of lead nitrate of two concentrations of 0.2 mg/l, 1.0 mg/l for 2 days under laboratory conditions The resultant histopathological changes in the gills, heart, dorsal muscles and liver were recorded by light microscope The observed changes in the treated groups were disintegration of secondary lamellae, atrophy, curling and shortening of secondary lamellae, swelling/ inflammation, desquamation, epithelial lifting, curling bend of secondary lamellae and necrosis in gills Atrophy and splitting

of muscle fibers are recognized as common changes recorded in the heart of experimental fish Atrophy in dorsal muscles and splitting of dorsal muscle fibers, necrotic damage and degradation of muscle fibers were an interesting observation in dorsal muscle tissue of experimental fish Examination of liver sections after exposure showed sinusoidal dilatation and leukocyte infiltration in central veins and in peripheral areas occurred after exposure The damages in histology of gills, heart, dorsal muscles and liver depend on the exposure concentrations tolead (II)-nitrate (Pb (NO3)2) As the exposure concentrations increased, the more adverse damage occurred

in the organs Therefore, the present investigation gives a brief account of the toxic effects of heavy metals on fish The present review illustrates that these histopathological alterations would contribute an important role in assessing the harmful effects of lead nitrate As such, fish are used as bio-indicators, providing useful purpose in monitoring heavy metals contamination Hence, implementation of regulations regarding the conservation of aquatic environments must be taken into consideration

Keywords: LEAD (II)-NITRATE (Pb (NO3)2), histopathology,

I also want to express my thanks to Dr Duong Van Thao, the second supervisor, for his supervision, encouragement, advice, and guidance in writing this thesis

Besides my supervisors, I would like to thank Ms Fadila Mutmainnah from Pascasarjana Program Sriwijaya University, Ms Mirna Fitrani, Ms Sefti Heza Dwinanti and Ms Ade Dwi Sasanti of Aquaculture Laboratory, Faculty of Agriculture, Sriwijaya University, Dr Imelda Mendoza Moreno, Mrs Aimee Ciriaco from Laguna State Polytechnic University, Siniloan Campus, Siniloan Laguna for providing me an additional knowledge about Lead nitrate, water quality and fish histology

In addition, formal thanks should be offered to the Rector of Sriwijaya University, Prof Badia Perizade, for granting my internship acceptance

I would also like to acknowledge with much appreciation to the Dean of Faculty of Medicine in Sriwijaya University, Dr Mohammad Zulkarnain M Med Sc, PKK., who gave the permission to use all required equipment and the necessary materials to conduct my research in Department of Anatomical Pathology, Faculty of Medicine, Sriwijaya University/Dr Mohammad Hoesin Public Hospital

Special thanks to Mrs Ana Nyayu, Mr Mohammad Zainuri, and other staffs and students in Aquaculture Laboratory, Faculty of Agriculture, Sriwijaya University for helping and providing me necessary equipment as well as knowledge for fish anatomy

I wish to thank the technicians who work in Department of Anatomical Pathology, Faculty of Medicine, Sriwijaya University/Dr Mohammad Hoesin Public Hospital for their help in tissue preparation namely: Mrs Fitri Faurianty and Madi Santoso, without them, this research could not be accomplished on time

My sincere thanks also go to Duong Hong Ngoc, Keraia Vince Geronimo, Phonevilay Soukhy, Tran Cong Phong, Nguyen Thi Van and Do Manh Dung, Jose Alberto Dunca for helping me finish this study

Of course, I would like to thank to my Indonesian friends – Rotua Febriani, Hendra Edison and others for their invaluable support and encouragement when I stayed in Palembang

Finally, special thanks to my family, my friends for their love and moral support throughout my study

Thai Nguyen, 20 th September, 2016

Student Mendoza, Jimlea Nadezhda A

Table of Contents

LIST OF FIGURES 1

LIST OF TABLES 2

PART I INTRODUCTION 3

1.1 Background and rationale 3

1.2 Objectives 5

1.3 Research questions and hypotheses 5

1.3.1 Research Questions 5

1.3.2 Hypotheses 6

PART II LITERATURE REVIEW 8

2.1 Lead Compound- Lead (II) Nitrate 8

2.2 Toxic effects of Lead on organisms 9

2.2.1 Toxicity 9

2.2.2 Histopathological Effects 12

2.3 Test species -Oreochromis niloticus 16

PART III MATERIALS AND METHODS 17

3.1 Time and Place 17

3.2 Materials 17

3.3 Equipment 17

3.4 Methods 19

3.4.1 Toxicity testing 19

3.4.2 Histopathological Examination 19

PART IV RESULTS 23

4.1 Histopathological observation of gills 23

4.2 Histopathological observations of heart 26

4.3 Histopathological observations of Dorsal muscle 27

4.4 Histopathological observations of liver 29

PART V DISCUSSION AND CONCLUSION 32

5.1 Discussion 32

5.2 Conclusions 41

PART VI REFERENCES 43

LIST OF FIGURES

Figure 1 Normal histological structure of gills

Figure 2 Comparison of the normal and treated gills

Figure 3 Histopathological changes observed in heart of experimental fish

Figure 4 Comparison of the normal and treated dorsal muscle

Figure 5 Comparison the normal and treated liver

Figure 6 Comparison of the normal and treated liver

LIST OF TABLES

Table 1.Physical and Chemical of Lead (II) Nitrate material

Table 2 Important routes of exposure to lead

PART I INTRODUCTION

1.1 Background and rationale

At present, the major challenge is to find ways to sustain activities, working procedures and management of industries towards economic development to promote human health and environmental protection The vulnerability of aquatic ecosystems

to heavy metal contamination has been recognized as a serious pollution problem Metals that are deposited in the aquatic environment may accumulate in the food chain and cause ecological damage posing threat to human health and sustainable food supply due to biomagnifications over time The anthropogenic pollutants from industrial, domestic and agricultural waste are ultimately absorbed by aquatic plants and animals Potential damage to ecosystem may originate from exposure to metal pollutants because of extent and location and current ecological services of the lake

as well These contaminants come sources such as from human activities that bring economic developments that deliver benefits to society (Molina, 2011) The heavy metal contamination of water bodies as one of the substantial environmental concerns was reported (Agrahari, 2009) and crucial harmful impacts on environmental quality, ecosystem integrity and health of humans have often been linked with improper management of chemical substances and the removal of hazardous materials Although some adverse health impacts of heavy metals have already been well known, heavy metal contamination is increasing in many parts of the world, mostly in developing countries, even though emissions have reduced in most developed countries over the last thousand decades (Järup, 2003) Bio-accumulation in various tissues of aquatic organisms and rising level of heavy metal toxicity poses serious threats to the

biodiversity of ecosystems and human health (Adeniyi et al., 2008; Vinodhini and Narayanan, 2008a; George et al., 2011)

Fishes have been considered as good bio-accumulators of organic and inorganic toxicants Fishes 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 (King and Jonathan, 2003).These days, a huge segment of the overall eating routine comprises of nourishments from fresh and salt water bodies This utilization has had a positive effect on job and livelihood for example, in instances of fish production and every year a huge assortment of goods made out of seafood items are becoming commercially available widely.Heavy metals concentrated

in water systems and expand through food chain and aquatic organisms especially fishes and being the primary consumers, they are largely influenced People on the other hand also are affected by eating fishes from those areas where main food is fish (Afshan et al., 2014) 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

The pollution might cause adverse effects on the ecosystem such as the health

of aquatic animals (Ashraj, 2005; Vosyliene and Jankaite, 2006; Farombi et al., 2007) Lead, like majority of the heavy metals are being discharged into surface waters affecting the health of aquatic organisms such as fishes Lead (Pb) is among the heavy metal and its contamination in the water systems has global effects that are harmful to human, health of the ecosystem and damage delivered to life in most aquatic resources especially fishes (Markus and McBratney, 2001).Generally, because of its low rate of

disposal, lead (Pb) is one of the most crucial toxins in our surroundings which accumulates in the body

As indicated by the results of past investigations, the present work was proposed to investigate the impact of lead nitrate on histological changes of the gills,

heart, liver and dorsal muscle of the tilapia, Oreochromis niloticus

This study is an important tool that evaluates the impacts of human activities and weighs the adverse effects to health of the people amidst the contributions of developments economically This study aims to evaluate the toxic effects of heavy

metals such as lead nitrate on the organs of Oreochromis niloticus (Tilapia) to support

of predicting potential health consequences to the human and health of fish which could serve as a scientific basis for decisionmaking and policy development and be able to determine the need for urgent measurements which should be done by concerned authorities to protect health of communities consuming fish products

The research aims to answer the follow questions;

- How does lead (II)-nitrate (Pb (NO3)2) affect the gills of Tilapia?

- How does lead (II)-nitrate (Pb (NO3)2) affect the liver of Tilapia?

- How does lead (II)-nitrate (Pb (NO3)2) affect the heart of Tilapia?

- How does lead (II)-nitrate (Pb (NO3)2) affect the dorsal muscle of Tilapia?

1.3.2 Hypotheses

Hypothesis 1:

HO (Null Hypothesis): Exposure to lead (II)-nitrate (Pb (NO3)2) contamination

will not result in changes in gills histology of Oreochromis niloticus

HA (Alternative Hypothesis): Exposure to lead (II)-nitrate (Pb (NO3)2)

contamination will result in changes in gills histology of Oreochromis niloticus

Hypothesis 2:

HO (Null Hypothesis): Exposure to lead (II)-nitrate (Pb (NO3)2) contamination

will not result in changes in liver histology of Oreochromis niloticus

HA (Alternative Hypothesis): Exposure to lead (II)-nitrate (Pb (NO3)2)

contamination will result in changes in liver histology of Oreochromis niloticus

Hypothesis 3:

HO: Exposure to lead (II)-nitrate (Pb (NO3)2) contamination will not result in

changes in heart histology of Oreochromis niloticus

HA: Exposure to lead (II)-nitrate (Pb (NO3)2) contamination will result in

changes in heart histology of Oreochromis niloticus

Hypothesis 4:

HO: Exposure to lead (II)-nitrate (Pb (NO3)2) contamination will not result in

changes in dorsal muscle histology of Oreochromis niloticus

HA: Exposure to lead (II)-nitrate (Pb (NO3)2) contamination will result in

changes in dorsal muscle histology of Oreochromis niloticus

PART II LITERATURE REVIEW

2.1 Lead Compound- Lead (II) Nitrate

Table 1 Physical and Chemical of Lead (II) Nitrate material

Chemical Name

LEAD NITRATE; Lead dinitrate; Lead(II) nitrate; Plumbous nitrate; Lead nitrate (Pb(NO3)2)

Product Names Lead(II) nitrate

Molecular Formula HNO3.1 or N2O6Pb

Chemical Formula Pb(NO₃)₂

In etanol: 0,04 g/100 mL

In metanol: 1,3 g/100 mL Vapor Pressure Negligible

2.2 Toxic effects of Lead on organisms

2.2.1 Toxicity

Table 2 shows the toxicity classification of lead via the designated routes of exposure

Table 2 Important routes of exposure to lead

Ingestion Ingestion of food contaminated by lead, water or alcohol

may pose risk for some populations Lead paint is the major source of lead exposure for children (ASTDR, 2005) As lead paint deteriorates, peels, chips, or is removed (e.g., by renovation), or pulverizes due to friction (e.g., in windowsills, steps and doors), house dust and surrounding area such as soil may become contaminated Lead then come inside the body through common hand-to-mouth gesture (Sayre et al 1974 as cited in AAP 1993)

Inhalation Inhalation is the second major pathway of exposure In

some countries abroad, the resulting emissions from leaded gasoline pose danger to the health of the public Inhalation could be the main route of exposure to some people working in industries that use lead Inhalation may be the main route of exposure for adults associated in home activities involving renovation procedures Lead is absorbed into the body, whereas from 20% to 70% of ingested lead is absorbed (with children usually absorbing a higher level than adults do) (ATSDR 2005)

Dermal Dermal exposure contributes to organic lead exposure

among workers, but is not considered a major pathway for the most of the population Organic lead could be absorbed through the skindirectly Organic lead (tetramethyllead) is more likely to be absorbed through the skin than inorganic lead People who work with lead are most likely to experience dermal exposure

Endogenous Exposure Endogenous exposure to lead could contribute significantly

to an individual’s latest blood lead level, and of particular harm to the developing fetus When taken up into the body, lead could be stored for long periods in mineralizing tissue (such as teeth and bones) The lead that has been stored may be released again into the bloodstream, particularly in times of calcium stress (e.g., pregnancy, lactation, osteoporosis), or calcium deficiency

(Source: ASTDR, 2007)

Lead (II) nitrate (Pb(NO3)2 ) is a toxic compound, and ingestion could cause acute lead poisoning, and also for all soluble lead compounds (USCG, 1999) Likewise, when dissolve in water, Lead (Pb) from Lead (II) nitrate (Pb(NO3)2, as one

of the dangerous chemical elements, lead could affect behavioral problems, high blood pressure, kidney damage, memory and learning difficulties and even reduced IQ of human being (Ernhart, 2006; Spivey, 2007) Lead could remain about 100 to 1,000 years in water aquatic systems; hence, lead (Pb) could have long term effects on plants and animals (UNEP, 2010) Lead is a common heavy metal found in the environment and is available from agriculture runoff, urban waste waters and discharges from industries The entering of heavy metals into the aquatic ecosystem produces water pollution problems due to their bio-accumulation and toxicity (Selvanathan, Vincent, and Nirmala, 2013)

Environmental pollution with heavy metals can pose serious risks such as abnormality in genes, behavioral disorders, physiological and biochemical problem in aquatic organisms (Scott et al., 2003) Among aquatic organisms occupying the polluted water bodies, fishes are sensitive to heavy metal contamination compared to other organisms (Alinnor, 2005) Lead could damage some body organs including the

nervous, reproductive and excretory systems (including kidneys), thus affecting functions of blood cells (Frumkin and Geberding, 2007; Vinodhini and Narayanan, 2008b)

Bioaccumulation is the capacity of fish to accumulate an element from water to

a level larger than that of its environment Hence, index of metal pollution in the aquatic system can be attributed in bioaccumulation of metals in fish (Javed, 2005; Karadede-Akin et al., 2007) When accumulation come to a significantly high level, heavy metals accumulated in the tissues of aquatic animals could become toxic (Yildirim et al., 2009) Significant amount of these metals may be accumulated in the organs of aquatic organisms when exposed to high concentrations

In fish, gills are regarded as dominant site for contaminant absorption provided their anatomical and/or physiological characteristics that boost absorption efficiency (Takarina et al., 2012) During the sub-lethal exposure, the amount of Pb absorbed by the fishes causes behavioral abnormalities due to disruptions in the integrative functioning of the medulla, cerebellum and optic tectum (Rademacher et al 2003).When fishes in aquatic environment are exposed to high level of metal ions, their tissues absorb these metal ions through various pathways from their surroundings There are two primary routs of metal acquisition; directly from the water and from the diet (Bury et al., 2003) After binding to the mucus layer, lead ions enter

in the body of fish through gills It is also taken up together with food and water and is finally absorbed in the intestine and other tissues (Kotze et al.,1999; Ay et al., 1999; Macdonald et al., 2002, Hensen etal., 2007) The gills are very sensitive to water-born metals and usually exhibit various metal induced abnormalities This allow osmotic

imbalance and also causes damage to the respiratory system function of the exposed fish (Jezierska and Sarnowski, 2002; Dobreva et al., 2008) However, the metal accumulation in tissues of aquatic animals will depend upon exposure concentration and period as well as some other factors namely salinity, temperature, interacting agents and metabolic activity of the tissue Lead also accumulates in other tissues of fish, such as skin and scales, gills, eyes, liver, kidneys and muscles (Rashed, 2001; Nussey et al., 2000; Alves et al., 2006; Spokas et al., 2006)

These studies contributes to greater understanding of adverse effects of Lead II nitrate on histopathology of organisms As a biomarker, histopathology could be performed to assess water quality as well as toxicity of pollutants

2.2.2 Histopathological Effects

There are several studies on histopathological effects of Lead II nitrate on organisms Histopathological alterations give a reliable, efficient measurable index of low-level toxic stress to a wide range of environmental contaminants The findings provide proof that tissue biomarkers, biochemical and bioaccumulation can be sensitive manifestations of mixed organic pollutants influencing surface waters (Osman, 2012)

According to results of the study by Banaee et al (2013), lead has a high tendency to accumulate in many tissues of fish, most particularly in soft tissues Hence, the lower the concentration level of lead in the muscle tissues, when compared with other tissues, seems acceptable The results of this study showed the linear

relationship between Pb (NO3)2 concentrations in water and bio-concentration of Pb in different tissues

Bioaccumulation of Lead in various organs of gold fish were studied by Banaee, Haghi and Zoheiri As stated in their findings, fishes were exposed to lead nitrate Pb (NO3)2 at a series of concentrations 0.0 mg/L (control group), 0.09, 0.15, 0.24, 0.3, 0.36 and 0.45 mg/l, which were equal to approximately 2, 3, 5, 6, 7 and 9%

of 96 h LC50 for 28 days of exposure Viscera had the highest lead bioaccumulation potential, followed by the gill The muscles were least target for detecting the bioaccumulation of Pb The authors reported that although lead was manifested in all tissues tested, Pb bioaccumulation potential is varying and depending on the structure

of the tissues (Banaee et al., 2013)

The presence of lead nitrate in water are toxic to fishes was investigated by Elumalai, Silvan and Mary Heavy metals are naturally present in the marine environment These heavy metals enter the water bodies by direct discharges from industrial and urban effluents The heavy metals cause damage to the aquatic environments, therefore, they conducted a study about toxic effects to assess the effect

of lead nitrate induced histological alterations in the gills of freshwater fish, Cirrhinus mrigala, which were treated in aqueous solution of lethal concentrations of lead nitrate for 24 days The histopathological impacts of lead nitrate in the gills, liver and muscles

of fish found are the following; complete fusion of gill lamellae and edema in the filamentary epithelium (Mary et al., 2014)

A study was performed by Ahmed and Bibi to evaluate the uptake and accumulation of waterborne lead (Pb) in various tissues of Catla catla when exposed

to lower doses Each group of single breed fingerlings (6-8 cm) of C catla was exposed to a sub-lethal dose of waterborne Pb at 0.0 µg/l, 1.0 µg /l, 2.5 µg /l, 5.0µg /l, 7.5 µg /l and 10.0 µg /l for 6 weeks The findings of the study indicated that the fishes living in aquatic system receiving industrial and city effluents were contaminated with heavy metals, had absorbed and accumulated in different tissues like skin, gills and intestine directly from water as well as from fish diet As the time passes by and the fishes grow, such harmful metals accumulated in several tissues like liver, muscles and intestine to a significantly high concentration not appropriate for human consumption (Ahmed and Bibi, 2010)

The acute toxicity effects of Pb2+ were studied on essential trace metals behavior and histopathology of Crucian carp (Carassius auratus gibelio) The findings reported the histopathological changes in the gills of treated fish were indicated by lamellar shrinkage, disruption of cartilaginous core, epithelial lifting, lamellar shortening with desquamation and curling of the secondary lamella Severe shrinkage of glomeruli, hypoplasias of hemopoietic tissue as well as mild glomerular and tubular necrosis were observed in kidney The structural changes in the liver were cellular edema, necrosis

of hepatocytes with nuclear degenerate on and pyknotic nuclei Then the brain exhibited severe proliferation of glial cells, cellular necrosis, severe perivascular edema and satellitosis These findings showed the detrimental effects of Pb2+ on trace metal metabolism and tissue architecture of Crucain carp (Khan et al., 2014)

Sharma and Tamot carried out the study to investigate the histopathological alterations in the liver and kidney of fresh water teleost, Channa striatus after exposure

to 28 mg/l (10% of 96 hrs.LC50) of lead nitrate for a period of 30 and 60 days under

laboratory conditions The most common alterations in the liver of fishes observed were severe loosening and necrosis of hepatic tissue, cytoplasmic vacuolation, expansion in cell size, eccentric and enucleated hepatocytes Their results illustrated that these histopathological changes would contribute in assessing the toxic impacts of lead nitrate (Sharma and Tamot, 2015)

The impacts of lead nitrate which caused histological alterations were investigated in the gills of freshwater fish, Channa striatus which were exposed to lead nitrate of three sub-lethal concentrations including 20mg/l, 30mg/l and 50mg/l for 15 and 30 days The histopathological lesions caused by Pb NO3 in the gills of fish observed were complete fusion of gill lamellae, edema in the filamentary epithelium, degeneration of lamellar epithelium, epithelial necrosis, rupture of gill epithelium and hemorrhage at primary lamellae and fusion of adjacent secondary lamellae Exposure

to sub-lethal concentrations of lead nitrate caused dose and duration dependent histopathological changes in the gills of Channa striatu (Parveen et al., 2013)

Studies about histology of different fish organs were performed to assess the effects of different types of stress including diet, heavy metals and pesticides stress (Arnold et al., 2000; Segnini et al., 2000; Jiraungkoorskul et al., 2002; Yang and Chen, 2003; Mekkawy et al., 2007, 2008, 2011a, 2012; Mobarak and Sharaf, 2011; Sayed et al., 2011, 2012a, b; Taweel et al., 2012; Bais and Lokhande, 2012) Most of these investigations highlighted the histopathological lesions occurred in the liver, kidney and other organs Different degrees of effects were evaluated depending on the dose, age and fish species

2.3 Test species -Oreochromis niloticus

Binomial name: Oreochromis niloticus (Linnaeus, 1758)

Oreochromis niloticus is one of the mostly common farmed aquatic species, provided its relative ease of culture and rapid reproduction rates Nile tilapia (Oreochromis niloticus) is a very important fish in aquaculture industries

PART III MATERIALS AND METHODS

3.1 Time and Place

This experiment was conducted in period of two months from March 2016 to April 2016 at Aquaculture Laboratory, Faculty of Agriculture, Sriwijaya University and Department of Anatomical Pathology, Faculty of Medicine, Sriwijaya University/Dr Mohammad Hoesin Public Hospital

3.3 Equipment

Equipment used in laboratory

The following materials and equipment were used for toxicity test and tissue processing

Materials used in laboratory:

Equipment used in laboratory:

- Automatic tissue processor

- Metal slides holder

- Histokinet

- LAB Line Barnstead 26025 Slide Warmer

- OLYMPUS BX51 microscope

- Shandon finesse 325 (microtome)

- Tissue embedding center

- Tissue floating bath

3.4 Methods

3.4.1 Toxicity testing

A total of 6 fish was chosen randomly for the experiment The test fish were divided into 3 chambers, each containing 10L of water with 2 fish and each Lead II nitrate concentration was represented by 2 replicates The control group was not exposed to the test chemical and other groups were exposed The concentrations used for toxicity estimation were 0.2 and 1 mgL-1 which were considered as test solutions During toxicity experiment fish were not fed Dead individuals were dissected for observing the histopathological alterations in the gills, liver, heart and dorsal muscles

3.4.2 Histopathological Examination

Dead fish were taken out and the portions of gills, heart, dorsal muscle and liver were excised out Procedures required in preparing tissues for light microscopy include following steps (Raphael, 1976; Bancroft& Gamble, 2002) These steps were conducted with the assistance of technicians in Department of Anatomical Pathology, Faculty of Medicine, Sriwijaya University/Dr Mohammad Hoesin Public Hospital

The gills, heart, liver and dorsal muscle tissues of the control and the experimental fish were dissected and placed into cassettes gently in order that even after death the shape, structure, relationship and chemical constituents of the cells and tissue were maintained The tissues were immediately fixed in 10 % neutral buffered formaldehyde solution pH 7.0 for more than 24 hours

Tissue Processing

Tissue processing is a long procedure with duration that is more or less for 24 hours Tissue processing was conducted mechanically and performed in several stages; dehydration, clearing, impregnating and embedding

Initially the tissues were taken from 10% formalin and then dehydrated by rinsed them for one hour each in a graded ethanol series of concentrations; e.g 50%, 70%, 90% and 100% After this, clearing process was conducted by immersed the min xylene for one hour The next step is impregnation with paraffin, which was performed

by rinse the tissues in paraffin wax at its melting point temperature of paraffin wax, which is 54-60oC Volume of wax should be about 25-30 times the volume of tissues and the duration of impregnation was about 4 hours At last, in a melting point of 60-62°C, tissues were embedded or blocked in paraffin wax as the last step of tissue processing Impregnated tissues are placed in a mould with their labels and then fresh melted paraffin is poured in it and allowed to settle and solidify Once the block has cooled sufficiently to form a surface skin it should be immersed in cold water to cool it rapidly

Sectioning

Thin sections of about 5µm were cut off from the paraffin block with the use of microtome followed by placing the ribbon onto a warm water (~45ºC) floating bath where bath temperature should allow for spreading of ribbon without melting The next step was carried out where the sections from water were mounted on clean glass slides smeared with Mayer’s egg albumin In order to dry the slides, they were placed

on a hot plate at about 50°C for 30 minutes The slides were ready for staining procedure

Staining procedure using Haematoxylin and Eosin

Initially, the slides containing paraffin sections were placed in a metal slide holder To deparaffinize and rehydrate sections, the slides were immersed in xylene, a hydrocarbon solvent, to dissolve paraffin by passing through various alcohol 95%, 90

%, 80 %, 70 %, 50 % and 30 % for 5 minutes each one after the other Then the tissues were rinsed in running tap water for 5 minutes and stained using haematoxylin for 10 minutes The slides afterward were washed in running tap water for 10 minutes After that, they were stained by Eosin from 15 seconds to 5 minutes The stained slides were dehydrated again by immersed them through an increasing alcohol levels 30%, 50 %,

70 %, 80 %, 90 % and 95 % in 3 minutes for each were performed While in 95% of alcohol and then, in 100% alcohol soak the slides for 2 to 3 times in 1 to 2 minutes each Next, the slides were placed in acetone and two changes were given for 3 minutes each After that, they were dipped in xylene (absolute alcohol with ratio of 1:1) and two changes were performed for each 3 minutes The slides after clearing with xylene were then mounted in Dibutyl Phthalate Xylene (DPX) medium by putting

a drop of medium on the slide using a glass rod, taking care in order that no bubbles could occur and gently put the coverslip onto the slideto allow the medium to spread beneath the coverslip, covering all the tissue section Then the slides were dried overnight Finally, sections were examined under microscope with camera attachment and were photographed at both high as well as low magnifications The nuclei stained

blue and cytoplasm in different shades of pink The stained slides were then

investigated for histopathological alterations

PART IV RESULTS

4.1 Histopathological observation of gills

Histologically, the gills of the control tilapia Oreochromis niloticus showed the

normal architecture of gill filaments such as primary lamellae, secondary lamellae with

mucus cells lying scattered on both sides (figure 1) (Gobinath et al., 2014)

Figure 1 Normal histological structure of gills The structure of gill of the control fish showing primary lamellae (PL), secondary lamellae (SL), epithelial cell (EC), and mucous cell (MC) Stained with H&E, magnification is 400x

SL

PL

MC

EC

Figure 2 Comparison of the normal and treated gills A-B normal gills C-D Curling (c), atrophy (a), disintegration of secondary lamellae (dis)and shorter gill lamellae (sh) E-F Swelling (s), desquamation (des), epithelial lifting (e.l), curling bend of secondary lamellae(c) and necrosis (nec) A-F are stained with H&E, magnification of A, C and