(Luận văn thạc sĩ) toxicological effect and histopathological alterations on fish after insecticide active compound contamination

THAI NGUYEN UNIVERSITY UNIVERSITY OF AGRICULTURE AND FORESTRY DONG THI LINH CHI TOPIC TITLE: TOXICOLOGICAL EFFECT AND HISTOPATHOLOGICAL ALTERATIONS ON FISH AFTER INSECTICIDE ACTIVE COMPOUND CONTAMINATION BACHELOR THESIS Study Mode : Full-time Major : Environmental Science and Management Faculty : International Training and Development Center Batch : 2011-2015 Thai Nguyen, September 2015 n DOCUMENTATION PAGE WITH ABSTRACT Thai Nguyen University of Agriculture and Forestry Degree Program : Bachelor of Environmental Science and Management Student name : Dong Thi Linh Chi Student ID : DTN 1153110007 Thesis Title : Toxicological Effect and Histopathological Alterations on Fish after Insecticide Active Compound Contamination Supervisor (s): Dr Arinafril Dr Ho Ngoc Son Abstract: Deltamethrin, a pyrethroid insecticide which is both very widely used and has a very wide range of applications in agriculture, animal health and public health, was investigated in the present study for histopathological studies in walking catfish fingerlings (Clarias batrachus) Fish were exposed to ml/L, 1.5 ml/L, 1.75 ml/L, ml/L and 2.5 ml/L deltamethrin The histopathological changes were studied in the gill, liver, and heart of the deltamethrin treated fish Clarias batrachus The results showed tissue specific alterations in the tissues Inflammation of the gill primary lamellae and desquamation of secondary lamellae were observed In the liver, there were an intrusion of fat, degradation of hepatocytes and sinusoidal dilatation Edema in cardiac muscles, splitting of muscle fibers and infiltration of blood cells were an interesting observation in heart tissue In conclusion, exposure to deltamethrin will result in histological changes in gills, liver and heart of Clarias batrachus The i n damages in histology of heart, liver and gills depend on the exposure concentrations to deltamethrin As the exposure concentrations increased, the more adverse damage occurred Keywords: Deltamethrin, histopathology, Clarias batrachus … Number of Pages: 43 pages Date of Submission: 30th September, 2015 Supervisor’s signature ii n ACKNOWLEDGEMENT From bottom of my heart, I would like to express my deepest appreciation to all those who provided me the opportunity to complete this research First and 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 I also want to express my thanks to Dr Ho Ngoc Son, the second supervisor, for his supervision, encouragement, advice, and guidance in writing this thesis Besides my supervisors, I would like to thank Ph.D Dr Krisna MURTI in Department of Anatomical Pathology, who kindly assisted me with the histopathological detection in this dissertation and was very patient with my knowledge gaps 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 Dr Mohammad Zulkarnain, who gave the permission to use all required equipment and the necessary materials to conduct my research in Laboratory of Department of Anatomical Pathology, Dr Mohammad Hoesin Public Hospital Special thanks to Ms Mirna Fitrani, Mrs Ana, Mr Abi, and other staffs in Laboratory of Aquacuture, Sriwijaya University and Ms Hafsah in Pesticide Toxicology Laboratory for helping and providing me necessary equipment as well as knowledge for fish anatomy iii n I wish to thank the technicians who work in Immunohistochemistry Laboratory of Anatomical Pathology Department for their help in tissue preparation and Mrs Zakinah Arlina who provided me fish for free Without them, this research could not be accomplished on time My sincere thanks also go to Ha, Thao, Linh, and Marisa for helping me finish this study Of course, I would like to thank to my Indonesian friends - Ria, Abdul, Veby, Ren, 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, 30th September, 2015 Student Dong Thi Linh Chi iv n TABLE OF CONTENTS LIST OF FIGURES LIST OF TABLES PART I INTRODUCTION 1.1 Background and rationale .3 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 Deltamethrin .7 2.2 Toxic effects of deltamethrin on organisms .9 2.2.1 Toxicity 2.2.2 Histopathological Effects 13 2.3 Test species - Clarias batrachus 17 PART III MATERIALS AND METHODS 18 3.1 Time and Place .18 3.2 Materials 18 3.3 Equipment .19 3.4 Methods 19 PART IV RESULTS 22 4.1 Histopathological observation of gills 22 4.2 Histopathological observations of liver 25 4.3 Histopathological observations of heart 29 PART V DISCUSSION AND CONCLUSION 32 5.1 Discussion .32 5.2 Conclusions 35 REFERENCES 37 v n LIST OF FIGURES Figure Stock solution of deltamethrin - Decis 18 Figure Normal histological structure of gills .22 Figure Histopathological Changes observed in gills (H&E x200) 24 Figure Normal histological structure of liver (H&E x400) 25 Figure Histopathological changes in liver of fish exposed to 1.0ml/L deltamethrin 26 Figure Histopathological changes in liver of fish exposed to 1.5ml/L deltamethrin 26 Figure Histopathological changes in liver of fish exposed to 1.75ml/L deltamethrin 27 Figure Histopathological changes in liver of fish exposed to 2.0ml/L deltamethrin 27 Figure Histopathological changes in liver of fish exposed to 2.5ml/L deltamethrin 28 Figure 10 Normal histological structure of heart 29 Figure 11 Histopathological changes observed in heart of experimental fish (H&E x200) .30 Figure 12 Histopathological changes in heart of fish exposed to a) 1.0 ml/L, b) 1.5 ml/L, c) 1.75ml/L, d) 2.0ml/L and e) 2.5ml/L of deltamethrin 31 n LIST OF TABLES Table 1.Physical and Chemical Properties of Deltamethrin Table Toxicity classification of Deltamethrin 10 Table Categories of Acute Toxicity for Fish and Aquatic Invertebrates 13 Table Equipment used in laboratory 19 n PART I INTRODUCTION 1.1 Background and rationale In the recent years, pesticides, in general, are used very widely in agriculture, forestry, public health and in veterinary practices which have resulted in the increased levels of toxic chemicals in the aquatic environment As a matter of fact, fish - a part of human diet, is contaminated either directly or indirectly by pesticide-related water pollution, it can lead to fish kills, reduce fish productivity, or even elevate concentrations of undesirable chemicals in edible fish tissue which can affect the health of humans consuming these fish (Velisek et al., 2011) Pyrethroids are synthetic analogues of pyrethrins, insecticidal substances obtained from the flowers of a species of chrysanthemum (Chrysanthemum cinerariaefolium) and exposure in insects is predominately through the insect cuticle (Casida, 1980) It can be rapidly absorbed, especially with pyrethroids containing halogen, insect neurotransmission can be disrupted and then can cause knockdown and possibly death within seconds to minutes (Wakeling et al., 2012) Pyrethroids are less noxious to mammals compared to insects by reason of mammals higher body temperature, larger body size, and reduced sensitivity of the ion channel sites (Bradberry et al, 2005; Ray et al, 2006) Deltamethrin is a broad-spectrum pyrethroid insecticide which is both very extensively used and has a broad range of applications in agriculture, animal health and public health (US EPA, 2004) Deltamethrin is a pyrethroid insecticide which kills insects on contact and through ingestion, with a mechanism of disrupting their normal nervous system function and therefore giving a quick knock-down effect (NPIC, 2010) n In laboratory conditions, deltamethrin is highly toxic for fish, aquatic arthropods, and honeybees, whereas, in field conditions, lasting adverse effects are not likely to occur under recommended conditions of use (WHO, 1990) As a member of pyrethroids, deltamethrin is most commonly introduced into aquatic systems via runoff and erosion as well as the drift from aerial sprays, consequently, it could be potentially dangerous to benthic and epi-benthic species, as well as fish and other organisms feeding on benthos (You et al., 2008) Due to high toxicity to aquatic organisms, deltamethrin must be used with extreme caution around water (Sharma et al., 2013) In addition, the lethal concentrations of most of the pesticides cause varying degrees of histopathological injuries to different organs in fish Therefore, histology and histopathology could be used as bio-monitoring tools or indicators of health in toxicity studies as they provide early warning 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 causes some changes in the metabolic functions The changes in the functions are initiated with the changes in the tissue and cellular level The response of biomarkers can be regarded as biological and biochemical effects after a certain toxicant exposure, hence, histopathological examinations have been recognized to be reliable biomarkers of stress in fish (Van der Oost et al., 2003) n 4.3 Histopathological observations of heart The heart of the control fish Clarias batrachus (Figure 11, Figure 12a) showed normal structure of cardiac muscle (CM) and nucleus (N) Figure 10 Normal histological structure of heart Heart from control fish showing cardiac muscle (CM) and nucleus (N) In treated fish, generally, edema and thinning in muscle bundle were observed Particularly, in fish exposed to 1ml/L deltamethrin (Figure 12a), the infiltration of blood cells (I), edema (E) between muscle bundles and splitting of muscle fibers (SF) were seen In fish exposed to 1.5ml/L and 1.75ml/L (Figure 12b and c), the splitting of muscle fibers and atrophy (A) of cardiac muscle were observed more clearly As Figure 12d showed heart with edema of cardiac muscle bundle and infiltration of blood cell Separation and degeneration of muscles, infiltration of blood cell were observed in Figure 12e 29 n a) Control b) 1.0ml/L e) 2.0ml/L d) 1.75ml/L c) 1.5ml/L f) 2.5ml/L Figure 11 Histopathological changes observed in heart of experimental fish (H&E x200) Figure 11 shows the alterations in histopathological structure of control and treated fish at different concentration of deltamethrin with magnification of 200 The structural changes can be clearly observed by looking at the distance among carrdiac muscles Edema and splitting of muscle fibers are regconized as common changes recoreded in the heart of experimental fish Figure 12 below shows the histopathological alterations observed in heart of treated fish at different concentraions from 1.0ml/L to 2.5 ml/L of deltamethrin with magnification of 400 Edema in cardiac muscles, splitting of muscle fibers and infiltration of blood cells were an interesting observation in heart tissue of experimental fish The damages in histology of heart depend on the exposure concentrations to deltamethrin As the exposure concentrations increased, the more adverse damage occurred in the heart tissue of fish 30 n a) 1.0ml/L b) 1.5ml/L I: Infiltration of blood cells, SF: splitting of fibers E: edema, A: atrophy of muscle bundles c) 1.75ml/L SF: splitting of fibers E: edema, A: atrophy of muscle bundles d) 2.0ml/L SF: splitting of fibers E: edema, A: atrophy of muscle bundles SF: splitting of fibers, I: infiltration of blood cells E: edema, A: atrophy of muscle bundles e) 2.5ml/L SF: splitting of fibers, I: infiltration of blood cells E: edema, A: atrophy of muscle bundles Figure 12 Histopathological changes in heart of fish exposed to a) 1.0 ml/L, b) 1.5 ml/L, c) 1.75ml/L, d) 2.0ml/L and e) 2.5ml/L of deltamethrin 31 n PART V DISCUSSION AND CONCLUSION 5.1 Discussion The histological changes in fish is a noteworthy and promising field to understand the extent to which changes in the structural organization are occurring in the organs due to environmental pollution Deltamethrin exposure did significantly alter tissue structure in gills, liver and heart of Clarias batrachus at the concentrations tested For instance, in gills, inflammation and desquamation in both primary and secondary lamellae are the most common changes recorded while fatty degeneration, atrophy of hepatocytes, and vessel congestion were found in liver There were alterations in cardiac muscle structure such as splitting of muscle fibers, edema and atrophy of cardiac muscle in Clarias Batrachus The histopathological changes observed in all the tissues indicate that deltamethrin is highly toxic to fish The gills, besides having important functions in the fish such as respiration, osmoregulation and excretion, remain in close contact with external environment, are particularly sensitive to changes in the quality of the water, and hence are considered the primary target of contaminants (Fernandes & Mazon, 2003; Camargo & Martinez, 2007) The gills of fish exposed to deltamethrin showed the loss of gill architecture with inflammation of primary lamellae and desquamation of secondary lamellae at different concentrations of test chemical (Figure a, b, c, d, f) Gill is in contact directly with pollutants, thus, any kind of damage to the gill tissue of fish might lead to disorder in the gas exchange process as well as the decrease of ion regulation efficiency via this organ (Fernandes & Mazon, 2003) Histopathology of gills is the appropriate bio-indicator for monitoring pollution (Reethamma, 2014) The treated gills of fish showed the damage to varying extent depending on the exposure 32 n concentrations As the exposure concentrations increased the degree of damage also increased The present results are in agreement with those observed in other fish species under the influence of different pollutants (Kakuta & Murachi, 1997; Olojo et al., 2005; Olurin et al., 2006; Yildirim et al., 2006; Cengiz, 2006; Yogita et al., 2013) Liver is the organ most associated with the detoxification, it is also one of the organs most influenced by contaminants in the water (Camargo & Martinez, 2007) The liver of the fish exposed to different concentrations of deltamethrin showed fatty degeneration, degradation of hepatic cells and dilation of sinusoid blood vessel (Figure 5, 6, 7, and 9) These changes may be attributed to direct toxic effects of pollutants on hepatocytes, since the liver is the site of detoxification of all types of toxins and chemicals Hepatic tissue of treated specimens showed varied degree of fatty change as evidence for the abnormal retention of lipids within a cell It might indicate an imbalance between the rate of synthesis of substances in the parenchymal cells and the rate of their release into the circulation system (Gingerich, 1982) The halogencontaining deltamethrin, which are highly lipophilic (Casida, 1980), interfere with cellular machinery involved in lipid metabolism Therefore, it can be considered as the reason why liver of treated fish showing fatty degeneration Besides this, oxygen deficiency is the most common cause of the cellular degeneration in the liver Rodrigues and Fanta (1998); Olojo et al (2005); Camargo and Martinez, (2007); Atamanalp et al (2008); Mohamed, (2009); Velisek (2009) and Reethamma (2014) have also reported analogous observations with pesticides in various fish Edema in cardiac muscles, splitting of muscle fibers and infiltration of blood cells were an interesting observation in heart tissue (Figure 12a, b, c, d, e) The histopathological alterations in the cardiac muscle are in agreement with those 33 n observations by many investigators who have studied the effect of different pollutants on fish’s muscle (Elnemaki & Abuzinadah, 2003; Patnaik et al., 2011; Jeheshadevi et al, 2014; Reethamma, 2014) Separation and degeneration of muscles, atrophy of muscle bundles and focal area necrosis were recorded in muscle tissue of Etroplus maculatus when exposed to pesticide Fluben Diamide (Reethamma, 2014) On exposure to sublethal concentration of lead and cadmium, marked thickening and separation of muscle bundles, haemolysis, necrosis, lesions with reduced compactness as well as intra-muscular oedema were observed in Cyprinus carpio communis (Patnaik et al., 2011) The changes in the cardiac muscle may be due to the blocking sodium channel mechanism of deltamethrin The function of sodium channels is essential for normal transmission of nerve impulses and this process is disrupted by binding of the deltamethrin, leading to edema in heart tissues As studied by Abriel (2007) and Wakeling (2012), Nav1.5, the main voltage-gated Na+ channel in the heart which have been linked to various cardiac phenotypes such as dilated cardiomyopathy, is highly expressed in the cardiac muscle and easily blocked by type II pyrethroids In the present study, the alterations noticed in histopathology of their gills, liver, and heart obviously showed that the risks of using pesticides Long term exposure of organisms to pesticides has a meaning of a continuous health hazard for the population Hence, human population is also at high risk by consuming these contaminated fish This implies that one should take the necessary precaution in the application of pesticides to protect the life of fish and other aquatic fauna Pesticide toxicity in fish has been studied and shown that at chronic level, it causes diverse effects including histopathological changes as well as nephrotoxicity, hepatotoxicity, 34 n mutagenesis and carcinogenicity (Sharma et al., 2013) It is essential to formulate stringent rules against indiscriminate use of this pesticide For a great public health and a safe application of pesticide, it is important to monitor regularly pesticide residues in foods and humans in order to assess the population exposure to pesticides as well as more experimental work should be performed to determine the concentration and time of exposure that not induce significant sub-lethal effects on fish 5.2 Conclusions Deltamethrin posed a potential risk to walking catfish It is obvious that this kind of insecticide has a direct impact on the histological alterations in Clarias batrachus Deltamethrin is known to impair the metabolic and the physiological activities of the organism and through repeated exposure deltamethrin tends to accumulate in its tissues The findings of this histopathological investigations demonstrate a direct correlation between exposure to deltamethrin and histopathological disorders observed in several tissues The current study reinforces the application of histopathology as a powerful tool for monitoring anthropogenic contamination within aquatic environments Furthermore, besides the investigation of the histopathological effects of deltamethrin in the gill, liver and heart of Clarias batrachus, the important finding is the mortality appeared in the short time after exposure The observed effects are serious when the concentration of the product used in the tests is safely recommendable concentrations according to a perspective of environmental safety Ecological consequences may be significant if the action of the pesticide persists Therefore, it is not only necessary to recommend that the allowed levels for pesticides 35 n should be lower, the exposure should be the shortest as possible to make them become more secure and moreover, different species should be tested in order to assess their sensitivity 36 n REFERENCES Abriel Hugues (2007) Roles and regulation of the cardiac sodium channel Nav1.5: Recent insights from experimental studies 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