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. 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 1 ml/L, 1.75 ml/L, 2 ml/L and 2. 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. 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. 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. 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 .1 LIST OF TABLES. Background and rationale.
Research questions and hypotheses. Toxic effects of deltamethrin on organisms. Test species - Clarias batrachus. MATERIALS AND METHODS.
Time and Place. Histopathological observation of gills. Histopathological observations of liver. Histopathological observations of heart.
DISCUSSION AND CONCLUSION .37 v n LIST OF FIGURES Figure 1. Stock solution of deltamethrin - Decis. Normal histological structure of gills. Histopathological Changes observed in gills (H&E x200).
Normal histological structure of liver. Histopathological changes in liver of fish exposed to 1. Histopathological changes in liver of fish exposed to 1. Histopathological changes in liver of fish exposed to 1.
Histopathological changes in liver of fish exposed to 2.0ml/L deltamethrin 27 Figure 9. Histopathological changes in liver of fish exposed to 2.5ml/L deltamethrin 28 Figure 10. Normal histological structure of heart. Histopathological changes observed in heart of experimental fish (H&E x200).
Histopathological changes in heart of fish exposed to a) 1.5ml/L of deltamethrin.31 1 n LIST OF TABLES Table 1.Physical and Chemical Properties of Deltamethrin. Toxicity classification of Deltamethrin. Categories of Acute Toxicity for Fish and Aquatic Invertebrates. Equipment used in laboratory.
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. 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.
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). 3 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. Due to high toxicity to aquatic organisms, deltamethrin must be used with extreme caution around water (Sharma et al. 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.
Objectives This study was designed to assess the toxicological effects of deltamethrin on Clarias batrachus (Walking catfish) as well as to reveal the histopathological alterations on its gills, heart and liver under different concentrations of deltamethrin. Research questions and hypotheses 1. Research questions - How does deltamethrin affect the gills of walking catfish? - How does deltamethrin affect the liver of walking catfish? - How does deltamethrin affect the heart of walking catfish? 1. Hypotheses Hypothesis 1: HO (Null Hypothesis): Exposure to deltamethrin will not result in changes in gills histology of Clarias batrachus.
HA (Alternative Hypothesis): Exposure to deltamethrin will result in changes in gills histology of Clarias batrachus. Hypothesis 2: HO: Exposure to Deltamethrin will not result in changes in liver histology of Clarias batrachus. 5 n HA: Exposure to deltamethrin will result in changes in liver histology of Clarias batrachus. Hypothesis 3: HO: Exposure to Deltamethrin will not result in changes in heart histology of Clarias batrachus.
HA: Exposure to deltamethrin will result in changes in heart histology of Clarias batrachus. Limitations As far as deltamethrin is considered, there is a lack of experimental results about the histopathological effects on heart tissues of fish of deltamethrin in the literature.Physical and Chemical Properties of Deltamethrin (1R,3R) [ α-cyano(3-phenoxyphenyl)methyl] 3-(2,2-dibrom Chemical Name o-ethenyl)-2,2-dim e thylcyclopropanecarboxylate (IUPAC) Common Name Deltam ethrin Butoflin®; Butox ®; Decis ®; K-Othrin®; K-Othrine ® Trade Names Dust; Striker® IEC insecticide Structural Formula Chemical Formula C22H19Br2NO3 CAS Registry 52918-63-5 Number Molecular Weight 505.053 gm per m L at 20° C (Decis®) Specific Gravity 0.5 gm per m L at 20° C (Technical Grade of AI, TGAI) Physical State White to beige crystalline powder (TGAI) Boiling Point Not applicable Soluble in acetone, dimethylformamide, dioxane, ethyl Solubility acetate, and toluene (all 23 - 39%), relatively insoluble in water (i.2 ppb in 24 hours) Vapor Pressure 1.5 x 10-8 mmHg at 25 ° C (> 90% AI) Octanol/Water 2.7 x 105 at 25° C Partition Coefficient Henry's Law 2.7 x 10-6 atm / m3 per m ole Constant pH 5.9 (in a 1% aqueous dispersion) (Source: ATSDR, 2003; MacBean, 2010) 7 n Deltamethrin [(S)-a-cyano-3-phenoxybenzyl(1R,3R)-3-(2,2-dibromvinyl)-2,2- dimethylcyclo propan-carboxylate], one of Type II synthetic pyrethroid insecticides, is similar in structure to natural pyrethrins derived from the chrysanthemum flowers. Deltamethrin was synthesised in 1974 and first marketed in 1977. Deltamethrin is considered the most powerful and therefore the most toxic of the pyrethroids, up to three orders of magnitude more so than some (ETN, 1995).
Deltamethrin causes neurotoxicity in insects and mammals by a similar mechanism of action, the modulation of nerve axon sodium channels. Generally, pyrethroids affect normal production and conduction of nerve signals in the nervous system and act on nerve membranes by delaying the closing of the activation gate for the sodium ion channel. It is commonly used to control caterpillars on apples, pears, and hops, and for the control of aphids, mealy bugs, scale insects, and whiteflies on greenhouse cucumbers, tomatoes, potted plants, and ornamentals (ETN, 1995). Deltamethrin is the active ingredient in Butoflin, Butoss, Butox, Cislin, Crackdown, Cresus, Decis, Decis- Prime, K-Othrin, and K-Otek and it is the first potent and photostable insecticide belonging to the type II pyrethroid group (ATSDR, 2003).
Worthing (1983) found that deltamethrin is extremely stable on exposure to air and temperature, and it is more stable in acidic as compared to alkaline media. All pyrethroids have at least four stereoisomers, with different orientation of the substituents on the cyclopropane ring (or the equivalent part of the phenylacetate) despite the fact that deltamethrin is marketed as a single isomer cis (WHO, 1990). The isomers have different biological activities. In the summers of 1991 and 1995, the pesticide caused massive eel (Anguilla anguilla) kills in Lake Balaton, Hungary 8 n following application for mosquito control.
In 1995, the presence of deltamethrin was demonstrated in several other fish species and in sediment samples taken from the lake (Balint et al.