Pesticide residues contamination analysis in water samples of Sreenagar in and malathion effects on the mortality rate of tilapia, Oreochromis mossambicus (Peters, 1852)

A thesis submitted to the Department of Fisheries, University of

in partial fulfillment of the requirements for the degree of

Master of Science (MS) in Fisheries

Submitted By Examination Roll: 811 MS session: 2015-16 Registration Number: 2011-212-777 Registration session: 2011-12

Department of Fisheries University of Dhaka, Dhaka-1000 February 2017 Dhaka University Institutional Repository

Declaration by Student

I hereby declare that the dissertation entitled “Pesticide residues contamination analysis in water samples of Sreenagar upazila in Munshiganj district and malathion effects on the mortality rate of tilapia, Oreochromis mossambicus(Peters, 1852)” submitted to the Department of Fisheries, University of Dhaka for the degree of Master of Science (MS) is based on self-investigation carried out under the supervisions of Dr. Kaniz Fatema, Professor and Chairperson, Department of Fisheries,University of Dhaka, Dhaka- 1000, Bangladesh and Dr. Md. Hasanuzzaman, Senior Scientific Officer, Institute of Food and Radiation Biology (IFRB), Atomic Energy Research Establishment, Bangladesh Atomic Energy Commission, Dhaka,Bangladesh.

I also declare that this or any part of this work has not been submitted for any other degree anywhere. All sources of knowledge used have been duly acknowledged.

Name:Md. Nasir Uddin Examination Roll:811 MS session:2015-16 Registration Number:2011-212-777 Registration session:2011-12 Department of Fisheries, Faculty of Biological Sciences University of Dhaka, Dhaka-1000, Bangladesh Dhaka University Institutional Repository

Certificate

We certify that the research work embodied in this thesis entitled “Pesticide residues contamination analysis in water samples of Sreenagar upazila in Munshiganj district and malathion effects on the mortality rate of tilapia, Oreochromis mossambicus(Peters, 1852)” submitted by Md. Nasir Uddin, roll number: 811, session: 2015-16, registreation number: 2011-212-777 has been carried out under our supervision.

This is further to certify that it is an original work and suitable for the partial fulfillment of the degree of Master of Science (MS) in Fisheries from the Department of Fisheries, University of Dhaka.

We wish every success in his life.

Name of supervisors

1. Dr. Kaniz Fatema Professor and Chairperson

Department of Fisheries, Faculty of Biological Sciences

University of Dhaka, Dhaka- 1000, Bangladesh

2. Dr. Md. Hasanuzzaman

Senior Scientific Officer

Institute of Food and Radiation Biology (IFRB)

Atomic Energy Research Establishment,

Bangladesh Atomic Energy Commission, Dhaka, Bangladesh Dhaka University Institutional Repository

ACKNOWLEDGEMENTS

I would like to express my gratefulness to Almighty ‘Allah’ for allowing me to complete this study successfully. I am also obliged to my dear parents for their continuous supports and encouraging words.

I am extremely delighted to express my heartiest gratitude and thanks to my honorable supervisors Dr. Kaniz Fatema, Professor and Chairperson, Department of Fisheries, University of Dhaka, Dhaka-1000, Bangladeshand Dr.Md. Hasanuzzaman, Senior Scientific Officer (SSO),Agrochemical and Environmental Research Division (AERD), Institute of Food and Radiation Biology (IFRB), Atomic Energy Research Establishment (AERE), Bangladesh Atomic Energy Commission (BAEC), Ganakbari, Savar, Dhaka for their sincere guidance, inspiration, encouragement and thoughtful suggestions throughout my study.

I would like to thanks our MS exam committee chairman Professor Dr Ghulam Mustafa, Departmentof Fisheries, University of Dhaka.And all the honorable teachers of Fisheries Department for their co-operations and inspirations during the study.

I would like to express my profound gratitude to Dr. Md. Amin Uddin, Head and Chief Scientific Officer (CSO), Agrochemical and Environmental Research Division (AERD) and Dr. Md. Khorshed Alam, Director, and Chief Scientific Officer (CSO), Institute of Food and Radiation Biology (IFRB), Atomic Energy Research Establishment (AERE), Ganakbari, Savar, Dhaka for giving me permission to conduct research in the institute.

Thanks to all Scientists of AERD. Special thanks to Md. Alamgir Kabir, Junior Experimental Officer (JEO); Md. Abdul Hamid and Gopal Majumder AERD, IFRB, AERE, Ganakbari, Savar, Dhaka for their continuous support.

I would like to thanks all staffs of my department for their co-operation during the study.

I would also like to thanks all of my friends for their help of my research work.

February, 2017 The Author Dhaka University Institutional Repository

ABSTRACT Different types of pesticides are widely used in Bangladesh. Pesticide residues pose harmful effect to the target and non-target organisms like fish and many other aquatic and terrestrial organisms including human either directly or indirectly through the food chain. The present study was undertaken to determine contamination analysisof some selected organochlorine (DDT, DDE and DDD), organophosphorus (malathion, diazinon and chloropyrifos) and carbamate (carbaryl) pesticide residues in water samples of different sources from Sreenagar upazila in Munshiganjdistrict using High Performance Liquid Chromatography (HPLC) equipped with Ultra Violate (UV) detector.Thirty onewater samples were collected from fish pond and lake to analyze the suspected pesticide residues. None of the tested water samples was found to be contaminated with malathion, diazinon, chlorpyriphos, carbaryl or DDT and its metabolites (DDE and DDD). Malathion is an organophosphate pesticide used in agriculture and houses to control the variety of insects, which may exert adverse effect on untargeted organisms, such as fish and other aquatic animals. The Mozambique tilapia (Oreochromis mossambicus) is very much sensitive to malathion. Lethal concentration of malathion in tilapia (Oreochromis mossambicus) and the LC50 value was calculated in exposure of (24, 48, 72, and 96) h malathion. The test was performed by following semistic (renewal) bioassay method in which the exposure medium was exchanged after every 24 h to maintain toxicant strength and level of dissolved oxygen, as well as minimizing the ammonia excretion levels during this experiment. Based on the experiment, Malathion seems to be highly toxic to tilapia (Oreochromis mossambicus). The 24 h, 48 h, 72 h and

96 h LC50 value is 3.236 ppm, 2.362ppm, 1.256 ppm and 0.690 ppm respectively. The results showed that LC50 values is decreased with increase in exposure duration, in other words in longer periods of exposure, lower concentrations of pesticide is caused 50% mortality in studied fish population and their mortality rate is dose dependent.

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CONTENTS

ABSTRACT ...... i LIST OF FIGURES ...... iv LIST OF TABLES...... v LIST OF PLATES ...... vi LIST OF APPENDIX TABLES...... vii LIST OF ABBREVIATIONS...... viii CHAPTER 1 ...... 1 INTRODUCTION ...... 1 1.1 Pesticide contamination ...... 1 1.1.1 Background ...... 1 1.1.2 Pesticide...... 5 1.1.3 Pesticides used in Bangladesh...... 8 1.2 Malathion toxicology in fishes...... 9 1.3 Review of Literature ...... 10 1.3.1 Pesticide contamination...... 10 1.3.2 Malathion toxicology in fishes ...... 16 1.4 Justification of the study ...... 17 1.5 Objectives...... 18 CHAPTER 2 ...... 19 MATERIALS AND METHODS ...... 19 2.1 Pesticide contamination analysis ...... 19 2.1.1 Study area ...... 19 2.1.2 Apparatus used ...... 22 2.1.3 Other Instruments ...... 23 2.1.4 Chemical and reagents used in laboratory...... 23 2.1.5 Sample sources ...... 24 2.1.6 Procedures ...... 24 2.1.7 Operation with HPLC...... 33 2.1.8 Analysis of pesticide residues ...... 37 2.2 Malathion toxicology in tilapia...... 38 2.2.1 Experimental fish ...... 38 2.2.2 Procedure...... 39 2.2.3 Analysis of malathion toxicity in tilapia ...... 42

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CHAPTER 3 ...... 44 RESULTS AND DISCUSSION...... 44 3.1 Pesticide residues analysis in water samples ...... 44 3.1.1 Organochlorines (OCs) Pesticide Residues...... 44 3.1.2 Organphosphorous (Ops) and Carbamate Pesticide Residues ...... 46 3.2 Malathion toxicity in tilapia...... 50 3.2.1 Lethal concentration of malathion in tilapia...... 50 3.2.2 Mortality of tilapia at different concentration of malathion for 24, 48, 72 and 96 h exposure period ...... 50 CHAPTER 4 ...... 59 CONCLUSIONS AND RECOMMENDATIONS ...... 59 4.1 Conclusions...... 59 4.2 Recommendations...... 60 REFERENCES ...... 61 APPENDICES ...... 71

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LIST OF FIGURES Figure 1.1 General classification of pesticides ...... 6 Figure 2.1 Map of Munshianj district including the study area of Sreenagar upazila.....20 Figure 2.2 Map of Sreenagar Upazila including the location of sample collection (unions)...... 22 Figure 2.3 The study area including sampling locations ...... 26 Figure 3.1 Exposure of 24 h malathion mortality rate in tilapia...... 51

Figure 3.2 Graph showing the relationship of probit of kill with log10 concentration of malathion used to deduce the 24 h LC50 ...... 51 Figure 3.3 Exposure of 48 h malathion mortality rate in tilapia...... 53

Figure 3.4 Graph showing the relationship of probit of kill with log10 concentration of malathion used to deduce the 48 h LC50 ...... 53 Figure 3.5 Exposure of 72 h malathion mortality rate in tilapia...... 55

Figure 3.6 Graph showing the relationship of probit of kill with log10 concentration of malathion used to deduce the 72 h LC50 ...... 55 Figure 3.7 Exposure of 96 h malathion mortality rate in tilapia...... 57

Figure 3.8 Graph showing the relationship of probit of kill with log10 concentration of malathion used to deduce the 96 h LC50 ...... 57

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LIST OF TABLES Table 1.1 Classification of pesticide on the basis of different type of pest control ...... 7 Table 1.2 Classification of pesticides based on hazards ...... 8 Table 2.1 Number of municipality, union, mouza and village of Munshiganj district (Banglapedia, 2015)...... 20 Table 2.2 Sampling locations (Unions) of Sreenagar Upazila (Banglapedia, 2015) ...... 21 Table 2.3 Source of water samples and locations (Unions)...... 25 Table 2.4 Operating condition of HPLC (UV mode)...... 34 Table 3.1 Organochlorine pesticide residues in water samples of Sreenagar Upazila ....44 Table 3.2 Organophosphorus and carbamate pesticide residues in water samples of Sreenagar Upazila...... 46 Table 3.3 Mortality of tilapia in exposure of 24 h malathion ...... 50

Table 3.4 24 h LC50 value of malathion in tilapia obtained from CALCULATION OF

LC50 USING PROBIT ANALYSIS software...... 52 Table 3.5 Mortality of tilapia in exposure of 48 h malathion ...... 52

Table 3.6 48 h LC50 value of malathion in tilapia obtained from CALCULATION OF

LC50 USING PROBIT ANALYSIS software...... 54 Table 3.7 Mortality of tilapia in exposure of 72 h malathion ...... 54

Table 3.8 72 h LC50 value of malathion in tilapia obtained from CALCULATION OF

LC50 USING PROBIT ANALYSIS software...... 56 Table 3.9 Mortality of tilapia in exposure of 96 h malathion ...... 56

Table 3.10 96 h LC50 value of malathion in tilapia obtained from CALCULATION OF

LC50 USING PROBIT ANALYSIS software...... 58

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LIST OF PLATES Plate 2.1 Chemical and Reagents used in laboratory ...... 24 Plate 2.2 Samples stored in freeze ...... 25 Plate 2.3 Hexane distillation plant ...... 27 Plate 2.4 Water distillation plant...... 27 Plate 2.5 Separating funnel ...... 29 Plate 2.6 Separation of aqueous layer ...... 29 Plate 2.7 Hexane extract was treated with 5-g anhydrous sodium sulphate to remove traces of water...... 30 Plate 2.8 Water free extract is ready to evaporate...... 30 Plate 2.9 Rotary vacuum evaporator ...... 31 Plate 2.10 Evaporated hexane is ready for nitrogen drying ...... 31 Plate 2.11 Nitrogen generator ...... 32 Plate 2.12 Solvent is drying by nitrogen generator...... 32 Plate 2.13 Drying solvent is stored in refrigerator prior to HPLC analysis ...... 33 Plate 2.14 HPLC (Water Company) with UV Detector...... 34 Plate 2.15 Column used in HPLC ...... 36 Plate 2.16 Experimental fish ...... 40 Plate 2.17 Aquarium sets ...... 40 Plate 2.18 Malathion contaminated fish...... 41

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LIST OF APPENDIX TABLES Appendix table 6.1 Concentrations and the Peak Areas of DDT...... 71 Appendix table 6.2 Concentrations and the Peak Areas of DDE...... 71 Appendix table 6.3 Concentrations and the Peak Areas of DDD ...... 71 Appendix table 6.4 Concentrations and the Peak Areas of Malathion ...... 71 Appendix table 6.5 Concentrations and the Peak Areas of Diazinon ...... 72 Appendix table 6.6 Concentrations and the Peak Areas of Chlorpyrifos ...... 72 Appendix table 6.7 Concentrations and the Peak Areas of Carbaryl...... 72 Appendix table 6.8 Initial test to lethal concentration of malathion for tilapia in exposure of 24 h...... 72 Appendix table 6.9 Initial test to lethal concentration of malathion for tilapia in exposure of 48 h...... 73 Appendix table 6.10 Initial test to lethal concentration of malathion for tilapia in exposure of 72 h...... 73 Appendix table 6.11 Initial test to lethal concentration of malathion for tilapia in exposure of 96 h...... 74 Appendix table 6.12 Mortality of fish exposed to each concentration of malathion in exposure of 24 h...... 74 Appendix table 6.13 Mortality of fish exposed to each concentration of malathion in exposure of 48 h...... 75 Appendix table 6.14 Mortality of fish exposed to each concentration of malathion in exposure of 72 h...... 75 Appendix table 6.15 Mortality of fish exposed to each concentration of malathion in exposure of 96 h...... 76

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LIST OF ABBREVIATIONS ADB Asian Development Bank AERE Atomic Energy Research Establishment BBS Bangladesh Bureau of Statistics BC Basement Count BDL Bellow Detection Limit BHC Benzene Hexachloride BT Bacillus Thuringensis BTs Butyltins DD Double Distilled DAD Diode Array Detector DAE Department of Agricultural Extension DDD 1 ,1-dichloro-2,2-bis(p-chlorophenyl) ethane DDE 1,1-dichloro-2,2-bis(chlorophenyl) ethylene DDT 1, 1, l -trichloro-2,2-bis(p-chloropheny1) ethane DEA Desethylatrazine DFG Deutsche Forschungsgemeinschaft ECD Electron Capture Detector EPA Environmental Protection Agency EU European Union FAO Food and Agriculture Organization Fig Figure FY Fiscal Year GDP Gross Domestic Product GC-MS Gas Chromatography-Mass Spectrometry GHS Globally Harmonized System GoB Government of the People’s Republic of Bangladesh HCH Hexachlorocyclohexane HPLC High Performance Liquid Chromatography HYV High Yielding Varieties IFRB Institute of Food & Radiation Biology IGRs Insect growth regulators IPM Integrated Pest Management ISO International Organization for Standardization KL Kilo Liter MAC Maximum Acceptable Concentration

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MoF Ministry of Finance MoEF Ministry of Environment and Forestry MH Moderately Hazardous Ml Milliliter MT Metric Ton NHMRC National Health and Medical Research Council OC Organochlorine OP Organophosphorus compound PD Parkinson’s Disease POPS Persistent Organic Pollutants Ppm Parts Per Million Ppb Parts Per Billion PPP Plant Protection Products PHP Public Health Pesticide. RT Retention Time SD Standard Deviation SE Standard Error UN United Nation UNCED United Nations Conference on Environment and Development UV Ultra-Violate Uni Union US United States WHO World health Organization WS Water Sample mg/L Milligram per Liter µg/L Micro gram per Liter

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CHAPTER 1

INTRODUCTION 1.1 Pesticide contamination 1.1.1 Background Water is a transparent and nearly colorless chemical substance that is the main constituent of Earth's streams, lakes, and oceans, and the fluids of most living organisms.

Its chemical formula is H2O, meaning that its molecule contains one oxygen and two hydrogen atoms, that are connected by covalent bonds. Water covers 71% of the Earth's surface (CIA, 2008). It is vital for all known forms of life. On Earth, 96.5% of the planet's crust water is found in seas and oceans, 1.7% in groundwater, 1.7% in glaciers and the ice caps of Antarctica and Greenland, a small fraction in other large water bodies, and 0.001% in the air as vapor, clouds formed of ice and liquid water suspended in air, and precipitation.Only 2.5% of this water is freshwater, and 98.8% of that water is in ice (excepting ice in clouds) and groundwater. Less than 0.3% of all freshwater is in rivers, lakes, and the atmosphere, and an even smaller amount of the Earth's freshwater (0.003%) is contained within biological bodies and manufactured products (Gleick, 1993).

Fish can’t live without water but water quality is decreasing day by day due to various reasons, pesticide is one of them. Bangladesh is agricultural country and 85% people depend on agriculture for their livelihood. Agriculture sector contributes about 17% to the country’s Gross Domestic Product (GDP) and employs more than 45% of total labor forces (BBS, 2015). Bangladesh has a population of about 152.25 million as of July 2012, based on population census (BBS, 2014). But the provisional estimated population is 157.90 million with growth rate of 1.36% in Fiscal Year (FY) 2014–15 (MoF, 2015).The density of population was 843 per km2 in 2001 which increased to 964 per km2 in 2011 (BBS, 2013).More than 75% of the population lives in rural areas (BBS, 2014). The main occupation of this people is agriculture. According to Chowdhury et al. (2011), 80% of the people depend on agriculture for their livelihood. Agriculture contributes about 18.70% of the total GDP of Bangladesh (BBS, 2013). The GDP in agriculture is 3.04 in FY 2014–15 with great contribution (22.60%) in total agricultural income by Fisheries alone (MoF, 2015).

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Agriculture is the single largest contributor to income in Bangladesh and provided jobs for about 36 million out of the 58 million people employed in 2000 (ADB, 2004). In Bangladesh, about 40% of the crop loss is caused by pests and insects attack which is a considerable loss (Bagchi et al. 2009). Worldwide, there is an estimation of 70,000 different pest species including insects and mites, plant pathogens and weeds causing an estimated 14, 13 and 13% agricultural crop damage, respectively (Pimentel, 2009a).For providing food security to the increasing population, different types of actions have been taken like introduction of high yielding variety but they are very much vulnerable to pests and as a result, different types of pesticides have been introduced in paddy and crop fields. Using pesticides, crop loss from pests can be reduced to 35–42% (Pimentel, 1997) although risks of using pesticides are serious as well (Pimentel, 2009b). For higher production of vegetables, pesticides are being used frequently in Bangladesh (Hossain et al. 2015). Farmers spray excessive amount of organophosphate and carbamate pesticides in the cultivated lands due to inadequate knowledge and instruction about the application of pesticide (Bhattacharjee et al. 2012). Pesticides are toxic material which acts not only on insect and other pests, but also against spread of diseases like malaria, typhoid, plague or sleeping sickness (Islam et al. 2007).

Bangladesh is rich in fish wealth. In the innumerable rivers, canals, tanks and other low lying and depressed areas and paddy fields that remain under water for about 6 months in a year and cover nearly 12 million acres, tropical fish of hundreds of varieties are cultivated (BBS, 2016). The freshwater inland aquaculture production in Bangladesh is the 4th highest in the world. There are 260 freshwater and 475 marine fish species in the country. About 12 exotic species are being cultured in the country. The total annual fish production is estimated at 36.84 lakh metric ton (MT) in 2014-15, of which 20.16 lakh MT (47.71%) are obtained from inland aquaculture, 10.24 lakh MT(34.45%) from inland capture fisheries and 5.99 lakh MT (17.84%) from marine fisheries. Fisheries sector contributes 3.65% to GDP and 23.84% to agricultural GDP and 2.73% to foreign exchange earnings by exporting fish products in 2015-16 (MoF, 2016).

The economy of Bangladesh is mainly based on agriculture. So, agriculture is her economic backbone with the production accounting for about one third of the gross domestic product. In Bangladesh, 40% of the crop loss can be attributed to attack by pests and insects which is a significant loss (Bagchi et al. 2008). The widely cultivated high yielding variety is highly vulnerable to pests and diseases. So, the use of pesticide is

Page | 2 CHAPTER 1 INTRODUCTION now an inherent part of agriculture for pest control. Thus, agrochemicals, including pesticides are considered a critical aid in improving agricultural production and the prevention of crop losses during pre and post-harvest (Rahman et al. 1995).

Due to use of pesticides for agriculture the water around the paddy fields may get contaminated. Although pesticide is beneficial for pest control but it also poses a harmful effect to our environment such as the pollution of water. After application of pesticide in the crop field the pesticide is degraded in the soil by the soil microorganisms in some extent but many of the toxic pesticide is transported into surface and ground water by agricultural runoff rain water from the crop field. Ultimately, the surface and ground water might be highly contaminated due to this agricultural runoff pesticide (Bagchi et al. 2008). Use of pesticides has to be controlled to avoid contamination of food supplies and ecological imbalance, but present measures taken in Bangladesh are inadequate and farmers rarely implement standards, allowing sale of unregistered pesticides and misuse (Matin et al. 1998).

Various types of pesticides, such as organochlorine, organophosphorous and carbamate have been extensively used by farmers in Bangladesh (Dasgupta et al. 2007). Since organochlorine pesticides have been banned in 1993 according to the Bangladesh Environment Conservation Act, 1995 due to their high toxicity (Matin et al. 1998), organophosphorous pesticides are widely used in agriculture. In Bangladesh, it is estimated that up to 64% of the crop-producing area is treated with carbamates, while up to 35% of the crop-producing area is treated with organophosphates (Chowdhury et al. 2012).

The United Nation has declared 2005–2015 as the ‘‘Water for Life’’ decade (WHO/NHMRC, 2006). For the indiscriminate use of pesticide, the surface and ground water bodies are easily being contaminated by runoff water or by rain. The widespread use of pesticides may contaminate the environment and freshwater fish (Rahman, 2000). It is unavoidable of entering the fractions of applied insecticides into the aquatic ecosystems because of the application techniques for crop protection (Van Wijngaarden et al. 2005). Due to normal agricultural usage like spray drift, runoff, and leaching, pesticide residues enter into the adjacent water bodies (Van de Zande et al. 2000).

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From water bodies, pesticide residues directly or indirectly pass through the food chain and ultimately cause harm to human and other aquatic or terrestrial organisms. The exposure of pesticides to human may cause severe problems. Recio et al. (2001) concluded that organophosphorous pesticides (OP) may affect sperm chromosome segregation and augment the risk for genetic syndromes. Exposure to pesticide residues in the environment is associated with breast cancer risk under specific situations (Leary et al. 2004).

Almost 10% of the total cancer patients are found to be caused due to pesticide poisoning alone (Gu and Tian, 2005). Chen et al. (2004) observed that the incidence of breast cancer was linearly correlated with the frequency of pesticide uses and organochlorine pesticide DDT and its derivative DDE is possibly responsible for breast cancer.

In 1955, persistent organic pollutants (POPs) were first used in Bangladesh and endrin was the first imported pesticide, of which 3 metric tons (MTs) were imported for agricultural pest control (Badruddin, 2004; Rahman, 2004). The government provided a 100% subsidy of pesticides until 1974 and were supplied free of cost to the farmers (Badruddin, 2004). The subsidy was reduced to 50% from April 1974 to April 1975 when the Department of Agricultural Extension (DAE) sold pesticides of various types at a 50% subsidy. The pesticides industry was privatized in 1979 when 100% price was imposed (GoB and MoEF, 2005).

At present, there are a total of 141 active ingredients of which 48 active ingredients are registered as public health pesticides (PHP) represented by 122 trade names and 93 active ingredients registered as agricultural pesticides (AP) represented by 303 trade names (GoB and MoEF, 2005). Out of the total pesticides use, over 80 pesticides are used in the rice fields (Ara et al. 2014). In Bangladesh from the year 1998 to 2012, the consumption of pesticides increased from 11610.66 to 40882.94 metric tons (MTs) comprising insecticides, fungicides, herbicide, rodenticides and public hygiene (BBS, 2013).

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1.1.2 Pesticide According to Food and Agriculture Organization (FAO) any substance or mixture of substances intended for preventing, destroying, or controlling any pest, including vectors of human or animal disease, unwanted species of plants or animals, causing harm during the production, processing, storage, transport, or marketing of food, agricultural commodities, wood and wood products or animal feedstuffs, or substances that may be administered to animals for the control of insects, arachnids, or other pests in or on their bodies (FAO, 2002).

Pesticides are substances meant for attracting, reducing, and then destroying any pest (EPA, 2007).

1.1.2.1 History of pesticide Since before 2000 BC, humans have utilized pesticides to protect their crops. The first known pesticide was elemental sulfur dusting used in ancient Sumer about 4,500 years ago in ancient Mesopotamia. The Rig Veda, which is about 4,000 years old, mentions the use of poisonous plants for pest control (Rao et al. 2007). By the 15th century, toxic chemicals such as arsenic, mercury, and lead were being applied to crops to kill pests. In the 17th century, nicotine sulfate was extracted from tobacco leaves for use as an insecticide. Two more natural pesticides were introduced in the 19th century, pyrethrum, which is derived from chrysanthemums, and rotenone, which is derived from the roots of tropical vegetables (Miller, 2002). Until the 1950s, arsenic-based pesticides were dominant. Paul Muller discovered that organochlorines such as DDT were dominant, but they were replaced in the U.S. by organophosphates and carbamates by 1975. Since then, pyrethrin compounds have become the dominant insecticide. Herbicides became common in the 1960s, led by "triazine and other nitrogen-based compounds, carboxylic acids such as 2,4-dichlorophenoxyacetic acid, and glyphosate" (Ritter, 2009).

The first legislation providing federal authority for regulating pesticides was enacted in 1910 (Goldman, 2007). However, decades later during the 1940s manufacturers began to produce large amounts of synthetic pesticides and their use became widespread (Daly et al. 1998). Some sources consider the 1940s and 1950s to have been the start of the "pesticide era (Graeme, 2005). Although the U.S. Environmental Protection Agency was established in 1970 and amendments to the pesticide law in 1972 (Goldman, 2007), pesticide use has increased 50-fold since 1950 and 2.3 million tonnes (2.5 million short

Page | 5 CHAPTER 1 INTRODUCTION tons) of industrial pesticides are now used each year (Miller, 2002). 75% of all pesticides in the world are used in developed countries, but use in developing countries is increasing (Miller, 2004). In the 1960s, it was discovered that DDT was preventing many fish-eating birds from reproducing, which was a serious threat to biodiversity. The agricultural use of DDT is now banned under the Stockholm Convention on Persistent Organic Pollutants, but it is still used in some developing nations to prevent malaria and other tropical diseases by spraying on interior walls to kill or repel mosquitoes (Lobe, 2006).

1.1.2.2 Classification of pesticide Pesticides are classified on the basis of their usage, chemical nature, mode of action and by hazards (Badruddin, 2004).

Figure 1.1 General classification of pesticides

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1.1.2.3 Classification on the basis of chemical composition According to chemical composition, pesticide may be classified as

a. Inorganic compounds: barium, sulphur, copper, chlorates, mercury, fluorine and borates b. Organic compounds: organochlorine, organophosphorous, derivatives of carbonic thio and carbamates etc. c. Synthetic and biological pesticides: bacterial and fungal preparations, antibiotics and phytocides

1.1.2.4 Classification on the basis of different type of pest control Table 1.1 shows the classification of pesticide on the basis of different type of pest control

Table 1.1 Classification of pesticide on the basis of different type of pest control

Type Action Algicides Control algae in lakes, canals, swimming pools, water tanks, and other sites Antifouling agents Kill or repel organisms that attach to underwater surfaces, such as boat bottoms Antimicrobials Kill microorganisms (such as bacteria and viruses) Attractants Attract pests (for example, to lure an insect or rodent to a trap). (However, food is not considered a pesticide when used as an attractant.) Biopesticides Biopesticides are certain types of pesticides derived from such natural materials as animals, plants, bacteria, and certain minerals Biocides Kill microorganisms Disinfectants and Kill or inactivate disease-producing microorganisms on inanimate sanitizers objects Fungicides Kill fungi (including blights, mildews, molds, and rusts) Fumigants Produce gas or vapor intended to destroy pests in buildings or soil Herbicides Kill weeds and other plants that grow where they are not wanted Insecticides Kill insects and other arthropods Miticides Kill mites that feed on plants and animals Microbial pesticides Microorganisms that kill, inhibit, or out compete pests, including

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insects or other microorganisms Molluscicides Kill snails and slugs Nematicides Kill nematodes (microscopic, worm-like organisms that feed on plant roots) Ovicides Kill eggs of insects and mites Pheromones Biochemicals used to disrupt the mating behavior of insects Repellents Repel pests, including insects (such as mosquitoes) and birds Rodenticides Control mice and other rodents

1.1.2.5 Classification on the basis of toxicity The World Health Organization (WHO) has developed toxicity classifiation for chemical pesticides based on hazard are given table 1.2

Table 1.2Classification of pesticides based on hazards

S/N WHO hazard class Statement 1 Class Ia = Extremely hazardous Very toxic 2 Class Ib = Highly hazardous Toxic 3 Class II = Moderately hazardous Harmful 4 Class III = Slightly hazardous Caution 5 Class IV = product unlikely to present acute hazard in normal use

1.1.3 Pesticides used in Bangladesh Pesticide was first introduced in Bangladesh in 1951. About 2 tons of pesticides were imported in 1956-57 and the quantity was raised to about 8.000 metric ton in 1993 (ESCAP, 1987). In Bangladesh, up to 64 % of the crop-producing area is treatedwith carbamates and up to 35 % of the crop-producing areais treated with organophosphates. These substances can betransmitted to freshwater fish and ultimately contaminatethe fish (Rahman, 2000). Many pesticides used in Bangladesh contain chlorpyrifos, carbofuran and carbaryl. Even small amounts of chlorpyrifos can cause neurological disorders, such as attention deficit hyperactivity disorder (ADHD), a developmental disorder in fetusesand children (Rauh et al. 2006). At present about 4 dozen of insecticides with over 150 trade names have been registered in Bangladesh According to a recent government

Page | 8 CHAPTER 1 INTRODUCTION survey, pesticide-related poisonings account for 8% of deaths in Bangladesh for people aged 15-49. 39 insecticides are being used in Bangladesh in agricultural and public health sector (Satter, 1985). According to Ahmed, (1985) Bangladesh has been using 20 insecticides, 18 fungicides, 3 herbicides and 2 rodenticides in agricultural and public health sector.

Insecticides used in Bangladesh can be grouped into three categories, based on their chemical nature (Rahman and Alam, 1997).

1. Organochlorines (Ocs) 2. Organophosphorous (Ops) 3. Carbamates

Organochlorines:Organochlorine pesticides are chlorinated hydrocarbons used extensively from the 1940s through the 1960s in agriculture and mosquito control. DDT, BHC, Aldrin, Heptachlor are examples of chlorinated hydrocarbon pesticides. All these organochlorine pesticides are banned in Bangladesh.

Organophosphorus: A phosphorus-rich organic compound that contain a halidewhich phosphorylates cholinesterase and irreversibly inhibits its activity. The term is used often to describe virtually any organic phosphorus (V)-containing compound, especially when dealing with neurotoxic compounds. Many of the so-called organophosphates contain C- P bonds. The organophosphate pesticides were developed in Germany in the 1940s and soon became an important defense against agricultural pests. A total of about 190 registered organophosphorus insecticides under different common names e.g. malathion, chlorpyrifos, dimethoate, diazinon etc. available for use in Bangladesh.

Carbamates: A carbamate is an organic compound derived from carbamic acid

(NH2COOH). Unlike organophosphates, carbamates are not structurally complex. This class includes a total of 46 registered carbamates available for use in Bangladesh.

1.2 Malathion toxicology in fishes Malathion is an organophosphate pesticide considered hazardless for mammals, and thus has been used more frequently than other pesticides (Pandey et al. 2005). Malathion (O,O-Dimethyl S-(1,2-bis(ethoxycarbonyl)ethyl)) is commonly used in agriculture and houses

Page | 9 CHAPTER 1 INTRODUCTION to control the variety of insects including aphids, beetles, pill bugs and scales. Malathion may occur in natural water sources through run-off from agricultural fields or directly through careless application, which may exert adverse effect on untargeted organisms, such as fish and other aquatic animals, since fish are often at the top of the aquatic food chain and capable to concentrate the pesticides (Tsuda et al. 1997; Sapozhnikova et al. 2004). Fishes are greatly affected by the indiscriminate use of these pesticides. Fish appear to pose the same biochemical pathways to deal with the toxic effects of endogenous and exogenous agents as mammalian species does (Lackner, 1998). Since the fish constitute an important link in food chain and their contamination by pesticides imbalance the aquatic system hence, it is important to examine the toxic effects of pesticides on them. Malathion is highly toxic for aquatic biota (Moore et al. 2011). In laboratory studies, malathion has been observed to cause different types of deformities in early life stages of fish, including deformed notochord, yolk sac edema, bent body (Lien et al. 1997). Fish exposed to malathion showed abnormal behavior and dose- and time- dependent increase in mortality were also observed (Pandey et al. 2005). With respect to the undesirable effects of this pesticide on aquatic organisms, especially fishes as economically important organisms, studying toxic effects of such chemicals on fish species of inland water bodies is very important.

1.3 Review of Literature 1.3.1 Pesticide contamination Many research works have done related with pesticide residues in water and its harmful effects on fish and human in all over the world which are related with the present study. Some of the previous works relates with this study is reviewed here.

A study was undertaken by Ara et al. (2014) to identify Organochlorine and Organophosphorous pesticides residues in water samples of some paddy fields, ponds and tubewells in Rangpur district (Taragong Thana) of Bangladesh by using High Performance Liquid Chromatography (HPLC). Among thirty samples, Organophosphorus (Malathion) pesticide was found in the twelve samples while Organocholorine was absent. The level of the pesticide residues (Malathion) were found in the range of 2 to 221 ppm, which were above the maximum acceptable levels of total and individual pesticide contamination.

Page | 10 CHAPTER 1 INTRODUCTION

Hasanuzzaman et al. (2016) identify and quantify Organochlorine and Organophosphorous pesticides residues in water samples of different sources from of Bangladesh by using High Performance Liquid Chromatography (HPLC). Among thirty samples, Organophosphorus (Malathion) pesticide was found in the seven samples while Organocholorine was absent. The level of the pesticide residues (Malathion) were found in the range of 42.58 to 922.8 µg/L, whereas diazinon was detected only one sample and the concentration was 31.5 µg/L.

Chowdhury et al. (2012a) investigated the presence of organophosphorus and carbamate residues in 24 surface water samples and five ground water samples from Pirgacha Thana, Rangpur district, Bangladesh using high-performance liquid chromatography. A number of samples of surface water from paddy fields were found to contain chlorpyrifos, carbofuran and carbaryl at concentrations ranging from 0-1.189, 0-3.395 and 0-0.163 µg/L, respectively. Surface water from the lakes had chlorpyrifos, carbofuran and carbaryl at concentrations ranging from 0.544-0.895, 0.949-1.671 and 0- 0.195 µg/L, respectively.

Chowdhury et al. (2012b) collected twenty seven water samples from both paddy and vegetable fields in the Savar and Dhamrai in Bangladesh and analyzed to determine the occurrence and distribution of organophosphorus (chlorpyrifos, malathion and diazinon) and carbamate (carbaryl and 33 carbofuran) pesticide residues. High performance liquid chromatography, equipped with a photodiode array detector, was used to determine the concentrations of these pesticide residues. Diazinon and carbofuran were detected in water samples collected from at 0.9 µg/L and 198.7 µg/L, respectively. Malathion was also detected in a single water sample at 105.2 µg/L from Dhamrai Upazila. Carbaryl was the most common pesticide detected in Dhamrai Upazila at 14.1 and 18.1 µg/L, while another water sample from Dhamrai Upazila was contaminated with carbofuran at 105.2 µg/L. Chlorpyrifos was not detected in any sample. Overall, the pesticide residues detected were well above the maximum acceptable levels of total and individual pesticide contamination, at 0.5 and 0.142 µg/L, respectively, in water samples recommended by the European Economic Community (Directive 98/83/EC).

Bhattacharjee et al. (2012) carried out a study on the water samples of paddy fields and Kaliganga River of which were analyzed to provide base line data on

Page | 11 CHAPTER 1 INTRODUCTION cypermethrin, chlorpyrifos and diazinon residue by using high performance liquid chromatography. Levels of Cypermethrin, chlorpyrifos and diazinon detected in the paddy field water samples were (0.505 ± 0.011 µg/L), (0.06 ± 0.001 µg/L) and (0.039 ± 0.002 µg/L), respectively. 0.11 ± 0.003 µg/L of cypermethrin and 0.012 ± 0.0006 µg/L of chlorpyrifos were also identified in the water samples of Kaligonga River. Diazinon residue was not detected in the river water samples. The detected concentrations of pesticide residues in the river water were below the accepted maximum residue limit (MRL) value of drinking water (0.1 µg/L) adopted by the FAO/WHO Codex Alimentarius Commission. Cypermethrin and chlorpyrifos were chosen for decontamination through rice bran, as it was found in river water. Two gm rice bran could easily decontaminated 95.6 % and 96.4 % of cypermethrin and chlorpyrifos. The result of this study showed that pesticide residue was detected in water samples were below the MRLs value, which can easily be decontaminated through absorption of rice bran.

Chowdhury et al. (2012c) investigated the occurrence and distribution of organochlorines such as aldrin, dieldrin, dichlorodiphenyldichloroethylene (DDE), dichlorodiphenyldichloroethane (DDD), dichlorodiphenyltrichloroethane (DDT), endrin, lindane and heptachlor insecticide residues in irrigated surface water samples collected from 22 districts in Bangladesh. The concentrations of the pesticides were determined using gas chromatography mass spectrophotometry. Water samples from five locations Nawabganj, Putia, Burichang and Chatak) were contaminated with DDT; the highest DDT concentration detected was 8.29 µg/L, and its metabolite, DDE, was detected at 4.06 µg/L. Water samples from four other locations (Natore, Sikderpara, Chatak and Rajoir) were contaminated with heptachlor residues, and the highest level detected was 5.24 µg/L, which is the above the maximum contaminant level recommended by the World Health Organisation. A water sample collected from Chatak, Sunamganj, was contaminated with both DDT and heptachlor pesticide residues. None of the water samples were contaminated with aldrin, DDD, dieldrin, endrin or lindane. It is concluded that continuous, long-term monitoring and essential steps to limit the use of the pesticides in Bangladesh are needed.

Hossain et al. (2014) in a study, collected water samples to monitor pesticide residues, and degradation of chlorpyrifos was also performed using soil bacteria. Identification of pesticide residues and determination of their levels were performed by high performance

Page | 12 CHAPTER 1 INTRODUCTION liquid chromatography with photodiode array detector. Among 12 samples, 10 samples were found contaminated with pesticides. Chlorpyrifos was detected in four tested samples and concentrations ranged from 3.27 to 9.31 µg/L whereas fenitrothion ranging from (Below Detection Limit 0.1 µg/L) to 33.41 µg/L in the tested samples. Parathion was found in two tested samples at the concentration of 0.73 and 6.23 µg/L. None of the tested samples was found contaminated with Methoxychlor, DDT and Ethion. Three soil bacterial isolates. Pseudomonas peli BG1, Burkholderia caryophylli BG4 and Brevundimonas diminuta PD6 degraded chlorpyrifos completely in 8, 10 and 10 days, respectively, when 20 mg/L chlorpyrifos was supplied as sole source of carbon. Whereas, BG1, BG4 and PD6 took 14,16 and 16 days, respectively, for complete removal of 50 mg/L chlorpyrifos. Chlorpyrifos degradation rates were found maximum by all three isolates at 2nd day of incubation for both tested concentrations.

Uddin et al. (2013) collected some water samples from some selected ponds of Meherpur region and examined for the presence of organophosphorus and carbamate pesticide residues. High performance liquid chromatography (HPLC) technique was used to determine the concentration levels of those residues. The results observed that slight contamination of some of the water samples with residues of diazinon, chlorpyrifos (organophosphorus insecticide) and carbofuran (carbamate insecticide). The residues level of diazinon and chlorpyrifos ranged from 0.033 to 0.079 ppm and 0.010 to 0.471 ppm respectively. Among carbamate pesticides, carbofuran identified in two samples ranged from 0.0143 to 0.0387 ppm, and carbaryl was not detected in any of the samples. Limit of detection (LOD) was 0.01 ppm. However, the residue level was also within the acceptable ranges according to the WHO guideline value of water quality.

In a study of Hasan et al. (2010) despite the worldwide ban, Persistent of Organic Pollutants (POPs) and other organochlorine pesticides are widely in use in developing countries including Bangladesh. The presence of POPs has been indicated in different matrices, such as poultry, fish, vegetables, lake water etc. This study detected POPs and organochlorine pesticides (HCB, O, P - DDT, DDD, 4, 4 -DDT, and α-HCH, β-HCH, and δ-HCH) in water samples from and around agriculture fields. The water samples were analyzed using Gas Chromatograph equipped with an Electron Capture Detector (GC-ECD). The highest extractable concentration among all the DDT analytes was 121.793X10-2 µgL-1 of 4, 4 -DDT. Among the HCH analytes, δ-HCH was present in all

Page | 13 CHAPTER 1 INTRODUCTION the samples. There is serious concern whether these bioaccumulative substances have now found their way into the fatty tissues of human body.

Islam et al. (2007) conducted a study to determine the organochlorine pesticides (aldrin, Para para-DDT, dieldrin, endrin, heptachlor and lindane) residues in surface water samples of the agricultural field of Bangladesh. Water samples were collected from different regions of Bangladesh using the administrative infrastructure of Department of Agricultural Extension (DAE) during 2005-2006. Gas Chromatography-Mass Spectrometry (GC-MS) techniques were used to determine the organochlorine pesticides residues in water samples. Results indicates slight contamination of some water samples of irrigated agricultural field samples with P, P-DT, heptachlor and lindane, while majority of the samples were found to be free from contamination. Aldrin, dieldrin and endrin were not found in any sample studied agricultural field.

An investigation was conducted by in Niger Delta, Nigeria (Upadhi and Wokoma, 2012). The levels of selected pesticides in surface waters, sediment and fish (Mudskipper) in the Elechi creek was carried out in two seasons and at three pre-determined sites. A higher concentration of 2, 4-diamine was observed in all stations and matrixes, while propoxur was least observed. Pesticide concentration in sediment is significantly different from that of water and fish tissue, which had the highest residual concentration. Concentration of pesticides ranged from 0.01 to 0.04 µg/L in water, 0.01 to 0.06 µg/L in sediment and 0.01 to 0.07 µg/L in fish, respectively. There was bioaccumulation of pesticides in fish samples. Observed residues are generally higher than stipulated limit of 0.01 µg/L by USEPA for pesticides of aquatic life, therefore, possess an ecological risk to the ecosystem and consequently human health.

A study conducted by Singh et al. (2010) mention that the cypermethrin (synthetic pyrethroids) showed strong pesticidal activity in freshwater teleost fish Colisa fasciatus for all the exposure periods (24h or 96h) in time as well as dose dependent manner. The

LC50 values decreases from 0.009 (24h) to 0.006 (96h) in winter season (water temp. 16°C) and 0.06 (24h) to 0.02 (96h) in summer season (water temp. 28'C). Sub-lethal doses (40 and 60% of LC50) of cypermethrin after 96h was also significantly alter the, levels of total protein, total free amino acid, in muscle and liver tissues, nucleic acids (DNA and RNA) in gonadal tissues and the activity of enzyme acetylcholinesterase

Page | 14 CHAPTER 1 INTRODUCTION

(AChE), lactic dehydrogenase (LDH) and succinic dehydrogenase (SDH) in nervous tissue of the freshwater teleost fish C. fasciatus in time and dose dependent manner.

Leena et al. (2012) reported that reports the concentration levels and distribution pattern of the persistent pesticides residues in water and bed sediments of the Ganga river at Bhagalpur stretch collected seasonally over a period of 2 years. The water and bed sediment were collected from three different sites (upstream, midstream and down- stream) and analyzed for their pesticide profile. The study has shown the presence of both organochlorine and organophosphorous pesticides in the water and sediment of River Ganga at Bhagalpur. We used Liquid-liquid extraction followed by GC-ECD for the determination of these compounds. Among the various pesticides analyzed, high concentration of Methyl parathion, Endosulfan and DDT were observed both in water and sediment samples of the river. The concentration of DDT in sediment was quite high which might be due to slow degradation of DDT in soil i.e. 75 - 100% in 4-30 years.

In a study of Kafilzadeh et al. (2012) investigated the levels of organochlorine (OC) pesticide residues in Lake Parishan have been investigated using water, sediment and fish (Barbus brachycephalus caspius) samples as a case study to find out the extent of pesticide contamination and accumulation in the lake. Six OC pesticides namely DDT, DDE, Lindane, Endosulfan, Heptachlor and Chlordane were analyzed in four sites at four seasons. Water samples were processed using a liquid-liquid extraction technique and gas chromatograph equipped with electron capture detector (GC-ECD). Soxhlet extraction was used for fish and sediment samples followed by clean up and gas chromatograph. DDE was the predominant residue in all the samples analyzed, at the mean concentrations of 0.055 ppb, 9.84 ppb and 4.86 ppb in water, sediment and fish samples, respectively. The lowest levels of OC pesticides were related to Heptachlor and Chlordane which none of them were found in water samples. Pol-e-Abgineh and Midstream sites had the highest and the lowest concentrations of OC pesticides, respectively.

In an investigation of Caldas et al. (2010) residues of four pesticides in groundwaters were surveyed during one year in an agricultural area in southern Brazil. The methods for separation, identification and quantification of the compounds included high- performance liquid chromatography with diode array detection (HPLCDAD) and liquid chromatography with electrospray interface tandem mass spectrometry (LC-

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ESIMS/MS). A pre-concentration step based on solid-phase extraction with 200 mg C18 cartridges was applied. All the analytical parameters were within the limits suggested for the validation of chromatographic methods. The LOQs for the methods, considering the 250-fold pre-concentration step, were 0.2 µg/L for all pesticides by HPLC-DAD, 4.0 ng L-l for clomazone, carbofuran and tebuconazole and 40.0 ng L-l for 2,4-D by LC-ESI- MS/MS. In groundwater samples, 2,4-D was not detected, and carbofuran, clomazone and tebuconazole were detected in concentrations ranging from 0.25 to 10.40 µg L-l, from 0.20 to 0.82 µg L-l and from 0.20 to 4.16 µg L-l, respectively. The methods proved to be adequate for the analysis of pesticides in groundwaters.

Manjunatha et al. (2015) investigated the extraction of chlorpyrifos in water and liver tissue of zebrafish (Danio rerio). Water was collected at 0, 24, 48, 72 and 96hrs after the addition of toxicant and the amount of chlorpyrifos present in the water and liver tissue. In the concentration level range of 200 µg/L for the extraction of chlorpyrifos from water and liver tissue. Amount of chlorpyrifos in water and liver tissue analyzed by HPLC to know the amount of residue left out in the water and zebrafish liver after addition of 200 µg/L of chlorpyrifos initially. The recoveries were the decrease of concentration chlorpyrifos in water and increase in liver with increase in time of exposure is an indication of the accumulation of the toxicant in the organism through uptake. This could be hazardous as it could make its way into the food chain.

1.3.2 Malathion toxicology in fishes Many research works have done related with malathion toxicity and its harmful effects on fish in all over the world which are related with the present study. Some of the previous works relates with this study is reviewed here.

The literature of Pickering et al. (1962) for Fathhead fish (25 ppm) and gold fish (0.79 ppm) shows that the value of Killifish (1.8 ppm) studied by Tsuda et al. (1997).Vittozi and De Angelis, (1991) summarized the 96 h LC50 values of malathion 0.091 to 22.09 ppm for different species.According to International Journal of Environmental

Monitoring and Analysis 2015; 3(5-1): 30-37 the 90h-LC50 of malathion for O. niloticus was (2.71 ppm).

Pathiratne and George, (1998) reported that the 96h LC50 for Nile tilapia was 2.2 ppm.

The 96 h LC50 value is 5 ppm for O. niloticus and 8.6 ppm for minnows documented by (Kandiel et al. 2014; Durkin et al. 2008). Al-Ghanim et al.(2012) reported a lower 96h

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LC50 value (1.06 mg/L) for O. niloticus.Malathion was toxic to Labeo rohita and the

LC50value 4.5 µg/L reported by Patil and David,(2008). As Malathion was toxic to C. gariepinus (LC50 8.22 mg/L) as recorded by Ahmad,(2012).

The difference in toxicity to the different fish species might be due to differences in the absorption of pesticides, their accumulation, biotransformation and excretion (Omitoyin et al.(2006).The magnitude of toxic effects of pesticides also depends on length and weight, corporal surface to body weight ration and breathing rate (Al-kahem et al. 1998).

Fishes are particularly sensitive to environmental pollution of water. Hence, pollutants such as insecticides may damage certain physiological and biochemical processes when they enter into the organs of fishes (John, 2007).Malathion was toxic to Colisa fasciatus and the 96 h LC50 value 2.12 ppm as reported by Singh et al.(2004). Reported by

Areechon and Plumb,(1990) 96 h LC50 of malathion was obtained 9.65 ppm.The 96 h

LC50 value for Labeo rohita is 9 ppm as reported by Pathiratne and George, (1998).Malathion was toxic to Oreochromis niloticus and the 96 h LC50 value 2 ppm reported by Patil and David, (2008).

According to Khangarot and Ray, (1988).Oryzias latipes 96 h LC50 of malathion was obtained 1.8 ppm.Malathion was toxic to Channa punctatus and the 96 h LC50 value is 6.61 ppm reported by Pandey,(2005).The 96 h LC50 value for Channa punctatus 1.61 ppm reported by Khangarot and Ray, (1988).The 96 h LC50 value for Primephales prmelas is 12.5 ppm and for Oncorhynchus mykiss is 0.1 ppm reported by Pickering et al. (1962).Malathion was toxic to Cyprinus carpio and the 96 h LC50 value is 2.1 ppm reported by Kaur and Dhawan, (1993).

1.4 Justification of the study Munshiganj District is agriculture based and Sreenagar Upazila is famous for rice production and other crops like wheat, potato etc. Various vegetables are also cultivated in this area. Bangladesh lacks sufficient information on pesticide use, even at the regional level. Pesticides have been used legally or illegally for various purposes. At present, information is not available about the level of pesticide residues in different ponds and lake water of Munshiganj. The fish ponds and lake water are used for fisheries sector. It is important to know the correct level of pesticide residues in different fish pond and lake water. The safe and effective use of these pesticides is essential to avoid contamination of the water and soil environments of these regions. This study is very

Page | 17 CHAPTER 1 INTRODUCTION important to monitor pesticides residues in water and fill gaps in current knowledge, to get early warning in the future problems. Therefore, the present study was carried out to determine pesticide residues in fish pond and lake water of Sreenagar Upazila in Munshiganj district and malathion effects on the mortality rate of tilapia, Oreochromis mossambicus. As the Mozambique tilapia (Oreochromis mossambicus) is a popular fish for aquaculture which is very much sensitive to malathion. Therefore, it is important to determine the toxicity of malathion in tilapia (Oreochromis mossambicus).

1.5 Objectives Sreenagar Upazila are famous for various types of crops. Consequently, pesticides have been used legally or illegally in considerable quantities in Sreenagar Upazila for various purposes. But now information is not available about the level of pesticide residues in the differen type of Samples (Fish pond, lake etc.) of in Sreenagar Upazila. Moreover, there are no substantioal works have yet been done for the determination of pesticide residues levels in the water samples of Sreenagar Upazila. The Mozambique tilapia (Oreochromis mossambicus) is very much sensitive to malathion, to determine the toxicity of malathion in tilapia find out the LC50 value.

Therefore, the present investigation has been undertaken to highlight the following objectives:

1. To identify the pesticide residues in fish pond and lake watersamples 2. To propose a sound recommendation of this region about the level of pesticide exposure and the quality of fish pond and lake water 3. To demonstrate the present status of pesticides used in this area 4. To investigate the malathion toxicity in tilapia

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MATERIALS AND METHODS 2.1 Pesticide contamination analysis 2.1.1 Study area The experiment comprising the study on the monitoring of pesticide residues in fish pond and lake water of 14 unions of Sreenagar Uazila at Munshiganj district in Bangladesh. For this reason, 31 water samples (16 pond water and 15 lake water)were collected from 14 unions of Sreenagar Upazila and brought to the Agrochemical and Environment Research Division (AERD), Institute of Food and Radiation Biology (IFRB), Atomic Energy Research Establishment (AERE), Savar, Dhaka. The water samples were processed for subsequent experiments and for necessary analysis.

2.1.1.1 Munshiganj district 2.1.1.1.1 Geographic Area and Location Munshiganj District () area 954.96 sq km, located in between 23°23' and 23°38' North latitudes and in between 90°10' and 90°43' East longitudes. It is bounded by Dhaka and Narayanganj districts on the north, Madaripur and Shariatpur districts on the south, Comilla and Chandpur districts on the east, Dhaka and Faridpur districts on the west (BBS, 2015).

2.1.1.1.2 Administration Munshiganj district consists of 6 upazilas, 67 unions, 662 mauzas, and 911 villages. The upazilas are Gazaria, Tongibari, Munshiganj Sadar, Lohajang, Sreenagar and Serajdikhan.

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Table 2.1 Number of municipality, union, mouza and village of Munshiganj district (Banglapedia, 2015)

Name of Area Municipality Union Mouza Village Upazila (sq km) Gazaria 130.92 - 8 114 133 Tongibari 149.96 - 12 113 156 Munshiganj 160.79 2 9 94 187 Sadar Lohajang 130.12 - 10 115 110 Sreenagar 202.98 - 14 102 148 Serajdikhan 180.19 - 14 124 177

2.1.1.1.3 Main Rivers The principal rivers of the district are Padma, Meghna, Dhaleshwari, Ichamati.

Figure 2.1 Map of Munshianj district including the study area of Sreenagar upazila

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2.1.1.2 Sreenagar Upazila 2.1.1.2.1 Geographic Area and Location Sreenagar Upazila (Munshiganj district) area 202.98 sq km, located in between 23°27' and 23°38' North latitudes and in between 90°10' and 90°22' East longitudes. It is bounded by Serajdikhan and Nawabganj (Dhaka) upazilas on the north, lohajang and shibchar upazilas on the south, Serajdikhan and Lohajang upazilas on the east, Nawabganj and Dohar upazilas on the west (BBS, 2015).

2.1.1.2.2 Administration Sreenagar Thana was turned into an upazila in 1983. The Sreenagar upazila of Munshiganj has 14 unions, 102 mouzasand 148 villages. The unions are Atpara, Kukutia, Kola Para, Tantar, Patabhog, Baghra, Baraikhali, Birtara, Bhagyakul, Rarikhal, Sholaghar, Shyamsiddhi, Sreenagar and Hasara union (BBS, 2015). The water samples of fish pond and lake water collected from these 14 unions (Figure 2.2)

Table 2.2 Sampling locations (Unions) of Sreenagar Upazila (Banglapedia, 2015)

Unions of Sreenagar upazila S/N Name of Unions GO Code 1 Atpara 06 2 Kukutia 54 3 Kola Para 47 4 Tantar 94 5 Patabhog 61 6 Baghra 13 7 Baraikhali 20 8 Birtara 33 9 Bhagyakul 27 10 Rarikhal 67 11 Sholaghar 81 12 Shyamsiddhi 88 13 Sreenagar 74 14 Hasara 40

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Figure 2.2 Map of Sreenagar Upazila including the location of sample collection (unions)

2.1.1.2.3 Main Rivers The principal rivers of the upazila are Padma, Ichamati.

2.1.2 Apparatus used a) Rotary Vacuum Evaporator (BUCHI, Switzerland) b) Freezer (General, ER-141F Japan) c) Ice box d) Desiccators e) Separating funnel set f) High Performance Liquid Chromatography (HPLC) (Waters Company, England)

g) Column C18 (Nova Pack) h) Clean up column Page | 22 CHAPTER 2 MATERIALS AND METHODS

i) Distillation Plant and j) Nitrogen Blower (Alfa Industry, England)

2.1.3 Other Instruments a) Beaker (100, 250 and 500 ml) b) Round bottom flask c) Burette (100 ml) d) Measuring cylinder (50 and 100 ml) e) Sample vial (10 ml) f) Conical flask (50, 100 and 250 ml) g) Funnel (50 and 100 ml) h) Burette (100 ml) i) Glass wool j) Micro syringe (1-100 pi) and (1-5 ml) k) Glass wear l) Distilled water m) Brush n) Gloves o) Cotton and tissue paper p) Detergents q) Aluminium foil r) Stand and holder s) Aquarium t) Aerator

2.1.4 Chemical and reagents used in laboratory In order to analyze the pesticides residues in water chemicals and reagents were used in the laboratory (plate 2.1) this are shown in the appendices.

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Plate 2.1 Chemical and Reagents used in laboratory

2.1.5 Sample sources In this study for detection and analysis of suspected pesticide residues in selected water bodies the samples were collected from two different sources. Here two sources were surface water body.

i. Fish pond water ii. Lake water

2.1.6 Procedures Fish pond and lake water were collected from 14 unions of Sreenagar upazila (Table 2.2) of Munshiganj District in Bangladesh. Total 31 samples from 2 different sources (fish pond and lake) of each union of Sreenagar upazila were collected in July, 2016 for the detection and analysis of pesticide residues and after collection the water samples were processed for subsequent experiments and for necessary analysis.

2.1.6.1 Collection and preservation of water samples The water samples of both fish pond and lake were collected in July, 2016. The water samples were collected randomly from 2 different sources from the study area in 500 ml plastic bottles and were labeled and immediately carried to the Pesticide Residue Analysis Laboratory of Agrochemical and Environment Research Division (AERD) at the Institute of Food and Radiation Biology (1FRB), Atomic Energy Research Establishment, Saver, Dhaka and kept in freezing condition (at -20°C) (plate 2.2) until extraction to avoid degradation (Table 2.3).

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Plate 2.2 Samples stored in freeze

Table 2.3 Source of water samples and locations(Unions)

Source of Sample

Locations/ Fish Pond-P Lake-L Unions

Atpara-1 1-A-P 1-A-L Kukutia-2 2-K-P 2-K-L Kola Para-3 3-KP-P 3-KP-L Tantar-4 4-T-P 4-T-L Patabhog-5 5-P-P 5-P-L Baghra-6 6-B-P 6-B-L Baraikhali-7 7-BA-P 7-BA-L Birtara-8 8-BI-P 8-BI-L Bhagyakul-9 9-BH-P 9-BH-L Rarikhal-10 10-R-P 10-R-L Sholaghar-11 11-S-P 11-S-L Shyamsiddhi-12 12-SH-P 12-SH-L Sreenagar-13 13-SR-P 13-SR-L Hasara-14 14-H-P 14-H-L

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Atpara=A, Kukutia=K, Kola Para=KP, Tantar=T, Patabhog=P, Baghra=B, Baraikhali=BA, Birtara=BI, Bhagyakul=BH, Rarikhal=R, Sholaghar=S, Shyamsiddhi=SH, Sreenagar=SR and Hasara=H

Figure 2.3The study area including sampling locations

= Sreenagar Upazila of Munshiganj District

= Unions of Sreenagar Upazila

2.1.6.2 Solvent Preparation in Laboratory Main purpose of solvent preparation was to remove impurities through solvent distillation plant. Science the boiling temperature of n-hexane is 65 C, its starts to vaporize at this temperature and after coming contract with continuous ⁰water supply, it concentrates and it was collected in round bottom flask that was connected with the stand (plate 2.3). By using this method, the organic solvent was made double distilled form. Water was also double distilled using distillation plant (plate 2.4)

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Plate 2.3 Hexane distillation plant

Plate 2.4 Water distillation plant

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2.1.6.3 Cleaning and Washing the Apparatus In the beginning of the extraction necessary glass wares were cleaned and rinsed by distilled water, detergent, and chemical (acetone and double distilled n-hexane). Then the glass wares were heated at 100 C in oven for 25-30 minutes to remove unexpected contaminants. After heating, the ⁰ apparatus were taken from the oven and kept in the basket to become normal in temperature. Then the glass wares were ready to be used.

2.1.6.4 Extraction of water The Extraction procedure was followed according to the method of Ara et al. (2014). Extraction of water sample (250 mL each) was performed with 100 mL double distilled hexane in a separating funnel with shaking for 5 minutes and then kept in stand for 10 minutes for settling down (Plate 2.5). Then the upper hexane and lower aqueous layer was collected in separate conical flask (Plate 2.6). The aqueous layer was re-extracted for two further extractions adding 50-mL solvent (DD-hexane) for each re-extraction and the solvent layer was collected. The combined hexane extract was treated with 5-g anhydrous sodium sulphate to remove traces of water (plate 2.7). The water free extract was evaporated by using rotary vacuum evaporator (Plate 2.9) to a small volume (Approx. 1 mL) and transferred to a cleaned and rinsed vial by mixing of double distilled hexane with a volume of approximately 5ml (plate 2.10). The sample of the vial was dried followed by complete evaporation of solvent (About to dried sample) under a mild stream of nitrogen (Plate 2.12). Prior to HPLC analysis the dried sample was stored in freeze (plate 2.13). Before HPLC analysis, the evaporated sample was dissolved in acetonitrile and then made to volume 1 mL for the analysis with high performance liquid chromatography (HPLC).

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Plate 2.5 Separating funnel

Plate 2.6 Separation of aqueous layer

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Plate 2.7 Hexane extract was treated with 5-g anhydrous sodium sulphate to remove traces of water

Plate 2.8 Water free extract is ready to evaporate

Page | 30 CHAPTER 2 MATERIALS AND METHODS

Plate 2.9 Rotary vacuum evaporator

Plate 2.10 Evaporated hexane is ready for nitrogen drying

Page | 31 CHAPTER 2 MATERIALS AND METHODS

Plate 2.11 Nitrogen generator

Plate 2.12 Solvent is drying by nitrogen generator

Page | 32 CHAPTER 2 MATERIALS AND METHODS

Plate 2.13 Drying solvent is stored in refrigerator prior to HPLC analysis

2.1.6.5 Precaution The following precautions were taken during clean-up procedure

 At first all kinds of apparatus were washed by detergent.  Apparatus were dried in oven.  Then washed 3 times with acetone and 3 times with double distilled hexane.  Fresh reagents were always used.  n-hexane was double distilled at 65 C (boiling point of hexane is 65-68°C).

⁰

2.1.7 Operation with HPLC The High Performance Liquid Chromatography (HPLC) equipped with Ultra Violet (UV) detector along with the output device was used (Plate 2.14) for determination of the level of organochlorine, organophosphorus and carbamate pesticide residues.

Page | 33 CHAPTER 2 MATERIALS AND METHODS

Table 2.4 Operating condition of HPLC (UV mode)

Type Waters Ltd.

Detector Type UV

Injection Technique Manual by Micro Syringe

Injection Volume 20 µl

Mobile Phase Acetonitrile 65%

Column Type C18 (Nova Pack)

Absorbance 254 nm

Plate 2.14 HPLC (Water Company) with UV Detector

Page | 34 CHAPTER 2 MATERIALS AND METHODS

2.1.7.1 Pump The role of the pump is to force a liquid (called the mobile phase) through the liquid chromatography at a specific flow rate, expressed in milliliters per min (mL/min). Normal flow rates in HPLC are in the 1 to 2-mL/min range. Typical pumps can reach pressures in the range of 6000-9000 psi (400-to 600-bar). During the chromatographic experiment, a pump can deliver a constant mobile phase composition (isocratic) or an increasing mobile phase composition (gradient).

2.1.7.2 Injector The injector serves to introduce the liquid sample into the flow stream of the mobile phase. Typical sample volumes are 5-to 20-microliters (µL). The injector must also be able to withstand the high pressures of the liquid system. An auto sampler is the automatic version for when the user has many samples to analyze or when manual injection is not practical.

2.1.7.3 Column Considered the “heart of the chromatograph" the column's stationary phase separates the sample components of interest using various physical and chemical parameters. The small particles inside the column are what cause the high back pressure at normal flow rates. The pump must push hard to move the mobile phase through the column and this resistance causes a high pressure within the chromatograph. There are various columns that are secondary to the separating column or stationary phase. There are several types of column like Guard, Derivalization, Capillary, Fast and Preparatory in HPLC.

Generally in HPLC Capillary columns (Cl8 Column) arc used (Plate 2.15). Capillary columns are very efficient than any other columns.

Page | 35 CHAPTER 2 MATERIALS AND METHODS

Column

Plate 2.15 Column used in HPLC

2.1.7.3 Detector The detector can see (detect) the individual molecules that come out (elute) from the column. A detector serves to measure the amount of those molecules so that the chemist can quantitatively analyze the sample components. The detector provides an output to a recorder or computer that result in the liquid chromatogram (i.e., the graph of the detector response).

2.1.7.4 Operation Technique An injector (sample manager or auto sampler) is able to introduce (inject) the sample into the continuously flowing mobile phase stream that carries the sample into the HPLC column. A high-pressure pump (solvent delivery system or solvent manager) is used to generate and meter a specified flow rate of mobile phase, typically milliliters per minute. The column contains the chromatographic packing material needed to affect the separation. This packing material is called the stationary phase because it is held in place by the column hardware. As a sample solution flows through a column with mobile phase, the components of that solution migrate according to the non-covalent interactions of the compound with the column. The mobile phase exits the detector and can be sent to waste, or collected, as desired.

Page | 36 CHAPTER 2 MATERIALS AND METHODS

2.1.8 Analysis of pesticide residues For the analysis of pesticide residues, the sample was injected by microsyringe into the High Performance Liquid chromatography (HPLC). The mobile phase was acetonitrile

65%. Column Cl8 (Nova Pack) along with the output device at 254 nm absorbance was used for determination of the level of organochlorine. organophosphorus and carbamate pesticide residues. Identification of suspected pesticide residues were done in relation to retention time (RT) of pure analytical standard of that insecticide (DDT, DDE, DDD, Malathion, Diazinon, Chloropyrifos and Carbaryl) which were purchased from GmbH (D-86199 Augsburg, Germany). Quantification of the suspected pesticide residues were done by formation of calibration curve from standard samples of different concentrations. The retention time featured is changed a little bit which of course does not make any problem for identification and 0.05% difference is acceptable.

2.1.8.1 Calibration curve formation The concentration of pesticides in the water samples were determined by creating a calibration curve that shows the relation between the pesticide concentration and the detector response. The calibration curves for malathion, diazinon, chlorpyrifos, carbaryl, DDT, DDE and DDD were prepared from different known concentrations of the respective standards. By diluting the known amount of the suspected standards with DD- hexanc, standard solutions were prepared. The standard solutions were then analyzed by HPLC and the concentrations were plotted against the areas in the chromatograms. From this a linear equation was obtained. The Limit of Detection (LOD) for each pesticide was determined as the lowest amount detected in the HPLC.

2.1.8.2 Calculation of Residues The residues “R" for water can be calculated from the following equation (DFG 1987):

R = FA. VEx.WSt. / FSt. VI. G

Where,

G = Sample volume (ml)

VEx = Volume of extract solution (ml)

VI = Portion of volume VEx, injected into the HPLC or volume of sample loop (µl)

WSt = Amount of pesticide injected with standard solution (ng)

Page | 37 CHAPTER 2 MATERIALS AND METHODS

2 FA = Peak area obtained from VI (mm ) and

2 FSt = Peak area obtained from WSt (mm )

2.1.8.3 Recovery Test Recovery is a measure of how much of an analyte that is lost during the clean-up procedure. For recovery test, 900 µg of each pesticide was diluted in distilled water and all the procedure were followed like the water samples and were calculated the residues using HPLC. The mean percentage recoveries for the various pesticides were calculated using the following equation using the following equation:

Pi = (Si / Ti) 100

Where, ×

Pi = Percent recovery

Si = Analytical results from the laboratory control standard and

Ti = Known concentration of the spike.

All the samples were analyzed for pesticide residues and finally were compared the data with the recommended level as assigned by different organizations.

2.2 Malathion toxicology in tilapia 2.2.1Experimental fish The experimental fish was tilapia (Oreochromis mossambicus) (palte 2.16)

Identification characterictics

a) B.XIV-XIX; D. XV-XVI/10-12; A. III(IV) 9-10; P.14-15; V.I./5; C.15; L.I. 29-33 b) Brownish body with 3-4 black spots on each lateral side. c) Ctenoid scale and single black dot present in the center of each scale. d) Upper lateral side of head is concave. e) Ending spine of dorsal fin is the largest and hard. (Shafi and Quddus, 2004)

Page | 38 CHAPTER 2 MATERIALS AND METHODS

Scientific classification

Kingdom: Animalia

Phylum: Chordata

Class: Actinopterygii

Order: Perciformes

Family: Cichlidae

Genus: Oreochromis

Species: O. mossambicus

2.2.2 Procedure Healthy specimens of Oreochromis mossambicus were obtained from local fish hatchery with an 8.5 cm average length and 14 gm average weight (plate 2.16). O.mossambicus was selected because of its ability to withstand stress and its high commercial value in the Kingdom. The fishes were stocked in 15-L glass aquaria containing tape water. The fish was acclimatized to this condition for 1 week before using in any trial during which the time they were provided with artificial feed was obtained locally. Water was changed daily. To find the proper range of concentrations conduct an initial test with (0, 1, 2, 3, 4) ppm concentration of malathion. 0 ppm concentration of malathion group set as control group. Each aquarium was stocked with 20 fishes. Low Concentration is the Concentration in the original range-finding test that produced mortality closest to 0. High Concentration is the Concentration that caused mortality closest to 100%. Then sets five different treatment groups of (0, 0.5, 1.0, 1.5, 2.0) ppm concentration of malathion in triplicate. The fish fed once daily. Feeding was ended while aeration continued during the 96 h test periods.

Page | 39 CHAPTER 2 MATERIALS AND METHODS

Plate 2.16Experimental fish

One control group with 0.0 ppm malathion concentration and four sets of 20 fishes each were subjected to serial dilutions of the stock solution of malathion (from 0.5 - 2 ppm) (plate 2.17).

Plate 2.17 Aquarium sets The test was performed by following semistic (renewal) bioassay method in which the exposure medium was exchanged after every 24 h to maintain toxicant strength and level

Page | 40 CHAPTER 2 MATERIALS AND METHODS of dissolved oxygen, as well as minimizing the ammonia excretion levels during this experiment. The aquarium water was aerated continuous with electric aerator. The fish was observed at 3 h intervals. All toxicity studies were conducted using technical grade malathion. And the malathion solution was added to the aquarium water just before the

96 h test periods. Fish were not fed during the experiment. For LC50 calculation, mortality of fish exposed to each concentration was recorded every 24 h for 96 h, dead fish being removed every 3- 8 h and by covered aluminum foil thrown it to the dustbin (plate 2.18). Observed three times for each concentration and take the mean value.

Plate 2.18 Malathion contaminated fish

Page | 41 CHAPTER 2 MATERIALS AND METHODS

2.2.3 Analysis of malathion toxicity in tilapia

2.2.3.1 Calculation of LC50 value

LC50 - The median lethal concentration or the concentration of a substance that kills 50 percent of the organisms tested in a laboratory toxicity test of specified duration (e.g., 96 hour LC50)

Calculated LC50value by probit analysis using computer software CALCULATION OF

LC50 USING PROBIT ANALYSIS (Raj, 2016)

The following steps are used in the calculation of LC50 in this method

1. Converting doses to log (10) doses (x) 2. Converting mortality to proportions 3. The proportions are corrected for control mortality if it is more than 10% Corrected mortality (p) = (% Responded – % Responded in Control)/(100 - Responded % in Control) x100 4. Converting corrected proportions (p) to empirical probits (y). 5. A dose response curve is drawn using the log(10) doses (x) and empirical probits (y) and the regression equation is derived. Empirical probits less than 1 and more than 7 are ignored as they have little and no significance in the estimation of LD or LC (Hayes, 2014). y=5+(x-µ)/s 6. From the equation of the curve and log10 doses, the expected probits (Yi) are derived 7. From the expected probits (Yi), expected mortality proportion followed by expected no.of animals are derived 8. The original mortality (Observed) and derived mortality (Expected) are used to calculate the Chi-Square test with (No. of log doses used -2) degrees of freedom. If the Chi-square test is non-significant, it indicates good curve fitting. 9. Z value is derived using the formula Z=1/(v2p)e(-1/2(Yi-5)^2) where Yi = Expected probits 10. The weighting coefficents (W) are derived using the formula W=Z^(2 )/PQ, where P = Expected proportion

Page | 42 CHAPTER 2 MATERIALS AND METHODS

Q= (1-P) 11. The weighted coefficients were used to calculate the standard error SE= s/v2nW where s = Standard deviation (1/slope) n= number of animals in each group W= Weighting coefficient 12. Working probits (Yw) are derived from the regression equation as follows Yw = Yi-(P/Z)-p/Z Y = Expected probits P = Expected proportion p = Observed proportion 13. The LD or LC values are derived from the curve drawn using working probits and log doses. Antilog of the dose corresponding to respective probit value. 14. 12. 95% Fiducial confidence limits are calculated using the formula Fiducial limits =Antilog (Log10 Dose ± 1.96 (SE)

Page | 43 CHAPTER 3

RESULTS AND DISCUSSION 3.1 Pesticide residues analysis in water samples Thirty one water samples of both fish pond and lake water were collected from 14 unions of Sreenagar Upazila in July, 2016. The water samples were analyzed for the detection of Organochlorine (DDT, DDD and DDE), Organophosphorous (Malathion, Diazinon and Chloropyrifos) and Carbamate (carbaryl) pesticide residues.

In this experiment, none of the tasted water samples amongthirty one were found to be the presence of pesticide residues. Pesticide residue was not found in fish pond and lake water in 14 unions of Sreenagar Upazila in july, 2016.

3.1.1 Organochlorines (OCs) Pesticide Residues Test of water samples for Organochlorine (DDT, DDD and DDE), pesticides were performed by High Performance Lquid Chromatography (HPLC). In this experiment, none of the tested water samples of Sreenagar Upazila in July, 2016 were found to contaminate with Organochlorine (DDT, DDD and DDE) pesticide residues (Table 3.1). Retention time (RT) of these organochlorine pesticides is given in Appendices. Standard chromatograms of DDT, DDD and DDE among the organochlorine pesticides are shown in appendices (Chromatogram).

Table 3.1 Organochlorine pesticide residues in water samples of Sreenagar Upazila

Water Source of Locations DDT DDE DDD Sample No. Water (Unions) (mg/L) (mg/L) (mg/L) Sample WS 1 1-A-P Atpara BDL BDL BDL WS 2 2-K-P Kukutia BDL BDL BDL WS 3 3-KP-P Kola Para BDL BDL BDL WS 4 4-T-P Tantar BDL BDL BDL WS 5 5-P-P Patabhog BDL BDL BDL WS 6 6-B-P Baghra BDL BDL BDL WS 7 7-BA-P Baraikhali BDL BDL BDL WS 8 8-BI-P Birtara BDL BDL BDL WS 9 9-BH-P Bhagyakul BDL BDL BDL

Page | 44 WS 10 10-R-P Rarikhal BDL BDL BDL WS 11 11-S-P Sholaghar BDL BDL BDL WS 12 12-SH-P Shyamsiddhi BDL BDL BDL WS 13 13-SR-P Sreenagar BDL BDL BDL WS 14 14-H-P Hasara BDL BDL BDL WS 15 15-SR-P Sreenagar BDL BDL BDL WS 16 16-R-P Rarikhal BDL BDL BDL WS 17 1-A-L Atpara BDL BDL BDL WS 18 2-K-L Kukutia BDL BDL BDL WS 19 3-KP-L Kola Para BDL BDL BDL WS 20 4-T-L Tantar BDL BDL BDL WS 21 5-P-L Patabhog BDL BDL BDL WS 22 6-B-L Baghra BDL BDL BDL WS 23 7-BA-L Baraikhali BDL BDL BDL WS 24 8-BI-L Birtara BDL BDL BDL WS 25 9-BH-L Bhagyakul BDL BDL BDL WS 26 10-R-L Rarikhal BDL BDL BDL WS 27 11-S-L Sholaghar BDL BDL BDL WS 28 12-SH-L Shyamsiddhi BDL BDL BDL WS 29 13-SR-L Sreenagar BDL BDL BDL WS 30 14-H-L Hasara BDL BDL BDL WS 31 15-B-L Baghra BDL BDL BDL WS = Water sample, P = Pond, L = Lake, Atpara=A, Kukutia=K, Kola Para=KP, Tantar=T, Patabhog=P, Baghra=B, Baraikhali=BA, Birtara=BI, Bhagyakul=BH, Rarikhal=R, Sholaghar=S, Shyamsiddhi=SH, Sreenagar=SR, Hasara=H, BDL = Below detection limit and the limit of detection (LOD) is 10 µg/L

Page | 45 CHAPTER 3 RESULTS AND DISCUSSION

3.1.2 Organphosphorous(Ops) and Carbamate Pesticide Residues Test of water samples for Organophosphorous (Malathion, Diazinon and Chloropyrifos) and Carbamate (Carbaryl) pesticides were performed by High Performance Lquid Chromatography (HPLC). In this experiment, none of the tested water samples of Sreenagar Upazila in July, 2016 were found to contaminate withOrganophosphorousand Carbamate pesticide residues (Table 3.2).Retention time and standard chromatograms of Malathion, Diazinon, Chloropyrifos among the organophosphorous and carbamate (carbaryl) pesicides are shown in Appendices (Chromatogram).

Table 3.2 Organophosphorus and carbamate pesticide residues in water samples of Sreenagar Upazila.

Water Source Locations Organophosphorus Carbamate Sample of (Unions) Malathion Diazinon Chloropyriphos Carbaryl No. Water (mg/L) (mg/L) (mg/L) (mg/L) Sample WS 1 1-A-P Atpara BDL BDL BDL BDL WS 2 2-K-P Kukutia BDL BDL BDL BDL WS 3 3-KP-P Kola Para BDL BDL BDL BDL WS 4 4-T-P Tantar BDL BDL BDL BDL WS 5 5-P-P Patabhog BDL BDL BDL BDL WS 6 6-B-P Baghra BDL BDL BDL BDL WS 7 7-BA-P Baraikhali BDL BDL BDL BDL WS 8 8-BI-P Birtara BDL BDL BDL BDL WS 9 9-BH-P Bhagyakul BDL BDL BDL BDL WS 10 10-R-P Rarikhal BDL BDL BDL BDL WS 11 11-S-P Sholaghar BDL BDL BDL BDL WS 12 12-SH- Shyamsiddhi BDL BDL BDL BDL P WS 13 13-SR- Sreenagar BDL BDL BDL BDL P WS 14 14-H-P Hasara BDL BDL BDL BDL WS 15 15-SR- Sreenagar BDL BDL BDL BDL P WS 16 16-R-P Rarikhal BDL BDL BDL BDL

Page | 46 CHAPTER 3 RESULTS AND DISCUSSION

WS 17 1-A-L Atpara BDL BDL BDL BDL WS 18 2-K-L Kukutia BDL BDL BDL BDL WS 19 3-KP-L Kola Para BDL BDL BDL BDL WS 20 4-T-L Tantar BDL BDL BDL BDL WS 21 5-P-L Patabhog BDL BDL BDL BDL WS 22 6-B-L Baghra BDL BDL BDL BDL WS 23 7-BA-L Baraikhali BDL BDL BDL BDL WS 24 8-BI-L Birtara BDL BDL BDL BDL WS 25 9-BH-L Bhagyakul BDL BDL BDL BDL WS 26 10-R-L Rarikhal BDL BDL BDL BDL WS 27 11-S-L Sholaghar BDL BDL BDL BDL WS 28 12-SH- Shyamsiddhi BDL BDL BDL BDL L WS 29 13-SR- Sreenagar BDL BDL BDL BDL L WS 30 14-H-L Hasara BDL BDL BDL BDL WS 31 15-B-L Baghra BDL BDL BDL BDL WS = Water sample, P = Pond, L = Lake, Atpara=A, Kukutia=K, Kola Para=KP, Tantar=T, Patabhog=P, Baghra=B, Baraikhali=BA, Birtara=BI, Bhagyakul=BH, Rarikhal=R, Sholaghar=S, Shyamsiddhi=SH, Sreenagar=SR, Hasara=H, BDL = Below detection limit and the limit of detection (LOD) is 10 µg/L

Organochlorine pesticides have been banned in 1993 according to Bangladesh Environment Conservation Act 1995 due to their high toxicity (Chowdhury et al. 2012b). Among the 31 water samples none of the water samples was detected to be contaminated with organochlorine (DDT, DDE and DDD) pesticide residues of Sreenagar Upazila. But in previous studies various organochlorine pesticide residues were reported in several regions of Bangladesh. In a study of Chowdhury et al. (2012c) a water sample collected from five locations (Feni, Nawabganj, Putia, Burichang and Chatak.) of Bangladesh, were contaminated with DDT; the highest DDT concentration detected was 8.29 µg/L, and its metabolite, DDE, was detected at 4.06 µg/L. Matin et al. (1998) investigated the water samples from the different regions of Bangladesh were studied for organochlorine insecticide residues (OCs) during 1992-1995, both before and after the banning of the use of OCs. The results indicate slight contamination of some of the water samples of both surface and underground sources with residues of DDT, heptachlor, lindane and

Page | 47 CHAPTER 3 RESULTS AND DISCUSSION dieldrin. But by the present study it may be concluded that the people of Sreenagar Upazila do not use Organochlorine pesticides during the period of time.

Organophosphorous pesticides are frequently used against pest because of their high insecticidal property low mammalian toxicity, less persistence and rapid biodegradability (Bhandare et al. 2011). These also affect non-target organisms either directly or indirectly. In rice land agroecosystem, all organisms including larvivorous fishes can be affected (Roger et al. 1990). At lower concentrations, physiological functions including the larvivorous potential are affected and results of the present study indicate the same. Indiscriminate and prolonged use may lead to mortality and depletion of the fish population (John et al. 2012).

The melathion was not detected in the water samples of Sreenagar Upazila,where the limit of detection (LOD) was 0.01 ppm. This is as good for the environmentas the farmers of this area do not use malathion indiscriminately. This is happened due to the consciousness of the people of the area as they are use the correct level of pesticides. But some other place in our country malathion was detected, so it’s necessary to control the appropriate level of pesticides. As malathion was detected in a single water sample at 105.2 µg/L from Dhamrai Upazila in saver in Bangladesh (Chowdhury et al. 2012b). A study was undertaken to identify Organochlorine and Organophosphorous pesticides residues in water samples of some paddy fields, ponds and tubewells in Rangpur district (Taragong Thana) of Bangladesh where the level of the Malathion pesticide residues were found in the range of 2 to 221 ppm (Ara et al. 2014). Another study conducted by Uddin et al. (2012) investigated that the pesticide residues in some pond water samples where the residue level of malathion ranged from 0.0241ppm (24. µg/L) to 0.0463 ppm (46.3 µg/L).

The Canadian maximum acceptable concentration (MAC) of 190 µg/L for malation (HC, 2014) and the European guideline value of 0.1 µg/L for individual pesticide (EU, 1998).The present study did not fixmalathion pesticide residues in the fish pond and lake water collected from Sreenagar Upazila which indicated that this is safe for human and other aquatic and terrestrial animal’s.

Page | 48 CHAPTER 3 RESULTS AND DISCUSSION

The amount of diazinon detected in some selected ponds of Meherpur region in Bangladesh ranged from 0.033 to 0.079 ppm (Uddin et al. 2013). Chowdhury et al. (2012) reported the presence of diazinon at the concentration of 0.9 µg/L in Saver. Diazinon was also detected at the concentration of 7.86 µg/L in the lake of Saver (Hossain et al. 2014), at the concentration of 0.027 µg/L in the paddy field of Manikganj (Bhattacharjee et al. 2012). The maximum acceptable concentration (MAC) of 20 µg/L for diazinon in drinking water as assigned by FAO/WHO (1980). The Canadian maximum acceptable concentration (MAC) of 20 µg/L for diazinon (HC, 2014) and the European guideline value of 0.1 µg/L for individual pesticides (EU 1998).The diazihon pesticide residue was not found in fish pond and lake water of Sreenagar upazila. So the fish pond and lake water of Sreenagar upazila is free from diazinon pesticide residues contamination.

During the study chlorpyrifos and carbaryl was not detected at any of the water samples collected from Sreenagar Upazila in July, 2016. From the results of the present study it has been observed that, Organophosphorus pesticides are not being used extensively in the Sreenagar Upazila during the study period. Where water samples were not found to be contaminated with organophosphorus pesticide residues. The concentrations of pesticides were much lower than the acceptable limit which indicates farmers are using Organophosphorus pesticides in most favorable condition in Sreenagar Upazila.

Pesticides often considered a quick, easy solution for controlling weeds and insect pest however, their use comes at a cost. Pesticides damage ecosystems and may damage or harm un-targeted animals. Pesticides decrease biodiversity and may cause a decline in populations or even cause extinction of species, "mess up" food chains/webs, disrupt the natural balance in ecosystems. The water samples of Sreenagar Upazila were collected in July, 2016. During this time the farmers was not use pesticides indiscriminately, so the selected pesticides is not available in fish pond lake and water of Sreenagar Upazila by leaching and surface runoff cultivated land water and any other ways The embankment of the fish ponds are well structured so that the cultivate land water cannot penetrate to the pond. During the time of collected water samples it was a rainy seasons so the rain water fill the lake full to the brim and farmers were not cultivated land like other seasons of the year and for this they was not use pesticide indiscriminately to control pest.

Page | 49 CHAPTER 3 RESULTS AND DISCUSSION

3.2 Malathion toxicity in tilapia 3.2.1 Lethal concentration of malathion in tilapia The present study found 0.0 ppm is the Low Concentration in the original range-finding test that produced mortality 5% and 2.00 ppm is the High that caused mortality 100%.The results shown in the appendices (Appendix table 6.8-6.11).

For LC50 calculation, mortality of fish exposed to each concentration was recorded every 24 h

3.2.2 Mortality of tilapiaat different concentration of malathion for 24, 48, 72 and 96 h exposure period 3.2.2.1 Exposure of 24 h malathion Table 3.3 shows the mortality of tilapia in exposure of 24 h malathion.

Table 3.3 Mortality of tilapia in exposure of 24 h malathion

S/N Concentration Fish Fish dead Fish alive Mortality of malathion exposed (%) (ppm) Control 0.0 20 0 20 0 1 0.5 20 0 20 0 2 1.0 20 2 18 10 3 1.5 20 4 16 20 4 2.0 20 6 14 30 *Mean value of triplicates

Page | 50 CHAPTER 3 RESULTS AND DISCUSSION

Exposure of 24 h malathion

Mortality rate

35 30 30 25 20 20 15 10 10 Mortality (%) Mortality 5 0 0 0 0 0.5 1 1.5 2 Concentration of malathion (ppm)

Figure 3.1Exposure of 24 h malathion mortality rate in tilapia

24 h LC50 value analysis

Calculating LC50 using Probit Analysis

4.50 4.48 4.45

4.40

4.35

4.30

4.25 Probit of kill Probit 4.20 4.16 y = 2.539x + 3.711 4.15 R² = 1

4.10 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 Log10 concentration of malathion (ppm)

Figure 3.2Graph showing the relationship of probit of kill with log10 concentration of

malathion used to deduce the 24 h LC50

Page | 51 CHAPTER 3 RESULTS AND DISCUSSION

Table 3.4 24 h LC50 value of malathion in tilapia obtained from CALCULATION OF

LC50 USING PROBIT ANALYSIS software

2 Function, R LC50 SD SE 95% Confidence level (ppm) Lower Upper y= 2.539x + 3.7113 3.236 0.398 0.102 2.045 5.121 R2=1

Table 3.3 shows the mortality of fish as a function of Malathion concentrations. The 24 h

LC50 value for Oreochromis mossambicus computed from the graph (Figure 3.2) constructed between Log10 concentrations of malathion in X axis and probit of kill in Y axis was expressed as 3.236 ppm (Table 3.4).

3.2.2.2 Exposure of 48 h malathion Table 3.5 shows the mortality of tilapia in exposure of 48 h malathion.

Table 3.5 Mortality of tilapia in exposure of 48 h malathion

S/N Concentration Fish Fish dead Fish alive Mortality of malathion exposed (%) (ppm) Control 0.0 20 0 20 0 1 0.5 20 2 18 10 2 1.0 20 4 16 20 3 1.5 20 6 14 30 4 2.0 20 10 10 50 *Mean value of triplicates

Page | 52 CHAPTER 3 RESULTS AND DISCUSSION

Exposure of 48 h malathion

Mortality rate

60 50 50

40 30 30 20 20 Mortality (%)Mortality 10 10 0 0 0 0.5 1 1.5 2 Concentration of malathion (ppm)

Figure 3.3Exposure of 48 h malathion mortality rate in tilapia

48 h LC50 value analysis

Calculating LC50 using Probit Analysis

6.00

5.00 5.00

3.72 4.48 4.00

3.00

Probit of kill Probit 2.00

1.00 y = 1.978x + 4.281 R² = 0.951 0.00 -0.40 -0.30 -0.20 -0.10 0.00 0.10 0.20 0.30 0.40 Log10 concentration of malathion (ppm)

Figure 3.4Graph showing the relationship of probit of kill with log10 concentration of

malathion used to deduce the 48 h LC50

Page | 53 CHAPTER 3 RESULTS AND DISCUSSION

Table 3.6 48 h LC50 value of malathion in tilapia obtained from CALCULATION OF

LC50 USING PROBIT ANALYSIS software

2 Function, R LC50 SD SE 95% Confidence level (ppm) Lower Upper y= 1.9783x + 4.2819 2.362 0.499 0.110 1.440 3.875 R2= 0.9519

Table 3.5 shows the mortality of fish as a function of Malathion concentrations. The 48 h

LC50 value for tilapia (Oreochromis mossambicus) computed from the graph (Figure 3.4) constructed between Log10 concentrations of malathion in X axis and probit of kill in Y axis was expressed as 2.362 ppm (table 3.6).

3.2.2.3 Exposure of 72 h malathion Table 3.7 shows the mortality of tilapia in exposure of 72 h malathion

Table 3.7 Mortality of tilapia in exposure of 72 h malathion

S/N Concentration Fish Fish dead Fish alive Mortality of malathion exposed (%) (ppm) Control 0.0 20 1 19 5 1 0.5 20 4 16 20 2 1.0 20 6 14 30 3 1.5 20 10 10 50 4 2.0 20 16 4 80 *Mean value of triplicates

Page | 54 CHAPTER 3 RESULTS AND DISCUSSION

Exposure of 72 h malathion

Mortality rate

90 80 80 70 60 50 50 40 30 30 Mortality (%)Mortality 20 20 10 5 0 0 0.5 1 1.5 2 Concentration of malathion (ppm)

Figure 3.5Exposure of 72 h malathion mortality rate in tilapia

72 h LC50 value analysis

Calculating LC50 using Probit Analysis

7.00

6.00 5.83

5.00 4.15 4.99 4.00

3.00 Probit of kill Probit 2.00

1.00 y = 2.511x + 4.844 R² = 0.898 0.00 -0.40 -0.30 -0.20 -0.10 0.00 0.10 0.20 0.30 0.40 Log10 concentration of malathion (ppm)

Figure 3.6Graph showing the relationship of probit of kill with log10 concentration of

malathion used to deduce the 72 h LC50

Page | 55 CHAPTER 3 RESULTS AND DISCUSSION

Table 3.872 h LC50 value of malathion in tilapia obtained from CALCULATION OF

LC50 USING PROBIT ANALYSIS software

2 Function, R LC50 SD SE 95% Confidence level (ppm) Lower Upper y= 2.5112x + 4.8446 1.256 0.386 0.081 0.870 1.813 R2= 0.8987

Table 3.7 shows the mortality of fish as a function of Malathion concentrations. The 72 h

LC50 value for Oreochromis mossambicus computed from the graph (Figure 3.6) constructed between Log10 concentrations of malathion in X axis and probit of kill in Y axis was expressed as 1.256 ppm (table 3.8).

3.2.2.4 Exposure of 96 h malathion Table 3.9 shows the mortality of tilapia in exposure of 96 h malathion

Table 3.9 Mortality of tilapia in exposure of 96 h malathion

S/N Concentration Fish Fish dead Fish alive Mortality of malathion exposed (%) (ppm) Control 0.0 20 2 18 10 1 0.5 20 8 12 40 2 1.0 20 12 18 60 3 1.5 20 16 4 80 4 2.0 20 20 0 100 *Mean value of triplicates

Page | 56 CHAPTER 3 RESULTS AND DISCUSSION

Exposure of 96 h malathion

Mortality rate

120 100 100 80 80 60 60 40 40 Mortality (%)Mortality 20 10

0 0 0.5 1 1.5 2 Concentration of malathion (ppm)

Figure 3.7Exposure of 96 h malathion mortality rate in tilapia

96 h LC50 value analysis

Calculating LC50 using Probit Analysis

7.00

6.00 5.82 4.73 5.00

4.00

3.00 Probit of kill Probit 2.00

1.00 y = 2.285x + 5.421 R² = 1 0.00 -0.40 -0.30 -0.20 -0.10 0.00 0.10 0.20 Log10 concentration of malathion (ppm)

Figure 3.8Graph showing the relationship of probit of kill with log10concentration of

malathion used to deduce the 96 h LC50

Page | 57 CHAPTER 3 RESULTS AND DISCUSSION

Table 3.10 96 h LC50 value of malathion in tilapia obtained from CALCULATION OF

LC50 USING PROBIT ANALYSIS software

2 Function, R LC50 SD SE 95% Confidence level (ppm) Lower Upper y= 2.285x + 5.4215 0.690 0.450 0.097 0.446 1.068 R2= 1

Table 3.9 shows the mortality of fish as a function of malathion concentrations. The 96 h

LC50 value for tilapia (Oreochromis mossambicus) computed from the graph (Figure 3.8) constructed between Log10 concentrations of malathion in X axis and probit of kill in Y axis was expressed as 0.690 ppm. (Table 3.10)

In the present study, 96h LC50 and 48 h LC50 of malathion was obtained 0.690 (0.446- 1.068) ppm and 2.362 (1.440-3.875) ppm for Oreochromis mossambicus. Vittozi and

Angelis, (1991) summarized the 96 h LC50 values of malathion 0.091 to 22.09 ppm for different species. The result is much lower than the According to Shahbazi et al. 2015

96hLC50of malathion was obtained 6.08 (5.22-7.18) ppm. for Capoeta damascina.

The results showed that LC50 values is decreased with increase in exposure duration, in other words in longer periods of exposure, lower concentrations of pesticide is caused 50% mortality in studied fish population. This situation had been observed in most studies as Malathion was toxic to Channa punctatus and the 96 h LC50 value is 6.61 ppm reported by Pandey, 2005 and the 96 h LC50 value for Channa punctatusis 1.61 ppm reported by Khangarot and Ray, 1988.

Present studies reportedthat 96h LC50 values for malathion in different fishes is ranged from 0.1 to 15 mg/L. The present study showed that.96h LC50 and 48 h LC50 of malathion is 0.690 (0.446-1.068) ppm and 2.362 (1.440-3.875) ppm.Thus, it can be concluded from the present study that tilapia is highly sensitive to malathion and their mortality rate is dose dependent.

Page | 58 CHAPTER 4

CONCLUSIONS AND RECOMMENDATIONS 4.1 Conclusions Modern agriculture is a technological enterprise that uses sophisticated science and engineering to produce crops from available resources of land, water, and energy. Bangladesh is one of the over populated country around the world and with the increase of population the food demand also increasing, the essential problem for agricultural research has become that of efficiency-how to wring yet more produce from the same or less resources. The development of pesticides has been one solution to this problem. For a relatively small additional cost, their use has resulted in greatly increased productivity.

The present study observed that the pond and lake water of Sreenagar upazilais free from pesticide residues contamination which is observed in this study. The use of organochlorine insecticides (except heptachlor) due to their persistence and bio- accumulative nature for all agricultural and public health purposes was banned in Bangladesh by late 1993 (Matin et al. 1998) so the Organophosphorous pesticides are widely used in agriculture. In Bangladesh, it is estimated that up to 64% of the crop- producing area is treated with carbamates, while up to 35% of the crop-producing area is treated with organophosphates (Chowdhury et al. 2012a). Organophosphorus pesticides are less damaging to the environment and they are less persistent than organochlorine pesticides. The effectiveness of organophosphorous pesticides, coupled with their relatively cheap cost encourages farmers to use more of these pesticides when growing their field crops.

Malathion seems to be highly toxic toOreochromis mossambicus. The 24 h, 48 h, 72 h and 96 h LC50 value is 3.236 ppm, 2.362ppm, 1.256 ppm and 0.690 ppm respectively.

The results showed that LC50 values is decreased with increase in exposure duration.In other words in longer periods of exposure, lower concentrations of pesticide is caused 50% mortality in studied fish population.Thus, it can be concluded from the present study that fish are highly sensitive to malathion and their mortality rate is dose dependent. So the farmer should not use malathion indiscriminately due to its toxicity. Application of relevant law to control haphazard use of pesticide and appropriate control measure should

Page | 59 CHAPTER 4 Conclusions and Recommendations be taken by government to reduce the risk of indiscriminate use of pesticide. The farmers should train and regular campaign should be taken to build public awareness against the hazardous effect of pesticides on public health and environment.

4.2 Recommendations The safe and effective use of these pesticides is essential to avoid contamination of the water and soil environments.It is important to know the correct level of pesticide residues in different sources.This study is very important to monitor pesticides residues in water and fill gaps in current knowledge. Further study recommended that pesticide consumption and its hazardous effect may be studied in the study area.

As the tilapia is a popular fish for aquaculture which is very much sensitive to malathion. Therefore, it is important to determine the toxicity of malathion in tilapia.

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Page | 70 APPENDICES Appendix table 6.1 Concentrations and the Peak Areas of DDT

Concentration 0.01 0.1 0.5 1 1.5 2 (mg/l) Peak Area 123 3953 18256 38285 59828 84297 (mm2)

Appendix table 6.2 Concentrations and the Peak Areas of DDE

Concentration 0.01 0.1 0.5 1 1.5 2 (mg/l) Peak Area 16652 55476 216192 448961 684572 942659 (mm2)

Appendix table 6.3 Concentrations and the Peak Areas of DDD

Concentration 0.01 0.1 0.5 1 2 4 (mg/l) Peak Area 5034 12453 36028 72782 148505 306178 (mm2)

Appendix table 6.4 Concentrations and the Peak Areas of Malathion

Concentration 0.01 0.1 5 10 15 30 (mg/l) Peak Area 2626 4275 18367 32527 51238 92675 (mm2)

Page | 71 APPENDICES

Appendix table 6.5 Concentrations and the Peak Areas of Diazinon

Concentration 0.01 0.1 0.5 2.5 5 10 (mg/l) Peak Area 1123 2243 4826 16368 32267 62868 (mm2)

Appendix table 6.6 Concentrations and the Peak Areas of Chlorpyrifos

Concentration 0.01 0.1 5 10 15 30 (mg/l) Peak Area 7351 9764 48925 100345 141086 259704 (mm2)

Appendix table 6.7Concentrations and the Peak Areas of Carbaryl

Concentration 0.01 0.1 0.25 0.5 1 2 (mg/l)

Peak Area 325678 12456723 324574955 64353619 150376273 296253678 (mm2)

Appendix table 6.8 Initial test to lethal concentration of malathion for tilapia in exposure of 24 h

S/N Concentration Fish Fish dead Fish alive Mortality of malathion exposed (%) (ppm) Control 0.0 20 0 20 0 1 1.0 20 2 18 10 2 2.0 20 6 14 30 3 3.0 20 10 10 50 4 4.0 20 14 6 70

Page | 72 APPENDICES

Appendix table 6.9 Initial test to lethal concentration of malathion for tilapia in exposure of 48 h

S/N Concentration Fish Fish dead Fish alive Mortality of malathion exposed (%) (ppm) Control 0.0 20 0 20 0 1 1.0 20 6 14 30 2 2.0 20 10 10 50 3 3.0 20 14 6 70 4 4.0 20 18 2 90

Appendix table 6.10 Initial test to lethal concentration of malathion for tilapia in exposure of 72 h

S/N Concentration Fish Fish dead Fish alive Mortality of malathion exposed (%) (ppm) Control 0.0 20 0 20 0 1 1.0 20 8 12 40 2 2.0 20 16 4 80 3 3.0 20 20 0 100 4 4.0 20 20 0 100

Page | 73 APPENDICES

Appendix table 6.11 Initial test to lethal concentration of malathion for tilapia in exposure of 96 h

S/N Concentration Fish Fish dead Fish alive Mortality of malathion exposed (%) (ppm) Control 0.0 20 1 19 5 1 1.0 20 12 8 60 2 2.0 20 20 0 100 3 3.0 20 All died 4 4.0 20 All died

Appendix table 6.12Mortality of fish exposed to each concentration of malathion in exposure of 24 h

S/N Concent Fish Fish dead Mortality ration of expose (%) malathi d on (ppm)

Replica 1 Replica 2 Replica 3 Mean

Control 0.0 20 0 0 0 0 0

1 1.0 20 0 0 0 0 0

2 2.0 20 2 1 3 2 10

3 3.0 20 4 3 5 4 20

4 4.0 20 6 5 7 6 30

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Appendix table 6.13 Mortality of fish exposed to each concentration of malathion in exposure of 48 h

S/N Concent Fish Fish dead Mortality ration of expose (%) malathi d on (ppm)

Replica 1 Replica 2 Replica 3 Mean

Control 0.0 20 0 0 0 0 0

1 1.0 20 2 1 3 2 10

2 2.0 20 4 3 5 4 20

3 3.0 20 6 5 7 6 30

4 4.0 20 10 9 11 10 50

Appendix table 6.14 Mortality of fish exposed to each concentration of malathion in exposure of 72 h

S/N Concent Fish Fish dead Mortality ration of expose (%) malathi d on (ppm)

Replica 1 Replica 2 Replica 3 Mean

Control 0.0 20 1 0 2 1 5

1 1.0 20 4 3 5 4 20

2 2.0 20 6 5 7 6 30

3 3.0 20 10 9 11 10 50

4 4.0 20 16 15 17 16 80

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Appendix table 6.15 Mortality of fish exposed to each concentration of malathion in exposure of 96 h

S/N Concent Fish Fish dead Mortality ration of expose (%) malathi d on (ppm)

Replica 1 Replica 2 Replica 3 Mean

Control 0.0 20 2 1 3 2 10

1 1.0 20 8 7 9 8 40

2 2.0 20 12 11 13 12 60

3 3.0 20 16 15 17 16 80

4 4.0 20 20 20 20 20 100

Chemicals used in the laboratory a) n-hexane-C6H14 (HPLC grade) (Merck, Germany) b) Acetone-((CH3)2CO) (extra pure, BDH, England) and malathion c) Anhydrous Sodium Sulphate-Na2S04 (Merck, Germany) d) Acetonitrile (Scharlau, Barcelona, Spain) HPLC grade e) Suspected Pesticide Standards (GmbH, D-86199 Augsburg, Germany)

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