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I am very grateful to my major advisor Dr.TY Boreborey her careful supervision, excellent guidance and encouragement from the very beginning of the thesis development up to the final thesis write up. I have a special respect and appreciation to her for her fatherly advice in all aspect and achievements of todays success. I am really glad to Dr. KUOK Fidero, chief of the Department of Chemical Engineering and Food Technology, who provided me with this opportunity to do research in ITC. I would like to thank Ms. BEANG Polingkong, the laboratory assistant. She permitted and assisted me to digest the fish samples in the laboratory as well as allowed me to manipulate the AAS instrument through the computer. I spread thanks also to SATREP project for allowing me to joint research group with great financial support throughout the period of the research. ABBREVIATIONS ATSDR Agency for Toxic Substances and Disease Registry AAS Atomic Absorption Spectrophotometer Conc. Concentration D.W Dry Weight EC Commission Regulation EU European FAO Food and Agricultural Organization Pb Lead TSL Tonle Sap Lake TF Transfer Factor PPM Part Per Million USFDA United State Food and Drug Administration WHO World Health Organization HYPERLINK l _Toc517678513 TABLE OF CONTENTS PAGEREF _Toc517678513 h vi HYPERLINK l _Toc517678514 LIST OF FIGURE PAGEREF _Toc517678514 h viii HYPERLINK l _Toc517678515 LIST OF TABLES PAGEREF _Toc517678515 h ix HYPERLINK l _Toc517678516 1. INTRODUCTION PAGEREF _Toc517678516 h 1 HYPERLINK l _Toc517678517 1.1 Background PAGEREF _Toc517678517 h 1 HYPERLINK l _Toc517678518 1.2 Objectives of the study PAGEREF _Toc517678518 h 3 HYPERLINK l _Toc517678519 1.3 Scope and limitation PAGEREF _Toc517678519 h 3 HYPERLINK l _Toc517678520 2. LITERATURE REVIEWS PAGEREF _Toc517678520 h 4 HYPERLINK l _Toc517678521 2.1 Heavy Metals in the Environment PAGEREF _Toc517678521 h 4 HYPERLINK l _Toc517678522 2.2 Distribution of Heavy Metals in the Aquatic Environment PAGEREF _Toc517678522 h 4 HYPERLINK l _Toc517678523 2.3 Accumulation of Heavy Metals in Fish PAGEREF _Toc517678523 h 6 HYPERLINK l _Toc517678524 2.4 Heavy Metal in the study PAGEREF _Toc517678524 h 7 HYPERLINK l _Toc517678525 2.4.1 Lead (Pb) PAGEREF _Toc517678525 h 7 HYPERLINK l _Toc517678526 2.4.2 Maximum limited level of Pb in freshwater fish PAGEREF _Toc517678526 h 9 HYPERLINK l _Toc517678527 2.5 Kind of studied fishes PAGEREF _Toc517678527 h 9 HYPERLINK l _Toc517678528 2.5.1 Trey Po PAGEREF _Toc517678528 h 9 HYPERLINK l _Toc517678536 2.5.9 Trey Chhkok PAGEREF _Toc517678536 h 13 HYPERLINK l _Toc517678537 2.5.10 Trey Broma PAGEREF _Toc517678537 h 13 HYPERLINK l _Toc517678538 2.6 Principle of Atomic Absorption spectrophotometer PAGEREF _Toc517678538 h 14 HYPERLINK l _Toc517678539 3. Methodology PAGEREF _Toc517678539 h 16 HYPERLINK l _Toc517678540 3.1 Description of Study Area PAGEREF _Toc517678540 h 16 HYPERLINK l _Toc517678541 3.2 Sampling and sample treatment PAGEREF _Toc517678541 h 16 HYPERLINK l _Toc517678542 3.3 Sample preparation PAGEREF _Toc517678542 h 17 HYPERLINK l _Toc517678543 3.4 Digestion procedure PAGEREF _Toc517678543 h 18 HYPERLINK l _Toc517678544 3.5 Instrumentations and Apparatus PAGEREF _Toc517678544 h 18 HYPERLINK l _Toc517678545 3.6 Reagent and standard PAGEREF _Toc517678545 h 18 HYPERLINK l _Toc517678546 3.7 Analysis of trace metals PAGEREF _Toc517678546 h 19 HYPERLINK l _Toc517678547 3.8 Transfer Factor PAGEREF _Toc517678547 h 20 HYPERLINK l _Toc517678548 4. Results and discussion PAGEREF _Toc517678548 h 21 HYPERLINK l _Toc517678549 4.1 Results PAGEREF _Toc517678549 h 21 HYPERLINK l _Toc517678550 4.1.1 The concentration of Pb in fishes collected on November 2017 PAGEREF _Toc517678550 h 21 HYPERLINK l _Toc517678551 4.1.2 The concentration of Pb in fishes collected on February 2018 PAGEREF _Toc517678551 h 24 HYPERLINK l _Toc517678552 4.1.3 The comparison of Pb concentration different organ of all fishes PAGEREF _Toc517678552 h 27 HYPERLINK l _Toc517678553 4.1.4 Transfer Factor PAGEREF _Toc517678553 h 28 HYPERLINK l _Toc517678554 4.2 Discussion PAGEREF _Toc517678554 h 29 HYPERLINK l _Toc517678555 4.2.1 Lead accumulation in fishes PAGEREF _Toc517678555 h 29 HYPERLINK l _Toc517678556 4.2.2 Lead accumulation in different organs of fishes PAGEREF _Toc517678556 h 30 HYPERLINK l _Toc517678557 4.2.3 Lead accumulation in different fish species PAGEREF _Toc517678557 h 31 HYPERLINK l _Toc517678558 4.2.4 Transfer Factor PAGEREF _Toc517678558 h 32 HYPERLINK l _Toc517678559 5. Conclusion PAGEREF _Toc517678559 h 33 HYPERLINK l _Toc517678560 References PAGEREF _Toc517678560 h 34 HYPERLINK l _Toc517678561 Appendices PAGEREF _Toc517678561 h 40 HYPERLINK l _Toc517678562 Appendix 1 Picture of fish samples in the study PAGEREF _Toc517678562 h 40 HYPERLINK l _Toc517678563 Appendix 1A Picture of studied fishes collected on November 2017 PAGEREF _Toc517678563 h 40 HYPERLINK l _Toc517678564 Appendix 1B Picture of studied fishes collected on November 2018 PAGEREF _Toc517678564 h 41 HYPERLINK l _Toc517678565 Appendix 2 Main materials used in the study PAGEREF _Toc517678565 h 42 HYPERLINK l _Toc517678566 Appendix 3 Standard concentrations of Pb and their absorption PAGEREF _Toc517678566 h 43 HYPERLINK l _Toc517678567 Appendix 4 Calibration Curve of Pb PAGEREF _Toc517678567 h 43 HYPERLINK l _Toc517678568 Appendix 5 Table of results PAGEREF _Toc517678568 h 44 LIST OF FIGURE TOC h z c Figure HYPERLINK Final thesis no MENDELEY.doc l _Toc517689941Figure 2.1 Anthropogenic sources of heavy metals flow in the environment PAGEREF _Toc517689941 h 5 HYPERLINK l _Toc517689942 Figure 2.2 The schematic diagram of AAS PAGEREF _Toc517689942 h 14 HYPERLINK Final thesis no MENDELEY.doc l _Toc517689943Figure 4.1 Pb in the fleshes of different fishes collected on November 2017 PAGEREF _Toc517689943 h 21 HYPERLINK Final thesis no MENDELEY.doc l _Toc517689944Figure 4.2 Pb in the gills of different fishes collected on Novemer 2017 PAGEREF _Toc517689944 h 22 HYPERLINK Final thesis no MENDELEY.doc l _Toc517689945Figure 4.3 Pb in inner organ of different fishes collected on November 2017 PAGEREF _Toc517689945 h 23 HYPERLINK l _Toc517689946 Figure 4.4 The average concentration of Pb in fishes collected on November 2017 PAGEREF _Toc517689946 h 24 HYPERLINK l _Toc517689947 Figure 4.5 Pb in the fleshes of fishes collected on February 2018 PAGEREF _Toc517689947 h 25 HYPERLINK Final thesis no MENDELEY.doc l _Toc517689948Figure 4.6 Pb in the gills of fishes collected on February 2018 PAGEREF _Toc517689948 h 25 HYPERLINK Final thesis no MENDELEY.doc l _Toc517689949Figure 4.7 Pb in inner organs of fishes collected on February 2018 PAGEREF _Toc517689949 h 26 HYPERLINK Final thesis no MENDELEY.doc l _Toc517689950Figure 4.8 The average concentration of Pb in fishes collected on February 2018 PAGEREF _Toc517689950 h 27 HYPERLINK Final thesis no MENDELEY.doc l _Toc517689951Figure 4.9 Pb in different organ of all fish species PAGEREF _Toc517689951 h 28 LIST OF TABLES TOC h z c Table HYPERLINK l _Toc517864997 Table 2.1 Permissible limit of Pb in freshwater fish PAGEREF _Toc517864997 h 9 HYPERLINK l _Toc517864998 Table 3.1 List of fish species, number, and size of fishes used in this study PAGEREF _Toc517864998 h 17 HYPERLINK l _Toc517864999 Table 3.2 Standard concentration of lead PAGEREF _Toc517864999 h 19 HYPERLINK l _Toc517865000 Table 4.1 Results of TF of Pb from water/sediment to fishes PAGEREF _Toc517865000 h 30 INTRODUCTION Background Tonle Sab Lake is known as the huge lake in Cambodia and the biggest freshwater body in Southeast Asia. It covered by an area of 100,000-1,600,000 ha (10,000-16,000km2) in the wet season and 250,000-300,000 ha (2,500-3.000km2) in the dry season with the water levels about 8m between the minimum in the dry season and the maximum in the rainy season (Matsui et al., 2006). This lake is bordered with five provinces including Kampong Cham, Pusat, Kampong Thom, Battambang, and Siem Reap. About one and half million Cambodian people are living in the greater Tonle Sap. They depend directly on the Tonle Sap, mostly fishers and farmers. 90 of peoples houses were constructed in the lake and only 10 are living in floating village. Most of them are depending on fishing and some agriculture for their livelihoods. This lake is an extremely useful natural resource of livelihood for Cambodian people as a source of food and income. It is one of the most productive freshwater ecosystems and the source of the commercial fishery in the world (Webb and Clements, 2011). Adding to this, The Tonle Sap is home to hundreds of kinds of fish. Every year, it provides yields about 235, 000 tons of fish, making it one of the worlds most productive freshwater ecosystems. Additionally, more than 200 species of fish have been documented in the Tonle Sap and over 500 species in the freshwaters of Cambodia. Along with the world standard, Cambodia is the fourth largest of freshwater capture fisheries production in the world after China, India, and Bangladesh. Cambodian is considered the highest of fish consumption in the world (Van Zalinge et al., 2001). Over the last few decades, Tonle Sap has been threatened by illegal fishing, medium and large commercial fishing, water pollution, hydroelectric dumping (e.g. the hydropower dams in Sesan II) and deforestation. Nowadays, we observe that Tonle Sap Lake is being polluted with different pollutants. Some of them are directly discharged from natural and domestic waste, industrial effluents, and others come from polluted runoff agricultural areas around the lake. Notably, the population in the Tonle Sap is being increased gradually from day to day. The population of Cambodian is rising rapidly at a rate of closely 2 a year (Chrish Nerdik, 2014). The pressure from the surrounding people is threatening to damage the environment in Tonle Sap, which can be caused by daily waste from community households, especially the 170 flooded villages. The increase of population leads to increasing more floating houses and more job activities in the lake. This is a concerning factor cause more pollutions in water quality and quantity that threaten fishs habitat as well the population in the lake (IUCN, 2013). The contamination of freshwater in Tonle Sap Lake with a wide range of pollutants has become a great concern over the last few decades. That water pollution can be mostly classified as organic, inorganic, suspended solids and sediments, radioactive, and heavy metals. Among those, heavy metals have been known as the most important forms of pollution, and they might accumulate in fishs tissues which are the top of the aquatic food chain (El-Moselhy et al., 2014 Malik et al., 2010). Fish is the most important food chain for Cambodian practically, people around Tonle Sap Lake. Fish may be contaminated with heavy metals released from domestic, industrial and other man-made activities, and they might be toxic for human consumption (Mol et al., 2010 Vinodhini and Narayanan, 2008). The effects of heavy metals on human health are the great interest today, especially for aquatic food products. Heavy metals have become a worldwide problem and scientific concern because the metals have toxic effects on organisms including animal and human who consumed food risked from heavy metal (Dural Eken et al., 2007). Likewise, there are many studies have been published on heavy metals in water, sediments and freshwater fish in others countries (Akan et al., 2012 Baharom and Ishak, 2015 El-Moselhy et al., 2014 Elnabris et al., 2013 Tzen, 2003 Vinodhini and Narayanan, 2008). The pollution of heavy metals in the environment becomes the most concern for human health. Heavy metals have been polluted almost everywhere in the world (including water, sediment, soil, plants, and fishes) that every scientist needs to think of. Studying on heavy metal in fishes become the great interest because fish are direct contact with water, so it constantly exposed to pollutants from contaminated water, they could be used as excellent biological markers of heavy metals in the aquatic ecosystem. Objectives of the study Scope and limitation The aims of the study were to determine the concentration of heavy metal (Pb) in different fishes from Tonle Sap Lake. The study was conducted in on November 2017 and February 2018. Eleven fishes of Tonle Sap Lake were chosen as samples in the study because they are the most common species in the area. The study was analyzed of three part of each fish such as fleshes, gills, and inner organs. Age of the sampled fish could not be established or controlled which would likely influence the concentrations of the metals to be obtained. It was limited regarding study all heavy metals because of time limitation and the ability of AAS machine. LITERATURE REVIEWS Heavy Metals in the Environment Heavy metals toxicity can result in damaged and decreased mental and mean anxious function, decrease energy levels, yet harm in conformity with blood composition, lungs, kidneys, liver, and mean integral organs toughness. Long-term exposure executes lead according to gradually prosperous physical, muscular, and neurological degenerative processes up to expectation imitate ailments namely multiple sclerosis, Parkinsons diseases, Alzheimers disorder then wide dystrophy. Repeated long-term publicity over half metals or their compounds may additionally motive cancer (Jaishankar et al., 2014). Distribution of Heavy Metals in the Aquatic Environment In the aquatic environment, heavy metals are furcate among a number of aquatic environmental booths (soil, water, suspended solids, sediments or aquatic animals. They might occur in solidified particulate and multipart form include Fe- and Mn-oxyhydrates, carbonates, clay minerals, sulfides and organic or biological substances (Helios Rybicka et al., 1995). Similarly, they are presented in aquatic systems as a result of the surviving of soils and rocks, from volcanic eruptions, and from a variety of human activities relating the mining, processing, or using metals or materials that comprised metal pollutants. Additionally, these metals enter the aquatic systems by direct releases via industrial and urban effluents, surface runoff and indirectly from floating fallout. Heavy metals transfer into water grant via domestic wastes or industrials, and even from acid rain breakage down soils and discharging strong metals into lakes, streams, rivers, or groundwater (Lenntech, 2018). Continuously, they can accumulate in fishes through the direct uptake of water and food which were contaminated with heavy metals. They are transferred into the fish through grills and distributed by the blood to different tissues in the body, where they may cause toxic effects before they are excreted via urine or farces, or across the gills (Langston and Bebianno, 1998). Dangerously, the extreme amount of them can build up to a toxic level (Karunanidhi et al., 2017). Heavy Metal is not only contained in the aquatic environment, they are also accumulated in the human body through the food chain (e.g. aquatic plants and aquatic animals). Therefore, there has been increasing anxiety, both in the developing countries and the developed countries in the world, about exposures, intakes and absorption of heavy metals by humans (Morais and Garcia, 2010). Generally, the cycle of anthropogenic sources of heavy metals in the surrounding is illustrated in the figure below as proposed by Chalermsupanimit (2006). Accumulation of Heavy Metals in Fish Our environment is becoming increasingly contaminated with heavy metals which is a result of mining, forestry, waste disposal and fuel combustion. The aquatic environment receives waste products from such activities and can be the last repository for heavy metals that repaired this new ally (Langston and Bebianno, 1998). Fishes have been recognized as a worthy accumulator of organic and inorganic contaminants (Eneji et al., 2011). They are used as bio-indicator regarding aquatic ecosystems for determination of heavy metals pollution and possible risk for human consumption. Accumulation of heavy metal in fish takes place directly from the water by gills and indirectly from food (Barron, 1990). Heavy metals feed fish through five major pathways (food or non-food particles, gills, water, and skin), followed by blood and are taken to a storage or liver for its transformation or storage (Weber et al., 2013). Generally, accumulation of metal in an organism depend on the interactions between physiological factor (growth, weight loss, absorption and accumulation), chemical factors (metal concentration, speciation and bioavailability) and environmental factors (temperature, pH, water hardness, conductivity, salinity and food concentration) (Casas and Bacher, 2006). Similarly, the degree of contamination depends on the type of pollution, the type of fish, the model location, the trophic level, and their eating behavior (Weber et al., 2013). Fish can uptake heavy metals across two main absorptions including the gills and gastrointestinal. Metal uptake in fish differs fundamentally from that in terrestrials because of the fact that fish have gills that are constantly submerged in a solution of metal ions. The gills are the main point of access for metal dissolving, so it becomes the most target for metal poisoning. The gills have the important physiological roles to take up oxygen from the water and release carbon dioxide. This physical constraint means that large amounts of metals are passed over the gills at any time, which enhances metal uptake. This physical barrier means that the amount of metals is passed over the gills at any times, which increases the absorption of metals (Langston and Bebianno, 1998). Another absorption is gastrointestinal that is an important route for metal absorption in fish. The most studies showed that inner organ is the most target organ for metals accumulation. Commonly, the relative importance of the intestine for the metal absorption rises with increased hardness and salinity of the water. These factors can decrease the bronchial absorption of many metals. Generally, metals absorbed into the fish body through the gills or intestinal wall then they are distributed by the circulation, bound to transport proteins, to the various tissues of the body. Another factor is that fish drink a lot of water, which means that metals can be taken not only by food, by the gastrointestinal tract, but also from water (Langston and Bebianno, 1998). In the same way, metal accumulation in fish can be influenced by the pollution and may differ for various fish species living in the same water body. Liver, kidney, and gills of fish are the most target organ for metals accumulation. If comparing to the other tissues, fish muscles usually contain the lowest levels of metals (Jezierska, 2006). Heavy Metal in the study Heavy metals include both essential and non-essential elements are significance toxic to ecosystems due to their toxicity, long persistence, bioaccumulation, and bio-magnification stresses in the food chain (Weber et al., 2013). The pollution of heavy metals in the aquatic environment has become a great concern in the world because these metals are indestructible, and most of them have toxic effects on organisms (Burchett, 2000). The most common Potential Toxic Elements (PTE) listed by the United States Environmental Protection Agency (USEPA) are arsenic (As), cadmium (Cd), chromium (Cr), copper (Cu), iron (Fe), lead (Pb) ,mercury (Hg), Manganese (Mn), nickel (Ni) and zinc (Zn). Some of these PTEs such as Cr, Cu, Fe, Mn, Ni, and Zn are essential for the metabolic activities of living organisms which require by organisms at a low level and become toxic at some higher levels. Whereas, elements as As, Cd, Hg, and Pb are known as non-essential metals and their toxic effect on human health even in trace amount s (Bury, 2003). fLead (Pb) Lead is a naturally occurring high toxic metal found in the Earths crust, accounting for 13 mg/kg of the earths crust. Several stable isotopes of lead occur in nature, including, in order of abundance, 208Pb, 207Pb, 206Pb, and 104Pb. It is a soft metal which has a melting point of 330oc (WHO, 1998). Moreover, Pb has no known biological function and there is no evidence that it is required, or otherwise beneficial, for life. Additionally, Pb is toxic even at a low level (Merlini and Pozzu, 1977). Nowadays lead is used in more than 900 industries, including mining, smelting, refining, battery manufacturing and so on. Its toxicity today is known as a main environmental health risk, with the most serious effects in young children, and it is also a common occupational health hazard in developing countries (Mohammadi et al., 2008). The frequent utilize of Lead has resulted in great environmental contamination, human contact and primary public health issues in many parts regarding the world (WHO, 2006). Lead is used in the production of lead-acid batteries and other electrical, refineries, fertilizers, plastics, pigments and paints, alloys and solders, biocides (pesticides, herbicides, preservations), catalysts, fuel, cable sheathing, rust inhibitors, ammunition and glazes (FAO, 1992 WHO, 1998). Natural and human activities are the major sources of Pb in the environment. Around 50 of atmospheric lead in the environment comes from natural sources. Forest fires, volcanic activity, weathering of soil, geochemical weathering and sea spray emissions, and remobilization of historical sources-such as Lead in water soil, and sediment come from mining area are natural sources of lead in the environment (WHO, 2010). Sources of lead that come from human activity include the discharge of ammunition, leaded fuel in light aircraft and the combustion of coal and wood, as well as various processes in metal production and manufacturing, and it contaminates crops, soil, water, food, air and dust, lead can enter the environment at any stage from its mining to its final use. Besides, lead can also directly enter aquatic environments from urban sources such as household uses, waste management, and sewage treatment plants, then it direct emissions to water from industrial sources during mining, manufacturing, and recycling. Lead can go into a body of water from a variety of aquatic, atmospheric and terrestrial routes and may be present in drinking water through leaching from lead-containing pipes, faucets, and solder frequently found in the plumbing of older buildings (CSEM, 2017). The most concern for the lead entering aquatic environments is from point-source releases related to Pb mining and industrial processing. Lead was tended to remove from the water by natural processes and deposit in sediments where its availability to pose harmful effects to aquatic organisms in very low. When established in sediment, it goes to bind or reacts with other chemical species and is transformed into forms that are largely insoluble such as adsorption onto manganese and iron-containing minerals and clay minerals, or in anoxic regions near the sediment/water interface, by conversion to lead sulfide (ila, 2018). After absorbed into the fish body through water, sediment, and foods, Pb is distributed in blood by the circulation, bound to transport proteins, to the various tissues of the body including bone, gill, kidney, spleen, and intestine of fish (Mager, 2011a). Pb is frequently presented highest concentration in the gill and kidney, but as these tissues are relatively little mass, their overall contribution to the whole body thus, Pb accumulation may be rather small. It was absorbed into fish by gills and excreted through gill too. Accordingly, it is probable that the gills may possibly act in a similar fashion to that of the skin, to excrete Pb within mucus, thereby implicating a potential role for bronchial goblet cells in the elimination of Pb (Mager, 2011b, pp. 213221). The gill is studied as the target organ for Pb accumulation in fish (El-Moselhy et al., 2014). Maximum limited level of Pb in freshwater fish The maximum limited level is the high level of inventory and quantities that must not exceed specific management. It can indicate the safety level of chemical substances in food and also the environment. For heavy metal, the maximum limited level is an important thing to think of. If there are over the maximum level of heavy metal in the environment, it will lead to cause high toxic and negative impact. The maximum limited level of Pb in freshwater fish was described below. (See table 2.1) Table STYLEREF 1 s 2. SEQ Table ARABIC s 1 1 Permissible limit of Pb in freshwater fish StandardPb concentration in fishReferencesFAO0.2 mg/kg (fresh weight) 0.5mg/kg (fresh weight) 2 ppm(FAO, 2003a) (FAO, 1983a) (FAO, 1983b)EC0.2-0.4 mg/kg (fresh weight)(Ec, 2000 EU, 1993)USFDA0.3 mg/kg (fresh weight)(USFDA, 1993)China0.5 mg/kg ww(China, 2015)Hong Kong6 mg/kg ww(Hong Kong, 2000) Kind of studied fishes Freshwater fishes of Tonle Sap Lake were used as samples to determine the assessment of Pb in their organs. Eleven different species fishes collected from Tonle Sap Lake were caught during November 2017 and February 2018. All fished were purchased from fishermen in Chhnok Tru floating village in Kompong Chhnang province. Seven different fish species were caught on November 2017 namely Trey Po, Trey Kai, Trey Prolung, Trey Prul, Trey Chhdor, Trey Klanghay, and Trey Chraking. Four other fish species were caught on the end of February 2018 such as Trey Broma, Trey Bra, Trey Chhdor, and Trey Chhkok. Trey Po Trey Prorlung Trey Prolung or Leptobarbus hoevenii (scientific name) is a kind of freshwater and omnivorous fish from family genus Leptobarbus that can grow up to 100 cm long with the common length about 50cm and the maximum weight 10kg. It is a non-migratory species found most often in free-flowing rivers and streams and seasonally on floodplains. This fish is usually occurring in the Mekong basin and found around the Tonle Sap River and Great Lake. It absolutely does contribute in local trophic migration to and from inundates forests and swim in the reef area and have been fishing in the Mekong River, especially during the seawater (January-February) and inland waterway migration (June-July). Besides, Juveniles feed on terrestrial insects, zooplankton, and tubificid worms. Adults consume more plant matter and known to eat on poisonous fruits in floodplain forests. Additionally, this type of fish eat mainly parenchyma and seeds of chaulmoogra trees (Hydnocarpus) fall into the stream was reported to become intoxicated and behaved strangely and believed to be toxic. Trey Prolung (Leptobarbus hoevenii) was reported from seven countries like Cambodia, Thailand, Viet Nam, Laos, Indonesia, and Malaysia (Bleeker, 1851 Rainboth, 1996, p. 74). Trey Prul Trey Prul or Cirrhinu microlepus (scientific name) is an important fishery species found in large rivers and lowland floodplains of Thailand, Cambodia, and Vietnam. This fish is the species of ray-finned fish in the genus Cirrhinus which can grow up to 65cm long with the maximum size 5kg. In adding, it most often takes place in riffles and deep slow reaches and always moves out into the flooded forest to find feeding. It is highly migratory that probably enters flooded floodplain forest in Cambodia. Its feeding habit is in a group of herbivore fish which feeds on leafy plant matter, phytoplankton and insects. Trey Prul (Cirrhinu microlepus) was found in 4 countries Cambodia, Thailand, Laos and Viet Nam. It mainly feeds on it feeds on leafy plant matter, phytoplankton and insects (Baird, 2013). Trey Chhdor Trey Chhdor is nasty and carnivore fish. It originated in the lake and other freshwater bodies in South Asia and Southeast Asia from India to Vietnam. In scientifically, it named Giant Snakehead or Channa micropeltes. This fish can grow up to 100cm with the maximum size 20kg. This kind of fish occurs in lowland rivers and swamps and usually associated with deep water bodies in the Mekong basin, and was found around the Tonle Sap River and Great Lake. In addition, this species lives standing or slowly flowing water from India to Indonesia and is common throughout Cambodia. It feeds mainly on fish but also takes some crustaceans while late juveniles of the species school and feed in packs. The species has a very varied Southeast Asian distribution, from the lower Mekong (Thailand, Lao PDR, Cambodia-including the Tonle Sap Lake and river) and the Mekong Delta in Viet Nam), Indonesia, the Malay Peninsula and the Chao Phraya and Maekhlong (Thailand) basins. Normally it was stated from 9 countries which are Cambodia, Thailand, Laos, Viet Nam, India, Myanmar, Singapore, Indonesia, and Malaysia (Allen, 2015 Cuvier in Cuvier and Valenciennes, 1831 Range, 2014). Trey Klanghay Trey Klanghay is a kind of omnivore fish which has a scientific name as Belodontiehthys truncates. This fish can grow up to 60cm long that is found in the Mekong (Viet Nam, Cambodia, Lao PDR and Thailand) and Chao Phraya (Thailand) basins, Mekong River drainage, around the Tonle Sap River and Great Lake. It occupies in big rivers with some flow and deep water pools, enters large streams to spawn in July and August, and then go back to the large rivers in November-December. It stays in deep water pools during the dry season and feeds on insects, shrimp, crabs, snails and especially fish. In addition, this fish usually travels upstream over the Khone Falls in June-July with other silurids and enters the flooded forest in July-October. It feeds mainly on cyprinids of the genus Henicorhynchus. Present evidence shows that it is still fairly widespread and abundant and it is quite a mutual species in northern Cambodia and southern Lao PDR that originated from Cambodia, Thailand, Laos, Viet Nam (Ng, 2015). Trey Bra Trey Bra or Pangasian odon hypophthalmus (scientific name) is a kind of freshwater, benthopelagic, and sark-catfish. This species found in large rivers of the Mekong and Chao Phrya basins, and now widely announced for aquaculture. Moreover, it is common in the lower Mekong, where the young are calm for rearing in floating fish cages. It is a group of omnivorous fish which feeds on fish and crustaceans as well as on vegetable. This fish is a migratory species which moving upstream of the Mekong from unidentified rearing areas to spawn in unidentified areas in May-July and going back to the mainstream once the river waters drop pursuing rearing habitats in SeptemberDecember. Trey Bra (Pangasianodon hypophthalmus) can grow at the maximum length 130 cm long with the maximum weight 44 kg. It distributed in Mekong, Chao Phraya, and Maeklong basins and introduced into additional river basins for aquaculture. Trey Bra ( HYPERLINK https//www.fishbase.de/summary/SpeciesSummary.phpgenusnamePangasianodonspeciesnamehypophthalmus Pangasianodon hypophthalmus) was reported from 10 countries including Cambodia, China, Laos, Myanmar, Philippines, Singapore, Taiwan, Thailand, Viet Nam and Bangladesh debris (Rainboth, 1996, p. 152). Trey Chhkok Trey Chhkok is known as a large and expensive freshwater fish in Cambodia. Its scientific name is Cyclocheilichthys enoplos (Bleeker, 1849). This fish is a high immigration species that occurs at midwater to bottom levels of rivers that have maximum length 74cm (common length 45cm) with the maximum weight up to 17kg. It mainly eats roots of plants, bivalves, and zooplankton. Young known feeding on green algae while adults prey also on crustaceans, insect larvae, and fish. It was found in the Lower Mekong Basin and seasonally adjusted from Tonle Sap Lake to northeastern Thailand between November and February, and then returns from June to July. Mostly, it spawns in July and August, probably on the floodplains or inundated riparian forests (Baird, et al., 1999). Trey Chhkok (Cyclocheilichthys enoplos) was reported from 6 countries which are Cambodia, Thailand, Laos, Viet Nam, Indonesia, and Malaysia. It distributes in Asia from Cambodia, Thailand, Laos and Viet Nam to Indonesia and Malaysia. (Base, 2004, p. 75) Trey Broma Trey Broma is a benthopelagic fish species which has the maximum length 100cm with the maximum weight 18 kg. Its scientific name is Boesemania Microlepis. It was found in found in parts of the Mekong River basin in Laos, Cambodia, Vietnam, and some connecting canals in Thailand (Baird, 2014). This fish occurs in flowing waters of large rivers and the deep-water pools of the Mekong River even during the dry season. It was classified in the group of carnivore fish which feed on crustaceans, shrimps, and fishes. The countries occurrence of this species is Cambodia, Indonesia (Kalimantan, Sumatera), Lao Peoples Democratic Republic, Malaysia (Peninsular Malaysia), Thailand, Viet Nam (Croaker and Baird, 2015). The principle of Atomic Absorption Spectrophotometer Atomic Absorption Spectrophotometer (AAS) is a very common procedure for detecting and metal and metalloids in samples, particularly environmental samples. It is very reliable to use and it has high ability to analyze over 62 elements, especially metals and metalloids. Moreover, it is an extremely sensitive technique, permitting concentrations as low as one part per billion to be measured. Atomic absorption spectrophotometer is also more rapid than conventional methods such as volumetric analysis. It is used to analyze the concentration of metals in various, for example, water, soils, food, plants, and blood. AAS is working depend on five basic components such as hollow cathode lamp, nebulizer, atomizer, monochromator, and detector. The schematic diagram of AAS was shown in Figure 2.2. There are of an atomic absorption instrument Figure STYLEREF 1 s 2. SEQ Figure ARABIC s 1 2 The schematic diagram of AAS Hollow cathode lamp is the main sources used for atomic absorption that an excellent, bright, stable line source for most elements. They are known as the most common sources of radiation in AAS that involve a tungsten anode and a hollow cylindrical cathode prepared by the element to be determined. Nebulizer has functioned as sucking up liquid samples at a controlled rate and creates a fine aerosol spray for introduction into flame. It mixes the aerosol and fuel and oxidant thoroughly forintroduction into flame. Atomizers Flame atomizer were used to create flame, we need to mix an oxidant gas and a fuel gas. In most of the cases, air-acetylene flame or nitrous oxide-acetylene flame is used. The flame atomizer is typically used for liquid or dissolved. Methodology Sampling and sample treatment 11 different species fishes were purchased directly from the local fisherman in Chhnok Tru floating village of Tonle Sap Lake during the wet season and dry season. We could not point to the reallocation of each sample, but we point out the location of the place we bought those samples. All sampled were immediately measured the weight and length, and they were packaged in plastic bags then stored with an ice block in order to maintain the freshness and later transported. Formerly, they were transferred to the laboratory where they were prepared and kept in a freezer at 19 oc until further analysis. Fishes were first identified by species, and then the total length (cm) and the body wet weight (g) of each specimen were measured. The detailed information is listed in Table 3.1. Table STYLEREF 1 s 3. SEQ Table ARABIC s 1 1 List of fish species, number, and size of fishes used in this study Khmer NameScientific nameNoFeeding habitWeightLength (November 2017)Trey PoPangasius larnaudii1Omnivore600g31.8cmTrey KraiChitala Ornate1Carnivore600g31.8cmTrey ProrloungLeptobarbus hoevenii1Omnivore430g23.5cmTrey PrulCirrhinu microlepus 1Herbivore740g42.4 cmTrey ChhdorGiant Snakehead 1Carnivore2500g53.8cmTrey KlanghayBelodontiehthys truncates2Omnivore1000g28.8cmTrey ChrakaingPuntioplites falcifer6Herbivore720g17.2cm (The end of February)Trey BromaBoesemania Microlepis2Omnivore2100g23.6cmTrey ChhdorGiant Snakehead1Carnivore1700g36.5cmTrey BraPagnasius djambal1Omnivore2600g68.5cmTrey ChhoukCyclocheilichthys enoplos 1Omnivore1200g47 cmSample preparation All samples were washed with distilled water to clean soil, dust and foreign materials. Each fish was separated into three parts include gills, flesh, and internal organ. All instrument which was used for preparing fish samples needs to be clean and make sure there was no contamination from each sample. Each part of fish were well separated, and they were cut into small pieces prior to dry. Prepared samples were individually freeze dry by freeze dryer (Christ Alpha 1-4 LD) for 24 hours with pressure 40 MPa at -20 oc for fleshes and 36 hours with pressure 40 MPa at -20 oc for gills and inner organs until reaching a constant weight. Digestion procedure Each dried samples were ground into a fine powder by using porcelain mortar and pestle or blender, and thereafter all powdered samples were prepared to digest the trace metals. According to FAO method, the powdered fish samples were carefully homogenized before exposing them to digestion and were digested using concentrated nitric acid and hydrogen peroxide with ratio 11 v/v (Daziel, 1983). 1g of dried powdered fish samples was weighed and transferred into 50 mL beaker. The mixture of 10 mL of concentrated HNO3 (65) and 10 mL of H2O2 (30) was added to each beaker. Each beaker was covered with an aluminum paper and left aside until occur the initial vigorous reactions. Then, the samples were heated on a heating Magnetic Stirrers to 130 oC until dissolution inside a fume hood to decrease the volume to 3-4 mL. After that, the samples were allowed to cool down at room temperature, were filtered and diluted to 50 mL in a volumetric flask with distilled water (Begum et al., 2009). Instrumentations and Apparatus All digestion works were carried out by using heating Magnetic Stirrers. Knife and chopping board were used for fish preparation. Ziplock size 16cm x 20 cm was used for packaging fish samples during the experimental procedure. The fish samples were dried by using Freeze Dryer (Christ Alpha 1-4 LD) and grounded by using blender into small particles. 50ml beaker, 50ml tube, Syringe, micro filter 45 m was used for digestion purpose. AA-7000 atomic absorption spectrophotometer was used to analyze for trace metals in the fish sample. Reagent and standard The chemicals standard was used as analytical reagent grade. HNO3 (3) was used for all dilutions and rinse equipment. Nitric acid, HNO3 (65), and hydrogen peroxide, H2O2 (30) were used in digestion procedure. Pb (1000 mg/L) stock standard was used for the standard solution. Working standard was prepared by diluting a concentrated stock solution of 1000 mg/L for Pb in HNO3 (3). Analysis of trace metals Lead concentration was determined in fish samples which were carried out by AA-7000 AAS (Shimadzu). Atomic Absorption Spectrophotometer (AAS) is a technic used to determinate the presence and concentrations of metals in both liquid and solid samples through the wavelengths of light. The accuracy of the atomic absorption spectrophotometer was determined by setting the calibration curves of each heavy metal. This also helps to confirm that the results of the determination were accurate and reliable. The calibration of the AA-7000 Atomic Absorption Spectrophotometer (Shimadzu) was made with standard solutions. Four working calibration standards were prepared by serial dilution of a concentrated stock solution of 1000 mg/L for Pb. These solutions were aspirated into AAS to analyze their concentration. The series of the concentrations of standard solutions and its correlation coefficient (R2) was used to calibrate the AA-7000 AAS were given in Table 3.8. For the calibration curves of each standard was shown in Appendix 3. After calibration, the samples were analyzed in triplicates, and the blank determinations in triplicates were also run in the same manner during the analysis. Table STYLEREF 1 s 3. SEQ Table ARABIC s 1 2 Standard concentration of lead MetalStandard conc. (ppm)WavelengthDetection limitThe correlation (R2)Pb0.5, 1, 2, 5283.13 nm1 g/L0.9998 The concentration of elements was calculated by following (Perkin Elmer Coorporation, 1996) as the formula below QUOTE Eq 3. SEQ Equation ARABIC 1 Where C the concentration of the element in the sample solution in mg/L V the volume of the undiluted sample solution in mL W the sample weight in grams df the dilution factor, if used, as described below Transfer Factor Transfer factor is the factor to indicate the main source of heavy metals pollution in fishes. Fish transfection factors from water systems, including water and sediment, were calculated according to (Rashed, 2001) and (Odokuma et al., 2010) as follows TF Mtissue / Msediment or water Eq 3.3 Where Mtissue is the metal concentration in fish tissue (ppm) Msediments is the metal concentration in sediment (ppm) Transfer factor can indicate whether heavy metal biomagnification takes place. A Transfer Factor greater than 1 indicates bioaccumulation. Example TF sedimends 0.001, TF water 1.908 so it indicated that fish contaminated HM from water rather than sediments. The transfer factor of water is greater than those of sediments and all transfer factor from sediment was less than 1, therefore this showed that fish bioaccumulate. Results and discussion Results The concentration of Pb in fishes collected in November 2017 Pb concentration in fish gills collected in November 2017 Lead concentration in fish gills from Tonle Sap lake was presented in Figure 4.2. The concentration of Pb varies from ND to 8.1425 mg/kg wet weight. Pb was not detected in the gill of Trey Po which indicates that this fish was not contaminated with Pb. The lowest level of Pb was found in the gill of Trey Krai with the quantity of 1.32 mg/kg wet weight while highest concentration was found in Trey Chrakaing (8.1425 mg/kg wet weight). The concentration of Pb in all fishes except Trey Po were higher than permissible level of international standard in the range from 0.2, 0.2-0.4, 0.3, 0.5 mg/kg fresh weight ( FAO, 2003b, Ec, 2000 USFDA, 1993 FAO, 1984). Pb concentration in fish inner organs collected in November 2017 The average concentrations of Pb in fishes in collected on November 2017 Analyzed results of Pb concentration in the three organs (fleshes, gills and inner organs) of four different fishes from the Great Lake collected in February 2018 were presented by range values and averages. The concentration of Pb in each organ of the four fishes is showed in Figure 4.5-4.8. All results are stated on a wet weight basic as mg/kg wet weight. The concentration of Pb was varied among the three organs of four different fish species which range from 3.556 to 10.0517 mg/kg wet weight. The lowest level of Pb is found in the fleshes of Trey Broma while the highest is in the inner organs of Trey Chhkok. The concentration of Pb in the flesh of different fishes collected in February 2018 The analyzed concentration of Pb in all fish fleshes collected from Tonle Sab Lake collected in February 2018 were shown in Figure 4.5. Four different fish fleshes of Tonle Sab Lake were chosen to be analyzed Pb concentration. The variation of Pb concentration in four different fish fleshes was from 3.556 mg/kg wet weight to 5.83 mg/kg wet weight. The study found that Trey Broma (Boesemania Microlepis) comprised the lowest level of Pb and Trey Chhkok (Cyclocheilichthys enoplos) contained the highest level of Pb. The results were rising gradually with the range of 3.556 mg/kg wet weight, 3.67 mg/kg wet weight, 4.126 mg/kg wet weight and 5.83 mg/kg wet weight in order of Trey Broma (Boesemania Microlepis), Trey Chhdor (Giant Snakehead), Trey Bra (Pagnasius djambal and Trey Chhkok (Cyclocheilichthys enoplos), respectively. Figure STYLEREF 1 s 4. SEQ Figure ARABIC s 1 5 Pb in the fleshes of fishes collected in February 2018 The concentration of Pb in the gills of different fishes collected in February 2018 The results of Pb concentration in fish gills from Tonle Sab collected in February 2018 in Figure 4.6. The study was conducted on four dissimilar fish species. The results show that the lowest level of Pb was found in Trey Brama (Boesemania Microlepis) gills with the value of 4.1625 mg/kg wet weight and the highest level of Pb was in Trey Chhkok (Giant Snakehead) gills that the value was reached to 7.43 mg/kg wet weight. The concentration of Pb in these fish gills were augmented intensely with the concentration of 4.1625 mg/kg wet weight, 4.5875 mg/kg wet weight, 5.1575 mg/kg wet weight and 7.43 mg/kg wet weight in order of in order of Trey Broma (Boesemania Microlepis), Trey Chhdor (Giant Snakehead), Trey Bra (Pagnasius djambal) and Trey Chhkok (Cyclocheilichthys enoplos), respectively. The concentration of Pb in the inner organs of different fishes collected on February 2018 Four different inner organs of fishes were conducted in this study to determine the level of Pb accumulation that the results were presented in Figure 4.7. After analyzed, the results show vary from 5.7414 mg/kg wet weight reach to 10.0517 mg/kg wet weight which the minimum concentration was found in Trey Bra (Pagnasius djambal) and the maximum concentration of Pb was in Trey Chhkok (Cyclocheilichthys enoplos). The level of Pb in the inner organ of fish was raised increasingly in order of Trey Bra (Pagnasius djambal), Trey Chhdor (Giant Snakehead), Trey Broma (Boesemania Microlepis) and Trey Chhkok (Cyclocheilichthys enoplos), individually. The average concentration of Pb in fishes collected in February 2018 Figure 4.8 represented the average level of Pb in different fishes of Tonle Sab Lake in February 2018. The concentration of Pb was found the higher amount in the inner organ of all fishes followed by gills and flesh. The calculated results of Pb concentration in the four fishes are compared in mg/kg wet weight given the following results Trey Broma 4.7475, Trey Chhdor 4.8617, Trey Bra 5.0083, Trey Chhkok 7.7410 which lead the following ranking Trey Chhkok (Cyclocheilichthys enoplos) Trey Bra (Pagnasius djambal) Trey Chhdor (Giant Snakehead) Trey Broma (Boesemania Microlepis). The comparison of Pb concentration different organ of all fishes Transfer Factor The results of TF of lead from water and sediments in each fishes body is shown in Table 4.1. The results showed that transfer factor of water were greater than those of sediments. All accumulation factors of water were greater than 1 except for Pb in Trey Po which is less than 1. The minimum value was measured in Trey Po while the maximum value of TF in water was in Trey Chrakaing. On the other hand, the transfer factor of sediment all fishes were found to be less than 1 except Trey Chrakaing. On the other hand, the transfer factor of Pb from sediment to fish species was found to be less than 1 except Trey Chakaing. The minimum value was found in Trey Po and the maximum value found in Trey Chrakaing. Table STYLEREF 1 s 4. SEQ Table ARABIC s 1 1 Results of TF of Pb from water/sediment to fishes Trey Chhdor collected on February 2018 Discussion Lead accumulation in fishes Fish are an important aquatic organism that is used as bioindicators of aquatic ecosystems for estimation of heavy metal pollution and risk potential for human consumption. When fish are exposed to increased iron levels in the rivers environment, they can absorb naturally occurring metals from the seeds through the seeds and the skin, or through the use of water and toxic food. In the body, metals are transmitted through the bloodstream and stored, transplanted or eliminated kidney, kidney or socks (WHO, 2004) and brought in contact with other organs and tissues (Van Der and Part, 1981). The understanding of the mechanisms or modes of action for the chemical concern is important such that the appropriate fish organs are selected and sampled. Selection of the appropriate fish organs can be specific to contaminants to discover their accumulation place in the fish body. This study was undertaken to investigate lead concentration in the fleshes, gills and inner organs of eleven fishes collected from Tonle Sab Lake, and to detect whether the level of Pb is potentially harmful to human health. Trey Po, Trey Krai, Trey Prolung, Trey Prul, Trey Chhdor, Trey Klanghay, Trey Chrakaing collected in November 2017 and Trey Broma, Trey Chhdor, Trey Bra, Trey Chhkok sampled at the end of February 2018, were selected because they are the most commonly consumed fish in Tonle Sap Lake. The levels of Pb were determined in the flesh, gill and inner organ of each species because they are important for human consumption and they can help to find the accumulation site of Pb in the fish body. This investigation showed that different fish species contained different concentrations of Pb in their fleshes, gills and inner organs. This is agreed with an arcticle that reported that different fish species accumulate metals in significantly different values in their tissue (Kalay et al., 1999). Furthermore, levels of heavy metals in fish vary in various species, foods, feeding habits whether herbivorous, omnivorous or carnivorous, whether pelagic or benthic fish, the level of water pollution, level of fish presence in water, and different aquatic environments (Canli and Atli, 2003 Kamaruzzaman et al., 2010). In addition, the ability of fish to accumulate heavy metals depends on ecological needs, metabolism, water and food, the degree of pollution in sediment, as well as salinity and temperature of water (Romo et al., 1999). Lead accumulation in different organs of fishes Pb is a part of the aquatic metal pollutions which common at remarkable levels of water system which may pose high toxicities on the aquatic organisms (Zhou et al., 2007). Tissue metal concentrations vary depending on the metal and organs. The concentration of metal differs depending on the metals and organs. The analyzed concentration of Pb found the highest in all the inner organ of all fish species both on November 2017 and February 2018 which followed by the gills and the fleshes. The inner organ includes liver, kidney, intestine, gonad, stomach, pancreas, reproductive organ, anus and wastes which are the accumulation site of heavy metal. Similarly, some study revealed that lead exposure resulted in various histopathological changes in the gills, liver, hepatopancreas, pancreas, stomach, and intestine of fish, and there are evidence of lead-induced histopathological changes in liver, gills, intestine, kidneys, and spleen of different fish models ( Aboud, 2010 Paul et al., 2014). Moreover, kidney and liver are known as the gateway for heavy metal detoxification and accumulation in the fish body (Vinodhini and Narayanan, 2008). Furthermore, Pb has elevated trends to bio-accumulate in fish kidney and liver tissue due to the similar function of the kidneys and the liver, which is associated with toxic detoxification processes. The presence of free protein and protein metallothionein linked to the kidneys and the liver form a strong bond with heavy metals (Maxwell and Iwegbue, 2008). The fish kidney located along the labyrinth of the body of the fish essentially contains excretory tissues, while the liver plays an important role in homeostasis (Dallinger, 1987). The intestines of fish compared to other organs act as a temporary place for metals metal in the fish body. The intestines involved in the absorption of small heavy metal fractions through food pipelines, in which heavy metals absorption rates are governed by specific transport systems through normal circulation throughout intestinal epithelium (Hasyimah et al., 2011). These are the reason for Pb high accumulation in the inner organ in the fish body. The gills of each fish species found that contained high level of the lead after inner organ. This well-known order was well found by other authors who established that this can be described by their respective roles in the fish life. This variation was may be due to the gill is the target organ for acute lead poisonousness and aspect little to whole body mass accumulation, which is clearly dominated by the lead content in the skeleton. Likewise, the gill is the important site for the entry of heavy metals that provokes lesions and gill damage (Vinodhini and Narayanan, 2008). In the other word, the gills have the important physiological roles to take up oxygen from the water and release carbon dioxide, and they are the main tissues of absorption of the metal contaminants for aquatic species. Moreover, the gills are the important point of access for metal dissolving, so it becomes the most targets organ for metal poisoning. This physical constraint means that large amounts of metals are passed over the gills at any time, which enhances metal uptake and also the absorption of metals (Langston and Bebianno, 1998). The lowest levels of Pb were found in the flashes of all fish species. The results are varied by different fish species. The flesh of fish is one the ultimate parts for heavy metal accumulation (Vinodhini and Narayanan, 2008). The heavy metals were consistently extent over the body muscles. Hence, the observed values were moderately lower than the other organs (gill and inner organ) (Vinodhini and Narayanan, 2008). Additionally, fish fleshes usually contain the lowest levels of metals if comparing to the other tissues because the flesh of fish is no known as the target organ for Pb accumulation ( Jezierska, 2006). Lead accumulation in different fish species The concentration of Pb was bioaccumulated at varying levels and was clearly distinguishable in different fish species of the Great Lake. In this present study, the level of Pb is depending on size and amount of fish. The results show the highest level of Pb mostly found in large fish and high amount of fish. Evidently, there was interesting to note that a large fish indicated very high concentrations of heavy metals ( El-Moselhy et al., 2014). They also suggested that feeding habit may be one reason of metal variation in the filter feeder which leads to accumulating relatively high concentrations of all metals in muscles. There are recommended that benthic fish, fish that likes to live near the bottom of the water, are likely to contain higher heavy metal concentrations than fish inhabiting the upper water column because they are in direct contact with the sediments and their greater uptake of heavy metal concentrations from zoobenthic predators ( Yi et al., 2011). Additionally, Carnivorous fish, which eat fish, shrimp, and zooplankton are generally known to have a high level of heavy metals in the body (Karadede et al., 2004). As well, Pb is mainly particulate bound with relatively low mobility and HYPERLINK https//www.sciencedirect.com/topics/chemistry/bioavailability o Learn more about bioavailability bioavailability in the environment, and its bioaccumulates mostly in organisms, in particular in biota feeding primarily on particles, e.g. worms and mussels (Al-Hossainy et al., 2017). Transfer Factor The presence of high levels of metals in the fish environment does not present a direct risk to fish if there is no greater collection of metal by fish tissues (Kamaruzzaman et al., 2010). Base on the calculated result of TF indicates that most fishes contaminated with Pb from the water. This is because the results of the TF value of Pb from water to fishes were greater than 1 except Trey Po. Even though, the TF of Pb from water to Trey Po was less than 1, but that value is still closed to 1 (0.8743) and the TF value of Pb from sediment to Trey Po was also far less than 1(0.0402), this lead to say that Trey Po still contaminated with Pb from water than sediment. On the other hand, all transfer factors from sediment were less than 1.00 except Trey Chrakaing which means that there is no bioaccumulation of any Pb occurred from sediment. However, the TF of Pb from sediment to Trey Chrakaing shows a bit higher than 1, it does not mean Trey Chrakaing contaminated Pb from sediment because the TF value of Pb from water to Trey Chrakaing shows highest in all fishes. Therefore, it leads to conclude that water is the bioaccumulation site of all studied fishes. Conclusion According to the result, the samples of the fleshes, gills and inner organs of eleven fish species obtained from Tonle Sap Lake during November 2017 and February 2018 were found to contain the concentration of lead at a different level. The results show that accumulation of Pb varied between organs and species depending on species-specific factors like feeding behavior, swimming patterns and genetic tendency, and/or other factors like age and geographical distribution that caused variation in metals accumulations between fish even from the same species. Comparing to the average concentration of lead in all fish samples, we found that Trey Po contained lowest level while Trey Chrakaing with the value ranges from 0.3448 to 8.1425 mg/kg wet weight. Moreover, this study found that the inner organ of each fish contained highest Pb levels compare with another organ which followed by gill and flesh. This indicates that inner organ is the favorite accumulation site of Pb in the fish body. The gill is also known as the target organ for Pb accumulation after the inner organ. Generally, fish flesh always contains less level of heavy metals, in particularly Pb. The accumulation of Pb in fishes was varied depending on the size of each fish. The large fish show contained a high concentration of Pb in their body. It is important to be careful on the inner organ and gill of fish, especially, large fish when consuming those fishes. Furthermost results show higher level than the international standard. This is maybe because of the size and the number of fishes. Some scientists stated that approximately 22 of the freshwater fish samples contained Pb levels that were marginally higher than the legal limit (0.4 mg/kg ww) set by the European Union. 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Appendix 1.B Picture of studied fishes collected in February 2018 Appendix 2 Main materials used in the study Appendix 3 Standard concentrations of Pb and their absorption A standard concentration of Pb and its Absorbance analyzed by flame AAS ConcentrationAbsorption0.5 ppm0.01471 ppm0.02672 ppm0.05125 ppm0.1191 Appendix 4 Calibration Curve of Pb Calibration curve of Pb for fish sample analysis by FAAS Appendix 5 Table of results The calculation from dry weight to wet weight Pb concentration in fishes collected in November 2017 (mg/kg wet weight) The result of Pb in water and sediment in November 2017 Sample IDWater (mg/L)Sediment (mg/kg)STJ3B0.40080STD3B0.40512.516PSKL0.33395.04PSD40.4055.78PSKK0.417541.292PSJ40.39660PSU40.41756.036SKU50.39666.816SKJ50.40080SKD50.41753.834KPU60.39244.998KPJ60.384116.668KPD60.371514.724CKD70.4053.834CKJ70.384110.314CKU70.40088.25STJ3B0.40080STD3B0.40512.516PSKL0.33395.04PSD40.4055.78PSKK0.417541.292PSJ40.39660PSU40.41756.036SKU50.39666.816SKJ50.40080SKD50.41753.834KPU60.39244.998Average0.39448.5698 PAGE MERGEFORMAT ii PAGE MERGEFORMAT iii PAGE MERGEFORMAT iv PAGE MERGEFORMAT viii PAGE MERGEFORMAT 47 Water Air Contaminated Heavy Metals Metals Aquatic Plant e.g. Water hyacinth Human Soil Aquatic Animals e.g. Fish, shrimp sail Human activities Sediment Figure STYLEREF 1 s 2. SEQ Figure ARABIC s 1 1 Anthropogenic sources of heavy metals flow in the environment Figure STYLEREF 1 s 4. SEQ Figure ARABIC s 1 1 Pb in the fleshes of different fishes collected in November 2017 Figure STYLEREF 1 s 4. SEQ Figure ARABIC s 1 2 Pb in the gills of different fishes collected in Novemer 2017 Figure STYLEREF 1 s 4. SEQ Figure ARABIC s 1 3 Pb in inner organ of different fishes collected in November 2017 Figure STYLEREF 1 s 4. SEQ Figure ARABIC s 1 6 Pb in the gills of fishes collected on February 2018 Figure STYLEREF 1 s 4. SEQ Figure ARABIC s 1 7 Pb in inner organs of fishes collected in February 2018 Figure STYLEREF 1 s 4. SEQ Figure ARABIC s 1 8 The average concentration of Pb in fishes collected on February 2018 Figure STYLEREF 1 s 4. SEQ Figure ARABIC s 1 9 Pb in different organ of all fish species Trey Krai Trey Prul Trey Por Trey Prolung Trey Chhdor Trey Klanghay Trey Chrakaing Trey Bra Trey Chhkok Trey Broma Trey Broma Blender Balance Tube 50 ml Heater magnetic AA-7000 Atomic Adsorption Spectrophometer (Shimadzu) Micro Filter (Nylon 0.45m) L
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