EDXRF ANALYSIS OF THE LEVEL OF HEAVY METALS IN MARINE FISHES FROM RAKHINE COASTAL REGIONS

PhD DISSERTATION

WIN WIN MAW

DEPARTMENT OF PHYSICS UNIVERSITY OF YANGON MYANMAR

SEPTEMBER 2004 2

CONTENTS

Page ACKNOWLEDGEMENT

ABSTRACT

CHAPTER I INTRODUCTION 1 1.1 General Features of Fishes 1 1.2 Types of Fish 2 1.2.1 Bony Fish and Cartilaginous Fish 3 1.3 Fish Shapes 4 1.4 Shellfish 4 1.5 Seafood 6 1.6 Seafood Quality and How to Check It 8 1.6.1 Smoked Seafood 9 1.6.2 Frozen Seafood 9 1.7 Shellfish and Packaging 10 1.7.1 Live Shellfish 10 1.7.2 Cooked Shellfish 11 1.8 Seafood Production, Consumption and Trade 13 1.9 Reducing Hazards with HACCP 17 1.9.1 Additional Protections 19 1.10 XRF - The Universal Metrology Method 20

CHAPTER II BACKGROUND THEORY OF X-RAYS 23 SPECTROSCOPY 2.1 Introduction of X-Ray 23 2.2 Nature of X-Rays 23 2.3 X-Ray Production 25 2.4 Properties of X-Rays 26 2.4.1 Fluorescence 27 2.4.2 Ionization 27 3

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2.4.3 X-Ray Diffraction 27 2.5 Interaction with Matter 28 2.5.1 Photoelectric Effect 28 2.5.2 Compton Effect 28 2.5.3 Pair Production 28 2.6 Application of X-Rays 29 2.6.1 Research Application 29 2.6.2 Industry Application 30 2.6.3 Medicine Application 31

CHAPTER III ANALYSIS OF X-RAY SPECTRA 33 3.1 Introduction 33 3.2 Spectrum Analysis 33 3.2.1 Continuous Spectrum 34 3.2.2 Absorption Spectra 36 3.3 Naming Transitions 37 3.4 Moseley's Law 38 3.5 Line Intensities 41 3.6 Satellite Peaks 42 3.7 Wavelength Shifts 43 3.8 Spectroscopic Analysis 46 3.9 WDS vs. EDS Detection 47 3.10 Calibration Curves 50 3.11 Energy Dispersive X-ray Fluorescence (EDXRF) Spectrometry 51 3.11.1 Performance and Advantages 52 3.11.2 Faster Analysis Time 52 3.11.3 Lower Cost of Analysis 53 3.11.4 Interferences of Elements 53 4

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3.11.5 Volatile Metals by EDXRF 54 3.11.6 EDXRF Hardware 54 3.12 Fundamental Parameters Analysis 56 3.12.1 General Bulk and Thin-Film Analysis 56 3.12.2 Analysis with or without Standards 57 3.12.3 Spectrum Calibration 57 3.12.4 Background Removal 58 3.12.5 Escape Peak and Sum Peak Removal 58 3.12.6 Smoothing 58 3.12.7 Intensity Extraction 58 3.13 Heavy metals 59 3.13.1 The Illnesses due to Heavy Metals 59 3.13.2 Absorbing Heavy Metals 61

CHAPTER IV EXPERIMENTAL PROCEDURE AND RESULTS 62 4.1 Samples Collection and Preparation 62 4.2 Energy Dispersive X-Rays Fluorescence 62 Spectrometer for Shimadzu EDX-700 4.3 Experimental Results 66

CHAPTER V DISCUSSION AND CONCLUSION 75 5.1 Discussion 75 5.2 Conclusion 95

REFERANCES

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ABSTRACT

Thirty-three marine fish samples were collected from Southern portion of Rakhine coastal strip of Myanmar. Twenty-one marine fish samples were collected from Thandwe Region and twelve marine fish samples were collected from Kyauk Gyi Region. The concentrations of heavy metals contained in these marine fish samples were analysed by using Energy Dispersive X-Ray Fluorescence (EDXRF) method and from these concentration results, the differences and varying of heavy metals concentrations between the two regions were analysed and discussed. The average concentrations of elements contained in each type of marine fish samples and the average concentrations of elements contained in the marine fish samples of each location are calculated. According to this research work, marine fish samples collected from Kyauk Gyi Region have higher concentrations of heavy elements than that of the Thandwe Region. Therefore, sea water of Kyauk Gyi Region has more heavy elements pollution than the sea water of Thandwe Region.

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CHAPTER I

INTRODUCTION

1.1 General Features of Fishes Fish diverse group of animals live and breathe in water. Fish are cold-blooded vertebrates, like reptiles, and have skeletons like mammals and reptiles. Some fish have skeletons made of bone. These include cod, plaice, and mackerel. Other fish have skeletons made from cartilage. These include sharks and rays. Their skeletons are softer and more bendy than animals with bone skeletons. The skin of most fish is protected by scales, which cover the surface of the skin. They also have gills, which enable them to live and breathe under water. Most fish have fins for swimming, scales for protection, and a streamlined body for moving easily through the water.

Fishes live in nearly every underwater habitat, from near-freezing Arctic waters to hot desert springs; from mud in dried-up tropical ponds to the deepest ocean abyss. Special antifreeze chemicals in the blood of Antarctic ice fish enable them to survive in water below 0° C (32° F). Desert pupfish found in hot springs of western North America live in temperatures higher than 40° C (100° F). Killifish release their eggs, or spawn, as the dry season begins in the tropics of South America and Africa, leaving their eggs to dry in the ground until the rains return six months later. In the deep ocean, where sunlight never reaches, many fishes cooperate with glowing bacteria to create their own light for communication and to attract mates and prey.

With approximately 25,000 recognized species, fishes make up the most diverse vertebrate group, comprising about half of all known vertebrate species. New fishes continue to be discovered and named at the rate of 200 to 300 species per year. With this vast number of different fishes comes a diversity of sizes and shapes, from huge whale sharks that reach 12 m (40 ft) in length to the smallest vertebrate, a tiny goby, measuring only 1 cm (0.4 in) long. 7

Fishes are generally streamlined with a pointed snout and pointed posterior and a broad propulsive tail. Unlike the shape of a human body, a fish‘s body shape is ideal for speeding through the water without creating excess resistance. This torpedo-shaped body is typical of the fastest-swimming fishes, the billfish and the tunas. One billfish, the sailfish, can swim in bursts of over 110 km/h (70 mph). Tunas are built for long-distance endurance as well as speed, swimming as fast as 50 km/h (30 mph) and migrating as far as 12,500 km (7700 mi) in only four months. Other fishes come in a wide variety of shapes. The snakelike eels, flat halibuts, and boxy puffers are all slower swimmers that have evolved distinctive bodies best adapted to their specific habitats. Unlike fishes that swim through the open water, these fishes have adapted to life in caves, on the ocean floor, and among coral reefs where speed is less important than camouflage or maneuverability

Some shellfish, like lobsters, crabs and prawns, have exoskeletons. That means their skeleton is on the outside of their bodies. These animals with exoskeletons are called crustaceans. Some shellfish are molluscs. They are protected from danger by withdrawing into a hard shell, which grows in size as the shellfish grows. Other molluscs have soft bodies with a shell inside, like squid and octopus.

There are more than 21,000 different kinds of seafood. Nearly half of these live in the sea and the rest in rivers and lakes. The species commonly eaten as seafood live at different depths, from near the surface to some of the deepest parts of the ocean. (12)

1.2 Types of Fish Fish are classified into two types: Demersal and Pelagic. Demersal species live on or near the seabed and are normally called white fish. This means that the oil in the fish is concentrated in its liver. They may have round bodies (cod), or flat bodies (plaice). The flat-bodied fish live on the seabed whilst round-bodied fish live close to the seabed. 8

Pelagic fish swim in large shoals and are found close to the surface. These are fish such as herring or mackerel which have oil stored in their flesh and are known as oil-rich fish. Tuna is a pelagic species that is found in warmer waters of the world.

Prawns are found in the seas and oceans of the world as well as the estuaries of rivers. In cold waters, they are caught in the sea by fishing boats with nets that are put out at either side of the boat.

In tropical countries, prawns are harvested from prawn farms, which may cover many hectares of coastal land or land in the very big river deltas such as the Ganges and Brahmaputra in Bangladesh.

Seafood feed on various foodstuffs which range from the smallest to the largest organisms. The type of food eaten in terms of nutrition is represented by what is called the food chain. This starts with the smallest organisms, single celled plants and animals which are collectively called phytoplankton or plankton. Plankton are tiny, and float in the sea in clouds.

The food chain progresses to grass and weed feeders, through zooplankton feeders and bottom feeders, to the carnivorous fish which eat smaller fish.

Phytoplankton feeders tend to have large mouths but no teeth, bottom feeders have their mouths on the underside of the head whereas grass feeders and carnivorous fish have mouths at the front of the head. There are exceptions to these generalisations. Sharks, which are carnivorous, have their mouths on the lower side of their head. (12)

1.2.1 Bony Fish and Cartilaginous Fish Bony fish are covered with scales, which are flat plates. Cartilaginous fish have scales which are spiny and make the surface rough to touch. They act as an extra barrier to protect the skin.

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Nearly all fish have a streamlined shape, which enables them to swim through the water with little resistance. The faster swimmers are more streamlined. Fish that sit in wait for their prey are not at all streamlined; the monkfish seems to be all mouth at the head end.

1.3 Fish Shapes Fish swim by bending their body so that their tail acts like a paddle, thrusting them through the water. Different fish have different shaped tails. The shape often indicates the speed at which they swim. The more pointed the tail, the faster it swims.

All fish have fins. The position and number of the fins varies. Fins on the back are called the dorsal fins, whilst on the underside is the single anal fin. In front of the anal fin are two sets of paired fins. The two sets of paired fins are called the pectorals, positioned just behind the head, and the pelvic fins, situated behind the pectorals.

Running along each side of the fish is a line known as the lateral line. This consists of special sensory cells used by the fish to detect food.

In the belly cavity are the various internal organs: liver, gut, kidneys and heart. The most important to us as consumers is the liver. It is in the liver that white fish store oil. (12)

1.4 Shellfish In contrast, the molluscan shellfish such as oyster, scallops and mussels, and the crustaceans such as lobster and crab, are both very different to fish. The majority of shellfish move around very little whilst crabs and lobsters walk around on their legs. A hard outer shell protects mussels, oysters and scallops. Lobsters and crabs have a similar hard outer covering but it is of a different substance. Internally, bivalve molluscs and crustaceans share the common features of any marine animal; for example, the heart, muscles, eyes and gills. 10

Giant moray Boxfish Leopard shark Harlequin grouper Clownfish

School Emperor angel fish Rock fish Black fish Dolphin

Skunk anenomefish Silver Ghost pipe fish Moray

Puffer Porcupine puffer fish

Shrimp Mantis shrimp Lobster Tentacles

Crab Porcelin crab Sea horse

Starfish Featherstar Cuttlefish

Scorpionfish Green turtle

Sea slug Clouded moray

Lionfish Fimbriated moray Sea snail Fig 1.1 Some Myanmar sea fishes 11

1.5 Seafood Seafood is a valuable food source. It contains protein, vitamins, minerals and fatty acids, which are all necessary for a balanced diet and a healthy lifestyle.

Protein Most vertebrate fish contain between 15% - 20% protein, slightly less than meat. The protein content of meat and fish is of a high biological value (HBV). The protein in seafood is mostly muscle protein made up of bundles of short fibres. The amount of connective tissue, in the form of soluble collagen, is low. Seafood also contains some of the amino acids that the body needs for growth and repair of body tissues. Seafood represents very good value for money as a protein source.

Vitamins Seafood is a good source of some B vitamins which are essential for the conversion of food to energy in the body's cells and for healthy nerve tissue. Oil-rich fish such as herring, mackerel, sardines and anchovy are also an important source of fat soluble vitamins A and D, which can also be extracted from the livers of white fish. Vitamin A prevents night blindness and Vitamin D prevents rickets.

Minerals Seafood is a good source of the minerals calcium and phosphorous. Trace minerals such as iodine, fluorine, magnesium, zinc and selenium are also present in seafood. White fish, in particular, can be a valuable source of iron because it is in a form which is relatively well absorbed.

Fatty acids

Fish oil Fish oils are known to be beneficial in the human diet. Oil-rich fish such as herring, sardines and mackerel are an important source of long chained 12 polyunsaturated fatty acids (PUFA) including eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), known collectively as Omega-3 fatty acids.

Land-based animals and plants cannot easily convert, by elongation, the parent Omega-3 PUFA alpha linoleic acid into EPA or DHA. However, the plankton on which seafood feed is able to do this. Fish are therefore a valuable source of EPA and DHA. Medical evidence suggests that the Omega-3 fatty acids have an important role in improving health.

Cholesterol is a type of fat that is naturally produced by our bodies and found in the diet. It is essential for life. The liver can produce cholesterol if dietary sources do not provide enough for the body's needs. If dietary sources provide enough, then the liver reduces production.

In a few people, this 'reduction' mechanism is faulty and a high intake of dietary cholesterol can lead to high levels of cholesterol in the blood. These are the key people who must be careful, and for these people, moderation is the key.

Too much cholesterol circulating in the bloodstream is a problem and it needs to be deposited. Cholesterol is usually deposited in the lining of the blood stream vessels, causing them to narrow. The heart then has to work harder to pump blood around the body. Blood clots form and block the blood vessels. This can shut off the blood and oxygen supply to the tissues. Tissues can become deprived of oxygen when the blood becomes blocked.

Two fatty acids (EPA and DHA) are known collectively as Omega-3. Oil-rich fish (such as herring, mackerel and sardines) are an excellent source of Omega- 3 fatty acids.

The special Omega-3 oils from the fish have been shown to have a lowering effect on blood fats. Omega-3 lowers a lipid in the blood known as 13 triglycerides. This may help reduce the risk of a heart attack and may also help to reduce blood pressure a little and keep the heart beat steady.

The Inuit people in Northern Canada and Greenland, and the inhabitants of fishing villages in Japan, have a high level of consumption of oil-rich fish. These communities have a very low level of coronary heart disease.

There is some evidence that fish oils help to prevent cancer cells progressing to the stage where they develop into a tumour. They also reduce inflammation and have provided relief for people suffering from rheumatoid arthritis. Studies have also suggested that consumption of fish oils helps maintain mental health and improves conditions such as eczema. (17,18,25)

1.6 Seafood Quality and How to Check It Seafood is a perishable commodity and should be presented to the buyer on ice in a cool clean display unit. The retailer should always be able to give advice about the products on display, and the variety of delicious seafood on offer increases every year. Not only should fresh seafood (wet fish), be considered but also frozen seafood from the freezer.

Wet fish means seafood that has been kept chilled and not frozen. The freshness of seafood is best judged by its appearance on the display counter. The eyes should be clear and bright and slightly bulging and not sunken. The colour of the gills should be bright red or pink with any slime present being colourless.

Fresh seafood has a unique, flavoursome 'sea fresh' aroma, sometimes described as 'sea-weedy'. The skin should be bright, clear and shiny. As the seafood ages, the surface eventually turns to a yellow colour. When pressed, the skin should be firm or elastic.

Where seafood is pre-packed, there should be a 'sell by' date and a 'best before' date on the label. The buyer -that's you! - should not buy packs after their 14 expiry date. Most seafood will keep in a refrigerator for two days when wrapped in polyethylene film. This prevents drip and also tainting of other foods.

With shellfish, it is more difficult to generalise. Many shellfish are sold live and oysters are brought to the table live and eaten raw. Bi-valve molluscs such as cockles, mussels, clams and oysters should have their shells tightly shut, or, close quickly if disturbed, (eg by a sharp tap). They should be stored at home as for wet fish.

Crustaceans should not show signs of black discolouration through the shell. This is due to a pigment formed through enzyme action and possibly indicates poor storage at some stage. However, there are occasionally black spots on crab shells, which do not affect the quality of the meat. If the crab or lobster is bought live, it should be cooked on the day of purchase.

1.6.1 Smoked Seafood The surface of the seafood should be glossy and free from 'smuts' and other debris. A dull or matt surface indicates poorer quality seafood may have been used, though freezing and thawing can also give this appearance. Crystals of salt on the surface may result from too heavy a brining process resulting in a very salty product when eaten.

The colour used to dye the seafood is normally a natural colorant such as carotene or turmeric. This is used especially in modern smoking processes, which are able to smoke the seafood in about 4 hours instead of the 10 - 12 hours used in traditional kilns.

1.6.2 Frozen Seafood The quality of frozen seafood offered for sale is usually very good. The biggest problem is in the storage of the frozen product. If there is a fluctuation in temperature, there is melting and re-freezing of some of the ice. This results in 15 the formation of large ice crystals in the flesh. These grow over a period of time and pierce the cell walls of the flesh. On cooking, the liquor in the cells is lost through these holes and this results in a tough chewy product. There is also water migration from the product. This is also due to fluctuating temperatures. Water moves from the surface of the product and is seen as ice crystals in the pack. Packs of shrimps or prawns sometimes show this effect.

To overcome the problems of water migration after freezing, the processor will spray cold water on to the surface of the prawns where it forms a shell of ice.

This ice is a sacrificial layer, which protects the prawn from the problems of dehydration in the freezer.

1.7 Shellfish and Packaging The waters surrounding the coasts of the UK produce some of the finest shellfish in the world. Some caught from the wild, some carefully cultivated, while more exotic species are imported from far flung shores.

Shellfish are available in a variety of forms: fresh, frozen, canned, bottled or often as part of ready prepared meals. Shellfish are all invertebrates (ie do not possess an internal skeleton) and are split into two main groups:

Molluscs have either an external hinged double shell (eg scallops, mussells), or a single spiral shells (eg winkles, whelks) or have soft bodies with an internal shell (eg squid, octopus).

Crustaceans have tough outer shells which act like armour and also have flexible joints which allow quick movement (eg crab, lobster).

1.7.1 Live Shellfish 1. Live shellfish should only be purchased from a reputable supplier or direct from a wholesale market or port where the catch is freshly landed. 16

2. They should be packed and sold in moist, cool conditions and be reasonably lively. 3. The shells of live molluscs such as oysters, clams and mussels should remain closed, or they should shut when rapidly tapped. 4. Live shellfish e.g., crabs and lobsters should be packed together to keep them moist. All paired claws should be present. 5. Lobster claws should be bound with elastic bands to prevent fighting and damage. 6. Lobster tails should spring back into place when uncurled. Storage 1. Keep the shellfish at a temperature of between +2°C and +10°C. 2. Keep shellfish in its packaging until use to avoid moisture loss. 3. Ensure that all shellfish remain moist. 4. Molluscs should be kept in a container embedded in ice. The round side of the shell should face downwards to help collect and retain the natural juices. 5. Check regularly to make sure that they are still alive. Reject any dead or dying specimens.

1.7.2 Cooked Shellfish Where the product has not been dressed or peeled: 1. The shells of cooked shellfish should be intact with no cracks. 2. The shells should not release copious amounts of liquid when shaken. 3. The cooked product should exhibit no signs of discolouration and there should be no odours indicative of spoilage. 4. Freshly cooked prawns and shrimps should be firm to the touch and should be chilled immediately. 5. Where frozen cooked shellfish is offered for sale, it should be labelled 'if thawed, do not refreeze'.

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Storage Cover and refrigerate and maintain temperature between 0°C and +4°C.

Packaging Packaging is necessary to protect the product but also has a wider role. Most frozen products are packaged in cartons, often with an internal container. This makes it easier to meet storage requirements. The product itself cannot be seen and an important part of the packet design is to promote the contents through an attractive display.

Many chilled products are packaged in trays and are gas-flushed when sealed to retard spoilage. This means that the product itself is on display, so it must have a good appearance. Labels or sleeves around the package carry legally-required information.

"Hot smoked" seafood is cooked as it is being smoked and therefore is 'ready to eat'. Most "cold smoked" seafood must be cooked before eating. (The exception is salmon.) The smoked seafood is placed in a laminated plastic bag, which has a very low permeability to gasses, and all the air is removed before the bag is sealed. The resulting pack has no air and this prevents the chemical oxidation of the fats and oils that produce rancid flavours. This type of packaging does not, however, prevent spoilage so this type of packaging should still be kept in chill storage conditions.

When looking at packs of seafood products, the same packaging material and container size can sometimes be seen for a whole range of seafood products. Manufacturers prefer new products that can be accommodated within existing package styles. They do not then have to modify production lines or invest in new equipment. Consistent package shapes and sizes also help to give a homogenous image to a range of products.

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Seventy percent of the world's catch of fish and fishery products is consumed as food. Fish and shellfish products represent 15.6 percent of animal protein supply and 5.6 percent of total protein supply on a worldwide basis. Developing countries account for almost 50 percent of global fish exports. Seafood-borne disease or illness outbreaks affect consumers both physically and financially, and create regulatory problems for both importing and exporting countries. Seafood safety as a commodity cannot be purchased in the marketplace and government intervenes to regulate the safety and quality of seafood. Theoretical issues and data limitations create problems in estimating what consumers will pay for seafood safety and quality. The costs and benefits of seafood safety must be considered at all levels, including the fishers, fish farmers, input suppliers to fishing, processing and trade, seafood processors, seafood distributors, consumers and government. Hazard Analysis Critical Control Point (HACCP) programmes are being implemented on a worldwide basis for seafood. Studies have been completed to estimate the cost of HACCP in various shrimp, fish and shellfish plants in the United States, and are underway for some seafood plants in the United Kingdom, Canada and Africa. Major developments within the last two decades have created a set of complex trading situations for seafood. Current events indicate that seafood safety and quality can be used as non-tariff barriers to free trade. Research priorities necessary to estimate the economic value and impacts of achieving safer seafood are outlined at the consumer, seafood production and processing, trade and government levels. (18,25,26,43)

1.8 Seafood Production, Consumption and Trade Estimates in the early 1970s predicted the potential for traditionally exploited marine species was about 100 million tonnes per year. Despite development of non-traditional species, marine fishery production by 1994 reached only 90 million tonnes with capture fisheries accounting for 84 million tonnes. In 1994, the world's 200 major fishery resources accounted for 77 percent of marine fish production. About 35 percent of these resources are showing 19 declining yields, about 25 percent are leveled at high exploitation rates, 40 percent are still developing and none remain at an undeveloped level. Thus, about 60 percent of the world's major fisheries resources are mature or declining, and there is a need to establish more effective management controls, reduce overall fishing effort and rebuild overfished stocks. In a few remaining areas of the world, it is possible to increase effort and landings. FAO estimates indicate that an increase of 10 million tonnes to a world total production of 93 million tonnes is possible only through management improvements and further fishery development of the capture sector. Another prediction indicates an additional 20 million tonnes is possible, but only if degraded resources are rehabilitated, under-developed resources are exploited further, overfishing is avoided in these as well as in those resources currently fully exploited, and discarding and waste are reduced. This creates important implications regarding the world's supply of food, since about 70 percent of the world catch of fish and fishery products is consumed as food.

On a worldwide basis, the total food supply of fish and fishery products was 74 million metric tonnes (live weight of fish) in 1993. This is the largest amount recorded, and represents an amount almost 2.5 times greater than in the early 1960s. Food fish and fishery product supply on a per capita basis was 13.4 kilograms (live weight of fish) in 1993. The 1991-1993 average was 13.0 kilograms, compared to the 1961-1963 average of 9.2 kilograms. Increases since the early 1960s have been steady and gradual, with the principal growth in demand for fish as food coming through population increases. For low- income food-deficit countries, per capita supply was 9.6 kilograms in 1993. The 1991-93 average of 9.0 kilograms per capita was slightly double the 1961- 1963 average of 4.3 kilograms per capita. On a relative basis, fish and fishery products has become a more important food product for low-income food- deficit countries. Fish represent 20.6 percent of all animal proteins and 4.7 percent of all proteins consumed per capita in low-income food-deficit countries. Fish and fishery products represent 15.6 percent of animal protein 20 supply and about 5.6 percent of total protein supply on a worldwide basis. This percentage has been stable over the last 33 years, ranging from a low of 13.8 percent to a high of 16.0 percent.

International trade in fish and fishery products has grown substantially over the last three decades. The value of fish entering the world export market has increased over twenty times from the 1968 value of US$2.2 billion to the 1993- 1995 average annual value of US$46.95 billion. Adjusting for inflation to measure the increase in real value terms, the 1993-1995 value is still five times greater that the 1968 value3. Today, more than 30 percent of the fish caught for direct human consumption enters international trade. Developing countries account for almost 50 percent of global fish exports. The share of exports from developing countries reached an all-time high of 51 percent with their net receipts from foreign exchange increasing from US$10.4 billion in 1990 to US$18.0 billion in 1995. Sixteen different countries averaged exports over US$1.0 billion annually from 1991 to 1993. These 16 countries accounted for 66 percent of world exports with 50 countries exporting 96 percent of the world total. Thailand was the leading exporter at US$4.0 billion. The others were as follows (all US$): United States, $3.3 billion; Norway, $2.7 billion; Denmark $2.3 billion; China Taiwan, $2.3 billion; China Mainland, $2.2 billion; Indonesia, $1.6 billion; Russian Federation, $1.6 billion; Chile, $1.4 billion; Korea Republic, $1.4 billion; Netherlands, $1.4 billion; Iceland, $1.2 billion; India, $1.2 billion; United Kingdom, $1.1 billion and Spain, $1.0 billion.

The 1993 survey of seafood consumers in the United States Northeastern and Mid-Atlantic areas also asked questions about the effect on seafood consumption behavior due to exposure to news related to finfish and shellfish. Fifty-seven percent of the consumers from both the finfish and shellfish consumer surveys had heard stories in the news media about seafood in 1992. Thirteen percent had heard only positive stories, 33 percent only negative stories and 43 percent both positive and negative stories. Seventy-three percent 21 said the stories had no effect on their seafood consumption. A higher percentage of the shellfish survey consumers (84 percent) had seen stories than had the finfish respondents (79 percent). Salmon received more negative publicly, with the amount of publicity decreasing in order listed for trout, hybrid striped bass and tilapia. More than 50 percent of the shellfish stories encountered were negative. Twenty-two percent of finfish consumers and 28 percent of shellfish consumers decreased their consumption due to the news stories.

1. The cost of implementing seafood HACCP or similar programmes to achieve various levels of reduced risk of contracting a seafood-borne disease or illness or enhancing seafood quality in processing plants for major fish and shellfish species or product forms must be determined. Industry-wide costs can then be determined and compared to the benefits of reduced risk and enhanced quality for major species or product forms to determine the net economic returns. The cost of implementing HACCP and similar processes at the fishing vessel level should also be determined (although HACCP applies generally to the processing level at this time) since HACCP will likely be applied at the fishing vessel level in the future.

2. Industry-wide seafood HACCP net economic benefits for major production regions or countries must be compared at the fisher, input supplier, and fish processing level, particularly among production regions or countries that compete with similar fish or shellfish species or product forms. This is critical in determining if seafood safety and quality programmes create competitive advantages for some production regions or countries.

3. Marginal benefit-cost analysis (or similar analytical techniques) should be performed for each Critical Control Point where HACCP is used. This will allow the determination of the most economically effective way to achieve a specified standard of risk reduction, and will allow HACCP to be used as a 22 business management tool at the processing plant level. All costs must be considered including training costs of employees.

4. HACCP implementation effects on the structure of the seafood industry in various production regions should be determined. Differing impacts on small versus large firms, or differing structural changes on industry among various production regions of the world which produce similar products must be determined. The public policy implications of mandated seafood safety and quality programmes that cause structural changes in the seafood industry must be predicted and evaluated. (18,25,26,43)

1.9 Reducing Hazards with HACCP Seafood can be exposed to a range of hazards from the water. Some of these hazards are natural to seafood's environment; others are introduced by humans. The hazards can involve bacteria, viruses, parasites, natural toxins, and chemical contaminants.

The HACCP system that seafood companies will have to follow will help weed out seafood hazards with the following seven steps:  Analyze hazards. Every processor must determine the potential hazards associated with each of its seafood products and the measures needed to control those hazards. The hazard could be biological, such as a microbe; chemical, such as mercury or a toxin; or physical, such as ground glass.  Identify critical control points, such as cooking or cooling, where the potential hazard can be controlled or eliminated.  Establish preventive measures with critical limits for each control point.  Establish procedures to monitor the critical control points. This might include determining how cooking time and temperatures will be monitored and by whom. 23

 Establish corrective actions to take when monitoring shows that a critical limit has not been met. Such actions might include reprocessing the seafood product or disposing of it altogether.  Establish procedures to verify that the system is working properly.  Establish effective recordkeeping.

Also, under FDA's HACCP regulations, seafood companies have to write and follow basic sanitation standards that ensure, for example, the use of safe water in food preparation; cleanliness of food contact surfaces, such as tables, utensils, gloves and employees' clothes; prevention of cross-contamination; and proper maintenance of hand-washing, hand-sanitizing, and toilet facilities.

In addition, molluscan shellfish handlers must follow a few additional rules; for example, they must obtain shellfish only from approved waters and only if they are properly tagged, which indicates that they have come from an approved source.

FDA estimates that more than half of the seafood eaten in this country is imported from almost 135 countries. The agency now requires for the first time that seafood importers take certain steps to verify that their overseas' suppliers are providing seafood processed under HACCP.

FDA periodically inspects seafood processors and warehouses. Required HACCP records will enable the agency to determine how well a company is complying over time.

The safety features of FDA's HACCP regulations are incorporated into the National Seafood Inspection Program of the Department of Commerce's National Oceanic and Atmospheric Administration. For a fee, NOAA inspects seafood processors and others, checking vessels and plants for sanitation and examining products for quality. The agency certifies seafood plants that meet federal standards and rates products with grades based on their quality. Seafood 24 processors in good standing with the program are free to use official marks on products that indicate the seafood has been federally inspected.

1.9.1 Additional Protections FDA promotes seafood safety in other ways, including: Setting standards for seafood contaminants. FDA has established a legally binding safety limit for polychlorinated biphenyls and guidelines for safety limits for six pesticides, mercury, paralytic shellfish poison, and histamine in canned tuna. (Histamine is the chemical responsible for scombroid poisoning.)

Administering the National Shellfish Sanitation Program, which involves 23 shellfish-producing states, plus a few non-shellfish-producing states, and nine countries. The program exercises control over all sanitation related to the growing, harvesting, shucking, packing, and interstate transportation of oysters, clams and other molluscan shellfish.

Lending its expertise to the Interstate Shellfish Sanitation Conference, an organization of federal and state agencies and members of the shellfish industry. The conference develops uniform guidelines and procedures for state agencies that monitor shellfish safety.

Entering into cooperative programs with states to provide training to state and local health officials who inspect fishing areas (for example, shellfish beds), seafood processing plants and warehouses, and restaurants and other retail places.

Working with NOAA to close federal waters to fishing whenever oil spills, toxic blooms, or other phenomena threaten seafood safety.

Sampling and analyzing fish and fishery products for toxins, chemicals and other hazards in agency laboratories. (18,25,26,43)

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1.10 XRF - The Universal Metrology Method XRF interprets the characteristic radiation emitted by the elements in the sample upon excitation, enabling accurate determination of the elemental composition - to sub-ppm concentration levels thanks to recent resolution and data processing advances.

XRF spectrometers fall into two groups: energy dispersive (EDXRF) and wavelength dispersive (WDXRF). The difference between the two systems is in the method of detection. EDXRF spectrometers have a detector that measures the different energies of the characteristic radiation coming directly from the sample, while WDXRF spectrometers use an analyzing crystal to disperse the different wavelengths. All radiation coming from the sample falls on the crystal, which diffracts the different wavelengths into different directions, similar to a prism dispersing different colors of light. XRD systems work with the interaction of X-rays with the crystalline phases of a sample, enabling the analysis of the structural phases present, which to a large extent determine the physical properties of the materials.

XRF systems and software are widely used for the analysis of amongst others cement, metals and steel, plastics, polymers and petrol, industrial minerals, glass, catalysts, semiconductors, thin films and advanced materials, pharmaceutical solids, recycled materials and environmental samples.

XRF instruments are themselves particularly suitable for semiconductor manufacture. One of the attractions of XRF is the wide variety of materials it can study. It determines layer composition, thickness, contamination, dopant levels and surface uniformity for virtually the complete range of semiconductor process films.

The excellent selectivity and precision of WDXRF makes it ideal for these applications. The Jade Wafer Analyzer is ‗Flagship‘ semiconductor instrument. It is virtually unique in that it characterizes all layers in advanced 26 semiconductors. It takes accurate non-contact measurements of thickness and composition, contamination, dopant levels and surface uniformity for a wide range of process films. The Jade family does this using simultaneous X-Ray fluorescence spectrometry. A 4 kW Super-Sharp Tube rapidly measures critical light elements like boron and nitrogen, which normally dictate overall measurement times. Up to 24 configurable measuring channels ensure rapid simultaneous determination of the elements of interest. Versions of the instrument optimized for specific applications are aimed at production environments, which need fast, dedicated equipment.

XRF also has the neat trick of being able to measure layers that are buried under a different material. Copper layers are often laid on top of tantalum nitride layers, which form an electrically conducting barrier to prevent copper from diffusing into the underlying silicon (hence destroying the devices). XRF can measure both layers simultaneously. The Ta-Lα signal is used to determine the TaN layer thickness, the N-Kα signal is used to determine the stoichiometry of the layer, while the Cu-Kα signal can be used to quantify the layer thickness of the Cu film.

Another great attraction of XRF is its reproducibility (measurements from the same wafer) and repeatability (measurements taken from successive wafers). An instrument‘s repeatability is the best and most practical measure of measurement quality. For the PQ Jade, it is down to an incredible ±0.3 Å – less than one third of an atom's diameter.

Energy-dispersive X-ray fluorescence (EDXRF) spectrometer with flexible capabilities for low-level determination of heavy elements are now available. Designed to meet environmentally-driven trace element analysis requirements, it is capable of detecting elements from Na to U in solid, pressed-powder, granule, liquid, thin-film and loose material samples. Trace analysis of heavy 27 elements such as As, Pb, Cd and the lanthanides, can be measured concentrations from 100% to sub-ppm levels in a single preparation. It supports applications in geology, agriculture, land reclamation, fossil and secondary fuel usage, emissions monitoring, recycling, electronics, catalyst production, and electrical appliance manufacture.

In this research work, thirty three marine fish samples were collected from Southern portion of Rakhine coastal strip of Myanmar. Twenty one marine fish samples were collected from Thandwe Region and twelve marine fish samples were collected from Kyauk Gyi Region, Southern portion of Rakhine coastal strip of Myanmar. The concentrations of heavy metals contained in this marine fish samples were analysed by using Energy Dispersive X-Ray Fluorescence (EDXRF) method and from these concentration results, the differences and varying of heavy metals concentrations between the two regions were analysed and discussed. (19) 28

CHAPTER II

BACKGROUND THEORY OF X-RAYS SPECTROSCOPY

2.1 Introduction of X-Ray X-ray, penetrating electromagnetic radiation, having a shorter wavelength than light, and produced by bombarding a target, usually made of tungsten, with high-speed electrons. X-rays were discovered accidentally in 1895 by the German physicist Wilhelm Conrad Roentgen while he was studying cathode rays in a high-voltage, gaseous-discharge tube. Despite the fact that the tube was encased in a black cardboard box, Roentgen noticed that a barium- platinocyanide screen, inadvertently lying nearby, emitted fluorescent light whenever the tube was in operation. After conducting further experiments, he determined that the fluorescence was caused by invisible radiation of a more penetrating nature than ultraviolet rays. He named the invisible radiation ―X-ray‖ because of its unknown nature. Subsequently, x-rays were known also as Roentgen rays in his honor. (22,35)

2.2 Nature of X-Rays X rays are electromagnetic radiation ranging in wavelength from about 100 Å to 0.01 Å (1 Å is equivalent to about 10-8 cm/about 4 billionths of an in.). The shorter the wavelength of the x-ray, the greater is its energy and its penetrating power. Longer wavelengths, near the ultraviolet-ray band of the electromagnetic spectrum, are known as soft x-rays. The shorter wavelengths, closer to and overlapping the gamma-ray range, are called hard x-rays. A mixture of many different wavelengths is known as ―white‖ x-rays, as opposed to ―monochromatic‖ x-rays, which represent only a single wavelength. Both light and x-rays are produced by transitions of electrons that orbit atoms, light by the transitions of outer electrons and x-rays by the transitions of inner electrons. In the case of bremsstrahlung radiation, x-rays are produced by the 29 retardation or deflection of free electrons passing through a strong electrical field. Gamma rays, which are identical to x-rays in their effect, are produced by energy transitions within excited nuclei.

Fig 2.1 Interaction of x-ray with matters

X-rays are produced whenever high-velocity electrons strike a material object. Much of the energy of the electrons is lost in heat; the remainder produces x-rays by causing changes in the target's atoms as a result of the impact. The x-rays emitted can have no more energy than the kinetic energy of the electrons that produce them. Moreover, the emitted radiation is not monochromatic but is composed of a wide range of wavelengths with a sharp, lower wavelength limit corresponding to the maximum energy of the bombarding electrons. This continuous spectrum is referred to by the German name bremsstrahlung, which means ―braking,‖ or slowing down, radiation, and is independent of the nature of the target. If the emitted x-rays are passed through an x-ray spectrometer, certain distinct lines are found superimposed on the continuous spectrum; these lines, known as the characteristic x-rays, represent wavelengths that depend 30 only on the structure of the target atoms. In other words, a fast-moving electron striking the target can do two things: It can excite x-rays of any energy up to its own energy; or it can excite x-rays of particular energies, dependent on the nature of the target atom. (9,16,22)

2.3 X-Ray Production The first x-ray tube was the Crookes tube, a partially evacuated glass bulb containing two electrodes, named after its designer, the British chemist and physicist Sir William Crookes. When an electric current passes through such a tube, the residual gas is ionized and positive ions, striking the cathode, eject electrons from it. These electrons, in the form of a beam of cathode rays, bombard the glass walls of the tube and produce x-rays. Such tubes produce only soft x-rays of low energy.

An early improvement in the x-ray tube was the introduction of a curved cathode to focus the beam of electrons on a heavy-metal target, called the anticathode, or anode. This type generates harder rays of shorter wavelengths and of greater energy than those produced by the original Crookes tube, but the operation of such tubes is erratic because the x-ray production depends on the gas pressure within the tube.

The next great improvement was made in 1913 by the American physicist William David Coolidge. The Coolidge tube is highly evacuated and contains a heated filament and a target. It is essentially a thermionic vacuum tube in which the cathode emits electrons because the cathode is heated by an auxiliary current and not because it is struck by ions as in the earlier types of tubes. The electrons emitted from the heated cathode are accelerated by the application of a high voltage across the tube. As the voltage is increased, the minimum wavelength of the radiation decreases.

31

Fig 2.2 Coolidge type X-ray tube

Most of the x-ray tubes in present-day use are modified Coolidge tubes. The larger and more powerful tubes have water-cooled anticathodes to prevent melting under the impact of the electron bombardment. The widely used shockproof tube is a modification of the Coolidge tube with improved insulation of the envelope (by oil) and grounded power cables. Such devices as the betatron are used to produce extremely hard x-rays, of shorter wavelength than the gamma rays emitted by naturally radioactive elements. (9,41)

2.4 Properties of X-Rays X-rays affect a photographic emulsion in the same way light does. Absorption of X radiation by any substance depends upon its density and atomic weight. The lower the atomic weight of the material, the more transparent it is to x-rays of given wavelengths. When the human body is x-rayed, the bones, which are composed of elements of higher atomic weight than the surrounding flesh, absorb the radiation more effectively and therefore cast darker shadows on a photographic plate. Another type of radiation, which is known as neutron radiation and is now used in some types of radiography, produces almost 32 opposite results. Objects that cast dark shadows in an x-ray picture are almost always light in a neutron radiograph. (6,16)

2.4.1 Fluorescence X-rays also cause fluorescence in certain materials, such as barium platinocyanide and zinc sulfide. If a screen coated with such fluorescent material is substituted for the photographic films, the structure of opaque objects may be observed directly. This technique is known as fluoroscopy.

2.4.2 Ionization Another important characteristic of x-rays is their ionizing power, which depends upon their wavelength. The capacity of monochromatic x-rays to ionize is directly proportional to their energy. This property provides a method for measuring the energy of x-rays. When x-rays are passed through an ionization chamber, an electric current is produced that is proportional to the energy of the incident beam. In addition to ionization chambers, more sensitive devices, such as the Geiger-Müller counter and the scintillation counter, can measure the energy of x-rays on the basis of ionization. In addition, the path of x-rays, by virtue of their capacity to ionize, can be made visible in a cloud chamber.

2.4.3 X-Ray Diffraction X-rays may be diffracted by passage through a crystal or by reflection (scattering) from a crystal, which consists of regular lattices of atoms that serve as fine diffraction gratings. The resulting interference patterns may be photographed and analyzed to determine the wavelength of the incident x-rays or the spacings between the crystal atoms, whichever is the unknown factor. X-rays may also be diffracted by ruled gratings if the spacings are approximately equal to the wavelengths of the incident x-rays. (6,16)

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2.5 Interaction with Matter In the interaction between matter and x-rays, three mechanisms exist by which x-rays are absorbed; all three mechanisms demonstrate the quantum nature of x-radiation.

2.5.1 Photoelectric Effect When a quantum of radiation, or a photon, in the x-ray portion of the electromagnetic spectrum strikes an atom, it may impinge on an electron within an inner shell and eject it from the atom. If the photon carries more energy than is necessary to eject the electron, it will transfer its residual energy to the ejected electron in the form of kinetic energy. This phenomenon, called the photoelectric effect, occurs primarily in the absorption of low-energy x-rays.

2.5.2 Compton Effect The Compton effect, discovered in 1923 by the American physicist and educator Arthur Holly Compton, is an important manifestation of the absorption of x-rays of shorter wavelengths. When a high-energy photon collides with an electron, both particles may be deflected at an angle to the direction of the path of the incident x-ray. The incident photon, having delivered some of its energy to the electron, emerges with a longer wavelength. These deflections, accompanied by a change of wavelength, are known as Compton scattering.

2.5.3 Pair Production In the third type of absorption, especially evident when elements of high atomic weight are irradiated with extremely high-energy x-rays, the phenomenon of pair production occurs. When a high-energy photon penetrates the electron shell close to the nucleus, it may create a pair of electrons, one of negative charge and the other positive; a positively charged electron is also known as a positron. This pair production is an example of the conversion of energy into mass. The photon requires at least 1.2 MeV of energy to yield the 34 mass of the pair. If the incident photon possesses more energy than is required for pair production, the excess energy is imparted to the electron pair as kinetic energy. The paths of the two particles are divergent. (9,40)

2.6 Application of X-Rays X-ray diffraction photograph has been a useful tool in understanding the structure of solids. The lattice of atoms in a crystal serves as a series of barriers and openings that diffracts x-rays as they pass through. The diffracted x-rays form an interference pattern that can be used to determine the spacing of atoms in the crystal. This photograph shows the pattern resulting from x-rays passing through a palladium coordination complex, a compound with a palladium atom at the center of each molecule.

X-rays in airport security machines use low energy x-rays to check luggage for dangerous items. Unless x-rays encounter a metal object, they will pass through the bag to a detector beneath the conveyor belt. An image is formed by the x- rays hitting the detector. The image is transmitted to a viewing screen where it is analyzed by security personnel for potential risk.

The principal uses of x-radiation are in the field of scientific research, industry, and medicine.

2.6.1 Research Application The hemoglobin molecule in red blood cells transports oxygen from the lungs to cells throughout the body. In the late 1930s Austrian-born British biochemist Max F. Perutz began examining the structure of this complex protein molecule by using a technique known as x-ray crystallography. By 1960 he had determined the three-dimensional structure of the protein. For this work, Perutz shared the 1962 Nobel Prize in chemistry. Perutz describes his study of the hemoglobin molecule in a 1964 Scientific American article.

35

The study of x-rays played a vital role in theoretical physics, especially in the development of quantum mechanics. As a research tool, x-rays enabled physicists to confirm experimentally the theories of crystallography. By using x-ray diffraction methods, crystalline substances may be identified and their structure determined. Virtually all present-day knowledge in this field was either discovered or verified by x-ray analysis. x-ray diffraction methods can also be applied to powdered substances that are not crystalline but that display some regularity of molecular structure. By means of such methods, chemical compounds can be identified and the size of ultramicroscopic particles can be established. Chemical elements and their isotopes may be identified by x-ray spectroscopy, which determines the wavelengths of their characteristic line spectra. Several elements were discovered by analysis of x-ray spectra.

A number of recent applications of x-rays in research are assuming increasing importance. Microradiography, for instance, produces fine-grain images that can be enlarged considerably. Two radiographs can be combined in a projector to produce a three-dimensional image called a stereoradiogram. Color- radiography is also used to enhance the detail of x-ray photographs; in this process, differences in the absorption of x-rays by a specimen are shown as different colors. Extremely detailed and analytical information is provided by the electron microprobe, which uses a sharply defined beam of electrons to generate x-rays in an area of specimen as small as 1 micrometer (about 1/25,000 in) square.

2.6.2 Industry Application In addition to the research applications of x-rays in physics, chemistry, mineralogy, metallurgy, and biology, x-rays are used in industry as a research tool and for many testing processes. They are valuable in industry as a means of testing objects such as metallic castings without destroying them. x-ray images on photographic plates reveal the presence of flaws, but a disadvantage of such inspection is that the necessary high-powered x-ray equipment is bulky 36 and expensive. In some instances, therefore, radioisotopes, which emit highly penetrating gamma rays, are used instead of x-ray equipment. These isotope sources can be housed in relatively light, compact, and shielded containers. Cobalt-60 and cesium-137 have been used widely for industrial radiography. Thulium-70 has been used in small, convenient, isotope projectors for some medical and industrial applications.

Many industrial products are inspected routinely by means of x-rays so that defective products may be eliminated at the point of production. Other applications include the detection of fake gems and the detection of smuggled goods in customs examinations. Ultrasoft x-rays are used to determine the authenticity of works of art and for art restoration.

2.6.3 Medicine Application X-ray photographs, called radiographs, and fluoroscopy are used extensively in medicine as diagnostic tools. In radiotherapy, x-rays are used to treat certain diseases, notably cancer, by exposing tumors to X radiation.

The use of radiographs for diagnostic purposes was inherent in the penetrating properties of x-rays. Within a few years of their discovery, x-rays were being used to locate foreign bodies, such as bullets, within the human body. With the development of improved x-ray techniques, minute differences in tissues were revealed by radiographs, and many pathological conditions could be diagnosed by means of x-rays. X-rays provided the most important single method of diagnosing tuberculosis when that disease was prevalent. Pictures of the lungs were easy to interpret because the air spaces are more transparent to x-rays than the lung tissues. Various other cavities in the body can be filled artificially with contrasting media, either more transparent or more opaque to x-rays than the surrounding tissue, so that a particular organ is brought more sharply into view. Barium sulfate, which is highly opaque to x-rays, is used for the x-ray examination of the gastrointestinal tract. Certain opaque compounds are 37 administered either by mouth or by injection into the bloodstream in order to examine the kidneys or the gallbladder. Such dyes can have serious side effects, however, and should be used only after careful consultation. The routine use of x-ray diagnosis has in fact been discouraged—by the American College of Radiology in 1982, for example—as of questionable usefulness.

A recent x-ray device, used without dyes, offers clear views of any part of the anatomy, including soft organ tissues. Called the body scanner, or computerized axial tomography (CAT or CT) scanner, it rotates 180° around a patient's body, sending out a pencil-thin x-ray beam at 160 different points. Crystals positioned at the opposite points of the beam pick up and record the absorption rates of the varying thicknesses of tissue and bone. These data are then relayed to a computer that turns the information into a picture on a screen. Using the same dosage of radiation as that of the conventional x-ray machine, an entire ―slice‖ of the body is made visible with about 100 times more clarity. The scanner was invented in 1972 by the British electronics engineer Godfrey N. Hounsfield, and was in general use by 1979.

X-ray fluorescence spectroscopy is useful for both qualitative and quantitative analyses of metallic elements, which emit x-rays at characteristic energies when bombarded by a high energy x-ray source. (9,40,45)

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CHAPTER III

ANALYSIS OF X-RAY SPECTRA

3.1 Introduction Spectroscopy, in physics and physical chemistry, the study of spectra. The basis of spectroscopy is that each chemical element has its own characteristic spectrum. This fact was recognized in 1859 by German scientists Gustav Robert Kirchhoff and Robert Wilhelm Bunsen. They developed the prism spectroscope in its modern form and applied it to chemical analysis. One of two principal spectroscope types, this instrument consists of a slit for admitting light from an external source, a group of lenses, a prism, and an eyepiece. Light that is to be analyzed passes through a collimating lens, which makes the light rays parallel, and the prism; then the image of the slit is focused at the eyepiece. One actually sees a series of images of the slit, each a different color, because the light has been separated into its component colors by the prism. The German scientists were the first to recognize that characteristic colors of light, or the spectra, are emitted and absorbed by particular elements. (4,5)

3.2 Spectrum Analysis Light is emitted and absorbed in minute units or corpuscles called photons or quanta. The energy e of a single photon is directly proportional to the frequency ν, and therefore inversely proportional to the wavelength λ. This is expressed by the simple formula e = h ν = h c / λ where h, the proportionality factor, is Planck's constant and c is the speed of light. The particular colors, or wavelengths (and thus energies), of the light quanta emitted or absorbed by an atom or a molecule depend in a rather complicated way on its structure and on the possible periodic motions of its constituent particles, because this structure and these periodic motions determine the total energy, potential plus kinetic, of the atom or molecule. The 39 constituent particles of an atom are its nucleus, which does not contribute to the emission and absorption of light because it is heavy and moves very sluggishly, and the surrounding electrons, which move about rapidly in distinct orbits; the atom emits or absorbs a quantum of light of a definite color when one of its electrons jumps from one orbit to another. The constituent parts of a molecule are the nuclei of the various atoms that compose the molecule and the electrons that surround each nucleus. The emission and absorption of light by a molecule are accounted for by its possible modes of rotation, by the possible modes of oscillation of its atomic nuclei, and by the periodic motions of its electrons in their various orbits. Whenever the mode of vibration or rotation of a molecule changes, changes also occur in its electronic motions, and the light of a definite color either is absorbed or is emitted.

Thus, if the wavelengths of the photons that are emitted by a molecule or an atom can be measured, considerable information can be deduced about the structure of the atom or molecule and about the possible modes of periodic motion of its constituent parts.

3.2.1 Continuous Spectrum The simplest form of spectrum, called a continuous spectrum, is emitted by a solid object that is heated to incandescence, or by a liquid or a very dense gas. Such a spectrum contains no lines because light of all colors is present in it, and the colors blend continuously into one another, forming a rainbow-like pattern. A continuous spectrum can be analyzed only by spectrophotometric methods. In the case of an ideal emitter, a blackbody, the intensities of the colors within the spectrum depend only on the temperature. Two of the laws relating to the distribution of energy in a continuous spectrum were discovered about 1890 by German physicist Wilhelm Wien and Austrian physicists Ludwig Boltzmann and Josef Stefan. The Stefan-Boltzmann law states that the total energy radiated per second by a blackbody is proportional to the fourth power of the absolute temperature; Wien's displacement law states that as the temperature is 40 raised, the spectrum of blackbody radiation is shifted toward the higher frequencies in direct proportion to the absolute temperature. In 1900 Planck discovered the third and most important law describing the distribution of energy among the various wavelengths radiated by a blackbody. In order to derive a law that interpreted his experimental findings, Planck argued that the thermodynamic properties of the thermal radiation emitted by matter must be the same regardless of the emission mechanism, and regardless of assumptions about the nature of atoms. These ideas led to the development of the quantum theory.

Fig 3.1 Continuous spectrum

The phenomena of fluorescence and phosphorescence result from the absorption of photons of a particular wavelength followed by the emission of photons of a longer wavelength. In both fluorescence and phosphorescence, the photon absorbed from the illuminating radiation excites an electron that is initially in the ground state to a higher state. This excited electron then falls to a 41 lower level, but not immediately back to the ground state, emitting a longer- wavelength photon than it absorbed. In fluorescence, the emission and absorption follow each other quite rapidly, so that fluorescence lasts only as long as the illuminating radiation is on. In phosphorescence, however, the emission occurs quite slowly and persists for a long time after the illuminating radiation has been turned off.

The sodium atom produces a more complex spectrum than does the hydrogen atom. The sodium atom has 11 orbital electrons: an inner group of 2, a middle group of 8, and an outer group of 1. If the spectrum of sodium is excited by the electric spark, many of these electrons can be responsible for production of lines; if it is excited by the electric arc, or by a flame, the outer electron is responsible for most of the lines, and to some extent, in its broad features, it behaves like the electron in the hydrogen atom. But complexities in the motion of this outer electron arise from its interaction with the ten core electrons that occupy the closed shells of the sodium atom. Moreover, the electron not only can move to orbits other than its own, but the orbits can have varying eccentricities, and in any orbit, the electron can have different orbital magnetic moments and different orbital angular momentum. These variations produce not only several series of lines, but also doublets and triplets, groups of two or three lines that differ only slightly in wavelength. The most important series of lines are called sharp, principal, diffuse, and fine (abbreviated S, P, D, and F in theoretical work; further series are abbreviated G and H without being named).

3.2.2 Absorption Spectra Most of the information that physicists have gained about the structure of the atom has been obtained through spectroscopy. Molecular spectra are similarly useful in elaborating the structure of molecules, which has even greater interest for chemists than for physicists. Most molecular spectra are characteristically band spectra; that is, the spectrum consists of a series of bright bands, each of which appears similar to a piece of the continuous spectrum, separated by dark 42 spaces. These bands are not continuous but consist of many closed spaced lines that can be resolved with high-resolution spectroscopes. The spacings of the lines in any series of molecular bands depend on whether the spectrum is rotational or vibrational. Because the rotational energy levels can be excited by small amounts of energy, and are thus close to one another, the lines in a rotational band are tightly packed with hardly any spacings. The vibrational levels, however, are much further apart, and the lines in a vibrational band are therefore much more widely spaced. The electronic energy levels of a molecule can also be excited, and the transitions of electrons between such levels give rise to the widely separated electronic lines in the molecular spectrum.

In addition to atomic absorption spectra, molecular absorption spectra also exist, which are obtained by passing continuous radiation through a molecular liquid or gas. This type of spectrum, consisting of dark bands separated by bright spaces, is the one that is most often used to study molecular structure. Other bands in molecular spectra are not resolvable into lines even by the most powerful instruments and are apparently continuous regions of absorption or emission of energy. (4,31,47)

3.3 Naming Transitions In theory, characteristic X-rays are named according to the shell being filled and the number of shells changed by the electron is indicated by  (1 shell),  (2 shells), or  (3 shells). However, spectrographic nomenclature was developed before atomic electronic structure was well-understood, producing many inconsistencies. In the example above, the 3d - 2p transition produces lines named L, L, and L; however, the L1 line does not represent a transition of 2 shells. In 1991, the Union of Pure and Applied Chemistry recommended using an energy-level-based nomenclature, but this is not widely used. According to the UPAC scheme, the K1 line would be called K-L3. The transitions shown in Fig 3.2 are for the element barium (Ba), but spectral lines produced by a given transition have the same name in all atoms. (34) 43

Fig 3.2 Electron energy level diagram for barium (Z = 56). The Greek letters show the nomenclature conventionally applied to the most probable electron transitions to the K, L and M shells, and the resulting characteristic x-ray emissions.

3.4 Moseley's Law Henry Moseley discovered that the wavelength (energy) of an x-ray depended upon the nuclear charge of an atom. In 1913, working at the University of Manchester, he photographed the x-ray spectrum of 10 elements that occupied consecutive places in the periodic table. He concluded that there was "a fundamental quantity which increases by regular steps as we pass from one element to the next." In 1920, Rutherford at Cambridge identified this quantity as the atomic number. The K lines shifted to higher energy with increased atomic number because the inner-shell electrons are more tightly bound by the higher number of protons in the nuclei (Figures 3.3 and 3.4). The energy of transition is proportional to the number of shells changed; K radiation is less energetic than K.

44

Fig 3.3 Energies of major x-ray emission lines observed below 10 keV.

Fig 3.4 Moseley's relation between wavelength and atomic number for the

K1, L1 and M1 spectral lines 45

Moseley's Law describes the relationship between atomic number and wavelength of a spectral line:

 = K (Z  )2 where K and  are constant for a given spectral line.

The constant σ (sigma) is equal to 1 for the K-lines and 7.4 for the more shielded L-lines. For energy this expression is approximately equivalent to: E(keV) = K (Z – 1)2 where Z = atomic number and K = 1.042  10-2 for K-shell K = 1.494  10-3 for L-shell and K = 3.446  10-4 for M-shell

Moseley's determination of this relationship provided a simple test of the order of the elements according to Z. It showed where elements were missing from the periodic table and led to the discovery of some of these elements. For example, hafnium (Hf), which chemically is almost identical to zirconium (Zr), was identified by D. Coster and G. von Hevesy in 1923 from its x-ray spectrum.

If the impinging electrons are insufficiently energetic to excite K radiation for a given element, they can excite L or M radiation, which require less energy to produce. This means that the excitation potentials (Ec) are higher for K-lines than L-lines or M-lines. For a given element, the energy of K radiation is greater than L radiation, which is, in turn, greater than M radiation. For example, Ec for Fe-K is 7.11 keV, whereas Ec for Fe-L is 0.71 keV. Thus, incident electrons of 5 keV energy, will not excite Fe-K, but can excite Fe-L. Often in analysis, one can use L-lines if K-lines are too energetic to 46 excite efficiently; however, the peak-to-background ratio is not as good for L- lines and M-lines as for K-lines. (27,32,34)

3.5 Line Intensities Line intensities are a function of transition probabilities and the rate at which the appropriate atomic shell is ionized by the incident electrons. The probability of ionization is a function of the effective target-atom ionization "cross-section," Q. The ionization cross-section is small (0.01 Å) compared with the typical atomic diameter (1 Å) and decreases with increasing atomic number. Thus, only a small proportion of atoms in the target material are ionized. Line intensities may be described by an empirical relationship: p I = C ib (E0 – Ec) where I = intensity of the line of interest, C = a constant,

ib = beam current

Ec = critical excitation potential of the line of interest,

E0 = accelerating voltage (keV) and

p = 1.7 for E0 < 1.7 Ec ( and smaller for higher values of E0)

Thus, the intensity of a line depends on the amount to which E0 exceeds Ec

(Figure 3.5). The ratio E0/Ec is termed the overvoltage. Optimal overvoltages may be calculated using the expression for the capture cross-section (Q) for a given shell. For example, for the K-shell this is: n b Q = 6.51  10-20 s s ln C U 2 S U Ec where ns = number of electrons in shell

bs, Cs = constant for particular shell

Ec = critical ionization energy for the shell (keV)

U = overvoltage (E/Ec) E = electron beam energy

47

Recommended values for the K-shell are bs = 0.9 and Cs = 0.65. Typical capture cross-sections for the K shell are on the order of 10-20 cm2, about a hundredth of the atomic diameter. Clearly, K-shell ionization has a very low probability. This function peaks at E0 of about 2.5 times Ec, thus this overvoltage for K-lines is optimal. With higher E0 and higher overvoltages, there is a greater chance of decreasing x-ray intensities through the production of satellite peaks.

Fig 3.5 Spectra of pure copper taken at accelerating voltages of 10 keV (above) and 20 keV (below). At 10 keV, only the L lines are

efficiently excited (Kab = 8.98 keV)

3.6 Satellite Peaks At high overvoltages more than one electron may be ejected from an atom. In this case, the overall structure of the electron shells is changed and x-rays are produced with slightly lower energies than those produced by single electron ionization. These x-rays appear as small satellite peaks near the characteristic 48 radiation peaks. Another important source of satellite peak production is the Auger process.

Generally, satellite peaks are not important, except when there is a large overvoltage. For example, using Ec of 15 keV to produce Fe-K is about optimum (15 / 7.112 = 2.1) but produces an eight-fold overvoltage for Si-K (15 / 1.838 = 8.2). This results in a larger intensity of Si satellite peak and decreased intensity of the Si-K line. In addition, satellite peaks lower the peak-to-background ratios because more x-rays fall outside PHA window. In routine analysis, the production of satellite peaks is only a potential problem for elements of Z < 20. However, this is not usually a significant problem. The intensity of the Si-K peak at E0 15 keV is satisfactory, because more Si-K x-rays are produced and detected than Fe-K x-rays, owing to higher abundances of silicon in geological materials and better spectrometer and crystal efficiencies for Si-K. (27,32,34)

3.7 Wavelength Shifts There are small but detectable changes in the energy of x-ray lines from pure element to various compounds of the element due to bonding and oxidation effects. Although x-ray lines are produced by inner-shell ionization and self- neutralization, the configuration of the inner shells is slightly influenced by the outer valence electrons (Fig 3.6). Peak shifts are most apparent in the outermost transitions (M-lines) and in low-Z elements (B through F for K, Al through Cl for K) where the x-ray-producing inner shells are less shielded from the valence electrons (Fig 3.7).

The oxidation state of an atom is reflected in the number of electrons in the valence shell and differences in the valence electrons produce changes in the overall electron cloud structure and minor wavelength shifts. For example, consider Fe2+ with 24 electrons and Fe3+ with 23 electrons; both have a nuclear charge produced by 26 protons. The fewer electrons of the Fe3+ atom 49 individually "feel" more of the nuclear charge and the configuration of the shells is slightly different than for Fe2+. The difference results in a small change in the energy difference between Fe-L and Fe-L. Attempts have been unsuccessful to exploit the wavelength shift to determine the oxidation state of

Fig 3.6 Dependence of the profile of the Al-K peak on the involvement of the Al valence electrons in chemical bonding. The K x-rays are produced by transitions from the 3s and 3p levels to the 1s level. In Al metal, the 3s and 3p electrons are not involved in chemical bonding and the transitions produce a relatively narrow band of

energies. In Al2O3, the valence electrons combine with the oxygen 2s and 2p electrons to fill molecular orbitals, spanning a wider energy band. The K emission becomes broader, asymmetric, and resolves into separate K and satellite K' peaks. 50

Fig 3.7 Chemical effects on the emission spectra of magnesium (Mg),

magnesium oxide (MgO), and magnesium fluoride (MgF2). Count rates are logarithmically plotted, and vertically shifted for easier comparison.

Fe in minerals. In part this is because the difference in the ratio of electrons to nuclear charge for the two oxidation states is small.

For lighter elements the wavelength shift is substantially larger, especially comparing metals with oxides. In minerals, the shift of S-K is appreciable when sulfides (S2-) are compared with sulfates (S6+). Standards should be the same oxidation state as the unknowns to avoid problems when analyzing sulfur. 51

The coordination of an atom in a mineral may also effect the electron cloud configuration and thus the resulting x-ray lines. In tetrahedral coordination, a cation has 4 nearest neighbors, whereas in octahedral coordination there are 6. As with the oxidation effect, equal numbers of nuclear protons pull on electrons, while, in this case, different numbers of oxygen atoms pull out. With more oxygen atoms pulling out (6-fold vs. 4-fold coordination), the electrons are pulled away from the nucleus resulting in larger transitions (higher energy). The coordination effects are also more easily observed in lighter elements than heavy elements. For example, Al-K lines shift to varying degrees relative the line produced from Al metal. In feldspars (AlIV) this shift is about 0.05 degrees toward higher energy; in kaolinite (AlVI) it is about 0.11 degrees. (23,34)

3.8 Spectroscopic Analysis When material is irradiated with x-rays, an electron in the inner shell orbit of the atom of the material is knocked out of its orbit, and a hole is created in the shell. The outer shell electron then falls into the hole. Accompanied with this transition, a fluorescent x-ray photon, whose energy is equal to the energy difference between these shells, is emitted. The energy of the fluorescent x-ray is characteristic to the element because energy levels are an inherent atomic property of the element. Therefore, fluorescent x-ray analysis is used for identifying trace elements (Fig. 3.8).

When analyzing heavy elements, only L and L lines can be observed if the x-ray energy is insufficient. However, in order to make an accurate quantitative analysis, K lines are needed. In high energy fluorescent x-ray analysis of heavy elements, such as tin, tungsten, lead and bismuth, is possible because high energy x-rays with narrow energy width are provided. Fig 3.9 shows the fluorescent x-ray spectra of standard arsenious acid produced in several countries. Based on the spectra, the material's site of origin can be determined. The trace element analysis can be applied to the study of ancient remains. (19,45) 52

Fig 3.7 Energy-levels and the transition of the outer shell electrons.

Fig 3.8 The fluorescent x-ray spectrum of arsenious acid according to its site of origin.

3.9 WDS vs. EDS Detection The energy dispersive (EDS) and wavelength dispersive (WDS) systems both have benefits and disadvantages. The main differences between the systems are 53 in detector efficiency and resolution. Schematic diagrams showing the components of the WDS and EDS systems are shown in Figure 3.9 and 3.10.

Fig 3.9 Schematic representation of a wavelength-dispersive spectrometer

Fig 3.10 Schematic representation of an energy-dispersive spectrometer (Goldstein et al. 1981).

The overall efficiency of collecting the x-rays produced is poor for both WDS (<0.2%) and EDS (><2%) with the EDS better because the detector is closer to the sample. Most x-rays produced from the sample do not make it to the detection systems. In a WDS system about 30% of the x-rays entering the 54 detector are actually counted; whereas, in an EDS system about 100% of the incident x-rays are counted. As a consequence, the minimum useful probe spot size is larger for WDS (about 2 µm). The greater efficiency of EDS permits the use of beam diameters as small as 50 Å (0.05 µm). However, the resolution of the WDS system is far superior (Fig 3.11).

Fig 3.11 Comparison of the resolution of the proportional counter, Si(Li) semiconductor detector, and several analyzing crystals. Note that the axes are logarithmic

Most x-rays produced from the sample do not make it to the detection systems. In a WDS system, about 30% of the x-rays entering the detector are actually counted; in an EDS system, almost 100% are counted. As a consequence, the minimum useful probe spot size for WDS is about 2 m; the greater efficiency of an EDS permits the use of beam diameters as small as 50 Å (0.05 m). 55

The instantaneous spectral acceptance range differs greatly between the two systems. WDS spectrometers can only examine the portion of the spectrum for which they are positioned, whereas in EDS entire useful energy range can be examined simultaneously. Finally, spectral artifacts produced by stray x-rays and electrons are more rare with WDS. The EDS detector, located close to the sample, receives many stray x-rays and electrons and suffers peak distortion, peak broadening, escape peaks, absorption, and internal Si fluorescence.(14,38)

3.10 Calibration Curves Calibration curves may be constructed which relate counts and concentration (Fig 3.12 and Fig 3.13). However, these require a large number of well- characterized standards with compositions bracketing the unknowns. The data used to make the curves must be taken at identical operating conditions (take- off angle, accelerating voltage and beam current) for all analyses.

Fig 3.12 Calibration curves for electron microprobe analysis of carbon in nickel steels

Calibration curves must be constructed for each mineral group, but, because unknowns approximate the standards in compositions, corrections are unnecessary. The requirement of a great number of standards is especially 56 difficult to satisfy especially with the accuracy required. In addition, this technique does not allow confident analysis of a truly unknown material.(53)

Fig 3.13 Calibration curve of counts per second vs. weight percent MgO for chemically analyzed biotite, chlorite, staurolite, chloritoid, and garnet. The scatter in the data results from absorption (or fluorescence) due to compositional variations in the sample of oxides other than MgO.

3.11 Energy Dispersive X-ray Fluorescence (EDXRF) Spectrometry Energy Dispersive x-ray Fluorescence (EDXRF) Spectrometry has been in use for nearly 20 years, and its use for analysis of environmental soils has grown rapidly in the last 12 years. Early instruments that were designed primarily for laboratory use, suffered from poor resolution, and could not easily resolve multiple elements in a single analytical run. These instruments required a highly trained operator. Recent advancements in detectors, filters, and microprocessor controls have made EDXRF spectrometers field worthy, portable, and user-friendly. 57

The use of EDXRF technology would apply to the analysis of heavy metals in soil and sediment. Although there are some advances that would allow the analysis of water and air, the majority of EDXRF projects in the environmental sector involve soil analysis.

3.11.1 Performance and Advantages It is clear that the future trend the environmental sector is in rapid site investigation and remediation using techniques that are quicker, cheaper, better. Mobile and Close Support Laboratories have been providing reliable near real time data mostly for organic contaminants for many years, however, the analysis of metals in a field setting has been difficult. Traditional techniques for metals analysis have not been easily adapted for field use due to time consuming and destructive sample preparation using concentrated acids, and analysis by instrumentation not well suited for field use.

The EDXRF technique is rapid and non-destructive to the sample, with sample preparation time consisting of microwave drying, and pulverizing of the sample with mortar and pestle. Because no dangerous reagents are used in the sample preparation and analysis, the technique is much safer to the operator. Aside from the sample, there are no additional waste streams generated during the analytical process.

3.11.2 Faster Analysis Time The benefit of EDXRF is that the data can be provided on a rapid turnaround basis. Generally, sample results can be provided to the client within 15 minutes of sample receipt. The detection limits for this approach are vary between 10 to 20 ppm for most elements and are much lower than the normal action levels for metals in soil and sediment.

Traditional analytical methods require extensive sample preparation techniques using concentrated acids to digest the soil sample (destructive technique). These techniques require twenty minutes to two hours for the acid digestion 58 prior to analysis by AA, GFAA, or ICAP. In addition, samples are batched when using traditional methods that may substantially increase turnaround time. The non-destructive sample preparation technique used for EDXRF analysis does not involve hazardous reagents, and allows the client to confirm the EDXRF results at a later date on the exact same sample used for the initial analysis.

3.11.3 Lower Cost of Analysis EDXRF is a very cost effective method for analyzing soil samples in the field. It is difficult to make a true comparison between the EDXRF and the traditional methods because the traditional methods cannot provide a result within 15 minutes, and generally require batching of samples during the digestion step that further increases turnaround time.

In addition to the analytical cost savings, a key factor in using EDXRF in the field is overall project savings due to improved decision-making and the reduced costs associated with the field effort. Using EDXRF to guide an excavation or site characterization will generally reduce the overall time required in the field due to the quick turnaround of the sample analysis.

3.11.4 Interferences of Elements Like many analytical methods, EDXRF suffers from a few limitations and interferences. Due to the production of a continuum of x-rays by the x-ray tube, scattered x-rays are produced by interaction with the rhodium anode, air, moisture, sample, and silicon/lithium detector. There is a constant level of radiation (bremsstrahlung) emitted from these interactions that can obscure elements, especially low Z elements. The use of primary filters can aid in reducing the background radiation and allow for better signal to noise ratio in the energy zones of interest. Because of the attenuation of produced x-rays as they interact with air in the sample chamber, low Z elements sensitivity (elements between sodium and phosphorus) is diminished. Detection of these 59 compounds can be enhanced if a vacuum source is connected to the analytical chamber.

There are two elements that are typically masked by high concentration elements; Cobalt‘s K line at 6.924 KeV is masked by high levels of Iron‘s K at 6.398 KeV. A more environmentally significant interference occurs between arsenic and lead. The lead L1 line at 10.550 KeV overlaps the position of the arsenic K line at 10.530 KeV, which causes the FP software to attempt to calculate arsenic from the Kb line. This quantitation is usually unsuccessful when the arsenic/lead concentration ratio is below 0.046. Due to this interference, the arsenic detection limit is set at 10% of the lead concentration for samples with high lead content. (14,47,48)

3.11.5 Volatile Metals by EDXRF Mercury, arsenic, and selenium are considered volatile elements and some care must be taken in their analysis, especially in the case of mercury. EDXRF in conjunction with the fundamental parameters software is an excellent method for rapid screening of mercury-contaminated soils. Certified soil reference standards for mercury are not readily available, so the quantitation for mercury (atomic number 80) is performed by using the calibration data for lead (atomic number 82). This quantitation is possible due to the proximity of the elements to each other on the periodic table of the elements and is referred to as a ‗standardless‘ calibration. The spectra for each element is easily distinguishable but the mass absorption coefficients for these elements are nearly the same (2.291 for Hg, 2.389 for Pb).

3.11.6 EDXRF Hardware EDXRF is relatively simple and inexpensive compared to other techniques. It requires and x-ray source, which in most laboratory instruments is a 50 to 60 kV 50-300 W x-ray tube. Lower cost benchtop or handheld models may use radioisotopes such as Fe-55, Cd-109, Cm-244, Am-241 of Co-57 or a small 60 x-ray tube. The second major component is the detector, which must be designed to produce electrical pulses that vary with the energy of the incident x-rays. Most laboratory EDXRF instruments still use liquid nitrogen or Peltier

Fig 3.14 The most common laboratory grade EDXRF configuration cooled Si(Li) detectors, while benchtop instruments usually have proportional counters, or newer Peltier cooled PIN diode detectors, but historically sodium iodide (NaI) detectors were common. Some handheld devices use other detectors such as mercuric Iodide, CdTe, and CdZnTe in addition to PIN diode devices depending largely on the x-ray energy of the elements of interest. The most recent and fastest growing detector technology is the Peltier cooled silicon drift detector (SDD), which are available in some laboratory grade EDXRF instruments.

After the source and detector the next critical component are the x-ray tube filters, which are available in most EDXRF instrument. There function is to absorb transmit some energies of source x-rays more than other in order to 61 reduce the counts in the region of interest while producing a peak that is well suited to exciting the elements of interest. Secondary targets are an alternative to filters. A secondary target material is excited by the primary x-rays from the x-ray tube, and then emits secondary x-rays that are characteristic of the elemental composition of the target. Where applicable secondary targets yield lower background and better excitation than filter but require approximate 100 times more primary x-ray intensity. One specialized form of secondary targets is polarizing targets. Polarizing XRF takes advantage of the principle that when x-rays are scattered off a surface they a partially polarized. The target and sample are place on orthogonal axis to further minimize the scatter and hence the background at the detector.

Fixed or movable detector filters, which take advantage of non-dispersive XRF principles, are sometimes added to EDXRF devices to further improve the instruments effective resolution or sensitivity forming a hybrid EDX/NDX device. (29)

3.12 Fundamental Parameters Analysis EDXRF can analyze up to 40 elements (could be configured for up to 100), as individual elements and/or compounds. Unanalyzed elements can be specified stoichiometrically bound with an analyzed element (e.g., oxides or carbonates). Elements can be analyzed in one or more compounds within the same analysis. One compound (or element) can be analyzed by difference. Any number of compounds (or elements) can be "fixed." For example, solutions, binders and/or hydrated crystals can be analyzed this way.

3.12.1 General Bulk and Thin-Film Analysis Any bulk, or single-layer (unsupported) thin-film, sample can be analyzed by either standardless or a calibration-with-standards FP approach. Each analysis may use up to 6 excitation conditions per analysis. Each excitation condition can vary almost any analysis setup, including the kV, acquire time, tube (or 62 secondary) target, detector type, detector or tube filter, source focusing optic, atmosphere (air, vacuum, helium), and spectrum processing (e.g., deconvolution type, background removal, sum & escape peak removal).

Optional software is available to handle multilayer samples up to 6 layers, for simultaneous film thickness and composition analysis using FP with standards.

3.12.2 Analysis With or Without Standards Many detectors and windows can be fully modeled. This allows analysis without any standards, with normalization to 100% (or any specified factor). This is only possible when a single excitation condition is used.

When more than one excitation is used, at least one of the elements for each condition must have been calibrated. Calibration factors may be generated using any type of standard (e.g., pure element or analytical "type" standard). A single "type" standard may be used, or the calibration may be done with a different standard for each element, or any combination of standards may be used. If some elements are calibrated and some are not, the latter can use calibration coefficients derived from the former group.

The mass thickness of the sample can either be specified or calculated. If the latter, then the analysis cannot be standardless. Several units are possible for thickness measurement, and the density can be calculated theoretically or specified, in the case of linear thickness calculations. Composition units may be ppm or wt%, with the additional output of atomic and mole percent.

3.12.3 Spectrum Calibration Using two known peaks in the spectrum, the software can calculate the effective gain (eV/channel) and offset (zero shift) for the spectrometer. These factors can then routinely be applied to subsequent spectra prior to other spectrum processing. It is vital that peaks are located at their expected energies, otherwise the spectrum processing cannot function correctly. 63

3.12.4 Background Removal This module uses iterative filtering to remove all peaks, leaving behind the spectral background. The background spectrum may be displayed or removed from the original spectrum.

Fig 3.15 Processed spectrum and removed background (blue curve)

3.12.5 Escape Peak and Sum Peak Removal Routines are available to optionally remove both detector escape and sum (pile- up) peaks.

3.12.6 Smoothing A specified number of 1:2:1 Gaussian smoothes can be applied to a spectrum.

3.12.7 Intensity Extraction Specified element peaks may be integrated over a fixed ROI (Region Of Interest), or the complete spectrum can be fit using synthetic Gaussians for every possible line in the regions of interest. One of six major lines (K, K, L, L, L, M) is selectable as the main analysis peak for intensity extraction. 64

All relevant lines required for deconvolution are then automatically included by the software for full overlap correction using a least-squares fitting procedure.

All required line energies and resolutions are calculated automatically from the specified analyte line. The Gaussian peak fitting can be done with a linear or non-linear least-squares approach. The latter allows constrained changes in the peak positions, intra-series line ratios, and peak widths, from their nominal starting points.

In addition to calculating elemental intensities, the software automatically calculates the estimated uncertainty and background values, which allows uncertainty and Minimum Detection Limit (MDL) calculations to be performed during the FP analysis. (13,50,52)

3.13 Heavy metals Heavy metals are natural constituents of the Earth's crust. Living organisms require trace amounts of some heavy metals, including cobalt, copper, iron, manganese, molybdenum, vanadium, strontium, and zinc. Excessive levels of essential metals, however, can be detrimental to living organisms.

Human activities have drastically altered the biochemical and geochemical cycles and balance of some heavy metals. Heavy metals are stable and persistent environmental contaminants since they cannot be degraded or destroyed. Therefore, they tend to accumulate in the soils and sediments. Excessive levels of metals in the marine environment can affect marine biota and pose risk to people consuming seafood. Nonessential heavy metals of particular concern to surface water systems are cadmium, chromium, mercury, lead, arsenic, and antimony.

 3.13.1 The Illnesses due to Heavy Metals Ingestion of metals such as lead, cadmium, mercury, arsenic, and chromium, may pose great risks to human health: 65

• Lead: Because of size and charge similarities, lead can substitute for calcium and be included in bone. Children are especially susceptible to lead because developing skeletal systems require high calcium levels. Lead that is stored in bone is not harmful, but if high levels of calcium are ingested later, the lead in the bone may be replaced by calcium and mobilized. Once free in the system, lead may cause nephrotoxicity (where a toxic agent inhibits, damages or destroys the cells and/or tissues of the kidneys), neurotoxicity (where a toxic agent or substance inhibits, damages or destroys the tissues of the nervous system, especially neurons, the conducting cells of the central nervous system), and hypertension (abnormally high blood pressure).

 • Cadmium: Cadmium may interfere with the metallothionein's ability to regulate zinc and copper concentrations in the body. Metallothionein is a protein that binds to excess essential metals to render them unavailable. When cadmium induces metallothionein activity, it binds to copper and zinc, disrupting the homeostasis (the maintenance of equilibrium, or constant conditions, in a biological system) levels.

 • Mercury: Mercury poses a great risk to humans. Mercury occurs naturally in the environment and it can also be released into the air through industrial pollution. Bacteria in the water cause chemical changes that transform mercury into methlmercury, which binds tightly to the proteins in fish tissue. Mercury tends to accumulate in the food chain so predatory fish species (eg shark) tend to have higher levels than non-predatory fish or species at lower levels in the food chain. High amounts of mercury can damage the nervous system of people, triggering such health problems as memory loss, slurred speech, hearing loss, lack of coordination, loss of sensation in fingers and toes, reproductive problems, coma, and possibly death.

 66

• Arsenic: Arsenic ingestion can cause severe toxicity through ingestion of contaminated food and water. Ingestion causes vomiting, diarrhea, and cardiac abnormalities.

 • Chromium: The presence of abundant chromium anions in the water is generally a result of industrial waste. The chronic adverse health effects are respiratory and dermatologic.

 3.13.2 Absorbing Heavy Metals Many organisms are able to regulate the metal concentrations in their tissues. Research has shown that aquatic plants and bivalves are not able to successfully regulate metal uptake, and as a result, bivalves tend to suffer from metal accumulation in polluted environments. Heavy metals may enter fishes and shellfishes in several ways. Small amounts are absorbed by these organisms directly from the water through their gills and other tissues.

However, most of the pollutants found in aquatic organisms arrive there through the food chain. First, phytoplankton, bacteria, fungi and other small organisms absorb these materials. In turn, these are eaten by larger animals, eventually ending up in the organisms eaten by people. (11,24,39,42,45)

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CHAPTER IV

EXPERIMENTAL PROCEDURE AND RESULTS

4.1 Samples Collection and Preparation In this research work, twenty-one marine fish samples were collected from Thandwe Region (Rakhine Coastal) and twelve marine fish samples were collected from Kyauk Gyi Region (Rakhine Coastal). The list of marine fish samples and their scientific names, common name, local names, family names, orders and classes are shown in Table 4.2 and Table 4.3. The locations of the samples collected area is shown in Fig 4.1. These marine fish samples were dried and grinded to find powders. These fine powders were made palettes by hydraulic press at Universities' Research Centre, University of Yangon.

4.2 Energy Dispersive X-Rays Fluorescence Spectrometer for

Shimadzu EDX-700 X-ray spectrometer permits simultaneous analysis of light elements to heavy elements. Instrument operation is simple and intuitive. The EDX-700 is used for determination of Na(11)-U(92).

X-rays fluorescence (XRF) uses x-rays to excite an unknown sample. The individual elements comprising the sample re-emit their own "Characteristic" x-rays. The EDX-700 is an Energy Dispersive XRF system, known as EDXRF. This indicates that the x-rays are detected using a semiconductor detector, which permits multielement, simultaneous analysis. This makes for the fastest possible analysis of a sample, even in the ppm range. The EDXRF design has a number of advantages. It is of a compact size and of easy operability over other types of XRF.

Analysis is a possible in the atmosphere because of the short distance between sample and the detector. Therefore almost all samples can be measured without 68

Fig 4.1 Location of Thandwe and Kyauk Gyi Regions

69 preparation for a vacuum environment. This allows the user to measure food samples without the necessary step of dehydration. The EDX-700 allows smaller parts of larger structures to be measure. Furthermore, analysis of waste materials iron machining and the identification of unknown materials are possible.

In present work, the samples were placed in cylinder shaped sample containers and covered 6mm thick myler. The sample containers were placed in the sample chamber of EDX-700 spectrometer which can measure sixteen samples at a time.

A large sample chamber with an automatic opening and closing door was used. After closing the door, the chamber was pumped up to vacuum. The vacuum pressure was about (38pa) and the detector temperature is about (-170 C), the EDX-700 was put into operation. Each sample was run for about 100 seconds and the spectra were analyzed in IBM/PC using EDX-700 software.

Main Specification of EDX-700 Spectrometer Method of measurement Energy dispersive x-ray analysis Sample type Solid, liquid or powers Energy range Na-U (EDX-700) Sample size 300 mm (dia) 150 (H) mm max

X-Ray Generator X-ray tube Rhodium (Rh) target Tube voltage 5-50kV Tube current 1-1000A Cooling method air cooling (with a fan) Exposure area 10mm dia (standard) (Automatic selection in 4 sizes of 1, 3, 5, 10mm dia) Primary filter Automatic selection from among 5 types of filter

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Detector Type Si (Li) detector

LN2 supply Only during measurement Dewar capacity 3 Liters

LN2 consumption Less than 1 liter per day Detection area 10mm2

Resolution 155eV (Mink, 1500cps)

MCA Resolution 2048ch Gain 10eV/ch and 20eV/ch

Sample Chamber Atmosphere in spectrometer Air, vacuum, He Sample change 8/16 samples turret, 8 samples turret with spinner

Evacuation system (for high-sensitivity analysis of light elements) Evacuation Oil rotary vacuum pump, directly connected Vacuum monitor With pressure sensor

Data Processing Unit Main unit IBM PC/AT compatible Memory More than 32MB Hard disk More than 2GB Floppy disk 3.5 inch x 1 CRT 17-inch color display Printer Color ink jet printer Operating system Windows 95, 98 or Windows NT

Software Qualitative analysis Software for measurement and analysis of measured data Quantitative analysis Calibration curve method, matrix correction, FP method, thin film FP method, background FP method. 71

Matching Software (intensity/content) Utility Automatic correction functions (intensity correction, energy correction, full width of half maximum correction)

4.3 Analysing of Concentrations of Elements Contained in Samples In this research work, concentrations of elements contained in thirty three palettes of marine fish samples collected from Thandwe Region (Rakhine Coastal) and Kyauk Gyi Region (Rakhine Coastal) were analysed using EDX-700 energy dispersive x-ray fluorescence system. The measuring time for each sample was 100 s. Fundamental Parameter (FP) method was used to determine the concentrations of elements contained in the marine fish samples from the acquired x-ray fluorescence spectra of the marine fish samples. These concentrations of elements contained in the marine fish samples are shown in Table 4.1. It is found that, heavy element P, S, Cl, K, Ca, Sc, Mn, Fe, Ni, Cu, Zn, Br, Sr, Zr and Pt are found in marine fish samples.

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Table 4.1 Concentrations of elements (ppm) contained in the marine fish samples Concentrations of elements (ppm) Element S01 S02 S03 S04 S05 S06 P 0 0 0 0 0 0 S 3450 4903 4981 4334 6276 6554 Cl 0 0 7365 0 0 0 K 7018 7422 8248 10030 7344 9356 Ca 7990 3507 7046 14620 13940 12850 Sc 0 0 215 0 0 0 Mn 0 0 0 0 0 0 Fe 259 210 391 796 364 373 Ni 0 0 229 100 0 0 Cu 0 0 0 100 0 0 Zn 75 0 0 94 112 0 Br 37 0 59 47 0 0 Sr 36 60 58 75 132 95 Zr 0 0 0 0 0 0 Pt 0 0 0 0 0 0

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Table 4.1 Continued

Concentrations of elements (ppm) Element S07 S08 S09 S10 S11 S12 P 0 0 0 0 0 0 S 4738 0 2699 7846 6476 6990 Cl 0 4522 0 8132 0 0 K 10790 7483 7112 9673 7376 8939 Ca 15780 28970 3913 11640 12650 31870 Sc 0 0 0 792 0 0 Mn 0 0 0 0 0 0 Fe 649 704 316 217 523 494 Ni 0 341 0 0 0 0 Cu 0 0 88 0 0 0 Zn 121 123 74 0 93 0 Br 0 0 40 0 0 0 Sr 106 245 39 74 77 167 Zr 0 15 0 0 0 0 Pt 0 0 0 0 0 0

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Table 4.1 Continued

Concentrations of elements (ppm) Element S13 S14 S15 S16 S17 S18 P 0 0 0 0 0 0 S 0 0 3556 4401 4451 6818 Cl 6277 0 0 0 0 8465 K 8187 9933 9305 6789 8457 11210 Ca 18590 3215 12320 5765 14330 16050 Sc 1185 0 0 0 0 482 Mn 0 0 0 0 0 0 Fe 310 291 415 287 346 420 Ni 0 0 0 0 0 0 Cu 0 0 0 0 104 0 Zn 106 89 100 97 108 509 Br 0 0 0 0 0 0 Sr 182 32 70 46 66 163 Zr 14 0 0 0 0 0 Pt 0 0 0 0 0 86

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Table 4.1 Continued

Concentrations of elements (ppm) Element S19 S20 S21 S22 S23 S24 P 0 0 0 0 0 0 S 4559 7102 5183 6481 6379 5123 Cl 0 7906 8465 5175 28220 0 K 7801 12310 9626 12830 6851 8512 Ca 16510 11200 13720 9348 12300 16240 Sc 0 530 0 330 891 0 Mn 0 0 0 0 0 0 Fe 363 305 281 387 319 482 Ni 0 0 0 0 0 0 Cu 102 0 0 0 0 0 Zn 87 0 0 130 0 93 Br 0 0 0 0 66 63 Sr 224 102 76 71 110 78 Zr 0 0 0 0 0 0 Pt 0 0 0 0 0 0

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Table 4.1 Continued

Concentrations of elements (ppm) Element S25 S26 S27 S28 S29 S30 P 0 5702 0 0 0 6666 S 7086 8818 7383 8174 5861 6380 Cl 0 0 0 0 7227 14050 K 9194 8896 9190 10330 15410 13660 Ca 22210 15220 7315 1492 4357 16350 Sc 1123 509 0 0 350 471 Mn 0 0 0 0 0 0 Fe 622 246 587 188 0 297 Ni 0 0 92 0 202 0 Cu 0 0 70 76 0 0 Zn 0 0 57 107 0 191 Br 84 91 69 44 0 81 Sr 172 123 64 29 53 106 Zr 0 0 0 0 0 0 Pt 0 0 0 0 0 0

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Table 4.1 Continued

Concentrations of elements (ppm) Element S31 S32 S33 P 0 4666 0 S 3247 4686 5279 Cl 0 17780 0 K 6723 5226 6092 Ca 6590 12370 10580 Sc 0 0 0 Mn 0 0 412 Fe 183 434 1681 Ni 0 0 0 Cu 89 164 222 Zn 97 125 254 Br 32 85 128 Sr 37 119 79 Zr 0 0 0 Pt 0 0 0 Table 4.2 List of the name of the sea fish sample of Thandwe Region (Rakhine Coastal)

Sample Scientific Name Common Name Local Name Family Order Class Code S01 Seriola songora Almaco jack Nga-htaw-but Carangidae Perciformes Actinopterygii S02 Cynoglossus lingua Long tongue sole Nga-khway shar Cynoglossidae Pleronectiformes Actinopterygii S03 Chanos chanos Milk fish Nga-tane Chanidae Clupeiformes Teleostomi S04 Formio niger Black pomfret Nga-moke-mei Carangidae Perciformes Teleostomi S05 Cataetyx messieri - Nga-saw-lone Brotulaidae Perciformes - S06 Dussumieria acuta Rainbow sardine Nga-kyaw-nyo Clupeidae Clupeiformes Teleostomi S07 Scarus rubroviolaceus Ember parrot fish Nga-kyet-tu-yway Scaridae Perciforms Teleostomi S08 Brotula maculata Blind fish Nga-sa-lone Brotulitae Perciformes Teleostomi S09 Pomadasys argyreus Silver grunter Nga-gone Pomadasyidae Perciformes Teleostomi S10 Platycephalus indicus Indian flathead Nga-kywere- padone Platycephalidae Scorpaeniformes Actinopterygii S11 Thrissocles purava - Nga-bar Engraulidae Clupeiformes Hamilton

73

S12 Cromileptes altivelis Humpback seabass Nga-tauk-tu Serranidae Symbranchiformes Actinopterygii S13 Gerres filamentosus Whipfin silver biddy Nga-se-ooe Gerreidae Perciformes S14 Muraenesox telabonides Indian pike conger Nga-shwe Muraenesocidae Cypriniformes Teleostomi S15 Labeo calabasu Orange fin labled Nga-nan-ma Cyprinidae Cypriniformes Teleostomi S16 Rastelliger kanagurta Rake gilled mackerel Nga-moe-thee Scombridae Perciformes Teleostomi S17 Caranx lugubris Black jack Nga-kyi-kan Carangidae Perciformes Actinopterygii S18 Tetrodon fluviatilus Estuarine blow fish Nga-pu-tin Tetraodontidae Tetraodontiformes Actinopterygii S19 Leiognathus equulus Common pony fish Nga-dinn-garr Leiognathidae Perciformes Actinopterygii S20 Siganus oramin - Nga-yan-shar Signidae Perciformes - S21 Chela sardinella Flying barb Nga-maw-taw Cyprinidae Cypriniformes Teleostomi

79

Table 4.3 List of the name of the sea fish sample of Kyauk Gyi Region (Rakhine Coastal)

Sample Scientific Name Common Name Local Name Family Order Class Code S22 Argyrops filamentosus Soldierbream Nga-se-ooe Sparidae Perciformes Actinopterygii S23 Pomadasys argenteus Lined silver grunter Nga-gone Pomadasys Perciformes Teleostomi S24 Serranus uscoguttatus Brown marbled reef cod Nga-tauktu-mei Serranidae Perciformes Actinopterygii S25 Mugil dussumieri Dussumier's mullet Nga-kin-kyin Mugilidae Mugiliformes Teleostomi S26 Lobotes surinamensis Brown triple tail Kyauk-ngapyae Lobotidae Perciformes Actinopterygii S27 Cromileptes altivelis Humpback grouper Nga-tauktu-phyu Serranidae Perciformes Actinopterygii S28 Loligo duvauceli Indian squid Yae-kyet/Kin-mon - - - S29 Anchoviella tri - Nga-phyu-lone Engraulidae Clupeiformes Actinopterygii S30 Stolephorus indicus Indian anchovy Nga-ni-tu Engraulidae Clupeiformes Actinopterygii S31 Sardinella lonigiceps Nga-kone-nyo Clupeidae Clupeiformes Teleostomi 74

Indian oilsardine S32 Metapenaeus dobsoni Golden shrimp Shwe-puzun - - - S33 Atrina pectinata Penshall Ma-yut(khayu) - - -

CHAPTER V

DISCUSSION AND CONCLUSION

Myanmar, a member of ASEAN has a long coastline of 2832 kilometers. It can be divided into three coastal regions: the Rakhine Coastal Region, the Ayeyarwaddy Delta and the Gulf of Moattama (Martaban) Coastal Region (from the Mawtin Point to the Gulf of Mottama, about 460 km in length) and Thanintharyi Coastal Region (from the Gulf of Mottama to the mouth of the Pakchan River, about 1200 km in length) in the Bay of Bangle and in the Anderman Sea. The total area of swamps along the coast is about 0.5 million hetares which provides a very good base for the development of aquaculture including shrimp and prawn culture. According to surveys in marine fisheries, the Maximum Sustainable Yield of Myanmar is estimated at about 1.05 million tons per year. Inland water bodies such as natural lakes, reservoirs, river systems etc. cover an area of about 8.2 million hetares. At present, flood fisheries produce about seventy thousand tons of fish and prawn annually.

5.1 Discussion Among the twenty-one marine fish samples (S01 to S21) collected from Thandwe Region, marine fish samples S01 to S13 are marine fish with scale and S14 to S21 are marine fish without scale. Among the twelve marine fish samples (S22 to S33) collected from Kyauk Gyi Region, marine fish samples S22 to S27 are marine fish with scale, S28 to S31 are marine fish without scale and S32 and S33 are shellfish. The following figures show the comparison of concentrations of heavy metals contained in the marine fish samples. 81

7000

6000

5000

4000

3000

2000

1000

0

S02 S03 S04 S05 S06 S07 S08 S09 S10 S11 S12 S13 S14 S15 S16 S17 S18 S19 S20 S21 S22 S23 S24 S25 S26 S27 S28 S29 S30 S31 S32 S33 S01 Thandwe Region Kyauk Gyi Region With scale With scale Without scale Without scale Shellfish

Fig 5.1 Comparison of concentrations of heavy metal Phosphorous (P) contained in the marine fish samples

82

9000

8000

7000

6000

5000

4000

3000

2000 1000

0

S02 S03 S04 S05 S06 S07 S08 S09 S10 S11 S12 S13 S14 S15 S16 S17 S18 S19 S20 S21 S22 S23 S24 S25 S26 S27 S28 S29 S30 S31 S32 S33 S01

Thandwe Region Kyauk Gyi Region With scale With scale Without scale Without scale Shellfish Fig 5.2 Comparison of concentrations of heavy metal Sulfur (S) contained in the marine fish samples

83

30000

25000

20000

15000

10000

5000

0

S01 S02 S03 S04 S05 S06 S07 S08 S09 S10 S11 S12 S13 S14 S15 S16 S17 S18 S19 S20 S21 S22 S23 S24 S25 S26 S27 S28 S29 S30 S31 S32 S33

Thandwe Region Kyauk Gyi Region With scale With scale Without scale Without scale Shellfish

Fig 5.3 Comparison of concentrations of heavy metal Chlorine (Cl) contained in the marine fish samples

84

16000

14000 12000 10000

8000

6000

4000

2000

0

S02 S03 S04 S05 S06 S07 S08 S09 S10 S11 S12 S13 S14 S15 S16 S17 S18 S19 S20 S21 S22 S23 S24 S25 S26 S27 S28 S29 S30 S31 S32 S33 S01

Thandwe Region Kyauk Gyi Region With scale With scale Without scale Without scale Shellfish

Fig 5.4 Comparison of concentrations of heavy metal Potassium (K) contained in the marine fish samples

85

35000

30000

25000

20000

15000

10000

5000

0

S01 S02 S03 S04 S05 S06 S07 S08 S09 S10 S11 S12 S13 S14 S15 S16 S17 S18 S19 S20 S21 S22 S23 S24 S25 S26 S27 S28 S29 S30 S31 S32 S33

Thandwe Region Kyauk Gyi Region With scale With scale Without scale Without scale Shellfish

Fig 5.5 Comparison of concentrations of heavy metal Calcium (Ca) contained in the marine fish samples

86

1200

1000

800

600

400

200

0

S01 S02 S03 S04 S05 S06 S07 S08 S09 S10 S11 S12 S13 S14 S15 S16 S17 S18 S19 S20 S21 S22 S23 S24 S25 S26 S27 S28 S29 S30 S31 S32 S33

Thandwe Region Kyauk Gyi Region With scale With scale Without scale Without scale Shellfish

Fig 5.6 Comparison of concentrations of heavy metal Scandium (Sc) contained in the marine fish samples

87

450

400

350

300

250

200

150

100

50

0

S01 S02 S03 S04 S05 S06 S07 S08 S09 S10 S11 S12 S13 S14 S15 S16 S17 S18 S19 S20 S21 S22 S23 S24 S25 S26 S27 S28 S29 S30 S31 S32 S33

Thandwe Region Kyauk Gyi Region With scale With scale Without scale Without scale Shellfish

Fig 5.7 Comparison of concentrations of heavy metal Manganese (Mn) contained in the marine fish samples

88

1800

1600

1400

1200

1000

800

600

400

200

0

S01 S02 S03 S04 S05 S06 S07 S08 S09 S10 S11 S12 S13 S14 S15 S16 S17 S18 S19 S20 S21 S22 S23 S24 S25 S26 S27 S28 S29 S30 S31 S32 S33

Thandwe Region Kyauk Gyi Region With scale With scale Without scale Without scale Shellfish

Fig 5.8 Comparison of concentrations of heavy metal Iron (Fe) contained in the marine fish samples

89

350

300

250

200

150

100

50

0

S01 S02 S03 S04 S05 S06 S07 S08 S09 S10 S11 S12 S13 S14 S15 S16 S17 S18 S19 S20 S21 S22 S23 S24 S25 S26 S27 S28 S29 S30 S31 S32 S33

Thandwe Region Kyauk Gyi Region With scale With scale Without scale Without scale Shellfish

Fig 5.9 Comparison of concentrations of heavy metal Nickel (Ni) contained in the marine fish samples

90

250

200

150

100

50

0

S01 S02 S03 S04 S05 S06 S07 S08 S09 S10 S11 S12 S13 S14 S15 S16 S17 S18 S19 S20 S21 S22 S23 S24 S25 S26 S27 S28 S29 S30 S31 S32 S33

Thandwe Region Kyauk Gyi Region With scale With scale Without scale Without scale Shellfish

Fig 5.10 Comparison of concentrations of heavy metal Copper (Cu) contained in the marine fish samples

91

600

500

400

300

200

100

0

S01 S02 S03 S04 S05 S06 S07 S08 S09 S10 S11 S12 S13 S14 S15 S16 S17 S18 S19 S20 S21 S22 S23 S24 S25 S26 S27 S28 S29 S30 S31 S32 S33

Thandwe Region Kyauk Gyi Region With scale With scale Without scale Without scale Shellfish

Fig 5.11 Comparison of concentrations of heavy metal Zinc (Zn) contained in the marine fish samples

92

140

120

100

80

60

40

20

0

S01 S02 S03 S04 S05 S06 S07 S08 S09 S10 S11 S12 S13 S14 S15 S16 S17 S18 S19 S20 S21 S22 S23 S24 S25 S26 S27 S28 S29 S30 S31 S32 S33

Thandwe Region Kyauk Gyi Region With scale With scale Without scale Without scale Shellfish

Fig 5.12 Comparison of concentrations of heavy metal Bromine (Br) contained in the marine fish samples

93

250

200

150

100

50

0

S01 S02 S03 S04 S05 S06 S07 S08 S09 S10 S11 S12 S13 S14 S15 S16 S17 S18 S19 S20 S21 S22 S23 S24 S25 S26 S27 S28 S29 S30 S31 S32 S33

Thandwe Region Kyauk Gyi Region With scale With scale Without scale Without scale Shellfish

Fig 5.13 Comparison of concentrations of heavy metal Strontium (Sr) contained in the marine fish samples

94

16

14

12

10

8

6

4

2

0

S01 S02 S03 S04 S05 S06 S07 S08 S09 S10 S11 S12 S13 S14 S15 S16 S17 S18 S19 S20 S21 S22 S23 S24 S25 S26 S27 S28 S29 S30 S31 S32 S33

Thandwe Region Kyauk Gyi Region With scale With scale Without scale Without scale Shellfish

Fig 5.14 Comparison of concentrations of heavy metal Zirconium (Zr) contained in the marine fish samples

95

90

80

70

60

50

40

30

20

10

0

S01 S02 S03 S04 S05 S06 S07 S08 S09 S10 S11 S12 S13 S14 S15 S16 S17 S18 S19 S20 S21 S22 S23 S24 S25 S26 S27 S28 S29 S30 S31 S32 S33

Thandwe Region Kyauk Gyi Region With scale With scale Without scale Without scale Shellfish

Fig 5.15 Comparison of concentrations of heavy metal Platinum (Pt) contained in the marine fish samples

96

According to Fig 5.1, only the marine fish samples S26 (with scale), S30 (without scale), S32 (Shellfish) collected from Kyauk Gyi Region have found the concentration of heavy element Phosphorous (P) and all the other marine fish samples of Thandwe Region and Kyauk Gyi Region have not found significant concentration of heavy element Phosphorous (P). Among the three marine fish samples, S26, S30, S32, the marine fish sample S30 (without scale), Nga-ni-tu (Indian ahchovy), collected from Kyauk Gyi region has the highest concentration of heavy element Phosphorous (P).

According to Fig 5.2, all the marine fish samples collected from Thandwe Region and Kyauk Gyi Region have found the concentration of heavy element Sulfur (S) except the marine fish sample S08 (with scale), S13 (with scale) and S14 (without scale) collected from Thandwe Region.. Among the marine fish samples, the marine fish sample S26 (with scale), Kyauk-ngapyae (Brown triple tail), collected from Kyauk Gyi Region has the highest concentration of heavy element Sulfur (S).

According to Fig 5.3, only the marine fish samples S03, S08, S10, S13, S18, S20, S21, collected from Thandwe Region and S22, S23, S29, S30 and S32, collected from Kyauk Gyi Region have found the concentration of heavy element Chlorine (Cl) and all the other marine fish samples of Thandwe Region and Kyauk Gyi Region have not found significant concentration of heavy element Chlorine (Cl). Among the marine fish samples, the marine fish sample S23 (with scale), Nga-gone (Lined silver grunter), collected from Kyauk Gyi Region has the highest concentration of heavy element Chlorine (Cl).

According to Fig 5.4, all the marine fish samples collected from Thandwe Region and that of Kyauk Gyi Region have found the concentration of heavy element Potassium (K). Among the marine fish samples, the marine fish sample S29 (without scale), Nga-phyu-lone, has the highest concentration of heavy element Potassium (K) and the marine fish samples S32 (shellfish), 97

Shwe-puzun, collected from Kyauk Gyi Region has the lowest concentration of heavy element Potassium (K).

According to Fig 5.5, all the marine fish samples collected from Thandwe Region and that of Kyauk Gyi Region have found the concentration of heavy element Calcium (Ca). Among the marine fish samples, the marine fish sample S12 (with scale), Nga-tauk-tu (Humpback seabass), collected from Thandwe Region has the highest concentration of heavy element Calcium (Ca) and the marine fish sample S28 (without scale), Yae-kyet/Kin-mon (Indian squid), collected from Kyauk Gyi Region has the lowest concentration of heavy element Calcium (Ca).

According to Fig 5.6, only the marine fish samples S03, S10, S13, S18, S19, collected from Thandwe Region, and S22, S23, S25, S26, S29, S30, collected from Kyauk Gyi Region have found the concentration of heavy element Scandium (Sc) and all the other marine fish samples of Thandwe Region and Kyauk Gyi Region had not found significant concentration of heavy element Scandium (Sc). Among the marine fish samples, the marine fish sample S13 (with scale), Nga-se-ooe (Whipfin silver biddy), collected from Thandwe Region has the highest concentration of heavy element Scandium (Sc).

According to Fig 5.7,only the marine fish sample S33 (shellfish), Mayut (Khayu) collected from Kyauk Gyi Region has found the concentration of heavy element Manganese (Mn) and all the other marine fish samples of Thandwe Region and Kyauk Gyi Region had not found significant concentration of heavy element Manganese (Mn).

According to Fig 5.8, all the marine fish samples collected from Thandwe Region and that of Kyauk Gyi Region have found the concentration of heavy element Iron (Fe). Among the marine fish samples, the marine fish sample S33 (shellfish), Ma-yut (Khayu), collected from Kyauk Gyi Region has the highest concentration of heavy element Iron (Fe) and the marine fish sample S29 98

(without scale), Nga-Phyu-lone, collected from Kyauk Gyi Region has the lowest concentration of heavy element Iron (Fe).

According to Fig 5.9, only the marine fish samples S03, S04, S08, collected from Thandwe Region and S27, S29, collected from Kyauk Gyi Region have found the concentration of heavy element Nickel (Ni) and all the other marine fish samples of Thandwe Region and Kyauk Gyi Region have not found significant concentration of heavy element Nickel (Ni). Among the marine fish samples, the marine fish sample S08 (with scale), Nga-sa-lone (Blind fish), collected from Thandwe Region has the highest concentration of heavy element Nickel (Ni).

According to Fig 5.10, only the marine fish samples S04, S09, S17, S19, collected from Thandwe Region and S27, S28, S31, S32, S33, collected from Kyauk Gyi Region have found the concentration of heavy element Copper (Cu) and all the other marine fish samples of Thandwe Region and Kyauk Gyi Region have not found significant concentration of heavy element Copper (Cu). Among the marine fish samples, the marine fish sample S33 (shellfish), Ma-yut (khayu), (Penshall), collected from Kyauk Gyi Region has the highest concentration of heavy element Copper (Cu).

According to Fig 5.11, all the marine fish samples collected from Thandwe Region and that of Kyauk Gyi Region have found the concentration of heavy element Zinc (Zn) except the marine fish samples, S02, S03, S06,S10, S12, S20, S21, collected from Thandwe Region and S23, S25, S26, S29, collected from Kyauk Gyi Region. Among the marine fish samples, the marine fish sample S18 (without scale), Nga-pu-tin (Estiarome blow fish), collected from Thandwe Region has the highest concentration of heavy element Zinc (Zn).

According to Fig 5.12, only the marine fish samples S01, S03, S04, S09, collected from Thandwe Region and all the marine fish sample collected from Kyauk Gyi Region except S22 and S29 have found the concentration of heavy 99 element Bromine (Br) and all the other marine fish samples of Thandwe Region and Kyauk Gyi Region have not found significant concentration of heavy element Bromine (Br). Among the marine fish samples, the marine fish sample S33 (shellfish), Ma-yut (khayu), (Penshall), collected from Kyauk Gyi Region has the highest concentration of heavy element Bromine (Br).

According to Fig 5.13, all the marine fish samples collected from Thandwe Region and that of the Kyauk Gyi Region have found the concentration of heavy element Strontium (Sr). Among the marine fish samples, the marine fish sample S08 (with scale), Nga-sa-lone, (Blind fish), collected from Thandwe Region has the highest concentration of heavy element Strontium (Sr) and the marine fish sample S28 (without scale), Yae-kyet/ kin-mon, (Indian squid), collected from Kyauk Gyi Region has the lowest concentration of heavy element Strontium (Sr).

According to Fig 5.14, only the marine fish samples S08 and S13 collected from Thandwe Region have found the concentration of heavy element Zirconium (Zr) and all the other marine fish samples of Thandwe Region and Kyauk Gyi Region have not found significant concentration of heavy element Zirconium (Zr). Among the three marine fish samples, the marine fish sample S08 (with scale), Nga-sa-lone (Blind fish), has the highest concentration of heavy element Zirconium (Zr).

According to Fig 5.15, only the marine fish sample S18 (without scale),Nga- pu-tin (Estiarome blow fish), collected from Thandwe Region has found the concentration of heavy element Platinum (Pt) and all the others marine fish samples of Thandwe Region and Kyauk Gyi Region have not found significant concentration of heavy element Platinum (Pt).

100

5.2 Conclusion Using the concentrations of heavy elements contained in the marine fish samples (Table 4.1), the average concentrations of elements contained in the each type of marine fish samples and the average concentrations of elements contained in the marine fish samples of each location are calculated and these data are shown in Table 5.1.

Table 5.1 Average concentrations (ppm) of heavy elements contained in the marine fish samples

Thandwe Region Kyauk Gyi Region Element With Without With Without Average Shellfish Average scale scale scale scale P 0 0 0 950 1667 2333 1650 S 4557 4509 4533 6878 5916 4983 5926 Cl 2023 3105 2564 5566 5319 8890 6592 K 8383 9429 8906 9246 11531 5659 8812 Ca 14105 11639 12872 13772 7197 11475 10815 Sc 169 127 148 476 205 0 227 Mn 0 0 0 0 0 206 69 Fe 431 339 385 441 167 1058 555 Ni 52 0 26 15 51 0 22 Cu 14 26 20 12 41 193 82 Zn 61 124 93 47 99 190 112 Br 14 0 7 62 39 107 69 Sr 104 97 101 103 56 99 86 Zr 2 0 1 0 0 0 0 Pt 0 11 6 0 0 0 0

101

100000 With scale (Thandw e) Without scale (Thandw e) With scale (Kyauk Gyi) 10000 Without scale (Kyauk Gyi) Shellfish (Kyauk Gyi) Thandw e Kyauk Gyi 1000

100 Concentration (ppm) Concentration

10

1 P S Cl K Ca Sc Mn Fe Element

1000 With scale (Thandw e) Without scale (Thandw e) With scale (Kyauk Gyi) Without scale (Kyauk Gyi) Shellfish (Kyauk Gyi) Thandw e 100 Kyauk Gyi

10 Concentration (ppm) Concentration

1 Ni Cu Zn Br Sr Zr Pt Element

Fig 5.15 Comparison of average concentrations of elements contained in each type of marine fish sample and that of each location

According to above figures and discussions, the marine fish samples with scale collected from Thandwe Region have higher concentration of heavy elements Ca, Ni, Sr and Zr than that of the other marine fish samples. The marine fish samples with scale collected from Kyauk Gyi Region have higher concentration of heavy elements S, K and Sc than that of the other marine fish samples. The marine fish samples (shellfish) collected from Kyauk Gyi Region have higher concentration of heavy elements P, Cl, Mn, Fe, Cu, Zn and Br than that of the other marine fish samples. 102

Generally, it can be said that, marine fish samples collected from Thandwe Region have higher concentration of heavy elements Ca, Ni, Sr and Pt and marine fish samples collected from Kyauk Gyi Region have higher concentration of heavy elements P, S, Cl, Sc, Mn, Fe, Cu, Zn and Br. So that, marine fish samples collected from Kyauk Gyi Region have higher concentrations of heavy elements than that of the Thandwe Region. Therefore, it can be said that sea water of Kyauk Gyi Region have more heavy elements contents than the sea water of Thandwe Region.

Although some heavy metals are important and essential trace elements, most can be, at high concentrations, such as those found in many environments today, most can be toxic to all branches of life, by forming complex compounds within the cell. Cells need to maintain certain cytoplasmic concentrations of these metals if they are to meet physiological requirements. To this end, microorganisms use a number of mechanisms to maintain the correct equilibrium, including the uptake, chelation and extrusion of metals.

Heavy metals are increasingly found our environment due to natural and industrial processes. Also, since the oxidation state of a metal ion may determine its solubility. There is also evidence of a correlation between tolerance to heavy metals and antibiotic resistance, a global problem currently threatening the treatment of infections in plants, animals, and humans. Overall, it is most important to remember that what we put into the environment can have many effects, not just on humans, but also on the environment and on the microbial community on which all other life depends.

Therefore, it is very important to know how much that heavy metals contents in our environments and energy dispersive x-ray fluorescence (EDXRF) technique is one of the beneficial technique to determine the level of heavy metals of our surrounding. 103

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