!

!!!!

!!!

LIVING INDICATORS OF!SEA POLLUTION: !

MUSSELS!!!

Candidate’s Name:!

Mert İNAN!

Candidate Session Number:!

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Word Count: 4475!

Advisor:!

Demet İZGÜ

T. E . D A n k a r a F o u n d a t i o n P r i v a t e H i g h S c h o o l • A n k a r a • 2 0 1 4

BIOLOGY EXTENDED ESSAY

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CONTENTSCONTENTS! i!

ACKNOWLEDGEMENT! iii!

1. Introduction! iv!

2. Hypothesis! vii!

Null Hypothesis! vii!

3. Method Development! viii!

3.1 MATERIALS & APPARATUS! xiv!

3.2 METHOD! xv!

5. Data Collection & Processing! xviii!

4. Conclusion & Evaluation! xx!

6. References! xxii!

Appendix A! xxiii!

Background Information About Mussels! xxiii!

Appendix B! xxiv!

Background Information About Dioxin! xxiv!

Appendix C! xxv!

Appendix D! xxvii!

Appendix E! xxviii! L i v i n g I n d i c a t o r s o f S e a P o l l u t i o n : M u s s e l s ! M e r t İN A N ! !

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!!!!"We are just an advanced breed of monkeys on a minor planet of a very average star. But we can understand the Universe. That makes us something very special."!

-Stephen Hawking!

!

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Figure 1: The Mytilus galloprovincialis mussel Source: http://www.productosecologicossininterme-diarios.es/Mussels-from-Galicia-Mytilus-galloprovincialis

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ACKNOWLEDGEMENT!

!Above all else , I would like to thank everybody that have aided or involved in this project. It was a pleasure to work in a scientific environment and collaborate with others to learn and try new things. Thank you a lot;!

• My dear biology teacher, Demet İZGÜ, who helped me on the way to write this ex-tended essay,!

• My father and my mother who helped me while researching for the topic,!

• My sister who was a great companion at all times,!

• My friends who elaborately listened and advised to my plans for this essay,!

• And lastly, to Mr. Hayrettin DUYUM, the chief of the Ankara Nutrient Analysis and Dioxin Measurement Laboratories, for easing my way at the laboratory...!

I hope that this project will enlighten people about their opinions about global sea pol-lution and how biotic indices can be used to measure these kinds of contaminations in-directly... Always remembering; prevention is better than cure.

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1. Introduction!! As Stephen Hawking said that "We are in danger of destroying ourselves by our

greed and stupidity. We cannot remain looking inwards at ourselves on a small and in-

creasingly polluted and overcrowded planet." he demonstrated the greedy human being

caring nothing but himself. Humans have always seen themselves above the nature,

however, in order to live in peace and prosper one should be part of the nature.!

! This greed and irresponsibleness of humans have led to a lot of catastrophes and

triggered the natural disasters to occur vastly and more numerously than they should

be in normal circumstances. But, it should not be forgotten that homo sapiens are always

affected adversely by doing these kinds of actions that contaminate the fabric of nature.!

! On the top of the list of unfavourable course of acts, there lies the pollution of the

seas. As they are the most valuable, delicate and fragile and mysterious parts of the

world, seas are very important ecosystems for us and other living organisms to thrive. I

was really concerned with this issue of global sea pollution. Especially, because I really

care about the environment protection, this issue had a personal involvement. One day,

while I was swimming in a sea I saw a lot of irrelevant materials inside it including

metal pins, glass bottles, organic wastes etc., however, I knew that those were the tip of

an iceberg, and that day was my first encounter with the visible sea pollution. I was a

little confused about the measurement of the pollution levels of a water body as I saw

news about the clearing of the Marmara Sea of any solid waste that polluted it. But

what was the meaning of "polluted"? Did it included only solid wastes?!

! After some research on the internet, I understood that the sea pollution wasn't

concerned only with the solid wastes that humans produced and threw away into the

depths of the seas, there were a lot of chemical compounds that were invisible to the

unaided eye. So did this meant that the sea pollution was directly proportional with the

invisible chemical compounds? I learned that it was...!L i v i n g I n d i c a t o r s o f S e a P o l l u t i o n : M u s s e l s ! M e r t İN A N ! !

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! To guarantee a "green" world for the future generations, we had to start from

somewhere and boost the public awareness about the global pollutions. One of these

were sea pollutions. I really wanted to measure them because of the conflicts mentioned

above. But how could I measure an invisible compound to the eye and how could I link

them with the pollution level?!

! First of all, I needed to clear my mind off these questions by finding them the

suitable answers. To find the answers, I have done more research and found different

answers: !

1. The meaning of pollution was the increased enrichment of a body of water by the

input of chemical compounds, !

2. It turned out that there were a lot of methods that can be used to measure the pollu-

tion of sea levels that will be criticized in the Method Development part of this arti-

cle.!

! Secondly, I had to precisely decide to my investigation topic. So, I had to narrow

down from sea pollution to a specific research question. As a result I decided to follow

these simple steps;!

1. Search for equipments that could help to observe quantitatively the pollution: I

found a lot of expensive methods involving a lot of procedure. And, one day, while I

was skimming my ESS (Environmental Systems and Societies) textbook, I found that

biotic indicator species could be used also to calculate the pollution levels. This was

the perfect fit for my experiment because they were cheap, practical and easy to col-

lect.!

2. Choose the indicator species that was relevant to my investigation: Mussels were

the best choice for this article thanks to their anatomy. I had also stumbled upon

some information regarding the mussels that they were the best way to observe the

sea pollution.!

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3. Choose the contaminant: Dioxin was the best polluting compound that showed ac-

curately the pollution of seas and because it can bioaccumulate in the mussels and

other organisms this was the best contaminant for this article. Further information

can be found on the following pages; Method Development and background infor-

mation about dioxin on Appendix B.!

! As a result, after following these steps I have specified my investigation. So in

order to better illustrate the picture of this article I would like to give some basic infor-

mation that I have found in the steps 1, 2 and 3.!

! To begin with, mussels are the main focus of this article because of their general

property of filtering the water. They are one of the most important indicator species of

sea pollution because of this property. They do this in order to prey on some planktons,

however, because they do not have an adapted mechanism to form a barrier to the en-

trance of toxic material, they also tend to filter the polluting compounds from the body

of water. As a result, they become the bank for the chemical polluting compounds in a

river. Thus, qualitatively investigating the toxic material inside them can give a wide

perspective about the levels of pollution in the seas. Mytilus edulis and Mytilus gallo-

provincialis are the widely known species of these pollution filters so one of these

species will be used as the indicator in this article. Hence, this article concentrates on the

mussels as biotic indicators of sea pollution. More information can be found on Appen-

dix A.!

! Furthermore, in order to relate a proportionality between the sea pollution and

the polluting compound I needed to choose an independent variable as a contaminant

called dioxin. Dioxin is one of the most toxic polluting organic compounds that is hard-

ly acknowledged by the people of the world. It is created as a by-product of some series

of reactions in the incinerators, and when the factories disposes these side products into

the seas they become invisibly polluted by theses persistent organic compounds. !

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! When a mussel filters out the water, it accidentally tarps dioxin in it and once the

compound enters its body there is nearly no way to get away from it and it starts to

bioaccumulate inside the mussel. So this type of compound is the most suitable one for

this essay because of this reason.!

! Dioxin can also cause serious diseases when it enters the human tissue, hence it

becomes indubitably important to calculate its level in a body of water to know the

probability of getting diseases caused by dioxin from either eating a mussel or any other

living organism inside the sea. As a result, I have chosen dioxin as my indicating cont-

aminant for the sea pollution. More detailed information concerning the dioxin, its gen-

eral properties, its detriments and bioaccumulation of it in Appendix B.!

! After all of this information, I think that one can have the adequate information

to dive deeper and create a specific question concerning the overall investigation. I will

be continuing to give more information about dioxin, mussels, measuring methods and

other important parts in the following pages...!

! As I have stated a quote from Stephen Hawking, I start to understand it better

now, because I saw that we love to create immense wastes but still we do not know how

to dispose them neatly. In consequence, every passing day adds another contaminating

particle into the very seas that we should be careful of. I had decided to investigate the

measurement of sea pollution at the beginning of this article. And, in this investigation,

I have focused on seas because they were the bodies of water that involved loads of our

daily activities and always contained intriguing beauties that may never be understood

by the human mind if we can protect them by any means there would be a more green-

aware communities in the world that would change the fate of the human kind.

! !

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! All in all, I have specified my investigation step-by-step by choosing the sea pol-

lution, then Turkish seas, then mussels and then dioxin at Tuzla, Istanbul, Amasra and

Izmir and created this research question:!

!Research Question!

! How does the values of the ratio of the mass of dioxin (an organic compound) to

the wet mass of mussels -measured in pgTEQ/gwet- changes when the habitat of

Mytilus galloprovincialis mussel (either Black sea, Aegean sea or Marmara sea)

changes, by calculating with DR CALUX (abbreviation for Dioxin Responsive Chemi-

cal-Activated Luciferase gene eXpression) method under nearly the same environmen-

tal conditions like weather, season, climatic conditions, distance to the lakeshore, depth

of the population?

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2. Hypothesis!! My research question was to investigate the amount of dioxin found in the tis-

sues of Mytilus galloprovincialis in different habitats of Black Sea, Aegean Sea and

Marmara Sea. This amount can depend on many different variables but the main reason

is by increased activity in the factories. !

! Over 220 million tons of plastic are produced each year according to the United

Nations facts and figures on marine pollution. However, the disposal of the not-useful

ones is a prime concern. They are tried to be destructed by burning generally. However,

this brings the issue of an organic side-product, dioxin, which triggers cancer mecha-

nisms in human body cells. Although, this process is not finished here, because of com-

panies that do not obey the rules of disposal because of economic reasons, dioxins end

up in seas and oceans. This results in an overall pollution of the marine ecosystem and

its components like the living beings in different trophic levels and also the human be-

ing at the top trophic level as the top predator.!

! In one of the food webs of the marine ecosystem, an animal called mussel exists,

and this mollusk is known for its capacity to filter the water of the marine ecosystem.

Because of this “dirty” job, it also collects some unneeded compounds like dioxin. In

addition to that, mussel accidentally and cumulatively holds this compound in its cells,

and when a predator preys on this animal, it directly gets the compound. !

! Because of this, mussels can be used as biotic indicators for the sea pollution, this

type of use is already present for Mytilus edulis (Blue Mussels) in the paper of Miyata

et al. that states as follows: Blue mussels accumulate a lot of polluting chemical com-

pounds and because of this reason, they are found to be the biological indicators of sea

pollution. The pollution of coasts are generally caused by population density and in-

dustrialization. (Miyata et al., 1987)!

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! As a result of these facts, it is important to know the outcomes of the sea pollu-

tion and observe the changes in the marine pollution, itself. This article acknowledges

these important facts and presents a hypothesis as follows: The fluctuations in the level

of pollution in marine ecosystems in Black sea, Aegean sea and Marmara sea containing

the population of Mytilus galloprovincialis are directly proportional with the amount

ratio of Dioxin compound that is present inside the mussels.!

Null Hypothesis!

! The levels of sea pollution in Black sea, Marmara sea and Aegean sea has no rel-

evance with the concept of dioxin and dioxin-like compounds inside the local popula-

tions of Mytilus galloprovincialis.

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!3. Method Development!

! While I was wandering around the seashore ad collecting seashells, I wondered

whether there was a reason for their shape. What were the causes of the patterns on

these shells? Then I thought that it was related with the average concentration of the

compounds in the sea: the more inorganic compound there is the more patterns are

present. After that, I thought of the sea pollution, and its possible effects on the patterns

of the shells of mussels. I and my family started to pick some seashells to observe the

patterns more clearly. Then we realized that there was no some patterns present, as a

result we concluded that the patterns were related to some causes other than the sea

pollution or inorganic compound concentration. Because they should have been the

same if they were related to those conditions. Therefore I needed to change my focus

onto another condition on mussels. I decided to keen on sea pollution because I was

pretty knowledgeable about those issues. After some research, I found that the real mys-

tery was not on the shells but the inside of the mussel. After receiving this kind of in-

formation, indeed to shift my observations to a whole new part: the inside of the mus-

sel.!

! One lucky day, I stumbled upon an internet article concerning the richness of the

heavy metals inside the mussels and their toxic effects on human tissues. Then I instant-

ly created a relation: if there is a high level of heavy metals in the seas, the seas should

be regarded as polluted and if the seas are highly polluted then the mussels - because of

their anatomy- should be polluted as well. Regarding this statement as a milestone I

started to research more about the mussels and the measurement of heavy metal pollu-

tion. Then, I have found a lot of research papers from different countries that aimed to

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find a relation between the sea pollution and mussels via calculating the amount of

heavy metals inside the mussels.!

! While my research was going on, my father found a laboratory that specialized at

heavy metal extraction from foods of animal and plant origin. When we went there to

have a small talk about my research, the coordinator of the laboratory suggested that I

should focus on an organic compound called dioxin to augment the originality of my

research. Because the same laboratory had a subgroup for experimenting on dioxin,

called Ankara Nutrient Analysis and Dioxin Measurement Laboratories, I thought that I

can finally measure the pollution of our seas and relate them with the mussels in there.!

Choosing the Indicator:!

Next step was to choose the indicator species. There were options like seaweed, algae,

fish, flea, Tubifex worms, microorganisms and mussels. However, catching the right fish

was a hardwork, there were too little contaminating compound found in the seaweed

and algae, there weren't the same species of fleas in each sea and the tests were to hard

for the microorganisms. As a result, best indicator was the mussels because they were

easy to be gathered, they contained a lot of contaminating compound because of their

anatomy, and they are regarded as delicacy food in a lot of countries thus can contain

unhealthy by-products.!

Choosing the Mussels:!

! There were plenty of species of mussels both in salty water and freshwater in Tur -

key. But I wanted to focus on the most abundant species in order to get the most data

available. I found Mytilus edulis, Mytilus galloprovincialis that were endemic to the

Turkish seas. I chose these species because the main aim was to see the pollution of seas

not the pollution of freshwater. Also these mussels are the ones that most of the local

people eat. In order to choose one of them, I have looked at their distributions in the

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seas.To my surprise, although Mytilus edulis was present in Mediterranean sea, it had

no connection with Turkey (Figure 4). As a result, the experiment started with Mytilus

galloprovincialis. (Figure 5) This was a wise choice because Food and Agriculture Or-

ganization of the United Nations mentions that " especial program is needed for the

maintenance of water quality, because an increase in pollution of the water could not

only increase the prevalence of parasites but also the levels of toxic substances, such as

heavy metals." (www.fao.org/fishery/culturedspecies/Mytilus_galloprovincialis/en).

After the collection of specimens I saw that there were three different sizes of the mus-

sels (Figure 6). For the sake of this experiment, small and intermediate mussels are dis-

carded. Figures can be found on Appendix D.!

Choosing the Contaminants:!

! While I was looking for global contaminants I found these; heavy metals

like mercury, lead etc., plastic substances, eutrophication-causing agents like ni-

trates and phosphates from detergents and fertilizers, bioaccumulating sub-

stances like DDT, Dioxin and other forms of organic pollutant compounds. I

needed to eliminate them, so I got some reasons to demote the substances to one.

Here they are;!• There were already some papers that were about mussels and heavy metals,!

• Plastic substances were irrelevant with mussels because they either drifted on the sur-

face of water or they were present in great volumes under the sea.!

• Although Turkey was one of the producers and appliers of DDT, it would take years

for a DDT compound to get inside to a mussel in small amounts because of the posi-

tions of agricultural fields according to the seas. The fields are too distant from the

seas.!

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• Small bodies of water are easy to eutrophicate but seas have rather small possibilities

of eutrophication, my choices didn't had eutrophication, thus looking at nitrate and

phosphate levels would not be meaningful.!

! As a result, because it has never been investigated before in Turkish seas and is a

clear indicator, dioxin was the best choice from polluting compounds.!

Choosing the Specimen Locations:!

! I would really like to measure the pollution of seas of the whole world but that

was a little improbable. So, I chose the ones near my country of Turkey. These were the

seas surrounding the country; Black Sea, Mediterranean Sea, Aegean Sea and Marmara

Sea. Some factors that helped my decision;!

• All seas were closed and they had merely no connection with an ocean that made

them the best place for accumulated contaminants.!

• Worldwide-going ships passed from these seas that showed high trading traffic.!

• They were the closest bodies of water to the Chernobyl disaster.!

! When I have asked to local fish traders about the whereabouts of the Mytilus gal -

loprovincialis, they replied that it was distributed in every sea except the Mediter-

ranean sea surrounding Turkey. Although I chose the seas, I didn't decided for the re-

gions that I would be taking the mussels from. I had to choose from cities of equal quali-ties to decrease the uncontrollable variables. I looked for major cities with a shore to these seas. These were Istanbul, Amasra, Izmir and Antalya. Istanbul is a big city, so I chose a region called Tuzla inside it with high factory facilities. And, when I went to these cities, they were all perfect for experiment except Antalya, because there weren't

any mussels found in there, so I excluded that city from the experiment. (Figure 7) In

addition, these places were selected because they were close to industrial organizations,

which aid the production of dioxins.!

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As a result, specimens were taken and transported with sterile and cold packages to

Ankara Nutrient Analysis and Dioxin Measurement Laboratories. The last step was to

extract the dioxins from them.!

Choosing the Method:!

! There were two alternative options called HRGC-HRMS and DR CALUX that

were used to detect the dioxin inside the tissue. The HRGC-HRMS (High Resolution

Gas Chromatography-High Resolution Mass Spectrometry) involved techniques that

were beyond the level of this essay and were too costly to use and required a lot of

equipments and a lot of calculations for Toxic Equivalency (TEQ) values. On the other

hand, DR CALUX method ( Figure 8) was both efficient and straightforward to calculate

the TEQ values. The procedure of analysis starts by growing BDS 'innovative' CALUX

cells that have been tailored to produce a radiation that can be detected by a luminome-

ter when dioxin binds to a receptor protein inside 96-well plates under standardized

conditions. Then, after sample collection, simple extraction (appendix 1) is used to ex-

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Figure 7: Habitats of taken specimens of Mytilus galloprovincialis A; Amasra. B; Tuzla, İstanbul. C; Karşıyaka, İzmir.

C

BA

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tract the dioxin. Following the clean-up procedure, the cells are exposed to the dioxin of

the mussels. Then the process (outlined in detail in the appendix 2) begins, which ends

up with the calculations of luminometer. As a result, the TEQ values of dioxin are com-

pared to the standard curve and the results are obtained.!

! All in all, this was the best method to be used in this research with its properties

like extreme sensitivity, rapidness, easy sample clean-up, small sampling size, reduced

cost and relevance to biological processes of cells to test the hypothesis. !

!

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Figure 8: This figure shows the schematic procedure of DR CALUX method.

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!3.1 MATERIALS & APPARATUS!

• 50 equally-sized (5±1 cm) Mytilus galloprovin-cialis mussels from each 3 locations!

• DR CALUX® Kit!

• 1 vial box with 49-cell partition!

• 15 vials of 60 mL!

• 5 vials of 1 mL!

• 100ml, 250ml, 500ml and 1000ml GL45 glass bottles!

• 4 x GL45 bottle caps!

• 2 litres of pure water!

• 200 mL of isopropanol!

• 800 mL of n–Hexane!

• 500 grams of 20 % acidic silica!

• 500 grams of 33 % acidic silica!

• 50 grams of sodium sulfate!

• 500 microliters of dimethlysulfoxide !

• 100 ml of phosphate buffer solution!

• 800 mg growth medium!

• 100 ml glass chromatography column!

• Nitrogen gas evaporator!

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a.

Figure 9: a, b, c: Getting ready for the exper-iments.

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• Five 96 well plates!

• Sensitive Weighing scale (Sartorius 1200)!

• Homogenizer (HST-HL1)!

• Ultrasonic water bath (ELMA Trassonic 460/H)!

• Evaporator (WT Tab40-W/Teknosem)!

• Laboratory shaker (Wise Shake SHO-2D)!

• Luminometer (Berthold/Orion II) !

• Vacuum manifold (Vakuum Brand)!

• CO₂ Incubator (Heal Force)!

• Vacuum furnace (Memmart)!

• Dessicator!

• Water bath (Nüve RT80)!

• Opposite phase microscope (Leica)

!3.2 METHOD!

This method requires a lot of attention and should be done under the supervision of a professional authority.!

On the field;!

1. Locate the colony and measure its distance to the lakeshore and the depth of the colony.!

2. Note down the pH of the sea, season of the year, time of extraction of the mussels and the temperature of the sea.!

!

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I. Mussels are gathered from specimen locations and transported to the laboratory with adequate supply.!

II. 30 Mussels of each location (90 in total) are taken out of their shells and put into a homogenizer, and 9 grams (weighed by sensitive weighing scale) of homogenized product is used for each trial.!

III. In order for the maximum extraction to occur needed amounts of substances are measured using the sensitive weighing scale according to the amounts in Table 1.!

IV. Required amounts of substances are gathered except n-Hexane in a 100 mL of GL45 glass bottle to start the procedure of DR CALUX test.!

V. Bottle is sealed with a cap and is put into the laboratory shaker for 10 minutes with 200 ± 20 beats per minute setting.!

VI. Adequate amount of n-Hexane is added (with reference to Table 1) to the mixture after 10 minutes and the it's quickly sealed again.!

VII. The mixture is then put into the laboratory shaker for an hour with the same set-ting.!

VIII. After the shaking process, enough time is given for the separation of the n-hexane phase under complete seal.!

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Table 1: This table shows the needed amounts of substances for the prepa-ration of mussels for DR CALUX test

Specimen Mass of the mussel

Volume of Pure water

Volume of isopropanol

Volume of n-Hexane

Volume of the bottle

Needed amounts of substances to enter DR

CALUX test

9 gr 15 ml 15 ml 30. ml 100. ml

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IX. The hexane part is transported into a clean 60 mL vial, and this solution is put into Nitrogen gas evaporator under 45°C.!

X. Repeat the procedure from steps VI to XI once more to extract the substances ade-quately.!

XI. The specimen with extracted substances runs into the process of clean-up. This pro-cedure can be found on the Appendix E.!

XII. The remaining solution after the process of clean-up is taken into a 1 ml vial via n-hexane and the residues in the chromatography column is vaporized using the ni-trogen gas evaporator. Then the n-Hexane is also evaporated.!

XIII. The remaining part is solved with 25 microliter of dimethylsulfoxide (DMSO). And, three and ten molar dilutions are made using this solution.!

XIV. These dilutions are then transferred into 350 microliter growth media each with 5.6 microliter.!

XV. The media are shaken for ten minutes.!

XVI. DR CALUX cell kit is opened up and the cells are planted into five 96 well plates of 100 microliters together with the growth medium with the extracted and cleaned-up solution. To the same well plate blank, reference and calibration cells are also planted without the extracted solution. After all of the planting process the well plates are closed and put into the incubator with 37 C°, 5% CO2 setting.!

XVII. After the incubation, growth medium is taken away from the well plates and washed with phosphate buffer solution.!

XVIII. In order to dismember the cell membranes of the DR CALUX cells a chemical substance from the kit called "lysis mix" is added to the solution and shaken for ten minutes. 2 injectors for the luminometer from the kit containing the "glow mix" that starts the reactions and the sodium hydroxide that finishes the reactions. !

XIX.The values are read from the luminometer in pg TEQ/wet mass units.!L i v i n g I n d i c a t o r s o f S e a P o l l u t i o n : M u s s e l s ! M e r t İN A N ! !

�x x

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XX. Steps from III to XIX is repeated for other trials and other specimens.!

XXI.Results of the experiment are gathered and shown in a table and a bar graph and the significance of the experiment is evaluated using the SPSS 10.0 software ANO-VA test. !

5. Data Collection & Processing!

!!!L i v i n g I n d i c a t o r s o f S e a P o l l u t i o n : M u s s e l s ! M e r t İN A N ! !

�x x i

Raw Data Table: This table shows the data collected for the mussels gathered from Istanbul, Izmir and Amasra, there are 15 trials in total with 5 for each city. The weights of each mussel is averagely present. There aren't any uncertainty for diox-in in the mussel showed by DR CALUX TEQ.

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Grafikler !

• Grafik 1’e bakıldığında Dioksin ve benzeri kirleticilerin en fazla Tuzla, İstanbul’da

olduğu görülmektedir. Bunun nedenleri, giriş kısmında da değinildiği gibi, fab-

rikaların kimyasal atıklarını denizlere boşaltması, sanayi gruplanmanın olduğu bu

gibi bölgelerde salınan gazların hem atmosferi hem suları kirletmesi ayrıca nüfus

yoğunluğunun getirdiği kontrol dışı kirlilik gibi insan kaynaklı kimyevi madde

salınımları olabilir.!

• İstanbul’un ardından dioksin seviyesinin en yüksek çıktığı ikinci bölgemizde Karşıya-

ka, İzmir’dir. Bunun sebepleri de İstanbul’un sebepleriyle aynıdır. Fakat, popülasy-

onun ve sanayi gruplaşmanın daha az görülmesi, doğal bir toksik süzgeci olan

midyelerden de anlaşılabileceği gibi, dioksin seviyesinin İstanbul’a göre daha az çık-

masına neden olmuştur.!

• Grafik 1’de de görüldüğü üzere Amasra’nın dioksin seviyesi diğer bölgere göre daha

az çıkmıştır. Bunun başlıca nedenleri sanayi kuruluşlarının azlığı ve insan yoğun-

luğunun diğer şehirlere göre azlığı olarak düşünülebilir.!

! Bu nedenler göz önünde bulundurulduğunda dioksin seviyelerinin bu şekilde

çıkması anlam kazanmıştır ve midyelerin de içlerinde barındırdığı dioksin ve benzeri

PCBs kirleticilerine bakılarak bu organik kirleticilerin tespit edilebileceği kanıtlanmıştır.!

• Grafik 2’ye bakıldığında hem iller arası farklılık hem de midye büyüklükleri arasın-

daki farklılık görülmektedir. Midyelerin büyüklüğü yaşlarıyla orantılıdır. Buradan da

anlaşılabileceği üzere midyelerin olgunluğu arttıkça barındırdığı dioksin ve benzeri

PCB toplamı da artar. Bu da giriş bölümünde de bahsedildiği gibi, biyoakümülasy-

onu kanıtlar niteliktedir.!

!L i v i n g I n d i c a t o r s o f S e a P o l l u t i o n : M u s s e l s ! M e r t İN A N ! !

�x x i i

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4. Conclusion & Evaluation

! Son yıllarda yapılan araştırmalar dioksinin hemen her yerde bulunabileceğini

göstermiştir. Gıdalarda, havada, toprakta ve suda bulunabilen dioksinin çok az mik-

tarının bile insan sağlığına olumsuz yönde etkilediği bilinmektedir. Dioksin bu nedenle

dünyada tehlikeli, toksik kimyasal atık olarak ilk sırada yer almaktadır.

Midyeler için yaşadıkları suyu filtre ederek beslendikleri için toksik kimyasalları

ve ağır metalleri içlerinde barındırdıkları bilinmektedir. Bu çalışmada üç farklı deniz

kıyımızdan 3 farklı boyutta toplanan midyelerde toksik kimyasallar olan dioksin ve

dioksin benzeri PCBs maddelerinin analizleri yapılmıştır. Deney sonuçları doğrul-

tusunda en yüksek dioksin ve PCBs düzeyleri İstanbul, Tuzla daha sonra İzmir,

Karşıyaka’da en düşük olarakta Amasra bölgelerinden çıkarılan midyelerde bulunmuş-

tur. Dioksin ve benzeri maddelerin şehirler arasındaki farkları istatistiksel olarak da an-

lamlı bulunmuştur. Aynı paralelde küçük, orta ve büyük boy midyelerdeki dioksin ve

benzeri madde düzeylerindeki farklılığa bakıldığında küçükten büyüğe gittikçe

biyoakümülasyon sonucunda dioksin ve PCBs miktarları artmaktadır. Her bölge için bu

fark da anlamlıdır. Bu verilerin doğrultusunda dioksin düzeylerine bağlı olarak kirlilik

Tuzla, İstanbul denizinde en yüksek, Karşıyaka, İzmir denizinde orta, Amasra

denizinde en düşük düzeyde tespit edilmiştir. İleride bu teknik kullanılarak kirliliğin

artıp azaldığı midyelerde dioksin düzeyi ölçülerek gösterilebilir. !

! Japonya kıyıları (Miyata ve ark., 1987), Baltık Denizi (Malmwarm ve ark., 2005),

Kanada doğu sahilleri (Hennigar ve ark., 2001), Kore kıyıları(Khim ve ark., 2000) ve

Avustralya kıyılarında (Haynes ve ark., 1995) Mytilus edilus (mavi midye)lerde yapılan

çalışmalarda da benzer sonuçlar bulunmuştur. !

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! Veriler üzerinden değerlendirme yapıldığında İstanbul, İzmir ve Amasra’dan

toplanan midyeler arasından en az dioksin miktarı Amasra’dan 0.30 pg/TEQ yaş ağırlık

olarak ölçülmüştür. En fazla dioksin miktarı ise İstanbul’dan 1.60 pg/TEQ yaş ağırlık

bulunmuştur. Bu değerler başka ülkelerde hafif değişiklikler göstermektedir. Örnek

olarak, Avustralya’da ölçülen en az dioksin miktarı 0.12 pg/TEQ yaş ağırlık’tır. Ölçülen

en fazla dioksin miktarı ise 2.3 pg/TEQ yaş ağırlık şeklindedir. (Muller ve ark.) Bu da

İstanbul’daki kirlilik düzeyinden yüksektir. Sonuç olarak, midyelerdeki dioksin düzeyi

çevre kirliliğinin biyolojik göstergesi olarak düşünülmüştür.!

! Bu çalışma Türkiye kıyılarında kara midyelerde dioksin düzeyini ölçerek deni-

zlerdeki kirliliği objektif olarak derecelendirmiştir ve bunun değişimi hakkında da bilgi

verme potansiyeli olduğunu göstermiştir. !

! Bu sebeplerden dolayı, bu çalışmanın sürdürülmesi ve midyelerin diğer kirleti-

cilerden olan ağır metaller yönünden incelenmesi de çevre kirliliğini azaltma yönünde

büyük bir yol kat ettirecektir ve daha sağlıklı, temiz çevrede, sağlıklı nesiller olarak

yetişmemize katkı sağlayacaktır.!

!!

!

L i v i n g I n d i c a t o r s o f S e a P o l l u t i o n : M u s s e l s ! M e r t İN A N ! !�x x i v

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6. ReferencesI. Hennigar P, Chase M, Harding G, Jones SH, Sowles J; Wells PG, (2001) Dioxins/Furans and

Chlorobiphenyls in Mytilus Edulis From The Gulf of Maine, Environment Canada - At-lantic Region, 138 !

II. http://www.chm.bris.ac.uk/motm/dioxin/dioxin-hp.html!

III. http://www.productosecologicossinintermediarios.es/Mussels-from-Galicia-Mytilus-gal-loprovincialis!

IV. Khim JS, Villeneuve DL, Kannan K, Hu WY, Giesy JP, Kang SG, Song KJ, Koh CH, (2000) Instrumental and bioanalytıcal measures of persistent organochlorines in blue mussels (mytilus edulis) from Korean coastal waters. Oceanography Bölümü, Seoul Devlet Üniver-sitesi Çevresel Kirleticiler ve Toksikoloji Arşivleri, 39(3), 360-8 !

V. Muller J, Muller R, Goudkamp K, Mortimer M, (2004), Dioxins in aquatic environments in Australia, Çevre ve Miras Bölümü, Avustralya Devleti Dioksin Programı, 6(B), 45 !

VI. Malmwarm A, Zebühr Y, Jensen S, Kautsky L, Greyerz E, Nakano T, Asplund L.,(2005) Identification of polybromınated dibenzo-p-dioxins in blue mussels (Mytilus Edulis) in Baltic Sea. Çevresel Kimya Bölümü, Stockholm Üniversitesi, Environmental Science & Technology dergisi, 1;39(21), 8235-42 !

VII. Shyu TS, Lee YH, Determination of PCDDS/DFS and dioxin-like PCBs in egg and fish feed samples in Taiwan by the Dr Calux bioassay!

VIII. Suzuki K, Yoshikawa H, Nakamura M, Kawashima H, Tamada M, Masunaga S, (2007) Bioaccumulation of dioxins in terrestrial food chain: Concentrations of Dioxin Congeners and Trophic Level, Kanagawa Çevresel Araştırma Merkezi, 69, 100!

IX. Şahbaz, F., Acar, J.,(1993) Dioksin ve Dioksinin Gıdalara Bulaşma Olasılıkları. Hacettepe Üniversitesi, Mühendislik Fakültesi Gıda Mühendisliği Bölümü GIDA Dergisi, 18 (4), 243-245!

X. Şenyuva H, Gilbert J,(2010), Dioksin/PCB Analiz Gerekliliği, Sincer Dış Ticaret Dergisi, 3!

XI. Shibamoto T, Yasuhara A, Katami T., (2007), Dioxin formation from waste incineration, Rev Environ Contam Toxicol., Department of Environmental Toxicology, University of Califor-nia, Davis, CA 95616, USA, 190:1-41!

L i v i n g I n d i c a t o r s o f S e a P o l l u t i o n : M u s s e l s ! M e r t İN A N ! !�x x v

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Appendix A!!Background Information About Mussels!

! Although mussels may be recog-

nized as shelled animals, they belong to

the phylum of mollusks because of their

inner structure. There are about 30.000

mussel species found on the Earth. One

of the significant species of mussels is

the Mytilus galloprovincialis (widely

known as the Mediterranean mussel).

This species is found in the waters rang-

ing from Black Sea to Aegean Sea, also encircling the country of Turkey. They have a

diet of plankton so they siphon and filter the water to catch them. At the posterior of

the mussel, there are two siphons; one for the intake and the other for the vacate of the

water called as the incurrent and excurrent siphons. The water passes from the gills

while moving from posterior to anterior, and the mouth catches the planktons. Because

most of the materials inside the water is filtered into the mussel, they are known as the

cleaners of the nautical environment. Therefore they can be used to investigate the level

of sea pollution. (Malwarm et al, 2005)

L i v i n g I n d i c a t o r s o f S e a P o l l u t i o n : M u s s e l s ! M e r t İN A N ! !�x x v i

Figure 2: The internal anatomy of Mytilus gal-loprovincialis Source: http://www.glf.dfo-mpo.gc.ca/folios/00012/images/fig_3_mussel-moule_2003-eng.jpg

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Appendix B!

Background Information About Dioxin!

! Dioxin, often known as 2,3,7,8,-Tetra-

chlorodibenzodioxin (TCDD) has hydrophobic

properties, decays really hard and is a stable pollut-

ing compound. Because they are stable they need a

lot of energy to be broken-down therefore they

are stored in the cells of living when they get in to

the organism. “Dioxins are known to be formed dur-

ing the combustion of industrial and domestic wastes

and to escape into the environment via exhaust gases

from incinerators.” (Shibamoto et al, 2007) Other known sources of dioxin include elec-

tricity generation, usage of fossil fuels, wildfires, production of chemical compounds

(pesticides, PVC and cosmetics etc.), production of leather, textile and paper, production

of limestone, asphalt and cement and production of metals. 90 % of the Dioxin-poison-

ing cases are caused via food. (Şahbaz and Acar, 1993)!

Experiments on Dioxin!

! Tests proved that even small amounts of dioxin can have toxic effects. Dioxins,

once got into the cell, bind with receptor proteins in the cytoplasm forming a complex,

then this complex travels to the nucleus to bind to the DNA and to render the protein

synthesis ineffective. In addition, they activate the aryl hydrocarbon receptors (AhR)

that may cause DNA mutation and increase the possibility of occurrence of devastating

diseases like cancer. Furthermore, according to a research in Japan babies and 8 years-

old children that were exposed to dioxin showed signs of mental deficiency and a

diminution in the abilities of comprehension. (Şahbaz ve Acar, 1993). !!!L i v i n g I n d i c a t o r s o f S e a P o l l u t i o n : M u s s e l s ! M e r t İN A N ! !

�x x v i i

Figure 3: 3D structure of the dioxin molecule!

Source: http://www.chm.bris.ac.uk/motm/dioxin/dioxin-hp.htm

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! Also a lot of researchers found that marine animals can store high amounts of

PCDD (Polychlorodibenzodioxin), another form of dioxin. For example, there has been

a research to quantitate the PCDD levels in the coasts of the mainland by using blue

mussels (Mytilus edulis). !

Detriments of Dioxin!

! Being exposed to dioxin or other dioxin-like polluting organic compounds can

show severe effects such as; cancer ( especially digestive system, liver and breast can-

cers), developmental disorders, lymphoid and gonadal atrophy, hepatotoxicity, cleft

palate, birth defects like defective kidney, immunotoxicity, neurotoxicity and cardiotoxi-

city, nausea, dyspnea, reproductive disorders, innate disability, hypertension and asth-

ma. (Şahbaz and Acar, 1993)! !

Appendix C!DR CALUX dioksin ve dioksin benzeri bileşenleri ayrı ayrı ölçmek yerine, bileşenlerin toksik etkilerine dayalı bir yöntem kullanmaktadır. Analizde toksik özelliğe sahip dioksinlere ve PCB’lere spesifik reseptör hücreler kullanılmaktadır. TEQ (WİKİPE-DİADAKİ BİLGİYİ EKLE http://en.wikipedia.org/wiki/Toxic_Equivalency_Factor) değerleri floresans sinyalleri ölçülerek belirlenebilmektedir. Bu teknikte, numune alma, standart yağ ekstraksiyonu, asit silika temizleme, karbondaki dioksin ve PCB’lerin ayrımı (%75/25, hekzan/toluen), 96 kuyucuklu plate’e uygulama, ışık emilimi yoluyla miktar tayini ve verilerin değerlendirilmesi basamaklarından oluşmaktadır. Bu yöntem yüksek seçiciliğe ve doğru, kesin ölçüm sonuçlarına sahiptir. (Şenyuva ve ark., 2010)!

! Mytilus galloprovincialis midyeleri, Karadeniz örneği olarak Amasra kıyılarından,

Marmara Denizi örneği olarak Tuzla kıyılarından, Ege Denizi örneği olarak İzmir

Karşıyaka kıyılarından toplandı (Şekil 3).

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Akdeniz örneği olarak düşünülen Antalya ve İskenderun kıyılarından midye örnekleri

bulunamadığından Akdeniz kıyıları araştırmaya katılamadı. Soğuk zincir içinde buz

aküleri ile korunarak ısı yalıtımlı kolilerde dioksin çalışılacak Ankara’daki laboratu-

vara nakledildi. Laboratuvarda koliler açılarak incelemenin yapılacağı tarihe kadar de-

rin dondurucuda (-20°C) tutuldu. !

İncelemenin yapılacağı tarihte her bölgeden toplanan midyeler büyüklüklerine göre 3

gruba ayırıldı; Küçük (3 cm’e kadar), Orta (5 cm’ye kadar) ve Büyük (5 cm’den büyük).

Her bir deneme grubunda, her alt gruptan 10 midye alınarak örnekler hazırlanmıştır.

Daha sonra midyeler fotoğraflanmıştır (Şekil 4).!

Ön Bilgi!

! Üç bölgeden alınan bütün grupların hepsinde Dioksin ve Dioksin benzeri bileşik-

lerin (PCDD/F ve dl-PCBs) analizi yapılmıştır. Deneyler 2 kez tekrar edilmiştir. Anali-

zler biyotespit yöntemi olarak bilinen DR CALUX (Dioxin Responsive Chemical-Acti-

vated Luciferase gene eXpression) (Şekil 5) sistemi ile yapılmıştır. TEQ değerleri flore-

sans sinyalleri ölçülerek belirlenmiştir. İlgili bileşikler (PCDD’ler, PCDF’ler, PCB’ler

gibi) ekstraksiyon solüsyonu (n-Hekzan:DEE) ile ekstrakte edilir. Materyaller için ek-

straksiyon solüsyonu ilave edilmeden önce su ve izopropanol ilave edilir. İzopropanol

su ile tabaka halindeki parçacık yüzeyini seyreltmek ve üç boyutlu protein yapısını

açarak ekstraksiyon solüsyonunun nüfus etmesini kolaylaştırmak için ilave edilir. Su ise

katı materyali askıda tutarak daha iyi çalkalanmasını sağlamak için ilave edilir. Ekstrak-

siyon solüsyonu ilave edildikten ve çalkalandıktan sonra, ekstraksiyon solüsyonunun

katmanını ayırmak için ekstra izopropanol ilave edilmiştir.

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Appendix D

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c.

Figure 4: This figure shows the distribution of Mytilus edulis in the world.!

©FAO Fishery Statistics, 2006

Figure 5: The distribution of Mytilus gal-loprovincilis is present in this schematic representation of the contents of Africa, Europe and Middle East.!

©FAO Fishery Statistics, 2006

b.

Figure 6: a,b,c: Photographs of the Mytilus gallo-provincialis with different sizes just before the exper-iments.

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Appendix E!! Firstly an acidic silica gel is prepared by; !

• preparing and cleaning the silica before a day,!

• pouring 20 % and 33% acidic silica into different glass bottles and shaking them in a shaker for 4 hours with 250 beats per minute.!

• getting a glass column and putting glass wool into it and then 5 grams of 33% and 5 grams of 20% acidic silica is added respectively into the glass tube. Then, because of it hydrophilic properties sodium sulfate is added for 1 cm to the column.!

• running 20 ml of extracted solution through the glass column. (Figure 10)

L i v i n g I n d i c a t o r s o f S e a P o l l u t i o n : M u s s e l s ! M e r t İN A N ! !�x x x i

Figure 10: Schematic display of the chromatography column.