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Biological analyses of dioxins

Globe Europe 17-20 April 2008

Javier Martinez Alarcon

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To analyze dioxins and similar substances Quick method

• Many samples can be analyzed at the same time• Less expensive• Not an alternative, but complementary to other

methods:– Positive samples are analyzed with GC-HRMS

Maximum values can be seen quicklyBased on modified genes in liver cells Cells give light under exposure to dioxins The quantity of light obtained is a measure of dioxins We do standard curve with 2,3,7,8-TCDD Semiquantitative method, we analyze mixtures.

Alternative is GC

We measure the biological effect of the dioxins in the cells

Bioassays for analysis of dioxin-like compounds

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Toxic equivalent factor (TEF)Aryl hydrocarbon receptor. Dioxin-like compounds

share a common mechanism of binding to the Ah-r

Each compound has a different TEF E.g. 2,3,7,8-TCDD has 1

If we know the concentration and TEF of each compound in a mixture, we will know the toxic equivalent (TEQ) of this mixture TEQ=Z x TEFz + Y x TEFy + N TEFn

We can transform analytical results into toxicological information

TEQ was introduced to estimate total toxicity of a mixture of pollutants. I.e. every compound which bind to Ah-r

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RNA RNA

Cytochrom p450Proteins

Luciferase

Light

PCDD,PCDFPCB, PAH

Ah-R

DRE

Biological effect of the dioxins

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BDS-DR.CALUX use a line of liver cells from rat to analyze dioxins

The cells give light under exposure with dioxinsCells are modified with a gene that produces luciferase Quantity of light given by cells is a measure of dioxins

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Global POP

Recruiting young scientists and increase motivation of the students

Increase knowledge about environmental issues among students

Schools from Arctic areas will be encouraged to participate. TEQ will be determined by NILU in fish that were caught by the students

Students will evaluate the results and possible sources and transport of the pollutants

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Global POP We have received 132 samples from 34 schools

But in the beginning there were 84 schools which were signed up

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Global POP

Gadus morhua, cod.

Perca fluviatilis, European perch.

Salmo trutta, trout.

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Day 1: Sample preparation

Fish, meat, egg, air, soils etc.

Extraction of fish with 3% ether-hexane.

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Day 1: preparing samples

Lipids are determined gravimetrically

Chromatography with acid silica

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Cell cultures are growing continously

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Day 2:Cells are exposed

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Day 3:Measurement of luciferaseactivity in a luminometer

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Standard curve with 2,3,7,8-TCDD

Each time we analyze a plate we must plot a standard curve

Afterwards our standard curve helps us to determine the other samples

2,3,7,8- TCDD are among the most poisonous compounds

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Lipid determination: we have determined 59 samples

The sample with 27% of fat is Anguilla anguilla, eel

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Recommendations by:WHO: 1-4 pg/Kg body weight/day

EU: 14 pg/kg body weight/week

We have got results for TEQ from 37 samples

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WHO: 1-4 pg/Kg body weight/day

0,68 pg/g If we eat 200 g of fish= 136 pg. 136/80= 1,7 pg / kg body weight With 4 pg /g: a person that weights 80 kg must eat 320 pg each day

To reach this quantity you must to eat 470,5 g of fish every day Who does that?

EU: 14 pg/kg body weight/week = 2pg/g /kg body /day

With 2 pg /g: a person that weights 80 kg must to eat 160 pg each day

To reach this quantity you must to eat 235,3 g of fish every day

Who does that?

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Foodstuff Maximum permitted level Action Limits for dioxinsMeat, bovine 3pg WHO-TEQ/g fat 2pg WHO-TEQ/g fatPoultry 2pg WHO-TEQ/g fat 1.5pg WHO-TEQ/g fatPig 1pg WHO-TEQ/g fat 0.6pg WHO-TEQ/g fatLiver and derived products 6pg WHO-TEQ/g fat 4pg WHO-TEQ/g fatFish muscle 4pg WHO-TEQ/g whole weight 3pg WHO-TEQ/g whole weight Milk 3pg WHO-TEQ/g fat 2pg WHO-TEQ/g fat Eggs 3pg WHO-TEQ/g fat 2pg WHO-TEQ/g fat Vegetable oil 0.75pg WHO-TEQ/g fat 0.5pg WHO-TEQ/g fat Fish oil 2pg WHO-TEQ/g fat 1.5pg WHO-TEQ/g fat Mixed animal fat 2pg WHO-TEQ/g fat 1.5pg WHO-TEQ/g fat

WHO 1-4pg/kg body weight/dayEU 14pg/kg body weight/week

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Conclusions :

Global POP samples have low levels of dioxins –like compounds. At least until now

We can eat fish, but the quantity depends on the weight of the person and intake per week/day

We will begin next week to give some results to the schools. The schools will have possibility to evaluate the results and possible sources of contamination

We will also give schools specific task to do, e.g. to calculate how much fish we can eat per day/week given the WHO/EU limits

At NILU we will attempt to have ready all samples in June