Biological analyses of dioxins

1. Biological analyses of dioxins Globe Europe 17-20 April 2008 Javier Martinez Alarcon

2. Bioassays for analysis of dioxin-like compounds • To analyzedioxins 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 quickly • Based 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

3. 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

4. PCDD,PCDF PCB, PAH Ah-R DRE RNA RNA Cytochrom p450 Proteins Luciferase Light Biological effect of the dioxins

5. BDS-DR.CALUX use a line of liver cells from rat to analyze dioxins • The cells give light under exposure with dioxins • Cells are modified with a gene that produces luciferase • Quantity of light given by cells is a measure of dioxins

6. 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

7. Global POP • We have received 132 samples from 34 schools • But in the beginning there were 84 schools which were signed up

8. Global POP Gadus morhua, cod. Perca fluviatilis, European perch. Salmo trutta, trout.

9. Day 1: Samplepreparation • Fish, meat, egg, air, soils etc. • Extraction of fish with 3% ether-hexane.

10. Day 1: preparing samples • Lipids are determined gravimetrically • Chromatography with acid silica

11. Cell cultures are growing continously

12. Day 2: Cells are exposed

13. Day 3: Measurement of luciferase activity in a luminometer

14. 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

15. Lipid determination: we have determined 59 samples The sample with 27% of fat is Anguilla anguilla, eel

16. 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

17. 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?

19. 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