Presentation on theme: "Www.nilu.no Biological analyses of dioxins Globe Europe 17-20 April 2008 Javier Martinez Alarcon."— Presentation transcript:
Biological analyses of dioxins Globe Europe April 2008 Javier Martinez Alarcon
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 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 Bioassays for analysis of dioxin- like compounds
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 TEF z + Y x TEF y + N TEF n 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
RNA Cytochrom p450 Proteins Luciferase Light PCDD,PCDF PCB, PAH Ah-R DRE Biological effect of the dioxins
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
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
Global POP We have received 132 samples from 34 schools But in the beginning there were 84 schools which were signed up
Global POP Gadus morhua, cod. Perca fluviatilis, European perch. Salmo trutta, trout.
Day 1: Sample preparation Fish, meat, egg, air, soils etc. Extraction of fish with 3% ether-hexane.
Day 1: preparing samples Lipids are determined gravimetrically Chromatography with acid silica
Cell cultures are growing continously
Day 2: Cells are exposed
Day 3: Measurement of luciferase activity in a luminometer
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
Lipid determination: we have determined 59 samples The sample with 27% of fat is Anguilla anguilla, eel
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
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?
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