Final Conference Safefoodera meeting Copenhagen, 7th June 2011

1. Bioavailability and risk assessment of polycyclic aromatic hydrocarbons (PAHs) and toxic elements (As, Cd, Hg and Pb) in processed meat and seafood products Final Conference Safefoodera meeting Copenhagen, 7th June 2011 Alejandro Barranco AZTI-Tecnalia, Food Research Division, Spain e-mail: abarranco@azti.es

2. Riskfoodcont Partners • IPIMAR (Portugal) • Portuguese Governmental Research Institute for Fisheries and Sea • Contaminants in seafood and risk analysis • INIA (Portugal) • National Institute of Agricultural Research • Contaminants in meat products • University of Maribor (Slovenia) • Department of Microbiology, Biochemistry, Molecular Biology and Biotechnology • Cell lines • AZTI-Tecnalia (Basque Country) • Detection system area • Zebrafish and biosensors © AZTI-Tecnalia

3. Outline • Objectives and structure • Why this project? • Objectives and workplan • Methodology • Main results • Seafood • Edible crab • Black scabbard fish • Meat products • Smoked meat products • Conclusions © AZTI-Tecnalia

4. Why RISKFOODCONT? • Human exposure to contaminants • They are in the environment (air, soil…) • Contact with contaminated materials • Residues in food Heavy metals PAHs © AZTI-Tecnalia

5. Why RISKFOODCONT? Heavy metals Number of notifications in UE. Source: RASFF anual report 2009 © AZTI-Tecnalia

6. Why RISKFOODCONT? Heavy metals Number of notifications in UE. Source: RASFF anual report 2009 © AZTI-Tecnalia

7. Why RISKFOODCONT? Heavy metals Cadmium: Meat, fish, cereals MRL: 0.05-3 mg/kg Lead: Milk, meat, fish, cereals MRL: 0.02-1,5 mg/kg Mercury: Fish and seafood MRL: 0.1-1 mg/kg Tin: Canned food MRL: 20-200 mg/kg Organo metallic compounds © AZTI-Tecnalia

8. Why RISKFOODCONT? Polycyclic aromatic hydrocarbons • Ubiquitous contaminants • Source: Combustion of organic materials with low amount of oxigen • >90% of exposure occurs via food residues • MRL: 1-10 ug/kg benzo[a]pyrene (edible oils and fats, smoked fish and meat, fresh fish and seafood) • Very lipophilic compounds • Toxic, carcinogenic and mutagenic by long term exposure © AZTI-Tecnalia

9. Why RISKFOODCONT? Polycyclic aromatic hydrocarbons Light PAHs Heavy PAHs © AZTI-Tecnalia

10. Objectives • Implement innovative research tools (e.g. pig and human cell lines, zebrafish embryo, toxicogenomic) to study the bioavailability and risk assessment of PAHs and heavy metals in processed food products, using smoked meat sausage and cooked edible crab and black scabbard fish as models • Identify specific toxicity biomarkers through the analysis of the toxicogenomic response to contaminant exposure • Develop rapid methods able to detect small amounts of toxicants in foodstuff even if masked by the coexistence of other compounds or specific food matrices © AZTI-Tecnalia

11. Workplan © AZTI-Tecnalia

12. Methodology Toxicological studies • Cell lines • 2 types of pig cell lines (PSI y CLAB) • Human cells (H4) Apical compartment • Polarised intestinal cell lines: • CLAb and PSI • H4 Basal compartment • Monocyte/macrophage cell lines: • Pom • TLT Microporous membrane © AZTI-Tecnalia

13. Methodology Toxicological studies. Zebrafish • Asiatic sweet water fish of ≈ 5 cm • Breed all year round. • Time of generation: 3-5 months. • Lay up to 300 eggs. Externally fertilized. • Share many characteristics with vertebrates • Its genome has been completely sequenced Zebrafish embryos and larvae • Embryos and larvae are transparent • Not considered animals Alternative test 5dpf 5hpf alternative tests

14. Methodology Toxicological studies. Zebrafish Biomarker genes/proteins Expresion profile Larvae 48 h exposure Validation of biomarkers qRT-PCR DNA chip Microarray © AZTI-Tecnalia

15. Methodology Bioavailability and culinary treatments • Protocoltosimulatethethingsoccuring at: • mouth • stomach • intestine Blackscabbardfishmuscle Raw (control) Steamed Grilled Fried Raw (control) Steamed Boiled 60 specimens © AZTI-Tecnalia

16. Methodology Risk assesment • Surveys about habits of consumption • Residues of target contaminants in food samples • Bioavailability and tosic effects • Several scenarios were proposed: • 150 g de fish/meal; 70 Kg adult • PTWI (Hg): 5 µg Hg/Kg/semana • Software: @RISK Biosensors • Identification of suitable biomarkers • Development of inmunoassays © AZTI-Tecnalia

17. Main reults • Toxic effects • Zebrafish. Metals Low concentration: 0.14 ppm CdCl2 + 9 ppb HgMetCl Medium concentration: 0.5 ppm CdCl2 + 50 ppb HgMetCl High concentration: 1.4 ppm CdCl2 + 100 ppb HgMetCl © AZTI-Tecnalia

18. Main reults • Toxic effects • Zebrafish. Metals 0.14 ppm CdCl2 + 9 ppb HgMetCl © AZTI-Tecnalia

19. 1ppb 10ppb Main reults • Toxic effects • Zebrafish. PAHs • No mortality was observed © AZTI-Tecnalia

20. Limit = 1.0 mg kg-1 Hg (mg kg -1) Cd (mg kg -1) Limit = 0.50 mg kg-1 Main reults Bioaccesibility and culinary treatments Black scabbard fish More Hg in grilled samples due to the loss of water Edible crab (Hepatopancreas) More Hg in grilled samples due to the loss of water © AZTI-Tecnalia

21. Main reults Bioaccesibility and culinary treatments % Mercury • Gastric step: 1.3 – 3.3% Hg • Gastrointestinal step : 5.8 – 14.5% Hg • Hg is more accesible at the intestine and in the case of raw fish © AZTI-Tecnalia

22. Main reults Bioaccesibility and culinary treatments • Gastric step: 45 - 72% Cd • Gastrointestinal step : 83 – 90% Hg • Cd is more accesible at the intestine and in the case of raw fish © AZTI-Tecnalia

23. Main reults • Bioavailability • Cell lines. Methylmercury • Very low amount (<4%) of Methylmercury goes through the cell lines • Transport lower with digested samples © AZTI-Tecnalia

24. Main reults • Bioavailability • Cell lines. Methylmercury • Higher bioavailabillity with real food samples © AZTI-Tecnalia

25. Main reults • Bioavailability • Cell lines. Cadmium • Up to 30% of Cadmium goes through cell lines • More transport with digested solutions © AZTI-Tecnalia

26. Main reults • Bioavailability • Cell lines. Cadmium • All samples did not show the same behaviour © AZTI-Tecnalia

27. Main reults • Surveys • Portugal. 800-2000 answers Consumption of smoked meat products Edible crab 97 % consume hepatopancreas © AZTI-Tecnalia

28. Main reults • Residues © AZTI-Tecnalia

29. Main reults • Risk assessment Edible crab Black scabbard fish PTWI = 5 g Hg/kg/week TWI = 2.5 g Cd/kg body weight Adults and boiled Hepatopancreas– 1:30 Whole– 1:80 Muscle – 1:641,000,000 Adults Children Despite the Hg content, low risk to exceed PTWI The consumption of hepatopancreas is not recomended . Fryed: 1:12,000,000 1:14,000,000 . Grilled: 1:3,000,0001:1,800,000 © AZTI-Tecnalia

30. Main reults • Biosensors • Mt and HSP70 genes in the case of metals • A strong induction of cytochrome P450 family C1 (CYP1A1 gene) has been observed in the case of PAHs • Design of an ELISA kit for the simultaneous of target biomarkers © AZTI-Tecnalia

31. Conclusions • Innovative and more realistic tools have been applied to evaluate risk assessment of single and multiple contaminants in food samples. • Zebrafish is a good model for investigating the effect of food contaminants as a preliminary screening. Intermediate toxocity assay between in vitro and in vivo tests • Excellent tool for identifying toxicity biomarker genes • It is important to know the mechanism of toxicity through our digestive system. In this sense, cell lines are an useful tool. • Contribute to set new limits for PAHs and heavy metals in food © AZTI-Tecnalia

32. Conclusions • Culinary treatments play an important role. In the case of mercury bioavailability was higher in raw fish • Despite the relatively high levels of Hg in cooked black scabbard fish, the risks of exceeding PTWI are extremely reduced • When consuming edible crab, consumers should reduce the brown meat portion to the lowest level and prefer the muscle • Risk benefit analysis • Dissemination • 3 national conferences /workshops • Articles (14) and congresses (9) © AZTI-Tecnalia

33. www.azti.es www.azti.es Thank you for your attention!!! Riskfoodcont team © AZTI-Tecnalia