Bioavailability and risk assessment of
polycyclic aromatic hydrocarbons (PAHs)
and toxic elements (As, Cd, Hg and Pb) in
processed meat and seafood products
Alejandro Barranco
AZTI-Tecnalia, Food Research Division, Spain
e-mail: abarranco@azti.es
Final Conference Safefoodera meeting
Copenhagen, 7th June 2011
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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
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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
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Why RISKFOODCONT?
Human exposure to contaminants
 They are in the environment (air, soil…)
 Contact with contaminated materials
 Residues in food
Heavy metals
PAHs
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Why RISKFOODCONT?
Heavy metals
Number of notifications in UE. Source: RASFF anual report 2009
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Why RISKFOODCONT?
Heavy metals
Number of notifications in UE. Source: RASFF anual report 2009
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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
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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
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Why RISKFOODCONT?
Polycyclic aromatic hydrocarbons
Light PAHs
Heavy PAHs
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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
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Workplan
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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
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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
Alternative test
•
•
Embryos and larvae are
transparent
Not considered animals
alternative tests
5hpf
5dpf
Methodology
Toxicological studies. Zebrafish
Larvae
48 h
exposure
ENDPOINTS
PARAMETER
Morphology
Malformation
Pigmentation
ANALYSIS
Microscopic
analysis
Development
Delay
Death
Molecular
Differential expression
analysis toxicity marker
genes
Expresion
profile
DNA chip
Microarray
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qRT-PCR
Biomarker
genes/proteins
Validation of biomarkers
qRT-PCR
Methodology
Bioavailability and culinary treatments
Black scabbard fish muscle
Protocol to simulate the things occuring at:
- mouth
- stomach
- intestine
Raw (control)
Steamed
Grilled
Fried
Raw (control)
Steamed
Boiled
60 specimens
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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
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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
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Main reults
Toxic effects
– Zebrafish. Metals
0.14 ppm CdCl2 + 9 ppb HgMetCl
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Main reults
Toxic effects
– Zebrafish. PAHs
– No mortality was observed
1ppb
10ppb
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Main reults
Bioaccesibility and culinary treatments
1.8
Black scabbard fish
1.4
Limit = 1.0 mg kg-1
1.2
1.0
More Hg in grilled samples due to
the loss of water
0.8
0.6
0.4
0.2
0.0
Raw
Fried
Grilled
Steamed
35
Cd (mg kg
-1)
Hg (mg kg -1)
1.6
30
Edible crab (Hepatopancreas)
25
20
More Hg in grilled samples due to
the loss of water
15
10
5
Limit = 0.50 mg kg-
0
1
Raw
Steamed
Boiled
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Main reults
% Mercury
Bioaccesibility and culinary treatments
 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
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Main reults
Bioaccesibility and culinary treatments
100
90
80
% Cadmium
70
60
50
40
30
20
10
0
Raw
Mouth step
Steamed
Gastric step
Boiled
Gastrointestinal step
 Gastric step: 45 - 72% Cd
 Gastrointestinal step : 83 – 90% Hg
 Cd is more accesible at the intestine and in the
case of raw fish
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Main reults
Bioavailability
– Cell lines. Methylmercury
 Very low amount (<4%) of Methylmercury goes through the cell lines
 Transport lower with digested samples
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Main reults
Bioavailability
– Cell lines. Methylmercury
 Higher bioavailabillity with real food samples
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Main reults
Bioavailability
– Cell lines. Cadmium
 Up to 30% of Cadmium goes through cell lines
 More transport with digested solutions
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Main reults
Bioavailability
– Cell lines. Cadmium
 All samples did not show the same behaviour
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Main reults
Surveys
– Portugal. 800-2000 answers
Edible crab
Consumption of smoked meat
products
97 % consume hepatopancreas
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Main reults
Residues
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Main reults
Risk assessment
Edible crab
Black scabbard fish
PTWI = 5 g Hg/kg/week
Adults
. Fryed:
. Grilled:
1:12,000,000
1:3,000,000
TWI = 2.5 g Cd/kg body weight
Children
Adults and boiled
Hepatopancreas– 1:30
Whole– 1:80
Muscle – 1:641,000,000
1:14,000,000
1:1,800,000
Despite the Hg content, low risk to
exceed PTWI
The consumption of hepatopancreas
is not recomended
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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
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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
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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)
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www.azti.es
Thank you for your attention!!!
Riskfoodcont team
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