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Faculty of Veterinary Medicine
Determination of Persistent Organic
Pollutants (POPs) in Fish Dipartimento di Scienze Veterinarie e Sanità Pubblica
(DIVET)
Prof. L.Chiesa, Dr. S. Panseri
-Prague, April 27, 2015
“Food safety is an assurance that a food will not cause harm to the consumer at the time of preparation and / or consumption” (Raspor, 2007)
Food security is affected by contamination by:
PHYSICAL agents (metals, glass ...)
CHEMICAL agents (toxins, residues)
Microbiological agents (bacteria)
Food Safety
Food safety is a shared responsibility from farm to table "FROM FARM TO FORK" (WHO)
OPERATIVE PROCEDURES
MONITORING SYSTEMS
Food Safety
QUALITY 'AND SAFETY IN THE SUPPLY CHAIN
AIR WATER
ENVIRONMENT FOOD
SOURCES OF POLLUTION ALONG THE SUPPLY CHAIN
Food Safety
REFERENCE STANDARDS (MRLs) -Residues pesticides-
•When placed in circulation in the EU products intended not contain pesticide residues exceeding the maximum residue limits (MRLs)
•The values of MRLs are set and evaluated for each substance at the time of their authorization
(CE) n. 396/2005 (CE) n. 2002/32
Food Feed
(CE) N. 1259/2011
Food Safety
Bioaccumulation and biomagnification of POPs
BIOACCUMULATION: The lipophilic active substances tend to accumulate in the adipocytes; this mechanism is to be considered a defense mechanism of the organism
BIOMAGNIFICATION: The process by which the concentration of the pesticide in the food chain increases to climb along the food pyramid
Pesticides
Common mechanisms POPs
(peristent organic pollutants)
POPs (persistent organic pollutants)
OCPs pesticides , PCBs
Low volatility
High stability
Lipophilic behavior
Persistent in the environment
Concentration in fatty foods
Chung et al., 2011 “Determination of organochlorine pesticide residues in fatty foods: A critical review on the analytical methods and their testing capabilities”, J.Chromatography A, 1218(33), 5555-5567
• OC Pesticides are a category of substances used particularly in the 50s and 60s
• Chlorinated molecules are extremely stable environmentally
• They are characterized by the phenomenon of bioaccumulation
• They are divided, according to the chemical structure into 3 classes
?? Research and
monitoring today
Organochlorine pesticides(OC) and Polychlorinated Byphenyls
(PCBs)
Cl Cl
Cl Cl
Cl
DDT
DDE
DDD
METOXYCHLOR
Cl Cl
Cl Cl
Cl Cl
Cl Cl
Cl Cl
OCH3
Cl
H3CO
Chemical Structures
Organochlorine pesticides (OCs)
ALDRIN
DIELDRIN
ENDOSULFAN I
O
O
SO
Cl
Cl
Cl Cl
Cl
Cl
ENDOSULFAN II
ENDOSULFAN SOLF.
α-CLORDANE
γ-CLORDANE
ENDRIN
Cl
Cl
Cl Cl
O
Cl
ClEPTACHLOR
EPTACHLOR EP.
Cl
Cl
Cl Cl
Cl
Cl
H
Cl
H
Cl
Cl
Cl
Cl Cl
Cl
Cl
Cl
ClO
O
SO
O
Cl Cl
Cl
ClCl
Cl
Cl
Cl
Cl Cl
Cl
Cl
O
HH
HH
HHO
O S
O
Cl Cl
Cl
ClCl
ClCl
Cl
Cl Cl
Cl
Cl
O
Cl
Cl
Cl
Cl
Cl Cl
Cl
Cl
Cl
ClCl
Cl
Cl
Cl
Organochlorine pesticides(OC)
Chemical Structures
Cl
Cl
Cl
Cl
Cl
Cl
EXACHLOROBENZENE
α-BHC Cl
Cl
Cl
Cl
Cl
Cl β-BHC ClCl
Cl
ClCl
Cl
δ-BHC
Cl Cl
Cl
Cl
Cl
Cl
γ-BHC
Cl
ClCl
Cl
Cl
Cl
Chemical Structures
Organochlorine pesticides (OC)
PHASE I: creation of extraction and purification method "in line" to extract POPs from fish tissue (OCs and PCB ICES-6) in a single analitycal step
PHASE II: application of the optimised method to evaluate the POPs content in different fish species
Aim of the Research
Consumer’s food safety
Thunnus thynnus, Pagellus bogaraveo, and Dentex dentex
MEDITERRANEAN SEA
“Eighty percent of the variance in an assay usually arises from the
sample preparation” (Stevenson R., Pittcon ‘98: Part 3 Sample Prep.: The place to make
the difference, American Laboratory, Vol. 30, No. 14 p.21, 1998).
Sample collection
Sample Storage
Sample Preparation
Analytes determination
Accelerated Solvent Extraction (ASE)– extraction and clean-up in
cell in a single step
EXTRACTION THECNIQUES
MATERIALS AND METHODS
QuEChERS
(Quick, Easy, Cheap, Effective, Rugged and Safe)
IDEAL FOR COMPLEX MATRIX (LIPID CONTENT)
GC-MS/MS (TRIPLE QUADRUPOLE QqQ)
The analyzes were carried out through the use of the gas chromatograph coupled with triple quadrupole detector (Thermo Scientific™ TSQ™ 8000 GC-MS system)
GC Parameters :
• Carrier gas: He 1mL/min
• Injector T (°C): 280 PTV
• Transfer line T (°C): 270
• Injecti on: 1μL Splitless
• Oven: 120°C x 3min, 120 to 300 2,5°C/min, hold 300°C 5 min
•Column: Rtx-5ms (30m, 0.25mm i.d., 0.25µm film tickness)
MS Parameters:
•Temp. Ion source (°C): 250
•Energy I.E. (eV): 70 SRM mode
MATERIALS AND METHODS
Accelerated Solvent Extraction - Conditions Extraction Solvent: n-Hexane : acetone (80:20 , v/v) Pressure: 1500 psi Temperature: 120°C Static Time:5 min Static Cycles: 2 Flush:60% Purge:90s Cell Size:33 mL
ASE PARAMETERS
3 g fish tissue and diatomeaceous heart
5 g fat retainer – silica gel
18 20 22 24 26 28 30 32 34 36 38 40 42Time (min)
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Rel
ativ
e A
bund
ance
34.83
41.6437.6339.70
30.35
32.74
41.44
30.96
32.9237.26
34.0340.2226.43 38.36 42.50
25.97 34.9529.40 32.1622.77 38.5317.92 20.4818.61 21.22 22.95 25.37 26.76 35.12
NL:8.88E5TIC MS 72456
TIC of pesticides in fortified fish tissue extract (10 ppb)
Compounds
α-BHC
Esacloroben.
β-BHC
γ-BHC
δ-BHC
Eptachlor
Aldrin
Eptachlor ep.
γ-Clordan
Endosulfan I
α-Clordan
Dieldin
p-p' DDE
Endrin
Endosulfan II
p-p' DDD
Endrin ald.
Endosul. Solf.
p-p' DDT
Metoxychlor
Fish tissue
LoD (mg/kg)
LoQ (mg/kg)
0.002 0.016
0.003 0.011
0.003 0.010
0.012 0.04
0.001 0.02
0.001 0.005
0.004 0.015
0.002 0.009
0.003 0.013
0.007 0.024
0.005 0.017
0.002 0.007
0.003 0.012
0.004 0.013
0.004 0.016
0.002 0.008
0.002 0.007
0.007 0.023
0.006 0.02
0.002 0.007
CV (%)
1.10– 3.45
LoD: 3*sb/m
LoD:10*sb/m
Results and discussion
Recovery (%)
>80% fo all compounds
Results and discussion
PCB ICES in fish samples
Results and discussion Ocs pesticides in fish samples
PCBs The concentration of all the PCBs (i.e. PCB 18, 28, 31, 44, 52, 101, 118, 138,149,180,194) were higher in samples obtained from Pagellus bogaraveo, whereas PCB 31, 52, 101, 118, 138, 180, 194 reached the minimum mean concentration in Dentex dentex, and PCB 18, 28, 44, 149 were present at lower levels in samples obtained from Thunnus thynnus. Pesticides Fish tissue analysis revealed maximum concentration of sum endosulfan (27.5), sum heptachlor (14.2), endrin aldehyde (70.25) in Thunnus thynnus, whereas sum DDT (254), sum HCH (7.56), aldrin (95.55), and endrin (3.55) were maximum in Pagellus bogaraveo and Dentex dentex respectively.
SMALL FISH SHOWED HIGHER CONCENTRATION
OF POPS BENTHOPELAGIC SPECIES
Results and discussion
In particular, benthopelagic fishes belonging to Fam. Sparidae (i.e. Dentex dentex and Pagellus bogaraveo) and inhabit the water just above the bottom, and pelagic fishes belonging to Fam. Scombridae (i.e. Thunnus thynnus) which live and feed away from the bottom in the open water column, represent a useful indicator of pollution of the environment in which they reside, since they could be subjected to possible contaminated sources for their location and hydrological characteristics.
Results and discussion
Fundamental work of monitoring of contaminants along supply chains (ensure the food safety for consumer)
• Methods of extraction was effective in the determination of POPs in
complex matrices (high-interference lipid matrix) in a single step (useful in routine laboratory analisys)
• muscle fish tissue is confirmed as an organ of bioaccumulation and biomagnification for OCs pesticides, PCBs and potentially for other POPs
Conclusions
