Determination of Persistent Organic Pollutants (POPs) in Fish
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1 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
2 Food Safety 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)
3 Food Safety Food safety is a shared responsibility from farm to table "FROM FARM TO FORK" (WHO) QUALITY 'AND SAFETY IN THE SUPPLY CHAIN OPERATIVE PROCEDURES MONITORING SYSTEMS
4 Food Safety SOURCES OF POLLUTION ALONG THE SUPPLY CHAIN AIR WATER ENVIRONMENT FOOD
5 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. 1259/2011 (CE) n. 396/2005 (CE) n. 2002/32 Food Feed
6 Pesticides 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 Common mechanisms POPs (peristent organic pollutants)
7 OCPs pesticides, PCBs POPs (persistent organic pollutants) Low volatility Persistent in the environment High stability Lipophilic behavior 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),
8 Organochlorine pesticides(oc) and Polychlorinated Byphenyls (PCBs) 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
9 Chemical Structures Organochlorine pesticides (OCs) DDT DDD DDE METOXYCHLOR H 3 CO OCH 3
10 ALDRIN DIELDRIN ENDOSULFAN I O O S O ENDOSULFAN II ENDOSULFAN SOLF. α-clordane γ-clordane ENDRIN O EPTACHLOR EPTACHLOR EP. H H O O S O O O H H H H H H O O S O O Organochlorine pesticides(oc) Chemical Structures
11 Organochlorine pesticides (OC) Chemical Structures α-bhc β-bhc δ-bhc γ-bhc EXACHLOROBENZENE
12 Aim of the Research 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 Thunnus thynnus, Pagellus bogaraveo, and Dentex dentex MEDITERRANEAN SEA Consumer s food safety
13 Sample collection Sample Storage Sample Preparation Analytes determination 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).
14 MATERIALS AND METHODS EXTRACTION THECNIQUES Accelerated Solvent Extraction (ASE) extraction and clean-up in cell in a single step IDEAL FOR COMPLEX MATRIX (LIPID CONTENT) QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe)
15 MATERIALS AND METHODS 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
16 ASE PARAMETERS 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 3 g fish tissue and diatomeaceous heart 5 g fat retainer silica gel
17 TIC of pesticides in fortified fish tissue extract (10 ppb) Relative Abundance Time (min) NL: 8.88E5 TIC MS 72456
18 Results and discussion Fish tissue LoD: 3*sb/m Compounds α-bhc LoD (mg/kg) LoQ (mg/kg) Esacloroben LoD:10*sb/m β-bhc γ-bhc δ-bhc Recovery (%) Eptachlor Aldrin Eptachlor ep >80% fo all compounds γ-ordan Endosulfan I α-ordan Dieldin p-p' DDE CV (%) Endrin Endosulfan II p-p' DDD Endrin ald Endosul. Solf p-p' DDT Metoxychlor
19 Results and discussion PCB ICES in fish samples
20 Results and discussion Ocs pesticides in fish samples
21 Results and discussion 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
22 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.
23 Conclusions 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 Fundamental work of monitoring of contaminants along supply chains (ensure the food safety for consumer)
24 THANK For the attention
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