Analysis of Halogenated Compounds in Fish Samples

The paper reports investigation on the presence of 13 halogenated compounds (six chlorinated compounds, nd-like PCBs and seven brominated compounds, polybrominated diphenyl ethers (PBDEs)), in five marine species which are part on Albanian consumer diet. The samples were extracted with ethyl acetate and treated with sulfuric acid. Extract was analyzed simultaneously using gas chromatography tandem mass spectrometry. Presence of PCBs was confirmed at1.4 percent of the samples. The most dominated congener found to be present were PCB 153 and PCB 138. Form the brominated compounds 2,2′,4,4′-tetra-bromodiphenyl ether was the only detected compound in 3 samples.


I. INTRODUCTION
Halogenated compounds are chemical compounds which contains in their chemical structure at least one halogen.Due to their long stability and toxicity these compounds are of a great concern for their impact on human health and environment as well.
Polychlorinated biphenyl represents a very known group of halogenated compounds which derived from the anthropogenic activity.They have been classified as persistent organic pollutants (UNEP 2009).Due to their long use and very high stability they are shown to be present worldwide in the environment.A very important class of PCBs are the so-called non dioxin-like PCBs.Different studies has already shown that PCBs have neurological, neuroendocrine, endocrine, immunological and carcinogenic effect in different species such as mammals, birds and fish (DeVito 2003).Maisano et al. 2016 suggested a potential onset of hepatic steatosis of PCBs (Maisano et al. 2016).
Another group of halogenated compounds of an increasing environmental concern are the Polybrominated Diphenyl Ethers (PBDEs).PBDE are part of Brominated flame retardants (BFRs) which are anthropogenic origin as well.They are wildly used in different chemical formulation in householder or office used products (electronic devices, textile industries and different building materials).The main formulation containing PBDEs are the following ones: penta-BDE (in which the concentration of BDE-47 in 39 % and BDE-99 is 49% Table I), octa-BDE (hexa-to deca-BDEs), and deca-BDE [(Mohamed Abou-Elwafa Abdallah 2009).penta-BDE and octa-BDE are classified as persistent organic pollutants (UNEP 2009) consequently their production is Ederina Ninga is with Food Safety and Veterinary Institute, Aleksandër Moisiu 82, Tirane, Albania ( email: ederina.ninga@yahoo.com).
Elvira Beli is with Faculty of Agriculture and Environment, Agricultural University of Tirana, Albania ( email: elvira.beli@yahoo.com) banned worldwide.Presence of PBDE in human body can cause disturbance of the thyroid hormone homeostasis and chronic neurotoxicity (Mehran Alaee 2002).Since polychlorinated biphenyl and polybrominated biphenyl ethers are wide spread in the environment, attention has been brought to their presence in marine organisms which are the main route of human exposure against PCBs and PBDEs.
Consequently, presence of these halogenic compounds in fish can bring long term risk to human health.

Instrument
GC/MS instrument.-Agilent 7890A GC system coupled to an Agilent 7000C Series Triple Quadrupole.
Centrifuge. -For the 50 mL/15 mL centrifuge tubes, an Eppendorf /5804R was used in order to make possible the separation between the solvent faze and the samples.
Vials and vessels.-50 mL and 15 ml fluorinated ethylene propylene (FEP) centrifuge tubes with ethylenetetrafluoroethylene screw closures were used.

Analysis of Halogenated Compounds in Fish Samples
Ederina Ninga, Elvira Beli, Elona Shahu, and Ilirjana Boci grade from WVR were used.Na2SO4 -Pesticides grade anhydrous Na2SO4 from WVR. Halogenated standards solution -Mix of PCBs standard solution from Sigma Aldrich, and Polybrominated Diphenyl Ethers (PBDE) from RoHS.

Samples
The determination of the halogenic compounds in fish was based in ethyl acetate extraction followed from quantitative determination of compounds in GC/MS/MS triple quadrupole.At total of 70 samples were randomly chosen from wild marine (38) fish and cultivated fish (32).
All the collected samples were kept in ice during the transportation to the lab the samples.They were kept in frozen condition in -20C until further analysis.
Analytical method Ten gram of fish muscle (edible part) was accurately weighted in a 50 ml centrifugal tube.PSA (0.5 g) and C 18 (0.5 g) were added to all the samples followed by 10 ml EtOAc.After 20 minutes' extraction in a horizontal shaker 5 gram of Na2SO4 anhydrous was added to each sample and another extraction step was performed for another 10 minutes.Extract was separated by centrifugation at 3200 rmp for 10 minutes at zero degree Celsius.After the centrifugation 5 ml extract was transferred in a 15 ml tube and then totally dried by using under nitrogen flow.After drying the samples were treated with 1 ml of sulfuric acid and the PCB/PBDE were reconstituted with 2 ml of hexane.The tubes were centrifuged in order to improve the separation between the acid and the solvent layer. 1 ml from the hexane layer was transferred to e glass vial and brought to dry.The samples were reconstituted with 0.1 ml ethyl acetate.

III. RESULTS
During this study the presence of halogenic compounds in fish from Adriatic and Ionian has been investigated.The research was conduct during three years (2014-2016).The fish species, number of samples, length and weight of samples are given in Table IV.In total were analyzed 70 samples, 38 of them were of sea origin (FAO37 region) and 32 of them marine cage culture farms in southern of Albania.Chlorinated compound investigated were the non-dioxine like PCBs (PCB-28, PCB-52, PCB-101, PCB138, PCB-180).
The presence of nd-PCB were detected in 50 samples.The calculated mean concentration range fall from 0.6 to 26 ng/g in w/w.There was no relation between the PCBs concentration and the fish species.The dominated congeners found to be present were hexa-chlorinated congeners, PCB 153 and PCB 138, followed by PCB 180, PCB 52.Presence of nd-PCB congeners has been previously shown in other studies from the Mediterranean areas.Seven Poly Brominated Diphenyl Ethers analyzed (PBDE-17, PBDE-28, PBDE -47, PBDE-99, PBDE-100, PBDE-153, PBDE-154) were analyzed simultaneously with nd-PCBs.Only 2,2′,4,4′-tetra-bromodiphenyl ether (PBDE-47) was found to be present only in three samples out of 70.

IV. CONCLUSION
The presence of halogenated compounds was investigated in this study.The presence of nd-PCBs were confirmed in 50 samples out of 70.The larger percent of samples was of sea origin which present an environmental contamination source.Lower concentrations were found as well to be present in fish from marine cage culture farms which of course comes from concentration of this compounds in fish feed chain.
Although the PCB finding levels were comparable with those reported in Europe within the Mediterranean area.PBDE 47 was found to be present in three samples.
Further investigation should include fishes from specific environmental area and other food products in order to have a better overview of the presence of these compounds in our environment and diet.

TABLE I :
COMPOSITION OF PENTA-BDE (MARK J. LA GUARDIA 2006)

TABLE II :
INSTRUMENTAL CONDITION

TABLE III :
LIST OF HALOGENATED COMPOUNDS AND THEIR RETENTION TIME, TRANSITION ION AND THEIR COLLISION ENERGY.

TABLE IV :
FISH SPECIES, SAMPLE ORIGIN, NUMBER OF SAMPLE , LENGTH AND WEIGHT OF SAMPLES ANALYZED