EJERS, European Journal of Engineering Research and Science

Vol. 4, No. 2, February 2019

Analysis of Halogenated Compounds in Fish

Samples
Ederina Ninga, Elvira Beli, Elona Shahu, and Ilirjana Boci

Abstract— The paper reports investigation on the presence of banned worldwide. Presence of PBDE in human body can
13 halogenated compounds (six chlorinated compounds, nd-like cause disturbance of the thyroid hormone homeostasis and
PCBs and seven brominated compounds, polybrominated chronic neurotoxicity (Mehran Alaee 2002).
diphenyl ethers (PBDEs)), in five marine species which are part
on Albanian consumer diet. The samples were extracted with TABLE I: COMPOSITION OF PENTA-BDE (MARK J. LA GUARDIA 2006)
ethyl acetate and treated with sulfuric acid. Extract was Structure Congener Name Concentration
analyzed simultaneously using gas chromatography tandem
2,2′,4,4′-tetra-
mass spectrometry. Presence of PCBs was confirmed at 71.4 BDE-47 38–42%
bromodiphenyl ether
percent of the samples. The most dominated congener found to
be present were PCB 153 and PCB 138. Form the brominated
,2′,3,4,4′-penta-
compounds 2,2′,4,4′-tetra-bromodiphenyl ether was the only BDE-85 2.2–3.0 %
bromodiphenyl ether
detected compound in 3 samples.

Index Terms— Chlorinated compounds, Polybrominated 2,2′,4,4′,5-penta-

45–49%
BDE-99 bromodiphenyl ether
diphenyl ethers, Fish.

2,2′,4,4′,6-penta-
I. INTRODUCTION BDE-100 7.8–13 %
bromodiphenyl ether

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
2,2′,4,4′,5,5′-hexa-
BDE-153 5.3–5.4 %
bromodiphenyl ether
2,2′,4,4′,5,6′-hexa-
BDE-154
bromodiphenyl ether
2.7–4.5 %
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.
II. MATERIAL AND METHODS
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.
Nitrogen Evaporator. — Thermo/Scientific, Reacti-
Therm, #TS18823 type was used for the solvent evaporation.
Vials and vessels. — 50 mL and 15 ml fluorinated ethylene
propylene (FEP) centrifuge tubes with ethylene–
tetrafluoroethylene screw closures were used.
Reagents
Ethyl Acetate and Hexane. —Pesticide residue analysis

Ederina Ninga is with Food Safety and Veterinary Institute, Aleksandër Elona Shahu is with Food Safety and Veterinary Institute, Aleksandër
Moisiu 82, Tirane, Albania ( email: ederina.ninga@yahoo.com). Moisiu 82, Tirane, Albania (email: elona_shahu@yahoo.com ).
Elvira Beli is with Faculty of Agriculture and Environment, Agricultural Ilirjana Boci is with Faculty of Natural Sciences, University of Tirana,
University of Tirana, Albania ( email: elvira.beli@yahoo.com ) Bulevard Zog I, Tirana, Albania (email: ilirjana.boci@fshn.edu.al ).

DOI: http://dx.doi.org/10.24018/ejers.2019.4.2.1099 37
 EJERS, European Journal of Engineering Research and Science
Vol. 4, No. 2, February 2019

grade from WVR were used. glass vial and brought to dry. The samples were reconstituted
Na2SO4 —Pesticides grade anhydrous Na2SO4 from WVR. with 0.1 ml ethyl acetate.
Halogenated standards solution — Mix of PCBs standard
solution from Sigma Aldrich, and Polybrominated Diphenyl TABLE II: INSTRUMENTAL CONDITION
Ethers (PBDE) from RoHS. GC Conditions
PSA and C18 sorbent — Primary Secondary Amine (PSA) Agilent DB-5 MS UI 60 m × 250 µm ×
Column 0.25 µm (p/n 122-5562UI) inlet PTV,
sorbent (40 mm particle size) and C18 from Agilent.
outlet vacuum
Samples Injection volume 1 µL
The determination of the halogenic compounds in fish was Solvent vent 45 °C (3 minutes), ramp at
based in ethyl acetate extraction followed from quantitative 720 °C/min to 320 °C Vent flow 100
determination of compounds in GC/MS/MS triple Injection mode mL/min pressure of 10 psi for 2.8
quadrupole. At total of 70 samples were randomly chosen minutes Purge flow was set to 1200
mL/min after 5 minutes.
from wild marine (38) fish and cultivated fish (32).
Carrier gas Helium
All the collected samples were kept in ice during the Carrier gas mode Constant flow
transportation to the lab the samples. They were kept in Column flow 1.5 mL/min
frozen condition in -20C until further analysis. 80 C for 1 min , then 15 C / min to
Oven program
Analytical method 300 for 3 min
Ten gram of fish muscle (edible part) was accurately Total run time 18.667 min
weighted in a 50 ml centrifugal tube. PSA (0.5 g) and C18 (0.5 MS Conditions
Electron ionization (EI), Multiple
g) were added to all the samples followed by 10 ml EtOAc. Operation mode
Reaction Monitoring (MRM)
After 20 minutes’ extraction in a horizontal shaker 5 gram of Transfer line temperature 280 °C
Na2SO4 anhydrous was added to each sample and another Source temperature 280 °C
extraction step was performed for another 10 minutes. Extract Quadrupole temperature 150 °C
was separated by centrifugation at 3200 rmp for 10 minutes
at zero degree Celsius. After the centrifugation 5 ml extract Data acquisition and analysis
was transferred in a 15 ml tube and then totally dried by using The data were acquired with Agilent MassHunter
under nitrogen flow. After drying the samples were treated Acquisition Software (B.07.02). Data analysis was performed
with 1 ml of sulfuric acid and the PCB/PBDE were with Agilent MassHunter Quantitative Analysis Software
reconstituted with 2 ml of hexane. The tubes were centrifuged (B.07.01).
in order to improve the separation between the acid and the
solvent layer. 1 ml from the hexane layer was transferred to e
TABLE III: LIST OF HALOGENATED COMPOUNDS AND THEIR RETENTION TIME, TRANSITION ION AND THEIR COLLISION ENERGY.

Name Type RT Mass Transition CE Mass Transition CE Mass Transition CE

PCB 28 ndl-PCB 10.88 255.8 -> 186.0 32 255.8 -> 151.0 50

PCB 52 ndl-PCB 11.34 291.8 -> 221.9 28 289.9 -> 219.9 28

PCB 101 ndl-PCB 12.48 327.9 -> 257.9 28 325.9 -> 291.0 12 325.9 -> 256.0 34

BDE 17 PBDE 13.10 408.0 -> 248.0 20 408.0 -> 246.0 20 245.0 -> 138.0 30

BDE 28 PBDE 13.31 408.0 -> 248.0 20 408.0 -> 246.0 20 245.0 -> 138.0 30

PCB 153 ndl-PCB 13.55 361.9 -> 291.8 28 359.8 -> 325.0 15 359.8 -> 289.9 28

PCB 138 ndl-PCB 13.90 361.9 -> 291.8 28 359.8 -> 325.0 15 359.8 -> 289.9 28

PCB 180 ndl-PCB 14.71 395.8 -> 325.8 30 393.8 -> 323.8 30 393.7 -> 359.0 15

BDE 47 PBDE 14.77 486.0 -> 327.9 20 486.0 -> 325.9 20

BDE 99 PBDE 16.13 564.0 -> 405.8 30 564.0 -> 403.9 25

BDE 100 PBDE 15.83 564.0 -> 405.8 30 564.0 -> 403.9 25

BDE 153 PBDE 17.75 643.6 -> 483.6 30 483.7 -> 374.9 45

BDE 154 PBDE 17.16 643.6 -> 483.6 30 483.7 -> 374.9 45

DOI: http://dx.doi.org/10.24018/ejers.2019.4.2.1099 38
 EJERS, European Journal of Engineering Research and Science
Vol. 4, No. 2, February 2019

III. RESULTS
During this study the presence of halogenic compounds in
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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.

DOI: http://dx.doi.org/10.24018/ejers.2019.4.2.1099 39