Page 11 - 825-IJMS-for Dr. Aldo Pacheco Ferreira

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International Journal of Marine Science 2013, Vol.3, No.26, 201-211
This region present its northern and eastern area
limited by the continent, a sandbank vegetation on
southern limit, and Ilha Grande Bay on the west. Its
greatest length is 42.5 kilometers from east to west
and its greatest width is 17.2 kilometers from north to
south, with a perimeter of 122 km. The tidal range is
approximately 1 m. predominantly north-easterly and
south-westerly winds activate thermal currents
between the bay and the ocean. The bay plays an
important role in regional aquatic ecology, mainly for
juvenile fishes that use the area as a rearing ground.
Its importance is unquestionable because it is a
breeding place for various fish species in its extensive
areas of mangroves and estuaries, and the fishing
industry is an important economic and social support
for the region (Perez et al., 2003). However, all these
activities are threatened by the presence of an
industrial complex composed of about 400 industries
(including metallurgical, petrochemical and smelting
companies, and also a leather tanning facility). These
industries release large amounts of chemicals that
reach the bay through the main rivers and by
atmospheric deposition (Souza et al., 2012).
3.2 Sampling
A total of 59 fishes (
Genidens genidens
, n=15,
Harengula clupeola
, n=22,
Haemulum steidachneri
Sphyraena sphyraena
, n=13) were captured
from Sepetiba Bay in July-August 2012 using two
procedures: a) manual fishing using multifilament net
(1.80 m in height, 9 m in width, and 5 mm mesh size);
and b) bottom trawling (using 20-mm mesh size
multifilament net with a 1.5 m opening and trawl
doors 60 cm in height).
Prior to analysis, each sample was categorized upon
its sample number, fish species, length (cm), weight (g)
and gender. The gender was determined macroscopically
by gonad observation after dissection; gonad stage
was determined according to Vazzoler (1996). The
head, tail fin and viscera were removed from the
fish, before analysis. The samples were sealed in
polyethylene bags and stored at
for posterior
3.3 Analytical procedures
PCBs and PCDD/Fs were extracted according to a
wide tested method (Ferreira, 2012). The samples
were analyzed by high-resolution mass spectrometer
(HRGC/HRMS) according to the USEPA 1613B
(1994), USEPA 8290A (2003), and USEPA 1668B
(2007) Methods, as quality control. 10 g of
freeze-dried combined pooled tissue samples from
each species were weighed and lyophilised. Dry
tissues were introduced in a steel extraction cell and
placed in the Accelerated Solvent Extractor (ASE 200,
Dionex). This machine using organic solvents
operates under high pressure and temperature
conditions (10 minutes at 125
and 1500 psi) and
permits the extraction of the different organic
compounds present from the biological matrix.
Subsequently being extracted, the samples were
concentrated using Kuderna-Danish, the extract
evaporated down to 1 mL, and the solvent was
transported to 10 mL of n-hexane.
Seventeen 2,3,7,8-substituted 13C-labeled tetra- through
octa-CDD and CDF congeners and 12 dioxin-like
PCBs (IUPAC-81, 77, 126, 169, 105, 114, 118, 123,
156, 157, 167, 189) were spiked. Moreover, aliquots
were treated with sulfuric acid (approximately 7-10
times) in a separation funnel. Then the hexane layer
with PCBs and PCDDs/DFs was rinsed with
hexane-washed water and dried by passing through
anhydrous sodium sulfate in a glass funnel. The
solution was concentrated to 2 mL and sequentially
subjected to silica gel, alumina, and silica
gel-impregnated activated carbon column chromatography.
Extracts were passed through a silica gel-packed glass
column (Wakogel, silica gel 60; 2 g) and eluted
with 130 mL of hexane. The hexane extract was
Kuderna-Danish concentrated and passed through
alumina column (Merck-Alumina oxide, activity grade
1; 5 g) and eluted with 30 mL of 2% dichloromethane
in hexane as a first fraction, which contained
multi-ortho-substituted PCBs. The second fraction
eluted with 30 mL of 50% dichloromethane in hexane,
containing non- and mono-ortho-PCBs and PCDDs/DFs,
was Kuderna-Danish concentrated and passed through
silica gel-impregnated activated carbon column (0.5 g).
The first fraction eluted with 25% dichloromethane in
hexane contained mono- and di-ortho-PCBs. The
second fraction eluted with 250 mL of toluene
containing PCDDs/DFs was concentrated and
analyzed using a high-resolution gas chromatograph
interfaced with a high-resolution mass spectrometer