Chemical analyses
Metals and organic pollutants in soft tissues of mussels were determined in freeze-dried,
homogenized whole samples. Levels of Ni, Mn, Zn, Cu, Cd, Pb, Hg, As, and Pb were
determined in acid-digested samples in a Perkin Elmer model Elan 6000 inductively
coupled plasma mass spectrometer (ICP-MS) (Barata et al. 2005). Samples of similar
weight of a certified reference material (dogfish muscle, NRCDORM-2; LGC
Promochem, Middlesex, UK) were digested during each analytical run. Analyses of
organic pollutants in mussel samples followed previous methods (Sánchez-Avila et al.
2011) and included fourteen Environmental Protection Agency (EPA) polycyclic
aromatic hydrocarbons (PAHs), seven polychloro biphenils (PCBs), DDT derivatives
and four altkylphenols (APs). PAHs were purchased from AccuStandard (New Haven,
CT, USA) as a solution mix at 1000 μg L−1 in methanol; phthalates were from Supelco
(Bellefonte, PA, USA) as a mix solution at 500 μg L−1 in methanol; PCBs (28, 52, 101,
118, 138, 153 and 180) were from Dr. Ehrenstorfer (Augsburg, Germany) as a mix
solution at 10 μg L−1 in iso-octane; DDT derivates were also obtained from Dr.
Ehrenstorfer GmbH; 4-Nonylphenol technical mixture (NP), 4-tertoctylphenol (OP), 4nonylphenol-mono-ethoxylate (NP1EO) and 4-nonylphenol-di ethoxylate (NP2EO)
from Dr. Ehrenstorfer GmbH. Stock standard solutions (100 mg L−1) of each compound
were prepared in absolute methanol and stored in the dark at −20 °C until use. These
solutions were used to spike the mussel samples and draw the calibration curves. The
surrogate standards (used for analyte quantification) for PAHs was a mix solution at
2000 μg L−1 in methanol containing anthracene D10, acenaphthylene D8,
benzo(a)pyrene D12, benzo(h,g,i)perylene D12, fluoranthene D10, naphthalene D8,
phenanthrene D10 and pyrene from Riedel-de Haën (Seelze, Germany), purchased as a
solid; PCBs surrogate standard was PCB 209 from Dr. Ehrenstorfer GmbH, used as a
solution at 10 μg L−1 in iso-octane; alkylphenols surrogate standard was 4-nnonylphenol-d8 and nonylphenol monoethoxylate D2 from Dr. Ehrenstorfer (Augsburg,
Germany) as a solution at 100 μg L−1 in acetone; anthracene d10 used as internal
standard, from Dr. Ehrenstrofer as a solution of 10 μg L−1 in cyclohexane. HPLC-grade
methanol, hexane, dichloromethane and acetone for organic trace analyses were
supplied by Merck (Darmstadt, Germany).
Extraction and clean up: surrogate standards (4-n-NP-D8, NP1EO-D2, 4.4-DDD
13C12, PCB 209, PAHs surrogates solution were added to a sample aliquot of (0.1 –
1g), to get a final concentration of 100 ng mL−1. Samples were homogenized and kept
at 4ºC overnight and subsequently extracted by sonication (10 min) one time with
hexane/dichloromethane (1:1, v/v) in a ratio of 100 mL per gram of dried mass. The
second part of the extraction (same ratio) was performed with a mixture of
hexane/acetone (1:1, v/v). After each extraction step, samples were centrifuged for 10
min and the extracts were combined and concentrated to approximately 1 mL under a
nitrogen current using a TurboVap LV system (Capiler Life Sciences, UK) at 25°C.
Extracts were subsequently cleaned up by solid phase extraction (SPE) cartridges with 5
g of florisil, previously conditioned with 20 mL of hexane/dichloromethane (1:1,v/v)
and 20 mL of hexane/acetone (1:1, v/v). The sample extract was eluted with 20 mL of
hexane/dichloromethane (1:1, v/v) and 20 mL of hexene/acetone (1:1, v/v). The eluent
was concentrated to a volume of less than 1 mL under a nitrogen current at room
temperature and reconstituted with ethyl acetate to a final volume of 200 µL, with
addition of 1 μg mL−1 of the internal standard Anthracene D10.
Final extracts were analyzed by gas chromatography – electronic impact – mass
spectrometry in tandem (GC-EI-MS/MS). Analyzes were performed using an Agilent
7890A GC System (Agilent Technologies, Palo Alto, CA, USA) interfaced to a 7000A
triple quadrupole mass spectrometer system (Agilent, USA). Separation and detection
method parameters were similar to those reported by (Sánchez-Avila et al. 2011). For
increased sensitivity and specificity, peak detection and quantification was performed in
Selected Reaction Monitoring (SRM) mode using two transitions per each compound.
Internal standard quantification was performed using the surrogate standards method.
Five points calibration curve were constructed at 100, 500, 1000, 5000 and 10000 ng/g
with good linearity over this concentration range (R2 > 0.994). The method limits of
detection (MLD) of the analytical method were calculated with spiked mussel samples
at 100 ng/g of native compounds. MLDs for alkylphenols varied from 26.3 to 125.1
ng/g; for PAHs from 7.3 to 58.3 ng/g; for PCBs from 3.8 to 19.1 ng/g; for
organochlorinated pesticides from 3.9 to 31.6 ng/g. Uncontaminated mussel tissue
doped with 100ng/g of a solution containing all tested native compounds was analyzed
along with samples to determine recovery rates. Sample blanks were rigorously
performed to eliminate any external source of contamination.