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100 RPM. After two, four, and six weeks of incubation at 15°C, the oil
remaining in replicate flasks (two at each sampling time) was extracted
and analysed as described below.
Additionally, replicate 100 g portions of sediment were placed into
1 liter stainless steel buckets. The containers were continuously
flushed with a solution of Rila marine salts supplemented with 10 ppm
KNO + 10 ppm KH PO . The height of the water level was adjusted to be
3 cm above the surface of the sediment layer. The flow rate was
adjusted to 10 ml/h. After two, four, and six weeks of incubation at
15°C the oil remaining in replicate sediment portions was extracted and
analysed as described below.
Analyses of ^in vitro Experiments
Residual oil was recovered from samples by extraction with
sequential portions of diethyl ether and methylene chloride. The
sediment was shaken at 200 RPM with repetitive portions of solvent. The
extracts were subjected to column chromatography to split the extracts
into aliphatic (f,) and aromatic (f~) fractions. Columns were prepared
by suspending silica gel 100 (E. M. Reagents, Darmstadt, W. Germ.) in
CH„C1„ and transferring the suspension into 25 ml burets with teflon
stopcocks to attain a 15 ml silica gel bed. The CH„C1 ? was washed from
the columns with three volumes of pentane. Portions of the extracts in
pentane were applied to the columns, drained into the column bed, and
allowed to stand for three to five minutes. The aliphatic fraction (f )
was eluted from the column with 25 ml pentane. After 25 ml pentane had
been added to the column, 5 ml of 20% (v/v) CH„C1„ in pentane was added
and allowed to drain into the column bed. Fraction f was 30 ml. The
aromatic fraction (f ) was eluted from the column with 45 ml of 40%
(v/v) CH 2 C1„ in pentane.
The fractions of each extract were then concentrated to about 5 ml
at 35°C and transferred quantitatively to clean glass vials. Fractions
f. and f were prepared for analysis by gas chromatography or gas
chromatography mass spectrometry. An internal standard, hexamethyl
benzene (Aldrich Chem. Co., Milwaukee, WI.), was added to each sample.
In fraction f . , hexamethyl benzene (HMB) was present at 12.6 ng/ml; in
fraction f„, HMB was present at 25.2 ng/ml.
Fraction f. was analyzed by GC on a Hewlett-Packard 5840 reporting
GC with FID detector. The column was a 30 m, SE54 grade AA glass
capillary (Supelco, Bellefonte, PA.). Conditions for chromatography
were injector, 240°C; oven 70°C for 2 min. to 270°C at 4°C/min. and hold
for 28 min.; FID, 300°C; and carrier, He at 25 cm/sec. A valley-valley
intergration function was used for quantitative data acquisition.
Response factors were calculated using n-alkanes, (C -C ) , pristane
and phytane standards.
Fraction f„ was analyzed with a Hewlett-Packard 5992A GC-MS.
Conditions for chromatography were injector, 240°C; oven 70°C for 2 min.
to 270°C at 4°C/min. and hold for 18 min. Data was acquired using a
selected ion monitor program. Thirteen ions were selected for
representative aromatic compounds. The ions monitored were 128, 142,