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, 



