Organics
201
fluorescence spectroscopy would give a reasonable estimation of the relative degree of
contamination of mussels by petroleum hydrocarbons.
8.2.2
Polyaromatic Hydrocarbons
Bjorseth et al. [574] described a capillary gas chromatographic method for determining PAHs in mussels. Up to 34 PAHs were identified. Dunn and Stich [575] have
described a monitoring procedure for PAHs, particularly benzo(a)pyrene in marine
organisms in coastal waters. The procedures involve extraction and purification of
hydrocarbon fractions from the sediments or organisms, and determination of compounds by thin layer chromatography and fluorimetry, or gas chromatography.
To avoid possible photodecomposition of P AHs, all extraction and purification
procedures were carried out under subdued yellow tungsten light. Between 20 and
40 g of tissue were placed in a 300 ml flask and 150 ml of ethanol, 7 g of potassium
hydroxide, boiling chips, and an aliquot of radioactive benzo(a)pyrene (either
1000 dpm i4C-benzo(a)pyrene, around 51lg or 25000 dpm 3H-benzo(a)pyrene,
around 0.1 ng) were added. The tissue was digested by refluxing gently for 1.5 h with
occasional swirling. The digest was added while hot to 150 ml of water in a 2-1
separatory funnel, and the digestion flask rinsed out with an additional 50 ml of
ethanol. The water-ethanol mix was extracted three times with 200 ml of iso-octane,
and the iso-octane extracts were combined and washed with 4 x 200 ml warm (60°C)
water. This extract was then passed down a Florisil clean up column. Polycyclic
aromatic hydrocarbons were eluted from the column with 3 x 100 ml benzene. The
combined eluate was reduced to 5 ml by rotary evaporation, 50 ml of iso-octane were
added, and the volume again reduced to 5 ml to remove the benzene.
P AHs were extracted from the iso-octane with 3 x 5 ml dimethyl sulphoxide. The
dimethyl sulphoxide extracts were combined with 30 ml of water, and the P AHs
extracted into 2 x 10 ml iso-octane. The iso-octane extracts were combined, washed
with water, and dried by passage through 10 g of sodium sulphate in a 15 ml coarse
fritted glass Buchner funnel. This extract was used for thin layer chromatography
benzo(a)pyrene being detected under long wavelength ultraviolet light.
The adsorbent at the position of the benzo(a)pyrene band was scraped off the plate
while still damp, and placed in a fine fritted Buchner funnel. The benzo(a)pyrene was
removed from the cellulose acetate by washing with 4 x 4 ml hot (65°C) methanol,
using gentle suction. The methanol was added to 10 ml of a solution of 20 % hexadecane in iso-octane, and the methanol and iso-octane were removed by rotary evaporation to leave the benzo(a)pyrene in 2 ml of hexadecane, ready for fluorimetry.
Benzo(a)pyrene was measured fluorimetrically in hexadecane using the baseline
technique of Kunte [576]. Samples and standards of 10-200 ng benzo(a)pyrene ml- i in
hexadecane were excited at 365 nm in an Aminco-Bowman spectrophotofluorimeter,
and the emission spectrum was recorded from 375 to 500 nm. An artificial baseline
was drawn between minima in the fluorescence spectrum occurring at 418 and
448 nm, and the height of the peak at 430 nm above this baseline was measured.
Where necessary, highly fluorescent samples were diluted with hexadecane to bring
their fluorescence within the range of the standards used.
201
fluorescence spectroscopy would give a reasonable estimation of the relative degree of
contamination of mussels by petroleum hydrocarbons.
8.2.2
Polyaromatic Hydrocarbons
Bjorseth et al. [574] described a capillary gas chromatographic method for determining PAHs in mussels. Up to 34 PAHs were identified. Dunn and Stich [575] have
described a monitoring procedure for PAHs, particularly benzo(a)pyrene in marine
organisms in coastal waters. The procedures involve extraction and purification of
hydrocarbon fractions from the sediments or organisms, and determination of compounds by thin layer chromatography and fluorimetry, or gas chromatography.
To avoid possible photodecomposition of P AHs, all extraction and purification
procedures were carried out under subdued yellow tungsten light. Between 20 and
40 g of tissue were placed in a 300 ml flask and 150 ml of ethanol, 7 g of potassium
hydroxide, boiling chips, and an aliquot of radioactive benzo(a)pyrene (either
1000 dpm i4C-benzo(a)pyrene, around 51lg or 25000 dpm 3H-benzo(a)pyrene,
around 0.1 ng) were added. The tissue was digested by refluxing gently for 1.5 h with
occasional swirling. The digest was added while hot to 150 ml of water in a 2-1
separatory funnel, and the digestion flask rinsed out with an additional 50 ml of
ethanol. The water-ethanol mix was extracted three times with 200 ml of iso-octane,
and the iso-octane extracts were combined and washed with 4 x 200 ml warm (60°C)
water. This extract was then passed down a Florisil clean up column. Polycyclic
aromatic hydrocarbons were eluted from the column with 3 x 100 ml benzene. The
combined eluate was reduced to 5 ml by rotary evaporation, 50 ml of iso-octane were
added, and the volume again reduced to 5 ml to remove the benzene.
P AHs were extracted from the iso-octane with 3 x 5 ml dimethyl sulphoxide. The
dimethyl sulphoxide extracts were combined with 30 ml of water, and the P AHs
extracted into 2 x 10 ml iso-octane. The iso-octane extracts were combined, washed
with water, and dried by passage through 10 g of sodium sulphate in a 15 ml coarse
fritted glass Buchner funnel. This extract was used for thin layer chromatography
benzo(a)pyrene being detected under long wavelength ultraviolet light.
The adsorbent at the position of the benzo(a)pyrene band was scraped off the plate
while still damp, and placed in a fine fritted Buchner funnel. The benzo(a)pyrene was
removed from the cellulose acetate by washing with 4 x 4 ml hot (65°C) methanol,
using gentle suction. The methanol was added to 10 ml of a solution of 20 % hexadecane in iso-octane, and the methanol and iso-octane were removed by rotary evaporation to leave the benzo(a)pyrene in 2 ml of hexadecane, ready for fluorimetry.
Benzo(a)pyrene was measured fluorimetrically in hexadecane using the baseline
technique of Kunte [576]. Samples and standards of 10-200 ng benzo(a)pyrene ml- i in
hexadecane were excited at 365 nm in an Aminco-Bowman spectrophotofluorimeter,
and the emission spectrum was recorded from 375 to 500 nm. An artificial baseline
was drawn between minima in the fluorescence spectrum occurring at 418 and
448 nm, and the height of the peak at 430 nm above this baseline was measured.
Where necessary, highly fluorescent samples were diluted with hexadecane to bring
their fluorescence within the range of the standards used.