198
Crustacea and Molluscs
performance liquid chromatography. Quantitation and identification of the individual
compounds are accomplished using gas chromatography and gas chromatographymass spectrometry. The nonvolatile polynuclear aromatic hydrocarbons which remain
in the homogenate after headspace sampling are solvent-extracted, and then analysed
by reversed phase liquid chromatography. The crustacea samples were kept at a low
temperature (-10°C) between sampling and analysis. Approximately 30 g tissue, 500 ml
hydrocarbonfree water, and 50 g of sodium hydroxide were combined in a flask,
together with aliphatic or aromatic hydrocarbon internal standards, and the mixture
homogenized. The tissue homogenate was heated to 70°C, and the headspace sampled
for 18 h at a nitrogen flow rate of 150 ml min-I. The headspace vapours were passed
into a Tenax GC packed stainless steel column. The homogenate solution remaining in
the flask after headspace sampling was extracted with pentane to remove non-volatile
polyaromatic hydrocarbons. This extract was concentrated by nitrogen purge, and
devolved in 1 ml acetonitrile for high performance liquid chromatography.
Following headspace sampling and drying, the Tenax GC was connected as part of
the injection loop of a liquid chromatograph and the organic compounds eluted with
pentane onto a Jl Bondepak NH2 clean-up column, the first 15 ml of eluate containing
the hydrocarbons. This fraction was reduced to 300 Jl by nitrogen purge and the
residue washed onto a Tenax GC column, the contents of which were thermally
purged onto a gas chromatographic column for analysis.
Recovery data for the aromatic and aliphatic compounds used as internal standards
in the tissue analyses are given in Table 8.13. Using an 18 h headspace sampling
period, recoveries from water for the higher molecular weight aromatic and aliphatic
components (i. e. trimethyl naphthalene, phenanthrene, MeC I6 and MeC 18 ) were nearly 100 %. Aliphatic hydrocarbon recoveries were found to be much lower than aromatic hydrocarbon recoveries in the headspace sampling of the tissue homogenate.
Using caustic digestion, recoveries from mussel tissue homogenate approached 100 %
for the higher aromatics but were only 30 % for the aliphatic components. It is
assumed that the aliphatic hydrocarbons were being retained in the lipophilic portion
of the tissue homogenate and that the partition coefficient for these hydrocarbons
between the headspace sampling gas and the lipophilic fraction was quite unfavourable. Recovery data for the complete analytical scheme indicate that some losses of the
internal standards also occur during the liquid chromatographic cleanup and concentration step. The losses that occur during the concentration step amount to 25 % for
mesitylene, 30 % for 2-methylundecane, 40 % for naphthalene, 11 % for 5-methyltetradecane, 5 % for trimethylnaphthalene, and less than 1 % for 7-methylhexadecane,
2-methylnaphthalene, phenanthrene, and hydrocarbons of higher molecular weight.
Since quantitation in these analyses was dependent upon an internal standard
added at the beginning of the analytical scheme, it was imperative to know whether
the internal standard components were recovered to the same extent as these components would be if incorporated in the tissue matrix. In a series of experiments with
live Mytilus (mussels) exposed to 14C-naphthalene and then analyzed using the 4-h
headspace sampling procedure and no HPLC cleanup, a HC recovery of 78 ± 12 %
was observed. In comparison, the recovery of nonlabeled naphthalene added as an
internal standard was found to be 66 ± 8 % for the same 4-h headspace sampling
procedure (see Table 8.13). Indications are, therefore, that at least in the case of
Crustacea and Molluscs
performance liquid chromatography. Quantitation and identification of the individual
compounds are accomplished using gas chromatography and gas chromatographymass spectrometry. The nonvolatile polynuclear aromatic hydrocarbons which remain
in the homogenate after headspace sampling are solvent-extracted, and then analysed
by reversed phase liquid chromatography. The crustacea samples were kept at a low
temperature (-10°C) between sampling and analysis. Approximately 30 g tissue, 500 ml
hydrocarbonfree water, and 50 g of sodium hydroxide were combined in a flask,
together with aliphatic or aromatic hydrocarbon internal standards, and the mixture
homogenized. The tissue homogenate was heated to 70°C, and the headspace sampled
for 18 h at a nitrogen flow rate of 150 ml min-I. The headspace vapours were passed
into a Tenax GC packed stainless steel column. The homogenate solution remaining in
the flask after headspace sampling was extracted with pentane to remove non-volatile
polyaromatic hydrocarbons. This extract was concentrated by nitrogen purge, and
devolved in 1 ml acetonitrile for high performance liquid chromatography.
Following headspace sampling and drying, the Tenax GC was connected as part of
the injection loop of a liquid chromatograph and the organic compounds eluted with
pentane onto a Jl Bondepak NH2 clean-up column, the first 15 ml of eluate containing
the hydrocarbons. This fraction was reduced to 300 Jl by nitrogen purge and the
residue washed onto a Tenax GC column, the contents of which were thermally
purged onto a gas chromatographic column for analysis.
Recovery data for the aromatic and aliphatic compounds used as internal standards
in the tissue analyses are given in Table 8.13. Using an 18 h headspace sampling
period, recoveries from water for the higher molecular weight aromatic and aliphatic
components (i. e. trimethyl naphthalene, phenanthrene, MeC I6 and MeC 18 ) were nearly 100 %. Aliphatic hydrocarbon recoveries were found to be much lower than aromatic hydrocarbon recoveries in the headspace sampling of the tissue homogenate.
Using caustic digestion, recoveries from mussel tissue homogenate approached 100 %
for the higher aromatics but were only 30 % for the aliphatic components. It is
assumed that the aliphatic hydrocarbons were being retained in the lipophilic portion
of the tissue homogenate and that the partition coefficient for these hydrocarbons
between the headspace sampling gas and the lipophilic fraction was quite unfavourable. Recovery data for the complete analytical scheme indicate that some losses of the
internal standards also occur during the liquid chromatographic cleanup and concentration step. The losses that occur during the concentration step amount to 25 % for
mesitylene, 30 % for 2-methylundecane, 40 % for naphthalene, 11 % for 5-methyltetradecane, 5 % for trimethylnaphthalene, and less than 1 % for 7-methylhexadecane,
2-methylnaphthalene, phenanthrene, and hydrocarbons of higher molecular weight.
Since quantitation in these analyses was dependent upon an internal standard
added at the beginning of the analytical scheme, it was imperative to know whether
the internal standard components were recovered to the same extent as these components would be if incorporated in the tissue matrix. In a series of experiments with
live Mytilus (mussels) exposed to 14C-naphthalene and then analyzed using the 4-h
headspace sampling procedure and no HPLC cleanup, a HC recovery of 78 ± 12 %
was observed. In comparison, the recovery of nonlabeled naphthalene added as an
internal standard was found to be 66 ± 8 % for the same 4-h headspace sampling
procedure (see Table 8.13). Indications are, therefore, that at least in the case of