Biomineral lipids in living and fossil molluscs
It has been proposed that geochemical and biomolecular palaeontological information can be obtained from biomineral associatedli pids. The location of lipidic material within the inorganic structureo f molluscan shells has previously been unknown, with important implications for long term survival of lipids and post-depositional contamination from the environment. Discrete experimental stages have been investigated and the different mechanical and chemical methods combined for the removal of contaminating material prior to the release and analysis of surficial, intercrystalline and intracrystalline lipids. Three extraction protocols have been compared using Recent Patella vulgata shells. Sequential stages of cleaning and extraction treatments identify n-alkanes, cholesterol (free and bound) and bound fatty acids. The n-alkanes are indigenous to the shell, but laboratory contamination can be significant, and highlights the need for experimental blanks. Bound fatty acids are extracted from intercrystalline and intracrystalline fractions. Cholesterol is extracted throughout the sequential methodology. The extraction of these compounds after extensive cleaning treatments illustrates the protective role of the inorganic biomineral. An experimental protocol for sequentially extracting protected lipids from the shells of Recent molluscs has been tested to distinguish the indigenous shell lipids from laboratory contamination and postdepositional ingress. The use of a calcium carbonate blank reveals the phthalate plasticisers extracted from the shells are due to laboratory contamination. Pristane, phytane and free fatty acids were rarely extracted which limits their use for interpretation. The n-alcohols, bound fatty acids, ß-hydroxy fatty acids, cholesterol and other steroids are extracted from the shells in higher yields than the calcium carbonate blanks and are considered indigenous to the shells. Multivariate statistical analysis is used to compare the distributions of bound fatty acids and steroids extracted from different shell locations with the reported fatty acids and steroids for the soft tissues of the same species. The reported values for the soft tissues were used to indicate the original shell lipid composition. The shells lack the unsaturated bound fatty acids reported in the soft tissues. The saturated bound fatty acids of Littorina littorea shells also differ in the carbon number distributions to the reported saturated fatty acids of the soft tissues. Surficial shell extracts are characterized by steroidal ketones, representing sterols which have been oxidised by the cleaning treatments used. The steroids from both intercrystalline and intracrystalline shell locations in Littorina littorea are most similar to the soft tissues. However, the intercrystalline steroids are different to the intracrystalline steroids which may indicate a different original composition. Potential Class level phylogenetic differences between the shells of Recent molluscs are revealed by their steroidal and bound saturated fatty acid compositions. The bivalves (n=3) have bound saturated fatty acids with a carbon number maximum of C16 whilst the gastropods (n=8) have a maximum of C16 or C18 and exhibit higher yields. ß-hydroxy fatty acids may indicate phylogenetic differences below the Class level for the Gastropoda. Principal component statistical analysis of the shell steroidal composition indicates differences at the Class level. Steroidal markers indicating the dietary intake have been found in the shells. The application of a methodology for the sequential extraction of lipids from molluscan shells has been used in a preliminary analysis of shell material for the presence of hydrocarbon pollutants. The shell nalkanes require comparison of carbon number distributions and yields with an experimental calcium carbonate blank to ensure indigeneity. Different n-alkane distributions within two Artica islandica shell samples are attributed to the different sampling locations. Differences between Patella vulgata and Littorina littorea shells from the same environment have also been observed, indicating different n-alkane uptake by different species. Polyaromatic hydrocarbons and sterane biomarkers reported to be present in the soft tissues of Patella vulgata exposed to the Braer oil spill have been searched for in the shells of an exposed sample. These compounds have not been detected. No increase in the shell n-alkane yields or similarity in carbon number distribution with the spilt oil is observed. This suggests no hydrocarbon incorporation or deputation pathway into the shell. Quaternary aged mollusc shells yield n-alkanes, n-alcohols, bound fatty acids and cholesterol. These have been extracted from both intercrystalline and intracrystal line locations within the shells. When compared with the extracts from Recent shells the yields of these lipids from fossil shells are significantly lower. The n-alkanes extracted from Quaternary shells are dominated by laboratory contamination, although some indigenous intracrystalline n-alkanes have been extracted. The bound fatty acids from intercrystalline sites within the fossils maintain their carbonn umber distribution but decreasein yields with increasinga ge; no diagenetic products were observed. The previously reported phylogenetic distinctions based on the bound fatty acids betweent he gastropodsa nd bivalves are maintainedf or fossils. However,t he information obtained from this analysis is limited by the small diversity of lipid distributions found in these fossil shells.