An Assessment of Sample Preservation Methods for the Determination of Stable Carbon and Nitrogen Isotope Ratios in Mollusks

Abstract

The aim of this study was to quantify the effects of preservation methods and preservation times on the stable carbon and nitrogen isotope values in the tissues of six mollusk species (Mytilus edulis, Crassostrea gigas, Ruditapes philippinarum, Acanthochiton seulschnochilon, Littorina brevicula, and Rapana venosa). To identify potential preservation effects on d 13 C and d 15 N values and to examine temporal changes in the effects of preservation, repeated analyses were carried out after 1 day, 5 days, 15 days, 1 month, 3 months, 6 months, and 12 months of preservation. The results showed that drying preservation was the most suitable method for preserving samples, while freezing and chemical preservation significantly affected the stable isotope values compared with those of the controls. The effects of preservatives on the tissues of different mollusk species were statistically significant for both d 13 C and d 15 N values. Shifts in the d 13 C and d 15 N values, due to freezing and chemical preservation, were higher in Acanthochiton seulschnochilon, Littorina brevicula, and Rapana venosa than in the other three species. The effects of preservatives on carbon isotope values were variable. In most cases, the shift of the d 13 C values from the control samples were less than 1% for those of the treated samples. The d 13 C and d 15 N data from the preserved samples could thus be used in food web reconstruction studies. Further studies will be necessary, however, in order to elucidate the effects of preservation type and time on other species.

Effects of ethanol-preservation on stable carbon and nitrogen isotopic signatures in marine predators

“…Such δ 15 N enrichment by ethanol was previously reported in several studies of fish (Sweeting et al 2004, Kelly et al 2006) and molluscs (e.g. Syväranta et al 2011, Liu et al 2013. Because ethanol could cause tissue hydrolysis and leaching (Sarakinos et al 2002), we infer that 70% ethanol effectively extracted certain constituents containing nitrogen from the muscle tissue in addition to lipids.…”

“…Sweeting et al 2004. Kelly et al 2006, Syväranta et al 2011, Liu et al 2013. We attribute this effect to the substance extraction process, since subsequent lipid extraction treatment (ET-LE) substantially reduced this variation.…”

“…However, we consider that the method presented in this study could also be applied to analyses of archived samples due to the following reasons; first the most significant effects of ethanol preservation on stable isotopic signatures generally occur within a short period, <1 day (e.g. Sweeting et al 2004, Liu et al 2013. Second, the additional treatment with highly-concentrated ethanol applied in this study is able to fully realise the potential of lipid extraction by ethanol, and thus is effective in offsetting the time-dependent effect of ethanol-based substance extraction.…”

Freezing and fractionation: effects of preservation on carbon and nitrogen stable isotope ratios of some limnetic organisms

“…Gathering samples from food‐web members presents some practical problems that complicate the interpretation of food‐web structure from isotopes. Drying samples immediately from a fresh state is the preferred method of sample preparation for isotopic analysis because it is free from preservative effects and specimens can be stored for extended periods of time in a dried state . However, conditions in the field often make immediate processing impossible, so sample preservation is usually required to prevent decomposition and associated isotopic fractionation of samples before they can be processed in the laboratory …”

“…Drying samples immediately from a fresh state is the preferred method of sample preparation for isotopic analysis because it is free from preservative effects and specimens can be stored for extended periods of time in a dried state. [2,9] However, conditions in the field often make immediate processing impossible, so sample preservation is usually required to prevent decomposition and associated isotopic fractionation of samples before they can be processed in the laboratory. [10,11] Considerable research has demonstrated that chemical preservatives (e.g., alcohol) and fixatives (e.g., formalin) can shift the isotope ratios of aquatic organisms but not in a systematic way.…”