Effects of preservatives and acidification on the stable isotope ratios (15N:14N, 13C:12C) of two species of marine animals

When animal tissues are prepared for stable isotope ratio analysis, they may or may not be treated with acid prior to analysis to remove carbonates and are loaded into tin or silver weigh boats for quantitative combustion. The effects of these methodological variations are poorly known. The effects of various preservation methods on isotopic compositions are also poorly known. We tested the effects of four preservation methods, (i) formalin, (ii) formalin followed by a transfer to ethanol (formalin/EtOH), (iii) saturated mercuric chloride solution, and (iv) freezing/freeze-drying, on the carbon and nitrogen isotopic composition of the muscle tissue of juvenile winter flounder (Pleuronectes americanus) and the tails (including exoskeleton) of mud shrimp (Crangon septemspinosa). Freezing and freeze-drying were the only preservation methods that did not affect stable isotope ratios of carbon and nitrogen. Formalin, formalin/EtOH, and saturated mercuric chloride solution produced significant increases in δ15N values (0.5-1.4‰) and decreases in δ13C values (0.6-2.3‰) compared with frozen samples. There was also an increase in the variability of δ15N and (or) δ13C values. We also tested the effects of acidification by comparing samples that were acidified either by fuming with concentrated HCl or by the direct application of 1 N HCl containing 1.0% platinum chloride (a combustion catalyst) to unacidified samples. Neither concentrated HCl fumes nor HCl/platinum chloride had a significant effect on the δ15N or δ13C values of either species compared with unacidified samples. Therefore, acidification may be unnecessary in the preparation of some marine animals. Finally, we compared the effects of two types of sample boats: tin and silver. We found no significant effect of boat material on the δ15N or δ13C values of either species.