Abstract
Stable isotope analysis has become one of the most widely used techniques in ecological studies. However, there are still uncertainties about the effects of sample preservation and pre-treatment on the ecological interpretation of stable isotope data and especially on Bayesian stable isotope mixing models. Here, Bayesian mixing models were used to study how three different preservation methods (drying, freezing, formalin) and two pre-treatments (acidification, lipid removal) affect the estimation of the utilized organic matter sources for two benthic invertebrate species (Limecola balthica, Crangon crangon) collected in the Baltic Sea. Furthermore, commonly used mathematical lipid normalization and formalin correction were applied to check if they were able to adjust the model results correctly. Preservation effects were strong on model outcomes for frozen as well as formalin preserved L. balthica samples, but not for C. crangon. Pre-treatment effects varied with species and preservation method and neither lipid normalization nor mathematical formalin correction consistently resulted in the desired model outcomes. Our analysis highlights that particularly small, not significant changes in stable isotope ratios introduced by different preservation and pre-treatments display a so far unrecognized source of error in stable isotope mixing models. We conclude that mathematical correction of benthic invertebrate stable isotopes data should be avoided for Bayesian mixing models and that previously unaddressed effects of sample preservation (especially those arising from preservation by freezing) have potentially biased our understanding of the utilization of organic matter in aquatic food webs.
Highlights
The development of stable isotope analysis (SIA) to study ratios of naturally occurring stable isotopes (SIs) of carbon (δ13C) and nitrogen (δ15N) has advanced the understanding of aquatic ecosystem functioning and in particular the knowledge about trophic interactions (Middelburg, 2014)
Our study confirmed the results of previous studies that the absolute preservation and pre-treatment effects on δ13C are high, while effects on δ15N are rather low
The strength of these effects is, not directly linked to their influence on the performance of Bayesian mixing models and previously developed guidelines about sample preservation and pre-treatment need to be re-evaluated for this purpose
Summary
The development of stable isotope analysis (SIA) to study ratios of naturally occurring stable isotopes (SIs) of carbon (δ13C) and nitrogen (δ15N) has advanced the understanding of aquatic ecosystem functioning and in particular the knowledge about trophic interactions (Middelburg, 2014). SIA has been successfully applied in ecological studies for over three decades, many uncertainties about the effects of sample preservation and treatment still remain (Lau et al, 2012; Schlacher and Connolly, 2014). Preservation and Pre-treatment in SIMMs. In principal, SIA consists of the complete combustion of dehydrated and pulverized samples in an elemental analyzer and transferring the gaseous products to CO2 and N2, which are subsequently detected in a mass spectrometer allowing for distinguishing between molecules containing 12C and 13C, as well as 14N and 15N. Samples must be preserved until the time of analysis. In case of organisms and sediments it is commonly done by freezing, since fluid preservation in formalin and/or ethanol is known to affect
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