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.

中文翻译：

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稳定同位素混合模型受样品保存和预处理选择的影响：对水生食物网研究的影响

稳定同位素分析已成为生态学研究中应用最广泛的技术之一。然而，样品保存和预处理对稳定同位素数据的生态解释，尤其是贝叶斯稳定同位素混合模型的影响仍存在不确定性。在这里，贝叶斯混合模型用于研究三种不同的保存方法（干燥、冷冻、福尔马林）和两种预处理（酸化、去除脂质）如何影响两种底栖无脊椎动物（Limecola balthica、 Crangon crangon) 收集于波罗的海。此外，还应用了常用的数学脂质归一化和福尔马林校正，以检查它们是否能够正确调整模型结果。对冷冻和福尔马林保存的 L. balthica 样品的模型结果的保存效果很强，但对 C. crangon 则不然。预处理效果因物种和保存方法而异，无论是脂质标准化还是数学福尔马林校正都无法始终如一地产生所需的模型结果。我们的分析强调，由不同的保存和预处理引起的稳定同位素比率的特别小的、不显着的变化显示了稳定同位素混合模型中迄今为止尚未识别的误差来源。