Effects of ethanol preservation and formalin fixation on amino acid stable isotope analysis (δ13C and δ15N) and its ecological applications

Kenny W.J. CHUA, Jia Huan LIEW, Kyung Hoon SHIN, Darren C.J. YEO*

*Corresponding author for this work

Research output: Journal PublicationsJournal Article (refereed)peer-review

12 Citations (Scopus)


Compound-specific isotope analysis of amino acids (CSIA-AA) is a promising nascent technique that alleviates many shortcomings of conventional bulk-tissue stable isotope analysis (“bulk SIA”) in ecological studies involving the tracing/reconstruction of carbon and nitrogen pathways. While CSIA-AA has been increasingly applied to preserved tissue samples (e.g., material in natural history collections), the effects of sample preservation on amino acid δ13C and δ15N profiles are poorly understood. It is therefore unclear if mathematical correction factors are necessary for interpreting isotopic profiles of preserved samples. In this study, we investigated effects of ethanol preservation and formalin fixation on amino acid δ13C and δ15N profiles. We also assess how these effects translate to two ecological applications of CSIA-AA: quantification of organic carbon sources, and estimation of trophic positions. Results from an 8-week controlled experiment on freshwater fish tissue show negligible preservation effects on most amino acid δ15N profiles, and results are similar for δ13C profiles of essential amino acids. Findings from mixing models using essential amino acid δ13C profiles similarly show that preserved samples can yield robust estimates of carbon source contributions. We also empirically demonstrate, for the first time, the use of amino acid δ13C profiles to enhance δ15N-based estimates of trophic position in food webs with multiple producers, and show that these estimates are not compromised by preservation effects. Overall, our findings support the view that amino acid δ13C and δ15N profiles from ethanol- and formalin-treated CSIA-AA samples can be directly used for addressing ecological questions.

Original languageEnglish
Pages (from-to)77-88
Number of pages12
JournalLimnology and Oceanography: Methods
Issue number2
Early online date23 Jan 2020
Publication statusPublished - 1 Feb 2020
Externally publishedYes

Bibliographical note

Funding Information:
We gratefully acknowledge funding support from the National Parks Board, Singapore (National University of Singapore (NUS) grant number: R-154-000-A55-490). KWJC is supported by the NUS President's Graduate Fellowship, while JHL was supported by an AcRF Tier 1 grant from the Singapore Ministry of Education (NUS grant number: R-154-000-A32-114). We thank Chris Yarnes from the University of California, Davis, for sample analysis; and the Associate Editor, Gordon Holtgrieve, and three anonymous reviewers for feedback that greatly improved the manuscript.


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