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

Abstract

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.

Errors associated with compound‐specific δ15N analysis of amino acids in preserved fish samples purified by high‐pressure liquid chromatography

“…Hetherington et al (2019) investigated shortterm (2 yr) effects of ethanol and formaldehyde in tuna and squid and the long-term (25 yr) effects of formaldehyde on two species of copepods. Although the study did observe substantial but variable N isotopic fractionation, the preservation effects were not significant, in line with four other studies (Hannides et al 2009;Ogawa et al 2013;Strzepek et al 2014;Chua et al 2020).…”

“…Nevertheless, of the five other published studies of preservative effects on δ 15 N in individual AAs, ours is the first to observe significant differences. For several species of freshwater fishes, Ogawa et al (2013) and Chua et al (2020) concluded that formaldehyde preservation had no effect. Similarly, Hetherington et al (2019) did not find significant changes for yellowfin tuna AAs preserved in ethanol and formaldehyde despite observing significant changes in bulk values.…”

Stable isotopes of amino acids from reef fishes uncover Suess and nitrogen enrichment effects on local ecosystems

“…Recent studies have indicated that it is possible to use fixed/ preserved museum specimens to reconstruct past trends in food web structure (Hannides et al 2009, Hetherington et al 2019. Indeed, δ 13 C AA used to track organic matter sources are not affected by fixatives or time under fixation (Chua et al 2020, Durante et al 2020b. Conversely, while δ 15 N AA values tend to vary between treatments, the authors found that for most fish, this variation is largely within the precision of the measurements and related to time between specimen collection and fixation.…”

Fluid‐preserved fishes are one solution for assessing historical change in fish trophic level

“…The sample sizes of the three studies that preceded our own were relatively small; the number of individuals used per fish species examined ranged between one and three. Chua et al (2020) accounted for small sample size by conducting a Bayesian analysis, which is robust to small sample The purpose of this study was to improve on the currently published research, but our study also has limitations. We did observe different enrichment/depletion patterns between our two species in response to fixation, but we are unable to determine whether these differences are related to the species being preserved differently, responding differently to the same fluid-preservation protocol, and/or having inherently different isotopic compositions.…”

“…Given these studies use various methods, cross-study comparison is inherently difficult. Interestingly, Chua et al (2020) and Hetherington et al (2019) (Simmons, 2014). Notably, these studies took before and after measurements, providing data to determine if a change occurred.…”