Effects of chemical preservation on bulk and amino acid isotope ratios of zooplankton, fish, and squid tissues

Abstract

Rationale It is imperative to understand how chemical preservation alters tissue isotopic compositions before using historical samples in ecological studies. Specifically, although compound‐specific isotope analysis of amino acids (CSIA‐AA) is becoming a widely used tool, there is little information on how preservation techniques affect amino acid δ15N values. Methods We evaluated the effects of chemical preservatives on bulk tissue δ13C and δ15N and amino acid δ15N values, measured by gas chromatography/isotope ratio mass spectrometry (GC/IRMS), of (a) tuna (Thunnus albacares) and squid (Dosidicus gigas) muscle tissues that were fixed in formaldehyde and stored in ethanol for 2 years and (b) two copepod species, Calanus pacificus and Eucalanus californicus, which were preserved in formaldehyde for 24–25 years. Results Tissues in formaldehyde‐ethanol had higher bulk δ15N values (+1.4, D. gigas; +1.6‰, T. albacares), higher δ13C values for D. gigas (+0.5‰), and lower δ13C values for T. albacares (−0.8‰) than frozen samples. The bulk δ15N values from copepods were not different those from frozen samples, although the δ13C values from both species were lower (−1.0‰ for E. californicus and −2.2‰ for C. pacificus) than those from frozen samples. The mean amino acid δ15N values from chemically preserved tissues were largely within 1‰ of those of frozen tissues, but the phenylalanine δ15N values were altered to a larger extent (range: 0.5–4.5‰). Conclusions The effects of preservation on bulk δ13C values were variable, where the direction and magnitude of change varied among taxa. The changes in bulk δ15N values associated with chemical preservation were mostly minimal, suggesting that storage in formaldehyde or ethanol will not affect the interpretation of δ15N values used in ecological studies. The preservation effects on amino acid δ15N values were also mostly minimal, mirroring bulk δ15N trends, which is promising for future CSIA‐AA studies of archived specimens. However, there were substantial differences in phenylalanine and valine δ15N values, which we speculate resulted from interference in the chromatographic resolution of unknown compounds rather than alteration of tissue isotopic composition due to chemical preservation.

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

“…Findings pertaining to the general robustness of compound‐specific isotope analysis of amino acids (CSIA‐AA) are largely in agreement with earlier works that preliminarily explored preservation effects on amino acid δ 15 N profiles (Hannides et al ; Ogawa et al ; Strzepek et al ; Hetherington et al ). While a small subset of amino acids were found to be more 15 N‐enriched in preserved samples, there appears to be as yet no consensus on the identity of these potentially more susceptible tracers (Table ).…”

“…Current study: n = 10; five species with 1–3 specimens each; Hannides et al (): n = 30; preserved and control treatments are of different taxa; Ogawa et al (): sample size not reported; two species; Strzepek et al (): n = 1; Hetherington et al (): n = 6; three species with two specimens each.…”

“…An important consideration for long-term (e.g., multidecadal) stable isotope studies using preserved specimens is the effect of preservation time on δ 13 C and δ 15 N profiles. Although this was not investigated in our 8-week CSIA-AA study, several studies using bulk SIA suggest that the majority of preservative-induced isotopic shifts occur within the first 3 weeks of preservation (Sarakinos et al 2002;Syväranta et al 2011;Hetherington et al 2019). Furthermore, a study comparing bulk SIA samples preserved over periods ranging from 1 to 15 years found no additional isotopic shifts with increased preservation time, suggesting that short-term preservation studies are likely sufficiently representative of effects over decadal time scales (Rennie et al 2012).…”

“…The effects of tissue preservation on CSIA-AA are comparatively less understood. Studies employing CSIA-AA generally assume that amino acid δ 13 C and δ 15 N profiles are robust against effects of chemical preservatives (Lorrain et al 2009;Blanke et al 2018), citing results from a small number of pioneering, albeit preliminary, primary investigations (Hannides et al 2009;Ogawa et al 2013;Strzepek et al 2014;Hetherington et al 2019). These studies are based on analyses with unspecified/very small sample sizes (n = 1-6) that sometimes involve indirect cross-taxa comparisons.…”

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

“…These samples have in most cases undergone some kind of chemical preservation, most commonly in formaldehyde or ethanol. Many studies have looked into the effects of such preservatives on bulk δ 15 N measurements and found variable and sometimes significant changes across a wide range of species (Rau et al 2003;Kelly et al 2006;Barrow et al 2008), occurring mainly during the first few weeks to months of preservation (Sarakinos et al 2002;Hetherington et al 2019). The mechanisms driving the δ 15 N fractionation are largely unknown, but likely the result of N containing compounds being solubilized and lost from preserved tissues since neither formaldehyde nor ethanol contains any N that could be added to the tissue (Bosley and Wainright 1999;Sarakinos et al 2002).…”