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

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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 δ15 N values.


We evaluated the effects of chemical preservatives on bulk tissue δ13 C and δ15 N and amino acid δ15 N 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.


Tissues in formaldehyde-ethanol had higher bulk δ15 N values (+1.4, D. gigas; +1.6‰, T. albacares), higher δ13 C values for D. gigas (+0.5‰), and lower δ13 C values for T. albacares (-0.8‰) than frozen samples. The bulk δ15 N values from copepods were not different those from frozen samples, although the δ13 C 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 δ15 N values from chemically preserved tissues were largely within 1‰ of those of frozen tissues, but the phenylalanine δ15 N values were altered to a larger extent (range: 0.5-4.5‰).


The effects of preservation on bulk δ13 C values were variable, where the direction and magnitude of change varied among taxa. The changes in bulk δ15 N values associated with chemical preservation were mostly minimal, suggesting that storage in formaldehyde or ethanol will not affect the interpretation of δ15 N values used in ecological studies. The preservation effects on amino acid δ15 N values were also mostly minimal, mirroring bulk δ15 N trends, which is promising for future CSIA-AA studies of archived specimens. However, there were substantial differences in phenylalanine and valine δ15 N 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.

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