Effects of fixatives on stable isotopes of fish muscle tissue: implications for trophic studies on preserved...

Isotopic ecology has been widely used to understand spatial connectivity and trophic interactions in marine systems. However, its potential for monitoring an ecosystem's health and function has been hampered by the lack of consistent sample storage and long-term studies. Preserved specimens from museum collections are a valuable source of tissue for analyses from ancient and pre-modern times, but isotopic signatures are known to be affected by commonly used fixatives. The aim of the present study was to understand the effects of fixatives on isotopic signatures of bulk tissue (δ¹³ C_m and δ¹⁵ N_m ) and amino acids (δ¹³ C_AA and δ¹⁵ N_AA ) of fish muscle and to provide correction equations for the isotopic shifts. Two specimens of each: blue cod (Parapercis colias), blue warehou (Seriolella brama), and king salmon (Oncorhynchus tschawytscha) were sampled at five locations along their dorsal musculature, at four time periods: (1) fresh, (2) after 1 month preserved in formalin, and after (3) 3 and (4) 12 months fixed in either ethanol or isopropanol. Lipid content was positively correlated with C:N ratio (r² = 0.83) and had a significant effect on δ¹³ C after treatments, but not on δ¹⁵ N. C:N ratio (for δ¹³ C_m ) and percent N (for δ¹⁵ N_m ) from preserved specimens contributed to the most parsimonious mixed models, which explained 79% of the variation due to fixation and preservation for δ¹³ C and 81% for δ¹⁵ N. δ¹³ C_AA were generally not affected by fixatives and preservatives, while most δ¹⁵ N_AA showed different signatures between treatments. δ¹⁵ N_AA variations did not affect the magnitude of differences between amino acids, allowing scientists to retrieve ecological information (e.g., trophic level) independently of time under preservation. Corrections were applied to the raw data of the experiment, highlighting the importance of δ¹³ C_m and δ¹⁵ N_m correction when fish muscle tissues from wet collections are compared to fresh samples. Our results make it possible to retrieve δ¹³ C_m , δ¹⁵ N_m , δ¹³ C_AA , and δ¹⁵ N_AA from museum specimens and can be applied to some of the fundamental questions in ecology, such as trophic baseline shifts and changes in community's food web structure through time.