Effects of preservation methods of muscle tissue from upper-trophic level reef fishes on stable isotope values (δ¹³C andδ¹⁵N)

Abstract

Research that uses stable isotope analysis often involves a delay between sample collection in the field and laboratory processing, therefore requiring preservation to prevent or reduce tissue degradation and associated isotopic compositions. Although there is a growing literature describing the effects of various preservation techniques, the results are often contextual, unpredictable and vary among taxa, suggesting the need to treat each species individually. We conducted a controlled experiment to test the effects of four preservation methods of muscle tissue from four species of upper trophic-level reef fish collected from the eastern Gulf of Mexico (Red Grouper Epinephelus morio, Gag Mycteroperca microlepis, Scamp Mycteroperca phenax, and Red Snapper Lutjanus campechanus). We used a paired design to measure the effects on isotopic values for carbon and nitrogen after storage using ice, 95% ethanol, and sodium chloride (table salt), against that in a liquid nitrogen control. Mean offsets for both δ13C and δ15N values from controls were lowest for samples preserved on ice, intermediate for those preserved with salt, and highest with ethanol. Within species, both salt and ethanol significantly enriched the δ15N values in nearly all comparisons. Ethanol also had strong effects on the δ13C values in all three groupers. Conversely, for samples preserved on ice, we did not detect a significant offset in either isotopic ratio for any of the focal species. Previous studies have addressed preservation-induced offsets in isotope values using a mass balance correction that accounts for changes in the isotope value to that in the C/N ratio. We tested the application of standard mass balance corrections for isotope values that were significantly affected by the preservation methods and found generally poor agreement between corrected and control values. The poor performance by the correction may have been due to preferential loss of lighter isotopes and corresponding low levels of mass loss with a substantial change in the isotope value of the sample. Regardless of mechanism, it was evident that accounting for offsets caused by different preservation methods was not possible using the standard correction. Caution is warranted when interpreting the results from specimens stored in either ethanol or salt, especially when using those from multiple preservation techniques. We suggest the use of ice as the preferred preservation technique for muscle tissue when conducting stable isotope analysis as it is widely available, inexpensive, easy to transport and did not impart a significant offset in measured isotopic values. Our results provide additional evidence that preservation effects on stable isotope analysis can be highly contextual, thus requiring their effects to be measured and understood for each species and isotopic ratio of interest before addressing research questions.

Preservation Methods Alter Carbon and Nitrogen Stable Isotope Values in Crickets (Orthoptera: Grylloidea)

“…However, we agree with other authors [20, 40, 47, 64, 69] that advise against correction factor extrapolation beyond the studied species SIA shifts caused by cricket preservation would confound critical ecological information, which suggests avoidance of sample preservation altogether.…”

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

“…This assumption is justified on at least three grounds. First, studies on teleost fishes suggest that the isotopic profiles of frozen samples are comparable with those from unpreserved samples (Kaehler and Pakhomov 2001;Stallings et al 2015;Wolf et al 2015). Second, purported effects of freezing on bulk SIA results are thought to result from the formation of ice crystals that cause the mechanical breakdown of cellular structures and leaching of isotopically enriched/depleted compounds (Dannheim et al 2007; Table 2.…”

Trophic overlap in mobulid rays: insights from stable isotope analysis

“…While there is no consensus on the effect of ethanol on δ 13 C and δ 15 N stable isotope values (Sarakinos et al 2002, Barrow et al 2008, Burgess & Bennett 2017, most preservation studies on fish muscle and fin tissue indicate there are negligible effects of ethanol on δ 15 N values, and the shift in δ 13 C values after longterm storage in ethanol is low (typically a mean increase of 0.5 to 1.5‰) (Kaehler & Pakhomov 2001, Sarakinos et al 2002, Kelly et al 2006, Vizza et al 2013, Stallings et al 2015. Additionally, Burgess & Bennett (2017) suggest that storage of elasmobranch tissues in ethanol mimics the effects of urea extraction, perhaps by acting as a solvent.…”