Transactions of the American Fisheries Society 142:1469–1476, 2013 American Fisheries Society 2013 ISSN: 0002-8487 print / 1548-8659 online DOI: 10.1080/00028487.2013.816366 NOTE The Effects of Ethanol Preservation on Fish Fin Stable Isotopes: Does Variation in C:N Ratio and Body Size Matter? Carmella Vizza,* 1 Beth L. Sanderson, Douglas G. Burrows, and Holly J. Coe National Marine Fisheries Service, Northwest Fisheries Science Center, 2725 Montlake Boulevard East, Seattle, Washington 98112, USA Abstract Although chemical preservation of stable isotope samples has been studied in a variety of species and tissue types, the effects of ethanol preservation on fish fin tissue have not been exam- ined. Using caudal fin samples from juvenile Chinook Salmon On- corhynchus tshawytscha and Rainbow Trout O. mykiss or steelhead (the anadromous form of Rainbow Trout), we investigated how storage time (2, 4, and 6 months), fin composition (C:N ratio), and fish body size (50–130 mm FL) influence preservation-induced changes in δ 13 C and δ 15 N. In both species, we found that treatment fins (frozen and later preserved in ethanol) exhibited higher δ 13 C than did paired reference fins (frozen). The changes in δ 15 N, how- ever, were smaller in magnitude and less consistent. Preservation- induced increases in fin δ 13 C, but not δ 15 N, were significantly cor- related with the change in C:N ratio (treatment–reference) in both species. In addition, these increases in δ 13 C were more highly corre- lated with body size in O. mykiss than in Chinook Salmon. Storage time had a significant effect on the shift in treatment fin δ 13 C and a small, but insignificant, effect on δ 15 N in O. mykiss. However, storage time was not a significant factor for explaining the iso- topic shifts observed in Chinook Salmon fin tissue. This is the first study to document variation in preservation-induced changes in δ 13 C within a species and to link this variation to C:N ratio. Future studies using species-specific and tissue-specific models to correct for preservation-induced shifts in stable isotope ratios should be aware that these models do not account for intraspecific variation in tissue composition. Stable isotope analysis is widely used for examining ecolog- ical questions about nutrient and energy flow, animal migration, and trophic interactions (Peterson and Fry 1987; Hobson 1999; Post 2002). While use of this technology has grown rapidly over the past decade (Mart´ ınez del Rio et al. 2009), questions linger about how to best collect and prepare samples for stable iso- tope analysis. Freezing is the preferred method of preservation for samples because it generally does not affect isotopic values *Corresponding author: cvizza@nd.edu 1 Present address: Department of Biological Sciences, University of Notre Dame, 292 Galvin Life Sciences Center, Notre Dame, Indiana 46556, USA. Received May 21, 2012; accepted June 5, 2013 Published online September 6, 2013 (Hobson et al. 1997; Arrington and Winemiller 2002; Sweeting et al. 2004; but see Syv¨ aranta et al. 2011). Unfortunately, this method can be problematic when samples are collected from remote sampling sites without access to freezers or dry ice. Chemical preservation, which is widely used for specimens in historical studies or archived collections, is a viable alternative for samples that must be transported long distances between the field and laboratory. Studies examining the effects of chemical preservation on stable isotopes have found inconsistent results. For example, Hobson et al. (1997) demonstrated that preservation media can differentially affect δ 13 C or δ 15 N (ratio of 13 C to 12 C or 15 N to 14 N relative to an international standard; Peterson and Fry 1987). Storing quail blood and muscle in ethanol had no sig- nificant effect on either δ 13 C or δ 15 N, while quail Coturnix coturnix japonica blood and muscle preserved in formalin ex- hibited lower δ 13 C signatures. Preservation techniques, such as formalin fixation and transfer to ethanol in natural history col- lections (Arrington and Winemiller 2002), and the length of time in which a sample is stored before analysis (Kaehler and Pakho- mov 2001; Sarakinos et al. 2002) can both have unique effects on stable isotope ratios. Furthermore, the effects of preservation may differ among taxonomic groups or species; Kaehler and Pakhomov (2001) found that the effect of preservation on δ 13 C varied largely between unrelated marine species (common kelp Ecklonia radiata, Kob Argyrosomus hololepidotus, and com- mon octopus Octopus vulgaris). These effects can differ within similar taxa, like ray-finned fishes (Actinopterygii; B. Kelly et al. 2006), and even among tissue types of an individual fish (Sweeting et al. 2004; B. Kelly et al. 2006). Differences between species and tissue types with respect to how chemical preservation affects stable isotope ratios may depend on the biochemical composition of the tissues analyzed 1469