Methods for identifying origin, movement, and foraging areas of animals are essential for understanding ecosystem connectivity, nutrient flows, and other ecological processes. Telemetric methods can provide detailed spatial coverage but are limited to minimum body size of specimen for tagging. In recent years, stable isotopes have been increasingly used to track animal migration by linking geophysical isotope landscapes called “isoscapes”. However, compared to telemetric methods, the spatial resolution of bulk stable isotopes is low.

Here we examined a novel approach by evaluating the use of compound-specific hydrogen and carbon stable isotopes of fatty acids (δ²H_FA and δ¹³C_FA) from liver, muscle, brain, and eye tissues for identifying site-specificity of fish from a sub-alpine river catchment. We analysed 208 fish (European bullhead, rainbow trout, and brown trout) collected in 2016 and 2018 at 15 different sites.

δ¹³C_FA values of these fish tissues correlated more amongst each other than those of δ²H_FA values. Both δ²H_FA and δ¹³C_FA values showed tissue-dependent isotopic fractionation, while fish taxa had only small effects. The highest site-specificity was for δ¹³C_DHA values, while the δ²H isotopic difference between LIN and ALA resulted in the highest site-specificity. Using linear discrimination analysis of FA isotope values, over 90 % of fish could be assigned to their location of origin, however, the accuracy dropped to about 56 % when isotope data from 2016 were used to predict the sites for samples collected in 2018, suggesting temporal shifts in site specificity of δ²H_FA and d13CFA. However, predictive results were still higher than site specificity compared to bulk tissue isotopes for a single time point. In summary, compound-specific isotope analysis of fatty acids may become a highly effective tool for assessing fine and large-scale movement and foraging areas of animals.