Influence of season, capture method, sample age and extraction protocols on the scale cortisol concentrations of three species of freshwater fish.

Scale cortisol concentration (SCC) is increasingly applied as a biomarker of chronic stress in fish, but knowledge gaps remain on how SCC is affected by the sampling season and method of fish capture, the time since sample collection, and the cortisol extraction protocol. Here, working with three freshwater fishes (common carp Cyprinus carpio, European chub Squalius cephalus and Northern pike Esox lucius), a robust extraction protocol was developed and then applied to identifying how scale cortisol levels can vary in fish populations according to aspects of the fish capture events. Across five scale cortisol extraction protocols, three provided relatively low yields, so their application would result in erroneously low SCC. Application of the extraction protocol providing the highest yields to scale samples indicated that fish sampled in winter have significantly lower SCC than those collected in spring and summer, while fish captured by angling have significantly lower SCC than fish collected from the same population by electric fishing. There were no significant differences in SCC measured from populations across 40 years, suggesting that archived scales potentially provide a valuable resource for measuring temporal changes in SCC. Future studies based on using scale cortisol for analyses of fish chronic stress should consider these issues in their study designs and evaluations to ensure measured differences in cortisol across time and space are due to differences in how the fish are responding to their environment rather than being an artefact of study design.