A Colorimetric Approach for Detecting a Selected Fecal Cortisol Metabolite as a Stress Biomarker in Atlantic Salmon

Atlantic salmon (Salmo salar) is a key aquaculture species in Norway, where maintaining optimal welfare and minimizing stress are crucial for sustainable production. Cortisol and its metabolites are widely recognized as reliable biomarkers for fish stress assessment. Although traditional cortisol measurement methods are effective, they typically require complex, labor-intensive laboratory procedures, limiting their feasibility for on-site aquaculture use. Conversely, colorimetry-based ultraviolet-visible (UV-vis) spectrophotometry presents a rapid and cost-effective alternative, yet its application for monitoring stress in fish remains underexplored. Therefore, this study aimed to develop a simple, rapid, and cost-effective colorimetric assay for detecting fecal tetrahydrocortisone (5β-THE), the predominant cortisol metabolite in Atlantic salmon feces, following the establishment of its baseline level under standard farming conditions. For this purpose, fecal samples collected over six months were analyzed using a validated LC-MS/MS method, revealing consistently low 5β-THE levels in nonstressed fish (average 533.81 ± 66.7 ng/g). Additionally, a blue-tetrazolium-based UV-vis spectrophotometric assay was developed for detecting 5β-THE. Key parameters, including solvent systems and reagent concentrations, were systematically optimized. The method exhibited excellent linearity (R ² = 0.997) and satisfactory detection limits (394.75 ng/mL for methyl tert-butyl ether and 498.54 ng/mL for tert-amyl alcohol systems). Although these detection limits were higher than expected in hydrolyzed fecal samples, the findings suggest the potential of this method for rapid, on-site stress assessment using raw samples, as 5β-THE concentrations in such samples exceeded the detection threshold. Future research will focus on optimizing the sample preparation process and validating the method.