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

## TL;DR

This study develops a quick and affordable colorimetric test to detect a stress-related cortisol metabolite in Atlantic salmon feces for on-site aquaculture use.

## Contribution

A novel colorimetric UV–vis spectrophotometric assay for detecting fecal tetrahydrocortisone in Atlantic salmon is developed and optimized.

## Key findings

- Baseline levels of 5β-THE in nonstressed salmon feces were consistently low (average 533.81 ± 66.7 ng/g).
- The developed colorimetric assay showed excellent linearity (R² = 0.997) and detection limits around 400 ng/mL.
- 5β-THE concentrations in raw fecal samples exceeded the detection threshold, suggesting feasibility for on-site stress assessment.

## Abstract

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.