Monitoring Farmed Fish Welfare by Measurement of Cortisol as a Stress Marker in Fish Feces by Liquid Chromatography Coupled with Tandem Mass Spectrometry

Abstract

The aquaculture industry has become a sustainable source of food for humans. Remaining challenges include disease issues and ethical concerns for the discomfort and stress of farmed fish. There is a need for reliable biomarkers to monitor welfare in fish, and the stress hormone cortisol has been suggested as a good candidate. This study presents a novel method for measurement of cortisol in fish feces based on enzymatic hydrolysis, liquid–liquid extraction, derivatization, and finally instrumental analysis by liquid chromatography coupled with tandem mass spectrometry. Hydrolysis and extraction conditions were optimized. Cortisol appeared to be mostly conjugated to sulfate and less conjugated to glucuronic acid in the studied samples of feces from farmed Atlantic salmon. The method was suitable for quantification of cortisol after enzymatic deconjugation by either combined glucuronidase and sulfatase activity, or by glucuronidase activity alone. The limit of detection was 0.15 ng/g, the limit of quantification was 0.34 ng/g, and the method was linear (R2 > 0.997) up to 380 ng/g, for measurement of cortisol in wet feces. Method repeatability and intermediate precision were acceptable, both with a coefficient of variation (CV) of 11%. Stress level was high in fish released into seawater, and significantly reduced after eight days.

Salting-Out Assisted Liquid-Liquid Extraction for UPLC-MS/MS Determination of Thyroxine and Steroid Hormones in Human Serum and Fish Plasma

“…However, it will be important to follow up on similar stress events in the future, as they could lead to decreased immune competence and be a sign of impaired health of the fish [48]. In future work, it will also be interesting to study the correlation with cortisol measurements in alternative sample materials, such as fish feces [49].…”

The Importance of Monitoring Cortisol in the Agri-Food Sector—A Systematic Review

“…In another study, Atlantic salmon released from aquaculture facilities into seawater cages experienced this as a stressful event. Still, cortisol levels decreased significantly, varying from 437 ± 293 ng/g after four days to less than 80 ng/g after eight days at sea [28].…”

“…A schematic of the analytical method is presented in Figure 2. For agri-food samples, the most common extraction solvents used are methyl tertiary-butyl ether (MTBE), methanol and ethyl acetate [13][14][15]17,18,26,28]. tially harmful to the operator, because it requires large amounts of toxic or flammable chemicals, resulting in a time-consuming, expensive and labor-intensive operation [31].…”

“…A schematic of the analytical method is presented in Figure 2. For agri-food samples, the most common extraction solvents used are methyl tertiary-butyl ether (MTBE), methanol and ethyl acetate [13][14][15]17,18,26,28]. SPE methods have been widely applied to overcome the drawbacks of LLE and implement greener solutions for sample preparation (Table 1).…”

“…In general, the concentration range of cortisol varies from 0.003 to 2720 ng/mL in samples from the agri-food sector (Table 1). The reported values obtained for the limit of detection (LOD) ranged between 0.04 to 1.11 ng/mL, whereas the limit of quantification (LOQ) varied from 0.025 to 3.70 ng/mL [14,28,29,34], as presented in Table 1.…”