Quanti¢cation of glucocorticoid (GC) levels in faeces has become an established method for the non-invasive assessment of adrenocortical activity. These hormones are frequently determined in plasma samples as parameters of adrenal activity and response to stress. Because GCs are metabolized and excreted with both intact hormone and their metabolites present in faeces, the concentration of GCs can be measured in excreta. Faecal samples present the advantages of easy collection, no stress to the animal and elimination of the issue of potentially misleading acute GC spikes. The aim of this study was to determine if an enzyme-linked immunosorbent assay (ELI-SA) for cortisol was appropriate for monitoring adrenocortical activity in faecal casts of ¢shes. Performance of the cortisol ELISAwas validated by comparison to high-performance liquid chromatography, which is an established method for measuring free GCs and GC metabolites in faeces. Parallelism and sample extraction e⁄ciency were compared for the two methods. Pearson's correlation across samples for these methods was 0.996. Results demonstrated that the ELISAwas an e⁄cient, sensitive and reliable method for cortisol measurement in faecal extracts, which should permit integration of non-invasive stress monitoring into studies of ¢sh behaviour and physiology.

This study determined effects of nitrate (NO3−)‐induced stress on faecal cortisol levels in koi (Cyprinus carpio). NO3− was presented in two formats: (1) bulk increase followed by dilution to assess cortisol response and recovery and (2) bulk increase followed by incremental increases to assess cortisol response limit. Fish were maintained group‐wise in treatment and control aquaria (n=6 per group), and 0.5 g L−1 NO3− (as NaNO3) was added to the water. Faecal samples were collected daily and blood samples were taken pretreatment and 72‐h posttreatment and were assayed for cortisol via ELISA. NO3− increased plasma and faecal cortisol 4.4‐fold and 3.9‐fold respectively. Plasma cortisol was not measured further. After a 74% NO3− decrease by dilution, faecal cortisol decreased to baseline within 24 h, and restimulation by NO3− (1.0 g L−1) elevated faecal cortisol to maximal study levels. In a separate experiment, exposure to 0.4 g L−1 NO3− increased faecal cortisol 6.9‐fold. However, three additional 0.4 g L−1 NO3− increments across 9 days did not further increase cortisol. This study demonstrates that faecal cortisol measurement in fish via an ELISA can be useful as an indicator of NO3−‐induced stress in the aquatic environment.

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