**Background:** Animal welfare assessment in captive cetaceans is limited, with few published empirical studies. Stress can lead to compromised health and poor welfare, but no single biological parameter adequately captures a stressful condition. This study aimed to validate sloughed skin scrape cortisol and acoustic activity as non-invasive tools to identify stressors in captive beluga whales (Delphinapterus leucas).
**Methods:** Three beluga whales (one adult female A, two adult males B and C) housed at the National Museum of Marine Biology and Aquarium (NMMBA) in Pingtung, Taiwan, were studied from March 2017 to March 2018. Skin scrape samples were collected weekly from the fluke (beluga A) or flipper (beluga B and C) using a sterilized wooden tongue depressor. Cortisol was extracted using a modified skin steroid extraction technique (ethanol/acetone, diethyl ether, acetonitrile/hexane) and quantified via a commercial enzyme immunoassay (Arbor Assays #K003-H1). Extraction efficiency was 41.6% ± 15.9%. Parallelism and matrix interference were assessed via F-test and spiking experiments. Acoustic activity was recorded for 5 minutes at 10:00 am daily using an SM4 transducer (96 kHz sampling rate). Calls were classified into whistles, pulses, combo calls, and unknown. Time lag between events and cortisol peaks was estimated by comparing event dates with cortisol peaks (defined as values > third quartile) across six candidate windows (28–32, 38–42, 48–52, 58–62, 68–72, 78–82 days). Eleven events were identified as potential stressors; five with high-quality recordings (event I: maintenance drilling noise; IV: enteritis in beluga C; VII: pool gate repair; VIII: fire drill; IX: impulsive sound from sea embankment) were selected for acoustic analysis.
**Key Results:** Cortisol concentrations in 92 scrape samples ranged from 0.213 ng/g to 8.55 ng/g (wet weight). No significant difference was found among the three whales (Kruskal-Wallis: H = 1.28, p > 0.05). Coefficients of variation were 67% (beluga A), 113% (beluga B), and 125% (beluga C). When outliers were excluded, CVs were 54%, 63%, and 41%, respectively. Flipper and fluke sampling locations showed similar cortisol concentrations (R² = 0.9685). The time lag analysis indicated that days 68–72 had the highest points, suggesting skin scrape cortisol reflects events occurring approximately two months prior. Six responses were observed in beluga A, five in beluga B, and four in beluga C. A total of 2650 calls were identified across the five selected events: 1830 pulse calls, 497 whistles, 311 combo calls, and 12 unknown. The number of calls plummeted on the day of the event for all five events. Chi-squared test of homogeneity (p < 0.05) and PCA showed that call distribution differed significantly across the five days surrounding each event. In events I, IV, and IX, pulse calls were in the majority on the event day, and call numbers on Day 5 remained lower than the mean of Days 1 and 2. During all event periods except event IV (enteritis), food intake was successful and no negative behaviors were observed by trainers.
**Clinical Implications:** This is the first study to estimate the time lag between stressful events and skin scrape cortisol peaks in beluga whales (68–72 days), consistent with the epidermal turnover rate of 70–75 days. The longer lag compared to bottlenose dolphins (45–60 days) may reflect thicker beluga epidermis. Skin scrape cortisol can detect dynamic changes even at low concentrations (median 1.14 ng/g), and sampling from flipper or fluke yields comparable results, offering flexibility for routine collection. Individual differences in cortisol responses were evident: beluga B showed stronger responses to events, beluga C responded only to significant stressors, and beluga A was relatively stable. Acoustic activity monitoring revealed decreased call numbers on event days even when no negative behaviors were observed, indicating that traditional behavioral observation may miss underlying stress. The authors recommend that aquaria consider incorporating skin scrape cortisol and acoustic activity monitoring into animal welfare standards.