Abstract
The black shinner Pseudopungtungia nigra is an endangered freshwater fish endemic to Korea, and standardized anesthetic protocols are needed for conservation-related hatchery handling. This study evaluated the effects of water temperature and MS-222 concentration on anesthetic induction and recovery responses in hatchery-reared juvenile P. nigra of approximately 3 cm total length. Juveniles were exposed to four MS-222 concentrations, 80, 100, 150, and 200 mg L−1, at three water temperatures, 21, 24, and 27 °C. Induction time, recovery time, and recovery success within 600 s were assessed using behavioral endpoints. The 80 mg L−1 treatment induced anesthesia within 600 s only at 27 °C, whereas fish exposed at 21 and 24 °C failed to reach the defined anesthetic stage within 600 s; therefore, this treatment was treated as a low-concentration induction-failure condition. In the main 3 × 3 factorial analysis using 100, 150, and 200 mg L−1, induction time decreased significantly with increasing MS-222 concentration and water temperature, with significant effects of temperature, concentration, and their interaction. In contrast, recovery time increased with increasing MS-222 concentration, indicating a clear trade-off between rapid induction and recovery stability. Although 200 mg L−1 produced the shortest induction times, it also resulted in the longest recovery times and delayed recovery at 24 and 27 °C. The 100 mg L−1 treatment showed stable recovery but required prolonged induction, especially at lower temperatures. Overall, 150 mg L−1 provided the most balanced behavioral response by substantially reducing induction time compared with 100 mg L−1 while avoiding the greater recovery burden observed at 200 mg L−1. These findings suggest that 150 mg L−1 MS-222 is a practical concentration for routine short-term handling of hatchery-reared juvenile P. nigra under the tested temperature and handling conditions. However, this recommendation should be interpreted as a behavioral handling guideline because physiological stress responses and long-term post-anesthetic outcomes were not evaluated.