Optimization of MS-222 Concentration for Short-Term Handling of Juvenile Pseudopungtungia nigra Based on Induction and Recovery Responses

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.