Comparing the toxicity of nitrate between different species of subterranean Niphargus amphipods

: This study investigates the sensitivity of subterranean amphipods of the genus Niphargus to nitrate pollution, focusing on understanding possible differences between cave-dwelling and spring-dwelling species. Five ecologically distinct subterranean crustacean species of the genus Niphargus were studied, three from springs at the interface between surface and subterranean ecosystems (Niphargus timavi, Niphargus spinulifemur, Niphargus sphagnicolus) and two from cave streams (Niphargus stygius, Niphargus podpecanus), together with Daphnia magna for comparative toxicity assessment. The organisms were exposed to nitrate for 72 h, 96 h and 21 days, followed by a 96-hour recovery period in a nitrate-free medium. In contrast to initial hypothesis, the results showed that nitrate did not induce immobility or mortality to any Niphargus species at concentrations higher than those environmentally relevant. The average 21 d LC50 values determined for different Niphargus sp. were 1735-4266 mg NO3- /L. No significant differences in nitrate sensitivity were found between cave-dwelling and spring-dwelling species. In addition, all species tested showed high recovery rates, regardless of their ecological origin. These results challenge the assumption that subterranean species are inherently more susceptible to chemical stressors due to their lower metabolic rate and lower detoxification capacities. This study highlights the importance of evaluating species-specific traits rather than relying solely on habitat-related generalizations. Although nitrate did not cause toxic effects in this context, potential long-term risks to groundwater ecosystems, such as effects on reproductive success and population dynamics, remain a concern. Comparing the sensitivity of Niphargus and D. magna after 72 h of exposure, N. timavi and N. stygius were more sensitive than D. magna, while N. sphagnicolus was similarly sensitive. These results suggest that tests on subterranean species should be performed to provide a more holistic understanding on the pollutant hazard for subterranean environment.