Groundwater and its ecosystems provide many ecosystem services to humans, including clean drinking water. Pollution poses a threat to these ecosystems and one of the most common pollutants is nitrate (NO3ˉ), mainly due to the widespread use of nitrogen fertilisers in agriculture. There are only a few ecotoxicological studies testing the effects of nitrate on groundwater organisms. In our MSc thesis, we therefore investigated the effects of nitrates on five species of subterranean amphipod crustaceans (Niphargus sp.) that differed in ecology (N. podpecanus and N. stygius were collected in subterranean waters, N. sphagnicolus, N. spinulifemur and N. timavi were collected in springs). The water at the sampling sites was not contaminated with nitrates, i.e., the concentrations were below 5 mg NO3ˉ/L at all sites. Adults of all species and juveniles of N. podpecanus and N. stygius were exposed to different concentrations of nitrates prepared from sodium nitrate (NaNO3) for 21 days after at > 4 weeks of acclimation at 12 ± 1 °C and darkness. We recorded daily (except on Saturdays and Sundays) the number of dead and visually affected animals. High nitrate tolerance was observed in all species (96-h EC50 from 1664 mg NO3ˉ/L to 5848 mg NO3ˉ/L, and 21-day LC50 from 1735 mg NO3ˉ/L to 4266 mg NO3ˉ/L). Among the adults, N. timavi proved to be the most sensitive species, with 96-h EC50 and 21-day LC50 values of 1664 mg NO3ˉ/L and 1735 mg NO3ˉ/L, respectively. We did not observe a higher or lower nitrate tolerance for the two subterranean species compared to spring-collected species. During the 96-hour post-exposure period in the control medium M4 a recovery of the test specimens was observed. Nitrate effects were also tested on N. podpecanus and N. stygius juveniles. Contrary to our expectation, we observed less affected juveniles than adults of the same species, however this is partly due to the methodology used. High nitrate tolerance was observed also for juveniles. Additionally, water fleas Daphnia magna were exposed to different nitrate concentrations for 72 hours according to the standard (OECD 202, 2004). A comparison between daphnids and Niphargus sp. adults was possible on a species level: N. timavi and N. stygius adults were more sensitive than D. magna after 72 h, but the sensitivity of N. sphagnicolus was the same. The use of high concentrations of nitrates in the experiments resulted also in the occurrence of microbial growth despite regular changes of test medium.
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