Acute and chronic toxicity of nitrate to the stygobiont amphipods Niphargus inopinatus and N. grandii.

Nitrate is the predominant groundwater pollutant within the European union (EU), despite comprehensive legislation. In this study, acute (up to 96 h) and time-independent toxicity of nitrate (NO3-) were investigated for two groundwater amphipod species, Niphargus inopinatus and N. grandii. Nitrate concentrations applied ranged from 10 to 5000 mg/L NO3-. Bulk stable nitrogen isotope analysis was used to assess the potential uptake and exchange of nitrate derived nitrogen into biomass. Furthermore, we challenged the hypothesis that amphipods via their gut microbiome contribute to nitrate removal from contaminated groundwater. Acute sublethal effects occurred after 24 h of exposure only at 5000 mg/L, the highest nitrate concentration tested. Mortality remained at 0% for both species after 96 h. The 96-h EC50 values were 397 mg/L for N. inopinatus and 1298 mg/L for N. grandii, whereas the time-independent ultimate EC50 were calculated with 127 mg/L and 164 mg/L NO3-, respectively, matching nitrate concentrations frequently found in contaminated groundwaters. These values were reached already after 6 and 10 days, respectively. A time-independent LC50 of 386 mg/L NO3- could be estimated for only N. inopinatus. The NOEC of NO3- after 4 weeks of exposure was 100 mg/L for both species. Chronic nitrate exposure, did not change the δ¹ ⁵N signature of test animals' bulk biomass. No evidence was collected for a nitrate reduction in groundwater mediated by the invertebrates. The present study underlines the need for further studies with stygobiont organisms, but also emphasizes its limitations.