| Summary: | Current standardized laboratory test protocols use model species that have limitations to accurately assess native species responses to stressors. We developed and tested a novel acute <i>in</i><i>situ</i> protocol for testing field-collected organisms. We used <i>Asellus aquaticus</i> and NaCl as a reference toxicant to test for the effects of location (laboratory vs. <i>in</i><i>situ</i>), medium (synthetic vs. field water), substrate (presence vs. absence), and protocol replicability. We further tested the protocol using groundwater-adapted isopods: <i>Proasellus assaforensis</i> for the effect of location,<i> P. cavaticus</i> of medium and <i>P.</i><i>lusitanicus</i> of substrate. Our results showed that <i>A.</i><i>aquaticus’</i> lethality obtained with the novel acute <i>in</i><i>situ</i> protocol did not significantly differ from those from laboratory testing. However, laboratory tested <i>P.</i><i>assaforensis </i>showed a higher sensitivity, suggesting that its acclimation to laboratory conditions might have pernicious effects. <i>A. aquaticus</i> and <i>P. cavaticus</i> showed a higher mortality using synthetic medium <i>in</i><i>situ</i> and under laboratory conditions, which overestimated the stressor’s effect. Besides, substrate use had no significant effect. The novel acute <i>in</i><i>situ</i> protocol allows the use of native species under realistic scenarios. It is particularly well adapted for assessing the risk of groundwater ecosystems but it can be applied to a wide range of ecosystems.
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