Multi-level effects of emerging contaminants on macroinvertebrates in Alpine streams: From DNA to the ecosystem

The aim of this study was to highlight the effects of water treatment plant (WTP) effluents on the macro -invertebrates of the Noce River (Italian Alps), from its headwaters (at TP_Up at 1792 m a.s.l. and TP_Dw at 1790 m a.s.l., which were 70 m upstream and 50 m downstream of the Tonal Pass WTP, respectively) to lowland reaches (at ML at 221 m a.s.l., 13 km downstream of one WTP and two reservoirs). The effects of five emerging contaminants (ECs; ibuprofen = IBU, furosemide = FUR, trimethoprim = TMP, triclocarban = TCC, and sucralose = SUCR) present in the effluents were evaluated seasonally (winter/summer) at multiple levels of biological organisation-from effects on the DNA (genotoxicity) to the organism (acute toxicity), community (alpha-diversity) and ecosystem (with environmental quality assessed using the STAR_ICMi and IBE indices). Eight insect species were selected for assessment: Baetis alpinus, Baetis rhodani, Diamesa cinerella, Dictyogenus alpinus, Perlodes intricatus, Pseudodiamesa branickii, Rhyacophila occidentalis, and Rhyacophila tristis. Responses to the five ECs were evaluated specific to species and sites. Among the tested ECs, IBU was the most harmful. While SUCR was less toxic, it was significantly genotoxic. According to the acute toxicity (96 h) and genotoxicity (Comet assay at sub-lethal concentrations) tests, D. cinerella exhibited the highest tolerance and B. alpinus the highest sensitivity to all tested ECs. Populations living at the TP_Dw, the site most contaminated by ECs in winter, accumulated the highest basal DNA damage but were more tolerant to chemical stressors. The negative effect of WTPs on biodiversity was highlighted with reduced alpha-diversity, which was the lowest at TP_Dw during winter when tourist influx was the highest and river discharge was the lowest. The general stress con-ditions identified by changes to species composition and both toxicity tests were not detected by the STAR_ICMi and IBE indices, with the ecological status reported as good/excellent at all sites during all seasons. This em-phasises how these indices, unlike biomarkers, are not good early warning indicators of the effects of trace ECs in running waters. The Comet assay has high potential for identifying stress in wild populations, even when ex-posed to environmental concentrations of ECs that are far lower than those required to cause acute toxicity.