Table_1_Benthic Diatom Communities in an Alpine River Impacted by Waste Water Treatment Effluents as Revealed Using DNA Metabarcoding.DOCX

Freshwater ecosystems are continuously affected by anthropogenic pressure. One of the main sources of contamination comes from wastewater treatment plant (WWTP) effluents that contain wide range of micro- and macropollutants. Chemical composition, toxicity levels and impact of treated effluents (TEs) on the recipient aquatic ecosystems may strongly differ depending on the wastewater origin. Compared to urban TEs, hospital ones may contain more active pharmaceutical substances. Benthic diatoms are relevant ecological indicators because of their high species and ecological diversity and rapid response to human pressure. They are routinely used for water quality monitoring. However, there is a knowledge gap on diatom communities’ development and behavior in treated wastewater in relation to prevailing micro- and macropollutants. In this study, we aim to (1) investigate the response of diatom communities to urban and hospital TEs, and (2) evaluate TEs effect on communities in the recipient river. Environmental biofilms were colonized in TEs and the recipient river up- and downstream from the WWTP output to study benthic diatoms using DNA metabarcoding combined with high-throughput sequencing (HTS). In parallel, concentrations of nutrients, pharmaceuticals and seasonal conditions were recorded. Diatom metabarcoding showed that benthic communities differed strongly in their diversity and structure depending on the habitat. TE sites were generally dominated by few genera with polysaprobic preferences belonging to the motile guild, while river sites favored diverse communities from oligotrophic and oligosaprobic groups. Seasonal changes were visible to lower extent. To categorize parameters important for diatom changes we performed redundancy analysis which suggested that communities within TE sites were associated to higher concentrations of beta-blockers and non-steroidal anti-inflammatory drugs in urban effluents vs. antibiotics and orthophosphate in hospital effluents. Furthermore, indicator species analysis showed that 27% of OTUs detected in river downstream communities were indicator for urban or hospital TE sites and were absent in the river upstream. Finally, biological diatom index (BDI) calculated to evaluate the ecological status of the recipient river suggested water quality decrease linked to the release of TEs. Thus, in-depth assessment of diatom community composition using DNA metabarcoding is proposed as a promising technique to highlight the disturbing effect of pollutants in Alpine rivers.