Characterization of wastewater effluents in the Danube River Basin with chemical screening, in vitro bioassays and antibiotic resistant genes analysis

Averaged 7-day composite effluent wastewater samples from twelve wastewater treatment plants (WWTPs) in
nine countries (Romania, Serbia, Hungary, Slovenia, Croatia, Slovakia, Czechia, Austria, Germany) in the
Danube River Basin were collected. WWTPs' selection was based on countries' dominant technology and a
number of served population with the aim to get a representative holistic view of the pollution status. Samples
were analyzed for 2248 chemicals of emerging concern (CECs) by wide-scope target screening employing LC-ESIQTOF-
MS. 280 compounds were detected at least in one sample and quantified. Spatial differences in the
concentrations and distribution of the compounds classes were discussed. Additionally, samples were analyzed
for the possible agonistic/antagonistic potencies using a panel of in vitro transactivation reporter gene CALUX®
bioassays including ERα (estrogenics), anti-AR (anti-androgens), GR (glucocorticoids), anti-PR (anti-progestins),
PPARα and PPARγ (peroxisome proliferators) and PAH assays. The potency of the wastewater samples to cause
oxidative stress and induce xenobiotic metabolism was determined using the Nrf2 and PXR CALUX® bioassays,
respectively. The signals from each of the bioassays were compared with the recently developed effect-based
trigger values (EBTs) and thus allowed for allocating the wastewater effluents into four categories based on their
measured toxicity, proposing a putative action plan for wastewater operators. Moreover, samples were analyzed
for antibiotics and 13 antibiotic-resistant genes (ARGs) and one mobile genetic element (intl1) with the aim to
assess the potential for antibiotic resistance. All data collected from these various types of analysis were stored in
an on-line database and can be viewed via interactive map at https://norman-data.eu/EWW_DANUBE.