Short-term stability of wastewater samples for storage and shipment in the context of the EU Sewage Sentinel System for SARS-CoV-2.

In the context of an EU-wide surveillance system for SARS-CoV-2 in wastewater, recommended by the European Commission, this study aims to provide scientific support to the adequacy of transport and storage conditions of samples both in terms of duration and samples temperature. Three laboratories in Slovenia, Cyprus and Estonia investigated the short-term, one-week, isochronous stability of wastewater samples by RT-qPCR based detection of SARS-CoV-2 genes. The results were tested for statistical significance to determine uncertainty of quantification and shelf-life, at testing temperatures of + 20 °C and - 20 °C, relative to reference at + 4 °C. Samples were collected from three urban wastewater treatment plant influents and analysed respectively for SARS-CoV-2 genes N1, N2 (Laboratory 1), N2, E (Laboratory 2) and N3 (Laboratory 3), with various analytical methods. For a period of 7/8 days at + 20 °C, decreasing trends of measured concentrations were observed for all genes resulting in instability according to the statistical analysis, while at - 20 °C the trend of variation was stable only for N1, N2 (Laboratory 1) and N3 (Laboratory 3). Trends for gene E concentrations at - 20 °C (Laboratory 2) could not be tested statistically for stability because of lack of data. Over a period of just 3 days at + 20 °C, the variation was statistically non-significant indicating stability for genes N1, E and N3 for laboratories 1, 2 and 3, respectively. Nonetheless, the outcome of the study presents evidence to support the choice of the selected temperature at which samples shall be preserved during storage before analysis or transport to the laboratory. The conditions (+4 °C, ∼ few days) chosen for EU wastewater surveillance are in accordance with these results, highlighting the importance of stability testing of environmental samples to determine the short-term analytical uncertainty.

Reduction of antibiotic resistance determinants in urban wastewater by ozone: Emphasis on the impact of wastewater matrix towards the inactivation kinetics, toxicity and bacterial regrowth.

This study investigated the impact of bench-scale ozonation on the inactivation of total cultivable and antibiotic-resistant bacteria (faecal coliforms, Escherichia coli, Pseudomonas aeruginosa, Enterococcus spp., and total heterotrophs), and the reduction of gene markers (16S rRNA and intl1) and antibiotic resistance genes (qacEΔ1, sul1, aadA1 and dfrA1) indigenously present in wastewater effluents treated by membrane bioreactor (MBR) or conventional activated sludge (CAS). The Chick-Watson model-predicted ozone exposure (CT) requirements, showed that higher CT values were needed for CAS- than MBR-treated effluents to achieve a 3-log reduction of each microbial group, i.e., ~30 and 10 gO3 min gDOC-1 respectively. Ozonation was efficient in inactivating the examined antibiotic-resistant bacteria, and no bacterial regrowth was observed after 72 h. The genes abundance decreased significantly by ozone, but an increase in their abundance was detected 72 h after storage of the treated samples. A very low removal of DOC was achieved and at the same time phyto- and eco-toxicity increased after the ozonation treatment in both wastewater matrices. The gene abundance, regrowth and toxicity results of this study may be of high environmental significance for comprehensive evaluation of ozone and may guide future studies in assessing these parameters for other oxidants/disinfectants.

Continuous ozonation of urban wastewater: Removal of antibiotics, antibiotic-resistant Escherichia coli and antibiotic resistance genes and phytotoxicity.

This work evaluated the removal of a mixture of eight antibiotics (i.e. ampicillin (AMP), azithromycin (AZM), erythromycin (ERY), clarithromycin (CLA), ofloxacin (OFL), sulfamethoxazole (SMX), trimethoprim (TMP) and tetracycline (TC)) from urban wastewater, by ozonation operated in continuous mode at different hydraulic retention times (HRTs) (i.e. 10, 20, 40 and 60 min) and specific ozone doses (i.e. 0.125, 0.25, 0.50 and 0.75 gO3 gDOC- 1). As expected, the efficiency of ozonation was highly ozone dose- and contact time-dependent. The removal of the parent compounds of the selected antibiotics to levels below their detection limits was achieved with HRT of 40 min and specific ozone dose of 0.125 gO3 gDOC- 1. The effect of ozonation was also investigated at a microbiological and genomic level, by studying the efficiency of the process with respect to the inactivation of Escherichia coli and antibiotic-resistant E. coli, as well as to the reduction of the abundance of selected antibiotic resistance genes (ARGs). The inactivation of total cultivable E. coli was achieved under the experimental conditions of HRT 40 min and 0.25 gO3 gDOC-1, at which all antibiotic compounds were already degraded. The regrowth examinations revealed that higher ozone concentrations were required for the permanent inactivation of E. coli below the Limit of Quantification (