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.

Sewage analysis as a tool for the COVID-19 pandemic response and management: the urgent need for optimised protocols for SARS-CoV-2 detection and quantification.

COVID-19 is an ongoing global pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). As of July 29th 2020, more than 16,6 million cases have been reported in more than 188 countries/territories, leading to more than 659000 deaths. One of the main challenges facing health authorities has been testing for the virus on a sufficiently comprehensive scale. The pandemic has been an impetus for the wastewater community as it has inspired scientists to look to wastewater to help fill in the gap of measuring the prevalence of SARS-CoV-2 within a given community. Testing the wastewater may serve as an early warning system allowing timely interventions. Although viral shedding varies among individuals and over the course of their infection, the sewage system can blend these variations into an average that represents the wider studied community. The urgent need has led to a lack of coherent reporting of data regarding the analysis, as these huge and remarkable efforts by the wastewater scientific community were made in a very short time. Important information on the analytical approach is often lacking, while there is still no optimisation of the methodology, including sampling, sample storage and concentration, RNA extraction and detection/quantification. This review aims at identifying the main issues for consideration, relating to the development of validated methodological protocols for the virus quantitative analysis in wastewater. Their inclusion will enable the methodological optimisation of SARS-CoV-2 wastewater analyses, transforming the wastewater infrastructure into a source of useful information for the health sector.

The role of operating parameters and oxidative damage mechanisms of advanced chemical oxidation processes in the combat against antibiotic-resistant bacteria and resistance genes present in urban wastewater.

An upsurge in the study of antibiotic resistance in the environment has been observed in the last decade. Nowadays, it is becoming increasingly clear that urban wastewater is a key source of antibiotic resistance determinants, i.e. antibiotic-resistant bacteria and antibiotic resistance genes (ARB&ARGs). Urban wastewater reuse has arisen as an important component of water resources management in the European Union and worldwide to address prolonged water scarcity issues. Especially, biological wastewater treatment processes (i.e. conventional activated sludge), which are widely applied in urban wastewater treatment plants, have been shown to provide an ideal environment for the evolution and spread of antibiotic resistance. The ability of advanced chemical oxidation processes (AOPs), e.g. light-driven oxidation in the presence of H2O2, ozonation, homogeneous and heterogeneous photocatalysis, to inactivate ARB and remove ARGs in wastewater effluents has not been yet evaluated through a systematic and integrated approach. Consequently, this review seeks to provide an extensive and critical appraisal on the assessment of the efficiency of these processes in inactivating ARB and removing ARGs in wastewater effluents, based on recent available scientific literature. It tries to elucidate how the key operating conditions may affect the process efficiency, while pinpointing potential areas for further research and major knowledge gaps which need to be addressed. Also, this review aims at shedding light on the main oxidative damage pathways involved in the inactivation of ARB and removal of ARGs by these processes. In general, the lack and/or heterogeneity of the available scientific data, as well as the different methodological approaches applied in the various studies, make difficult the accurate evaluation of the efficiency of the processes applied. Besides the operating conditions, the variable behavior observed by the various examined genetic constituents of the microbial community, may be directed by the process distinct oxidative damage mechanisms in place during the application of each treatment technology. For example, it was shown in various studies that the majority of cellular damage by advanced chemical oxidation may be on cell wall and membrane structures of the targeted bacteria, leaving the internal components of the cells relatively intact/able to repair damage. As a result, further in-depth mechanistic studies are required, to establish the optimum operating conditions under which oxidative mechanisms target internal cell components such as genetic material and ribosomal structures more intensively, thus conferring permanent damage and/or death and preventing potential post-treatment re-growth.