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.

Environmental DNA metabarcoding for benthic monitoring: A review of sediment sampling and DNA extraction methods.

Environmental DNA (eDNA) metabarcoding (parallel sequencing of DNA/RNA for identification of whole communities within a targeted group) is revolutionizing the field of aquatic biomonitoring. To date, most metabarcoding studies aiming to assess the ecological status of aquatic ecosystems have focused on water eDNA and macroinvertebrate bulk samples. However, the eDNA metabarcoding has also been applied to soft sediment samples, mainly for assessing microbial or meiofaunal biota. Compared to classical methodologies based on manual sorting and morphological identification of benthic taxa, eDNA metabarcoding offers potentially important advantages for assessing the environmental quality of sediments. The methods and protocols utilized for sediment eDNA metabarcoding can vary considerably among studies, and standardization efforts are needed to improve their robustness, comparability and use within regulatory frameworks. Here, we review the available information on eDNA metabarcoding applied to sediment samples, with a focus on sampling, preservation, and DNA extraction steps. We discuss challenges specific to sediment eDNA analysis, including the variety of different sources and states of eDNA and its persistence in the sediment. This paper aims to identify good-practice strategies and facilitate method harmonization for routine use of sediment eDNA in future benthic monitoring.

Expanding ecological assessment by integrating microorganisms into routine freshwater biomonitoring.

Bioindication has become an indispensable part of water quality monitoring in most countries of the world, with the presence and abundance of bioindicator taxa, mostly multicellular eukaryotes, used for biotic indices. In contrast, microbes (bacteria, archaea and protists) are seldom used as bioindicators in routine assessments, although they have been recognized for their importance in environmental processes. Recently, the use of molecular methods has revealed unexpected diversity within known functional groups and novel metabolic pathways that are particularly important in energy and nutrient cycling. In various habitats, microbial communities respond to eutrophication, metals, and natural or anthropogenic organic pollutants through changes in diversity and function. In this review, we evaluated the common trends in these changes, documenting that they have value as bioindicators and can be used not only for monitoring but also for improving our understanding of the major processes in lotic and lentic environments. Current knowledge provides a solid foundation for exploiting microbial taxa, community structures and diversity, as well as functional genes, in novel monitoring programs. These microbial community measures can also be combined into biotic indices, improving the resolution of individual bioindicators. Here, we assess particular molecular approaches complemented by advanced bioinformatic analysis, as these are the most promising with respect to detailed bioindication value. We conclude that microbial community dynamics are a missing link important for our understanding of rapid changes in the structure and function of aquatic ecosystems, and should be addressed in the future environmental monitoring of freshwater ecosystems.