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

S Tavazzi; C Cacciatori; S Comero; D Fatta-Kassinos; P Karaolia; I C Iakovides; P Loutsiou; I Gutierrez-Aguirre; Z Lengar; I Bajde; T Tenson; V Kisand; P Laas; K Panksep; H Tammert; G Mariani; H Skejo; B M Gawlik

doi:10.1016/j.jece.2023.109623

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

J Environ Chem Eng. 2023 Apr;11(2):109623. doi: 10.1016/j.jece.2023.109623. Epub 2023 Mar 2.

Authors

S Tavazzi¹, C Cacciatori¹, S Comero¹, D Fatta-Kassinos^{2

3}, P Karaolia², I C Iakovides^{2

3}, P Loutsiou², I Gutierrez-Aguirre⁴, Z Lengar⁴, I Bajde⁴, T Tenson⁵, V Kisand⁵, P Laas⁵, K Panksep⁵, H Tammert⁵, G Mariani¹, H Skejo¹, B M Gawlik¹

Affiliations

¹ Ocean and Water Unit, Joint Research Centre, European Commission, via E. Fermi 2749, T.P. 120, I-21027 Ispra, Italy.
² Nireas-International Water Research Centre, University of Cyprus, P.O. Box 20537, 1678 Nicosia, Cyprus.
³ Department of Civil and Environmental Engineering, School of Engineering, University of Cyprus, P.O. Box 20537, 1678 Nicosia, Cyprus.
⁴ National Institute of Biology, Vecna Pot 111, SI-1000 Ljubljana, Slovenia.
⁵ Institute of Technology, University of Tartu, Nooruse 1, E-50411 Tartu, Estonia.

Abstract

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.

Keywords: Isochronous stability testing; SARS-CoV-2; Wastewater samples; Wastewater-based epidemiology.