Wastewater sample storage for physicochemical and microbiological analysis

Abstract

Wastewater-based epidemiology (WBE) is a crucial tool for health and environmental monitoring, providing real-time data on public health indicators by analysis of sewage samples. Ensuring the integrity of these samples from collection to analysis is paramount. This study investigates the effects of different cold-storage conditions on the integrity of wastewater samples, focusing on both microbiological markers (such as extractable nucleic acids, SARS-CoV-2, and crAssphage) and physicochemical parameters (including ammonium, orthophosphate, pH, conductivity, and turbidity). Composite samples from the combined raw wastewater influent from five wastewater treatment works in South Wales, UK, were stored at 4°C, −20°C, and −80°C, and subjected to up to six freeze-thaw cycles over one year. The study found significant effects of storage temperature on the preservation of certain WBE markers, with the best yield most frequently seen in samples stored at −80°C. However, the majority of WBE markers showed no significant difference between storage at −80°C or at 4°C, demonstrating that it may not always be necessary to archive wastewater samples at ultra-low temperatures, thus reducing CO 2 emissions and laboratory energy costs. These findings underscore the importance of optimized storage conditions to maintain sample integrity, while ensuring accurate and reliable WBE data for public health and environmental monitoring. • Most wastewater markers show no significant difference between 4°C and −80°C storage. • Wastewater storage at 4°C maintains sample integrity for most markers for up to 12 weeks. • Freeze-thaw cycles significantly degrade RNA viruses like SARS-CoV-2 in wastewater. • CrAssphage DNA remains stable in frozen wastewater for up to one year of storage. • Extractable RNA from wastewater increases in frozen samples, due to bacterial lysis.

Topics & Concepts