Evaluating Viral Pollution in Wastewater and Mediterranean Ecosystems

Abstract

Increasing water scarcity and climate-related extremes are amplifying the need for safe water reuse, however, wastewater treatment plants (WWTPs) may not fully remove viral pathogens, posing potential risks to freshwater environments. This study assessed human enteric and respiratory viruses using (RT)-qPCR, along with faecal indicators using culture-based methods, at WWTPs in two Mediterranean protected areas (Albufera Natural Park and the Ebro River region) across three campaigns. A total of influent (n = 33), reclaimed wastewater (n = 33), biosolids (n = 32), surface water (n = 33), and sediment (n = 29) samples were analysed. Influent wastewater contained a wide range of viral and faecal indicators, with biosolids showing similar levels. Although reclaimed water demonstrated reductions in Escherichia coli and somatic coliphages, these reductions remained below the ≥ 5 and ≥ 6 Log₁₀ threshold required under EU water reuse regulations, respectively. In surface waters, human enteric viruses were detected at low concentrations, with human norovirus genogroup II (HuNoV GII) showing the highest incidence (36.36%). SARS-CoV-2 was detected in 15.15% of samples, while influenza A virus (IAV) was found in only one sample. Sediments yielded viral signals in 10–35% of samples, whereas E. coli and somatic coliphages were detected in 6.9% and 10.3% of samples, respectively. Hepatitis E virus (HEV) genotype 3f exhibited a clear winter trend, whereas faecal indicators were consistently detected across matrices. In contrast, E. coli and somatic coliphages showed no correlation with viral detections. As the first comprehensive survey of viral presence in these Mediterranean systems, this study provides a baseline for monitoring and managing viral contamination risks during and after extreme climate events, which may exacerbate pathogen mobilization and dissemination. Additionally, the detection of zoonotic viruses, including IAV and HEV, highlights the value of wastewater and environmental surveillance (WES) in biodiversity-rich environments.