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The circulation of SARS-CoV-2 in the environment has been confirmed numerous times, whilst research on the bioaccumulation in bivalve molluscan shellfish (BMS) has been rather scarce. The present study aimed to fulfil the knowledge gap on SARS-CoV-2 circulation in wastewaters and surface waters in this region and to extend the current knowledge on potential presence of SARS-CoV-2 contamination in BMS. The study included 13 archive wastewater and surface water samples from the start of epidemic and 17 influents and effluents from nine wastewater treatment plants (WWTP) of different capacity and treatment stage, sampled during the second epidemic wave. From that period are the most of 77 collected BMS samples, represented by mussels, oysters and warty venus clams harvested along the Dalmatian coast. All samples were processed according to EN ISO 15216-1 2017 using Mengovirus as a whole process control. SARS-CoV-2 detection was performed by real-time and conventional RT-PCR assays targeting E, N and nsp14 protein genes complemented with nsp14 partial sequencing. Rotavirus A (RVA) real-time RT-PCR assay was implemented as an additional evaluation criterion of virus concentration techniques. The results revealed the circulation of SARS-CoV-2 in nine influents and two secondary treatment effluents from eight WWTPs, while all samples from the start of epidemic (wastewaters, surface waters) were negative which was influenced by sampling strategy. All tertiary effluents and BMS were SARS-CoV-2 negative. The results of RVA amplification were beneficial in evaluating virus concentration techniques and provided insights into RVA dynamics within the environment and community. In conclusion, the results of the present study confirm SARS-CoV-2 circulation in Croatian wastewaters during the second epidemic wave while extending the knowledge on wastewater treatment potential in SARS-CoV-2 removal. Our findings represent a significant contribution to the current state of knowledge that considers BMS of a very low food safety risk regarding SARS-CoV-2.

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Norovirus, a genus in the family Caliciviridae, is a leading cause of viral gastroenteritis in humans and responsible for many outbreaks worldwide. Filter-feeding shellfish are important vehicles for transmission of foodborne pathogens, including enteric viruses such as norovirus, when grown in sewage-polluted water. In this study, we investigated a norovirus outbreak linked with consumption of oysters and mussels. In January 2019, a gastroenteritis outbreak was identified involving eight patients with symptoms of gastroenteritis. Norovirus was diagnosed in stool samples using immunochromatographic test RIDA® QUICK Norovirus, and confirmed with real-time PCR. Of four shellfish (oysters, mussels) samples analyzed using real-time PCR, three were norovirus GI-positive and GIIpositive, while one sample was only GII-positive. Six stool samples were collected, two of which were norovirus GI-positive and GII-positive, while three were GII-positive only. Following phylogenetic characterization of the human stool viruses, five out of eight belonged to the GII.6 cluster. Shellfish collected during this outbreak investigation contained the same GII.6 sequence. This is the first norovirus outbreak connected with shellfish consumption in Croatia. Understanding the transmission routes and vehicles of norovirus outbreaks is of great public health importance, and these results imply the co-circulation of GII.6 norovirus in people and oysters in Croatia.