Coronavirus in water media: Analysis, fate, disinfection and epidemiological applications

Considerable attention has been recently given to possible transmission of SARS-CoV-2 via water media. This review addresses this issue and examines the fate of coronaviruses (CoVs) in water systems, with particular attention to the recently available information on the novel SARS-CoV-2. The methods for the determination of viable virus particles and quantification of CoVs and, in particular, of SARS-CoV-2 in water and wastewater are discussed with particular regard to the methods of concentration and to the emerging methods of detection. The analysis of the environmental stability of CoVs, with particular regard of SARS-CoV-2, and the efficacy of the disinfection methods are extensively reviewed as well. This information provides a broad view of the state-of-the-art for researchers involved in the investigation of CoVs in aquatic systems, and poses the basis for further analyses and discussions on the risk associated to the presence of SARS-CoV-2 in water media. The examined data indicates that detection of the virus in wastewater and natural water bodies provides a potentially powerful tool for quantitative microbiological risk assessment (QMRA) and for wastewater-based epidemiology (WBE) for the evaluation of the level of circulation of the virus in a population. Assays of the viable virions in water media provide information on the integrity, capability of replication (in suitable host species) and on the potential infectivity. Challenges and critical issues relevant to the detection of coronaviruses in different water matrixes with both direct and surrogate methods as well as in the implementation of epidemiological tools are presented and critically discussed.

Aqueous media, in particular wastewater, can be directly contaminated with CoVs via infected feces. Aqueous media have been thus considered as a potential transmission carrier (Gundy et al., 2008;Lodder and de Roda Husman, 2020;Wigginton et al., 2015;Ye et al., 2016). CoVs have been reported to persist in aqueous media from few hours to few weeks, though their viability and infectivity strongly depend on several factors (vide infra).
Given that it is difficult to evaluate the impact of waterborne viral infections, the role of these infections is often underestimated as was the case during the SARS epidemic in Hong Kong in 2003. Bioaerosols, generated from the aeration in sewer pipelines and not subject to specific disinfection treatments, were identified to be source of the SARS-CoV-1 spreading in Amoy Gardens, a private housing estate in Hong Kong (Hung, 2003;McKinney et al., 2006;WHO, 2003). The promiscuous sharing of ponds by ducks, pigs and humans was indicated as origin and hot spots of resurgence of the influenza A virus subtype H5N1: an enveloped virus with spikelike proteins on surface similar to those of CoVs (Matsui, 2005). The spillover transmission of H5N1 virus to humans occurred from ducks, the original host species for the virus, through pigs as an intermediate species (Li et al., 2004;Matsui, 2005). Animals have also been proven to be J o u r n a l P r e -p r o o f reservoirs for the novel SARS-CoV-2. In fact, the virus has been found to be efficiently replicant in cats and ferrets and comparatively and poorly replicant in dogs, pigs, chickens and ducks (Shi et al., 2020).
Conversely, data on the occurrence of SARS-CoV-2 traces in wastewater can be used by epidemiologists and government authorities for evaluation of the extent of circulation of the virus in the population associate to a water sanitation network. At the moment of writing of these manuscript, several countries, such as the Netherlands, Australia, Finland, France, Italy, Portugal The presence of the virus in wastewater constitutes a potential hazard, due to its possible propagation through this medium, and its detection provides a powerful tool in the hands of the scientific, health and government communities, as an epidemiological indicator of the spread of the virus, including the number of asymptomatic infections. In this context, it is important the knowledge of the state-of-the-art on the methods for detection, quantification and determination of infectivity of the virus in aqueous matrices. The detection of the virus in wastewater is not directly correlated to the infectivity. Therefore, particular attention has been dedicated in the manuscript to the method for determination of the viable SARS-CoV-2 virions and the estimation of the potential risks associated. The current consolidated methods for molecular detection of the virus in wastewater, based on the amplification of viral genome, suffer in the case of the assay of the SARS-CoV-2 of some limitations, which were identified and discussed in this manuscript. The necessity to concentrate the SARS-CoV-2 traces present in wastewater, the absence of a robust and validated protocol for sample processing and the open challenges in this field are also critically analyzed and discussed. In addition, new methodologies and technologies, based on the detection of the SARS-CoV-2 RNA as well as of the proteins and other viral vestiges in wastewater are emerging and here examined. The development of these J o u r n a l P r e -p r o o f technologies could allow a more rapid and effective implementation of wastewater-based epidemiology. Fundamental is the exact knowledge on behavior and persistence of the SARS-CoV-2 in all aqueous media. This information combined with the knowledge on the efficacy of the methods for treatment of the water media for the disinfection allows establishing real risks and the correct strategy for control the spread of virus through the water environment (see Figure   1).

Viable virions and quantification and of coronaviruses in water media
The detection of pathogens, particularly viruses, in water media and wastewater plays a pivotal role for developing mitigation measures and health and safety plans (Xagoraraki and O'Brien, 2020) Viruses can be considered as supramolecular assemblies of biological small-and macromolecules. They cannot reproduce by themselves and require host living cells for the replication.
In fact, the debate of the living or non-living nature of viruses is old and still open (Gortner, 1938). Therefore, the determination of viral infectivity and concentration in a contaminated sample must be done using the culture in a suitable host organism and molecular techniques detecting the genetic material (DNA or RNA) or proteins of the virus.
The test for determination of the number of viable virus particles provides information on its state of integrity or inactivation by determining its ability to replicate when cultured in a suitable host species. The determination of the period during which the virus results viable, and thus capable of replication in determined conditions, provides information on its stability and persistence. However, detection of viable viruses in a sample does not necessarily implies infectivity: the route of transmission of the virus through that medium can be ineffective for the initiation of the actual infection in humans or other species targeted by the virus.
Assay of bacteria, that are aquatic host microorganisms for viruses, have been proposed as a simple and rapid indicator for the determination of the fate of viruses in water. In fact, the assay of bacteria in water media does not requires the "complex" sample pre-treatments and the molecular methods necessary for virus quantification (vide infra). viruses and bacteria. Considered that CoVs do not infect bacteria or other aquatic microorganisms, it is impossible to quantify their presence using other indicators. In addition, the stability of these viruses in water media significantly differs from that of other viruses, typically infecting these environments, invalidating also the association of other viral indicators to CoV persistence. However, the fate of CoVs in water and wastewater and the efficacy of their inactivation methods can be estimated by analysing the fate of other enveloped viruses with higher resistance in wastewater.

Determination of viable coronavirus particles in water media
Detection of CoV RNA molecules in a sample does not necessarily implies infectivity of that specimen. RNA molecules or their fragments can be detected from damaged, and thus inactive, viral particles. Unaltered virus particles, thus potentially infective, can be quantified with molecular techniques (e.g. using quantitative polymerase chain reaction (qPCR); see section 2.2.) targeting specific gene sequences or other molecular sections of the virus susceptible to fragmentation, or by enumeration of enlarged gene sequences (Ho et al., 2016;Polston et al., 2014;Wu et al., 2019). Capsid-integrity PCR is a recent technique that quantifies intact virions by excluding from the molecular analysis genomic material coming from damaged ones. RNA or DNA molecules coming from fragmented viruses are covalently functionalized with specific J o u r n a l P r e -p r o o f molecular markers before the molecular analysis (PCR) that, in this way, quantifies only the genomic material coming from intact virions (Leifels et al., 2021;Leifels et al., 2020).
The infectivity of a virus can be determined by plaque assays, a quantitative method of measuring infectious viruses by quantifying the plaques formed in cell cultures upon infection with serial dilutions of the virus specimen (Baer and Kehn-Hall, 2014) or by tissue culture infectious dose-50 (TCID 50 ), which detects the presence or absence of cytopathic effect in cells infected with serial dilutions of a virus specimen.
Considering that other pathogens, particularly those present in wastewater, can induce CPE on CoV host cells, confirmatory tests via nucleic acid extraction, amplification and sequencing (vide infra) are necessary in case of positive results. Laboratories with qualified Biosafety Level 3 (BSL-3) are required for the handling of those viruses.
In addition, the number of infectious virus particles can be calculated by tissue culture infectious dose (TCID 50 ) assay. The virus sample is serially diluted and added to cells, selected to show J o u r n a l P r e -p r o o f cytopathic effect, placed in a multiwell plate. The infection with the virus results in cell morphological changes or mortality, and each well is classified as infected or not infected. Automated readings, colorimetric or fluorometric, are also possible. The calculation of the TCID 50 is thus done identifying the dilution at which 50% of the wells show a CPE. The Spearman-Karber or the Reed-Muench methods are typically applied, and the viral loading is expressed as TCID 50 per volume of sample (Ramakrishnan, 2016;Reed and Muench, 1938;SPEARMAN, 1908).
Although the SARS-CoV-2 RNA has been detected worldwide in wastewater (see Table 1), results of viral culture experiments, used for the determination of the viable virions, have up to now been negative (Ge et al., 2020;Rimoldi et al., 2020a;Rimoldi et al., 2020b;Wang et al., 2020c;Westhaus et al., 2021). Wang et al analysed, without finding evidence of viable SARS-CoV-2, both the inlets and outlet sewages of pre-processing disinfection pool, as well as the final outlet of the sewage disinfection pool, of an hospital in China hosting COVID-19 cases . Similar negative results were found by Ge et al. analysing sewage samples from insolation wards during hospitalization of COVID-19 patients in the Hospital of Zhejiang University, China. The samples which were collected from the inlets and outlets of preprocessing disinfection equipment, were found positive for SARS-CoV-2 RNA detection and negative to the assay of viable virus particles carried out in Vero E6 cell line (Ge et al., 2020).
Rimoldi et al analysed influents and effluents of municipal WWTPs and water of river used for the discharge of the final effluents, during the emergence of COVID-19 epidemics in March 2020 in Milan, Italy (Rimoldi et al., 2020a;Rimoldi et al., 2020b). The infectivity of wastewater, both untreated and treated in WWTP, was investigated by Westhaus et al. culturing the specimen in Caco-2 cells in Germany (Westhaus et al., 2021). CPEs for this cell line can be observed by J o u r n a l P r e -p r o o f optical microscopy as morphological modifications of the Caco-2 cells (Hoehl et al., 2020).
CPEs were not observed in any wastewater sample analyzed in this study (Westhaus et al., 2021). These findings provide important information on the real risk associable with the presence of SARS-CoV-2 in wastewater, indicating a very limited stability of this virus in this kind of aquatic medium. However, the possibility of false-negative results in the test of viable CoVs cannot be ruled out. As matter of facts, for SARS-CoV-1, which is the most proximal virus to SARS-CoV-2 (Andersen et al., 2020), 5-log of virus titer reduction was determined to occurs within 2 and 14 days in wastewater at 20 and 4 °C, respectively (see Table 3) (Wang et al., 2005b;Wang et al., 2005c). A similar persistence was determined for SARS-CoV-2, 1-and 2log of virus titer reduction were observed respectively within the ranges of 1.4-3.3 and 2.9-6.5 days in frozen and thawed wastewater at room temperature (see Table 3) .

Concentration and detection methods of coronaviruses in water media
During an outbreak event, the loading of a virus in wastewater can be correlated with the contents in human stool or urine samples (Ye et al., 2016). (WHO, 2020a). Some of those analytical methods were applied also for detection of CoVs in water and wastewater samples (see Table 1 and 2).
Typically, the abovementioned molecular techniques employed to ascertain the occurrence of viruses in unconcentrated wastewater and surface water (Corpuz et al., 2020) yield results below the detection limit of the methods. Consequently, it is mandatory to concentrate a large volume of water sample before the analysis (Ahmed et al., 2020d;Bofill-Mas and Rusiñol, 2020;Corpuz et al., 2020;Rusiñol et al., 2020;Ye et al., 2016). Concentration methods of CoVs in water media have been addressed in ongoing research (Ahmed et al., 2020d). These studies were J o u r n a l P r e -p r o o f recently reviewed by La Rosa et al.(La Rosa et al., 2020a) and Corpuz et al. (Corpuz et al., 2020).
Water samples can be pre-treated and concentrated using various processes, such as centrifugation/ultracentrifugation, virus adsorption-elution (VIRADEL), membrane (electropositive or electronegative) filtration, centrifugal ultrafiltration or precipitation with suitable coagulating agents (see Table 1 and Figure 2) (Ahmed et al., 2020d;Bofill-Mas and prior to seeding of the virus (Wang et al., 2005b). Therefore the currently observed scarce resistance of SARS-CoV-1 in wastewater could be even overestimated.
CoVs, like the other enveloped viruses, are sensitive to pH variations, with an optimal stability at slightly acidic pH range of 6.0-6.5 c.a. (Sattar et al., 2009). Nevertheless, SARS-CoV-2 have been demonstrated to exhibit sufficiently high stability in a wide range of pH values (Chin et al., 2020). This information should be taken into account in the case of application of VIRADEL techniques for the recovery of CoVs from wastewater, where the virus is captured by filtration on glass wool or with electropositive or electronegative membranes at strongly alkaline or acidic pHs, respectively, and then eluted from the filter. The acidification of the retained viruses with acidic solutions leads to the protonation of the capsid or of the envelope, depending on the virus typology, which assumes a positive charge and can be absorbed onto electronegative membranes. On the contrary, treatment with alkaline solutions causes the viruses to become negatively charged, therefore electropositive filters should be used for the recovery of the virus.
After absorption onto filter the virus can be recovered by elution with buffered solutions eventually containing suitable agents for the virus desorption. previous study also showed that enveloped viruses, such as MHV, tend to be adsorbed to the solid fraction of wastewater (Ye et al., 2016). In the latter study, it was shown that a higher percentage of enveloped viruses (MHV: 26%, ϕ6: 22%), compared to non-enveloped viruses (MS2: 6%, T3: <5%), was adsorbed to wastewater solids at equilibrium.
Those considerations should be taken into account during the sampling and storage of wastewater samples, as well as during the treatment for the detection, quantization and particularly in the determination of the viable virus particles. The development of a standard method for concentration of wastewater contaminated with SARS-CoV-2 should be based on this information. Currently, several procedures have been applied with success for the detection of the SARS-CoV-2 in wastewater, although scarce information is available on the actual efficacy of the recovery of this virus (Table 1).
Sample pre-treatments include conventional filtration, centrifugation or settling for removal of raw suspended solids and debrides. As discussed above, these treatments could affect the J o u r n a l P r e -p r o o f quantification of the virus by excluding from the molecular detection viral particles adsorbed onto particulate. Pasteurization at 56-60°C for 60-90 min has been also applied for the inactivation of viruses and other pathogens for safe handling of the sample. Centrifugal ultrafiltration has been extensively applied for the concentration of the wastewater by using centrifugal filters with different molecular weight cut-offs (MWCO) (Ahmed et al., 2020a;Ahmed et al., 2020b;Balboa et al., 2020;Bar Or et al., 2020;Crits-Christoph et al., 2020;Manupati et al., 2020;Medema et al., 2020a;Medema et al., 2020b;Nemudryi et al., 2020a;Nemudryi et al., 2020b;Sherchan et al., 2020;Trottier et al., 2020;Westhaus et al., 2021;Wu et al., 2020b). Ultracentrifugation is also a convenient method for the rapid concentration of wastewater samples (Ampuero et al., 2020;Prado et al., 2020;Wurtzer et al., 2020;Yaqub et al., 2020). The direct flocculation (Mlejnkova et al., 2020)  al., 2020) have been also adopted for the precipitation of the virus. These latter techniques were optimized for the precipitation of proteins and particularly utilized for the recovery of nonenveloped viruses with protein capsid exposed, such as enteric viruses (Corpuz et al., 2020).
However, CoVs presents spike proteins on the viral envelope therefore this method of wastewater concentration works well also for this kind of viruses. VIRADEL methods were also largely employed for concentration of SARS-CoV-2 in wastewater, principally by using electronegative charged membranes (Ahmed et al., 2020a;Ahmed et al., 2020b;Curtis et al., 2020;Haramoto et al., 2020;Hong et al., 2020;Miyani et al., 2020;Sherchan et al., 2020). In J o u r n a l P r e -p r o o f absence of treatment for the concentration of the samples cannot be obtained reliable quantitative information on the virus concentration in wastewater (Table 1).
The application of the recent high-throughput sequencing methods will allow fast and reliable determination of viral parameters in water matrices (Bofill-Mas and . The genomic material of the viruses is often overshadowed by the host and bacterial genomes (Nieuwenhuijse and Koopmans, 2017). Therefore, automating the processing for analysing and interpreting viral genomic data is being investigated with great interest by virologists, environmental engineers, and bioinformatics.

RNA-based emerging and alternative methods
Previous studies of SARS-CoV-2 in wastewater focused on detection and quantification of the viral RNA through RT-PCR and RT-qPCR methods. The study of the different strains of the virus present in the wastewater and in the corresponding community is also important. High throughput sequencing has been used to study different strains of the virus to monitor its mutation . In a recent study by Crits-Cristoph et al., high-throughput sequencing was used to study the different SARS-CoV-2 genotypes circulating in wastewater systems in California (Crits-Christoph et al., 2020).
Another emerging approach in the detection and quantification of the SARS-CoV-2 RNA is digital PCR (dPCR), which is identified to be less affected by PCR inhibition (Sidstedt et al., 2020). This is an advantage when detecting viruses from wastewater, which is a matrix that has several possible PCR inhibitors. It is also noted that the dPCR has a lower limit of detection and has been reported to be more sensitive than qPCR (Ahmed et al., 2020b;Barceló, 2020;Falzone J o u r n a l P r e -p r o o f et al., 2020; Suo et al., 2020). The detection limit of dPCR is reported to be 10 times lower compared to that of the RT-qPCR (Barceló, 2020). Suo et al. (2020) showed that the limit of detection of an optimized droplet digital PCR (ddPCR) is at 500 (maximum) times lower than the RT-qPCR in analytes with low-level SARS-CoV-2 load (Suo et al., 2020). This may be favourable for detecting even low concentrations of SARS-CoV-2 in influents of wastewater in areas with low COVID-19 prevalence and in treated wastewater. The dPCR has been previously applied for clinical diagnostics, and its advantages show its potential as tool for detecting and quantifying SARS-CoV-2 in wastewater.
The ddPCR, an improvement of the RT-PCR, has been reported to be useful in quantification of very low target concentrations of nucleic acids in samples that are contaminated (Taylor et al., 2017). Most of the studies that quantified SARS-CoV-2 in wastewater samples utilized the RT-qPCR method. However, a few studies have so far utilized the ddPCR method to not only detect but also quantify the SARS-CoV-2 genome in wastewater (Ahmed et al., 2020b;Gonzalez et al., 2020;Jun-Bo et al., 2020)]. RT-ddPCR's main advantages over the RT-qPCR include i) the direct absolute quantification without the reliance on a calibration curve , and ii) the less effect of PCR inhibitors (Deiana et al., 2020;Kuypers and Jerome, 2017). However, it also has its disadvantages, notably low reaction mixture volume and smaller dynamic range (Kuypers and Jerome, 2017). A one-step ddPCR assay was found by Graham et al. to be more sensitive than RT-qPCR in detecting and quantifying SARS-CoV-2 in primary settled solids from wastewater treatment plants, due to the reduced effect of PCR inhibitors (Graham et al., 2021).

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based assays for SARS-
CoV-2 RNA have also been recently developed for clinical applications (Broughton et al., 2020;Hou et al., 2020). Broughton et al. (2020) reported a limit of detection of a CRISPR-based assay J o u r n a l P r e -p r o o f at 10 copies/µL input, which is higher than that of the US Centers for Disease Control and Prevention (CDC) RT-qPCR assay (1 -3.2 copies / µL input) (Broughton et al., 2020).
However, in these recent studies by Broughton et al. and Hou et al., the total assay times for the CRISPR-based assays were reported to be shorter at 40-45 minutes compared to the 4 hours spent in the RT-qPCR assay. There is no published study that has yet reported the use of CRISPR-based assays for detection of SARS-CoV-2 RNA in wastewater. However, its sensitivity and rapidity can be advantageous for wastewater-based epidemiological studies, where real-time and accurate information about SARS-CoV-2 circulating in wastewater is important.
It is to be noted that as of this time of writing, there is no established standard assay for the detection of SARS-CoV-2 in environmental samples, such as in wastewater and surface waters (La Rosa et al., 2020b). Recent studies that detected SARS-CoV-2 RNA in wastewaters utilized assays that target different regions of the SARS-CoV-2 genome, which include the following: i) N-gene encoding nucleocapsid protein, ii) S-gene encoding spike protein, iii) E-gene encoding envelope protein, iv) ORF1ab (La Rosa et al., 2020bRosa et al., , 2021, v) RNA-dependent RNA polymerase (RdRP), and vi) M-gene encoding membrane protein. Table 2 shows the different assays used in the studies on the detection of SARS-CoV-2 in wastewater and the corresponding limits of detection. The study of Ahmed et al. (2020a) showed that the N_Sarbeco assay was more sensitive than the NIID_2019-nCOV_N in detecting the SARS-CoV-2 RNA in wastewater (Ahmed et al., 2020a). In the study of Medema et al., (2020), the CDC_N1 and CDC _N3 assays were shown to be more sensitive than CDC_N2 (Medema et al., 2020b). This agreed with the results of the study of Ahmed et al. (2020b), where CDC_N1 assay was the most sensitive (limit of detection: 1 copy/ µL RNA template) compared to other assays targeting the N-gene. In this J o u r n a l P r e -p r o o f study, it was also shown that the least sensitive was the E_Sarbeco assay (limit of detection: 5 copies/ µL RNA template) (Ahmed et al., 2020b). La Rosa et al. (2020b) noted the low sensitivity of the RNA-dependent RNA polymerase (RdRp) assay, which led to no positive results in the RT-qPCR in their study. However, the authors in the latter study noted that the RdRP assay displayed a higher sensitivity than the S-gene assay and that it was able to reduce PCR inhibitor concentrations to below the acceptable levels (median at 29%). In the study of Westhaus et al. (2021), it was shown that the assay targeting the RdRP gene (compared to assays targeting the N, E, and M genes) had the highest specificity to SARS-CoV-2 RNA since no signals were detected for CoV-229E and SARS-CoV-1 (Westhaus et al., 2021).
Most of the studies that quantified SARS-CoV-2 in wastewater samples utilized the RT-qPCR method. However, a few studies have so far utilized the ddPCR method to not only detect but also quantify the SARS-CoV-2 genome in wastewater (Ahmed et al., 2020b;Gonzalez et al., 2020). RT-ddPCR's main advantages over the RT-qPCR include i) the direct absolute quantification without the reliance on a calibration curve , and ii) the less effect of PCR inhibitors (Deiana et al., 2020;Kuypers and Jerome, 2017). However, it also has its disadvantages, notably low reaction mixture volume and smaller dynamic range ( These multiplex assays could be useful in using different well-established target SARS-CoV-2 genes; however, they have not yet been used in environmental samples.
J o u r n a l P r e -p r o o f

Non-RNA-based emerging and alternative methods
Most of the recent studies on the presence and abundance of SARS-CoV-2 in wastewater samples have been performed through detection and quantification of the viral RNA. However, measurements of the SARS-CoV-2 RNA in wastewater have their own limitations. This includes the stability of the SARS-CoV-2 RNA in the wastewater at varying conditions such as higher temperature as discussed in Section 2.1 and will be discussed in Section 3. Detection of low levels of RNA in some wastewater samples is also a challenge, which means that significant levels of amplification are needed for the PCR-based methods (Neault et al., 2020).
Barceló suggested that the detection of biomolecules, which have already been used in clinical diagnostics, can be used to study the presence of SARS-CoV-2 in wastewater aside from detection of the viral RNA (Barceló, 2020). Among these biomolecules are the proteins of SARS-CoV-2, which may provide information that is supplementary to those obtained from detection and measurement of the viral RNA (see Figure 3). An advantage of the detection of the viral proteins over the viral RNA is the reduction of the number false positive tests from the process of amplification since proteins could not be directly amplified . Neault et al. studied the presence of SARS-CoV-2 in samples from primary sludge and PEGprecipitated influent wastewater solids by detecting the structural proteins of the virus (Neault et al., 2020). In the latter study, the detection and quantification of proteins was achieved thanks to higher stability and higher number of gene copies of the proteins than the viral RNA.
Immunoblot analysis was applied to detect the SARS-CoV-2 structural proteins, which include showed that the proteins measured by MPAD produced higher signals (64 to 208-fold in primary sludge samples, and 20 to 128-fold in influent solids samples) than the ones generated by viral RNA measured by RT-qPCR. In recent studies on the detection of SARS-CoV-2 in clinical samples, the target proteins were the N and S structural proteins . The N protein was chosen due to its higher abundance during infections (Ihling et al., 2020;Nikolaev et al., 2020a). On the other hand, the S protein is chosen in other studies due to its being highly immunogenic and its reported specificity compared to the other SARS-CoV-2 structural proteins (Mavrikou et al., 2020;Seo et al., 2020). It was noted that a longer S protein was observed to be encoded by SARS-CoV-2 compared to the other coronaviruses (Caruana et al., 2020). Thus, the S protein of SARS-CoV-2 potentially enhances the specific detection of the virus (Seo et al., 2020). J o u r n a l P r e -p r o o f However, Feng et al. noted that the content of proteins in a specimen is very low, the detection could be challenging and thus would require very sensitive methods . One of these methods that can be used is mass spectrometry (MS). This technique has recently been applied to detect SARS-CoV-2 proteins for RT-qPCR M-gene 200 copies/ reaction (Westhaus et al., 2021) clinical applications. Peptides that are unique to the SARS-CoV-2 N protein, which is identified as the most abundant protein in the virion, were identified using recently-developed MS-based assays applied to clinical samples (Ihling et al., 2020;Nikolaev et al., 2020b). The use of MS-based methods to detect SARS-CoV-2 proteins could be extended for wastewater samples. The detection of infectious viruses in wastewater samples using proteins has been previously carried out by Ye et al. (Ye et al., 2019). This was accomplished using an integrated cell culture-mass spectrometry (ICC-MS), in which strain-specific viral peptides of reovirus were identified. Noteworthy, in this method, mass spectrometry eliminated the step of primer design, which is characteristic of PCR-based methods. Although reovirus was the only virus studied/detected, the ICC-MS could be potentially used for detection of SARS-CoV-2 proteins in wastewater. The use of mass-spectrometry could also be potentially used for quantification of SARS-CoV-2 proteins in wastewater samples . Barceló (2020) suggested that environmental proteomics could be a tool complementary to PCR-based methods for use in WBE (Barceló, 2020). Mass spectrometry for the characterization and quantitation of the SARS-CoV-2 viral proteins might be able to provide more insight on the fraction of the SARS-CoV-2 load that is capable of infecting cell culture. As discussed, SARS-CoV-2 has the structural proteins N, S, M, and E. SARS-CoV-2 infection is mediated by the S protein, which plays key roles in the attachment, fusion, and entry of the virus into host cells (Duan et al., 2020). Being able to obtain measurements of the N, S, M, and E proteins in different samples in the future could potentially deliver more information on the relationship between viral load and infectivity. However, there are still challenges in this approach as proteins cannot be directly amplified and consequently, direct detection of viral proteins still lack sensitivity with current analytical instruments .
The detection of SARS-CoV-2 proteins using biosensors suitable for clinical applications has also been recently explored (Mavrikou et al., 2020;Seo et al., 2020). Mavrikou et al. developed a biosensor that detects S protein of SARS-CoV-2, which is reported to be more specific to the virus than other structural proteins and is responsible for the binding of the virus to the human cellular receptor (Mavrikou et al., 2020). In the latter study, the binding of the SARS-CoV-2 S protein to a membrane-bound antibody, specific to this protein, produced changes in the bioelectric properties of the engineered cell membrane. A device based on the

J o u r n a l P r e -p r o o f
Bioelectric Recognition Assay measured the changes in the properties of the membrane cell real-time. Another biosensor that relies on the principle of the binding of SARS-CoV-2 S protein to a specific antibody was developed by Seo et al. (Seo et al., 2020). A field-effect transistor (FET)-based biosensor with graphene sheets was coated with antibodies specific to the S protein. These studies showed that immunological methods can be used to detect SARS-CoV-2 rapidly and accurately, even without sample pretreatment. The latter studies showed the use of detection of SARS-CoV-2 proteins, particularly N and S proteins, for clinical diagnosis. This could be explored further for use in detection of SARS-CoV-2 in wastewater as demonstrated by Neault et al. (Neault et al., 2020). However, there are also challenges presented by the detection of SARS-CoV-2 proteins such as cross-reactivity, specifically for N protein, and the availability of antibodies specific to the proteins . Another approach on molecular diagnosis of SARS-CoV-2 is the use of aptamers instead of antibodies to target the SARS-CoV-2 proteins Song et al., 2020).  (Chung et al., 2019). The latter study shows the potential of paper-based sensors for detection and quantification of SARS-CoV-2 from wastewater. It is to be noted that these biosensors are meant to be complementary tools to other detection and quantification methods.

Environmental stability of coronaviruses
The understanding of the environmental persistence of pathogens and the effectiveness of applicable disinfection methods make it possible to assess hazards associated to a contamination.     a Overall virus inactivation rate coefficient estimated from log reduction and the corresponding time.

Persistence of CoVs on inanimate surfaces
Inanimate surfaces have been shown to be potential sources of CoV contamination, both directly via fomite transmission and indirectly, via water media which came into contact with the infected surface (Peyrony et al., 2020). The aerosolization of fomites has also been demonstrated to be effective in the spreading of viruses such as the Influenza A virus (Asadi et al., 2020b). In this context, it is thus important to ascertain the persistence of CoVs on various surfaces and the adequacy of disinfection tools used to treat them (Bhardwaj and Agrawal, 2020). Human CoVs can remain infectious on inanimate surfaces from 2 hours up to even several weeks, depending on the environmental conditions (see Table 3) (Casanova et al., 2009;Chan et al., 2011;Chan et al., 2004;Chin et al., 2020;Duan et al., 2003;Geller et al., 2012;Gundy et al., 2008;Lai et al., 2005;Rabenau et al., 2005;Sattar et al., 2009;Sizun et al., 2000;van Doremalen et al., 2020;van Doremalen et al., 2013;Wang et al., 2005b;Ye et al., 2016).

J o u r n a l P r e -p r o o f
The infectivity of SARS-CoV-1 on porous and non-porous surfaces, such as those of cloth, filter paper, glass, mosaic, metal and plastic, persisted for 60-72 (5-log reduction of virus titer), as has been assessed by analysing the CPE of the infected surface in Vero cells (Duan et al., 2003). Up to 9 days were necessary for a 5-log reduction of the infectivity of the virus on plastic surfaces (polystyrene of Petri dishes) (Rabenau et al., 2005).
A limited persistence of infectivity, less than 6 hours, was found in autoclaved soil. The sterilization was applied to avoid the effect of concurrently present microorganisms on the inactivation of the virus (Duan et al., 2003). A similar stability was observed for MERS-CoV: 72 hours were found necessary for 6-log of virus titer reduction on plastics and steel at 20°C (van Doremalen et al., 2013). SARS-CoV-1 and SARS-CoV-2 showed similar stability when compared under the same environmental conditions (van Doremalen et al., 2020). A significant reduction of infectivity, of c.a. 3 log, was found for SARS-CoV-2 after 72 hours on plastic and steel by TCID 50 assay (van Doremalen et al., 2020). Under the same conditions, the persistence of SARS-CoV-1 was similar (see Table 3). A previous study showed that the stability of SARS-CoV-1 strongly depended on temperature and humidity conditions. At 22-25 °C and relative humidity of about 40-50 %, the virus infectivity persisted up to 4 weeks on a laboratory multi-well plate made of plastics: 1-log loss of titre was observed in 5 days and a progressive reduction of the titre of 5-log was found in 4 weeks (Chan et al., 2011). The SARS-CoV-2 titer reduces by c.a. 3-log in 4 h on copper surfaces and in 24 hours on cardboard, whereas SARS-CoV-1 requires 8 h on the same surfaces (van Doremalen et al., 2020). Another recent study on the environmental persistence of infectivity of SARS-CoV2 showed a virus titer reduction of 4.7-log in 3 h on printing and tissue papers, while from 2 up to 7 days were necessary to achieve 3-6-log of virus titer reduction both on porous and non-porous surfaces, such as banknote, wood, cloth, respiratory mask, glass, stainless steel, and plastics (Chin et al., 2020).

Persistence of CoVs in water media
Human beings are continuously in contact with the water for their daily activities, such as drinking, personal hygiene, washing, irrigation, food production, and recreational purposes (see Figure 4). Used water, including stormwater and runoffs, is ultimately collected as wastewater. Feces, urine, or vomit infected with pathogens enter the human sewage; this can then affect the urban water cycle. The knowledge of the persistence of those J o u r n a l P r e -p r o o f microorganisms in water media allows to define accurately the levels of hazards for humans and environment.
As matter of facts, the generation of bioaerosols in aeration basins in wastewater treatment plants constitutes a risk of infection in particular of enteric viruses (Mirskaya and Agranovski, 2018). Studies have demonstrated that field workers are exposed to infections from bioaerosols (Masclaux et al., 2014). On February 5, 2020, the U.S. Occupational Safety and Health Administration (OSHA) released a wastewater worker guidance, which stated that the current disinfection techniques used in WWTPs, such as oxidation with hypochlorite or peracetic acid and inactivation by ultraviolet (UV) irradiation, were sufficient to protect the health of wastewater plant operators and the public (OSHA, 2020;WEF, 2020). This recommendation was based on CoV disinfection data obtained from the healthcare settings, which conforms to OSHA's position on the susceptibility of CoV to disinfection. WHO and United States Environmental Protection Agency (EPA) have not raised particular concerns on the current procedures of water sanitation with regard to the SARS-COV-2 (EPA, 2020; WHO, 2020b).
Representative CoVs, such as human CoV 229E (cause of a common cold) and the feline infectious peritonitis virus (FIPV), were examined determining the T 99.9 , i.e. the time required for the virus titer to decrease of 3-log (99.9%). That virus titer reduction was observed in dechlorinated and filtered tap water in 10 days at 23 °C and in 130 days at 4 °C. The inactivation of those CoVs occurred faster in wastewater with a T 99.9 of only 2−4 days.
Under the same conditions, a non-enveloped virus, such as the poliovirus 1, was more stable than CoV 229E and FIPV (Gundy et al., 2008). CoV 229E presented a similar stability with 5-log of titer reduction within 9 days when suspended in minimum essential medium (MEM) containing antibiotics (penicillin and streptomycin), both in the presence and absence of 10% of fetal bovine serum (FBS). In a dried form, deposed on a polystyrene petri dish, 72 h were needed (Rabenau et al., 2005).
The assay of viable virions of two surrogate CoVs, TGEV and MHV, was evaluated in reagent grade water, lake water, and pasteurized settled sewage by determining the T 99 , i.e the time required for a reduction in virus titer of 99%, in turn corresponding to 2 log) (Casanova et al., 2009). Thus, 2-log of virus titer reduction were observed at 25°C for TGEV and MHV in reagent grade water respectively in 22 and 17 days, whereas at 4 °C no significant reduction of the infectious titer for both viruses was observed for 49 days. In a lake water sample J o u r n a l P r e -p r o o f at 25°C, the same infectivity reduction was observed for TGEV and MHV respectively in 13 and 10 days. At 4 °C, 1-log of titer reduction of TGEV was achieved in 14 days, whereas the MHV resulted in comparison more stable. In pasteurized settled sewage at 25°C, 9 and 7 days were necessary for 2-log of TGEV and MHV titer reduction, respectively.
The spontaneous inactivation of SARS-CoV-1 in water media, feces, and urine was investigated in vitro (Wang et al., 2005b;Wang et al., 2005c). The virus titer was found reduced of 5-log after 2 days at 20 °C in dechlorinated tap water, domestic sewage, or hospital sewage; after 3 days in feces and 17 days in urine.
Reducing the temperature to 4 °C the infectivity of SARS-CoV-2 persisted for over 14 days in those media. Wet human specimens (blood serum, sputum, stool and urine) and biological media for cell and virus culture preserve quite well the infectivity of SARS-CoV-1 (Duan et al., 2003;Rabenau et al., 2005;Wang et al., 2005b;Wang et al., 2005c) and that of other human CoVs (see Table 3). The virus titer was reduced by 5-log in serum and sputum in 96 h and in urine in 72 h (Duan et al., 2003). A slow rate of reduction of the titer at room temperature for SARS-CoV-1 of 0.5-log reduction over 9 days was observed in serum-free cell culture medium, whereas under the same conditions, CoV E229 lost its infectivity completely. Phosphate-buffered saline (PBS) solution at pH 7.4 was proven to assure good stability to CoVs. E229 and OC43 persisted in this medium over 3 days (Sizun et al., 2000), while SARS-CoV-1 over 14 days were necessary for 5-log of virus titer reduction (Wang et al., 2005b;Wang et al., 2005c). Another more recent study on the environmental persistence of infectivity of SARS-CoV2 demonstrated that the virus is highly stable at 4 °C in virus transport medium: 0.7log of titre reduction was observed after 14 day (Chin et al., 2020).
CoVs can survive for 2-4 days in sewage and wastewater at room temperature and for a more prolonged time at lower temperatures like that of the winter (Casanova et al., 2009;Duan et al., 2003;Gundy et al., 2008;Wang et al., 2005b;Wang et al., 2005c;Ye et al., 2016). The novel SARS-CoV-2 was detected and found infective in human stool samples (Cheung et al., 2020;Tian et al., 2020;Wang et al., 2020d) and detected worldwide in wastewater and surface water receiving wastewater (see Table 1). The RNA of the virus was found in sewage during and even before the emergence of COVID-19 cases (La Rosa et al., 2021;Medema et al., 2020b).
However tests for the determination viable viruses in WWTP influent and effluent wastewater (Rimoldi et al., J o u r n a l P r e -p r o o f 2020a; Rimoldi et al., 2020b;Westhaus et al., 2021), as well as in hospital wastewater (Ge et al., 2020; and in river receiving contaminated wastewater, (Rimoldi et al., 2020a;Rimoldi et al., 2020b) confirmed the limited resistance of the virus in such water media. The results indicate that the latter water medium is particularly aggressive for SARS-CoV-2 (see paragraph 2.1, Table 1 and Table 3 and for further explanations paragraphs 4.4, 4.5 and 4.6). Infecting wastewater with SARS-CoV-2, Bivins et at. determined a persistence of 1.4-3.3 days for 1-log and of 2.9-6.5 days for 2-log of inactivation of the virus in this media . However, in the same study, a similar resistance was observed for the virus in tap water. In fact, at room temperature the spontaneous reduction of the virus titre of 1-log and 2-log occurred within 1.8-2.2 and 3.6-4.4 days, respectively (see Table 3). This further confirms the limited persistence of this virus in water media.

Methods for inactivation of coronaviruses
Lipid-enveloped viruses with high hydrophobicity, such as CoVs, are less stable in water when compared to non-enveloped viruses. In principle, the performance of water sanitation methods for CoVs can be evaluated J o u r n a l P r e -p r o o f based on the inactivation data of non-enveloped viruses with higher resistance. Unfortunately, the evaluation of infectivity based solely on determination of viable CoVs in wastewaters is considerably simplified.
The performance of these disinfection methods in regard to CoVs and particular regard to SARS-CoV-2, form drinking water to wastewater effluent, is discussed below.

Thermal inactivation
Temperature is considered to be one of the most influential parameters for enveloped virus inactivation (Darnell and Taylor, 2006;Duan et al., 2003;John and Rose, 2005;Kariwa et al., 2006;Lamarre and Talbot, 1989;Rabenau et al., 2005). Membrane and capsid proteins are sensitive to heat-induced denaturation. Aqueous foods, such as milk and fruit juice, as well as drinking water, can be treated in pasteurization processes at moderate (56-65°C for 30 min) or high temperature (80-135 °C for 1-4 s), in order to destroy or deactivate microorganisms, enzymes and viruses. The disinfection of SARS-CoV-1 (-6-log) was achieved at 56 °C, 67 °C, and 75 °C, respectively within 90, 30, and 30 min (see Table 4) (Duan et al., 2003). Similar results were reported on thermal inactivation of SARS-CoV-1 in vitro in media for cell or virus culture (Darnell and Taylor, 2006;Kariwa et al., 2006;Rabenau et al., 2005). In another study, a non-human CoV, the canine coronavirus (CCoV), was identified to be infectious even at 56 °C; however, it was inactivated at temperatures higher than 65 °C (Pratelli, 2008).
MERS-CoV was found sensible to temperature variations: the virus titer was reduced by 5-log in 72 hours at 20°C and in 24 h at 30°C, both on plastics and steel (van Doremalen et al., 2013).
The debate concerning the effects of environment temperature on the rate of transmission of SARS-CoV-2 is still open (Auler et al., 2020;Tobías and Molina, 2020). Nevertheless, the disinfecting effect of heat on the virus is undoubted. The thermal effect on the infectivity of SARS-CoV2 in virus culture media was investigated at 56 and 70 °C, demonstrating that treatments of 30 min and 5 min were respectively sufficient for achieving 6-log of virus deactivation (Chin et al., 2020). In wastewater 2-log of virus removal were obtained at 50°C in 28-34 min, while at 70°C were found sufficient 3.7-5.7 min .
J o u r n a l P r e -p r o o f

Inactivation by ultraviolet radiation
The inactivation of microorganisms by ultraviolet (UV) radiation is currently extensively applied for disinfection of surfaces, drinking water, as well as in tertiary treatments of wastewater for the abatement of the loading of highly resistant species. A relatively large number of the viruses, typically enteric ones, such as noroviruses, rotaviruses, reoviruses, sapoviruses, astroviruses, enteroviruses, adenoviruses and JC viruses, persist in the effluent from full-scale WWTPs with less than 2-log of loading removal (Qiu et al., 2018). Unlike the abovementioned viruses, enveloped viruses are more vulnerable to UV exposure. A prolonged exposure time of 40−60 min was necessary to achieve a satisfactory degree of inactivation for SARS-CoV-1 in vitro (Darnell and Taylor, 2006;Duan et al., 2003;Kariwa et al., 2006). Kariwa (Sabino et al., 2020). The removal of 3-log of viruses was observed in less than 3 s of irradiation, whereas for 5-log of inactivation were necessary almost 50 s (see Table 4).
Due to the lipid acidic envelope, CoVs were found to be sensitive to the variation of pH and the greater virus stability was found at slightly acidic pH levels of 6-6.5. (Geller et al., 2012) On the contrary, SARS-CoV-2 was found highly stable in a wide range of pH values (3-10) at room temperature (Chin et al., 2020).
Based on the data obtained from other viral indicators, the use of chlorine for water disinfection is the most effective and economical solution for this problem. Chlorine effectively inactivates the virus by destroying the viral envelope or capsid (Thurman and Gerba, 1988). In particular, free chlorine has been proven to affect directly the proteins present in the viral envelope, rather than the less reactive lipidic material and the RNA core (Ye et al., 2018). Chlorine can also react with the ammonia present J o u r n a l P r e -p r o o f in wastewater to form combined chlorine (e.g., chloramines). These compounds have the capability to disinfect; however, during disinfection, they are less active and behave differently from free chlorine. It is thus important for every wastewater treatment facility to examine the chlorine species and their relative abundance during the disinfection process.
Studies of the treatment of municipal water and wastewater using chlorine and its derivatives have reported significant inactivation efficiencies for SARS-CoV-1. In hospital wastewater, domestic sewage, and dechlorinated tap water, the virus remained active only for 2 days. Moreover, SARS-CoV-1 became more susceptible to disinfectants compared to E. coli and f2 phage. For SARS-CoV-1 inactivation, free chlorine was more relatively more effective compared to chlorine dioxide. Free residual chlorine exceeding 0.5 mg/L or chlorine dioxide exceeding 2.19 mg/L in wastewater ensured the complete inactivation of SARS-CoV (Wang et al., 2005b). Furthermore, extreme pH levels (pH > 12 or pH < 3), formalin, and glutaraldehyde were determined to inactivate SARS-CoV-1 quite well (Darnell et al., 2004). Peracetic acid has been determined to have the ability to destroy some nonenveloped viruses (e.g., norovirus), which are known to be more resistant to chemical agents compared to enveloped viruses (Dunkin et al., 2017).

J o u r n a l P r e -p r o o f
These studies have shown that the virus tends to exhibit limited resistance in wastewater . In general, investigations on viable SARS-CoV-2 in real influents and effluents, both from domestic and hospital sewages, have resulted in negative results (Ge et al., 2020;Rimoldi et al., 2020a;Rimoldi et al., 2020b;Wang et al., 2020c;Westhaus et al., 2021) albeit the RNA molecules of the virus have been shown to be highly persistent, even up to 50 days in wastewater at room temperature . Concerns have also been raised due to the ecological risks associated to an excessive use of disinfectants for wastewater, such as the sodium hypochlorite that produces high levels of disinfection by-product residuals (Zhang et al., 2020a).

Effects of wastewater holding
Solvents, detergents and disinfectants, normally present in wastewater, can compromise the viral envelope of CoVs (see Table 4). In addition, during the biological treatment stage in WWTPs, the presence of antagonistic microorganisms can enhance the inactivation rates of many viruses (John and Rose, 2005).
As mentioned above, the assay of bacteriophages provides a suitable indicator for the fate of enveloped viruses in sewages (Adcock et al., 2009;Worley-Morse et al., 2019;Ye et al., 2016). The spontaneous inactivation of bacteriophage Φ6 was considered as a potential model for the survival and inactivation of enveloped human viruses. This virus undergoes inactivation of 5-log in 2 and 6 days at 22 and 30 °C, respectively. Longer holding times, for precaution, should be adopted at lower temperatures (Casanova and Weaver, 2015). Even for a more aggressively enveloped virus, such as Ebola, WHO recommended holding wastewater in a reservoir for one week prior to further handling or transport.

J o u r n a l P r e -p r o o f
Without disinfection treatments, SARS-CoV-1 have been shown to preserve its infectivity in municipal and hospital wastewater up to 2 days at 22 °C and for over 14 days at 4 °C (Wang et al., 2005b). A reduced persistence of the virus was observed in hospital wastewater where is typically present a high content of disinfectants (Wang et al., 2005b;Wang et al., 2005c). Sewage from two hospitals with SARS patients in Beijing, China was examined by Wang et at. (Wang et al., 2005c). Electropositive filter media were used to concentrate the virus in the sample, whereas cell culture and RT-PCR techniques were utilized to verify viable virions and detect the amount of virus. The viral RNA was detected even 8 days after disinfection with chlorine, though the virus itself was inactive. SARS-CoV-1 was inactivated by the high concentration of disinfectants used in hospitals. The total residual chlorine was in the range of 01.0 mg/L and 3.012.5 mg/L, and the free residual chlorine was 00.5 mg/L and 1.55.0 mg/L, respectively for the two hospitals studied. Similar resistance was observed for SARS-CoV-2 in wastewater at room temperature, where up to 6.5 days were necessary for an attenuation of 2log of the titer of viable virus . On the contrary for a reduction of 2-log the RNA content of the virus in wastewater were found necessary over than 50 days.
Notwithstanding the limited resistance of the SARS-CoV-2 in sewages, faecal-to-oral route and aerosolization of these media could concur in the spreading of the virus. The aerosolizations of infected urines and faeces from sewage pipelines, as well as during the washing of urinals and toilettes, have been indicated as potential routes of SARS-CoV-2 transmission (Ding et al., 2020;El Baz and Imziln, 2020;Ji-XiangWang et al., 2020;McDermott et al., 2020;Patel, 2020;Yun-yunLi et al., 2020).
As matter of facts, this route of transmission was found effective for the spreading of SARS-CoV-1 in 2003 in Amoy Gardens, a private housing estate in Hong Kong (Hung, 2003;McKinney et al., 2006;WHO, 2003).
J o u r n a l P r e -p r o o f

Sedimentation and inactivation in bioreactors.
Suspended solids and particulate organic matter in both water and wastewater provide contribute to the physical protection of viruses, which can prolong the infectivity of CoVs (Chaudhry et al., 2015;Gundy et al., 2008). When the virus is adsorbed on the porous surface of the particulate, probably, it is sterically protected from the attack of antagonistic microorganisms. In fact, the high levels of suspended solids and organic matter in primary wastewater guarantee prolonged viral infectivity with respect to secondary wastewater effluents. However, the removal of suspended solids together with the adsorbed viruses by sedimentation ensures the reduction of infectivity. As a reference, CoVs inactivation in filtered tap water resulted greater than that in unfiltered samples (Gundy et al., 2008).
Membrane bioreactors (MBRs) have an important role in the removal of particulate matter, including viruses. Enveloped viruses can be effectively inactivated in MBRs (Bodzek et al., 2019;Chaudhry et al., 2015;Jumat et al., 2017;Lv et al., 2006;Simmons et al., 2011). Suspended solids and viruses can be retained using membrane filtration in the presence of antagonist microorganisms and adverse physicochemical conditions (e.g., aeration and chemical dosing) in the MBRs; this retention leads to the efficient inactivation of enveloped viruses, such as CoVs (Bodzek et al., 2019;Chaudhry et al., 2015;Lv et al., 2006).

Wastewater-based epidemiology of SARS-CoV-2: current status
The concentration of substances stable in wastewater, excreted by humans or associable to their activities, can be used to back-estimate the initial occurrence in the serviced population. This constitutes the basis of the concept of the wastewater-based epidemiology (WBE). WBE has been J o u r n a l P r e -p r o o f utilized as an instrument for the real-time generation of information on the consumption and abuse of drugs, both legal and illegal, in a population (Lorenzo and Picó, 2019;Polo et al., 2020). WBE approach can be extended to other challenging purposes, such as the determination of the level of exposure of a population to some chemical and biological agents, such as pesticides, pollutants and pathogens, or even for gaining information on the incidence of specific diseases, such as diabetes, allergies and cancer. A consistent epidemiological surveillance in WWTPs, even in the time of inconspicuous infections, constitutes a sensitive tool for monitoring pathogen circulations in the public.
proposed the use of wastewater sludge as a leading indicator of community outbreak dynamics (Peccia et al., 2020a;Peccia et al., 2020b). As already mentioned, enveloped viruses such as SARS-CoV-2 tend to be adsorbed on or otherwise retained by colloidal and high molecular weight fractions of effluent organic matter, notably abundant in wastewater. Kitamura et al. showed that SARS-CoV-2 RNA concentrations in the solid fraction were higher than in the corresponding supernatant of wastewater samples (Kitamura et al., 2021). The concentration in the solid fraction ranged from 1.6 x 10 2 -1. CoV-2 N1 target and SARS-CoV-2 N2 target concentrations, on a per mass basis, were higher (N1: ~100, N2:~1000 times higher) in the primary settled solids than in the corresponding influent samples of wastewater treatment plants (Graham et al., 2021). As discussed in Section 2.2, enveloped viruses were shown to be adsorbed to wastewater solids in equilibrium.

J o u r n a l P r e -p r o o f
The discussed studies demonstrate the potential of WBE-based surveillance of SARS-CoV-2 in wastewater or sludge to determine the level of circulation of viruses in the corresponding community, and the pattern of spread of the disease among communities. WBE also has the potential to act as an early warning tool that predicts the emergence or re-emergence of a disease outbreak. The detection of higher viral concentration in wastewater samples as compared to the expected value based on the number of confirmed positive cases may also indicate that undetected asymptomatic cases are prevalent in the affected area. A study recently measured the SARS-CoV-2 concentration in the wastewaters of a commercial passenger aircraft and a cruise ship. The latter study suggested that data on the virus RNA concentration in wastewater systems can be used not only to determine the viral load of communities but also as an additional tool for prioritization of clinical testing and contact tracing (Ahmed et al., 2020b). Thus, the knowledge of the concentrations of SARS-CoV-2 in wastewater/sanitation systems may be utilized as a complementary tool to help in determining decisions related to public health.
However, the estimation of the number of infected persons in a community from the viral titres in wastewater involves uncertainties from the assumptions in the calculations. This includes the uncertainties in the used rate of viral shedding in feces of infected persons.  reported a maximum SARS-CoV-2 shedding rate of 10 5.8 copies/mL fecal sample of infected patients (Zhang et al., 2020a). On the other hand, Jeong et al., (2020) reported a range of 10 0.85 to 10 1.49 SARS-CoV-2 genome copies/mL of stool samples from COVID-19 positive patients (Jeong et al., 2020). It can be noted that not all stool samples from COVID 19 patients are positive for SARS-CoV-2, indicating that not all infected persons shed the virus in their feces . The use of information on SARS-CoV-2 viral load in untreated wastewater should also consider the flow rate since variations due to rainfall or other weather conditions can significantly impact the resulting viral titres (Sims and Kasprzyk-Hordern, 2020). The collection of grey and rainwater together with human stools and urine in wastewater sanitation network affects the concentration of viruses and, in turn, the potential association of the virus concentration with the number of infected persons for epidemiological analyses.
These uncertainties must be accounted for in WBE-based models used to correlate the SARS-CoV-2 viral loads in wastewater or in sludge and the prevalence of COVD-19 cases in corresponding communities. This will ensure that accurate information from WBE, combined with clinical testing data, can be used as a tool to help determine approaches needed to manage the pandemic.

Wastewater-based epidemiology of SARS-CoV-2: limits, challenges and perspectives
The precision of the correlation between the number of infected people and the content of viral RNA in wastewater is influenced by numerous factors. First of these factors is the correct determination of the viral content in wastewater, as extensively discussed in prior sections of this article. In turn, the viral genome content in wastewater can be influenced by external factors, such as dilution due to the conveyance of rainwater or by seasonal and periodic variations of grey and industrial water in the influx. The knowledge of the degree of dilution as a result of rainfall and a periodic monitoring of the wastewater flow, followed by a statistical analysis, would allow to minimize these effects.
Alternatively, it is possible to normalize the viral concentration data with respect to an "internal standard" in wastewater. Such standard can be a chemical compound, resistant in wastewater, whose modality of release is known and, for this reasons, it can act as an indicative analyte that can be related to the concentration of the virus. For this purpose, some molecules present in foods, drugs, cosmetics and personal care products, whose consumption by the population is well predictable, can be used.
Caffeine, nicotine, creatinine, cholesterol, coprostanol, cortisol, 5-hydroxyindoleacetic acid (metabolite J o u r n a l P r e -p r o o f of the serotonin) and androstenedione have been proposed as endogenous and exogenous biomarkers for those applications (Polo et al., 2020;Sims and Kasprzyk-Hordern, 2020).
The detection of SARS-CoV-2 in wastewater could be even anticipated. Other significant sources of uncertainty on the viral load in wastewater are the titer losses produced during sample acquisition and processing steps (Ahmed et al., 2020a). Notwithstanding the RNA of SARS-CoV-2 was found to be persistent in this media with a known decay rate (Ahmed et al., 2020c;Bivins et al., 2020), a study by Ahmed et al.(Ahmed et al., 2020c) showed that storage temperature can affect the final determination of the loading of the virus in the sample. Hence, the storage of the sample at 4°C and the subsequent rapid analysis was suggested in order to limit side effects of the decomposition of the virus. These aspects should be considered also during the phase of sampling and transport to the laboratory for analysis. WWTPs are often equipped with automated samplers that allow the withdrawal of aliquots at predetermined periods for the obtainment of reproducible composite specimens with predetermined characteristics. Unfortunately, this WWTP equipment is less typically refrigerated with resulting limitation of the sample stability. In fact, Hart and Halden, showed that seasonal changes in temperature is also a source of uncertainty in the detection and quantification of SARS-CoV-2 RNA in wastewater, which subsequently affects the calculation of the viral load in the corresponding community (Hart and Halden, 2020).
The most difficult factor to address in the context of WWTP-based epidemiological observations is to determine the number of infected people associable to the wastewater (Peccia et al., 2020a). In an infected population, in addition to the confirmed COVID-19 cases, i.e. persons with symptoms of the J o u r n a l P r e -p r o o f disease, there is an unknown number of asymptomatic infected people. Efforts have been made by the countries affected by the pandemic to identify the active cases, but few investigations have established the exact level of circulation of the virus in a population, as performed by Lavezzo et al. via extensive investigation of nasopharyngeal swabs of the population exposed to SARS-CoV-2 in the municipality of Vo', a small town in Veneto, Italy (Lavezzo et al., 2020a;Lavezzo et al., 2020b). The assessment of this parameter is complicated by the number of asymptomatic cases infected by the virus, largely excluded from the counting of the infected persons. The concentration of SARS-CoV-2 RNA in sewage samples therefore has the potential to reflect the actual circulation of the virus in the population since even the viruses shed by undocumented asymptomatic individuals are measured. Investigations on hospital sewages can help to overcome the issue of the association of the number of infect people with the SARS-CoV-2 concentration in wastewater. The number of COVID-19 cases in a hospital can be known with certainty and thus associable to the viral load in the relate sewage. However, this correlation needs further refinement for real epidemiological applications in a population wider than that of the number of cases in a hospital. However, the virus excretion from asymptomatic infect people differs from than of COVID-19 active cases and complicates the projection of such indicator in a "real" population different to that of a hospital. (Cardillo et al., 2021;Hasanoglu et al., 2020) J o u r n a l P r e -p r o o f 6. Final remarks 1. All coronaviruses, including the recently emerged SARS-CoV-2, present a limited persistence in water media: 2-5 days in tap water and 2-6 days in (frozen and thawed) wastewater were found sufficient for 2-log reduction of SARS-CoV-2 titer.
2. The assay of viable virus particles provides crucial information on the potential infectivity of a 3. An increasing number of studies have demonstrated that coronaviruses are transmitted primarily via airborne pathways and the hazards associated to the possible water-mediated transmission of the SARS-CoV-2 appears to be of low epidemiological significance.
4. Transmission of SARS-CoV-2 could be however possible via fomites, faecal-oral route and aerosolization of infected sewages from urinals, toilets and sewage pipeline. Therefore, the precautionary alerts raised by the scientific community since the detection of SARS-CoV-2 in water media and in particular in wastewater still deserve attention and further detailed examination.
5. SARS-CoV-2 is characterized by a fragile structure and is susceptible to conventional disinfection methods that have been shown to be highly effective for its inactivation. Ca. 5 min of exposure to sodium hypochlorite (1%), ethanol (70%), iodine (7.5%), soap solution and other J o u r n a l P r e -p r o o f common disinfectants were sufficient for affording 7-8-log of SARS-CoV-2 titer reduction.
Thermal inactivation, like the pasteurization process, is effective in SARS-CoV-2 deactivation: 30 min at 56 or 5 min at 70°C were sufficient for achieving the complete depletion of the infectivity. SARS-CoV-2 is susceptible to sunlight and rapidly inactivated by UV radiation.
UV-C with wavelength of 254 nm and intensity of 2.2 mW/cm 2 affords 3-log of SARS-CoV-2 titer reduction in less than 3 seconds of irradiation.
6. Excessive utilization of disinfectants has been associated with environmental and human health issues, therefore for SARS-CoV-2 disinfection conventional doses of disinfectants are recommended for sanitation and for wastewater treatment. 10. Sampling (e.g. representative site, type of sampling, storage of samples) and processing (e.g. concentration and quantification methods) affect the measure of the concentration of coronaviruses included SARS-CoV-2 in wastewater. This is necessary to exploit and implement the significant potential of the WBE as an effective tool of surveillance. 24-h composite sampling and replicated analyses are recommended for robust and consistent results.
11. Alternative analytical methods, based on technologies different from the molecular amplification of the SARS-CoV-2 RNA, are currently emerging. These methods could improve the rapidity and the cost-efficacy and extend the number of rapidly processable samples.

The WBE-derived correlations between levels of contagion in a population and virus
loading in the wastewater produced by the exposed population should be refined and made more accurate. Virus concentrations in wastewater are susceptible to dilution effects; thus, the results must be normalized with respect to a specific population, opportune bio-markers and networking conditions by using numerical models.
13. The progress that can be reached during this pandemics in the WBE-based monitoring can be extended to other applications and monitoring of pathologies, such as diabetes, obesity and hypertension.
14. An accurate knowledge of the behavior of virus in the environment can be implemented in a precise and informative model that can also use WBE-derived data. Such model can help