key: cord-0909775-wcn6o3fd
authors: Foladori, Paola; Cutrupi, Francesca; Segata, Nicola; Manara, Serena; Pinto, Federica; Malpei, Francesca; Bruni, Laura; La Rosa, Giuseppina
title: SARS-CoV-2 from faeces to wastewater treatment: What do we know? A review
date: 2020-06-24
journal: Sci Total Environ
DOI: 10.1016/j.scitotenv.2020.140444
sha: 8534f76e218dd659365743572e9401ffd1d391c4
doc_id: 909775
cord_uid: wcn6o3fd

Abstract SARS-CoV-2, the virus that causes COVID-19, has been found in the faeces of infected patients in numerous studies. Stool may remain positive for SARS-CoV-2, even when the respiratory tract becomes negative, and the interaction with the gastrointestinal tract poses a series of questions about wastewater and its treatments. This review aims to understand the viral load of SARS-CoV-2 in faeces and sewage and its fate in wastewater treatment plants (WWTPs). The viral load in the faeces of persons testing positive for SARS-CoV-2 was estimated at between 5·103 to 107.6 copies/mL, depending on the infection course. In the sewerage, faeces undergo dilution and viral load decreases considerably in the wastewater entering a WWTP with a range from 2 copies/100 mL to 3·103 copies/mL, depending on the level of the epidemic. Monitoring of SARS-CoV-2 in sewage, although no evidence of COVID-19 transmission has been found via this route, could be advantageously exploited as an early warning of outbreaks. Preliminary studies on WBE seem promising; but high uncertainty of viral loads in wastewater and faeces remains, and further research is needed. The detection of SARS-CoV-2 in sewage, based on RNA sequences and RT-PCR, requires a shared approach on sample pre-treatment and on-site collection to ensure comparable results. The finding of viral RNA in stools does not imply that the virus is viable and infectious. Viability of CoVs such as SARS-CoV-2 decreases in wastewater - due to temperature, pH, solids, micropollutants - but high inactivation in WWTPs can be obtained only by using disinfection (free chlorine, UVC light). A reduction in the quantity of disinfectants can be obtained by implementing Membrane-Bioreactors with ultrafiltration to separate SARS-CoV-2 virions with a size of 60–140 nm. In sludge treatment, thermophilic digestion is effective, based on the general consensus that CoVs are highly sensitive to increased temperatures.

J o u r n a l P r e -p r o o f 5 SARS-CoV-2 in faeces and wastewater? (ii) how large is the viral load of SARS-CoV-2 in faeces and its capacity of active replication? (iii) how large is the concentration of SARS-CoV-2 in municipal wastewater and is there evidence of faecal-oral transmission? (iv) is wastewater monitoring useful in the developing field of Wastewater Based Epidemiology (WBE) for early-warning surveillance of the spread of the virus? (v) what is the role of wastewater treatment plants?

Since the recent onset of the COVID-19 pandemic, some of the investigations reviewed here is preliminary and/or ongoing. However, relevant publicly available papers not yet peerreviewed have been included.

In general, viruses detected in faeces can derive from: (1) swallowing of respiratory secretions from the upper respiratory tract; in this case the virus can be damaged by the gastric acidity in the stomach, but protection when mixed with food or potential resistance to low pH may allow its passage in the intestine; (2) residues of infected antigen-presenting immune cells; (3) virus replication in intestinal cells (Gu, Han, and Wang 2020; Xiao et al. 2020) , considering that both avian and human influenza viruses are able to replicate in human intestinal epithelial cells (Minodier et al. 2015) .

SARS-CoV and MERS-CoV, which caused the previous outbreaks, were associated with gastrointestinal symptoms in a significant proportion of patients (Zhou et al. 2017; Yeo, Kaushal, and Yeo 2020) . During the two SARS-CoV epidemic outbreaks, in 2002 and 2003, up to 73% of patients had gastrointestinal symptoms during the development of the disease, and the presence of SARS-CoV RNA was demonstrated in the stool specimens (WHO 2003; Zhou et al. 2017) . For a small percentage of patients, viral RNA was still present after 30 days of illness (K. H. Chan et al. 2004 ). The ability of active SARS-CoV to replicate was J o u r n a l P r e -p r o o f 6 identified in the intestine specimens. It was consequently speculated that the human gastrointestinal tract could be an infection site of SARS-CoV (Ding et al. 2004; Zhou et al. 2017) . During the 2012 MERS-CoV outbreak, a quarter of patients reported symptoms, such as diarrhoea or abdominal pain, before severe respiratory symptoms (Assiri et al. 2013;  Mackay and Arden 2015) and MERS-CoV RNA was found in 14.6% of stool samples (Corman et al. 2016) . Zhou et al.(2017) demonstrated that intestinal epithelial cells were highly susceptible to MERS-CoV and could support viral replication.

The new SARS-CoV-2 can affect not only the respiratory system with fever, cough, rhinorrhea, dyspnea or severe pneumonia, but may also cause other clinical symptoms like lethargy, muscle ache, headache, neurologic manifestation or gastrointestinal symptoms such as diarrhoea (Guan et al. 2020; L. Pan et al. 2020) . Among the first studies from China, Guan et al.(2020) extracted data on 1099 patients with laboratory-confirmed COVID-19 from 552 hospitals in mainland China through January 29, 2020, and among varying degrees of illness, diarrhoea was considered uncommon (3.8% of 1099 patients). Subsequent observations on 204 patients with COVID-19 from January 18 th to March 18 th , 2020, highlighted that onethird of patients reported diarrhoea (L. , indicating that digestive issues may be a common early symptom of the disease. The differences between the two studies matches the findings of Cheung et al. (2020) who observed a significant heterogeneity among the studies and a significant subgroup difference in the data from and outside Hubei Province. In a meta-analysis of 60 studies and 4243 patients, the pooled prevalences of all gastrointestinal symptoms and viral RNA positive stool were 17.6% and 48.1% (Cheung et al. 2020 ).

Therefore, SARS-CoV-2 positivity can be observed also in faeces of persons in the absence of gastrointestinal symptoms or diarrhoea: in particular, live SARS-CoV-2 was found in the stool of two patients without diarrhoea (W. Wang et al. 2020 ).

J o u r n a l P r e -p r o o f 7 A synthesis of the incidence of viral RNA positivity in the stools of patients infected with SARS-CoV-2 is reported in Table 1 . In addition, Table 2 reports the loads of SARS-CoV-2 measured in the faeces detected positive by real-time RT-PCR.

< Table 1 . Gastrointestinal (GI) symptoms, diarrhoea and viral RNA positivity in stools of patients infected with SARS-CoV-2. > W. Wang et al.(2020) investigated the biodistribution of SARS-CoV-2 among different tissues of patients with confirmed COVID-19 (bronchoalveolar lavage fluid, blood, sputum, faeces, urine, and nasal samples). In faeces, the positive specimens were 44 of 153, corresponding to 29% of the collected specimens. The mean cycle threshold value for stool specimens was 31.4 (st. dev. = 5.1), indicating a viral load < 2.6·10 4 copies/mL, which was significantly lower than the nasal swabs where the copy number was 1.4·10 6 copies/mL (Ct mean 24.3) (W. Wang et al. 2020) . Lin et al. (2020) tested faecal samples of 65 hospitalised patients and approximately one half were positive, including cases with and without gastrointestinal symptoms. The authors concluded that the gastrointestinal tract may be a potential transmission route and target organ of SARS-CoV-2. With regard to this latter observation, SARS-CoV-2 uses the ACE2 protein as a receptor (Wan et al. 2020) , which is present not only in the respiratory epithelium, but also in the gastro-enteric mucosa (Hamming et al. 2004 ). Xiao et al.(2020) examined the viral RNA in faeces from 73 hospitalized patients with SARS-CoV-2 infection and found that a percentage of 53.4% tested positive for viral RNA in stool. Furthermore, more than 20% of patients with SARS-CoV-2 continued to remain positive in faeces, even after showing negative results in respiratory samples ).

J o u r n a l P r e -p r o o f 8 Another epidemiological and clinical investigation on ten paediatric SARS-CoV-2 infection cases highlighted that some patients were persistently positive on rectal swabs even after their nasopharyngeal testing had become negative ).

Y. , investigating faecal samples from 74 patients, observed that the faecal samples of over half of patients remained positive for SARS-CoV-2 RNA for a mean of 11.2 days after the respiratory tract samples became negative. In certain cases, this duration in faeces persisted for nearly 5 weeks after the respiratory samples tested negative. Y. suggest that the virus may be actively replicating in the gastrointestinal tract even after viral clearance in the respiratory tract. investigated the faeces of 23 patients, finding 83% positive, with a median duration of virus shedding of 22.0 days for faeces. During this period, the mean virus titre in faeces was 5623 copies/mL, but the highest titre at the peak reached 10 5.8 copies/mL (N. Zhang et al., 2020) .

As far as we know, only one study -Cheung et al.(2020) -tested viral RNA in stool collected on the day of hospitalization. In this study viral RNA was detected in 15.3% of the patients.

In particular, viral RNA was found in 38.5% of patients with diarrhoea and in 8.7% of patients without diarrhoea, confirming again that the presence of SARS-CoV-2 RNA in faeces is not necessarily correlated with diarrhoea. A median viral load of 10 4.7 copies/mL (range 10 3.4 -10 7.6 ) was found in the stool of 9 positive patients (Cheung et al. 2020). The stool viral load (median values) was 10 5.1 and 10 3.9 copies/mL in the presence or absence of diarrhoea, respectively. Frequent measurements of viral RNA concentration were performed on the stool of nine hospitalized patients with COVID-2019 during the course of the disease by Wolfel,Corman and Guggemos (2020) , who found high viral RNA concentrations in initial samples, with a peak during the first week of symptoms. The viral content then declined gradually over time, J o u r n a l P r e -p r o o f 9 but stool samples remained RNA-positive for three weeks in six of the nine patients, in spite of full resolution of symptoms (Wolfel, Corman, and Guggemos 2020) . Maximum viral load over 10 7 RNA copies/g faeces was measured in some cases; then a progressive decrease by 2-3 orders of magnitude occurred in the subsequent weeks (Wolfel, Corman, and Guggemos 2020) . These results indicate that the viral load in faeces may be highly variable in the range 10 3 -10 7 RNA copies/g faeces, depending on the day of sampling post onset. To change units from #/mL to #/g, the density of wet human faeces, i.e. about 1.06-1.09 g/mL (Penn et al.

In a meta-analysis of 60 studies and 4243 patients by Cheung et al. (2020) , 70.3% of the stool samples were positive for SARS-CoV-2 even after respiratory specimens tested negative.

Regarding urine, many cases reported negative samples (L. Wang et al. 2020; Yifei Chen et al. 2020; F. Yu et al. 2020; Lescure et al. 2020; Young et al. 2020; Lo et al. 2020; W. Wang et al. 2020 ). Among the rare positive cases ), viral RNA was found to be still present in urine specimens after throat swabs were negative . Wolfel, Corman and Guggemos (2020) observed that the swallowing of respiratory secretions could not be the only passive origin of the virus in the intestine, because a much higher presence of SARS-CoV-2 RNA was found in stool compared to MERS-CoV during its outbreak. This suggested again a potential active replication in the gastrointestinal tract.

The detection of SARS-CoV-2 in the gastrointestinal tract raises the question of a potential faecal-oral transmission (W. Wang et al. 2020; Guan et al. 2020; Xiao et al. 2020; Gu, Han, and Wang 2020; Yuen et al. 2020 ; Amirian and Susan Amirian 2020). The route begins with the transport of the virus in the sewerage and treatment plants, or in pit toilets used in lowincome countries for human excreta disposal, or spread through the practice of "open defecation" which concerns about 900 million people worldwide (WHO 2017). Inadequate sanitation may be a source of contamination by viruses in soil and groundwater.

In previous outbreaks, the prolonged presence of SARS-CoV and MERS-CoV in the environment suggested that faecal excretion, environmental contamination and fomites might contribute to the viral shedding (Yeo, Kaushal, and Yeo 2020; Zhou et al. 2017; Goh, Dunker, and Uversky 2013) . Hence, also the SARS-CoV-2 could be transmitted via this kind of route, but at present no faecal-oral transmission cases have been documented according to (WHO 2020b) . A framework of possible SARS-CoV-2 faecal-oral transmission routes is described in Heller et al. (2020) , who unpack the different pathways that may transmit viruses from faeces to mouth. The critical points of the potential ramifications of the COVID-19 pandemic on waste and wastewater services was highlighted by Nghiem et al. (2020) .

In wastewater treatment plants (WWTPs), the current need is to understand the fate of SARS-CoV-2 considering the removal in the treatment stages and the emissions in: (i) effluent wastewater that may become reclaimed water; (ii) excess sludge that may become biosolids;

(iii) other by-products; (iv) microbial aerosol originated by forced aeration, mixing, pumping, etc. In these complex systems, a prerequisite for the virus to cause infection is its ability to retain viability. The current knowledge is that CoVs viability decreases in wastewater -due to not optimal temperature, acidic pH, light exposure, high content of particulate solids and pollutants -and this gives confidence that the viral infectivity may be attenuated from faeces to sewage, then to WWTPs and then in the environment (La Rosa et al., 2020) . However, as J o u r n a l P r e -p r o o f of June 2020, given the limited information on SARS-CoV-2 in sewage and WWTPs, it would be premature to draw any conclusion.

Other routes, such as solid waste or aerosol from toilets to the sewerage, can be hypothesized to originate fecal-oral transmission of SARS-CoV-2. From the state of the art, it is unlikely that these matrices will become an important transmission pathway for SARS-CoV-2, but they direct attention to the need for much more research in this field.

The presence of SARS-CoV-2 in human samples is confirmed by the detection of specific viral RNA sequences that are unique to SARS-CoV-2 by qPCR. The viral genes included the nucleocapsid protein gene N, the envelope protein gene E, the spike protein gene S, and the RNA-dependent RNA polymerase gene RdRP (also reported as Orf1ab) ("Laboratory Testing Specific pre-treatments steps are normally performed, in particular during wastewater tests, in order to purify and/or concentrate the virus and thus improve the detection efficacy.

However, there is still a lack of agreement on a standard protocol. Different viral enrichment J o u r n a l P r e -p r o o f 13 approaches used with wastewater samples in recent SARS-Cov-2 investigations and PCR assays used to detect SARS-CoV-2 RNA are included in Table 3 .

< Table 3 

Faeces reach the sewerage system and undergo a large dilution. Raw wastewater contains organic matter, particulate solids, micropollutants and many pathogens, especially enteric.

Viruses contained in faeces may undergo several transformations along the sewer network and possibly a reduction of number and viability, as an effect of solid deposition, decreasing pH, temperature and other factors. Table 4 summarises the rare studies that have quantified the concentration of SARS-CoV-2 (expressed in copies/mL) in municipal wastewater. F. The virus copies in wastewater are largely diluted in comparison to the viral load in the faeces. According to Section 2, viral copies in the faeces of persons testing positive for SARS-CoV-2 varied from 5·10 3 to 10 7.6 copies/mL (N. Zhang et al., n.d.; Cheung et al., 2020) . The dilution of positive faeces in wastewater causes a drop in the concentration by 4-5 orders of magnitude or more. This dilution is due to various factors: the daily flow rate discharged into the sewerage by a person (that is about 80% of the average daily supply of drinking water per capita and makes an approximate 10 3 fold dilution), stormwater or infiltration of parasite inflow in the sewer network. Moreover, not the whole population contributes to the viral load and this depends on the percentage of positive cases among the population served by a WWTP. By way of example, the number of cases in Lombardy, one of the Italian regions most affected to date, was 560 cases in 100,000 people on the 10 th of April, 2020; this produced a further dilution of 5.6 ·10 3 fold.

When capillary and frequent individual testing in a population is not possible, aggregate information about the level of the outbreak could be useful for monitoring its evolution and the effectiveness of containment measures. Together with other relevant approaches, additional information could be extrapolated from the viral load in municipal wastewater. This is the focus of the developing field of WBE, which in broad terms means "the application and development of using the quantitative measurement of human biomarkers in sewage to evaluate lifestyle, health and exposure at the community level" (https://scorecost.eu/). It has been so far quite extensively used in studies related to drugs or pharma consumption, but also for poliovirus (Nakamura et al. 2015) and infectious disease surveillance and early warning (Sims and Kasprzyk-Hordern 2020). A detailed proof-of-concept study of the WBE approach has been described by Ahmed et al.(2020) , who tested 9 wastewater samples, collected from two WWTPs and a pumping station for a period of six days. This study quantified the SARS-CoV-2 RNA copies in raw wastewater, with the aim of estimating, via Monte Carlo simulation, the number of infected individuals in the catchment area. The work by Ahmed et al.(2020) draws attention to the uncertainty of some input parameters. In fact, the viral load in the stool of infected persons is not constant as described in Section 2, and it appears to be a critical parameter. The model of Briefly, a simplified theoretical approach of WBE methodology starts from the concentration of SARS-CoV-2 (converted into copies/m 3 ) measured in municipal wastewater taken along the network or at the inlet of a WWTP, but at a point that represents a known urbanized area drained by the sewer system. The daily viral load in wastewater (expressed in copies/d) is then calculated by multiplying the concentration by the daily flow rate of wastewater (expressed in m 3 /d). This load is then compared with the viral copies in the faeces of persons testing positive for SARS-CoV-2, to obtain an estimation of the number of positive cases in the urbanised area. Unfortunately, analytical data have demonstrated that the viral load in faeces is highly variable. It is so for up to 4 orders of magnitude, from 5·10 3 to 10 7.6 copies/mL (see Section 2), and further research is needed to propose reasonable values that can be used as a reference.

Despite these difficulties, wastewater monitoring could be proposed also as a semiquantitative detection system or at worst for detecting presence/absence in the early surveillance of COVID-19 diffusion (Nghiem et al., 2020) .

In synthesis, WBE could be a promising tool for COVID-19 surveillance, but extensive and highly coordinated studies are required, including the quantification of individual virus load in stool and during the disease, because this information is very uncertain at the moment but is fundamental for obtaining accurate estimations. At present, no comprehensive studies on the fate of SARS-CoV-2 along the entire chain of a WWTP including digested sludge or biosolids are available. Two recent studies reported the investigation of SARS-CoV-2 in treated wastewater (Wurtzer et al. 2020; Randazzo et al., 2020) ; main findings are summarised in Table 5 . In particular, in the study of (Wurtzer et al. 2020 ), 6 out of 8 samples of treated wastewater were positive for SARS-CoV-2; the viral load was 1-10 2 copies/mL and was reduced by 100 times compared to the raw wastewater entering the plant (Wurtzer et al. 2020) . In this study, some results were near the quantification limit These findings indicate that secondary wastewater treatment may contribute to reduce the virus concentration in wastewater, thanks to the adverse environmental conditions that the virus encounters, but removal is largely variable and thus, to enhance the level of virus inactivation in WWTPs, for example for reuse, disinfection has an important role.

Current knowledge about SARS-CoV-2 in WWTPs is largely based on what is known from the similar CoVs (Nghiem et al., 2020) , which are severely affected by several environmental J o u r n a l P r e -p r o o f 20 factors (i.e. temperature, solids, pH) or disinfectants. There is evidence that CoVs are less stable in the environment than enteric viruses -such as adenoviruses, norovirus, rotavirus and hepatitis A -for which a wide literature exists in WWTPs (Simmons and Xagoraraki 2011; Ye et al. 2016; Gundy, Gerba and Pepper, 2009 ). In wastewater, T 90 ranges from days to months for nonenveloped viruses, whereas it is several hours or days for enveloped viruses (Ye et al., 2018) . Viruses of avian influenza, SARS, MERS, Ebola and SARS-CoV-2 are enveloped (Bibby, Aquino de Carvalho and Wigginton, 2017) . At the moment, the mechanisms explaining the higher susceptibility of enveloped viruses to be inactivated in aqueous environments are mostly unknown in the literature (Ye et al., 2018) . Some environmental factors that may affect the stability of SARS-CoV-2 in water are summarised in Table 6 .

Disinfection treatments implemented in WWTPs are based on hypochlorous acid (free chlorine), chlorine dioxide, ozone and peracetic acid or UV radiation (Naddeo and Liu, 2020). Due to the phylogenetic similarities between SARS-CoV-1 and SARS-CoV-2, disinfection technologies adopted during the SARS epidemic can be implemented also for the inactivation of SARS-CoV-2 in wastewater (J. Wang et al., 2020) . The enveloped viruses, having a fragile outer membrane, are more susceptible to oxidant disinfectants, such as chlorine, than nonenveloped human enteric viruses (WHO, 2020c). Among chemical disinfectants, free chlorine proves more effective in inactivating SARS CoV than chlorine dioxide (Wang et al., 2005b) , but a continuous determination of the residual chlorine content should be implemented on the effluent, to adjust the disinfectant dosage accordingly. In fact, J o u r n a l P r e -p r o o f a free chlorine residual is important to ensure the removal of the virus, but excessive disinfectant applications may cause potential adverse environmental effects, for example on ecosystems or in agriculture (Bruins and Dyer, 1995) .

A reduction in the quantity of disinfectants and by-products can be obtained implementing Membrane-Bioreactors equipped with ultrafiltration (UF). The absolute pore size (defined according to Simmons and Xagoraraki, 2011) of UF is from 0.01 μm, permitting to separate SARS-CoV-2 virions with size of 60-140 nm (Table 6 ).

Water contaminated with sewage discharged from combined sewer overflows (CSO) during events of heavy rainfall, is another issue that poses potential risks, including the definition of specific exposure scenarios (Bibby, Aquino de Carvalho, and Wigginton 2017). During CSO, the flow rate that is above a threshold is discharged directly into the receiving water bodies in order to reduce the impact on public health, since the mix of sewage and rainfall may contain pathogenic microorganisms and other pollutants.

The recent global outbreak of SARS-CoV-2 has highlighted scant knowledge about CoVs in the field of sewage and WWTPs. This review has collected the scientific literature currently available on the route of SARS-CoV-2 from faeces to WWTPs, although the research available in this field is very limited, fragmented or still in the early stage of development.

A percentage from 15 to 83% of patients infected with SARS-CoV-2 have detectable viral RNA in faeces, even in the absence of gastrointestinal symptoms or diarrhoea. Patients may continue to remain positive in the stool, even when respiratory tract samples become negative. Conversely, urine is often negative. The load of SARS-CoV-2 in faeces is highly variable, in the range 5·10 3 -10 7.6 RNA copies/mL, depending on various factors (i.e. time from onset). This viral load decreases remarkably when faeces are diluted in municipal J o u r n a l P r e -p r o o f 22 wastewater, where the concentration of SARS-CoV-2 drops to a range from 2 copies/100 mL to 3·10 3 copies/mL, depending on the level of the epidemic. Quantitative data on viral load in faeces and wastewater and their relationship, currently uncertain, are fundamental for WBE applications to be used for the early warning of outbreaks. In particular, the large uncertainty in the viral load in faeces makes it difficult to determine a typical value that could be extremely useful in WBE.

The fate of SARS-CoV-2 in WWTPs, because of the actual scarcity of analytical data, is predicted on the basis of similar CoVs that are severely affected by environmental factors (e.g. temperature, solids, pollutants, pH). However, current findings indicate that faecal-oral transmission is not proven. By analogy with the previous studies on SARS and MERS outbreaks, there are reasons to conclude that the viral content may be controlled in WWTPs. 153 ----44/153 29 -live SARS-CoV-2 was observed in the stool sample from 2 patients without diarrhoea -the total number is referred to specimens instead of patients -scarcity of detailed clinical information available (Wolfel, Corman and Guggemos, 2020) 9 > 10 7 (peak) (range 10 3 -10 7 ) -viral load highly variable, depending on the day of sampling post onset. -peak during the first week from onset (Zhang et al., no date) 23 5623 (mean) 10 5.8 (peak) -faecal samples detected by rRT-PCR targeting ORF1ab, N and S genes -Ct values of ORF1ab gene from ⁓25 to 43 -Negative samples for Ct value of 43 (limit of detection) -Ct values of 37. 6, 32.64, 29.22, and 25 .77 corresponding to 1×10 3 , 1×10 4 , 1×10 5 , and 1×10 6 copies/ mL, respectively.

( Cheung et al., 2020) 59 10 4.7 (median) (range 10 3.4 -10 7.6 ) -10 5.1 copies/mL with diarrhoea) -10 3.9 copies/mL without diarrhoea (W. Wang et al., 2020) (Wang et al., 2005)  at 70°C for 5 min, SARS-CoV-2 is inactivated (Chin et al., 2020)  The decline in infectivity of CoVs (surrogates for SARS-CoV) followed a typical first-order kinetic at rate of 1.5-2.0 log per week at 25°C, while at 4°C the rate was slower and approximately 0.  SARS-CoV in wastewater is more susceptible to sodium hypochlorite and chlorine dioxide than Escherichia coli (Wang et al., 2005b)  to control the virus, the dosage of hypochlorous acid (free chlorine) should ensure a free residue chlorine over 0.5 ppm, to confirm that chlorine has not been completely depleted and ensure complete inactivation of SARS-CoV (Wang et al., 2005)  Free residue chlorine over 2.2 mg/L is needed for chlorine dioxide for complete inactivation of SARS-CoV (while E. coli is not completely inactivated)  Inactivation is due to the reaction with proteins in the nucleocapsid instead of genome or membrane lipids (Ye et al., 2018) Treatment by UV disinfection (UVC light)  Enveloped viruses do not seem to have a higher susceptibility to UVC light than non-enveloped viruses (Wigginton and Boehm, 2020)  Inactivation primarily targets the genome, while lipid membrane do not protect viruses from the radiation (Wigginton and Boehm, 2020) Treatment by primary and secondary settling  26% of enveloped viruses adsorbed to solids, compared to 6% of nonenveloped (Ye et al., 2016)  a significant part of CoVs, is likely to be present in the primary or secondary sludge  inactivation in sludge is similarly as in wastewater (3 log  The hydrophobicity of the viral envelope makes enveloped viruses less soluble in water and they tend to adhere to solids and to concentrate in sludge (Gundy, Gerba and Pepper, in 2-4 d) (Gundy, Gerba and Pepper, 2009) 2009) Treatment by mesophilic and thermophilic anaerobic digestion  Human CoV were detected in sludges pre and post anaerobic digestion (Bibby, Viau and Peccia, 2011; Bibby and Peccia, 2013)  SARS-CoVs infectivity is lost at 56°C for 90 min (temperature of thermophilic anaerobic digestion) (Duan et al., 2003)  in anaerobic digestion at 28°C, poliovirus lose infectivity and ammonia may act as a virucidal agent (Ward and Ashley, 1979)  higher inactivation of CoVs is expected in anaerobic digestion because CoVs are much more sensitive to warm temperature than poliovirus  thermophilic aerobic digestion is much more effective against nonenveloped viruses than mesophilic digestion

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