key: cord-0689496-53nfudkm
authors: Collivignarelli, Maria Cristina; Collivignarelli, Carlo; Miino, Marco Carnevale; Abbà, Alessandro; Pedrazzani, Roberta; Bertanza, Giorgio
title: SARS-CoV-2 in sewer systems and connected facilities
date: 2020-07-04
journal: Process Saf Environ Prot
DOI: 10.1016/j.psep.2020.06.049
sha: 0a895361d8e504c8381b6bde1560e23a287d5bdb
doc_id: 689496
cord_uid: 53nfudkm

As for the SARS coronavirus in the 2003 epidemic, the presence of SARS-CoV-2 has been demonstrated in faeces and, in some cases, urine of infected people, as well as in wastewater. This paper proposes a critical review of the state of the art regarding studies on the presence of SARS-CoV-2 in wastewater and sewage sludge, the factors affecting its inactivation and the main proposed treatments. In-vitro tests demonstrated low resistance of SARS-CoV-2 to high temperature, while even significant changes in pH would not seem to determine the disappearance of the virus. In wastewater and in sewage sludge, to date studies on the influence of the different parameters on the inactivation of SARS-CoV-2 in real matrices are not available. Therefore, studies involving other HCoVs such as SARS-CoV and HCoV-229E have been also considered, in order to formulate a hypothesis regarding its behaviour in sewage and throughout the steps of biological treatments in WWTPs. Finally, SARS-CoV-2 in wastewater might track the epidemic trends: although being extremely promising, an effective and wide application of this approach requires a deeper knowledge of the amounts of viruses excreted through the faeces and the actual detectability of viral RNA in sewage.

Several studies have shown the presence of the SARS-CoV-2 in faeces and urine of infected patients (Chen N. et al., 2020; Gu et al., 2020; Huang et al., 2020; Tian et al., 2020; Xiao et al., 2020) . In Table 1 , the results of recent clinical studies are reported. There is literature agreement about the possible finding in faeces of infected subjects, while the occurrence in the urine has not been always confirmed. For instance, Lescure et al. (2020) followed five patients admitted to French hospitals. In this case, the virus was detected in the faeces of two patients while no urine samples appeared positive.

From a critical analysis of literature, some fundamental aspects can be highlighted. The first concerns the low level of SARS-CoV-2 RNA shed in the first phase, after illness onset, demonstrated by a rRT-PCR (Real-Time Reverse

Transcriptase-Polymerase Chain Reaction) cycle (Ct) value generally in a range between 30 and 40 (considering 40 as positivity threshold) Zhang et al., 2020) , quite higher than that deriving from swabs of the respiratory tract (> 20) (Zou et al., 2020) . While the viral load in the respiratory tract tends to run out more quickly, several studies have demonstrated that the virus can be found in the stool of an infected subject even after a prolonged period Lescure et al., 2020) . Zhang et al. (2020) observed that the median duration of virus emission was 10 days in swabs from respiratory tracts and up to 22 days in faeces. Despite the negativity of respiratory swabs, in 5% of cases, the faeces were tested positive also 26 days after discharge. Gupta et al. (2020) examined 26 articles on the presence of SARS-CoV-2 in faeces and revealed its occurrence up to 30 days after the onset of the first symptoms in infected patients (in a low number of cases, up to 47 days). Bonato et al. (2020) detected viral RNA in faecal samples of 53.4% hospitalized patients; faeces remained positive in 23.3% of people being tested negative for oropharingeal swabs.

Although, at the moment, a path of oral-faecal contamination has not been demonstrated yet and further studies are required, this aspect is still very important because it could determine a greater probability of the presence of viral fragments in WW, also in areas where the infection is still in its early stages, thus allowing to follow the approach of the wastewater based epidemiology. The answer entails the availability of a standard technique for extracting and quantifying the virus in such a complex matrix, and, in particular, for assessing its pathogenicity. As reported above, SARS-CoV-2 can reach the sewerage via faeces, and, possibly, urine, beside other human excreta (Table 2) . However, the survival capacity of the virus in WW remains very uncertain and, to date, the results available on the SARS-CoV-2 are limited. Ahmed et al. (2020) researched the presence of the virus in the WW of the city of Brisbane (Australia) in the period at the time of the epidemic peak. Although it was detected in two samples, the viral concentration were very low (12 and 1.9 copies (100 mL) -1 , respectively) and therefore the quantification cycle (Cq) values were very high (37.5 and 39, respectively) and comparable to those that the virus has in the faeces and urine. F. Wu et al. (2020) They quantified an average number of 42.7 (± 32.9) SARS-CoV-2 RNA copies mL -1 with peak of more than 100 copies mL -1 . During the epidemic peak, Wurtzer et al. (2020) measured to 10 6 -10 7 unitSARS-CoV-2 RNA mL -1 in French sewage, while Randazzo et al. (2020b Randazzo et al. ( , 2020a monitored the viral RNA in WW collected from two Spanish regions. The latter, quantified SARS-CoV-2 (5.38 ± 0.21 log genomic copies L -1 ) also in samples collected in La Murgia, an area, where

CoViD-19 had a very low impact. Between February and April, in Milan and Rome 12 samples were collected from WWTPs and analysed by La Rosa et al. (2020b) . SARS-CoV-2 RNA was evidenced in 50% of samples. Two aspects are particularly noteworthy: first, viral fragments were detected in Milan only few days after the first Italian case, was officially released (in the town of Codogno, about 60 km South of Milan). As suggested by La Rosa et al. (2020b) this means that the analysis of WW may lead to a more accurate estimation of the actual period of CoViD-19 onset.

Secondly, they started from the standard WHO procedure for Poliovirus surveillance (WHO, 2003) and designed a new SARS-CoV-2 specific primer set for molecular analysis, in order to search the SARS-CoV-2. The development of a standardized procedure of SARS-CoV-2 RNA detection in WW is a crucial aspect in order to obtain reliable and comparable results. However, to date there is no univocal method, and this represents a significant gap in the research, regarding both the phases of extraction (from such a complex matrix) and quantification.

These studies clearly confirm that the virus can therefore be present in WW, but many points remain uncertain. For instance, it is mandatory to investigate the impact of external conditions, such as temperature, pH and retention time, on the survival of SARS-CoV-2 in the aquatic environment. This requires the execution of tests aimed at assessing the virus vitality, practically almost never performed, until now. Rimoldi et al. (2020) which revealed that virus present in WW and river water collected in Milan did not exhibit cytopathic effect. In this case, only a limited number of samples was examined in a very short period of pandemic disease, therefore further research is required. 

Although the coronaviruses survival in the environment can differ according to the specific type (Casanova et al., 2009; Wigginton et al., 2015) , these indications allow to formulate some preliminary considerations. Based on the abovementioned findings (Section 4.1.1.), SARS-CoV-2 might be present in WW, but its survival is likely extremely low, especially when the temperature of the WW remains stably above 20 °C. To date, the real influence of other parameters, like pH, sunlight and disinfectant agents, on survival time is still uncertain. Nonetheless, presuming a behaviour similar to that of other coronaviruses, this pathogen should rapidly be inactivated (WHO, 2020c). The samples were taken immediately after the treatment phases and therefore other factors that could accelerate the disappearance of the virus in the hypothesis in which they are spread on the soil such as for example pH variations, solar irradiation, and heat were not taken into consideration. HCoV-HKU1 was again identified by Bibby and Peccia (2013) in anaerobically digested SS. They expressed concern due to the high load of virus in treated SS that could be transmitted by the aerosols emitted during the land application. However, also in this case, the infectivity of the viruses, the influence of the anaerobic digestion temperature and meteorological conditions on the resistance of the virus were not defined.

As also recognized by U.S. National Research Council and Italian National Institute of Health (INIH, 2020a; USNRC, 2002) , the presence of the viruses in the SS is not directly indicative of a potential hazard of the matrix as an effective transmission capacity of the pathogen is not proven. Moreover, SARS-CoV manages to resist in the external environment for a limited time if subjected to heat and solar radiation (Darnell et al., 2004) and there are currently no studies on its possible survival on soil.

Generally, there are three main aspects that can improve the removal of viruses: (i) high retention time, (ii) high values of temperature and (iii) high/low values of pH. Recent studies show that also SARS-CoV-2 is particularly sensitive to these three aspects. Chin et al. (2020) have demonstrated how the resistance of the virus from 22 °C to 70 °C dropped from 7 d to 1 min, highlighting its sensitivity of this virus towards high values of temperature. This result is very similar to the previous findings in case of SARS-CoV by Rabenau et al. (2005) , who observed greater stability compared to

HCoV-229E in the external environment, but a rapid inactivation under temperature conditions of 56-60 °C. On the other hand, it has been shown that even significant changes in pH (from 3 to 10) would not seem to determine the disappearance of SARS-CoV-2 (Chin et al., 2020) .

Although the studies on SARS-CoV-2 must be considered preliminary, as they are carried out only in vitro, they provide interesting insights that can enable the selection of the most appropriate treatments, in order to ensure complete inactivation of SARS-CoV-2 in the SS. Taking into account its sensitivity for temperature values greater than 50 °C, conventional and advanced biological thermophilic processes (e.g. anaerobic digestion (Leite et al., 2017) and

aerobic/anaerobic membrane bioreactors (Collivignarelli et al., 2017) , respectively), and thermal drying treatments (Richard, 2019) However, none of the aforementioned studies on the influence of the different parameters on the inactivation of SARS-CoV-2 and SARS-CoV focus specifically on SS but it is very likely that, beside temperature (responsible for protein inactivation and extracellular enzymes activity increase), also the presence of bacteria, protozoa, metazoa, and, more generally, organic matter affect the virus survival (Gundy et al., 2009; Pinon and Vialette, 2018; Rzeżutka and Cook, 2004) . Therefore, an increase in retention time might be proficuous. Anyway, owing to the total absence of literature J o u r n a l P r e -p r o o f

From the analysis of the literature, the lack of data about the influence of parameters like temperature, pH and retention time on the survival of the virus in WW and SS is evident. Since ad hoc studies on SARS-CoV-2 are currently scarcely available, information can be inferred from experiments conducted in the past years on SARS-CoV, mostly in vitro. As confirmed by several authors Kitajima et al., 2020; La Rosa et al., 2020a) , part of the lack of results on this issue is attributable to the difficulty in finding SARS-CoV-2 RNA within these particular matrices, as there is currently no officially recognized univocal method. Sewage and sludge contain hundreds of molecules (namely, fulvic and humic acids, fats, proteins, metal ions, detergents), liable to interfere with polymerase chain reaction: new techniques of molecular biology, such as the digital PCR and the next generation sequencing, might allow overcoming this issue. Furthermore, the adoption of different commercial kits for performing the PCR leads to the obtainment of results only partially comparable, owing to the variability of extraction efficiency Sims and Kasprzyk-Hordern, 2020) . Moreover, La Rosa et al. (2020b) , regarding the monitoring of wastewater, highlight the inadequacy of the current techniques for concentrating and recovering non-enveloped enteric viruses. Therefore, identifying and sharing protocols from sample pre-treatment to viral particles quantification would allow simplifying, accelerating and stimulating the analysis of these matrices, before the issuing of a standardized technique.

The authors suggest evaluating the effect of the different parameters, possibly simultaneously, on the inactivation of SARS-CoV-2 in real matrices, like sewage, sludge, soil, freshwater (both surface and groundwater). A deeper knowledge of the actual occurrence and persistence of the infectious virus is mandatory. These aspects would also allow predict the behaviour of SARS-CoV-2 in the different phases of the WWTP, to identify possible exposure risks for workers. Furthermore, the possibility to track precisely the virus vitality (if any) from sewers to the WWTPs throughout the subsequent phases of treatment efficacy will help to optimize process conditions and to select more adequate technologies (with particular focus on disinfection).

In order to track dynamics of the epidemic and to get an early warning in case of possible outbreaks, the adoption of the WBE approach represents an interesting initiative. For example, the INIH (2020) identified the presence of SARS-CoV-2 RNA in the wastewater of Milan and Turin in samples dating back to December: over 2 months before the first official case in the end of February. Although WBE might reveal a precious tool for the early warning of future outbreaks and tracking the epidemic geographically and over time (Barcelo, 2020; Kitajima et al., 2020; Lodder and de Roda Husman, 2020; Sims and Kasprzyk-Hordern, 2020; Wurtzer et al., 2020) (Fig. 1) , its thorough and diffused application still requires the acquisition of further information on crucial elements, which represent the input data of possible simulations. It is worth underlining the need of deeper studies about the virus shedding by infected asymptomatic and paucisymptomatic people, the actual stability of viruses and virions in sewage and, J o u r n a l P r e -p r o o f definitely, the possibility to exploit, in parallel, other biomarkers/pharmaceuticals to track the outbreak, in order to integrate the available information and obtain a more precise assessment for surveillance purposes.

The authors declare no conflicts of interest J o u r n a l P r e -p r o o f

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