Persistence of SARS-CoV-2 RNA in wastewater after the end of the COVID-19 epidemics.

Although the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been widely detected in wastewater in many countries to track the COVID-19 pandemic development, it is still a lack of clear understanding of the persistence of SARS-CoV-2 in raw sewage, especially after the end of the COVID-19 pandemic event. To fill this knowledge gap, this study conducted a field trial on the SARS-CoV-2 presence in various wastewater facilities after the end of the COVID-19 epidemics in Beijing. The result showed that the wastewater treatment facility is a large SARS-CoV-2 repository. The viral RNA was still present in hospital sewage for 15 days and was continually detected in municipal WWTPs for more than 19 days after the end of the local COVID-19 epidemics. The T90 values of the SARS-CoV-2 RNA in raw wastewater were 17.17-8.42 days in the wastewater at 4 ℃ and 26 ℃, respectively, meaning that the decay rates of low titer viruses in raw sewage were much faster. The results confirmed that the SARS-CoV-2 RNA could persist in wastewater for more than two weeks, especially at lower temperatures. The sewage systems would be a virus repository and prolong the presence of the residual SARS-CoV-2 RNA. The study could enhance further understanding of the presence of SARS-CoV-2 RNA in raw wastewater.

Introduction

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is an enveloped, positive-sensed virus belonging to the genus Betacoronavirus that has caused a severe coronavirus disease 2019 (COVID-19) pandemic (Coronaviridae Study Group of the International Committee on Taxonomy of Viruses, 2020). This virus has spread worldwide and infected approximately 218 million people by October 2021. Droplets and aerosols have been suggested as the main routes for the spread of SARS-CoV-2 in direct human-to-human transmission (Morawska et al., 2020), but the SARS-CoV-2 RNA was also detected in the excrements of infected individuals with a load of 102 - 1010 copies per gram (Han et al., 2020, Pan et al., 2020, Xiao et al., 2020), which are discharged into the wastewater. Detectable SARS-CoV-2 in wastewater has been reported in many countries, including Australia, Japan, Italy, Netherlands, Spain, and the United States (Ahmed et al., 2020a, Haramoto et al., 2020, La Rosa et al., 2020b; Medema, 2020; Randazzo et al., 2020; Sherchan et al., 2020), suggesting that the virus survived in the wastewater was a potential source of transmission. Most of the research has focused on establishing detection methods for SARS-CoV-2 RNA, optimizing the virus concentration tools, achieving higher detection sensitivity, tracing the sources of the virus spread, and assessing wastewater-based epidemiology (WBE) to monitor the infections in communities (Ahmed et al., 2020b, Ahmed et al., 2020d, Graham et al., 2020, Kocamemi et al., 2020, Rimoldi et al., 2020). However, little is known about the possible distribution and persistence characteristics of SARS-CoV-2 in the sewage systems after the end of the COVID-19 pandemic. This information is crucial for the WBE to monitor potential infected individuals in sewerage systems.

In previous studies, SARS-CoV-2 was detected in wastewater, river water, and sludge from wastewater treatment plants (WWTPs) (Balboa et al., 2021; Bibby and Peccia, 2013; Kocamemi et al., 2020; Peccia et al., 2020). SARS-CoV-2 contamination would amount up to 7 × 106 copies/L in sewage discharged from feces, 3.19 × 106 copies/L in river water, and 4.6 × 108 copies/L in sludge (Patel et al., 2021). Sowing with high initial titers of SARS-CoV-2 remained infectious in the wastewater even after 7 days (Bivins et al., 2020). The occurrence of SARS-CoV-2 in sewage systems and other water sources indicated a possible spread pathway of the virus through the water (Heller et al., 2020, Hindson, 2020, Patel et al., 2021). Hospital wastewater, in particular, can significantly increase the risk of transmission to the environment, as a large number of infected cases are received in hospitals (Achak et al., 2021). In addition, due to the encapsulation of solids and insufficient disinfection, SARS-CoVs survive in the effluent, even after adequate disinfection measures (Wang et al., 2005b, Zhang et al., 2020). Therefore, inefficiently disinfected wastewater is likely to be a source of the long-term presence of SARS-CoV-2 and needs to pay more attention.

Many studies suggest that viruses can persist in wastewater for several to a dozen days. Furthermore, the persistence and disintegration of the virus strongly depends on the ambient temperature (Ahmed et al., 2020c, Gundy et al., 2009, Hokajärvi et al., 2021, Wang et al., 2005a, Ye et al., 2016). In addition, the lipid bilayer membrane structure that covers the protein capsid makes enveloped viruses more susceptible to the changes of environmental factors such as pH, salinity, temperature, and organic matters (Kumar et al., 2020a, Ye et al., 2018, Ye et al., 2016). Earlier studies have shown that SARS-CoV can persist in wastewater for 3 days at 20 ℃ and more than 14 days at 4 ℃ (Wang et al., 2005b). Recently, further research found that the decay rate constants of SARS-CoV-2 RNA in untreated wastewater under 37, 25, 15 and 4 ℃ were 0.286, 0.183, 0.114 and 0.084/day (Ahmed et al., 2020c). On the other hand, it was almost unchanged when the wastewater samples were stored for 58 days spiked with SARS-CoV-2 inoculum prepared from nasopharyngeal swabs of a COVID-19 patient at − 20 ℃ and − 75 ℃ (Hokajärvi et al., 2021). However, the virus titers in these studies were often achieved by spiking exogenous viruses, where the final concentration was far from those in the raw sewage (106 copies/mL vs 103 copies/L). What’s more, the integrity of the viral structure may be better than in raw wastewater. Therefore, while the data revealed a specific decay pattern of viruses in the wastewater, there may be a difference in the persistence properties of the relatively lower titers (~ 103 copies/L) of SARS-CoV-2 in raw wastewater (Ahmed et al., 2020c, Bivins et al., 2020, Sala-Comorera et al., 2021).

The spread of the COVID-19 epidemic in China has been well-controlled through effective and provident measures. This offers a special but real scenario without the continuous release of SARS-CoV-2 particles into aqueous environments as there is no uninterrupted source of infection. So, it provides us an opportunity to investigate the potential persistence of SARS-CoV-2 residues in wastewater after the end of the local epidemic. In this work, we tracked the presence of SARS-CoV-2 RNA in wastewater after the end of the COVID-19 epidemics in Beijing to assess the persistence of viral RNA in the field wastewater discharged from hospital and municipal sewage systems, as well as in rivers. Further investigations into the decay properties of SARS-CoV-2 in raw sewage were carried out at various temperatures. The study could enhance further understanding of the presence of SARS-CoV-2 RNA in the real water system.

Materials and methods

Samples collection

Two small-scale population infections of the COVID-19 epidemic occurred in Beijing before the autumn of 2020. The first wave of the epidemic befell from January 20th to March 23rd, which resulted in 416 indigenously infected cases. Another 335 infections’ epidemic spread from a wholesale market of agricultural and sideline products on June 11th and ended on July 6th after the last infected case was reported. During this period, sewage from the sewage systems, including hospital discharge, was tested for SARS-CoV-2. The information from each sampling campaign, including the location and time, is shown in Fig. 1. The data on existing cases and new confirmed cases were from the official website of the Beijing Center for Disease Prevention and Control (https://www.bjcdc.org/ColumnAction.do?dispatch=getEjPage&id=4473&cID=4493). The wastewater samples were taken for the first time on April 30th since the end of the first wave of the COVID-19 epidemic, from the wastewater treatment facilities of six hospitals that had ever received infected cases. Afterward, the samples from the municipal influent of WWTPs, sewer, and rivers were also fetched before and after the second-round of the local COVID-19 epidemic in Beijing. In each sampling campaign, 1000 mL of wastewater samples or 50 mL of sewer sediment were collected by grabbing and 0.2% diethyl pyro-carbonate (DEPC) was added to the collected samples. All of the samples were transported to the BSL-2 laboratory on ice within 5 h.

Fig. 1

Maps of the sampling location (116˚32′06.56″N - 116˚23′50.37″N; 39˚53′12.93″E - 39˚56′38.28″E) and the COVID-19 epidemic trend in Beijing. A) Samples collection site of hospital wastewater treatment facilities (red dots) and municipal WWTPs (green dots). The capacities of the municipal WWTPs are 5 × 103, 1.6 × 105, 1.1 × 105 and 4 × 104 m3/day, respectively. B) Original site of the SARS-CoV-2 transmission for the second wave of the COVID-19 epidemic (raindrops) and the samples collection location along the river (ball stick). C) Trend of COVID-19 cases diagnosed in Beijing, showing the end date of two pandemics (solid lines) and the sampling schedule (arrows).

Wastewater concentration and RNA extraction

Phi6, an enveloped bacterial virus, was seeded as process control and evaluated the method recovery efficiency of ultrafiltration with 103 PFU/mL. Samples were processed without any pasteurization to inactivate the virus in the wastewater to reduce the loss of virus. Large suspended particulate matter was removed by a continuous centrifuge at 5000 g for 30 min 500 mL of supernatant were filtered through a Centrion® Plus-70 ultrafilter with a cut-off of 30 kDa (Millipore, Amsterdam, the Netherlands) by centrifugation at 4 ℃, 2480 g for 15 min. The supernatant needs to be concentrated in batches until the final volume of the concentrates is up to 300–600 μL, and the concentrations were collected by inverted centrifugation at 900 g for 3 min with a retentate cup.

For the sewer sediment samples, 50 mL of solid sediment were mixed with 500 mL of deionized water and stirred for 10 min. The mixtures were treated as the wastewater samples mentioned above.

The extraction of viral RNA from concentrated samples was carried out with the RNeasy PowerMicrobiome Kit (Qiagen, Hilden, Germany) in the light of the manufacturer’s specification. Finally, purified RNA was obtained by eluting with 100 μL RNase-free water.

SARS-CoV-2 RNA detection and quantification

Detection and quantification of viral RNA by TaqMan quantitative real-time polymerase chain reaction (RT-qPCR) on a ViiA™ 7 instrument (Applied Biosystems). In this study, two primer/probe sets were selected: The E set responded to the envelope protein (E) gene, and the N1 set came from a fragment of the nucleocapsid (N) gene ( Table 1). Each RT-qPCR reaction contained 5 μL extracted viral RNA template, 4 μL of 5 ×RT-qPCR reaction master mix (Roche Diagnostics, Almere, The Netherlands), 500 nM of forward and reverse primers, 250 nM of Taqmen probes, and supplementation of RNase-free water to a final volume of 20 μL. The detection procedure was set as 60 ℃ for 15 min for reverse transcription of RNA, 95 ℃ for 5 min for pre-degeneration, and the following 45 cycles (95 ℃ 15 s, 55 ℃ 30 s) for targets amplification and fluorescence signal acquisition.

Table 1

Primer/probe sets of RT-qPCR assays used in this study.

Assay	Name	Sequence	Reference
E_Sarbeco	Forward primer	5’-ACAGGTACGTTAATAGTTAATAGCGT-3’	(Corman et al., 2020)
	Reverse primer	5’-ATATTGCAGCAGTACGCACACA-3’	(Corman et al., 2020)
	TaqMan probe	5’-VIC-ACACTAGCCATCCTTACTGCG CTTCG-BHQ1–3’	(Corman et al., 2020)
CDC_N1	2019-nCoV_N1-F	5’-GACCCCAAAATCAGCGAAAT-3’	(US_CDC., 2020)
	2019-nCoV_N1-R	5’-TCTGGTTACTGCCAGTTGAATCTG-3’	(US_CDC., 2020)
	2019-nCoV_N1-P	5’-VIC-ACCCCGCATTACGTTTGGTGGACC- BHQ1–3’	(US_CDC., 2020)

Each sample was tested with three technical replicates, and RNase-free water was set as the no template control. The measurements were valid only if the positive control was positive and the no template control was negative. SARS-CoV-2 target gene copies were determined using a standard calibration curve and a 10-fold serial dilution (ranged from 1 × 103 to 1 × 108 copies/mL) of a standard plasmid synthesized by Sangon Biotech, which cloned the target sequences on pUC57 vector. The standard material was quantified by an ultra-micro analyzer (NanoDrop Onec) and verified by the fluorescence (Qubit 2.0) method. The log10-linear regression of copy number per mL and corresponding Cycle threshold (Ct) values measured in triplicate were used to generate the standard curve and 95% confidence intervals (Bustin et al., 2009).

The sample was determined as positive as long as one replicate was positive. For each sample replicate, the SARS-CoV-2 RNA concentration within the linear range of the standard curve was used to quantify the vial concentration, and the final quantification was the mean of three replicates of virus copies. The assay limit of detection in this study was determined with previous method (Verbyla et al., 2016).

SARS-CoV-2 persistence in wastewater at various temperature

The decay properties of SARS-CoV-2 RNA in wastewater were investigated using the positive samples collected from the influent of municipal WWTPs. The SARS-CoV-2 positive wastewater sample was divided into 200 mL portions with a 250 mL serum bottle. The divided positive samples were incubated statically at low and normal temperature variety (4 ℃ and 26 ℃) in the dark to assess the temperature effect on the decay of SARS-CoV-2 RNA in raw wastewater. A 200-mL triplicate portion was condensed to 300 μL every two days. The concentrates were stored at − 80 ℃ before all divided portions were concentrated. The RNA extraction, detection, and quantification of the concentrates were carried out according to the method mentioned above.

Decay features of SARS-CoV-2 RNA in raw wastewater at various temperatures

The properties of the SARS-CoV-2 RNA in raw sewage were described with ln-linear models of one order, and the decay rates were calculated with Eq. (1) (Chick, 1908). The virus titer and the corresponding time points were used to determine the decay rate constants of the first-order kinetics. The T 90 value (time required to reach each 90% reduction) was obtained using Eq. (2). Where k is the slope of the linear regression equation and also the constant first-order decay rate, C 0 and C are the viral loads in the initial assays and at time t, respectively. The associated 95% confidence intervals of the decay rate constants were calculated by linear regression analysis. All linear fitting and statistical analyses were performed using GraphPad Prism Version 8.0.2 software (GraphPad Software, La Jolla, CA, USA).

Results

RT-qPCR performance

The recovery efficiency of enveloped bacterial phage phi6 by ultrafiltration was 33.5 ± 5.6% with the double agar plaque assay (Table S1). The slopes of the standards for E_Sarbeco and CDC_N1 primer/probe sets were − 3.051 and − 3.257, respectively. Y-intercept values were 41.006 (for E_Sarbeco primer/probe set) and 42.239 (for CDC_N1 primer/probe set). The amplification efficiencies for these two assays were 112.69% (for E_Sarbeco primer/probe set) and 102.78% (for CDC_N1 primer/probe set). The values of the correlation coefficient (R 2) for E_Sarbeco and CDC_N1 were 0.995 and 0.996, respectively (Fig. S1). Both of these tow primer/probe ALOD were 3 copies/reaction (Fig. S2). The quantification of the SARS-CoV-2 titers was determined using RT-qPCR in the range of 8.5 × 101 - 8.8 × 103 copies/L for the sewage system and 8.5 × 101 - 9.52 × 102 copies/L for river water. The total viral load was at a lower level in comparison with previous reports that were between 102 and 106 copies/L (Ahmed et al., 2020a, Fongaro et al., 2021, Patel et al., 2021).

Detection of SARS-CoV-2 RNA in sewage

Detection of SARS-CoV-2 RNA in hospital wastewater treatment facilities

Residual SARS-CoV-2 RNA in hospital wastewater was initially examined after the first wave of the COVID-19 epidemic in Beijing. After the first round of the local COVID-19 epidemic ended 15 days, a total of 6 influent, 5 secondary, 12 tertiary treated effluent water, and 2 sewer sediment samples were taken from the wastewater treatment facilities of hospitals. Positive samples were confirmed for Ct values below 40 and the RNA concentrations were calculated using the standard plasmid. Three out of six hospitals influent were detected the SARS-CoV-2 RNA concentration at 1.60 × 102 – 9.61 × 102 copies/L. Three secondary treated effluents were determined to be positive for viral RNA and quantified as 3.7 × 102 – 2.34 × 103 copies/L wastewater. Unexpectedly, we also found the SARS-CoV-2 RNA in tertiary effluent samples (8/12) from hospitals with titers in the range from 4.64 × 102 – 8.62 × 102 copies/L, as well as ~ 4 × 103 copies/L in sewer sediment (2/2) 15 days after the end of the COVID-19 epidemic ( Fig. 2).

Fig. 2

Viral load detected in hospital wastewater treatment facilities. Detection of SARS-CoV-2 RNA in influent, secondary treated effluent, tertiary treated effluent from hospital sewage treatment facilities and sediment in drainage system of hospital after the first COVID-19 epidemic wave in Beijing.

The results seem to conflict with the general knowledge that the SARS-CoV-2 RNA concentration in influents was lower than effluents in the field assays (Fig. 2). A high concentration of virus that persisted in the activated sludge in the biochemical tank (Balboa et al., 2021, Graham et al., 2020, Peccia et al., 2020) is a possible factor, which leads to the high exposure of virus in the effluent the effluents. Previous studies found that the load of SARS-CoV-2 in solid is much higher than that in wastewater. Besides, the randomness of grab sampling is another possible reason to be considered (Achak et al., 2021; Rafiee et al., 2021).

Detection of SARS-CoV-2 RNA in municipal WWTPs

A total of 15 influent samples were taken from 4 municipal WWTPs between 14 days before and 33 days after the end of the second wave of the local COVID-19 epidemic. The samples were processed using the ultrafiltration method, and the SARS-CoV-2 RNA was tested with a one-step RT-qPCR assay. As summarized in Fig. 3, SARS-CoV-2 RNA was detected in 6 (40%, 6/15) wastewater samples. Consistent with many previous reports, SARS-CoV-2 RNA was detected in wastewater during the COVID-19 epidemic. In addition, SARS-CoV-2 RNA was still detected in wastewater 19 days after the end of the COVID-19 epidemic, as shown in Fig. 3.

Fig. 3

A) SARS-CoV-2 RNA detection in municipal WWTPs in Beijing from 14 days before to 33 days after the end of the second round of local epidemic. B) New confirmed COVID-19 cases per day in different districts. The zero-point indicated that where there was no longer any clinical infection, WWTPs correspond to the affiliating district with the same symbol color.

Detection of SARS-CoV-2 RNA in sewer

The sewer sampling points are located 1.5 km downstream from the wastewater discharge outlet of the agricultural and sideline wholesale market, which was the major spreading source of the second wave of the COVID-19 epidemic in Beijing. The wholesale market was closed immediately, and a thorough disinfection treatment was performed after the outbreak. As shown in Fig. 4, four sewage samples were collected in the sewers from 17 days before to 19 days after the end of the second round of the COVID-19 epidemic. The results showed that two samples were determined as positive during the epidemic and no viral RNA was detectable in the sewers after the end of the pandemic.

Fig. 4

Detection of SARS-CoV-2 RNA in the sewer from 17 days before to 19 days after the end of the COVID-19 epidemic. The light-colored area represents the second wave of the local outbreak in Beijing. The horizontal axis indicates the time relative to the day that the local epidemic was over.

Detection of SARS-CoV-2 RNA in river

River water samples were also taken 17 days before to 19 days after the end of the second wave of the COVID-19 epidemic. A total of 9 samples were taken at the three locations from upstream to downstream of the river, which is 8–10 kilometers from the wholesale market for agricultural and sideline products. It should be emphasized that the first-time water samples were taken after a heavy rain event during the pandemic. The results showed that 33.3% (3/9) of all river samples were considered as positive, and the quantitation of viral RNA in river samples collected during the COVID-19 epidemic ranged from 9.7 × 101 to 9.52 × 102 copies/L. All of the first samples collected from three sites were positive, but none of the samples acquired after the COVID-19 epidemic ended were found to be positive ( Fig. 5).

Fig. 5

SARS-CoV-2 RNA detected in the river 17 days before to 19 days after the end of the second wave of the COVID-19 epidemic. The light-colored area represents the second wave of the local outbreak in Beijing. The horizontal axis indicates the time relative to the day that local epidemic was over.

Estimated constants of the first-order decay rates at various temperatures

The temperature was controlled to remain stable at 4.0 ± 1 ℃ and 26 ± 2 ℃ throughout the experiment. The initial loading of SARS-CoV-2 RNA positive sample, which was used to assess the influence of temperature on the persistence of SARS-CoV-2 in raw sewage, was ~ 5 × 103 copies/L. The 200-mL portions were concentrated to 300 μL at each sampling point using the ultrafiltration method. However, further tests could no longer be carried out after 4 days because the viral load was too low to conduct effective detection. The declining loads of SARS-CoV-2 RNA in raw wastewater at 4 ℃ and 26 ℃ are shown in Fig. 6. The average first-order decay rate constants (k) were 0.134/day at 4 ℃, which is much less than that of 0.274/day at 26 ℃ with R 2 values of 0.743 and 0.994, respectively (Fig. 6 and Table 2). In addition, the mean T 90 values (time for 90% virus reduction) of the SARS-CoV-2 RNA in raw wastewater were calculated in the range from 7.68 to 25.87 days, corresponding to 26 ℃ and 4 ℃, respectively.

Fig. 6

Mean decay ln-linear regression curves of SARS-CoV-2 over time (days) in raw sewage at 4 ℃ (A) and 26 ℃ (B). The red dosh lines are 95% confident intervals.(For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Table 2

Decay rate constants (k/day) and T90 values of SARS-CoV-2 RNA in raw wastewater with low-titers SARS-CoV-2 virus.

Temperatures (℃)	k [95% CI slop]	R2	T90 days
4 ℃	-0.134 [− 0.314–0.046]	0.743	17.17
26 ℃	-0.274 [− 0.314 to − 0.234]	0.994	7.68

Discussion

SARS-CoV-2 RNA occurrence in the urban water system

In previous studies, large variations in SARS-CoV-2 concentration exposure in the urban water system have been reported. For examples 1.9 × 101 - 7.0 × 106 copies/ L in wastewater (Ahmed et al., 2020a, Ahmed et al., 2020b, Ampuero et al., 2020, Arora et al., 2020, Fongaro et al., 2021, Mlejnkova et al., 2020, Peccia et al., 2020, Randazzo et al., 2020, Wurtzer et al., 2020a, Wurtzer et al., 2020b), 1.6 × 102 - 2.51 × 105 copies/L in secondary treated wastewater (Haramoto et al., 2020, Randazzo et al., 2020, Sherchan et al., 2020), 2.0 × 101 - 1.0 × 105 copies/L in tertiary treated effluent (Kumar et al., 2020b, Sherchan et al., 2020, Wurtzer et al., 2020a, Zhang et al., 2020), 1.3 × 103 - 4.6 × 108 copies/L in sludge (Balboa et al., 2021, Kocamemi et al., 2020), 2.0 × 102 - 3.19 × 106 copies/L in river water (Guerrero-Latorre et al., 2020, Haramoto et al., 2020, Peccia et al., 2020). In this study, a relatively low SARS-CoV-2 RNA exposure was detected in comparison with the literature previously reported (shown in Fig. 7).

Fig. 7

Detection of SARS-CoV-2 in sewerage systems and river water on this study in comparison with the data previously reported in the literatures. Black boxes and red violins symbolize present the SARS-CoV-2 titers reported in the literatures and our study results, respectively.(For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Many factors will impact the viral concentration detected in wastewater. Environmental factors such as pH, temperature, organic matter, retention time, the sampling and concentration methods all affect the detection of the virus (Achak et al., 2021; Ahmed et al., 2020c; Rafiee et al., 2021; Wang et al., 2005b). Additionally, many studies estimated that the load of SARS-CoV-2 RNA in wastewater is consistent with the prevalence (Haramoto et al., 2020; Medema, 2020; Randazzo et al., 2020). In this study, only a few infected cases occurred in Beijing, and most of the samples were collected after the local epidemics ended. These may be the key factors that only small amounts of SARS-CoV-2 RNA were detected in the sewage system. The strict social controls and timely epidemiological follow-up investigations significantly decreased the discharged source of SARS-CoV-2 RNA.

Solids in sewage system were a major SARS-CoV-2 repository

It is widely believed that infectious coronaviruses are unlikely to persist in wastewater for long periods of time because these viruses contain a lipid bilayer membrane (Achak et al., 2021; Ye et al., 2016). However, while the local epidemic was over almost 20 days, SARS-CoV-2 RNA was still detected in the influent of municipal WWTPs (Fig. 3A). Similar results were also obtained in influent, secondary, and tertiary treated wastewater of the hospital, since no infection occurred 15 days later (Fig. 2). In addition, about 4 × 103 copies/L of the SARS-CoV-2 RNA were detected in the sediment of the drainage pipe that receives the wastewater discharged from the upstream hospital (H3). This means that the sewage systems would be a virus repository and prolong the residuals of SARS-CoV-2.

Many previous studies have revealed that high concentrations of SARS-CoV-2 RNA could be detected in sewage sludge (Balboa et al., 2021; Kocamemi et al., 2020). Katherine's study showed that the concentration of SARS-CoV-2 RNA per mass of solid was about 1000 times higher than that in influent (Graham et al., 2020, Peccia et al., 2020). Similarly, the studies by Kocamemi and Peccia also suggested that the concentration of SARS-CoV-2 RNA in the primary and secondary tank sludge was around 1.7 × 103-4.6 × 105 copies/mL, which is three orders of magnitude higher than the values reported in the wastewater (Kocamemi et al., 2020, Peccia et al., 2020). Therefore, the suspended and solid sediment would be the attached repository of the SARS-CoV-2 for a long time, and the virus would slowly be released into the liquid phase.

The transport of SARS-CoV-2 in sewers

Since the sediment in the sewage systems could be a repository of SARS-CoV-2 and release viruses into the aqueous phase, the SARS-CoV-2 could persist in sewage for a long time. This would prolong the fate of SARS-CoV-2 in the surrounding sewage systems. Furthermore, due to the encapsulation and barrier effect of solid particles on SARS-CoV-2, the normal disinfection dose for hospital wastewater could not effectively destroy all SARS-CoV-2 virus particles in wastewater, especially those with large suspended solids. The present study demonstrates that the SARS-CoV-2 RNA can still be detected even after the disinfection treatment of hospital wastewater (Fig. 2). The inadequate disinfection of the contained wastewater discharged into the municipal drainage pipe would increase the possibility of SARS-CoV-2 spreading in the sewage system. In this study, we also detected SARS-CoV-2 RNA in river water after a heavy rain event on June 19th, 2020 during the second epidemic period. The repository of SARS-CoV-2 in the sewer would be the major virus source for the river via the sewer overflow during a heavy rain event. Therefore, sewer would be a potential virus transport source for urban water systems.

The decay SARS-CoV-2 in wastewater

The persistence and decay of SARS-CoV-2 in wastewater were strongly dependent on temperature variables in the environment (Gundy et al., 2009, Hokajärvi et al., 2021, La Rosa et al., 2020a). The decay rates of the SARS-CoV-2 RNA were measured at low and normal temperatures in wastewater that covered the ambient temperature variation range. At both low and high temperatures, the decay rates of low titer viruses in raw sewage were much faster than that spiked with high titer exogenous viruses (k values were 0.134 vs 0.084 at 4 ℃ and 0.274 at 26 ℃ vs 0.183 at 25 ℃) (Ahmed et al., 2020c). This may be due to the incomplete viral structure in the wastewater that makes viral RNA easier to degrade. A previous study discovered several forms of SARS-CoV-2 RNA present in wastewater, including genomic RNA within an infected virion, in incomplete virus particles, and free genomic RNA, with an integrity-based RT-qPCR assay (Wurtzer et al., 2021). In this study, the SARS-CoV-2 RNA was detected in wastewater 19 days after no new confirmed cases were reported. Although the SARS-CoV-2 RNA could persist in wastewater for a long time, it may also be shed by the possible missing asymptomatic infected individuals during extensive screening. In addition, the SARS-CoV-2 RNA might also be excreted by recovered individuals who were able to protract the shedding of viral RNA in feces for days to 3 months after full recovery (Gupta et al., 2020, Li et al., 2020, Zheng et al., 2020). Therefore, considering early warning of outbreaks or surveillance of epidemics through WBE, the low concentration persistence of SARS-CoV-2 RNA in the sewage systems may cause misjudgment by WBE in assessing the small scale of infection just after the end of the epidemic.

The decay of infectious SARS-CoVs in wastewater was also reported with the seeding of SARS-CoV-2 virus inoculum isolated from nasopharyngeal swabs of the COVID-19 patients. Documented evidence suggests that SARS-CoV remained infectious after 14 days in wastewater at 4 ℃, while it was only 2 days at 20 ℃ (Wang et al., 2005b). A recent study also reported that spiked viable SARS-CoV-2 remained infectious for 7 days during the higher titer of 105 TCID50 /mL experiments and for 3 days at the lower titer of 103 TCID50 /mL in wastewater, which was performed in the highest biological safety level laboratories (BSL-4) (Bivins et al., 2020). This means that the SARS-CoV-2 virus may no longer be infectious after several days in the wastewater. Infectious SARS-CoV-2 in raw wastewater was not detected by cell culture-based assays (Rimoldi et al., 2020, Westhaus et al., 2021). Therefore, the SAR-CoV-2 in wastewater is likely not infectious.

Conclusions

The study conducted a field trial on the SARS-CoV-2 presence in various wastewater facilities after the end of the COVID-19 epidemics in Beijing. When the local COVID-19 epidemics were over, SARS-CoV-2 RNA was still detected in hospital wastewater treatment facilities for more than 15 days and in municipal WWTPs for 19 days. The sewage systems were possible a large SARS-CoV-2 repository, and the virus was able to persist in the sewage systems for a long time after the end of the COVID-19 epidemics. Additionally, the virus could transmit into the river from sewer through the sewer overflow event. The sewage systems were a potential source of viral transport for a long time, especially in a low-temperature environment.

CRediT authorship contribution statement

Shaolin Yang designed and performed the experiment, analyzed the results, wrote, revised and edited the manuscript. Qian Dong, Siqi Li, Zhao Cheng, Xiaofeng Kang, Daheng Ren and Chenyang Xu performed the experiments. Xiaohong Zhou, Peng Liang, Yi Qian, Yi Liu and Jiuhui Qu improved the language of writing. Lingli Sun, Jianhong Zhao, Yang Jiao and Taoli Han provided safety training and experimental guidance. Yanchen Liu and Xia Huang were involved in conceptualizing, writing original draft, revising and editing the manuscript.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.