Wild Type and Variants of Sars-Cov-2 in Parisian Sewage: Dynamics in Raw Water and Fate in Wastewater Treatment Plants

Syndicat Interdepartemental l'Assainissement l'Agglomeration Parisienne: SIAAP Abstract The presence of SARS-CoV-2 RNA has been extensively reported at the inuent of wastewater treatment plants (WWTPs) worldwide and its monitoring has been proposed as a potential surveillance tool to early alert of epidemic outbreaks. However, the fate of the SARS-CoV-2 RNA in the treatment process of WWTP has not been widely studied yet; therefore in this study, we aimed to evaluate the eciency of treatment processes in reducing SARS-CoV-2 RNA levels in wastewater. The treatment process of three WWTPs of the Parisian area in France were monitored on six different weeks over a period of two months (from April 14 th to June 9 th 2021). SARS-CoV-2 RNA copies were detected using digital polymerase chain reaction (dPCR). Investigation on the presence of variants of concern (Del69-70E484 and L452R) was also performed. Additionally, SARS-CoV-2 RNA loads in the WWTPs inuents were expressed as the viral charge per population equivalent and showed a good correlation with French public health indicators (incidence rate). SARS-CoV-2 RNA loads were notably reduced along the water treatment lines of the three WWTPs studied (2.5-3.4 log). Finally, very low SARS-CoV-2 RNA loads were detected in euents (non-detected in over half of the samples) which indicated that the potential health risk of the release of wastewater euents to the environment is probably insignicant, in the case of WWTPs enabling an ecient biological removal of nitrogen.


I. Introduction
Surveillance of different markers in wastewater in uents, has enabled in the past to characterize emerging chemicals, illicit drug use patterns and food consumption patterns (nitrogen and phosphorus) ( Kumar et al. 2021). The e ciency of WWTP treatment processes and the potential health risks associated with the release of wastewater e uents containing SARS-CoV-2 into the environment remain to be veri ed. As the COVID-19 epidemy continues to spread all over the world, new variants of the SARS-CoV-2 virus are being detected. Those variants might be more transmissible or capable of evading immune response or their mutations might suppress diagnostic detection (Wurtzer et al. 2020 The present work intended to (i) provide an insight of the evolution of the main variants present in the raw waters coming from three different urban catchments and (ii) evaluate the presence of SARS-CoV-2 RNA using the new method of dPCR through the treatment process of three majors Parisian WWTPs.

Parisian WWTPs monitored in this study
The three studied plants are located upstream and downstream of the Parisian conurbation and their ows vary between 50,000 and 600,000 m 3 per day ( Figure 1). All of them are operated by the Greater Paris Sanitation autority (SIAAP) in charge of collecting, transporting and treatmenting wastewater produced by close to 9 millions of inhabitants. Different technologies are used for the treatment of water and sludge.The SEG plant uses the bio ltration process for water treatment, the Seine Valenton plant (SEV) uses activated sludge treatment for water, and the Seine Morée plant (SEM) uses membrane ltration for water treatment. These WWTPs are designed to e ciently reach the European standards (« Directive 91/271/EEC » 1991; « Directive 2000/60/EC » 2000).
These three plants are fed by raw water coming from urban catchments presenting constrasted characteristrics and differe mainly in their design capacity and treatment processes, as brie y described in Table 1. The catchment area of SEG (located in the northwest area) is characterized by a very high urbanization level; by contrast,SEV and SEM (located in the southeast and east area) have a moderate urbanization level.

Sewage sampling and analytical procedures
Samples of raw (in uent), settled and treated (e uent) waters were collected continuously (24-h). More precisely, a rst campaign occured from April 14th to April 28th a second campaign from May 26th to June 9th, corresponding to contrasting SARS-CoV-2 incidence levels (French National Health Agency). Sampling was performed using autosampler devices, during dry-weather conditions, from 7 am to 7 am the next day. To screen for important SARS-CoV-2 variants, particular molecular signatures were developed using a 5-plex assay that takes full advantage of the ve detection channels available on the QIAcuity One 5plex, QIAcuity Four and QIAcuity Eight instruments. The 5-plex assay uses one probe to detect SARS-CoV-2 wild type N1 region     Table   S1. The normalized SARS-CoV-2 RNA concentration (per 100 000 PE) and the incidence rate (per 100 000 hab.) for the departments corresponding to the catchment area of each WWTP are also presented in Table 2. The incidence rate data of departments 75 (Paris), 92 (Hauts-de-France), 93 (Seine-Saint Denis) and 94 (Val-de-Marne) were collected from the open data portal of the French National Health Agency. As broadly discussed in other studies, the correlation between the SARS-CoV-2 RNA concentration in raw wastewater and the incidence rate (r 2 = 0.61, Figure  S1), con rms that SARS-CoV-2 RNA monitoring in wastewater is a good candidate indicator of the epidemic spread, as recently shown by (Wurtzer et  Besides the quanti cation of SARS-CoV-2, we determined the presence of variants of concern within the population: Alpha (Del69-70), Beta-Gamma (E484), Delta (L452R). The proportion of Del69-70 and E484 variants detected during the rst sampling period is in good agreement with open data weekly released by French National Health Agency.
However, the Delta variant L452R was not a variant of concern during the sampling period, therefore, no data related to it were published at that time. It can be noted that the L452R variant was already present on in uent samples from April 28th 2021 in the Parisian region; moreover, similar proportions of L452R variant (23 %) were reached among sequenced patients swabs only from June 20th 1-27th 2021, in the Parisian region. These results indicate that SARS-CoV-2 RNA isolated from WWTP in uents is also a reliable tool to detect the introduction of variants of concern in the local population weeks before they appear at signi cant levels in either clinical or screening swab samples.
2. E ciency of treatment process from WWTPs SARS-CoV-2 RNA average removal e ciency of the global treatment process as well as of the settling and biological treatment steps between April, 14th and April, 28th are shown in Figure 2.a. The e ciency of WWTPs on reducing the values of main physico-chemical parameters is presented in Figure 2.b.
in Parisian WWTPs, for the rst sampling campaign period between April, 14th and April, 28th The three WWTP allows an e cient removal of particules, organic matter and nitrogen. Indeed, the treatment water lines enables the removal of 97-99% of suspended solids, 96-100% of BOD 5 and 73-85% of total nitrogen, as shown by Figure 2b. In these operating conditions, the average reduction of SARS-CoV-2 RNA was of of 2.4-3.3 log reduction (i.e. 85-97%) as it can be seen in Figure 2.a. Regarding the settling step, low to moderate reductions of SARS-CoV-2 RNA levels were observed (0.01-0.4 log) whereas for the biological treatment step higher removals were obtained (2.4-2.9 log). Low concentrations (<50 UG/ml) of SARS-CoV-2 RNA were detected in outlets of the studied WWTPs, in all cases except on April 28th in SEV (Table 2).

Iv. Conclusions
The presence of SARS-CoV-2 RNA in raw wastewater (in uent), decanted and treated water (e uent) was quanti ed using RT-dPCR technique. SARS-CoV-2 RNA loads on in uent showed good correlation with the incidence rate of COVID-19 in the Parisian area. The presence of variant L452R (Delta) was detected in samples from the present study from April 18th, a few weeks prior to its inclusion as a variant of concern by the French National Health Agency, which con rms the interest of using sewage analysis as a complementary approach to early detection of epidemics outbreaks.
Finally, the three standard sewage treatment processes (activated sludge, bio ltration or membrane bioreactor) in the Parisian area operated by SIAAP, with a complete treatment of carbon and nitrogen (removals of 96-100% of BOD5 and 73-85% of total nitrogen), are very e cient in eliminating SARS-CoV-2 RNA, with average removals of 2.4-3.5 log.
Declarations Figure 1 Location and nominal ow rate of WWTPs , in grey, plants sharing catchment area with the 3 studied in white Figure 2 Removal of a.) Sars-CoV-2 (n=3) and b.) SS, BOD5 and Total Nitrogen (TN) (n=14) in Parisian WWTPs, for the rst sampling campaign period between April, 14th and April, 28th