key: cord-0749470-znn2keid authors: Balboa, S.; Mauricio-Iglesias, M.; Rodriguez, S.; Martinez-Lamas, L.; Vasallo, F. J.; Regueiro, B.; Lema, J. M. title: The fate of SARS-CoV-2 in wastewater treatment plants points out the sludge line as a suitable spot for incidence monitoring date: 2020-05-26 journal: nan DOI: 10.1101/2020.05.25.20112706 sha: 3fe7c9a52b166f11fa4ca615aee25dac55371070 doc_id: 749470 cord_uid: znn2keid SARS-CoV-2 genetic material is detectable in the faeces of a considerable part of COVID-19 cases and hence, in the urban wastewater. This fact was confirmed early during the spread of the COVID-19 pandemic and prompted several studies that proposed monitoring its incidence by wastewater. This paper studies the fate of SARS-CoV-2 genetic material in wastewater treatment plants using RT-qPCR with a two-fold goal: i) to check the safety of the water effluent and also of the sludge produced and ii) based on the understanding of the virus particles fate, to identify the most suitable spots for detecting the incidence of COVID-19 and monitor its evolution. On the grounds of the affinity of enveloped virus towards biosolids, we hypothesized that the sludge line acts as a concentrator of SARS-CoV-2 genetic material. Sampling several spots in primary, secondary and sludge treatment at the Ourense (Spain) WWTP showed that, in effect, most of SARS-CoV-2 particles cannot be detected in the water effluent as they are retained by the sludge line. We identified the sludge thickener as a suitable spot for detecting SARS-CoV-2 particles thanks to its higher solids concentration (more virus particles) and longer residence time (less sensitive to dilution caused by precipitation). Although more studies will be needed for confirmation, these results contribute to clarify the role of WWTPs in COVID-19 mitigation. Urban wastewater can be a vector for the spread of viral diseases, especially viruses that are transmitted through the faecal-oral route. There are numerous descriptions of virus detection in wastewater plants, including Norovirus, Sapovirus, Hepatitis A virus, Adenovirus, Poliovirus or Enterovirus among others (Ehlers et al., 2005; Sassi et al., 2018; Symonds et al., 2016; Taboada-Santos et al., 2020) . Current knowledge regarding the behaviour of SARS-CoV-2 in wastewater is very limited, although its RNA has been detected in faeces of symptomatic individuals (Holshue et al., 2020; Woelfel et al., 2020) and in urban wastewater in different countries (Medema et al., 2020; Randazzo et al., 2020a Randazzo et al., , 2020b Rosa et al., 2020; Wu et al., 2020; Wurtzer et al., 2020) . Although it is assumed that the enveloped viruses are not excreted in high concentrations and that their survival in water is limited, there is little experimental evidence to confirm these hypotheses in wastewater; in fact, transmission of these viruses through wastewater was identified as responsible for an outbreak of severe acute respiratory syndrome (SARS) in Hong Kong in 2003 (Yu et al., 2004 . Urban wastewater constitutes a complex matrix which includes suspended solid materials, colloidal and dissolved biodegradable organic matter, nutrients, pathogens, etc. In wastewater treatment plants (WWTP) most of solids are separated from the water to the so-called sludge line. The vast majority of WWTPs have a first stage of homogenization and separation of solid (primary settler) and a secondary settler where the activated sludge is separated from the clarified water. Finally, the two types of sludge (primary and secondary) are concentrated in the thickener from where they are sent to the sludge treatment unit. Pollutants of hydrophobic nature are mostly retained in primary or secondary sludge (Prado et al., 2014) , a phenomenon described already many decades ago (Wellings et al., 1976) . It is known that enveloped viruses, due to the presence of a lipid bilayer surrounding the protein capsid, have a different affinity to non-enveloped viruses, . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 26, 2020 . . https://doi.org/10.1101 with a greater tendency to adsorb to solid and/or colloidal particles (Ye et al., 2016) . This was experimentally proved to occur for two enveloped viruses: Murine coronavirus MHV (murine hepatitis virus) and Pseudomonas phage ϕ6 (Ye et al. 2016). Therefore, most probably, SARS-CoV-2 and particles thereof are indeed hydrophobic and, accordingly, they would be associated to the solids and/or colloidal material. Yet, most of the current literature concerning SARS-CoV-2 or its genetic material in wastewater deals with their presence in the water phase and very little attention has been paid to their fate in the sludge line. Another aspect of concern for water boards and utilities is the potential transmission of SARS-CoV-2 in WWTP and their effluents. Actually, what is known so far about the transmission of SARS-CoV-2 is not particularly worrisome, given that WWTP operation is already intended to avoid the transmission of potential pathogens present in wastewater. Being sludge and the water effluents the main outflows from a WWTP, it is important to ascertain whether SARS-CoV-2 can be detected in these streams, even keeping in mind that the presence of SARS-CoV-2 genetic material does not imply that it is in an infective state. Hence, this manuscript pays special attention to the sludge in WWTPs. The sludge treatment is very heterogeneous in WWTPs as large plants often feature anaerobic digestion treatment, usually at moderate temperatures (35-40 ºC) and long residence times (10-20 days), which would help to inactivate the possible viral load. In contrast, in smaller plants sludge can receive a mere heat drying treatment before being shipped to an authorized manager, or even just centrifugation to reduce its water content. The goal of this manuscript is two-fold: first, to shed some light on the fate of SARS-CoV-2 in WWTPs by examining the detection of its genetic material along the water and sludge lines and second to check whether the hydrophobic nature of SARS-CoV-2 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 26, 2020 . . https://doi.org/10.1101 make the sludge line, and particularly the thickened sludge, as a suitable spot for monitoring its incidence in the WWTP catchment area. Wastewater and sludge samples were taken from Ourense WWTP in north-western Spain (characteristics and sampling points in Table 1 ). The 250 ml samples were taken twice a week from April 6 to April 21 2020, kept at 4ºC before being sent in less than 24 h to the Universidade de Santiago de Compostela (USC) facilities to be concentrated. is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 26, 2020. . https://doi.org/10.1101/2020.05.25.20112706 doi: medRxiv preprint DIN 38405-9), total nitrogen (kit equivalent to ISO 11905-1) and phosphorus (spectrophotometry, 4500-P). beef extract) was added to 50 ml of sludge, incubated for 2h at 4ºC to detach viruses bound to organic material. Samples were then centrifuged at 8 000 xg during 30 min and filtered through a 0.45 μm polyethersulfone (PES) membrane to remove . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 26, 2020. . https://doi.org/10.1101/2020.05.25.20112706 doi: medRxiv preprint eurkaryotic and prokaryotic cells. Then, viruses were precipitated by adding 1:5 (v/v) of PEG 8000 (80g/L) and NaCl (17.5 g/L) during an overnight shacking (150 rpm) at 4ºC and a centrifugation at 13 000 xg during 90 min. Samples were then resuspended in PBS buffer pH 7.4 and stored at -80ºC for further analysis. Concentration control was performed with bacteriophage MS2, by inoculating each sample with 250 l the virus (5.5 x 10 6 viral particles/ml). RNA extraction and RT-qPCR was carried out at the department of Microbiology of Complexo Hospitalario Universitario de Vigo. Nucleic acid extraction from both water and sludge concentrated samples was performed using MicrolabStarlet IVD using the STARMag 96 x 4 Universal Cartridge Kit (Seegene, Seoul, South Korea) according to manufacturer specifications. Viral RNA was detected and quantified by a one-step multiplex RT-qPCR Allplex system™ 2019-nCoV (Seegene, Seoul, South Korea). The assay is designed to detect RNA-dependent polymerase (RdRP) and nucleocapsid (N) genes specific to SARS-CoV-2, and a region conserved in the E gene of the structural protein envelope for the detection of pan-Sarbecoviruses including SARS-CoV-2. The test uses internal RNA control for sample preparation and control of the PCR amplification process. For the RT-PCR, the CFX96 system was used ™ (Bio-Rad Laboratories, Hercules, CA, USA). The analysis of the results was performed using the specific Seegene viewer software 2019-nCoV. In parallel, a SARS-CoV-2 EDX standard (Bio-Rad, Hercules, CA) containing synthetic RNA transcripts of five SARS-CoV-2 genetic targets (genes E, N, ORF1ab, RdRp and S) of known concentration was used to establish a linear regression curve and obtain the concentration in copies/ml. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 26, 2020. . https://doi.org/10.1101/2020.05.25.20112706 doi: medRxiv preprint The characterisation of the sample in terms of chemical oxygen demand (COD), suspended solids and, for water samples, total nitrogen is shown in Table 2 . It can be seen how the influent has a very variable composition, which is most probably caused by precipitation events. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 26, 2020. . https://doi.org/10. 1101 Indeed, the influence of rain on sewerage streams is a hurdle to the use of the WWTP influent as an epidemiological indicator. However, the sludge streams tend to have a more steady content of biosolids as it reflects the mass flow of solids and COD entering the WWTP. Particularly, solid concentration in thickeners seems to be much more stable than the characteristics of the primary influent. A total of 15 samples of water and 35 samples of sludge collected from April 6 to April 21 (2020) were tested for the presence of SARS-CoV-2 RNA. All samples were positive for our internal control, bacteriophage MS2, although with variable efficiency (33.3 % ± 15.6). Such a variation has been described before (Petterson et al., 2015; Silva-Sales et al., 2020) , probably due to the complexity and variability of sewage samples. Table 3 (complete samples in table A1) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 26, 2020. . https://doi.org/10.1101/2020.05.25.20112706 doi: medRxiv preprint Table 3 . RT-PCR as mean amplification cycles for RNA-dependent polymerase (RdRP) and nucleocapsid (N) SARS-CoV-2 specific genes, a conserved region in of the structural protein envelope for the detection of pan-Sarbecoviruses (E). ND stands for a non-detected gene. Only positive samples are shown in this . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 26, 2020. . https://doi.org/10.1101/2020.05.25.20112706 doi: medRxiv preprint Following the presence of SARS-CoV-2 genetic material along the WWTP allows inferring its fate in the different processes. For that purpose, samples were quantified using commercial standards, as explained in M&M section, leading to the results shown in Table 4 . SARS-CoV-2 RNA was systematically detected in the influent to the primary settler (between 7.5 and 15 cp/ml) but not in the secondary treatment effluent, confirming that the effluent is safe for reuse and discharge to water bodies, as other studies have also reported (Randazzo et al., 2020b) . Another potential mechanism of transmission of airborne is the production of aerosol in secondary treatment, particularly if . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 26, 2020. . https://doi.org/10.1101/2020.05.25.20112706 doi: medRxiv preprint aeration is provided by horizontal rotors or surface turbines (Gotkowska-Płachta et al., 2013; Sánchez-Monedero et al., 2008) . Given the rare occurrence of SARS-CoV-2 RNA in the inflow to the secondary treatment, the potential of dispersion by aerosols created during aeration can be ruled out. As for the sludge line, it appears that SARS-CoV-2 RNA is mainly retained at the primary settler (10 -40 cp/ml) and only detected in one occasion in the biological sludge (7.5-10 cp/ml), which suggest that, as hypothesized, the virus particles have a higher affinity for the sludge and therefore, it is mostly diverted to the sludge line. Interestingly, its concentration increases in the thickeners which have a longer retention time, (approximately 24 hours) and a higher solid content. However, no genetic material is detected in the digested sludge, which is surely related both to the severe temperature undergone during thermal hydrolysis and to the long residence time in the anaerobic digesters. Therefore, the results confirm the safety of the sludge after thermal treatment and anaerobic digestion. However, in smaller WWTPs is only treated by volume reduction methods with no thermal treatment, the safety of sludge disposal remains to be verified. It is seen that the concentration of SARS-CoV-2 genetic material is systematically higher in some sludge sampling spots ( particularly at primary sludge and thickened sludge) compared to the influent samples (Table 4 ). This result confirms one of the hypothesis of this work, namely that the affinity of virus particles for biosolids would divert the genetic material of SARS-CoV-2 towards the sludge line. Although the number of samples analysed in this work is limited and replication in other WWTPs is required, this finding suggests that monitoring COVID-19 incidence in the population in the sludge might have a higher sensitivity than in the wastewater. In this WWTP, the primary settler and the sludge thickeners would act in effect as "concentrators" of . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 26, 2020 . . https://doi.org/10.1101 SARS-CoV-2 genetic material. Furthermore, the retention times in sludge thickeners (~24h) are usually higher than in primary settlers (~1-2 h). This higher retention time results in dampening the potential variations of SARS-CoV-2 particles in wastewater in an even more effective way than taking composite samples. Such a buffering is not helpful when the phenomenon to be monitored has fast dynamics, but in the case of COVID-19 population incidence, the desired monitoring dynamics would be in the order of days, making it a suitable sampling spot. The affinity of SARS-CoV-2 by biosolids was seen to govern to a large extent its fate in WWTPs by being associated to sludge streams. As a consequence, SARS-CoV-2 genetic material was not detected in the water effluent, confirming its safety. The combined treatment of thermal hydrolysis and anaerobic digestion also prevented the detection of SARS-CoV-2 in sludge leaving the plant. The primary sludge and mostly the thickened sludge showed higher and steadier concentrations, which suggests that COVID-19 incidence could be monitored preferably in the sludge line rather than, or in addition to, the raw wastewater. Longer residence times and higher solid concentrations in sludge thickeners would make it a more robust sampling spot, which merits being further investigated. Group (ED431C2017/029). All these programs are co-funded by ERDF (EU). . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 26, 2020. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 26, 2020. . https://doi.org/10.1101/2020.05.25.20112706 doi: medRxiv preprint The supplementary material contains the results of the RT-PCR and physicochemical characteristics of the water and sludge for all the sampling points and days. Table A1 . Full collection of samples, showing results of RT-PCR as mean amplification cycles for RNA-dependent polymerase (RdRP) and nucleocapsid (N) SARS-CoV-2 specific genes, a conserved region in of the structural protein envelope for the detection of pan-Sarbecoviruses (E). The (number) after the sampling spot corresponds to figure 1. ND stands for a non-detected gene. April . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 26, 2020 . . https://doi.org/10.1101 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. 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