key: cord-0929360-7llnvqif
authors: Alahdal, Hadil M.; Ameen, Fuad; AlYahya, Sami; Sonbol, Hana; Khan, Anas; Alsofayan, Yousef; Alahmari, Ahmed
title: Municipal wastewater viral pollution in Saudi Arabia: effect of hot climate on COVID-19 disease spreading
date: 2021-06-17
journal: Environ Sci Pollut Res Int
DOI: 10.1007/s11356-021-14809-2
sha: 5234874634725a1be960f2e8445b0508bf5f841f
doc_id: 929360
cord_uid: 7llnvqif

The viral RNA of SARS-Coronavirus-2 is known to be contaminating municipal wastewater. We aimed to assess if COVID-19 disease is spreading through wastewater. We studied the amount of viral RNA in raw sewage and the efficiency of the sewage treatment to remove the virus. Sewage water was collected before and after the activated sludge process three times during summer 2020 from three different sewage treatment plants. The sewage treatment was efficient in removing SARS-CoV-2 viral RNA. Each sewage treatment plant gathered wastewater from one hospital, of which COVID-19 admissions were used to describe the level of disease occurrence in the area. The presence of SARS-CoV-2 viral RNA–specific target genes (N1, N2, and E) was confirmed using RT-qPCR analysis. However, hospital admission did not correlate significantly with viral RNA. Moreover, viral RNA loads were relatively low, suggesting that sewage might preserve viral RNA in a hot climate only for a short time.

The contamination of wastewaters with viruses causing diseases is an emerging threat that the global climate change seems to worsen (El-Sayed and Kamel 2020). The new coronavirus disease COVID-19 causing the global pandemic is needing great efforts to be controlled. It has also been speculated that the SARS-CoV-2 virus will circulate over the world like seasonal influenza ). Therefore, it seems that we will need preventing actions for a long time in the future. The disease symptoms appear 4 to 14 days after exposure; meanwhile, the person can further spread the virus. Viruses usually spread through the air, but they can spread through wastewater as well. The virus can be transmitted from sewage through the workers that are exposed to the virus (Elsamadony et al. 2021 ). This transmission pathway was recommended to be studied in a recent review (Bogler et al. 2020) .

Wastewater surveillance has been shown to be helpful in assessing the disease spreading (Mohapatra et al. 2020; Polo et al. 2020 ; Thompson et al. 2020; Zhu et al. 2021) . Disease incidence has correlated with viral RNA, which can be analyzed using the molecular method of quantitative real-time PCR (RT-qPCR). Viral RNA has been observed to correlate strongly positively with local hospital admission; a correlation coefficient (r) has been higher than 0.9 (Medema et al. 2020; Peccia et al. 2020) . The efforts to utilize wastewater surveillance to control the disease has already been made throughout the world, for instance, in China ( Bar-Or et al. 2020) , Australia (Ahmed et al. 2020) , Netherlands (Medema et al. 2020) , Japan (Haramoto et al. 2020) , Turkey (Kocamemi et al. 2020) , and France (Wurtzer et al. 2020) . Several reviews have already been published (Saawarn and Hait 2020; Hasan et al. 2021; Ihsanullah et al. 2021; Zhou et al. 2021) . The wastewater approach has scarcely been studied in Arabic countries. We found only one study in UAE (Hasan et al. 2021) . It is vital to study sewage and sewage treatments locally because the environmental conditions, most of all temperature, are known to affect the survival of viruses (Aboubakr et al. 2020) . The survival of SARS-CoV-2 virus in sewage is still not well known. Coronavirus causing an epidemic in 2003 SARS-CoV was found in sewage for 2 days at 20°C and for 14 days at 4°C (La . Recently, high temperatures have been shown to inhibit the spreading of COVID-19 disease (Guo et al. 2021; Notari 2021) . It can be assumed that the high temperature (around 50°C) prevailing in the Arabian Gulf countries during summer is destroying the virus more rapidly than cool or moderate temperatures.

Our aim was to assess how SARS-CoV-2 viral RNA persists in sewage systems in a hot climate. High temperatures prevailing in Saudi Arabia may destroy the virus, and thus, the spreading of the virus may not be significant. We sampled untreated wastewater from three sewage treatment plants three times and compared the results to reported disease cases in the hospitals nearby. We also sampled treated sewage to analyze the efficiency of the treatment procedure to remove viral RNA.

Three replicated untreated and treated (cleaned) wastewater samples (2 L) were collected to sterilized glass bottles from three sewage treatment plants situated in Riyadh city three times monthly in 15th June, 17th July, and 16th August 2020. The sewage plants were DQ sewage water treatment facility (SW1), KFSHRC water treatment plant (SW2), and NWC strategic water systems (SW3) (Fig. 1) . The samples were collected after activated sludge treatment before the disinfection step. The temperatures of sewage were 35°C, 38°C, and 40°C ± 2°C in June, July, and August, respectively. The samples were immediately cooled at 4°C, and the analyses were carried out within 48 h.

The wastewater was concentrated using the flocculation method using 3.0% w/v beef extract solution (Sigma-Aldrich, St. Louis, USA), pH 9.5 in 0.05 M glycine buffer. Each sewage water (SW1, SW2, and SW3) sample (500 mL) was acidified (Michael-Kordatou et al. 2020 ) at pH 3.5 ± 0.1 and 10 mL of the beef extract was agglomerated by the addition of 0.1 M HCl at pH 3.0 followed by stirring for 10 h. The stirred suspension was centrifuged at 10,000×g for 30 min at 4°C. The pellet was dissolved in 8 mL of phosphate buffered saline (PSB) and the RNA was extracted using NucliSENS® miniMAG® system (BioMerieux, Marcy l Etoile, France) according to the manufacturer's instructions.

RT-qPCR was carried out using Allplex COVID 19 RT-PCR kit (Seegene Inc) specific for SARS-CoV-2. RNA was amplified using R-Biopharm AG RIDA®CYCLER system with initial activation (2 min) at 25°C, reverse transcription at 50°C for 15 min, initial denaturation at 95°C for 2 min, annealing with 45 cycles at 95°C for 10 s, and extension at 55°C for 15 s. The fluorescence analysis was carried out using Light Cycler 480 (Roche Molecular Diagnostics, Mannheim, Germany). The length of the PCR product was analyzed with high-resolution automatic electrophoresis (Agilent, Santa Clara, USA). Viral load was determined spectrophotometrically (277 nm) as described by Rostislav et al. (2021) . Cycle threshold value (Ct), which is inversely related to the viral load, was reported to assess the SARS-CoV-2 viral load as recommended by Tom and Mina (2020) . A rise of three in the Ct value indicates a tenfold reduction in viral load, and Ct < 29 indicates strong, Ct 30-37 moderate, and Ct 38-40 weak or negative load (Tom and Mina, 2020).

The epidemic was at a relatively high level in Saudi Arabia during the study period. The 14-day notification rate per 100,000 population varied between 57 and 160 during the study period (ECDC 2020). In Riyadh, the disease incidence was higher; the 14-day notification rate per 100,000 population was 100 in May, 650 in June, 633 in July, and 413 in August (MOH 2020).

Patients analyzed to have the COVID-19 disease in hospitals near the sewage treatment plants were used to assess disease incidence. The data from three hospitals were used. Each sewage treatment plant collected the wastewater of one hospital. The hospitals were King Khalid University Hospital; King Faisal Specialist Hospital & Research Center; and King Salman Bin Abdulaziz Hospital corresponding SW1, SW2, and SW3 sewage treatment plants (Fig. 1) . The hospital admission data were divided into 2 weeks period starting 1 month before the first sewage sampling and ending 2 weeks after the last sewage sampling. 

Spearman rank correlation between the Ct value and the disease cases during 2 weeks after the sampling was calculated. Paired t-test was used to analyze the difference between untreated and treated sewage. P < 0.05 was considered significant.

The levels of viral RNA load in untreated sewage differed between the treatment plants SW1 having the highest load. Inversely related to viral load, the Ct values varied between 14 and 22 for all three target genes (N1, N2, E) during the 3 months. For all genes, lower Ct values (14-16), indicating higher viral load, were observed in June and August than in July (Ct = 21) (Fig. 2a) . In SW2 and SW3, the Ct values were at a slightly higher level in general (Fig. 2b, c) . In SW2, the June samples were the highest, over 30, indicating only moderate viral load. In SW3, the months did not differ much. The three treatment plants showed different trends during the sampling SW1 and SW2 differing from SW3 where the Ct values were almost equal each month.

A total of 2374 (SW3), 3499 (SW1), and 5761 (SW2) disease cases were reported in the hospitals. In May, almost no cases were reported and the rapid spreading of the disease started in June when the first samples were taken (Table 1) . Disease cases and target gene Ct values did not correlate (Fig.  3) . Spearman rank correlation between Ct values and disease cases (data of all hospitals combined, n = 9) were not significant, and the correlation coefficients (R S ) were 0.42, 0.37, and 0.42 for N1, N2, and E, respectively.

The sewage treatment procedures removed viral RNA efficiently in all three treatment plants. The means of the viral loads of the 3 months varied between 70 and 85 copies mL −1 before the sewage treatment (Fig. 4) . Viral load was significantly (paired t-test, p < 0.05) lower in treated sewage than in untreated sewage. Viral load varied between 3 and 10 copies mL −1 in treated sewage.

Wastewater surveillance has been shown to give an early indication about the starting epidemic of COVID-19 disease (Medema et al. 2020; Randazzo et al. 2020b) . Disease incidence and SARS-CoV-2 viral RNA load have been shown to correlate strongly positively already many times, as reviewed by Mandal et al. (2020) . A predictive regression model has been published (Vallejo et al. 2020) . A high correlation (r > 0.9) between viral RNA and hospital admission was observed in the USA (Peccia et al. 2020) . Lower correlations have also been reported, for instance, r = 0.4-0.5 in Japan (Hata et al. 2021). In some studies, no correlation was found (Trottier et al. 2020; Wu et al. 2020 ). In our study, no significant correlation was observed between the disease cases reported in hospitals during 2 weeks after the sampling of sewage and the Ct values of the three genes. One explanation is that the strength of the correlation depends on the variation in the data set, which was low in our case. Moreover, we had only nine observations that we sampled during 3 months from three hospitals when the disease incidence was relatively high, the 14-day notification rate per 100.000 population varying between 57 and 160 during the study period (ECDC 2020). Unfortunately, we were not able to sample in April when disease incidence was very low (<10). In general, the monitoring of viral load has been assessed as a reliable tool to give an early warning about the spreading of the disease (Polo et al.

2020; Thompson et al. 2020) . However, wastewater surveillance might not be sensitive enough to detect small changes in the epidemic at the high level of disease incidence, as was the case in our study. One aspect to be taken into consideration in our surveillance results is the hot temperature that prevailed in Riyadh during the sampling period. Viral RNA is known to be destroyed in high temperatures. Average temperatures in Riyadh were 42°C, 43°C, and 43°C in June, July, and August, respectively. The maximum temperature reached 45°C during July and August. Despite the high temperatures, disease incidence was high in Riyadh. However, the viral loads seemed to be at a relatively low level in sewage compared to previously reported values. Our RNA loads of N1, N2, and E genes varied between 70 and 85 copies per mL. In previous reports from sewage, when the prevalence of COVID-19 disease increased, the RNA load increased from 2.6-30 gene copies to 790-2 200 gene copies per mL (N1-N3 genes) (Medema et al. 2020) . Elsewhere, a maximum of 3000 copies per mL has been reported in untreated sewage (Wurtzer et al. 2020) . In the feces of COVID-19-positive people, the viral RNA load has been 10 3 -10 8 gene copies per mL (Foladori et al. 2020) . It may be that the high temperature of sewage (35-40°C) had destroyed viruses in Riyadh. A study from a neighboring country UAE in the Arabian Peninsula reported viral load from 0.75 to 340 gene copies per mL and 0.3 to 2.9 from untreated wastewaters (Hasan et al. 2021) . These values are relatively low and support our suggestion that the hot climate might have destroyed viral RNA. However, comparing viral loads with previous studies is difficult because of the slightly different procedures used in viral RNA analyses. A notable observation was that viral RNA seemed to be concentrated in activated sludge as compared to influent (Carrillo-Reyes et al. 2021) . This indicates the need to standardize the procedures, which has been recognized in several studies already (Michael-Kordatou et al. 2020) . The summary interpretation of our results in Riyadh is as follows. Disease incidence was high during the hot summer 2020 and the viral RNA was largely destroyed by the high temperature of sewage. However, due to relatively small number of samples in our study and small changes in disease incidence during the study period, the results must be interpreted with caution.

The risk of disease spreading from wastewater has been assessed to be low (Rimoldi et al. 2020; Dada and Gyawali 2021) . In contrast, many authors suggest that wastewater may be a relevant transmission route for the disease (Amoah et al. 2020; Bogler et al. 2020; Gormley et al. 2020) . Viral RNA has been found from fecal samples up to 5 weeks and viable up to 2 days in urine samples, as reviewed by Langone et al. (2020) .

Several studies have shown that enteric viruses are destroyed in different wastewater treatment procedures ). However, SARS-CoV-2 RNA has been studied scarcely from this point of view. Moreover, viral loads and treatment procedures have often been reported insufficiently in published studies. An unidentified treatment removed SARS-CoV-2 RNA from sewage in Italy (Rimoldi et al. 2020) . The RNA was removed from sewage by thermal hydrolysis and anaerobic digestion (Balboa et al. 2021 ) as well as by conventional wastewater treatments followed by a tertiary disinfection step with peracetic acid or high-intensity UV lamps in Spain (Randazzo et al. 2020a) . Free chlorine is known to be an especially efficient disinfectant (Wang et al. 2005; Tran et al. 2020 ) (Tran et al. 2020) . In India, up-flow anaerobic sludge blanket technology reduced SARS-CoV-2 RNA remarkably: inlet sewage had a Ct value of 30, whereas in the final effluent, the values were zero . The published studies show that common sewage treatments were mostly able to remove viral RNA. However, it is possible that some viruses might be left after the treatment. Metagenomic data revealed high diversity of respiratory viruses, including coronaviruses, to be present in anaerobic digester effluent in the USA (Bibby and Peccia 2013) . We found one article where SARS-CoV-2 RNA presence was confirmed with RT-qPCR method in treated sewage. After an unidentified wastewater process in Paris, viral RNA was found at a concentration up to 0.11 copies per mL (Wurtzer et al. 2020 ). The sewage treatment had reduced viral load to a hundredth part. In our study, while the primary sewage had the RNA load of 70-85 copies per mL, the treated sewage had 3-10 copies per mL. The removal of viral RNA was assessed as efficient but treated sewage still contained some SARS-CoV-2 RNA.

Coronaviruses are generally thought to survive viable for a few days depending on the environment (Kampf et al. 2020) . However, some studies have found that coronaviruses might survive in wastewater for a week (Casanova and Weaver 2015) . When SARS-CoV was inoculated into sewage, it remained infectious for 2 days at 20°C (Masaaki et al., 2020) . A recent review concluded that coronaviruses would survive a maximum of 3 days at 20°C in wastewater (Amoah et al. 2020) . Wastewater composition (organic matter), temperature, and pH are among the most important factors controlling the survival of viruses in wastewater (Medema et al. 2020) . Because it is known that higher temperatures destroy viruses, our observation is notable. Although we interpret that most viruses had been destroyed by the high temperature, a part of SARS-CoV-2 RNA survived in wastewater at 35-40°C

. However, the viability of the virus has not been studied previously or in our study, which should be the next step in our research.

Several potential treatment technologies to remove SARS-CoV-2 RNA were presented in two recent reviews (Bhatt et al. 2020; Lesimple et al. 2020) . For instance, membrane bioreactors have been suggested to remove viral RNA efficiently (Naddeo and Liu 2020) . We showed that the activated sludge process without any disinfection step removed SARS-CoV-2 RNA efficiently. The removal was also reported from Mexico recently (Carrillo-Reyes et al. 2021) . However, in our Riyadh case, the high temperature possibly had a remarkable effect, and more studies about the efficiency in different environments are needed.

The persistence of SARS-CoV-2 viral RNA in untreated sewage was confirmed in a hot climate in Saudi Arabia. However, the high temperature prevailing in Riyadh seemed to destroy viruses relatively rapidly. The sewage treatment procedures used mostly destroyed the viral RNA, and the spread of the disease through wastewater and sewage treatment plant workers is assessed as minimal. The wastewater surveillance has now been suggested as a routine approach to help in controlling COVID-19, and it seems to work in low-prevalence areas (Black et al. 2021; Rooney et al. 2021; Tiwari et al. 2021) . However, in a hot climate, the efficiency of wastewater surveillance is not proved, and we can only make tentative conclusions. The hot climate, such as in Saudi Arabia, may affect the epidemiological factors of SARS-CoV-2 virus, and the effect of climate should be studied further.

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Acknowledgements The authors extend their appreciation to the re-