Identification coronavirus (SARS-CoV-2) and physicochemical qualities in various water sources and the efficiency of water treatment plants in their removal- case study: Northwest region of Iran

The presence of SARS-CoV-2 virus in water resources and the transmission of diseases caused by it is one of the factors threatening the quality of water resources. This study for the first time concentrates on the presence of SARS-CoV-2 in water resources an urban location. In the present study, the samples were collected from known depth (30–50 cm) of rivers, dams and lakes. In each sample of water collected, different parameters such as residual chlorine, pH (phenol red), turbidity, total dissolved solids and temperature were also measured. Out of 267 samples, two samples were detected to be positive which their Ct values were 34.2 and 35.67. The existence of viable form of this virus in water and wastewater may be associated with issues for providing public health and difficulties in implementation of pandemic control strategies, and this situation can be exacerbated in developing countries that do not have adequate access to sanitation and safe water.


Introduction
The presence of viruses in water resources and the transmission of diseases caused by it is one of the factors threatening the quality of water resources (Mancuso et al. 2021). With the outbreak of coronavirus in 2019 in the world and its identification in municipal wastewater, the possibility of the presence of this virus in water sources and services is raised (García-Ávila et al. 2020). Coronaviruses are RNA-positive viruses and belong to the coronavirus family and the order Nidovirales. These viruses are widespread in human and mammalian species (Karami et al. 2021a, b). The severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) are the disease, which has been two large epidemics due to coronaviruses (Dargahi et al. 2021a;Dargahi et al. 2021b). At the end of 2019, another disease, which has been become pandemic a novel mutation of the coronavirus (categorized as SARS-CoV-2), was recognized and rapidly spread in the world (Lahrich et al. 2021;Vosoughi et al. 2021). According to reports, the number of infected people with this new mutation, till June 2021, was more than 180.4 million individuals affected, among which more than 3.9 million people have passed away [https:// www. world omete rs. info/ coron avirus/]. This disease has been associated with many huge disasters so that control of this infection has not been possible in the even with the best healthcare systems (Langone et al. 2021). The economic damage of trillions of dollars and the unclear future have created an urgent need for new solutions to such new infections (Zandian et al. 2021). Human viral pathogens transmitted by water can be associated with moderate to high health significance; these viral pathogens, based on the WHO, are adenovirus, astrovirus, hepatitis A and E, rotavirus, norovirus and other enteroviruses (La Rosa 2020). Studies have been confirmed that a number of family of coronavirus can be survived in water systems, and clarified that viral loads (depending to population infection rates) can be presented in untreated wastewater . As known COVID-19 is a respiratory disease, however, large amounts of SARS-CoV-2 RNA have been found in patients' feces (Wu et al. 2020), as well as in raw sewage (Fongaro et al. 2021;La Rosa et al. 2020), sewage sludge (Peccia et al. 2020) and surface water (Guerrero-Latorre et al. 2020;Prado et al. 2021). Detecting the viable SARS-CoV-2 in urine (Sun et al. 2020) and feces (de Oliveira et al. 2021;W. Wang et al. 2020a, b) of patients has led to increasing the concerns about the possibility of transmission of COVID-19 by the fecal-oral or fecal-nasal routes. The existence of viable form of this virus in water and wastewater may be associated with issues for providing public health and difficulties in implementation of pandemic control strategies (Vickers 2017); this situation can be exacerbated in developing countries that do not have adequate access to sanitation and safe water. So far, credible evidence of transmission of COVID-19 through contaminated water has been achieved (La Rosa et al. 2020). Nonetheless, the need for research about the resistance of SARS-CoV-2 on environmental matrices, e.g., surface water and wastewater, has been emphasized by the World Health Organization (Organization 2020). One of the important sectors to protect the human health during such current pandemic is water services (Shutler et al. 2020b). Finding the SARS-CoV-2 in fecal samples as in untreated wastewater can be indicative of the possibility of its fecal-oral transmission. This possibility has been resulted in worries toward the transmission of this coronavirus into the environment recently. These worries is rising when the untreated or inadequately treated wastewater is released to environment since it may rise the risk infection with SARS-CoV-2 in waters (Arslan 2020;Cahill and Morris 2020). The water which is used for human consumption should be treated by conventional methods and chlorine-based disinfection correctly should be used to provide a residual chlorine level higher than 0.5 mg/L in the distribution network; this can help to fight SARS-CoV-2 (Wang et al. 2020a, b).
The results of studies have shown that coronavirus is able to survive in human feces for 3 days and in raw water without chlorine and hospital wastewater for 2 days at a temperature of 2 °C, and if the wastewater is not treated, the virus can enter surface and groundwater, and through it, it can cause environmental pollution and disease in humans (Lodder and de Roda Husman 2020; Medema et al. 2020). Coronaviruses are important pathogens in humans and animals. Previous research has proved the presence and survival of a number of virus types in groundwater resources (24-21). Therefore, COVID-19 can cause pathogenicity if it enters the water through various ways such as cemetery sewage, hospital effluent and domestic sewage. In 2020, Naddeo et al. conducted a study on the fate of the coronavirus in the urban water cycle. The results showed that COVID-19 virus can easily enter surface water sources through sewage and cause disease in a large number of people (Dargahi et al. 2021a, b;Naddeo and Liu 2020).
Providing safe water to protect human health is essential during the spread of all infectious diseases, including coronavirus 2019 . In terms of the prevalence of coronavirus (SARS-CoV-2) in the world and its identification in municipal wastewater, the possibility of the presence of this virus in water sources and services is raised. Therefore, examining the presence of coronavirus (SARS-CoV-2) in water resources and services is a new topic and can be studied from different aspects and is considered important in terms of quality control of water resources and services. Identifying it in water sources and services can also be an important aid in detecting the virus in the environment. Therefore, this study aimed to identify coronavirus and determine physicochemical properties including pH, TDS, residual chlorine, turbidity and temperature in surface water (rivers, dams and lake), ground water (well water, springs), water distribution networks and water treatment plants (WTP) in Ardabil province.

Materials and methods
In this descriptive analytical study, the water resources and services of North West Iran were collected. Samples were selected for the prevalence of coronavirus (SARS-CoV-2) in the water resources and services of Ardabil province. Methodology was based on collecting available information from various sources (reports, articles and databases) to determine the main and important rivers, lakes and dams in Ardabil province. In the present study, water resources included water transmission lines, storage tanks, drinking water distribution network, groundwater resources as well as water treatment plants in Ardabil province. For this purpose, out of 6 sub-basins in Ardabil province, 2 sub-basins (Darrehe-Rood and Balharud) related to Aras's catchment and 2 sub-basins (Ghezel Ozan and Hiruchay) related to Ghezel Ozan catchment (4 basins in total), it was selected to cover most of the cities of the province. The rivers associated with the four basins identified in this study included: Balkhali Chai, Qarasu, Ghezel Ozan, Hiro Chai, Aras, Khiavchai, Balharud, Givi Chai and Garmi Chai, Mill Moghan, Givi, Gilarloo, Yamchi and Sabalan dams, and Ardabil, Bilesvar, Jafarabad, Roh Kennedy and Parsabad water treatment plants were selected examples of surface water resources. Transmission lines, water canals, storage tanks and drinking water distribution network of Ardabil cities of Ardabil province (Ardabil, Pars Abad, Garmi, Bilesvar, Aslandooz, Meshkinshahr, Nir, Namin, Khalkhal, Jafarabad, Ingut) and suburban villages in this study were considered. Also, wells and springs in the cities of Ardabil province were selected sources of groundwater. 5 L per sample was collected in special glass containers in the package. Samples will be collected using the combined sampling method (by sampling from a specific point at different times and combining them together). The samples will be transferred to the laboratory on ice and the samples will be stored at 4 °C until analysis. Finally, distribution and qualitative zoning map of surface water resources were done through GIS software (Fig. 1). In the present study, 267 samples from different water sources in northwest of Iran (Ardabil province) were examined to identify SARS-CoV-2 virus, and the results are presented in Table 1. In each sample of water collected, different parameters such as residual chlorine (VAHEB, VE611, DPD) pH (VAHEB, VE611) phenol red (VAHEB, VE611, DPD), turbidity (AQUA LVTiCAL250T-IR), total dissolved solids (TDS) (DMT-20) and temperature (DMT-20) were also measured. The physicochemical results of samples related to water sources of Ardabil province are presented in Table 1. Sampling was performed in August, September and February 2020 to March and April 2021. Filtered samples were prepared by consecutively filtering through with 0.45 μm and 0.22 and 0.1 μm pore size filters. The samples will then be centrifuged for 12 min at 12,000 rpm. The supernatant is discarded and extracted into the remaining micro tubes. Cutoff Ct value in several kits was used to identify SARS coronavirus-2 in water samples < 40. In addition, a schematic of coronavirus transmission pathways in water sources is presented in Fig. 2. In this figure, the possibility of transmitting coronavirus to water sources has been determined.

RNA extraction
Viral RNA was extracted from sample material and collected in elution buffer, using the High Pure Viral Nucleic Acid Kit at 200 µl. PCR amplification was performed using the Super-Script™ III One-Step RT-PCR System with Platinum™ Taq DNA Polymerase (Invitrogen, USA).

Data analysis method
After collecting the samples and analyzing them by PCR, the data obtained by EXEL and SPSS software version 20 were analyzed with similar articles and existing guidelines.

Results and discussion
In the present study, after collecting 267 samples from different water sources in northwest of Iran (Ardabil province), they were tested to detect COVID-19 virus, the results of which are presented in Table 1. For the collected samples, measurement of the amount of parameters, e.g., residual chlorine, pH, turbidity, total dissolved solids (TDS) and temperature, was also taken. The results related to physicochemical characteristics of samples taken from water sources of Ardabil province are shown in Table 1. Sampling was taken in two months of 2020 (August and September) and three months of 2021 (February, March and April). In the present study, two samples were obtained to be "positive" and three replicate were considered for them. Out of 267 samples, two samples were detected to be positive, which their Ct values were 34.2 and 35.67. In the present study, the samples were collected from known depth (30-50 cm) of rivers, dams and lakes. Also, in Fig. 1, the general zoning map for the presence of COVID-19 virus has been shown.
Several studies have been performed in this area and have confirmed the presence of coronavirus in aquatic sources, while some studies have not been able to find coronavirus in aquatic sources. The presence and resistance of the coronavirus in the environment are a very important issue for researchers. The presence of coronavirus in human feces 1 3 89 Page 4 of 17

Table 1
Coronavirus (SARS-CoV-2) outbreak on water sources and treatment plants in northwest of Iran (n = 267). and sewage has been proved (Langone et al. 2020;Mancuso et al. 2021). Sewage is generated in corona treatment centers or residential areas where infected people live. One of the important points in detecting the coronavirus in water sources is the amount and method of collecting the virgin virus. The results of this study showed that there were two positive samples in water sampling that according to Table 1, both samples were taken from rivers in different cities. In studies that collect genetic material, it is more difficult to detect them (Rimoldi et al. 2020;Shutler et al. 2020a). Unlike bacteria, it is difficult to detect viruses due to the lack of cell culture to detect them. In the present study, the lack of detection of more positive samples may be due to the above. The reasons that can cause the virus undetectable in the water depends on various factors that cause the virus to eliminate. Among the effective factors in the elimination of viruses in water are the process of absorption, adhesion, sedimentation, inactivation due to temperature, sunlight, pH changes, salts and minerals (Giacobbo et al. 2021;Tran et al. 2020). On the other hand, viruses in water and wastewater come in contact with a variety of substances, including drugs, chemicals and detergents, which make the virus less detectable than it actually is .

Sampling location Type of Water Source
Obtaining information on viral contamination of water and sewage can help diagnose the spread of the disease. In many cases, infected people have no symptoms but can be a source of virus production and excretion (Shutler et al. 2020a). Therefore, this information can be used as an epidemiological identification of the disease. On the other hand, analyzing the obtained data and also finding the relationship between the concentration and frequency of the virus in water and wastewater in different areas can help to predict critical and sensitive points (Adelodun et al. 2021). The coronavirus is present in human feces, but its amounts are unknown (Bilal et al. 2020). If the virus enters surface water through household wastewater disposal, it can contaminate surface water ). If contaminated surface water is used for drinking, there is a risk of infection. This risk becomes significant when the contaminated water is not treated. Contaminated water can also enter water reservoirs and contaminate them on a large scale (Shutler et al. 2020a). In many developing cities, water treatment does not take place or water treatment is done in a basic way, and there is still the possibility of contamination. The World Health Organization recommends an effective dose of 5 mg/L to kill viruses in water. According to Iranian standard, the amount of residual chlorine for disinfection of water is between 0.2 and 0.8 mg per liter. In some places, according to the values mentioned, viruses may survive despite chlorination. Therefore, it is recommended to inspect different areas for chlorination in these conditions (Tran et al. 2020). If wastewater is disinfected in places where there are infected people (such as hospitals), many viruses survive with covering with other materials and the Fig. 2 Schematic of SARS -CoV-2 transmission pathways in water sources effect of chlorine on them is somewhat ineffective (Giacobbo et al. 2021;Tran et al. 2020). In this study, the main parameters of water quality were also tested. In the present study, the measured parameters were turbidity, pH, total dissolved solid (TDS) and temperature along with the contamination of water with coronavirus that mean values of these parameters are shown in Table 2. The relationship between the various parameters is shown in Table 3. Environmental factors may affect the survival of viruses in the aquatic environment. Due to the small number of positive samples in the present study, it was not possible to establish a statistical relationship between positive cases and environmental parameters. However, the values of positive environmental samples in the river for residual chlorine, pH, turbidity, total dissolved solid and temperature were 0 mg/L, 6.4, 60 NTU, 172 mg/L and 3.2 °C, respectively. Also, the environmental parameters of the positive samples in the tap for residual chlorine, pH, turbidity, TDS and temperature were 0.5 mg/L, 7.4, 0 NTU, 126 mg/L and 6.5 °C, respectively. Due to the small number of positive samples in our study, the effect of environmental factors is ambiguous and more studies are needed. On the other hand, some limited studies have been done in the environment. According to the obtained statistical test results, there was a significant relationship between pH and other water quality parameters studied in this study. TDS also had a standard relationship with other environmental parameters. Chlorine also had a statistically significant relationship with other environmental parameters. But no significant relationship was found between temperature and turbidity. Most studies have confirmed the relationship between residual chlorine and pH (Yang and Cheng 2007). Given that chlorine may be the most important factor in killing viruses in the aquatic environment, controlling pH and chlorine helps control viruses and other microorganisms in the aquatic environment, because the amount of residual chlorine is dependent on BPH, so that at lower pHs, its disinfection power increases.
A study by (Adelodun et al. 2021) showed that SARS-CoV-2 virus can be found in the environment with varying degrees of resistance and survival. In their review article, they did not definitively confirm the transmission of the virus through food, water and other environmental components. In the present study, the persistence and status of viruses in water have not been studied. In some cases mentioned in Table 1, positive samples have been found. de Oliveira et al. in their study pointed to water and sewage contamination with the coronavirus, which may cause concerns through fecal-oral transmission (de Oliveira et al. 2021). However, this study noted that fecal-oral transmission was not established. Although the transmission of the virus from water is ambiguous, but considering that in our study in the two cities of the province, the sample of coronavirus has become positive, it is better to take serious health recommendations. Believe that in areas where sanitation is not done properly, viruses can reach the main body of water resources and survive for a long time. Their study also showed that low seasonal temperatures increase the risk of transmitting the virus through water. But the survival of viruses depends on the environment. Our study was conducted at ambient temperature, because our samples were taken from the environment. Studies have been performed on the survival of viruses at different temperature changes. Bivins et al. (2020) reported that the SARS-CoV-2 virus survives in water and wastewater for 1.5 and 1.7 days, respectively. At 50 and 70 °C, T 90 (the time for 90% reduction) decreased relative to ambient temperature. In fact, as temperatures rise, viruses' resistance decreases. In our previous study, we investigated SARS-CoV-2 in municipal wastewater treatment plant, collection network and hospital wastewater that found some positive samples in effluent of wastewater treatment (Dargahi, et al. 2021a, b). Kumar et al. (2021) reported that viruses lose their infectious ability to deal with wastewater that may contain alcohol-based detergents. In some areas where rainwater mixes with municipal wastewater, it may also become contaminated, although it will help dilute the wastewater. Tran et al. (2020) have stated that in addition to viral contamination of water sources by mixing them with sewage, contaminated masks of patients left in the environment can also be another cause of contamination of water sources with coronavirus. The results of their study showed that the survival of coronavirus in water sources is strongly related to temperature, water properties and concentration of suspended solids, pH and concentration of disinfectants. The World Health Organization considers conventional water purification and chlorination processes sufficient to kill viruses and bacteria. de Oliveira et al. (2021) also considered the influence of environmental factors to be effective. Their study found that temperature had a significant effect on the resistance and survival of the virus in the aquatic environment. They showed T 90 levels of 7.7 and 5.5 for river water and wastewater at 4 °C, respectively. But with increasing temperature, this resistance has decreased. Rimoldi et al. (2020) reported that they found SARS-CoV-2 viral RNAs in water sources. However, the tests for infectivity in the laboratory were negative. This indicates a low risk of water pollution caused by SARS-CoV-2. Mancuso et al. stated that since most fresh water is used for agriculture, the quality of water for irrigation should be ensured. Coronaviruses can infect agricultural products and cause contamination via irrigation (Mancuso et al. 2021). Different results have been reported in the studies. In a study by Mahlknecht et al. (2021) that carried out at the height of the corona pandemic took samples from the water sources of Monterrey. Their results were analyzed by PCR which revealed that 44% of sources near the city were positive with a virus load of 2.6 to 38.3 Copies/ml. Also, 12% of the samples taken from the dams and 13% of the samples taken from the rivers were positive. A total of 50% of their samples were positive for coronavirus. Our study showed that 2 cases of the all taken samples were positive regarding the presence of SARS-CoV-2. Positive samples were taken from river and tab.

Conclusions
The existence of viable form of SARS-CoV-2 in water is associated with the public health measures. Among the effective factors in the elimination of viruses in water are the process of absorption, adhesion, sedimentation, inactivation due to temperature, sunlight, pH changes, salts and minerals. Chlorine and pH, which were significantly associated in this study, are one of the factors that can control the coronavirus in aquatic environments. Difficulties in implementation of pandemic control strategies can be exacerbated in developing countries that do not have adequate access to sanitation and safe water. The results of this study showed that there were two positive samples in water sampling that according to, positive samples were taken from rivers and tab in different cities. Although the transmission of the virus from water is ambiguous, but considering that in our study in the two cities of the province, the sample of coronavirus has become positive, it is better to take serious health recommendations. Environmental health engineering believes that in areas where sanitation is not done properly and viruses can reach the main body of water resources and survive for a long time.
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