key: cord-0836236-euy9v2sh
authors: Gholipour, Sahar; Mohammadi, Farzaneh; nikaeen, Mahnaz; shamsizadeh, Zahra; Khazeni, Atefeh; Sahbaei, Zohreh; Mousavi, Seyed Mohammad; ghobadian, Mojtaba; Mirhendi, Hossein
title: COVID-19 infection risk from exposure to aerosols of wastewater treatment plants
date: 2021-01-23
journal: Chemosphere
DOI: 10.1016/j.chemosphere.2021.129701
sha: 26efce3666497f4fd13a0de1eefc949b55749df7
doc_id: 836236
cord_uid: euy9v2sh

Fecal shedding of SARS-CoV-2 from COVID-19 patients and presence of the viral RNA in wastewater have extensively been reported. Some wastewater treatment plant (WWTP) processes generate aerosols which have the potential to transmit pathogenic microorganisms and present a health risk for exposed individuals. We analyzed the presence of viral RNA of SARS-CoV-2 in raw wastewater and air samples of WWTPs. The risk that may arise from exposure to virus-contaminated aerosols of wastewater was estimated by developing a quantitative microbial risk analysis (QMRA) method. SARS-CoV-2 was detected in 9 of 24 (37.5%) wastewater samples with a concentration about 10(4) genomic copies L(-1). The viral RNA was also detected in 40% (6/15) of air samples. QMRA analysis showed a relatively high risk of SARS-CoV-2 infection for wastewater workers via exposure to the viral aerosols. The estimated annual infection risk ranged from 1.1 × 10(-2) to 2.3 × 10(-2) per person per year (PPPY) for wastewater workers which was higher than the reference level recommended by WHO (10(-3) pppy). However, due to the lack of data on survival of SARS-CoV-2 in wastewater and its fate in aerosolized state, more research is needed to determine the importance of wastewater in transmission of COVID-19.

Recently, a novel coronavirus, SARS-CoV-2, emerged as the etiological agent of a respiratory disease called COVID-19. COVID-19 was rapidly spread worldwide and the World Health Organization (WHO) declared the coronavirus outbreak a public health emergency of international concern (WHO, 2020a). Similar to severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS), SARS-CoV-2 belongs to the Betacoronavirus genus. Betacoronaviruses are singlestranded, positive sense, enveloped RNA viruses which are responsible for respiratory infections (Yeo et al., 2020) . The virus is primarily transmitted from person-to-person by exposure to respiratory droplets from infected patients (Kampf et al., 2020) . However, some studies have been reported survival of human coronaviruses in the gastrointestinal tract and shedding in feces of infected patients (Kitajima et al., 2020) . Information from researches on human coronaviruses especially SARS-CoV and MERS-CoV shows respiratory and gastrointestinal symptoms such as diarrhea after viral infection (Yeo et al., 2020) . Intestinal infection in COVID-19 patients has also been reported and approximately 2-10% of cases with confirmed disease presented with diarrhea (Yeo et al., 2020) . Some studies have reported detection of COVID-19 viral RNA in fecal samples of infected patients (Kitajima et al., 2020) . During peak shedding, a high number of virus particles is released per gram of fecal matter of COVID-19 patients which could lead to widespread environmental dissemination of the virus through contaminated water and wastewater (Yeo et al., 2020) . There are several reports of the molecular detection of SARS-CoV-2 in wastewater (Ahmed et al., 2020; Lodder and de Roda Husman, 2020; Medema et al., 2020; Nemudryi et al., 2020; Wurtzer et al., 2020) with concentrations of a maximum of over 10 6 copies per liter of untreated (Kitajima et al., 2020; Wurtzer et al., 2020) . It has been proposed that the analysis of wastewater could be a powerful tool for COVID-19 surveillance.

However, the documented presence of SARS-CoV-2 in wastewater raises the question regarding the potential transmission risk of this virus through wastewater, especially for those working with human waste and wastewater (Foladori et al., 2020; Yang et al., 2020; Gormely e al., 2020; El Baz and Imziln, 2020; Gwenzi, 2021 et al., 2006) . Structural similarities between the SARS-CoV and SARS-CoV-2 (Corpuz et al., 2020) , in combination with relatively high stability of SARS-CoV-2 in the aerosol state (Fears et al., 2020) , calls for WWTPs to be considered as a potential source for transmission of COVID-19 through bioaerosols produced by wastewater treatment processes (Corpuz et al., 2020; Kitajima et al., 2020; Gwenzi, 2021 activated sludge plants at a height of 1.5 m above the ground level. Air samples (3500-4500 L) were collected using portable pumps at a flow rate of 7.5-8.5 L min -1 and then transferred to the laboratory in an insulated box with cooling packs.

Before detection of SARS-CoV-2, 200 ml of wastewater samples were concentrated by aluminum hydroxide adsorption-precipitation method as described in Standard Methods for the examination of water and wastewater (APHA, 2012).

However, for increase of detection sensitivity, some samples were more concentrated by application of polyethylene glycol (PEG) (Wu et al., 2020) . Air samples were also concentrated by application of PEG (Wu et al., 2020 against of a 10-fold serial dilution of a quantified plasmid. RNA of SARS-CoV-2 was amplified by RT-PCR and then used for DNA cloning. Plasmids containing the SARS-CoV-2 insert were purified using AccuPrep Plasmid Mini Extraction Kit (Bioneer, Korea), quantified and a ten-fold serial dilution was prepared. The limit of detection (LOD) resulted as 10 genomic copies per reaction.

Wastewater is containing pathogenic microorganisms such as viruses which could be aerosolized during wastewater treatment processes. In the study, a QMRA model was used to assess the risk of infection associated with inhalation of SARS-CoV-2 contaminated aerosols/droplets produced from wastewater treatment processes.

Sewage workers may expose to pathogenic microorganisms from biological aerosols produced during wastewater treatment processes. Exposure to SARS-CoV-2 aerosols/droplets in WWTPs could pose a health risk for workers (Corpuz et al., 2020; Kitajima et al., 2020) . Different stages of COVID-19 outbreak, in terms of percentage of infected population, can affect the concentration of viral particles in wastewater, aerosols emitted from WWTPs, and consequently estimated risk.

Therefore, it may be better to use the human fecal shedding method for estimating 

Aerosols are generated by some units at WWTPs such as pumping station and aeration tanks. High fractions of the aerosols have diameters 10 (88% of produced aerosols are less than 4 in diameter), which are considered respirable, could be deposited in the respiratory tract, and may reach the alveolar region of the lungs (EPA, 2011).

The daily dose (TCID 50 d -1 ) of SARS-CoV-2 aerosols inhaled by the WWTP workers is given by (Barker, 2014) :

Where C w is the concentration of SARS-CoV-2 in wastewater (TCID 50 L -1 ); PC wa is the microbial water-to-air partitioning coefficient (L m -3 ); IR is the average inhalation rate (m 3 h -1 ); t exp is the daily exposure duration (h) which was considered 8 h for occupational exposure, and RR is the aerosol retention rate in the lungs which is calculated by equation (2) (Schoen and Ashbolt, 2011) :

Here, f i 1 is the fraction of aerosols of size range i, and f i 2 is the fraction of the aerosols of size range i that are deposited in the lower respiratory tract (Schoen and Ashbolt, 2011) . We assumed that WWTPs could generate inhalable-size aerosols (smaller than 10 microns) which may carry the virus.

For C w, we used the best available data to estimate densities of infectious SARS-CoV-2 in the wastewater. The concentration of SARS-CoV-2 in wastewater is estimated by equation (3) , 2020) . F is the daily fecal production by infected patients (g feces per person per day), Q is the flow rate of WWTP (m 3 d -1 ) and CF is the conversion factor of genomic copy number to TCID 50 and dt is 46 days.

No pathogen decay was considered during aerosolization or transport through air to a receptor.

By the dose-response model, the probability of infection is estimated due to the inhaled dose. Because of the structural similarities of SARS-CoV-2 with SARS-CoV and human coronavirus and the lack of a dose-response model for SARS-CoV-2 we used exponential dose-response models which have been suggested by Watanabe et al., (2010) Where n is the frequency of exposure (the number of days per year on which a worker may be exposed to SARS-CoV-2 aerosols). We considered 20 working days per each month and an exposure period of 12 months (one year).

The values and statistical distributions of the variable parameters introduced in the study are presented in Table 2 . Monte-Carlo simulation technique with 10000 random sampling from each distribution input was used to run the model to develop a distribution of likely risk and incorporate the uncertainty and variability around each parameter. All modeling and analysis were implemented using RStudio version 1.3.959. Sensitivity analysis was performed using Spearman's rank order correlation to identify those variable input parameters contributing to the uncertainty of estimated infection risk and have the greatest influence on the estimated risk.

Since the concentration of SARS-CoV-2 in wastewater and subsequently in 

All of other parameters in QMRA model were kept to their average value.

Monitoring of pathogenic microorganisms in wastewater is a promising approach to understand the prevalence and circulation of the microbial diseases in a community. In the study, we detected SARS-CoV-2 in 9 of 12 (37.5%) wastewater samples. Viral RNA was detected in 5 of 12 and 4 of 12 samples of raw wastewater from WWTP A and B, respectively (Table 3) (Table 3) .

ly (Table   erent countries We found relatively consistent result for the viral load in WWTPs as quantified by the real-time PCR assay with which was calculated by the QMRA model (C w ) (Fig. 3) . However, some authors have reported much higher concentrations of the viral RNA in wastewater than the confirmed cases of COVID-19 in the community (Randazzo et al., 2020; Wu et al., 2020) . GII. Adenoviruses (4/16), NV GI (6/16), FRNA bacteriophages GIII (3/16), and enteroviruses (3/16) were also detected, but at lower frequencies (Matsubara and Katayama, 2019).

Presence of SARS-CoV-2 RNA in wastewater samples and wastewater aerosols could pose a health concern for WWTPs workers.

The results of this study showed a median infection risk (with illness as endpoint of response, Eq. 4) of 2.3 10 -2 (95% CI: 1.65 10 -4 -4.9 10 -1 ) and 1.1 10 -2 (95% CI: 8 10 -5 -2.9 10 -1 ) per person per year (pppy) for workers of WWTP A and B, respectively. Fig. 4 compares the levels of estimated infection risk with the 10 -4 and 10 -3 pppy reference levels proposed by EPA (EPA, 2011) and WHO (Mara et al., 2007) , respectively. As shown in Fig. 4 , the estimated annual infection risk of SARS-CoV-2 for wastewater workers was about 1 log higher than the WHO guideline threshold of 10 -3 pppy. The estimated risk was also higher than the tolerable infection risk of 5.5 10 -4 pppy has been recommended by Zaneti et al. (2021) for SARS-CoV-2.

The results showed a higher infection risk for workers of WWTP A which is related to the higher prevalence of COVID-19 in the region served by WWTP A (Fig. 3) . Study of Zaneti et al. (2021) showed an infection risk of SARS-CoV-2 from 2.6 10 -3 to 1.3 10 -2 from accidental ingestion of sewage by WWTP workers while performing routine activities. They reported that the estimated risk was above the tolerable infection risk for SARS-CoV-2 of 5.5 10 -4 pppy, thus reinforcing the concern about wastewater as a potential source of COVID-19 transmission. A QMRA analysis showed a high infection risk from adenoviruses for wastewater workers from exposure to bioaerosols from influent and biological oxidation tanks The results s he resu for >3 min exposure time (Carducci et al., 2018) . Pepper and Gerba (2018) reported an infection risk of greater than 10 -4 for exposure to spray irrigation of reclaimed water when the number of Legionella in the water exceeded 1000 colony forming units (CFU) per mL.

As shown in Fig. 4 , risk assessment analysis by equation 5 with death as endpoint of response, showed lower risk of SARS-CoV-2 for workers from exposure to wastewater aerosols.

We assumed that the load of SARS-CoV-2 genomes in wastewater correlated with the number of symptomatic patients with a mean fecal shedding duration of 11.89 days and shedding rates from 6.3 10 5 to 1.3 10 8 RNA copies per gram (Table 2 ). Some studies have reported the detection of SARS-CoV-2 in fecal samples of non-symptomatic individuals as well as lower shedding rate about 10 2 -10 3 RNA copies per gram of fecal matter of symptomatic patients (Kitajima et al., 2020) . It has been reported that viral RNA could be detected in the feces of 81.8% COVID-19

cases even with a negative throat swab result (Ling et al., 2020) . In an investigation, shedding of SARS-CoV-2 in a cluster of 9 cases was 10 7 RNA copies g -1 faeces one

week after symptom onset which decreased to the 10 3 RNA copies g -1 three weeks after symptom onset (Medema et al., 2020) . Therefore, due to the lack of sufficient information about this emerging pathogen, the estimated risk may be over-or underestimated.

Sensitivity analysis was performed to determine the influence of variation in the input parameters on the estimated infection risk. As expected, the sensitivity analysis suggested that the infection risk of SARS-CoV-2 is greatly affected by the number of cases of COVID-19 in the population (Pi) (Fig. 5) . However, the is also an important factor affects the infection risk (Fig. 5) . Although it has been reported that coronaviruses may remain infectious in water and sewage for days (Qu et al., 2020) , no accurate data is available for conversion of genomic copy numbers of SARS-CoV-2 to TCID 50 in aquatic environments. Therefore, we used a wide WWTP to the predicted concentration in wastewater. Estimated PC wa was one order of magnitude greater than values recommended for estimating bacterial transfer, in particular legionella, from air-to-water (Bauer et al., 2002; Chattopadhyay et al., 2017) . However, the estimation of bioaerosol concentrations generated in WWTPs exhibits very high uncertainty for microbial pathogens (EPA, 2011). Type and surface properties of pathogenic microorganisms and wastewater quality could affect the water-to-air partitioning coefficient of pathogens (Chattopadhyay et al., 2017) .

On the other hand, bioaerosolized viral particles may considerably dilute by air currents or inactivated by environmental factors. Pyankov et al. (2018) reported that the decay of airborne MERS-CoV virus is much higher for hot and dry air conditions, with only 4.7% survival over 60 min. However, we assumed no dilution or loss for viral particles which may overestimate the infection risk of COVID-19.

Although our findings suggest that wastewater may contribute to COVID-19 transmission, the estimated risk for wastewater workers still presents significant challenges due to the knowledge gaps about this emerging pathogen.

The lack of a dose-response model for SARS-CoV-2 is a critical limitation for conducting QMRA for this pathogen (Kitajima et al., 2020; Corpuz et al., 2020 Although in a recent study it was reported that SARS-CoV-2 aerosols could maintain their infectivity for up to 16 h (Fears et al., 2020) , detection of SARS-CoV-2 RNA in wastewater and wastewater aerosols dose not necessarily indicate viability and infectivity of the viral particles (Foladori et al., 2020) . In other words, the lack of data on the environmental stability of SARS-CoV-2 and its viability and fate in wastewater is a major uncertainty affects the potential risk of exposure to wastewater aerosols. Most researches are based on the molecular detection of SARS-CoV-2 which do not indicate the presence of infectious virus (Corpuz et al., 2020) .

Therefore, more researches are needed to perform molecular detection of SARS-

CoV-2 along with cell culture isolation to validate infectivity of the virus and role of wastewater in transmission of COVID-19.

Furthermore, our bioaerosol information for driving water-to-air partitioning coefficient (PC wa ) of viral particles is very limited. This highlights the importance of further research to develop more accurate data for estimating viral emission from wastewater to air.

Further improvements in information about the fecal shedding rate and duration of SARS-CoV-2 in infected patients are also necessary to reduce uncertainties associated with the risk outcome.

As the COVID-19 pandemic continues, preventive measures must be taken everywhere there is a potential risk for transmission of the viral particles. 

Our outbreak and its potential transmission through environments underscore the need to obtain more reliable information on the survival and fate of SARS-CoV-2 in wastewater. Epidemiological evidence is also required to validate the COVID-19

transmission through wastewater aerosols.

This research was supported by the Vice Chancellery for Research at the Isfahan University of Medical Sciences (Grant No. 198237 f the Spear Spearman's ra man's unded ded by the 95% by the Po r n r r n a l n a r n r n r n J o u r n a l P r e -p r o o f P r P r P r 

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