Introduction

The wastewater reuse is a key and strategic factor for agricultural activities in countries encountered with water scarcity, limited access to fresh water and rainfall variability1,2. In addition, climate changes and extreme weather events along with exponential population growth has amplified this issue; it makes the authorities to reuse wastewater as rich source of nutrient for plant growth and crops irrigation3. Nevertheless, untreated wastewater or fecal sludge with inadequate and poor management has been extensively used for irrigation purposes, especially, in developing countries as an economic way4. It is estimated that more than 18 million hectares of land all over the world are irrigated with untreated wastewater5. However, the main concern imposed on wastewater irrigation is human health risk6. The crops irrigation with untreated and faecal sludge can spread the pathogens and accordingly pose the threat to public health, specially the sensitive groups of ages7. Exposure to faecal contamination and pathogens in untreated wastewater cause a variety of symptoms and diseases including gastroenteritis, environmental enteric dysfunction, and stunting8. According to World Health Organization (WHO) report (2015), 1.7 billion cases of pediatric diarrhea and 525,000 deaths are attributed to diarrheal disease, especially, among the children9. Furthermore, it is estimated that annually approximately 38.6 million illness in U.S are detected due to exposure to with known pathogens, of which, 30.9 million (80%) of illness cases are attributed to viruses10. Although a desirable percentages of various human pathogens are removed by secondary and tertiary treatment in wastewater treatment plant (WWTPs); viruses commonly resistant to these treatment processes can spread into environment and present a risk for public health1. Furthermore, the efficient reduction of faecal indicators (faecal coliforms (FC) or Escherichia coli (EC)) below the certain standards don’t ensure the safety of effluent discharge from WWTPs4. Enteric viruses with high resistant to conventional wastewater treatment and their long time survival are known as major etiological agents for gastrointestinal illnesses (GI)11. Enteric viruses are observed in the feces of infected person with a concentration of 105−1011viral particles per gram of stool; it has low-infectivity doses and is the leading cause of a spectrum of symptoms from diarrhea to death12. Although the relative importance of different exposure pathway to enteric viruses are site-specific13, the presence of rotavirus (RoV) and Norovirus (NoV) as main viral causes of gastroenteritis in wastewater used for irrigation of agricultural crops such as vegetables can threat the human health2. NoV are known as responsible for gastroenteritis outbreaks associated with consumption of contaminated water3. In addition, due to extensive presence of NoV in wastewater, non-enveloped structure and resistance to environmental degradation, it is selected as reference virus for estimation of quantitative microbial risk assessment (QMRA)14. QMRA is a popular model framework for evaluating and quantifying health risk associated with exposure to photogenes15. Briefly, QMRA translates the pathogen dose that a person is exposed via ingestion or inhalation pathway in a particular scenario into probability of infection (Pinf) and disease burden (DB)16. Generally, QMRA is usually performed in a four step steps: (i) hazard identification, (ii) exposure assessment, (iii) dose-response modeling, and (iv) risk characterization3. Disability Adjusted Life Years (DALYs) is known as recommended metric by world health organization (WHO) for comparison the DB of different diseases and disabilities associated with exposure to a certain pathogen17. The recommended DALY (per person per year) or tolerable DALY for water exposure is 10−6loss per person per year (pppy)2. The Monte Carlo simulation technique and probabilistic-based risk model is commonly used to estimate the uncertainty and variability of different influencing environmental parameters on pathogen exposure18. Such risk assessment can aid the regulatory bodies and authorities to make a decision on the safety of wastewater reuse in agricultural activities and crop irrigation19. Over the recent years, many studies have focused on QMRA and DALY attributed to RoV and NoV emitted from wastewater treatment plant (WWTP)20and wastewater used for irrigation of agricultural crops1,2,6. For instance, our previous study indicated that the medium calculated DALY based on viral load emitted from aeration tank in WWTP for NoV (1.23 × 10 −1) and RoV (5.76 × 10−2) were significantly higher the recommended valued by US.EPA (10 −4 DALY pppy) and WHO (10 −6DALY pppy)20. Eloy Gonzales-Gustavson and et al. (2019) studied the QMRA attributed to Nov and adenovirus in wastewater for irrigation of lettuce in Catalonia. The authors reported that DB for NoV and adenovirus were higher than the WHO recommendation of 10−6DALYs for both viruses2. Therefore, the present study was developed to comprehensively investigate the health risk attributed to two main viral agents of gastroenteritis (RoV and NoV) in high-consumption vegetable irrigated with outlet from different stages of wastewater treatment process in the largest wastewater treatment plant (WWTP) in middle east.

Methods and materials

Study area description and sampling procedure

The present study focused on the QMRA attributed to RoV and NoV in different stages of wastewater treatment train in the largest WWTP in the middle east. The south Tehran wastewater treatment plant (STWWTP) is located at south of Tehran, Iran, covering 2,100,000 inhabitants. STWWTP was established in 2011 with 4 modules, equipped with activated sludge for treatment of 450,000 m3/day wastewater that are collected from different districts of Tehran. The effluents are employed for irrigation of crops and agricultural land uses located at the downstream plains of Tehran. After the biological process, the sewage is passed the chlorination tank in order to inactivate the pathogenic agents before discharge into environments. A schematic diagram of different stages of wastewater treatment train and STWWTP is illustrated in Fig. 1. In order to perform a comprehensive QMRA for different scenarios based on possible failures in wastewater treatment process and direct discharge of sewage for crops irrigation, four sampling points within the wastewater treatment train were selected (See Fig. 1). Raw wastewater, primary sedimentation tank outlet, secondary clarifier outlet and effluent samples were withdrawn monthly from October 2020 to September 2021. All the grab samples were withdrawn on the same day in different months of a year. Sampling was done using a pre-sterilized 50- mL Falcon tube and was immediately transferred at 4 °C to the virology laboratory. The contents of the Falcon tubes was stored at −70 ° C until further diagnostic tests and experiments for quantitative concentration of the viruses.

Fig. 1
figure 1

A Schematic representation of STWWTP and sampling points (1: Screening, 2: Grit Chamber, 3: Primary Settling, 4: Aeration Tanks, 5: Secondary Clarifier, 6: Tricking Filter, 7: Disinfection Process). * sampling Points.

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Detection and quantitative concentration of viral pathogen

Samples were centrifuged at 4000 rpm for 25 min at 4 °C, and the resulting supernatant was then transferred to a new tube Then 0.39 ml Dextran, 0.35 ml NaCl, and 0.28 ml polyethylene glycol 6000 were added to each 50 ml of the supernatant. Then the supernatant was removed, and the pellet was suspended in phosphate buffer saline(PBS), and was used for viral RNA isolation using the synthesis cDNA kit (YTA, Yekta Tajhiz Azma, Iran). The cDNA was kept at −70 °C until further analysis. One-step real-time RT-PCR was performed using an Applied Biosystems 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA) and a commercial TaqMan EZ RT-PCR Core Reagent Kit. The following primers were used: forward primer, RotaNVP3-F (50 - ACCATCTACACATGACCCTC-30); reverse primer, RotaNVP3-R (50 -GGTCACATAACGCCCC-30); and Rota TaqMan probe, FAM-ATGAGCACAATAGTTAAAAGCTAACACTGTCAATAMRA. The mixture was pre-heated for 2 min at 50 C. Reverse transcription was performed for 30 min at 60 C, followed by an enzyme inactivation step for 5 min at 95 C. Subsequently, 45 amplification cycles were performed at 94 C for 5 s and 60 C for 1 min.

For quantification of NoV, we used the primers which target norovirus sequences at the ORF1-ORF2 junction, a highly conserved region of the norovirus genome As previously described by Jathikumar et al. (2005)21. PCR master mixture contained TaqMan one-step 2x master mix(10 µl), forward primer(1 µl), reverse primer(1 µl), probe(0.5 µl), and double-distilled water(2.5 µl). Then a 15 µl master mixture was mixed with a 5 µl sample, in a total volume of 20 µl. The mixture were amplified by 40 cycles at 94 °C and 60 °C for 10 s and 30 s at the steps of denaturation and annealing-extension, respectively.

Statistical analysis

The Minitab software (Version 17.0) and Kruskale-Wallis statistical test was used to determine the difference between concentrations of RoV and NoV in different seasons of the year, as well as to examine differences between the average concentration levels viruses in the four sample sites. Results with P-value < 0.05 were selected as a meaningful level.

Estimating the probabilities of infection and illness attributed to exposure to RoV and NoV due to consumption of high-consumption vegetable (lettuce) irrigated with Raw wastewater, primary sedimentation tank outlet, secondary clarifier outlet and effluent from STWWTP

The microbial risk assessment method developed by Haas22was used to quantitatively estimate the exposure level of Tehran citizens to two viruses of interest (RoV and NoV) due to consumption of high-consumption vegetable (lettuce) or leafy green vegetable irrigated with various types of raw wastewater, primary sedimentation tank outlet, secondary clarifier outlet and effluent. QMRA is known as a general framework to calculate the probabilities of infection (Pinf) and illness (Pill) following exposure to environmental pollutants8. Unlike most studies which mainly focus on the probabilities of infection as the endpoint of exposure to contaminants (it can somehow magnify the exposure risk assessment), an additional factor called as infection-to-illness ratio was used in the present study to translate the probabilities of infection to illness in order to make the data closer to reality. Overall, QMRA translate the concentrations of pathogenic agents to Pinf and Pill via four main steps (Hazard Identification; Exposure Assessment; Dose- Response; Risk Characterization)23,22.

Hazard identification

It is assumed that citizens of Tehran consuming vegetables irrigated with raw wastewater, primary sedimentation tank outlet, secondary clarifier outlet and effluent are at risk of ingestion the RoV and NoV. RoV and NoV are known as main viral causes of gastroenteritis in wastewater used for irrigation of agricultural crops such as vegetables can threat the human health. Therefore, the focus of the study is on these two main pathogenic agents of gastroenteritis in Iran.

Exposure assessment

Exposure assessment was performed considering the number of genomes of RoV and NoV each person received due to consumption of lettuce vegetables irrigated with raw wastewater, primary sewage tank outlet, secondary clarifier outlet and effluent of STWWPT in one day and one year. In addition, based on the time and frequency of exposure a person consuming the high-consumption vegetable infected with RoV and NoV, daily and annual risk assessment of infection and illness was calculated.

In this study, a simplified approach without consideration the age of exposed people was utilized to estimate the Pinf and Pill; all people were assumed to be susceptible to infection and illness (Susceptible fraction of the population = 1). However, some factors, including the volume of irrigation water captured by lettuce, the withholding time between the last wastewater irrigation event and harvest, lettuce daily consumption by the individual, and the rate of virus reduction due to washing with water can affect the level of exposure of people, so equations for the rate of virus loss should also be considered.

Exposure calculation

The ingested daily dose of RoV and NoV on lettuce surface (dls) (Virus/person) by a person was calculated using the following formula:

$$\:{\text{d}}_{ls}={C}_{eff}\times\:{V}_{surf}\times\:\:{e}^{-kt}\times\:M\times\:{10}^{-Rwash}$$
(1)

Ceff: concentration of RoV and NoV in samples withdrawn from different stages of wastewater treatment train in STWWTP (Virus/mL).

Vsurf: the volume of irrigation water captured by lettuce (mL/g).

K: Virus kinetic decay constant (day−1).

t: the withholding time between the last wastewater irrigation event and harvest (day).

M: lettuce daily consumption (g/person.day).

Rwash: the rate of virus reduction due to washing with water (Log unit)6.

Dose-response assessment

The dose-response model describes the quantitative correlation between the probability of illness and a certain level of microorganism during the exposure process. Dose- response models depending on specific microbial species has been suggested in many studies24. In this study, the exponential model was used to describe the dose-response model of RoV and NoV, which is mentioned in the following equation:

$$\:{\text{P}}_{\text{inf}}\left(\text{d}\right)\text{=1-}\text{exp}\left(\text{-}\text{rd}\right)$$
(2)

where Pinf denoted the probability of infection for an individual when exposed.

to a single dose of pathogen “d”; r is infectivity constants of viruses in exponential models. The specific parameters for two pathogenic viruses studied in the present study are given in Table 1. As RT-PCR quantification of viruses does not give accurate information about the effectiveness of viruses, we used the dose harmonization coefficient in this study25 (See Table 1).

Table 1 species-specific parameter values and dose harmonization for RoV and NoV.
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The daily and annual risk of infection due to exposure to RoV and NoV was calculated as per Eq. 320:

$$\:{\text{P}}_{\text{inf}\left(\text{D}:\text{A}\right)}\left(\text{d}\right)=1-[1-{\text{P}}_{\text{i}\text{n}\text{f}}\left(\text{d}\right){]}^{\text{n}}$$
(3)

Where Pinf (A, D) is the probability daily and annual risk of infection arising from daily exposure n = 1 and annual exposure: n = 130.

Risk characterization

The risk characterization was performed according to viral concentration of two pathogens to which individual are exposed. Pill, as conditional of exposure and infection was calculated using following Eq. 

$$\:{\text{P}}_{\text{ill}}\text{=}{\text{P}}_{\text{inf}\left(\text{A:D}\right)}\left(\text{d}\right)*{\text{P}}_{\raisebox{1ex}{\text{ill}}\!\left/\:\!\raisebox{-1ex}{\text{inf}}\right.}$$
(4)

Where, Pill/inf, or the ration of illness to infection for RoV and NoV were 0.576927and 0.528, respectively.

The specific potential of disease burden attributed to gastrointestinal illness due to exposure to RoV and NoV was estimated based on Eq. 5:

$$DB\hspace{0.17em}=\hspace{0.17em}Pill\:\left(A\right)*\:S\:*\:DBPC$$
(5)

where DB is burden of disease, expressed in per person per year (pppy), S denoted the susceptible fraction of population (as earlier mentioned in the present study, all individuals are assumed to be susceptible for gastrointestinal illness and DBPC is disease burden per case (DALY/year), depending on both premature mortality and loss of healthy years due to morbidity. TableS1summarizes the values for illness outcomes, duration, severity and DBPC for both RoV and NoV, obtained from different studies29:

Model implementation

Generally, four categories of parameters are commonly employed in a model: constant (C), variable (V), uncertainty (U) and variable and uncertainty (VU). In this study, eight influencing parameters were initially identified and categorized. The detailed information on different categories of parameters used in the present model are described in Table 2. The best and most appropriate mathematical distribution for the concentration of RoV and NoV was selected based on Goodness-of-fit tool in the Crystallball software. The evaluation model was simulated using the two-dimensional Monte Carlo (MC2D) technique simulation Package and run for 10,000 iterations for each distribution input parameter. A 95% confidence interval was considered to illustrate the effects of U and VU parameters and their impact on disease burden (DB). All analysis of the model was done in R software. Sensitivity analysis based on tornado plot was performed to understand the contribution of each parameter on variation of DB associated with exposure to RoV and NoV present in high-consumption vegetables (lettuce) irrigated with raw wastewater, primary sewage tank outlet, secondary clarifier outlet and effluent from STWWTP. The Input parameters and their corresponding appropriate distribution are summarized in Table 3.

Table 2 Viral concentration in the different sampling dates.
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Table 3 QMRA model input parameters and their distributions.
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Results and discussion

Quantification of viruses

The viral load of two viruses of interest (RoV and NoV) in different stages of wastewater treatment in STWWTP were monthly withdrawn and measured using the RT-PCR. Table 2 summarizes the concentration levels of RoV and NoV in different samples taken from raw wastewater, primary sedimentation tank outlet, secondary clarifier outlet and effluent. It is important to note that no NoV were identified in raw wastewater, primary sedimentation tank outlet, secondary clarifier outlet and effluent.

As summarized in Table 2, the activated sludge process leads to a significant decrease in the concentration of RoV during the treatment process; the average concentration of RoV in raw wastewater (234 Virus.mL−1) decreased after the primary sedimentation processes (136 Virus.mL−1) and secondary clarifier (53 Virus.mL−1), and the minimum concentration of viruses (12 Virus.mL−1) was observed in the effluent. In addition, statistical analysis showed that there is a significant difference between the concentrations of RoV in different seasons of a year (P-value < 0.005). The highest concentration of RoV in raw wastewater (299 Virus.mL−1) and the lowest concentration of RoV (187 Virus.mL−1) were observed in winter and autumn, respectively. Lower temperatures provide better conditions for the growth and survival of viruses in various wastewater treatment plant processes20; it can increase the risk of infection and illness in people consuming high-consumption vegetables (lettuce) irrigated with the effluent from different stages of wastewater treatment in STWWTP31investigated the viral profile (RoV, astroviruses and NoV) in three WWTP in Beijing, China. The authors reported that RoV are the most dominant type of virus (32.3%, 31/96) at different stages of the process train, and human NoV were observed in only 3% of samples (3/96). In addition32, surveyed the presence of RoV in different stages of wastewater treatment in one of WWTP in China. The authors concluded that, RoV were observed in 67%, 47% and 14% of the output samples from the primary sediment tank, secondary clarifier tank and effluent, respectively.

RoV reduction in different stages of wastewater treatment in STWWTP

The RoV reduction in primary sedimentation tank outlet, secondary clarifier outlet and effluent in STWWTP was calculated using Eq. 6:

$$\:\text{Log Reduction}={\text{Log}}_{\text{10}}\text{}\frac{{\text{N}}_{\text{0}}}{{\text{N}}_{\text{t}}}$$
(6)

Where, N0 is RoV measured in raw sewage and Nt denotes the concentration of RoV in different stages of the treatment train. The logarithmic decrease in the concentration of RoV at different stages of wastewater treatment process in STWWTP is summarized in Table 4.

Table 4 Logarithmic decrease in the concentration of NoV at different stages of wastewater treatment.
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As shown in Table 4, the average logarithmic decrease in different stages of wastewater treatment in STWWTP were as follows: effluent (chlorination Tank) > Secondary sedimentation Tank > Primary sedimentation Tank. Although virus removal is not considered a design parameter for municipal WWTP due to the lack of regulations and legislation related to viruses, many studies have focused on virus removal in primary and secondary treatment stages of WWTP as well as membrane bioreactors (MBR)33. Viruses are commonly inactivated using disinfectants in tertiary treatment before discharged into the environment or reuse34. The highest presence of the virus in the wastewater treatment plant is expected to be in biosolids or sludge35. The formation of flakes in the sludge and the subsequent settlement are the major mechanism for viruses removal in WWTP. In addition, Eloy Gonzales - Gustavson and et al. (2019) showed that the natural wetland led to a 3.9 and 2.8 logarithmic decrease of NoV GII and HAdV, respectively. While, the logarithmic decrease of NoV GII and HAdV in activated sludge process were estimated to be 1.9 and 2.5, respectively2.

The probability of infection and illness by QMRA

The Gamma distribution with the highest fitness to RoV concentration was used to estimate the pertinent parameters to QMRA and DALY index. Given the average concentration of RoV measured in raw wastewater, primary sedimentation tank outlet, secondary clarifier outlet and effluent in STWWTP, the probability of infection (Pinf), probability of annual infection (Pinf.A), the probability of disease (Pill) and burden of disease (DB) were estimated for Tehran citizens consuming high-consumption vegetables (lettuce).

Pinf, Pinf.A, and pill due to consumption of high-consumption vegetables infected with RoV

In this study, taking into account the different conditions and scenarios of irrigation of agricultural products, the Pinf and Pinf.A were estimated following consumption of high-consumption vegetables (lettuce). Table S2 summarizes the Pinf and Pinf.A of RoV following the consumption of high-consumption vegetables irrigated with raw wastewater, primary sedimentation tank outlet, secondary clarifier outlet and effluent from STWWTP.

As shown in Table S2, the 95% confidence interval of Pinf attributed to RoV for citizens consuming high-consumption vegetable irrigated with the raw wastewater, primary sedimentation tank outlet, secondary clarifier outlet and effluent from STWWTP were 3.06 × 10 −6 − 2.20 × 10 −4, 8.47 × 10 −7 − 2.91 × 10 −4, 1.64 × 10 −9 − 3.59 × 10 −4, 8.15 × 10 −13 − 4.23 × 10 −4, respectively. As summarized in Table S2, the median Pinf experienced a decreasing trend when irrigated with raw wastewater to effluent disinfected with chlorination in STWWTP; the median Pinf attributed to consumption of high-consumption vegetable irrigated with raw wastewater decreased from 3.18 × 10 −5 to 5.88 × 10 −6 when irrigated with effluent disinfected with chlorination process. In addition, the 95% confidence interval of Pinf.A attributed to RoV for citizens consuming high-consumption vegetable irrigated with the raw wastewater, primary sedimentation tank outlet, secondary clarifier outlet and effluent from STWWTP were 0.000397–0.028214, 0.00011–0.03714, 2.14 × 10 −7 − 4.56 × 10 −2, and 1.06 × 10 −10 − 5.35 × 10 −2, respectively. As summarized in Table S2, the median Pinf.A experienced a decreasing trend when irrigated with raw wastewater to effluent disinfected with chlorination in STWWTP; the median Pinf.A attributed to consumption of high-consumption vegetable irrigated with raw wastewater decreased from 0.004126 to 7.35 × 10 −4 when irrigated with effluent disinfected with chlorination process.

Fig. 2
figure 2

illustrates the comparison between the Pinf.A attributed to RoV present in consumed vegetables irrigated with the raw wastewater, primary sedimentation tank outlet, secondary clarifier outlet and effluent from STWWTP and reference levels recommended by US EPA (10−4 pppy) and WHO (10−6 pppy).

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Figure 2. The boxplot of estimated Pinf.A (pppy) of RoV for Tehran citizens consuming high-consumption vegetable irrigated with the raw wastewater, primary sedimentation tank outlet, secondary clarifier outlet and effluent from STWWTP (10−4 and 10−6 are the recommended values by EPA and WHO, respectively).

In many studies, the Pinf is considered as the endpoint of QMRA, which magnifies the probability of risk. In the present study, in order to complete the process of QMRA, the probability of illness (Pill) due to exposure to RoV following ingestion high-consumption vegetables with the raw wastewater, primary sedimentation tank outlet, secondary clarifier outlet and effluent from STWWTP were estimated. The Pill was estimated by taking into account the ratio of infection to disease (ill/inf: 0.5769) due to exposure to rotavirus and simulated using the Monto Carlo method in R software. The annual Pill for people consuming high-consumption RoV-infected lettuce following irrigation with the raw wastewater, primary sedimentation tank outlet, secondary clarifier outlet and effluent from STWWTP are summarized in Table S3.

As shown in Table S3, the 95% confidence interval of Pill attributed to RoV for citizens consuming high-consumption vegetable irrigated with the raw wastewater, primary sedimentation tank outlet, secondary clarifier outlet and effluent from STWWTP were 0.000161–0.011427, 4.46 × 10 −5 − 1.50 × 10 −2, 8.66 × 10 −8 − 1.85 × 10 −2, and 3.92 × 10 −11 − 1.99 × 10 −2. As summarized in Table S3, the median Pill experienced a decreasing trend when irrigated with raw wastewater to effluent disinfected with chlorination in STWWTP; the median Pill attributed to consumption of high-consumption vegetable irrigated with raw wastewater decreased from 0.001671 to 2.83 × 10 −6 when irrigated with effluent disinfected with chlorination process. Figure 3 shows the comparison between the Pill attributed to RoV present in consumed vegetables irrigated with the raw wastewater, primary sedimentation tank outlet, secondary clarifier outlet and effluent from STWWTP and reference levels recommended by US EPA (10−4 pppy) and WHO (10−6 pppy).

Fig. 3
figure 3

The boxplot of estimated Pill (pppy) of RoV for Tehran citizens consuming high-consumption vegetable irrigated with the raw wastewater, primary sedimentation tank outlet, secondary clarifier outlet and effluent from STWWTP (10−4 and 10−6 are the recommended values by EPA and WHO, respectively).

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In the present study, QMRA was used to estimate the Pinf and Pill following exposure to RoV in vegetable with outlet from different stages of STWWTP. Generally, as shown in Figs. 2 and 3, the median Pinf.A and Pill caused by exposure to RoV following ingestion the high-consumption vegetables (lettuce) irrigated with raw wastewater, primary sedimentation tank outlet, secondary clarifier outlet and effluent from STWWTP exceed the recommended values by WHO (10−6 pppy) and USEPA (10−4pppy)36. D. D. Mara et al. (2007) estimated the risk assessment of infection caused by RoV following consumption of wastewater-irrigated vegetables and concluded that Pinf for consumption of agricultural products irrigated with effluent from sewage treatment plants in Mexico was 1.14 × 10 −2pppy37. In addition, Duncan Mara and Andrew Sleigh (2010) investigated the QMRA by the Monte Carlo method to estimate the risks of NoV infection for wastewater - irrigated lettuce. The results indicated that Pinf due to exposure to NoV (10−3 pppy) exceeded the standard limit (10−6pppy); it requires a reduction of 5 log to meet the standard17.

Estimation of disease burden (DB) due to consumption of high-consumption vegetables infected with RoV

To calculate and simulate the DB for RoV-related disease, the DBPC parameter, which includes a range of outcomes (See Table S1) was included in the corresponding Eq. 38. The 95% confidence interval for the DB of rotavirus-related illness due to ingestion of high-consumption vegetables (lettuce) irrigated with the raw wastewater, primary sedimentation tank outlet, secondary clarifier outlet and effluent from STWWTP are summarized in Table 5.

Table 5 DB for Tehran citizens consuming high-consumption vegetable infected with RoV.
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DALY is a global indicator for measuring the burden of disease following a global and regional outbreak10. The median DALY index calculated in the present study for citizens in case of consumption of high-consumption vegetable irrigated vegetables with the raw wastewater, primary sedimentation tank outlet, secondary clarifier outlet and effluent were 3.68 × 10 −4, 2.98 × 10 −4, 1.22 × 10 −4, and 5.14 × 10 −5, respectively. The median DALY for all scenarios were higher than the values recommended by WHO (10−6) and USEPA (10−4)36. The DB calculated in present study are comparable with other studies focused on exposure to RoV in aquatic environments 27402941. Moazeni and et al. (2017) estimated the health effects due to exposure to enteroviruses following the use of sewage from two sewage treatment plants in Isfahan, Iran for irrigation of agricultural land. The results indicated that the estimated DALY were approximately 10−3pppy, exceeding the recommended value by WHO and EPA38. In addition, Helena Sales-Ortells and et al. (2015) surveyed the health risk due to consumption of lettuce irrigated with secondary and tertiary sewage. The authors reported that DALY attributed to NoV in lettuce irrigated with secondary and tertiary sewage were 7.8 × 10 −4 and 3.9 × 10 −4pppy, respectively4. This is the first study to examine the presence of RoV in various stages of the sewage treatment plant process train in Iran, introducing an estimated QMRA and DALY index for citizens consuming high-consumption vegetables (lettuce). This comprehensive and transparent results derived from DALY and QMRA can help decision makers and governing bodies to justify priorities for dealing with wastewater compared to other hazards in the environment40.

Sensitivity analysis and limitations

Sensitivity analysis and the role of influencing parameters on DB related to the RoV in consumed vegetables (lettuce) irrigated with the raw wastewater, primary sedimentation tank outlet, secondary clarifier outlet and effluent from STWWTP are shown in Fig. S1 (A –D). As shown in Fig. S1 (A –D), variation in RoV concentration and the time between last irrigation and consumption as a UV parameter relative to other parameters have the greatest impact on the DB attributed to exposure to RoV in vegetables.

Moazeni and et al. (2017) reported that amount of lettuce consumption and concentration of enteroviruses had the greatest impact on variation of DB in lettuce irrigated with sewage, which are in line with our results38. In addition, Helena Sales-Orells et al. (2015) estimated the risk of NoV in wastewater-irrigated lettuce in Catalonia, Spain and the results indicated that NoV concentrations in wastewater and amount of lettuce consumption played the greatest role in variation of Pinf and DB4. Risk estimation using QMRA tools are influenced by some limitations; these can affect the accuracy of risk assessment in some way. In the present study, due to the lack of sufficient data to model the probability of infection and illness and in addition, disease burden in the form of a log-normal distributive function, Gamma distributive functions were employed. Another hypothesis that could not indicate the actual expression of the status quo is the lack of data classification based on age and gender in the study area. Children under 5 years old and the elderly are epidemiologically more susceptible to gastrointestinal disease compared to other groups of ages in a society.

Conclusion

In The present study, the concentration of RoV as the main cause of gastrointestinal disease in various stages of the wastewater treatment train in STWWTP were measured using the RT-PCR method for one year. The results obtained from the present study indicated that although activated sludge process leads to the reduction and elimination of RoV, the presence of viruses in the effluent can still be considered as a risk factor for the consumption of vegetables irrigated with this type of sewage. QMRA confirm the high Pinf and Pill for citizens consuming high-consumption vegetables (lettuce) irrigated with raw wastewater, primary sedimentation tank outlet, secondary clarifier outlet and effluent. The Pinf and DB attributed to RoV-related disease for citizens consuming high-consumption vegetables (lettuce) irrigated with raw wastewater, primary sedimentation tank outlet, secondary clarifier outlet and effluent from STWWTP exceeded the values recommended by WHO and USEPA; it indicates a serious risk to various members of society, especially children and the elderly. The sensitivity analysis test showed that the RoV concentration, the time between last irrigation and consumption, and the amount of high-consumption vegetables (lettuce) as UV parameters had the greatest impact on variation of DB compared to other influencing parameters. Overall, QMRA analysis indicated that STWWTP with activated sludge process is not efficient to meet the standard recommended WHO (10−6 pppy). This indicates additional treatment methods should be considered before effluent discharge to environment, especially, crops irrigation. The preventive measurement that can be considered to reduce the viral load and consequently Pinf and Pill attributed to exposure to RoV in recycled water include: improving the decontamination and UV system, application another water source instead of sewage in the last irrigation phase, and extending the time interval between the last irrigation and vegetable consumption. The results of this study indicated that Pinf and Pill due to lack of appropriate demographic information including gender and age classification appear to be overestimated. However, this is the first study in the field of QMRA in wastewater treatment train (different stages) and help decision makers and authorities to find new approaches to reduce the health risk attributed to infected sewage-irrigated vegetables.