








key: cord-338243-njkhwkwk
authors: Zhang, Dayi; Ling, Haibo; Huang, Xia; Li, Jing; Li, Weiwei; Yi, Chuan; Zhang, Ting; Jiang, Yongzhong; He, Yuning; Deng, Songqiang; Zhang, Xian; Wang, Xinzi; Liu, Yi; Li, Guanghe; Qu, Jiuhui
title: Potential spreading risks and disinfection challenges of medical wastewater by the presence of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) viral RNA in septic tanks of Fangcang Hospital
date: 2020-06-23
journal: Sci Total Environ
DOI: 10.1016/j.scitotenv.2020.140445
sha: 
doc_id: 338243
cord_uid: njkhwkwk

Abstract The outbreak of coronavirus infectious disease-2019 (COVID-19) pneumonia raises the concerns of effective deactivation of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) in medical wastewater by disinfectants. In this study, we evaluated the presence of SARS-CoV-2 viral RNA in septic tanks of Wuchang Cabin Hospital and found a striking high level of (0.5–18.7) × 103 copies/L after disinfection with sodium hypochlorite. Embedded viruses in stool particles might be released in septic tanks, behaving as a secondary source of SARS-CoV-2 and potentially contributing to its spread through drainage pipelines. Current recommended disinfection strategy (free chlorine ≥0.5 mg/L after at least 30 min suggested by World Health Organization; free chlorine above 6.5 mg/L after 1.5-h contact by China Centers for Disease Control and Prevention) needs to be reevaluated to completely remove SARS-CoV-2 viral RNA in non-centralized disinfection system and effectively deactivate SARS-CoV-2. The effluents showed negative results for SARS-CoV-2 viral RNA when overdosed with sodium hypochlorite but had high a level of disinfection by-product residuals, possessing significant ecological risks.

The outbreak of coronavirus infectious disease-2019 (COVID-19) pneumonia since 2019 is caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) (Lai et al., 2020; Li et al., 2020; Ralph et al., 2020) , and it has rapidly spread throughout 202 countries around the world. Till 20 th June 2020, there are over 8.0 million confirmed cases and around 450,000 deaths globally, and the number is still increasing rapidly (WHO, 2020a) . There is clear evidence of human-to-human transmission of SARS-CoV-2 (Chan et al., 2020; Chang et al., 2020; Li et al., 2020; Poon and Peiris, 2020) . Besides direct contact and respiratory routes (Carlos et al., 2020; Lai et al., 2020; Wu et al., 2020) , stool transmission might be an alternative route owing to the survival of SARS-CoV-2 in patient's stools (Holshue et al., 2020; Ling et al., 2020; Tian et al., 2020; Xiao et al., 2020; Xing et al., 2020; Zhang et al., 2020c) . As municipal wastewater pipe network receives huge amounts of wastewater from asymptomatic patients and treated sewage from hospitals, SARS-CoV-2 from non-or inefficient-disinfected wastewater might persist for a prolonged time in pipe network, becoming a secondary spreading source (Zhang et al., 2020a) . It brings urgent requirement and careful consideration of disinfection strategies to prevent SARS-CoV-2 from entering drainage pipe network.

Disinfection is of great importance to eliminate or deactivate pathogenic microorganisms. Traditional disinfection strategies include ultraviolet germicidal irradiation and biocidal agents, e.g., gaseous ozone, alcohol, formaldehyde, hydrogen peroxide, peroxyacetic acid, povidone iodine and chlorine-based disinfectant (Kampf et al., 2020; Tseng and Li, 2008; Tseng and Li, 2007; Walker and Ko, 2007) . Some disinfectants are intensively used in hospitals for nurse personal care (Dumas et al., 2019; Ioannou et al., 2017) . Among them, chlorine-based disinfectants are widely J o u r n a l P r e -p r o o f used for their broad sterilization spectrum, high inactivation efficiency, low price, and easy decomposition with little residue (How et al., 2017) . Nevertheless, overuse of chlorine-based disinfectants brings concerns of disinfection by-products (DBPs) which are harmful to ecosystems and human health (Bull et al., 2011; Richardson et al., 2007; Wang et al., 2014) . More than 600 kinds of DBPs have been observed (Richardson, 2011) , such as trihalomethanes (THMs), haloacetic acids (HAAs), halogen acetonitriles (HANs), halonitromethanes (HNMs) and haloacetamides (HAcAms) (Ding et al., 2020; Kozari et al., 2020; Luo et al., 2020; Zhai et al., 2014) .

Some of them are reported attributable for bladder cancer (Evlampidou et al., 2020; Li and Mitch, 2018) and adverse reproductive outcomes (Nieuwenhuijsen et al., 2000) .

For effective centralized disinfection, World Health Organization (WHO) has suggested free chlorine ≥ 0.5 mg/L after at least 30 minutes of contact time at pH<8.0 (WHO, 2020b). Additionally, China has launched a guideline for emergency treatment of medical sewage containing SARS-CoV-2 on 1 st February 2020, requiring free chlorine of ≥ 6.5 mg/L and contact time of ≥1.5 hour in disinfection units (China-MEE, 2020) . Unfortunately, the performance of chlorine-based disinfectants on SARS-CoV-2 in real medical wastewater treatment system is not clear yet.

In this work, we studied the presence of SARS-CoV-2 viral RNA in septic tanks of Wuchang Cabin Hospital (Wuhan, China) to evaluate the disinfection performance and optimize disinfection strategies to prevent SARS-CoV-2 from spreading through drainage pipelines. Further analysis of DBPs evaluated the potential ecological risks in the effluents.

J o u r n a l P r e -p r o o f

Wuchang Cabin Hospital was a temporary hospital designated for COVID-19 patients, formerly Wuchang Stadium located in Wuhan (longitude, E114°20'05''; latitude, N30°32'46'') . It was open from 5 th February as the first cabin hospital in Wuhan and closed on 10 th March, in total receiving 1,124 COVID-19 patients ( Figure 1 ). It had eight separate toilets and all sewage from toilets and showers were combined, with daily volume of wastewater ranging from 60 to 200 m 3 . The wastewater was firstly pumped from toilets and showers into the first preliminary disinfection tank, and sodium hypochlorite was unregularly added to a final concentration of 800 g/m 3 when it was 90% full. Afterwards, the first preliminary disinfection tank was temporarily enclosed and the wastewater was then pumped into another disinfection tank and disinfected in the same way. On each day, septic tanks outside the hospital were emptied at 8 am to discharge the effluents into pipe network and wastewater treatment plants and received wastewater from preliminary tanks at 10 am. Sodium hypochlorite was supplemented for 1.5 hr contact and the mixing was conducted by continuous pumping and stirring. Wastewater was then pumped into three septic tanks. Before

March 5 th , the dosage of sodium hypochlorite in septic tanks was 800 g/m 3 and it increased to 6700 g/m 3 after 5 th March to secure complete deactivation of SARS-CoV-2.

Influent and effluent samples were collected from septic tanks of Wuchang Cabin Hospital on 26 th February, 1 st March and 10 th March, 2020 (Table 1) . On these sampling days, the number of COVID-19 patients in Wuchang Cabin Hospital was J o u r n a l P r e -p r o o f stable, ranging from 200 to 400. Around 2.0 L of water was directly collected in a plexiglass sampler and transferred into a sterile plastic bag for biological analysis and a brown glass bottle for DBPs analysis. As we found obvious settlement of stool particles and suspended solids in septic tanks, a stratified plexiglass sampler was used to obtain samples from different layers of septic tanks to assess the levels of viruses and DBPs along depth, designated as top-layer (0-50 cm) and bottom-layer (50-100 cm) water.

Free chlorine was measured on site using PCII58700-00 (HACH, USA). DBPs measurements were carried out on a GCMS-QP2020 (Shimadzu, Japan) equipped with Atomx purge, trap autosampler (Teledyne Tekmar, USA) and an SH-RTX-5MS

[(5%)-phenyl-(95%)-methylpolysiloxane] fused silica capillary column (30 m × 0.25 mm, 0.25 μm film thickness). The autosampler operating conditions were as follows:

purge for 11 min at 30 °C with high-purity nitrogen gas at a flow rate of 40 mL/min, dry purge for 1.0 min at 20 °C with the flow rate of 40 mL/min, pre-desorption at 180 °C and desorption for 2.0 min at 190 °C, and bake for 6.0 min at 200 °C.

Collected water samples were placed in 4 °C ice-boxes and immediately transferred into laboratory for RNA extraction. After centrifugation at 3,000 rpm to remove suspended solids, the supernatant was subsequently supplemented with NaCl (0.3 mol/L) and PEG-6000 (10%), settled overnight at 4 °C, and centrifuged at 10,000 g for 30 minutes. Viral RNA in pellets was extracted using the EZ1 virus Mini kit 

One-way ANOVA was used to compare the difference between samples and p-value less than 0.05 refers to significant difference. All data were presented in mean ± standard deviation (n=3).

J o u r n a l P r e -p r o o f

For all influents of septic tanks received from Wuchang Cabin Hospital, there was no positive result for SARS-CoV-2 viral RNA. On 26 th February and 1 st March, SARS-CoV-2 viral RNA was (14.7±2.2)×10 3 and (7.5±2.8)×10 3 copies/L in the effluents of septic tanks, respectively (Table 1) The absence of SARS-CoV-2 viral RNA in the influents of septic tanks suggested that preliminary disinfection in Wuchang Cabin Hospital was satisfactory to remove SARS-CoV-2 in aqueous phase after 1.5-hour contact. In septic tanks, disinfection achieved free chlorine > 6.5 mg/L for 1.5 hours when the dosage of sodium hypochlorite was 800 g/m 3 , meeting well with the guideline for emergency treatment of medical sewage containing SARS-CoV-2 suggested by China CDC. However, SARS-CoV-2 viral RNA was surprisingly positive in the effluents after 12 hours when free chlorine declined to nondetectable. It might be explained by the release of embedded SARS-CoV-2 viral RNA from stool particles in septic tanks. SARS-CoV-2 has been found in patients' stools in many previous studies (Wu et al., 2020; Xing et al., 2020) , which could escape from disinfection and slowly release into aqueous phase. Suspended particles as small as 7 mm can protect viruses from UV exposure and dwindle their vulnerability to direct sunlight inactivation, and 0.3-mm sized particles can shield viruses from disinfection for their prolonged survival (Templeton et al., 2005) . As stools are rich in organic compounds and form numerous suspended solids containing SARS-CoV-2, they are of high risk as the source releasing viruses in septic tanks. It is also evidenced by more SARS-CoV-2 RNA copy numbers in upper-layer waters, explained by more stool residuals and suspended solids in the bottom of septic tanks absorbing SARS-CoV-2 from aquatic water. Our results suggested that current recommended disinfection dosage could effectively remove aqueous SARS-CoV-2 viruses in wastewater containing limited suspended solids, like wastewater treatment plants (La Rosa et al., 2020; Zhang et al., 2020b) . but it might be not enough for embedded viruses in suspended solids and require higher level of chlorine-based disinfectant supplement. Septic tanks can behave as a long-term source J o u r n a l P r e -p r o o f to release SARS-CoV-2 viral RNA into waters when disinfection is not sufficient and challenges public health via potentially spreading viruses in drainage pipelines.

The surprising presence of SARS-CoV-2 viral RNA after disinfection with sodium hypochlorite suggested that free chlorine > 0.5 mg/L after 1.5-hour contact time cannot completely removing SARS-CoV-2 viral RNA, and 800 g/m 3 dosage of sodium hypochlorite might be not enough to secure a complete disinfection of medical wastewaters, particularly for those from cabin, temporary or non-centralized hospitals containing huge amount of stool debris and suspended solids. From the negative results of SARS-CoV-2 viral RNA (Table 1) , the complete deactivation of SARS-CoV-2 was achieved when the dosage of sodium hypochlorite was 6700 g/m 3 .

As the number of COVID-19 patients were similar on the three sampling days, they produced similar wastewater discharge and viral load. The change of SARS-CoV-2 in effluents was therefore attributing to the increasing sodium hypochlorite supplement for disinfection. Nevertheless, it was an over-dosage and resulted in a significant level of DBPs in the effluents, which was about 15 times higher than other hospital wastewater (Luo et al., 2020) . The four detected chloroform and trihalomethanes accounted to the majority of total DBPs in wastewaters (Furst et al., 2019) and their composition was similar as previous studies (Luo et al., 2020; Zhong et al., 2019) .

They show high ecological risks and challenge the surrounding environment receiving disinfected medical wastewater, possessing threats to ecological system and human health (Ding et al., 2020; Li et al., 2019 give initial information about the potential risks of viral spread and DBPs residues during disinfection processes for medical wastewater containing SARS-CoV-2.

Our study for the first time reported an unexpected presence of SARS-CoV-2 viral RNA in septic tanks after disinfection with 800 g/m 3 of sodium hypochlorite and current disinfection guideline by WHO and China CDC might not secure a complete removal of SARS-CoV-2 in medical wastewater. SARS-CoV-2 might be embedded in patient's stools, protected by organic matters from disinfection, and slowly release when free chlorine declines. Septic tanks in non-centralized disinfection system of Journal Pre-proof J o u r n a l P r e -p r o o f cabin hospitals or isolation points potentially behave as a secondary source spreading SARS-CoV-2 in drainage pipelines for a prolonged time. Disinfection strategy is of great urgency to improve and the ecological risks of DBPs need to be carefully considered.

J o u r n a l P r e -p r o o f 

Potential carcinogenic hazards of non-regulated disinfection by-products: Haloquinones, halo-cyclopentene and cyclohexene derivatives, N-halamines, halonitriles, and heterocyclic amines

Novel Wuhan (2019-nCoV) Coronavirus

A familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster

Epidemiologic and Clinical Characteristics of Novel Coronavirus Infections Involving 13 Patients Outside Wuhan, China

Technical solution for emergency treatment of medical sewage contaminated by novel coronavirus

Developmental toxicity of disinfection by-product monohaloacetamides in embryo-larval stage of zebrafish

Association of Occupational Exposure to Disinfectants With Incidence of Chronic Obstructive Pulmonary Disease Among US Female Nurses

Trihalomethanes in Drinking Water and Bladder Cancer Burden in the European Union

Disinfection Byproducts in Rajasthan, India: Are Trihalomethanes a Sufficient Indicator of Disinfection Byproduct Exposure in Low-Income Countries?

First Case of 2019 Novel Coronavirus in the United States

Organic chloramines in chlorine-based disinfected water systems: A critical review

Biomarkers of end of shift exposure to disinfection byproducts in nurses

Persistence of coronaviruses on inanimate surfaces and their inactivation with biocidal agents

Formation potential of emerging disinfection by-products during ozonation and chlorination of sewage effluents

Coronavirus in water environments: Occurrence, persistence and concentration methods -A scoping review

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and coronavirus disease-2019 (COVID-19): The epidemic and the challenges

Early Transmission Dynamics in Wuhan, China, of Novel Coronavirus-Infected Pneumonia

Drinking Water Disinfection Byproducts (DBPs) and Human Health Effects: Multidisciplinary Challenges and Opportunities

Occurrence and ecological risk assessment of disinfection byproducts from chlorination of wastewater effluents in East China

Persistence and clearance of viral RNA in 2019 novel coronavirus disease rehabilitation patients

Disinfection by-products formation and acute toxicity variation of hospital wastewater under different disinfection processes

Chlorination disinfection byproducts in water and their association with adverse reproductive outcomes: a review

Emergence of a novel human coronavirus threatening human health

2019-nCoV (Wuhan virus), a novel Coronavirus: human-to-human transmission, travel-related cases, and vaccine readiness

Disinfection By-Products: Formation and Occurrence in Drinking Water. Encyclopedia of Environmental Health

Occurrence, genotoxicity, and carcinogenicity of regulated and emerging disinfection by-products in drinking water: A review and roadmap for research

Inactivation of particle-associated viral surrogates by ultraviolet light

Review article: Gastrointestinal features in COVID-19 and the possibility of faecal transmission

Inactivation of surface viruses by gaseous ozone

Inactivation of viruses on surfaces by ultraviolet germicidal irradiation

Effect of ultraviolet germicidal irradiation on viral aerosols

Disinfection technology of hospital wastes and wastewater: Suggestions for disinfection strategy during coronavirus Disease 2019 (COVID-19) pandemic in China

Analytical and Toxicity Characterization of Halo-hydroxyl-benzoquinones as Stable Halobenzoquinone Disinfection Byproducts in Treated Water

COVID-19) Situation Report, 2020a. WHO. Water, sanitation, hygiene and waste management for COVID-19: Technical brief

Clinical Characteristics of Imported Cases of COVID-19 in Jiangsu Province: A Multicenter Descriptive Study

Evidence for gastrointestinal infection of SARS-CoV-2

Prolonged presence of SARS-CoV-2 in feces of pediatric patients during the convalescent phase

Formation of Brominated Disinfection Byproducts during Chloramination of Drinking Water: New Polar Species and Overall Kinetics

SARS-CoV-2 spillover into hospital outdoor environments

Ultra-fast and onsite interrogation of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) in environmental specimens via surface enhanced Raman scattering (SERS)

Fecal specimen diagnosis 2019 Novel Coronavirus-Infected pneumonia

Disinfection byproducts and their toxicity in wastewater effluents treated by the mixing oxidant of ClO2/Cl-2



