Virucidal efficacy of hypochlorous acid water for aqueous phase and atomization against SARS-CoV-2

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Abstract

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Coronavirus disease 2019 (COVID-19) is an infectious viral disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that emerged at the end of 2019. SARS-CoV-2 can be transmitted through droplets, aerosols, and fomites. Disinfectants such as alcohol, quaternary ammonium salts, and chlorine-releasing agents, including hypochlorous acid, are used to prevent the spread of SARS-CoV-2 infection. In the present study, we investigated the efficacy of ionless hypochlorous acid water (HOCl) in suspension and by spraying to inactivate SARS-CoV-2. The virucidal efficacy of HOCl solution in tests against SARS-CoV-2 was evaluated as 50% tissue culture infectious dose. Although the presence of organic compounds influenced the virucidal efficacy, HOCl treatment for 20 s was significantly effective to inactivate Wuhan and Delta strains in the suspension test. HOCl atomization for several hours significantly reduced the SARS-CoV-2 attached to plastic plates. These results indicate that HOCl solution with elimination containing NaCl and other ions may have high virucidal efficacy against SARS-CoV-2. This study provides important information about the virucidal efficacy and use of HOCl solution.

HIGHLIGHTS

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• The virucidal effect of ionless hypochlorous acid water (HOCl) was unaffected by SARS-CoV-2 variants.
• The anti-SARS-CoV-2 effect of HOCl was attenuated by organic compounds in the virus solution.
• The anti-SARS-CoV-2 effect of HOCl was confirmed by HOCl atomization.
• Use of HOCl atomization against anti-SARS-CoV-2 effect can be expected to reduce the burden of cleaning in hospital rooms, daycare centers, schools, etc.

atomization, coronavirus disease 2019 (COVID-19), hypochlorous acid water (HOCl), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), suspension test

ABBREVIATIONS

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ACCavailable chlorine concentrationCOVID-19coronavirus disease 2019HOClhypochlorous acid waterMPOmyeloperoxidaseROSreactive oxygen speciesSARS-CoV-2severe acute respiratory syndrome coronavirus 2TCID5050% tissue culture infectious dose

INTRODUCTION

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The new pathogen severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative virus of coronavirus disease 2019 (COVID-19), which emerged at the end of 2019 and has since spread rapidly worldwide (Zhu et al. 2020). The clinical symptoms of COVID-19 vary widely from asymptomatic to severe, and it can be fatal (Chen et al. 2020; Guan et al. 2020). As of 28 August 2023, the World Health Organization (WHO) confirmed 770 million cases and >6.9 million deaths (WHO 2023).

SARS-CoV-2 is an enveloped positive-sense single-stranded RNA virus, which can spread through asymptomatic and symptomatic individuals through ejected respiratory droplets and aerosols when breathing, coughing, sneezing, and speaking under insufficient ventilation (Hu et al. 2021; Leung 2021). The virus can survive from a few hours to several days on various material surfaces, such as glass, plastic, natural rubber, and metal (Riddell et al. 2020; van Doremalen et al. 2020; Hirose et al. 2021; Wiktorczyk-Kapischke et al. 2021). Therefore, the droplet infection and fomite transmission are feasible routes of infection. Consequently, to prevent human-to-human viral transmission, it is important to inactivate SARS-CoV-2 in the form of released droplets or on the surface of fomites. In addition, handwashing and hand disinfection are also important to prevent SARS-CoV-2 infection.

For this purpose, the use of chemical disinfectants and sanitizers can reduce the high risk of pathogen transmission. We have explored or developed various disinfectants and sanitizers to prevent infectious diseases. The main components used in disinfectants and sanitizers contain alcohol, quaternary ammonium salts, and amphoteric surfactants (McDonnell & Russell 1999; Tischer et al. 2012; Gerba 2015; Tsujimura et al. 2015; Falk 2019; Xiao et al. 2022). At the beginning of the COVID-19 pandemic, the demand for alcohol disinfectants increased. Therefore, alternative disinfectants were evaluated in Japan. Ionless hypochlorous acid water (HOCl) is one of the several reactive oxygen species produced by phagocytes, such as macrophages and neutrophils, playing a key role in disinfection of bacteria, fungi, protozoa, and viruses (Aratani 2018). HOCl is a strong oxidizing agent with antipathogen efficacy. HOCl has rapid and broad-spectrum antimicrobial efficacy against clinically relevant microorganisms in vitro and in vivo (Wang et al. 2007). HOCl was reportedly superior to hypochlorite or hydrogen peroxide in terms of therapeutic index, which is an indicator of the safety margin of a disinfectant (Wang et al. 2007). HOCl can inactivate Gram-negative and positive bacteria, fungi, and viruses (WHO 2021). HOCl exhibits high virucidal efficacy by interacting with viral structural proteins (capsid or surface compound), lipid envelope, and DNA/RNA (Block & Rowan 2020; Hawkins & Davies 2021; Qiao et al. 2022). HOCl can react better with thiol-containing compounds, such as cysteine, methionine, and glutathione, than other cellular components and therefore induces protein unfolding and aggregation (Gray et al. 2013; da Cruz Nizer et al. 2020). Hawkins & Davies (2002) reported that HOCl-induced DNA damage. HOCl reacts with unsaturated fatty acids and cholesterol and induces a change in membrane properties (Spickett et al. 2000; da Cruz Nizer et al. 2020). Previous studies have reported the viricidal efficacy of HOCl solution against influenza virus, herpes simplex virus, human immunodeficiency virus 1, and coronavirus in the aqueous phase (Tachikawa et al. 1999; Miyaoka et al. 2021). According to Hatanaka et al. (2022) and Takeda et al. (2021), HOCl solution containing 28 and 51–56 ppm of chlorine concentration is efficient against SARS-CoV-2. A study reported that >35 ppm of HOCl is efficient against SARS-CoV-2 infection (NITE: National Institute of Technology and Evaluation 2022). In the study, each experimental condition by five facilities was different, for example, the producing method of HOCl solution (electrolyzed or not electrolyzed), the concentration of fetal bovine serum (FBS; 1–5%) in the virus solution, and reaction rates (1:9 or 1:19) with HOCl and virus solution.

Atomization with HOCl reduces bacteria on surfaces in vitro and in the hospital room (Hakim et al. 2016; Miyazaki et al. 2022). The virucidal efficacy of HOCl atomization was reported against norovirus, avian influenza virus, and SARS-CoV-2 on surfaces (Park et al. 2007; Hakim et al. 2015; Urushidani et al. 2022). Details of the appropriate use of HOCl against SARS-CoV-2 in the aqueous phase and under atomization are unclear.

In the present study, we investigated whether HOCl treatment would influence the infectivity of SARS-CoV-2 to susceptible cells in the presence of the different concentrations of organic compounds and variants. We also explored whether HOCl atomization would provide effective disinfection to prevent SARS-CoV-2 infection.

MATERIALS AND METHODS

Cell culture

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VeroE6 cells expressing the transmembrane serine protease TMPRSS2 (VeroE6/TMPRSS2: JCRB1819) were purchased from the National Institutes of Biomedical Innovation, Health and Nutrition (JCRB Cell Bank, Osaka, Japan). VeroE6/TMPRSS2 cells were cultured in Dulbecco's modified Eagle's medium (D-MEM) GlutaMAX (Thermo Fisher Scientific, Tokyo, Japan) containing 5% heat-inactivated FBS (Vitromex, Vilshofen, Germany), 100 units/mL penicillin, and 100 mg/mL streptomycin (Nacalai Tesque, Inc., Kyoto, Japan), and 1 mg/mL G-418 solution (Roche Diagnostics GmbH, Mannheim, Germany) under biosafety level 2.

Virus

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The Pango lineage A virus strain that was prevalent in the early stages of the COVID-19 pandemic (JPN/TY/WK-521) was provided by the National Institute of Infectious Diseases. To compare more pathogenic and transmissible variants of concern with Pango lineage A virus, the Pango lineage B.1.617.2 virus strain (KUH355) was isolated clinically and provided by the Kitasato University Hospital. Both viruses were used to infect VeroE6/TMPRSS2 cells for virus expansion, and virus-infected cells were cultured in D-MEM containing 2% FBS. After 48 h of incubation, each virus-infected cell culture supernatant was collected and centrifuged at 3,000 rpm for 10 min at room temperature (23 °C) to eliminate cells and debris. Culture supernatants were divided and stocked at −80 °C until use. All viruses were treated in the biological safety cabinet class II type A2 (LAL-1500XPA2 + : Oriental Giken Inc., Tokyo, Japan) of biosafety level 3 (BSL3).

Reagents

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HOCl was provided by NIPRO Corporation (Osaka, Japan). HOCl was produced by the electrolysis of the saturated sodium chloride (NaCl) solution. The products were purified by reverse osmosis filtration to remove ions, such as Na+. The conductivity and pH of 500 ppm HOCl used in this study were 280.1 mS/cm and 6.24, respectively.

Suspension test

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The JPN/TY/WK-521 (2 × 107 pfu/mL) and KUH355 (1.5 × 107 pfu/mL) strains were mixed with 1–200 ppm of HOCl or water for injection (Otuska Pharmaceutical Co., Ltd, Tokyo, Japan) at a ratio of 1:9 and 1:99 for 20 s and 5 min, respectively. Three viral solutions were treated with the same HOCl concentration or water for injection. After treatment, the reaction was stopped by adding 10% sodium thiosulfate (FUJIFILM Wako Pure Chemical Corporation, Tokyo, Japan) to the virus solution and HOCl mixture. Ten-fold serial dilutions of these solutions were then prepared using D-MEM. Each diluted mixture was then added to VeroE6/TMPRSS2 cells. After 3 days of incubation, culture supernatants were eliminated. Then, VeroE6/TMPRSS2 cells were fixed with the cold methanol (Nacalai Tesque) and stained with the 0.5% methylene blue (FUJIFILM Wako Pure Chemical Corporation) solution for calculation of the tissue culture infectious dose 50 (TCID50) using the Behrens–Karber method (Behrens & Karber 1953). Live cells were stained blue and macroscopically observed, whereas dead cells from SARS-CoV-2 infection were not stained:

The average value of TCID50 was used as the viral infectivity. All experiments were performed in the biological safety cabinet of BSL3.

Spray test

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The test chamber used for the spray tests was placed into the biological safety cabinet in BSL3. KUH355 (1.5 × 107 pfu/mL) virus solutions diluted 10- and 100-fold with double distilled water (ddw) were placed in plastic plates and placed on a wet towel in the test chamber (Figure 3). 30–500 ppm and 10–300 ppm of HOCl or distilled water were sprayed for 5 h using two ultrasonic atomizers. Each of the three plastic plates was treated with HOCl or distilled water at the same time. Plastic plates were collected at 1, 3, and 5 h, and 0.95 mL of recovery liquid (D-MEM) was added to the plastic plates, and the virus was suspended. Ten-fold serial dilutions of the virus solutions were prepared using D-MEM. Each diluted mixture was added into VeroE6/TMPRSS2 cells. After 3 days of incubation, the culture supernatants were eliminated. Then, VeroE6/TMPRSS2 cells were fixed with cold methanol and stained with 0.5% of methylene blue solution for the calculation of TCID50 by the Behrens–Karber method. All experiments were performed in the biological safety cabinet of BSL3.

Statistical analysis

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Statistical analyses were performed with GraphPad Prism 8.2 (GraphPad Software, La Jolla, CA). Statistical significance was analyzed using one-way analysis of variance (ANOVA) with the Bonferroni post hoc test. Differences were considered statistically significant at P < 0.05.

Ethical approval statement

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All infectious experiments were reviewed and approved by the Biosafety Committee of Kitasato University (approval numbers: A06-116 and 199) and the Kitasato University Medical Ethics Organization (approval number: COVID-19-CT001).

RESULTS

Evaluation of HOCl virucidal efficacy against SARS-CoV-2 by suspension test

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Virucidal efficacies of HOCl against SARS-CoV-2 are shown in Figure 1. Virus stocks containing 2% FBS were mixed with 10, 30, 50, 100, and 200 ppm HOCl solution for 20 s and 5 min at room temperature at a ratio of 1: 9. In the presence of 0.2% FBS, the HOCl solution with the concentration not less than 30 ppm was significantly effective against the Pango lineage A virus strain (JPN/TY/WK-521), resulting in a 5.2–5.4 log10 decrease. In contrast, the 10 ppm HOCl solution had no anti-SARS-CoV-2 effect (Figure 1(a)). Using the Pango lineage B.1.617.2 virus strain (KUH355), the HOCl solution with the concentration not less than 30 ppm was significantly effective against SARS-CoV-2, resulting in a 1.3–5.8 log10 decrease. In contrast, the HOCl solution at 10 ppm did not exert any SARS-CoV-2 virucidal efficacy (Figure 1(b)). The cytopathic effects of 200 ppm HOCl and Na2S2O3 stop solution on VeroE6/TMPRSS2 cells were not observed (Supplementary Figure 1).

Figure 1

Evaluation of HOCl virucidal efficacy against SARS-CoV-2. SARS-CoV-2 virus solution was treated with various concentrations of ionless hypochlorous acid water, as described in Materials and Methods. (a) Pango lineage A virus (JPN/TY/WK-521) strain treated with 10 (white bar), 30 (light gray bar), 50 (gray bar), 100 (dark gray bar), and 200 (black dot bar) ppm of HOCl solution. (b) Pango lineage B.1.617.2 virus (KUH355) strain treated with 10 (white bar), 30 (light gray bar), 50 (gray bar), 100 (dark gray bar), and 200 (black dot bar) ppm of HOCl solution. The ddw (black bar) indicates that the virus solution was treated with water for injection. Na2S2O3 (gray dot bar) indicates that 200 ppm HOCl solution pretreated with 10% Na2S2O3 solution was mixed with the SARS-CoV-2 virus solution. Data were analyzed using one-way ANOVA with the Bonferroni post hoc test. **** p < 0.0001.

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Evaluation of HOCl virucidal efficacy against SARS-CoV-2. SARS-CoV-2 virus solution was treated with various concentrations of ionless hypochlorous acid water, as described in Materials and Methods. (a) Pango lineage A virus (JPN/TY/WK-521) strain treated with 10 (white bar), 30 (light gray bar), 50 (gray bar), 100 (dark gray bar), and 200 (black dot bar) ppm of HOCl solution. (b) Pango lineage B.1.617.2 virus (KUH355) strain treated with 10 (white bar), 30 (light gray bar), 50 (gray bar), 100 (dark gray bar), and 200 (black dot bar) ppm of HOCl solution. The ddw (black bar) indicates that the virus solution was treated with water for injection. Na2S2O3 (gray dot bar) indicates that 200 ppm HOCl solution pretreated with 10% Na2S2O3 solution was mixed with the SARS-CoV-2 virus solution. Data were analyzed using one-way ANOVA with the Bonferroni post hoc test. **** p < 0.0001.

Virucidal efficacies of HOCl against SARS-CoV-2 in the presence of 0.02% FBS are shown in Figure 2. In the presence of a 0.02% FBS, the HOCl solution with concentration not less than 10 ppm was significantly effective against both the JPN/TY/WK-521 and KUH355 strains of SARS-CoV-2, resulting in a 5.3 and 6.7 log10 decrease, respectively. In contrast, 1 ppm HOCl solution had no virucidal effect on SARS-CoV-2 (Figure 2). The virucidal efficacy of HOCl in the presence of 0.02% FBS was 10 times greater than that in the presence of 0.2% FBS; 200 ppm HOCl pretreated with 10% Na2S2O3 did not observe the virucidal efficacy against SARS-CoV-2 in the presence of 0.2 and 0.02% FBS (Figures 1 and 2).

Figure 2

Influence of organic compounds on the SARS-CoV-2 virucidal efficacy of HOCl. SARS-CoV-2 virus solution was treated with various concentration of ionless hypochlorous acid water, as described in Materials and Methods. (a) JPN/TY/WK-521 strain treated with 1 (white bar), 30 (light gray bar), 50 (gray bar), and 100 (dark gray bar) ppm of HOCl solution. (b) KUH355 strain treated with 1 (white bar), 30 (light gray bar), 50 (gray bar), and 100 (dark gray bar) ppm of HOCl solution. The ddw (black bar) indicates that the viral solution was treated with water for injection. Na2S2O3 (gray dot bar) indicates that 200 ppm HOCl solution pretreated by 10% Na2S2O3 solution was pretreated with SARS-CoV-2 virus solution. Data were analyzed using one-way ANOVA with the Bonferroni post hoc test. **** p < 0.0001.

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Influence of organic compounds on the SARS-CoV-2 virucidal efficacy of HOCl. SARS-CoV-2 virus solution was treated with various concentration of ionless hypochlorous acid water, as described in Materials and Methods. (a) JPN/TY/WK-521 strain treated with 1 (white bar), 30 (light gray bar), 50 (gray bar), and 100 (dark gray bar) ppm of HOCl solution. (b) KUH355 strain treated with 1 (white bar), 30 (light gray bar), 50 (gray bar), and 100 (dark gray bar) ppm of HOCl solution. The ddw (black bar) indicates that the viral solution was treated with water for injection. Na2S2O3 (gray dot bar) indicates that 200 ppm HOCl solution pretreated by 10% Na2S2O3 solution was pretreated with SARS-CoV-2 virus solution. Data were analyzed using one-way ANOVA with the Bonferroni post hoc test. **** p < 0.0001.

Evaluation of virucidal efficacy of HOCl atomization against SARS-CoV-2

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Although the virucidal efficacy of HOCl was dependent on the presence of organic compounds, the suspension test indicated sufficient SARS-CoV-2 virucidal efficacy. To investigate the SARS-CoV-2 virucidal efficacy of atomized HOCl, we conducted the spray test in the test chamber (Figure 3). When sprayed with 500 ppm HOCl, no clear change was observed in the virus titer of the KUH355 strain solution containing 0.2% FBS after spraying for 1 h, but it was significantly suppressed after spraying for 3 and 5 h, by 2.3 log10 and 5.7 log10, respectively (Figure 4(a)). Next, the KUH355 strain solution containing 0.02% FBS was atomized by 10, 30, and 300 ppm HOCl solution for 1, 3, and 5 h. When sprayed with 300 ppm HOCl, the virus titer of KUH355 strain solution containing 0.02% FBS was significantly suppressed after spraying for 1, 3, and 5 h, by 5.1 log10, 4.2 log10, and 4.1 log10, respectively (Figure 4(b)). In the presence of 0.02% FBS, the atomized HOCl solution at 30 ppm was significantly effective for 3 and 5 h, resulting in a 2.8 and 4.1 log10 decrease, respectively (Figure 4(b)), suggesting that HOCl atomization showed dose- and time-dependent effectiveness despite the presence of organic compounds. The virus titer of the control group showed a gradual decrease in a time-dependent manner for KUH355 strain (Figure 4(a) and 4(b)). We also continuously monitored the change in relative humidity and atmospheric available chlorine concentration (ACC) in the test chamber. When 10, 30, 300, and 500 ppm HOCl was atomized in the same way as for the spray test, ACC remained constant as relative humidity of about 90% was maintained in the chamber during the test. ACC at 5 h after spraying with 10, 30, 300, and 500 ppm HOCl showed 0.01 ± 0.008, 0.051 ± 0.012, 0.175 ± 0.050, and 0.311 ± 0.029 ppm, respectively (Supplementary Figure 2).

Figure 3

Diagrammatic representation of the chamber. (a) Schema of the chamber used for the spray test. (b) Photo graph of the chamber.

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Diagrammatic representation of the chamber. (a) Schema of the chamber used for the spray test. (b) Photo graph of the chamber.

Figure 4

Evaluation of SARS-CoV-2 virucidal efficacy of atomized HOCl. SARS-CoV-2 virus solution was treated with various concentrations of ionless hypochlorous acid water, as described in Materials and Methods. (a) KUH355 virus strain in the presence of 0.2% FBS condition atomized with 30 (gray bar), 300 (dark gray bar), and 500 (dot bar) ppm of HOCl solution. (b) KUH355 strain in the presence of 0.02% FBS condition atomized with 10 (gray bar), 30 (dark gray bar), and 300 (dot bar) ppm of HOCl solution. The control (white bar) indicates the sequential reduction of the viral titer during the spray test. The stock (black bar) indicates the viral titer of a virus stock solution. The ddw (light gray bar) indicates that the virus solution was atomized with double distilled water. Data were analyzed using one-way ANOVA with the Bonferroni post hoc test. * p < 0.05. ** p < 0.005. **** p < 0.0001.

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Evaluation of SARS-CoV-2 virucidal efficacy of atomized HOCl. SARS-CoV-2 virus solution was treated with various concentrations of ionless hypochlorous acid water, as described in Materials and Methods. (a) KUH355 virus strain in the presence of 0.2% FBS condition atomized with 30 (gray bar), 300 (dark gray bar), and 500 (dot bar) ppm of HOCl solution. (b) KUH355 strain in the presence of 0.02% FBS condition atomized with 10 (gray bar), 30 (dark gray bar), and 300 (dot bar) ppm of HOCl solution. The control (white bar) indicates the sequential reduction of the viral titer during the spray test. The stock (black bar) indicates the viral titer of a virus stock solution. The ddw (light gray bar) indicates that the virus solution was atomized with double distilled water. Data were analyzed using one-way ANOVA with the Bonferroni post hoc test. * p < 0.05. ** p < 0.005. **** p < 0.0001.

DISCUSSION

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This study was performed to determine whether HOCl, produced by electrolysis of saturated chloride solution and filtrated through a reverse osmosis membrane to remove ions, had antiseptic efficacy against SARS-CoV-2 infectivity in the presence of organic compounds and under conditions of atomization. In the suspension test, we demonstrated a reduction of SARS-CoV-2 infectivity by contact with the HOCl solution for 20 s and 5 min. We found that the presence of organic compounds affected SARS-CoV-2 infectivity depending on the level of the ACC in the HOCl solution. We also observed that HOCl atomization significantly reduced SARS-CoV-2 infectivity in the spray test. The presence of organic compounds, including FBS, greatly affected the virucidal efficacy against SARS-CoV-2 in the suspension test and atomization test.

HOCl, a reactive chlorine species similar to ClO− and Cl2, is a high-potency oxidizing agent (Deborde & von Gunten 2008). In the innate immune system, HOCl is generated from hydrogen peroxide and chloride ions by myeloperoxidase (MPO) in phagocytes (Pattison et al. 2012; Winterbourn & Kettle 2013). Then, neutrophils and monocytes utilize HOCl to eliminate invading microorganisms (Gray et al. 2013; Da Cruz Nizer et al. 2020). MPO-knockout mice physiologically demonstrate increased susceptibility to infection with Candida albicans and Klebsiella pneumoniae in comparison with wild-type mice (Hirche et al. 2005; Homme et al. 2013). The HOCl solution shows powerful antibacterial efficacy against Gram-positive bacteria (Bacillus spp., Clostridium spp., Staphylococcus spp., Streptococcus spp.) and Gram-negative bacteria (Bordetella spp., Campylobacter spp., Vibrio spp., Pseudomonas spp.); Actinomycetaceae (Corynebacterium spp.); and Enterobacteriaceae (Escherichia spp., Shigella spp., Yersinia spp.), as well as virucidal efficacy against DNA (Adenoviridae, Herpesviridae) and RNA (Orthomyxoviridae, Coronaviridae) viruses (Tachikawa et al. 1999; Tagawa et al. 2000; Huang et al. 2008; Taharaguchi et al. 2014; Tamaki et al. 2014; Takeda et al. 2020; Miyaoka et al. 2021).

Previous studies using the suspension test have reported that treatment with HOCl solution can inactivate SARS-CoV-2 in cultured cells, such as VeroE6/TMPRSS2 cells. Hatanaka et al. (2022) have reported that 28.1 ppm HOCl solution achieved a 4-log reduction of SARS-CoV-2 infectivity within 10 s. In addition, 29.7 and 59.4 ppm HOCl solution achieved a 3.7–3.9- and 5-log reduction of SARS-CoV-2 infectivity within 10 s to 3 min in the presence of 0.1% FBS. Takeda et al. (2021) have reported that a residual chlorine concentration of 51–56 ppm in HOCl solution achieved >4-log reduction of SARS-CoV-2 infectivity within 20 s in the presence of 0.05% FBS. NITE summarized the results of the virucidal efficacy against SARS-CoV-2 at five facilities. They showed that a 19–200 ppm HOCl solution achieved a 3- and 4-log reduction of SARS-CoV-2 infectivity within 20 s to 5 min in the presence of 0.05–0.25% FBS. These studies have reported that viral solutions were prepared in the viral growth medium containing 1–5% FBS. The reaction rates of the virus solution and HOCl were 1:9 or 1:19. Then, they evaluated virucidal efficacy at a final concentration of 0.05–0.25% FBS. Moreover, previous studies have reported that the amount of protein in saliva, which is the cause of the droplet infection, is usually 100 times lower than that in plasma. Saliva contains 2% of proteins and 98% of water (Nunes et al. 2011; Kang & Kho 2018). Therefore, we chose the concentration of 0.2% and 0.02% FBS. In the present study, we found that 30 ppm HOCl solution achieved >5-log reduction of JPN/TY/WK-521 strain infectivity within 20 s in the presence of 0.2% FBS. In contrast, we demonstrated that 30 ppm HOCl solution achieved a 1.1-log reduction in KUH355 strain infectivity within 20 s in the presence of 0.2% FBS. This difference in the virucidal efficacy might affect the Delta variant, which is more transmissible than the original variant (Mistry et al. 2022). We also indicated that 10 ppm HOCl solution achieved sufficient reduction (>5 log) of JPN/TY/WK-521 and KUH355 virus strains infectivity within 20 s in the presence of 0.02% FBS. Thus, 30 ppm HOCl solution for 20 s may be sufficient to prevent the infection of the original SARS-CoV-2 strain. In contrast, the more transmissible mutant SARS-CoV-2 strain may require 50 ppm HOCl solution for > 20 s.

To reduce the risk of airborne transmission of pathogens, the bactericidal and virucidal efficacies of HOCl solution have been previously evaluated using atomization. Hakim et al. (2016) have reported that 50 and 100 ppm HOCl solution achieved an approximately 4-log reduction of Escherichia coli and Salmonella infantis in dry conditions within 5 min of atomization. Benedusi et al. (2022) have also reported that 300 ppm HOCl solution showed 69–99.9% reduction of E. coli, Staphylococcus aureus, and Pseudomonas aeruginosa with 10 min of atomization, although bactericidal activities were affected by the differences in material surfaces, such as semi-porous, flat, and porous. They also showed that 300 ppm HOCl solution achieved >95% reduction of human coronavirus 229E and adenovirus with 5–10 min of atomization. Urushidani et al. (2022) intermittently sprayed 250 or 8,700 ppm HOCl solutions for 2.5–5 s (each 4 min) in the test chamber and reported that 250 ppm HOCl solution did not neutralize air-dried SARS-CoV-2 infectivity after 16 min HOCl exposure. However, 8,700 ppm HOCl solution reduced air-dried SARS-CoV-2 infectivity to decrease the limit of detection within 12 min for 16 min HOCl exposure. In the present study, we found that atomized HOCl at 300 and 500 ppm for 3 and 5 h achieved a 1.4–2.3 and 2.3–5.7 log reduction of KUH355 virus solution infectivity in the presence of 0.2% FBS. We also observed that 30 ppm HOCl atomization for 3 and 5 h achieved 2.8–4.1 log reduction of KUH355 viral solution infectivity in the presence of 0.02% FBS. Moreover, SARS-CoV-2 infectivity in the presence of 0.02% FBS was reduced by >4-log by 300 ppm of HOCl atomization for 1, 3, and 5 h. Our results suggest that the HOCl solution may have effective virucidal efficacy against SARS-CoV-2, although a longer atomization time compared with a previous report may be needed. Our data also showed that atmospheric ACC is constantly maintained at <0.40 ppm in 200-L test chamber after atomization with 500 ppm HOCl. Our test chamber was almost twofold in volume as that reported in another study, in which the chlorine concentration was approximately 10 ppm with 8,700 ppm atomization. Therefore, differences in chlorine concentration in the experimental chamber may have influenced the virucidal efficacies against SARS-CoV-2 we observed in this study.

The WHO does not recommend the disinfection of COVID-19 by fogging environmental surfaces with disinfectant, as it may increase the risk of adverse effects to the eyes, respiratory tract, and skin. Because HOCl reportedly did not cause these adverse effects, the adverse effects of HOCl are probably related to the chlorine concentration and pH of the exposed HOCl. In addition, no adverse events were observed by drinking 20–60 ppm HOCl water in animal studies, washing wounds with 200 ppm HOCl solution, and mouth washing with 80 and 200 ppm HOCl solution in clinical studies (Morita et al. 2011; Kubota et al. 2015; Block & Rowan 2020; Alzahrani et al. 2022; Sevinç Güll et al. 2022). Although the effect of the high purity of HOCl on adverse events is unclear, HOCl should be atomized under controlled conditions at the appropriate concentration. Our results, which prove the relationship between the virucidal efficacy and the ACC of HOCl, suggest that HOCl atomization for disinfection may be useful for cleaning unmanned hospital rooms, schools, daycare centers, and various facilities at night.

CONCLUSIONS

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We have clarified that both aqueous and atomized HOCl solutions are effective in neutralizing SARS-CoV-2 infectivity. The virucidal efficacy of HOCl was not influenced by the difference in SARS-CoV-2 variants (Pango lineage A and B virus strains). In addition, we showed the virucidal efficacy of HOCl atomization against SARS-CoV-2. HOCl atomization may reduce the burden of cleaning and disinfection work in various facilities such as hospital rooms, daycare centers, and schools.

ACKNOWLEDGEMENTS

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The authors thank laboratory staffs of Kitasato University and NIPRO Corporation for technical and experimental supports.

FUNDING

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This study was supported by the COVID-19 Kitasato Project Study and Research on Emerging and Re-emerging Infectious Diseases and Immunization on Health, Labour and Welfare Policy Research Grants (grant number 21HA2017). This study was funded by a research grant from NIPRO Corporation.

FINANCIAL INTERESTS

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Author M.K. received research support from NIPRO Corporation. All authors except M.K. have no financial interests.

CONTRIBUTIONS

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M.K., R.E., H.F., N.S., and N.K. performed the study concept and design. M.K. and R.E. performed writing the original draft. M.K., R.E., S.M., H.F., N.S., N.K., H.K., and H.H. performed the development of methodology and writing, review, and revision of the manuscript. M.K., R.E., H.F., and N.S. provided acquisition, analysis and interpretation of data, and statistical analysis. H.F., N.S., and N.K. provided reagents, materials, test chamber, and instrumentations. N.K., H.K., and H.H. supervised and provided the financial support for this project. All authors have read and approved the final manuscript.

DATA AVAILABILITY STATEMENT

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All relevant data are included in the paper or its Supplementary Information.

CONFLICT OF INTEREST STATEMENT

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M.K. received research support from NIPRO Corporation. All authors except M.K. have no financial interests.

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© 2024 The Authors

This is an Open Access article distributed under the terms of the Creative Commons Attribution Licence (CC BY 4.0), which permits copying, adaptation and redistribution, provided the original work is properly cited (http://creativecommons.org/licenses/by/4.0/).

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James F. Walsh;

Deanna P. Scher;

Jane R. de Lambert;

Anita C. Anderson

J Water Health (2024) 22 (3): 612–626.

https://doi.org/10.2166/wh.2024.361

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Abstract

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In a recent monitoring study of Minnesota's public supply wells, Cryptosporidium was commonly detected with 40% of the wells having at least one detection. Risk factors for Cryptosporidium occurrence in drinking water supply wells, beyond surface water influence, remain poorly understood. To address this gap, physical and chemical factors were assessed as potential predictors of Cryptosporidium occurrence in 135 public supply wells in Minnesota. Univariable analysis, regression techniques, and classification trees were used to analyze the data. Many variables were identified as significant risk factors in univariable analysis and several remained significant throughout the succeeding analysis techniques. These factors fell into general categories of well use and construction, aquifer characteristics, and connectedness to the land surface, well capture zones, and land use therein, existence of potential contaminant sources within 200-feet of the well, and variability in the chemical and isotopic parameters measured during the study. These risk categories, and the specific variables and threshold values we have identified, can help guide future research on factors influencing Cryptosporidium contamination of wells and can be used by environmental health programs to develop risk-based sampling plans and design interventions that reduce associated health risks.

HIGHLIGHTS

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• The influence of multiple factors on the presence of Cryptosporidium in public wells is examined.
• The human-caused and naturogenic risk factors, both well-understood and novel, are identified.
• The range of influential factors reflects varying pathways and sources of Cryptosporidium
• Findings can help drinking water managers assess risk and target monitoring efforts.

Cryptosporidium, drinking water, groundwater contamination, microbial risk factors, public water supply wells, water monitoring

INTRODUCTION

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The enteric parasite Cryptosporidium is the leading cause of waterborne disease among people in the United States (CDC 2023). Those with weakened immune systems are most at risk of developing serious and sometimes fatal illness (CDC 2023). Historically, Cryptosporidium has not been considered a contaminant of concern in groundwater sources of drinking water unless surface water is entering the wells (groundwater under direct influence, GWUDI). As a result, Cryptosporidium (and other protozoa) have rarely been included in groundwater monitoring studies (Hynds et al. 2014; Murphy et al. 2017), and U.S. regulations designed to prevent and monitor for Cryptosporidium contamination do not exist for groundwater, except GWUDI.

From 2014 to 2016, the Minnesota Department of Health (MDH) conducted a study to monitor for a diverse suite of microbiological contaminants in Minnesota, USA public water supply wells (Stokdyk et al. 2020). A total of 145 community and noncommunity wells representing the major aquifer types across the state were repeatedly sampled over 1 or 2 years and tested for viral, bacterial, and protozoan pathogens and other indicators of fecal contamination using quantitative polymerase chain reaction (qPCR). Surprisingly, Cryptosporidium was found to be common in the public supply wells, with 40% of wells having at least one detection. Concentrations ranged from 0.05 to 246 gene copies per liter with a mean of 10.4 gene copies per liter. Gene sequencing to determine species was successful for 45 of the samples, with 41 identified as Cryptosporidium parvum, 2 as Cryptosporidium andersoni, and 2 as Cryptosporidium hominis, suggesting a range of possible sources, including humans, cattle, and other mammals. Of interest, Cryptosporidium detections were not found to be associated with surface water influence (Stokdyk et al. 2019). This finding is at odds with current risk and regulatory paradigms that assume limited occurrence of Cryptosporidium in groundwater. More recently, quantitative microbial risk assessment was used to quantify population-level health risk for nine viral, bacterial, and protozoan pathogens in the Minnesota study (Burch et al. 2022). Cryptosporidium was found to be a major contributor to health risk, making improved identification of wells susceptible to Cryptosporidium contamination a priority.

Risk factors for potential groundwater contamination by Cryptosporidium, beyond surface water influence, remain poorly defined (Chique et al. 2020). To our knowledge, no studies have systematically examined a wide range of hydrogeologic and anthropogenic factors that may influence Cryptosporidium occurrence in public or private wells across a diversity of hydrogeologic conditions. Most studies on potential risk factors have been targeted environmental investigations, prompted by human outbreaks of cryptosporidiosis, which are narrow in scope (Rose 1997; Watier-Grillot et al. 2022).

A better understanding of risk factors is especially imperative for Cryptosporidium, as routinely analyzed coliform indicator bacteria have been shown to be a poor predictor for the presence of enteric protozoa (Rose 1997; Stokdyk et al. 2020). Furthermore, once contamination occurs, the oocysts have a high survival rate (more than 24 months at 20°C) and a high resistance to disinfection (Betancourt & Rose 2004; Fradette et al. 2022). The primary objective of this work was to evaluate hydrogeologic and anthropogenic factors and routine general water chemistry parameters that may be associated with Cryptosporidium detections in public supply wells. The goal was to improve identification of risk factors for Cryptosporidium contamination, which would allow drinking water programs to target monitoring and interventions to reduce associated health risks.

METHODS

Study design and sample collection

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The study was statewide in scope and included 145 wells from 123 public water systems (88 community wells and 57 noncommunity wells) in Minnesota (Figure 1). The wells were sampled every other month for one of two study phases (typically six samples per well per phase). The 89 wells in the first phase (May 2014–April 2015) were randomly selected from among non-disinfecting, year-round, community and nontransient noncommunity systems in Minnesota. For the second phase (May 2015–May 2016), 85 wells potentially vulnerable to contamination based on geologic setting, well construction, and past monitoring or groundwater flow modeling were prioritized, without regard to disinfection status, including 29 wells from Phase 1 (typically sampled 6 times per phase for a total of 12 samples). The two-phase selection process produced a sample of wells that generally represented the aquifers and geology of wells statewide, with a slight overemphasis on more vulnerable geologic settings.

Figure 1

Location of 145 sampled public water system wells.

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Location of 145 sampled public water system wells.

Samples were collected by dead-end ultrafiltration (Smith & Hill 2009) using Hemodialyzer Rexeed-25s filters (Asahi Kasei Medical MT Corp., Oita, Japan). Groundwater volume sampled ranged from 140 to 1,783 L (mean, 728 L). All samples were collected prior to treatment (including disinfection) from sampling taps that were disinfected using a flame or alcohol wipes. Filters were shipped on ice and processed within 48 h.

Since pathogen occurrence was found to be sporadic in these study wells (Stokdyk et al. 2019, 2020), and additional studies have also found Cryptosporidium to occur intermittently in U.S. groundwater (Hancock et al. 1998), the outcome of interest in this analysis is any detection of Cryptosporidium in the well identified through the repeated sampling.

Study well characteristics

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Well depth ranged from 20 to 630 feet, and age, casing length, and open interval length also varied (Table S1). Fifty-seven percent of the study wells drew from Quaternary glacial sand and gravel deposits; other aquifers included sandstone, fractured crystalline rock, carbonate rock, mixed sandstone, carbonate rocks, and shale (Table S2). The geologic sensitivity ratings of study wells, reflective of the estimated vertical time of travel for water moving from the land surface to the aquifer in question, also varied. Study wells were considered representative of the ranges observed for all Minnesota public wells for these characteristics, with a slight bias towards wells with higher geologic sensitivity ratings.

Laboratory analysis

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Analytical methods have been previously described (Stokdyk et al. 2016, 2019, 2020). Briefly, the samples were tested for Cryptosporidium by qPCR, targeting the 18S gene using a LightCycler 480 instrument (Roche Diagnostics, Mannheim, Germany) and following procedures and reaction conditions described in Stokdyk et al. (2016, 2019, 2020). qPCR was performed in duplicate, with the average of positive replicates reported.

In addition to the samples collected by ultrafiltration, samples were also collected for a variety of chemical and isotopic parameters indicative of wastewater influence and/or groundwater residence time and field measurements were taken for water temperature, specific conductance, pH, dissolved oxygen, and redox potential. Total coliform, Escherichia coli, total organic carbon, nitrate/nitrite nitrogen, ammonia, bromide, chloride, and boron were analyzed by the MDH Public Health Laboratory using standard methods (Table S3). Analyses of tritium and the stable isotopes of water (oxygen-18 and deuterium) were conducted at either the University of Waterloo Environmental Isotope Laboratory or Isotope Tracer Technologies. Deviation of stable isotope samples from the meteoric water line for central Minnesota (Landon et al. 2000) was determined using the line-conditioned excess approach (Landwehr & Coplen 2004).

Potential risk factors

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A total of 79 hydrogeologic and landform/land use/land cover variables were selected for risk factor analysis based on their: (1) likelihood of affecting pathogen occurrence and transport at the land surface and in the subsurface, (2) function as indicators of groundwater residence time or human-impacted water quality, and (3) availability in Minnesota datasets. Several data sources were consulted to create the final set of potential predictors (Table S4). These variables were grouped into five general themes: Well use and construction; Aquifer characteristics, connectedness between aquifer and land surface; Well capture zone and land use within capture zone; Potential contaminant sources in the Inner Wellhead Management Zone (IWMZ); and Chemical and isotopic parameters (Table 1). Land-use variables within the well capture zones were derived from the National Land Cover Database and include various types of development (low-, medium-, and high-intensity residential, commercial, and industrial) as well as agricultural land uses. Potential pathogen sources within the IWMZ included septic systems (tanks and drainfields), sewer lines, and sewage lift stations. Chemical and isotopic variability was evaluated by assessing an average of six rounds of samples collected on a bimonthly basis over a 12-month period. Variable definitions are found in Tables S5–S9.

Table 1

Hydrogeologic and anthropogenic factors (n = 79) assessed by themea

aSee Tables S5–S9 for variable definitions, descriptive statistics, and univariable analysis results. bTT=travel time.

Statistical analysis

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Analyses were limited to wells with at least three samples available (n = 135). Descriptive statistics were compiled for each potential risk factor in Table 1. Next, visual plots and univariable statistical tests looked for relationships between the potential predictors and Cryptosporidium detection. For continuous variables, the Mann-Whitney U test was selected, as the explanatory variables of interest typically showed skewed distributions. The Chi-square test was used for categorical variables. For categorical variables that contained groups with small sample sizes, categories were combined into larger groupings for testing when logical and feasible. The Cochran–Armitage trend test was applied to ordinal variables.

In addition to serving a prescreening role for multivariable modeling, univariable analysis results were considered independently for two reasons. First, several risk factors could not be included in multivariable modeling because they had a high degree of missing data which would have reduced the statistical power to evaluate other risk factors. Second, some variables had to be removed prior to or during multivariable modeling due to their collinearity or overall interrelatedness with other covariates.

Multivariable modeling was conducted to allow each potential risk factor to be evaluated for its independent effect and strength of association in the presence of other factors. Explanatory variables with >20% missing data were excluded. To reduce the number of potential predictors in Table 1 to a feasible size for multivariable modeling, only variables with a strength of association of p ≤ 0.2 in univariable analysis were included. The study phase (Phases 1, 2, or both) was included as an independent variable. This factor not only accounted for potential differences in climatological conditions between the sampling periods, but also differences in the number of observations per well. Six samples were available for most wells sampled in Phase 1 or Phase 2, whereas wells sampled during both phases typically had 12 samples, which could affect (i.e., bias) the corresponding probability of pathogen detection for the well. Prior to building the regression model, Pearson correlation coefficients were assessed to check for collinearity between continuous variables. Inclusion/exclusion decisions were made when high correlation was found (>0.75).

A modified Poisson regression model with robust error variance was used to estimate the relative risk of Cryptosporidium detection using the generalized estimating equations-based method of Zou (2004). The model uses a log link function with a Poisson distribution. This approach was taken because odds ratios in logistic regression can overestimate relative risk when the outcome is common (McNutt et al. 2003) and in this case, there is a high prevalence of wells with a Cryptosporidium detection (40%). A modified Poisson regression model is also considered more robust to outliers and avoids non-convergence issues compared to a log-binomial model for common binary outcomes (Chen et al. 2014). A backward variable selection process was used based on p-value and reduction in the Quasi-likelihood under the independence model criterion statistic (analogous to Akaike's Information Criterion). Variables with a p-value ≤.05 were ultimately retained in the final model. Once the independent variables for the multivariable model were identified, a screening process for interaction terms among these variables was undertaken. Only interactions deemed plausible and relevant were assessed. Cook's distance, leverage, and residual plots were examined to identify influential points and assess model fit.

A classification tree, a type of machine learning method, was also created as an alternative approach to identify predictors of Cryptosporidium detection (Breiman et al. 1984). Compared to regression, these models have fewer restrictions on the type of data that can be included, better accommodate missing data, and are not confined to the implicit assumptions of regression. The splits may also suggest strategies for drinking water managers to tailor monitoring and interventions if they show adequate predictive accuracy. In building the tree, entropy was used to assess candidate splits for each node. As with the regression model, variables with >20% missing values were excluded; the remaining missing values were assigned to the most popular node. To help prevent overfitting, and to create a simpler tree, the tree was pruned back with cost-complexity pruning (Breiman et al. 1984) and a minimum leaf size of five. Cost-complexity analysis plots were used to select the final subtree. Ten-fold cross-validation was performed rather than hold-out validation due to the relatively small size of the dataset.

All analyses were performed using SAS version 9.4 (SAS Institute Inc., Cary, NC, USA) with proc GENMOD procedure used for the Modified Poisson regression models and the HPSPLIT procedure used for the classification tree.

RESULTS AND DISCUSSION

Univariable analysis

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Descriptive statistics, visual plots, and statistical test results for the 79 predictor variables are found in Tables S5–S9, with the findings summarized in Table 4.

In the ‘Well use and construction’ theme, three variables met the criteria (p-value ≤0.2) for retention in further analyses (Table 2 and Table S5). Shallower well depth, depth cased, and smaller saturated casing values were more likely among wells with a Cryptosporidium detection. Several of the well characteristic variables had a high percent of missing data (>20%) and were excluded from further analysis; notably, grouted yes/no, grout material, percent casing grouted, percent grout saturated, annular space, and casing jointing method. Of these, a higher proportion of Cryptosporidium-positive wells was visually apparent among wells that were not grouted, constructed with B group (bentonite) grout material, and with larger annular space (Table S5).

Table 2

Risk factors for Cryptosporidium detection in univariable analysis with select visual plots (n = 135)a

aVariables with p-values ≤.20 were included in further analysis. Continuous data were compared between groups using the Mann-Whitney U or Kruskal–Wallis test. The Chi-square test was used to perform intergroup comparisons. The Cochran–Armitage trend test was used for ordinal variables. Due to space considerations, not all figures are shown. See Tables S5–S9 for full results.

bA high percent of missing data prevent further testing of this variable in multivariable analysis.

cAquifer porosity type: 1 = primary unconsolidated, 2 = primary consolidated within 50 feet of the land surface, and 3 = secondary.

dOther sewage treatment types could not be tested due to small counts.

In the ‘Aquifer characteristics, connectedness between aquifer and land surface’ theme, Cryptosporidium-positive wells were more likely to have porosity characteristics of unconsolidated sediments or fractured crystalline bedrock, higher land surface elevation, lower drawdown (which had a high percent of missingness), and water quality characteristics reflecting modern groundwater age (Table 2 and Table S6). These characteristics include detectable tritium, evidence for human impact based on elevated chloride/bromide ratios and evidence of rapid recharge based on fluctuating water quality parameters. While not statistically significant, the proportion of wells with detections increased from lowest, middle, to highest categories of geologic sensitivity (Table S6).

Several ‘Well capture zone and land use within capture zone’ variables met the criteria (p-value ≤0.2) for retention in further analyses (Table 2 and Table S7). Cryptosporidium-positive wells had lower levels of low, medium, and high development intensity within the 1-year travel time (1-yr TT) well capture zone. These development variables were not below the significance cut-point for 10-year travel time (10-yr TT), but the same trend was seen (Table S7). A higher proportion of Cryptosporidium-positive wells were located where ‘development is mostly agriculture’ (1-yr TT) versus not, and Cryptosporidium-positive wells had a higher percent of open water or wetland in the well capture zones, with both the 1- and 10-yr TT capture zones below the ≤0.2 p-value threshold. Wells with detections were also more likely to have larger runoff catchment areas and a higher percent of impervious surfaces in the runoff catchment area.

For the ‘Potential contaminant sources in the IWMZ’ theme, the infrequent presence of contamination sources within the IWMZ for several variables (right-censored at >200 ft) resulted in re-classification into ordinal categories for analysis (Table S8). While 61% of wells were missing sewage treatment information, a higher proportion of wells with detections were seen for gravity trenches compared to mound systems and pressurized septic systems (Table 2 and Table S8). Within the IWMZ, increasing distance to septic/sewage systems was associated with decreasing likelihood of detections. While not meeting the threshold for further analysis, wells with Cryptosporidium detections had lower mean and median design flows and were more common for septic versus municipal sewer types.

Several coefficients of variation (CVs) within the ‘Chemical and isotopic parameters’ theme had p-values ≤0.2 (Table 2 and Table S9). Higher bromide, chloride, chloride/bromide ratio, boron, nitrate, total organic carbon, specific conductance, and δ2H CV values were seen among wells with Cryptosporidium detections.

Multivariable regression model

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Variables meeting the p < .20 criteria in univariate analysis were included in regression analysis, with p < .05 used as a cut-point for retention in the final model. The final adjusted multivariable model is shown in Table 3. Increased runoff catchment area-percent impervious, nitrate CV, and bromide CV were significantly associated with increased risk of Cryptosporidium detection. Shallower well depth was also associated with higher risk of detection (i.e., increasing well depth showed a protective effect). Of note, saturated casing value (tertiles, with ‘missing’ as the fourth group) was significant in the model, but only when well depth was excluded, and vice-versa. Well depth was retained due to fewer missing data. Lower percent low-intensity development was also associated with higher risk of detection. In univariable analysis, the other development intensities (medium and high) showed the same direction of effect, suggesting Cryptosporidium risk may be related to generally undeveloped land.

Table 3

Adjusted relative risks (95% CIs) for Cryptosporidium detection from the modified Poisson regression model

aN = 135. Relative risks are shown as exponentiated coefficients. Models adjusted for phase of study (Phases 1, 2, or both).

Classification tree

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Entering all predictor variables with p-values <.20 in univariable analysis into a classification tree model with a minimum leaf size of five, a tree with seven leaves best balanced cost complexity and misclassification. Depth of well, bromide CV, runoff catchment area-percent impervious, and nitrate CV ranked comparably in importance, followed by percent open water/wetland (1-yr TT) and runoff catchment area (Table 4). The model incorrectly predicted the outcome for 20% of wells, with a higher misclassification rate in the cross-validation (44%), indicating that the model may not generalize well to new data. The model area under the curve (AUC) was 0.82, which is considered acceptable discrimination.

Table 4

Classification tree output for Cryptosporidium detection

aAUC = The area under the receiver operating characteristic curve. A model that fits the data perfectly would have an AUC of 1.

The first split was based on well depth, with wells <118 feet approximately twice as likely to have a Cryptosporidium detection (61% of wells) compared to 32% of wells ≥118 feet deep (Figure 2). For wells ≥118 feet, all wells with a runoff catchment area of ≥0.83 acres had a detection, but the sample size was small (n = 5). Of those with a runoff catchment area <0.83 acres, 86% of wells had detections when bromide CV was ≥32% (n = 7) compared to 23% of wells with a bromide CV <30%. Of these 79 wells, if nitrate CV was ≥130%, 83% had detections (n = 6) compared to those with nitrate CV <130%, in which 18% had detections.

Figure 2

Classification tree: risk factors for Cryptosporidium detection. Notes: + = percent of wells with a Cryptosporidium detection. The color of the bar in each leaf node indicates the most frequent Cryptosporidium detection outcome (detection = red, no detection = blue) and represents the classification assigned to all observations in that node. The height of the bar indicates the proportion of wells in the node that have the most frequent outcome.

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Classification tree: risk factors for Cryptosporidium detection. Notes: + = percent of wells with a Cryptosporidium detection. The color of the bar in each leaf node indicates the most frequent Cryptosporidium detection outcome (detection = red, no detection = blue) and represents the classification assigned to all observations in that node. The height of the bar indicates the proportion of wells in the node that have the most frequent outcome.

Of wells <118 feet, all wells with a runoff catchment area ≥0.83 acres had detections, but the sample size is small (n = 5). Of wells with a runoff catchment area <0.83 acres and open water or wetland <1%, 27% had a detection compared to those with open water or wetland >1% in which 68% had detections.

Synthesis of findings

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In a state-wide surveillance study, Cryptosporidium detection was unexpectedly common in Minnesota public supply wells. Adding to this concern, the incidence of cryptosporidiosis is reported to be increasing, with rates highest in northern Midwest states (CDC 2019). Cryptosporidium's previously reported lack of correlation with coliform bacteria (Rose 1997; Stokdyk et al. 2020), low threshold for infectivity (DuPont et al. 1995), and high tolerance to chlorine (Betancourt & Rose 2004; Fradette et al. 2022) make it critical to fill current gaps in knowledge regarding risk factors for Cryptosporidium in drinking water wells. Climate change also heightens the urgency of this work, as extreme precipitation events can intensify pollutant runoff and pathogen infiltration and subsequently increase the risk of waterborne disease outbreaks, including cryptosporidiosis (Curriero et al. 2001; Ikiroma & Pollock 2021). In addition, its large size may allow Cryptosporidium to serve as a bellwether for smaller pathogens such as bacteria and viruses. For these reasons, we explored potential risk factors for Cryptosporidium presence in public drinking supply wells.

Univariable analysis findings were considered independently in addition to serving as a prescreening for multivariable modeling. Certain physical and chemical factors across all five themes were found to significantly differ between wells with and without a Cryptosporidium detection in univariable analysis. This demonstrates that contamination risk cannot be solely attributed to one type of risk factor, such as well construction characteristics. Shallower well and casing depths and lower saturated casing values were significant risk factors. Riskier aquifer characteristics included unconsolidated glacial sediments or bedrock aquifers dominated by secondary porosity and the presence of modern water based on tritium detection. An increase in the proportion of wells with detections with increasing geologic sensitivity was visually apparent as well. While Stokdyk et al. (2020) found that aquifer type and geologic sensitivity were not significant predictors of overall pathogen risk, when looking only at protozoa detections, sand and gravel aquifers and fractured crystalline rocks were noted as showing the greatest proportion of positive wells, with the same being said of the wells with the highest geologic sensitivity ratings. Indicators of human-impacted water quality based on elevated chloride/bromide ratios, and/or high temporal variability reflective of relatively rapid recharge, were also indicative of higher risk. In addition, smaller well drawdowns, suggestive of higher aquifer transmissivities, were identified as significant; however, hydraulic conductivity and aquifer thickness were also evaluated separately and not found to be significant factors. Similarly curious is the finding of higher land surface elevation being a factor in Cryptosporidium occurrence.

Our analyses also noted the importance of landform, land use, and land cover characteristics in relation to Cryptosporidium risk. For example, the absence of commercial, industrial, or residential development within the 1- or 10-year TT capture areas for wells, and/or the development within these areas consisting primarily of agricultural use, was related to elevated Cryptosporidium risk, as was the presence of open water or wetlands within these areas. Similarly, the presence of relatively large surface water catchments in the immediate vicinity of study wells, and the presence of a relatively large proportion of impermeable surfaces such as roadways and rooftops within these catchments, increased Cryptosporidium risk. These findings may reflect on the prevalence of potentially zoonotic Cryptosporidium species such as C. parvum in this study, rather than human-specific species, although the observations described above related to linkage with indicators of human-impacted water quality are at odds with this finding, as is the observation of higher risk associated with proximity and density of sewage treatment systems within the IWMZ, a finding similar to that described by Borchardt et al. (2021). Taken together, these sets of findings may reflect on the range of scenarios that may increase Cryptosporidium risk at wells, spanning the relative absence of development, where presumably zoonotic sources of Cryptosporidium predominate, to the presence of at least minimal development in terms of nearby septic systems or impervious surfaces that may impart associated indicators of human-impacted water quality such as elevated chloride/bromide ratios.

The final multivariable regression model included well depth, runoff catchment area, runoff catchment area-percent impervious, bromide CV, and nitrate CV as statistically significant predictors. Other general chemistry parameter CVs were significant in the multivariable model in the absence of each other, indicating redundancy, and the final model retained the CVs with the ‘strongest’ effects. Variability in these chemical parameters may reflect dynamic responses to recharge events (Walsh et al. in press) and/or other transient hydrologic factors such as well pumping. These insights can support monitoring efforts that include time-series or continuous data collection of well water chemistry to help better assess microbial risk. Each of these CV relationships was positive, with greater values indicating higher risk.

Classification trees are well suited to the study of environmental data because they can accommodate complex interactions among variables, allowing for the observation of moderating effects that might be missed when using traditional model-based approaches. Classification trees also provide easy to follow visualizations of the predictive process and the hierarchical importance of the variables from the top to the bottom of the tree, and therefore do not suffer from the ‘black box’ effect of more complicated machine learning approaches. The ability of classification trees to efficiently segment a ‘population’ of wells into meaningful subsets can help drinking water managers identify potentially high-risk wells, or their inverse, so that resources (e.g., monitoring) can be targeted accordingly. For this reason, we intentionally created a simple tree model using cost complexity pruning and limiting the number of nodes to make the tree useful for public health decision making. The cross-validation fit showed higher specificity than sensitivity, with wells less likely to have detections having 1) well depth >118 feet and a runoff catchment area-percent impervious <81%, unless bromide CV was >32%. For shallower wells (<118 feet), smaller runoff catchment areas with lower percent open water or wetland decreased the likelihood of detections. While classification trees and regression models are different algorithms, we found that both the regression and classification tree models identified nitrate and bromide CVs, well depth, and runoff catchment area (or runoff catchment area-percent impervious) as influential in Cryptosporidium occurrence, increasing confidence in these findings.

The protective effects of increasing well depth, less potential for surface water runoff around the well, and the absence of high bromide variability reflective of rapid or changing recharge conditions make intuitive hydrogeologic sense and are generally substantiated in groundwater literature. For example, numerous studies have described correlation between shallower well depths and microbial contamination (e.g., Gonzales 2008; Maran et al. 2016; O'Dwyer et al. 2018). While none of these settled on the same primary cutoff depth noted here (118 feet), those studies were focused on microbial contaminants other than Cryptosporidium and likely included wells of differing characteristics, such as depth ranges and construction features, than those in this study. Variability in chemical parameters has been recognized as reflective of rapid recharge and degraded water quality, but again, not specific to Cryptosporidium risk or to bromide or nitrate variability. For example, Jacobson & Langmuir (1974) found CVs of many ionic parameters >15% to be reflective of rapid groundwater flow conditions in conduit karst, with slightly lower values seen in diffuse flow systems, and Dhar et al. (2008) found that wells deeper than 30 m showed significantly less temporal chemical variability than shallower wells. Bexfield & Jurgens (2014) found that seasonal variability in water quality at public supply wells was influenced by well usage patterns, and Aisopou et al. (2015) found that pesticide concentrations in production wells were dependent on variables such as pumping rate and the hydrogeology of the aquifer. Stokdyk et al. (2019) also looked at variability in chemical and isotopic parameters for the same data set used in this analysis on the basis that such variability likely reflected rapid recharge and microbial risk. However, the authors found that Cryptosporidium-positive wells were no more likely to show evidence of rapid recharge or surface water influence, based on the evaporative signature of water isotopes, than wells that lacked detections. These conclusions are likely at odds with the current analysis because the 2019 analysis utilized arbitrarily determined threshold values for establishing variability, while including the evaporative signature analysis, whereas the current analysis simply looked at the full range of observed values for all chemical and isotopic parameters to look for association with Cryptosporidium occurrence. Relationships between surface water runoff catchment area and impervious surfaces and pathogen risk are less substantiated in the literature beyond the effects of well flooding (Andrade et al. 2018), and the observation that extreme climatic events have been associated with waterborne disease outbreaks more generally (Curriero et al. 2001; Auld et al. 2004; Cann et al. 2013), implying possible correlation with runoff mechanisms. While apparently novel, the observations described in this study about association with surface water runoff make intuitive sense, given that the flow or ponding of surface water that might be contaminated with Cryptosporidium in the vicinity of drinking water wells could exploit macropores or other fast flow pathways into the subsurface.

While this study focused on public wells, there are important takeaways for private wells, which are estimated to serve 17% of the U.S. population (Murray et al. 2021). The observed importance of well depth as a predictor of Cryptosporidium risk is concerning given that private wells are generally shallower than public wells. For example, the average private well depth in Minnesota is 138 feet (n = 415,638) compared to 179 feet (n = 20,171) for public wells (Minnesota Well Index database, as of 1/12/2023). In addition, full-length grouting is not required for private wells, and well siting considerations may be less rigorous than those used for public wells, or not considered at all for wells drilled before the Minnesota Well Code took effect in 1974. Finally, private wells have no water quality testing requirements after the time of construction in Minnesota as well as most other states and are unlikely to be sampled repeatedly or for the parameters deemed useful in this analysis, outside of special studies.

There are several unique features of this study. First, Cryptosporidium occurrence in wells is an understudied area of research, making this work an important contribution. Secondly, a relatively large number of wells were monitored multiple times (typically 6 times, up to 12) and this greater sampling frequency was important since positive wells were often found to have a few positive detections accompanied by multiple negative results. The application of multiple data analysis approaches involving a wide range of potential risk factors is also a strength.

There are also some limitations to this analysis. First, the focus of this work was on Cryptosporidium occurrence, not concentration, which may have a unique set of risk factors. Second, the analysis did not consider potential meteorological predictors such as preceding precipitation events, which have been shown to be influential. In a separate Minnesota study, springtime, the season with the highest amount of rainfall in Minnesota, was associated with the greatest frequency of microbial contamination (MDH 2023, in press). However, the longitudinal collection strategy was designed to capture results across seasons. Certain variables could not be adequately assessed due to a high frequency of missing data. Univariable analyses suggest some of these variables, such as percent grout saturated, annular space, drawdown, and septic system design flow, may be important predictors of Cryptosporidium detection. However, the lack of available data indicates that these characteristics are not often available to drinking water programs, making their utility in identifying at-risk wells limited from a practical standpoint.

There are reasons why these findings should be interpreted with caution. Both multivariable regression models and tree models can be unstable and result in overfitting when the number of observations is small relative to the number of predictor parameters. We also do not know whether these findings are generalizable to other areas within or outside the U.S.; additional studies conducted in a variety of settings are needed. Despite these caveats, the results suggest that more groundwater systems should be monitored for Cryptosporidium based on the risk and protection factors identified here and highlighted in other relevant reports. Based on the findings of this work, MDH intends to bolster the predictive outcome of its own existing well vulnerability scoring rubrics by adding additional variables (such as bromide variability and runoff/impervious area around wells), assigning heavier weight to variables that passed more rigorous tests, and using specific cutoff values for the parameters identified here.

CONCLUSIONS

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Risk of Cryptosporidium occurrence in wells was associated with multiple anthropogenic and natural factors, suggesting both a wide array of influential factors and diverse range of mammalian hosts of the different species of this organism. Shallower well depth and depth cased, lower saturated casing values, potential for surface water runoff around the well, and variability in chemical parameters reflective of rapid or changing recharge, along with certain land use and land characteristics, were consistent predictors of occurrence across data analysis methods. Drinking water programs can work to minimize the risk of cryptosporidiosis by considering the factors identified here when developing predictive scoring rubrics, well monitoring strategies, and well construction and siting plans. The findings should also be considered when assessing the adequacy of current drinking water regulatory standards for Cryptosporidium.

ACKNOWLEDGEMENTS

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Funding was provided by the Minnesota Clean Water, Land, and Legacy Amendment Fund. The authors wish to thank the public water suppliers who participated in the study and the MDH team responsible for field duties including Dane Huber, Jared Schmaedeke, Trisha Sisto, Mike Sutliff, and Nathan Gieske. Alycia Overbo of MDH provided communications support. Laboratory assistance and insight were provided by Aaron Firnstahl (USGS), Joel Stokdyk (USGS), Susan Spencer (USDA-ARS retired) and Mark Borchardt (USDA-ARS retired).

AUTHOR CONTRIBUTIONS

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James Walsh contributed to the conceptualization, project administration, investigation, writing the original draft, and funding acquisition. Deanna Scher contributed to the formal analysis, visualization, and writing the original draft. Jane de Lambert contributed to the conceptualization, data curation, writing the original draft, and also reviewed and edited the manuscript. Anita Anderson contributed to the conceptualization, writing the original draft, and also reviewed and edited the manuscript.

DATA AVAILABILITY STATEMENT

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Data cannot be made publicly available; readers should contact the corresponding author for details.

CONFLICT OF INTEREST

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The authors declare there is no conflict.

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Abstract

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This paper explores the socio-cultural and gender-based dynamics associated with place values, and their implications for women's access to water through case studies of upland and riverine communities in southern Nigeria. We used a range of fieldwork methods including public meetings, focus group discussions, in-depth interviews, keen observations, key informants and other secondary sources. Our findings show that drinking water sources are a part of the many forms of visible material structures that embody and generate automatic reproduction of gender-based beliefs, attitudes, feelings and practices. The outcome of such practices affects men and women differently in relation to access, workload and capacity for hygiene and other socio-economic practices. In discussing access to essential public goods, social and economic capacities take priority focus over the impact of ‘place values’ either as standalone or intersectional elements. Research should be expanded to incorporate these elements and their intersectional perspectives in shaping access to water.

HIGHLIGHTS

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• Water sources are loaded with values and specific worldviews.
• Such values shape how water sources are accessed and managed.
• Men and women are affected differently under such circumstances.
• Tradition and resource scarcity value reinforce determination for domination and control of places.
• Strong public intervention can minimise challenges of access and achieve inclusive and universal access.

access, gender, Nigeria, places, twin bearing mothers, WASH challenges

INTRODUCTION

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Research findings widely demonstrate and document the many barriers women face in achieving improvement in access to water for sanitary and hygienic living. Among such barriers are powers of places. Places are never neutral; they are loaded with values and specific worldviews, and imply different consequences depending on context of analysis (Macintyre et al. 2002). Places can be produced, reproduced and transformed in specific ways depending on factors and processes including historical, material and political practices, administrative processes, environmental/geographical circumstances, social norms, religious beliefs, resource and scarcity values, among others. Underpinning these factors and processes are the dynamics of power relationships – the determining factors of decision processes that shape access to resources and opportunities in and around places. According to Cummins et al. (2007) geography, public regulations and intervention and relational network play a critical role in shaping access to public goods. In their submissions, ‘access to goods, services and other assets may be dependent partly on the geographical disposition of facilities and their jurisdictions but also on social networks and social power, interventions of various actors and degrees of regulation which produce layers of resources accessible to different members of local populations in different ways…’ (p. 1828). Religious factors and traditional norms seem to be missing in this consideration, yet they are critical to shaping access to public resources depending on ‘place’ realities.

Places have hosted traces of meanings, values and ideologies, allowing for tangible expression of power through symbolic traces and transformative practices (Anderson 2010). Such values are reinforced through intentional acts of ordering and bordering and other forms of cultural trace-making, providing justification to protect and defend against transgressional practices. Non-human actors also exert influence through existential, ecological, socio-economic and other forms of support. Geographical/environmental aspects of water resource availability and distribution impact differently on men and women in relation to capacity for access and challenges of every day management routines (van Wijk et al. 1996; Ivens 2008; Akpabio & Brown 2012; Wang et al. 2019). Akpabio & Brown (2012) and Wang et al. (2019) have, in their separate studies, highlighted the challenges women face in accessing water in ecologically difficult or fragile locations (e.g., high mountains, marshy soils, flood risk locations, etc.). The sanitary risks associated with managing waste water at the domestic/private places have also been emphasised.

Places and their meanings have enhanced and reinforced our understanding of gender-based ideologies and their various translations. Who makes decisions on who should have access to (or manage) which places/facilities and their resources, and why, are everyday aspects of gender-based politics of control and domination. Place values and associated exercise of power have regularly featured in everyday acts of access and management of water resources. As Akpabio et al. (2017) explains, ‘while men have absolute control and entitlements to all productive resources of nature, including land and water; women's rights to such resources … are subsumed in a broader web of their roles as wives, which entitles them to the collection, use and domestic management of water …’ (p.63).

Traditions and power relationships are widely perpetuated and expressed through water management practices in view of the perceived role of water in connecting humans with the supernatural. Women have been reported to be severely affected. In Nigeria, Zimbabwe, Rwanda and Congo, women gain access to water on terms set by their men: ‘in our land, tradition forbids a woman in her menstrual period from going to stream or river to have her bath …’ (Akpabio et al. 2017: 64). Place contexts and values mediate and shape community behaviours and associated distribution of power. This carries different implications for men and women. Under such circumstances, women's capacity to secure improvement in sanitary living for themselves and their households is severely limited.

Access to water is accepted as a human right and is critical to achieving the sustainable development goal (SDG 6) and will impact on other elements of good health and wellbeing (SDG 3), empowering women and girl children by minimising everyday pressure to secure household water, while freeing up time for studies (SDG 4), contributing to the goal of achieving gender equality (Goal 5), among other ancillary and public health benefits. Sub-Saharan Africa failed to achieve the millennium development goals (MDGs) targets of 2015, and is currently moving at a slow pace towards the target of achieving universal access to clean water and sanitation by 2030 as set by the SDG. Part of the challenges to achieving the SDG target are related to deep-seated, disproportionate workload imbalances in the course of provision and management of household water (Sorenson et al. 2011; Geere et al. 2018; WBG & GWSP 2019; Dickin & Caretta 2022). This paper intends to contribute to the growing literature by exploring the socio-cultural and gender-based issues associated with place values, and their implications for women's access to water, in addition to providing highlights on the forms of progress that are necessary for improved and inclusive access through case studies of two ecologically distinct communities in Nigeria, namely, riverine and upland locations.

Study area and research methods

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Our project was conducted in two communities: Mbiabet Ikot Udo and Isotoyo in upland northern and riverine southern Akwa Ibom State, Nigeria, respectively (Figure 1).

Figure 1

Study locations: Mbiabet Ikot Udo (Ini Local Government Area) and Isotoyo (Eastern Obolo Local Government Area). Inset: Akwa Ibom State and Nigeria.

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Study locations: Mbiabet Ikot Udo (Ini Local Government Area) and Isotoyo (Eastern Obolo Local Government Area). Inset: Akwa Ibom State and Nigeria.

Mbiabet Ikot Udo is an upland settlement of about 1,200 people in Ikpe clan in Ini local government area of Akwa Ibom State. It has extensive wetland resources which support rice cultivation although other crops such as cucumber, maize, cassava, pepper, Okra, cocoyam, and water yam are additional sources of food and occasional commercial crops especially during the rainy season with high incidence of waterlogging. Two seasonal extremes (a highly flooded rainy season and a much drier season with desiccated soils) dictate farming practices and water supply dynamics, giving rise to wetland farming and an upland agriculture mostly dominated by oil palm production, which happens to serve as the only source of coping for the local farmers. Mbiabet Ikot Udo is, however, endowed with a first-order stream which, at its source, is characterised by gentle slope, low water volume, low velocity and narrow stream width. The stream, which goes by the name ‘idim Affia’, is the only source of water for drinking and other uses. Its main channel is blocked due to deposition of eroded materials of sandy component from the undulating plains. Disruption in its normal flow has rendered the stream stagnant and muddy, encouraging the growth and spread of water hyacinth and algae. It is one such place where women encounter multiple challenges in accessing water, resulting in a dire need for progress in several domains. For instance, the stream is not accessible to women and girls during their menstrual period and by twin bearing mothers. We learned that if this custom is violated, the stream will secrete some irritating substances, making the water not suitable for any use until a sacrifice or traditional incantation is done to appease the gods. Violation of this norm attracts fines involving a goat or other payable items.

Isotoyo is a coastline village settlement in Amasaba clan off the Atlantic Ocean. Amasaba clan is made up of eight villages, namely Isotoyo, Okromobolo, Omobikem, Ama Nglass, Amangbiyi, Ozugbo, Amodula and Bethlehem. It is instructive to note that the present location of Amasaba community is traditionally the home of one of the villages – Isotoyo. The other seven had to relocate from their traditional homes since 2008 having been displaced due to conflict. They speak the Obolo language, though most of the people understand the English language (at least Pidgin) and Ibibio. Over 90% of the people live in houses with the walls and roofs made of thatch mats. Most of the adult men are involved in fishing, a negligible few are employed by oil servicing companies as labourers at oil rigs, while the women are involved in small-scale subsistence farming, periwinkle picking, mat and sheet making, among others, to augment family income. The community depends on a single stagnant pond, which is occasionally affected with high tides, in which case introducing some amount of salt water from the sea. Menstruating and lactating women (of about 3 months of childbirth) are traditionally forbidden from accessing the pond as they are considered spiritually unclean for the gods of the pond. Violation of this tradition will lead to the drying up of the pond until traditional rites are performed to bring it back. Just like in Mbiabet Ikot Udo, the affected women depend on children and relatives to meet their daily WASH needs (Akpabio 2012). When support is not available at the time of need, they are likely to depend on unclean sources and risky reuse of wastewater, exposing them to water-based, water-borne and water-related diseases (White et al. 1972).

While Mbiabet Ikot Udo, to a large extent, depends on pit latrines or open defaecation depending on circumstances, Isotoyo largely depends on various forms of open defaecation on account of poor/marshy soils which do not support the digging of pits, in addition to the absence of publicly funded drinking water facilities. They depend on hanging toilets (designated separately for men and women) which discharge directly into the river. Others defaecate with the aid of locally made wooden boats. Open defaecation is common in the day and night. Women bear the greatest burden because of high cultural values placed on their privacy, imposing on them the daily routine of having to negotiate and engage in risky choices to cater for their sanitation needs (Akpabio & Brown 2012). Our fieldwork interest was focused on the various challenges women face accessing water, sanitation and hygiene (WASH) services and possible remedial options. We explored these issues in a multi-level fieldwork activity that lasted for about 7 weeks beginning from 8 April, 2023, and involved village meetings, in-depth and semi-structured interviews, field observations, focus group discussions, review of secondary and grey literatures and a final workshop and Media chat on the 19 May 2023 and 3 August 2023, respectively. Our first public meetings at Mbiabet Ikot Udo did not achieve much in terms of inclusivity of voices. A similar experience was noticed on the 25 August 2023 at our enlarged community meeting with representatives of Amasaba clan in Eastern Obolo. The meeting at Isotoyo was 98% dominated by men. It was an open session of stories, narratives, clarifications, questions and answers. We later went out in groups on visits to sources of water and toilet facilities. This afforded us the opportunity to get in-depth in our conversations with individuals and groups including women. At the end, 27 individuals were interviewed (18 women and 9 men). Of the 18 women interviewees, seven were mothers of twins. We equally engaged some key informants to have a comprehensive perspective of the issues under investigation. Our workshop involved the representatives of the fieldwork communities, academics, policymakers, the press and the general public. The workshop was used to share the findings, broaden the conversations and seek further inputs from the stakeholders. There were many follow-up meetings and conversations for the two study communities to conclude our month-long fieldwork activities.

Ethics and gender-based power relationship

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Unlike Mbiabet Ikot Udo with a relatively fairer representation of about 42% of women, there is strong gender-based domination and control at Isotoyo with 98% male participants. Women's voices are most likely to be suppressed under such conditions. These were reflected during our various conversations and our interest was to seek to extend a further invitation to women. In an enlarged general meeting, women participants were relatively less vocal and open, in small group contexts their voices became clearer, and on one-on-one basis, they were much more open in their communication of their challenges and concerns at Mbiabet Ikot Udo. At Isotoyo, they were highly supervised. Only one woman was nominated to participate in our conversation. Few of our female team members were however granted permission to interview few women. The information gathered was shared, triangulated and collaboratively discussed. Further clarifications were made through the local informants and phone conversations in follow-up interviews. Although our team members tried their best to gain some insights into the issues under study, there were however some ‘no-go’ areas which were explicitly emphasised by the village authorities. For instance, some aspects of the consequences and ritual practices in response to ‘perceived desecration’ and violation of traditional norms as well as managing the perceived consequences were ‘red flags’. All ethical procedures of anonymity, informed consent, confidentiality and ability to withdraw from participation at any time were observed. Additionally, our team had applied and obtained due ethical approval from the University of Uyo Ethics Committee before proceeding with the study.

Study limitations

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It is important to acknowledge the many limitations encountered during the fieldwork and data collection processes. First, we were unable to achieve free and equal participation of women across the study areas. Women's, participation level was relatively better at Mbiabet Ikot Udo (about 42%), where some women were able to contribute on general issues. Only one woman participated in our meeting with the community at Isotoyo though without any contribution. Our request to increase the number of women participants was turned down. Following some negotiations with the village authorities, our female members in the team were granted limited access to few women for in-depth interviews, though with some level of supervision. Such opportunity helped us gain relatively deeper insights into some aspects of cultural and gender-based traditions that influence access to WASH practices. In all cases across the communities, we noticed that we were able to obtain relatively detailed information at individual rather than general group levels.

Second, there were some ‘red flags’ especially related to details of ritual practices and consequences of traditional infractions. While we were able to achieve relatively open discussions of some consequences of breach of restrictive cultural traditions at Mbiabet Ikot Udo, such areas were ‘red flags’ at Isotoyo: ‘… these are the traditional rules and we can't go beyond that …’, stated one of the clan elders. Sensitive and related issues of this category were addressed through local informants and diaspora natives who participated in some of our meetings and follow-up discussions.

Health issues and outcomes are central to WASH practices. Our research did not explicitly focus on collecting data on health issues, and surveys particularly on endemic diseases and consequences. We focused strictly on socio-cultural and gender-based factors shaping access to WASH practices. Although we did not explicitly focus on health outcomes of WASH practices, some respondents were able to mention and discuss some health issues associated with WASH practices, which have been reflected in our various discussions.

Finally, our study focused on two communities with contrasting ecologies (riverine and upland). The two communities are of different ethnicities and speak different languages though they are broadly identified as belonging to the Ibibios. Given these geographical differences, our study findings cannot adequately represent the complex and highly culturally diverse state of Nigeria. However, they are useful signposts to further research.

FINDINGS

The case of Mbiabet Ikot Udo

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Central to WASH access and associated workload is a single and relatively stagnant stream locally known as ‘idim Affia’ (reddish brown with impurities), the nature of the hydrogeology (static water level is at >55 meters deep before accessing groundwater), and socio-cultural tradition.

Idim Affia- the main drinking water source at Mbiabet Ikot Udo

‘Idim Affia’, literally translated as ‘white stream’, connotes purity and cleanliness even when this qualification is contradicted by its colour transition. The stream is relatively stagnant, encouraging the growth of algae and build-up of debris yet it is the only source of water for drinking, bathing, washing and laundry. Ninety-eight percent of the respondents disapproved of the colour and taste of the stream.

A range of ‘dos’ and ‘don'ts’ for accessing ‘idim affia’ exists such as prohibition from entry in certain market days, restricting women in their menstrual cycle from gaining entry, permanent prohibition of mothers of twin children from entry and use, demarcating different sections for different use purposes. Enforcing these rules, standards and controls as well as managing consequences of breach depend on threat of sanctions, ritual practices, demonstration of evidence of consequences, appealing to the spirit, regular reminders and emphasis on negative consequences and the use of taboos to enforce behavioural conformity. All the mothers of twins interviewed expressed unhappiness over their fate in relation to the ancestral tradition that prohibits their entry and use of the stream. However, they are unable to help themselves.

Ordering and bordering practices as highlighted above produce different consequences for men and women in relation to access, use and associated workloads. Men are traditionally entitled to full access for drinking and sanitary living; women are not: ‘mothers of twins are not allowed to enter ‘idim Affia’ since the time of our forefathers. If there is violation of such arrangement, the colour of the water normally changes…’ this superstitious belief stems from the fear that the spirits associated with twins could wreak havoc on the entire community if their mothers come into contact with the (sacred) stream. Consequently, these women are denied access to the primary water source, forcing them to rely on alternative, often more unsafe, sources. A mother of twins confided and revealed how they manage the situation: ‘This issue can bring quarrels and fights, especially in a household where the male gender is not a cooperative type as you will likely ask him to help you if your children are too young to go to stream, or if you ask him for money to pay someone to help fetch water… but we have to pay, you forgo one and pay for water or you remain like that…’.

Two perspectives on the potability of ‘idim affia’ were observed. The relatively educated respondents expressed concerns on the quality of the stream as their main drinking water source, and in the absence of improvement, they depend on self-remedies including the use of disinfectants to safeguard against water-borne diseases: ‘our water is not clean all the time, so we need to use dettol to disinfect it as we store water in clay pots and jerry cans of different sizes…’, noted a woman in her late 40s who is a teacher in one of the public elementary schools. This was corroborated by a few other respondents with relatively educated and exposed backgrounds. On the contrary, a couple of the respondents rarely questioned the quality of the stream as their drinking water source: ‘this is our main source of drinking water since we settled here…our forefathers depended on this and never had problem…if we were to have problem, you would not see people here…so we are under divine care…God is wonderful!’, noted a male respondent who should be in his early 70s. The believe that the stream can be managed through spiritual and ritual practices support some normative rules and traditional barriers which have the unintended consequences of limiting certain categories of women from enjoying full access. Physical ‘pollution’, ‘dirt’ and every other material practices that are likely to affect the quality of the water rarely attract concerns as the spiritual sources of pollution encapsulated in perceived sanitary taboos of periodic menstrual flow and the perceived abnormality of multiple child birth. These are perceived to be spiritual sources of pollution to the stream's deities.

Toilet systems revolved around pit latrines and open defaecation. Over 90% of the respondents depend on pit latrines and occasionally on open defaecation. Ownership of pit latrines is tied to the availability of land portion within the vicinity of the compound. The smaller the land portion, the closer the pit latrine to human dwellings/kitchens. Over 80% of the compounds visited maintained their pit latrines between 3 and 5 meters away from the house depending on the available land extent: ‘…if the compound is small, you may or may not decide to build a pit latrine…’. In one of the compounds, we observed a pit latrine located a little less than three meters away from the kitchen with makeshift structures. When the landlords were asked about the possibility of disease outbreaks, their responses were mostly based on spiritual explanation and personal experiences: ‘…we have lived here under this condition for years and no problem…God protects us…’, argued a landlord in his late 50s. A single pit latrine could serve as many as three households within a compound. Pressure to use the latrine varies depending on the hours of the day. Early morning queues encourage open defaecation especially in emergency cases and for women who are mostly concerned with protecting their privacy.

The case of Eastern Obolo

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Access to WASH is much more challenging for the Isotoyo (Amasaba) community in Eastern Obolo on account of poor soils, salinity problem, locational disadvantage (a small island) and absence of publicly funded WASH infrastructures as well as poor socio-economic condition of the population. All villagers depend on stagnant sources of water (pond) for drinking. The soil is marshy, and it will be extremely difficult and costly to sustain ground water services and pit latrine. By all indications, most of the available natural sources of water regularly experience salinity problems, while the only source of drinking water (pond) is of deplorable quality: dark reddish in colour and can hardly qualify for use in cleaning and laundry yet it is what passes as a drinking water source.

Stagnant pond at Eastern Obolo

One of the village Chiefs had this to say: ‘…the miracle is that you [referring to us] cannot drink this water and survive…and we have been drinking these sources without any problem…is God not wonderful…’ Occasionally, the available pond gets inundated by salt water from the sea, according to the interviewees.

Different forms of open defaecation were observed including hanging toilet that discharge directly onto the river.

One of the designated toilet places at Eastern Obolo

But in all these men and women maintained separate spots across the muddy soils for defaecation. Rain water and tidal movement occasionally inundate those spots with water, transferring human excreta and other forms of wastes to further pollute the available sources of drinking water: ‘…that is what we drink…in the rainy or dry seasons…we have been suffering on account of poor-quality water and sanitation…’, noted an elderly man. Marshy and poorly developed soils make it difficult and extra costly to build and maintain pit latrines and boreholes. As the villagers noted, it would be very costly to build and maintain water and sanitary structures on account of the soil conditions, and also the cost of transporting materials from the town across the river to the settlements. Any makeshift arrangements for toilets can easily be overwhelmed during heavy rain and tidal flood. On this account, strong public intervention is quite necessary and urgently needed.

Just as in Mbiabet Ikot Udo (Ini LGA), women in their menstrual flow are not allowed access to the available drinking sources of water. Furthermore, lactating mothers can only access those drinking water sources after 3 months of child birth. They insist it is their tradition and must be upheld. Violation of this norms will cause the drinking water to dry off, and can only be remedied through some rituals and traditional sacrifices, which they were not comfortable explaining further to our team. Just as in Mbiabet Ikot Udo, women victims of these traditional norms depend on children, husbands, relatives or villagers to gain access to drinking water while under the period of restriction. The villagers noted they have many other taboos that mediate access to those ponds but were uncomfortable disclosing further to our team. We gathered as well as inferred that women and children perform over 97% of the daily tasks of provision, utilisation and management of water and sanitation. Unlike Mbiabet Ikot Udo, there is strong gender-based domination and control with women's voices most likely to be highly suppressed and participation in such meetings highly supervised. These were reflected during our various conversations and our interest was to seek to extend the invitation to women to participate in the meeting.

Workload, financial and health implications of findings

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Spiritual meanings, cultural values, perceptions and traditional regulations surrounding drinking water sources and places carry different implication for men and women in the perspectives of disproportionate workloads, financial burden, health outcomes, sanitary and hygiene conveniences and family conflicts among other challenges (Table 1).

Table 1

Distribution of water provision, sanitation and hygiene management roles and responsibilities

The bulk (roughly over 80%) of tasks and responsibilities related to provision, use and management of water, sanitation and hygiene rests on women and the girl children while men and boy children dominate in construction, regulation and enforcement practices (Table 1). Water fetching responsibilities belong to women (64%), followed by girls (20%) and boys (13%), and only 3% claimed men are also involved in complementary contexts and critically needy situations. As one woman in her early 40s argued: ‘…it is not the responsibility of men to fetch water…they can only support where there is no help…or in other circumstances…we the women and our girls are responsible…the boy child is also involved especially when others are busy and or no female child…’ (translated).

The weight of rubber varies from 20 to 25 l of water for each person, and many rounds of fetching are needed depending on the size of the household. On average we learnt about 5 rounds of water could be fetched in an hour depending on location and congestion at the source. Specifically, we were informed women spend about 1 h fetching about five rounds of water, but the frequency of fetching rounds could be reduced if men offered to help, but the children can go for as many rounds as possible, which usually works against the possibility of their engaging in academic and other social activities. A round-trip to obtain water from ‘idim affia’ (Mbiabet Ikot Udo) can take about 50 min and can be significantly higher depending on time, season and congestion challenges. For Eastern Obolo, about 8 min for a round-trip. The stream size (as small as less than a square meter) and the need to wait for the settling of impurities stirred up in a single collection makes unusual queues and congestion inevitable: ‘…at this point, we can wait for ages as each person takes turn…and usually there has to be a gap to allow for the settling of the impurities before another turn…’ noted a woman in her early 40s.

Other tasks and responsibilities within the exclusive preserve of women involve the use and management of domestic water, sanitation and hygiene. As can be seen in Table 1, the major tasks include domestic water use and management, trash emptying, dish washing/washing and maintenance of water storage facilities, house cleaning/cleaning of toilets and bathrooms, laundry, child hygiene management and care for the elderly and sick. In most cases, these tasks and responsibilities can be delegated to the girl child, and in limited cases to the boy child depending on circumstances. Constructional, regulatory and enforcement roles/tasks are primarily within the masculine domain, and can be delegated to boy children as a form of mentoring/training responsibilities. Table 1 shows 97, 92, 89 and 41% of responsibilities for providing water-related storage facilities, toilet/bathroom construction and maintenance, regulation/enforcement and maintenance of drinking water sources respectively rest on men. The practice of gendering roles/responsibilities is inherently a patriarchal norm where girl and boy children are prepared and socialised into clearly defined roles and responsibilities, which are reproduced over generations. Being a man carries the mindset of funding the family and performing tasks that are considered energy demanding and risky. It may look odd to find men taking up responsibilities that are the exclusive preserve of the women and vice versa. Tasks and responsibilities exclusively preserved for men or women can only be encroached in circumstances where a single gender dominates in a household.

The financial implications for securing access to drinking water were quantified mostly for women who opted to buy water for drinking and those traditionally excluded from accessing the available sources of water. What we arrived at are the equivalent of daily, weekly, monthly and yearly spend on securing water barely enough for drinking, sanitation and hygiene. For Mbiabet Ikot Udo, the respondents reported an average of as high as NGN150 (USD 0.33)1 daily expenditure, translating to weekly spend of NGN1050 (USD 2.30) and a monthly expenditure of about NGN4500 (USD9.83). In a year, an average of NGN54000 (USD117.90) would have been spent to secure water for drinking, sanitation and personal hygiene. For Eastern Obolo, the figure is slightly higher: about NGN200 (USD0.44), NGN1400 (USD3.06), and NGN5600 (USD12.23) respectively for daily, weekly and monthly expenditures. By implication, about NGN67200 (USD146.7) would have been spent yearly on water for drinking, sanitation and hygiene in a country with a monthly minimum wage of about NGN30,000 (USD65.5). In most cases, this presents a huge financial burden for women without any form of assistance and support. Over 90% of the respondents claimed they cannot afford such a huge spend, which imposes the necessity for economical use of available water even to the detriment of achieving basic sanitation and hygiene needs (Langford et al. 2017).

Poor access to improved sources of water, sanitation and hygiene potentially carries serious health consequences. Although our research did not focus exclusively on health consequences, we were able to infer a number of issues and attitudes through our various discussions including popular perception linking poor sanitation to cholera (utoro) outbreak and their variances. No specific instances of morbidity and mortality were discussed as associated with the water they drink or their toilet system. During our public meetings, extremely few speakers were able to link (in)appropriate WASH practices with health outcomes, with cholera and dysentery (unan) attracting frequent mention: ‘…were you to stay here, you would not drink that water and survive…it is only God that protects us…’, noted an elderly man in his 70s. On further interview at Mbiabet Ikot Udo, two women acknowledged having occasionally used Dettol to protect their families against possible water-borne diseases. Our analysis shows that speakers and respondents with some level of education and exposure were able to discuss the link between WASH practices and health outcomes. However, such discussions depended on our probing especially at Mbiabet Ikot Udo. At Isotoyo, there was some level of awareness among participants of possible health impact. Such awareness probably has to do with the benefit of widespread environmental activism and campaigns against ecological and health impact of petroleum products exploration. Activists probably use the health impacts of occasional oil spillage and other forms of water pollution to inform the community and wider public to reinforce calls for compensation. As in Mbiabet Ikot Udo, they were fatalistic - depending on the supernatural being as the only source of protection: ‘…we have lived here for many years and no one dies for drinking water from these sources…it is only God that protects…’, argued a male respondent in his late 50s. One important indicator of dissatisfaction with the condition of WASH across the study areas was repeated call on public authorities and charity organisations for some interventions. As we inferred, this probably had more to do with the necessity to minimise physical suffering and improve on conveniences than address health related risks: everyone seems to be either talking about spending many hours having to look for water or not having enough water to meet daily needs.

DISCUSSIONS

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Our findings have demonstrated that drinking water sources (e.g., Idim affia, Mbiabet Ikot Udo and other ponds in Eastern Obolo) are a part of the many forms of visible material structures that embody and generate automatic reproduction of gender-based beliefs, attitudes, feelings and practices to support cultural control. Women's biological circumstances and processes of menstruating and birthing are shrouded and associated with spiritual pollution, dirt and uncleanliness, and should not be allowed close touch with material structures (idim affia and others) that also command spiritual and existential values (Douglas 1966). According to Akpabio (2007, 2012), local ideas and perceptions of water sources depend, to a certain extent, on cultural values and spiritual meanings. A menstruating or lactating woman gaining entry into a perceived ‘sacred place’ is seen as deviating from established norms and constitutes a source of pollution (Douglas 1966). These, in addition to morbid fear of consequences by the victims, over the years, have served to achieve spontaneous consent to make life easy probably for the dominant authorities (Gramsci 1971). We have noted in our introductory section that places are saturated with meanings and have hosted traces of values and ideologies, allowing for tangible expression of power through symbolic traces and transformative practices. The traces and narratives around idim affia and the rest collectively symbolise an aspect of the various processes for institutionalising patriarchal structures and norms and reproducing the dominating influence and power of men over women.

The character and power of places are directly and indirectly shaped by social and historical practices tied to a range of political, economic, ethnic and religious histories (Anderson 2010). This validates the assumption that place-making is never neutral as societies consciously and intentionally engage in acts of inscribing and reproducing values and ideologies. By taking and making idim affia and the rest in line with their values, feelings and beliefs, they continuously work to perpetuate, reproduce and reinforce the ideology of patriarchy. As an act of power, the making and reproduction of values points to who controls the decision power and processes of defining and setting terms. Historically, patriarchal societies mirrored in our study communities invest enormous decision and managerial powers in the male gender, which enables them to inscribe and reproduce their version of realities.

Men command near-absolute control and entitlements to almost all productive resources of nature, while women's rights are applicable by virtue of being wives2 mostly at the domestic and private arena. Interestingly, women have been forced to accept their position as naturally given and the possibility of change seems highly impossible. In essence, women are forced to conform to such practices for fear of consequences accompanying breaches including excessive and prolonged bleeding and possible loss of the only source of drinking water. In another perspective, appeal to history has also been the basis for compliance. During our discussions, most of the respondents claimed they inherited the practices and values and must be sustained: ‘…this is what we came to see…and must be sustained…’, argued a female respondent in her early sixties. Dominating traces and pattern of behaviours that face no competing alternatives or resistance become normalised, enable spontaneous consent, and according to Jordan & Wheedon (1995; 5), ‘it is through these [dominating traces] that we learn what is right and wrong, good and bad, normal and abnormal, beautiful and ugly. It is through them that we come to accept that men are better leaders than women…’

Idim affia has historically inserted its existential value to the communities in context of persistent water scarcity and absence of public water services. To allow the continued performance of its ecological, spiritual and existential functions, the necessity for control, policing and protection seems to support cultural acts of ordering and bordering which affect men and women differently. Obviously, the scarcity challenge has reinforced a range of spiritual attachment and cultural practices that are intentionally designed to protect the only sources of drinking water for the people. Water resources availability or lack there of remains critical to the flourishing of social and traditional institutions with a mission to provide the necessary structures for the reproduction of values, ideologies and practices. Obviously, the study communities are confronted with everyday challenges associated with limited water resources availability. According to Ghosh & Bandyopadhyay (2009) and Akpabio (2012), it is the scarcity value of water that commands user interest and encourages intentional mechanisms of management and control. Water scarcity forces communities to invent regulatory control practices and develop norms to protect their resources and sustain their services/functions. According to Saleth (2004), water institution is shaped and largely dependent on the nature and characteristics of the available water resources. Drawing from these theoretical positions, we argue that limited availability of water in time, space, quantity and quality forces the two communities to mobilise all suitable social and cultural resources to regulate access and sustain their capacity for existential and ecological functions. We have noticed that the evolving social and traditional institutions disproportionately affect women than men. Such differences have been explained through the patriarchal perspective.

Possible areas for intervention

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Access to water in the right quantity and quality is critical to improvement in sanitation and hygiene for improved health outcomes. The SDG envisages full access to water and sanitation by 2030, and our findings mean there are many barriers to cross. What forms of progress and interventions do we need and whose duty is it to provide water, minimise gender-based discrimination and promote inclusive access? At the moment, it may be difficult to achieve universal and inclusive access unless there is strong public intervention through massive and sustainable infrastructures. It is highly capital-intensive to develop sustainable and enduring water infrastructures due to high water level, marshy soils and vulnerability to salt intrusion for the riverine locations. The communities can ill afford such capital-intensive investment. The appropriate sub-national government can leverage on the corporate social responsibility (CSR) plans of the oil multinationals operating in the region, in addition to private interest to explore the possibility of building water transfer infrastructures and functional boreholes for the affected communities.

The second angle depends on the software approach, namely legal structures and remedies. Currently local communities depend on social and traditional institutions for managing available resources. Modern laws and policies on gender mainstreaming have not been in place. Rural areas tend to face difficult challenges relative to the urban areas where public and private interests in water services are relatively stronger. There is an urgent need to evolve appropriate laws that promote inclusivity in access to available water resources and services. However, our study findings equally note that laws and policies will hardly produce the desired results unless systematic efforts are in place to cultivate and develop water supply infrastructures to reap the social and economic benefits of improved and universal access (Hutton et al. 2007).

Still related to the software angle is the need for massive awareness campaigns and public education on the public health values of improved drinking water, proper sanitation and sound hygiene. Poor knowledge of WASH–public health nexus limits the capacity of the affected communities to seek improvement options. As the interviewees rarely linked access to improved WASH services with improved health outcomes, the scope for behavioural changes will continue to be narrow. Intense public engagement and media campaigns are clearly necessary to weaken some discriminatory cultural tradition and improve access to water, sanitation and hygiene for women.

CONCLUSIONS

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Places have different meanings and values, and shape perception and attitudes to their uses and management. Our study sets out to explore how socio-cultural and gender-based values are reproduced in places, and their impact on access, use and management of available resources and associated externalities, namely water and sanitation with specific reference to women in Nigeria. Our findings reinforce the idea of non-neutrality of places as physical and material entities. Places are produced, reproduced and transformed in line with specific cultural values and worldviews and this shapes how resources and associated externalities are perceived, used and managed. We situate our analysis in relation to gender-based factors shaping access to water places. Our findings demonstrate that women face different levels of challenges and barriers negotiating access to water for domestic and personal use purposes on account of socio-cultural traditions that reinforce unequal gender-based power relationships. The biological circumstances of lactation and menstruation, and the socio-cultural ideology of ‘being under men's control’ entail different layers of barriers which impact their daily workloads, personal hygiene, finance and health. Finally, in discussing access, interest is excessively focused on capacity to gain benefits but less on the impact of place attributes and values: physical and social distance effects, ecological circumstances and social meanings. Place contexts, social values and public investment intersect to influence who gains access to what facilities and resources. Research should be expanded to incorporate these elements and their intersectional perspectives in shaping access to water.

ACKNOWLEDGEMENTS

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The IHE-Delft Water and Development Partnership programmes, financed by the Dutch Ministry of Foreign Affairs, provided support to our WASH-Gender project. Our ideas received substantial inputs during our special session at the XVIII IWRA World Water Congress, Beijing, China (11–15 September 2023). We acknowledge the benefits of their inputs and suggestions in strengthening this paper. We appreciate the following individuals and communities for their effort and cooperation that enhanced the success of our fieldwork activities. They include Unyime Saturday, Eme Eteke, Joan Imaisong, Karen Abraham, Itohowo Sam, Akanimo Ekpo, Saviouir Udoh, Victory Dennis as well as Mbiabet Ikot Udo and Isotoyo community members. We thank the anonymous reviewers for their efforts and insights that contribute to further strengthening the quality of this manuscript. The ideas are, however, those of the authors.

1

As at the time of our fieldwork, 1USD translated to NGN458 at the official rate

2

As wives, every aspect of domestic water management-food production and preparation, care of domestic animals, household hygiene, washing and disposal-depends on them and in most cases this comes at a cost to other socio-economic engagements.

DATA AVAILABILITY STATEMENT

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All relevant data are included in the paper or its Supplementary Information.

CONFLICT OF INTEREST

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The authors declare there is no conflict.

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© 2024 The Authors

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