Safe drinking water: to what extent are shallow wells reliable?

A stable and sustainable supply of water is still a challenge in rural areas. According to Mooney et al. (2020), groundwater resources are significant sources of drinking water. In Brazil, data from the National Agency of Water and Sanitation (ANA, acronym in Portuguese) stressed the importance of these resources for drinking water supply, because 52% of the Brazilian municipalities make use of them (ANA 2010).

Due to the hydric crisis that the Brazilian population has been facing since 2015, the search for alternative solutions is increasing. As groundwaters have been used extensively as sources of drinking water (Foster et al. 2022; Rama et al. 2022), their microbiological quality should be monitored, especially for those wells that are shallow and unprotected. Brazilian legislation establishes that all drinking water sources are liable to quality control and surveillance through quality monitoring, including those that are off grid which are named as alternative solutions for water supply (Brasil 2021).

In Brazil, according to our legislation (Brazil 2021), all sources of drinking water that are not connected with the distribution network are denominated as alternative solutions. There are two types of alternative solutions: individual and collective. The first is addressed to supply drinking water to single-family dwellings, while the latter is addressed to provide a collective drinking water supply for human consumption. Despite the frequent usage of a collective supply of drinking water in rural areas, including schools and leisure spots, its quality is largely overlooked compared to surface waters, while it can be easily contaminated by fecal matter from both point and nonpoint sources.

Field studies and outbreak investigations indicate that groundwater systems can become significant sources of enteric pathogens (Daniels et al. 2018; Huang et al. 2023), because groundwaters are vulnerable to microbiological contamination through the intrusion of infected feces from livestock by surface runoffs (Wallender et al. 2014; Moreira & Bondelind 2017) and by the inadequate disposal of solid waste, sewage sludge, and domestic sewage, mainly in areas with lack of sanitation along with social vulnerability.

It is well recognized that groundwater sources for drinking water play an important role in transmission of several waterborne pathogens (Woolf et al. 2023): bacteria as pathogenic Escherichia coli, Salmonella, Shigella, and Campylobacter; virus such as enterovirus, rotavirus, and hepatitis A; and parasite protozoa such as Giardia and Cryptosporidium (Gilpin et al. 2020; Stokdyk et al. 2020). Outbreak investigations also suggest that groundwater systems can be a relevant source of these protozoa (Daniels et al. 2018; Chique et al. 2020).

In Brazil, Razzolini et al. (2011) evaluated the probability of infection by Giardia in the consumption of water from shallow wells in a peri-urban area of the Metropolitan Region of São Paulo (RMSP), in which cysts occurred in 62.5% of the samples with concentrations ranging from <0.1 to 36.1 cysts/L. The results demonstrated the vulnerability of people residing in these areas and exposure to waterborne pathogens, with a high risk of infection for Giardia. Santos et al. (2012) and Fletcher et al. (2014) pointed out that giardiasis and cryptosporidiosis are more prevalent in children and also adults associated with environmental quality, more specifically lack of water quality sources. Daniels et al. (2018), in India, demonstrated that groundwater sources for drinking water contaminated by Giardia and Cryptosporidium have a significant impact on children's health in a rural area, even at low concentrations. The authors also pointed out that when there is a lack of protection of certain drinking water sources other waterborne pathogens can be transmitted.

Therefore, the aim of this study was to investigate the level of fecal contamination by searching for the bacterial indicator E. coli and the occurrence of Cryptosporidium oocysts and Giardia cysts in well water samples from a low-income rural area of the Metropolitan Region of Sao Paulo (MRSP), which mainly supply water for an elementary school and a fishing area. According to the WHO & UNICEF (2022), many children are submitted to ill-equipped schools to provide safe drinking water and, consequently, they are exposed to infections due to a lack of a proper drinking water supply. Regarding our study, we extended our concern to leisure areas where families get together for bonding as part of development and well-being. Thus, we believe this study includes Sustainable Development Goals (SDGs) #4 and #6, the first addressed to provide a safe learning environment and the second to ensure access to safe drinking water (6.1), as highlighted by the WHO and the UNICEF report (WHO & UNICEF 2022).

Two specific shallow wells were selected for monitoring the quality of drinking water – one located at an elementary school and the other at a fishing spot intended for leisure (Figure 1). The selection of wells was based on two strategic locations: an elementary school attended by children under 5 years old and a fishing spot, a recreational area that attracts various people. The elementary school represents a sensitive location due to the vulnerability of young children to waterborne contamination, while the fishing spot is a point of population concentration, increasing potential exposure to health risks related to water quality. Besides this, there are animals present in the vicinity. At the school, it was observed that the water from the well passed through a storage tank before reaching a tap that supplies drinking water. Therefore, this point was also included in the monitoring process as the water tank could potentially be a source of contamination.

The hydrogeology of the municipality of Santa Isabel is marked by the presence of fractured aquifers, where groundwater is found in fissures and fractures of the rocks, being crucial for the supply of rural areas. Human activities in Santa Isabel include agriculture, livestock, and rural tourism, which can impact groundwater quality due to the use of fertilizers, pesticides, and the presence of animals near the wells. The development of groundwater in the region is limited by inadequate infrastructure, increasing the dependence on shallow wells for potable water supply. It is emphasized that, in this context, only 44.41% of the municipality of Santa Isabel has access to sanitation services. In comparison, the average for the state of São Paulo is 92.18%, while the national average is 66.95% (SNIS 2021).

Sample collection occurred in two moments because of the COVID-19 pandemic: from September 2019 to March 2020 and from October 2021 to April 2022. The total of water samples collected was 69, as follows: 23 samples were from the well of the elementary school (WES), 23 were from the drinking fountain tap at the elementary school (TES), and 23 samples were from the well of the fishing area (WFA). Fortnightly, 20-L water samples were collected for Giardia and Cryptosporidium according to the USEPA 1623.1 method (USEPA 2012), kept at 4–8°C during transportation, and processed within 24 h. For the fecal indicator E. coli, a 250 mL sample was collected according to APHA (2017) guidelines. Thirteen bovine fecal samples were also collected around the study area (radius <1 km). These samples were stored in a freezer (−20°C) until genotyping analysis.

The enumeration of E. coli was carried out through a chromogenic assay using the Colilert kit (Iddex) according to the Standard Methods for the Examination of Water and Wastewater, Section SM 9223 B (APHA 2017). The results are expressed in MPN/100 mL.

Protozoan parasites were quantified using immunomagnetic separation – immunofluorescence assay following the USEPA 1623.1 method (USEPA 2012). Water samples (20 L) were concentrated through FiltaMax (Idexx^®) and immunomagnetic separation was performed with Dynal Dynabeads^®Crypto-Giardia Combo reagents and equipment. The dissociation of the complex beads, cysts, and oocysts was made by heat dissociation. The slides were stained using fluorescein-conjugated monoclonal antibodies for Giardia and Cryptosporidium (Waterborne, New Orleans/USA) and DAPI (4′, 6-diamidino-2-phenyl-indole, Sigma). The cysts and oocysts were counted by immunofluorescence reaction and confirmed by DAPI fluorescence and differential interference contrast (DIC) microscopy using an Olympus B52 epifluorescence microscope equipped with bright field, phase contrast DIC, and epifluorescence optics. Negative and positive control slides were also prepared. The recovery efficiency of the method was determined by spiking 10 L of purified water samples with EasySeed, according to the instructions of the manufacturer (BTFbio, Australia). The theoretical detection limit (DL) was calculated based on the assumption that at least one (oo)cyst could be detected by the microscopic examination of the slides. A 20-L water sample was concentrated, and the entire packed pellet was examined, so the DL was 1 (oo)cyst in 20 L or 0.05 (oo)cyst per liter. The accuracy and initial precision recovery according to the USEPA method 1623.1/2012 as part of the quality control of the quantification analysis of Cryptosporidium oocysts and Giardia cysts presented initial recovery of 45% for Cryptosporidium oocysts and 48% for Giardia cysts. For the matrix recovery, the values were 48 and 52% for Cryptosporidium oocysts and Giardia cysts, respectively.

The scraping of Cryptosporidium oocysts and Giardia cysts from positive and negative slides was adapted based on the protocols outlined by Di Giovanni et al. (2006) and Nichols et al. (2006). The scraping of samples from the slides was performed using bacteriological loops and heated phosphate-buffered saline tween for washing. Fecal samples were directly submitted for DNA extraction.

Genomic DNA was extracted from Cryptosporidium oocysts and Giardia cysts using the QIAamp DNA Stool Mini^® kit for fecal samples and the Dneasy PowerSoil Kit (Qiagen, Venlo, the Netherlands) for the scraped material from water samples' slides, following the manufacturer's instructions with modifications in the heating time during buffer lysis. Specifically, all samples were heated at 95°C for 10 min. The purified DNA was then stored in a freezer at −20°C until the time of use.

For identifying the zoonotic and anthroponotic species of the parasites, quantitative PCR (qPCR) was carried out for all fecal and water samples. The chosen genes were SSU rRNA for Giardia and 18S rRNA for Cryptosporidium, both widely conserved and with multiple copies in each organism's genome. The primers and probe used for the joint detection of Cryptosporidium parvum, Cryptosporidum hominis, and Cryptosporidum meleagridis (Cry-HPM) were described by Araújo et al. (2018), while modified primers and probes by Yu et al. (2009) were used for the detection of Giardia duodenalis complex. The primers and probes' sequences and qPCR cycling parameters are shown in Table 1. The data collection and analysis were performed using the StepOne Plus Real-Time PCR System (Applied Biosystems™). The reaction were performed in triplicate (20uL) and the parasites were amplified using the following components: 4.9 μL of ultrapure water, 10 μL (1×) of TaqMan^® Environmental 2.0 Master Mix (Applied Biosystems™), 0.16 μM of each primer (Exxtend, Brazil), 0.15 μM of TaqManMGB probe, 2 μL of 10× Internal Positive Control (IPC) Mix, 0.4 μL of 50× IPC DNA (Applied Biosystems™), and 2 μL of template DNA. The amplification protocol consisted of a pre-PCR step at 50°C for 2 min, initial denaturation, and activation step at 95°C for 10 min, followed by 40 cycles of amplification, denaturation at 95°C for 15 s, and annealing and extension at 60°C for 1 min. Real-time PCR was performed in accordance with the MIQE guidelines (Bustin et al. 2009). The inhibition of DNA samples was assessed using TaqMan™ Exogenous Internal Positive Control (Applied Biosystems™) in every experiment. The sample data set and the IPC were compared in each analysis to reduce the risk of false-negative results due to the presence of inhibitors in the samples. Positive controls were included using feces contaminated with (oo)cysts, and negative controls consisted of eukaryotic microorganisms.

The concentrations of (oo) cysts of Cryptosporidium and Giardia in the samples from both periods are shown in Table 3. Regardless of the period of interruption, the frequencies of Cryptosporidium oocysts and Giardia cysts in samples from the well at the elementary school (WES) were 13% (3/23) and 26% (6/23), with concentrations ranging from <0.05 to 11.95 oocysts/L and <0.05 to 32.85 cysts/L, respectively. In the samples from the elementary school drinking water fountain (TES), the frequencies were 21.7% (5/23) for Cryptosporidium and 30.4% (7/23) for Giardia with concentrations ranging from <0.05 to 1.15 oocysts/L and <0.05 to 5.5 cysts/L, respectively. For the shallow well used as a drinking water supply in the fishing area (WFA), Cryptosporidium oocysts in 43.8% (10/23) and Giardia cysts were detected in 47.8% (11/23), with concentrations ranging from <0.05 to 0.2 oocyst/L and <0.05 to 2.65 cysts/L, respectively.

Table 2 shows the results of the quantification of the parasites and frequencies as well. For Giardia, the occurrence frequency was higher than Cryptosporidium in the well and tap at the elementary school at both moments of the study. In the fishing area, both parasites had the same frequency of 83.3% during the first moment of the study. The frequency of occurrence of these pathogens in the wells highlights the imperative for heightened attention and enhanced protection.

There was a particular day (February 17, 2020) in the week leading up to the collection when a significant rainfall occurred within a 24-h period, according to the National Institute of Meteorology (Inmet), the highest for a February month in 37 years. Such rainfall during that period may have influenced the high load contamination in the collected load and the presence of the target genotypes of Cryptosporidium and Giardia.

It is interesting to note that the highest frequency of lower DL values for the parasites were obtained in the second moment of the study, possibly because of the non-use of the wells during the COVID-19 pandemic, which has altered the demanding routine of the shallow wells, resulting in water stagnation and the settling of oocysts. It is worth noting that the potential for infection from both protozoa can cause significant morbidity in childhood diarrhea even at low levels of concentration when present in water sources (Daniels et al. 2018). The frequency of E. coli in the well samples from the elementary school was 47.8%, showing high concentrations ranging from <1 to 1.2 × 10³ MPN/100 mL, while in the drinking water fountain it was 14.3% (3/21) with concentrations varying from 0.1 to 10² MPN/100 mL. In the well at the fishing area, the frequency of the fecal indicator was 61.9%, with concentrations ranging from <1 to 1.0 × 10⁸ MPN/100 mL. Genter et al. (2021) demonstrated in a systematic review and meta-analysis that shallow wells groundwater in low-and middle-income countries (LMIC) are more susceptible to contamination by fecal indicator bacteria such as E. coli, which increases the susceptibility of alternative water supply solutions being more prone to contamination compared to network-connected water sources.

Considering the results by periods of sample collection, the level of contamination by the parasites and indicator E. coli was significant in the first moment of the study for all collection points (Table 3). Such variability may have occurred due to factors such as precipitation, lack of protection of the water distribution systems, and intermittence in the use and circulation of animals in the vicinity of the water reservoirs. In the water fountain tap, perhaps the lack of periodic cleaning is a factor that compromised the quality of the water for consumption. As cattle is a reservoir for diarrheagenic E. coli (DEC), the high frequency of E. coli in wells nearby animal feces makes this contamination worrisome. In Bangladesh, Saima et al. (2023) carried out a study that demonstrated the presence of DEC in 62 out of 169 isolates (37%) at the point-of-drinking water samples, while in the public domain sourced water from 240 isolates, 109 (45%) were identified as DEC.

The municipal health authorities were informed of each positive result for the presence of parasites and/or fecal indicator contamination. The measure was a filter installation in the school's drinking tap water to minimize deficiencies in the quality of water consumed by children, teachers, and school staff. It seems to have been an efficient measure since the frequency of parasites occurrence decreased for a period. Nevertheless, preventive periodic maintenance of the filter was not carried out properly, and after a short period, the parasites and E. coli were detected even in the filtered water. Maintenance measures are important to prevent the formation of biofilms containing pathogens and, at a given moment, their release into the drinking water.

The high frequency of both parasites even with variation of concentrations (Table 3) emphasizes the fragility of the drinking water supply chain. Analyzing the four dimensions of water security issued by UN-Water (Marcal et al. 2021), one of them – drinking water and human health well-being – is clearly compromised, which means children's and other users' health might be jeopardized by the consumption of this water.

Our results revealed the presence of anthroponotic species of Cryptosporidium and G. duodenalis in drinking water, indicating a serious concern. Specifically, in the well water at the elementary school (WES), the target gene 18S rRNA for Cry-HPM was detected in 52.2% (12/23) of the samples while the SSU rRNA gene for G. duodenalis complex was detected in 13% (3/23). Regarding taps at the elementary school (TES), the presence of Cry-HPM was detected in 43.5% (10/23) of the samples, and G. duodenalis in 4.34% (1/23). In the well at the fishing area (WFA), the genetic target for Cry-HPM was detected in 30.4% (7/23) of the samples, and G. duodenalis was detected in 8.71% (2/23) of the samples. It is worth reinforcing that at the municipality of Santa Isabel the predominant land use is pasture (54.28%), which is an important factor for the dissemination of pathogens. Breternitz et al. (2020) showed that municipalities with lower sewer connections, larger urban areas, and livestock activities exhibited high concentrations of Cryptosporidium and Giardia in body waters. Lal et al. (2013) demonstrated the influence of environmental factors on the risk of transmission of (oo)cysts, highlighting the role of land use in the incidence of cryptosporidiosis and giardiasis.

These data reinforce the fragility of the whole water supply system. According to the review on waterborne outbreaks by Moreira & Bondelind (2017), Cryptosporidium was one of the etiological agents associated with groundwater contamination events, with the main causes of contamination being the intrusion of animal feces or wastewater due to heavy rainfall.

In this study, the findings reveal the presence of (oo)cysts of Cryptosporidium and Giardia in the wells and drinking tap water at the elementary school (Table 2). Studying shallow well water quality (n = 96) in rural area of India, Daniels et al. (2018) found Cryptosporidium oocysts in 5% and Giardia cysts in 17% of samples analyzed. In rural areas, the gravity of the issue escalates due to the insufficiency of proper sanitation infrastructure. The incidence of diarrheal diseases can be attributed to the shortcomings in sanitation services and systems, which include management aspects such as water supply, sanitary facilities, waste management, stormwater control, vector proliferation, and subpar housing conditions (NSS 2021). According to the data from the studied municipality, there have been 471 hospital admissions related to environmental sanitation deficiencies in Santa Isabel from 2007 to 2021 (DataSUS 2020).

In the fecal cattle samples, which were collected nearby the shallow wells, Cry-HPM was detected in 84.6% (11/13) of the samples, while G. duodenalis complex was detected in 46.1% (6/13). The presence of these pathogens in cattle feces near shallow wells raises concerns about the possible contamination of water sources and the subsequent risk of transmission to humans and other animals. This situation highlights the importance of monitoring and controlling these parasites to prevent potential outbreaks and protect public health in rural areas such as Santa Isabel, SP. Ryan et al. (2016) emphasize the One Health issue of understanding the transmission routes of zoonotic species of Cryptosporidium that are connected to animal health and the environment as well, which is accentuated by poor sanitation. Moreover, Ryan et al. (2016) stated that the lack of diagnosis in LMIC has resulted in limited knowledge of the burden of cryptosporidiosis leading to ineffective public health management of Cryptosporidium. The high prevalence of diarrheal diseases represents a considerable public health challenge, especially in LMIC (Khalil et al. 2018). In Brazil, cases of infectious diarrheal disease account for a considerable proportion of young children, and the protozoa Cryptosporidium and Giardia represent a significant proportion of these infections (Castro et al. 2015).

Brazilian law mandates quality control and surveillance, including monitoring, for all drinking water sources, even off-grid sources referred to as alternative water supply solutions. In rural regions like the municipality of Santa Isabel, with no water distribution network, no sanitary sewage collection, and the presence of animals that are potential reservoirs of (oo)cysts is a critical situation.

According to Wallender et al. (2014), more than 36 million people in the world use an untreated groundwater source as their primary drinking water supply. As observed in this study, shallow wells intended for human consumption are not considered safe based on the results of the presence of feces indicator anthropic species of protozoa parasites. The detection of Cryptosporidium hominis-parvum-meleagridis and G. duodenalis in waters from wells used for drinking water supply in a low-income rural area of the MRSP, which mainly supplied an elementary school and a fishing area, raises an alert due to their potential for infection in humans, especially for children in the elementary school who consume this water.

Our results suggest that runoff significantly impacted the microbial quality of water during the rainy months of November and December (Table 3) as indicated by elevated concentrations of parasites, particularly Giardia. Additionally, the presence of livestock in the vicinity of shallow wells may have further contributed to the degradation of water quality. To enhance water quality, a coordinated approach involving various health surveillance sectors is essential. This includes the regulation and monitoring of wells by environmental surveillance authorities and the reinforcement of sanitary barriers in accordance with WHO recommendations (WHO 2018).

Our study contributes relevant information about a possible transmission chain between the animals present in the rural area, groundwater for consumption, and children, tourists, and residents of the municipality of Santa Isabel, SP. Based on our findings, we offer significant contributions to the Water Safety Plans elaboration in vulnerable regions with vulnerable people, particularly concerning sanitary surveillance. We propose several critical steps to improve future research and water safety interventions. First, expanding the geographical and temporal scope of studies is essential. Future research should encompass a greater number of water sources and be conducted over longer periods to account for seasonal variations in contamination levels. This approach will enhance the robustness of the results in order to provide evidence-based recommendations to ensure the safety of drinking water.

Anthropic species of Cryptosporidium and Giardia were detected in groundwater sources and a tap, utilized for drinking, situated in a primary school and a fishing area within a rural area of Santa Isabel municipality, São Paulo. The findings underscore the urgent need for effective public policy strategies and interventions to mitigate the exposure to these pathogens. This includes ensuring proper waste disposal; implementing protection barriers to the water supplies to minimize contamination sources; and establishing regular surveillance of drinking water quality. Public education and awareness about the risks associated with consuming contaminated water and the importance of proper hygiene practices are also crucial. By implementing these public policy strategies, the risks posed by waterborne pathogens in rural regions such as Santa Isabel can be effectively addressed, contributing to a safer and healthier community.

We would like to thank FAPESP grant #2018/26246-0 São Paulo Research Foundation (FAPESP) for the financial support. This study was financed in part by the Conselho Nacional de Desenvolvimento Científico e Tecnológico – Brasil (CNPq) – (168983/2018-4).

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

The authors declare there is no conflict.