Insufficient Chlorination as the Critical Determinant of Microbiological Non-Compliance in Rural Water Systems: First Integrated Assessment in Azuero, Panama
Astrid Moreno 
* 1, Michelle Aguirre 1, Alexis De La Cruz L 1
* 1, Michelle Aguirre 1, Alexis De La Cruz L 11 University of Panama, Azuero Regional University Center, Faculty of Natural, Exact and Technological Sciences, Panama.
michelle.aguirre@up.ac.pa
alexis.delacruz@up.ac.pa
Correspondence author: astrid-y.moreno-g@up.ac.pa
ABSTRACT
Access to safe drinking water in rural communities depends heavily on day-to-day system operation and sustained disinfection, both of which are often fragile. Although global reports describe widespread microbiological contamination of rural water supplies, there remains a lack of integrated studies that pinpoint the specific drivers of failure in regions such as Azuero, Panama. In this cross-sectional study, we evaluated rural water systems in the municipalities of Chitré, La Villa de Los Santos, and Santa Ana, using a combination of microbiological and physicochemical indicators. Total coliforms, Escherichia coli, and nematodes were assessed by membrane filtration and microscopic examination, while pH and free residual chlorine were measured in situ.
Nematodes were not detected in any sample. In contrast, total coliforms were detected at all sampling points, with concentrations ranging from 1 to 4,556 CFU/100 mL, clearly above the regulatory limit (<1 CFU/100 mL). E. coli was found at three sites (47–110 CFU/100 mL). Although pH values complied with national standards, free residual chlorine was frequently absent or below the recommended range. No significant differences in bacterial loads were observed between tanks and wells (p > 0.05), but a weak negative correlation between residual chlorine and total coliforms (r = –0.25) suggests that inconsistent chlorination is the main factor associated with microbiological non-compliance.
This work provides the first integrated assessment of rural water systems in the Azuero region, jointly analysing microbiological, physicochemical, and operational information. The results show that inadequate chlorination—rather than infrastructure type—undermines water quality, and they point to an urgent need to reinforce community-level disinfection practices, preventive maintenance, and continuous monitoring in rural Panama.
Keywords: Drinking water quality, Rural aqueducts, Total coliform bacteria, Escherichia coli contamination, Chlorination failure, Free residual chlorine, Microbiological water monitoring, Community water systems, Waterborne pathogens, Panama
INTRODUCTION
Access to microbiologically safe drinking water is still a major public health challenge, particularly in rural areas where supply systems often operate with limited resources and technical support. The World Health Organization (WHO) estimates that about 2 billion people worldwide rely on drinking-water sources contaminated with fecal matter, which contributes to the burden of diarrhoeal disease, dysentery, and cholera1. Escherichia coli is widely used as the primary indicator of recent fecal contamination, and its detection in drinking water suggests the possible presence of enteric pathogens2. The WHO Guidelines for Drinking-Water Quality therefore specify that both coliform bacteria and E. coli must be absent in 100 mL samples intended for human consumption1.
In many low- and middle-income countries, rural water systems frequently fail to meet these microbiological criteria. Recent global assessments indicate that up to 70% of such systems show some degree of contamination5,17, mainly due to weaknesses in disinfection, lack of routine maintenance, and the vulnerability of small-scale infrastructure6,7. The United States Environmental Protection Agency (EPA) likewise emphasises the role of free residual chlorine as the principal barrier against microbial contamination in decentralized supplies, recommending that residual concentrations be maintained within a narrow range to ensure effective inactivation of bacteria3,15.
Panama has adopted national regulations such as DGNTI-COPANIT 21-2019 to govern drinking-water quality, but applying these standards in community-managed rural systems remains difficult in practice. Studies carried out in Azuero have reported the presence of coliforms and fluctuations in residual chlorine in local aqueducts, where populations are supplied mainly through wells and storage tanks that do not always receive systematic technical supervision8. Similar situations have been described elsewhere in Latin America, where microbiological contamination is often linked to irregular chlorination, leaky or precarious connections, and the absence of regular cleaning of storage infrastructure9,18.
Against this backdrop, the present study is novel in two ways. First, it represents the first integrated assessment of rural water systems in the Azuero region, simultaneously examining microbiological, physicochemical, and operational variables. Second, it explicitly tests the hypothesis that the key determinant of regulatory failure is not the type of infrastructure (tanks versus wells), but inadequate operational management, particularly insufficient free residual chlorine. By combining field measurements with a detailed comparison with international standards, this work generates robust, actionable evidence to inform public health policy and community water-safety interventions.
MATERIAL AND METHODS
Study Design
A descriptive, cross-sectional study was conducted to evaluate physicochemical and microbiological water quality in rural supply systems located in the Azuero region of Panama. Sampling was performed during a single field campaign and included community-managed storage tanks and wells from the localities of La Arena, Chitré, Monagrillo, Santa Ana, and Los Santos. The study focused on detecting total coliforms, Escherichia coli, and nematodes, and on measuring pH and free residual chlorine.
Sampling Sites
Five representative rural communities were selected based on population relevance, accessibility, and their dependence on decentralized water systems. Coordinates were recorded following GPS verification.
Table 1. Sampling points in the Azuero region.
Sample Collection
Two volumes were collected at each sampling point using sterile sampling bottles:
· 300 mL for nematode detection.
· 100 mL for bacteriological analysis of total coliforms and E. coli.
Samples were collected following aseptic procedures, ensuring no contact between bottle, hands, or external surfaces. Each bottle was labeled with the sample code, date, site, and time of collection.
During sampling, pH and free residual chlorine were measured in situ using a DPD colorimetric kit (Hach® or equivalent), following manufacturer instructions and standards for drinking water field monitoring.
Samples were transported to the Environmental Analysis Laboratory of Chitré in insulated coolers at 4 °C and processed within 6 hours of collection.
Microbiological Analysis
Detection of Nematodes
A 300 mL water sample was filtered through a 0.45 µm cellulose membrane using a sterile filtration system.
The membrane was then placed into a sterile container with distilled water and examined under optical microscopy (10× and 40× objectives) to determine the presence or absence of nematodes.
The membrane was then placed into a sterile container with distilled water and examined under optical microscopy (10× and 40× objectives) to determine the presence or absence of nematodes.
Total Coliforms and Escherichia coli
A 100 mL sample was filtered through a 0.45 µm sterile membrane, which was transferred onto a chromogenic medium (Colilert®, m-ColiBlue24®, or equivalent). Plates were incubated at 37 °C for 24 hours.
Colonies of total coliforms and E. coli were counted and expressed as CFU/100 mL, following color-based differentiation outlined in the chromogenic protocol.
Colonies of total coliforms and E. coli were counted and expressed as CFU/100 mL, following color-based differentiation outlined in the chromogenic protocol.
Physicochemical Analysis
pH and free residual chlorine were measured in the field immediately after sample collection.
Results were compared against the national drinking water standard DGNTI-COPANIT 21-2019, which establishes:
Results were compared against the national drinking water standard DGNTI-COPANIT 21-2019, which establishes:
· pH: 6.5–8.5
· Free residual chlorine: 0.3–0.8 mg/L
Statistical Analysis
Data processing was performed using standard statistical procedures:
· Shapiro–Wilk test was used to assess data normality.
· Student’s t-test compared microbiological values between tanks and wells.
· Pearson correlation coefficient (r) evaluated the association between free residual chlorine and total coliform levels.
A significance threshold of p < 0.05 was applied.
Ethical Considerations
No human subjects or laboratory animals were involved in this study.
Water samples were collected from public rural water systems following environmental monitoring practices and did not require institutional ethics approval.
Water samples were collected from public rural water systems following environmental monitoring practices and did not require institutional ethics approval.
RESULTS
Sampling Locations
Five rural communities in the Azuero region were evaluated. Their coordinates and geographic positions are shown in Table 1, and their spatial distribution is illustrated in Figure 1.
Figure 1. Geographic distribution of the five rural sampling sites evaluated in the Azuero region of Panama.
The map shows the locations of La Arena, Chitré, Monagrillo, Santa Ana, and Los Santos, where physicochemical and microbiological water quality parameters were assessed. All sites exhibited microbiological non-compliance, highlighting a widespread and systemic issue in rural water systems.
Physicochemical Parameters
Most physicochemical parameters complied with DGNTI-COPANIT 21-2019 requirements, except for free residual chlorine, which exhibited substantial variability and recurrent non-compliance.
In storage tanks, pH ranged from 6.8 to 7.8 (mean 7.13), while free residual chlorine ranged from 0 to 3.0 mg/L (mean 0.5066 mg/L).
In wells, pH was slightly more stable (6.8–7.2; mean 6.96), and free residual chlorine levels were consistently low (0–0.2 mg/L; mean 0.12 mg/L).
In wells, pH was slightly more stable (6.8–7.2; mean 6.96), and free residual chlorine levels were consistently low (0–0.2 mg/L; mean 0.12 mg/L).
These results are detailed in Table 2.
Table 2. Physicochemical parameters in tanks and wells compared with DGNTI-COPANIT 21-2019 standards.
Microbiological Parameters
Microbiological analysis revealed universal contamination.
Total coliforms were detected in 100% of samples, exceeding the permitted limit (<1 CFU/100 mL) in all locations.
Total coliforms were detected in 100% of samples, exceeding the permitted limit (<1 CFU/100 mL) in all locations.
- Tanks: 1–4,556 CFU/100 mL
- Wells: 110–387 CFU/100 mL
Escherichia coli was detected at three sampling points:
- Minimum: 47 CFU/100 mL (well 2)
- Maximum: 110 CFU/100 mL (tank 9)
- Overall median: 88 CFU/100 mL
Mesophilic heterotrophs exceeded the DGNTI-COPANIT 77-2007 limit in one tank (581 CFU/mL).
A consolidated summary is provided in Table 3.
Table 3. Summary of microbiological results in rural water systems (Azuero region).
Comparison with Regulatory Standards
All water sources exceeded DGNTI-COPANIT 21-2019 microbiological limits.
Regulatory thresholds are shown in Table 4.
Regulatory thresholds are shown in Table 4.
Table 4. Microbiological regulatory limits (DGNTI-COPANIT 21-2019; DGNTI-COPANIT 77-2007).
Nematode Analysis
Microscopic examination revealed no presence of nematodes in any of the 300 mL samples.
Statistical Analysis
Normality was confirmed using the Shapiro–Wilk test for total coliforms and E. coli.
A Student's t-test indicated no significant differences between tanks and wells (t = 0.50465; p = 0.61993).
Pearson's correlation demonstrated a weak negative association between free residual chlorine and total coliform levels (r = –0.2505).
This relationship is visually represented in Figure 2.
Figure 2. Scatterplot showing the relationship between free residual chlorine and total coliform concentrations.
DISCUSSION
The findings of this study show that all analysed samples contained total coliforms and that E. coli was detected at several sites, indicating clear non-compliance with WHO recommendations, which require the complete absence of these indicators in water intended for human consumption1,2. This pattern mirrors reports from other rural settings around the world, where microbial contamination of small systems is common and is frequently linked to shortcomings in disinfection and day-to-day operation5,17.
In our study, pH values remained within acceptable limits, suggesting that the main problem is not related to major physicochemical imbalances but to the instability of free residual chlorine. Residual chlorine was often absent or below the recommended range, particularly in wells, which is consistent with the EPA's view of chlorine as the primary defence against microbial contamination in decentralized systems3,15. Studies from Africa, Asia, and Latin America have similarly documented that decreases in residual chlorine are associated with higher levels of coliforms and E. coli in rural supplies6,7,18.
The presence of E. coli at three sampling points is particularly relevant from a public health perspective, as it reflects recent fecal contamination and indicates an increased risk of waterborne disease. Global estimates attribute millions of cases of gastrointestinal illness each year to untreated or poorly disinfected rural water systems10,17. The absence of significant differences between tanks and wells in bacterial load suggests that infrastructure type is not the main driver of risk. Instead, what appears to matter most is how systems are operated—whether chlorination is consistent, whether tanks are cleaned, and whether distribution networks are protected from intrusions6,7. This interpretation is consistent with previous observations in Panama and other Latin American countries, where community management often lacks clear, standardised protocols for chlorine handling and infrastructure maintenance8,18.
A key contribution of this study is to demonstrate that inadequate chlorination is the critical factor associated with microbiological non-compliance in the evaluated systems. International assessments have shown that even when pH and turbidity values meet guideline levels, microbiological quality may still fail if residual chlorine is not maintained within recommended ranges1,3,5. The negative correlation between residual chlorine and total coliforms observed here, although modest, aligns with studies describing highly variable conditions in rural systems, where sediment accumulation, biofilm development, infiltration, and lack of cleaning can all reduce the effectiveness of chlorine6,7,18.
Finally, the absence of nematodes in the samples analysed is consistent with the low prevalence typically reported for deep or semi-protected supply systems. Nevertheless, targeted surveillance of these organisms remains relevant in areas where water sources may be exposed to surface runoff or other environmental inputs1,7.
Implications for policy and practice
The results of this study have clear implications for the management of rural water services in Panama and similar contexts. Demonstrating that tanks and wells show comparable microbial loads challenges the common assumption that the source itself is the main determinant of risk. Policies focused solely on constructing new wells or storage structures are therefore unlikely to solve the problem if operational practices do not improve at the same time. Given limited resources, priority should be placed on strengthening community operators' skills and ensuring a reliable supply of disinfectants.
Identifying insufficient chlorination as the central weakness enables targeted, measurable interventions. Rather than generic calls to "improve water quality", the evidence supports three concrete lines of action: (i) standardised training programmes based on WHO Water Safety Plans, emphasising correct dosing, verification of residual chlorine and corrective actions; (ii) simplified community monitoring, using affordable DPD kits so that users can check residual chlorine daily; and (iii) mandatory preventive maintenance, including regular cleaning and disinfection of storage tanks to limit sediment and biofilm accumulation that consume residual chlorine and shelter bacteria.
CONCLUSIONS
In summary, this study shows that the microbiological quality of water in the evaluated rural systems in Azuero is inadequate, with universal detection of total coliforms and sporadic occurrence of Escherichia coli. Basic physicochemical parameters such as pH met national standards, but residual chlorine was frequently absent or below the recommended range. Taken together, the findings indicate that the main problem is not the type of infrastructure but inconsistent chlorination and insufficient operational management of community systems. Strengthening disinfection protocols, standardising chlorination procedures, ensuring periodic cleaning of tanks and wells, and establishing continuous monitoring programmes are essential steps to secure safe water, reduce the burden of waterborne disease, and improve local stewardship of rural water supplies in the Azuero region.
Author Contributions
Conceptualization, A. De La Cruz; methodology, A. De La Cruz; sample collection, A. Moreno and M. Aguirre; laboratory analysis, A. Moreno and M. Aguirre; data curation, A. Moreno; formal analysis, A. De La Cruz; writing—original draft preparation, A. Moreno and M. Aguirre; writing—review and editing, A. De La Cruz; visualization, A. Moreno; supervision, A. De La Cruz.
All authors have read and approved the final version of the manuscript.
All authors have read and approved the final version of the manuscript.
Funding
This research was financially supported by the Environmental Analysis Laboratory of Chitré (Operating Notice No. 8-707-1000-2019-633249).
The APC was also funded by this institution under the BioNatura Institutional Publishing Consortium (BIPC).
The APC was also funded by this institution under the BioNatura Institutional Publishing Consortium (BIPC).
Institutional Review Board Statement
Not applicable. This study did not involve human participants or laboratory animals. All water samples were collected from rural community water systems following standard environmental monitoring practices.
Informed Consent Statement
Not applicable. No human subjects were involved in the study.
Data Availability Statement
All data generated in this study were obtained directly from field sampling and laboratory analyses conducted by the research team.
No external datasets were used. Additional details are available upon reasonable request from the corresponding author.
No external datasets were used. Additional details are available upon reasonable request from the corresponding author.
Acknowledgments
The authors express their gratitude to the Environmental Analysis Laboratory of Chitré for providing technical support, laboratory facilities, and supplies throughout the development of the physicochemical and microbiological analyses.
The authors also acknowledge community water board members in La Arena, Chitré, Monagrillo, Santa Ana, and Los Santos for granting access to the sampling sites.Conflicts of Interest
The authors also acknowledge community water board members in La Arena, Chitré, Monagrillo, Santa Ana, and Los Santos for granting access to the sampling sites.Conflicts of Interest
The authors declare no conflict of interest.
The funders had no role in the design of the study, in the collection, analysis, or interpretation of data, in the writing of the manuscript, or in the decision to publish the results.
The funders had no role in the design of the study, in the collection, analysis, or interpretation of data, in the writing of the manuscript, or in the decision to publish the results.
Data and AI Disclosure
All figures and tables were created by the authors using data obtained through field sampling and laboratory analyses described in this manuscript.
Figures 1–3 were generated, edited, or redesigned using author-supervised AI-assisted workflows (OpenAI), strictly for visualization and language editing purposes. No AI system generated scientific data, processed raw measurements, or produced original scientific interpretations.
Figures 1–3 were generated, edited, or redesigned using author-supervised AI-assisted workflows (OpenAI), strictly for visualization and language editing purposes. No AI system generated scientific data, processed raw measurements, or produced original scientific interpretations.
Generative artificial intelligence was used only for language polishing, grammatical correction, and formatting standardization, always under complete human supervision. The authors independently verified all scientific results, interpretations, and conclusions in accordance with BioNatura Journal’s policy on AI-assisted content (https://bionaturajournal.com/artificial-intelligence--ai-.html).
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Received: 20 Sep 2025 / Accepted: 27 Nov 2025 / Published (online): 15 Dec 2025 (Europe/Madrid)
Citation: Moreno A, Aguirre M, De La Cruz A. Insufficient chlorination as the critical determinant of microbiological non-compliance in rural water systems: first integrated assessment in Azuero, Panama. BioNatura Journal. 2025; 2(4): 15. https://doi.org/10.70099/BJ/2025.02.04.15
Additional Information
Correspondence should be addressed to: astrid-y.moreno-g@up.ac.pa
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