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Article

From Quality to Purpose: Rethinking Groundwater Microbiological Standards for Emergency Urban Water Use

by
Pedro Teixeira
1,2,*,
Sílvia Costa
1,
João Brandão
2,3,† and
Elisabete Valério
2,3,†
1
Municipal Directorate for Environment, Green Infrastructure, Climate and Energy, Bromatology and Water Laboratory, Lisbon Municipality, Avenida Cidade do Porto S/N, 1700-111 Lisbon, Portugal
2
cE3c—Center for Ecology, Evolution and Environmental Changes, Faculty of Sciences of the University of Lisbon, 1749-016 Lisbon, Portugal
3
Department of Environmental Health, National Institute of Health Doutor Ricardo Jorge, Avenida Padre Cruz, 1649-016 Lisbon, Portugal
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Water 2025, 17(22), 3329; https://doi.org/10.3390/w17223329
Submission received: 17 October 2025 / Revised: 13 November 2025 / Accepted: 18 November 2025 / Published: 20 November 2025
(This article belongs to the Special Issue Urban Water Pollution Control: Theory and Technology, 2nd Edition)
Versions Notes

Abstract

Climate change and increasing water scarcity are driving the need for resilient and fit-for-purpose urban water management. This study presents a case from Lisbon, Portugal, where twenty-one groundwater sources were evaluated as potential alternative supplies for emergency drinking and non-potable uses. Between 2018 and 2022, 127 samples were analyzed for microbiological (Escherichia coli, enterococci, fecal coliforms, heterotrophic plate count, Pseudomonas aeruginosa and Legionella pneumophila, physicochemical and fungal parameters (filamentous and yeast), alongside with microbial source tracking (MST) to determine contamination origins. Most sites showed exceedances of fecal indicators and heterotrophic bacteria, making water unsuitable for direct consumption without treatment, while fungi were ubiquitous and often above proposed guidance levels, highlighting a major regulatory gap. MST results indicated that human-derived contamination was rare and highly localized. Physicochemical parameters generally met legal thresholds, although occasional nitrate or salinity elevations reflected agricultural or coastal influences. Several sources were considered suitable for irrigation (EF, CC, AB, VF, and BS) whilst a subset met the criteria for potable supply with minimal treatment for risk management (CG, MM, CC, QC, EB, GR, PO, and MS). The findings of this study demonstrate that systematic, multiparametric assessment supports adaptive water allocation and emergency planning, aligning with EU regulations and advancing Sustainable Development Goal 6. The study argues for reconsideration of current microbiological standards, to improve public health protection in urban water reuse and emergency supply strategies.

1. Introduction

The latest report on climate change from the Intergovernmental Panel on Climate Change [1] summarizes the current knowledge of its widespread impact, risks, mitigation, and adaptation [1]. Approximately half of the world’s population currently faces severe water scarcity for at least a part of the year due to climatic and non-climatic drivers [1]. In urban settings, impacts on infrastructures like water and sanitation have been observed, associated with factors such as increasing mean temperatures, sea level rise, extreme precipitation events, and drought [1]. Climate responses and adaptation must therefore necessarily include efficient water resources management. In this context, groundwater represents a significant but often underutilized source of freshwater in urban areas that can supplement surface-water supplies, buffer the impact of droughts or infrastructure failure, and enhance urban water systems resilience [2,3,4].
In this study, we present the case of water resources management in Lisbon, the capital city of Portugal. Lisbon has about 576,000 inhabitants [5] and consumes annually about 50 million of m3 of drinking water [6]. Most of this water is transported from over 100 km away, from the Castelo do Bode reservoir (a large artificial lake). In addition to the distance from any freshwater sources, challenges for this urban center include the need to expand the urban green areas and manage the pressure of a growing population and economy. Lisbon Municipality is not only the largest water consumer in the metropolitan area but also uses most of its water for non-potable purposes, particularly for the irrigation of green spaces [6]. Ensuring sustainable water resources management requires thus a continuous search for alternative water supplies within efficient urban planning, always guided by a fit-for-purpose approach.
The concept of the study presented in this article was conceptualized in 2018, to support the municipal civil protection services in developing an emergency plan for the supply of drinking water in Lisbon, in the event of extreme water scarcity or as result of a catastrophe. Since then, what was originally designed as an emergency response based on hypothetical climate change scenarios has turned into a reality, with water shortages becoming increasingly frequent [7]. The initial objective of the study expanded to incorporate not only possible groundwater abstraction sites suitable for human consumption, but also to supply non-potable uses, such as irrigation and industrial use or street cleaning. We have selected several groundwater sources from Lisbon region (twenty-one different ones) that could potentially be used as alternative water sources to comply with the emergency plan and evaluated relevant microbiological and chemical parameters in order to evaluate their suitability.
Detecting Fecal Indicator Bacteria (FIB), such as Escherichia coli (E. coli) and Enterococcus spp., is usually the focus of routine water quality assessment [8,9,10,11,12,13,14,15]. However, this approach has several limitations: (1) FIB presence is not always well correlated with pathogen existence [16,17,18,19,20,21]; (2) FIB can survive and grow in sediments [19,22,23,24]; (3) FIB is mainly excreted in the feces of all warm-blooded animals, which does not allow to draw conclusions on the source of contamination and apply the adequate correction measures [25,26,27,28]; and (4) fungi are not part of any water quality current regulation, except for drinking water in Sweden [29]. In water bodies, pathogenic agents may appear as the result of fecal contamination, posing significant risk for human health and therefore impairing water use for a wide array of purposes. Sources of fecal contamination may include wastewater discharge and stormwater runoff [30,31,32], wildlife and domestic animals [33,34,35,36] wastewater treatment plants and draining systems failure [33,37], or septic systems leakages [38]. Besides fecal pollution of water bodies, contamination with pathogenic agents may occur with other microorganisms indigenous to aquatic environments such as Legionella spp., Pseudomonas aeruginosa, Vibrio spp., Helicobacter pylori, Aeromonas spp., Mycobacterium avium [12,39,40], and several fungi [29,41].
To overcome this gap, microbial source tracking (MST) tools targeting host-associated molecular marker genes have also been developed to resolve fecal contamination of different sources such as human dogs, chicken, cattle, pigs, seagulls, possums, ducks, horses [42,43], but also virus, bacteria, fungi, or protozoa [44,45,46,47]. Considering that human fecal contamination presents a greater risk to human health, many MST methods focus on this kind of contamination [48,49,50].
A very significant number of fungal species have been detected in drinking-, surface-, and groundwater, in Europe alone, over the past 30 years [29]. Opportunistic fungal pathogens are among some of the fungi detected in water, including the highly relevant for public health Aspergillus fumigatus, Candida parapsilosis, and Candida albicans. Some fungal species tend to populate or generate biofilms in water surfaces and are frequently responsible for invasive fungal infections, particularly in susceptible patients. Annually, invasive aspergillosis affects over 2.1 million people, particularly those with Chronic Obstructive Pulmonary Disease, in intensive care, or with blood or lung cancer, resulting in a high 85.2% mortality rate. Chronic pulmonary aspergillosis impacts 1.84 million people, with 18.5% mortality rate (340,000 deaths), and Candida spp. infections cause nearly 1 million deaths yearly, affecting 1.57 million people globally, with a 63.6% mortality rate [51].
The World Health Organization’s “Guidelines for Drinking-Water Quality” emphasize the importance of water safety plans and proactive measures to ensure a reliable and safe water supply, particularly during emergencies. While the document does not explicitly require municipalities to identify alternative water sources, it highlights the critical need for comprehensive emergency response planning, which includes securing these sources to safeguard public health and safety during disasters [15].

2. Materials and Methods

2.1. Sampling

Field work was conducted between October 2018 and November 2022. During this period, samples were collected at irregular intervals while ensuring adequate spatial and temporal coverage of the study area. A total of 127 samples collected at 21 sites (MC, CG, EF, CV, MM, VG, CC, QC, MP, TN, SC, CA, AB, VF, CR, EB, GR, PO, MS, BS, PB) which were selected following an initial assessment of potential groundwater abstraction points. The selected sites correspond to locations currently or formerly used for local groundwater supply, presenting good to acceptable conservation conditions and adequate safety and accessibility for sampling activities. The sampling did not follow a regular temporal frequency. Instead, sampling campaigns were conducted opportunistically over the study period, depending on field accessibility, weather conditions, and logistical constraints. All groundwater samples were collected using sterile buckets in all sampling campaigns to ensure aseptic conditions and transferred to sterile polyethylene containers. Sodium thiosulfate (a dechlorination agent—Na2S2O3) was added following the standard procedure of the certified laboratory responsible for sample collection and analysis. This reagent is routinely used when the chlorine content or treatment status of the water is unknown, to neutralize any potential residual disinfectant and ensure accurate microbiological assessment. The samples were stored at 4 °C, for less than 2 h until chemical and microbiological determinations were initiated. Time lapse between sample collection and laboratorial processing did not exceed 24 h.

2.2. Physical and Chemical Assessment

Temperature and pH were both determined (Thermo Scientific.Orion.3-Star BenchtoppHMeter, Thermo Fisher Scientific, Waltham, MA, USA) according to standard methods [52]. Conductivity was measured according to Standard Guideline NP EN 27888:1996 [53] (MeterLab CDM 210) and Mohr’s Method was performed to assess chlorides. pH, temperature, and conductivity were measured in situ. Samples were also analyzed for nitrite (NO2−), nitrate (NO3−), and ammonium (NO4+) according to APHA methodologies [52]. Nitrite was determined by the Colorimetric Method (Hitachi U-2000), nitrate was analyzed by the Cadmium Reduction Method (Hitachi U-2800), and ammonium was determined by the Phenate Method (Hitachi U-2800). Oxidability was determined by the Kübel and Tiemann’s method [54], and total hardness determined by the EDTA Titrimetric Method (NP 424:1966). Total iron (phenanthroline method, SpectroquantTM NOVA 60, Merck, Darmstadt, Germany,), sulfates (turbidimetric method, SpectroquantTM NOVA 60, Merck, Darmstadt, Germany,), total and dissolved solids were also determined according to standard methods [52].

2.3. Microbiological Analysis

Detection of total coliforms, E. coli, P. aeruginosa, L. pneumophila, enterococci, and fecal coliforms were performed using Colilert, Pseudalert, Legiolert, and Enterolert with Quanti-Tray (IDDEX Laboratories, Westbrook, ME, USA). Samples were processed according to manufacturer’s instructions. These microorganisms were selected because they are standard indicators of water quality and potential health risks, encompassing both general bacterial contamination and specific opportunistic pathogens relevant to environmental monitoring. For heterotrophic plate count at 22 °C and 37 °C, agar inclusion technique was performed using 1 mL aliquots of the water sample and adding 15 mL of Yeast Extract agar (VWR Chemicals, Radnor, PA, USA), and incubated at 36 °C for 44 h and 22 °C for 68 h. After the incubation period at each temperature, all colonies were quantified for each case. Values not enclosed by the detection limits were converted, i.e., values “<1 CFU/100 mL” were converted into “0.1 CFU/100 mL”, values “>300 CFU/100 mL” were converted into “301 CFU/100 mL”, and values “>2419.6 CFU/100 mL” were converted into “2420 CFU/100 mL”; this method is part of the laboratory’s standardized data treatment protocol and ensures consistency across all reported datasets.
For fungi analysis, 10 mL aliquots of water samples were filtered through 0.45 μm pore size nitrocellulose membranes (Merck Millipore, Burlington, MA, USA), and filters incubated for up to 7 days at 30 °C in Sabouraud Chloramphenicol Agar (Biokar Diagnosis, Allone, France) for filamentous fungi quantification and in Dichloran Rose Bengal Chloramphenicol (Biokar Diagnosis, France) for yeast quantification. One is aware that incubating at 37 °C would be the most appropriate for selecting pathogenic fungi from environmental matrices; however, we did not intend to be so restrictive due to ailments other than invasive infections. Many allergenic and invasive fungal species are a constant presence in underground water sources, such as dematiaceous molds (Exophiala spp., Cladophialophora spp., Alternaria spp., Phialophora spp., Curvularia spp.). Also, some dermatophytes can cause superficial infections, cannot withstand 37 °C, and are often shed by rodents and other wild animals. Many allergenic species grow only at lower temperatures, such as Fusarium spp., Stachybotrys spp., Trichoderma spp., and most Penicillium spp. Lastly, many mycotoxin-producing taxa do not grow at 37 °C (such as Fusarium spp. and Claviceps spp.).

2.4. Microbial Source Tracking Assays

2.4.1. DNA Extraction from Water Samples

The 33 samples from the 2022 campaign were also analyzed for MST analyses.
The first step was to filter 1 L of the water sample, using a 0.4 μm polycarbonate filter membrane (Whatman, Maidstone, UK), which was placed in the filtration station using sterile tweezers. Subsequently, the filter membrane was picked up at opposite edges using two sets of sterile forceps. The filter was folded to obtain a cone and inserted inside the tube, with the top side facing inwards. DNA extraction was performed using the Aquadien Kit from Bio-Rad according to the manufacturer’s instructions [55]. The DNA was preserved at −20 °C until use.

2.4.2. Microbial Source Tracking (MST) Analyses

The presence of species-specific Bacteroidales was determined by conventional Polymerase Chain Reaction (PCR), using the primers previously selected and validated for Human (HF183F + BacR287), Ruminants (RUM_CF128F + RUM_Bac708R), Dogs (DF113F + DF472R) [56], and Lactobacillus spp. for domestic birds and waterfowl [57]. PCR reactions of 25 μL contained 1x PCR buffer (Invitrogen, Waltham, MA, USA), 1 U of Taq polymerase (Invitrogen), 3 mM of MgCl2, 1 mM of dNTPs, 1 μM of each primer pair, and 20–30 μg of DNA template. PCR negative controls (without sample DNA) were also included in each set of PCR reactions to monitor contamination. Simultaneously, DNA extracted from fecal samples was also included as positive control. PCR cycling conditions are as follows: initial denaturation at 95 °C for 5 min; 40 cycles of (1) denaturation step at 94 °C for 45 s, (2) annealing step for each primers pairs, at temperatures indicated in [57,58] for 45 s, and (3) elongation step at 72 °C for 1 min; and a final extension at 72 °C for 5 min. The PCR products were visualized in 1% agarose gels, stained with GelRed® Nucleic Acid Stain (Biotium, Fremont, CA, USA), and using a 100 bp DNA ladder (PanReac AppliChem, ITW Reagents, Barcelona, Spain). The visualization of the gel was performed under a UV-light transilluminator (UVITEC Cambridge, Cambridge, UK).

3. Results

Table 1, Table 2 and Table 3 summarize the median values of results of all water quality parameters analyzed per sampling point, alongside with the regulatory thresholds established by national legislation for drinking water and irrigation purposes. Physical parameters (temperature, turbidity, and conductivity) provide insight into the general characteristics of the water and allow identification of deviations from reference values. Chemical parameters include most of the ions, nutrients, and other relevant compounds of compliance with legal thresholds and identification of potential risks for potable and non-potable uses. Color codes indicate compliance levels with water quality standards. For drinking water, red represents values above the admissible limit and orange values above the recommended level. For irrigation water, purple indicates the maximum admissible values, while yellow corresponds to the recommended limit.
The Portuguese water quality legislation derives directly from the European Union (EU) legal framework, ensuring alignment with EU environmental and public health standards. For drinking water, the applicable national regulation, Law-decree 152/2017 [58] of 7 December, which transposes the European Council Directive 98/83/EC on the quality of water intended for human consumption, later updated by the Directive (EU) 2020/2184, on the quality of water intended for human consumption [59]. Regarding water intended for irrigation, the quality criteria are defined in Law-decree 236/98 of 1 August [60], which transposes several EU directives, including the Water Framework Directive (2000/60/EC) [61]. These legislative instruments establish and recommend admissible concentration limits for microbiological, physical, and chemical parameters, ensuring that Portuguese standards are fully consistent with the European regulatory framework and contribute to the sustainable management and protection of water resources. This comprehensive presentation enables an integrated evaluation of water quality from microbiological, physical, and chemical perspectives, supporting further interpretation in the context of water safety and sustainable urban-water management.

3.1. Physicochemical Results

Conductivity values were generally below 1000 µS cm−1, indicating low to moderate mineralization characteristic of groundwater. pH and total hardness remained within recommended limits for potable and irrigation uses. Median nitrate concentrations were mostly below the 50 mg L−1 limit for drinking water, though occasional exceedances were recorded. Nitrite and ammonium levels were low or sporadic, while chloride and sulfate concentrations stayed within the admissible limits for both drinking water and irrigation standards. Total dissolved solids and oxidability values varied slightly among sites but were mostly within the expected range for Lisbon’s groundwater.

3.2. Microbiological Results

Indicators of fecal contamination, including E. coli and enterococci, total coliforms, and heterotrophic plate counts (HPC), frequently exceeded the admissible limits for drinking water established in Law-Decree 152/2017. Fecal coliforms were detected in several samples, though typically below the recommended values for irrigation. P. aeruginosa was detected at six sites, and L. pneumophila at three sites. Overall, the presence of these microorganisms indicates varying microbiological quality among sampling points.

3.3. Fungal Parameters

The exploratory analysis performed in this study regarding the fungi detection and enumeration showed that yeasts and filamentous fungi were detected in almost all samples, often with confluent growth. Although fungal parameters remain unregulated, the European Commission’s Joint Research Center has issued a recommendation for their inclusion in water-quality assessments (Recast Drinking Water Directive—Guidance Note) [62].
Overall, these preliminary results indicate that while most physicochemical parameters complied with the established regulatory limits, several sites exhibited microbiological non-conformities. These findings support the subsequent interpretation of water safety and suitability in the Discussion Section.

3.4. MST Markers

The human marker was found occasionally at sites MM, MS, and SC, while other host-associated markers (Rum, Dogs, Birds) were not detected. These results suggest that contamination from domestic wastewater was rare and localized within the monitored groundwater sites.

4. Discussion

4.1. Physical and Chemical Assessment

Physicochemical parameters (chlorides, electrical conductivity, total hardness, pH, nitrates, nitrites, ammonium, oxidability, total dissolved and suspended solids, iron, and sulfates) also varied spatially. Median conductivity values, typically below 1000 mS cm−1, reflected low to moderate mineralization characteristic of groundwater, though isolated exceedances suggested possible saline intrusion or anthropogenic inputs—consistent with Lisbon’s coastal hydrogeology [63]. Similar spatial variability in conductivity and chloride concentrations has been reported in other coastal urban aquifers, where moderate mineralization and occasional exceedances were linked to saline intrusion and human activities [64]. These findings support the interpretation that coastal groundwater systems often display chemical signatures influenced both by marine proximity and by localized anthropogenic recharge. The pH and total hardness generally remained within recommended limits for potable and irrigation uses, indicating stable carbonate equilibria.
Nitrate, nitrite, and ammonium exhibited distinct behaviors in the groundwater samples. Nitrite concentrations were generally low, indicating minimal recent contamination, as nitrites are rapidly oxidized to nitrates under typical aquifer conditions. Nitrate levels varied, with occasional samples approaching or exceeding the drinking water limit of 50 mg L−1, reflecting possible inputs from agricultural activities and fertilizer use [65]. Ammonium was detected very sporadically, also suggesting an absence of recent organic pollution. Chloride median concentrations were moderate, never exceeding the threshold limit for drinking water (250 mg L−1), but sometimes exceeding the 70 mg L−1 recommended limit for irrigation. Total dissolved and suspended solids mirrored the mineralization patterns, while iron levels were generally very low. Oxidability values, representing organic matter content, were mostly within acceptable limits but showed occasional elevation, with reduced concentrations possibly associated with minor surface inputs. Median results for sulfates concentrations were generally moderate across the sampling points, remaining well below the drinking water limit of 250 mg L−1 and recommended irrigation limits, with relatively low spatial variability. This suggests that sulfate in the groundwater is primarily of natural origin, derived from mineral dissolution within the aquifer, rather than from anthropogenic inputs. The observed concentrations are not associated with health risks but may have minor esthetic or operational implications.

4.2. Microbiological Quality

The microbiological summary shown in Table 1, Table 2 and Table 3 reveals a clear and consistent pattern: indicators of fecal contamination—E. coli and enterococci—total coliforms and heterotrophic plate counts (HPC) frequently exceed the admissible limits established for drinking water (Law-decree 152/2017) [58], which is incompatible with potable use without treatment. This pattern is consistent with findings from other urban groundwater studies, where FIB is frequently detected in groundwater [66,67,68]. Fecal coliforms were also detected in many samples, although in reduced concentrations, mostly below the recommended value for irrigation. These results indicate direct fecal contamination of the source or distribution points.
At a lower frequency, P. aeruginosa was also detected at several sites (n = 6), and L. pneumophila was detected at 3 sites. Although fecal contamination represents the main source of water contamination, monitoring of disease-causing organisms such as P. aeruginosa or L. pneumophila remains important, as these microorganisms are indigenous to aquatic environments [12,39], but are still able to cause severe human infections. Yeasts and filamentous fungi were detected in almost all samples, often with confluent growth. Although these organisms are not necessarily pathogenic, their detection provides useful insight into the overall microbial stability of the system. However, overall microbiological non-conformities might constitute a potential public health concern, specifically if the water is to be used for human consumption.
Regarding the MST assays, MM site held a positive (n = 1) and a negative result (n = 1) for the human marker, and a positive result for the Birds marker (n = 1). At site MS, a positive (n = 1) and a negative result (n = 1) for the HF183 marker were also observed. For site SC, the human marker showed positive results for the two out of three water samples. No positive results were detected at any of the other sites investigated for human, Rum, Dogs, or Birds markers. MST analysis showed that the human-associated HF183 marker was detected only sporadically, with occasional positive results at sites MM, MS, and SC. These findings indicate that contamination from domestic wastewater is very rare and localized and does not constitute a major source of microbial contamination in the twenty-one groundwater sites investigated.

4.3. Potential Uses and Suitability of the 21 Underground Sampling Points

Overall, these exploratory monitoring results revealed exceedances of the parametric limits for several key water quality indicators in groundwater samples with potential use for drinking and irrigation purposes. For samples potentially intended for human consumption, exceedances were observed for Total Coliforms (84%), E. coli (47%), enterococci (68%), and P. aeruginosa (15%), indicating microbiological contamination likely associated with surface infiltration or fecal pollution sources. Among the physicochemical parameters, occasional exceedances were detected for Conductivity (1%), pH (1%), Nitrate (12%), Nitrite (1%), Ammonium (1%), and Oxidability (7%), which may reflect localized anthropogenic inputs or natural geochemical variability. In groundwater with potential use for irrigation, random exceedances were recorded for Fecal Coliforms (5%), Nitrate (12%), Conductivity (22%), and pH (1%). The differentiation between drinking and irrigation suitability also considered the MST results, along with the overall microbiological and physicochemical profiles of the samples. Sites showing sporadic or no human-associated markers, lower levels of microbiological contamination, with values within or close to legal limits, and where no relevant pathogens such as L. pneumophila or P. aeruginosa were detected, were considered the most suitable for potential drinking water use after minimal treatment. Sources with slightly higher microbial indicators or animal-associated markers were regarded as more appropriate for irrigation purposes. A differentiated assessment based on median values, proximity to legal thresholds, and MST results suggests that several sampling points could be safely used for irrigation or adapted for potable supply after a minimal treatment. In practical terms, this minimal treatment would primarily consist of chlorination, which is effective in inactivating the microbiological contaminants identified (e.g., E. coli, enterococci, or P. aeruginosa) and ensuring residual disinfection during storage or distribution. In some cases, basic filtration may also be recommended to remove suspended solids or reduce turbidity prior to disinfection. Overall, such simple and widely applicable treatments are considered adequate to address the contaminants found and to ensure the safe use of these sources under emergency conditions. The study also clearly shows that this kind of assessment is highly relevant to identifying emergency water sources for which purposes.

4.4. Drinking Water Use

The analysis of physicochemical and microbiological parameters indicates that sites with E. coli < 10 CFU/100 mL and low or undetected enterococci present favorable conditions for potable use following minimal treatment. At these sites (CG, MM, CC, QC, EB, GR, PO, and MS), conductivity is below 2500 mS cm−1, nitrate concentrations are below the 50 mg L−1 limit (except for sites MM and GR), pH, total hardness, and chlorides are within recommended limits for almost all samples, and sulfate levels are moderate, well below the 250 mg L−1 threshold. Ammonium and nitrite concentrations were very low or rarely detected, indicating minimal recent organic contamination. Importantly, L. pneumophila and P. aeruginosa were absent at these sites, reinforcing suitability for potable use, as both are opportunistic pathogens. MST analysis further confirms that contamination from domestic wastewater is very rare and highly localized, suggesting that microbial loads most probably reflect natural or diffuse sources.
Looking at fungi, it is very clear that many samples exceed the recommendation of 100 CFU/100 mL proposed by Novac-Babic et al. [29] and integrated in the Recast Drinking Water Directive—State of Play: Guidance Note for the Analysis of Microbiological Parameters [62]. However, this kind of water is exposed to natural environmental fungal habitats, and the relevance of this parameter may need to be reassessed at an incubation temperature of 37 °C, which selects fungi that have the potential to cause invasive infections.

4.5. Irrigation Use

Sites EF, CC, AB, VF, and BS present fecal coliform concentrations below 100 CFU/100 mL and physicochemical parameters within the recommended or permitted limits defined in the Law-decree 236/98 [60], including conductivity generally below 1000 mS cm−1, nitrate < 50 mg L−1, and moderate chloride and sulfate levels. These sites are therefore suitable for irrigation, provided that standard operational and exposure control measures are applied. In contrast, although sites MC, CV, VG, MP, and CA also comply with most chemical and microbiological thresholds, the detection of P. aeruginosa and/or L. pneumophila indicates potential health risks, particularly through aerosol or dermal exposure during irrigation activities. Both species are opportunistic pathogens capable of causing infections in humans, especially in vulnerable individuals. Consequently, additional disinfection or restricted use (without aerosol production and contact) is recommended for these sources to ensure safe use. This approach supports safe and efficient water use, while aligning with circular economy and public health protection principles.
This fit-for-purpose allocation maximizes available water resources, promotes circularity, and strengthens Lisbon’s resilience to scarcity and climate variability while protecting public health. This analysis is based on median values, which summarize central tendencies. Incorporating these data into Water Safety Plans will ensure that each source use (potable or non-potable) is supported by evidence-based risk management.

5. Conclusions

This study provides an integrated assessment of the microbiological and physicochemical quality of twenty-one groundwater sources in Lisbon, supporting the identification of fit-for-purpose uses under current European and national water quality standards. The results reveal that while fecal indicators such as E. coli and enterococci frequently exceed admissible limits for drinking water, several sites meet the requirements for irrigation or could be adapted for drinking supply following minimal treatment. The detection of opportunistic pathogens (P. aeruginosa, L. pneumophila) at a few locations underscores the need for continuous microbiological monitoring and risk-based management to ensure health protection.
The combination of conventional indicators with MST demonstrated that human-derived contamination is rare and localized, providing additional evidence for safe use and supporting the development of site-specific Water Safety Plans. Physicochemical data confirmed overall moderate mineralization, stable carbonate equilibria, and limited anthropogenic influence, consistent with the coastal hydrogeological context of Lisbon.
The integrated evaluation of physicochemical, microbiological, and MST results allowed the identification of groundwater sources with potential for differentiated uses—specifically drinking or irrigation. Sites showing low or non-human-derived contamination, low microbiological contamination, compliance with or proximity to legal limits, and absence of relevant pathogens (L. pneumophila, P. aeruginosa) can be considered suitable for potential drinking water use after minimal treatment like chlorination. Sources presenting slightly higher microbial indicators or animal-associated markers would be more appropriate for irrigation. These findings demonstrate that simple and rapid assessments can effectively support the identification of underground sources for emergency water supply, guiding their safe and fit-for-purpose use. With these integrative analyses, this study demonstrates how local groundwater resources can contribute to water security and resilience in urban settings. The proposed fit-for-purpose allocation framework maximizes resource availability while safeguarding public health, aligning with the principles of circular economy and sustainable water governance.
This research contributes directly to the advancement of Sustainable Development Goal 6—ensuring the availability and sustainable management of water and sanitation for all—and supports the European Commission’s Water Resilience Strategy, by providing practical evidence on how alternative sources can safely complement conventional networks during crises and under climate stress. Implementing these findings within Water Safety Plans operationalizes preparedness, efficiency, and adaptive governance, strengthening Lisbon’s—and Europe’s—long-term water resilience in a changing climate.

Future Directions

Given the exploratory nature of this study, our main goal was to summarize key parameters and identify underground sites with potential for further investigation and emergency water use. Future work will include more detailed statistical analyses—such as spatial and temporal variability and correlations among hydrochemical and microbiological parameters—once the most promising sites are now selected for continued monitoring. Methodological improvements, namely for fungi enumeration and quantification, are also envisioned, including performing immediate serial dilutions to ensure more accurate quantification and comparability of the results.

Author Contributions

Conceptualization, P.T., J.B. and E.V.; methodology, P.T., E.V., S.C. and J.B.; investigation, P.T., E.V., S.C. and J.B.; writing—original draft preparation, P.T. and E.V.; writing—review and editing, all authors; project administration, P.T., E.V. and J.B.; funding acquisition, P.T. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by Fundação para a Ciência e Tecnologia (FCT) through CE3C—Center for Ecology, Evolution, and Environmental Changes unit funding (UID/00329/2025) and CHANGE LA/P/0121/2020, (https://doi.org/10.54499/LA/P/0121/2020).

Data Availability Statement

The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding author.

Acknowledgments

The authors would like to thank their colleagues from the Lisbon Municipality, namely from the Bromatology and Water Laboratory, the Municipal Civil Protection Service, and the Department of Green Infrastructure, for their collaboration and support throughout this study.

Conflicts of Interest

The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Abbreviations

The following abbreviations are used in this manuscript:
CFUColony Forming Units
FIBFecal Indicator Bacteria
HPCHeterotrophic Plate Counts
LODLimit of Detection
MPNMost Probable Number
MSTMicrobial Source Tracking
NDNot Detected
PCRPolymerase Chain Reaction

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Table 1. Microbiological, physical, and chemical results (median) for underground water samples collected at 7 (1–7/21) different sites in Lisbon. MPN—Most Probable Number, CFU—Colony Forming Units. ND—Not Detected. n = Total Analyzed Samples. LD (Limit of Detection) = 1 MPN/100 mL. Admissible and recommended values from Portuguese legislation (Law-decree 152/2017 and Law-decree 236/98). Color codes indicate compliance levels with water quality standards. For drinking water, red represents values above the admissible limit and orange values above the recommended level. For irrigation water, purple indicates the maximum admissible values, while yellow corresponds to the recommended limit.
Table 1. Microbiological, physical, and chemical results (median) for underground water samples collected at 7 (1–7/21) different sites in Lisbon. MPN—Most Probable Number, CFU—Colony Forming Units. ND—Not Detected. n = Total Analyzed Samples. LD (Limit of Detection) = 1 MPN/100 mL. Admissible and recommended values from Portuguese legislation (Law-decree 152/2017 and Law-decree 236/98). Color codes indicate compliance levels with water quality standards. For drinking water, red represents values above the admissible limit and orange values above the recommended level. For irrigation water, purple indicates the maximum admissible values, while yellow corresponds to the recommended limit.
SiteMCCGEFCVMMVGCCDrinking Water ConcentrationsIrrigation Water Concentrations
ParameterAdmittableRecommendedAdmittableRecommended
Total Coliforms (MPN/100 mL)2.0E+03(n = 6)9.0E+00(n = 7)9.6E+01(n = 7)1.1E+03(n = 6)1.9E+01(n = 8)2.4E+03(n = 7)1.4E+03(n = 7)0--
E. coli (MPN/100 mL)4.0E+00(n = 6)<1(n = 7)9.0E+00(n = 7)3.0E+00(n = 6)<1(n = 8)2.3E+01(n = 7)<1(n = 7)0--
Fecal Coliforms (MPN/100 mL)7.5E+00(n = 6)<1(n = 8)2.0E+00(n = 7)1.5E+00(n = 6)<1(n = 8)5.2E+01(n = 7)<1(n = 7)---100
Enterococcus (MPN/100 mL)3.7E+01(n = 6)<1(n = 7)7.5E+00(n = 7)7.2E+01(n = 6)<1(n = 8)4.4E+01(n = 7)3.1E+01(n = 7)0---
Legionella pneumophila (NMP/mL)<1(n = 1)<1(n = 2)<1(n = 2)1.0E-01(n = 3)<1(n = 2)5.5E-01(n = 3)<1(n = 1)----
Pseudomonas aeruginosa (NMP/100 mL)2.0E+00(n = 3)<1(n = 2)<1(n = 2)1.0E-01(n = 3)<1(n = 2)2.0E+00(n = 1)<1(n = 2)0---
HPC37 °C (CFU/mL)301(n = 6)120(n = 7)12(n = 7)301(n = 6)11(n = 8)301(n = 7)301(n = 7)-20--
HPC22 °C (CFU/mL)301(n = 6)152(n = 7)43(n = 7)301(n = 6)41(n = 8)301(n = 7)301(n = 7)-100--
Yeasts CFU/10 mL45(n = 1)2(n = 4)2(n = 6)Confluent growth(n = 5)1(n = 6)7(n = 3)Confluent growth(n = 5)----
Confluent growth(n = 3)Confluent growth(n = 1)Confluent growth(n = 2) ----
Filamentous Fungi CFU/10 mL9(n = 1)2(n = 4)2(n = 6)Confluent growth(n = 5)1(n = 6)5(n = 2)Confluent growth(n = 5)----
Confluent growth(n = 3)Confluent growth(n = 1)Confluent growth(n = 2)----
Chlorides (mg Cl−/L)30(n = 6)221(n = 7)36(n = 6)65(n = 6)155(n = 7)36(n = 6)38(n = 7)250 -70
Conductivity (mS/cm at 20 °C)417(n = 6)1404(n = 7)579(n = 6)774(n = 6)1253(n = 7)722(n = 6)541(n = 7)2500 -1000
Total Hardness (mg CaCO3/L)150.0(n = 6)336(n = 6)273(n = 6)354.5(n = 6)504(n = 7)366(n = 6)290(n = 7)-150–500--
pH (units)7.4(n = 6)7(n = 7)7.7(n = 6)7.1(n = 6)7.2(n = 7)7.4(n = 6)7.7(n = 7)≥6.5 e ≤ 9.0-≥4.5 e ≤ 9.86.5—8.4
Nitrate (mg NO3−/L)2.8(n = 6)9(n = 7)11(n = 6)26(n = 6)121.7(n = 7)23(n = 6)7(n = 4)50--50
Nitrite (mg NO2−/L)<0.02(n = 6)<0.02(n = 7)<0.02(n = 6)<0.02(n = 6)<0.02(n = 7)<0.02(n = 6)<0.02(n = 7)0.5---
Ammonium (mg NH4+/L)<0.06(n = 6)<0.06(n = 7)<0.06(n = 6)<0.06(n = 6)<0.06(n = 7)<0.06(n = 6)<0.06(n = 7)0.5---
Oxidability (mg O2/L)1.0(n = 6)1(n = 7)0.6(n = 6)0.59(n = 6)1(n = 7)1(n = 6)1.69(n = 7)5---
Total Dissolved Solids (mg/L)272(n = 6)989(n = 7)392.0(n = 7)531(n = 6)969(n = 7)481(n = 6)342(n = 6)---640
Total Suspended Solids (mg/L)0(n = 6)0(n = 7)<2(n = 7)2(n = 6)<2(n = 7)<2(n = 6)2(n = 7)---60
Iron (mg Fe/L)<0.8(n = 6)<0.8(n = 7)<0.8(n = 7)<0.8(n = 6)<0.8(n = 7)<0.8(n = 6)<0.8(n = 6) --5
Sulfates (mg SO42−/L)40(n = 6)194(n = 7)56(n = 7)68(n = 6)125(n = 7)57(n = 6)23(n = 7)250--575
Table 2. Microbiological, physical, and chemical results (median) for underground water samples collected at other 7 (8–14/21) different sites in Lisbon. MPN—Most Probable Number, CFU—Colony Forming Units. ND—Not Detected. n = Total Analyzed Samples. LD (Limit of Detection) = 1 MPN/100 mL. Admissible and recommended values from Portuguese legislation (Law-decree 152/2017 and Law-decree 236/98). Color codes indicate compliance levels with water quality standards. For drinking water, red represents values above the admissible limit and orange values above the recommended level. For irrigation water, purple indicates the maximum admissible values, while yellow corresponds to the recommended limit.
Table 2. Microbiological, physical, and chemical results (median) for underground water samples collected at other 7 (8–14/21) different sites in Lisbon. MPN—Most Probable Number, CFU—Colony Forming Units. ND—Not Detected. n = Total Analyzed Samples. LD (Limit of Detection) = 1 MPN/100 mL. Admissible and recommended values from Portuguese legislation (Law-decree 152/2017 and Law-decree 236/98). Color codes indicate compliance levels with water quality standards. For drinking water, red represents values above the admissible limit and orange values above the recommended level. For irrigation water, purple indicates the maximum admissible values, while yellow corresponds to the recommended limit.
SiteCCQCMPTNSCCAABVFDrinking Water ConcentrationsIrrigation Water Concentrations
ParameterAdmittable RecommendedAdmittable Recommended
Total Coliforms (MPN/100 mL)(n = 7)1.1E+02(n = 6)1.6E+03(n = 7)3.4E+02(n = 6)6.1E+02(n = 7)7.3E+02(n = 5)1.7E+02(n = 4)2.0E+03(n = 3)0--
E. coli (MPN/100 mL)(n = 7)<1(n = 6)3.0E+00(n = 7)2.5E+00(n = 6)2.0E+00(n = 7)<1(n = 5)1.5E+01(n = 4)9.0E+00(n = 3)0--
Fecal Coliforms (MPN/100 mL)(n = 7)<1(n = 6)2.0E+00(n = 7)<1(n = 7)1.1E+00(n = 7)<1(n = 5)1.5E+01(n = 4)7.0E+00(n = 3)---100
Enterococcus (MPN/100 mL)(n = 7)2.5E+00(n = 6)6.8E+01(n = 7)2.0E+00(n = 6)2.0E+00(n = 7)2.0E+00(n = 5)2.3E+01(n = 4)1.5E+02(n = 3)0---
Legionella pneumophila (NMP/mL)(n = 1)<1(n = 2)<1(n = 1)<1(n = 1)5.5E-01(n = 3)<1(n = 1)<1(n = 1)<1(n = 1)----
Pseudomonas aeruginosa (NMP/100 mL)(n = 2)<1(n = 2)1.2E+03(n = 2)<1(n = 3)<1(n = 2)2.0E+00(n = 1)<1(n = 2)<1(n = 1)0---
HPC37 °C (CFU/mL)(n = 7)79(n = 6)301(n = 7)120(n = 6)57(n = 7)255(n = 5)63(n = 4)270(n = 3)-20--
HPC22 °C (CFU/mL)(n = 7)159(n = 6)301(n = 7)234(n = 6)134(n = 7)>300(n = 5)>300(n = 4)>300(n = 3)-100--
Yeasts CFU/10 mL(n = 5)1(n = 6)Confluent growth(n = 7)4(n = 2)3(n = 4)23(n = 2)10(n = 2)15(n = 1)----
Confluent growth(n = 2)Confluent growth(n = 1)Confluent growth(n = 1)----
Filamentous Fungi CFU/10 mL(n = 5)1(n = 6)Confluent growth(n = 5)2(n = 2)2(n = 4)17(n = 2)17(n = 2)14(n = 1)----
Confluent growth(n = 2)Confluent growth(n = 1)Confluent growth(n = 1)Confluent growth(n = 1)----
Chlorides (mg Cl−/L)(n = 7)71(n = 6)33(n = 7)94(n = 6)107(n = 6)60(n = 5)63(n = 4)45(n = 3)250 -70
Conductivity (mS/cm at 20 °C)(n = 7)963(n = 6)899(n = 7)1499(n = 6)1164(n = 6)778(n = 5)877(n = 4)865(n = 3)2500 -1000
Total Hardness (mg CaCO3/L)(n = 7)424(n = 6)477(n = 7)518(n = 5)536(n = 6)338(n = 5)425(n = 4)397(n = 3)-150–500--
pH (units)(n = 7)7.0(n = 6)7.0(n = 7)7.4(n = 6)6.8(n = 6)7(n = 5)7(n = 4)7(n = 3)≥6.5 e ≤ 9.0-≥4.5 e ≤ 9.86.5—8.4
Nitrate (mg NO3−/L)(n = 4)38(n = 6)5.9(n = 6)3(n = 6)28(n = 6)27(n = 5)34(n = 4)47(n = 3)50--50
Nitrite (mg NO2−/L)(n = 7)<0.02(n = 6)<0.02(n = 7)0.02(n = 6)<0.02(n = 6)<0.02(n = 5)<0.02(n = 3)0.03(n = 3)0.5---
Ammonium (mg NH4+/L)(n = 7)<0.06(n = 6)<0.06(n = 6)<0.06(n = 6)<0.06(n = 6)<0.06(n = 5)<0.06(n = 4)<0.06(n = 3)0.5---
Oxidability (mg O2/L)(n = 7)0.5(n = 6)1.5(n = 7)0.5(n = 6)0.8(n = 6)0(n = 5)1(n = 4)1(n = 3)5---
Total Dissolved Solids (mg/L)(n = 6)696(n = 6)625(n = 7)1139(n = 7)856.5(n = 6)535(n = 5)618(n = 4)626(n = 3)---640
Total Suspended Solids (mg/L)(n = 7)<2(n = 6)6(n = 7)<2(n = 7)<2(n = 6)<2(n = 5)<2(n = 4)<2(n = 3)---60
Iron (mg Fe/L)(n = 6)<0.8(n = 6)<0.8(n = 7)<0.8(n = 7)<0.8(n = 6)<0.8(n = 5)<0.8(n = 4)<0.8(n = 3) --5
Sulfates (mg SO42−/L)(n = 7)118(n = 6)104(n = 7)471(n = 6)148(n = 6)52(n = 5)56(n = 4)63(n = 3)250--575
Table 3. Microbiological, physical, and chemical results (median) for underground water samples collected at additional 7 (15–21/21) different sites in Lisbon. MPN—Most Probable Number, CFU—Colony Forming Units. ND—Not Detected. n = Total Analyzed Samples. LD (Limit of Detection) = 1 MPN/100 mL. Admissible and recommended values from Portuguese legislation (Law-decree 152/2017 and Law-decree 236/98). 4. Color codes indicate compliance levels with water quality standards. For drinking water, red represents values above the admissible limit and orange values above the recommended level. For irrigation water, purple indicates the maximum admissible values, while yellow corresponds to the recommended limit.
Table 3. Microbiological, physical, and chemical results (median) for underground water samples collected at additional 7 (15–21/21) different sites in Lisbon. MPN—Most Probable Number, CFU—Colony Forming Units. ND—Not Detected. n = Total Analyzed Samples. LD (Limit of Detection) = 1 MPN/100 mL. Admissible and recommended values from Portuguese legislation (Law-decree 152/2017 and Law-decree 236/98). 4. Color codes indicate compliance levels with water quality standards. For drinking water, red represents values above the admissible limit and orange values above the recommended level. For irrigation water, purple indicates the maximum admissible values, while yellow corresponds to the recommended limit.
SiteCREBGRPOMSBSPBDrinking Water Concentrations Irrigation Water Concentrations
ParameterAdmittableRecommendedAdmittableRecommended
Total Coliforms (MPN/100 mL)5.9E+01(n = 6)2.9E+01(n = 6)2.4E+03(n = 5)<1(n = 7)3.0E+00(n = 8)2.4E+03(n = 4)1.3E+03(n = 6)0--
E. coli (MPN/100 mL)1.5E+00(n = 6)<1(n = 6)<1(n = 5)<1(n = 7)<1(n = 8)1.5E+01(n = 4)2.5E+01(n = 6)0--
Fecal Coliforms (MPN/100 mL)2.0E+00(n = 6)<1(n = 6)<1(n = 5)<1(n = 7)2.0E+00(n = 7)2.1E+01(n = 4)4.5E+01(n = 6)---100
Enterococcus (MPN/100 mL)<1(n = 6)<1(n = 6)5.0E+00(n = 5)<1(n = 7)<1(n = 8)5.7E+01(n = 4)3.9E+01(n = 6)0---
Legionella pneumophila (NMP/mL)<1(n = 1)<1(n = 1)<1(n = 1)<1(n = 1)<1(n = 1)<1(n = 1)<1(n = 1)----
Pseudomonas aeruginosa (NMP/100 mL)<1(n = 1)<1(n = 1)<1(n = 1)<1(n = 1)<1(n = 1)<1(n = 1)2.0E+00(n = 1)0---
HPC37 °C (CFU/mL)67(n = 6)6(n = 6)>300(n = 5)10(n = 7)3(n = 7)>300(n = 4)>300(n = 6)-20--
HPC22 °C (CFU/mL)105(n = 6)60(n = 6)>300(n = 5)15(n = 7)6(n = 7)>300(n = 4)>300(n = 6)-100--
Yeasts CFU/10 mL7(n = 3)4(n = 3)11(n = 1)3(n = 7)13(n = 5)Confluent growth(n = 4)7(n = 1)----
Confluent growth(n = 3)Confluent growth(n = 5)----
Filamentous Fungi CFU/10 mL6(n = 3)9(n = 3)12(n = 1)1(n = 7)8(n = 5)Confluent growth(n = 4)11(n = 1)----
Confluent growth(n = 3)Confluent growth(n = 5)----
Chlorides (mg Cl−/L)79(n = 6)84(n = 6)84(n = 5)175(n = 6)48(n = 6)66(n = 4)148(n = 6)250 -70
Conductivity (mS/cm at 20 °C)652(n = 6)887(n = 6)959(n = 5)1212(n = 6)962(n = 6)761(n = 4)1256(n = 6)2500 -1000
Total Hardness (mg CaCO3/L)272(n = 5)379(n = 5)425(n = 5)556(n = 6)513(n = 6)362(n = 4)541(n = 6)-150–500--
pH (units)7.5(n = 6)7(n = 6)7(n = 5)7(n = 6)7.3(n = 6)7(n = 4)7(n = 6)≥6.5 e ≤9.0-≥4.5 e ≤ 9.86.5—8.4
Nitrate (mg NO3−/L)8(n = 5)27(n = 6)156(n = 5)19(n = 6)13(n = 6)37(n = 4)5(n = 6)50--50
Nitrite (mg NO2−/L)0.05(n = 6)<0.02(n = 6)<0.02(n = 5)<0.02(n = 6)<0.02(n = 6)0(n = 4)<0.02(n = 6)0.5---
Ammonium (mg NH4+/L)<0.06(n = 6)<0.06(n = 6)<0.06(n = 5)<0.06(n = 6)<0.06(n = 6)<0.06(n = 4)<0.06(n = 6)0.5---
Oxidability (mg O2/L)2(n = 6)0.5(n = 6)1(n = 5)1(n = 6)0.4(n = 6)1(n = 4)1(n = 6)5---
Total Dissolved Solids (mg/L)456(n = 6)592(n = 6)767(n = 5)854(n = 6)745(n = 7)536(n = 4)940(n = 6)---640
Total Suspended Solids (mg/L)3(n = 6)<2(n = 6)<2(n = 5)<2(n = 6)<2(n = 7)<2(n = 4)4(n = 6)---60
Iron (mg Fe/L)<0.8(n = 6)<0.8(n = 6)<0.8(n = 5)<0.8(n = 6)<0.8(n = 7)<0.8(n = 4)<0.8(n = 6) --5
Sulfates (mg SO42−/L)28(n = 5)52(n = 5)86(n = 5)111(n = 6)202(n = 7)48(n = 4)246(n = 5)250--575
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Teixeira, P.; Costa, S.; Brandão, J.; Valério, E. From Quality to Purpose: Rethinking Groundwater Microbiological Standards for Emergency Urban Water Use. Water 2025, 17, 3329. https://doi.org/10.3390/w17223329

AMA Style

Teixeira P, Costa S, Brandão J, Valério E. From Quality to Purpose: Rethinking Groundwater Microbiological Standards for Emergency Urban Water Use. Water. 2025; 17(22):3329. https://doi.org/10.3390/w17223329

Chicago/Turabian Style

Teixeira, Pedro, Sílvia Costa, João Brandão, and Elisabete Valério. 2025. "From Quality to Purpose: Rethinking Groundwater Microbiological Standards for Emergency Urban Water Use" Water 17, no. 22: 3329. https://doi.org/10.3390/w17223329

APA Style

Teixeira, P., Costa, S., Brandão, J., & Valério, E. (2025). From Quality to Purpose: Rethinking Groundwater Microbiological Standards for Emergency Urban Water Use. Water, 17(22), 3329. https://doi.org/10.3390/w17223329

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