Investigating microbial pollution in karst groundwater

Many GWSs are well aware of the susceptibility of sources in karst areas to contamination events, including rapid changes to water quality following heavy rainfall. Such changes are difficult to monitor, proving both costly and time-consuming conventional microbiological methods. Hydrogeologist, Luka Vucinic, has been researching monitoring options, resulting in the publication of two research studies on the topic. In this article, Luka discusses what and his fellow researchers have learned.

Groundwater from karst aquifers, through springs and wells, is a major source of drinking water worldwide. There is nothing that highlights the importance of karst groundwater in Ireland better than the fact that around 40% of the island of Ireland is underlain by limestone (the most significant karstifiable rocks). Consequently, karst, with its landforms and features represents a significant aspect of the Irish landscape.

Unfortunately, groundwater in these areas can be easily contaminated due to the lack of thick soil coverage above karstified limestone rocks as well as the presence of swallow holes that allow water and pollutants to quickly enter the subsurface.

Human wastewater effluent from on-site domestic wastewater treatment systems (DWWTS) (predominantly septic tanks) is identified as significant threat to groundwater quality in such lowland Irish karst environments since approximately one-third of the population in Ireland relies on them.

Many incidences of waterborne diseases can be associated with contamination of surface water and groundwater due to failing on-site DWWTS.

Therefore, being able to detect rapid changes in faecal bacteria concentrations at karst springs used for water supply is extremely important. It is also crucial to establish the sources of the pollution, so that appropriate measures can be taken, both in terms of immediate protection of human health and the management of karst aquifers.


This research study, recently published in the Journal of Contaminant Hydrology, was focused on investigating whether multiple different fluorescence approaches can be used to identify rapid changes in microbial water quality at karst springs associated with rainfall events, and whether such variations in microbial indicators can be linked to contamination by on-site domestic wastewater treatment systems effluent.

This involved three continuous sampling campaigns targeted across rain events at two karst springs. Elmvale spring (County Clare) was continuously sampled on two occasions in December 2018 and in January 2020, and Ballindine spring (County Mayo) once in January 2020. All samples collected during the three continuous sampling periods were analysed for flow cytometric total cell counts, total coliforms, faecal indicator bacteria (E. coli and enterococci), turbidity, and presence of fluorescent whitening compounds (well-known indicators of human wastewater contamination since their origin is mostly from laundry detergents).

In addition to these analyses, tryptophan-like fluorescence was measured in samples collected during two sampling periods. This is a fluorescence occurring from a range of compounds that has been used successfully as an indicator of faecal contamination in drinking water supplies and groundwater. Escherichia coli (E. coli) cells have been proven to directly emit this type of fluorescence and to excrete compounds that fluoresce similarly.

This study builds upon earlier published research (in Water Resources Research journal), with the previous study primarily evaluating the effectiveness of using flow cytometry (and different parameters that can be measured with this method) in predicting changes in concentrations of faecal indicator bacteria at nine karst springs in the west of Ireland (in three counties: Clare, Galway, and Mayo) over a 14-month period.

An overview of the most important sources and pathways for faecal bacteria and domestic wastewater contaminants in rural environments (from

Key findings

Flow cytometry is a method, first developed in the 1960s, that is used routinely in biology and medicine but now is emerging as a very promising technique in environmental microbiology.
Given that conventional and advanced microbiological methods are usually either very expensive, complex, labour-intensive and/or time-consuming, flow cytometry is very attractive. This is because it is a significantly cheaper alternative method that can produce results within minutes (in comparison to more conventional methods that can produce results within 24-48 hours), and can be automated, if necessary.

This research has shown that flow cytometry can rapidly provide measurements of total cell counts that can be very useful to indicate rapid changes in faecal indicator bacteria and microbial water quality in general.

Additionally, the results suggest that monitoring of tryptophan-like fluorescence concentrations at karst springs can be also very useful for detecting rapid changes in concentrations of traditional faecal indicators. For that reason, authors suggest that direct on-site, real time flow cytometric and tryptophan-like fluorescence monitoring hold great promise in karst environments. It was also found that the frequency of detection of positive fluorescent whitening compounds signals may reveal useful information about the magnitude of human wastewater effluent impacts on karst aquifer systems.

Correlation matrix heatmap for both monitoring events at Elmvale spring. TCC (total cell counts), TC (total coliforms), ENT (enterococci), FWCs (fluorescent whitening compounds) and turbidity (from:

Both springs during the three continuous 24-hour sampling periods showed marked responses to rainfall events for all of the microbial parameters measured. Elmvale spring showed faster responses to rainfall events than Ballindine spring (a 3 to 4 hour lagged response compared to the 12 to 13 hour lag during winter periods when the trials were carried out). This is because Ballindine catchment is a lot smaller in size but has thicker layer of soil and no swallow holes or estavelles while Elmvale catchment is highly karstified with more pathways enabling rapid and turbulent groundwater flows.

These studies confirmed the importance of monitoring of microbiological water quality at karst springs, given the resulting substantial increases in bacterial concentrations following rainfall events.

Luka has completed a PhD on this topic in 2021 and is a primary author of two papers that have been published on foot of his research, while a few other papers are expected to be published in the near future. “Multiple fluorescence approaches to identify rapid changes in microbial indicators at karst springs” is available at: “Flow Cytometry and Fecal Indicator Bacteria Analyses for Fingerprinting Microbial Pollution in Karst Aquifer Systems” is available at:

This article originally featured in the most recent edition of the Rural Water News magazine. To read the full edition and to sign up to our magazine mailing list, click here.