Karstic aquifers supply 25% of all drinking water in the world, but they are highly vulnerable to contamination, and their preservation is constantly threatened in developing countries (Wilkinson, et al., 2022).

In densely populated and environmentally sensitive areas, the vulnerability of groundwater is in and, more recently, there is concern regarding contamination by pesticides or pharmaceuticals (including their metabolites) and their further spatial distribution question (Wachniew, et al., 2016; Douglas, et al., 2018). The risk of contamination in karst areas can be considered one of the most significant. The results of previous studies explicitly conducted in karst areas have confirmed the considerable flow rate of distribution of substances in the water-soil-geological system (Huang, et al., 2021).

Pesticides and pharmaceuticals belong to a group of chemical compounds called emerging organic contaminants (EOCs) (García, et al., 2020). EOCs are substances potentially harmful to the environment (Chopra and Kumar 2020; Mali, et al., 2023). Many EOCs can accumulate in aquifers through a process known as leaching, which involves their movement into deeper soil layers and eventually into groundwater sources such as aquifers (Zemann, et al., 2016; Pérez-Lucas, et al., 2019). The accumulation of pesticides in aquifers poses serious environmental and health risks, as these water sources are used for drinking water worldwide. Severe effects on human health resulting from significantly prolonged exposure and accumulation are possible (Zhang, et al., 2006; Bhandari, et al., 2020; Perera-Rios, et al., 2022; Ruomeng, et al., 2023). EOCs are widely used and can accumulate in the environment through various sources. The predominant entry points of EOCs into the environment are via wastewater discharge, agricultural and aquacultural practices, and industry and hospital inputs (Alderton, et al., 2021). The transportation of microorganic pollutants to aquifers is determined by their characteristics and subsurface conditions, such as hydrogeological factors, groundwater residence time, redox processes, soil type, and chemical properties. In addition, other processes, such as ion exchange, adsorption or desorption onto minerals and organic matter, biodegradation and chemical transformations, may control their migration into aquifer systems (Stoppiello, et al., 2020). Many global studies and research papers have revealed the findings pesticides and their metabolites in surface and groundwaters, including karst waters and drinking water sources (Eurostat, 2018; Ferrando and Matamoros, 2020; Baran, et al., 2021; Syafrudin, et al., 2021; Troldborg, et al., 2022).

Treating water contaminated by new artificial chemical compounds, mainly from pharmaceutical and personal care products, is a significant concern (Metcalfe, et al., 2011). Pharmaceutical findings are prevalent in surface waters (Bexfield, et al., 2019; Chopra and Kumar 2020) and also identified in karst aquifers (Morasch, 2013; Dodgen, et al., 2017).

The European Water Framework Directive 2000/60/EC (European Council, 2000) aims to elevate all surface waters to “good” status, involving a range of biological, hydrological and chemical metrics. An Environmental Quality Standard (EQS) is a chemical metric used to assess status. Chemicals selected for EQS derivation are deemed to present a Europe-wide risk, and these EQSs are applied across all European Union countries. The EQSs represent legally binding maximum surface water concentrations, and drive regulatory action to reduce the concentration of chemicals in surface waters (Leverett, et al., 2021).

The occurrence of pesticide residues is repeatedly confirmed in agricultural soils (Karasali, et al., 2016; Hvězdová, et al., 2018; Silva, et al., 2019; Geissen, et al., 2021; Bakanov, et al., 2023; Riedo, et al., 2023), but no limits have yet been set.

This study aims to determine the vulnerability of the hypogene karst study area to pesticide leaching and potential accumulation. We employed advanced analytical methods, supported by geological and topological knowledge, to evaluate the transfer of emerging organic pollutants into hypogene karstic waters.

The data obtained within this study revealed contaminants, such as pesticides, pesticide metabolites, and pharmaceuticals, in the waters of the Hranice Karst. Moreover, bisphenol A (BPA) and DEET were also occasionally found. Pesticides or their metabolites banned in the EU were found in all types of waters (Tables 3–5). Box plot of all EOCs in the groups surface water (S), groundwater (G), and thermal karst waters (T) in ng·L⁻¹ was created (Figure 3). Box plot of all EOCs parent compounds and EOCs metabolites in S, G and T groups shows Figure 4. The sum of the contents of the pesticide parent compound and their metabolite shows Figure 5 and the sum of the contents of the pharmaceutical parent compound and their metabolite shows Figure 6. The chart values represent the sums of EOCs substances measured during individual samplings at all monitored locations. Upper whisker: max value of the dataset; lower whisker: min value of the dataset; the box boundaries from top to bottom are as follows: top: 75th percentile; first bar: median; bottom: 25th percentile. The chart shows the cross-arithmetic mean; stars: outliers.

From the above results, variable inputs of primary substances and metabolites into the system are evident. The results indicate that the highest concentrations of pesticides, their metabolites, and pharmaceuticals (reaching up to µg·L⁻¹) were identified in surface waters group. These sites encompasses surface and shallow subsurface waters, including drainage waters and shallow wells. Group surface waters represents areas where pollution from both non-point and point sources enters the hydrological system. These sites are typically situated in intensively cultivated agricultural landscapes, where pesticides and industrial fertilisers are substantially used. In addition, the influence of human habitation in these areas might contribute to potential contamination through wastewater discharge. Contaminants enter the hydrological system mainly via surface runoff infiltration, leaching through soil layers and from wastewater since none of the sewage treatment plants in the area of interest have technology for removal. The degree of pesticide contamination in water is affected by the properties of the pesticide (e.g., solubility), characteristics of the soil (e.g., organic matter content within the soil, soil pH or content of clay and coarse particles) (Park, et al., 2020), site conditions and the application and management of pesticides (Syafrudin, et al., 2021). Shallow groundwater is easily contaminated with pesticides, and their distribution is linked to land use and crop growth (Andrade and Stigter, 2009). Land management practices, such as irrigation and application, together with intensive rainfall events, can increase a pesticide’s mobility (Menchen, et al., 2017).

Elevated concentrations of EOCs, also in the µg·L⁻¹ range, have been detected in groundwater (Figure 4), specifically in wells ranging from 10 s to 100 m deep. These deep wells contained pesticide metabolites, indicative of contamination originating from agricultural activities, and pharmaceuticals and BPA, both likely derived from wastewater contamination. Given the permeable substrate in these areas, characterised by locally permeable sandy soils and underlying limestone, the EOCs can disperse from these deep groundwater sources. The sandy soils and limestone substrate facilitate the migration and dispersion of these contaminants in the groundwater and allow deep karst mineral waters contamination.

The occurrence of organic micropollutants in the groundwater, such as pesticides, has attracted global attention because of the potential negative effects on human health (Luo, et al., 2014). The threshold value of 0.1 μg·L⁻¹ (European Comission, 2006b) was repeatedly exceeded in all monitored groundwater samples, with some contaminants surpassing this limit up to tenfold. The sole exception is well G-7, which did not exceed this threshold throughout the observation period. The geological profile of the borehole G-7 is composed of fluvial gravels and sandy loam sediments with high permeability down to a depth of 6.8 m; below that, to a depth of 20.0 m, it consists of clays and claystones with low permeability. In addition, the limit of 0.5 μg·L⁻¹ was surpassed in wells G-1, G-3, G-4, G-6, and G-8 (Supplementary Table S3).

The geological profile of well G-1 at depths of 0–18 m consists of alternating highly permeable and impermeable rocks, specifically Neogene sands and sandstones, or possibly clays. It comprises tectonically fractured and karstified Palaeozoic limestones at 18–66 m depth. Both rock types generally exhibit high permeability, so the studied substances may be transported from greater distances or villages opposite the water flow. The flow here is considered to be conformal with the direction of the river’s current. The geological profile of well G-3 up to a depth of 6.8 m consists of fluvial gravels and sandy loam sediments of high permeability and up to 40 m by weathered and karstified limestones. Well G-4 is formed up to a depth of 4.8 m by sandy and clayey fluvial gravels in the profile. From a depth of 4.8 m onwards, it consists of limestones that show intense weathering, karstification and the presence of cave spaces. The geological profile of well G-6 up to a depth of 8.6 m is made of fluvial gravels and sandy loam sediments of high permeability. At depths of 8.6–28.5 m, there are clays and claystones of minimal permeability. Beneath these clays, karstified Paleozoic limestones have been confirmed down to a depth of 100 m. The geological profile of well G-8 is led by Paleozoic limestones, which exhibit high permeability. The interaction between land use and water quality is essential worldwide, as agriculture has been proven to exert considerable pressure on the quality of groundwater and surface waters due to leaching and erosion processes (Kronvang, et al., 2020).

In wells G-6 and G-4, BPA was repeatedly detected, with concentrations reaching up to 655 ng·L⁻¹ in G-4 and 268 ng·L⁻¹ in G-6 (Table 4).

Ibuprofen tends to be detected in various environmental samples: wastewater, sludge, soil, surface water and groundwater. In Europe, groundwater concentrations of ibuprofen occur between 3 ng·L⁻¹ and 395 ng·L⁻¹ (Chopra and Kumar 2020). As part of the groundwater quality monitoring, wells located within the built-up areas of the villages Černotín and Teplice nad Bečvou (S-6, S-7, S-8, and S-11) were also included; contamination by pharmaceuticals (diclofenac, ibuprofen) as well as metabolites of pesticides in concentrations reaching up to 1 μg·L⁻¹, was detected. The source of this contamination is likely wastewater leaks from sewer systems (or sewage treatment plants with infiltration, but these should not be present) in family homes built above the sampled sites. Once in the groundwater, these pollutants attenuate very slowly and can persist for long periods because of unfavourable redox and degradation conditions (e.g., anaerobic conditions and limited microbial activity) (Sorensen, et al., 2015; Sorensen et al., 2015; Dueñas-Moreno, et al., 2022). Dueñas-Moreno, et al. (2022) pointed out the high-risk BPA represents for human health once introduced into the groundwater.

Negligible to trace concentrations of EOCs (sum of all found substances), on the order of tens to hundreds of ng·L⁻¹, were detected at the TKW sites (Figure 3), which were presumed to be predominantly of hypogenic origin and hence were not expected to contain EOC-type substances. The threshold values of 0.1 μg·L⁻¹ and 0.5 μg·L⁻¹ were exceeded at TKW sites only in the case of T-11, located precisely at the Hranice Abyss and in T-6. From the TKW group, significant findings of EOCs were observed in the balneological wells T-1 and T-3 (Table 3, Supplementary Table S3). These findings included trace amounts of diclofenac, ibuprofen, metazachlor ESA, metolachlor ESA, and chloridazon-DESPH. These monitoring results refute the initial hypothesis that deep hydrothermal karst waters are ancient and uncontaminated.

Among all the monitored pesticides and their metabolites within the scope of the Hranice Karst monitoring (refer to our table), metazachlor ESA and metolachlor ESA were among the substances with the highest GUS indices (Table 2), with values of 6.18 and 7.22, respectively. GUS is a technique to predict the ability of chemical compounds to contaminate groundwater and is also used for assessing the leaching potential of pesticides. GUS is an experimentally calculated value that relates pesticide half-life and sorption potential (from laboratory data). The monitoring results demonstrated that, within the karst system, EOCs with elevated GUS indices can contaminate wells at depths of 60 m and 101 m. When utilising pesticides, particularly within karst terrains and on permeable soils, there should be thorough monitoring of the propensity of chemical compounds to contaminate groundwater. Subsequently, application strategies should be adjusted based on these observations to mitigate contamination risks.

The GUS may be used to rank pesticides for their potential to move toward groundwater (Gustafson, 1989). Pesticides with a short half-life, low water solubility and high sorption potential may have less potential to move through soil. Pesticides with a long half-life, high water solubility and low sorption potential possess a more significant potential to move through soil (Wauchope, et al., 1992).

Chloridazone is an herbicide widely used in agriculture to control beets and corn. The primary chloridazon metabolites are desphenyl-chloridazon (DPC) and methyl-desphenyl-chloridazon (Me-DPC), which can be a threat to surface and groundwater resources (Vidal, et al., 2022). Chloridazon inhibits the photosynthesis process in annual broad-leaf weeds and has been widely used in the past several decades because it was considered relatively harmless (Cioni and Maines, 2010; Jakiene, et al., 2015). Due to the polarity and solubility of metabolites, they are regarded as mobile compounds which cause surface water and groundwater pollution.

Chloridazon-DESPH also possesses a notably high GUS index of 5.64, indicating a significant potential for movement toward groundwater. Furthermore, it exhibits a high Screening Concentration In Ground Water (SCI-GROW) index of 9.25 μg·L⁻¹, signifying a pronounced likelihood of appearing in groundwater due to its propensity for leaching and mobility in the soil–water environment. These indices collectively suggest a need for careful management and monitoring of chloridazon-DESPH to prevent groundwater contamination.

SCI-GROW is a screening model which the Office of Pesticide Programs in the US Environmental Protection Agency frequently uses to estimate pesticide concentrations in vulnerable groundwater. The model provides an exposure value to determine the potential risk to the environment and human health from drinking water contaminated with the pesticide. The SCI-GROW estimate is based on the environmental fate properties of the pesticide (aerobic soil degradation half-life and linear adsorption coefficient normalised for soil organic carbon content), the maximum application rate, and existing data from small-scale prospective groundwater monitoring studies at sites with sandy soils and shallow groundwater (USEPA, 2016).

In the lakes within the Zbrašov Aragonite Caves (T-10, T-11), trace amounts of atrazine and its metabolites have been confirmed (Table 5). In the slopes above the caves, intensively farmed fields are located to the south and west. The land use of this area has not changed significantly over the long term. Atrazine is a triazine herbicide extensively used against various dicotyledonous weeds, but it has been banned in the European Union since 2005 due to its environmental and health implications. Despite the ban, the persistence and mobility of atrazine in the environment can result in its continued detection in various water bodies, reflecting its historical use and long-term environmental persistence. These findings highlight the necessity for ongoing monitoring and assessment of water quality within the cave environment to understand the environmental distribution and fate of banned or restricted substances. Although atrazine use was banned in France in 2003, both atrazine and its metabolite desethylatrazine were detected in French groundwater years after its removal from the market (Baran, et al., 2008). The GUS leaching potential index of atrazine is 2.57 (moderate potential for movement toward groundwater), and the GUS of desethylatrazine is 3.24 (high potential for movement toward groundwater). Atrazine has a high potential to contaminate groundwater, especially in environments with porous soil profiles (de Albuquerque, et al., 2020), as confirmed by the research results in the Hranice Karst. Many studies are published yearly on atrazine’s presence in the environment, especially for its metabolites (Kolpin, et al., 1998; Fava, et al., 2007; Iker, et al., 2010; Jablonowski, et al., 2011) modelled atrazine concentrations in shallow groundwater (concentrations ranged from <0.004 to 4.7 μg·L⁻¹) and found that atrazine concentrations are controlled mainly by the history of atrazine use in relation to the groundwater age.

Despite banned application, triazines and acetochlor are still detected (Baran, et al., 2021). Furthermore, in the lakes within the Zbrašov Aragonite Caves, trace amounts of chloridazon-DESPH, chloridazon-DESPH-ME, alachlor ESA, and the pharmaceutical diclofenac have been detected (Supplementary Table S3). Although intensively farmed fields are located on the slopes above the caves, pharmaceuticals must be attributed to historical infiltration of wastewater or leaks from sewage systems in the past. Recently, a modern sewage system was built in the village. The detection of these compounds, even in trace amounts, indicates the presence of anthropogenic contaminants within the karstic aquifer, necessitating continued vigilance and monitoring to assess the environmental health and potential risks associated with these residues.

EOCs were also detected in the deep waters of the Hranice Abyss (T-6, T-7, T-8, T-9, and T-11). Noteworthy are the findings of BPA (242 ng·L⁻¹ in T-9), DEET (132 ng·L⁻¹ in T-6), chloroacetanilide pesticides and their metabolites (including alachlor ESA, metolachlor oxanilic acid (OA), metolachlor ESA, metazachlor OA, and metazachlor ESA), as well as compounds from the pyridazone group (chloridazon-DESPH, chloridazon-DESPH-ME) (Table 5).

The highest concentrations of the pollutants were identified in well T-11 (Table 5). Given the lower water conductivity values’ decreased concentrations of bicarbonates (HCO₃), calcium (Ca), and magnesium (Mg) compared to the other inputs (T-9 and T-6), it can be inferred that the water in T-11 is not purely mineral water. Instead, a mixing process appears to have occurred, involving surface waters (which are likely contaminated) blending with the deeper, mineral-rich waters in this location. This mixing introduced pollutants into the deeper water strata, which warrants careful and continuous monitoring to understand the extent and implications of such contamination for the water quality in the area adjacent to Hranice Abyss.

The detection of BPA indicates contamination from wastewater sources. Dueñas-Moreno, et al. (2022) reported that BPA has been globally detected in groundwater reservoirs and landfills. Sewage sludge and wastewater are the primary BPA sources of groundwater pollution. BPA pollution is caused by anthropogenic activities, including manufacturing processes, the distribution, use, consumption, treatment and disposal of devices and products containing phthalates and BPA, resulting in long-term transport from the environment to groundwater reservoirs. Their physicochemical properties condition the transport and fate of phthalates and BPA, the type of pollution sources, the type of matrix (air, water, and soil) and the environmental conditions (pH, temperature, and pressure) (Tran, et al., 2022).

The continued detection of alachlor ESA in both caves and the abyss (Supplementary Table S3), despite its ban since 2007, raises significant considerations. Despite their prohibited use, many pesticides and their metabolites are still found in water and other areas (Jablonowski, et al., 2011; Baran, et al., 2021; Kruć-Fijałkowska, et al., 2022). The water in the specified areas may exhibit slow movement or circulation dynamics, allowing contaminants introduced before the ban to remain in situ for extended periods. Slow-moving or stagnant water bodies can act as reservoirs for these compounds, releasing them gradually (Zhu, et al., 2017). The unique environmental conditions in these areas might hinder the degradation process of alachlor and its metabolites. The environmental persistence of certain compounds can be extended under conditions that are not conducive to their breakdown, resulting in prolonged detectability (Mouvet, et al., 1997). Although alachlor has been banned, illegal or unregulated use might still be occurring, or sources of contamination might not have been identified or mitigated. This continued input can maintain the presence of alachlor and its metabolites in the environment. The persistence of alachlor ESA and its metabolites in the water demands further investigation to understand the dynamics of water movement, the degradation pathways of these compounds under local environmental conditions and potential unidentified sources of contamination.

Trace amounts of the NSAID diclofenac and the antibiotic azithromycin were detected in the waters of the Hranice Abyss. Notably, diclofenac was found at its highest concentration of 47.4 ng·L⁻¹ in T-3. Azithromycin was detected in T-7 (73.8 ng·L⁻¹) (Table 5).

The presence of pharmaceuticals like diclofenac and azithromycin in aquatic environments is often attributed to the incomplete metabolism of these drugs in humans and animals and the insufficient removal of these compounds during wastewater treatment processes (Jones, et al., 2022). Once released into the environment, these pharmaceuticals can persist and be transported through water bodies, leading to their detection in remote and deep-water locations like the Hranice Abyss.

Active pharmaceutical ingredients are emitted into the natural environment during manufacture, use and disposal. There is evidence that environmental exposure to pharmaceuticals has deleterious effects on the health of ecosystems and humans (e.g., by selecting antibiotic-resistant bacteria, feminising fish and increasing the susceptibility of fish to predation) (Kidd, et al., 2007; Brodin, et al., 2013; Wellington, et al., 2013; Horký, et al., 2021).

Diclofenac has thus been detected in trace concentrations in the balneological wells within the ZAC and in the Hranice Abyss (Supplementary Table S3). The limit recommended by European Commission in surface water is 5.4 ng·L⁻¹ resp. 230 ng·L⁻¹ to protect birds from diclofenac secondary poisoning through the food chain (Peters, et al., 2022). The limit of 230 ng·L⁻¹ was exceeded at two of our monitored sites. In S-8 and S-9, the maximum concentration of diclofenac was 549 ng·L⁻¹ and 231 ng·L⁻¹ (Table 3, Supplementary Table S3). The less strict limit of 5.4 ng·L⁻¹ was exceeded in group S (S-3, S-6, S-9, and S-10), group G (G-1, G-2, G-4, G-5, and G-6) and group T (T-1, T-3, T-4, T-9, and T-10) (Supplementary Table S3).

The correlation between land use and concentrations of EOCs (specifically pharmaceuticals) was also confirmed by other studies (Schäfer, et al., 2016; Chen, et al., 2023). In particular, there is a pattern of positive correlation between contaminant concentrations and land use characterised by cropland and impermeable surfaces (when considering horizontal distribution) and a negative correlation between concentrations and areas covered with vegetation.

The monitoring of the Hranice Hypogenic Karst showed the presence of EOCs in all types of waters, including trace concentrations in deep TKW and balneological wells. The detected EOCs were represented by pesticides, pharmaceuticals and their metabolites; BPA and DEET were occasionally found. Thus, the results refute the original hypothesis that deep hydrothermal karst waters are ancient and uncontaminated. The excessive permeability of the karst system leads to an enhanced vulnerability for retention and spread of contaminants. The results generally confirm that current land use is associated with pollution in the karst system.

In groundwater and the Zbrašov Aragonite Caves lakes, trace findings of atrazine and its metabolites were confirmed. In the warm HA springs and Zbrašov Aragonite Caves lakes, trace concentrations of alachlor metabolites were found. In both cases, these are substances whose application has been banned in the European Union for more than 10 years. The unique environmental conditions in these areas might hinder the degradation processes of these substances, and slow-moving or stagnant water bodies can thus act as reservoirs for these compounds.

When using pesticides, especially in karst locations and on permeable soils, one should monitor the ability of chemical compounds to contaminate groundwater. In agricultural practice, this means monitoring the values of the GUS index of the substances used and adapting one’s application to these values. This karst area is a highly vulnerable system to anthropogenic activities and it is recommended to be monitored beyond legal requirements. In the future, more extensive sampling of source and ground waters through concerted campaigns or deployed samplers and higher resolution weather and land use measures would bring more certainty to understanding contamination of karst aquifers and their environmental, drinking water and mineral water risks.