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Article

Does Microplastic Pollution in the Epikarst Environment Coincide with Rainfall Flushes and Copepod Population Dynamics?

by
Lara Valentić
1,
Tanja Pipan
1,2 and
Nataša Ravbar
1,2,*
1
Research Centre of the Slovenian Academy of Sciences and Arts, Karst Research Institute, Novi trg 2, 1000 Ljubljana, Slovenia
2
UNESCO Chair on Karst Education, University of Nova Gorica, Glavni trg 8, 5271 Vipava, Slovenia
*
Author to whom correspondence should be addressed.
Sustainability 2024, 16(22), 10123; https://doi.org/10.3390/su162210123
Submission received: 23 October 2024 / Revised: 11 November 2024 / Accepted: 14 November 2024 / Published: 20 November 2024
(This article belongs to the Special Issue Karst Groundwater Sustainability)
Browse Figures
Versions Notes

Abstract

:
Karst areas are characteristic landscapes formed by the dissolution of soluble rocks, whose hydrology is largely influenced by rapid infiltration through the karst massif. These areas are often hotspots of biodiversity, especially for epikarst and cave fauna. The epikarst, the uppermost layer of the unsaturated zone, plays a crucial role in regulating water flow in karst aquifers. The aim of this study was to investigate the extent of microplastic (MP) pollution, its relationship with precipitation and its correlation with copepod populations in karst areas. The study was conducted between April 2021 and October 2022 in the Postojna–Planina karst area in SW Slovenia at surface and underground sampling sites to determine the pathways of MP pollution from the surface to the depth of the karst massif. The results indicate that heavier rainfall flushes out more MP contaminants from the surface and epikarst environment. The transport dynamics of MP pollution are similar to the dynamics of copepods, which are the baseline organisms for the food chain in caves. One MP sample contained only polyamide particles, which could indicate clothing as a possible source of pollution, but the results are inconclusive. With this study, we provide the first insight into the transport of MP pollution from the surface environment to deeper karst massifs.

Graphical Abstract

1. Introduction

Karst areas are characteristic landscapes formed by the dissolution of soluble rocks such as limestone, dolomite and gypsum, resulting in unique topographical features such as dolines, caves and sinking streams. A key feature of karst areas is their particular hydrology, which is largely influenced by rapid infiltration and percolation through the unsaturated zone, as well as through the complex network of voids, fissures and conduits of the saturated zone [1]. Compared to non-karstified areas, water in karst systems moves in unpredictable ways, often resulting in rapid and complex subsurface drainage patterns [2]. The epikarst, which is the uppermost layer of the unsaturated zone, is typically highly weathered and fractured, and is characterised by enhanced porosity and permeability. It thus plays a crucial role in regulating the flow of water from the surface to deeper aquifers, as it may store significant amounts of water [3,4]. It can be of considerable environmental concern because it is easily affected by contamination, and its weathered and fractured characteristics prevent the contaminants from being decayed or diluted efficiently [5].
Furthermore, the biodiversity within the epikarst is important, as studies of epikarst fauna have shown that small voids and fissures are highly diverse [6,7]. Even at a large scale, and where intensive sampling efforts were undertaken [8], the epikarst fauna was rich in species, but yet undersampled [9]. Based on sampling at different depths in caves, including deep sites and sites close to or in epikarst, Culver and Pipan [6] suggest that epikarst terrestrial fauna may be more diverse than cave terrestrial fauna. Moreover, many animals seen in caves, especially cave pools, are animals that have literally fallen out of the epikarst via ceiling drips [6], which makes the monitoring of obligate cave-dwelling invertebrates (aquatic stygobionts and terrestrial troglobionts) challenging, e.g., [10]. Additionally, many species of concern are rare, which makes the effective censusing of these animals rather difficult [11].
Numerically, the most abundant animals are copepods, which enter caves in relatively large numbers: one copepod per drip per day in Organ Cave in West Virginia [6]. These copepods include stygobionts, facultative subterranean dwellers (stygophiles) and widespread surface-dwelling species [6]. Many other groups of organisms have been collected in epikarst drips [12], including both surface-dwelling species and stygobionts. Stygobiotic amphipods are of special interest, as they are frequently collected directly from drips or from isolated drip-fed pools, e.g., [13].
In parallel to biological concerns, environmental pollution presents a significant challenge, with plastic pollution accounting for 60% to 80% of all human waste [14]. Plastic pollution is the result of the wear and tear of plastic products, combined with poor waste management in the past and present. Microplastic (MP) particles are typically 1 µm–5 mm in size, although the study of MP particles that are <50 µm in size is currently challenging due to the limitations of analytical methods [15,16,17]. They are either produced in small size (primary MP) or are the result of the wear and tear of larger plastic products (secondary MP), which is the main reason for most MP found in environmental samples.
In general, the most common plastic polymers found in the environment are polyethylene (PE), polypropylene (PP), polyethylene terephthalate (PET), polystyrene (PS), polyvinyl chloride (PVC), and polyurethane (PU), as they are the most produced plastic polymers [18,19]. PE and PP have lower density than water; therefore, they float on the water surface, while PET and PS sink. PE and PP can also sink due to water turbulence or the biofouling of particles with algae, microorganisms, or the larvae of various organisms [19,20].
The first indication that MP pollution could have serious consequences in karst environments was the study by Ivar do Sul and Costa [21], which confirmed a sharp decline in copepod feeding when MPs were present. Only in recent years have studies on MPs in karst received the attention they deserve, e.g., [22,23,24,25,26,27,28] and others. However, due to the complexity and heterogeneity of the karst surface and subsurface environment in different karst regions, appropriate sampling methods and protocols for MP sampling in karst are still under development. Therefore, it is difficult to draw general conclusions that apply to all karst regions worldwide.
Building on the research of Valentić et al. [22], who found high concentrations of MP in rimstone pools, we formulated the following hypotheses (H) to investigate MP pollution in epikarst:
Hypotheses H1:
Epikarst is contaminated with MP due to leaching from the surface;
Hypotheses H2:
MP concentrations correlate with rainfall flushing through epikarst;
Hypotheses H3:
The highest MP concentrations coincide with the highest copepod populations in the same samples.
The aim of our study was to investigate the extent of MP loading, its relationship with rainfall and its correlation with copepod populations in the upper part of the unsaturated zone of the karst aquifer. We conducted field studies and laboratory analyses, focusing on sampling and identifying MP particles, analysing their patterns in relation to precipitation and investigating their co-occurrence with copepods in caves.

2. Materials and Methods

The study took place in the Postojna–Planina karst area in SW Slovenia (Postojna coordinates: 45°46′33.11″ N 14°12′49.18″ E), which is characterised by exemplary geomorphological features and has become an ideal location for the study of karst landscapes due to a rich history of pioneering and research [29,30]; see Figure 1. The surface observation sites are located on the gentle slopes of the Javorniki Mountains at an altitude of between 530 and 640 m. The relief is typically karstic with rounded hills and karst depressions: so-called dolines. Lithologically, Jurassic and Cretaceous limestones predominate, which are highly karstified [31]. Underground, there is a very extensive Postojna–Planina cave system (PPCS), through which seepage water and sinking rivers flow, some of which drains into the regionally important Malenščica water source. Both caves in the PPCS are known tourist caves.
Climatically, the study area is located in the transition zone between the Mediterranean and Continental climate, with an average annual air temperature of 9.3 °C and almost 1500 mm of precipitation [32,33]. The limestone is generally covered by thin Rendzic Leptosol or Chromic Cambisols, with predominant semi-natural Dinaric silver fir–beech forests.
To sample MP pollution, we set up four surface (R1-4) and two underground (POJA, JEZ) sampling sites. On the surface, we chose sampling points that were located as directly as possible above underground sampling points. Based on the results of Valentić et al. [22], all rainfall samples at the surface sampling sites were cumulative samples taken over the entire observed rainfall events in November 2021 and September–October 2022. We collected rainfall water into 20 L glass bottles protected with plastic cases and with stainless-steel funnels to concentrate the rainfall in the bottles. Two surface points were located in a forest regeneration stand (later also referred to as the forest sample), while the other two were positioned in a meadow. Rainfall water was later filtered in the laboratory over glass filters and analysed for MP particles using FTIR-ATR.
Several dozen metres underground, we selected two rimstone pools in the well-known PPCS with different water volumes and water flow characteristics, where we sampled MP pollution. The selected test area is part of the LTER (Long-Term Ecological Research) network and serves as an incubator for testing new monitoring methods and innovations [34]. As these are tourist caves, with Postojna cave also being a world-renowned tourist attraction with an underground train and about 826,000 visitors per year [35], the selected sampling points in both caves were rather easily accessible.
Underground, we monitored the flow of cave seepages at Postojna sampling site POJA and the rimstone pool water level at Planina sampling site JEZ in the period between April 2021 and October 2022 [36]. In both cases, water temperature and electrical conductivity at 30 min intervals were measured. The monitoring protocols were designed separately for each sampling point, taking into account the microlocal characteristics of the water flow and geomorphology. Dripping water was measured using a Stalagmate drop counter (Driptych) placed in a funnel, from which water was collected in a flow-through cup. In the rimstone pool, an Onset Hobo U20 diver was used to monitor the water level. Onset Hobo U24 divers were used to measure water temperature and electrical conductivity. Discharge was measured occasionally at various hydrologic conditions to define the stage–discharge relations. Based on these relations and water level measurements, discharge was defined. Half-hour precipitation data from the Postojna meteorological station were obtained from the Slovenian Environment Agency (ARSO) [32].
The sampling of MP pollution at these underground sampling sites was conducted in rimstone pools located directly beneath active epikarst drips that had reacted with rainwater. Sampling was conducted using a hand-held 60 μm mesh net; we attempted to filter (and concentrate) the entire volume of water from the rimstone pools (see Figure 2).
The underground sampling of MP pollution had a different sampling frequency compared to hydrological and copepod sampling, and was determined in regard to the characteristics of each cave site. Based on the findings of Valentić et al. [22], who found that most MPs are present in rimstone pools due to the accumulation of percolating water over long periods of time, we took only one sample in May 2021 from each cave. During the November 2021 rain event, we tested the assumption that the first flush of rain through epikarst also contains the highest concentration of MP pollution; therefore, we sampled the MPs as soon as the underground sampling sites reacted to the rainfall. During the 2022 rain event, we sampled on different days (before rainfall, immediately after the reaction, in the middle of the event and close to the end of the event) to see if the dynamics of MP pollution in the water samples changed.
Statistical analyses were performed for MP pollution using the PAST [37] programme, using a significance value of p = 0.05 for all tests. The normality test, performed with a Shapiro–Wilk test, showed non-normally distributed data (for cave samples: W = 0.47, p = 4.14 × 10−4; for rainfall samples: W = 0.69, p = 0.002). Therefore, the data were further analysed using the Mann–Whitney U test and the Spearman’s correlation coefficient (rs) to determine the relationship between two sets of samples (cave MP samples vs. rainfall MP samples).
The epikarst fauna in rimstone pools, filled with water from drips, was sampled using a 60 μm mesh net and then preserved for further sorting and identification. During sampling, the water in the pools was vigorously agitated to dislodge particles from the bottom, where most of the organisms were concentrated. We pumped various quantities of the pool water at different sampling points and filtered it through a 0.060 mm net. The total volume of water filtered at each site ranged from approximately 25 to 50 L. The sampling sites were located away from any stream passages to ensure consistency. We followed the detailed sampling techniques and procedures outlined by Brancelj et al. [12]. In the laboratory, organisms were sorted under a stereomicroscope at 40× magnification and stored in 70% ethanol. Before dissection, specimens were placed in a 1:1 mixture of glycerol and 70% ethanol, which was later replaced with pure glycerol. Dissections were carried out at 100× magnification using a Nikon Eclipse 600 microscope. Further processing and the identification of specimens took place under a compound microscope (Nikon Eclipse 600) at 400× to 1000× magnification. Specimens that were not dissected were stored in labelled tubes. Identification to the species level was achieved using various taxonomic keys, including [38,39,40,41,42,43]. Only copepods were used in this analysis, but they represented the vast majority of aquatic crustaceans and other invertebrates present in the samples. A summary of all the used sampling campaigns is presented in Table 1.
Due to obtaining normally distributed data for both discharge and abundance, as indicated by the Shapiro–Wilk test (for discharge: W = 0.96, p = 0.77; for abundance: W = 0.95, p = 0.71), we applied the Pearson correlation coefficient (r). Pearson’s method measures the linear relationship between two variables, which aligns well with normally distributed data. The Pearson correlation coefficient (r) was calculated to evaluate copepod abundance with respect to discharge. Statistical analysis was undertaken, and the significance of environmental variables was tested using PAST [37].

3. Results

3.1. Hydrological Conditions Prior to and During Sampling

In 2021, 70 mm of rain fell in the first half of April, followed by 82 mm between 29 April and 3 May. On 30 April, the discharge in the JEZ pool rose from 0.4 to 22.3 L/s, but fell back to almost the initial value on the same day. After the rainfall on 2 May, it rose again slightly. At the same time, the electrical conductivity (EC) value fell sharply from 318 to 297 μS/cm, followed by a moderate increase compared to the initial value. This behaviour indicates that a large amount of fresh, less saturated rainwater flowed rapidly through the unsaturated zone, which is confirmed by an increase in pool temperature values after the rainfall (not presented in Figure 3).
The water characteristics at POJA after the April rains also showed an extremely rapid response. The drip water flow rate increased to 36.2 mL/min by 30 April and then immediately decreased again. On 4 May, it began to rise again due to further rainfall. After the rain, the EC value of the water rose slightly from 460 to 480 μS/cm, but then fell to 440 μS/cm. After further rainfall, the values rose again and fell significantly by 4 May, indicating an influx of less saturated rainwater.
After a rather dry June, only 36 mm of rain fell on July 4. The flow values in the JEZ pool did not change, but the EC values decreased and the temperature increased. The POJA site showed very low flow values, while the EC and temperature values increased.
In early October, 48 mm of rain fell, followed by a 12.3 mm rain event on 21 October, and nearly 70 mm of rain fell between 1 November and 4 November. In the JEZ pool, the water level rose from 0.6 to 3.6 L/s on 2 November and dropped almost to the initial value the following day. After the rainfall on 3 November, it rose to 26.7 L/s within 12 h and then fell back to the initial value. At the same time, the EC value increased slightly, while the temperature initially decreased by one degree. At the POJA site, flow rates increased from very low on 3 November to 60 mL/min the next day, and fell back to baseline by 6 November. During the period of increased flow rates, the EC value of the water gradually decreased from 440 to 310 μS/cm. The temperature values did not change.
In the second half of September 2022, 80 mm of rain fell between 15 and 17 September and more than 170 mm fell between 24 and 30 September. At the end of the month, during the sampling, the discharge in the JEZ pool rose from 2.2 L/s to 50 L/s within a few hours and fell back to the initial values one day later. Similar values were reached between 29 and 30 September. During this time, the EC of the water rose from 355 to 373 μS/cm. Simultaneously with the decrease in flow values, both the EC and temperature decreased, indicating the outflow of stored water. The drip at the POJA site increased to 44 mL/min on 26 September and to 57 mL/min one day later. The temperature remained constant, and the EC gradually increased from 400 to 450 μS/cm.

3.2. Copepod Sampling Results

Six copepod species from the group Harpacticoida were collected (Speocyclops infernus, Bryocamptus balcanicus, Elaphoidella cvetkae, Moraria poppei, Morariopsis scotenophila, and Parastenocrais nolli alpina), as well as one amphipod species from the genus Niphargus; all but one were stygobionts. These are all species previously reported from the cave system [6]. While copepods were numerically common, amphipods were not and only three individuals were found (see Table 2).
The rate that animals come out of epikarst into the Postojna–Planina Cave System is shown in Figure 4 and Table 3. Just considering copepods, which allows comparison with other sites and caves, there were a total of 54 copepods collected, or 0.30 copepods per day. Cumulative analysis of the entire cave system reveals a correlation between copepod abundance and the average discharge of dripping water (Figure 4).
The Pearson correlation coefficient between discharge and copepod abundance is approximately 0.94, indicating a strong positive correlation. The p-value is approximately 0.057, which is slightly above the conventional significance threshold of 0.05, suggesting that the correlation is not statistically significant at the 5% level, or can be described as moderately significant (borderline significant) because the p-value is close to 0.05.

3.3. Microplastic Sampling Results

Rainfall samples for MP pollution were taken during two autumn rainfall events, one in 2021 and one in 2022. The rainfall event in 2021 had 70 mm of rain, with six and seven MP particles found in forest samples from the Postojna and Planina region, respectively (see Figure 5). The meadow samples from November 2021 had significantly lower MP amounts. A similar trend is observed for the Planina samples from a rainfall event in 2022. This event had 170 mm of rain, and the highest number of MP particles in the Planina samples was found in the forest sample. As for rainfall in the Postojna region, the forest rainfall sample from 2022 was lost during sampling due to a faulty 20 L glass bottle. Therefore, we cannot draw any reliable conclusions for the Postojna region, but it is possible that the results would show a similar trend.
During the November 2021 rainfall event (in the period between 1 November 2021 and 4 November 2021), MP pollution inside the caves was sampled daily from 2 November 2021 to 5 November 2021, when the water levels inside the caves returned to the baseline. In the Postojna cave, the drips at the POJA site reacted on 4 May 2021, and that sample did not contain any MP particles. On the other hand, the sampling site JEZ in the Planina cave reacted earlier in the same rainfall event. By far, the largest number of determined MP particles was found in the sample from JEZ in the Planina cave on 5 November 2021, when we determined six MP particles. That day had flow rates already in recession. Other sampled days had only one to two MP particles present in each sample (see Figure 6). The same rainfall event also resulted in the highest number of found copepods. The autumn rains in 2022 did not result in many MP particles being found. The highest amounts of MP particles were found in the cave samples from the sampling site in the Planina cave (site JEZ) and POJA on 26 September 2022, when sampling was performed after heavy rainfall. That day also had very high flow rates, but soon started to recess (see Figure 3).
Most MP polymers were found in the samples from Postojna in the form of polyamide (PA, which is mainly used in clothing), PET, polybutylene terephthalate (PBT) or a combination of PET and PBT (Figure 7). Other polymers were mostly found at random. PBT is mainly used as an insulating material or general engineering plastic and has a similar composition to PET [43], which is mainly used as a packaging material.
As for the rainfall event in 2022, it was the strongest rainfall event sampled in this study; more than 170 mm of rain fell between 24 September and 30 September, and MP pollution was sampled on 26 September and 30 September. Interestingly, the highest number of determined MP particles was found in the JEZ sample from 26 September (Figure 8): this sample contained 80 particles (only PA), which could indicate clothing as a source of pollution. The sample from the JEZ sampling site from 30 September contained only one MP particle. On the other hand, samples from POJA in September 2022 contained much lower numbers of determined MP particles, but they stayed rather consistent: on 26 September, we determined nine MP particles, and on 30 September, we determined six MP particles.
Based on all gathered results, we performed the Shapiro–Wilk test to determine the normality of the distribution. This test showed non-normally distributed data (for cave samples: W = 0.47, p = 4.14 × 10−4; for rainfall samples: W = 0.69, p = 0.002); therefore, we further tested the data using the Mann–Whitney U test, comparing the cave samples with the rainfall samples (U = 23, p = 0.35), and found that the results are not statistically significant. The Spearman’s correlation coefficient (rs) also showed that data from both sets of samples are weakly negatively correlated and not significant (rs = −0.09, p = 0.81).

4. Discussion

The epikarst habitat itself is a highly vulnerable and important one. As first described by Mangin [44] and Williams [45], epikarst holds a considerable reservoir of subterranean water that is poorly integrated both vertically and horizontally. This makes it very vulnerable to any spills of pollutants either on the surface or from leaking underground storage tanks. Because of the poorly integrated nature of epikarst, contaminants tend to persist [46,47].
Caves are not exempt from the impacts of anthropogenic pollution, including MPs. Although the PPCS is a tourist cave, it is a globally exceptional site of subterranean biodiversity with 105 troglobiotic animal species [10], of which 47 species have been scientifically described from the PPCS [48]. Above the cave passage is a rich epikarst community, with a total of 22 copepod species having been found in the 20 drips sampled [6]. Among the six copepod species found in this study, the most abundant was the cyclopoid Speocyclops infernus, followed by the harpacticoids Bryocamptus balcanicus and Moraria poppei. May and November, the months with the highest precipitation, led to increased discharge in the cave, filling the pools, which coincided with a higher abundance of animals. Interestingly, site JEZ had a significantly greater abundance of organisms compared to site POJA. There is considerable heterogeneity in the number of species among drips, as some had no copepods and few were quite rich in them. Based on an analysis of Slovenian cave data [6], drips contained on average two more stygobiotic copepod species than the associated pools. In any case, epikarst fauna is rich and highly endemic, but also vulnerable to anthropogenic pressure.
Ecosystems are increasingly exposed to multiple stressors, notably chemical pollution and global warming, stemming from human activities. These stressors can negatively affect biodiversity, ecosystem functioning, and ecosystem services. While there has been progress in understanding the effects of chemical stressors, including microplastics, their interactions remain complex and challenging to quantify. The impacts of microplastics can vary considerably due to factors like environmental variability, making their ecological effects unpredictable.
This study aims to explore the relationship between microplastic pollution and rainfall, adding to the understanding of the interaction between these factors. To the best of our knowledge, this is the first study to investigate the transport of MP particles through the epikarst deeper into a karst system. The study by Valentić [25] showed that karst springs contain very low concentrations of MP pollution due to efficient dilution and that heavy rainfall flushes more MP particles out of the karst system. Therefore, it is not far-fetched to assume that epikarst has similar dynamics of MP pollution transport—possibly even more pronounced, as the permeability of epikarst varies greatly. The results pertaining to MP pollution in our study indicate a possible correlation between rainfall and MP particles found in karst caves, which consequently also points to a possible MP reservoir in epikarst. Our results also confirmed that the majority of MP pollution is flushed out of the karst system mostly during strong rainfall events. On the other hand, Spearman’s correlation coefficient showed a weak negative correlation between the measured MPs in the rainfall samples and cave samples that was not significant. Nevertheless, the concentrations of the MPs found in the study by Valentić [25] were still rather negligible (less than one particle per m3), despite conducting continuous sampling over one-week periods for an entire year; therefore, we would need to sample other strong rainfall events on a daily basis to be confident in our claims.
When we try to draw conclusions about the potential sources of the MP polymers in our samples, we need to take into consideration that the surface sampling sites were located in relatively remote, forested areas and meadows that did not have many nearby anthropogenic sources of MP pollution. For the underground sampling sites, cave visitors and percolating rainfall water were the most prominent sources of pollution.
Rainfall samples from the Postojna region mostly contained PA particles—this could indicate that MP pollution comes from far-away pollution sources due to wind transport, e.g., [49,50]. One rainfall sample in the Postojna region (taken from a meadow in November 2021) also contained two PET/PBT particles, which could have originated from rare visitors around the sampling site. On the other hand, the results of the rainfall samples above the Planina cave indicated that the forest regeneration stand rainfall samples contained more MP pollution than the meadow rainfall samples. This could be due to the wash-off of MP pollution that was dry-deposited on tree leaves before the rainfall event. But to obtain conclusive results, we would need a longer period of comparison for both types of samples.
From Figure 7, we can see that the majority of PET and PBT polymers in the Postojna samples were found inside the cave. This may indicate pollution from the mass tourism in the Postojna cave, as the POJA sampling site is located rather close to the tourist railway path inside the cave. The samples from the Planina region contained mostly PA and PE/PET/PBT blends of MP particles (Figure 8), both in the caves and on the surface. PA particles indicate a possible source of pollution from clothing (i.e., a distant anthropogenic source of pollution), transported with wind and later with rainfall flushing through the epikarst. Generally speaking, MP pollution at the JEZ sampling site peaked during high flow rates after heavy rainfall events, when the flow rates were already in recession. This is a rather surprising result, as many studies, e.g., [51,52], have shown that the first flush of rainfall after longer drought brings the most pollution to karst springs.
In general, the abundance of copepods collected in drip water was positively correlated with flow rates. We found that copepod abundance was potentially positively correlated with microplastic (MP) concentration, as expected. High precipitation has resulted in high discharge, leading to both high copepod abundance and elevated MP concentrations. We also observed a higher abundance of organisms at the JEZ site—this site had the highest MP concentration, with a confirmed correlation of MP pollution and precipitation. In the case of the copepods in the sampled pools, their source habitat is in the epikarst above. There is no way to determine which drips will be best (although species richness in other caves has been correlated with some aspects of water chemistry, as well as ceiling thickness [6]), nor which sampling times are best.

5. Conclusions

With this study, our aim was to investigate the extent of MP loading in epikarst environments, as this type of research is still in its infancy stages. We conducted extensive field campaigns to try and determine relationships between MP pollution, rainfall and copepod populations within one large cave system (PPCS). Based on all conducted sampling campaigns and laboratory analyses, we can summarise the results as follows.
Hypotheses H1 (epikarst is contaminated with MP by leaching from the surface) and H2 (MP concentrations correlate with rainfall flushing through the epikarst) can only be partially confirmed, as we do not have enough data from the Postojna cave system to draw conclusive statements, with the statistical analysis contradicting the collected results. It is possible that the conduit network in the epikarst environment above the Postojna cave is more complex compared to the Planina cave, as the results showed that the strongest rainfall event in 2022 resulted in a more uniform MP load in the water sampled from the Postojna cave compared to the other rainfall events. In the Planina cave, the correlation between rainfall events and MP pollution is strong: MP pollution was detected in the cave water samples approx. 2 days after a rainfall event.
H3 (the highest MP concentrations coincide with the highest copepod populations in the same samples) is confirmed, as copepod dynamics had a strong positive correlation to rainfall events that was borderline statistically significant.
To the best of our knowledge, this is the first study of MP pollution dynamics in the epikarst environment; therefore, we need more experimental data about MP pollution in different rainfall events and how the pollution transports through epikarst. These data are important to facilitate a better understanding of MP pollution in vulnerable karst environments, especially where karst aquifers are used as a main source of potable water. These karst aquifers have a stable year-round water supply due to the epikarst regulating the water flow from the surface. Therefore, pollution with MPs further raises the question of the health of the ecosystem, as the water entering the PPCS from the epikarst is critical to the continued health of the stygobiotic community. This highlights the potential impact of MP contamination on the community of living organisms in cave habitats. Although the impact of MP on organisms in caves has not yet been studied, it is known that a new amphipod species called Eurythenes plasticus has been discovered in the Pacific Ocean that has MP particles in its digestive system [53]. This leads to a possible transmission of MP via the food chain. The monitoring and management of MP pollution is essential to ensure the sustainability and health of these unique ecosystems. Further research is needed to fully understand the long-term effects of MPs on these communities and to develop effective mitigation strategies.

Author Contributions

All authors participated significantly and equally to the conceptual and methodological preparation of the paper in terms of data collection, analysis and interpretation, as well as graphical production. Writing was undertaken by each author for their area of expertise: L.V. for the microplastic part of the study, T.P. for the biodiversity part and N.R. for the hydrological part. The project was led by N.R., while funding acquisition was undertaken by T.P. and N.R. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Slovenian Research Agency for financial support within the projects: Infiltration processes in forested karst aquifers under changing environment (No. J2-1743); Ecohydrological study of spatio-temporal dynamics in karst critical zones under different climate conditions (No. NK-0002); and the Research Programme: “Karst Research” (No. P6-0119). Several EU projects are also acknowledged: eLTER Preparatory Phase Project (eLTER PPP); eLTER Advanced Community Project (eLTER PLUS); and »Development of research infrastructure for the international competitiveness of the Slovenian RRI space—RI-SI-LifeWatch« and LifeWatch ERIC.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in the study are included in the article; further inquiries can be directed to the corresponding author/s. The raw data supporting the conclusions of this article are deposited in IZRK MetaData Portal at https://metadata.izrk.zrc-sazu.si/ (accessed on 23 October 2024), and will be made available by the authors on request.

Acknowledgments

The authors acknowledge the help of Franjo Drole, Blaž Kogovšek and Cyril Mayaud for cave hydrological data acquisition.

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.

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Figure 1. Location of the study area, as well as surface and underground sampling sites.
Figure 1. Location of the study area, as well as surface and underground sampling sites.
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Figure 2. Example of MP sampling at sampling site JEZ in the Planina cave (photo: N. Ravbar).
Figure 2. Example of MP sampling at sampling site JEZ in the Planina cave (photo: N. Ravbar).
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Figure 3. This graph shows the precipitation and hydrochemical conditions in the underground stations of JEZ and POJA during the individual sampling periods.
Figure 3. This graph shows the precipitation and hydrochemical conditions in the underground stations of JEZ and POJA during the individual sampling periods.
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Figure 4. Relationship between abundance of copepods and average discharge during particular sampling periods in PPCS.
Figure 4. Relationship between abundance of copepods and average discharge during particular sampling periods in PPCS.
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Figure 5. Distribution of microplastic particles found in rainfall samples from above Postojna–Planina cave system.
Figure 5. Distribution of microplastic particles found in rainfall samples from above Postojna–Planina cave system.
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Figure 6. Distribution of found microplastic particles in water samples from Postojna–Planina cave system.
Figure 6. Distribution of found microplastic particles in water samples from Postojna–Planina cave system.
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Figure 7. Number of MP polymers found in samples from Postojna cave and rainfall above Postojna cave.
Figure 7. Number of MP polymers found in samples from Postojna cave and rainfall above Postojna cave.
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Figure 8. Number of MP polymers found in samples from Planina cave and rainfall above Planina cave.
Figure 8. Number of MP polymers found in samples from Planina cave and rainfall above Planina cave.
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Table 1. Summary of sampling methods for different parameters.
Table 1. Summary of sampling methods for different parameters.
Sample TypeSampling MethodSampling PeriodSampling Site
Epikarst faunaNet filtrationMay, July, October, November 2021JEZ
Epikarst faunaNet filtrationMay, July, October, November 2021POJA
Microplastic pollutionNet filtrationMay and November 2021, September 2022POJA, JEZ
Microplastic pollutionRainfall collectionNovember 2021, September 2022R1-4
Discharge, electrical conductivity, temperatureDrip counter and HOBO diverMay and November 2021, September 2022JEZ, POJA
Table 2. Distribution of copepod and amphipod species found during four sampling events at two sites (POJA and JEZ) in the Postojna Planina Cave System.
Table 2. Distribution of copepod and amphipod species found during four sampling events at two sites (POJA and JEZ) in the Postojna Planina Cave System.
Sampling SiteDateSpeocyclops infernusBryocamptus balcanicusElaphoidella cvetkaeMoraria poppeiMorariopsis scotenophilaParastenocrais nolli alpinaNiphargus stygiusTotal
POJA4 May 20211 2 1 4
POJA8 July 2021
POJA29 October 2021 2 12 5
POJA16 November 2021 1 1
JEZ4 May 202173 21 316
JEZ8 July 202111 1 3
JEZ29 October 202134 1 8
JEZ16 November 202192252 20
Total 211251051357
Table 3. Number of copepods in different sites during the four sampling periods.
Table 3. Number of copepods in different sites during the four sampling periods.
DatePOJAJEZ
4 May 2021413
8 July 202103
29 October 202158
16 November 2021120
TOTAL1044
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Valentić, L.; Pipan, T.; Ravbar, N. Does Microplastic Pollution in the Epikarst Environment Coincide with Rainfall Flushes and Copepod Population Dynamics? Sustainability 2024, 16, 10123. https://doi.org/10.3390/su162210123

AMA Style

Valentić L, Pipan T, Ravbar N. Does Microplastic Pollution in the Epikarst Environment Coincide with Rainfall Flushes and Copepod Population Dynamics? Sustainability. 2024; 16(22):10123. https://doi.org/10.3390/su162210123

Chicago/Turabian Style

Valentić, Lara, Tanja Pipan, and Nataša Ravbar. 2024. "Does Microplastic Pollution in the Epikarst Environment Coincide with Rainfall Flushes and Copepod Population Dynamics?" Sustainability 16, no. 22: 10123. https://doi.org/10.3390/su162210123

APA Style

Valentić, L., Pipan, T., & Ravbar, N. (2024). Does Microplastic Pollution in the Epikarst Environment Coincide with Rainfall Flushes and Copepod Population Dynamics? Sustainability, 16(22), 10123. https://doi.org/10.3390/su162210123

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