Microplastic pollution in karst systems is still poorly studied, despite the presence of protected species and habitats, and important water reserves. Vulnerable key species hosted in these habitats could consume or assimilate microplastics, which can irreversibly damage management efforts, and thus ecosystems functionality. This can be particularly true for subterranean water habitats where microplastic pollution effects on wildlife management programs are not considered. The aim of this study is to provide a case study from the Classical Karst Region, which hosts peculiar habitats and key species protected at European level, such as the olm Proteus anguinus. As this area has been deeply exploited and modified over time, and is adjacent to highways, roads and railways, which could contribute to pollution within the karst system, threatening the ecosystems, it provides a perfect model system. In this study we collected and investigated water and sediment samples from aquatic environments of surface and subterranean habitats hosting several subterranean environment-adapted organisms. Examined particles were counted and characterized by size, color and shape via visual identification under a microscope, with and without UV light. Furthermore, spectroscopic analyses were carried out in order to identify microplastics typology. Microplastics were found in all examined habitats. In water, microplastics concentration ranged from 37 to 86 items/L, in sediments from 776 to 2064 items/kg. Fibre-shape was the main present, followed by fragments and beads, suggesting multiple sources of pollution, especially textile products. Most of the particles were fluorescent under UV light and were mainly transparent, while not-fluorescent ones were especially black, blue or brown. Samples contained especially polyesters and copolymers. These results highlight intense MP pollution in karst areas, with significant impacts on water quality, and potential effects on subterranean environment-dwelling species. We stress the importance of monitoring pollution in these critical environments for biodiversity and habitat conservation: monitoring in karst areas must become a priority for habitat and species protection, and water resources management, improving analyses on a larger number of aquatic surface and subterranean habitats.

A new worrying micropollutant threathens natural environments: the microfibres (MFs). Natural, regenerated and synthetic MFs have been detected in different environments, as well as in organisms. While synthetic MFs are generally detected in microplastic analyses, natural and regenerated MFs are not taken into account, or are wrongly considered plastics. They are generally considered biodegradable even if their degradation processes in ecosystems are poorly known. Their potential faster degradation could release toxic compounds, and their characteristics could led to a long-term accumulation in the environment. Understanding their dangerousness and the possible impact they could have on ecosystems is fundamental for environment conservation. We collected and investigated water and submerged sediment samples in different caves and springs of the Classical Karst Region (NE Italy), rich in protected habitats and species. MFs were analysed via microscopy and spectroscopy. MFs were found in all samples, highlighting pollution in surface and subterranean habitats of the karst system. MF concentration was higher in submerged sediments respect to waters, highlighting an accumulation of MFs over time. Big microfibres were less abundant, and MF amount increased with the decrease in the considered size. More than 80% of fibres were fluorescent under UV light. Fluorescent MFs were especially transparent, while non-fluorescent ones were mainly black and blue. Most MFs were cellulosic, and synthetic MFs represent only 15-22%, highlighting a significant gap between the MF composition detected in natural environments and the global production of synthetic textiles in recent times. Synthetic MFs were more abundant in waters. Our results improve the knowledge on micropollutants in karst environments, laying the foundations for future research. MF pollution monitoring in karst areas must become a priority for species protection, habitat conservation, and waters management, improving analyses on a larger number of aquatic environments, taking into account the ecological connections between surface and subterranean habitats.

Stygobiont species show common, typical traits derived from their adaptation to subterranean life. Due to the general absence of light in cave environments, the majority of them are eyeless. Although the absence of eyes generally does not allow them to perceive luminous stimuli, some stygobionts still present phototaxis. Previous studies determined that different species of the eyeless amphipod crustaceans of the genus Niphargus are able to react to light; this has been interpreted as an adaptation to avoid dangerous surface habitats, even if recent studies suggest that this could also be an adaptation to exploit them when a situation is less dangerous (i.e., during the night). Niphargus thuringius is a stygobiont amphipod that can also be observed in spring environments despite possessing all the main morphological features of subterranean organisms, such as depigmentation and a lack of eyes. In the present study, we test how the species respond to light stimuli according to the light cycle and predation risk experienced during a conditioning period. We assessed the reactions to light stimuli of adult individuals of N. thuringius after 30 days of rearing in microcosms with different conditions of light occurrence (total darkness or a light/darkness daily cycle) and predation risk (without predators, with one predator, and with two predators). Both light stimuli during the test and rearing conditions affected the behavior of Niphargus thuringius. With light stimuli, individuals presented a strong photophobic response. Moreover, individuals reared in conditions of high predation risk preferred a more sheltered environment during behavioral tests than individuals reared in safe conditions. Our results add a new species to those of stygobiont amphipods known to display negative phototaxis, confirming that this pattern is widespread and conserved in the field. N. thuringius could be a good candidate model to perform further studies aiming to assess if differences occur between spring populations and populations present in deeper groundwater.

Springs are environments that can provide general insights into factors favouring diversity in ecotones, but they are often neglected in freshwater studies. One of the challenging processes acting in ecotones is the landscape of fear (LOF), the space–time variation of perceived predation risk. Spring exploitation often involves species that are mesopredators in surface fresh water and that can become apex predators in ground water, as in the case of the fire salamander larvae (Salamandra salamandra). Here, we aim to determine whether the activity and foraging patterns of the fire salamander in springs are affected by LOF. We surveyed the night‐ and daytime abundance of fire salamander larvae in 15 springs to assess predator occurrence. We also reared 48 salamander larvae with and without non‐lethal exposure to predators within tanks simulating groundwater or surface freshwater light features. Before and after a month of rearing, we tested larva efficiency in catching prey when exposed to predator chemical cues, both in light and dark conditions. In the field, the number of active fire salamander larvae was significantly higher during the night. At night, the number of active larvae across the transition area between ground water and surface water was higher in plots closer to the surface. Testing and rearing conditions significantly affected larva behaviour, and prey capture was significantly more effective in light conditions. It was less successful in larvae reared with predator chemical cues and in the presence of predators. Moreover, larvae reared with predators under light conditions were slower than those raised with predators in dark conditions. Our results show that LOF can interact with extant environmental features and constitute a significant behavioural pressure for mesopredator species living in freshwater ecotones.

Ecologists and zoologists have worked for centuries to understand the spreading, extent, and regulation of biological rhythms among eukaryotes living in arhythmic ecosystems. Investigating whether the long‐standing hypothesis that biological clocks went lost in species inhabiting arhythmic environments can thus have broad implications in assessing evolutionary and ecological pathways allowing colonization of new and physiologically challenging environments. Subterranean habitats generally lack temporal variation in environmental conditions. However, chronobiological research in such communities remains hindered by methodological, ethical, and logistical constraints, particularly for field‐based studies. Here, we propose a flexible, noninvasive framework to assess temporal variation in the activity of animals using detection probability as a proxy for biological rhythms. Our approach is based on Bayesian occupancy and abundance models, which can incorporate quantitative (count), qualitative (presence/absence), or semiquantitative (abundance classes) data derived from repeated surveys. This framework is adaptable to a variety of taxa, monitoring strategies, and environmental conditions. It is particularly well‐suited for sensitive ecosystems, as it minimizes disturbance and allows for standardized, repeatable analysis of activity patterns. We illustrate the utility of the framework through three case studies ( Proteus anguinus Laurenti 1768, Niphargus sp. Schiødte 1849, and Spelaeomysis bottazzii Caroli 1924) encompassing different data types and showing its potential to detect rhythmicity even in stable subterranean environments. By facilitating robust in situ assessments of temporal activity, our method provides a novel and accessible tool for exploring biological rhythms in arhythmic environments, and can thus support the conservation of highly specialized, often vulnerable, fauna.

Springs are ecotones between groundwater and surface water, important for a variety of both surficial and subterranean organisms. However, their use by amphibians has been poorly assessed. This is evident considering estavelles, typical ecotones functioning not only as spring but also as sinkholes. Here we aim to assess the use of estavelles by amphibians in the Classical Karst Region. From June 2020 to January 2025, we surveyed 61 springs, both during day and night. We visually recorded the occurrence of amphibians, along with abiotic and biotic features—including the presence of pikes (Esox cisalpinus), water flow level, drying events, and substratum heterogeneity. Forty-three springs exhibited estavelle-like hydrological behavior at least once. We identified five amphibian species. The use of springs was frequent for Proteus anguinus, Rana latastei, and Pelophylax sp. Amphibians’ occurrence was significantly linked to non-perennial estavelles characterized by low water flow, habitat heterogeneity, and absence of pike. Overall, reproductive activity in estavelles was limited; breeding likely occurs in nearby damp lentic habitats connected to the springs. Our results provide the first herpetological assessment of estavelle spring habitats in the Classical Karst Region, emphasizing their role as shelters for epigean species and feeding patches for stygobionts.