Microplastic pollution calls for urgent investigations in stygobiont habitats: A case study from Classical karst.

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.

Microplastics in caves: A new threat in the most famous geo-heritage in the world. Analysis and comparison of Italian show caves deposits.

Microplastic pollution represent a worldwide concern, however, in karst areas is still largely unknown, especially in underground environments. Caves are the most important geological heritage worldwide, rich in speleothems, unique ecosystems custodians of important drinking water reserves, and a significant economic resource. Thank to their relatively stable environmental conditions, they can preserve information for a long time such as paleontological/archaeological remains, however, these characteristics make caves vulnerable environments too, easily damaged by climate variations and pollution. To increase the current knowledge of microplastic pollution, the deposits of different Italian show caves were investigated, improving the method for microplastic separation. Microplastic were identified and characterised using MUPL automated software, observed with and without UV light under a microscope, and verified under µFTIR-ATR, highlighting the importance of combine different methods. Microplastics were present in sediments of all examined caves, and were always greater along the tourist route (an average of 4300 items/kg) than the speleological areas (an average of 2570 items/kg). Microplastics less than 1 mm dominated the samples and the amount increased with the decrease in the size considered. Fibre-shaped dominated the samples and 74% particles was fluorescent under UV light. Analysed sediment samples contained especially polyesters and polyolefins. Our results highlight the presence of microplastic pollution in show caves, giving useful information to assess risks posed by microplastics in show caves and emphasizing the importance of pollutants monitoring in underground environments to define strategies for the conservation and management of caves and natural resources.

Preliminary investigations of microplastic pollution in karst systems, from surface watercourses to cave waters.

Microplastic pollution in different environments has increasingly been documented in detail in recent times, but it is still poorly studied in caves and karst aquifers. To deepen the knowledge of microplastic pollution, for karst environment protection and conservation purposes, we collected and investigated different water samples from a karst area of Italy, considering connected surface and cave waters. Microplastics were extracted from water samples by filtration and subjected to organic matter removal with 15% hydrogen peroxide solution. Microplastics on filters were counted and characterised (size, colour, shape) via visual identification under a microscope, with and without UV light, exploiting fluorescence given by fluorescent whitening additives contained in plastic materials. Finally, spectroscopic analyses were carried out on 10% of the microplastics observed on each filter. The concentration of microplastics in cave waters varied from 12 to 54 items/L, with a mean value of 28 items/L. In the surface water of a tributary, it was of 23 items/L, and in the downstream, it was 29 items/L. Fibres represented the most abundant shape (95.1%) in the karst system waters, and most microplastics (82.9%) were smaller than 1 mm. The majority of the microplastics were fluorescent under UV light (84.8%), and most fluorescent particles were transparent (46%). However, black microplastics (68%) were more common among the non-fluorescent ones. Polyethylene (51.7%) was the main type of microplastics found in the karst system waters. Our results show the presence of microplastics in karst systems and provide useful information for future research. Karst aquifers are open systems, subjected to possible contamination by surface pollutants. Microplastics in karst systems can be consumed by animals, damage ecosystems and contaminate water resources; surface karst areas and underground environments should therefore be monitored and protected, especially regarding the management of water resources. To further understand the sources and transport of microplastics within a karst system, analyses on a greater range of surface and subterranean waters throughout the world are required.

Automated method for routine microplastic detection and quantification.

Microplastics (MPs) are a heterogeneous group of solid polymers with dimensions <5 mm, which are a widespread contaminant of the environment. Their ubiquitous presence grabbed researchers' attention in the last decade, and the problem of MPs detection and quantification is currently a topic of utmost importance. Most identification and quantification protocols are still based on the visual count, which is an extremely time-consuming and error-prone task due to operator subjectivity. To address such an issue, different software analysis procedures are available, but they mainly rely either on the use of optical microscopy, covering a minimal area for each sample (mm2 size), or they allow only the identification of the largest particles (>1 mm). Here, a semi-automatic innovative image processing method for quantifying and measuring microplastics on filter membrane substrates is presented and validated, comparing results with data obtained using visual counting performed by an experienced operator. The algorithm was tested with artificially generated microplastic images and samples taken from natural environments. Samples of Borgio Verezzi show cave sediment and Po River water were filtered on a glass filter membrane, and photographs were taken under 365 nm illumination, both without and with Nile Red staining. The proposed image analysis method, implemented in an easy-to-use Python script, was quite accurate and fast (about 10 s/image average processing time), showing an average deviation below 10 %, which is further reduced to about 8 % if the samples are stained with Nile Red.

Microplastic pollution in show cave sediments: First evidence and detection technique.

Microplastic particles are a global problem, which has been widely found in marine and terrestrial environments. However, microplastic pollution in caves and karst aquifers is still poorly studied. To improve the current knowledge of microplastic pollution, we investigated the sediments of a show cave in Italy. We developed a methodology based on a cave-adapted version of the methods used in several studies to detect microplastics from sediments of different environments and with various laboratory tests. The microplastics were extracted from sediments via density separation and subjected to organic matter removal. Filters were observed with and without UV light under a microscope, before and after organic matter removal, and the microplastics were characterised according to shape, colour, and size, with visual identification. About 55% of the fibres observed under the microscope on filters were removed via organic matter removal. An average of 4390 items/kg dry weight was calculated for the touristic zone and 1600 items/kg dry weight for the speleological/research section. Fibre (84.9%) was the most abundant shape, and most microplastics were smaller than 1 mm, accounting for 85.4%, of which 58.4% were shorter than 0.5 mm. The highest microplastic abundance was fluorescent under UV light (87.7%); however, 12.3% of the microplastics observed on filters were not fluorescent. Most fluorescent fibres were transparent (84%), whereas blue (46.1%) and black (22.4%) fibres were more common for the non-fluorescent ones. Our results highlight the presence of microplastics in show caves, and we provide a valid non-invasive and non-expensive analytical technique for the preparation and isolation of microplastics from cave sediments, giving useful information for evaluating the environmental risks posed by microplastics in show caves.