Open AccessReview

Lost in the Dark: Current Evidence and Knowledge Gaps About Microplastic Pollution in Natural Caves

Manuela Piccardo

and

Stanislao Bevilacqua

Department of Life Sciences, University of Trieste, 34127 Trieste, Italy

Author to whom correspondence should be addressed.

Environments 2024, 11(11), 238; https://doi.org/10.3390/environments11110238

Submission received: 20 September 2024 / Revised: 22 October 2024 / Accepted: 25 October 2024 / Published: 29 October 2024

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Abstract

In this study, a systematic review of the scientific literature was carried out to summarize the emerging evidence on microplastic pollution in natural caves. After the screening of 655 papers on the topic from a combined search on the Web of Knowledge and the Scopus databases, we found only 14 studies reporting quantitative data on microplastics from a total of 27 natural caves. Most of the assessments focused on water and sediment, with very limited investigations concerning the cave biota. Overall, the most common types of particles found in caves were small (<1 mm) fibers (~70–90% of items), transparent or light-colored, mostly made of polyethylene and polyethylene terephthalate. Anthropogenic cellulosic materials, however, represented a non-negligible portion of particles (i.e., ~20–30%). Microplastic concentrations in caves varied between 0.017 and 911 items/L for water and 7.9 and 4777 items/kg for sediment, thus falling within the levels of microplastic pollution found in other terrestrial, freshwater, and marine environments. Levels of microplastic pollution appear largely variable among caves, stressing the need to extend the geographic and environmental ranges of the assessments, which are currently concentrated on Italian caves on land, with very few case studies from other regions of the world and from marine caves. Despite their putative isolation, natural caves have a high vulnerability to microplastic contamination, requiring much more research effort to understand the potential risk that plastics pose to these fragile ecosystems.

Keywords:

micro-particles; fibers; cellulose; underground ecosystems; pollution; sediments; groundwater

Graphical Abstract

1. Introduction

From 2012 until 2022, worldwide plastic production increased by about 40%, rising from 288 Mt to 400 Mt [1,2]. Mainly due to poor waste management, the estimated global leakage to the environment (terrestrial and aquatic) was 22 Mt in 2019, and this value is projected to double, reaching 44 Mt by 2060 [3]. Once in the environment, plastics can accumulate near the source of emission or be carried by winds and currents, traveling long distances before contaminating remote areas [4]. Several agents, including solar radiation, heat, oxidants, and mechanical stress (e.g., from winds and waves), among others, begin to degrade plastic litter once it enters the environment [5]. Some plastic materials are inherently stronger and more persistent than others, potentially taking thousands of years to degrade, though in any case, the degradation process produces so-called macro-, micro-, and nanoplastics [5].

Several definitions of the term ‘microplastic’ exist, one of the most common defining microplastics as plastic particles ranging in size from 1 µm to 5 mm [6,7]. Microplastics can have industrial origins or derive from the fragmentation of larger plastic items, which leads to the classification of sources into two main categories: primary and secondary [5,7]. Primary microplastics are typically small microbeads intentionally manufactured by the plastics industry for use in cosmetics, personal care products, detergents, and paints. Some authors also include the synthetic fibers used to produce garments as primary microplastic [5]. Secondary microplastics are irregular plastic fragments unintentionally produced from the fragmentation of larger plastic items, such as bags, bottles, ropes, and nets [8]. Microplastics are now considered ubiquitous contaminants, with their presence reported both in anthropized areas and in remote regions of the globe, from isolated tropical islands [9,10] to high mountain ecosystems [11,12] and poles [13]. Due to their widespread presence and durability in the environment, microplastics are recognized as environmental hazards, with the potential impacts associated with microplastic contamination being still largely unexplored and unclear [14].

Although scientific research has made significant strides in studying the presence, abundance, and distribution of microplastics, underground environments such as natural caves remained largely unexplored despite their high ecological, economic, social, and scientific value. Caves host unique organisms with specific adaptations and specialized to survive in these extreme environments [15], some of which are endemism listed as Vulnerable in the IUCN Red List of Threatened Species (https://www.iucnredlist.org/, accessed on 15 September 2024), such as the olm Proteus anguinus (Laurenti 1768), the first obligate subterranean vertebrate described [16]. Show caves are important tourist attractions around the world, with a global commercial value of approximately EUR 2 billion per year [17]. Underground systems, through groundwater, also supply essential ecosystem services to humans by satisfying the daily freshwater needs of 2.5 billion people globally [18]. Caves also have cultural and scientific importance: they have always been considered spiritually significant by indigenous communities, and some represent true archaeological heritage [19]. Furthermore, as the most accessible underground ecosystems, caves offer a window into the subterranean domain, making them of great interest to the scientific community, particularly for paleoclimatic studies [20].

Despite the pervasiveness of plastic pollution, which has been recognized as a threat to important and fragile underground environments like natural caves for a long time, e.g., [21], quantitative assessments of microplastics in these environments are fragmented and limited to recent years, requiring the integration of available data that can serve as a reference for current and forthcoming assessments of microplastics pollution in these environments. Here, we attempted to synthetize the existing evidence on microplastic pollution in caves in order to achieve the following:

(i): Critically examine technical aspects such as sampling strategies, extraction protocols, and chemical analyses, with a matrix-by-matrix focus on water, sediment, and biota;
(ii): Provide a detailed identification of the most representative plastic particles;
(iii): Outline the levels of pollution by geographical areas and compare them with other ecosystems;
(iv): Summarize the potential sources of contamination;
(v): Discuss the ecological implications and outline future research avenues in this field.

2. Research Methodology

This systematic review followed the standards of the Preferred Reporting Items for Systematic Review and Meta-Analysis (PRISMA) Statement [22]. Studies used for this review were drawn from Web of Knowledge Core Collection (WoS) and Scopus databases. The terms ‘plastic(s)’, ‘microplastic(s)’, and ‘micro-plastic(s)’ were searched in combination with ‘cave(s)’ to generate a list of relevant peer-reviewed papers. We restricted the search to three search fields, including title, abstract, and author keywords, and to articles in English. No limitations were placed on the date of publication. However, the search date for the review ended on 10 September 2024. Studies were searched all over the world, without limitations. The assessment of each potentially relevant record found in the databases was performed by two reviewers, and only research articles (no proceeding papers) were included. After removing duplicate records, papers were screened for eligibility. Papers were excluded when (1) quantitative data were absent (e.g., papers reporting only the presence of microplastics), (2) the studied environment was not a true natural cave (e.g., studies on wetland underground systems), (3) in case of inappropriate indexing and absence of field data (e.g., studies on protocol validation or laboratory tests). For each record, we retrieved data on the year, geographic position, investigated environmental matrix (i.e., water, sediment, and biota), sampling and protocol strategy, characteristics, and the amount of microplastics found in the study.

The literature search produced 389 results from WoS and 446 from Scopus; the duplicate check allowed us to exclude 140 results, leaving 695 results to be subjected to further screening (Figure 1).

The exclusion phase, according to the eligibility criteria, limited the number of studies to 14 (Table 1), which were subjected to in-depth reading and analysis to meet the objectives of this systematic review. Furthermore, in order to facilitate interpretation of table and figures, a numeric identifier (ID) was assigned each publication (Table 1).

3. Results and Discussion

The first work published on the topic dates back to 2022 (for a total of three publications for that year). In 2023, seven papers were published on the topic, reaching four papers in the first 7 months of 2024. The most studied continent is Europe, with 10 studies, of which 8 concentrated on the Italian peninsula, 1 in Greece, and 1 in Slovenia. The other part of the world contributed two studies from the USA and two studies from Asia (Korea and China). A total of 27 natural caves were assessed in the selected studies. The investigated caves can be classified into three groups: tourist, therefore frequented by the general public or speleologists; non-tourist; and submarine caves (only two studies). The preferred environmental matrix for assessing microplastic contamination levels was water, followed by sediment and biota.

3.1. Sampling, Protocol of Extraction, and Chemical Analysis

Table 2 shows, case by case, details on the sampling strategy, the protocol of extraction, and the chemical analysis used in each study.

3.1.1. Waters

Water was analyzed in 10 caves, taking a broad sample size range from 1 L to 1150 L collected in paddles and pools and using grab samples or pumps or auto-samplers. In all cases, filtration was an essential step in the extraction protocol, alone or in combination with other steps such as digestion and density gradient separation. The pore size of the filters in most cases was found to be <2 µm, rarely between 8 and 20 µm. More specifically, digestion was chosen in 6 out of 11 cases using 10–15–30% hydrogen peroxide as a digesting solution. For the density-separation step, the authors undoubtedly preferred a hypersaline solution of sodium chloride (NaCl). A tough alternative and less conventional solution made of sodium bromide (NaBr) and lithium tungstate (LiWO₄) was also tested. The particles extracted were often chemically analyzed using the micro Fourier-transform infrared spectroscopy (µ-FTIR) and, rarely, the micro Raman (µ-Raman) technique or fluorescence microscopy. When specified (only in half of the cases), the percentage of particles included in this final step was 10% (3 cases), 15% (1 case), 22–42% (1 case), and all >100–200 µm (2 cases). The level of contamination was expressed using three different units: items/L, items/kg, or items/m³.

3.1.2. Sediments

The quantity of sediment, mostly superficial (upper 2–5 cm layer) and rarely submerged, ranged from a minimum of 150 g to a maximum of 1 kg and was sampled via grab sampling. In five out of eight cases, the first step of the extraction protocol began with drying, often followed by separation using a density gradient in hypersaline solutions of sodium chloride. In half of the cases, the samples required a digestion phase in hydrogen peroxide (at 15–30%) for the removal of organic matter. Finally, the filtration of the supernatant was performed with filters of variable pore sizes between 0.45 µm and 20 µm (a preferable pore size of 1.2 µm). All studies but one (i.e., [23]) benefited from chemical characterization on a subset of extracted particles in order to refine estimates of microplastics and exclude particles of natural origin. The preferential technique for the chemical analysis of particles from cave sediments was micro Fourier-transform infrared spectroscopy (µ-FTIR) on >10% of the total particles extracted. The level of contamination was expressed both in items/kg and in items/m³.

3.1.3. Biota

Only three studies investigated the presence of microplastics in biological matrices, focusing on groundwater fauna [24], monk seal feces [25], and agglutinated foraminifera [26]. In these cases, samples were taken using grab sampling or by means of brushes, nets, and pumps. Digestion was always performed (in 10% KOH or 10–30% H₂O₂), followed by density separation (in NaCl or NaI solutions) and filtration (1.2–1.6 µm of pore size). µ-FTIR and Attenuated Total Reflection (ATR)-FTRI were chosen for the chemical analysis of the particles. The level of contamination was expressed as items per dry or wet weight.

3.2. Methodological Considerations

For the investigation of microplastic presence in cave systems, different matrices were selected—water, sediments, and biota—analyzing them individually or simultaneously. The size of samples, the number of replicates, and spatial scale varied depending on the characteristics of the site under investigation, the ease of access to the cave, and the scientific questions posed by the researchers. While these choices are justified and reasonable, the adopted experimental designs have rarely taken into account the spatial heterogeneity of cave environments arising, for instance, from variations in groundwater flow regimes or cave topography. Since the natural variability in environmental factors, such as the sediment grain size and organic matter content, may influence microplastic occurrence especially in complex ecosystems like karst systems [37,38], preliminary assessments of intra-cave spatial and environmental heterogeneity should be carried out to ensure the representativeness of sampling and to prevent under- or overestimation of microplastics in the investigated matrices.

The protocols adopted have often been inspired by published and validated sector studies focused on marine matrices, where microplastic research is undoubtedly more advanced. The adopted protocol was often multi-step, frequently involving the use of an NaCl brine. The use of simple table salt is widely accepted within the scientific community because it provides the best cost–benefit ratio, but it is ineffective for extracting high-density polymers [39]. The level of detail is generally high, with a minimum cut-off, dictated by the pore size of the filters, of just a few microns. In almost all cases, the analysis concluded with chemical characterization, often using micro Fourier-transform infrared spectroscopy techniques. While the use of chemical characterization techniques helps reduce the bias introduced by the operator’s subjectivity, the percentage of extracted particles subjected to this step remains relatively low (often less than 15%). This is justified by several reasons: high costs of purchase, maintenance, and use of FTIR; the need for qualified personnel to perform the analyses; and the relatively long time required to scan the filters (on the order of days per filter). In light of this, to balance cost–benefit considerations and minimize potential over- or underestimation errors, it is recommended, on a case-by-case basis, to pay particular attention to the strategy for selecting the extracted particles to be subjected to such analysis and to clearly and transparently share the reasons for these choices. Additionally, this underscores the need to adopt all possible precautions and strategies to make visual analysis more effective, objective, and sensitive. Specific protocols developed and proposed in the reviewed studies represent considerable improvements in this direction and are strongly recommended for the assessment of microplastic particles in cave sediments. These included the use of UV lamps to facilitate the visual identification of fluorescent particles [23], a semi-automatic image-processing method for quantifying and measuring microplastics on filter membrane substrates [40], and the adoption of a standardized size and color sorting (SCS) system for categorizing microplastic [5].

3.3. Microplastic Identikit

In this section, data were analyzed and shown matrix by matrix in order to create an identikit of the most representative micro-particles.

3.3.1. Water

In the analysis of the publications screened in this review, fibers represent the most abundant type of micro-particles (70%), followed by fragments (20%) and film (10%, Figure 2). The polymer type of items was quite variable with five different plastic polymers, among which polyethylene is the most frequent (35%), followed by polyethylene terephthalate (20%), polypropylene, polyvinylchloride, and polyester (10% each). Anthropogenic cellulose, referring to non-plastic man-made materials based on natural cellulose (e.g., rayon and viscose) with trace of chemical processes (e.g., dyes), accounted for 20% of particles. The typical color of particles is characterized by a transparent or clear color (50% of cases altogether), whereas a lower portion of particles was blue, black, or defined generically with the term ‘colored’ (respectively 10% of cases for each type). In 20% of studies, the information on color was not available. From the point of view of the size, most of the particles (80%) were smaller than 1 mm.

3.3.2. Sediments

The pattern of micro-particles extracted from sediments collected in natural caves presents similar characteristics to that from water samples. Fibers were the most abundant type (88%), followed by fragments (12% of cases, Figure 3). The array of polymer types partially overlaps between sediments and water, sharing polyethylene, polypropylene, and polyethylene terephthalate. A copolymer was found exclusively in sediments, whereas polyester and PVC were only found in water samples. Anthropogenic cellulosic materials represented a non-negligible portion of particles (29%) in sediments. The most frequent polymer in sediments, excluding anthropogenic cellulose, was polyethylene (21%), followed by copolymer and PET (14% each) and polypropylene (7%). As for water, in sediments, the typical particle was characterized by a transparent/clear color (50%), followed by blue particles (25% of cases), with 25% of case studies lacking color characterization. Most of the particles found in sediments were smaller than 500 µm.

3.3.3. Biota

The data relating to this section are scarce and refer, in one case, to terrestrial underground fauna [24] and, in two cases, to samples collected in underwater marine caves [25,26]. In the former study, Sforzi et al. 2024 [24] found cellulosic pellets smaller than 1 mm as the most abundant typology of particles extracted from groundwater invertebrates. In the other cases, Hernandez-Milian et al. 2023 [25] found blue fibers of polyamide smaller than 3 mm in monk seal excrements, and Bergamin et al. 2024 [26] only reported the presence of polyethylene inside the body of agglutinated foraminifera.

3.4. Fibers: The Most Common Type

In 2020, global fiber production reached a total volume of 120 million tons, equating to nearly 16 kg per capita [41]. Textiles contribute approximately 14% of plastic waste production by sector, making them the second-largest source of plastic pollution after packaging [42]. Microfiber release occurs throughout various stages of textile and garment production, as well as during normal wear, laundering, emissions from outdoor textile equipment, and from discarded textiles. Among these sources, domestic and commercial laundry has been identified as a primary contributor to microfiber pollution [43], with studies reporting that around 700,000 microfibers (about 0.5 g in weight) can be released with every wash cycle [44]. Rivers serve as the main transport pathway for these mishandled microfibers, which flow downstream and are eventually discharged into the ocean [45] and underground systems.

Recently, the issue of anthropogenic microfibers has also been recognized in karst systems. In this review, it is evident that fibers represent the dominant type (>70%), typically transparent or light-colored, often less than 1 mm in size (thus classifiable as mini-microplastics, [5]), and composed of anthropogenic cellulose or PE. Anthropogenic cellulose was the most abundant polymer in 20% of the cases in water and 29% of the cases in sediment samples. However, it must be specified that it cannot be included as a plastic. Fibers, in fact, can be classified into three subcategories [1,2]: synthetic/plastic (made of plastic polymers), anthropogenic/natural man-made cellulose (natural cellulose with the presence of chemicals such as dyes), and natural (e.g., cotton, linen, modal, or Lyocell). Therefore, it is essential to clearly distinguish the chemical nature of such particles to avoid misunderstanding. However, although natural or regenerated fibers do not strictly fall into the category of microplastics, they potentially represent a risk to ecosystems due to the presence of toxic compounds such as resins, dyes, and flame retardants or simply because they are capable of causing physical damage. For example, Kim et al. 2021 [46] compared the toxicity of natural (Lyocell) and synthetic (PET and PP) microfibers (both prepared without the use of additives) on depuration capacity of Daphnia magna, food intake, growth, mortality, and immobilization rate. The study demonstrated that synthetic fibers have a greater impact on immobilization than natural fibers. It was also shown that the strongest effects on mortality, growth inhibition, and gut damage during depuration occurred after the exposure to the naturally derived microfibers, probably as a result of the imbalance in nutrient uptake and reductions in metabolic activity that followed gut damage caused by the ingestion of microfiber. Although unique in its kind, the study confirms how even natural microfibers can have adverse effects on aquatic organisms, stressing the need for future ecotoxicological research.

3.5. Variations in Pollution Levels

3.5.1. Geographic Variations

The accumulation of microplastic in the environment varies geographically with locations, hydrodynamics conditions, environmental pressure, and time [47]. Natural caves are not an exception to this pattern since global pollution levels appear extremely variable. However, the geographic distribution of data is highly biased toward specific areas, making any generalization on global scale patterns of variation in pollution levels still premature.

Undoubtedly, Europe, particularly the Italian and Slovenian Karst regions, leads the study of this phenomenon. These areas exhibit higher levels of contamination, with concentrations significantly exceeding those found in other parts of the world: more than 2000 items/kg in sediment (Figure 4a) and up to 911 items/L in water (Figure 4b). Sediment seemed to be the most polluted matrix, with a wide range of contamination levels. The lowest level of micro-particles was reported in [31], with 7.8 items/kg, whereas the most impacted cave was identified in [32], showing 4777 items/kg. The karst system of North-western Italy had a maximum average contamination level of 4390 items/kg in sediments and 64 items/L in water (Figure 5, blue box). In this area, different caves, including those in Piedmont (Bossea Cave) and Liguria (Borgio Verezzi and Toirano Caves), were analyzed, all of which are open to the public. Due to this, contamination levels were investigated in both tourist and non-tourist sections. The most contaminated cave was Bossea, with average values of 4390 items/kg of dry weight in tourist sections and 1600 items/kg in non-tourist sections. Bossea Cave, Italy’s first show cave opened to the public in 1874, is very popular, attracting about 12,000 tourists annually [23]. The cave has a single entrance and extends for about 2800 m, representing the terminal sector of a large karst system. Furthermore, the cave is traversed for approximately 1.5 km by a subterranean river (Mora River) and hosts several underground scientific laboratories. This suggests multiple potential sources of microplastic contamination, both internal—such as the installation and maintenance of the electrical system, tourist activity, and scientific research—and external, including surface pollution penetrating the subsurface via air currents or underground watercourses.

Research on microplastics in cave systems in the remaining parts of the world is limited to America and Asia, for a total of six investigated caves. The only two studies conducted in the Americas, both in Cliff Cave, Missouri, reported maximum levels of 843 items/kg in sediments [33] and 81 items/L in water [33,34]. Overall, the level of contamination in this cave appears limited (Figure 4), likely due to the cave’s specific characteristics and location. Cliff Cave runs primarily beneath a forested area (Cliff Cave County Park), although parts of it also extend beneath a residential area and border commercial zones and highways to the west of the park. Additionally, the cave is fenced off to reduce the spread of white-nose syndrome among bats, with only a limited number of visitors permitted during summer guided tours. The cave’s topographical features and proximity to human activities suggest potential sources and processes of contamination, including surface pollution combined with atmospheric deposition and flood-related processes.

Asian studies have focused exclusively on water samples collected in the Dragon Palace Cave (Guizhou Province, [35]) and four different caves in the Donghae and Samcheok areas of Korea [36]. The levels of pollution ranged from 0.014 to 9.50 items/L of water. In the case of the Dragon Palace Cave, a popular tourist attraction with high visitor density during peak season, the analysis of extracted particles suggested various primary sources of pollution in karst groundwater, including personal care products, domestic wastewater, agricultural irrigation, plastic waste, organic manure, and fishing activities. Once in the environment, micro-particles can reach the underground through air deposition, soil erosion, surface runoff, and soil penetration. In the Korean study, the investigated area was characterized by a complex landscape with nine different land uses, including agriculture, industry, road/railroad networks, and residential/commercial zones. Despite this complexity, the maximum pollution level in the cave samples was only 0.3 items/L. According to the authors, the high proportion of plastic fragments collected may be attributed to the widespread use of non-fiber plastic products in agricultural activities within the study region. These products include plastic mulch, pesticide bottles, fertilizer bags, greenhouses, sprinklers, plastic crates, pesticide sprayers, insulation materials, plastic casings, and plastic drainage pipes. However, some sources of micro-particles may not be directly linked to agricultural activities in the immediate vicinity but could result from long-distance transportation through the atmosphere.

The karst system of North-eastern Italy exhibits similar levels of contamination, with a maximum of 4777 items/kg in sediments and 163 items/L in water (Figure 5, purple box). However, the only study conducted on the Slovenian side (Figure 5, red box), which is closely connected to the Italian side as it is part of the same Classical Karst system, shows a low level of contamination, with 0.017 items/L in water and 35.3 items/kg in sediment. These differences may be attributed to various factors, including differences in the protocols used and the small number of samples (only four in the Škocjan Caves system in Slovenia), which may have resulted in potential issues related to sample representativeness.

In the central area of the Italian peninsula, Buca del Vasaio Cave in Tuscany stands out as the most polluted site, with a maximum contamination level of 911 items/L. However, the marine caves studied by Romano et al. (2023) [31] and Bergamin et al. (2024) [26] did not reveal significant contamination issues, with only 17.2 items/kg and 7.8 items/kg, respectively. In these cases, the primary sources of contamination are likely linked to maritime traffic, land-based activities, the presence of divers, and, to a lesser extent, fibers from tourists’ clothing. In fact, the first study focused on the middle and northern branches of the cave, which are frequented by tourists only for short periods. Interestingly, this is the only geographical area where studies on underground/underwater fauna have been conducted (see Section 3.3.3).

3.5.2. Comparison with Other Ecosystems

Plastic pollution has reached every environmental compartment, extending even to the most remote areas of the globe. The amount and size of particles, their type, and polymer composition can be largely variable both within and among different environments [48]. Such an idiosyncratic distribution, concentration, and main features of microplastics depend on the main sources of contamination, transport, and sinking patterns characterizing the different environments, the investigated environmental matrix, and the closeness to human activities and settlements [48]. Moreover, the heterogeneity in sampling, analysis of particles, and counting methods throughout the bulk of microplastic assessments strongly affect estimates of microplastic pollution, so comparisons among environmental compartments should be taken with caution.

In Figure 6, we summarize the results from the most recent and comprehensive reviews on microplastic pollution across marine, freshwater, and terrestrial environments (see the figure caption for references). Fibers and fragments made of polyethylene and polypropylene were the most common particles across all compartments. Small particles (<1 mm) were the dominant size in sediments, irrespective of the compartment, whereas large particles (>1 mm) seemed to be more common in aquatic media, especially in marine and river waters. Colored particles were mostly found in surface waters (marine and freshwaters), while transparent/clear particles were the dominant type in ground waters (including cave waters) and sediments from all environmental compartments.

As expected, the microplastic concentration is higher in sediments than in water, as sediments represent the ultimate destination of particles, whatever the environmental compartment (Figure 6). For water, the highest maximum concentration was found in rivers, followed by lakes and seawater (including brackish and estuarine systems). The highest concentration recorded in groundwater (aquifers and wells) was 4–5 times higher than in other aquatic media, although the average concentration of particles was generally lower than 100 particles/L [49]. The range of microplastic pollution in sediments was comparable across environments, with the exception of sediments in lakes, which showed lower maximum and mean concentrations.

Figure 5. Geographical distribution of studies performed on microplastics in cave systems. A special focus on the Italian and Slovenian Karst regions (the most studied areas). Data are shown as percentage of contamination with respect to the maximum (for sediments: Balestra et al. 2024b [32], reference ID = 10; for waters: Sforzi et al. 2024 [24], reference ID = 2). The graphs have been placed inside boxes of different colors, corresponding to the different sub-regions. Numbers in the global map and in bar plots refer to paper ID in Table 1.

Figure 6. Ranges (min–max) of particle concentrations in water and sediment from marine (including brackish and estuarine systems), lake (including lakes, ponds, and reservoirs), riverine (including canal, streams, and rivers), soil (water: groundwater including aquifer and wells; sediment: agricultural, urban, and rural soil), and cave environmental compartments. Numbers in brackets indicate the approximate average concentration. For each environmental matrix in each compartment, the most common type of particles, polymers, and colors are also shown (for types of particles and colors, two symbols indicate an almost equal contribution). Data are from the following sources—marines: [48,50,51,52,53,54,55,56,57,58]; lakes: [48,51,59,60,61]; rivers: [48,51,58,60,61,62,63,64]; soil: [48,49,51,65,66,67,68]; caves: this study.

Based on the available data in the literature, the maximum level of microplastics found in caves was 4777 items/kg in sediments and 911 items/L in waters, with an average concentration of 1394 items/kg and 184 items/L, respectively. These values are aligned with concentrations reported in other environmental compartments, especially for water, suggesting that caves are as vulnerable to microplastic pollution as other superficial environments. The reasons for this are apparent when considering the nature of such systems. Karst aquifers are characterized by the presence and development of dissolution features in soluble rock, such as caves, conduits, and sinkholes, which result in high connectivity between the surface and subsurface [36]. This unique architecture leads to complex water flow paths in karst environments and facilitates the introduction of surface contaminants, such as microplastics. Further assessments are needed to gain a better global perspective on the phenomenon, understand the scale of the problem, and hypothesize the potential implications for ecosystems and human health.

4. Knowledge Gaps and Future Research Needs

Natural caves are confined systems characterized by harsh environmental conditions in terms of low availability of trophic resources, low oxygen concentration, and a lack of light. As a result, cave fauna tend to have long lifespans, slow metabolism, low reproduction rates, and naturally small population size, similar to those in the deep ocean. The same characteristics that allow these organisms to survive also make them vulnerable to changes in their peculiar habitat. Plastic litter may cause significant alterations to these systems, threatening these fragile underground environments in several ways. For example, microplastics can be ingested by cave animals, with effects that remain unpredictable. Sforzi et al. (2024) [24] found microplastics in every groundwater taxon analyzed (i.e., Isopoda, Harpacticoida, Cyclopoida, and Ostracoda), reporting pellets as the most abundant type and cellulose as the most abundant polymer. The initial evidence in biota is emerging, but no attempts have been made to quantify the effects of microplastics on cave organisms. Much more research efforts are needed before a risk assessment can be made, even though the high concentrations of microplastic reported in the reviewed studies raised wide concerns about possible detrimental effects on cave communities. Several studies highlighted toxic effects, histopathological consequences, and oxidative stress in aquatic organisms exposed to environmentally relevant microplastic concentrations in water (e.g., 40 particles/L [69]; 100–1000 particles/L [70]; 10–100 particles/L [40]) comparable to those found in caves. Also, as found for many invertebrate species in marine and freshwater environments [71,72], the ingestion of microplastic could severely impact on growth, reproduction, and behavior of cave fauna, ultimately affecting their populations, with potential consequences propagating to the whole cave food web, which can be relatively simple and not so functionally redundant to absorb a substantial change in community structure. Microplastics, by polluting aquifers, may also threaten human health, as the presence of large dissolution openings in karst terrains allows for the rapid transport of water through these systems, facilitating the introduction of surface contaminants into subsurface habitats and groundwater reservoirs for human uses [36]. Finally, plastics can irreversibly compromise speleothems and paleontological or archaeological finds through physical damage and deposition, threatening the historical, geological, and cultural heritage of caves.

A major issue limiting our comprehension of microplastic pollution in natural caves concerns the narrow spatial distribution of the investigated caves. The geographical coverage of studies is poor and patchy, with no studies from South America, Africa, or Oceania. Half of the assessments were conducted in Italy, where the studied caves exhibited the highest levels of contamination, with concentrations significantly exceeding those found in the other parts of the world (>2000 items/kg in sediment and up to 911 items/L in water). Globally, plastic contamination levels vary greatly—by 3 to 4 orders of magnitude—depending on the cave’s topographical features, its connections to the epigean environment, and the intensity and type of anthropogenic activities in the area, from tourism-related activities in show caves to surface activities such as agriculture, industry, and vehicular traffic. Extending the assessments to other geographical areas and types of caves is therefore a priority in this research field. Specifically, future assessments should focus, whenever possible, on caves that are rarely visited by humans or recently discovered in order to establish a baseline of microplastic contamination. Such information could allow for estimating the contribution of indirect sources of contamination and clarify the actual role of direct human frequentation in determining the level of microplastic pollution in natural caves. Due to the complexity of cave environments and the large discrepancies in the amount of microplastics among caves, quantifying the within-cave variability of microplastic pollution at a range of spatial and temporal scales is also strongly recommended in order to implement robust sampling designs and achieve reliable estimates of microplastic pollution despite possible small-scale patchy distributions of particles.

Sediments were found to be more contaminated than water, potentially acting as long-term sinks and therefore representing the preferred matrix for microplastic monitoring plans. Irrespective of the matrix, small and transparent fibers were the dominant type. This reinforces the need for appropriate identification strategies (e.g., the use of UV lamps) and chemical characterization to correctly classify their chemical nature; most studies have analyzed less than 10–15% of the extracted particles due to costs, though much more effort should be devoted to increasing this percentage. The chemical characterization of particles is of critical importance in these environments since a relevant fraction of particles found in caves may be represented by non-plastic polymers (i.e., natural or regenerated cellulosic materials), potentially leading to overestimating plastic pollution levels. It is worth noting, however, that the information on the presence and amounts of non-plastic polymers should not be discarded as, similarly to microplastics, cellulosic particles may also negatively affect the biota [73,74].

Above all, the scientific literature on plastic pollution is still sparse and mostly focused on show caves, which are more easily accessible to researchers and raise relatively minor difficulties for field works. However, this may lead only to a partial picture of plastic pollution in natural caves, preventing a deeper insight into the problem. From this view, major attempts to promote citizen science initiatives involving industry professionals and speleologists—who, with their skills and courage, may assist scientists in accessing otherwise unreachable sites—can be instrumental in collecting new invaluable data essential to enhancing the research in the field and to implementing specific protection and mitigation measures for these isolated and critical environments.

Author Contributions

Conceptualization, M.P. and S.B.; methodology, M.P. and S.B.; formal analysis, M.P.; writing—original draft preparation, M.P.; writing—review and editing, S.B. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Data Availability Statement

The raw data supporting the conclusions of this article will be made available by the authors on request.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. PRISMA 2020 flow diagram adopted for the systematic review presented [22].

Figure 2. Identikit of the most representative micro-particles detected in water samples collected in natural caves according to the (a) type, (b) polymer (anthropogenic cellulose = natural cellulose with the presence of chemicals such as dyes, PE = polyethylene, PP = polypropylene, PET = polyethylene terephthalate, PVC = polyvinyl chloride, polyester), (c) color, and (d) class of size. na = not available. Graphs created with Infogram (https://infogram.com/, accessed on 20 October 2024).

Figure 3. Identikit of the most representative micro-particles detected in sediment samples collected in natural caves according to the (a) type, (b) polymer (anthropogenic cellulose, PE = polyethylene, PP = polypropylene, PET = polyethylene terephthalate, copolymer), (c) color, and (d) class of size. na = not available. Graphs created with Infogram (https://infogram.com/, accessed on 20 October 2024).

Figure 4. Maximum levels of micro-particle/plastic pollution in (a) sediment and (b) water collected worldwide in cave systems. For the correct interpretation of the reference ID, refer to Table 1.

Table 1. List of publications analyzed in the present review.

ID	Reference	DOI	No. of Caves Assessed
1	Balestra and Bellopede (2022) [23]	http://dx.doi.org/10.1016/j.envpol.2021.118261	1
2	Sforzi et al. (2024) [24]	http://dx.doi.org/10.3390/su16062532	2
3	Hernandez-Milian et al. (2023) [25]	http://dx.doi.org/10.1016/j.marpolbul.2023.115227	1
4	Bergamin et al. (2024) [26]	https://doi.org/10.1016/j.rsma.2024.103547	1
5	Valentic et al. (2022) [27]	http://dx.doi.org/10.3986/ac.v51i1.10597	4
6	Balestra et al. (2023) [28]	http://dx.doi.org/10.1016/j.jconhyd.2022.104117	1
7	Balestra and Bellopede (2023) [29]	http://dx.doi.org/10.1016/j.jenvman.2023.118189	3
8	Balestra et al. (2024a) [30]	http://dx.doi.org/10.1016/j.jenvman.2024.120672	3
9	Romano et al. (2023) [31]	http://dx.doi.org/10.1016/j.marpolbul.2022.114452	1
10	Balestra et al. (2024b) [32]	https://doi.org/10.1016/j.chemosphere.2024.142811	3
11	Hasenmueller et al. (2023) [33]	http://dx.doi.org/10.1016/j.scitotenv.2023.164690	1
12	Baraza and Hasenmueller (2023) [34]	http://dx.doi.org/10.1016/j.watres.2023.120204	1
13	An et al. (2022) [35]	http://dx.doi.org/10.3390/ijerph192214751	1
14	Jeong et al. (2023) [36]	http://dx.doi.org/10.1016/j.scitotenv.2023.165006	4

Table 2. For each publication (ID refers to papers listed in Table 1), the type of matrix investigated, the sampling strategy adopted, as well as some details about the protocol of extraction and the related chemical analysis are reported. na = not available; when the same study explores different matrices, the respective information is separated by a semicolon.

SAMPLING STRATEGY		PROTOCOL OF EXTRACTION				CHEMICAL ANALYSIS
Matrix	Volume of Samples and Sampling Technique	Procedural Steps	Density Seperation	Digestion	Filtration (Pore Size)	Analysis Performed?	Technique	% Items Analysed	Reference ID
sediment	300 g of sediment from the upper 5 cm of soil	drying, density separation, settling, filtration, drying, filter digestion	NaCl	15% H₂O₂	1.2 µm	NO	-	-	[1]
sediment	150 g of sediment	drying, density separation, settling, filtration, drying, filter digestion	NaCl	15–30% H₂O₂	1.2 µm	YES	µ-FTIR	<10%	[7]
water	water collected from spring with an automatic sampler	filtration	not performed	not performed	0.45 µm	YES	µ-FTIR	10%	[12]
water	500 L of water collected with a peristaltic pump	filtration, digestion, filtration	LiWO4	not performed	20 µm	YES	µ-FTIR	na	[14]
water	2 L of grab samples	filtration, digestion, density separation, settling, filtration	NaCl	30% H₂O₂	1 µm	YES	µ RAMAN	na	[13]
water	flowing water from pools and springs	filtration, drying, digestion	na	15% H₂O₂	0.8 µm	YES	µ-FTIR	10%	[6]
sediment and water	400 g of sediment from the upper 2 cm of soil; 1 L of grab sample or water collected with a plankton net (100 µm mesh) used for 60 min	density separation, settling, filtration; filtration	NaCl	not performed	8 µm	YES	µ-FTIR	all > 200 µm	[9]
sediment and water	250 g of sediment from the upper 5 cm of soil; 500–1150 L of water bulk samples from springs	drying, digestion, drying, density separation, settling, filtration, drying	NaCl	30% H₂O₂	1.2 µm	YES	µ-FTIR	15%	[8]
sediment and water	0.5–1 kg of sediment from the upper 5 cm of soil; water from puddles and pools	drying, 2 mm sieving, density separation, filtration; filtration	not performed	not performed	12–20 µm	YES	µ-FTIR	na	[5]
sediment and water	submerged sediment; 1 L of water bulk sample from puddles and still waters	drying, digestion, drying, density separation, settling, filtration, drying	NaCl	30% H₂O₂	1.2 µm	YES	µ-FTIR	<10%	[10]
sediment and water	sediment from the upper 5 cm of soil; 1 L of water	density separation, settling, filtration; filtration	NaCl	not performed	0.45 µm	YES	µ-FTIR	22–42%	[11]
water and biota	max 2 L of grab sample of water; for biota: brushes, net, immersion pump, and net	digestion, density separation, settling, filtration; digestion, filtration, Nile red	NaCl	30% H₂O₂	1.2 µm	YES	µ-FTIR flµorescence microscopy	na	[2]
sediment and biota	400 g of grab sample of sediment	digestion, filtration, density separation, filtration	NaBr	10% H₂O₂	1.6 µm	YES	µ-FTIR	all	[4]
biota	grab samples of feces	sieved, digestion, density separation	NaCl and NaI	10% KOH	na	YES	ATR-FTIR	na	[12]

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Piccardo, M.; Bevilacqua, S. Lost in the Dark: Current Evidence and Knowledge Gaps About Microplastic Pollution in Natural Caves. Environments 2024, 11, 238. https://doi.org/10.3390/environments11110238

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Piccardo M, Bevilacqua S. Lost in the Dark: Current Evidence and Knowledge Gaps About Microplastic Pollution in Natural Caves. Environments. 2024; 11(11):238. https://doi.org/10.3390/environments11110238

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Piccardo, Manuela, and Stanislao Bevilacqua. 2024. "Lost in the Dark: Current Evidence and Knowledge Gaps About Microplastic Pollution in Natural Caves" Environments 11, no. 11: 238. https://doi.org/10.3390/environments11110238

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Piccardo, M., & Bevilacqua, S. (2024). Lost in the Dark: Current Evidence and Knowledge Gaps About Microplastic Pollution in Natural Caves. Environments, 11(11), 238. https://doi.org/10.3390/environments11110238

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