Supported data and manuscript "Identification of asbestos fibres from soil sediments in the Pilsen region of the Czech Republic and the impact of these minerals on the health of the local population"

CORRECTED PROOF Vacuum xxx (xxxx) 114343 Contents lists available at ScienceDirect Vacuum journal homepage: www.elsevier.com/locate/vacuum Identification of asbestos fibres from soil sediments in the Pilsen region of the Czech Republic and the impact of these minerals on the health of the local population Stepanka Jansova⁎, Zdenek Jansa, Pavel Calta, Veronika Vavrunkova, Lucie Nedvedova, Jan Minar New Technologies –Research Centre, University of West Bohemia, Plzeň, Czech Republic ARTICLE INFO Handling Editor: Oleg Malyshev ABSTRACT Asbestos is the term for silicate minerals with a typical fibrous structure that crystallises as separable fibres that can be released into the environment due to natural processes and anthropogenic activities. There is a need to intensify geo-environmental monitoring of the occurrence of natural asbestos on a global scale. The study of this material is essential to clarify the impact of asbestos on public health and to have an accurate knowledge of the requirements for asbestos replacement materials. The technical and ecological reasons for switching to these fibres are complex, as asbestos replacement materials are subject to considerable technological and economic demands, as well as demands for their biological safety. The main objective of this paper is to establish a suitable methodology for detecting asbestos in soil sediments and accurately identify the different types from a range of samples. Samples were analysed by electron microscopy and X-ray diffraction and compared with standards or available literature. The measurements demonstrated the presence of asbestos in the site sediments and identified specific types of asbestos. The conclusion of this work is confirming the presence of asbestos in all samples, including its most dangerous types, which can cause severe diseases. In this context, the mechanism of asbestos-related diseases will be further addressed, which is linked to the size and shape of the individual fibres, the chemical composition of the asbestos types and the links between their basic structural units. 1. Introduction Asbestos is a naturally occurring fibrous mineral widely used, particularly in the 20th century. These minerals are united by a major common property: a fibrous structure. However, in recent decades, considerable research has focused on the strong association between fatal diseases and exposure to asbestos-like minerals present in various environmental matrices. Asbestos is a hazardous substance that, after years of widespread use in industry and construction, has been identified as a carcinogenic substance with mutagenic effects that pose a risk to the human body and should be treated cautiously. When asbestos is disturbed, it releases fibres in the form of dust, which can pose a serious health risk. Detailed analytical procedures have been established to determine the amount and type of dust particles in the air. However, analyses in soil sediments still leave room for finding the correct and refining the whole procedure to identify the different types of asbestos accurately [1–3,6]. Asbestos is a subgroup of fibrous minerals - silicates. Asbestos minerals consist of 40–60 % silicon, the remainder being oxides of iron, magnesium and other metals. A common feature of all asbestos is its fibrous structure, with the length of the fibres being many times their diameter [4,5,10]. Asbestos is divided into two main categories: serpentines and amphiboles (Fig. 1), whose basic building unit of the silicate structure is the silicon-oxygen tetrahedron [SiO4]4-. Asbestos minerals that grow in two or three directions instead of breaking into smaller pieces (fragments) are classified as 'non-asbestiform' type asbestos, while these minerals may still have the same chemical formula as the 'asbestiform' type. The term 'asbestiform' type of asbestos refers only to silicates in poly filament bundles and consists of extremely flexible fibres of relatively small diameter and long length. These fibres are easily separable This article is part of a special issue entitled: EVC-17/ECOSS-37 published in Vacuum. ⁎Corresponding author. E-mail address: [email protected]cu.cz (S. Jansova). https://doi.org/10.1016/j.vacuum.2025.114343 Received 26 September 2024; Received in revised form 24 February 2025; Accepted 15 April 2025 0042-207/© 20XX Note: Low-resolution images were used to create this PDF. The original images will be used in the final composition. CORRECTED PROOF S. Jansova et al. Vacuum xxx (xxxx) 114343 Fig. 1. Two basic types of asbestos fibres; electron microscope images: amphibole type, (b) serpentine type. from the host matrix or can be cleaved into thinner fibres. Therefore, a specific characteristic of asbestos is its tendency to form long, thin, fibrous structures that tend to split along their entire length [3–5,10,17]. A representative of serpentines is chrysotile, which appears as a white fibre. It is obtained from rocks commonly found throughout the world. Chrysotile fibres are wavy and flexible and tend to form clusters, making it possible to spin and weave them into fabrics [7,9,[21]]. The second group of asbestos, the amphiboles, has five members (crocidolite, amosite, tremolite, anthophyllite and actinolite) whose fibres are smooth and have pointed ends (needle-shaped). The most dangerous of the amphibole group is crocidolite, which is often referred to as blue asbestos [1,2,5]. The aim of this paper is, therefore, not only to determine the appropriate me[37]thodology for detecting the presence of asbestos in soil sediments and the subsequent determination of the exact types from the samples but also to summarise the current knowledge on the extensive issue of asbestos occurrence and to highlight its impact on public health. The effort to emphasise the need for focused mapping of the natural occurrence of asbestos and its inclusion in the laws and decrees of the Ministry of the Environment is essential to this publication. 2. Sample preparation and experimental methods The samples were collected in the exact location around the Plaska Dam in the northern part of the Pilsen Region in the Czech Republic (Fig. 2). Due to the increased incidence of cancer in the area, an analysis of soil sediments was requested to verify the presence of asbestos or to determine their exact species. Care had to be taken to avoid contamination of the tools, instruments and especially the samples themselves with each other (see Fig. 3). The samples were air-dried in a laminar box for several days. The subsequent grinding phase had two steps. The first step was sieving the samples, which allowed the removal of coarse additional materials from the sample (e.g., plant fragments, etc.). The second grinding step was the milling. This involved hand grinding in an agate mortar, which reduced the difference in sample size classes and allowed us to homogenise the sample. Since our experiment was a qualitative analysis and not a quantitative one, the chosen sample preparation steps allowed us to accept a partial loss of the native information of each sample. Even so, the grinding time itself had to be considered, which, especially for the amphibole group, could be a problem if grinding was carried out for more than 30 s [19]. Samples of both series were annealed at temperatures up to 530 °C for 4 h. The sediment samples were placed in corundum pellets in a CarFig. 2. Map showing soil sediment sampling locations. Plaská Dam, Pilsen Region, Czech Republic [40]. bolite horizontal vacuum tube annealing furnace. The annealing rate of 10 °C/min was reduced to 5 °C/min after reaching 300 °C. After reaching an annealing temperature of 530 °C, the temperature was stable for 4 h. The samples cooled naturally in the air. The annealing was performed to remove the organic components of the sampled material [16], which facilitated the search for asbestos fibres and the associated analysis by scanning electron microscope. The serpentine group - specifically chrysotile - changes to meta chrysotile in the temperature interval 550–750 °C and then above 800 °C rapid recrystallisation occurs, with forsterite and enstatite (non-fibrous structure) being the main transformation products. For the amphibole group, thermal decomposition occurs at temperatures above 900 °C and around 1100 °C. Generally, this decomposition is less described in the literature and depends on the amphibole type. Therefore, the selected annealing temperature was set at 530 °C to avoid the thermal decomposition of the as2 CORRECTED PROOF S. Jansova et al. Vacuum xxx (xxxx) 114343 Fig. 3. Diagram of the annealing process. bestos phases of interest. The chosen annealing temperature is a suitable method to simplify the given phase system of the samples without affecting the crystallinity and morphology of the asbestos minerals (Fig. 9). Based on the comparison of measurements of the same samples before and after annealing, we can say that the organic parts of the samples were burned and that the sintering of the others did not occur at the selected temperature. Loss of native information concerning the minerals of interest was impossible in this case [19]. The entire sample preparation, including the collection itself, was carried out under strict safety measures (use of sealed and leak-proof bags for storing the collected samples, protective masks and clothing, cleaning of the sampling instruments after each collection, breathing mask with the required level of filters …etc.). Asbestos is a proven human carcinogen and must be handled with great care. For this reason, the need to find a proper and unique procedure for soil sample preparation and subsequent analysis is all the more important because working with such a hazardous material causes severe diseases, which will be discussed later in this article. 3. The impact of asbestos from a medical perspective Asbestos is an occupational and environmental pollutant. Its fibres tend to split along their length, producing microscopic fibres. If these fibres become airborne, they become flammable [12].Due to their microscopic size, asbestos fibres can remain in the air for several days after being released from their fundamental (natural or artificial) matrix until they eventually settle in soil or water [11]. Asbestos dust particles have a significant negative impact on human health, especially on the respiratory system. However, the negative effect of asbestos on health is mainly due to mechanical irritation of internal tissues and chemical action. It is, therefore, a combination of irritant, chemical and biological effects on the organism [13].According to the World Health Organization (WHO), all types of asbestos are classified as carcinogenic, and their fibres are considered 'respirable' and hazardous if they are less than 3 μm in diameter, more than 5 μm in length and have a length to fibre ratio greater than 3:1 [8]. No amount of inhaled or ingested asbestos is safe, meaning there is no safe exposure threshold for asbestos. Health studies report that the long-term presence of asbestos fibres in the human body causes severe diseases, with the primary organs affected being the lungs, pleura or peritoneum, and in other cases, the heart, digestive tract or ovaries. Inhalation of asbestos dust can result in simple dusting of the lungs, called asbestosis, in which healthy lung tissue is replaced by connective tissue. This disease is usually asymptomatic in the long term and is often found quite incidentally on X-ray examination because of the increased deposition of calcium in the connective tissue. Asbestosis can be lived with, like hyalinosis of the pleura (connective tissue changes in the pleura). But a far more serious disease is bronchogenic lung cancer, a malignant disease more common in smokers than non-smokers. Another serious disease caused by asbestos is pleural or peritoneal mesothelioma (cancer of the pleura or peritoneum) [12–15]. Asbestos fibres can be inhaled or ingested. The toxicity of a mineral fibre is closely related to the size and shape of the fibre and its chemical composition. These factors influence whether inhaled fibres penetrate the respiratory tract to the alveolar space and whether the stability of the fibres is such as to cause an increase in their toxicity. At this point, macrophages, or natural immunity cells, play a vital role in the immune response. The macrophage is formed by transformation from monocytes. These are formed in the bone marrow from haemopoietic stem cells and are washed into the bloodstream. The monocytes circulate in the blood for about 8 h, then enter the tissues and change into macrophages. Tissue macrophages then show numerous heterogeneities depending on the tissue. The basic function of the macrophage is phagocytosis. This process ensures the engulfment and processing of foreign, non-functional, dead or diseased cells and other corpuscular material (material size over 100 nm). It is the oldest immune process; it can be found in lower animals [14,20[22]]. Alveolar macrophages phagocytose particles such as dust and microorganisms and remove them from the surface of the alveoli. They also attempt to remove asbestos fibres through phagocytosis. Relatively short fibres appear to be completely encapsulated and removed from the lung compartment by phagosomes (a membrane-bounded vesicle in the cytoplasm that already contains a foreign particle engulfed by the cell during phagocytosis) so that fibres less than five μm in length are not retained in the lungs and do not cause chronic inflammation. In contrast, macrophages imperfectly phagocytosed longer fibres, leading to "frustrated phagocytosis" (Fig. 4) and remaining in the lungs for longer periods. Long phagocytosed asbestos fibres are associated with carcinogenesis because they activate the pyrin domain of the NOD-like receptor containing 3 (NLRP3) inflammasome and trigger the production of inflammatory interleukin-1β(IL-1β). Damaged and necrotic cells release inflammatory proteins such as high-mobility group box-1 protein (HMGB1), which induce macrophage accumulation and activation of NLRP3, amplifying the inflammatory response and secretion of tumour necrosis factor-⍺. Inflammatory cells release reactive oxygen species (ROS) and reactive nitrogen species (RNS) that are capable of causing DNA damage. Thus, phagocytosed asbestos in macrophages causes a mutagenic microenvironment rich in ROS and HMGB1, which increases mutations in mesothelial cells. Chronic inflammation with pleiotropic effect is generated, resulting in malignant transformation. ROS is shown to mediate asbestos-induced DNA damage mutagenesis in human hybrid cells [14,20]. The histological diagnosis of asbestos-related disease toxicity requires the presence of an asbestos fibre 'core' coated with ironcontaining materials. Macrophage necrosis occurs repeatedly along with lysosomal cell death, and ferroptosis could create a mutagenic Fe (II)-rich microenvironment. Excess iron is involved in mesothelioma carcinogenesis (e.g., 27 % of the total weight of the amphibole is Fe; in serpentine, it is 1–3 %). Since alveolar macrophages transport substances into the extracellular space or blood, macrophages may be responsible for transporting asbestos fibres into the pleura and other parts of the human body. For this reason, asbestos can also cause serious diseases in different organs, such as the digestive tract, ovaries, etc. [14,20]. 4. Results The fibre morphology of the samples was studied using a Quanta 200 thermoemission scanning microscope with an EDS detector in ESEM mode for non-conductive samples without the need for plating. This analysis was used primarily in the initial phase of the experiment to give a basic idea of the structure of the samples and to confirm, if 3 CORRECTED PROOF S. Jansova et al. Vacuum xxx (xxxx) 114343 Fig. 4. Model of mesothelioma carcinogenesis in an asbestos-induced mutagenic microenvironment. Phagocytosed asbestos induces a mutagenic Fe-rich microenvironment. Mutation of BAP1 contributes to the suppression of mesothelial cell death and the accumulation of other mutations associated with mesothelioma carcinogenesis. Abbreviations: IL-1β: Interleukin-1β; ROS: reactive oxygen species; HMGB1: high mobility group box-1 protein; BAP1: BRCA1-associated protein [14]. necessary, the presence of fibres that would correspond morphologically to those of asbestos minerals. In the next phase of the experiment, this analysis was then used to examine in detail the fibres found and the structure of the samples as a whole. The fine-grained fraction of the soil samples in their original state without annealing was analysed, and after annealing, the fibres found were compared with asbestos-type standards and with the available literature. The above morphological investigation of the first batch of samples showed the presence of fibres whose morphology was consistent with asbestos fibres in all four samples of the first batch in the preand postannealing conditions. Smooth fibres with pointed ends were found in the first three samples of the 1st series, corresponding to the amphibole group (Fig. 6). In the fourth sample of Series 1, smooth fibres from the amphibole group and wavy, flexible fibres from the serpentine group (Fig. 5), which tend to clump together, were found. Morphological analysis of these samples was challenging given the low percentage of occurrence of the fibres of interest in the sediment studied overall. SEM analysis was easier to perform after annealing to remove the organic components of the material collected. In the second series, fibres whose morphology was consistent with asbestos fibres were demonstrated in all eight preand post-annealing samples. In this series, however, the morphological investigation did not reveal fibres corresponding to chrysotile or serpentine-type asbestos fibres. However, 'only' smooth, straight fibres with pointed ends (very sharp ends) corresponded to the amphibole group. Elemental composition was measured repeatedly for all samples, and an attempt was made to focus on areas with visible fibrous segments or, where possible, individual fibres. This involved both area and point collection. C and O were not quantified in the EDS examination for all samples evaluated, as these elements are challenging to quantify; contamination of the sample from the environment and the fixation media used would have biased the measured data. Identifying asbestos fibres from natural sources is impossible based on elemental composition analysis alone, precisely because their composition reflects minerals' natural diversity and variability. Thus, EDS analysis guided us in roughly classifying serpentines or amphiboles concerning certain elements that distinguish different types of asbestos. The measured elemental compositions provided input for the X-ray analysis (Fig. 7). Image analysis of the images was carried out using the NIS Elements software, which provided valuable information regarding the exact length and width of the fibres found. The search was initially for fibres that met the WHO requirements for "respirable" fibre size, and almost all fibres found met these requirements after sizing. Therefore, they were considered 'respirable' and highly hazardous to the human body. Measurements by X-ray diffraction analysis played a crucial role in this thesis's experiment. The study was performed on a PANalytical X'Pert Pro powder diffractometer, and a copper X-ray tube with a wavelength of Kα1 = 0.154 nm was used as the X-ray source. An identical diffractometer setup was used to perform the experiment to ensure the measurements' objectivity and the results' comparability. All samples were measured at normal atmospheric pressure and room temperature, and a standard symmetric geometry with a Bragg-Brentano arrangement was used for the measurements. The measurement range of all samples was identically chosen to be between 17 and 85° [2θ]. Within this range, all the most muscular diffraction lines of the expected phases occur, possibly asbestos and the accompanying phases, such as SiO2. All samples (before annealing) show an increased background, especially in the front part of the diffraction record. This is an increase due to the presence of organic components in the soils and the diffraction of air molecules present in the cavities and pores of the samples. This effect decreases and disappears with increasing angle 2θ. After annealing, there is a partial reduction of the background present and a reduction of some phases sensitive to higher temperatures. Asbestos phases are not Fig. 5. Fibers found whose morphology, according to the comparison, corresponded to serpentine-type fibres. 4 CORRECTED PROOF S. Jansova et al. Vacuum xxx (xxxx) 114343 Fig. 6. Found fibres whose morphology, according to the comparison, corresponded to amphibole-type fibres. Fig. 7. a) Example of the elemental composition spectrum of sample No. 3 from the 1st series and b) the elemental composition of the found fibre of the same sample. affected due to their resistance to the maximum annealing temperature used. Due to the hazardous nature of the samples (possible drift of asbestos fibres into the diffractometer or the laboratory), all precautions had to be taken to prevent drift (safe handling of the sample holder, covering the bulk sample until the diffractometer door was closed, use of a disposable protective suit, rubber gloves and respirator). The advantage of using the X-ray diffraction method is that the machine itself does not mechanically stress the sample, it is not manipulated in any way during the measurement process, and the process of dispersion of the sample into the air is thus minimised. However, the associated safety precautions must be taken. The diffraction line record shows the presence of each phase in the samples, and it is clear that the presence of asbestos fibres was confirmed by diffraction analysis in all samples in both series (Fig. 8). We can confidently say that chrysotile from the serpentine group is present in the first, third and fourth samples of Series 1 and Series 2. This is the least hazardous form of asbestos. It can also be said that in all samples of both series except sample 6′, the presence of anthophyllite from the amphibole group is confirmed. In the fourth sample of series 1, actinolite (also from the amphibole group) is confirmed. Almost all the measured fibres from the samples before and after annealing met the WHO definition and were classified as "respirable" and highly hazardous. However, more significant portions of asbestos minerals were also found, from which, due to cleavability, it is possible to separate smaller, possibly respirable portions and fibres. In addition to chrysotile, there are confirmed occurrences of amphibole types of asbestos, i.e. types 5 CORRECTED PROOF S. Jansova et al. Vacuum xxx (xxxx) 114343 Fig. 8. Asbestos minerals identified in both Series 1 and Series 2 samples by Xray diffraction analysis. that are considerably more dangerous from a health point of view than chrysotile. Powder X-ray diffraction is a suitable method for determining the phase composition of soil sediment samples and the possibility of determining the exact type of asbestos mineral[24–27,29–38]. However, other phases are present in asbestos-containing soil sediments, and diffraction of these phases can result in many overlaps in the diffraction maxima of the asbestos phases observed. This makes the evaluation itself difficult. This is due to the very similar elemental composition of the different phases. The problem is solved by successive refinement of the measured diffraction record of the investigated sample with the help of standards databases [28]and in cooperation with accurately determining the elemental composition from SEM with EDS. This procedure makes it possible to accurately determine the individual phases present and thus determine the type of asbestos minerals in soil sediment samples.[39] 5. Discussions Although asbestos in soil and water can pose a serious health risk, there has been little research and regulation of asbestos minerals. Several factors influence the pathogenicity of asbestos minerals, the most important of which are the morphology and chemical composition of the fibres. Macrophages cannot completely absorb fibres longer than 10 μm; therefore, fibre length is still considered the main (although not the only) cause of adverse biological effects [18,23]. Disturbance of asbestos-containing building materials (often through renovation, demolition or reclamation) has been identified as the dominant risk pathway for potential human exposure to asbestos worldwide. However, the significant hazards posed by geologically occurring asbestos and other carcinogenic mineral fibres should also be highlighted. Environmental exposure or dust release during earthmoving activities in areas with geologically occurring asbestos or similar minerals has been less studied; as a result, few management strategies are currently in place [18,23]. Previously, the standard did not count fibres shorter than 5 μm and were not considered critical. However, their health hazards are not ruled out, and considerable research and studies are underway on the possibility of the riskiness of their chemical composition. The source of contamination of free air with asbestos fibres, especially actinolite in the Pilsen region, has been demonstrated and officially confirmed for many years. For this reason, further detailed investigations of Plzeň and its surroundings are needed due to the significant ecological contamination by asbestos in the air and now in soil sediments, which must be clarified and further investigated to achieve better and more objective results and conclusions to which other specialists from the medical and geological fields are gradually being invited. Fig. 9. Example of simplification of the phase system of sample 3 from the first series: a) diffraction record of the sample in the original state, (b) diffraction record of the sample in the post-annealing state, (c) comparison of diffraction records in the original state and in the post-annealing state. Increased urban development may disturb asbestos-containing rock outcrops or soil containing these and other types of carcinogenic minerals, leading to more exposures, and it is, therefore, important to establish safe protocols for the identification, extraction, transport and disposal of hazardous soil contaminated with mineral fibres. Thus, all areas near populated areas that may contain asbestiform minerals based on geological studies should be investigated to quantify the risk posed and, if necessary, establish restrictions and procedures to protect construction workers and the general public from exposure [18,23]. 6 CORRECTED PROOF S. Jansova et al. Vacuum xxx (xxxx) 114343 Therefore, rocks and soils naturally containing asbestos are also sources of airborne fibres. Environmental concentration can vary considerably due to human activities, land use and natural factors. Secondary sources of asbestos, such as soils and waters with natural or anthropic contamination, can release significant amounts of fibres into the air due to this and under certain conditions (i.e. land extraction, slope reclamation, tunnelling, construction activities, …). 6. Conclusion The secondary sources of asbestos fibres mentioned above have been and are very little studied, and this situation needs to change. Developing sampling, detection and quantification techniques of asbestos in soil is important for assessing sites where contamination is suspected or confirmed. However, there is a lack of standardised analytical methods that provide data on the procedure for collecting these soils and waters for analysis, on a uniform and specific procedure for preparing and processing these samples, and on the analyses of these samples, leading to information on the presence of carcinogenic minerals at a given site [18,23]. In this paper, we studied samples obtained from a site in the Pilsen region, where the presence of asbestos was suspected due to an increased incidence of cancer in residents. The samples were prepared for the experiment according to the abovementioned procedure, morphologically examined and compared with standards, and analysed by electron microscopy and X-ray diffraction analysis. This procedure confirms the presence of several types of asbestos in the region occurring in soil sediments. Specifically, the following types of asbestos were found: anthophyllite, chrysotile, and actinolite. The presence of asbestos fibres was confirmed in all samples according to Fig. 8, and the measured fibres from the samples after annealing met the WHO definition and were therefore determined to be respirable. The asbestos found in our samples is hazardous to the human body and may increase cancer. Previous studies [39] show that the source of air and soil contamination by asbestos fibres from actinolite, which occurs in proterozoic metamorphosed altered basalts (traditionally called "spilites") in the Pilsen region, has been demonstrated and officially confirmed. It is, therefore, appropriate to encourage further research and mapping of this area, which contains our confirmed minerals, chrysotile, anthophyllite and actinolite, and to find ways of integrating this knowledge into the global problem of asbestos and its impact on public health. The main conclusion of this work is to establish a straightforward procedure for the preparation and measurement of soil samples, identify the different types of asbestos, and warn about its impact on public health. From these findings, there is an acute need for further targeted mapping of the natural occurrence of asbestos and working with this issue at the level of law. CRediT authorship contribution statement Štěpánka Jansová: Writing –review & editing, Writing –original draft, Supervision, Resources, Methodology, Formal analysis. Zdeněk Jansa: Writing –review & editing, Formal analysis. Pavel Calta: Methodology. Veronika Vavrůňková: Methodology. Lucie Nedvědová: Methodology. Ján Minár: Supervision. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgement This publication was supported by the project Quantum materials for applications in sustainable technologies (QM4ST), funded as project No. CZ.02.01.01/00/22_008/0004572 by Programme Johannes Amos Commenius, call Excellent Research. Data availability Data will be made available on request. 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