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Article

Microstructural and Microbiological Properties of Peloids and Clay Materials from Lixouri (Kefalonia Island, Greece) Used in Pelotherapy

by
Paraskevi Lampropoulou
1,*,
Petros Petrounias
1,2,
Aikaterini Rogkala
1,
Panagiota P. Giannakopoulou
1,
Eleni Gianni
1,2,
Spiridon Mantzoukas
3,
Ioannis Lagogiannis
4,
Nikolaos Koukouzas
2,*,
Sophia Hatziantoniou
5 and
Dimitrios Papoulis
1
1
Section of Earth Materials, Department of Geology, University of Patras, 26504 Patras, Greece
2
Chemical Process & Energy Resources Institute, Centre for Research & Technology Hellas (CERTH), 15125 Athens, Greece
3
Department of Agriculture, University of Ioannina, 45100 Ioannina, Greece
4
ELGO-Demeter, Plant Protection Division of Patras, 26442 Patras, Greece
5
Section of Pharmaceutical Technology, Department of Pharmacy, University of Patras, 26504 Patras, Greece
*
Authors to whom correspondence should be addressed.
Appl. Sci. 2023, 13(9), 5772; https://doi.org/10.3390/app13095772
Submission received: 18 February 2023 / Revised: 11 April 2023 / Accepted: 4 May 2023 / Published: 7 May 2023
(This article belongs to the Section Environmental Sciences)
Browse Figures
Versions Notes

Abstract

:
Clays have been applied for centuries by people for many uses. From pottery to medicine, clays and clay minerals constitute part of individuals’ daily routines. Natural fine-grained earth material, commonly found at Xi beach coastal area of Lixouri in Kefalonia island, was used during the last few decades by local people as well as from tourists for pelotherapy, even though this material has never been examined for its suitability for this use. This work was conducted aiming to characterize this material and determine if it is harmless and beneficial for therapeutic purposes or skin care. Field work revealed that the material is not homogenous and, therefore, some of its parts could be characterized as suitable, non-suitable or even harmful. The collected samples from Kefalonia were characterized according to their petrographic and chemical features using Scanning Electron Microscopy (SEM), X-Ray Diffraction (XRD), X-Ray Fluorescence (XRF) and Transmission Electron Microscopy (TEM). Moreover, the microbial burden of the material was assessed analyzing the bacterial and fungi load. This study indicates that this geomaterial can be suitable for natural mud/peloid applications, but only in some parts of this area.

1. Introduction

Kefalonia Island, one of the larger islands in Greece and the largest in western Greece, belongs to the Ionian Islands. It is a famous vacation spot that hosts thousands of tourists every year. One activity that visitors enjoy on this island is the “well-known” mud bath, using local geomaterial with clay minerals in Xi Beach, located in the Lixouri area. More specifically, Kefalonia island became a member of the International parks of Unesco (geopark) in the last few years, receiving extended visibility from visitors looking for points of interest such as these pelotherapy spots.
Clay minerals focused the interest of the researchers during the last decades due to their special characteristics. As they are natural materials with advanced properties and low cost, their exploitation increased, with the main aim being their use in industrial applications [1]. Greece contains clay minerals deposits and occurrences that were extensively investigated for their characteristics as well as their industrial applications, which differ from one mineral to another [2,3,4]. A variety of clay minerals are used in the cosmetics industry due to their several optimum physical, as well as physicochemical, properties, such as specific surface area, high adsorption capacity, swelling capacity and reactivity to acids [5,6].
Clay minerals and their microstructural characteristics, as mentioned, are the subject of numerous studies in various fields, such as geology, cosmetology, materials science, pharmacy and medicine. They were used as traditional folk remedies for more than 2000 years because of their health benefits. Nowadays, they are utilized in traditional healing therapies and other natural skin care products [7,8,9]. Vali and Bachmann [10] related the ultrastructure of the colloidal dispersions of clay to the rheological properties affecting peloid application. However, peloid microstructure was previously specifically and systematically studied by very few authors, such as Gámiz et al. [11].
Numerous researchers studied the use of clays in the preparation of peloids and dermocosmetic products [5,12,13,14,15,16,17,18,19,20,21], and they found that the main phyllosilicates presented are smectites, kaolinite, illite, illite–smectite mixed layers, and chlorite in different proportions [6]. A comparative physico-chemical composition study of muds from different areas in the Homogeneous Euganean Hills Hydromineral Basin (B.I.O.C.E.), Italy, reported the composition of peloids as “clayey-silt” (65.42% silt and 24.62% clay) and “silty-clay” (64.37% clay and 34.41% silt). Their heavy metal content was studied via comparison with commercial cosmetic mud and was found to be higher than in commercial mud; however, no allergic reactions were detected. A proposal to establish a protocol for effective control of these types of natural products was proposed in [22].
The use of natural cosmetic products is increasing around the world. Despite their natural origin, the risks of intoxication, allergic processes, prolonged chemical exposure, or presence of other side effects are significant. Mud baths with peloids were traditionally used for cosmetic or therapeutic purposes. Peloids are wet or dry fine-grained geomaterials composed of clay minerals, such as smectite, muscovite and kaolinite, as well as non-clay minerals, i.e., quartz, calcite and feldspars, in several amounts and with or without organic matter [23]. Generally, peloids derived from bentonite that have chemical and rheological properties, such as appropriate heat dissipation and high swelling potential, as well as operability properties, may be considered as suitable for pelotherapy. However, pelotherapy results depend on a complex of various parameters, such as physical skin characteristics, the temperature of skin or peloid, duration of external application and light exposure, amount of coated peloid paste on skin, etc.
There are numerous published studies regarding the biological fraction of peloids; among them, studies worth highlighting include those on the Euganea basin muds in the Spa area of Abano Terme, Italy. Thus, Ceschi-Berrini et al. [24] described the presence of the genus Phormidium in thermal waters of the Euganea basin and subsequently identified the presence of acylglycerolipids produced by the aforementioned cyanobacteria, which appeared to confer therapeutic and cosmetic properties on the mud [25]. Moro et al. [26], when studying the microbial diversity in the same area, described a new species of Cyanoprokaryote called Cyanobacterium aponinum, which was found in the microbial mats of Euganean thermal springs. Accordingly, Poli et al. [27] described a thermophilic bacterium in the mud from this thermal basin called Anoxybacillus thermarum, which provides an idea of the special characteristics of the biological composition of these muds. Auxiliary studies by Moro et al. [26] expanded the biodiversity of these muds to species of the genus Leptolyngbya and Spirulina (now Arthrospira), proposing that the cyanobacterial composition of phototrophic mats in the rather unusual environment of the Euganean Thermal District is variable, depending on the physico-chemical features of the different thermal spa waters. It should be noted that studies performed on 90 thermal spas showed that the cyanobacterial diversity might be related to thermal mud processing in the different maturation tanks with thermal waters at different temperatures [28].
The microstructure, physicochemical, mineralogical and textural characteristics of the geomaterial when applied on the skin are some of the main factors related to the treatment efficacy and health risks [29,30]. Clay minerals’ type, as well as their concentration in peloids, must be examined on detail, since clay minerals affect major parameters during treatment, such as the dispersity, dermal absorption/adsorption, and toxicity of elements [29,30,31].
It is necessary for natural materials used as peloids to be tested for their presence and concentration in microorganisms as they could be more susceptible to containing microorganisms like fungi, which may cause severe health and dermatological problems. It should be noted that natural peloids should be free of pathogenetic microbiological content in order to be safe for skin application. Moreover, mud and peloids derived from sedimentary rocks are often characterized through higher amounts of organic matter compared with those from volcanic rocks [30]. The presence of known bioactive organic compounds (e.g., lipids, carbohydrates and humic acids) may be desirable due to the increase in the balneological value of peloids. Despite this fact, a detailed description of organic matter is required.
In the present study, seaside geomateriald from the Lixouri area (Kefalonia, Greece) were examined for their mineralogical, physical and microbiological characteristics. The extensive description of this material is crucial as visitors to Kefalonia Unesco Geopark encourage future tourists to perform traditional mud baths (using geomaterial of this area) during their visit to the island by affirming the “well-known” beneficial properties on the skin. Apart from the empirical results of this treatment, the key question of this research seeks to investigate, from the point of view of microstructure, geochemical, mineralogical and microorganism characterization, whether this natural material is efficient or even appropriate for use as peloid or just constitutes a placebo perception, and if this activity can continue in the future as the existing designated number three geopark is disturbed. It should be noted that this is a preliminary study, since a high number of samples and analyses are required for the investigation of the extensive area.

2. Geological Setting

Kefalonia Island, which is one of the Ionian Islands, is located in the external edge of the Hellenides fold-and-thrust system, which was created in response to Cenozoic continental collision following the closure of the Tethys Ocean [32,33,34] (Figure 1). This island is mainly separated through alpine formations (Paxoi and Ionian geotectonic units) and Plio–Quaternary sediments [32,35]. The Paxoi unit composed of Triassic–Middle Miocene carbonates and clay-clastic sequence of flysch from the Middle Miocene–Early Pliocene, including alternations of marls, clays and mudstones, while the Ionia unit composed of Triassic evaporitic series and Jurassic–Cretaceous limestones and slates [35]. Pliocene–Calabrian and Middle-Upper Pleistocene–Holocene series make up the Plio–Quaternary sediments, which characterized through the presence of a marine sequence, yellowish marls with siltstones, sandstones, sands and coarse calcarenites. Pliocene–Calabrian series prevail in the Lixouri area, where the marine sequence is observed as an uplifted coastal zone with 2 to 10 km width range. Such formations have a thickness variation between 200 and 500 m, and are developed transgressively over Paxoi unit [35]. The studied field region focusses on Plio–Quaternary sediments, which are extended in Xi bay to the south of Lixouri, and specifically on those sediments which cover the coastal area of Xi beach. (Figure 1 and Figure 2; Table 1). These are lithological formations about 300 m thick and were formed in a relatively deep marine environment, the evolution of which was linked to intense tectonic events during the Pleistocene. In the clayey sediments of Xi beach, we observe erosive forms (structures) created via sea water, air and atmospheric precipitation.

3. Sampling and Field Features

In the present study, surface and core drilling sampling was carried out at Xi beach, in the south area of Lixouri, on Kefalonia island, which is defined using the geographical coordinates presented in Table 1. Our sampling strategy was based on the main macroscopic differences of fine-grained sediments in the outcrop in areas to which visitors mainly have easy access, making it possible for us to easily take a piece of rock for use. According to those macroscopic characteristics, the collected surface samples (~2 kgr each sample) were classified in three groups: 1, 2 and 3. The S1 and S2 samples of group 1 were collected from the surface of an ungraded seaside slope with low angle (Figure 3a). They appear with a gray-to-yellow color and a dry brittle structure (Figure 3a). S3 and S4 included surfaced samples from group 2, which were characterized with the presence of yellow or light gray color and a friable and slightly soapy texture (Figure 3b). Group 3 samples (S6, S7) exhibited a relatively brittle texture with a hue of light yellow-to-gray color (Figure 3c). After core drilling was conducted up to a depth of 20 m in the study area, a specimen from a depth of 10 m was collected (S5) with the aim of identifying the extent of surface appearance (Figure 3d,e). This sample presented similar texture characteristics to surfaced samples, while reflecting the same rock formation in depth (Figure 3f). After sampling, the specimens were dried, homogenized and pulverized at −200 mesh in the laboratory prior to further analysis.

4. Materials and Methods

The mineralogical phases of the investigated samples were determined using X-ray diffractometry (XRD) with the aid of a Bruker D8 Advance Diffractometer (Bruker, Billerica, MA, USA), with Ni-filtered CuKα radiation. The <2 µm clay fraction was separated by settling and dried on glass slides. The clay fraction samples were scanned from 2 to 30° 2θ for each <2 μm specimen and identified from three XRD patterns (after being air-dried at 25 °C, ethylene glycol treatment and being heated for 2 h at 490 °C). The mineral phases were found using DIFFRAC plus EVA 12® software (Bruker-AXS, Billerica, MA, USA) based on the ICDD Powder Diffraction File of PDF-2 2006, while the semi-quantitative analyses were performed with the diagnostic peak areas of each phase using the same software and its “area” tool. The detection limit of XRD analyses is ~2–3%. Moreover, the morphology of phases was examined using the JEOL JEM-2100 SEM at an acceleration voltage of 200 kV. Transmission Electron Microscopy (TEM) analysis was conducted on a JEOL JEM-2100 system, which was operated at 200 kV. Images and SAED patterns were recorded using an Erlangshen CCD Camera (Gatan Model 782 ES500W). The specimens were prepared via dispersion in water and spread onto a carbon-coated copper grid (200 mesh).
The moisture content (w) was determined (AASHTO T255 [37] standard). Furthermore, sample loss on ignition (LOI) was measured (ASTM D7348-13 [38] standard). Moreover, an X-Ray fluorescence spectrometer (XRF) and a sequential spectrometer were used for the determination of the major and trace elements of the studied clays. An amount of 0.8 g of dried ground sample was mixed with 0.2 g of wax and pressed to a pellet under 15 tones. Pressed pellets were analyzed with a RIGAKU ZSX PRIMUS II spectrometer, which was equipped with Rh-anode.
The grain size distribution of samples (Table 2) was carried out using a Malvern Mastersizer Hydro 2000 M laser granulometer, while the lithological classification of sediments was based on the plotting of sand, silt and clay percentages at the ternary classification diagrams, based on the work of Folk et al. [39]. The plasticity limit was determined according to the ASTM D4318-05 [40] standard test method.

Sampling—Isolation—Identification of Microorganisms Presents

Six samples containing soil were selected from the four study areas in the region of Xi beach, where the surface litter was removed and the soil was dug to a depth of 10 cm with a soil core borer. The samples, which weighed 300 g each, were collected from Lixouri, Kefalonia island. The soils were then placed in plastic bags and refrigerated at 4 °C until they were transferred to the laboratory for further processing.
The soil was air-dried for three days at 30 °C before being pulverized and sieved (metal, 2 mm 1 mm, Aggelis Equipment, Athens, Greece). The Soil–Plate Method was used to isolate fungi. [41,42]. In Petri plates with PDA, 0.05 g of dry soil was spread and incubated at 25 °C. After 3 days, fungal colonies formed, and a tiny sample of mycelium from each colony was moved to a different plate to maintain a pure culture for each fungus for incubation. Total viable count (TVC), total coliforms, E. coli, enterococci, S. aureus, P. aeruginosa, sulfite-reducing clostridia and fungi were chosen as indicator criteria.
For 24 h, the bacterial strains were grown in liquid lysogeny broth (LB) at 37 °C and 170 rpm. The liquid culture medium consisted of NaCl 1%, Tryptone 1% and yeast extract 0.5%. For the solid medium, 1.5% agar was added to the liquid broth. All the ingredients, which were provided by PanReacAppliChem, were diluted in distilled sterile water via stirring. Strains were maintained at −80 °C in 20% (v/v) glycerol in LB broth. Six liquid cultures were prepared and incubated at 37 °C and 170 rpm. Every 20–30 min, one culture was measured using a spectrophotometer (SHIMADZU, UV-900) through setting the OD at 600 nm. After serial dilutions of the measured culture, 50 μL were plated in LB agar petri dishes and incubated for 24 h at 37 °C. After incubation, the colony forming units per ml (CFU/mL) were calculated [42]. In this way, inocula suspensions were prepared with approximately 1–2 × 108 CFU/mL. This bacterial concertation was prescribed based on the Broth Microdilution Method [43]. Fungi were cultured in Sabouraud Dextrose Agar (SDA) petri dishes for fourteen days at 25 °C. For the preparation of SDA, 64 g of medium (PanReacAppliChem) was diluted in 1 L distilled and sterilized water [44].
The Wizard Genomic DNA extraction kit (Promega) was used to extract genomic DNA from the soil samples. The ITS1 (5′-TCC GTA GGTGAA CCT GCG G-3′) and ITS4 (5′-TCC TCC GCT TAT TGA TAT GC-3′) primers, U341F (5′-CCT ACG GGR SGC AGC AG-3′) and 805R (5′-GAC TAC CAG GGT ATC TAA T-3′) primers, and NEBNext 2x High-Fidelity Master Mix were used to amplify regions of the 18S rDNA gene for fungi and 16S rRNA gene for bacteria, respectively. The used thermocycling protocol consisted of 30 s at 98 °C, 25 cycles of 10 s each at 98 °C, 58 °C, and 72 °C, and a final step of 5 min at 72 °C. Following Illumina’s instructions, a second PCR was used to add index Illumina adapters. The enhanced pieces were sanitized using Agencourt’s Ampure globules (USA). Mr. DNA Lab performed next-generation sequencing, and online platforms for metagenomic analysis were used to analyze the results.
This study took into account the principles of the Scientific Committee on Consumer Safety (SCCS). Cosmetics were divided in two categories, based on the recommended limits of their microbial content. Category 1 contained products intended for children under three years, or to be used in the eye area or mucous membranes, while Category 2 included all other products. The total viable count for aerobic mesophilic microorganisms for Category 1 and 2 products was 102 and 103 CFU/g (or mL) of the product. Moreover, potential pathogens (Escherichia coli, Pseudomonas aeruginosa, Staphylacoccus aureus and Candida albicans) were not be detectable in 1 g or ml of either Category 1 or 2 products.

5. Results and Discussion

The paper study presents, for the first time, the results from a multifaceted as well as systematic study of the microstructure of seaside sediments from Xi beach, where tourists have enjoyed the use of coastal geomaterial for mud baths and pelotherapy for at least the last fifty years. A similar study was conducted by Bergamaschi et al. [22] on mud from Bacino Idrominerario Omogeneo dei Colli Euganei, Italy. However, extended studies into the microstructure of peloids remain limited. The present study can essentially answer the crucial question of whether this use is really beneficial to humans or simply wastes the geoenvironment for no reason. The study area is particularly well-known and popular among visitors to the island, something that was proven through analysis of a database of more than 846 Trip Advisor reviews. The mud bath treatments available in this area are strongly advertised by visitors and via the tourist search database of Trip Advisor, with more than 346 reviews characterizing the area as Excellent [45]. It should be also noted that the Xi beach area holds the distinctive title of “Travelers’ Choice for 2021”, placing it in the top 10% of total relative candidates. As can be seen, Geosite 3 of Kefalonia Geopark is a very popular and special destination precisely because of this geosite, which visitors use a lot.
However, the main question that the present research wishes to answer is whether the extensive and sustained use of fine-grained natural material on Xi beach by tourists is harmless and beneficial for therapeutic purposes or skin care, since there is a bibliographic gap about this free human activity in Kefalonia Island. For this scope, a preliminary study regarding to the geochemical, mineralogical and textural characteristics of collected samples is displayed and discussed in this section, in accordance with their contamination analyses.

5.1. Microstructure Analysis

According to the macroscopic description of the geomaterials studied during the field study and drilling, they exhibited fine grain size textures and are classified as silt–clay materials. Laboratory analyses of studied samples indicated similar qualitative mineralogical compositions, while the predominate pelitic texture is also confirmed. The representative XRD analysis results are presented in Figure 4 and Figure 5 and Table 2. They demonstrate that the samples mainly consisted of phyllosilicates, quartz, calcite, feldspars, pyrite, gypsum, dolomite and opaque minerals. Illite, vermiculite, chlorite and mixed-layers chlorite–smectite clay minerals were detected after clay fraction analyses (Figure 6). The majority of the minerals display clastic origin (e.g., quartz, feldspar, illite/muscovite) derived from the weathered rocks of the broaden area, while others, such as calcite, are clastic or authigenetic [46]. Pyrite appears in framboidal agglomerates (Figure 6a,b), which are formed after sulfate reduction and release of H2S. The last process can react to iron ions derived from detrital material during flood phenomena [47]. Since pyrite and other phases are usually found in small particles in the bond of the examined samples (<5 μm of spot size), they prohibit accurate quantitative analyses via SEM; thus, only EDS spectra were selected to be presented as providing approximate stoichiometric (FeS2), while the other elements were attributed to the nearby micro region influence. Nevertheless, in this work, special care was taken to present phase analyses using the more suitable method of XRD analyses for these fine grain size materials. Additionally, quantitative mineral variations, especially in terms of samples derived from different areas, were detected (Table 3). This heterogeneity, especially in the amounts of phyllosilicate minerals, indicates that visitors who collect random specimens use material with rather different properties that obviously different results in each time. Since there are not standard official regulations about the mineral contents or element concentrations demanded in the natural materials used as peloids, our results are comparable to those in literature [29,31,48,49] as well as to a commercial natural clay material used for cosmetic purposes (Reference sample named S0). In this way, a comparison between the Kefalonia material and materials that are on the market and/or were tested regarding their effectiveness is possible. Previous published studies revealed that the content of phyllosilicates in peloids, especially smectite, muscovite/illite and kaolinite, should exceed the 30–40 wt% of bulk composition [29,48]. These minerals often exhibit low particle size and platy morphology, high surface area, high adsorption/absorption/moisture capacity, ion exchange ability and high thermal retention, while also being very soft with high plasticity after being mixed with water. These characteristics favor the easy coating of peloid paste on the skin, providing hydration or removing toxins and microbes from the skin, while they can offer nutritional ion metals with therapeutic benefits in human dermatitis, rheumatism etc. [30]. Natural materials from coastal areas, such as S1 and S2 (group 1), were used by the visitors of the island more frequently, since they are located near to the sea (Figure 2).
The results of the plasticity limits were 15.50, 15.93 and 20.52 for samples 3, 6 and 1, respectively (Table 2). Therefore, according to the Jenkins soils classification [50], all samples were characterized as high-plasticity soils. Samples of group 1 (S1, S2) were characterized through the presence of slightly higher amounts of phyllosilicates (50–52 wt%) and lower of non-phyllosilicate minerals compared to the other crystalline ones (48–50 wt%). Even though they do not contain pure smectite, which exhibits higher adsorption/absorption capacity and ion exchange ability among clay minerals, they contain significant amounts of mix layer chl–sm and pure vermiculite (26–28 wt%) clay minerals. Moreover, samples from group 1 consist of 24 wt% illite/muscovite, which further contributes to the hydration of dry skin [29]. Sometimes mica is altered to halloysite (mineral of the kaolinite group), as shown in Figure 7, which is not only biocompatible to normal human dermal fibroblasts, but also enhances the wound healing processes. A similar structure with halloysite minerals was also reported by Papoulis et al. [51,52]. Using TEM analysis, the halloysite nanotubes that occupy a high surface area are observed, as can be seen in Figure 7b. In the literature, peloids usually contain low or minor amounts of carbonates [31]. However, the S1 and S2 specimens consist of significant micritic content (18–19 wt%), though it is not considered to act negatively. On the contrary, micritic calcite particles can act as a peeling constituent [31,53]. Previous researchers stated that using peloids with high amounts of nano silicon oxide and cristobalite should be avoided, since these phases are linked to silicosis phenomena or other diseases, such as carcinogenesis after exposure to silica dust [54,55,56]. On the contrary, the microcrystalline or amorphous quartz was shown not to be dangerous for human and pelotherapy, especially when this phase coexists with clay minerals [53,56]. In S1 and S2 samples, silicon oxide was largely found in the form of microcrystalline quartz (>100 nm in crystal size, Figure 8a), as well as in skeletons of diatom micro-organisms homogeneously dispersed in the matrix (Figure 8b,c); it was less common in nano crystalline quartz (<100 nm in size). Τhe presence of diatoms improves the efficacy of peloids due to their thermophilic and anti-inflammatory properties [6], something that may remedy pain through reducing inflammation of the skin.
In fact, the toxicity of silicon oxide phases in humans depends on multiple parameters (e.g concentration, use, time of exposure), and it is difficult to predict the effect. Nevertheless, it is recommended that this material is only used in mud paste form and removed when dry, avoiding harmful inhalation. Framboidal pyrite detected in these samples is not expected to influence the peloids behavior, since they occur in low amounts in texture microareas (lower to the XRD detection limit of about 2–3 wt%), whilst toxic trace element-free pyrite is used in cosmetics. It is reasonable to state that in the samples, toxic arsenic was detected in very low levels (Table 4), reflecting negligible release of such ions from pyrite crystals. Gypsum (2 wt%), feldspar (4–5 wt%) and dolomite (4 wt%) were detected in S1 and S2 as commonly presenting in peloids, doing so without changing their properties. Nevertheless, these minerals do not negatively affect human skin during the application of peloid paste due to their low amounts of toxic trace elements compared to harmless composition and low-to-moderate hardness (2–6 in Mohs scale). Samples of group 2 (S3, S4) present dense and fine textures, with low amounts of clay minerals (23–24 wt%) in their bulk composition compared to group 1 and commercial samples (S0). Moreover, this group could be characterized through the presence of increased percentages of quartz (31 wt%) and calcite (35 wt%) (Table 3). A lower amount of clay minerals in their bulk composition downgrades their efficiency in cosmetic or other therapeutic applications. Clay minerals of smectite group are lacking from their matrix since illite/muscovite coexist with vermiculite or chlorite. Diatoms are absent or occur seldomly. Additionally, since the distribution of quartz grains in the microstructure increases, the possibility of undesirable skin abrasion due to their high hardness (7 in Mohs scale) also increases. Thus, this fact was one of the parallel reasons for studying the materials of this region, which are widely used by numerous of tourists each year. These samples are also differentiated from the other peloids in the literature [53], as well as the S0 sample that was analyzed in this study (Table 3), which acted as the standard sample. Based on the bulk mineralogical composition criterion, earth materials at Xi beach similar to those of group 2 did not cover basic requirements and are not proposed for pelotherapy, as they do not present any of the necessary clay minerals required to improve or heal skin conditions. On the other hand, the samples of group 3 (S6, S7) that were collected farthest from the sea contained intermediate amounts of phyllosilicates (19–26 wt%) compared to the other samples, followed by lower amounts of quartz (29–32 wt%), calcite (27–29 wt%), feldspars (6–8 wt%), dolomite (5–13 wt%) and gypsum (3–5 wt%). Regarding the core sample (S5), petrographic features do not differentiate it strongly from the respect surface samples (samples of group 2 and 3, Table 3), confirming the extension of the in-depth studied horizon. Based on the aforementioned results, it is obvious that the clay fraction in the studied samples is significantly varied (it ranges between 19 and 52%), revealing the necessity for further investigation in this area and more systematic sampling in order to delineate micro-areas that could be used for pelotherapy in the future.

5.2. Geochemistry

Representative geochemical data for the surface of the tested materials are presented below (Table 4). They reflect the coexistence of silica and non-silica minerals in their bulk compositions. The concentrations of the majority of the main elements are comparative with those of the commercial product (S0, Table 4), as well as with similar products mentioned in the literature as being used for pelotherapy [31]. The percentages of the major silicon, calcium, and aluminum oxides detected were 44.03–45.41, 12.50–17.01, and 9.44–14.10 wt%, respectively, in the studied samples. Calcium ions participating mainly in carbonates, as well as in clay minerals, were spotted. However, even these increased percentages of calcium ions are likely to have a beneficial effect, with calcium participating in clay lattices, which can easily penetrate the skin during the pelotherapy treatment due to the high ion exchange ability of these minerals, contributing positively to the osteoporosis phenomena [57] and constituting a strong health problem for thousands of people. Lower amounts of iron (4.33–5.99 wt%), magnesium (2.98–3.96 wt%), potassium (1.95–2.31 wt%) and sodium oxide (1.19–1.34 wt%) complete the bulk oxide compositions of analyzed specimens. In regard to the trace elements, the analyses revealed increased concentrations of Ba (222–241 ppm), Zr (99–105 ppm), Sr (251–369 ppm) and Cr (177–220 ppm). As there are no established limits for element concentrations in natural mud materials used for cosmetic or other external therapeutic purposes, the analyses of this study compared well with those in the literature [31] as well as the S0 product. The results revealed that, in most cases, they are under acceptable levels when compared with the Canadian health products guides [53,58]. Ba and Zr are characterized as elements with low risk for cosmetics, while Cr is classified as partially toxic if it exceeds the 1100 ppm in the bulk composition; this issue is something that does not occur in the analyzed samples. The limit values of Sr are not provided; however, it is characterized as a less hazardous element, while according to the guidelines high concentrations should be taken into account in peloid therapy. Combining the geochemical and mineralogical results, the relatively high concentrations of sulfur in S1 sample can be attributed to both the detected pyrite in framboids form and gypsum. Generally, sulfur rich peloids are strongly supported as treatment in skin conditions, such as psoriasis [59].
According to the European Regulation (Annex II) [60] and US Pharmacopoeia, the amount of Pb in clays should not exceed 40 ppm [61]. The average values (ppm) relative to Pb of each sample were compared with these reference values in order to understand to what extent the concentration of the metal exceeds this limit. According to the standards of the Pharmacopoeia, the levels of Pb remain within the acceptable limits, since they are all lower than 40 ppm (Table 4).
Table
Table 4. Representative geochemical analysis (XRF) of studied samples, major elements in wt%, and trace elements in ppm. Abbreviations: Applsci 13 05772 i001, acceptant levels; n.t., not toxic, l.h., less hazardous; l.r., low risk.
Table 4. Representative geochemical analysis (XRF) of studied samples, major elements in wt%, and trace elements in ppm. Abbreviations: Applsci 13 05772 i001, acceptant levels; n.t., not toxic, l.h., less hazardous; l.r., low risk.
wt%S1S2S6S5S0Acceptant Levels
SiO244.0345.4144.6345.4044.02Applsci 13 05772 i001 [31]
TiO20.620.530.580.540.72Applsci 13 05772 i001 [31]
Al2O314.109.4410.0110.0513.68Applsci 13 05772 i001 [31]
Fe2O35.994.334.944.545.93Applsci 13 05772 i001 [31]
MnO0.070.090.080.070.09Applsci 13 05772 i001 [31]
MgO3.962.983.223.423.98Applsci 13 05772 i001 [31]
CaO12.5017.0116.6316.3512.98Applsci 13 05772 i001 [31]
Na2O1.251.341.291.191.53Applsci 13 05772 i001 [31]
K2O2.311.952.082.182.77Applsci 13 05772 i001 [31]
P2O50.130.100.110.090.18Applsci 13 05772 i001 [31]
SO31.830.360.780.580.66Applsci 13 05772 i001 [31]
Cl-0.040.020.020.030.01Applsci 13 05772 i001 [31]
LOI13.1116.0115.3415.3612.91
Ppm
As14.110.812.311.39.3
V125891039863
Cr17721022018198n.t. [31,53,58]
Co20.317.819.320.38.7
Ni14512013812555
Cu352628259
Zn7984757062
Rb10783988862
Sr251369321311195l.h. [31,53,58]
Y20.219.118.919.916.8
Zr10010510299163l.r. [31,53,58]
Ba241226238222315l.r. [31,53,58]
Hf2.32.82.62.13.8
W1.41.01.01.01.5
Pb1211131215n.t. [60,61]
La2621242428
Ce50.345.248.046.157.3
Th8.86.87.37.18.3

5.3. Microganisms Analyses

According to the fungi analyses results from S1 and S7 samples, the largest number of isolations (11 and 7, respectively) were recorded. Samples S2, S3 and S6 resulted from two isolates per sample. Finally, no isolation was obtained from samples S4 and S5. Skin-disease-causing dermatophyte fungi were not isolated from our samples.
From the five soil samples, 24 isolates were morphologically identified. S1, S2, S3, S6 and S7 were identified as Metarhiziumanisopliae (Metchnikoff), Sorokin (Hypocreales: Clavicipitaceae), Beauveria bassiana (Bals.-Criv.) and Vuill. (Hypocreales: Cordycipitaceae). Occurrence of EPF (entomopathogenic fungi) in the soil depends also on the soil type, plant species and cultivation practices of the surrounding study area (Figure 9). The fungi also exhibit very low mammalian toxicity [62,63,64,65]. Furthermore, these fungi are not damaging to plants and are non-threatening to animals and people [64,65,66,67,68].
Muds in pelotherapy applications are used for their thermal properties, with people who receive mud treatment either being covered with mud wholly or locally, or entering pools containing mud and applying the mud on their bodies. Therefore, it was pointed out that parameters that might impair the hygienic conditions of peloids and thermal waters are mainly coliforms and fecal coliforms, as well as bacteria of the groups Pseudomonas and Staphylococcus [69]. P. aeruginosa is a gram-negative, aerobic and ubiquitous environmental bacterium, being present in moist areas and surface waters and a possible cause of exposure to the bacteria [70,71].
However, there are no international quality standards for mud or peloid applications, except in a few countries, such as Germany, France and Cuba, who follow national microbiological specifications [72]. Nevertheless, the low fungi load in the studied samples is not expected to significantly influence the adsorption/absorption clays capacity during mud therapy on the human skin.
Total coliforms refer to a large class of bacteria present in nature, whereas E. coli refers to recent fecal infection. Coliforms are most likely present in virgin mud as natural colonizers that remain in the culture plant because of maturation tanks’ permissive thermal water temperatures. Two samples (S3, S5) were found to be positive for E. coli and P. aeruginosa (Table 5). Their presence may be due to either immaculate contamination of virgin mud or a defective regeneration operation in used mud. The hygienic quality of Kefalonia samples is of core importance. However, presence E. coli and P. aeruginosa in Kefalonia samples was very low in CFU/g. Members of the Staphylococcus genus can be either saprophytic environmental species or microorganisms transmitted from the patient’s skin; hence, the lack of opportunistic pathogen species S. aureus was particularly notable in our findings. The other bacterium class included in the examined criteria was Anaerobic clostridia, which are often found in the environment. Mud is an anoxic matrix that promotes the growth of A. clostridia, and their varying abundance is most likely determined via the mud cultivation and mixing procedures used at each facility. Skin disease-causing dermatophyte fungi were sporadically isolated in five R samples. The most likely source was a direct transfer from the patient’s skin to the utilized mud.

5.4. Geoheritage Implications

For the global geological community, Greece and more specifically Kefalonia island is an open geological museum and natural laboratory, where intense geodynamic processes, along with archaeological and mineral cultural wealth, create a location deserving of preservation and promotion. Specifically, the coastal part could use the geoheritage in a more socially acceptable way for touristic purposes. Some regions of this island are already been included in the Geopark of UNESCO; Xi beach is one of these areas, being known as geosite number 3. On the other hand, at Xi beach, Lixouri, mud baths and pelotherapy activities are included in the touristic attractive activities. According to the above frame, it becomes obvious that an irrational use of earth material from the coastal geological environment of the study area should be avoided in order to protect it for sustainable conservation, given that is visited by thousands of tourists every year. However, this activity can continue under a frame of proper use of natural material and geotourism development. The results of this study can help in this direction, creating a positive point of sensitization for local citizens and tourists. In this study, it is suggested to de-limit mud bath activity in areas of the seaside zone where higher quality peloids exist.
Nevertheless, it should be highlighted that for systematic therapeutic applications under a greater strike frame in the future, a higher amount of collected samples will be demanded, while further analyses regarding the physicochemical parameters and the microbial load of the sediments of Lixouri area will be needed in order to increase our knowledge of these peloids.

5.5. The Importance of Microstructural and Microbiological Properties

In order to better understand the analysis pattern followed in this study, it is useful to summarize the investigations that should be carried out on peloids derived from Xi beach (Kefalonia) used for pelotherapy in order to characterize them from several points of view and identify possible characteristics. Mineralogical characterization is a fundamental analysis. Understanding the mineralogical composition of the samples and their special microstructure could be the starting point for formulating a cosmetic product for domestic use. Microstructure seems to determine the entire basis of mud properties, as well as the final effect on human skin. Furthermore, the mineralogical composition influences the final chemical composition of each mud sample. Regarding the chemical characterization, matrices should firstly be characterized through the presence of pH, always taking into account use on the external surface of the body where the skin has a good buffering capacity [20]. These muds must be analyzed periodically to guarantee quality before and during their application in human skin. However, it is known that, quite often, only the major elements are analyzed; thus, it is important to also analyze the trace elements and their microstructure when developing peloids for cosmetic uses. Multiple scientists, such as the authors of [73], tried to evaluate the pharmaceutical and cosmetic applications of peloids, and they devised conclusions similar to ours.
There are many differential components in a peloid; given that each mineral–medicinal water is unique, researchers need to study the type of micro-organisms present therein. The micro-organism composition in seawater differs due to latitudes that are unlike the composition of seawater, which is similar at all latitudes; hence, researchers need to study the type of micro-organisms present in a particular environment. Thus, understanding the nature of the biological matrix is important to associate the therapeutic effect with precise mud types, which is why also characterizing the matrix from a microbiological point of view is fundamental to understanding their possible therapeutic effects.
At this point, it should be noticed that, in the future, the mechanisms via which natural clay minerals may kill bacteria, such as that found by Morrison et al. [74], should be investigated to determine the least expensive cure modalities and, specifically, to maximize effective antibacterial agents.

6. Conclusions

The present study presents, for the first time, a preliminary compositional and textural characterization of clay materials of Xi beach in Lixouri area of Kefalonia island that are used for pelotherapy. We quantified mineralogical variations present among the studied samples. The seaside samples indicate higher amounts of clay minerals in their matrix compared to those of further away from the coast and, according to their compositional characterization, they were suggested as more suitable for mud therapy. The hazardous metals in studied samples were detected in acceptable levels compared to those in the literature and commercial sample analyzed in this study. Low loads of non-hazardous fungi (other bacteria) for humans were detected in examined samples.
According to the above, this human activity of pelotherapy in the studied area is safe and can be continued in the future under a simultaneous framework of sustainable use and geotourism development. Since Xi beach is included in the geopark of UNESCO, it is suggested that this activity be delimited nearby the sea. Nevertheless, more samples should be analyzed from the whole area in the future in order to enrich our knowledge of peloids in this region, while also providing a better depiction of appropriate strike zones for use. Moreover, according to the results of this preliminary study, basic directions for proper use and desirable human effects of this activity are arisen. To sum up, this preliminary research (of a broad area) can constitute a trigger for further research and sustainable use of this material. According to the above, more samples can be analyzed, and more experiments can be carried out for an optimum material characterization. Nevertheless, the mechanisms through which natural clay minerals kill bacteria should be more thoroughly investigated in the future in order to find out the least expensive cure modalities and, specifically, to maximize effective antibacterial agents.

Author Contributions

Conceptualization, P.P.; methodology, P.L., A.R., P.P.G., E.G., S.M., I.L., S.H. and D.P.; software, P.L., P.P., A.R., E.G. and D.P.; validation, P.P. and N.K.; formal analysis, P.L.; investigation, P.L., P.P., A.R., P.P.G., N.K. and D.P.; resources, P.L., A.R., S.M., I.L. and S.H.; data curation, P.L., E.G. and I.L.; writing—original draft preparation, P.L., P.P., A.R., P.P.G., N.K. and D.P.; writing—review and editing, P.L., P.P., S.H. and D.P.; visualization, P.L. and A.R.; supervision, P.P.; project administration, P.P. and D.P. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Acknowledgments

We thank M. Kalpogiannaki for her assistance in the construction of the geological map. Special thanks are given to P. Avramidis for his assistance during the grain analyses, whick took place at Sentimentlogy Labs of Geology Department, University of Patras. Moreover, we would also like to express our thanks to Nama Lab S.A for their assistance during the drilling.

Conflicts of Interest

The authors declare no conflict of interest.

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Applsci 13 05772 g001 550
Figure 1. Geological map of Kefalonia (after IGME) [36].
Figure 1. Geological map of Kefalonia (after IGME) [36].
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Figure 2. Geological map with sampling area of Lixouri City, Kefalonia.
Figure 2. Geological map with sampling area of Lixouri City, Kefalonia.
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Figure 3. Representative images of sampling area of: (a) S1, S2 samples; (b) S3, S4 samples; (c) S6, S7 samples. (d,e) Drilling for core sampling. (f) S5 core sample.
Figure 3. Representative images of sampling area of: (a) S1, S2 samples; (b) S3, S4 samples; (c) S6, S7 samples. (d,e) Drilling for core sampling. (f) S5 core sample.
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Applsci 13 05772 g004 550
Figure 4. Representative images of sampling areas: (a) S1, S2 samples; (b) S3, S4 samples; (c) S6, S7 samples. (d) Drilling for core sampling. (e) S5 core sample. Abbreviations: Cc, calcite; Dol, dolomite; Fsp, feldspar; Gyps, gypsum; Qz, quartz; CL, clay minerals.
Figure 4. Representative images of sampling areas: (a) S1, S2 samples; (b) S3, S4 samples; (c) S6, S7 samples. (d) Drilling for core sampling. (e) S5 core sample. Abbreviations: Cc, calcite; Dol, dolomite; Fsp, feldspar; Gyps, gypsum; Qz, quartz; CL, clay minerals.
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Figure 5. Representative diffractogram of the clay fraction of S1 sample. Abbreviations: Chl-Sm, mix layer chlorite–smectite; I, illite; M, muscovite; V, vermiculite.
Figure 5. Representative diffractogram of the clay fraction of S1 sample. Abbreviations: Chl-Sm, mix layer chlorite–smectite; I, illite; M, muscovite; V, vermiculite.
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Figure 6. SEM image of S1 sample showing (a) aggregates of pyrite forming framboids, and (b) pyrite crystals smaller than 1 μm and (c) chemical composition of pyrite crystals smaller than 1 μm given by the EDS spectrum. Impurities of other elements are attributed to broadened micro area composition (spot size of beam ~5 μm).
Figure 6. SEM image of S1 sample showing (a) aggregates of pyrite forming framboids, and (b) pyrite crystals smaller than 1 μm and (c) chemical composition of pyrite crystals smaller than 1 μm given by the EDS spectrum. Impurities of other elements are attributed to broadened micro area composition (spot size of beam ~5 μm).
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Figure 7. (a) SEM image of S2 sample probably showing muscovite partially altered at edge of halloysite nanotubes (shown by the blue arrow); (b) TEM image of a probable halloysite nanotube of S2 sample, scale bar = 50 μm.
Figure 7. (a) SEM image of S2 sample probably showing muscovite partially altered at edge of halloysite nanotubes (shown by the blue arrow); (b) TEM image of a probable halloysite nanotube of S2 sample, scale bar = 50 μm.
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Figure 8. SEM image showing (a) microcrystaline quartz with conjunctival fractures in microstructure of S1 sample and (b,c) fractured diatoms with clay minerals in S1and S2 samples.
Figure 8. SEM image showing (a) microcrystaline quartz with conjunctival fractures in microstructure of S1 sample and (b,c) fractured diatoms with clay minerals in S1and S2 samples.
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Figure 9. (a,b) Infected larvae in Petri dishes with a filter paper impregnated with dH2O; (c,d) isolates of Beauveria bassiana from samples S1 and S6; (e,f) petri dishes with soil of samples after three days (Soil Plate method); (g,h) isolates of Metarhiziumanisopliae from sample S7.
Figure 9. (a,b) Infected larvae in Petri dishes with a filter paper impregnated with dH2O; (c,d) isolates of Beauveria bassiana from samples S1 and S6; (e,f) petri dishes with soil of samples after three days (Soil Plate method); (g,h) isolates of Metarhiziumanisopliae from sample S7.
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Table
Table 1. Coordinates of tested samples.
Table 1. Coordinates of tested samples.
SampleXY
S1185,723.514,229,421.364
S2185,619.184,229,419.617
S3185,560.7494,229,412.35
S4185,529.6444,229,596.075
S5186,705.954,229,931.50
S6184,741.9334,230,746.108
S7185,601.8034,230,392.747
Table
Table 2. Representative particle size analyses and plasticity limits of studied samples.
Table 2. Representative particle size analyses and plasticity limits of studied samples.
Sample Sand (%)Silt (%)Clay (%) After Folk and WardPlasticity Limit
S10.26086769.7137730.16857Silt (Z)20.52
S30.32422359.5601835.63327Mud (M)15.50
S60.20563773.0638426.6753Silt (Z)15.93
Table
Table 3. Semi quantitative mineralogical compositions of studied samples. Abbreviations: Cc, calcite; Chl–Sm, mix layer chlorite–smectite; Dol, dolomite; Fsp, feldspar; Gyps, gypsum; I, illite; Sm, smectite; V, vermiculite; Qz, quartz.
Table 3. Semi quantitative mineralogical compositions of studied samples. Abbreviations: Cc, calcite; Chl–Sm, mix layer chlorite–smectite; Dol, dolomite; Fsp, feldspar; Gyps, gypsum; I, illite; Sm, smectite; V, vermiculite; Qz, quartz.
Sample%Phases
V, Sm, Chl-Sm, Chl M/I FspGypsCcDolQz
S128245218418
S226244219421
S313107-35431
S412126-35431
S513129724431
S616106529529
S710983271332
S012308-18626
Table
Table 5. Results of microbiological analysis in Kefalonia samples.
Table 5. Results of microbiological analysis in Kefalonia samples.
Soil SampleMicrobial Load (CFU/g *)
MicroorganismS1S2S3S4S5S6S7
Bacteria
Escherichia colind<1ndnd<1ndnd
Pseudomonas aeruginosandndnd<1<1ndnd
Staphylacoccus aureusndndndndndndnd
Other bacteria2632ndndndndnd
Fungi
Candida albicansndndndndndndnd
Aspergillus nigerndndndndndndnd
Cordycipitaceae family **7890200ndndndnd
nd: not detected; * colony formation unit per gram soil; ** non-pathogenic fungi.
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Lampropoulou, P.; Petrounias, P.; Rogkala, A.; Giannakopoulou, P.P.; Gianni, E.; Mantzoukas, S.; Lagogiannis, I.; Koukouzas, N.; Hatziantoniou, S.; Papoulis, D. Microstructural and Microbiological Properties of Peloids and Clay Materials from Lixouri (Kefalonia Island, Greece) Used in Pelotherapy. Appl. Sci. 2023, 13, 5772. https://doi.org/10.3390/app13095772

AMA Style

Lampropoulou P, Petrounias P, Rogkala A, Giannakopoulou PP, Gianni E, Mantzoukas S, Lagogiannis I, Koukouzas N, Hatziantoniou S, Papoulis D. Microstructural and Microbiological Properties of Peloids and Clay Materials from Lixouri (Kefalonia Island, Greece) Used in Pelotherapy. Applied Sciences. 2023; 13(9):5772. https://doi.org/10.3390/app13095772

Chicago/Turabian Style

Lampropoulou, Paraskevi, Petros Petrounias, Aikaterini Rogkala, Panagiota P. Giannakopoulou, Eleni Gianni, Spiridon Mantzoukas, Ioannis Lagogiannis, Nikolaos Koukouzas, Sophia Hatziantoniou, and Dimitrios Papoulis. 2023. "Microstructural and Microbiological Properties of Peloids and Clay Materials from Lixouri (Kefalonia Island, Greece) Used in Pelotherapy" Applied Sciences 13, no. 9: 5772. https://doi.org/10.3390/app13095772

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