The Soil Chemical Properties Influencing the Oribatid Mite (Acari; Oribatida) Abundance and Diversity in Coal Ash Basin Vicinage

The samples of two technogenic sediments (MOS, coal ash sediment; MOD, a mixture of ash and natural soil) and two natural soils (MOM, meadow; MOF—forest; both Cambisol Dystric) in the vicinage of the coal ash basin were studied. We evaluated risk element concentrations and select-ed chemical and microbiological parameters to determine their influence on the community structure of Oribatida. High concentrations of various toxic elements, alkaline pH, and low hu-midity negatively affected the abundance of oribatid mites. The microbial indicator values showed that the soil microbial community formed in technogenic sediments could effectively use organic carbon. However, considering the wide C/N ratio of the substrates, the process of soil organic matter (SOM) decomposition was slowed down, and thus nutrients were less available for the mites, which could be among the reasons why we did not find any individual of oribatid mite in MOS. In MOD, however, we found representatives of three species, but only Tectocepheus velatus sarekensis established an abundant community at the highly contaminated site. Anthropogenic pressure resulted in the selection of r-strategists, which became dominant and reduced the whole community’s species’ diversity. Therefore, we consider it a suitable indicator of improper human intervention in the ecosystem.


Introduction
Global changes in climate and land use are affecting the biodiversity of soil arthropods in many ecosystems [1]. Oribatid mites represent species-rich and highly abundant soil mites inhabiting the soil's upper layers [2]. There is increasing interest in their reaction to environmental conditions such as metal pollution because of their essential role in detrital food webs. They are actively involved in the soil decomposition process, increasing soil fertility by recycling nutrients out of dead organic matter. The low reproduction and long life-cycle rates suggest that oribatid mites may be sensitive to environmental change [3]. They are not passive inhabitants of ecosystems; instead, they are strong interactors. Despite being generally sensitive to disturbance, oribatid mites show a considerable variation in sensitivity among taxonomic groups. The various scientific papers declare that the number of species is declining toward the more significant disturbance, and, at the same time, the composition of their entire community is changing [4,5]. The large number of factors determine the distribution and abundance of soil mites. Among the abiotic factors, soil pH and soil organic matter (SOM) represent the most significant drivers concerning soil pH variation on soil arthropods' presence [6,7] and arthropods' role in SOM degradation. Highly increased levels of metals, wide C/N ratio, and pH values were recognized as the structuring forces that influence the distribution, diversity, and richness of oribatid species, whereas the soil reaction (pH) influences these differences fundamentally [8]. Changes in the value of the soil reaction mostly come from pollution from industrial production and mining. Its value affects the soil's chemical, physical, and biological properties and the solubility of organic carbon and increases the bioavailability of various toxic elements, mainly metals. Soils around heating power plants are often enriched in metals due to ash blowout of refuse heaps, and toxic metals tend to accumulate in high concentrations in the topsoil near point sources [9].
Oribatid mites proved to have a high capacity to colonize postindustrial dumps [10]. With an increasing metal load of the soil, sensitive species may be replaced by resistant ones without changing the total number of mites. One of the critical reasons for reducing the abundance and species' richness of Oribatida at highly contaminated sites may be explained by toxic effects through the uptake of toxic metal-loaded microorganisms, especially fungi [11]. In general, oribatids and their communities are relatively tolerant of toxic metal exposure [12,13]. Many studies have measured their responses to environmental pollution at the community level [14,15]. The studies proved that heavy pollution exposure decreases both abundance and species' diversity, but species vary in their sensitivity to pollution, and some species may even benefit from moderate pollution levels [14,16]. Oribatida includes some species with resistance to metals to maintain their populations in even highly contaminated soils [15][16][17]. The findings mentioned above suggest that the oribatid mites have a great potential to be used as bioindicators of changing soil environment.
Therefore, it is crucial to understand how the mite community responds to ongoing soil ecosystems' changes. Soil microarthropods play a vital role in the nutrient cycle due to the litter's fragmentation, increasing the microbial action area and stimulating microorganisms' growth [18]. They accelerate the circulation of elements within ecosystems, either directly or indirectly, by stimulating soil microbes and contributing to soil fertility maintenance [19]. Their essential role in litter decay as detritivores suggests that any change in their community structure in response to global environmental change is likely to have further consequences for ecosystem functioning.
The hypotheses were as follows: (1) Anthropogenic pressure results in the selection of r-strategists, which will become dominant and reduce the whole community's species' diversity. (2) Oribatids can be considered appropriate indicators of improper human intervention in the soil ecosystem, reflecting extremely low abundance compared to their abundance in undisturbed habitats.
This study's main goal was to characterize the oribatid mite community, which inhabits the coal ash basin's extreme environment and its close surrounding. Knowing the biological parameters and the properties of soil and contaminants acting in situ is crucial for proper ecological risk assessment. Concerning coal ash disposal sites, representative data about toxicity on oribatid mites are lacking. Therefore, the aim was to detect the influence of environmental load caused by coal processing to the Oribatida community as a change in species' occurrence. Based on the hypotheses we made, we targeted identifying species with negative responses under stress and those prospering in deteriorating living conditions.

Study Area
This investigation aimed to study the species' composition and diversity of Oribatid mites and microbiological activity in a power plant ash pond, the dam, and surrounding meadow and forest soil located in Zvolen-Môt'ová, Slovakia (Central Europe), and the relationship between biological parameters of such soils and their chemical properties ( Figure 1). The ash pond's dam is 10 m high, and the whole area reaches approximately 7 hectares. The sedimentation of ash sediment with permanent storage in the basin was carried out from 1991 to 2005. This storage represents more than 320,000 m 3 of stored ash. At present, it is operated in a cyclical manner-the period of flotation and ash export alternates [20].
( Figure 1). The ash pond's dam is 10 m high, and the whole area reaches approximately 7 hectares. The sedimentation of ash sediment with permanent storage in the basin was carried out from 1991 to 2005. This storage represents more than 320,000 m³ of stored ash. At present, it is operated in a cyclical manner-the period of flotation and ash export alternates [20]. Individual sites at which samples were taken represent four biotopes with gradually decreasing exposure to the contaminant. They include (1) ash pond sediment-ash (MOS), (2) forming Technosol (MOD)-a heterogeneous mixture of ash and natural soil, (3) nearby meadow (MOM) and surrounding forest soil (MOF), both representing soil type Dystric Cambisol according to the World Reference Base for Soil Resources WRB Classification System [21]. The sites were not separated from each other by any physical barrier ( Figure 2). The ash sediment was sampled in two lines of a dry surface of the impoundment: the ash deposit's central part and the dam. Sites MOM and MOF represent the closest natural biotopes where the concentrations of any risk elements do not exceed the statutory limit. Individual sites at which samples were taken represent four biotopes with gradually decreasing exposure to the contaminant. They include (1) ash pond sediment-ash (MOS), (2) forming Technosol (MOD)-a heterogeneous mixture of ash and natural soil, (3) nearby meadow (MOM) and surrounding forest soil (MOF), both representing soil type Dystric Cambisol according to the World Reference Base for Soil Resources WRB Classification System [21]. The sites were not separated from each other by any physical barrier ( Figure 2). The ash sediment was sampled in two lines of a dry surface of the impoundment: the ash deposit's central part and the dam. Sites MOM and MOF represent the closest natural biotopes where the concentrations of any risk elements do not exceed the statutory limit.

Soil Sampling and Analyses
We took two sets of samples (one set for microbiological analyses, one set for soil fauna extraction purpose) from each study site from the 0-10-cm depth interval. Soil samples were obtained using soil cores: five soil cores per field, from which eight samples

Soil Sampling and Analyses
We took two sets of samples (one set for microbiological analyses, one set for soil fauna extraction purpose) from each study site from the 0-10-cm depth interval. Soil samples were obtained using soil cores: five soil cores per field, from which eight samples (with a total volume of 0.20 dm 3 each) were combined to give one composite soil sample. Afterwards, the samples were placed in sterile plastic bags and put into the cooling box. The fresh soil samples were taken to the laboratory immediately. Once in the laboratory, one part of the samples was ground and passed through a 2-mm sieve; the other part was put in the Tullgren apparatus. Before the necessary chemical and physical analysis procedures, the samples were air-dried. The soil samples intended for microbiological analyses were kept field moist and stored until the analyses were performed. We measured the soil moisture content by the gravimetric method. A subsample of a fresh, sieved composite sample was weighed, oven-dried (at 105 • C) until there was no further mass loss, then reweighed. The moisture content in this paper was expressed as a mass of water per mass of dry soil. Soil pH was determined with a glass electrode (Sentix 980) in suspension with distilled water and 1 M KCl, respectively, using 1:2.5 weight ratio (20 g of sample, 50 mL of H 2 O or KCl). Electrical conductivity of the ash sediment was measured in the water-saturated paste with TetraCon 925 conductivity probe. The total amount of organic carbon and nitrogen was determined by a method based on the Pregl-Dumas principle using the NCHSO "FLASH 2000" Elemental Element Analyzer from Thermo Scientific. Samples were analyzed for the content of potentially toxic elements by a combination of inductively coupled plasma (ICP) emission spectrometry and ICP mass spectrometry. Samples were pretreated by hot acid digestion. Content of 40 elements was determined (Mo, Cu, Pb, Zn, Ag, Ni, Co, Mn, Fe, As, U, Th, Sr, Cd, Sb, Bi, V, Ca, P, La, Cr, Mg, Ba, Ti, Al, Na, K, W, Zr, Ce, Sn, Y, Nb, Ta, Be, Sc, Li, S, Rb, Hf). The analysis of potentially toxic elements in studied samples was carried out in ACME Analytical Laboratories Ltd. (Vancouver, BC, Canada).

Microbiological Analyses
Microbial biomass carbon was measured using the fumigation extraction procedure described by Vance et al. [41]. Soil respiration rate was measured as carbon dioxide evolution over 24 h, according to Schinner [42]. We calculated the microbial indicators, microbial metabolic quotient (qCO 2 ), as the amount of basal carbon dioxide produced per unit microbial biomass carbon [43] and substrate availability index (SAI) as the potential for basal production of carbon dioxide ratio according to Cheng et al. [44]. Microbial quotient (qMic) was determined from the microbial biomass carbon to total organic carbon ratio MBC/Corg [45]. This paper's microbial measurements are the mean of triplicate determinations and expressed on a soil-oven, dry-weight basis.

Statistical Analyses
The statistical estimation was carried out with PAST version 4.03 [46]. Abundance (A) is expressed by the number of individuals in dm 2 of the soil surface (ind./dm 2 ). Species' number (SN) in a sample was defined as the number of species to 1.6 dm 2 of soil surface. The species' dominance (SD) was expressed as the percentage of a particular species' individuals to the total number of oribatid mites. According to Tischler [47], a modified categorization into five dominance classes was used to identify dominance. Diversity (Shannon-Weaver's (H')) (S-total number of species; p i -the proportion of species i relative to the total number of species) [48], Margalef Richness Index in Biodiversity [49] (S-total number of species; n-total number of individuals in the sample), and equitability (J) (H-Shannon diversity; N-species number) [50] indices were used to evaluate the species' richness and evenness of oribatocoenoses.
All relationships between soil chemical and biological variables were evaluated using Pearson's correlation coefficients. Differences at p < 0.05 level were deemed significant. Hierarchical clustering (UPGMA: unweighted pair group method with arithmetic mean) was used to visualize the relationship between individual species and coenoses. A matrix of Bray-Curtis distances between coenoses was calculated from the original data matrix based on individual species' dominance values.

Results
The main polluting factor on the site was the exceeding concentration of various risk elements, among which eight elements (As, Ba, Cd, Co, Cr, Cu, Ni, Zn) exceeded the permitted limits in the ash sediment MOS and its dam MOD, while arsenic and copper concentrations reached the indication value for remediation ( Table 1). The MOM and MOF areas represented natural soils (both Cambisol Dystric), in which the concentrations of risk elements did not exceed the reference value. The technogenic sediment of the basin (MOS) and the dam (MOD) showed an alkaline soil reaction. In contrast, the meadow (MOM) and the nearby forest's pH (MOF) showed weakly acidic values. The results of electrical conductivity (1008 µS.cm −1 ) measurement in the ash sediment suggested that the risk of mobilization of detected toxic elements from the tailings and the adverse effects on biota was not significant. The percentage of organic carbon was high in all monitored samples; the lowest value was found in the MOD sample. The carbon-to-nitrogen ratio, an essential indicator of SOM decomposition rate, was extensive (43)(44)(45) in the MOS and MOD samples; in the other samples, this ratio was meaningfully lower (Table 2). We also measured significantly lower humidity in the MOS (11.76 %) and MOD (13.64 %) samples compared to all monitored samples. Microbial biomass carbon (MBC) values were very high in all examined samples and ranged from 2.37 to 2.68 mg C/g dry matter. Both basal and potential respiration values were low in MOS and MOD samples. We recorded slightly higher basal respiration in the MOM and MOF, but the potential respiration values were many times higher than in the MOS and  Table 2). The microbial quotient (qMic) refers to the microbial activity potential to mineralize organic matter in the soil. This quotient's lower values are usually associated with the reduced availability of organic material for the microorganisms. The microbial quotient was low in the samples (Table 2). On the contrary, the metabolic quotient (qCO 2 ) values were surprisingly lower in MOS and MOD than in the meadow and forest samples ( Table 2). The metabolic quotient expresses the efficiency of carbon utilization by the microbial community. Its values increase when the microbial community in the soil is exposed to stress. On the other hand, low qCO 2 values signal optimal carbon consumption by the soil microorganisms. We found a bit higher values of this parameter in the MOM and MOF. The substrate availability index (SAI), indicating better microorganisms' ability to utilize the readily available organic substances provided by the technogenic substrate, was lower in MOS and MOD compared to natural soils MOM and MOF, which was entirely unexpected ( Table 2). Table 2. Chemical and microbiological properties of soils (MOS, ash sediment; MOD, heterogeneous mixture of ash and natural soil; MOM, meadow; MOF, forest); pH (H 2 O/KCL), negative log of the hydrogen ion concentration; w, water content in the sample; Corg, total organic carbon; Ntot, total organic nitrogen; MBC, microbial biomass carbon (dry weight); CO 2 B, basal respiration (per 24 h); CO 2 P, potential respiration (per 24 h); SAI, substrate availability index (CO 2 P/CO 2 B); qCO 2 , metabolic quotient 100× (CO 2 B/MBC); qMic, microbial quotient (MBC/Corg); C/N, total organic carbon-and-nitrogen ratio. A total of 32 species of oribatid mites were recorded (MOF 62.5%, MOM 74.9%, MOD 9.4%, MOS 0.0%). In the sample MOS, we did not find any representative of the oribatid mites. In the sample MOD, we recorded only three species. Opiella (Moritzoppia) translamellata (Willmann, 1923) and Poecilochthonius italicus (Berlese, 1910) were found in the sample MOD in very low abundance, while the eurytopic species Tectocepheus velatus sarekensis (Trägårdh, 1910) was numerous. Oribatocoenoses MOF and MOM were formed mainly by species of moist meadow and forest habitats (Table 3).  Overall, 37.5% of the species showed a tendency to come freely into the neighboring habitat and create numerically stable and comparable communities except Platynothrus peltifer (C.L. Koch, 1839), which showed a strong preference for MOF, and species Hypochthonius rufulus (C.L. Koch, 1840) and Scheloribates laevigatus (C.L. Koch, 1836), preferring MOM. We surprisingly found O. (M.) translamelata, a moisture-loving species, in the MOD sample, but as it belongs to the species that prefer forest ecosystems, its long-term remaining in the meadow habitat is questionable. Its co-occurrence with Poecilochthonius italicus (Berlese, 1910), which prefers the habitat's meso-xerophilic nature, indicates one (or both) of these two species' accidental occurrence in the MOD mite community. The MOM and MOF oribatid The correlation analysis outlined that the abundance and diversity of oribatid mites increase with decreasing the substrate's pH. There was a positive correlation between mite abundance and diversity and soil moisture, and the same was observed with soil microbial respiration. Interestingly, the highest was the soil carbon-to-nitrogen rate and the lowest was mite diversity and abundance. We also found a negative correlation with mite abundance and increased arsenic, nickel, cobalt, and chromium concentrations. The risk elements demonstrably negatively affected mite diversity, primarily cobalt and nickel (Table 4). Table 4. The correlation between chemical and microbiological attributes and oribatid mites' diversity and abundance. A, mite abundance; D, mite diversity; pH (H 2 O/KCl), negative log of the hydrogen ion concentration; w, water content in the sample; MBC, microbial biomass carbon (dry weight); SMA, soil microbial activity (respiration); C/N, total organic carbon-and-nitrogen ratio. Significance labels: * p < 0.01; ** p < 0.05; ns, non-significant. Punctoribates punctum (C.L. Koch, 1839) and Xenillus tegeocranus (Hermann, 1804) had a similar position MOF locality. P. italicus and T.v. sarekensis, occurring at the MOD site, did not belong to any of the mentioned clusters; moreover, their joint occurrence appeared to be accidental (Figure 3). preferring a forest-habitat type, of which only O. (M.) Translamellata occurred in a recent position at the MOD site. Grouping C represented a recent to subrecent species bound mainly to the meadow character of the MOM locality. Species Punctoribates punctum (C.L. Koch, 1839) and Xenillus tegeocranus (Hermann, 1804) had a similar position MOF locality. P. italicus and T.v. sarekensis, occurring at the MOD site, did not belong to any of the mentioned clusters; moreover, their joint occurrence appeared to be accidental (Figure 3).

Discussion
Reduction of the abundance and species' richness of oribatid mites in soils close to industrial factories is a well-known phenomenon. The effects of metal pollution from different sources on the community structure of soil arthropods have been demonstrated by many researchers [53][54][55][56]. The authors underlined that oribatids generally seemed to be more susceptible to toxic metals than most of the other arthropods [12,[57][58][59][60]. A generally observed pattern was that communities were impoverished with increasing metal concentration since most species decrease in abundance, although some species may survive even the most polluted sites [61,62]. We can confirm this claim as we detected very low mite abundance at contaminated sites with one dominating species, T. v. sarekensis. The reduced number and low abundance of some species in the oribatid community at the MOD might indicate that this community is resistant, with reduced self-regulation possibilities. There are more reasons why oribatids' abundance and diversity were strongly affected at the polluted sites. First, many pollutants quickly accumulate in the soil, which is the living space for many oribatid species. Second, fungal hyphae, known to accumulate metals effectively, are the primary food source for many species [63]. In MOS and MOD samples, we repeatedly tried to cultivate microscopic fungi, which are considered a required component of juveniles' diet of Oribatida, but without success. The pond's and dam's sediment did not favor the development of microscopic fungi, which also reflected in the abundance of oribatid mites. The MOS and MOD samples showed meaningfully lower diversity and equitability than meadow and forest samples. On the other hand, even though the diversity values were much higher in the natural soil than in the technogenic sediments, we can consider the oribatocoenosis at this site relatively poor. The abundance of mites and species' number of Oribatida were also highly reduced in the MOS and MOD, gradually increasing toward the natural, unpolluted sites, MOM and MOF. To a certain degree, the soil protects mites against pollutants and, therefore, whole absence of Oribatida was observed only in highly polluted ash sediments. Our study revealed that the recorded values of pollutants (especially the concentrations of As, Cu, Ni, Co) influenced the mite communities' structure in the ash pond and its dam ( Table 4).
T. v. sarekensis was found to resist a wide range of toxic metal pollution. It was the only species that thrived in high abundance in heavily polluted samples of forming Technosol MOD. According to Siepel [63], it was the only oribatid species observed as comparatively abundant at highly polluted sites. T. v. sarekensis is considered an opportunistic herbofungivorous species. It is also well known for belonging to a group of parthenogenetic r-selected organisms, which can recolonize areas where sexual reproduction may be hindered by environmental conditions [64]. Its life strategy and high tolerance allow it to occupy free niches after the displacement of other species and acquire a highly dominant position in the community [4,[65][66][67][68]. Most oribatid species are, although defined as Kselected organisms with low dispersion ability [69]. They cannot adapt to new conditions, escape easily from the stress of disturbance, or quickly recolonize disturbed habitats. Only a few eurypotent species from oribatocoenoses, usually Oppiidaea and Brachychthoniidae, can survive in permanently stressed biotopes.
The technogenic substrates often form an unfavorable microhabitat for mites because of the low amount of SOM, low humidity, and wide C/N range. At optimal humidity, the oribatids can survive even in poor substrates with a minimum SOM content; in substrates with a wide C/N range, they are limited mainly by the lack of SOM immobilized by soil microorganisms. We found a low (none) oribatid abundance and diversity in samples of MOS and MOD, where the C/N range was extensive even though the amounts of MBC and respiration suggested a good functioning microbial community. Moreover, microbial indicators' values showed that despite the reduced organic matter available to the microorganisms, the microbial community could efficiently utilize the organic carbon, even more than in both unpolluted sites. The wide range of C/N, on the other hand, suggests that in the nitrogen-poor substrates, MOS and MOD decomposition processes were slowed down due to microbial immobilization of organic matter. This could be the other reason for the total absence or reduced numbers of mites in these kinds of substrates.
Manu [17] demonstrated that, even though the heavy metal pollution in the study site was high, the total number of species and numerical abundance reached high values due to the high humidity and acidity of the substrate, which provided a more favorable habitat. We also confirmed a positive correlation between humidity and mite abundance (r = 0.84, p < 0.01). The abundance was meaningfully lower in MOD samples, where we measured significantly lower substrate moisture than in the natural soils. Therefore, it seems that the availability of soil water highly influences the performance of soil fauna. Depending on the species, different strategies can be used to cope with low soil-water potential. Soil microarthropods may be resistant to desiccation by reducing the water permeability of their integument. This strategy has been adopted by many epedaphic (surface-dwelling) species, whereas euedaphic (soil-dwelling) species have little or no control of evaporation across the integument [70]. In general, soil moisture's positive effect on soil arthropod communities' abundance has been emphasized [71,72]. Holmstrup et al. [72] presented a study that showed that the abundance of microarthropods in copper-contaminated soil was stimulated by enforced drought in a field experiment. It was expected that the combination of copper and drought would harm soil microarthropods. We confirmed a negative correlation between mite diversity and copper (Table 4), which, connected with the low humidity of the technogenic substrates MOS and MOD, could have had a considerable effect on the mite community.
Soil acidity unlikely affects oribatid mite communities directly since most oribatid mite species tolerate even very acidic conditions [62,73]. Furthermore, soil microarthropods' community structure has been relatively resistant to substantial soil pH changes [74]. The soil pH affects the colonization of forests by soil macrodecomposers, which affects oribatid mites via changes in the thickness of the litter layer since soil acidity may affect calcium availability. A negative correlation was observed between pH and mite abundance and diversity (Table 4). Although we cannot ignore the impact of drought and various toxic metals on the mite community, our observations showed that, with increasing pH, the diversity of oribatid decreases. However, in the case of MOS and MOD samples, we are inclined to believe that the very low diversity values were caused by a combination of critical factors, e.g., higher pH, low humidity, and high concentrations of some risk elements.

Conclusions
The results obtained in this study provide additional knowledge of the community structure of oribatid mites in the disturbed soil environment around a coal ash basin. The results confirm that the impoundment's technogenic sediment and its dam contain high concentrations of various toxic elements, which, in connection with alkaline pH, low substrate humidity, and wide C/N range, has a negative effect on the abundance of oribatid mites. Even though the substrate's microbial community seems to utilize the accessible organic carbon efficiently and survive without energy loss, the organic compound stays immobilized for other soil organisms. This suggestion is supported by changes in oribatid mite community composition, which was negatively reflected in their abundance and percentage of dominances in acarocoenoses. Despite such a substrate's unsuitable properties, some oribatid species can withstand critical metal concentrations and establish an abundant community. Compared to the community's composition in natural soils, there was an observed reduction in species' diversity, exchanging long-lived species for short-lived ones. T. v. sarekensis was found to survive in a disturbed habitat in a small population and ultimately increase its abundance. In conclusion, we confirmed that high values of diversity and equitability occurring in natural soil habitats decrease significantly in localities with regular anthropogenic intervention. When protecting soil ecosystems, there must be the biodiversity of soil invertebrates, emphasizing oribatid mites. The differential response of T. v. sarekensis to adverse conditions makes it a suitable indicator for assessing the degree of ecosystem vulnerability.

Institutional Review Board Statement:
The authors declare that the accepted principles of ethical conduct have been followed strictly. Since, in the case of soil mite study, no ethical issues are required so far, our knowledge is clear.

Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.

Conflicts of Interest:
The authors declare no conflict of interest.