Living with Contamination: Insights into an Epigeic Macrofaunal Community in an Area Extremely Polluted by Risk Elements

Abstract

Five meadows along the Litavka River highly contaminated with heavy metals from mining were sampled for insects and spiders using pitfall traps. The sites are in the Příbram region of central Bohemia, Czechia, which was previously reported as one of the most polluted areas in Europe due to intensive mining and metal processing. The determination of pseudo-total risk elements in soil revealed that all the sampled sites were contaminated with heavy metals, especially As, Cd, Pb, and Zn, with levels ranging from moderate to extreme. The trap results showed a total of 1142 beetles from 21 species, 946 ants from 16 species, 548 spiders from 28 species, and 96 harvestmen from 3 species. With the exception of the scabrous ground beetle, Carabus scabriusculus, all captured specimens were species common to the wet meadows and forest edges of Czechia. With respect to species richness, the dominant spider and beetle taxa were less abundant at the heavily polluted locations than at the moderately polluted locations. In the case of ants, however, there was no relationship between contamination level and abundance or species richness. Thus, it is worthwhile recording and analyzing the differential changes in abundance of the dominant spider and beetle species in polluted areas.

1. Introduction

Soil is one of the most important natural resources, crucial to life on Earth, and plays a central role in current environmental challenges including food security and global climate change [1]. Similar to other natural resources, the soil faces a variety of threats associated with human activities. Globally, soil contamination by heavy metal risk elements (REs) represents a serious and growing problem. These metals occur naturally in the Earth’s crust at either comparatively harmless or non-hazardous concentrations, but they can be toxic at the high concentrations associated with mining and ore processing.

Rivers have often been contaminated with REs due to historic and modern mining and industrial activities, and the soils on the riverbanks are some of the most hazardous environments in terms of heavy metal contamination. The first problems arising from RE contamination most likely appeared with the mining of metal ores in the Middle Ages, but pollution rapidly increased during the Industrial Revolution of the 19th century with the rise of metal-processing, smelting, and chemical plants [2]. Little was done at that time to prevent the dispersal of REs into the environment [3]. In central and eastern Europe, the contamination of river systems by REs peaked between the1950s and 1970s because of the intensification of industry and agriculture during the Communist period [4].

Soil supports a very complex ecosystem accommodating a high biodiversity, and research into the effect of heavy metal contamination on soil fauna is of particular interest. Such organisms are dependent on the soil, at least in part, and any changes in it can fundamentally affect them. The sensitivity of soil fauna to soil composition is very high, and they are good indicators of soil stress [5]. Recent research on the type, accumulation, and mobility of REs revealed that contamination occurs mainly in the upper 30 cm of the soil profile [6,7]; however, traces of Zn and Cd were found even at depths of 2 m [8]. Studies have shown that invertebrates inhabiting the upper levels of soil such as earthworms, springtails, mites, and nematodes can be used as bioindicators to assess the degree of soil RE pollution [9]. Only a few reports on the effects of RE pollution on terrestrial arthropods are available, however [10,11,12,13].

To contribute to this knowledge base, our study aimed to monitor the diversity and abundance of epigeic macrofauna at several sites selected near the Litavka River in the Příbram district of Central Bohemia, Czechia. This area was previously found to be among the most polluted floodplains in Europe as a result of historical mining and smelting combined with flooding [14,15,16]. In this work, we assessed the relationship between the arthropod community and soil RE contamination.

2. Materials and Methods

The epigeic macrofauna was sampled at four permanent grasslands on the banks of the Litavka River at the following locations: (1) 49.678 N, 13.975 E; (2) 49.705 N, 13.983 E; (3) 49.710 N, 13.988 E; (4) 49.718 N, 14.013 E; and (5) 49.784 N, 13.987 E (Figure 1). The altitude of the study sites ranged from 377 to 511 m above sea level with a mean annual precipitation of 700 mm, and a mean annual temperature of 6.5 °C [17]. The soil types included weakly acidic modal cambisols with less frequent fluvisols and gleysols [18]. The locations are characterized in detail in the previous paper [19], where the ability of insects from the families, Coleoptera and Hymenoptera, to bioaccumulate REs was evaluated.

The arthropods were collected during July, August, and September 2019, using pitfall traps made of plastic cups (0.5 L) filled with 5% aqueous formalin solution as a preservative. Four traps were distributed at each sampling site separated from each other by a minimum distance of 40 m to maintain independence. The traps were collected every 14 days from 1 July to 23 September 2019. All species that were captured were identified. The ants and beetles were identified under a stereomicroscope (Nikon SMZ-25, Tokyo, Japan) using entomological keys [20,21,22,23,24]. The spiders and harvestmen were identified according to Nentwig et al. [25], with nomenclature based on the World Spider Catalog [26]. The captured arachnids and beetles were counted and stored in 80% ethanol. The ants were preserved as pinned specimens.

All soil samples for RE content analyses were collected from soil removed during the setting of the pitfall traps at depths of 0–25 cm. In addition, eight soil subsamples were taken and mixed together at each location. These composite soil samples, (~5 kg each), were transported to the laboratory for chemical analyses. Seven selected elements (As, Cd, Cu, Cr, Ni, Pb, Zn) were extracted with aqua regia (Cl₃H₄NO₃) and pseudo-total concentrations were determined. An inductively coupled plasma-optical emission spectrometer (ICP-OES, Agilent 720, Agilent Technologies Inc., Santa Clara, CA, USA), equipped with a two-channel peristaltic pump, a Struman–Masters spray chamber, and a V-groove pneumatic nebulizer made of inert material was used for the determination of REs in the soil samples. The experimental conditions were power of 1.2 kW, plasma flow of 15.0 L/min, auxiliary flow of 0.75 L/min, and nebulizer flow of 0.9 L/min. Except for location 1, the values have been published by Mukthtorova et al. [19].

One-way ANOVA was used to evaluate RE levels from the monitored locations. After obtaining significant results (p < 0.05), multiple comparisons using the Tukey HSD test were applied to identify significant differences for each RE between locations. These analyses were performed using Statistica ver13 (Statsoft, Tulsa, OK, USA). The program, Canoco 5 [27] was used for Canonical Correspondence Analysis (CCA) with supplementary variables (locations), for establishing the relationships between individual plots, species, and REs.

3. Results

3.1. Risk Elements

The levels of REs in soils are summarized in

Table 1. Concentrations of risk elements (µg/g of soil) at the monitored locations determined using AAS. The letters indicate significant differences based on the post-hoc Tukey test, assuming p < 0.05.

	As	Cd	Cu	Ni	Pb	Zn	Cr
1 *	55.4 ± 22.2 a	2.7 ± 12 a	44.3 ± 17.1 a	21.6 ± 3.5 ab	1350.7 ± 493.2 c	455.7 ± 193.5 a	40.5 ± 5.0
2 *	663.0 ± 14.1 b	19.5 ± 0.5 abc	97.1 ± 2.6 bc	37.8 ± 0.9 c	2169.8 ± 178.5 b	3304.8 ± 65.9 b	58.8 ± 0.8
3 *	628.6 ± 191.7 b	43.9 ± 26.3 c	127.2 ± 35.0 c	29.8 ± 10.4 cb	3898.3 ± 329.1 d	6309.8 ± 1724.6 c	60.5 ± 55.1
4 *	226.2 ± 128.3 a	28.4 ± 12.9 bc	61.9 ± 30.7 ab	22.9 ± 8.3 ab	2682.2 ± 460.3 b	3866.4 ± 1146.2 b	40.4 ± 5.7
5	18.5 ± 9.5 a	1.6 ± 1.3 ab	16.4 ± 3.2 a	14.6 ± 2.4 a	241.4 ± 145.5 a	273.7 ± 177.8 a	30.1 ± 1.9
p	>0.001	>0.001	>0.001	>0.001	>0.001	>0.001	0.356
F	43.28	9.42	17.75	8.39	62.29	38.44	1.168

* The data have been previously published by Mukhtorova et al. [19].

. Regarding the pollution rate at the monitored locations, significant differences were found for all monitored REs with the exception of Cr. The lowest RE contamination was detected at Loc. 5, about 10 km from the most seriously polluted sites. The RE pollution at Loc. 1 was lower in comparison with the other studied locations. The highest pollution of Cd, Cu, Pb, and Zn was recorded at Loc. 3, next to an extremely polluted mine

3.2. Soil Macrofauna

In total, 1142 beetles from 21 species, 946 ants from 16 species, and 644 arachnids (548 spiders + 96 harvestmen) from 31 species (28 spiders + 3 harvestmen) were collected from pitfall traps. The highest number of arthropods (1022 individuals) was captured at location 1, where the species richness (35 species) was the lowest among the study sites. At locations 2, 3, and 5, 47 different species were recorded. The highest diversity (48 species) was detected at location 4. Excluding location 1, where beetles were the dominant group of the captured arthropods, the majority of specimens at the monitored sites belonged to spiders (16–18 species), followed by beetles, ants, and harvestmen (

Table 2. Total number of arthropod species at each location (richness).

Arthropods	Number of Species at Each Location
	1	2	3	4	5
Spiders (Aranae)	11	18	20	19	16
Harvestmen (Opiliones)	3	2	1	2	3
Ants (Formicidae)	6	10	10	10	10
Beetles (Coleoptera)	14	15	13	13	13
Total	35	47	47	48	47

).

Among ants, Lasius niger and Lasius umbratus were the only two species that were detected at all studied sites. Formica cunicularia, F. fusca, L. platythorax, Myrmica scabrinodis, and Tetramorium spp. were found to be present at four locations. The most abundant species varied among the locations. While Myrmica sabuleti dominated among the captured specimens at location 1, Formica pratensis was dominant at location 3, and Lasius niger at locations 2, 3, and 5 (Table S1, Figure 2).

In the case of beetles, the results showed that four beetle species were present at all five monitored locations: Philonthus decorus, Pterostichus melanarius, and Poecilus cupreus. Amara aenea, Europhilus fuliginosum, and Otiorhynchus sulcatus were captured at four out of five studied sites (Table S2). Similar to ants, the abundance of captured beetle specimens varied among the sites. Philonthus decorus was the dominant species at locations 1 and 4. The beetles of the genus Philonthus were also dominant at location 2. Amara genea and Otiorhynchus sulcatus were the most abundant beetle species captured at location 3. Lastly, the majority of the catch from location 5 was Silpha obscura. Interestingly, the species that were very abundant in the less polluted locations (1 and 5) such as P. decorum, S. obscura, or P. cupreus occurred in much lower numbers at locations with higher RE levels. At locations 2 and 5, the occurrence of Carabus scabriusculus, which is included on the Red List of the Czech Republic [28] as vulnerable, was recorded. The species distributions in relation to RE content as derived from CCA are shown in Figure 3.

The presence of Piratula hygrophila, Pardosa lugubris, P. prativaga, P. pullata, and Diplostyla concolor was detected at all five monitored locations (Table S2). However, all these species were more abundant at the less polluted locations, 1 and 5. Similarly, Piratula latitans was the dominant species at locations 1 and 5, but was absent from locations 3 and 4 (Figure 4). Ground spiders (Lycosidae) were rarely caught in the pitfall traps at the study sites and no clear trend in relation to RE pollution could be determined. In the case of harvestmen, Paranemastoma quadripunctatum was captured at all the sites. Lacinius ephippiatus was the most abundant species from 2 and 4, but was absent from location 3. The occurrence of the last harvestmen, Rilaena triangluaris, was detected at two locations (1 and 5), where this was also the most abundant harvestmen species (Table S3).

4. Discussion

In this study, the composition of epigeic fauna was monitored in the vicinity of the Litavka River. At all of the monitored locations, our analysis showed very high levels of REs, exceeding the EU limits for feeding animals (EU Commission Regulation No. 574/2011) as well as the Czech limits for growing fruits and vegetables (Government Regulation No. 75/2015 Coll.). The detection of high concentrations of REs confirmed that this area was extremely polluted, and supported the outcome of this study in providing insights into differences in macrofauna composition under conditions of heavy metal pollution. The soil RE levels exceeded, in most cases, the preventive and/or indicative values of REs according to the Czech Public Notice No. 153/2016 [29], characterizing conditions for the protection of agricultural soil quality in the Czech Republic.

Arthropods such as ants, beetles, and spiders can respond rapidly to environmental changes, are also easy to capture, and their taxonomic history is well documented [30,31]; their potential use in the biomonitoring of areas contaminated by heavy metals warrants further investigation. The effects of extreme environments, including very high levels of soil REs, on communities of terrestrial arthropods such as ants, beetles, spiders, and harvestmen are still not well understood. The available data are scarce and sometimes contradictory; therefore, more research on this topic is highly relevant to fill in the gaps in the literature.

4.1. Ants

Ants are considered to be useful bioindicators of ecosystem conditions, and sampling of ants by pitfall trapping can be used to evaluate land management decisions such as grazing, logging, and prescribed fires and to assess the influence of global climate change on ecosystems according to Hůrka et al. [31]. They may also be potential indicators for various pollutants including REs. Recent studies on RE levels in ants proved that they can accumulate REs and respond to soil contamination with respect to species, colony status, behavior, and immunity [32,33,34,35]. Furthermore, the contamination might also affect the ants’ morphology. For instance, Skaldina et al. [36] reported that workers of Formica lugubris collected from polluted areas had lower body mass and less melanized heads than those from the reference site. Regarding the effect of RE pollution on species richness, the data obtained by this study support the findings of other authors that soil RE levels are not a deciding factor in the composition of ant communities, but that they affect ants mainly through habitat changes. According to Eeva et al. [37] forest age and foraging sources were more important determinants than the pollution level in Formica ants. No significant effect of soil contamination with Pb and Sb on the abundance of Formicidae was observed by Migliorini et al. [38]. Belskaya et al. [39] found the highest species richness, diversity, and abundance of ants in the transitional zone between moderately polluted forest habitats and industrial waste areas on the eastern slopes of the Southern Urals. Grześ et al. [10] even reported increased species diversity with increasing metal pollution. The community composition of ant species in our study is similar to those seen by other authors, showing no relevant relationship with RE. The greater abundance of workers in the trap may be explained by its being close to the ant nest. In comparison with Czech myrmecofauna, the ant species identified here at the polluted locations are those commonly present in damp habitats and woodland edges in the Czech Republic [40,41].

4.2. Beetles

As the most abundant order of insects, beetles inhabit the majority of terrestrial habitats; therefore, they are useful as bioindicators of environmental conditions. Compared with other epigeic invertebrates, the ability of beetles to take up and accumulate REs is low [42] and depends on foraging strategy, developmental stage, sex, and breeding type [43]. However, some studies to assess the relationship between beetles and heavy metal pollution have been undertaken, for example, by determining species richness in areas contaminated by REs [44,45,46].

In our study, the Carabidae were the most abundant and diverse family of coleopterans. This is in accord with Baranová et al. [46] who reported that carabids dominated insect assemblages in metal mining spoil heaps. Carabids were also found to be the dominant epigeic beetle species by Lock et al. [45], who showed no significant correlation between species richness or activity of carabid beetles and zinc concentrations in ancient mining areas in Belgium. Skalski et al. [44] observed that Geotrupidae, Carabidae, and Silphidae were more abundant in the reference sites than the polluted sites, whereas members of the Curculionidae and Staphylinidae families were more abundant on heavily contaminated sites, which matches the trend seen in this study: Silpha obscura was missing at heavily polluted locations, while it was the most abundant species at the least contaminated locations. Moreover, Pterostichus melanarius and Poecilus cupreus were less abundant at locations with higher levels of REs. Our data on Staphylinidae and Curculionidae are in line with Skalski et al. [44] for Otiorhynchus sulcatus and Medon sp., while the abundance of S. decorus was lower with increasing RE pollution. In the case of Medon sp. and Otiorhynchus sulcatus, the affinity of these species for more polluted soil was also confirmed by CCA. In contrast, both species shown to be negatively correlated with REs (O. helopioides and C. granulosus) are carabids, which were reported to be resistant to this type of pollution because their metabolism has an effective detoxification mechanism [47]. However, because of the relatively short period of monitoring and the low number of captured specimens, no strong conclusions can be drawn from this finding. To improve the statistical analysis, long-term monitoring is essential.

4.3. Spiders and Harvestmen

Arachnids are abundant and diverse predators inhabiting both natural and disturbed terrestrial ecosystems, and, thus, they are an important part of the epigeic macrofauna. Due to their short life cycle and high mobility, both spiders and harvestmen can respond rapidly to small changes in their environment [48]. Therefore, the composition of spider communities is considered an effective tool for monitoring and comparing the species richness in various habitats, as well as to evaluate the effects of anthropogenic activities on biodiversity. According to Marc et al. [49], spiders are known to be involved in the biological magnification of a variety of soil contaminants, including REs, and ground spiders were shown to contain significantly higher levels than web spiders. The ability of ground spiders to accumulate REs can vary. Unlike ants, whose capacity to accumulate REs varies even within individual nests at the same location [35], certain species-specific patterns of spiders have been confirmed; thus, arachnid species diversity and abundance may be used as pollution indicators when assessing environmental quality. Regarding RE pollutants, Jung et al. [50] stated that, in general, communities of spiders were not sensitive enough to prove RE contamination in soil, but analysis of the composition and abundance of populations of the Linyphiidae (sheetweb weavers) allowed researchers to distinguish moderately polluted sites from unpolluted ones. Pardosa astrigera and P. laura (Lycosidae) seemed to be promising RE indicators and Oedothorax insulanus (Linyphiidae) showed good potential as a heavy metal sentinel. In comparison with unpolluted sites, Jung et al. [50] observed a decrease in species diversity and an increase in the abundance of Lycosidae in moderately polluted locations. In our study, however, exactly the opposite trend was observed. The highest abundance of Lycosidae (especially Pardosa and Pilatula) and the lowest species richness of spiders were observed at the less polluted location (Loc. 1). It must be pointed out, however, that the RE content in the soil in our study was much higher than that of Jung et al. [50], who sampled species from the banks of the Singil stream between the cities of Ansan and Sihueng, South Korea. Another study conducted by Vorobeichik et al. [51] reported that the species richness and abundance of arachnids decreased with the pollution gradient, which thus partly agrees with our findings. Our study confirmed that the Lycosidae make up the dominant species in highly RE-polluted habitats of the temperate climate as was previously reported [50,52]. However, in Russia, the family Linyphiidae dominated at both slightly and heavily polluted sites [53]. This difference could be explained by the fact that Linyphiidae is known to represent the dominant species in the ground layer of northern areas, in general [54], and the composition of spider communities on polluted sites did not differ significantly from those on uncontaminated soil. With regard to Czech arachnids, the captured spiders were obtained on open bright meadows, bright areas in forests, and forest edges [55,56].

Regarding harvestmen, the lowest values of species diversity and abundance in our study were recorded at Loc. 3, which had the heaviest pollution among the sampled sites. As pollution levels increased, the species richness decreased. However, Lacinius ephippiatusi was abundant at Loc. 2, where contamination from As, Cd, and Cu was similar to that at Loc. 3, but where this species was missing. Due to the comparatively low number of captured specimens in our study, no clear conclusions can be drawn from the harvestmen data. However, the decline in diversity or the absence of harvestmen in heavily contaminated areas was previously reported by Zolotarev et al. [57] from the impact zone of a copper smelter in central Russia. These authors explained the lack of harvestmen by their reproduction and foraging strategies. Unlike spiders, harvestmen lay their eggs in soil and consume the integument of their prey, which may be contaminated by absorbed metals; thus, prey from a polluted area can be toxic to harvestmen.

5. Conclusions

The analysis of RE contents in soil samples showed high to extremely high contamination of the soil at all the sampled meadows at the Litavka riverside. While statistical analysis showed no significant linkage between RE levels in soil and species richness of ground macrofauna, the abundance seemed to be a better indicator of metal pollution. For instance, a decrease in the abundance of dominant spiders and beetles with increasing soil RE was noted. In comparison with ants, the communities of beetles and spiders appeared to be more promising as indicators of RE pollution. However, further long-term monitoring is necessary to verify this hypothesis and to select the most suitable orders or families of arthropods for this purpose.