Carabid Beetle (Coleoptera: Carabidae) Response to Soil Properties of Urban Wastelands in Warsaw, Poland

Urban wasteland is of special interest to city planners. However, to integrate such areas into city space management with consideration of nature conservation aspects, a sound assessment of their ecological potential is necessary. The aim of this paper was to analyze whether carabid beetle assemblages of the wastelands are affected by soil parameters, particularly trace element contamination. Therefore, we studied the carabid fauna in relation to selected soil parameters on 56 ampling plots situated in 24 wastelands located in the city of Warsaw (Poland). The results have confirmed our assumptions that the number of species, as well as the number of individual carabid beetles, are negatively affected by an increasing amount of pollutants in the soil. Particularly, the trace elements Pb, Cu, and Cd showed a significantly negative impact. The results are of value when it comes to the use of urban wastelands in the context of sustainable city development. Future use of urban wastelands will be faced with trade-offs between the use for public interests (e.g., housing space) and ecological interests. Phytoremediation and entomoremediation may be included in decontamination measures. The results of studies, such as the one conducted by us, may help to select the respective wastelands for certain purposes.


Introduction
Cities are subjected to permanent economic, social, and political changes, which influence the transformation of spatial urban structures [1]. With a rapid increase in urban population, environmental, social, and economic challenges are growing as well. Currently, cities are being redesigned to become more sustainable [2]. However, sustainable management of urban space is a complex task that requires a many-sided approach. Researchers [3] have emphasized that there is a need for a framework for creating new policies and encouraging more strategically organized efforts in sustainable environmental planning.

Study Areas and Sampling Plots
In order to elaborate data on carabid beetles and soil parameters, 24 wastelands ( Figure 1) were included in the presented study. At each site, based on vegetation characteristics, characteristic vegetation units were identified, and selected soil parameters were elaborated, in each of which independent sampling plots for the collection of carabid beetles were established. Based on the obtained results, sampling plots were assigned to the respective vegetation units and soil parameters. As a result, 56 sampling plots were chosen for the analyses (Table 1).
With respect to carabid beetles, it is generally observed that young stages of succession exhibit a high number of species and individuals, compared with advanced stages of succession [22][23][24]. In order to be able to exclude a bias concerning this matter, each sampling plot was assigned to either a young or advanced stage of succession (Table 1). Table 1. Phytosociological characterization and the stage of succession of the sampling plots, as well as numbers of collected carabid beetle species and individuals (100 trap-days). The first number of the sampling plots denotes the area (numbers of the areas, as in Figure 1), the second number is the respective sampling plot in the area.

Sampling Plot
Phytosociological Units Stage of Succession Species Individuals

Field and Laboratory Methods
Carabid beetles were collected using pitfall traps [25]. At each sampling plot, two traps were installed with a maximum distance of 3 m from each other. The traps were glass jars topped with a funnel (upper diameter of about 10 cm), set flush with the soil surface. A roof was suspended a few centimeters above the funnel and ethylene glycol was used as a killing agent and preservative. The traps were placed at least 15 m from the edge of the plots. However, this was not possible in every case, due to the shape of some plots (1-2, 1-3, 1-4, 2-3, 3-3, 4-1, 9-1, 12-1, 12-2, 13-2, 15-1, 17-5). Carabids were sampled from mid-May to late September in 2018. The traps were controlled for proper performance during some field visits, and exchanged in July.
Determination and nomenclature of the individuals collected were carried out in accordance with the literature [26]. All individuals were identified to the species level by the first author. Voucher specimens of each species were deposited in a collection of the first author at the Warsaw University of Life Sciences-SGGW. We characterized the species with respect to the functional traits, habitat preference, trophic specialization, and breeding type, based on the literature [11,[26][27][28][29][30][31].
Soil samples were picked with the use of an Egner stick, up to a depth of 20 cm. The soil was sampled in triplicates at each wasteland , with an area of 5000-10,000 m 2 . A single replicate was a pooled sample of 20-25 individual ones. To exclude any variation in the physical and chemical properties of the soil, the averages obtained for the whole area represented all plots within the given area. The soil samples for pH, electrical conductivity (EC), soil water content (SOC), and organic matter (Org) were collected in mid-June (Term I), July/August (Term II), and mid-September (Term III), while the determination of trace element concentrations occurred once in mid-September.
Soil samples were stored in a refrigerator prior to analysis for up to 24 h. A quantitative analysis of the content of selected trace elements (TE) (Cu, Zn, Pb, Cd, Cr, Ni) in the soil was carried out at the end of September 2018. The soil samples were mineralized in a mixture of concentrated HNO3 and HClO4, and then the TE were analyzed by atomic absorption spectrometry (AAS) (Perkin Elmer AAnalyst 800). The analyses were performed in the accredited laboratory of the National Chemical-Agricultural Station in Warsaw, in accordance with the station's research procedure 27 (6th edition, dated 14 February 2011). The analysis of soil pH and electrical conductivity (EC, mS·cm −1 ) was carried out as follows: soil samples were treated with distilled water in a 1:2 ratio and then analyzed with the use of a pH Meter/Conductometer (inoLab, Conbest). In order to analyze organic matter (% of

Field and Laboratory Methods
Carabid beetles were collected using pitfall traps [25]. At each sampling plot, two traps were installed with a maximum distance of 3 m from each other. The traps were glass jars topped with a funnel (upper diameter of about 10 cm), set flush with the soil surface. A roof was suspended a few centimeters above the funnel and ethylene glycol was used as a killing agent and preservative. The traps were placed at least 15 m from the edge of the plots. However, this was not possible in every case, due to the shape of some plots (1-2, 1-3, 1-4, 2-3, 3-3, 4-1, 9-1, 12-1, 12-2, 13-2, 15-1, 17-5). Carabids were sampled from mid-May to late September in 2018. The traps were controlled for proper performance during some field visits, and exchanged in July.
Determination and nomenclature of the individuals collected were carried out in accordance with the literature [26]. All individuals were identified to the species level by the first author. Voucher specimens of each species were deposited in a collection of the first author at the Warsaw University of Life Sciences-SGGW. We characterized the species with respect to the functional traits, habitat preference, trophic specialization, and breeding type, based on the literature [11,[26][27][28][29][30][31].
Soil samples were picked with the use of an Egner stick, up to a depth of 20 cm. The soil was sampled in triplicates at each wasteland , with an area of 5000-10,000 m 2 . A single replicate was a pooled sample of 20-25 individual ones. To exclude any variation in the physical and chemical properties of the soil, the averages obtained for the whole area represented all plots within the given area. The soil samples for pH, electrical conductivity (EC), soil water content (SOC), and organic matter (Org) were collected in mid-June (Term I), July/August (Term II), and mid-September (Term III), while the determination of trace element concentrations occurred once in mid-September.
Soil samples were stored in a refrigerator prior to analysis for up to 24 h. A quantitative analysis of the content of selected trace elements (TE) (Cu, Zn, Pb, Cd, Cr, Ni) in the soil was carried out at the end of September 2018. The soil samples were mineralized in a mixture of concentrated HNO 3 and HClO 4 , and then the TE were analyzed by atomic absorption spectrometry (AAS) (Perkin Elmer AAnalyst 800). The analyses were performed in the accredited laboratory of the National Chemical-Agricultural Station in Warsaw, in accordance with the station's research procedure 27 (6th edition, dated 14 February 2011). The analysis of soil pH and electrical conductivity (EC, mS·cm −1 ) was carried out as follows: soil samples were treated with distilled water in a 1:2 ratio and then analyzed with the use of a pH Meter/Conductometer (inoLab, Conbest). In order to analyze organic matter (% of dry matter), the soil samples were heated in a preheated muffle furnace (P330, Nabertherm) for 5 h at 450 • C, and the organic matter content was calculated as a difference between the initial and final sample weight. The soil water content (SWC) (% w/w) was determined by the gravimetric method. The soil samples were dried in a convection oven at 105 • C, and water content was calculated as the ratio of water weight to the weight of air-dried soil.
The maximum permissible levels of trace elements in light soils (mg·dm −3 ) were taken from the Polish national regulation, the Decree of the Minister of Environment of 1 September 2016 [32].

Statistical Methods
For each sampling plot, the data of the two traps and the sampling periods were pooled and the total number of species was calculated. Since the collecting period differed between the sampling plots by some days, the number of individuals was adjusted to 100 trapping days per one trap (100 trap-days) for each sampling plot.
In the first step, the data were bias tested regarding the species numbers and numbers of individuals due to stages of succession. In order to do so, the species numbers and numbers of individuals (100 trap-days) were compared between the young stages of succession and the advanced ones. As normal distribution was rejected by Kolmogorov-Smirnov tests, we used a Mann-Whitney U test with IBM SPSS Statistics, version 25.
Canoco for Windows 4.56 [33,34] was used to carry out nonparametric multivariate regression analyses on species numbers and numbers of individuals, using the following soil parameters selected as independent variables: trace elements (TE mg·kg −1 dry matter: Pb, Cd, Ni, Cr, Cu, Zn), soil pH (pH I-Term I, pH II-Term II, pH III-Term III), electrical conductivity (EC I-Term I, EC II-Term II, EC III-Term III), organic matter (Org I-Term I, Org II-Term II, Org III-Term III), and soil water content (SWC I-Term I, SWC II-Term II, SWC III-Term III). The significance of the individual variables was tested using Monte Carlo permutation tests (unrestricted, 1999 permutations), first, for each variable separately in order to study the variance explained by each variable separately, and then using an automatic forward selection of variables (reduced model) in order to study additionally explained variance after already adding variables to the ordination model [34,35].
Canoco for Windows 4.56 was also used to conduct a constraint ordination analysis in order to determine the major pattern in variation in relationship with the environmental factors. Detrended Canonical Correspondence Analysis (DCCA) was first used to select the appropriate statistical model based on the longest gradient [35] and then Canonical Correspondence Analysis (CCA) was carried out. Dominance values (percentage share of the respective species in a sample) for the carabid species at the different sites were used. The analyses were performed using inter-sample distance scaling, Hill's scaling, and unweighted data for each of the species. Since dominance values were used, the data were not transformed.

Results
From the 56 sampling plots altogether, 3994 individual carabid beetles belonging to 76 species were collected (Table S1). Both numbers of species and numbers of individuals varied strongly among the sampling plots ( Table 1). The highest number of both species and individuals was collected in a plot with regenerative woodlands of Salicetum albo-fragilis with an advanced stage of succession (Sampling Plot 19-1). In contrast, no beetles were collected in a plot with regenerative woodland with Populus × canescens association and a regenerative woodland with Acer negundo association, both located in the same study area (Sampling Plots 16-1 and 16-2).
For both the numbers of species (Mann-Whitney U test, p = 0.735) and individuals per 100 trap-days (Mann-Whitney U test, p = 0.175) the Mann-Whitney U test did not reveal any significant difference, hence, a bias of the data with respect to succession stage could be neglected.
The pH of the soils ranged from 4.12 to 8.5, that is, slightly acidic to alkaline, while the salt concentration ranged from 0.08 to 6.90 mS·cm −1 (Appendix A, Table A1). In most wastelands, the salt concentration was relatively low and within the concentration range typical of urban soils in recreational locations (parks, lawns). Within several locations, a high variability of this parameter was noted. The highest electrical conductivity was recorded in the soils sampled from Plots 8-1 and 8-2.
The content of organic matter ranged between 0.44% and 19.61% dry matter. The lowest actual soil moisture (% w/w) was noted in Study Area 13, whereas the highest one in Study Area 8.
The obtained results of trace element concentrations (Appendix A, Table A1) have confirmed that the permissible limits of trace elements in the topsoil were exceeded in light soils of agricultural areas by Pb, Cu, and Zn, only in some wastelands. Concentrations of Pb were above the limits in Study Areas 16 and 12, Cu in Study Areas 11 and 12, and Zn in Study Areas 9 and 15. Trace concentrations of Pb were also found in Study Areas 1, 10, 19, and 22, while Cu was in Study Area 22. The least contaminated area among the studied wastelands was Study Area 10, where the only recorded trace concentrations were of Pb, Ni, and Cr. For the remaining three elements (Cd, Cu, Zn), the concentrations did not exceed the limits even for protected areas.
When testing the impact of the soil parameters on the number of carabid species separately, Pb and Cu had a significant impact. Using a forward selection of variables, significant results were received for Pb, Cd, Org III, and Cr (Table 2). Regarding the impact of the parameters on the number of collected individual carabid beetles, the variables separately tested revealed a significant impact of Pb. Using a forward selection of variables, besides Pb, the variables Cd and EC II had an additional significant impact (Table 3).  The first canonical axis of the CCA (Figure 2) explained 5.7% of the variation in species data and 16.4% of that in the species-environment relationship. The second canonical axis explained 3.9% and 11.1%, respectively. Almost all environmental factors pointed to the top left from the origin of the diagram, with the exception of EC I and EC II pointing to the top right, and Cr, SWC I, and SWC III pointing to the bottom left. Sampling plots that best fit into the ordination space located in the left from the origin are 15-1 (top left), and 14-2 and 2-1 (bottom left). All others are located to the right of the origin of the diagram. Species that best fit into the ordination space positively related to the environmental factors are Stomis pumicatus and Licinus depressus (Pb, Zn, EC III), Pterostichus melanarius (Cr, SWC I, SWC III) and Harpalus rufipes, Harpalus affinis, and Calathus melanocephapus (EC I, EC II). However, a majority of species that best fit into the ordination space is rather negatively related to the environmental factors (Calathus fuscipes, Calathus erratus, Syntomus truncatellus, Poecilus lepidus, Badister peltatus, Harpalus anxius).

Discussion
The results have confirmed our assumptions that numbers of species, as well as numbers of carabid beetle individuals, are negatively affected by increasing concentrations of trace elements in the soil. Particularly, the trace elements Pb, Cu, and Cd showed a statistically significant negative impact.
Our study has revealed some clear differences in soil parameters, including the trace elements. When it comes to the degree of soil pollution at an individual locality, historical aspects, as well as the present situation of the study areas and sampling plots, are crucial. For example, Study Areas 15 and 16, which showed high pollutions with respect to some trace elements, are closely located to railway tracks and a cement plant [36].
Carabid beetles are known to react to soil conditions [11], as also shown by a significant effect of organic matter (Org III) and electrical conductivity (EC II). Particularly, organic matter plays an important role, because the organic layer plays an essential role for many species, for instance, as a refugee after habitat disturbances, or as a place for foraging or laying eggs [37][38][39].
The effects of trace elements on carabid beetles observed by us correspond with the results obtained previously by other researchers [40][41][42]. They suggested that trace elements in the soil significantly and negatively affect carabids. Carabid beetles are one of the most intensively studied groups of epigeic arthropods regarding their relation to trace element accumulation and their use as a bioindication of environmental pollution. Responses of organisms to trace element pollution are greatly dependent on several factors, such as species, form and concentration of metals, time of exposure, and pH conditions, which are closely related to the bioavailability of metals [43,44]. Furthermore, Cu, Zn, and Mn are micronutrients needed for the growth of organisms, while Cd and Pb are harmful even in small amounts [45]. The indicatory potential of carabid beetles can manifest itself not only as an absence or presence of species, but also as altered physiological and/or morphological characteristics [46]. These authors had reported severe gut degeneration as a result of Cd, Ni, and Zn pollution; however, the contribution of these metals to the development of the symptom was different. Other studies showed a highly sensitive immune response to environmental pollution in the preimaginal stages [47] and reduced size of individuals collected in polluted areas [48].
How strongly an individual species is affected depends on its autecological characteristics. Undisputedly, feeding habits have an important impact on how much of the toxic substances are accumulated, as demonstrated by researchers [49], especially for Cd. Researchers [16] showed that autumn breeding species seemed to be more sensitive than spring breeders in the case of Zn and Pb pollution. Feeding behavior of the respective species may also be of importance [15]. In our study, however, the CCA (Figure 2) did not reveal a clear distinction with respect to the breeding type or trophic specialization of the species (Table S1). However, the degree of pollution in the respective area can be also relevant. Studies have indicated that in highly polluted habitats, carabid beetles showed a very high accumulation potential for Cd, Pb, and Zn, whereas in habitats of low pollution they were able to regulate the concentration of trace elements in their body via detoxification mechanisms.
In conclusion, carabid beetles as entomoremediators may be useful in the decontamination of soils extremely polluted by trace elements [50].
Even if we could reject differences between the study sites with respect to the stage of succession as a bias, other factors might have an influence on numbers of species and individuals, too. Some studies have proven a decrease in species numbers from rural to urban forested sites [51], and accordingly, the location of the sampling plots in the city space might have had some impact. The degree of anthropogenic pressure may also depend on the location of the plots. Toxic pollutants and other environmental factors often show interactions [18]. The combined effect of nickel and chlorpyrifos was temperature-dependent both in the adults and larvae of Pterostichus oblongopunctatus [52,53]. Since pitfall traps measure the activity density of the carabids, particularly, numbers of individuals may be also affected by factors influencing their mobility. For example, dense vegetation might impede the movements of carabid beetles ("Raumwiderstand") [54,55]. Locomotory activity also depends on the feeding state of the individuals [56].
Even if the numbers of species and individuals of carabid species in individual sites depend on a complex set of different biotic and abiotic environmental factors, our study has indicated the impact of soil contamination and is in accordance with previously published data. The results also corroborate the usability of carabid beetles as indicators for the environmental impact of soil pollution. Thus, the increased levels of soil pollutants negatively impact the studied areas by both degrading the soil quality itself and causing a reduction of carabid beetle species diversity. The results are of value when it comes to the use of urban wastelands in the context of sustainable city development. Some obvious tasks include solid assessments of contamination and ecological potential, since the reclamation of wastelands demands to take into account natural processes, plants, animals, physical factors, nutrients, and toxicities [57]. Future use of urban wastelands is going to be faced with trade-offs between the use for public interests (e.g., housing space) and ecological interests. Already in the 1990s, the idea of 'rotating wastelands' was discussed, that is, to maintain a certain number of wastelands by building on some wastelands only when new ones appear [58]. A different approach proposes a unifying framework for urban wilderness as a social-ecological system [9]. Researchers [59] raised awareness of the need for a more theoretically nuanced and historically grounded focus on the intersections between urban ecology and culture in the contemporary city. Other researchers [5] emphasized that urban wastelands, after not having been used for many years, have spontaneously generated their own social and natural values, which should be taken into consideration at different levels of planning. In order to increase the potential of the areas, the strategy of phytoremediation, that is, the use of plant species to combat alterations in environmental conditions, can be of interest [60]. Phytoremediation seems to be among the most effective measures in order to restore contaminated areas. As mentioned above, the carabid beetles themselves may even participate as entomoremediators. Yet, the results of studies, such as the one conducted by us, may help to select the respective wastelands for certain purposes.

Conflicts of Interest:
The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.