Soil contamination with toxic substances is a severe and persistent threat to terrestrial ecosystems. Springtails (Collembola) are widely used as bioindicators of soil health. Along a pronounced pollution gradient near the Middle Ural Copper Smelter in Russia, the springtail Parisotoma notabilis maintains high population density, even in heavily contaminated areas. We hypothesized that this resistance is facilitated by the occupation of distinct parts of the gradient by different intraspecific genetic lineages. From springtail samples collected across the pollution gradient, molecular analysis of the 28S rRNA gene identified four lineages: L1, L2, L4-Hebert, and a newly discovered lineage designated L-East. Their distributions differed significantly along the pollution gradient: L1 dominated the most heavily polluted area, L2 was restricted to the uncontaminated forest, and L4-Hebert and L-East occurred throughout the gradient. Our findings demonstrate that a comprehensive understanding of biotic responses to toxic stress will require a shift from the species level to the intraspecific genetic level, and emphasize the critical importance of accounting for intraspecific diversity in standardizing ecotoxicological bioassays. These findings also highlight that the use of genetically heterogeneous laboratory cultures may yield unrepresentative or inconsistent results. Therefore, the development of new standards mandating the use of certified, specific genetic lineages of test organisms is advisable. For environmental monitoring, P. notabilis, particularly its L1 lineage, shows high potential, as an increase in L1 frequency reliably indicates a decline in soil health.

Despite its potential significance for male fertility, bilateral testicular asymmetry in vertebrates remains poorly understood. Fluctuating asymmetry (FA), which serves as an indicator of stochastic developmental variation, constitutes a crucial yet frequently overlooked aspect of phenotypic variability. Although FA is commonly examined in morphological traits, its measurement in functional testicular traits has not been previously conducted due to methodological constraints. We analyzed FA in epididymal sperm motility (curvilinear velocity, VCL) and sperm concentration (SEC) in reproductively mature male bank voles (Clethrionomys glareolus, n = 61) from wild populations using Computer-Aided Sperm Analysis (CASA). Parallel measurements of "soft" (testis and kidney mass) and "hard" (mandibular dimensions) morphological traits were performed for comparison. CASA showed reliable reproducibility (VCL: 0.89; SEC: 0.73), enabling accurate FA quantification and confirming the absence of directional asymmetry or antisymmetry. Functional traits exhibited remarkably high FA values: 12% of the mean trait value for VCL and 30% for SEC, which is an order of magnitude greater than that of morphological traits. FA accounted for one-quarter (VCL) to one-half (SEC) of the total variation, representing two to three times the corresponding proportions of morphological traits. These elevated FA levels suggest that within-individual left/right differences may surpass between-individual variation. Our findings underscore the importance of (1) conducting repeated bilateral measurements for accurate FA quantification in testicular function and (2) utilizing averaged data from both testes for population-level analyses. These findings have significant practical implications for physiological and ecotoxicological research using rodent models, in which epididymal sperm analysis is standard practice, highlighting the necessity of considering functional asymmetry to prevent biased conclusions.

Ecotoxicological studies predominantly rely on artificially contaminated soils and fieldwork on contaminated soils remains scarce. This study focuses on the Kargaly site in the Orenburg region near the southern Urals, where a rare instance of monometallic soil pollution has occurred with copper (Cu). We established Cu toxicity thresholds for Dendrobaena veneta, a European nightcrawler, using soils collected along a Cu toxicity gradient (total Cu content of 121-10,200 mg kg-1) in a chernozem (Mollisol) agricultural field. Earthworm survival in the reproduction bioassay was an unreliable predictor of Cu toxicity. However, the number of juveniles in the reproduction bioassay and earthworm avoidance behavior were sensitive indicators of Cu toxicity. While total soil Cu strongly predicted earthworm responses, the effect of soluble (0.01 M CaCl2-extractable) Cu on earthworm responses was not statistically significant. Similarly, the Cu content in earthworm tissues was an unreliable predictor of Cu toxicity in D. veneta. The effect concentrations at 25% (EC25) and 50% (EC50) of total soil Cu for earthworms were 177 and 407 mg kg-1, respectively, for the reproduction bioassay, compared with 783 and 1,603 mg kg-1, for earthworm avoidance behavior. This study is among the few that estimate Cu toxicity thresholds for earthworms in real-world contaminated soils rather than artificially spiked ones. This is the first report of the Cu toxicity threshold for the genus Dendrobaena, highlighting the novelty of this study.

Ecotoxicological research often relies predominantly on artificially contaminated soils, with studies on real-world contaminated soils remaining scarce. This study focuses on the Kargaly site in the Orenburg region of Russia, a rare instance of monometallic soil pollution by copper (Cu). The similarity of other elements in the soil samples to background levels highlights the uniqueness of this monometallic contamination. We established Cu toxicity thresholds for soil microorganisms and earthworms using soils collected along a Cu toxicity gradient in a chernozem (Mollisol) agricultural field. The total soil Cu predicted earthworm responses as effectively as did 0.01 M CaCl2-extractable Cu. While total soil Cu strongly predicted microbiological responses, 0.01 M CaCl2-extractable Cu was a poor predictor for microorganisms. The effective concentrations of total soil Cu for earthworms at 25% (EC25) and 50% (EC50) were 480 and 1005 mg kg-1, respectively, compared to 4,570 and 7,797 mg kg-1 for microorganisms, respectively. Similarly, the EC25 and EC50 of 0.01 M CaCl2-extractable Cu were 14 and 46 mu g L-1 for earthworms, respectively, although 0.01 M CaCl2-extractable Cu did not predict microbial toxicity well. Overall, earthworms were more sensitive to Cu than were microorganisms. This study is among the few that estimate Cu toxicity thresholds in real-world contaminated soils rather than artificially spiked soils.

This review focuses on the issue of metal antagonism in soils contaminated by multiple metals as a result of industrial emissions. Building upon previous findings in aquatic ecosystems, the potential of zinc to mitigate copper toxicity in more complex soil systems is explored. A range of studies investigating the role of zinc in reducing copper toxicity to plants and microorganisms in soils contaminated by copper mining in central Chile are examined. The mechanisms underlying metal interactions in soils, including the terrestrial biotic ligand model and the intensity/capacity/quantity concept, are thoroughly discussed. Furthermore, the review underscores the pressing need for future studies to enhance our understanding and develop effective strategies for mitigating copper toxicity in industrially contaminated soils.

The bait-lamina test (BLT) is a widely used method for assessing the feeding activity of soil detritivores. However, one of its limitations is the ambiguity in the interpretation of results, as it is difficult to determine which taxa or size groups consumed the bait. To address this issue, we propose a modification of the BLT called the differential BLT, which allows for the estimation of the contribution of macrofauna to the total feeding activity of soil detritivores. In the differential BLT, the size of the holes is reduced to prevent bait ingestion by macrofauna. The values obtained from the large holes represent the bait consumption by all groups, while the difference between values obtained from large and small holes is attributed to macrofauna alone. Through a laboratory experiment with defaunated soil subsequently inoculated with earthworms and using the strips with various hole sizes, we identified the threshold aperture size of 0.2-0.3 mm that excludes earthworms from the bait. In an experiment with laboratory-cultured enchytraeids, we demonstrate that these aperture sizes do not prevent bait consumption by mesofauna. We also conducted a field experiment in the southern taiga coniferous forests, where we verified the applicability of the differential BLT in situ and estimated that macrofauna contributes two-thirds to the total feeding activity. The partitioning of the total feeding activity of soil detritivores using the differential BLT can be a valuable tool for soil ecologists to assess the functioning of below-ground animal communities.

We have tested the hypotheses that the satyrs Aphantopus hyperantus and Coenonympha arcania accumulate metals in higher concentrations near the Middle Ural Copper Smelter than in the background area and that metal accumulation in the body of adult individuals is negatively correlated with the wing length but positively correlated with the fluctuating asymmetry of the wing length. We measured the length of the forewing and individual concentrations of Zn, Cu, Pb, and Cd in the body of adults captured at different distances from the copper smelter (Revda city, Russia). The metal content reaches very high levels, with Zn concentrations being higher than Cu and Pb concentrations by an order of magnitude and Cd concentration by two orders of magnitude. In both species, metal accumulation is higher in males than in females. Maximum concentrations of Zn, Cu, and Cd have been recorded near the smelter. The wing length either did not differ between the sites or was higher near the smelter. The statistically significant negative relationship between Cu concentrations and the wing length has been recorded only for females of one of the species ( A. hyperantus ). In both species, the fluctuating asymmetry of the wing length did not differ between samples from different sites and did not depend on metal concentrations at the individual level.

The effect of individual trees on soil and litter respiration in forests polluted with heavy metals from copper smelter emissions was investigated for the first time. We tested the hypothesis that the portion of spatial variability in soil respiration explained by the distance from the tree trunk decreases on polluted plots in comparison with the background area. The study was performed in the southern taiga spruce–fir and birch forests under long-term pollution from the Middle Ural Copper Smelter in the Revda City, Sverdlovsk region, Russia. Measurement points were located near spruce and birch trees at different distances from tree trunks (tree-base plot, the middle of the crown projection, and canopy gap). The total CO2 emission, litter respiration, litter contribution to soil respiration, specific respiratory activity of litter, and litter stock were measured at each point. In the background area, soil respiration decreased from the tree trunk to the canopy gap. The hypothesis was only partially confirmed: the portion of respiration variance explained by the distance from tree trunks decreased in polluted areas in comparison with the background areas in spruce forests but did not change in birch forests. The observed change in spruce forests was related to a drop in specific respiratory activity of litter, though litter stock was considerably higher near the tree trunk than in the canopy gap. We propose to locate measurement points in the middle of the crown projection, i.e., at a sufficient distance from tree trunks and away from the canopy gaps, to reduce potential bias in soil respiration estimates.

ABSTRACT A significant portion of the current knowledge regarding the use of iron nanoparticles for remediating metal-contaminated soils is derived from laboratory experiments, leaving several unanswered questions. This article presents a field experiment comparing the efficacy of magnetite nanoparticles and microparticles for the immobilization of metals and the growth of plants in metal-contaminated soils. This study aimed to investigate the effects of magnetite particle size on metal immobilization and plant growth in soils exposed to airborne pollution from the Middle-Urals Copper Smelter in the southern taiga subzone near Revda, Russia, 50 km from Ekaterinburg. Magnetite nano- and microparticles were added to forest litter at a 4 % w/w dose. The total metal contents in litter from the study plots were 1-2 orders of magnitude higher than background metal concentrations. The magnetite nanoparticle treatment was found to decrease the concentration of exchangeable copper in soil and improve the growth of red fescue (Festuca rubra L.) on polluted soil compared to the control. In contrast, magnetite microparticles did not show any statistically significant effects. These findings are in line with laboratory results that demonstrated the superior metal adsorption properties of magnetite nanoparticles compared to microparticles. However, this study was limited in duration (2 months), and longer field studies would be necessary to confirm the role of iron particle size in the rehabilitation of metal-contaminated soils.

During soil recolonization by macrofauna in areas previously defaunated by industrial pollution, non-typical humus forms are produced. Given that the evidence of zoogenic activity cessation with increased forest litter depth in these humus forms, we tested the hypothesis that the lower organic layers are more toxic than the upper ones. The studies were conducted in the southern taiga, near the Middle Ural Copper Smelter (Revda city, Russia), in spruce-fir and birch forests. We investigated the series of degraded humus forms at different recovery stages, including those without signs of regradation, as well as at the initial and advanced recovery stages. In the organic layers, each of which were 1-2 cm thick and 6-8 cm in total, we measured the following parameters: pH(water), total acidity, the content of exchangeable Ca2+ and Mg2+, acid-soluble and exchangeable metals (Cu, Pb, Fe, Cd, and Zn), organic carbon, and total nitrogen. Simultaneously, we diagnosed the degree of zoogenicity of the organic layers following the European morpho-functional classification of humus forms. Concentrations of the metals increased with forest litter depth, reaching a maximum at the boundary between the organic and organic-mineral horizons (the difference exceeded an order of magnitude). In the same direction, the acidity increased, but the saturation of the exchange complex with Ca2+ and Mg2+ decreased. Within a particular forest litter profile, metal concentrations and acidity were lower in the layer with the highest zoogenicity compared to the layer with the lowest zoogenicity. Based on the metals, pH(water), and exchange complex, the accuracy of the predictions of the degree of layer zoogenicity within the OF horizon in the discriminant analysis reached 100 %. These findings suggest that the vertical gradient of toxic burden persisting in the forest litter after pollution cessation can explain the recovery pattern of humus forms in the contaminated areas.

Since the late 1980s, long-term monitoring of terrestrial ecosystems in metal-contaminated areas near the Middle Ural Copper Smelter has been carried out in the Central Urals. As a part of these monitoring programmes, the data on species diversity, community composition and abundance of soil macroinvertebrates continue to be gathered. The dataset (available from the GBIF network at https://www.gbif.org/dataset/61e92984-382b-4158-be6b-e391c7ed5a64) includes a 2004 census for soil macroinvertebrates of spruce-fir forests along a pollution gradient in the Central Urals. The dataset describes soil macrofauna’s abundance (the number of individuals per sample, i.e. the density) and community structure (list of supraspecific taxa, list of species for most abundant taxa and supraspecific taxa or species abundance). Seventeen sampling plots differed in the levels of toxic metal (Cu, Zn, Pb, Cd and Fe) soil contamination from air emissions of the Middle Ural Copper Smelter (heavily polluted, moderately polluted and unpolluted areas). The dataset consists of 340 sampling events (= samples corresponding to upper and lower layers of the 170 soil monoliths) and 64658 rows (2907 and 61751 for non-zero and zero density of taxa, respectively). Arachnida (Araneae and Opiliones), Carabidae (imagoes), Elateridae (larvae), Chilopoda, Diplopoda, Gastropoda, Staphylinidae (imagoes) and Lumbricidae were identified to species level. In contrast, Mermithida, Enchytraeidae, Lepidoptera larvae, Diptera larvae, Hemiptera, Hymenoptera and some other insects were identified to family or order levels. In total, 8430 individuals of soil macroinvertebrates were collected in two soil layers (organic and organic-mineral horizons), including 1046 Arachnida (spiders and harvestmen), 45 Carabidae, 300 Elateridae, 529 Myriapoda, 741 Gastropoda, 437 Staphylinidae, 623 Lumbricidae and 4709 other invertebrates. The presence-absence data on each taxon are provided for each sampling event. An overwhelming majority of such absences can be interpreted as “pseudo-absences” at the scale of sampling plots or study sites. The dataset contains information helpful for long-term ecotoxicological monitoring of forest ecosystems and contributes to studying soil macrofauna diversity in the Urals.

In recent decades, atmospheric emissions from industrial enterprises have been reduced in many countries, which makes it possible to analyze the patterns of ecosystem recovery. The review provides an annotated list of studies of natural recovery (i.e., without involving any reclamation measures) of terrestrial ecosystems near industrial plants that have ceased or significantly reduced their emissions. Seventy-three studies of biota recovery (70 publications) performed near 22 plants (mainly metallurgical ones) have been identified; other 18 and 14 studies deal with analysis based on repeated records of the dynamics of the content of pollutants in plants and animals and in soils, respectively. Numerous gaps in the knowledge of natural recovery have been revealed: uneven study of different biomes and ecosystem types, fragmentariness (absence) of data on many taxa, the prevailing number of single-component studies within a specific area, and dominance of relatively short observation series with a small number of time points. These gaps make it so far impossible to generalize data on a global scale. Shortcomings in the presentation of results in publications (incomplete data on the dynamics of emissions and dates of material collection) also make it difficult to generalize data.

The bait-lamina test (BLT) is widely adopted for assessing the feeding activity of soil detritivores. The downside of the BLT is that it leads to ambiguity in the interpretation of results; it is unclear which taxa or, at least, which size groups consumed the bait. We propose a novel differential BLT to partition the individual contribution of macro-detritivores (earthworms, dipteran larvae, millipedes, woodlice, and mollusks) and meso-detritivores (enchytraeids, collembolans, and oribatids) to the total feeding activity. The main idea of the differential BLT is alternating large and small apertures within the same strip. The miniaturization of the holes mechanically prevents bait ingestion by macrofauna. Thus, the bait losses from small holes estimate only the mesofauna feeding activity, while the values in large ones represent the bait consumption by both groups. The effects of competition between macrofauna and mesofauna were negligible, therefore, the difference between values for large and small holes is attributed to macrofauna alone. In the laboratory experiment with defaunated soil subsequently inoculated with earthworms, and using the strips with various hole sizes, we identified the threshold aperture size (<0.4 mm) to exclude earthworms from the bait. In the experiment with laboratory cultured enchytraeids, we show these aperture sizes did not prevent bait consumption by meso-detritivores. In the field experiment in the southern taiga coniferous forests, we verified the applicability of the novel differential BLT in situ and estimated the contribution of macrofauna to the total feeding activity as two-thirds. After testing many variants, we propose the following modifications to strips: add three 0.3 mm width rectangle apertures to the left, right, and top of each standard round hole. Differential BLT allows researchers to partition the macro- and mesofauna contributions to the total feeding activity of soil detritivores, providing a valuable tool for soil ecologists performing assays of below-ground invertebrate communities.

The potential use of zero-valent iron (ZVI) nanoparticles (i.e., <100 nm in size) for the remediation of metal contaminated soils has sparked a flurry of research in recent years. However, even reading a large number of these papers cannot completely dispel doubts that ZVI nanoparticles are indeed superior to ZVI microparticles (e. g., iron powder or grit) in immobilizing metals and metalloids in soils. Our primary objective was to compare the adsorption properties of iron-based amendments (ZVI micro-and nanoparticles, natural iron oxides) supplied in a biochar matrix in soils contaminated by a copper-nickel (Cu/Ni) smelter on the Kola Peninsula in Russia. The following iron-containing amendments were added to the studied soil: a composite of ZVI nanoparticles and biochar (synthesized by pyrolysis of iron-impregnated biochar), a mixture of iron powder (i.e., ZVI microparticles) with biochar, and a mixture of iron oxides (from natural ferromanganese nodules) with biochar. Perennial ryegrass (Lolium perenne L.) was grown in pots on untreated and amended soils for 21 days under laboratory conditions. In our time-limited study, ZVI nanoparticles did not prove superior to ZVI microparticles or natural iron oxides at immobilizing metals in copper-and nickel-contaminated soil. In other words, ZVI particles size was irrelevant under the experimental setup of this study in its effects on exchangeable metal concentrations, foliar elemental concentrations, and plant growth.

The effect of toxic metals on roots is mainly studied in laboratory single-species experiments. Data for multi-species plant communities obtained in natural conditions are needed to understand ecosystem functioning under pollution. However, information on the industrial emission effects on the below-ground biomass is fragmentary and contradictory. This study aims to analyze the fine and coarse root mass changes along strong pollution gradients. We hypothesize that long-term soil contamination from copper smelter emissions decreases root mass in forests. We assessed the root mass in the forest litter and upper mineral soil layer along two pollution gradients caused by emissions from two copper smelters: in the Middle Urals (coniferous forests) and the Southern Urals (deciduous forests). We divided roots into two diameter fractions (0.5–2.0 mm and 2.1–5.0 mm). Only the fine root mass in the mineral soil in deciduous forests, but not the total root mass, decreased 2.2-fold near the smelter compared to uncontaminated areas. However, this effect is much weaker than for above-ground biomass, and it does not manifest at all in coniferous forests. The percentage of roots localized in the forest litter was negligible (7–10% in the coniferous forests and 2–5% in the deciduous forests) and remained unchanged along the pollution gradients. The absence of a pronounced effect of metal contamination from copper smelter emissions on the fine and coarse root mass may be regarded as evidence of plant communities’ resistance to long-term pollution through a shift in species composition toward tolerant species with high below-ground phytomass, particularly grasses.

The bait-lamina test is one of the few available methods for measuring the functional activity of soil animals, which was standardized for soil health assessment by ISO 18311. The bait consumption is measured visually on discrete scales. A two-point scale (0 or 1) is used more often, but the threshold for bait consumption scores varies from study to study: 1 point is assigned either to a hole that has been perforated to any extent, or to a hole that has been at least half perforated, or to a fully empty hole. Less often, a three-point scale (0, 0.5, and 1) or a five-point scale (0, 0.25, 0.5, 0.75, and 1) can be used. We investigated how much the use of different scales can influence statistical inferences. We have established that a point on a five-point scale is directly proportional to the proportion of the mass consumption of bait, so in this scale the value of feeding activity is an acceptable surrogate for the bait consumption rate. We analyzed outcomes of the bait-lamina test for 7 measurement rounds at the control area and an area highly polluted with metals near the copper smelter. We assumed the most accurate five-point scale to be the reference and compared it to six less precise scales with fewer points (two three-point and four two-point scales). The difference between the reference and such rough scales depends on the percentage of intermediate points (0.25, 0.5, and 0.75). It turned out that intermediate values are by no means rare (from 11% to 100% of all non-zero values), and in the polluted area they are almost twice as common as in the control area (66% and 28%, respectively). Because of this, the relative difference between the reference scale and rough scales is 2–4 times greater at the polluted site than at the control site. This can lead to a bias in the effect size index: for the rough scales, the log Response Ratio can either double or decrease by one-third relative to the reference scale. This increases the probability of type I and II errors in statistical hypothesis testing. Thus, the outcomes of the bait-lamina test are not invariant relative to the measuring procedure. The three-point scale and ISO 18311 two-point scale are the least biased relative to the reference scale. The use of other rough scales carries a risk of artifacts and should be avoided.

Scientists around the world have long been searching for effective strategies to reduce the bioavailability of metals in contaminated soils. In case of metal-spiked soils, some studies have proposed gypsum as a soil amendment to alleviate metal phytotoxicity. However, for real field-collected soils, evidence on the efficacy of gypsum as a metal phytotoxicity amendment is limited. Therefore, the present study was designed to examine the effect of gypsum on plant growth in soils polluted by a copper smelter. We grew perennial ryegrass on untreated and gypsum-treated soils (at a dose of 3% by weight) under laboratory conditions. We found that gypsum had no effect on alleviating metal phytotoxicity in our soils. We also demonstrated - for the first time - that gypsum increased the concentrations of soluble metals in the soil, enhancing metal uptake by plants. The calcium ions from gypsum displace metals in the soil exchangeable complex; however, the metals do not get immobilized in soils because gypsum is a neutral salt. While our results contrast with the Terrestrial Biotic Ligand Model, that Model has never been tested on real industrially polluted soils but only on metal-spiked soils. Our main conclusion is that gypsum is ineffective in alleviating metal phytotoxicity in real industrially polluted soils and, moreover, its use is inappropriate as a soil remediation method, because it increases the environmental hazard rather than reducing it. Our study is the very first attempt to recognize that gypsum is a hazardous material when used to ameliorate soils polluted by metals.

Chemically contaminated soils are not included in the European morpho-functional classification of humus systems and forms. However, they differ from the uncontaminated analogs in biological, chemical, and physical features. We investigated the topsoils contaminated by long-term sulfur dioxide and metal emissions from a copper smelter (southern taiga; conifer and deciduous forests; the Middle Urals, Russia). The smelter has been in operation for more than 80 years, but ten years ago, emissions almost ceased which initiated the recovery of adjacent ecosystems. We performed 1,155 humus profile descriptions on 231 sampling plots in highly contaminated, moderately contaminated, and background sites. In the contaminated sites, we found 21 non-typical humus forms that do not fit the European classification. Earthworms’ extinction caused by soil toxicity resulted in a Mull-to-Mor transformation of humus profiles. Recent recolonization of poorly decomposed litter by earthworms and other soil macrodetritivores after emission cessation triggered a Mor-to-Mull shift. Because different topsoil layers react to environmental changes with unequal rates, signs of several stages of humus evolution can be imprinted in one profile. More rapid degradation of organic horizons than organic-mineral ones results in non-typical Mor forms when the thick non-zoogenic OF combines with zoogenic A horizon. Zoogenic O horizons’ heterogeneity due to non-zoogenic inclusions, or inversion of the litter layers sequence, or lagging of A horizon recovery behind the O horizons, represents the discrepancies between non-typical regraded and typical humus forms. Mor-to-Mull recovery can occur with and without passing the Moder forms. In the first path, all intermediate stages at least fitted to one of the known humus systems. In the second path, intermediate humus forms are represented by non-zoogenic OF conjoining highly zoogenic OF and lacking OH horizon (we named such forms Mormull). In both paths, the recovery starts from the upper litter layer and spreads downward, i.e., the signs of the zoogenic activity change in the opposite direction than within the natural Mor profile. We consider the non-typical humus forms as non-equilibrium topsoil states that are ephemeral on the forest succession scale. We described non-typical humus forms in detail, proposed their evolutionary diagram, nomenclature, and preliminary typology, and extended the Humus Index with non-typical forms.