Life in a Contaminated Environment: How Soil Nematodes Can Indicate Long-Term Heavy-Metal Pollution

A combination of Cobb sieving and decanting (Cobb, 1918) and a modified Baermann technique (van Bezooijen, 2006) were used for extracting nematodes from 100 g of fresh soil in aqueous soil suspensions using a set of two cotton-propylene filters. One or two filter trays were used for each sample to prevent material from exceeding 0.5 cm in depth above the filter. Aqueous suspensions were removed after 48 hr of extraction at room temperature, and nematodes were counted under a stereomicroscope (40× magnification). Excessive water was removed, and the nematodes were fixed with a hot solution of 4% formaldehyde and 1% glycerol (Seinhorst, 1962). At least 100 nematodes were identified at the species level based on the original species descriptions and accessible taxonomic keys of nematode genera and groups using an Eclipse 90i light microscope (Nikon, Tokyo, Japan) at standard magnifications.

Nematode abundance in each sample was expressed as the number of individuals per 100 g of dry soil. Nematode genera were assigned to trophic groups as described by Yeates et al. (1993), adjusted and supplemented by following Sieriebriennikov et al. (2014), and assigned to 17 functional guilds integrating nematode feeding strategies (trophic groups) and the nematode colonizer-persister (c-p) scale (Bongers and Bongers, 1998). The six nematode trophic groups were: bacterivores (Ba), fungivores (Fu), predators (P), omnivores (Om), plant parasites (Pp), and insect parasites. The Pp group included both obligatory and facultative plant parasites that may attack plants or fungi. Colonizer-persister groups characterizing nematode life strategies are classified on a scale of 1 to 5 (Bongers, 1990). C-p1 represents “r-strategists” (colonizers), with short life cycles, small eggs, high fecundity, high colonization ability, and high tolerance to disturbance, eutrophication, and anoxybiosis. Colonizers generally live in ephemeral habitats. At the other end of the scale, c-p5 nematodes represent “k-strategists” (persisters), with the longest generation times, largest bodies, lowest fecundities, and the highest sensitivity to disturbance. Persisters are never dominant in a sample and generally live in stable habitats (Bongers, 1990). C-p scaling allows the calculation of the basal maturity index (MI) for non-parasitic nematodes, the plant-parasitic index (PPI) for plant parasites only (Bongers, 1990), and the MI2–5 MI (Yeates, 1994) for c-p2-5 nematode taxa. Functional guilds allow the calculation of the enrichment index (EI), the structure index (SI), the basal index (BI), and the channel index (CI) proposed by Ferris et al. (2001). The genus diversity index (H´gen) defined by Shannon and Weaver (1949) was also calculated. All indices (except H´gen) and total biomass were calculated using the NINJA online program (Sieriebriennikov et al., 2014); (https://shiny.wur.nl/ninja/), including graphic depictions of the conditions of the soil food webs, the “faunal profile” (Ferris et al., 2001), which was modeled separately for each sample, and the functional metabolic footprint (Ferris, 2010), which was modeled for each site.

The soil contents of hazardous elements in the area of our study were naturally higher than in other areas of the Czech Republic due to the specific composition of the parental rocks. The bedrocks consisted mainly of schists, sandstones, graywackes, and quartzes. These rocks were mostly covered in the alluvia by non-calcareous alluvial sediments. Minerals rich in potential risk elements include galenite (PbS), sphalerite (ZnS), boulangerite (Pb₅Sb₄S₁₁), and antimonite (Sb₂S₃) (Borůvka and Vacha, 2006). These minerals are consistent with the data obtained from two of the alluvial meadows in our study. As, Pb, and Zn contents were relatively high (exceeding the admissible limits of harmful substances in soils) at control site A 3.6 km upstream from the main source of pollution and at site E 13.3 km downstream from the source.

Surveys conducted in the last two decades (Vaněk et al., 2005; Žák et al., 2009; Dlouhá et al., 2013; Kotková et al., 2019; Kebonye et al., 2021) have indicated that the floodplain area of the Litavka River was highly polluted by As, Pb, and Zn, which agrees with our records. The soils of the alluvial meadows in the vicinity of the waste-sedimentation ponds and mine-waste heaps had considerably higher amounts of Zn, As, and Pb compared with the control site and with the site farthest from the sources of pollution. The soils at these highly polluted sites in our study were also strongly acidic, consistent with the findings reported by Kebonye et al. (2021).

Our assumption that the differences in the nematode communities between our surveyed meadows could be attributed to the effects of Zn, Pb, and As contamination and higher soil acidity was thus reasonable. Nisa et al. (2021) reported that nematode abundance decreased from alkaline to acidic soils at 10 sites along the Kashmir Valley, which is also supported by our results. The meadows in our study heavily contaminated by Pb, Zn, and As from anthropic activities had acidic soils and significantly fewer nematodes and lower abundances of most nematode trophic groups and functional guilds relative to the meadows with neutral soil pHs and lower contents of hazardous elements. Similar findings were reported by Park et al. (2011) from very acidic (pH 2.8) soils polluted by Pb and Zn around an abandoned mine in South Korea and by Pen-Mouratov et al. (2008) from the Almalyk mining and metallurgical complex in the Republic of Uzbekistan. The soils in the study by Pen Mouratov et al. (2008), however, were weakly alkaline (pH 8.1) and polluted by Cu, Pb, and As, in contrast with the results of Nisa et al. (2021) and our observations. Pb and Zn contents were not correlated with the total number of nematodes in neutral soils (pH 6.8–7.1) in the tailings of the Baoshan Pb/Zn mine in China (Shao et al., 2008). Extremely alkaline soils (pH 9.0) heavily contaminated by magnesium (Mg) from the processing of Mg ore also had total nematode abundances similar to those from neutral soils less polluted by Mg (pH 6.9–7.4) and farther from the source of pollution (Šalamún et al., 2014).

In our study, the Ba_1,2 genera Rhabditis, Mesorhabditis, Cephalobus, and Plectus; the Fu_2,4 genera Aphelenchoides and Dorylaimoides; the Om_4,5 genera Eudorylaimus, Mesodorylaimus, and Aporcelaimellus; and the P_4,5 genera Mylonchulus, Prionchulus, and Oxydirus were the most sensitive to higher soil contents of Pb, Zn, and As. In contrast, Pp₃ obligate plant parasitic nematodes did not appear to suffer from the effects of contamination by Pb, As, and Zn in the highly polluted meadows.

Our findings partially agree with those of previous investigations of the impacts of heavy metals on particular nematode taxa but contradict with some data. For example, Korthals et al. (2000) reported high sensitivities of Alaimus (Ba₄), Thonus (Om₄), and Aporcelaimellus (Om₅) and the tolerance of Aphelenchoides (Fu₂), Tylenchorhynchus, Rotylenchus, and Pratylenchus (Pp₃) to experimental additions of Cu and Zn to the soil. Smit et al. (2002) observed the dominance of Aphelenchoides and Mesodorylaimus (Om₄) in soils that received the highest amounts of Zn in comparison to lower amounts. Sánchez-Moreno et al. (2006) found that Alaimus, Boleodorus, and Tetylenchus (Pp₂); Criconemoides, Hemicycliophora, Heterodera, and Hoplolaimus (Pp₃); and Monhystera (Ba₂), Panagrolaimus (Ba₁), and Dorylaimus (Om₄) were the most sensitive to contamination with Pb, Cu, Zn, and Ni in an affected riparian zone 2 yr after a large mine spillage. Similarly, Pen-Mouratov et al. (2008) reported that the abundances of Chiloplacus, Plectus, Dorylaimus, Eudorylaimus, and Nygolaimus (P₅) and Aphelenchoides, Tylenchorhynchus, Pratylenchus, and Tylenchus (Pp₂) were negatively correlated with As, Cu, Zn, and Pb pollution. Rodríguez-Martín et al. (2014), however, reported that Aphelenchoides and Aphelenchus (Fu₂); Eudorylaimus and Ecumenicus (Om₄); Nygolaimus, Discolaimus, and Discolaimoides (P₄); and Helicotylenchus were tolerant to high Pb contents. These contradictory findings for the sensitivity vs. tolerance of particular nematode taxa to heavy-metal loads among previous studies and our findings were probably due to differences in the heavy metals and their combinations in the soils. The total contents of the elements measured in these studies were also highly variable, which may also have been an important factor contributing to the contradictory results. For example, iron (Fe) in a study by Martinez et al. (2018) was the main soil pollutant in a mining area in the southern Philippines (2,300–2,600 mg/ kg), where Pb and Zn contents were very low (20– 60 mg/kg). This study found that some genera of predacious and omnivorous nematodes were most abundant at sites with elevated heavy-metal contents, especially Fe, even though these nematodes were generally expected to be sensitive to both chemical pollution and physical disturbance (e.g., Ironus and Eudorylaimus). In contrast, the Pb and Zn contents in our study reached maxima of 4,030 mg/kg and 3,867 mg/kg, respectively, at the heavily polluted sites. The genera Eudorylaimus, Mesodorylaimus, and Aporcelaimellus (Om_4-5) were only sporadically present at the sites with lower amounts of Pb and Zn, suggesting that the Pb and Zn contents were negatively correlated with the abundances of these Om nematodes.

The lower abundances of the majority of the taxa, and the absence of several taxa, at the polluted sites than the control site indicated changes in the community structures and led to changes in the community indices. The abundances of these taxa were significantly lower at the most heavily polluted sites, and reductions of these indices were less pronounced with increasing distance from the sources of pollution. Our results are in accordance with the findings by Sánchez-Moreno and Navas (2007), Shao et al. (2008), Park et al. (2011), and Šalamún et al. (2014). These studies also found that heavy-metal pollution strongly affected the structures of the soil nematode communities, indicated by lower values of the maturity indices (MI and MI2–5) or SI. In contrast, EI in two of these studies (Park et al., 2011; Šalamún et al., 2014) did not differ significantly between polluted and unpolluted sites, suggesting that heavy-metal contamination may have disturbed the soil ecosystem by suppressing biological activity.

Our findings for obligate plant parasitic nematode taxa were consistent with the results reported by Shao et al. (2008) from mine tailings 12 yr after dwindling yields caused the cessation of Pb and Zn mining in 1996. This study also found that plant-feeding nematodes were not negatively affected by heavy-metal pollution and that their abundance increased with the recovery of vegetation at disturbed sites, consistent with the findings by Háněl (2004) from waste dumps of industrial pyrite and calcium. The abundance of plant-feeding nematodes is mostly determined by community structure, biomass, and the vigor of plants (Bongers and Ferris, 1999). The sites investigated in our study were typical alluvial meadows with no physical injuries and dominated by various species of grasses and herbs, suggesting that the effects of heavy metals on plant-feeding nematodes can be mitigated by the presence of plants with rich root systems that serve as sources of food. In contrast, Park et al. (2011) reported that the abundance of plant-feeding nematodes was considerably lower at sites contaminated with heavy metals than at uncontaminated sites; the species of plants at the study sites differed little, but their growth was poorer at the contaminated than the uncontaminated sites.

The relatively large number of nematode taxa recorded in the alluvial meadows of the Litavka River demonstrated the capacity of local soils to harbor diverse nematode fauna in this area. Some of these soils, however, have been affected by heavy metals from ponds that sediment mine waste and from waste heaps, spread repeatedly by contaminated flood waters, and our analyses indicated that these pollutants have adversely affected the community of soil nematodes at the target sites. Pb, Zn, As and, to a lesser extent, Cu were the main pollutants responsible for the observed impact. Nematode abundance, number of taxa, and consequently nematode biomass, as well as the trophic complexity of the nematode communities, were significantly reduced at the heavily polluted sites. Interestingly, the abundances of all functional guilds among the bacterivores (Ba_1,2,3,4), predators (P_3,5), omnivores (Om₄,₅), plant parasites (Pp₂), and fungivores (Fu_2,4) were sensitive to heavy metals, and the abundance of Pp₃ nematodes did not indicate heavy-metal contamination. H´gen, MI, MI2–5, EI, SI, and BI were the most valuable community indices for assessing the impact of heavy metals. Our results thus support the general view that communities of soil nematodes provide highly responsive metrics and sensitivities as bioindicators of heavy metal pollution in the environment.