**3. Direct toxic effects of industrial emissions – A case study at Kovohuty JSC Krompachy**

#### **3.1. Results**

**Trace elements**

28 Emerging Pollutants in the Environment - Current and Further Implications

Difference Test (P<0.05)

**2.3. Data analysis**

permutation [25].

the results from these two areas separately.

**Sampling site K1 K2 K3 K4 Limit**

*Total concentration (HNO3 extraction) Arsenic (As)* 78.654±15.287a 19.574±5.402b 24.377±5.685b 5.609±1.306c 5 *Cadmium (Cd)* 1.494±0.181a 1.061±0.478b 0.339±0.062c 0.161±0.013c 0.3 *Chrome (Cr)* 5.781±7.019a 1.458±0.159a 1.475±0.206a 1.012±0.021a 10 *Copper (Cu)* 257.643±38.452a 175.398±80.216b 65.796±14.021c 18.322±2.129c 20 *Nickel (Ni)* 2.617±0.262b 3.598±0.349a 0.627±0.314c 1.021±0.027c 10 *Lead (Pb)* 25.140±8.380a 29.038±11.551a 8.216±3.761b 5.195±0.089b 30 *Zinc (Zn)* 80.62±18.019a 60.253±29.127a 8.067±4.449b 3.201±0.290b 40

*Mobilisable fraction (Na2EDTA extraction)*

abcd Means followed by the same letters on the same rows are not statistically different according to Least Significant

Limit - limits posted by The Decree of the Ministry of Land Management of the Slovak Republic No. 531/1994-540 on the

Spearman's nonparametric correlation coefficient (rs) was calculated using STATISTICA v. 9.0 to test the relationships between the characteristics of a nematode community and the concentrations of mobilisable heavy metals at a site [25]. Correlations at *P*<0.05 and *P*<0.01 were considered significant. Differences in the mean traits and indices of a community amongst sites were tested by Duncan's tests. We used a constrained ordination redundancy analysis (RDA) in CANOCO 5 to analyse the ecological distances between sites (nematode community and soil parameters). The significance of an axis was tested by Monte Carlo

The effects of contamination on soil ecosystems can be categorised as direct or indirect. Alterations in the soil communities near Kovohuty JSC Krompachy were likely due to direct toxicity from the high levels of heavy metals in the soil samples. The contamination acted mostly indirectly near SMZ JSC, altering the basic soil properties. We will present and discuss

**Table 2.** Total and mobilisable concentration of trace elements in sampling sites from Krompachy (mg.kg-1) [1].

admissible values of harmful substances in uncontaminated soil. n.a. – not available

*Arsenic (As)* 4.779±0.688a 2.870±0.690b 1.074±0.494c 0.105±0.021d n.a. *Cadmium (Cd)* 0.407±0.101a 0.266±0.097b 0.066±0.016c 0.023±0.003c n.a. *Chrome (Cr)* 0.081±0.02a 0.065±0.031a 0.073±0.036a 0.010±0.003b n.a. *Copper (Cu)* 76.660±23.554a 46.945±18.585b 14.320±3.788c 4.380±0.011c n.a. *Nickel (Ni)* 0.54±0.115a 0.568±0.112a 0.167±0.197b 0.101±0.008b n.a. *Lead (Pb)* 18.565±2.744a 16.293±7.951a 5.413±1.681b 4.055±0.337b n.a. *Zinc (Zn)* 63.285±17.193a 26.945±12.502b 3.600±1.377c 0.643±0.085c n.a.

#### *3.1.1. Heavy metals*

The total heavy metal content (except Pb and Ni) along the Krompachy transect was highest at the K1 site and decreased downwind (Table 2). The patterns of the decreases in concentra‐ tion, however, varied amongst the metals. As, Cd, and Cu decreased significantly (*P*<0.05) and relatively continuously towards K4. The concentrations of Ni, Pb, and Zn were relatively high at sites K1 and K2 but were significantly lower at K3 (*P*<0.05). Cr content did not vary significantly (*P*>0.05) along the transect. The mobile fractions of all metals had similar de‐ creasing trends (Table 2). As, Cd, Cu, and Zn had high total concentrations, exceeding the limits for uncontaminated soils in Slovakia [26] (Table 2). Corg and pH were likely the most important factors influencing the mobility of the metals, and the RDA (Figure 2) indicated that they were negatively correlated with the concentrations of all heavy metals except Cr.

#### *3.1.2. Composition of the nematode fauna: trophic and c-p groups*

A total of 58 genera were identified, including 20 bacterivores, five fungivores, 10 omnivores, eight predators, and 15 plant feeders (Table 3). The most common genera in the communities were *Acrobeloides*, *Aphelenchoides*, *Geocenamus*, *Helicotylenchus*, *Paratylenchus*, *Pratylenchus*, and *Rhabditis* (Table 3). The mean number of genera (17.75) in the replicates was lowest near the industrial plant (K1), which also had the lowest mean population density of 386.5 per 100 g of soil (Table 4). Both parameters increased with distance from the plant to 35.75 and 833.25, respectively, at K4. Ninorg. was significantly correlated (*P*<0.01) with generic richness, and soil pH (*P*<0.01) and Ninorg. (*P*<0.05) were significantly correlated with nematode abundance.

The industrial complex affected the densities of the trophic groups. The trophic structure differed at each sampling site (Table 4). Plant feeders were the most abundant trophic group, with a proportion in the community near 66% at the most contaminated site, K1. Bacterivores were the second most common group, representing more than 50% of the total nematode populations at K2 and K3. This group was positively correlated with Corg and pH (*P*<0.01) (Table 5). Fungivores, considered relatively insensitive to disturbances, including heavy metal pollution, occurred in relatively low densities, except at K2 with 8.98% of the total population. Omnivores and predators were most affected by the pollutants, with very low densities at K1- K3. The low resistance of these two groups to disturbances is in agreement with their negative correlation (*P*<0.01) with Cr (predators) or with all heavy metals (omnivores). Both trophic groups were also negatively correlated with Ninorg., and soil moisture (Table 5). The distribution of the c-p groups near the plant was consistent with their life strategies and survival abilities. General opportunistic nematodes, c-p 2, were most common at the most polluted sites (K1- K3), followed by c-p 1. The other three c-p groups represented only ca. 10% of the identified nematodes. The numbers of c-p 4 and 5 nematodes were significantly higher at the most remote site, K4, where representatives of c-p 4 were the most abundant within the nematode com‐ munity (Table 4).

#### *3.1.3. Ecological indices*

Mean H′ and generic richness were highest at K4, indicating the richest composition of nematode taxa (Table 4). MI2-5 and SI, measures of ecosystemic maturity, showed analogous development (Figure 2), with stable values from K1 towards K3 and steep increases at K4 (Table 4). EI values indicated that K2 stored the highest amounts of organic matter available for degradation, but K1 had the highest rate of degradation, as indicated by the lowest CI value (*P*<0.05) (Table 4).



*3.1.2. Composition of the nematode fauna: trophic and c-p groups*

30 Emerging Pollutants in the Environment - Current and Further Implications

munity (Table 4).

(*P*<0.05) (Table 4).

**Nematode genera**

*3.1.3. Ecological indices*

A total of 58 genera were identified, including 20 bacterivores, five fungivores, 10 omnivores, eight predators, and 15 plant feeders (Table 3). The most common genera in the communities were *Acrobeloides*, *Aphelenchoides*, *Geocenamus*, *Helicotylenchus*, *Paratylenchus*, *Pratylenchus*, and *Rhabditis* (Table 3). The mean number of genera (17.75) in the replicates was lowest near the industrial plant (K1), which also had the lowest mean population density of 386.5 per 100 g of soil (Table 4). Both parameters increased with distance from the plant to 35.75 and 833.25, respectively, at K4. Ninorg. was significantly correlated (*P*<0.01) with generic richness, and soil pH (*P*<0.01) and Ninorg. (*P*<0.05) were significantly correlated with nematode abundance.

The industrial complex affected the densities of the trophic groups. The trophic structure differed at each sampling site (Table 4). Plant feeders were the most abundant trophic group, with a proportion in the community near 66% at the most contaminated site, K1. Bacterivores were the second most common group, representing more than 50% of the total nematode populations at K2 and K3. This group was positively correlated with Corg and pH (*P*<0.01) (Table 5). Fungivores, considered relatively insensitive to disturbances, including heavy metal pollution, occurred in relatively low densities, except at K2 with 8.98% of the total population. Omnivores and predators were most affected by the pollutants, with very low densities at K1- K3. The low resistance of these two groups to disturbances is in agreement with their negative correlation (*P*<0.01) with Cr (predators) or with all heavy metals (omnivores). Both trophic groups were also negatively correlated with Ninorg., and soil moisture (Table 5). The distribution of the c-p groups near the plant was consistent with their life strategies and survival abilities. General opportunistic nematodes, c-p 2, were most common at the most polluted sites (K1- K3), followed by c-p 1. The other three c-p groups represented only ca. 10% of the identified nematodes. The numbers of c-p 4 and 5 nematodes were significantly higher at the most remote site, K4, where representatives of c-p 4 were the most abundant within the nematode com‐

Mean H′ and generic richness were highest at K4, indicating the richest composition of nematode taxa (Table 4). MI2-5 and SI, measures of ecosystemic maturity, showed analogous development (Figure 2), with stable values from K1 towards K3 and steep increases at K4 (Table 4). EI values indicated that K2 stored the highest amounts of organic matter available for degradation, but K1 had the highest rate of degradation, as indicated by the lowest CI value

*Bacterial feeders Acrobeloides* 2 44.00±26.15 84.25±25.13 202.3±39.8 30.00±13.04 *Alaimus* 4 1.00±2.00 0.50±0.58 0.75±1.50 15.25±12.74

**K1 K2 K3 K4**

**c-p Abundance Abundance Abundance Abundance**


**Table 3.** C-p values, the average abundance (±SD), and dominance of nematode genera in individual sampling sites from Krompachy [1].

Several significant relationships between the heavy metal contents in the soil and the ecological indices were observed across all sampled sites (Table 5). As an indicator of diversity, generic richness was negatively correlated (*P*<0.05 or <0.01), with nearly all heavy metals except Ni, similar to H′ (except Ni and Pb). SI and MI2-5 were negatively correlated (*P*<0.01) with Cr content.


abc Means followed by the same letters on the same rows are not statistically different according to Least Significant Difference Test (P<0.05)

**Table 4.** Percentage of individual nematode trophic groups and average of ecological indices values calculated for Krompachy sampling sites [1].

#### **3.2. Discussion**

**Nematode genera**

from Krompachy [1].

**K1 K2 K3 K4**

**c-p Abundance Abundance Abundance Abundance**

*Mesodorylaimus* 5 - - 1.50±3.00 2.00±1.63 *Microdorylaimus* 4 - - - 3.75±2.63 *Prodorylaimus* 5 - - - 1.00±2.00

32 Emerging Pollutants in the Environment - Current and Further Implications

*Anatonchus* 4 - 0.50±1.00 - 2.25±3.30 *Clarkus* 4 - 4.50±9.00 0.25±0.50 14.25±12.50 *Mononchus* 4 1.75±2.36 30.25±23.77 3.25±4.03 0.75±1.50 *Mylonchulus* 4 - 1.75±2.87 - 8.75±5.32 *Nygolaimus* 5 - - - 1.00±2.00 *Oxydirus* 5 - - - 17.25±16.09 *Prionchulus* 4 - 5.25±8.54 - 0.25±0.50 *Tripyla* 3 - 2.00±2.45 0.75±1.50 15.75±7.41 *Plant feeders Aglenchus* 2 5.50±1.73 27.50±21.55 6.00±3.74 31.50±21.58 *Aphelenchoides* 2 8.75±7.80 147.5±74.9 68.75±23.43 10.50±13.10 *Axonchium* 5 1.00±2.00 - 0.75±1.50 - *Boleodorus* 2 10.00±9.06 20.75±9.64 15.00±10.10 17.25±3.30 *Coslenchus* 2 - - 0.50±1.00 1.25±1.89 *Criconema* 3 - 0.75±0.96 16.25±30.51 1.25±2.50 *Geocenamus* 3 149.8±129.8 2.00±2.83 5.00±3.92 2.75±1.26 *Helicotylenchus* 3 1.25±0.50 73.75±100.1 34.00±34.81 218.8±208.6 *Heterodera* 3 1.25±2.50 - - 20.75±25.18 *Macrotrophurus* 3 - - - 0.50±1.00 *Malenchus* 2 0.50±1.00 2.25±4.50 2.25±3.86 7.50±11.21 *Paratylenchus* 2 14.50±14.15 31.25±30.61 159.0±177.9 21.25±19.10 *Pratylenchus* 3 66.00±69.33 94.00±54.30 89.00±94.70 53.00±52.35 *Rotylenchus* 3 - - - 0.50±1.00 *Tylenchus* 2 - - 0.50±1.00 0.50±1.00

**Table 3.** C-p values, the average abundance (±SD), and dominance of nematode genera in individual sampling sites

Several significant relationships between the heavy metal contents in the soil and the ecological indices were observed across all sampled sites (Table 5). As an indicator of diversity, generic

*Predators*

Heavy metals can both directly and indirectly affect soil environments, directly by modulating the physiology and behaviour of the soil fauna and flora, or indirectly by altering environ‐ mental conditions such as pH or resource availability [27, 28]. The solubility of heavy metals in soil, for which substrate pH is the main driver, determines their toxicity to soil biota [3]. The soil conditions (mostly pH) in the Krompachy area suggest that the solubility of heavy metals could be relatively low, but only the solubilities of As and Cr were low. The solubility and thus the accessibility of the other heavy metals with >20% potential mobility at all sites (K1-K4) could be considered a consequence of chronic contamination of the soil profile and/or a low ability of the soil to bind these elements. The values of the soil parameters and the trend of heavy metal contamination along the transect ruled out the impact of natural geological and/ or physicochemical properties. Kovohuty JSC Krompachy was thus the most likely source of the higher heavy metal levels. This conclusion is in agreement with previous data collected from this area [17, 29, 30].

The influence of polymetallic pollution is evident in all aspects of soil ecosystems, including communities of soil nematodes and their trophic structures. Bacterivores and plant feeders were the most resistant to the adverse effects of pollution, consistent with the findings of a previous study in this area [31]. On the other hand, acidic soil combined with heavy metal pollution can positively influence mostly fungi and decrease bacterial densities [32, 33], but our results do not support these effects. We found relatively low proportions of fungivores and high proportions of bacterivores in the acidic soils at most sites. Numerous studies [34, 35, 36, 37] have described differences in the survival of soil nematodes with similar ecological and life strategies under harsh abiotic circumstances, including heavy metal contamination. Fungivores, such as representatives of the genus *Aphelenchus* tolerant to contamination [38, 39], should thus be more abundant under such environmental contamination. The density of *Aphelenchus*, however, was significantly higher only at K2, which could indicate that interact‐ ing environmental factors (e.g. moisture and texture) might have an important impact on the population density of this trophic group [40].



could be relatively low, but only the solubilities of As and Cr were low. The solubility and thus the accessibility of the other heavy metals with >20% potential mobility at all sites (K1-K4) could be considered a consequence of chronic contamination of the soil profile and/or a low ability of the soil to bind these elements. The values of the soil parameters and the trend of heavy metal contamination along the transect ruled out the impact of natural geological and/ or physicochemical properties. Kovohuty JSC Krompachy was thus the most likely source of the higher heavy metal levels. This conclusion is in agreement with previous data collected

The influence of polymetallic pollution is evident in all aspects of soil ecosystems, including communities of soil nematodes and their trophic structures. Bacterivores and plant feeders were the most resistant to the adverse effects of pollution, consistent with the findings of a previous study in this area [31]. On the other hand, acidic soil combined with heavy metal pollution can positively influence mostly fungi and decrease bacterial densities [32, 33], but our results do not support these effects. We found relatively low proportions of fungivores and high proportions of bacterivores in the acidic soils at most sites. Numerous studies [34, 35, 36, 37] have described differences in the survival of soil nematodes with similar ecological and life strategies under harsh abiotic circumstances, including heavy metal contamination. Fungivores, such as representatives of the genus *Aphelenchus* tolerant to contamination [38, 39], should thus be more abundant under such environmental contamination. The density of *Aphelenchus*, however, was significantly higher only at K2, which could indicate that interact‐ ing environmental factors (e.g. moisture and texture) might have an important impact on the

*As Cd Cr Cu Ni Pb Zn pH Moisture Ninorg. Corg*

*Bacterial feeders* 0.162 0.135 0.338 0.141 0.138 0.124 0.035 0.664\*\* 0.1 -0.088 0.699\*\* *Fungal feeders* -0.384 -0.321 -0.215 -0.313 -0.022 -0.19 -0.409 0.34 0.802\*\* 0.611\* 0.352 *Omnivores* -0.753\*\* -0.846\*\* -0.693\*\* -0.857\*\* -0.748\*\* -0.831\*\* -0.796\*\* -0.178 -0.069 0.475 -0.035 *Predators* -0.469 -0.41 -0.723\*\* -0.44 -0.074 -0.28 -0.475 0.03 0.522\* 0.628\*\* 0.276 *Plant feeders* 0.303 0.329 0.003 0.35 0.209 0.297 0.444 -0.54\* -0.529\* -0.479 -0.600\* *c-p 1* 0.576\* 0.629\*\* 0.185 0.612\* 0.768\*\* 0.662\*\* 0.588\* 0.012 0.479 0.074 0.102 *c-p 2* 0.329 0.318 0.612\* 0.276 -0.1 0.191 0.297 0.275 -0.509\* -0.609\* 0.169 *c-p 3* -0.015 0.112 -0.232 0.091 0.226 0.079 0.206 -0.494 -0.212 -0.176 -0.727\*\* *c-p 4* -0.615\* -0.679\*\* -0.674\*\* -0.671\*\* -0.506\* -0.532\* -0.656\*\* -0.143 0.224 0.594\* 0.116 *c-p 5* -0.804\*\* -0.746\*\* -0.854\*\* -0.777\*\* -0.507\* -0.737\*\* -0.712\*\* -0.153 -0.092 0.433 -0.221 *Genera richness* -0.836\*\* -0.736\*\* -0.845\*\* -0.771\*\* -0.409 -0.601\* -0.765\*\* 0.144 0.267 0.656\*\* 0.085 *Abundance* -0.329 -0.182 -0.162 -0.168 -0.029 -0.018 -0.176 0.680\*\* 0.444 0.529\* 0.297 *Enrichment Index* 0.382 0.468 0.018 0.468 0.753\*\* 0.521\* 0.444 0.059 0.662\*\* 0.288 0.104 *Structural Index* -0.447 -0.474 -0.750\*\* -0.459 -0.25 -0.341 -0.453 -0.303 0.2 0.553\* -0.081

from this area [17, 29, 30].

**Indices, trophic & c-p groups**

population density of this trophic group [40].

34 Emerging Pollutants in the Environment - Current and Further Implications

**Table 5.** Correlations among heavy metal concentrations, soil characteristics, nematode community structure, and selected ecological indices from Krompachy [1].

Omnivores and predators are generally considered the most sensitive to any disturbances and stresses [41, 42, 43]. Our data supported the high sensitivity of these two trophic groups and their preference for more eco-friendly conditions over polluted environments. In addition, only these two groups were strongly negatively correlated (*P*<0.01) with heavy metal pollution. Specifically, omnivores correlated negatively with all heavy metals (*P*<0.01), and predators strongly correlated negatively (*P*<0.01) with Cr. Nagy et al. [44] reported similar findings from a field experiment in Hungary, where the lowest concentration of the Cr mobile fraction that produced an observable effect was approximately 0.5 mg kg-1. The presence of the more toxic and mobile Cr6+, which our assay could not detect, may have been responsible for the signifi‐ cant influence of Cr on these two trophic groups, despite the lower Cr concentrations in our study. A synergistic contribution of other heavy metals may also have been responsible for the stress response of these trophic groups.

**Figure 3.** Structure and Enrichment Index graphic representation (Krompachy) [1]. A - disturbed food web, bacterial decomposition; B - maturing food web, balanced decomposition; C - structured food web, fungal decomposition; D degraded food web, fungal decomposition [11].

Community characteristics and ecological indices (H′, MI2-5, and SI) indicated that the soil conditions at our sites were significantly stressed by the contaminants. Generic richness and H′ were lower under higher loads of heavy metals. The lightly contaminated K4 site had the highest diversity of nematode genera, belonging to the entire c-p scale, including opportunists (e.g. *Rhabditis*, *Acrobeloides*, and *Aphelenchus*) and persisters (e.g. *Dorylaimus*, *Oxydirus*, and *Nygolaimus*). The proportion of persisters with higher c-p values rapidly decreased with increasing pollution, which consequently led to lower nematode diversity and a simplification of the food chain. Diversity was mostly negatively correlated with the degree of pollution, but nematode abundance increased at moderately contaminated K2 and K3, but rapidly decreased under a heavy load at K1. Sánchez-Moreno et al. [39] reported similar findings in southern Spain after the Aznalcollar mining spill. A higher generic diversity reflects a higher resilience of ecosystems and complexity of relationships at all trophic levels [45, 46]. A decrease in generic diversity may cause an ecosystem to regress to an earlier successional stage or even to collapse. On the other hand, an increase in total density under a low or moderate degree of pollution does not necessarily indicate an uncontaminated environment but rather better survival conditions, as in our study (the growth in the population of opportunistic nematodes). Contamination can in some cases act as a direct or indirect stimulus to a particular component of the ecosystem and lead to inaccurate assessments of the actual environmental state [4]. Nevertheless, the generic composition of the nematode communities and several of the ecological indices (EI, SI, and MI2-5) suggest that a certain amount of regression is occurring in the study area closer to the pollution source.

Georgieva et al. [42] have studied the combined effect of heavy metals. The maturity of an ecosystem, measured by MI2-5, was negatively affected more under significantly lower polymetallic contamination than if the heavy metals were applied separately. A nematode community was highly insensitive to Cd, showing no significant changes, at soil concentrations up to 160 mg kg-1 [47]. Similar insensitivities in nematode communities were also described for increased levels of As [48]. The toxicity of some elements, though, can increase in combi‐ nation with other heavy metals and may have negative impacts on nematodes [42]. This synergistic effect could thus be responsible for the decrease in MI2-5 and SI towards the pollution source in our study, despite their nonsignificant correlation with the heavy metals.

The analysis of SI and EI at Krompachy indicated significant differences amongst the sites in structural complexity and resource availability of the ecosystem. The graphic representa‐ tion of the SI and EI parameters (Figure 3) mapped K4 in quadrat B (maturing ecosystem with low or moderate disturbance), K1 and K2 in quadrat A (ecosystem under high disturbance and with a disturbed food web), and K3 in quadrat D (stressed ecosystem with a degraded food web). These results indicated differences in the composition of the structural component (c-p groups 3-5) rather than the enrichment component (c-p 1) [49]. The shift in the ecosystem in our study under heavy metal pollution corresponded well with the conditions reported by [50, 51, 52].

The decomposition of organic matter can also have a large impact on the development of a soil ecosystem and its maturity. The rate of breakdown of organic matter depends on soil condi‐ tions and the participation of a variety of decomposers [11]. We studied decomposition using CI and found that the breakdown of organic matter followed distinct degradation pathways at the different sites. EI indicated high levels of energy resources at all sites except K3, but CI indicated that the quality of these resources (C:N ratio) differed: K1 and K3 had mainly bacterial pathways of degradation, dominant in N-enriched conditions, but the other sites had a slower breakdown, with an increasing importance of fungi breaking down organic matter with a higher C:N ratio. The change from fast to slow decomposition was also apparent in the nematode community structure and the substitution of bacterial enrichment opportunists (mainly *Rhabditis*) by more efficient general opportunists (*Cephalobus* and *Acrobeloides*).
