Nematodes as soil stress indicators for polycyclic aromatic hydrocarbons: A review

The most important factor for the degradation of PAHs in freshly contaminated soils is their hydrophobicity. The more soluble nature of LMW PAHs and their often higher concentrations in soil solutions compared to HMW PAHs influences the interstitial mesofauna directly through oral or transcuticular nutrient intake (Kammenga et al., 1994). Nematodes that are sensitive to toxicants (e.g. predators or omnivores) and have a relatively permeable cuticle are exposed to this risk immediately after the introduction of PAHs into the soil ecosystem. On the other hand, representatives of stress-resistant nematode genera (e.g. mostly bacterivores and fungivores) may benefit from the introduction of PAHs through, for example, a decrease in predation pressure or an increase in available food resources. The shift towards stress-resistant species in the nematode community structure has been well documented by Moreno et al. (2009) and Soto et al. (2017) in localities heavily polluted with PAHs, where nematode genera well-adapted to disturbed conditions significantly dominated. The replacing of sensitive nematode species by resistant ones can affect the food web from both bottom-up (food availability, overpopulation, etc.) and top-down effects (predation, pests’ control, etc.) and ultimately result in a lower ecosystem biomass despite constant population densities (Soto et al., 2017). Chen et al. (2009) also observed the decline of the environmental maturity level and simplification of the soil food web under the higher pressure of PAHs contaminants in the soil.

The toxic effect of contaminants on nematodes, which have a relatively permeable body surface may be reinforced by ingestion of food containing risky substances. Except omnivores and nematodes that consume the soil substrate itself, such as some diplogasterids, or Daptonema spp. (Yeates et al., 1993), which are under the direct risk of digesting soil organic matter particles, the pollutants may also act as a bottom-up limiting factor to the food web. Li et al. (2005) described experimentally various physiological responses and behavioural changes in Aphelenchus and Acrobeloides (fungivorous and bacterivorous nematodes, respectively) exposed to benzo(a)pyrene and phenanthrene. Even though the trophic preferences of these genera are different, they were similarly sensitive to PAHs. The development of both taxa was delayed after the addition of benzo(a)pyrene, despite the fact they are classified as genera with well-developed physiological and behavioural adaptations to stress conditions. Aside from the direct, lethal effect on organisms, PAHs can also act as narcotics in lower dosages and affect essential physiological processes, including the growth, reproduction and development of organisms (Menzel et al., 2005). The development delay may be the result of interference between the PAHs and nutrient uptake and metabolism ultimately leading to the organism’s “physiological starvation” (Postma & Davids, 1995). Reproduction processes seem to be even more sensitive to PAHs than survival and development processes of nematodes. Benzo(a) pyrene significantly affected the egg size and hatch rate in representatives of both Acrobeloides and Aphelenchus genus (Li et al., 2005). Swain et al. (2010) suggest that organisms exposed to PAHs may have to expend a greater amount of their available energy for survival, which is then reflected in lower fertility and offspring production. In this study, the exposure of nematodes to fluoranthene probably caused a switch in the energy metabolism from carbohydrates to proteins, resulting in a high amount of free amino acids (as degradation products) found in nematode cells. Similar responses have been observed in other organisms subjected to PAHs, where re-synthesis of degraded key structural proteins during growth, development and egg production went hand in hand with a considerable energetic cost to the organism and the delaying or disrupting of these processes (Jones et al., 2008; Swain et al., 2010). A decline in the populations of key organisms responsible for various important processes (nutrition and energy flow, pathogens control, etc.) in the soil ecosystem can gradually lead to the overall decrease of ecosystem production and loss of its functions. This negative trend could subsequently mean the beginning of a negative spiral heading to the lower maturity of the ecosystem.

Fig. 2

Caenorhabditis elegans is the nematode most commonly used as a model in ecotoxicological studies. Individuals of this species exposed to PAH substances at concentrations between 2.7 and 5.2 mg.L^-1 in the soil showed acute mortality rates from 56 to 99 percent (Cofield et al., 2008). Lower doses of PAHs used in experiments did not appear to have a lethal effect, but they caused numerous developmental defects, such as inhibition of growth, fertility and reproduction in this species (Höss et al., 2009). Similar results (inhibition of reproduction in C. elegans) were observed under field conditions with a similar level of soluble PAHs contamination in freshwater sediments (Höss et al., 2007). The level of the PAHs contamination seems to be the most important value in assessing the toxic effects of these contaminants on nematodes, and it is relatively independent of other environmental factors, such as the physico-chemical properties of the substrate. However, as nematodes correlate strongly with the labile dissolved PAHs fraction (Cofield et al., 2008), the differences in substrate-binding capacity and composition of PAHs applied in the studies may decrease/increase their original concentration to comparable levels of the soluble PAH fraction to which the nematodes were exposed in both studies.

On the molecular level, PAHs induce multiple detoxification responses in nematodes, including the expression of cytochrome P450 genes responsible for the detoxification of xenobiotics. A concentration-dependent relationship was found between the intensity of expression of these genes and the benzo(a)pyrene added to the soil (Menzel et al., 2005). An even stronger induction of the P450 genes family was found in the use of fluoranthene (Menzel et al., 2001). Saint-Denis et al. (1999) pointed out that benzo(a) pyrene may also be activated by cytochrome P450-independent metabolic pathways. Alternative metabolic pathways of PAHs may include the generation of free radicals or the formation of reactive oxygen species as metabolic by-products, leading to oxidative stress (Penning, 2014). Wu et al. (2015) came to a similar conclusion in their study on oxidative stress in C. elegans exposed to benzo(a)pyrene. Laboratory studies provide important information on the potential direct effect of PAHs on living organisms, and even though they do not accurately simulate the influence of PAHs under field conditions, they provide important insights for future studies.

The introduction of PAHs into an ecosystem can indirectly cause both positive and negative responses in nematode communities. The positive effects can be channeled through the ability of bacteria and fungi (decomposers, primary food sources for nematodes) to utilize various PAH compounds, including the most common PAHs – naphthalene, phenanthrene and pyrene – as sole carbon sources (Duan et al., 2015). This process is primarily controlled by the bioavailability of these compounds to microorganisms, even without the need for PAHs to be present in the soil solution (Alexander, 2000; Zhang et al., 2012). Depending on the nature of the PAHs introduced into the soil ecosystem and the decomposer channels used for their breakdown, soil communities respond distinctively by changes in the internal structure of their community. Bacteria can use LMW PAHs as a direct source of energy (Sack et al., 1997), but only fungi are able to degrade HMW PAHs, despite their recalcitrant and hydrophobic nature (Cerniglia, 1992). The increase of decomposer populations (bacteria and fungi) in the soil may act as a positive stimulus for other trophic groups of micro-organisms, such as fungivorous or bacterivorous nematodes, and later for higher levels of the food web. Blakely et al. (2002) observed the delayed response to a prospering decomposers community under sufficient food availability. Both bacterial biomass and bacterivorous nematodes flourished in soils contaminated by LMW PAHs, while the fungi population in the system was attenuated, which was illustrated by the continual presence of fungivores in the soil (Blakely et al., 2002). Furthermore, the increase of bacterivorous nematodes grazing on PAH-degrading bacteria could even accelerate the dissipation of these organic pollutants (Sun et al., 2017). According to Zhou et al. (2013), the reason for this contradiction between increasing pollutant degradation and predation could be the more intense in activity of native bacteria that can degrade PAHs. The trigger action of such enhancement could be the direct selective pressure of contaminants and predators or increased nutrient mineralization and nutrient cycling provided by bacterivorous nematodes (Sun et al., 2017). In the latter case, the addition of a key nutrient to the system may significantly enhance the degradation, especially in a contaminated environment with limited nutritient resources (Yu et al. 2005).

Apart from this, the grazing of bacterivores keeps the population of soil bacteria at a reasonable level, thus preventing the inhibition of their growth caused by substrate shortage or the accumulation of toxic metabolites originating from the degradation. On the other hand, Näslund et al. (2010) found in marine sediments that, in the case of higher mesofaunal densities, naphthalene mineralization, as well as, the number of naphthalene-degrading bacteria, decreased. However, the authors pointed out that this phenomenon could be due to the higher predation pressure during the experiment in combination with significantly different bacterial diversity in treatments with different mesofaunal abundances.

The negative effects of PAH contamination on nematodes may be reflected, for example, in the availability of suitable microhabitat conditions. Given that the distribution of nematodes in the soil is largely dependent on their feeding habits and body size (Blakely et al., 2002), the intra-aggregate pore space could be an important determining factor for nematodes distribution. Therefore, increasing the bulk density of the soil by tightly bind PAHs to soil particles could significantly alter the actual conditions of microhabitats. This change may be a significant barrier to large-sized nematodes (e.g. Dorylaimida, or other omnivores and predators) using the intra-aggregate space as a refuge from other predators, or as a preferential place for reproduction and development (Briar et al., 2011). The decrease in soil pores diameter also means less oxygen and nutrient transportation through the soil microhabitats (Blakely et al., 2002). A lack of nutrition flow and suitable habitats for larger nematodes could lead to their decrease in the soil community. As larger nematodes represent mainly omnivores and carnivores, their decrease may significantly influence the top-bottom pressure in the habitat (Blakely et al., 2002; Sun et al., 2017). Nevertheless, Snow-Ashbrook and Erstfeld (1998) reported high abundance of omnivores/carnivores accompanied by a slight stimulatory effect of PAHs on the overall invertebrate community. Even though the genuine reason for the increased occurrence of sensitive nematodes in the most contaminated plots could not be explained from the study, Erstfeld and Snow-Ashbrook (1999) hypothesized that bottom-up effect regulation by the stimulated microflora, together with physico-chemical characteristics (organic carbon, soil moisture, pH and grain size) at the study sites, may play the key role.

In the toxicological data collection and assessments of the impact of soil contamination, toxicological tests predominantly rely on a single-species laboratory test. For nematodes, the Caenorhabditis elegans toxicity test is the most commonly used (Wu et al., 2015). The adaptation of nematodes to life in the soil has resulted in a great diversity of their life strategies and different nematode traits, including morphology, physiology, food preferences and behaviour. Therefore, even though single-species tests are important for characterizing potential acute and subacute impacts of contaminants under controlled conditions (Bejarano & Michel, 2016), they are not able to provide enough insight into the ecosystem from an ecotoxicological perspective and describe all interactions that may occur among nematodes, contaminants and other components of the food web. Preferring information obtained on the community level, the monitoring framework might gain robustness, higher resolution and additional knowledge about the ecosystem and the processes running within it (organic matter degradation, nutrient and energy flow, availability of food resources, etc.).

As shown above, the outcomes of experimental studies can sometimes contradict each other, which means that no clear and relatively reasonable conclusions can be drawn about the impact of pollutants on nematodes. To obtain clear results under laboratory conditions, concentrations of pollutants beyond compare with real field conditions are often used. High levels of contamination indeed have an acute impact on nematode community structure, but this represents only a relatively short and partial effect of contaminants on the soil ecosystem, while contaminants availability in the system is still relatively high. On the other hand, the chronic phase lasting much longer (superseding the acute phase, when the availability of contaminants drops to a certain level) has possibly a stronger effect on the nematode community structure. In this phase, contamination does not shape the community directly but rather indirectly influences various physiological and behavioural aspects of nematode life.

Another problem closely associated with the chronic effects of contamination is the duration of exposure. Under controlled conditions, nematodes are exposed to pollutants for days or weeks, usually capturing the reaction of mostly a few following generations. The subacute exposure to foreign compounds leads to a transgenerational changes in nematode fitness (Yu & Liao, 2016). The induced multigenerational effects include additional mechanisms (cumulative damage, acclimatization, adaptation) that shape the nematode community and divert the direction of its development from that expected. Therefore, it is essential to consider the multigenerational effects to highlight the possible impacts of contamination over multiple generations and to include them in development predictions.

Although the understanding of soil ecosystem functioning has improved significantly in the last decades, due to the high heterogeneity and dynamics of the soil ecosystem and the absence of baseline input data, comparison and evaluation is often a challenge. One step towards improving the robustness of the experimental data obtained and the applicability of nematodes as a tool for bioindication of soil stress is the harmonization of different methods and approaches used in ecological studies of ecosystem pollution. Obstacles to harmonization of biological methods often lie in the requirement for “fresh” samples of nematode communities for analyses and the lack of reference control material as a data source (Faber et al., 2013). Therefore, standard methodological approaches, as can be found in other scientific fields, are usually not applicable in the field of soil biology. Instead, the way to compare the relative efficiency and reliability of different biological methods is in their application under the same experimental conditions, i.e. concurrently on the same set of plots or samples. The harmonization step is necessary as part of the effort to achieve standardized methods at national and international level and to make progress in the use of native soil indicators in actual monitoring.

Another issue hampering the engagement of nematodes in soil monitoring is the extremely time consuming taxonomic determination using traditional morphological methods (Donn et al., 2012). However, the recent increase in the development and use of molecular approaches, which are capable of processing large numbers of samples with high sensitivity in a relatively short time, should reduce the difficulties of identification based on morphology alone (Stone et al., 2016). The low resolutions of morphological methods often led to misconceptions in taxa distribution, through numerous cryptic species that are relatively site specific (Taylor et al., 2006). These subtle differences ultimately resulted in the incorrect assessment of the nematode community structure as well as an inability to recognize the uniqueness of each community and its proper reactions towards environmental conditions or stress (Stone et al., 2016)

However, it should be noted that molecular methods are still under development and may not currently be able to fully incorporate nematode identification and generate accurate representations of nematode community diversity in a single step. Therefore, it is necessary to employ more than one technique to obtain valid results (Lott et al., 2014). Stone et al. (2016) suggested that methods such as T-RFLP, which allow for quick but rather coarse analysis of large sample sets, could act as a sieve for selecting those to be analyzed in more detail at a later stage using additional methods. The main advantages of this approach lie in using rapid and cost-effective screening platforms without the considerable data processing and storage space requirements of most next-generation sequencing technologies, while yielding comparable resolution of community structure (Pilloni et al., 2012). Rapid analysis opens up the possibilities of studying nematode assemblages and their spatial and temporal dynamics in sample-intensive studies not only in polluted soils, but also in natural or disturbed soils and in different agricultural environments. Although the entire nematode community is used in soil monitoring, for specific purposes, these methods are able to restrict the analysis to groups of interest or to be expanded and applied to the entire food web or eukaryotic faunal community of the soil (Donn et al., 2012).