9. Effects Assessment
9.3. Choice of standard laboratory test methods for microorganisms
9.3.1. General considerations for microbes
A description of the available test methods for microorganisms is reported in AppendixI. Available ISO standards with potential relevance for soil microbes are listed in Appendix G.
While general toxicity-test systems using single microbial species or strains (e.g. Johnson et al., 2009; Dijksterhuis et al., 2011) can give information both on direct effects on the test organism and on specific toxicity mechanisms or degradation pathways, they are of limited use in prospective risk assessment for microbes in general. Their main disadvantage is the quite low representativeness of one species to the vast microbial genotypic and phenotypic diversity of soils. Also, a big majority of microorganisms currently cannot be cultured as pure isolates, but can only be studied in the context of more or less complex natural communities (e.g. Wagner, 2004). Hence, using field samples in mesocosm or microcosm experiments with entire communities has substantially higher environmental relevance and has recently been advocated (Puglisi, 2012); (ECHA, 2014; Karpouzas et al., 2014).
In community-level tests using (semi)field studies with natural soil, longer term chronic effects induced by pesticides can be determined, taking taxonomic and functional shifts in microbial communities into account. Endpoints can relate to function (activities or processes), biomass (total biomass, or biomass for taxonomic or functional sub-groups) or structural properties (community structure or diversity) (see Appendix I).
The big progress in development and adaption of molecular methods has lately facilitated descriptions of different aspects of in situ microbial communities, particularly concerning their structural properties. One established method is PLFA (phospholipid fatty acid analysis), where the PLFA composition of a sample gives a picture of the microbial community structure. Nucleic acid-based methods have bigger potential to give detailed information on the structure and diversity of microbial communities and the presence and abundance of specific phylogenetic or functional groups of organisms. In recent years, metagenomic approaches and 454 sequencing have been broadly introduced to studies of dynamics in soil microbial community structure (e.g. Feld et al., 2015). Besides giving information on the overall community structure or phylogenetic diversity, molecular methods can also provide data related to functional properties. For instance, quantitative PCR-based methods can be used to determine the abundance of particular functional genes (i.e. independently of the phylogenetic affiliation of the organisms carrying the genes), and hence subgroups of the microbial community potentially able to perform a specific process (e.g. Fang et al., 2014; Anzuay et al., 2015).
Additionally, functional gene arrays (e.g. geochip based analyses) can give information on the
presence/absence of a large number of functional genes, and thereby give a fingerprint of the metabolic potential of microbial communities (He et al., 2012).
There is no doubt that modern molecular methods have a huge potential to yield valuable information on effects of environmental changes and stressors on microbial communities. However, performing the analyses and interpreting the large data sets often produced is scientifically complex and so far no such methods have been evaluated for use in a regulatory context.
Chemical pesticides (and other organic pollutants) can influence microorganisms in two opposite ways: (i) the compound is toxic to the microbe; and (ii) for a microbe exhibiting tolerance to the compound, it may act as a nutrient and energy source and, hence, stimulate growth of those organisms. A consequence of the possible dual effects of pesticides on different metabolic and functional groups of microbes is that spiking soil with pesticides commonly results in increases in microbiological parameters. Accordingly, in the terrestrial data set of the systematic literature review of responses of microorganisms to pesticides (Puglisi, 2012; external procurement funded by EFSA), a substantial part of the entries in the database represented significant increases. The summary evaluation of the data was reported separately for type of substance (fungicides, herbicides and insecticides) and type of assay (based on activity, biomass or community structure). Regarding effects on microbial activities, 39–47% of entries showed no significant effects, 21–32% significant decrease and 11–22% significant increase. For assays based on biomass, corresponding numbers were 26–55%
(no effect), 11–23% (decrease) and 14–44% (increase). In the material analysed by Puglisi (2012), increases in response were more common for insecticides than for either fungicides or herbicides, both for the activity- and biomass-based endpoints. In the analysis of effects on endpoints based on community structure (using PCR-DGGE, PLFA, BIOLOG®, etc.), a different and qualitative approach was taken using three classes: significant changes, transient significant changes and no significant changes. The majority (80–95%) of the entries showed significant changes in community structure, while the remainder showed either no or transient effects.
It is important to keep in mind that, in community-based tests, a no-effect outcome in a functional endpoint after pesticide spiking to soil is a net response, and not proof that no populations have been affected in either a negative or a positive way. In community-based measurements of chronic effects, functional redundancy – i.e. if organisms performing a certain function are inhibited or eliminated, other organisms take over and fill the niche – can also hide effects on specific populations within functional groups, thereby complicating the interpretation of test outcomes.
9.3.2. Prospects for improving test strategies for microorganisms
Phylogenetic or functional groups as well as individual species of microorganisms contribute to several of the different ESs. To begin with, all soil microorganisms principally contribute to the ESs genetic diversity, biodiversity, cultural services and food-web support. Apart from this, many microbial groups also contribute to several other ESs. Taking the functional (and polyphyletic) group denitrifying bacterial taxa as an example, these bacteria will often also contribute to the ESs nutrient cycling, natural attenuation and possibly pest and disease control. Additionally, although knowledge regarding both the structural and functional properties of soil microbes is growing rapidly, for many functions and activities performed by soil microbes (especially common functions performed by many taxonomical groups), confining the functions to certain phylogenetic units is still a challenge.
Based on the above it is concluded that, for selection of the most relevant community-based microbial test systems, the best strategy seems to be to consider assays that cover as wide a range as possible of processes and key drivers, while it is hardly realistic to design a set-up containing specific assays for specific key drivers. The currently required community-based functional tests of effects on N transformation (the EU and North America) and on general microbial activity (CO2 evolution; North America) take the activity of a wide selection of microbes into account, but are considered comparatively coarse and insensitive. MicroResp® is a recent development of the BIOLOG® method that has potential to give more detailed information regarding degradation capacity of a soil, and the effect of pollutants on degradation (Campbell et al., 2007). In contrast to the N-transformation and respiration tests, MicroResp® yields data on the degradation capacity in the sample of a number of different types of substrates. It describes the functional/physiological capacity for degradation and measures microbial activity as CO2 evolution directly from, e.g., soil separately for each included substrate. So far, MicroResp® has not been used to study effects of pesticides on soil microbial processes but it is recommended that its capacity in that respect is determined in future research.
The aim of finding broad microbial tests that cover as much as possible of phylogenetic and functional variation may need to be balanced against the aim of sensitivity. For illustration, functional redundancy can be expected to contribute less to insensitivity when studying more specialised functions. One example is that potential nitrification has been found to be relatively sensitive to pollutants (Pell et al., 1998), since the ammonia-oxidising bacteria mediating the first step represent a small group of fastidious, slow-growing lithotrophic bacteria. In recent years, however, it has become evident that some archaeans can also oxidise ammonia in soil (Leininger et al., 2006). Redundancy is more likely for functions that are widely distributed among many microbial groups, meaning that if inhibitory effects are seen for such functions, it is likely that many different microorganisms have been affected.
Currently, there is no data requirement related to mycorrhizal fungi. Their importance in providing several important ecosystem services is, however, well known and reported in the literature. In addition, mycorrhizae provide specific functions (e.g. soil formation) that represent an exclusive trait of mycorrhizae in general and arbuscular mycorrhizal fungi in particular.
A standardised test with arbuscular mycorrhizal fungi was proposed by ISO (ISO/TS 10832:2009) where the acute effect of chemicals (or contaminated matrices) on spore germination of the arbuscular mycorrhizal fungal species Funneliformis mosseae (formerly Glomus mosseae) (strain BEG12) is assessed. The test is conducted over 14 days at 24°C on sand or OECD artificial soil, with the percentage of germinated spores, in relation to the number of recovered spores, being the measured parameter.
The test species (F. mosseae formerly G. mosseae) has been proposed as a model species to assess the toxicity of different chemicals due to its widespread distribution around the world and its high abundance to crop systems (Smith and Read, 2008). When testing different PPPs, however, Giovannetti et al. (2006) found that spore germination was only a sensitive parameter towards some of the fungicides tested but not to the tested herbicides. On the other hand, mycelium growth (also at the asymbiotic phase) proved to be a much more sensitive parameter, being inhibited by all fungicides and herbicides tested.
These results could indicate that, when using arbuscular mycorrhizal fungi as a test species, parameters other than spore germination could be assessed, increasing the informative value of the assessment. This could indicate some improvements in the existing protocol to accommodate the measurement of extra parameters. In a recent study, Mallmann (2016) demonstrated the practicability and reproducibility of the ISO protocol, together with its ability to be used with other arbuscular mycorrhizal fungi species (five species were tested) and to measure other parameters, including the growth of the asymbiotic mycelium. Moreover, since the sensitivity to some chemicals can vary between strains of the same species, studies like this one that focus also on evaluating inter- and intraspecies variation in sensitivity should be performed and the more appropriate test species and strain should be selected.
Conclusion
From the above and from AppendixI it can be concluded that a number of methods exist to measure soil microbial properties of soil in terms of abundance, activities/functions as well as community structure. A number of interpretation problems are identified, e.g. effects on functional endpoints might be diluted by functional redundancy; regarding structural endpoints, the interpretation in the sense of their connection with certain functions is hard to define.
Novel methods are continuously developed that are able to record a broad spectrum of functional and structural endpoints. These methods need further adjustment and standardisation for use in risk assessment of pesticides, but may become useful tools in the future.
Since the present test aimed at N transformation covers a number of processes, it is considered a relevant indicator, most obviously for the functions nutrient cycling and food-web support. In the present test design for agrochemicals, it is prescribed to test two dosages and to determine at different points in time whether effects are > 25%. When after 28 days effects are > 25%, the test can be prolonged to 100 days. For non-agrochemical substances, however, a dose–response design is prescribed. In order to assess whether the effects fit with the magnitude and the temporal scale mentioned in the SPG chapter, it is recommended to use a dose-effect design for agro-chemicals as well.
Activities of protozoa and many fungi are, to a substantial extent, covered by the N-transformation test, whereas, mycorrhizae are not incorporated. It is therefore recommended to add a test with mycorrhizal fungi. The ISO test with Funneliformis mosseae (formerly Glomus mosseae) allows for
adaption of the endpoints measured or to test other species/strains. Further research and development is needed to improve the test design.