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Ecological Sensitivity Distribution (ESD): integrating molecular-based structural and functional microbial community responses in a new tool for environmental risk assessment of chemicals

Periodic Reporting for period 1 - MicroERA (Ecological Sensitivity Distribution (ESD): integrating molecular-based structural and functional microbial community responses in a new tool for environmental risk assessment of chemicals)

Reporting period: 2016-10-01 to 2018-09-30

Aquatic ecosystems shelter microbial communities, a key compartment implied in many ecosystem functions such as biogeochemical cycles or pollutants degradation and crucial for aquatic ecosystems proper functioning. However, these communities are currently exposed to agricultural and industrial chemicals which may alter their structure (abundance, composition) and their functional activity. To protect environmental communities from chemicals, the European community applies many risk assessment methods of various levels of environmental relevance which are mainly based on structural responses. Moreover, most of those methods suffer from several drawbacks such as their incompatibility to consider responses at the community level and especially the microbial ones. Thus, this project aimed to propose a new generation of risk assessment tool (ESD: Ecological Sensitivity Distribution) by revisiting the Species Sensitivity Distribution (SSD) concept for microbial communities and by considering the protection of functions. The SSD approach (used in the context of several directives) aims to derive thresholds that protect environmental communities to chemical-induced effects from the cumulative distribution of many species sensitivity data obtained from monospecific bioassays under laboratory conditions. One of the interests of the project was to consider several levels of responses, by combining functional measures on different levels of biological complexity, namely molecular (=transcriptomics, metabolomics) and apical/classical endpoints (e.g. growth, photosynthesis) in order to assess the added-value of the omics technologies in ecological risk assessment (ERA).
When aiming to integrating data on different levels in an ESD comparable effect thresholds are needed. The microERA project led to the development of a turnkey tool called DRomics (Dose-Response for omics, available as an R package and an online interface This tool manages molecular responses obtained in a dose-response framework and allows in fine integrating molecular responses (e.g. genes, metabolites) in ERA. The results highlighted that omics responses were more sensitive than classical endpoints and consequently, genes and metabolites involved in a function showed higher sensitivity than the direct measurement of the functions (e.g. energy metabolism pathway vs. photosynthesis). In conclusion, the use of cumulative sensitivity distributions of omics data could reinforce risk assessment procedures and enable the integration of functions and community studies in ERA.
First, the DRomics tool was developed in order to manage molecular responses obtained in a dose-response framework. As the microERA project necessitates dealing with this kind of data, we needed a reliable procedure able to manage the data obtained during the experiment. This tool 1/ detects responding data (e.g. genes, metabolites), 2/ finds out the best model to describe the response, 3/ builds dose-response curves even in the case of complex responses (e.g. biphasic), 4/ derives an effect concentration (here, benchmark dose) and 5/ builds a cumulative distribution of gene or metabolite sensitivity.
Second, in a proof-of principle study, the functionality of the tool was demonstrated for an existing dataset, describing the response of the chlorophyte Scenedesmus vacuolatus to the biocide triclosan (6 concentrations including control). Responses were observed at the molecular level (untargeted transcriptomics (microarrays) and metabolomics) and apical/classical endpoints (growth, photosynthesis). This step allowed to first reach our goals on a model organism with a lower biological complexity compared to communities and to identify potential limitations to be solved for consecutive experiments on periphytic communities. Using DRomics, we observed that apical endpoints were less sensitive than most of molecular endpoints and that the well-known sigmoid response was more the exception than the rule at the molecular level. An ESD was built on both transcriptomics and metabolomics responses and clearly highlighted importance of the consideration of omics responses in ERA and reinforced our initial assumptions.
Third, an experiment was performed to investigate the responses of stream periphytic communities to the herbicide diuron on different functional levels. Responses at the molecular level (transcriptomics=sequencing of RNA, metabolomics) were investigated after 1 hour of exposure. Moreover, the same experiment was performed in parallel on communities pre-exposed for one month to diuron in order to investigate potential tolerance acquisition mechanisms on functional responses. A strong effort was also put in the optimization of a protocol to extract RNA in a sufficient quality and quantity for the downstream analysis. Quality control of the extraction and the sequencing of RNA were satisfying. The annotation of the sequencing is still in treatment. An ESD build on community functional responses in the light of metabolic pathways annotation (e.g. KEGG pathways) is under development considering tolerance mechanisms.
The microERA project highlighted the importance to base ecotoxicological studies on different endpoints belonging to various levels of biological complexity but also to implement molecular responses in ERA procedures. It demonstrated the necessity of using a dose-response experimental design for molecular endpoints to derive effect thresholds, useful for ERA and therewith moved ERA towards the definition of a new generation of protective thresholds. The ESD, dealing with functions (here, metabolic pathways), allowed going further by identifying and ranking sensitive/risky pathways to the targeted chemicals. From the concept of the ESD and based on the output of the DRomics tool (workflow publicly available), this work exemplified how to consider functional endpoints obtained at community-level in ERA and in fine in decision making in order to reach the Water Framework Directive goals. This approach can be used for any organisms and stressor meaning that it can be applied in a large variety of contexts and corresponds to a wide audience, from the academy to the regulators and the industry.