The proposed work will be divided into four work packages (WP) where data and experiences will be shared between the different WPs.
Figure 1. Overview of individual work packages and their internal relationship
WP1 - Identification of ED targets
Work package 1 aims to develop a knowledge database for D. magna based on identification of 1) potential ED targets, 2) develop predictive computational models for compound interactions with these targets and 3) experimentally verify potential interactions between the environmental compounds and their ED targets.
WP2 - Adverse outcome pathways (AOPs)
The linkage of the interaction of an ED with their molecular targets and the adverse effect relevant for regulatory accepted endpoints is achieved through the identification of a series of key molecular/biological events that describe the toxicity pathway from the chemical interaction with biological targets to the production of an adverse outcome (figure 2). Work package 2 aims to 1) build conceptual ED AOPs in D. magna, 2) expand and evaluate the conceptual AOPs through experimental approaches and 3) assemble the resulting theoretical and experimental data into a complex AOP network. As the knowledge of key events increase, the network of AOPs will gradually develop from a few key molecular events that may reflect the MoA to complete and detailed understanding of all key events representing the full mechanism of action (the complete AOP network).
Figure 2. Conceptual approach to establish Adverse outcome Pathways for endocrine disruption in D. magna.
WP3 - Robustness and applicability
Work package 3 aim, through a series of exposure studies, to evaluate the robustness and applicability of the developed conceptual AOPs for ED. These studies will assess 1) the ability to address combined toxicity (join toxicity), and 2) ability to accommodate exposure to multiple stressors.
WP4 - Regulatory contribution
Work package 4 aims to establish a platform for collaboration between experts from research, industry and regulatory agencies to expand and disseminate the knowledge generated by the project and facilitate implementation of AOPs within a regulatory framework by 1) supporting development of AOPs within an open access knowledge database, 2) Develop computational (in silico) tools to assist predictive risk assessment, 3) assist in test guideline development.
Computational and experimental methods will be used to identify possible ED targets in D. magna and identify novel EDCs in crustaceans by targeted in vitro bioassays (figure 3). The ED mode of action will be investigated by in vivo studies by toxicogenomics, functional assays and ecotoxicity tests to link perturbations of key molecular events to adverse outcomes of regulatory relevance.
Figure 3. Approach to develop Adverse Outcome Pathways of endocrine disruption in D. magna.