Abstract
Algae are key components of aquatic food chains. Consequently, they are internationally recognised test species for the environmental safety assessment of chemicals. However, existing algal toxicity test guidelines are not yet optimized to discover molecular modes of action, which require highly-replicated and carefully controlled experiments. Here, we set out to develop a robust, miniaturised and scalable Chlamydomonas reinhardtii toxicity testing approach tailored to meet these demands. We primarily investigated the benefits of synchronised cultures for molecular studies, and of exposure designs that restrict chemical volatilisation yet yield sufficient algal biomass for omics analyses. Flow cytometry and direct-infusion mass spectrometry metabolomics revealed significant and time-resolved changes in sample composition of synchronised cultures. Synchronised cultures in sealed glass vials achieved adequate growth rates at previously unachievably-high inoculation cell densities, with minimal pH drift and negligible chemical loss over 24-h exposures. Algal exposures to a volatile test compound (chlorobenzene) yielded relatively high reproducibility of metabolic phenotypes over experimental repeats. This experimental test system extends existing toxicity testing formats to allow highly-replicated, omics-driven, mode-of-action discovery.
Highlights
Algae are internationally established test organisms in chemical risk assessment [1,2,3]
Following bioassay with inclusion of ante-natal, neonatal, adolescent, middle aged, old aged and pregnant animal introduction of cell cycle synchronisation, we aim to reduce the duration of exposure, and adjust subjects, and tissue samples from allfor of high-throughput these life-stages to infer a chemical mode of action patterns ofpooling sampling time-test duration screening, to enable (MoA)
We designed an alternative closed-vial chemical exposure system that allows for sufficient growth of C. reinhardtii for a metabolomics assay, while minimising test duration and chemical volatilisation
Summary
Algae are internationally established test organisms in chemical risk assessment [1,2,3]. One application of NAMs is to increase confidence in cause–effect analyses by providing mechanistic evidence on the harmful effects of chemicals, e.g., in the form of rapid and large-scale discovery of molecular key events (mKE) in an adverse outcome pathway (AOP) framework [5]. This transition, from traditional whole organism endpoint-based to molecular pathway-based (eco)toxicology, is motivated by the application of high-content technologies such as transcriptomics and metabolomics, which have demonstrated a capability to discover biological processes [6]. A robust, scalable test guideline for omics-driven discovery of toxicological key events in algae first requires a rigorously characterised algae culturing and exposure protocol with biological
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