Mode of action assessment in Raphidocelis subcapitata by complementing the OECD test guideline 201 with transcriptomics

The growing number of chemicals worldwide increases the need for risk assessment and investigation of their environmental effects to protect ecosystems. Understanding molecular effects of substances in environmental organisms increasingly contributes to hazard assessment. The combination of ecotoxicological tests and transcriptome analysis enables to identify molecular effects and to understand the modes of action of substances at gene expression level. This study aimed to extend the ecotoxicity test according to OECD Test Guideline 201 with transcriptome analysis to identify gene expression changes associated with modes of action of the photosynthesis-inhibiting herbicide bentazone and the zinc oxide nanoparticle (ZnO NP) NM-110 in Raphidocelis subcapitata. Acute toxicity tests showed observable effects of both substances on R. subcapitata over 72 h. A newly established RNA extraction protocol enabled subsequent transcriptomic analyses at subacute exposure levels. These revealed significant numbers of differentially expressed genes at EC5 and EC20 of both substances compared to unexposed controls. Overrepresentation analysis using an eggNOG annotated R. subcapitata genome revealed that the observed molecular effects partially aligned with the established modes of action for each substance, yet differed from one another. However, both shared a common link to the generation of reactive oxygen species and oxidative stress. These findings contribute to a more detailed understanding of molecular effects of bentazone and ZnO NP NM-110 in R. subcapitata. The established workflow for toxicogenomic analysis in R. subcapitata represents a New Approach Methodology (NAM) to record molecular profiles of substances in the future in order to support environmental hazard assessment.