RESUMO
Maintaining constant exposure concentrations during ecotoxicological studies while testing rapidly degradable substances is a challenge. To achieve stable concentrations during exposure, flow-through systems are used. To assess the impact of substances on higher aquatic plants, the 14-day macrophyte water-sediment Myriophyllum spicatum growth inhibition test (Organisation for Economic Co-operation and Development [OECD, 2014a] test guideline 239) only includes a static or a semistatic test design. The main aim of our study was to investigate the applicability of a flow-through system for M. spicatum. The standard OECD test design was miniaturized, and a flow-through system with spill-over was developed to achieve stable exposure concentrations of a rapidly degrading substance. The main endpoints were total shoot length and fresh and dry weight. Photosynthetic activity was used as an endpoint for the identification of early effects using the noninvasive Image-Producing Pulse Amplitude Modulation (IMAGING-PAM) procedure. Atorvastatin (AV; fast degrading) and bentazone (BT; photosynthesis inhibitor) were used as model substances to observe differences of the effect concentration depending on the test design. At higher exposure levels of AV, stronger necrosis combined with lower effect concentrations was observed in the flow-through test compared with the semistatic test, indicating the applicability of the flow-through test for evaluating degradable substances. The test with BT demonstrated a concentration-dependent decrease in the photosynthetic yield (Y(II)) from day 3 onward even before macroscopically visible changes occurred. Our results show that the flow-through system in the macrophyte growth inhibition test (OECD test guideline 239; 2014a) is a suitable alternative when one is testing rapidly degradable substances such as AV. In addition, we showed that photosynthetic yield can serve as a supplementary endpoint, when one is testing substances with photosynthesis inhibition as a mode of action. Environ Toxicol Chem 2024;00:1-12. © 2024 The Author(s). Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.
RESUMO
In environmental risk assessment of substances, the 14-day growth inhibition test following OECD test guideline 239 is employed to assess toxicity in the macrophyte Myriophyllum spicatum. Currently, this test evaluates physiological parameters and does not allow the identification of the mode of action (MoA) by which adverse effects are induced. However, for an improved ecotoxicity assessment of substances, knowledge about their ecotoxic MoA in non-target organisms is required. It has previously been suggested that the identification of gene expression changes can contribute to MoA identification. Therefore, we developed a shortened three-day assay for M. spicatum including the transcriptomic assessment of global gene expression changes and applied this assay to two model substances, the herbicide and photosynthesis inhibitor bentazone and the pharmaceutical and HMG-CoA reductase inhibitor atorvastatin. Due to the lack of a reference genome for M. spicatum we performed a de novo transcriptome assembly followed by a functional annotation to use the toxicogenomic results for MoA discrimination. The gene expression changes induced by low effect concentrations of these substances were used to identify differentially expressed genes (DEGs) and impaired biological functions for the respective MoA. We observed both concentration-dependent numbers and differentiated patterns of DEGs for both substances. While bentazone impaired genes involved in the response to reactive oxygen species as well as light response, and also genes involved in developmental processes, atorvastatin exposure led to a differential regulation of genes related to brassinosteroid response as well as potential metabolic shifts between the mevalonate and methyl erythritol 4-phosphate pathway. Based on these responses, we identified biomarker candidates for the assessment of MoA in M. spicatum. Utilizing the shortened assay developed in this study, the investigation of the identified biomarker candidates may contribute to the development of future MoA-specific screening approaches in the ecotoxicological hazard prediction using aquatic non-standard model organisms.