Establishment of a Flow‐Through System for the Macrophyte Growth Inhibition Test (OECD 239) Including Photosynthetic Activity Measurement to Determine Early Effects

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.