Nepetalactone (NL) is the main bioactive constituent of Nepeta cataria essential oil (EO). Although its biocidal potential is well established, defining potential impacts on non-target aquatic organisms is important for environmental risk assessment. This study evaluated the ecotoxicity of pure cis–trans NL with that of N. cataria EO (72% NL), using the freshwater microalga Raphidocelis subcapitata, and Daphnia pulex, following OECD 201 (algal growth inhibition) and OECD 202 (Daphnia acute immobilisation) guidelines. Negative controls and a methanol solvent control were included; algal tests were run at five concentrations in triplicate, while Daphnia were exposed to twelve concentrations, and all endpoints were based on analytically confirmed exposure levels. For R. subcapitata, pure NL produced an ErC50 (growth-rate inhibition) of 41.78 ppm and an EyC50 (yield inhibition) of 21.11 ppm. The EO was substantially more toxic, with an ErC50 of 1.40 ppm NL equivalents (1.94 ppm EO) and an EyC50 of 11.14 ppm NL equivalents (15.46 ppm EO). In D. pulex, pure NL yielded a 48 h EC50 of 54.93 ppm (175.81 ppm at 24 h). Again, the EO showed higher toxicity, with a 48 h EC50 of 8.38 ppm NL equivalents (22.75 ppm at 24 h, NL equivalents). Overall, acute effects across two trophic levels at concentrations that may be environmentally relevant were shown, but the EO consistently exceeded the toxicity of isolated NL. This points to contributions from minor constituents and/or synergistic interactions within the EO, underscoring that botanical biopesticide risk assessments should evaluate whole extracts rather than relying only on active-ingredient data. In parallel, docking of NL to environmentally relevant targets (estrogen and androgen receptors, and TRPA1) is underway using validated workflows (AutoDock Vina, Avogadro, PubChem) to explore plausible binding interactions.

Acknowledgments: EFSA funding under the EU-FORA Programme (EUBA-EFSA-2024-ENREL-01).