With the rapid increase in the synthesis and application of graphene oxide (GO), questions have emerged about its inadvertent entry into aquatic habitats and the ecological consequences associated with such exposure While several studies have addressed the acute effects of GO, knowledge on its chronic impacts across multiple trophic levels remains limited. In this study, we assessed the chronic toxicity of a well-characterized GO product using model organisms representing three trophic levels: the bioluminescent marine bacterium Aliivibrio fischeri, unicellular green algae (Chlamydomonas reinhardtii, Chlorella vulgaris, Desmodesmus subspicatus), the cyanobacterium Synechococcus elongatus, and the freshwater cladoceran Daphnia magna. Endpoints included bioluminescence inhibition in bacteria, growth inhibition in photosynthetic primary producers, and reproduction and refined physiological parameters (heart rate, feeding activity) in D. magna. Our results demonstrated clear concentration-dependent chronic effects of GO, with A. fischeri, the applied photosynthetic primary producers and D. magna exhibiting significant inhibition of bioluminescence, growth, delayed onset of reproduction, and reduced fitness parameters, respectively. Based on the collected data, a comprehensive ecotoxicological risk assessment was carried out, revealing that pristine GO may pose negligible hazard to aquatic ecosystems under environmentally relevant exposure scenarios. The outcomes clearly demonstrate the relevance of incorporating chronic and multi-trophic effects when evaluating the ecological risks of emerging nanomaterials such as GO.

As the exposure of the aquatic ecosystem to graphene oxide (GO) increases with its growing production and use, understanding the structure–property–toxicity relationships becomes increasingly critical in the development of effective safe design guidelines. An appropriate testing methodology is crucial in ecotoxicity assessments to accurately characterize the environmentally relevant toxicity of nanoparticles, particularly for GO, where the physicochemical properties fundamentally determine their interactions and toxicity toward aquatic organisms. Many ecotoxicological methods require the heat sterilization of samples as a preliminary treatment prior to analysis. To investigate changes in toxicity profiles induced by wet heat sterilization pretreatments (autoclaving and Tyndall treatment) of a well-characterized GO product, a comprehensive ecotoxicological evaluation was performed with Daphnia magna. This included conventional lethality and immobilization tests, along with sublethal endpoints such as heart rate and feeding activity, supplemented with the analysis of oxidative stress biomarkers. Physicochemical alterations in GO due to sterilization were examined with dynamic light scattering, ultraviolet-visible, and thermogravimetry/mass spectrometry. Sublethal endpoints were shown to be more sensitive indicators of toxicity than conventional methods, with feeding activity and heart rate inhibition demonstrating time and concentration-dependent effects. Heat-sterilized GOs exhibited greater ecotoxicity compared to pristine GO, as evidenced by elevated ROS levels and increased oxidative stress biomarkers (GPx and GST activities), implicating oxidative stress as a central mechanism of toxicity. Despite the subtle differences observed in the physicochemical properties, the impact of heat sterilization on toxicity is clear. Our research underscores the critical importance of adopting appropriate testing and evaluation methodologies for comparing GO ecotoxicity results under axenic and non-axenic conditions as well as a multimarker approach to accurately evaluate the risks posed by GO.