There is an urgent need to evaluate the toxicity of xenobiotics and environmental mixtures for preventing loss in water quality for the sustainability of aquatic ecosystems. A simple prebiotic chemical pathway based on malate formation from pyruvate (pyr) and glyoxalate (glyox) is proposed as a quick and cheap screening tool for toxicity assessment. The hypothesis of this simple assay is that chemicals hindering the formation of biochemical precursors (malate) in conditions of prebiotic Earth before the appearance of structured life forms could form the basis of toxicity. Moreover, this early pro-metabolic pathway relies principally on the aldol condensation reaction. Hence, this assay is based on the pyr and glyox reactions in the presence of Fe(II) as a source of electrons using temperatures between 40 and 70^oC and leading to biologically relevant precursors such as oxaloacetate and malate. Malate and oxaloacetate are important intermediates of energy metabolism including sugars and lipids. Incubation of pyr and glyox at 40–70 ◦C in the presence of reduced iron Fe(II) led to malate formation following the first 3 h of incubation. The addition of various xenobiotics/contaminants (silver, copper, zinc, cerium IV, samarium III, dibytlphthalate, 1,3-diphenylguanidine, carbon-walled nanotubes, nanoFe2O3 and polystyrene nanoparticles) led to inhibitions in malate synthesis at various degrees. Based on the concentration inhibiting malate concentrations by 20% (IC20), the following potencies were observed: silver < copper ~ 1.3-diphenylguanidine ~ carbon-walled nanotube < zinc ~ samarium < dibutylphthalate ~ samarium < Ce(IV) < nFeO3 < polystyrene nanoplastics. The IC20 values were also significantly correlated with the reported trout acute lethality data, suggesting their potential as an alternative toxicity test. The pyr-glyox pathway was also tested on surface water extracts (C18), identifying the most contaminated sites from large cities and municipal wastewater effluents' dispersion plume. The inhibition potencies of the selected test compounds revealed that not only prooxidants but also chemicals hindering enolate formation, nucleophilic attack of carbonyls and dehydration involved in aldol condensation reactions were associated with toxicity. The pyr-glyox pathway is based on prebiotic chemical reactions during the emergence of life and represents a unique tool for identifying toxic compounds individually and in complex mixtures.