In this study, the toxic effects of binary mixtures of Pharmaceutical Active Compounds (PhACs) that are observed in the
effluents from a wide range sewage treatment plants and surface water bodies were investigated using the bioluminescent
bacterium Aliivibrio fischeri (Microtox® test). The selected chemicals were the nonsteroidal anti-inflammatory drug (NSAID)
diclofenac sodium [DCF] and the anti-epileptic carbamazepine [CBZ]. In addition, caffeine [CFF], a psychoactive stimulant of
the central nervous system, was also included in the study. Binary combinations were prepared at a predefined ratio that
corresponded to the individual IC50 values of the investigated compounds (equitoxic ratio). The experimental results were
compared with those obtained using the two most frequently used predictive models in aquatic toxicology: the Concentration
Addition (CA) and Independent Action (IA) models. The results indicated that both models predict the observed mixture toxicity
of the DCF-CBZ and DCF-CFF mixtures quite well. However, in the case of CFF-CBZ, both models slightly overestimated the
experimental results, suggesting the presence of a potential antagonistic effect. The application of the Combination Index (CI)
method, which allows us to identify and quantify the nature of the interactions between the chemicals present in a mixture
(synergistic, additive or antagonistic effects), confirmed the additive behaviour of the DCF-CBZ and DCF-CFF combinations and
the slightly antagonistic effect observed for the binary mixture of CFF-CBZ.