Exploring α-Terpineol–Antibiotic Synergistic Combinations as an Environmentally Safer Antimicrobial Strategy for Freshwater Systems

Background: This study evaluated the ecotoxicity of five synergistic antimicrobial combinations of terpineol-antibiotic (SACTA), developed to reduce antibiotic (ABX) doses while maintaining antimicrobial efficacy. The ABXs tested were gentamicin (GTM), chloramphenicol (CHL), tetracycline (TC), and florfenicol (FF). Methods: Median effective concentrations (EC50) were determined using Aliivibrio fischeri and Daphnia magna. In addition, the effects of SACTAs on the metabolic profile of an indigenous riverine microbial community were assessed using Biolog EcoPlates™. Results: α-Terpineol (TER) alone showed EC50 values of 21.49 and 17.64 µg/mL for D. magna and A. fischeri, respectively. The toxicity of SACTAs varied depending on the antimicrobial combination, exhibiting synergistic, additive, or antagonistic effects. Among the tested combinations, TER-CHL was the most toxic with EC50 values of 7.84 µg/mL for D. magna and 12.75 µg/mL for A. fischeri. In contrast, TER-FF and TER-GTM, with lower TER proportions, were the least toxic, with EC50 values exceeding 30 µg/mL for D. magna and 60 µg/mL for A. fischeri. Although SACTAs generally showed higher toxicity than ABXs tested individually, their EC50 values remained below the minimum inhibitory concentrations (MICs) determined in single-species antimicrobial assays. In contrast, riverine microbial communities displayed greater resilience, with only additive or antagonistic effects observed. Among the tested combinations, TER-FF produced the strongest effects on the microbial community, reducing metabolic fitness and decreasing the utilization of carbohydrates and polymers. Importantly, all SACTAs exhibited toxicity values equal to or lower than those of ABXs tested alone at their respective MICs, based on changes in community growth and metabolic profiles. Conclusions: Overall, these findings suggest that SACTAs may represent a promising strategy for reducing ABX use while minimizing additional ecotoxicological risks to freshwater ecosystems.