Mechanistic insights into synergistic and antagonistic effects of nanoplastics on antibiotic toxicities via a Biospectroscopy-Bioreporter Coupling (BBC) approach

• Polystyrene nanoplastics exhibited antagonism as concentrators and synergism as nanocarriers. • Raman spectroscopy and Random Forest algorithm recognized individual toxicants. • Successful toxicity source-apportionment from antibiotics and polystyrene Nanoplastics. • Aging altered nanoplastic morphology and their interactions with antibiotics. The co-occurrence of nanoplastics (NPs) and antibiotics poses emerging and complex toxicological challenges. In this study, we combined whole-cell bioreporter ( Acinetobacter baylyi ADPWH_recA) and Raman biospectroscopy to evaluate cytotoxicity and genotoxicity induced by three representative antibiotics of amoxicillin, ciprofloxacin, and tetracycline at environmentally relevant concentrations (0.01–1 mg/L), in the presence of 80-nm pristine (PS), UV-aged (uvPS), and freeze–thaw-aged (ftPS) polystyrene NPs. Amoxicillin and tetracycline alone induced cytotoxicity, whereas ciprofloxacin exhibited pronounced genotoxicity and elevated intracellular reactive oxygen species. Raman biomarkers ( e.g. , 1122, 1210, 1386 and 1700 cm −1 ) revealed distinct mechanisms involving membrane disruption, DNA damage, and lipid disorder. PS-NPs modulated antibiotic toxicity in a concentration- and type-dependent manner, acting as either adsorbents (antagonism) or nanocarriers (synergism). For cytotoxic antibiotics such as amoxicillin and tetracycline, PS-NPs primarily functioned as adsorbents, reducing bioavailability and mitigating toxicity. In contrast, for ciprofloxacin, which targets intracellular components, PS-NPs enhanced cellular uptake and exacerbated genotoxicity. A Random Forest model integrating spectral features achieved a classification accuracy of over 93 % and enabled the source-apportionment of toxicity, elucidating the dual roles of PS-NPs as both adsorbents and nanocarriers. These findings provide mechanistic insights into the interplay between nanoplastics and antibiotics, demonstrating the efficacy of the Biospectroscopy-Bioreporter coupling (BBC) strategy for risk assessment of emerging contaminants.