Early-life ciprofloxacin exposure induces carry-over metabolic and developmental responses in soybean under single and combined glyphosate exposure

Environmental contaminants frequently occur as complex mixtures in agricultural systems, yet their biological effects may persist even when residues are no longer detectable in harvested soybean seeds. This study evaluated whether a single soil exposure to ciprofloxacin (CIP), applied alone or in combination with glyphosate (G), induces persistent metabolic and developmental responses in soybean (Glycine max). Soybean plants were exposed at sowing to environmentally relevant concentrations of CIP (0.10 and 1.00 mg L⁻¹), either alone or combined with G. Ciprofloxacin residues were not detected in harvested seeds. However, integrated Ultra-High-Performance Liquid Chromatography (UHPLC), Gas Chromatography-Flame Ionization Detector (GC-FID), and ^1H Nuclear Magnetic Resonance (NMR) analyses revealed treatment-dependent alterations in folic acid, isoflavones, carbohydrate-related metabolites, and selected fatty acids. Exploratory multivariate analyses revealed distinct treatment-associated patterns and identified the low-dose combined exposure treatment as the most distinct metabolic profile. Although germination remained unaffected, progeny seedlings exhibited treatment-dependent differences in primary root development, indicating carry-over effects associated with parental exposure. The observed responses were particularly evident under combined exposure conditions, suggesting metabolic modulation associated with combined exposure. Collectively, these findings indicate that early-life contaminant exposure following a single application at sowing can be associated with persistent biochemical and developmental responses. These results highlight the importance of integrating metabolomic approaches with conventional residue analysis to improve the assessment of biological responses to emerging contaminants and contaminant mixtures in agroecosystems.