Transformation of β-Lactam Antibacterial Agents during Aqueous Ozonation: Reaction Pathways and Quantitative Bioassay of Biologically-Active Oxidation Products

Reactions of ozone (O3) with the beta-lactam antibiotics penicillin G (PG) and cephalexin (CP) have previously been found to yield products retaining antibacterial activities. These products are unequivocally identified here as the stereoisomeric (R)-sulfoxides of each parent molecule and characterized by a combination of chemical analysis and an antibacterial activity assay. PG-(R)-sulfoxide, which is approximately 15% as potent as PG itself, is formed in approximately 55% yield, whereas CP-(R)-sulfoxide, which is 83% as active as CP, is formed with a maximum 34% yield. PG-(R)-sulfoxide is recalcitrant toward further oxidation by O3, but readily transformed by hydroxyl radical (HO*) (kHO*,app"=7.4x10(9) M(-1) s(-1), pH 7), resulting in quantitative elimination of its antibacterial activity. In contrast, CP-(R)-sulfoxide is degraded by both O3 and HO* (kO3,app"=2.6x10(4) M(-1) s(-1) and kHO*,app"= 7.6x10(9) M(-1) s(-1), pH 7), leading to quantitative elimination of its antibacterial activity. During ozonation of a secondary municipal wastewater effluent sample (pH 8.1, CDOC=4.0 mg/L, [alkalinity]=3.6 mM as HCO3-) spiked with [PG]0=1 microM, PG-(R)-sulfoxide yields did not exceed 0.15 microM for O3 doses up to 100 microM (4.8 mg/L), but reached 0.47 microM with 10-mM t-BuOH added as a HO* scavenger. In contrast, CP-(R)-sulfoxide yields did not exceed 0.1 microM for the same wastewater spiked with [CP]0=1 microM in either the presence or absence of t-BuOH, indicating that CP-(R)-sulfoxide transformation is governed primarily by direct reaction with O3. These findings suggest that, for a given degree of parent compound transformation, PG-(R)-sulfoxide yields would likely be greatest during ozonation of wastewaters characterized by low O3 demands and high HO* scavenging rates, whereas CP-(R)-sulfoxide yields would be less matrix-dependent. In general, complete deactivation of penicillins during wastewater treatment will likely require higher O3 exposures than necessary for deactivation of cephalosporins.