Decay dynamics of pBI143 compared to bacterial and viral fecal contamination indicators in wastewater and river water

• The decay behavior of pBI143 was compared with that of MST and ARG markers. • The average T 90 of pBI143 in wastewater ranged from 1 to 12 days. • PEG precipitation yielded the highest pBI143 concentrations. • pBI143 matched to HF183-TaqMan/ gyrB , but differed from CrAssphage and ARGs. • intI 1 and sul 1 showed rebound patterns in river water at 15 °C on day 5. Fecal contamination remains a public health concern; however, traditional indicators such as Escherichia coli lack the specificity needed for accurate source tracking. Emerging human-associated markers, such as plasmid pBI143, offer improved accuracy, although their behavior in water systems remains poorly understood. In this study, we evaluated the environmental stability of pBI143 in comparison to traditional microbial source tracking (MST) and antibiotic resistance gene (ARG) markers in river water and untreated wastewater. Additionally, the performance of polyethylene glycol (PEG) precipitation was compared with that of electronegative membrane filtration (EMF) for sample processing. Water samples were incubated at 4 °C, 15 °C, and 25 °C for 7 days (river water) and 14 days (wastewater) to demonstrate temperature variations. Quantified targets included human-associated markers (pBI143, gyrB , and HF183-TaqMan), intI 1, sul 1, CrAssphage, and HAdVs. The results revealed that pBI143 concentrations were highest in wastewater (8.8 ± 0.1 log 10 copies/L). In river water, PEG precipitation methods showed a maximum of 6.8 log 10 copies/L for pBI143, while EMF yielded comparable results. No significant differences were observed between EMF and PEG precipitation methods (Wilcoxon test, p > 0.05). The average time required for 90 % reduction (T 90 ) of pBI143 ranged from 1 to 12 days in wastewater, 2–12 days in downstream river water, and 3–10 days in upstream river water. These T 90 values resembled those of HF183-TaqMan and gyrB with short-term decay, whereas contrasted with CrAssphage, intI 1 , and sul 1 that demonstrated longer persistence. The findings support the utility of pBI143 as a reliable human-associated fecal marker, with consistent performance across temperatures and water types. Conversely, the unexpected rebound of intI 1 and sul 1 emphasizes the need for enhanced monitoring of downstream river environments, where ARGs may persist or re-emerge. Future research should explore the role of moderate temperatures in sustaining the long-term presence of MST and ARGs in aquatic environments.