High-risk compounds and transformation products in wastewater treatment plants: A non-targeted analysis workflow...

Wastewater treatment plants (WWTPs) are critical barriers against various contaminants. Rapid screening of high-risk compounds is therefore essential, yet current data-intensive methods laborious and slow, hindering timely identification before environmental and human harm occurs. This study provides a rapid screening method for identifying high-risk compounds in WWTPs that have received minimal attention to date, utilizing a non-targeted analysis (NTA) workflow combined with a risk prioritization strategy. Molecular network was then employed to explore the transformation products (TPs) of high-risk compounds, and toxicological prioritization index (ToxPi) scores were used to prioritize the risks of substances with transformation relationships, aiming to achieve a more comprehensive risk screening profile. The NTA workflow extracted 3377 positive molecular features and 5976 negative molecular features from effluent samples of the main treatment units at a large municipal WWTP in eastern China. Process tracking revealed the biological tank had the best removal efficiency, but identified a major blind spot: poor removal of low molecular weight (200-300 Da) compounds. Among 4521 tentatively identified compounds, four high-confidence (identification scores >7) 'Danger' compounds (phytosphingosine, 3-methyl-2-oxobutyric acid, catechol, m-cresol) were prioritized. The "identification score >7" refers to the matching score between the acquired MS/MS spectrum and the spectral library in MS-DIAL (score range 0-10). A higher score indicates greater spectral similarity. Molecular network analysis of their TPs showed that, except for catechol, TPs had higher ToxPi scores than parent compounds. Our findings not only expose a key limitation in current treatment processes but also provide a novel perspective for more comprehensive environmental risk assessment and management of high-risk compounds.