High-Throughput Effect-Directed Analysis of Androgenic Compounds in Hospital Wastewater: Identifying Effect Drivers through Non-Target Screening Supported by Toxicity Prediction

The increasing number of contaminants released into the
environment
necessitates innovative strategies for their detection and identification,
particularly in complex environmental matrices like hospital wastewater.
Hospital effluents contain both natural and synthetic hormones that
might significantly contribute to endocrine disruption in aquatic
ecosystems. In this study, HT-EDA has been implemented to identify
the main effect-drivers (testosterone, androsterone and norgestrel)
from hospital effluent using microplate fractionation, the AR-CALUX
bioassay and an efficient data processing workflow. Through nontargeted
screening, over 5000 features (ESI+) were initially detected, but
our workflow’s prioritization based on androgenic activity
prediction reduced the number of features requiring further analysis
by over 95%, significantly streamlining the workload. In addition,
the semiquantitative nontarget analysis allowed for the calculation
of the contribution of an identified compound to the total activity
of the sample without the need for reference standards. While this
contribution was low (∼4.3%) and applicable to only one compound
(1,4-androstadiene-3,17-dione), it presents the first approach for
calculating such contributions without relying on standards. Compared
to the available alternatives our workflow demonstrates clear environmental
relevance by enhancing HT-EDA for more efficient identification and
prioritization of effect-drivers in hospital effluents, and it can
be adapted to address other environmental threats in complex mixtures.