Triethyl Phosphate: An Emerging Environmental Stressor Inducing Locomotor Dysfunction in Crucian Carp of the Yellow River Estuary – Novel Insights from in Situ Ecotoxicoproteomic Profiling

With the ongoing degradation of aquatic ecosystems, the identification of environmental stressors presents a significant scientific challenge, constrained by methodological limitations. This study proposes an integrative strategy combining ecotoxicoproteomics, in vitro / in vivo bioassays, and advanced chemical analyses to identify stressors and assess their hazards through biomarker discovery and adverse outcome (AO) validation. When applied in the Yellow River Estuary, this approach revealed key findings: Ecotoxicoproteomic analysis of sentinel species, crucian carp ( Carassius auratus ), identified membrane receptor integrin α v β 3 as a key biomarker linked to neurotoxicity-related AOs. An in vitro competitive binding assay revealed higher integrin α v β 3 binding activity in mainstream versus tributary samples. Comprehensive chemical analysis identified triethyl phosphate (TEP) as the primary causative toxicant, accounting for over 46% of integrin α v β 3 binding activity. In vivo studies confirmed that TEP induced dose-dependent neurodevelopmental toxicity and locomotor dysfunction in crucian carp, with a benchmark dose lower confidence limit of 120 ng/L, aligning with environmental concentrations of 87.52–328.84 ng/L in the mainstream. This study substantiates TEP as an emerging stressor that induces locomotor dysfunction in fish within the study area. By establishing a novel identification strategy for environmental stressors, it provides a scientific foundation for mitigating aquatic ecosystem degradation in the Yellow River Estuary.