Endocrine Disruption in Freshwater Cladocerans: Transcriptomic-Network Perspectives on TBOEP and PFECHS Impacts in Daphnia magna

Abstract Freshwater cladocerans such as Daphnia magna are keystone grazers whose hormone‑regulated life‑history traits make them sensitive sentinels of endocrine‑disrupting chemicals (EDCs). The organophosphate flame‑retardant tris(2‑butoxyethyl) phosphate (TBOEP) and perfluoroethylcyclohexane sulfonate (PFECHS) now co‑occur at ng L⁻¹–µg L⁻¹ in surface waters, yet their chronic sub‑lethal impacts on invertebrate endocrine networks remain unclear.We analysed two publicly available 21‑day microarray datasets (TBOEP: GSE55132; PFECHS: GSE75607) using Gene Ontology enrichment, STRING protein‑interaction networks, Drosophila phenotype mapping and KEGG(Kyoto Encyclopedia of Genes and Genomes) ‑anchored frameworks to build putative adverse outcome pathways (AOPs) for D. magna. Differentially expressed genes were clustered into functional modules and hub nodes ranked by degree and betweenness.TBOEP suppressed molting and growth, altering 1 157 genes enriched for metabolism and membrane processes; hubs VRK1, MIB2 and adenylosuccinate synthetase formed a muscle anatomical development sub‑network. PFECHS down‑regulated vitellogenin and shifted 879 genes dominated by oxidative‑stress and glutathione‑metabolism signatures; central nodes UBC9, eIF4A‑III, Tra‑2α and HDAC1 linked meiotic‑cycle, oogenesis and cyclic‑compound binding. Despite chemical dissimilarity, both compounds converged on Wnt‑signalling nodes—TBOEP via presenilin‑1, PFECHS via CK1ε/CK2—thereby reducing TCF/LEF‑dependent transcription. Predicted outcomes include impaired oocyte maturation, reduced fecundity and stunted body size, consistent with observed decreases in length and vitellogenin protein.Our network analysis indicates that environmentally relevant concentrations of TBOEP and PFECHS destabilise endocrine, developmental and metabolic pathways in D. magna without overt lethality, foreshadowing population‑level repercussions for freshwater plankton dynamics. Hub genes and Wnt‑mediated key events emerge as sensitive biomarkers for monitoring mixed EDC exposure.