Montmorillonite-fungal biomass hybrid as sustainable sorbent in rotating-disk sorptive extraction coupled to GC-MS for trace-level monitoring of estrogens in hospital wastewater.

In this study, we pioneered a bio-inorganic hybrid material by combining montmorillonite (MMT) with Pleurotus ostreatus mycelium. Moving beyond routine extraction phases, we focused on the preparation, physicochemical characterization, and application of this unique biohybrid for rotating-disk sorptive extraction (RDSE). The engineered material successfully blends the structured mesoporosity and high surface area of pristine clay with the dense, functionalized biopolymer network of fungal hyphae. By deconvoluting the sorptive mechanism, we found that estrogen retention is driven by a highly cooperative network of non-covalent forces. At a near-neutral wastewater pH (6.9-7.2), where the target steroids remain neutral, mass transfer is governed by localized hydrogen bonding with both clay edge sites (Si-OH, Al-OH) and cell-wall functionalities (NH2, -OH), supplemented by stabilizing cation-π interactions involving interlayer Ca2+ ions. When we applied this method to determine four estrogens (E1, E2, EE2, and E3) via gas chromatography-mass spectrometry (GC-MS) in complex hospital wastewater, our MMT-fungus sorbent significantly outperformed conventional primary-secondary amine phases. The protocol yielded low detection limits (1.1-9.1 ng L-1), quantitative recoveries (95-100%), and narrow intra-laboratory precision (%RSD ≤ 8.5%; Horwitz ratio [HorRat], 0.12-0.35). Authentic effluents revealed critical steroidal micropollutant levels between 195 and 853 ng L-1. Finally, multi-metric sustainability tools confirmed a highly favorable environmental profile (AGREEprep = 0.75, VIGI = 80, BAGI = 52.5), confirming that high-performance analytical chemistry can actively coexist with eco-friendly material engineering.