Microstructure and ecotoxicological response of Portland cement composites incorporating coal fly ash

• Incorporating 10–20 wt% fly ash improved cement matrix density and microstructure. • Fly ash addition reduced E. coli and P. aeruginosa biofilms by up to 2 log₁₀ CFU/cm². • FTIR revealed portlandite and calcite reduction after E. coli exposure. • Ecotoxicity tests showed <10 % inhibition for D. subspicatus and A. salina . • Slight phytotoxicity (∼70 % GI) occurred only at 1 g/L with no fly ash effect. Utilization of coal fly ash (FA) as a Portland cement (PC) admixture offers a promising yet underexplored approach to reduce PC’s environmental impact and enable sustainable reuse of industrial by-products. This study addresses the gap concerning the technical performance and environmental safety of FA-containing binders (PC-FA). FA characterization via XRD, XRF, EDS, and SEM revealed dominant crystalline phases (quartz, mullite, magnetite, rutile) and a heterogeneous microstructure. Incorporating 10 wt.% and 20 wt.% FA into PC produced denser matrices, refined microstructures, and enhanced pozzolanic activity without compromising mortar integrity (compactness, absence of microcracks, voids and segregation). Bioassays at environmentally relevant concentrations (1 g/L, 0.1 g/L, 0.01 g/L) were carried out on prokaryotic ( Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa ) and eukaryotic ( Desmodesmus subspicatus, Artemia salina, Sinapis alba ) models. No significant inhibition of bacterial growth was observed. Biofilm formation on all PC surfaces was significantly lower than on polystyrene (reference), with strain-specific effects: FA increased S. aureus biofilm formation, while E. coli and P. aeruginosa formed less biofilm on FA-doped samples than on PC-REF by up to 2 log 10 (CFU/cm 2 ). FTIR of PC composites incubated with E. coli indicated reduced portlandite and calcite contents, suggesting a lower internal pH and possible chemical disruption under bacteria-favouring conditions. Low toxicity (<10 %) of FA or PC-FA toward D. subspicatus and A. salina was determined. Low phytotoxicity (∼70 % germination index) was observed in S. alba at high exposure (1 g/L) for all PC types, without FA-related effect. These results support PC-FAs´ potential in sustainable construction applications.