Responses of zebrafish to pristine and UV-aged polyethylene microplastics: Single and combined effects with galaxolide (HHCB) in an aquatic microcosm

Microplastics (MPs) and synthetic musk compound galaxolide (HHCB) are widely prevalent new pollutants in aquatic environments, yet their combined toxic effects have not been fully elucidated. This study utilized an aquatic microcosm system to simulate real aquatic ecosystem and explored the toxic effects of polyethylene microplastics (PE-MPs) (2, 20 mg/L) and HHCB (2.5, 62.5 μg/L) on zebrafish ( Danio rerio ) under single and combined exposure conditions. PE-MPs include both pristine and UV-irradiated aged polyethylene microplastics (p-PEs and a-PEs). After 28 days of exposure, transcriptomic analysis indicated that p-PEs exposure upregulated genes in the cardiac muscle contraction pathway, while a-PEs interfered with mitosis by downregulating the spdl1 gene, hindering individual growth and development. Combined exposure of PE-MPs and HHCB significantly upregulated proteasome subunit genes ( psmb13a and psmb9a ), suggesting potential immunoproteasome activation that has been associated with inflammatory processes in mammalian studies, though disease relevance in fish requires further investigation. Increased PE-MPs concentrations downregulated genes related to phototransduction signaling pathways (such as opn1mw3 , opn1mw4 , rpe65a , rgs9a ), potentially leading to circadian rhythm disorders. Higher concentrations of a-PEs (20 mg/L) also caused more severe metabolic disturbances and signal transduction abnormalities. The addition of HHCB affected cytokine-cytokine receptor interaction, possibly resulting in inflammatory responses and even carcinogenic risks. Furthermore, increased concentrations of a-PEs and the addition of HHCB disrupted normal cardiac muscle contraction functions, causing myocardial damage. Biological activity assays further demonstrated that PE-MPs and HHCB induced oxidative stress, neurotoxicity, and disrupted osmoregulation and glycogen metabolism in zebrafish. This study provides critical data for ecological and health risk assessment of combined pollutants in actual aquatic ecosystem, calling for attention to the single and combined effects of PE-MPs and organic pollutants in aquatic ecosystem. • Aged polyethylene microplastics (a-PEs) are more toxic than pristine ones (p-PEs) • Co-exposure to PEs and HHCB impaires proteasome function • An increase in PEs concentration causes rhythm and metabolic disorders • The addition of HHCB leads to inflammatory responses and even cancer development • PEs and HHCB disrupt osmoregulation and glycogen metabolism