Chronic exposure to Poly(hexamethylenebicyanoguanide-hexamethylenediamine) hydrochloride impairs molting and physiological functions in Daphnia magna across generations

• PHMB exerts dose and generation-dependent reduction in D. magna growth • PHMB significantly reduced molting frequency and reproductive output in D. magna • PHMB reduced behavioral indices in a concentration and generation-dependent manner • PHMB altered gene expression related to molting, reproduction, and cardiac function • PHMB induces toxicity across multiple generations of D. magna Poly(hexamethylenebicyanoguanide-hexamethylenediamine) hydrochloride (PHMB) is widely used in the environment and industries due to its antimicrobial properties. However, its effects on non-target organisms in aquatic ecosystems, especially on Daphnia magna across multiple generations remain poorly understood. Therefore, we aimed to comprehensively evaluate the multigenerational toxicity of PHMB under continuous exposure conditions by assessing its effects on the growth, cardiotoxicity, neurotoxicity, molting, and reproduction of D. magna across three successive generations (F0, F1, and F2). We conducted a multi-level, 21-day chronic exposure study for each generation of D. magna using 25, 50, and 100 nM PHMB. We systematically evaluated multidimensional indicators encompassing physiological, life history, and neurobehavioral traits, along with qPCR analysis of target genes to elucidate the underlying molecular mechanisms. PHMB induced progressive, dose-dependent toxicity across successive generations. Chronic exposure severely suppressed cardiovascular and motor functions, impaired ecdysis, and ultimately culminated in a catastrophic reproductive collapse by the F2 generation. These multi-level phenotypic impairments were molecularly underpinned by significant transcriptional dysregulation in genes governing ecdysteroid signaling, reproduction, cardiac stability, and neurotransmission. Exposure to PHMB causes cumulative multigenerational toxicity in D. magna , potentially leading to extensive physiological impairment and population-level reproductive decline. These findings underscore the critical necessity of incorporating multigenerational and mechanistic evaluations into ecological risk assessment paradigms to prevent underestimating the long-term threats of persistent polymers in aquatic environments.