Common antimicrobials disrupt early zebrafish development through immune-cardiac signaling

Abstract

The global production and use of antimicrobial chemicals surged during and after the COVID-19 pandemic, yet their developmental toxicity in aquatic organisms at environmentally relevant concentrations remains poorly understood. Here, we investigate and compare the developmental effects of two restricted antimicrobial chemicals—triclosan (TCS) and triclocarban (TCC)—and three alternative antimicrobials—benzalkonium chloride (BAC), benzethonium chloride (BEC), and chloroxylenol (CX)—on zebrafish embryos (Danio rerio) at concentrations of 0.4, 4, and 40 μg L⁻¹. We find that BAC induces the most severe reduction in hatching rates, followed by TCS, TCC, BEC, and CX. BAC also exhibits the strongest inhibition of heart rate, with toxicity levels comparable to those of TCS and TCC. All tested chemicals, except CX, cause significant teratogenic effects. Transcriptomic analysis reveals substantial disruptions in immune-related coagulation cascades and mitogen-activated protein kinase signaling pathways. Further validation via protein-protein interaction network analysis and real-time quantitative polymerase chain reaction confirms that altered expression of key hub genes in these pathways impacts bone and heart development, as well as immune system function, potentially driving developmental toxicity. This study provides the first systematic comparison of developmental toxicity among currently used antimicrobials at environmentally relevant concentrations, revealing that the alternative antimicrobial BAC poses greater developmental risks than the banned TCS and TCC. These findings raise concerns about the safety of BAC as a widespread substitute and highlight the necessity for more rigorous environmental risk assessments of alternative antimicrobials before their large-scale application.