Role of humic acid in modulating the combined toxicity of polylactic acid microplastics and diflubenzuron: Impacts on mitochondrial genotoxicity in Daphnia magna

The proliferation of biodegradable plastics like polylactic acid (PLA) necessitates a deeper understanding of their ecotoxicological impacts, particularly their interactions with co-existing pollutants in aquatic ecosystems. This study investigates the combined toxicity of PLA microplastics (MPs), the insecticide diflubenzuron (DFB), and the influence of natural organic matter (NOM) on the freshwater cladoceran Daphnia magna . Acute 48-hour exposures were conducted to assess mortality, while sublethal impacts were evaluated by measuring oxidative stress biomarkers (reactive oxygen species, malondialdehyde), mitochondrial DNA (mtDNA) copy number, and cellular ATP levels. The interaction between PLA MPs and humic acid (HA) led to the formation of bridge-like structures. The presence of HA generally alleviated the enhanced acute toxicity resulting from the combined exposure to PLA MPs and DFB. At sublethal concentrations, the combination of high DFB (50 ng L⁻¹), HA (5 mg L⁻¹), and PLA MPs (1 mg L⁻¹) resulted in a reduction in mtDNA copy number, which in turn triggered enhanced ATP content as a compensatory mechanism at the cellular and molecular levels. These findings indicate that NOM, acting as an environmental modifier, significantly influences the toxicity of biodegradable MPs and pesticides at various biological levels, emphasizing the need to integrate such natural factors into the risk assessment frameworks for these emerging contaminants. • Surface morphology and elemental composition of PLA MPs were altered through interaction with HA. • The addition of HA mitigated the acute toxicity of the PLA MP and DFB co-exposure in D. magna . • At 50 ng L⁻¹ DFB, PLA–HA heteroaggregates decreased mtDNA copy number in D. magna . • The PLA–HA and DFB (50 ng L −1 ) combination induced a compensatory increase in ATP content.