TOXICITY ASSESSMENT OF ZINC OXIDE NANOPARTICLES AND ORGANIC POLLUTANTS ON ZEBRAFISH USING BLISS INDEPENDENCE

Background: Zinc oxide nanoparticles (ZnONPs) and chlorhexidine (CHX), a persistent organic pollutant (POP), are widely used engineered nanomaterials (ENMs) that may threaten freshwater ecosystems. While ZnONPs are considered safer than metal salts, they can still induce oxidative stress, membrane damage, and ion release. The co-occurrence of CHX and ZnONPs in aquatic systems raises concerns over combined toxic effects, which conventional risk assessment methods may not adequately address. Aim: To evaluate the individual and combined acute toxicity of ZnONPs and CHX on zebrafish (Danio rerio) embryos, and to characterize their interaction using the Bliss Independence model. Materials and Methods: Zebrafish embryos (<3 h post-fertilization) were exposed for 72 h to ZnONPs, CHX, and their binary mixtures (1:1) at IC₂₀ and IC₅₀ concentrations in 24-well plates under controlled conditions. ZnONPs were synthesized and characterized by UV–Vis spectroscopy and dynamic light scattering (DLS). Endpoints assessed included mortality, heart rate reduction, teratogenicity, and spinal deformities. Interaction effects were analyzed using the Bliss Independence model, with statistical evaluation by one-way ANOVA and Tukey’s post hoc test. Results: The 72-h LC₅₀ values were 4.5 ppm (ZnONPs) and 3.9 ppm (CHX). Mixture toxicity varied with ratio and concentration: antagonism predominated at IC₂₀ and IC₅₀ (CI > 1) in 1:1 and 1:2 ratios, while CHX-rich mixtures showed synergism (CI < 1) at IC₇₀. CHX-heavy mixtures exhibited the highest lethality and teratogenicity, whereas ZnONP-rich mixtures reduced CHX toxicity, likely via decreased bioavailability or ROS scavenging. Conclusion: Mixture toxicity of ZnONPs and CHX is ratio- and endpoint-dependent, highlighting the need for interaction-based ecological risk assessments.