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Background: Zinc oxide nanoparticle (nZnO) and chemicals with different mode of action (MOA, i.e., narcotic and reactive) were frequently detected in the Yangtze River. Organisms are typically exposed to mixtures of nZnO and other chemicals rather than individual nZnO. Toxicity of nZnO is caused by the dissolution of Zn2+, which has been proved in the field of single toxicity. However, it is still unclear whether the released Zn2+ plays a critical role in the nZnO toxicity of nZnO–chemicals mixtures. In the present study, the binary mixture toxicity of nZnO/Zn2+ and chemicals with different MOA was investigated in acute (15 min) and chronic (12 h) toxicity test upon Vibrio fischeri (V. fischeri). The joint effects of nZnO and tested chemicals were explored. Moreover, two classic models, concentration addition (CA) and independent action (IA) were applied to predict the toxicity of mixtures. Results: The difference of toxicity unit (TU) values between the mixtures of Zn2+–chemicals with those of nZnO–chemicals was not significant (P > 0.05), not only in acute toxicity test but also in chronic toxicity test. The antagonistic or additive effects for nZnO-chemicals can be observed in most mixtures, with the TU values ranging from 0.75 to 1.77 and 0.47 to 2.45 in acute toxicity test and chronic test, respectively. We also observed that the prediction accuracy of CA and IA models was not very well in the mixtures where the difference between the toxicity ratios of the components was small (less than about 10), with the mean absolute percentage error (MAPE) values ranging from 0.14 to 0.67 for CA model and 0.17–0.51 for IA model, respectively. Conclusion: We found that the dissolved Zn2+ mainly accounted for the nZnO toxicity in the mixtures of nZnO–chemicals, and the joint effects of these mixtures were mostly antagonism and additivity. CA and IA models were unsuitable for predicting the mixture toxicity of nZnO–chemicals at their equitoxic ratios. [ABSTRACT FROM AUTHOR]