In addition to the promoting effect on plant morphological and physiological responses, nCu is essential for animal nutrition. Several studies have revealed that the dose of 2 mg nCu/l is a threshold limit value added to drinking water of mice [2, 3], although administration of intestinal epithelial cells with 2 mg nCu/l results in cell injury and toxicity (27). Differently, nCu treatment of chicken at the dose of 5 to 15 mg/l in drinking water has no effect on Cu content in the blood plasma (8). Our results denoted that supplementation with 2 mg nCu/l in the drinking water did not show any apparent changes in mice as proved by unaltered parameters including accumulation of Cu in liver and blood circulation, hepatic and renal biochemical markers and infiltration and activation of splenic immune cells as compared to the control group; therefore, nCu concentration of 2 mg/lL would be used for further applicable biomedicine.