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Urea is a widely applied fertilizer to enhance crop yields. Ecological risks associated with the excessive application of urea fertilizer threaten the paddy fields' sustainable agriculture and biodiversity preservation. There are no practical thresholds based on proven data on microbial communities. Protozoa are nitrogen-sensitive organisms. For the first time, this study conducted acute and chronic urea toxicity tests on eight species of organisms. The results indicate that Blepharisma sp. is the most sensitive species to urea exposure and is a suitable indicator for determining the safe threshold of urea. This study estimated the predicted no-effect concentration using species sensitivity distribution curves. Subsequently, it established the threshold for urea application in rice fields based on the fields' area and the surface water's height. The short-term safety threshold for urea in the studied paddy field with black soil is 87.7 mg/L, equivalent to 43.85 kg of urea per hectare for a single nitrogen fertilizer application. The long-term safety threshold is 5.02 mg/L, representing the concentration for re-applicating urea. The biodiversity-safeguarding application threshold provides the basis for developing a urea fertilizer reduction protocol to safeguard the paddy fields' biodiversity., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Li Wang, Chunyu Hou, Benchao Jiang, Xuming Pan, Liangzi Cao, Tianyi Shi, Mei Han, Wenyuan Wang has patent #ZL 202310462078.4 licensed to Harbin Normal University. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)