*Result*: Comprehensive evaluation of the toxicological effects of biochar on rice physiology, enzymology, and transcriptome.

*Biochar (BC) application in soil remediation has raised concerns about its phytotoxicity, yet existing research predominantly focused on phenotypic characteristics and physiological changes while the underlying molecular mechanisms remains poorly understood. Therefore, this study investigated the phytotoxic mechanisms of perishable waste biochar (PWB) and rice straw biochar (RSB) with different pyrolysis temperatures (i.e., 350, 500, and 650 °C) on rice seedlings by combining multiple indices of physiology, enzymology and transcriptome. Results demonstrated that PWB and RSB pyrolyzed at low temperature (i.e., 350 °C) significantly affected the germination, root elongation, shoot length, and chlorophyll contents (Chl a/Chl b) of rice seedlings. However, while PWB pyrolyzed at higher temperatures (i.e., 500 °C and 650 °C) significantly impacted all above parameters, RSB at these higher temperatures significantly affected the germination rate, shoot length, and chlorophyll contents, however, the root length was unaffected. The water-soluble components dominated the inhibitory effects of BC on seed germination and rice root/shoot growth, accounting approx. 44.0 % of germination inhibition, with contributing ∼100 % to root/shoot growth inhibition. Low temperature (i.e., 350 °C) BCs activated antioxidant and lipid peroxidation levels of rice seedlings, while higher temperature (i.e., 500 °C and 650 °C) biochar had little effects on the level of lipid peroxidation. Correlation analysis linked the oxidative damage to the growth inhibition and chlorophyll contents, suggesting that oxidative stress might be the key mechanism of phytotoxicity on rice seedlings by BCs. Transcriptome analysis showed that photosynthesis, signal transmission, and amino acids related metabolism were the main enriched metabolic pathways in rice under BC exposure. The enrichment of Mitogen-Activated Protein Kinase (MAPK) signaling pathway in low temperature PWB and carotenoid biosynthesis pathways in low temperature RSB regulated their toxicity effect on rice seedlings. These findings provide novel insights into the ecotoxicological risks of BC, emphasizing the previously neglected contribution of its water-soluble components, thereby facilitating more accurate risk assessments and the development of more effective mitigation methods.
(Copyright © 2026. Published by Elsevier Inc.)*

*Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.*