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Biochars have drawn wide attention as adsorbents, carbon sequesters and soil re-mediators. However, these substances are ambiguous regarding their effects on the motility, phenotypic changes and potential adaptative mechanisms of soil organisms. This study investigated how peanut shell biochar (PBC) affects the C. elegans model via a one-choice selection test and RNA-seq analysis. The results showed that C. elegans were able to select either PBC or a water control, and a clear preference for PBC was observed after 48 h of exposure, with the chemotaxis index (CI) reaching approximately 1.0. The nematode preferences for PBC vs sterile PBC/graphite were not significant, which demonstrated that initial microorganisms and appearances were not the reasons for the worms' selection, but the selection behaviour was instead determined by volatile odours. The treatments also showed that biochar amendment significantly decreased the body length, brood size and superoxide dismutase (SOD) activity of C. elegans to 960.20 ± 15.23 μm, 173.22 ± 4.56, 165.81 ± 3.82 U/mL SOD, respectively. Then, a possible molecular mechanism of PBC-induced developmental and reproductive effects on C. elegans was explored. Differential gene expression analysis was performed, and 1625 genes (1425 up- and 225 downregulated genes) were regulated in response to PBC treatment. The top 20 regulated genes were col genes (col-129 ; col-81 ; col-139 ; col-71), bli-6 , perm-4 and his-24 , which indicated that cuticle collagen synthesis, eggshell formation and/or heterochromatin in postembryonic growth may be disrupted following exposure to PBC. Therefore, our study suggested that quality standards be used to test nematode preferences and responses to biochar amendment, with the aim of safe application in soils, seedling substrates or fertilizers. Unlabelled Image • C. elegans has a clear migration to PBC by volatile odours • C. elegans became shorter and had fewer off-springs by exposed to PBC • 84.65% DGEs genes were upregulated in response to PBC. • Regulating col- , bli-6 , perm-4 and his-24 genes can affect the growth and reproduction of C. elegans. [ABSTRACT FROM AUTHOR]