Phytotoxic effects of methylene blue dye on mustard (Brassica juncea L.): insights from germination bioassays and multivariate analysis

Abstract

Methylene blue (MB), a commonly employed cationic dye, contaminates aquatic and terrestrial ecosystems. Although its presence in polluted environments has been documented, little is known about its phytotoxicity to terrestrial agricultural crops. This study was conducted to assess the effect of MB on the germination and seedling development of a major oil crop, mustard (Brassica juncea L.), in a controlled hydroponic system. MB was found to significantly affect germination and seedling development. This effect was concentration-dependent. Germination was reduced by 50% compared to the control at higher concentrations. Similarly, root growth, shoot growth, seedling vigour index, and biomass were significantly reduced. On the other hand, relative toxicity and phytotoxicity increased substantially. To move beyond descriptive phenotyping, a predictive multivariate statistical analysis was employed to assess correlations between variables and establish physiological tipping points. Principal component analysis, hierarchical cluster analysis, and heatmap correlation analysis revealed a strong negative correlation between MB concentration and growth. Toxicity increased significantly with increasing MB concentration. This study revealed that MB significantly affects germination and seedling development and poses a potential risk to agricultural ecosystems, particularly during the early stages of crop establishment. Furthermore, by statistically identifying radicle elongation and seedling vigour as the most sensitive parameters, this study establishes them as reliable, early-warning biomarkers for agricultural risk assessment. Further field studies are needed to better understand its long-term impact on plant growth and productivity.