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Publication Analysis of phytotoxicity and plant growth stimulation by multi-walled carbon nanotubes(2016) Zaytseva, Olga; Neumann, GünterNanotechnology is a rapidly expanding area of science and technology, which has gained a great interest due extraordinary properties of nanomaterials with numerous potential fields for practical application. Meanwhile, carbon nanotubes (CNTs) are among the ten most-produced engineered nanomaterials worldwide with applications in automotive industry, building and construction, electronics, and many other industrial sectors, showing also a great potential for integration into environmental and agricultural applications. However, during the last decade it has been demonstrated that nanomaterials can exert significant and extremely variable effects also on living organisms. In higher plants, both, positive and negative responses on growth and development have been reported but the related mechanisms are still not entirely understood. This study presents a systematic assessment of CNT effects on representative crops under standardized conditions with special emphasis on interactions with plant nutrition. After the introductory background (Chapter 1), presenting a comprehensive literature review on carbon nanomaterials with special emphasis on plant responses, environmental and agricultural applications, Chapter 2 describes the impact of selected multi-walled carbon nanotubes (MWCNTs) on seed germination and early seedling development of different crops (soybean−Glycine max, maize−Zea mays, and common bean−Phaseolus vulgaris). In face of highly variable plant responses to CNT treatments reported in the literature, the study was designed as a systematic analysis under standardized growth conditions, dissecting the effects of one single type of MWCNTs, depending on plant species, MWCNT dosage, duration of exposure to MWCNT treatments, and plant-developmental stage, including imbibition, germination and seedling development. Short-term seed treatments (36 h) with MWCNTs reduced the speed of water uptake particularly by soybean seeds, associated with an increased germination percentage and reduced formation of abnormal seedlings. However, during later seedling development, negative effects on fine root production were recorded for all investigated plant species. Inhibition of root growth was associated with reduced metabolic activity of the root tissue and a reduction of nitrate uptake, which could be mainly attributed to the smaller root system. The results demonstrated that even under standardized growth conditions largely excluding external factors, plant responses to MWCNT exposure exhibit differences, depending on plant species but also on the physiological status and the developmental stage of individual plants. Soybean was selected as a model plant for further studies since both, positive and negative effects of the same dose of MWCNTs (1000 mg L-1) could be observed even in the same individual plants. Chapter 3 investigates effects of short-term soybean seed exposure (36 h) to MWCNTs on seedling development, depending on the nutrient availability of the substrate. At 8 DAS stunted growth and poor fine root production were first detectable in seedlings germinating on moist filter paper without additional nutrient supply. This effect was preceded by reduced metabolic activity of the seedling tissues detectable by vital staining already at 2 DAS. Root growth inhibition was a long-lasting effect, detectable in soil culture up to 38 DAS. More detailed investigations revealed zinc (Zn) deficiency as a major growth-limiting factor. The growth of affected soil-grown plants was recovered by foliar application of ZnSO4 or by cultivation in nutrient solution supplied with soluble ZnSO4. A more detailed investigation of the physiological mechanisms related with the inhibitory effects of MWCNTs on plant growth is presented in Chapter 4. Oxidative stress was identified as a major factor determining MWCNT-induced root growth inhibition in soybean, demonstrated by recovery of root development after external supplementation with antioxidants. Induction of oxidative stress by MWCNT application was detectable already after the 36 h imbibition period particularly in the tips of the radicle as indicated by accumulation of superoxide anions, reduced triphenyltetrazolium chloride vital staining, and induction of superoxide dismutase activity. The expression pattern of the oxidative stress indicators coincided with preferential accumulation of MWCNTs in the cells of the root tip and was reverted by external application of proline as antioxidant. MWCNT-induced plant damage could be reverted by external supplementation of micronutrients (Zn, Cu, Mn) as important cofactors for various enzymes involved in oxidative stress defense (SOD, biosynthesis of antioxidative phenolics). Accordingly, SOD activity increased in seedling roots after Zn supplementation. During germination, the CNT treatments inhibited particularly the Zn translocation from the cotyledons to the growing seedling, and CNTs exhibited a selective adsorption potential for Zn and Cu, which may be involved in internal immobilization of micronutrients. Therefore, this study demonstrated for the first time that phytotoxicity of CNTs is linked with disturbances of micronutrient homeostasis during seedling development. Implications for environmental phytotoxicity assessment of MWCNTs and their potential applications in agriculture are discussed in a final overview presented in Chapter 5.