## 3 Results and Discussion
### Growth, Lignocellulosic Composition and Metal Uptake by Vetiver Grass Grown in Stamp Sand
The stamp sand and the control soil had significantly different levels of PTMs, including copper, using [PERSON]'s \(t\)-test with \(\alpha\) = 0.05. The physico-chemical properties of stamp sand indicate that it is not favorable for plant growth due to elevated metals, low water holding capacity, and low organic matter content [37; 38]. Stamp sand analyzed by sequential extraction revealed that the largest fraction (other than residual) for Cu was the acid-soluble fraction. This raises environmental and health concerns due to the high potential of the PTMs to leach into surface and groundwater. This concern is supported by the TCLP data, which revealed high concentrations of Cu far exceeding those recommended by the USEPA [39]. The reducible forms of Cu were also predominant, which allows for mobilization of metals through the formation of soluble metal salts from metal oxides [6]. The rich mineral content of stamp sand could also lead to the inhibition of soil microbiota, resulting in low SOM from decomposition [40]. Nitrate, phosphate and sulfate content of the stamp sand was also analyzed. Stamp sand has low phosphate (7 mg/kg), and although the nitrate concentration in stamp sand was adequate (222 mg/kg), nitrate uptake is likely to be severely reduced due to the inhibition of nitrate reductase from exposure to Cu toxicity [41]. The phenotypic differences were compared between the control and stamp sand grown vetiver after 60-days to determine the impact of metals on plant growth (Figure 1). The initial (day zero) biomass of each vetiver plant used for the stamp sand treatments was 19.8 g and each vetiver plant used for the control soil treatments was 15 g. The average root length for the vetiver plants used for the stamp sand treatments was 34.4 cm, and that of the control vetiver plants was 37.4 cm. The shoots were all trimmed to 30 cm length. The average chlorophyll content of the vetiver plants at day zero was 18.8 mg/kg for the stamp sand treatments and 22.0 mg/kg for the control treatments. The differences between the experimental and control groups for changes in mass of whole plant and root and shoot lengths after 60 days were significantly different with \(p\) = \(<\)0.0001, \(p\) = 0.0408, and \(p\) = 0.003, respectively (Figure 1). There was no significant difference between the chlorophyll content between the experimental and control groups (\(p\) = 0.0940) (Figure 1), however, the impact of Cu on chlorophyll was visible, with the stamp sand-grown plants showing much paler yellowish green leaves compared to the dark green leaves in the control plants (Figure S4). Chlorophyll biosynthesis in plants is not directly impaired by Cu toxicity, but is affected by elevated levels of Mg, Mn, and/or Fe [41]. Rates of various photosynthetic processes, however, can be impaired directly due to Cu toxicity. At high Cu concentrations, biosynthesis of photosynthetic proteins such as Cu-containing plastocyanin PetE, are reduced [42]. Within plant cell organelles such as the chloroplast and nucleus, Cu (II) is often reduced to Cu (I), a reactive species that leads to oxidative stress [42; 43; 44; 45; 46; 47]. Chloroplasts are a major storage location for Cu ions in plant cells [44; 47]. Copper (I)  
Figure 1: Effect of stamp sand on the growth of vetiver grass compared to control soil. Error bars represent standard deviation with n = 6. Statistical significance was determined using [PERSON]’s \(t\)-test and with \(\alpha\) = 0.05. (*) denotes \(p\)-value \(<\) 0.05.  
ions readily bind the thylakoid membrane, and impact photosystems I and II, inhibiting the function of chloroplasts in general. In the nucleus, Cu (I) binds to DNA in the presence of hydrogen peroxide and further reacts to form Cu (II) and hydroxylated-DNA, which damages the chromatin [43]. The reduced rate of photosynthesis leads to stunted growth in both aerial and root structures [42; 46] and can explain the observed significant decreases in whole plant mass, shoot length, and root length.