*2.4. Nitrogen and Cadmium Analysis in Ryegrass Shoots and Roots*

After harvesting, ryegrass shoots and roots were separated, washed with D.I. water, and oven-dried to constant weight at 105 ◦C, and their weight was recorded. Dried plant materials were ground using a mechanical grinder and further analyses were followed. Ground plant materials were analyzed for Cd using nitric acid digestion and TN using dry combustion and determination via LECO, as previously described for soil. For Cd in plant tissues, 0.5 g of ground plant materials were predigested for 1h with 10 mL of trace metal grade HNO3 in the HotBlock™ Environmental Express block digester (Environmental Express, 2345A Charleston Regional Parkway, Charleston, SC, USA), and the digestion products were then heated to 115 ◦C for 2 h and diluted with D.I. water to 50 mL [28]. Finally, the digested samples were filtered and analyzed for Cd by an ICP-AES.

The N and Cd concentration determined in plant tissues (uptake) was multiplied by their respective biomass to eliminate any dilution effect caused by over yield and the N and Cd in ryegrass shoots and/or roots were then treated as "removed" (shoots, when harvested) and/or "accumulated": *N*/*Cd removed*/*accumulated* = (*N*/*Cd concentration* × *Yield*).

The Cd transfer factor (TF) was calculated by considering the Cd removal and accumulation following: *TF* = (*Cd removed in shoots* ÷ *Cd accumulated in roots*).
