Abstract: Before the measurement of stable carbon isotope signatures (δ¹³C), plant samples should be well homogenized. Using a ball-mill fitted with poly tetra fluoroethylene (PTFE) plastic tubes is one of the most efficient and convenient methods. However, sample-tube plastic might contaminate plant samples during milling. In this study, a two-factor experiment was carried out using four growth chambers, with different relative humidity of the air (50% and 80%) and δ¹³C of the air (¹³C depleted and enriched). Leaf samples of Cleistogenes squarrosa (C4) were milled and homogenized using a ball-mill fitted with PTFE tubes and measured for δ¹³C, and the results were compared with that of leaf samples milled using metal tubes. Due to plastic contamination, ¹³C discrimination (Δ¹³C) of the two groups of plants, which were grown in CO₂ with different δ¹³C and could be assumed as replicates, were significantly different (with an offset of 4.8‰ on average). The contamination led to errors in δ¹³C of individual leaves up to 8‰. Given the lower Δ¹³C value of C4 plants (normally about 1‰-8‰) compared with that of C3 plants, such an error caused by plastic contamination far exceeded the acceptable error range. By using a two-member mixing model that was similar to the ‘Keeling plot', such errors could be effectively eliminated, and the δ¹³C of plant samples and pollutants were accurately estimated. Our results showed that the widely applied method of using PTFE tubes for ball-mill homogenization is not suitable for examining Δ¹³C of C4 plants, as it might lead to large errors. For studies with high precision requirements, samples should be milled in stainless-steel tubes.

Key words: ball-mill, plastic contamination, stable carbon isotope, two-member mixing model, water use efficiency, pretreatment of isotope samples