Sample Extraction, Digestion, and Purification

For all extractions, digestions, and matrix separation procedures, distilled nitric (HNO₃) and hydrochloric acid (HCl) were used. In addition, commercially available hydrofluoric acid (HF, 47–51% trace metal grade, Fisher Chemical) and hydrogen peroxide (H₂O₂, 30%, Suprapur, Merck) were used for sample digestion, while “analytical” grade salts were used for Cd soil spiking and soil fertilization. All laboratory work was conducted in the clean lab facilities of ISTerre Grenoble. For plant watering and laboratory work, deionized water was used (>18.2 MΩ). Samples were digested and evaporated in PFA beakers. Before analysis, samples were stored in polypropylene tubes (Metal-Free Centrifuge Tubes, Polypropylene, VWR).

To determine the Ca(NO₃)₂ extractable Cd fraction of the soil, 30 ml 0.05 M Ca(NO₃)₂ was added to an equivalent of 3 g of dry soil; the samples were shaken for 16 h, centrifuged (3500 g) for 20 min, and then filtered (Rotilabo KH55.1 0.45 μm, Carl Roth). Prior to storage in the fridge, 1 ml 14 M distilled HNO₃ was added to the samples. One hundred milligrams of soil powder was digested in 6 ml distilled 14 M HNO₃ + 2 ml HF. For root and shoot samples, 200 mg of powder were predigested for >12 h in 7 M HNO₃, evaporated, and digested in 6 ml 14 M HNO₃ + 2.5 ml H₂O₂ + 0.5 ml HF for >48 h (Wiggenhauser et al., 2021).

After digestion, the samples were purified for isotope analysis by removing matrix elements from the sample using resin anion exchange chromatography. To this end, the samples were evaporated, refluxed in 7 M HCl for >12 h, evaporated, and then resolubilized in 2 M HCl. The sample was then split into two aliquots, of which a small aliquot was used to measure the Cd concentration in unpurified samples, while the large aliquot of the sample was purified for isotope analyses. For the purification, we slightly adapted a method that was developed for environmental samples that are Cd contaminated (Cloquet et al., 2005; Wei et al., 2015). Polypropylene columns (Poly-Prep, Bio-Rad) were filled with 2 ml resin (AG-MP-1). After cleaning the resin using 30 ml 0.5 M HNO₃, the resin was preconditioned using 4 ml H₂O followed by 4 ml 2 M HCl. The sample that was dissolved in 4 ml 2 M HCl was then loaded on the resin. Matrix elements were subsequently eluted from the column using 8 ml 2 M HCl, 12 ml 0.3 M HCl, and 14 ml 0.012 M HCl. Finally, Cd was eluted into Teflon beakers using 22 ml 0.0012 ml HCl.

For speciation analysis, the cryomilled and frozen samples were pressed to pellets. To this end, the sample powder was transferred into a precooled die set (plunged in liquid N₂), quickly transferred into a hydraulic press, and back into a container that contained liquid N to remove the pressed pellet from the die set. The pressed pellets had a diameter of 5 mm and a thickness of 1–4 mm. During storage, transport to the synchrotron facilities, and mounting samples on the sample holder at the synchrotron facilities, samples were kept in −80°C or liquid N to ensure no changes in Cd speciation.