*2.4. Determination of Inorganic Elements in Soil, Forage, and Milk by ICP-MS*

Procedures to quantify inorganic elements were based on official methods for trace element determination by ICP-MS: ISO/TS 16965:2013, EN 17053:2018 and ISO 15151:2018 for soils, animal feed, and milk, respectively. All of them were self-optimized and validated in our laboratory by using different European reference materials (Joint Research Centre, EU): loam soil (ERM CD-141), rye grass (ERM CD-281), and skimmed milk powder (ERM BD-151). Accuracy, reproducibility, and optimization of these procedures are detailed in Appendix A.

Samples of soil (0.1 g), forage (0.5 g), or milk powder (0.5 g) were digested in 8 mL of aqua regia (HCl:HNO3 3:1) in closed polytetrafluoroethylene vessels using an Ethos One microwave digestion system (Milestone Srl., Sorisole, BG, Italy), as described in Table A2. The digested solution was filtered through a 0.22 μm syringe filter (Merck Millipore, Billerica, MA, USA), then diluted to 40 mL with ultrapure water in the case of forage and milk samples [overall dilution, 1:80 (*w*/*v*)] or 20 mL in the case of soil samples. An aliquot (1 mL) of diluted soil samples was then diluted to 10 mL [overall dilution, 1:2000 (*w*/*v*)].

Standard metal solutions were prepared daily. Solutions of Na, K, Mg, and Ca were prepared from a multi-elemental stock solution (1000 μg mL<sup>−</sup>1), while solutions of Cr, Zn, Fe, Cu, As, Se, Cd, and Pb were added from individual stock solutions (1000 μg mL−1). All standards were prepared in 1% HNO3. Internal standards (HPS, North Charleston, Charleston, SC, USA) were as follows: 45Sc for Na, K, Ca, and Mg; 72Ge for Cr, Fe, Cu, Zn, Se, and As; 103Rh for Cd; and 193Ir for Hg and Pb. Samples were analyzed per duplicate.

The content of inorganic elements in the soil and forage samples was calculated in terms of dry weight, while the content of inorganic elements in the milk was calculated in terms of wet weight after applying a correction factor based on mean water content (88%) [25]. The assay procedures were validated per triplicate for the three sample matrices using the Certified European Reference Materials, as set out in Section 2.1. Elements whose concentrations were not reported in the reference materials were spiked into the materials. Recoveries in ryegrass ranged from 93% for Cd to 114% for Ca, while those in skimmed milk powder ranged from 88% for Pb to 113% for Cr (Table A2).
