Digiblock

The analysis was carried out on tissues from 5 control mussels and exposed mussels. DigiBlock, a sample preparation technology, was used to digest the samples (LabTech, MA, Hopkinton, USA). In a Teflon jar, a quarter-gram sample (0.1000 g) was placed. Digestion was performed using 10 mL of Optima-grade Nitric acid (HNO₃) and 3 mL of 30% Optima H₂O₂. Reconstituted samples were placed in 10 mL of 18.2 MΩ cm at 25 °C H₂O with 2% nitric acid (both optima-grade). ICP-QMS quadrupole inductively coupled plasma-mass spectrometry was used to quantify metals (820 MS, Bruker). Calibration solutions were made from multielement standard stock solutions containing 20.00 mg/L of each element. Nine calibration solutions were used to generate calibration curves. In order to control reagent purity and laboratory equipment, reagent blanks containing ultra-pure water were also tested. A mix solution of internal standard (6 Li, 45 Sc, 72 Ge, 89 Y, 103 Rh, 159 Tb, 165 Ho, 209 Bi) of 10 µg/L, on-line aspired using a T union with the sample and standard solution, was used for determination. Every ten samples, a blank sample and at least two standard samples were tested to confirm the instrument calibration constants. A verified reference of NIST bovine liver was examined before and after each session of sample analysis to control the correctness of the analysis.

Aliquots of the supernatants were taken for the determination of the Al concentration in nutrient solutions. The roots and shoots were dried at 50 °C, combined into one sample for each pot, and ground to a powder. All samples were digested in a mixture of concentrated HNO₃ and 38% H₂O₂ at 70 °C using the digestion system DigiBlock (LabTech, Sorisole, Italy). The content of Al and the nutrient elements (Ca, Fe, K, Mg, Mn, P, S, and Zn) in the digested samples was determined using an inductively coupled plasma emission spectrometer (ICPE-9000, Shimadzu, Tokyo, Japan), according to the manufacturer’s instructions. At the end of the experiments, a precipitate (residue) formed in the pots, which in previous studies with peas and PGPR was collected and analyzed and contained precipitated Al [36 (link)]. In this work, the amount of Al in this residue was calculated as the difference between the amount of Al introduced into the pot and its total amount in the plants and the solution.

The dried plant shoots (leaves, stems, and pod walls) and seeds were ground to a powder. Nitrogen concentration and atom % ¹⁵N in the ground samples were determined using the elemental analyzer (FlashEA 1112, Thermo Scientific, Italy) coupled with the isotope ratio mass spectrometer Delta V Advantage (Thermo Fisher Scientific, Dreieich, Germany) and the continuous flow interface ConFlo III following manufacturer’s instructions. To determine Cd and nutrient (Ca, Fe, K, Mg, Mn, S, P, and Zn) concentrations, the ground shoot samples were digested in a mixture of concentrated HNO₃ and 38% H₂O₂ at 70 °C using DigiBlock digester (LabTech, Sorisole, Italy). Elemental concentrations of digested plant samples were determined using an inductively coupled plasma emission spectrometer ICPE-9000 (Shimadzu, Kyoto, Japan).