Laboratory mill

Four plants from nine Brussels sprout cultivars each (except ‘Esperal’ which was not available for this experiment) were cultivated in the greenhouse and after three months transferred to the field as a potted plant. In the field, all plants were covered with a net (0.5 × 0.5 mm mesh size; Filbio PA, Hartmann–Brockhaus, Pfaffenhofen-Wagenhofen, Germany), and during the first two weeks, an additional sunscreen was applied to avoid sunburn. After two months, three leaves were cut off from two levels (upper level as first fully grown leaves and mid-level as not senescent leaves of middle part) of the plant. Both mid-vein and stem were removed from the leaves, and then transferred into a Falcon tube and finally flash frozen in liquid nitrogen. The samples were stored at −20 °C and later lyophilised using a Christ Alpha 1–4 LSC freeze drier (Christ Gefriertrocknungsanlagen GmbH, Osterode, Germany). The dry leaf material was then ground with a laboratory mill (IKA-Werke GmbH, Staufen, Germany) to a fine powder, and a sample transferred to an Eppendorf cup for glucosinolate analyses. Glucosinolate concentrations were determined as desulfo-glucosinolates using a modified method according to DIN EN ISO 9167-1, which is described in full detail in Wiesner et al. [26 (link)].

ICST and LC-MS/MS methods were used to determine the ZEN concentrations of extraction solutions of 202 samples in total. Firstly, a laboratory mill (IkaWerke, Staufen, Germany) was used to grind the wheat samples into 20 mesh fine powder.
Then 10 g fine powder of each sample was collected and mixed with 40 mL extracting solution (acetonitrile: water: acetic acid = 79:20:1v/v/v) and centrifuged for 30 min with the speed of 180 rpm [24 (link)]. After being centrifuged for 10 min with the speed of 3000 rpm, 0.5mL final extract of each sample was diluted with 0.5 mL extracting solution (acetonitrile: water: acetic acid = 79:20:1v/v/v). Subsequently, the mixture passed through a nylon filter which had the diameter and pore size of 13 mm and 0.22 μm separately and finally flowed into an auto sampler vial to be analyzed by LC-MS/MS [25 (link)–27 ].
With regard to the colloidal-gold immunochromatographic strip, 0.01 M PBS (pH 7.4) was utilized to dilute 0.5 mL filtrate of each sample into 2 times volume and 100 μL of above mentioned solution was added onto the sample pad. Five minutes later the result could be read. The tests on natural and spiked samples were both conducted in triplicate.

Plants were harvested after 1 d, rinsed with deionized water, and separated into leaves, stem, and root. After 7 and 60 d, leaves were designated as apical (1 ≤ LPI ≤ 6), medial (7 ≤ LPI ≤ 18), and basal (LPI > 18) on the basis of LPI. Roots were washed with 10 mM CaCl₂ to remove mineral elements adsorbed to the root surface. Plant organ samples were dried in a forced-circulation oven at 65 °C and ground with a laboratory mill (IKA-Werke GmbH & Co.KG, Staufen, Germany). Ground samples (0.2 g) were digested with 5 mL HNO₃ followed by 1 mL of HClO₄. The resulting solution was analyzed using an atomic absorption spectrometer (AAnalyst 200, Perkin-Elmer, Waltham, MA, USA). Two analytical reference standards for Cd, Zn, and manganese (Mn) were used as control (WEPAL IPE, Wageningen University): Daucus carota (L.) leaf (307 ± 71 µg kg⁻¹ of Cd, 25.0 ± 2.93 mg kg⁻¹ of Zn and 42.1 ± 4.53 mg kg⁻¹ of Mn, certified concentrations) and shoot (3070 ± 491 µg kg⁻¹ of Cd, 185.0 ± 16.4 mg kg⁻¹ of Zn and 427.0 ± 40.6 mg kg⁻¹ of Mn, certified concentrations).