2.3.3. Nitrate

Nitrate content was measured with the salicylsulphuric acid method [40]. 10 mg of oven-dried samples (80 ◦C for 48 h) were suspended in 10 mL of distilled water and left in agitation for 2 h. After that, 20 μL of sample were added to 80 μL of 5% salicylic acid in sulphuric acid and to 3 mL of NaOH 1.5 N. Samples were cooled at room temperature and the spectrophotometer readings were performed at 410 nm. Nitrate content was calculated referring to a KNO3 standard calibration curve. Data were expressed on a fresh weight (FW) basis considering the fresh weight/dry weight ratio.

#### 2.3.4. Mineral Composition

For assessing the mineral composition, oven-dried samples (80 ◦C for 48 h) were ground and digested with nitric acid, and elements were measured using inductively coupled plasma mass spectroscopy (ICP-MS). Data were expressed on an FW basis considering the fresh weight/dry weight ratio.

#### *2.4. Contribution to Mineral Dietary Intake and Health Risk Assessment*

The estimated dietary intake (EDI, mg/day) of mineral elements possibly resulting from the consumption of micro/baby greens of the studied species was calculated by the following formula:

$$\text{EDI} = \text{C}\_{\text{metal}} \times \text{(SP/1000)}\tag{1}$$

where,

$$\mathbf{C\_{metal}} = \text{the element concentration (mg/kg FW) in the produce} \tag{2}$$

$$\text{SP} = \text{a supposed portion of 20 g of micro/baby greres} \tag{3}$$

For evaluating the contribution of microgreens and baby greens to human mineral requirements, EDI was expressed as percentage (EDI%) of the recommended dietary intake (RDI, mg/day) (for Ca, P, Mg, Fe, Cu, Zn, Mo, and Se) or adequate intake (AI, mg/day) (for Mn and Cr) as defined by Italian Society of Human Nutrition (SINU), considering RDI and AI values referred to an adult male [41].

In order to assess the possible health risk due to the intake of metals related to micro/baby greens consumption, the health risk index (HRI) was calculated for Fe, Cu, Zn, Mn, Cr, Se, Mo, Co, Ni, As, and Cd according to the following formula:

$$\text{FIRI} = \text{EDI}\_{\text{BW}} / \text{RfD} \tag{4}$$

where,

> EDIBw = EDI (as defined above) per kg of body weight (BW) (5)

$$\text{RfdD (mg/kg BW/day)} = \text{oral reference dose} \tag{6}$$

which is an estimate of the daily exposure of humans to heavy metals having no hazardous e ffect during the lifetime according to US-EPA [42]

As BW an average body weight for an adult was considered and assumed to be 55.9 kg as in previous studies [43]. Since RfD is not available for Al and Pb, the possible health risk was evaluated on the basis of Al tolerable weekly intake (TWI; mg/kg BW/week) according to EFSA [44], and of Pb *Codex Alimentarius* maximum level (ML; mg/kg FW) (maximum concentration of a contaminant in a food commodity recommended by the FAO/WHO *Codex Alimentarius* Commission to be legally permitted in that commodity) [45].

#### *2.5. Statistical Analysis*

Yield and composition data were subjected to a two-way ANOVA (3 species × 2 stages of harvest) according to a randomized block experimental design with three replicates, by using CoStat Statistics

Software. Significant differences among means were determined by using Duncan's Test at *p* < 0.05. A principal component analysis (PCA) was also performed on composition data by using the software STATISTICA for Windows. Before performing PCA, all values of considered variables were replaced by standardized values, which were computed as follows:

$$\text{Standardized value} = \text{(raw value -- mean)/Std. deviation} \tag{7}$$
