**4. Discussion**

For the three different growing periods, the accumulated Cd in the edible parts of LSC and PCW grown in CK were in the ranges of 4.5–6.9 and 8.2–8.9 mg/kg, respectively (Figures 1 and 2). These values were 1.7–3.5 and 1.1–1.5 times higher than LSS and PCC, respectively. Further results revealed that the accumulating capacity of Cd of LSC and PCW was stronger than LSS and PCC. Moreover, the lettuce and pak-choi accumulated almost constant concentration of Cd in the edible parts regardless of the growing period once planted in the Cd-contaminated soils. The experimental results of this study are in agreement with Lai and Chen [49].

BCF was used to evaluate the accumulation of Cd in the edible parts of two leafy vegetables at D49 (Table 3). BCFR and BCFS are the ratio of root and shoot concentration to soil concentration, respectively. The LSC accumulated more Cd than LSS, and the BCFR of LSC at D49 was 6.1 and 2.6 times higher than LSS grown in CK and BC, respectively. All the BCFR and BCFS of lettuce grown in CK were less than 0.9, and there was a 25–68% and 32–58% decrease in BCFR and BCFS, respectively, under BC treatment in comparison with CK. Whether applying BC or not, the BCFS of LSC was 1.1–1.8 times higher than that of LSS. This phenomenon reveals that LSC can accumulate higher Cd concentrations in the edible parts and also shows higher risk through oral consumption than LSS.

The PCW accumulated more Cd than PCC, and the BCFR of PCW at D49 was 1.9 and 2.3 times higher than PCC grown in CK and BC, respectively. All the BCFR and BCFS of pak-choi grown in CK were less than 0.9, and there was a 27–55% and 45–50% decrease in BCFR and BCFS, respectively, under BC treatment compared with CK. Even though the BCFR of PCW was higher than PCC, the BCFS of PCW grown in different treatments was 57–75% that of PCC. This phenomenon reveals that the Cd accumulated in the roots of PCW was not efficiently upwardly transferred to shoots and thus had lower BCFS compared with PCC.

Besides BCF, TF was used to evaluate the transfer of Cd from roots to edible parts of lettuce and pak-choi (Table 3). Approximately all the TF values of LSC and PCW, with higher BCFR and BCFS under different growth periods and treatments, were below unity in general. For LSS and PCC, however, TF values were higher than LSC and PCC, respectively, and were in the ranges of 0.3–1.8 and 0.4–2.1, respectively. Further results reveal that the Cd uptake by LSC and PCW grown in CK were mainly accumulated in the non-edible parts, regardless of growing periods. However, the TF values of LSS and PCC grown in BC at D42 and D49 reached 1.1–2.1. This result reveals that the accumulated Cd would transfer to shoots more easily than the other two varieties, even grown in BC. Except for LSS-D49, the BC treatment was efficient in increasing the TF of LSS and LSC in comparison with CK, regardless of growth periods. This result revealed that even the BC decreased the accumulated Cd

concentration in the roots; on the contrary, BC increased the upward translocation of Cd from roots to shoots in two lettuce varieties because of Cd's high mobility compared with other PTEs.


**Table 3.** The bioconcentration factor (BCF), transfer factor (TF), average daily dose (ADD), and hazard quotient (HQ) calculated using three different methods 1.

<sup>1</sup> The same lowercase letter indicates no significant difference between treatments for the same leafy vegetable. <sup>2</sup> The meanings of abbreviations are the same as in Table 1. <sup>3</sup> BCFR: ratio of root conc. to soil conc.; BCFS: ratio of shoot conc. to soil conc.; TF: ratio of shoot conc. to root conc. <sup>4</sup> ADDv and HQv are vegetable-induced ADD and HQ based on the total concentration, chemical form, and bioaccessible fraction of Cd, respectively.

In comparison with CK, the BC treatment did not have significant effects on changing the 0.1 N HCl and 0.05 N EDTA extractable Cd concentrations (Table 1), and also the chemical form of Cd compartmentalized in the shoots of lettuce and pak-choi (detail data not shown). However, the accumulated Cd concentration in the roots and shoots of lettuce and pak-choi grown under BC treatment at different growth periods were decreased or significantly decreased (*p* < 0.05) compared with CK (Figures 1 and 2).

Antoniadis et al. [50] reported that the vegetable-induced average daily dose (ADDv) and vegetable-induced hazard quotient (HQv) can be calculated using Equations (1) and (2), respectively, where Cp is the Cd concentration (mg/kg) in the edible parts of vegetables. The mean individual daily vegetable consumption (MIDVC) in Taiwan during 2013–2016 was 0.133 kg/day based on the Report on the Nutrition and Health Survey and vegetable calorie counts, which can be used to calculate Cd intake daily per person from vegetables. The tolerable daily intake (TDI) of Cd set by the European Food Safety Authority (EFSA) was 0.36 μg/kg·BW/day. Nonetheless, food was the dominant source of Cd exposure of humans and accounts for approximately 90% of the intake [51]. Among all foods, approximately 26% was from vegetables [52], which means that the TDI from vegetables (TDIv) is 0.084 μg/kg·BW/day.

$$\text{ADD}\_{\text{V}} = \frac{\text{C}\_{\text{P}} \times \text{MIDVC}}{\text{kg} \cdot \text{BW}} \tag{1}$$

$$\text{HQ}\_{\text{V}} = \frac{\text{ADD}\_{\text{V}}}{\text{TDI}\_{\text{V}}} \tag{2}$$

Blanching is the most common method in Taiwan for cooking leafy vegetables, and it also decreases the concentration of PTE in thoroughly cooked vegetables. Based on the findings of Lam et al. [47], approximately 50% of the Cd accumulated in the water spinach was leached into boiling water. In this study, three methods based on total concentration (TC), chemical form (CF), and bioaccessible fraction (BF) of Cd in the edible parts of vegetables were used to calculate the HQv. The FE and FW were considered to have a higher mobility than other chemical forms and were easily leached into boiling water. Therefore, the sum of the proportion of the other four chemical forms, i.e., FNaCl, FHAc, FHCl, and FR, was used to calculate the HQv, coded as HQv-CF. Furthermore, approximately 32–55% (average is 44%) of the accumulated Cd in water spinach could be metabolized by in vitro digestive fluids [47], which reveals that approximately 44% of the Cd is bioaccessible and can be absorbed by the human body, coded as HQv-BF.

Regardless of treatments, the HQv-TC, HQv-CF, and HQv-BF values of usedleafy vegetables at D49 were in the ranges of 2.4–7.9, 0.01–0.3, and 0.5–1.8, respectively (Table 3). Because more than 73% of the accumulated Cd was compartmentalized in the FE chemical form, which could be leached out of vegetable tissues during blanching, the HQv-CF values were less than 0.3 in general. The application of BC significantly decreased the HQv of pak-choi at D49, and the HQv-TC, HQv-CF, and HQv-BF was 20–89% in comparison with the CK. However, BC's effect on the HQv of lettuce was contrary to that of pak-choi because the HQv increased compared with CK. The HQv of lettuce and pak-choi used in this study was lower in comparison with water spinach grown in artificially Cd-spiked soils with a total concentration of 2.8–3.1 mg/kg [53]. According to the calculated results of HQv-CF and HQv-BF, except for pak-choi grown in CK, oral intake of these four leafy vegetables has a low risk even though the soil Cd concentration was 2 to 3 times beyond the control standard of farmland, i.e., 5 mg/kg, based on the SGWPR Act of Taiwan.
