**1. Introduction**

Many illegal factories arbitrarily discharge wastewater into irrigation channels, where farmers unknowingly draw contaminated water for paddy rice cultivation, causing many farmlands in Taiwan to be contaminated with potential toxic elements (PTEs) [1]. If the total concentration of PTEs in the soil is above the control standard stipulated by the Soil and Groundwater Pollution Remediation Act (SGWPR Act) of Taiwan, crops should be removed to prevent human consumption [2]. However, when the total concentration of PTEs in the soil is slightly above the monitoring standard and below the control standard, crop safety must be emphasized, especially concerning the pathway of oral intake of vegetables.

Cadmium (Cd), a non-essential PTE, is one of the most toxic substances to plants and animals because it is easily transferrable from soil to plant [3,4]. The accumulation of Cd in the edible tissues of crops poses serious concerns to the trophic risk of Cd transfer along a food chain [5]. Because of the carcinogenic and adverse effects of Cd on biological processes, the International Agency of Research on Cancer and the United States Environmental Protection Agency classified Cd into Group 1 and Class B, respectively [6]. Excessive concentration of Cd damaged mesophyll cells and epidermal cells in wheats [7] and resulted in the reduction of photosynthesis and chlorophyll content [8,9]. Once the roots uptake PTEs, plants can decrease the toxicity of PTEs by cell wall deposition and vacuolar compartmentation [10–13]. Cell walls are mainly composed of pectin, protein, cellulose, and semi-cellulose [14], and functional groups in cell walls could restrict PTEs into the cytoplasm, which prevents the protoplast from being poisoned [15].

The chemical forms can be of help in understanding the tolerance and mechanisms of PTE detoxification in plants. These chemical forms include inorganic (FE), water-soluble (FW), pectate- and protein-integrated (FNaCl), undissolved phosphate (FHAc), oxalate (FHCl), and residual (FR) forms [16,17]. Among them, FE and FW have high mobility and thus are easily translocated to other plant organs [18]. Many studies revealed that FNaCl was related to adjusting Cd stress in ramie, cabbage and American pokeweed [18–20]. There was also a positive linear correlation between Cd concentration in the FNaCl and FHAc forms, and that in the shoot, which revealed that chemical forms can be used to predict the accumulation of Cd in the shoot [21].

Many soil amendments (e.g., compost, phosphate fertilizer, and organic matter) have been used for remediation of PTE-contaminated soil [22,23]. For instance, Liu et al. [24] found that the co-application of vermicompost and selenium could alleviate the Cd accumulation in rice. The presence of organic matter redistributed the PTE to less-available forms and thus ameliorated its toxicity to plants [25]. The solubility of PTE is high in an acidic pH, whereas addition of lime and biochar (BC) can increase the soil pH and thus can decrease the bioavailability [26–28]. Biochars are porous, low density, and carbon-rich solid products from the pyrolysis of waste biomass. Xu et al. [29] revealed that adding BCs to contaminated paddy soil improved the transformation of Cd from the acid-soluble fraction to the oxidizing and residual fractions. BCs also had a great capacity to reduce the percentage of inorganic and water-soluble fractions in lettuce roots grown in Cd-contaminated soil [30]. Additionally, BCs mainly increased undissolved cadmium phosphate and thus increased the Cd accumulation in pokeweed root [31].

In this study, different varieties or cultivars of lettuce and pak-choi were grown in soil dominantly contaminated with Cd. Husk BC was applied to replenish nitrogen to the recommended amount and to decrease the soil acidity. The objective of this study was to understand the effect of the application of husk BC on the accumulation, translocation, and chemical forms of Cd in two leafy vegetables. In addition, the vegetable-induced hazard quotient (HQv) was calculated via the chemical form and artificial digestant extractable concentration of Cd in the blanched edible parts to assess the risk from oral intake.

#### **2. Materials and Methods**

Experimental soil samples were collected from the surface layer (0–30 cm) of a Cd-contaminated site in central Taiwan because the soil Cd resulted from the use of irrigation water mainly accumulated in the top 30 cm [32]. Soil samples were air-dried, ground, passed through a 10-mesh stainless steel sieve, and then homogenized before analysis. Their basic properties were then analyzed, including pH [33], electrical conductivity (EC) [34], content of organic carbon (OC) [35], cation exchange capacity (CEC) [36], texture [37], and total concentration of Cd, chromium (Cr), nickel (Ni), and zinc (Zn) [38]. Other soil samples were homogenized and used for a pot experiment. The total concentrations of nitrogen, phosphorus, and potassium in the BC were analyzed in accordance with the methods described by Bremner [39], Kuo [40], and Helmke and Sparks [41], respectively. Two treatments used in this study were CK (control without applying biochar) and BC, which applied husk BC to replenish nitrogen to the recommended amount. For lettuce and pak-choi, 46,920 and 98,000 kg/ha of BC was applied, respectively. In total, 1.0 kg of soil samples or mixture was added to each pot, and seeds of two pak-choi—*Brassica chinensis* L. var. Chinensis (PCC) and *Brassica chinensis* L. cv. Wrinkled leaf (PCW)—and two lettuce—*Lactuca sative* L. cv. Chinese (LSC) and *Lactuca sative* L. var. Sative (LSS)—were sown. The above vegetables were selected because of their importance in human diets [42]

and they are the most commonly consumed leafy vegetables in Taiwan [43]. The pots were translocated to a phytotron (25.1 ± 0.4 ◦C, relative humidity 61.2 ± 5.7%). Soil moistures were determined every two to three days, and remained at 60–80% of water-holding capacity by replenished deionized water.

All leafy vegetables were harvested 35, 42, and 49 days (D35, D42, and D49) after sowing and divided into roots and shoots. At this time, the shoot heights were measured and the chlorophyll content of the largest extended leaf was determined using a Konica Minolta SPAD-502 and recorded as SPAD (soil plant analyzer development) readings. Fresh plant tissues were rinsed with tap water and then deionized water. To remove the adsorbed Cd, the roots were soaked in 20 mM of Na2-EDTA for 15 min. The Cd compartmentalized in different chemical forms in the shoots was analyzed using a sequential extraction in accordance with the methods described by Lai [21]. Six chemical forms were extracted in the following sequence, with the corresponding agents: inorganic Cd (FE) extracted by 80% alcohol, water-soluble Cd (FW) extracted by deionized water, pectate- and protein-integrated Cd (FNaCl) extracted by 1 M NaCl, undissolved Cd phosphate (FHAc) extracted by 2% CH3COOH, Cd oxalate (FHCl) extracted by 0.6 M HCl, and residual Cd (FR) digested with aqua regia. All the other plant tissues were oven-dried at 65 ◦C for 72 h and then weighed (dry weight) before grinding (Rong Tsong Precision Tech. Co., Taichung, Taiwan), and then digested with HNO3/HClO4 (*v*/*v* = 3/1). The Cd concentration in the digestant was determined using a flame atomic absorption spectrophotometer (FAAS, Perkin Elmer AAnalyst 200, Waltham, MA, USA).

Soil samples of different treatments were air-dried, ground, passed through a 10-mesh stainless steel sieve, and then homogenized before analysis. The available concentration of Cd in the soils was extracted with 0.1 N HCl [44] and 0.05 N EDTA [45]. A BCR sequestration extraction based on the European Community Bureau of Reference [46] was also conducted to understand the Cd distribution in the different fractions. These four fractions include the exchangeable fraction (F-I), organic matter-bounding fraction (F-II), oxide-bounding fraction (F-III), and residual fraction (F-IV).

Statistical analysis was performed using the Statistical Package for the Social Sciences (SPSS, Armonk, NY, USA). Analysis of variance (ANOVA) was used to test the effect of different treatments on the different growth exhibitions and soil properties. The least significant difference (LSD) test was used to compare the significant differences of pH, EC, available Cd concentration, Cd distribution in the different fractions, four growth exhibitions, Cd concentration in the roots and shoots, bioconcentration factor (BCF = ratio of root concentration to soil concentration), and transfer factor (TF = shoot concentration to root concentration) between treatments (*p* < 0.05).

## **3. Results**

#### *3.1. Soil Properties*

The total concentrations of Cd, Cr, Ni, and Zn were in the ranges of 10–13, 51–60, 126–146, and 61–68 mg/kg, respectively. For the four PTEs analyzed, only Cd and Ni were beyond the control standard (Cd 5 mg/kg, Ni 130 mg/kg) of farmland based on the SGWPR Act of Taiwan and could cause the samples to be regarded as Cd- and Ni-contaminated soils. However, only Cd accumulation in the lettuce and pak-choi was discussed in this paper because of following reasons. First, the total concentration of Cd was beyond not only the Canadian soil quality guideline (1.4 mg/kg) but also the Soil Contamination Warming Limit (4 mg/kg) and Counterplan Limit (12 mg/kg) of Korea for agricultural lands. Moreover, Cd has higher bioaccessibility in comparison with Ni [47] and is also the only one PTE regulated by the Office of the Journal of the European Union [48] among four soil PTEs analyzed in this study. The BC used in this study had very strong alkalinity and high EC. Its pH and EC were 10.54 ± 0.06 and 1.82 ± 0.08 dS/m, respectively. Total concentrations of nitrogen, phosphorus, and potassium in the BC were 0.24 ± 0.05%, 0.61 ± 0.10%, and 1.59 ± 0.09%, respectively. For the four PTEs analyzed in this study, the BC only had 1.40 ± 1.99 and 10.42 ± 2.25 mg/kg of Cd and Zn, respectively, and the total concentrations of Cr and Ni were not detectable.

The application of BC was able to significantly increase the soil pH and EC in comparison with CK. The BC treatments raised or significantly (*p* < 0.05) raised pH from 6.6–6.8 and 6.9–7.0 to 6.8–7.1 and 7.6–8.0 for the lettuce and pak-choi cultivated soils, respectively (Table 1). Because of the high EC of BC, the soil EC also increased or significantly (*p* < 0.05) increased from 0.03–0.08 (CK) to 0.06–0.18 dS/m under the BC treatment.

**Table 1.** Effect of different treatments on the pH, electrical conductivity, two chemical agents extractable Cd concentrations, and Cd's distribution in the different fractions.


<sup>1</sup> Mean ± standard deviation; ND: not detectable; The same lowercase letter indicates no significant difference between treatments for the same soil property and the same leafy vegetable. <sup>2</sup> PCC, PCW, LSC, and LSS are *Brassica chinensis* L. var. Chinensis, *Brassica chinensis* L. cv. Wrinkled leaf, *Lactuca sative* L. cv. Chinese, and *Lactuca sative* L. var. Sative, respectively; CK: control without applying husk biochar; BC: applying 46,920 and 98,000 kg/ha of husk biochar for lettuce and pak-choi, respectively. <sup>3</sup> F-I, F-II, F-III, and F-IV are exchangeable fraction, organic matter-bounding fraction, oxide-bounding fraction, and residual fraction, respectively.
