*3.3. Cadmium Transfer Factor*

Table 5 shows the Cd transfer factor (TF) from ryegrass roots to shoots. The TF of accumulated Cd was similar to that observed by Antonangelo and Zhang [11] when evaluating the simple Cd uptake TF. Therefore, the TF decreased as the application of both biochars increased (Table 5). A lower TF indicates higher Cd accumulation in roots and lower transference to aboveground plant parts, which lowers the risk to the primary consumer [33,39]. Other studies have also reported the preferential accumulation of Cd in the roots of grasses rather than in shoots, such as ryegrass [31] and rice [32]. In our study, it is evident that the accumulated Cd was higher in the roots of ryegrass than in the shoots even for the control. On average, TF as a function of PLB application was higher than that of SGB, which is consistent with the fact that ryegrass shoots yielded better under the PLB amendment; which was also closely related to the N removal, thus resulted in higher Cd removal aboveground when compared to SGB. The maximum Cd TF was reached when <sup>206</sup> ± 38 kg N ha−<sup>1</sup> was removed in ryegrass shoots (Figure 3). This is close to a TF of 0.5 obtained between the control and the lowest rate of PLB application rate (0.5%).

**Table 5.** The transfer factor (TF) of accumulated Cd from ryegrass roots to shoots as a function of biochar (B) application rates (R).


Different lowercase letters in columns (within biochar rates) and uppercase letters in rows (same biochar rate) are significantly different at *p* < 0.05 (Tukey). SGB: switchgrass-derived biochar. PLB: poultry litter-derived biochar. *TF* = *Cd removal in shoots* ÷ *Cd accumulated in roots*.

**Figure 3.** Linear and quadratic regression models (gray dashed lines) for Cd transfer factor and N removal in ryegrass shoots as a function of applications of switchgrass (SGB) and poultry litter-derived biochars (PLB). The black solid line represents a linear regression with a plateau model combining both biochar-treated soils (SGB + PLB). The black dashed vertical line indicates the joint point (plateau point) of N removal where the Cd transfer factor ceases to increase. \*\*: *p* < 0.01. \*\*\*: *p* < 0.001.

The linear with plateau model was converged from the whole dataset of measurements and for both biochars combined, which is probably a result of the simple linear regression obtained for SGB and a quadratic regression for PLB (Figure 3). The highest point of which the linear line reaches its maximum in the SGB is close to the maximum point reached with the quadratic line drawn in the PLB before the Cd TF values begin to reduce. Therefore, a maximum point (plateau or joint point), was obtained for both biochars combined thus indicating an optimum N removal in ryegrass shoots in which Cd removal also reached its limit (Figure 3).

#### **4. Discussion**

Our research demonstrated that Cd accumulation in ryegrass plant parts is proportionally affected by N uptake and ryegrass yields. Nitrogen fertilization may indeed change the phytoavailability of Cd in the growth medium and its absorption by plants [40]. Additionally, several studies have shown that N fertilization increases Cd concentration in plant tissues [16,41,42]. One of the reasons may rely on a possible synergic effect between nitrate (NO3 −) and Cd [42–45] while the ratio between NO3 − and ammonium-NH4 <sup>+</sup> might influence the accumulation of Cd in plants as well [46]. Commonly, NH4 <sup>+</sup> easily undergoes the nitrification process in aerobic environments and forms NO3 −, which is taken up by plants, and such a process releases H+ in the soil solution thus reducing its pH and increasing the Cd bioavailability. In this sense, more research is encouraged to test nitrate and ammoniacal sources of nitrogen fertilizers to evaluate their impact on toxic elements removal. Additionally, evaluating if biochars produced from different feedstocks are a better source of NO3 − or NH4 <sup>+</sup> would contribute to the better understanding of metal remediation practices if biochars can immobilize the metal and at the same time increase the toxic element removal by providing N efficiently while keeping metal concentration below the threshold for animal grazing.

A higher accumulation of Cd was found in the ryegrass roots as compared with the shoots. Since the root is the first plant part in contact with Cd in contaminated soil and its structural component likely accumulates the largest amount of Cd present in the plant tissues [47,48]. This is a physiological strategy in which plants phytostabilize the metal in the roots to prevent toxic elements from reaching the xylem, being transported to the shoots, and damaging the photosynthetic apparatus of plants [46] This prevention of Cd transport to the shoots may occur through the synthesis of chelants or even physical barriers that prevent the Cd movement in the apoplast [49]. Therefore, more attention must be paid to plants metabolism and molecular mechanisms to reveal the direct role of organic amendments, such as biochar, in future phytoremediation studies, as pointed out by Liu et al. [50]. For example, the recent study of Peco et al. [51] has found *Biscutella auriculata* L., a wild herbaceous species that grows on pastureland, as a new Cd-tolerant plant capable of activating efficient metal-sequestering mechanisms in the root surfaces and leaves, and of inducing phytochelatins in both parts, besides stimulating antioxidative defenses in roots.

## **5. Conclusions**

Cadmium removal was the highest at the 1% PLB rate accompanied by the highest ryegrass yield. However, the Cd concentration in grazable forage remained acceptable. The Cd transfer factor from ryegrass roots to shoots increased when up to <sup>206</sup> ± 38 kg N ha−<sup>1</sup> was removed in ryegrass shoots. Application of up to 1% PLB is a viable option, since it is a practical rate for handling operations requiring less volume of material than SGB.

**Author Contributions:** Conceptualization, J.A.; methodology, J.A. and H.Z.; validation, J.A. and H.Z.; formal analysis, J.A.; investigation, J.A. and H.Z.; resources, H.Z.; data curation, J.A.; writing original draft preparation, J.A.; writing—review and editing, J.A. and H.Z.; visualization, J.A.; supervision, H.Z.; project administration, H.Z.; funding acquisition, H.Z. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by the Oklahoma Agricultural Experiment Station.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Raw data were generated at an environmentally controlled growth chamber located at CERL (Controlled Environmental Research Lab) Central, Oklahoma State University, main campus. Derived data supporting the findings of this study are available from the corresponding author J.A. on request.

**Conflicts of Interest:** The authors declare no conflict of interest.
