Comparison of As(V) and As(III) removal by A. calamus L.
The removal efficiencies of As(V) and As(III) by A. calamus L. were compared during a 31-day exposure. As shown in Fig. 1, the remaining concentrations of As(V) and As(III) in water showed a trend of continuous decrease. Differently, the remaining concentration of As(III) dropped more quickly than that of As(V). For As(V) treatment, the arsenic removal efficiency increased first slightly for 5 or 7 days and then sharply from 28.39%±0.72% (200 µg L‒1), 6.13%±1.60% (500 µg L‒1), 10.66%±2.14% (1,000 µg L‒1) to 96.50%±0.33%, 95.03%±0.13%, 97.09%±0.36%, respectively. For As(III) treatment, the removal efficiency of arsenic presented a linear increase since the beginning of the experiment and then kept nearly invariant between the 13th and 31st day. The removal efficiency of arsenic reached 97.10%±0.67% (200 µg L‒1), 97.41%±0.08% (500 µg L‒1), and 95.45%±0.40% (1,000 µg L‒1), respectively. In general, A. calamus L. could remove more than 95% of As(V) or As(III) from water. Compared with As(V), As(III) could be removed by A. calamus L. more efficiently.
Arsenic is a non-essential metalloid that is highly toxic to organisms including plants. Phytoremediation is a sustainable, natural, and eco-friendly approach to environmental decontamination (Amiri et al., 2020). Thus, more information is needed to identify the suitable plant species for the phytoremediation of arsenic-contaminated water. Aquatic macrophytes can be a good candidate the for phytoremediation of metals. Several floating macrophytes have been investigated for the removal of metals. Pistia stratiotes L. had the highest total Mn removal from the water (Hua et al., 2012). Lemna minor and Eichhornia crassipes were also effective in the removal of metals (i.e., Cu) (Rezania et al., 2016). Azolla pinnata could remove 65‒95% of applied metals (Zn, Cu, Pb, Cr, and Cd) from wastewater (Akhtar et al., 2021). Compared to floating macrophytes, little information has been gathered to date regarding arsenic removal by emergent macrophytes. In this study, more than 95% of the total arsenic was removed from As(V)-/As(III)-contaminated water. Nevertheless, de Souza et al. (2019) reported that Lemna valdiviana was able to remove 82% of its initial As(III) concentration under optimal conditions. It was suggested that A. calamus L. could be an effective plant for phytoremediation. On the other hand, the pre-oxidation of As(III) to As(V) is a desirable process for the effective removal of iAs from water by using physicochemical methods. Our results showed that A. calamus L. could efficiently remove As(III) as well as As(V) without a pre-oxidation process. Accordingly, A. calamus L. could be suitable for iAs phytoremediation.
Accumulation and transformation of arsenic species by A. calamus L.
Accumulation of arsenic in A. calamus L. was presented in Fig. 2. After 31-day exposure, the total arsenic in different vegetative organs of A. calamus L. increased with the increase of initial As(V) concentration. About 34.24±0.49 mg kg‒1 (200 µg L‒1), 46.76±5.39 mg kg‒1 (500 µg L‒1), and 292.77±36.18 mg kg‒1 (1,000 µg L‒1) of arsenic have been found to be accumulated in the underground part (root) of A. calamus L.. Only 1.31±0.06 mg kg‒1 (200 µg L‒1), 6.12±0.10 mg kg‒1 (500 µg L‒1), and 3.71±0.15 mg kg‒1 (1,000 µg L‒1) of arsenic were accumulated in the aboveground part (stem and leaf). Thus, it can be seen that the arsenic contents were much higher in the root than in the stem and leaf. After As(III) treatment, A. calamus L. accumulated 82.14±11.51 mg kg‒1 (200 µg L‒1), 26.08±6.36 mg kg‒1 (500 µg L‒1), and 62.38±7.97 mg kg‒1 (1,000 µg L‒1) of arsenic in the roots, respectively. Similarly, a small amount of arsenic was accumulated in the stem and leaf under As(III) exposure. It was indicated that iAs was mainly accumulated in the root of A. calamus L.. This conclusion was supported by the observation that the translocation factors (TFs) of As(V) and As(III) were no more than 0.044 (see Table 1).
Various arsenic species were identified and quantified in the vegetative organs of A. calamus L. (Fig. 3). For As(V) treatment, As(V) and As(III) were found in the whole plant, whereas DMA (0.09‒0.13 mg kg‒1) was only present in the aboveground part. As(V) was the main species in the As(V)-exposed plants (45.86%‒70.21%). Both the content and percentage of As(III) significantly increased with the increase in the concentration of As(V) treatment. But the percentage of As(III) was no more than 51.23%. It was suggested that some of the As(V) absorbed into the root system was reduced to As(III) and further methylated to DMA in the stem and/or leaf. For As(III) treatment, As(V) and As(III) were also identified in the whole plant, but DMA (0.06 and 0.07 mg kg‒1) was detected in the leaf under the treatment of 500 and 1,000 µg As(III) L‒1. Correspondingly, As(III) was the main species in the stem and leaf of As(III)-exposed plants (55.76%‒85.52%), while As(V) was still dominated in the root under 200 and 500 µg As(III) L‒1 treatment (64.8% and 58.42%). Although the As(V) content increased with increasing the concentration of As(III) treatment, the percentage of As(V) decreased. It was indicated that a large amount of As(III) absorbed could be oxidized to As(III) in the root system, and its oxidation efficiency was limited by the As(III) content in cells. The methylation of As(III) to DMA seems to occur mainly in the leaf of emergent macrophyte.
Table 1
Bioaccumulation factors (BCF) and translocation factors (TF) of A. calamus under different arsenic concentrations.
| Treatment concentration (µg L‒1) | BCF | TF |
As(Ⅴ) | 200 | 1.32 | 0.043 |
500 | 1.44 | 0.036 |
1000 | 0.81 | 0.044 |
As(Ⅲ) | 200 | 8.30 | 0.007 |
500 | 1.90 | 0.044 |
1000 | 2.29 | 0.032 |
In this study, iAs was more accumulated in the root than in the stem and leaf of A. calamus L. at each treated concentration. Our result was similar to the results of several submerged macrophytes. Vallisneria natans (Lour.) Hara accumulated more arsenic in the roots (187.11‒248.65 mg kg− 1) than in the leaves (19.10‒39.52 mg kg− 1) after 14 days of treatment (Li et al., 2018). Arsenic was also more accumulated in the roots of Myriophyllum alterniflorum DC. (156±22 mg kg− 1) than in its shoots (104±12 mg kg− 1) (Krayem et al., 2016). Hydrilla verticillata (L.f.) Royle has been shown to accumulate > 73% of arsenic in the root (Xue and Yan, 2011). Additionally, the highest concentration of arsenic was found in the roots of two free-floating macrophytes (Eichhornia crassipes (Mart.) solms and Ipomoea aquatica Forsk) and one emergent macrophyte (Typha angustifolia L.) growing in the wastewater treatment ponds (Nateewattana et al., 2010). It was proved that the root of aquatic macrophytes acted as a barrier to limit arsenic translocation to the stem and leaf. This idea was further evident from the low TF values (0.007‒0.044) of arsenic in A. calamus L..
Some organisms, such as bacteria, archaea, fungi, algae, and animals (including humans), are able to methylate As(III) to various oAs species (e.g., monomethylarsonic acid (MMA) and DMA) (Zhao and Wang, 2020). In organisms, As(V) can be first reduced to As(III), followed by methylation to MMA, and then methylation to DMA (Wu et al., 2015). However, few higher plants appear to have the ability to methylate As(III). In this study, DMA could be detected in the aboveground part (i.e., leaf) of A. calamus L. at each concentration of As(Ⅴ)/As(III) treatment. This appears to be the first demonstration of DMA formed in higher plants. DMA is a major metabolite of iAs in many organisms including humans (Cullen et al., 2016). In general, methylation of iAs has been regarded as a detoxification mechanism because oAs is supposed to be less toxic than iAs (Zhen et al., 2020). The presence of DMA in the aboveground part (i.e., leaf) of A. calamus L. was considered to be a means of detoxification.
iAs-induced changes in growth status of A. calamus L.
The effect of iAs concentration on the growth status of A. calamus L. was studied to evaluate its stress resistance. As illustrated in Table 2, the height growth rates of As(V)-exposed plants were lower than the control group, but the weight gain rates were slightly higher than the control group (except for the 500 µg As(V) L‒1 condition). On the contrary, the height growth rates of As(III)-exposed plants were higher than the control group, while the weight gain rates were lower than the control group. These results indicated that As(V) had a little inhibitory effect on height growth, and As(III) could slightly inhibit the weight gain. In addition, A. calamus L. could keep its green leaves during the 31 days of iAs exposure (Fig. 4), suggesting that low content of iAs in leaves had no obvious inhibitory effect on the chlorophyll synthesis of emergent macrophyte. A. calamus L. has a certain resistance to arsenic pollution in water.
Table 2
Effect of iAs concentration on growth status of A. calamus L. during 31 days of exposure.
Concentration (µg L‒1) | Height0 (cm) | Height1 (cm) | Height growth rate* (%) | Weight0 (g) | Weight1 (g) | Weight gain rate* (%) |
As(Ⅴ) | 0 | 30.3 | 73.3 | 142 | 6.91 | 16.68 | 142 |
200 | 30.7 | 71.0 | 131 | 6.75 | 16.39 | 143 |
500 | 31.3 | 61.1 | 96 | 6.59 | 12.45 | 89 |
1,000 | 28.8 | 67.0 | 132 | 6.50 | 16.13 | 148 |
As(Ⅲ) | 0 | 41.5 | 71.3 | 72 | 10.93 | 20.97 | 92 |
200 | 39.5 | 69.4 | 76 | 11.14 | 19.75 | 77 |
500 | 40.3 | 70.4 | 75 | 10.53 | 19,82 | 88 |
1,000 | 37.4 | 68.4 | 83 | 10.72 | 19.97 | 86 |
*Height growth rate=[(Height1‒Height0)/Height0]×100% |
Weight gain rate=[(Weight1‒Weight0)/Weight0]×100%
In nature, few plant species are capable of accumulating or detoxifying extraordinarily high levels of arsenic (Souri et al., 2017). Arsenic can affect the morphological, physiological, biochemical, and metabolic attributes of plants like root-shoot length and biomass, chlorophyll content, and photosynthetic rate (Bali & Sidhu, 2021). Singh et al. (2019) reported As(V) treatments (100 and 200 µM) led to significant reduction in root and leaf biomass. Arsenic treatment also reduced the fresh weight by 25.8% and dry weight by 31.0% in Artemisia annua L. as compared to the respective control (Naeem et al., 2020). In this study, As(V) was found to slightly decrease the height growth rate of A. calamus L.. This may be related to the decrease of nutrients (e.g., Ca, Mg, and P) uptake and transport caused by As(V) (Gusman et al., 2013). However, As(III) was more inclined to slightly reduce the weight gain rate of A. calamus L.. This phenomenon may be caused by the reduction in the photosynthesis rate under As(III) treatment, which leads to reduced organic matter content (Gupta et al., 2021).
iAs-induced changes in lipid peroxidation and oxidative stress of A. calamus L.
MDA, a marker of lipid peroxidation, was determined in the As(V)/As(III)-exposed plants. As demonstrated in Fig. 5a&d, the MDA concentrations in the root were significantly higher than those in the stem and leaf. For As(III) treatment, the MDA concentrations showed an upward trend with the increase of treatment concentration. For As(V) treatment, the MDA concentrations in A. calamus L. with 500 µg As(V) L‒1 treatment were the highest. H2O2 is a normal by-product of cellular metabolism that in higher concentrations can induce oxidative stress. Similarly, the concentrations of H2O2 were the highest in the root of As(III)-exposed plants in each treated concentration (Fig. 5f). But the amount of H2O2 generated in response to As(V) was lower than that responding to As(III). CAT is involved in H2O2 catabolism, which is important in defense against oxidative stress. The activities of CAT in the As(III)-treated groups were the lowest in the leaf, and increased with the increase of the As(III) concentration (Fig. 5e). The highest CAT activity in As(V)-exposed plants was found in the root at 500 µg As(V) L‒1 (Fig. 5b).
Arsenic is a redox-active element that causes oxidative stress by generating reactive oxygen species (ROS) in the plant tissues from its conversion of As(V) to As(III), leading to non-specific oxidation of biomolecules, enzyme inactivation, and membrane damage (Adhikary et al., 2022). ROS are reactive derivatives of O2 metabolism, including superoxide radical (O2·‒), singlet oxygen (1O2), hydroxyl radical (·OH), and H2O2. Out of all the ROS molecules, H2O2 has the longest stability (half-life of 10‒3 s) within plant cells (Huang et al., 2019). H2O2 is considered to be an important signaling molecule that responds to various stimuli. In A. calamus L., the accumulation of H2O2 was greater in the root than in the stem and leave. The concentration of H2O2 was obviously related to the concentration of arsenic absorbed. On the other hand, plants have evolved a suite of antioxidant system to protect against oxidative stress by scavenging ROS (Singh et al., 2017). CAT is one of the crucial antioxidant enzymes that mitigates oxidative stress to a considerable extent by metabolizing H2O2 to H2O and O2. Corresponding to H2O2, CAT activity increased significantly upon As(III) treatment. As described by Liu et al. (2017), a higher activity of antioxidant enzyme generally exhibited better resistance to heavy metal stress. It was suggested that A. calamus L. could well resist As(III) stress. In addition to the enzymatic antioxidant defense systems, plants synthesize the non-enzymatic antioxidants such as MDA and glutathione (GSH) which are also involved in scavenging ROS (Ahire et al., 2021). MDA is generated by the peroxidation of membrane polyunsaturated fatty acids, which is the most commonly used marker for the determination of the antioxidant status. In this study, both As(V) and As(III) stress caused a significant increase in MDA levels in the root of A. calamus L.. At the same time, the level of MDA increased with increasing arsenic concentration. These results were in agreement with the findings of Banerjee and Roychoudhury (2022) and Chu et al. (2022), suggesting that A. calamus L. was equipped with an efficient antioxidant mechanism against iAs-induced oxidative stress.