Removal of perchlorate by a lab-scale constructed wetland using achira (Canna indica L.)


 Achira (Canna indica L.) has not yet been tested for its potential in removing perchlorate (ClO4−) from wastewater. In this study, constructed wetlands with and without achira were used to investigate the removal efficiency and removal mechanism of perchlorate. The results showed that more ClO4− was removed by the wetlands with achira relative to those without. Perchlorate removal in the wetlands without achira decreased with time, whereas perchlorate in the wetlands with achira was stably removed. In terms of ClO4− content, the achira tissues were in the descending order of: leaf > aerial stem > flower or rhizome > root. Perchlorate was concentrated in leaves (more than 55.8%) rather than in root (less than 0.67%). Mass balance calculation showed that plant uptake accounted for 5.81–7.34% of initial ClO4− input, while microbial degradation accounted for 29.39–62.48%. The wetlands with achira were favorable for soil microorganism growth and proliferation and in turn ClO4− biodegradation. Furthermore, the effluent pH increased in achira wetland columns and in turn promoting ClO4− removal. The results indicating that the wetlands with achira promote ClO4− removal by improving the rhizosphere environment.


Introduction
Used in such industrial productions as reworks, rubber, and paint manufacturing, perchlorate has emerged as a contaminant in soil, water, and food. United State Environmental Protection Agency (U.S. EPA) reported that perchlorate concentrations up to 2000 mg/kg, 3700 mg/L, 120 mg/L, and 811 μg/L have been documented in soil, groundwater, surface water, and drinking water, respectively (U.S. EPA, 2005), as well as airborne particulate matter (Wang et al. 2017). In addition, perchlorate has also been detected in food stuffs, breast milk, baby formulas, soft drinks, and human body uids (Her et  .This has caused a major concern due to the health effects of perchlorate, which is documented to inhibit iodine uptake by the thyroid gland, decrease thyroid production, and lead to thyroid disease (Abt et  Phytoremediation is a bioremediation technology where perchlorate is reduced via plant uptake, phytodegradation, and rhizo-degradation (Yifru and Nzengung 2008; Bhaskaran et al. 2013). Plant uptake and phytodegradation are slow processes, whereas rhizo-degradation is rapid. Utilizing dissolved organic carbon as a carbon and energy source, PRB can rapidly reduce perchlorate to nontoxic chloride (Dahan et al. 2017; Yifru and Nzengung 2008).
Constructed wetlands are established mainly for wastewater treatment (Sehar and Nasser 2019; Abdel-Mohsein et al. 2020). As constructed wetlands are easy for operation and maintenance, environmentally friendly, and e cient, they have been widely used in treating domestic wastewater (Bilgin et

Materials And Methods
Preparation of constructed wetland columns Wetland columns were prepared using cylindrical ceramic pots (30 cm height and 26 cm inner diameter) ( Fig.  1). For each pot, there was a side opening at the bottom connected to a tube for effluent collecting. Each pot was first filled with a 5-cm layer of cobble (4 kg/pot) and then a 20-cm layer of soil (8 kg/pot). The cobbles, white 1-3 cm diameter, purchased from Goldstone Powder Materials Co., LTD (China), which had been washed with tap water before use. The soil was collected from the surface layer of a paddy eld in the Experimental Farm of the South China Agricultural University, air dry and through 2 mm sieve before use. The main properties of the soil are shown in Table 1.

Plant species
The wetland plant species, achira, was selected for its large biomass and high tolerance to perchlorate (He et al. 2013). Plants of the same size at their three-leaf stage were collected from a local nursery. The seedlings height were about 10-15 cm.

Experimental details
Constructed wetland columns with or without plants (achira) were prepared to treat waters containing 0 (C 0 ), 40 (C 40 ), or 100 mg/L (C 100 ) perchlorate. For the three treatments with achira, two achira seedlings at three-leaf stage were transplanted to each wetland column.
Before the experiment started, the columns were rst ooded with 6 L tap water for 7 days, drained, and let stand for one day. Then, they were fed with 6 L tap water from the top containing 0, 40, or 100 mg/L perchlorate. Water was added every day for seven days to compensate for evaporation loss. Then, e uents were collected on the 8th day, and the wetland columns were let Plant and bulk soil samples were collected at the end of the experiment. During the whole experiment (53 days), the 18 wetland columns were sheltered from rain. The properties of the tap water used in the experiment are shown in Table 2.

Sample preparation
The collected e uent samples were stored in plastic bottles and kept in a refrigerator until analysis. The plants including roots were washed with tap water and blotted dry. After plant height, root length, and fresh weights of shoot and root were measured, shoots and roots were dried, ground into powder, and sieved through a 35-mesh (0.5 mm) sieve. Soil samples were freeze-dried, ground, and homogenized with a 35-mesh (0.5 mm) stainless steel sieve.
For ClO 4 concentration determination, the soil and plant samples were processed as described by He et al. (2013). Brie y, 2.0 g of plant powder were added into a 50-mL conical ask with 30 mL Milli-Q water (Milli-Q Water System, Millipore Corporation, Bedford, MA), shaken at 200 rpm and room temperature for 3 h, and centrifuged at 6 000 rpm for 25 min. The supernatant was vacuum ltered through a 0.22 μm polyethersulfone lter (Millipore Corp., Bedford, MA) and passed through a pre-conditioned ENVI-18 solid-phase extraction (SPE) cartridge (Supelco, Bellefonte, PA) to remove organic materials. The rst 1.0 mL ltrate is discarded and the remaining aliquots are used for analysis. Soil sample (5.0 g) was weighed into a 50-mL conical ask with 20 mL deionized water, shaken at 200 rpm and room temperature for 3 h, and centrifuged at 6 000 rpm for 25 min. The supernatant was ltered through a 0.22 μm polyethersulfone membrane lter, an OnGuard H cartridge, and an OnGuard RP successively to remove solid substances, metallic ions, and hydrophobic compounds, respectively.

Perchlorate quanti cation
Chromatographic column IonPac AG20/AS20 was used at 30 o C, the mobile phase was 30 mM KOH at 1.0 mL/min, and the injection volume was 10 μL. After ltration through a 0.22-μm lter, the sample solution was injected into the system. The retention time of ClO 4 − was 15.5 min. The concentrations of ClO 4 − in the samples were quanti ed by external calibration. Standard curves were calculated from injection of 50 to 50,000 μg/L calibration standards. A calibration standard was run along with every 10 samples, while a laboratory reagent blank, a laboratory forti ed blank, and a laboratory forti ed duplicate were run long with every 20 samples. Based on injection of 10 μL injections, the instrument detection limit was 20 μg/L. Quality control samples were prepared as the e uent, plant, or soil samples. The mean recovery of ClO 4 − was 100 ± 10%.

Enumeration of soil microbial populations
Populations of culturable bacteria, fungi, and actinomycetes in the rhizosphere samples were estimated using the serial dilution technique. Ten gram soil were mixed with 100 mL sterile water, shaken for 20 min, let stand for 3-5 min, serially diluted, and plated onto 1/10-strength beef-protein agar (BPA), potato dextrose agar (PDA), and starch nitrate agar (SNA) for enumeration of bacteria, fungi, and actinomycetes, respectively.

Statistics analysis
The experiment adopted a completely randomized design in triplicates. Data were analyzed using the analysis of variance (ANOVA) and Duncan's multiple range tests with SPSS-13 statistical package (SPSS Inc., Chicago, IL, USA) at a signi cant level of P < 0.05. Results are presented as mean ± standard error. Perchlorate removal rate was computed as follows: where C 0 is the initial perchlorate concentration (mg/L); V 0 is the initial volume (L); C i is perchlorate concentration at day i (mg/L); and V i is the volume at day i (L).

Results And Discussion
Perchlorate removal by the constructed wetland columns Differences in perchlorate removal were observed in the different treatments (Fig. 2 Plant uptake Plant height, and plant biomass in columns C 40 and C 100 were signi cantly lower as compared with column C 0 (  (Table 4). These ndings are in line with previous ndings for other wetland plants (Seyfferth and Parker 2008).
Perchlorate content in the above-water tissues (aerial stem, leaf, and ower) of achira was much higher than that in the underwater tissues (root and rhizome). The order of perchlorate content in different tissues of achira was: leaf > aerial stem > ower or rhizome > root. Perchlorate content in achira leaf was 2 868.51 and 10 441.06 mg /kg DW in column C 40 and C 100 , respectively. In contrast, lower perchlorate content in root was observed, 141.03 and 703.51 mg/kg DW in column C 40 and C 100 , respectively. It is evident that perchlorate mainly accumulated in leaf (more than 55.8%) rather than in root (less than 0.67% where M in is total ClO 4 input to the wetland system (g), M out is ClO 4 output with the e uent from the wetland system (g), M p is the part absorbed by the plants (g), M m is the part degraded by microbes (g), and M s is the part adsorbed by the soil and gravels.
As shown in Table 5, less than 6.32% of ClO 4 input was adsorbed by the soil or gravels in any of the treatments, about 5.81-7.34% was absorbed by achira in the planted columns, and more than 29.39% was degraded by microbes in any of the treatments. A signi cantly higher percentage of the initial ClO 4 input was biodegraded in the planted columns than in the unplanted columns. Of the initial ClO 4 input, 62.48% and 43.27% was biodegraded in the planted C 40 and C 100 treatments, respectively. In comparison, 45.90% and 29.39% was biodegraded in the unplanted C 40 and C 100 columns, respectively. Interestingly, achira greatly increased the proportion of perchlorate being biodegraded, indicating that plants play an important role by creating a more favorable environment for microbial growth and activity. In this study, no external carbon was supplied to the wetland columns. Carbon required by the perchlorate-reducing microbes must be self-supplied in the wetland systems. Therefore, it is expected that plant would play a more important role with time in such wetland systems as it provides both habitat and food for the indigenous perchlorate-reducing microorganisms.
Microbial populations in the constructed wetlands As shown in Table 6, the CFUs of culturable bacteria, fungi, and actinomycetes in rhizosphere decreased with running time of the wetland systems, indicating that microbial growth and reproduction was inhibited by perchlorate and the inhibiting effect became more prominent with time. Sha et al. (2020) also showed that microbial community changed when exposed to perchlorate. For a same perchlorate input level, bacterial, fungal, and actinomycete counts were greater in the planted columns than in the unplanted ones, suggesting that achira helped to improve microbial growth in the wetland systems. Correlation analysis showed that there was a signi cant positive correlation between perchlorate removal and the microbial counts (Table 9).
Additionally, bacteria were prevalent over fungi and actinomycetes in the wetland columns no matter with plants or not. Taken together, contaminants are removed in constructed wetlands under the synergistic action of plant-soil-microorganism, and the symbiont interaction between microorganisms and animals and plants plays a central role (Lee et al. 2009). Microbial species and population in a constructed wetland directly affect the performance of the system.

E uent pH value in the constructed wetlands
As shown in Table 7, the differences in e uent pH between treatments became bigger with running time, especially after the 44th day, when e uent pH in the planted columns was signi cantly higher than that in the unplanted columns, suggesting that achira helped to raise pH value in the wetland systems. Furthermore, in the later periods, the e uent pH values of the columns, planted or not, were in the order of: C 0 > C 40 > C 100 .
Correlation analysis showed that there was a signi cant positive correlation between perchlorate removal and e uent pH ( ). In the present study, NO 3 concentration in the e uent decreased gradually with the operation continuing, but increased with increasing ClO 4 concentration in both the unplanted and planted columns (Table   8). E uent NO 3 concentration in the planted columns was signi cantly lower than that in the unplanted columns after 17 days of operation (Table 8). And as shown in Fig.2

Conclusions
In summation, we demonstrated the constructed achira wetlands have the potential to treated ClO 4 contaminated wastewaters. Microbial degradation and pH raise played a major role in overall removal of perchlorate, plant uptake and transformation only contributed to a relatively small portion, but planting achira could greatly improve ClO 4 biodegradation in this wetland system. As plants can provide a continuous carbon source and support medium for microbe growth, plants will play a more important role in long term wetland treatment systems.    The data are presented as mean ± standard error (n = 3). Different lowercase letters in a same column indicate signif differences between treatments (Duncan's multiple range tests, P < 0.05)      Percent removal of perchlorate in different constructed wetland columns.