Effects of arsenate and phosphate on their accumulation by an arsenic ...

Plant and Soil 249: 373–382, 2003. © 2003 Kluwer Academic Publishers. Printed in the Netherlands.

373

Effects of arsenate and phosphate on their accumulation by an arsenic-hyperaccumulator Pteris vittata L. Cong Tu1 & Lena Q. Ma2 Soil and Water Science Department, University of Florida, Gainesville, FL 32611-0290, USA 1 Present address: Department of Plant Pathology, North Carolina State University, Raleigh, NC 27695-7616, USA. 2 Corresponding author∗ Received 8 April 2002. Accepted in revised form 4 October 2002

Key words: accumulation, arsenate, hyperaccumulator, interaction, phosphate, Pteris vittata L.

Abstract Arsenate and phosphate interactions are important for better understanding their uptake and accumulation by plant due to their similarities in chemical behaviors. The present study examined the effects of arsenate and phosphate on plant biomass and uptake of arsenate and phosphate by Chinese brake (Pteris vittata L.), a newly-discovered arsenic hyperaccumulator. The plants were grown for 20 weeks in a soil, which received the combinations of 670, 2670, or 5340 µmol kg−1 arsenate and 800, 1600, or 3200 µmol kg−1 phosphate, respectively. Interactions between arsenate and phosphate influenced their availability in the soil, and thus plant growth and uptake of arsenate and phosphate. At low and medium arsenate levels (670 and 2670 µmol kg−1 ), phosphate had slight effects on arsenate uptake by and growth of Chinese brake. However, phosphate substantially increased plant biomass and arsenate accumulation by alleviating arsenate phytotoxicity at high arsenate levels (5340 µmol kg−1 ). Moderate doses of arsenate increased plant phosphate uptake, but decreased phosphate concentrations at high doses because of its phytotoxicity. Based on our results, the minimum P/As molar ratios should be at least 1.2 in soil solution or 1.0 in fern fronds for the growth of Chinese brake. Our findings suggest that phosphate application may be an important strategy for efficient use of Chinese brake to phytoremediate arsenic contaminated soils. Further study is needed on the mechanisms of interactive effects of arsenate and phosphate on Chinese brake in hydroponic systems.

Introduction Arsenic (As) is toxic whereas phosphorus (P) is essential for plants. They are both Group VA elements and thus have similar electron configurations and chemical properties. In soil, therefore, arsenate and phosphate will compete with each other for soil sorption sites, resulting in a reduction in their sorption by soil and an increase in solution concentrations (Livesey and Huang, 1981; Manning and Goldberg, 1996; Smith et al., 2002). For example, Livesey and Huang (1981) found that phosphate significantly suppressed the sorption of arsenate by soils. Gao and Mucci (2001) have recently reported similar competitive effects between arsenate and phosphate. They observed ∗ FAX No: +1-352-392-3902. E-mail: [email protected]

that increasing arsenate in solution results in the enhanced competition between phosphate and arsenate for sorption sites and a subsequent decrease in the amount of phosphate sorbed. Similarly, it may be difficult for plants to distinguish between arsenate and phosphate. Thus uptake of arsenate and phosphate by plants is very likely to be competitive. Furthermore, after entering a plant, arsenate may replace phosphate in ATP synthesis, and/or in various phosphorolysis reactions, thus interfering with phosphate metabolisms and causing toxicity to a plant (Dixon, 1997). In contrast, phosphate may be able to alleviate arsenate toxicity by improving phosphate nutrition (Sneller et al., 1999). Arsenate competes with phosphate as a substrate for the phosphate uptake system in many species, in-

374 cluding angiosperms (Asher and Reay, 1979; Jacobs and Keeney, 1970), mosses (Wells and Richardson, 1985), lichens (Nieboer et al., 1984), fungi (Beever and Burns, 1980) and bacteria (Silver and Misra, 1988). Many studies have shown that arsenate reduces phosphate uptake by plants (Asher and Reay, 1979; Jacobs and Keeney, 1970). Since the plant uptake system has a higher affinity for phosphate, only mild inhibition of arsenate on phosphate plant uptake is observed. Also, such competitive inhibition may be insignificant as arsenate is toxic to plants at higher levels. In recent years some studies have also shown that at low levels arsenate can increase phosphate uptake (Burlo et al., 1999; Carbonell et al., 1998). These authors assumed that such enhancement of plant phosphate uptake resulted from a physiological phosphorus deficiency caused by low arsenate, since arsenate can substitute for phosphate within the plants but is unable to carry out phosphate’s role in energy transfer. Phosphate have long been reported to suppress plant uptake of arsenate (Asher and Reay, 1979; Khattak et al., 1991; Meharg and Macnair, 1991; Rumberg et al., 1960; Woolson et al., 1973). In a hydroponic solution containing 50 µM arsenate, sufficient phosphate will alleviate arsenate toxicity and improve plant growth. Plant arsenate uptake rate is reduced by 75% at 0.5 mM phosphate (Meharg and Macnair, 1991). A molar P/As ratio of at least 12 is needed to protect plants against arsenate toxicity (Walsh and Keeney, 1975). Nonetheless, plant arsenate uptake and toxicity depends on both the P/As ratio and phosphate nutrition levels. A hydroponic study has shown that at the same P/As ratio arsenate is much less toxic at high phosphate levels since more arsenate is taken up by the plants at low phosphate levels (Sneller et al., 1999). In soil, however, the influence of phosphate on arsenate phytotoxicity varies. This is because soil properties affect the availability of phosphate and arsenate. With 1100 µmol kg−1 arsenate supply, additions of up to 9700 µmol kg−1 phosphate do not influence arsenate toxicity on a silt loam soil (Jacobs and Keeney, 1970; Woolson et al., 1973). This can possibly be attributed to the soil having a high phosphate fixation capacity and available phosphate probably does not increase much after phosphate addition. However, applying the same amount of phosphate actually enhances arsenate toxicity in a sandy soil, which is due to the displacement of sorbed arsenate from the soil by phosphate. When sufficient phosphate is added to maintain an available phosphate/arsenate molar ratio of about 16, phosphate improves plant yields

(Woolson et al., 1973). At very high levels of added arsenate (13 mmol arsenate kg −1 ), however, phosphate does not overcome arsenate toxicity even at a molar phosphate/arsenate ratio of 24. Chinese brake (Pteris vittata L.) is a newlydiscovered arsenic hyperaccumulator (Ma et al., 2001). It accumulates 160–853 µmol As kg−1 in its aboveground parts from uncontaminated soils containing 6.3–100 µmol As kg−1 , and takes up as much as 310 mmol As kg−1 when grown in soil spiked with 20 mmol kg−1 arsenate (Ma et al., 2001). Addition of 670 µmol kg−1 arsenate to a sandy soil increases the fern biomass by 107%. Moreover, this fern can tolerate up to 6.7 mmol kg−1 arsenate in a sandy soil and removed up to 26% of the added arsenate after 18 weeks (Tu and Ma, 2002). Unfortunately, there is no information available about arsenate and phosphate interactions in Chinese brake. The objectives of this study were to (1) examine the effects of arsenate and phosphate interactions on fern biomass production and uptake of arsenate and phosphate, and (2) determine the molar ratios of phosphate to arsenate in both soil and plant for better fern growth. The results will provide critical information for better understanding arsenate hyperaccumulation by Chinese brake and optimizing soil conditions for arsenate phytoextraction.

Materials and methods Soil characterization The soil used in this study is a sandy soil (sandy, siliceous, hyperthermic grossarenic paleudult) from Gainesville, Florida. The soil pH was measured using a 1:1 soil to water ratio; cation exchange capacity (CEC) was determined by an ammonium acetate method (Thomas, 1982); organic matter content was measured by the Walkley Black method (Nelson and Sommers, 1982); and particle size was measured by the pipette method (Day, 1965). Selected physical and chemical properties of the soil are presented in Table 1. Experimental design Our previous study showed that Chinese brake produced the highest biomass when 670 µmol kg−1 arsenate was added (Tu and Ma, 2002). Thus arsenate concentrations were chosen at 1×, 4×, and 8× that concentration, i.e., 670, 2670, and 5340 µmol arsenate kg−1 , and referred to as low (AsL ), medium (AsM ) and

375 Table 1. Selected properties of the soil used in this study Property pH (1:1 soil/water ratio) Organic matter content (g kg−1 ) CECa (cmol(+) kg−1 ) Total P (mmol kg −1 ) Water-soluble P (µmol kg −1 ) Total As (µmol kg −1 ) Water-soluble As (µmol kg −1 ) Sand (g kg−1 ) Silt (g kg−1 ) Clay (g kg−1 )

This soil 7.3 11.0 4.4 20.8 96.8 9.2 0.27 882 91 27

a Cation exchange capacity of soil.

high (AsH ) levels, respectively. In loamy soils, phosphate was found to have no further benefit for plants at greater than 3200 µmol kg−1 phosphate (Jacobs and Keeney, 1970; Woolson et al., 1973). Therefore, phosphate concentrations were selected as 0.25, 0.5, and 1× the maximum phosphate concentration, i.e., 800, 1600 and 3200 µmol phosphate kg−1 , referred to as low (PL ), medium (PM ), and high phosphate (PH ). One control (As0 P0 ) without adding arsenate or phosphate was also included for reference purposes.

for chemical analysis. Soil samples were collected from each pot at transplanting the ferns for measuring water-soluble arsenate and phosphate. Chemical analysis Plant (∼0.1–0.5 g) and soil (0.5–1.0 g) samples were weighed into a 120-mL Teflon pressure digestion vessel, mixed with 10 mL of nitric acid (concentrated trace-metals grade), and digested using USEPA Method 3051 with a CEM MDS-2000 microwave sample preparation system (CEM, Matthews, NC). After cooling, the solution was filtered and diluted to a volume of 100 mL. Water-soluble phosphate and arsenate were extracted with deionized water at 2:20 soil to solution ratio by shaking for 2 h at 25 ± 1 ◦ C. Phosphorus concentrations in solution were measured on a ICP-MS unit (Perkin-Elmer ELAN 6000, Norwalk, CT). Arsenic was determined using a graphite furnace atomic absorption spectrophotometer (Perkin-Elmer SIMMA 6000). Statistical analysis The data (excluding As0 P0 ) were statistically evaluated with a two-way analysis of variance using SAS programming (SAS Institute Inc., 1996). Means were separated using the Duncan multiple range test at a 5% level of probability.

Greenhouse experiment Soil (1.5 kg) was mixed with the designated amounts of arsenate and phosphate, 200 mg N kg−1 and 100 mg K kg−1 , and placed in a 2.5-L plastic pot. Arsenate and phosphate were added as Na2 HAsO4 and NaH2 PO4 , respectively. Potassium was provided as KNO3 and N as both KNO3 and NH4 NO3 . There were four replicates for each treatment using a complete randomized experimental design. After 2 weeks of equilibrium, one healthy fern plant with five to six fronds was transplanted into each pot. Plants were allowed to grow for 20 weeks in the greenhouse (temperature ranged 14–30 ◦ C; average photosynthetically active radiation was 825 µmol m−2 s−1 ) and were watered daily or as needed. At the end of the experiment, the ferns were harvested. Each individual fern was further separated into roots (including rhizomes) and young, mature and old fronds based on their ages. They were all washed thoroughly with tap water, and then rinsed quickly with 0.1 M HCl solution followed by several rinses with deionized distilled water. All samples were ovendried for 3 days at 65 ◦ C, and ground to a fine powder

Results and discussion Soil water-soluble arsenate and phosphate It is well known that water-soluble fractions of soil arsenic and phosphorus are readily available for plant uptake. In the present experiment, arsenic speciation did not show the existence of other arsenic species in solution (unpublished data). Thus, the changes in their water-soluble fractions are important for understanding interactive effects of arsenate and phosphate on plant. Water-soluble arsenate and phosphate in the soil were first determined (Figure 1). After two-weeks of incubation following arsenate and phosphate applications, arsenate and phosphate interactions in the soil significantly affected their water-soluble fractions (Figure 1 and Table 2). Soluble arsenate was significantly slightly increased only by higher phosphate application compared to the low phosphate levels (Table 2 and Figure 1a, c). However, water-soluble phosphate was greatly enhanced by arsenate additions

376 Table 2. Results of the two-way ANOVA and Duncan tests for the effects of arsenate and phosphate on their contents in soil and Chinese brake and the dry biomass of the plant Source of variation

ANOVA F values As rate P rate As rate × P rate

Dry biomass (g plant−1 ) 390.0∗∗∗a 44.9∗∗∗ 37.6∗∗∗

Duncan multiple range test As rate 10.0 bb AsL c AsM 10.6 a 4.8 c AsH P rate PL 7.2 b 8.9 a PM PH 9.2 a

Soluble As Soluble P —–(µmol kg−1 soil)—– 2198.9∗∗∗ 9.2∗∗ 5.0∗∗

78.2∗∗∗ 37.0∗∗∗ 2.1NS

Root As Frond As Root P Frond P ————-(mmol kg−1 d.w.)————216.1∗∗∗ 2.0NS 5.1∗∗

124.2∗∗∗ 1.5NS 2.8NS

4.9∗ 2.4NS 5.8∗∗

8.3∗∗ 6.7∗∗ 3.6∗

100.5 c 589.3 b 1339.0 a

580.7 c 1091.4 b 1510.8 a

7.7 c 23.4 b 34.4 a

38.3 c 118.5 b 159.4 a

168.4 a 159.1 ab 145.8 b

112.4 a 117.4 a 104.3 b

646.8 b 659.7 b 722.3 a

731.1 c 1080.7 b 1371.0 a

23.0 a 20.5 a 21.9 a

113.0 a 100.0 a 103.1 a

160.1 a 164.3 a 148.9 a

106.9 b 108.9 b 118.2 a

a NS – not significant F ratio (P < 0.05), ∗ , ∗∗ , and ∗∗∗ significant at P < 0.05, 0.01, and 0.001, respectively. b Treatment means from the ANOVA test. Values followed by the same letter, within the same source of variation, are

not significantly different (P < 0.05), Duncan multiple range test.

c As , As and As = 670, 2670 and 5340 µmol kg−1 arsenate, P , P , and P = 800, 1600 and 3200 µmol kg−1 L M H L M H

phosphate, respectively.

at all three phosphate levels (Table 2 and Figure 1b, d). Within experimental arsenate levels, there was a positive correlation between arsenate rates and soluble phosphate (coefficient = 0.992–1.00). Compared to low arsenate treatments, water-soluble phosphate increased by 70–106% and 164–197% at medium and high arsenate levels, respectively. A closer examination of arsenate and phosphate availability in soil showed that 22–123% of the applied phosphate was water-soluble while only 13–26% of the applied arsenate was water-soluble (Table 3), indicating adsorption of more arsenate than phosphate by the soil. This is also supported by increased watersoluble phosphate/arsenate molar ratios of 0.8–7.2 (P molar concentrations in the soil divided by As molar concentrations in the soil) compared to the added phosphate/arsenate molar ratios of 0.1–4.8 (Table 3). Arsenate and phosphate interactions in soil have previously been shown to increase their concentrations in soil solution by competing for sorption sites (Gao and Mucci, 2001; Smith et al., 2002). For example, application of phosphate fertilizer to arseniccontaminated soils has resulted in the displacement of about 77% of the total arsenic in the soil and redistribution of arsenic to lower depths in the soil profile (Woolson et al., 1973). Similarly, phosphate sorption by soil is decreased by arsenate as demonstrated by

Table 3. Percentages of water-soluble arsenate and phosphate concentrations in total addition and the phosphate/arsenate molar ratios in soil Treatment

% of water-soluble/ total added P As

As0 P0 AsL /PL c AsL /PM AsL /PH AsM /PL AsM /PM AsM /PH AsH /PL AsH /PM AsH /PH

NAb 34 26 22 69 62 43 123 99 63

NA 13 15 17 19 23 24 25 25 26

P/As molar ratioa Water-soluble 320 4.3 5.3 7.2 1.3 1.8 2.3 0.8 1.2 1.5

Total added NA 1.2 2.4 4.8 0.3 0.6 1.2 0.1 0.3 0.6

a P molar concentrations in the soil divided by As molar concentra-

tions in the soil. b Not applicable. c As , As and As = 670, 2670 and 5340 µmol kg−1 arsenate, L M H PL , PM , and PH = 800, 1600 and 3200 µmol kg−1 phosphate,

respectively.

Gao and Mucci (2001) who reported that phosphate sorption decreased from 57 to 48% and 57 to 42% following the addition of 8.7 and 22 µM arsenate, respectively. In our experiment, when 5,430 µmol kg−1

377

Figure 1. Effects of different arsenate and phosphate levels on water-soluble arsenate and phosphate concentrations in a soil in the greenhouse experiment. The treatment As0 P0 (
). The levels of added phosphate were 800 (), 1600 (♦), and 3200 (×) µmol kg−1 . The levels of added arsenate were 670 (), 2670 (), and 5340 () µmol kg−1 . Bars represent standard deviations of four replicates.

arsenate and 800–1600 µmol kg−1 phosphate were concurrently added to the soil, all the phosphate was essentially water-soluble (Table 3), i.e., no phosphate was adsorbed by the soil. Biomass of Chinese brake fern For a hyperaccumulator, biomass is a key factor for phytoremediation practices. Moreover, it is also an overall measurement of plant health. Our previous experiment showed that the biomass of Chinese brake was greatly enhanced by arsenate up to 1330 µmol kg−1 and it survived even at 6700 µmol kg−1 (Tu and Ma, 2002). The enhancement of fern biomass by arsenate was also observed in this study up to 2670 µmol kg−1 arsenate (Figure 2 and Table 2). This arsenate rate was two times greater than previously reported (Tu and Ma, 2002). Addition of medium arsenate levels increased fern biomass by 10–24% as compared to that in low arsenate. However, phosphate only increased fern biomass at the high arsenate level. At 5340 µmol kg−1 arsenate, the fern biomass was greatly increased by 1600 µmol kg−1 phosphate compared to that at 800 µmol phosphate kg−1 addition,

though it was still lower than that of As0 P0 . However, no further biomass enhancement was observed at 3200 µmol kg−1 phosphate. These findings indicate that phosphate has an alleviating effect on arsenate phytotoxicity to Chinese brake only at high arsenate levels (Figure 2). Interestingly, the molar ratios of water-soluble phosphate to arsenate in the soil were 0.8, 1.2 and 1.5 for 5340 µmol arsenate kg−1 treatments with 800, 1600 and 3200 µmol phosphate kg−1 , respectively (Table 3). It seems that phosphate plays a minor role in inhibiting arsenate phytotoxicity at a soluble P/As molar ratio of greater than 1.2. This value was much lower than the results obtained from nonhyperaccumulators (Hurd-Karrer, 1939; Woolson et al., 1973). Phosphate effect on arsenate uptake and accumulation in Chinese brake Chinese brake is very efficient in absorbing arsenic from soil and translocating it from roots to shoots (Ma et al., 2001; Tu and Ma, 2002). Arsenic concentrations in the plant are generally much higher in the fronds than in the roots, with concentrations increasing with

378

Figure 2. Effects of different arsenate and phosphate levels on the biomass of Chinese brake grown in a soil in the greenhouse experiment. The levels of added phosphate were 0 (
), 800 (), 1600 (×), and 3200 (♦) µmol phosphate kg−1 . Bars represent standard deviations of four replicates.

soil arsenate. These findings were also observed in the present study (Figure 3), with the highest arsenate concentrations found in the fern growing in the soil receiving high arsenate (5340 µmol arsenate kg−1 ). At low and medium arsenate levels (≤2670 µmol kg−1 arsenate and water soluble P/As = 1.3–7.2), phosphate slightly but not significantly increased arsenic concentrations in the roots and fronds of the fern (Table 2 and Figure 3c, d). Interactive effects of phosphate and arsenate was observed on root arsenic (Table 2). However, at high arsenate levels (5340 µmol kg−1 ), phosphate at 1600 µmol kg−1 decreased arsenic concentrations by 23–25% in the roots and fronds (Figure 3c, d). This appears to be due to the dilution effects from greater biomass production, which resulted from alleviation of arsenate phytotoxicity by phosphate (Figure 2). Previous studies showed that the effect of phosphate on plant arsenate uptake depends on plant growing conditions (Asher and Reay, 1979; Jacobs and Keeney, 1970; Khattak et al., 1991; Meharg and Macnair, 1991; Pickering et al., 2000; Woolson et al., 1973). In a hydroponic system, phosphate at 500 µM reduced arsenate uptake by 75% in both tolerant and non-tolerant plant genotypes of soft grass (Holcus lanatus L.) grown in 50 µM arsenate solution (Meharg and Macnair, 1991). Alfalfa (Medicago sativa L.) shoot arsenate concentrations were also decreased by phosphate (Khattak et al., 1991). Even for Indian mustard (Brassica juncea L.), a hyperaccumulator, grown in 500 µM arsenate hydroponic solution with phosphate addition at 1000 µM, a reduction of arsenate uptake by 55–72%

over the control was reported (Pickering et al., 2000). Since arsenate and phosphate are generally transported by the same uptake system, which has a much greater affinity for phosphate than arsenate (Asher and Reay, 1979; Meharg and Macnair, 1990; Meharg et al., 1994), phosphate can effectively reduce the arsenate uptake and toxicity of plants. In the soil systems, however, phosphate may either reduce arsenic concentrations in the plant through competition and/or enhanced plant growth by alleviating arsenate phytotoxicity, or increase arsenate uptake by the plant and hence phytotoxicity, depending on soil conditions and/or relative phosphate/arsenate levels (Creger and Peryea, 1994; Jacobs and Keeney, 1970; Woolson et al., 1973). This is because addition of phosphate usually increases arsenate concentrations in soil solution by replacing arsenate sorbed by soil particles (Smith et al., 2002). Plant arsenic accumulation takes both arsenate uptake and plant biomass into consideration, providing a better indication of phosphate effects on arsenate phytoextraction (Table 4). Compared to low phosphate level treatments, applying more phosphate fertilizer enhanced total arsenate accumulation by the fern from the soil, especially at high arsenate levels (5340 µmol kg−1 ), where adding 1600–3200 µmol kg−1 phosphate resulted in increases up to 286% in arsenate accumulation with biomass contributing more than arsenate concentration. The highest arsenate accumulation (1042 µmol arsenate plant−1 ) by the fern was recorded in AsM PH treatment (water soluble P/As molar ratio = 2.3), accounting for 26% of initial total soil arsenic. These findings have great implications for optimizing phytoextraction of soil arsenic. Arsenate effect on phosphate accumulation by Chinese brake Phosphorus concentrations in plants normally range from 96 to 160 mmol kg−1 of dry matter during the vegetative stage, with the highest in the young and lowest in the old parts of the plants (Marschner, 1995). In the present study, phosphorus concentrations in the fronds were 71–175 mmol kg−1 , which are within the normal ranges of plant phosphorus contents. The distribution of phosphorus in the fern is also similar to that of nonhyperaccumulators, with greater concentrations in the roots than in the aboveground biomass, and greater in young fronds than in old fronds. Phosphorus concentrations in the fern were little influenced by phosphate rates (Table 2 and Figure 4). These results suggest

379

Figure 3. Effects of different arsenate and phosphate levels on arsenate concentrations in roots and fronds of Chinese brake grown in a soil in the greenhouse experiment. The treatment As0 P0 (
). The levels of added phosphate were 800 (), 1600 (♦), and 3200 (×) µmol kg−1 . The levels of added arsenate were 670 (), 2670 (), and 5340 () µmol kg−1 . Bars represent standard deviations of four replicates. Table 4. Arsenic accumulation (µmol plant−1 ) in Chinese brake as influenced by different arsenate and phosphate levels in soil

Treatments

Roots

Fronds

As0 P0 AsL /PL a AsL /PM AsL /PH AsM /PL AsM /PM AsM /PH AsH /PL AsH /PM AsH /PH

0.21±0.1b 21.2±6.9 22.4±7.1 30.6±3.7 70.9±11 66.3±5.5 67.2±9.8 17.8±0.6 71.5±1.4 78.2±1.5

0.76±0.1 267.9±9.9 283.1±3.7 285.7±10 856.7±50 870.5±9.5 974.5±63 165.4±16 582.9±33 629.4±44

Total 0.97 289.1 305.5 316.2 927.6 936.8 1041.7 183.2 654.5 707.6

% of soil As 7.0 28.4 30.0 31.0 23.1 23.3 25.9 2.3 8.2 8.8

a As , As and As = 670, 2670 and 5340 µmol kg−1 arsenate, L M H PL , PM , and PH = 800, 1600 and 3200 µmol kg−1 phosphate,

respectively. b Mean ± SE (standard error).

that Chinese brake fern is unable to hyperaccumulate phosphate from soil. In contrast, arsenate rates and the interaction of arsenate and phosphate significantly influenced phosphorus concentrations in both roots and

fronds of the fern (Table 2). At high rates, arsenate decreased phosphate accumulation in the roots and fronds, obviously due to phytotoxicity (Table 2 and Figure 4a, b), but increased phosphorus concentrations in young fronds at medium rates of arsenate (Data not shown). This result may help us to explain the enhancement of fern biomass by arsenate at medium rates. The molar ratio and bioaccumulation preference of arsenic to phosphorus in Chinese brake The above results clearly showed that phosphate and arsenate interaction influenced the growth of and arsenate and phosphate uptake by Chinese brake. So, P/As molar ratios, P molar concentrations in the plant divided by As molar concentrations in the plant, can be a good index for their relative abundance and roles in the plants. Generally, the molar P/As ratios in the fern were higher in the roots (3–29) than in the fronds (0.5–3.8) (Table 5). Much higher results have previously been reported for non-hyperaccumulating plants grown in elevated arsenate environments such as corn (Zea mays L. subsp. mays) seedlings with 102–4400

380

Figure 4. Effects of different arsenate and phosphate levels on phosphate concentrations in roots and fronds of Chinese brake grown in a soil in the greenhouse experiment. The treatment As0 P0 (
). The levels of added phosphate were 800 (), 1600 (♦), and 3200 (×) µmol kg−1 . The levels of added arsenate were 670 (), 2670 (), and 5340 () µmol kg−1 . Bars represent standard deviations of four replicates.

(Woolson et al., 1973), tomato (Lycopersicon esculentum Mill.) with 500–4200 (Burlo et al., 1999), and salt-water cordgrass (Spartina alterniflora L.) with 800–8000 (Carbonell et al., 1998). This further indicates that Chinese brake hyperaccumulates arsenate in plant biomass. At high arsenate, the P/As ratios of the fronds increased by applying more phosphate. By looking at the fern biomass (Figure 2) and the corresponding P/As ratios in the fronds (Table 5), it can be inferred that the P/As molar ratios of greater than 1.0 in the fronds seems to be necessary for normal growth of the fern. Bioaccumulation preference (BP) of arsenate to phosphate by the plant measures the selectivity of the fern in taking up arsenate from soil as compared to phosphate, i.e., a higher number indicates a greater preference of the fern for arsenate uptake. The BP was calculated using the following equation:

Table 5. The molar ratio and bioaccumulation preference (BP)a of arsenate to phosphate in Chinese brake grown in a soil with different arsenate and phosphate levels Treatment

As0 P0 AsL /PL c AsL /PM AsL /PH AsM /PL AsM /PM AsM /PH AsH /PL AsH /PM AsH /PH

P/As molar ratiob Roots Fronds 2272 28.8 24.5 16.7 7.9 5.8 6.0 3.2 5.8 4.1

763 3.8 2.7 2.7 1.0 1.0 1.0 0.5 0.7 0.8

Roots 0.1 0.1 0.2 0.4 0.2 0.3 0.4 0.3 0.2 0.4

BP Fronds 0.4 1.1 1.9 2.7 1.3 1.8 2.3 1.7 1.7 1.8

a Bioaccumulation preference:

BP BP =

As concentrations in plant tissue/As contents in the soil P concentrations in plant tissue/P contents in the soil

The BP values were found to be greater than 1 in the fronds but less than 0.5 in the roots with an exception shown in the control (Table 5). Therefore, arsenate was preferentially accumulated in the aboveground

=

As concentrations in plant tissue/As contents in the soil P concentrations in plant tissue/P contents in the soil

b P molar concentrations in the plant divided by As

molar concentrations in the plant.

c As , As and As = 670, 2670 and 5340 µmol kg−1 , L M H PL , PM , and PH = 800, 1600 and 3200 µmol kg−1 ,

respectively.

381 parts over phosphate, especially in old fronds. However, in the control soil where arsenate was very low, arsenic distribution in the aboveground biomass was similar to that of phosphorus, i.e., both arsenic and phosphorus were the highest in the young fronds and lowest in the old fronds (data not shown). Based on our data, it seems that phosphorus and arsenic distributions were similar in the plant at low soil arsenate levels, but they were transported to different parts of the plant at elevated concentrations. In conclusion, synergistic interactions of arsenate and phosphate were observed in the soil. In the plant, phosphate addition to soil increased arsenate accumulation by Chinese brake, especially at high soil arsenate concentrations by alleviating arsenate phytotoxicity. Moderate addition of arsenate increased phosphate uptake by the fern. To improve the fern growth, the P/As molar ratios were required to be at least 1.2 in soil solution or 1.0 in the fronds. The present findings suggest that phosphate application may serve as a feasible strategy for more efficient phytoremediation of arsenic contaminated soils using Chinese brake fern. The interactions of arsenate and phosphate in the hyperaccumulator need further study in hydroponic systems.

Acknowledgements This research was partially supported by the National Science Foundation (Grant BES-0086768 and BES0132114). The senior author gratefully thanks Ms. Karen M. Parker at the Department of Plant Pathology, North Carolina State University, for improving the English of the manuscript. The thoughtful comments by Prof. Dr. A. A. Meharg, the Editor, and two anonymous reviewers are highly appreciated.

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