Arsenic Tolerance, Accumulation and Elemental Distribution in Twelve Ferns: a Screening Study

AUSTRALASIAN JOURNAL OF ECOTOXICOLOGY Vol. 11, pp. 101-110, 2005

Arsenic tolerance and accumulation in ferns Sridokchan et al

ARSENIC TOLERANCE, ACCUMULATION AND ELEMENTAL DISTRIBUTION IN TWELVE FERNS: A SCREENING STUDY

Weeraphan Sridokchan1, Scott Markich2 and Pornsawan Visoottiviseth1* 1Department of Biology, Mahidol University, Bangkok 10400, Thailand. 2Aquatic Solutions International, PO Box 3125 Telopea, NSW 2117, Australia. Manuscript received, 15/11/2004; resubmitted, 22/12/2004; accepted, 24/12/2005.

ABSTRACT Twelve species of ferns were screened for their ability to tolerate and hyperaccumulate arsenic (As). Ferns were exposed to 50 or 100 mg As L-1 for 7 and 14 days using hydroponic (soil free) experiments. The fronds and roots were analysed for As, selected macronutrients (K, Ca, Mg, P and S) and micronutrients (Al, Fe, Cu and Zn). Five fern species (Asplenium aethiopicum, Asplenium australasicum, Asplenium bulbiferum, Doodia heterophylla and Microlepia strigosa) were found to be sensitive to As. However, only A. australasicum and A. bulbiferum could hyperaccumulate arsenic up to 1240 and 2630 µg As g-1 dry weight (dw), respectively, in their fronds after 7 days at 100 mg As L-1. This is the ﬁrst known report of ferns that are sensitive to As, yet are As hyperaccumulators. All As tolerant ferns (Adiantum capillus-veneris, Pteris cretica var. albolineata, Pteris cretica var. wimsetti and Pteris umbrosa) were from the Pteridaceae family. P. cretica and P. umbrosa accumulated the majority of As in their fronds (up to 3090 µg As g-1 dw) compared to the roots (up to 760 µg As g-1 dw). In contrast, A. capillus-veneris accumulated the majority of As in the roots (1190 µg As g-1 dw) compared to the fronds (370 µg As g-1 dw). The root uptake of K, P and S was signiﬁcantly (P ≤ 0.05) reduced when ferns were exposed to As, but the translocation factor, or the movement of nutrients from the roots to the fronds, increased in most ferns to maintain nutrient requirements and ion balance. P. cretica and P. umbrosa may be useful for phytoremediating As contaminated sites because of their ability to hyperaccumulate and tolerate high As levels. Key words: Pteridaceae; fern; arsenic; hyperaccumulator; tolerance.

INTRODUCTION (water) concentration, > 10, (ii) a translocation factor (TF), Arsenic is ubiquitous in trace amounts in the natural defined as the ratio of As frond concentration to As root environment (WHO 2001). It commonly combines with Fe concentration, > 1 and (iii) the capability of accumulating and S to form arsenopyrite (FeAsS), the most abundant arsenic >1000 µg As g-1 dw in fronds (Cai and Ma 2003). Pteris mineral (Mandal and Suzuki 2002). The use of arsenical vittata has a BCF > 10 and can accumulate > 20 000 µg As pesticides is also a primary source of As contamination in g-1 dw in fronds. More recently, several other ferns in the many countries (WHO 2001). Cropping of long-term irrigated order Pteridales (Pityrogramma calomelanos, Pteris cretica, soils subjected to As contaminated ground water may pose Pteris longifolia, Pteris umbrosa and Pteris nervosa) have a significant risk to animal and human health through soil- been identified as As hyperaccumulators (Visoottiviseth et crop transfer of As. In addition, consumption of excess As in al. 2002; Zhao et al. 2002; Chen et al. 2003). However, not groundwater/potable water has caused human health problems all fern species in this Order hyperaccumulate As. Meharg in several countries (Chen and Ahsan 2004). Arsenic levels in (2003) reported that Pteris straminea and Pteris tremula did soil and water from As-contaminated environments may reach not hyperaccumulate As. 3000 µg As g-1 dry weight (dw) and 10 mg L-1, respectively The ability of some As tolerant ferns to hyperaccumulate (WHO 2001). As may offer a practical means of in situ remediation of As Arsenic is a non-essential element for plants. Biomass contaminated soils and waters, since ferns generally have production and yields are reduced significantly at elevated As higher biomass and bioaccumulation factors than other As concentrations, often leading to death (Carbonell-Barrachina non-tolerant plants (Cai and Ma 2003). For large areas of et al. 1997). Threshold values of 10-100 µg As g-1 dw have contamination, phytoremediation is generally considered to been reported for As non-tolerant plants (Kabata-Pendias be of economic benefit, compared to ex-situ chemical/physical and Pendias 2000). The first report of an As hypertolerant extraction techniques (Bondado and Ma 2003). However, plant was the Chinese brake fern (Pteris vittata), with a phytoremediation is plant-specific and not a rapid process. toxicity threshold of about 10 000 µg As g-1 dw in fronds Researchers are currently improving the efficiencies of some (Tu and Ma 2002; Wang et al. 2002). This fern was also plants (e.g. application of microorganisms to the rhizosphere) the first identified As hyperaccumulator (Ma et al. 2001); to enhance their ability to phytoremediate contaminated it was collected from a contaminated (chromated-copper- sites (Fitz and Wenzel 2002). The aim of this study was arsenate) area of Florida, USA. Arsenic hyperaccumulators to screen a large range of ferns for their ability to tolerate are defined as having (i) a bioconcentration factor (BCF), and hyperaccumulate As, and to potentially find a new As- defined as the ratio of As frond concentration to As soil hyperaccumulating species that may offer a practical means of remediating As contaminated environments.

*Author for correspondence, email: [email protected] 101 AUSTRALASIAN JOURNAL OF ECOTOXICOLOGY Vol. 11, pp. 101-110, 2005

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MATERIALS AND METHODS Plant and water analyses Whole plants were thoroughly rinsed with tap water, followed Plants by deionised water (Milli Q; 18 M cm-1). The frond and Twelve fern species (with two varieties of Pteris cretica), Ω root of each plant was separated and dried at 45°C to a ubiquitous and abundant throughout Australia, New Zealand constant measured weight. The dried fronds and roots were and south-east Asia (McCarthy 1998; Nakaike 2002), were sliced into small pieces and ground in a mortar and pestle. A acquired from a fern nursery in Sydney, Australia (Table homogenised sub-sample (200 mg) of the fronds and roots 1). Most were one-year-old sporelings, with the exception from each plant was digested with 10 mL of concentrated of two species (Asplenium australasicum and Asplenium nitric acid (AR grade; BDH) and 1 mL of hydrogen peroxide bulbiferum) that were approximately two years old. The ferns (AR grade; BDH) at 95°C, until the solution was clear. were acclimatised in a greenhouse for seven days, after which The digest solution was filtered, adjusted to 100 mL with time healthy plants were selected for use in experiments. deionised water and stored at 4°C. The digest solutions Soil was carefully removed from the growing roots, and were analysed for aluminium (Al), As, copper (Cu) and zinc the whole plants were then thoroughly rinsed in tap water, (Zn) using inductively coupled plasma mass spectrometry followed by deionised water (Milli Q; 18 MΩ cm-1). Since (ICPMS; Hewlett Packard 4500). Potassium (K), calcium the bioavailability of As to plants growing in soil is dependent (Ca), magnesium (Mg), phosphorus (P), sulfur (S), and iron on the bioavailability of dissolved As in soil water (Adriano (Fe) were analysed using inductively coupled plasma atomic 2001), hydroponic (soil-free) experiments, where ferns were emission spectrometry (ICPAES, Perkin Elmer Optima exposed to As in nutrient solutions, were used in this study. 3000 DV). This minimised a number of soil chemistry variables (e.g. clay, iron oxide and organic matter content) that may influence Sub-samples of test solutions from each concentration were As bioaccumulation and toxicity (WHO 2001). collected before and after nutrient renewal and stored at 4°C prior to analyses. Arsenic was determined using ICPMS. For Culture Conditions both plant and water analyses, an As calibration standard Individual ferns were maintained in 450 mL of modified and a reagent blank were analysed with every ten samples to Hoagland nutrient solution (¼ strength macronutrients, full monitor signal drift. For all samples and all metals, the signals strength micronutrients and 1 mg P L-1; Hoagland 1944) in changed by less than 9%, but typically 3-5%. Where ICPMS acid-cleaned polyethylene containers (600 mL) at pH 6.5 ± was used, gallium was employed as an internal standard to 0.3, for 14 days prior to the start of experiments, as well as correct for non-spectral interferences. The mean percentage during the experimental period. Sodium arsenate (analytical coefficient of variation (CV) for duplicate samples was 8%. reagent grade; BDH) was added to the nutrient solution to achieve concentrations of 0.01 mg As L-1 for the controls and Speciation modelling 50 or 100 mg As L-1 for the treatments. The nutrient solutions The speciation of As in the test solutions (pH 6.5) was were renewed twice a week to minimise nutrient and As calculated using the thermodynamic geochemical speciation depletion. Individual plants of each species were exposed to code HARPHRQ (Brown et al. 1991). The input parameters each As concentration in triplicate using a randomised block were based on physico-chemical data measured in the test design (i.e. 3 plants x 3 As concentrations x 13 species (two solutions (pH and As concentrations). Stability constants were varieties of one species) = 117 plants) for 7 or 14 days. taken from Martell et al. (2004). Plants were exposed to a 12 h light:12 h dark cycle (40 W cool Statistical analysis white fluorescent lighting, 120 µmol photons PAR m-2 s-1) at A t-test was used to determine if elemental concentrations in 27 ± 1°C. The pH of the test solutions was measured daily the fronds and roots of As exposed ferns (50 or 100 mg As in one replicate container from each treatment, and adjusted L-1) were significantly (P ≤ 0.05) different from the controls (± 0.3 pH units) when required using 5 mM NaOH or HCl. (0.01 mg As L-1). Plants were harvested after 7 or 14 days of As exposure, or immediately if ferns displayed advanced symptoms of dying (severe withering and/or necrosis of fronds), to minimise RESULTS changes in the elemental content and distribution. The Arsenic speciation biomass of each fern was determined to the nearest 0.1 g at The mean measured concentrations of As in the test solutions the beginning and end of the experiment. Any changes in were within 10% of their nominal concentrations, but usually biomass after 7 or 14 days were reported as a percentage of within 5%. Speciation calculations of the test solutions the initial biomass. showed that dissolved As was present as As5+ or arsenate 2- - The concentrations of the two As treatments (50 or 100 mg As (27% HAsO4 and 83% H2AsO4 ) at all As concentrations. L-1) are considerably enhanced relative to naturally-occurring The toxicity of arsenic is dependent on its speciation, with As background levels for soil water and water (< 0.01 mg inorganic arsenicals, such as arsenate, thought to be more toxic L-1), as well as those reported for As contaminated sites (< than organic forms (Tamaki and Frankenberger 1992). 10 mg L-1) (Mandal and Suzuki 2002). They were chosen to specifically select for fern species that would be highly Arsenic tolerance and accumulation tolerant, and thus, serve as efficient As hyperaccumulators. Table 1 summarises the results of the As tolerance of selected ferns. All species of the genus Asplenium (A. aethiopicum,

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Figure 1. Translocation factor (TF) of As in ferns classiﬁed by their ability to tolerate and accumulate As. Sen-non: As sensitive and non- hyperaccumulating ferns; Sen-acc: As sensitive and hyperaccumulating ferns; Tol-non: As tolerant and non-hyperaccumulating ferns; Tol-acc: As tolerant and hyperaccumulating ferns. Error bars indicate standard error of the mean.

A. australasicum and A. bulbiferum), as well as Doodia Tables 2 and 3 show the As concentrations in the roots and heterophylla and Microlepia strigosa, withered soon (4 - 6 fronds, respectively, of ferns exposed to 50 or 100 mg As L-1 days) after exposure to 50 or 100 mg As L-1; mean biomass for 7 days. There are four fern species which may be grouped decreased 30 to 42% (Table 1). Ferns grouped as moderately as As hyperaccumulators in the fronds. Two are classified as tolerant to As included Cyrtomium falcatum, Nephrolepis As sensitive (A. australasicum and A. bulbiferum), while two cordifolia, Onychium japonicum and Pteris ensiformis. are As tolerant (Pteris cretica and P. umbrosa). The other eight Their mean biomass decreased (4 - 20%) when exposed to species of ferns were not able to hyperaccumulate As (non- 50 mg As L-1 and they withered (mean biomass decreased 19 hyperaccumulators), including A. capillus-veneris, which is - 25%) at 100 mg As L-1 within 14 days. All ferns classified highly tolerant to As. In this species of fern, most of the As as highly tolerant to As were from the Family Pteridaceae (70%) was accumulated in the roots (1190 µg As g-1 dw; Table (Adiantum capillus-veneris, Pteris cretica var. albolineata, 2). Only 30% of As (371 µg As g-1 dw) was transported to the P. cretica var. wimsetti and P. umbrosa). They survived both fronds (Table 3). Like the other As non-hyperaccumulating of the As treatments and exposure times. Fern biomass was ferns, A. capillus-veneris concentrated the majority of As in only slightly reduced (2 - 4%) after 14 days’ exposure to the roots. 100 mg As L-1. Based on the results of As tolerance and accumulation, the Ferns classified as being “healthy” (Table 1) exhibited no twelve ferns were classified into four groups (Figure 1). The visible toxic symptoms when exposed to As. Conversely, the first group is the As sensitive and non-hyperaccumulating fronds of ferns that were visibly “withered” exhibited varying ferns, A. aethiopicum, D. heterophylla and M. strigosa. The symptoms of As toxicity. The fronds of A. aethiopicum, M. second group is the As sensitive and hyperaccumulating strigosa and N. cordifolia were noticeably withered and ferns, A. australasicum and A. bulbiferum. The third group curled, but not necrotic. Their fronds remained green but is the As tolerant and non-hyperaccumulating ferns, C. highly dehydrated, causing frond structural damage. However, falcatum, N. cordifolia, O. japonicum, P. ensiformis and the fronds of A. australasicum, A. bulbiferum, D. heterophylla, A. capillus-veneris. The last group is the As tolerant and C. falcatum, O. japonicum and P. ensiformis were noticeably hyperaccumulating ferns, P. cretica var. albolineata, P. cretica withered and necrotic. Necrosis started from the tip and var. wimsetti and P. umbrosa. margins of the frond, turning them yellow/brown.

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The effect of As on element uptake and DISCUSSION translocation in ferns Arsenic tolerance and accumulation The accumulation of K, P and S in the roots of As All As hyperaccumulating ferns reported so far belong to hyperaccumulating ferns was significantly (P 0.05) reduced ≤ the Family Pteridaceae, Order Pteridales, Genera Pteris following exposure to 50 or 100 mg As L-1 for 7 days, and Pityrogramma (Ma et al. 2001, Visoottiviseth et al. except S in A. bulbiferum and P. cretica var. albolineata 2002; Zhao et al. 2002), and are typically highly tolerant to (Table 2). Conversely, the concentrations of Ca and Mg As. This study establishes that two ferns (A. australasicum significantly (P [ 0.05) increased in many of these ferns. The and A. bulbiferum) from the Family Aspleniaceae are also concentrations of K and S in the fronds of ferns exposed to able to hyperaccumulate As – but are sensitive to As. These As were not significantly (P > 0.05) different from those in As sensitive ferns (Table 1) showed As toxicity symptoms the controls. However, the P concentration in the As tolerant similar to other As sensitive plants. Therefore, due to the and hyperaccumulating ferns (P. cretica var. wimsetti and relative sensitivity of A. australasicum and A. bulbiferum P. umbrosa), and three of the non-hyperaccumulating ferns to As, they are not practical for use in remediating As- (D. heterophylla, C. falcatum and O. japonicum) were contaminated environments, at least at As concentrations significantly (P 0.05) lower in the controls than those ≤ in water or soil water > 50 mg L-1. In contrast to As tolerant exposed to As (Table 3). The Fe concentration in the roots and hyperaccumulators, they lack a mechanism to detoxify As. fronds of most ferns was typically not affected by exposure Unlike A. australasicum and A. bulbiferum, A. aethiopicum to As (Tables 2 and 3). However, the concentrations in the did not hyperaccumulate As. Similarly, P. ensiformis did fronds and roots of O. japonicum decreased in As-exposed not hyperaccumulate As, even though it belongs the same plants, whilst the Fe concentration in the roots of A. capillus- Order as P. cretica and P. umbrosa, which were efficient As veneris increased. The extremely high Fe concentration (4410 hyperaccumulators (Table 3). This finding is consistent with g As g-1 dw) in the root of A. capillus-veneris was strongly µ that of Meharg (2003), who found that other species of Pteris correlated with the highest As concentration (1190 g As g-1 µ (P. straminea and P. tremula) were not As hyperaccumulators. dw) (Table 2). The concentrations of Al, Cu and Zn in the These data suggest that the ability of ferns to hyperaccumulate roots and fronds were not significantly (P > 0.05) different As is highly specific to the species, rather than the genus. between the controls and As-exposed ferns (Table 2 and 3). Meharg (2003) used a phylogenetic basis to suggest that The concentrations of these metals were up to 10-fold higher the highly-evolved ferns, like Pteridales, have an improved in the roots than the shoots. ability to tolerate and accumulate As compared with other The translocation factor (TF) was calculated by dividing fern groups. the element concentration in the frond by its concentration Arsenate (As5+) and arsenite (As3+) are the inorganic in the root. The TF describes the ability of the plant to phytoavailable forms of As in soil solution (Meharg and transport elements from the roots to the shoots. Although Hartley-Whitaker 2002). However, microbes, which can the accumulation of K, P and S in the fronds of most ferns methylate and demethylate arsenic species in soils, may decreased when exposed to As, the TF values actually transform inorganic As species to organic species and increased, especially for K and P (Table 4). Conversely, the vice versa (Turpeinen et al. 1999). Dissolved As in the test TFs of Ca, Mg and Fe decreased in several ferns exposed solutions was calculated to be present as arsenate (27% to As. Therefore, the translocation of these elements was HAsO 2- and 83% H AsO -) at all As concentrations. The inhibited by the presence of As. The TFs for Al, Cu and Zn 4 2 4 toxicity of As to plants is dependent on its speciation, were not significantly (P > 0.05) different between control with inorganic arsenicals, such as arsenate, reported to be and As-exposed ferns (data not shown). The mean TFs for more toxic than organic forms (Tamaki and Frankenberger As were extremely high in the As hyperaccumulating ferns, 1992). Thus, As was present in the test solutions in its most (i.e. 7.4 and 3.2 for As sensitive hyperaccumulators and As bioavailable and phytotoxic form. tolerant hyperaccumulators, respectively; Figure 1). The mean TFs were not significantly (P > 0.05) different amongst the As The As concentrations used in this study (50 and 100 mg As non-hyperaccumulators (Figure 1). For A. capillus-veneris, L-1) were environmentally high and the P concentration was the As TF significantly decreased (P ≤ 0.05) when exposed low (1 mg P L-1), giving an As: P ratio of 50-100. The typical to As. This fern had the highest measured As concentration range of total P in soils is 0.20-2.0 g P kg-1 (McGrath 1994). in the roots (1190 µg As g-1 dw) of all ferns and showed a However, dissolved P is typically less than 0.1 percent of the 40-fold increase in As concentration relative to the control total soil P and usually exists as orthophosphate, inorganic exposure (29 µg As g-1 dw; Table 2). A similar result was also polyphosphates and organic P (Magette and Carton 1996). observed for Fe with this species. As a consequence, the TF Thus, a concentration of 1 mg P L-1, as used in this study, of Fe was substantially reduced (i.e. from 0.31 to 0.02 in the is consistent with that typically occurring in soil water. -1 100 mg As L exposure). Phosphate (PO4), a chemical analogue of arsenate (AsO4), is well known to reduce As uptake and toxicity in plants via competition, at elevated levels. Hence, the low P, high As concentrations used in this work were selected to enhance any potential As toxicity to the selected species of ferns. Meharg (2003) reported Nephrolepis cordifolia and Pteris cretica to have a similar As tolerance when grown in contaminated soil

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Arsenic tolerance and accumulation in ferns Sridokchan et al . A The effect of As on physical condition and biomass of selected ferns, their arsenic tolerance classiﬁcation As on physical of The effect Table 1. Table

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Table 2. Mean element concentrations in roots of ferns exposed to As for 7 daysA.

(100 mg As kg-1), but was unable to determine the dissolved As tolerance of these two species, and may be an advantage As concentration to which each species was exposed. In when screening ferns for As tolerance in the field. contrast to the findings of Meharg (2003), N. cordifolia was The root uptake of K, P and S significantly (P ≤ 0.05) classified as moderately As tolerant and P. cretica highly As decreased, whilst Ca and Mg increased, when ferns were tolerant, based on results from the present study. The chemical exposed to As. However, most of the macronutrients in the conditions used in the present work (i.e. uniform dissolved As frond of As exposed ferns were not significantly (P > 0.05) concentrations) may have assisted in better separating the true

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Table 3. Mean element concentrations in fronds of ferns exposed to As for 7 daysA.

different from the controls, except for P. To offset the reduced The mechanisms of arsenic tolerance in ferns uptake of K, P and S, some fern species, particularly those that Plants have two basic defence mechanisms for protection from are both As tolerant and As hyperaccumulators (P. cretica and metal(loid) toxicity: (1) exclusion or (2) hyperaccumulation P. umbrosa), increased their TF of these elements to maintain (Hall 2002; Meharg and Hartley-Whitaker 2002; Cobbett a stable levels in the fronds and maintain ion balance. These 2003). Most As tolerant plants exclude As via decreased findings are consistent with those of Carbonell-Barrachina et uptake of arsenate due to suppression of the high-affinity al. (1998) for Spartina alterniflora exposed to As. phosphate uptake system (Meharg and Macnair 1992; Meharg

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Table 4. Translocation factors of elements in fernsA.

and Hartley-Whitaker 2002). In this study, A. capillus-veneris One proposed mechanism of As tolerance in P. vittata is is a good model of an As excluding plant with a high As chelation followed by sequestration. According to this tolerance. This species can store high levels of As in its hypothesis, arsenate is ﬁrst reduced to arsenite in cytoplasm, root (1190 µg As g-1 dw) without displaying phytotoxic and then chelated by ligands (to form phytochelatins and effects. The high levels of Fe also found in the roots of this glutathione) to avoid the consequences of cellular toxicity. species may have some role in ameliorating As toxicity, and Arsenic complexes are eventually sequestrated into vacuoles deserve further study. Hyperaccumulation is an alternative to be stored. This hypothesis is supported by energy dispersive defence mechanism, whereby As is internally sequestered. X-ray microanalysis, which reveals that As is primarily Hyperaccumulators typically protect themselves from the located in the upper and lower epidermal cells, probably in toxic effects of metal(loid)s by transporting them from the the vacuoles (Lombi et al. 2002). Francesconi et al. (2002) roots to the shoots and transforming them to a non-toxic form, found a high proportion of arsenate (95-97%) in the root either via (a) a change in chemical form (i.e. oxidation state) and a high proportion of arsenite (up to 70%) in the frond. and/or (b) complexation with proteins, such as phytochelatins, Once inside the plant, arsenate is reduced to arsenite either metallothioneins and glutathione, and storing the metal(loid) through the action of reducing agents such as glutathione or complex in the vacuole (Zenk 1996; Vatamaniuk et al. ascorbic acid, or more likely through the action of arsenate 2000). To date, the mechanism of hypertolerance and reductase (Meharg and Hartley-Whitaker 2002). Then hyperaccumulation in As hyperaccumulating plants, such arsenite is transported to the shoot and is present primarily as Pteris spp. and Pityrogramma calomelanos has not been as aqueous arsenite species in the frond (Wang et al. 2002; resolved. Webb et al. 2003). Wang et al. (2002) concluded that As

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Arsenic tolerance and accumulation in ferns Sridokchan et al is primarily present in inorganic forms in the fronds of P. Cai Y and Ma LQ. 2003. Metal Tolerance, Accumulation, vittata, although the possibility of complexation could not and Detoxification in Plants with Emphasis on Arsenic in be excluded. However, no simple organo-As compounds (i.e. Terrestrial Plants. In Biogeochemistry of environmentally arsenobetaine, arsenocholine, arsenosugars, and methylated important trace elements, Cai Y and Braids O (Eds), Oxford forms), which have been detected in other terrestrial plant University Press, London, UK, pp. 95-114. species at low levels (Geiszinger et al. 2002; Meharg and Carbonell-Barrachina AA, Aarabi MA, DeLaune RD, Hartley-Whitaker 2002), were present in the frond extracts Gambrell RP and Patrick WH Jr. 1998. Arsenic in wetland (< 0.5%). vegetation: Availability, phytotoxicity, uptake and effects on Zhao et al. (2003) found that phytochelatins play a limited role plant growth and nutrition. Science of the Total Environment (1 - 3%) in the hypertolerance of As in P. vittata exposed to 217, 189-199. elevated As concentrations. This finding is consistent with that Carbonell-Barrachina AA, Burlo F, Burgos-Hernandez of Raab et al. (2004), who found that As in P. cretica is present A, Lopez E and Mataix J. 1997. The influence of arsenite mainly in non-bound inorganic forms with only 1% as As- concentration on arsenic accumulation in tomato and bean phytochelatin complexes. The arsenic-induced thiol group in plants. Scientia Horticulturae 71, 167-176. P. vittata was purified and analysed by Zhang and Cai (2003) and found to be a phytochelatin with two subunits (PC2). Chen T, Huang Z, Huang Y, Xie H and Liao X. 2003. Cellular More recently, Zhang et al. (2004) reported an unknown distribution of arsenic and other elements in hyperaccumulator As species, other than arsenite (As3+) or arsenate (As5+) in Pteris nervosa and their relations to arsenic accumulation. leaflets of P. vittata. The chromatographic behaviour of this Chinese Science Bulletin 48, 1586-1591. unknown As species, its stability at different pHs and charge Chen Y and Ahsan H. 2004. Cancer burden from arsenic in states, suggest that it unlikely to be an As3+–PC2. Uncovering drinking water in Bangladesh. American Journal of Public an As tolerance mechanism in As hyperaccumulating ferns Health 94, 741-744. is essential to understand As hyperaccumulation and the evolution of this unique capacity. Cobbett C. 2003. Heavy metals and plants - model systems and hyperaccumulators. New Phytologist 159, 289-293. CONCLUSIONS Fitz WJ and Wenzel WW. 2002. Arsenic transformations in The major outcome of this screening study was the discovery the soil-rhizosphere-plant system: fundamentals and potential of two species of ferns (A. australasicum and A. bulbiferum) application to phytoremediation. Journal of Biotechnology that were able to hyperaccumulate As in their fronds, yet be 99, 259-278. classified as As non-tolerant. This apparent paradox is the Francesconi K, Visoottiviseth P, Sridokchan W and Goessler first known report of this phenomenon and requires further W. 2002. Arsenic species in an arsenic hyperaccumulating fern, experimental work, particularly at lower As concentrations. Pityrogramma calomelanos: A potential phytoremediator of No new species of As tolerant and hyperaccumulating ferns arsenic-contaminated soils. 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