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 Arsenic tolerance and accumulation in ferns Sridokchan et al 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.