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Arsenic biomonitoring using a hyperaccumulator fern (Pteris vittata)

Vincenzo Minganti,*a Laura Cornara,b Massimo Piana,c Antonio Corallob and Mauro Giorgio Mariottic
a

Dipartimento di Chimica e Tecnologie Farmaceutiche ed Alimentari, Universita ` di Genova, Via Brigata Salerno (ponte), 16147 Genova, Italy. E-mail: minganti@dictfa.unige.it; Fax: 139-0103532684; Tel: 139-0103532605 b Dipartimento per lo Studio del Territorio e delle sue Risorse, Universita ` di Genova, Corso Dogali 1M, 16136 Genova, Italy c Dipartimento di Produzione Vegetale, Universita ` di Milano, Via Celoria 2, 20133 Milano, Italy
Downloaded on 03 October 2010 Published on 03 December 2003 on http://pubs.rsc.org | doi:10.1039/B307981C
Received 11th July 2003, Accepted 30th October 2003 First published as an Advance Article on the web 3rd December 2003

Samples of Pteris vittata L. (brake fern or ladder brake) collected in Genova and in areas outside urban centres, have been analysed for arsenic content in order to assess if hyperaccumulating plants are suitable for monitoring purposes. Hyperaccumulation ability of the Ligurian fern populations was evaluated by analysing specimens grown with hydroponic media added with As(V). Arsenic concentrations in the range 2310 mg g21 dry weight have been measured in samples collected in different sites along the Ligurian coast. Arsenic concentrations in fern fronds correlate with the estimated arsenic emission in the area, verifying the applicability of P. vittata as an arsenic biomonitor.

1. Introduction
Arsenic is a naturally occurring element, widely distributed in the environment. In the Earths crust the most important arsenic containing ores are arsenopyrite (FeAsS), orpiment (As2S3) and realgar (AsS). Arsenic concentration in groundwater reects the content of soil and rock with which water comes in contact. Sources of arsenic are both natural (soil and rock erosion, dissolution of minerals and ores, volcanic activity and biological activity) and anthropogenic (municipal wastes incineration, coal and oil red power plant, smelting and roasting of non-ferrous metals, cement works, lead and copper alloy production, electronics industries, use of arsenic-containing pesticides and herbicides). Anthropogenic sources exceed natural sources by a factor of 3 to 1. Copper, nickel and zinc smelting and coal combustion account for about 60% of anthopogenic input of arsenic into the environment, so the atmosphere is the most important form of transport for arsenic of anthropogenic origin.13 The use of biomonitors in environmental studies originate from the need of integrating (over time or over space) the signal (contaminant concentrations) and by simplifying the analytical method required owing to the enrichment of the pollutant in the biomonitors tissue. Therefore the screening of new organisms suitable for use as biomonitors is a target of various environmental studies. For this purpose, we have concentrated our attention on Pteris vittata L. (brake fern or ladder brake), a newly discovered arsenic-hyperaccumulator.46 Due to its constitutive hyperaccumulation,7 both in contaminated and uncontaminated soils,8 and given its widespread distribution, this plant species is a prominent candidate for use in phytoremediation.9,10 This plant has both considerable biomass and ability to concentrate arsenic in its above-ground parts.11 Moreover, P. vittata is a perennial and fast-growing species, present as indigenous or naturalized in many warm temperate zones of the world, where it is also capable of growing in highly urbanized areas, with a minimum amount of soil substrate.12 P. vittata actually spreads and colonizes various countries and habitats and in some regions it is considered an exotic invasive plant. In Italy it has

rapidly invaded many localities along the Tyrrhenian coasts from Sicily to Liguria, in a variety of habitats including steep slopes and stone or concrete walls. Several potential toxic trace elements including As, Cd, Cr, Pb, Mn and Zn can be found in the aerosols and deposited dusts encountered in the urban environment as a consequence of various anthropic activities.13 These pollutants are subsequently transferred to soil due to rainwater percolation. Since 1985, Ho and Tai12 investigated the potential use of roadside fern (Pteris vittata) in the biomonitoring of aerial metal deposition, in particular with regard to lead pollution. This paper reports on the As level in the fern P. vittata growing both in urban and rural areas with the aim to verify the possible use of this species as arsenic-biomonitor.

2. Materials and methods

Fronds of P. vittata, assessed to belong to the tetraploid cytotype (n ~ 58), were collected from eight sites along the Ligurian coast (Fig. 1), four of which are within the urban area of the city of Genoa. The sites are located at various distances from the sea and from main industrial plants. Sample collection was made during two consecutive years, in late spring of 2001 and 2002. Due to the orography of the region (a mountain range close to the sea) the most important transfer processes possible are in the EastWest or WestEast directions. Predominant wind directions14 measured in the town of Genoa (Fig. 1) are from North-East (toward the sea) or from the South-East (from the sea), therefore winds should play a secondary role in pollutant transfer along the region. After harvesting, two mature fern fronds from each of the different sites were used to measure the amount of As by the method described below. A series of hydroponical experiments were carried out for testing the hyperaccumulation ability of the fern sampled in Liguria. No attempt to establish a dose-concentration relationship was done in this work since it was beyond the aim of the study and, moreover, data on this subject are already available in literature.7,10
J. Environ. Monit., 2004, 6, 2325
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DOI: 10.1039/b307981c

This journal is The Royal Society of Chemistry 2004

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Downloaded on 03 October 2010 Published on 03 December 2003 on http://pubs.rsc.org | doi:10.1039/B307981C

Fig. 1 Site localisation along the Ligurian coast and in the city of Genoa. Predominant wind direction and mean speed (m s21), by ARPALCMIRL (www.meteoliguria.it/vento.html), are plotted according to their probability; going from 1% (inner line) to 0.1%, 0.01%, 0.001% and 0.0001% (outer line).

Part of the samples collected in their natural habitat were grown on a general-purpose compost to obtain the production of new fronds, that was achieved within three months. At the end of this period these plants were washed carefully with water to remove adhering compost and transferred to 500-ml pots (one plant per pot) containing Hoagland nutrient solution that was aerated continuously and renewed weekly. During the hydroponic culture, ferns were kept inside a controlled environment green-house with 25 uC/20 uC day/night temperature, and 6070% relative humidity. After 1 week, Na2HAsO4 was added to the nutrient solution to give 100 mg As(V) mL21. Plants were grown for 3 months and during this period samples of the fronds were collected in order to evaluate the As concentration in tissues. Five pinnae from the median zone of the frond were harvested and the spores separated. Laminae were then oven dried and processed by ICP-AES analysis. Samples of dried tissue were obtained from the plants collected in their natural habitat and from those grown in the greenhouse. Analysis were done on unwashed samples. No standard procedure for washing plant samples has yet been proposed (deionised or distilled water, detergents, dilute acids, chloroform, with or without sonication, dry brushing and/or blowing are all methods used in literature). The need of washing is under discussion.15 Unwashed plant samples have been frequently preferred for environmental monitoring.15 These samples (0.1 g) were mineralised using 5 mL of 65% (m/m) nitric acid in a closed PFA Teon vessel heated in a MDS 2000 (CEM Corporation, Matthews, NC, USA) microwave digestion system. After cooling, the solutions were transferred into 25 mL volumetric asks and diluted to volume. Ultra pure water with specic resistivity w18 MV cm (Elgastat UHQ, Elga Ltd., High Wycombe Bucks, UK) was used for all solutions and for rinsing all glassware. Nitric acid was puried in a quartz sub-boiling apparatus. Arsenic concentration were measured by atomic emission spectrometry with an inductively coupled plasma torch (ICP-AES) using a J.Y. 24 apparatus (Jobin-Yvon, Longjumeau, France) equipped with a Cetac U-5000AT1 ultrasonic nebulizer (Cetac Technologies Inc., Omaha, Nebraska, USA). Calibration was done
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using aqueous standard solutions obtained by dilution of a commercial stock solution (Merck, Darmstadt, Germany). Arsenic was measured using the 193.759 nm wavelength, and the limit of detection (three times the standard deviation of blank solutions, n ~ 4) was 2 mg g21. The accuracy of analytical determinations was assessed by analysing a Certied Reference Material (CRM). The CRM used was TORT-2 (Lobster Hepatopancreas Reference Material for Trace Metals, National Research Council of Canada, Ottawa, Ontario, Canada) because of its high arsenic content. The result obtained on the CRM (20.8 0.7 mg g21 dry weight, n ~ 3) is in good agreement with the certied value (21.6 1.8 mg g21 dry weight).

3. Results and discussion

Results obtained with the accumulation test are reported in Fig. 2, proving that the ligurian P. vittata populations are able

Fig. 2 Arsenic concentration in P. vittata fronds grown in hydroponical system with (r) or without (%) the addition of Na2HAsO4 (100 mg As(V) mL21).

J. Environ. Monit., 2004, 6, 2325

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Table 1 Arsenic concentrations in Pteris vittata sampled in Liguria (North-West Italy) Sampling site 1 2 3 4 5 6 7 8
a

Coordinates 43u4702.8@ 44u0253.3@ 44u2522.4@ 44u2508.9@ 44u2448.4@ 44u2504.3@ 44u2433.0@ 44u2012.4@ 7u3318.7@ 8u1319.4@ 8u4404.6@ 8u5130.2@ 8u5508.1@ 8u5509.5@ 8u5534.8@ 9u1259.8@

Elev./m 75 2 60 6 1 20 1 18

Dist./m 164 390 90 540 0 380 0 140

As/mg g21 2 28 2 209 130 57 310 16 1 2 1 25 16 7 37 3

Ventimiglia (Mortola) Albenga (railway station) Crevari (cemetery) Sestri Ponente (Calcinara) Genova (Ponte dei Mille) Genova (Principe railway station) Genova (Expo `) S. Margherita Ligure (railway station)

Coordinates are given according the World Geodetic System 1984 (www.wgs84.com). Elevation on sea level, and distance from sea are also given. Arsenic concentrations are the mean of two analysis and the standard deviation is reported. Concentrations are given in mg g21 dry weight.

Downloaded on 03 October 2010 Published on 03 December 2003 on http://pubs.rsc.org | doi:10.1039/B307981C

The obtained results indicate that P. vittata can effectively be used as an arsenic bioindicator, considering its high ability to concentrate arsenic and its capability of growing in highly urbanised areas.

Acknowledgements
This research was funded by the Universities of Genova and Milano (FIRST 2001-2003). We thank Dr Irene Santi for the technical assistance and Dr Lidia Badalato and Dr Vincenzo Parisi (Regione Liguria) for providing data about As pollution in Liguria.

Fig. 3 Arsenic concentrations in fronds of P. vittata vs. arsenic emission classes. Labels represent the sampling sites (see Table 1). Arsenic emission classes are: class A from 0 to 0.01 kg year21; class B from 0.01 to 0.5 kg year21; class C from 0.5 to 1 kg year21; class D from 1 to 50 kg year21 and class E w50 kg year21.

References
1 2 3 J. Matschullat, Arsenic in the geosphere a review, Sci. Total Environ., 2000, 249, 279312. B. K. Mandal and K. T. Suzuki, Arsenic round the world: a review, Talanta, 2002, 58, 201235. P. L. Smedley and D. G. Kinniburgh, A review of the source, behaviour and distribution of arsenic in natural waters, Appl. Geochem., 2002, 17, 517568. L. Q. Ma, K. M. Komar, C. Tu, W. Zhang, Y. Cai and E. D. Kenneley, A fern that hyperaccumulates arsenic, Nature, 2001, 409, 579. T. Chen, C. Wei, Z. Huang, Q. Huang, Q. Lu and Z. Fan, Arsenic Hyperaccumulator Pteris vittata L. and its arsenic accumulation, Chin. Sci. Bull., 2002, 47, 902905. E. Lombi, F. J. Zhao, M. Fuhrmann, L. Q. Ma and S. P. Mc Grath., Arsenic distribution and speciation in the fronds of the hyperaccumulaor Pteris vittata, New Phytol., 2002, 156, 195203. F. J. Zhao, S. J. Dunham and S. P. McGrath, Arsenic hyperaccumulation by different fern species, New Phyt., 2002, 156, 2731. A. A. Meharg, Arsenic and old plants, New Phyt., 2002, 156, 14. P. Visoottiviseth, K. Francesconi and W. Sridokchan, The potential of Thai indigenous plant species for the phytoremediation of arsenic contamined land, J. Environ. Pollut., 2002, 118, 453461. W. Zhang, Y. Cai, C. Tu and L. Q. Ma, Arsenic speciation and distribution in an arsenic hyperaccumulating plant, Sci. Total Environ., 2002, 300, 167177. C. Tu and L. Q. Ma, Effects of Arsenic Concentations and Form on Arsenic Uptake by the Hyperaccumulator Ladder Brake, J Environ. Qual., 2002, 31, 641647. Y. B. Ho and K. M. Tai, Potential use of a roadside fern (Pteris vittata) to biomonitor Pb and other aerial metal deposition, Bull. Environ. Contam. Toxicol., 1985, 35, 430438. P. K. Hopke, R. E. Lamb and D. F. S. Natusch, Multi-elemental characterization of urban roadway dust, Environ. Sci. Technol., 1980, 14, 164172. ARPAL-CMIRL: www.meteoliguria.it/vento.html. R. Bargagli, Trace Elements in Terrestrial Plants, Springer-Verlag, Berlin, Heidelberg, New York, 1998. Regione Liguria, Dipartimento Tutela dellAmbiente, ed Edilizia, Settore Politiche e Programmi Ambientali, 1999, personal communication.

to hyperaccumulate arsenic in agreement with literature data about a Central Florida (USA) population.4 The remarkable difference between As-treated samples and controls shown in Fig. 2 indicate that P. vittata can be a good bioindicator since this fern may magnify the environmental As level. Results obtained for monitoring purposes are reported in Table 1. The geographical distribution of arsenic in fern fronds is summarised in Fig. 1 with circles representing the concentrations measured. Samples collected in urban sites (stations No. 47) show an arsenic concentration (176 108 mg g21) signicantly (p v 0.05, t-test) higher than values measured outside the town (12 13 mg g21). No correlation exists between arsenic concentrations in fern fronds and elevation on sea level or distance from the sea, indicating that the sea is not an important arsenic source. Atmospheric arsenic emission in the area studied have been evaluated by Regione Liguria16 taking into account the inventory of possible sources: industries, transports, and power plants. From such values, ve classes have been calculated considering both diffused and local (weighted for distance from sampling site) emissions. Low emissions (00.01 kg year21) are attributed to class 1, and emissions w50 kg year21 are class 5. The emission classes proposed show a good correlation (r ~ 0.78, p v 0.05) with the arsenic concentrations measured in fern fronds as shown in Fig. 3. We hypothesise that the arsenic concentration within the fern fronds mirrors the arsenic presence in the environment, due to both atmospheric fallout and soil availability of this trace element. Moreover the As soil concentration is likely related to atmospheric deposition and subsequent percolation processes

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