Arsenic biomonitoring using a hyperaccumulator fern (Pteris vittata)

Abstract

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 2–310 µg g⁻¹ 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.

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1. Introduction

Arsenic is a naturally occurring element, widely distributed in the environment. In the Earth’s crust the most important arsenic containing ores are arsenopyrite (FeAsS), orpiment (As₂S₃) and realgar (AsS). Arsenic concentration in groundwater reflects 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 fired 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.^1–3

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 biomonitor's 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.^4–6 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.¹²P. 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 East–West or West–East 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.

Fig. 1 Site localisation along the Ligurian coast and in the city of Genoa. Predominant wind direction and mean speed (m s⁻¹), by ARPAL-CMIRL (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).

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

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 °C/20 °C day/night temperature, and 60–70% relative humidity. After 1 week, Na₂HAsO₄ was added to the nutrient solution to give 100 µg As(V) mL⁻¹. 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 Teflon vessel heated in a MDS 2000 (CEM Corporation, Matthews, NC, USA) microwave digestion system. After cooling, the solutions were transferred into 25 mL volumetric flasks and diluted to volume. Ultra pure water with specific resistivity >18 MΩ cm (Elgastat UHQ, Elga Ltd., High Wycombe Bucks, UK) was used for all solutions and for rinsing all glassware. Nitric acid was purified 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-5000AT⁺ ultrasonic nebulizer (Cetac Technologies Inc., Omaha, Nebraska, USA). Calibration was done 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 µg g⁻¹. The accuracy of analytical determinations was assessed by analysing a Certified 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 µg g⁻¹ dry weight, n = 3) is in good agreement with the certified value (21.6 ± 1.8 µg g⁻¹ 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 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.

Fig. 2 Arsenic concentration in P. vittata fronds grown in hydroponical system with (◆) or without (□) the addition of Na₂HAsO₄ (100 µg As(V) mL⁻¹).

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. 4–7) show an arsenic concentration (176 ± 108 µg g⁻¹) significantly (p < 0.05, t-test) higher than values measured outside the town (12 ± 13 µg g⁻¹). 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.

Table 1 Arsenic concentrations in Pterisvittata sampled in Liguria (North-West Italy)

	Sampling site	Coordinates	Elev./m	Dist./m	As/µg g^−1
a 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 µg g^−1 dry weight.
1	Ventimiglia (Mortola)	43°47′02.8″ 7°33′18.7″	75	164	2 ± 1
2	Albenga (railway station)	44°02′53.3″ 8°13′19.4″	2	390	28 ± 2
3	Crevari (cemetery)	44°25′22.4″ 8°44′04.6″	60	90	2 ± 1
4	Sestri Ponente (Calcinara)	44°25′08.9″ 8°51′30.2″	6	540	209 ± 25
5	Genova (Ponte dei Mille)	44°24′48.4″ 8°55′08.1″	1	0	130 ± 16
6	Genova (Principe railway station)	44°25′04.3″ 8°55′09.5″	20	380	57 ± 7
7	Genova (Expò)	44°24′33.0″ 8°55′34.8″	1	0	310 ± 37
8	S. Margherita Ligure (railway station)	44°20′12.4″ 9°12′59.8″	18	140	16 ± 3

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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, five classes have been calculated considering both diffused and local (weighted for distance from sampling site) emissions. Low emissions (0–0.01 kg year⁻¹) are attributed to class 1, and emissions >50 kg year⁻¹ are class 5. The emission classes proposed show a good correlation (r = 0.78, p < 0.05) with the arsenic concentrations measured in fern fronds as shown in Fig. 3.

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

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

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.

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