Fresenius Envir Bull 9: 275 - 280 (2000) © Editors, Freising-Weihenstephan/FRG 1018-4619/2000/5-6/275-06 DM 20 or 3.50/p

PHYTOREMEDIATION OF THE POLLUTED SOILS AFTER THE TOXIC SPILL OF THE AZNALCOLLAR MINE BY USING WILD SPECIES COLLECTED IN SITU A. de Haro1*, A. Pujadas2, A. Polonio1, R. Font1, D. Velez3, R. Montoro3 and M. del Rio1. l 2 3

I.A.S. - CSIC. Av. Alameda del Obispo s/n.E-14080 Cordoba. Spain.

University of Cordoba. Av. Menendez Pidal s/n. E-3048. Cordoba. Spain.

IATA. CSIC. Apartado de Correos 73. E-46100. Burjasot. Valencia. Spain. (*) Corresponding author. E-mail: [email protected]

ABSTRACT The accident of the Aznalcollar mine on April 1998 in the proximity of the Donana National Park (southern Spain) le.d to the contamination of the Guadiamar river and the adjacent agricultural areas (5000 Ha). After physically removing the sediments the soils have remained polluted by heavy metals such as Pb, Cu, Zn, Cd, Tl, Sb and metalloids as As. Periodical field surveys have been made in the affected land in order to identify the metal tolerant species that are spontaneously growing in the polluted soils. From the ninety six different plant species collected, Amaranthus blitoides (accumulation of As, Pb and Cu), Erodium moschatiim (accumulation of Zn) and Lavatera cretica (accumulation of Cd) highlight as the most promising species to be used with phytoremediation purposes. Key words: Phytoremediation, heavy metals, arsenic, Erodium Amaranthus blitoides

moschatiim, Lavatera

cretica,

INTRODUCTION After the pioneer work of Minguzzi and Vergnano1 showing the hyperaccumulation of nickel by Alyssum bertolonu from the Impruneta region near Florence, more than 400 plants5that hyperaccumulate a given heavy metal,have been described2. These plant species, endemic to metalliferous soils, are able to concentrate metals to extraordinarily high levels (>1% dry weight) in contrast to normal concentrations in plants. The possibility of using this kind of plants to remove pollutans from the environment or to render them harmless was first

examined by Chaney3 and later reviewed by Baker and Brooks4, this

technology being called phytoremediation. On 25 April 1998 a pyrite slurry occurred in the Guadiamar river (Aznalcollar, Seville, Southern Spain) as a result of the collapse of a dam in a pond containing pyrite slurry and waste water with toxic elements from the Anzalcollar mine activities. An area of 40 Km. in length and 0,5 Km. wide, including agricultural exploitations was covered with a thick layer of pyrite slurry. After the considerable effort made to physically remove the slurries, the soils have remained polluted by heavy metals,such as Pb, Cu, Presented at the 10th International Symposium of MESAEP in Alicante, Spain, 2 - 6 Oct. 1999

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Zn, Cd, Tl, Sb and metalloids as As. Only few months after cleaning the affected lands, an important number of wild species were able to grow

in the contaminated area, in spite of this remaining

contamination. The aim of this paper is to present the first data of the wild plants species collected in periodical field surveys in the polluted soils, in order to identify metal tolerant and metal accumulator species that could be used for phytoremediation purposes.

MATERIAL AND METHODS

1. Sites description and sampling From October 1998 to July 1999 fourteen expeditions were undertaken in three different locations of the affected area (Fig. 1). The sampling sites were chosen by the type of soil and its variability for the remaining contamination (Table 1).

Fig. 1. Map of the zone affected by spill (from Simon et al. 6 ) with the location of the sampling sites.

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A. de Haro et al. Table 1. Soil characteristics and remaining contamination at the sampling sites (mg Kg"1 dry weight) Locations

1 pH I

soil

j Pb ] Zn j Cu | Cd j As

Typic 1. Soberbina Acid

4.1 1 Haploxeralf | 676 j 950 | 162 1 0.16 | 90 !

2. P241

7.4

Calcic Haploxeralf

| 103

1411 413

5

35 \

Typic 3. Quema

6.1 I Xerofluvent -3231 • 2956

646

10

1226

(Analytical data from Prof Aguilar, Dr. Simon and Dra. Bernal)

2. Chemical analysis of plants Several specimens from each species of plant were collected, botanically identified3 and weighed. Shoots were thoroughly washed with tap water and given a final rinsing with deionized water (0,2 % non phosphate detergent solution) and then dried at 80°C for 48 hours in an oven. The dried samples were ground in a mill, analysing them separately for Pb, Zn, Cu, Cd and As. To prepare the samples for heavy metal measurement, dry material (ca 250 mg) was digested with 3ml. of nitric acid in a conical flask at a temperature of 130°C and then with 1 ml. of perchloric acid at 230°C. After cooling, H 2 0 was added to the acid solution until a volume of 15 ml. The heavy metal concentrations were determinated using flame atomic absorption spectroscopy (Perkin Elmer 1100B) and expressed as mg Kg"1 dry weight of plant tissue. To prepare the samples for arsenic analysis, the lyophilized sample (0.25 ±0.01 g) was weighed, and 1 mL of ashing aid suspension and 5 mL of 50% (v/v) HN0 3 were added, and the mixture was evaporated on a sand bath until total dryness. The ash from the mineralized samples was dissolved in 5 mL of 50% (v/v) HCl and 5 mL of reducing solution (Kl-ascorbic acid). After 30 minutes,the resulting solution was diluted to volume with 50% (v/v) HCl and filtered through Whatman No. 1 filter-paper into a 25 mL calibrated flask. The arsenic concentration was determinated by flow injection analysis-hydride generation-AAS (FIA-HG-AAS)7. RESULTS AND DISCUSSION

Field surveys and sampling at the three locations allowed the collection of six hundred and ninety three plants, that have been identified as belonging to one of ninety six different plant species (Table 2).

A. de Haro et al.

278 Table 2. Plants collected at contaminated soils. Family Aceraceae Amaranthaceae

S£ecies Acer negudo Amaranthus albus Amaranthus blitoides Amaryllidaceae Narcissus pseudonarcissus Boraginaceae Anchusa azure a Echium planiagineum Heliotropium europaeum Heliotropium supinum Caryophilaceae Portulaca oleracea Chenopodiaceae Chenopodium album Chenopodium murale Compositae Anacyclus clavatus Anacyclus radiatus Calendula arvensis Carthamus lanatus Centaurea sp. Chamaemelum fuscatum Chamaemelum mixtum Chrysanthemum coronarium Chrysanthemum sagetum Cichorium intybus Coleostephus myconis Conyza canadensis Crepis vesicaria Galactites tomentosa Helianthus annuus Lactuca serriola Leontodon taraxacoides Picris echioides Pulicaria sp. Scolymus hispanicus Senecio vulgaris Silybum marianum Sonchus asper Sonchus oleraceus Sonchus amensis Convolvidaceae Convolvidus althaeoides Convolvulus arvensis Cniciferae Capsella bursa-pastoris Coronopus squamatus Diplotaxis erucoides Diplotaxis virgata Hirschfeldia incana Raphanus raphanistrum Sinapis alba Cucurbitaceae Ecballium elaterium Cyperus rotundus Cyperaceae

Table 2. (Continued). Family Equitaceae Euphorbiaceae

Species Equisetum ramosissimum Euphorbia helioscopia Euphorbia peplus Mercurialis annua Fumariaceae Fumaria sp. Geraniaceae Erodium malacoides Erodium moschatum Arundo donax Gramineae Avena sp. Bromus sp. Cynodon dactylon Echinochloa sp. Hordeum sp. Lolium multiflorum Phalaris sp. Phragmites sp. Piptatherum miliaceum Sorghum halepense Astragalus cicer Leguminoseae Lotus tetraphylus Lupinus angustifolius Medicago minima Medicago orbicularis Medicago sativa Ononis sp. Psoralea bituminosa Scorpiurus sp. Trifolium sp. Vicia lutea Lavatera cretica Malvaceace Malva nicaeensis Fraxinus angustifolia Oleaceae Oro banc he crenata Orobanchaceae Oxalis pes-caprae Oxalidaceae Papaver roheas Papaveraceae Plantago coronopus Plantaginaceae Polygonum aviculare Polygonaceae Rumex pulcher Anagalis arvensis Primulaceae Ouenopodiaceae Beta vulgaris Reseda sp. Resedaceae Scrophulariaceae Kickxia lanigera Datura stramonium Solanaceae Solanum nigrum Tamarix africana Tamaricaceae Foeniculum vulgare Umbelliferae Viola sp. Violaceae Tribidus terrestris Zygophyllaceae

A. de Haro et al.

279

Compositae (30 species), Leguminoseae (15), Gramineae (9), and Cruciferae (8) were the most frequent Botanical Families represented in the collection. The vegetation at location 1 (Soberbina Acid) was very scarce and;therefore7the proportion of plants collected at this site only represent

7% of the

total. This fact, together with the high content in heavy metals and arsenic found in most of the plant collected at this place,could be explained by the low pH, lack of calcium carbonate and, consequently, the high bioavailability of the remaining heavy metals and arsenic. The Pb content in all the plants analysed ranged from 0 to 435.5 mg kg"1 dry weight, and the species showing the highest accumulation of this metal was Amaranthus hlitoides (mean in contaminated soil: 126.32 mg kg"1, in non contaminated soil: 7.71 mg kg"1). The Cu content in the plants analysed ranged from 1.18 to 151.90 mg kg"1, and the species showing the highest accumulation of this metal was Amaranthus hlitoides (mean in contaminated soil: 55.40 mg kg"1, in non contaminated soil: 8.89 mg kg"1). The Zn content in the plants analysed ranged from 16.21 to 977.23 mg kg"1, and the species showing the highest accumulation of this metal was Erodiam moschatum (mean in contaminated soil: 542.53 mg kg"1, in non contaminated soil: 50.96 mg kg"1). The Cd content in the plants analysed ranged from 0 to 8.12 mg kg"1, and the species showing the highest accumulation of this metal was Lavatera cretica (mean in contaminated soil: 6.21 mg kg'1, in non contaminated soil: 0 mg kg"1). The As content in the plants analysed ranged from 1.77 to 114.16 mg kg"1, and the species showing the highest accumulation of this metal was Amaranthus hlitoides (mean in contaminated soil: 82 mg kg"1, in non contaminated soil: 0.26 mg kg"1). Taking into account its ability to concentrate several pollutans of the affected area (Pb, Cu and specially As) and to produce an important amount of biomass (mean in contaminated soil: 158 g/plant fresh weight), Amaranthus hlitoides highlight as one of the most promising species to be used in the remediation of this area. Seeds and/or propagules of the selected species are being collected in order to preserve and multiply this germplasm, and to carry out studies under controlled conditions that allow us to explore the whole potential of these species for phytoremediation purposes.

ACKNOWLEDGEMENTS The authors thank Consejo Superior de Investigaciones Cientificas (CSIC) and Consejeria de Medio Ambiente (Junta de Andalucia) for supporting this research, and Gloria Fernandez Martinez (Institute de Agricultura Sostenible, CSIC, Cordoba) for her help in performing the analyses of plants. The authors also wish to thank Prof. Aguilar, Dr. Simon and Dra. Bernal for their contribution in soil composition data.

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REFERENCES 1- Minguzzi, C , and Vergnano, O. 1948. II contenuto di nichel nelle ceneri di Alyssum bertolonii. Atti della Societd Toscana di Scienze Naturale 55, 49-74. 2- Baker, A.J.M. 1995. Metal hyperaccumulation by plants: our present knowledge of the ecophysiological phenomenon. In: Will plants have a role in bioremediation?. 14 th Annual Symp. Current Topics in Plant Biochemistry, Physiology and Molecular Biology. Columbia, MO, pp. 7-8. 3- Chaney, R.L. 1983. Plant uptake of inorganic waste constitutes. In "land Treatment of Hazardous Wastes", edited by J.F. Parr, P.B.Marsh, and J.M. Kla. Noyes Data Corp., Park Ridge, pp. 50-76. 4- Baker, A.J.M. and Brooks, R.R. 1989. Terrestrial higher plants which hyper-accumulate metal elements - A review of their distribution, ecology and phytochemistry. Biorecovery 1, 81-126. 5- Valdes, B., Talavera, S., Fernandez-Galiano., E. (eds.).1987. Flora Vascular de Andalucia Occidental. Vol. 1 (485 pp.), Vol. II (640 pp.) and Vol III (556 pp.). Ketres Ed. Barcelona. Spain. 6- Simon, M., Ortiz, I., Garcia, E., Fernandez, J., Dorronsoro, C , Aguilar, J. 1999. Pollution of soils by the toxic spill of a pyrite mine (Aznalcollar, Spain). Set Total Environ. 242: 105-115. 7- Ybariez, N.; Cervera, M. L.; Montoro, R.; De la Guardia, M. 1991. Comparison of dry mineralization and microwave-oven digestion for the determination of arsenic in mussel products by platform in furnace Zeeman-effect atomic-absorption spectrometry. J. Anal At. Spectrom. 6, 379-384.

Received for publication:

February 03, 1999

Accepted for publication:

May

08, 2000