MINERVA BIOTEC 2001;13:33-5

Phytoremediation of a metal contaminated area in Southern Spain ESTEBAN ALCÁNTARA, ROSARIO BARRA***, MANUEL BENLLOCH*, ALEXANDER GINHAS*, JESÚS V. JORRÍN** J. A. LÓPEZ***, ÁNGEL LORA***, MARIA A. OJEDA*, MANUEL PUIG****, ANTONIO PUJADAS***, RAQUEL REQUEJO** JAVIER ROMERA, JUAN RUSO**, ENRIQUE D. SANCHO****, STEFAN I. SHILEV****, MANUEL TENA**

The EMIR-UCO is a multidisciplinary group including agronomists, botanists, soil scientists, plant physiologists, microbiologists and biochemists at the Agronomy and Forest Science High Technical School, University of Córdoba, involved since 1998 in research projects directed at developing and evaluating phytoremediation techniques for metal contaminated soils, initially related to the multicomponent metal contamination caused by the Aznalcollar (Southern Spain) toxic spill. The main objectives and related activities are to use plant and microorganisms as bioindicators of toxic metal contamination, to carry out botanical surveys directed at identifying and classifying autoctonous plant species growing in heavily contaminated areas, evaluating, by using field and greenhouse experiments, the tolerance to toxic metals in crops and wild species, developing either continuous and induced phytoextraction protocols adapted to both hyperaccumulators and high biomass producer plants, isolating and characterizing rizospheric bacteria and their effect on plant growth, tolerance to toxic metals and their ability to accumulate them, and characterizing plant responses to toxic metals at the molecular level, with special emphasis on metal adsorption, translocation and accumulation, and synthesis of stress metabolites (i.e. secondary metabolites, antioxidants). Plant model systems include crops (sunflower, maize, chickpea, thistle) and herbaceous wild species (Nerium oleander, Canna sativa, wild sunflower relatives, etc.). Most relevants results, so far obtained, will be described. This multidisciplinary approach has 15-18 November 2000, Sorrento (Italy). InterCOST Workshop on Bioremediation. This research is being supported by the Consejería de Medio Ambiente de Andalucía and FEDER (Project 1FD97-2101).

Address reprint requests to: M. Benlloch, ETS Ingenieros Agónomos y Montes, Departamento de Agronomía Apdo. 3048, 14080, Cordoba, Spain. E-mail: [email protected]

Vol. 13 - No. 1

From the Department of Plant Physiology *Department of Agronomy **Department of Biochemistry and Molecular Biology ***Department of Botany ****Department of Microbiology EMIR-UCO (Multidisciplinary Research Project on Phytoremediation - University of Cordoba) Agronomy ad Forest Science High Technical School University of Cordoba, Cordoba, Spain

proved specially useful in building a greater understanding of the many and varied processes involved in phytoremediation techniques. Botany - Microbiology - Biochemistry - Metals, heavy - Bioremediation - Soil. KEY WORDS:

oxic metal contamination is nowadays one of the

T most serious problems with great incidence on

human health and the environment. As a consequence of mining and other industrial activities, municipal wastes and sewage, toxic metals and metaloids such as Cu, Zn, Pb, Cd, Hg, As, etc. are being accumulated over toxic levels in soil and water. Phytoremediation by using plants and microorganisms as metal accumulators is one of the most promising strategies 1 problem. to solve this In April 1998, the ponds which stored the sterile residues from the mining bed of Aznalcollar (Sevilla, Spain), broke open. Some part of those multicomponent toxic residues (Table I) C3.6 Hm 3 of polluted water and 0.9 Hm 3 of tailings) spilled into the Agrio and Guadiamar river basin, spreading 40 km. 2

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PHYTOREMEDIATION OF A METAL CONTAMINATED AREA IN SOUTHERN SPAIN

ALCÁNTARA

TABLE I. -Element composition and concentration (mg kg-') in spil-

led contamination at Aznalcollar (Sevilla, Southwestern Spain) on 1998. 2

Element

Mn Zn

Pb Cu As . Co

Cd Ni Hg

Sludge

Contaminated soil

Non-contaminated soil

0-10 cm

10-30 cm

0-10 cm

845.5 230.8

787.7

681.9

721.9

7187.0 7996.1 1993.2 3113.5

747.9 370.4

457.6

47.3 29.4 20.3 3.3

15.9

132.8 127.0 2.2 29.1 0.4

10-30 cm

816.1 194.7

176.6

41.8

39.5

112.6 52.1

42.2

43.1 21.0

15.0 1.6 29.5 0.3

18.1 14.4 0.5 32.1 0.4

15.8 0.4 29.6 0.4

EMIR-UCO is a multidisciplinary group including agronomists, botanists, soil scientists, plant physiologists, microbiologists and biochemists at the Agronomy and Forest Science High Technical School, University of Córdoba, involved in research projects directed at developing and evaluating phytoremediation techniques for metal contaminated soils. Since 1998, EMIR-UCO is focused in the development of induced and continuous phytoextraction techniques applied in the recovery of soils affected by Aznalcollar toxic spill. Main objectives and related activities are to use plants and microorganisms as bioindicators of toxic metal contamination, to carry out botanical surveys directed at identifying and classifying autoctonous plant species growing in heavily contaminated areas, evaluating, by using field and greenhouse experiments, the tolerance to toxic metals in crops and wild species, developing either continuous and induced phytoextraction protocols adapted to both hyperaccumulators and high biomass producer plants, isolating and characterizing rhizospheric bacteria and their effect on plant growth, tolerance to toxic metals and their ability to accumulate them, and characterizing plant responses to toxic metals at the molecular level, with special emphasis on metal adsorption, translocation and accumulation, and synthesis of stress metabolites (i.e. secondary metabolites, antioxidants).

Materials and methods Botanical surveys were made during spring and fall by using square pots (20x20, 20x5 m) covering

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most ecosystems affected. Species collected were stored in herbarium and samples for each one have been evaluated for metal accumulation. Several methods to determine plant response to metal contamination have been studied, involving different sludge concentrations in combination with quelant (EDTA) addition, by using controlled soil and hydroponic systems. 3 Bacterial isolates coming from microbiological surveys in contaminated area on soils and rhizospheres have been evaluated for heavy metal tolerance, and selected isolates added in soil and hydroponic systems. Total soluble phenolics were extracted from liquid medium in the hydroponic system, root and leaves of plants used in each experiment. Growth (stem diameter, height, leaf area index, fresh and dry weight) was measured and toxicity symptoms visually evaluated, metal content in roots and leaves (Fe, Mn, Cu, Zn, Cd, and Pb) determined by atomic absorption spectrophotometry, and phenolic content (phenolics, isoflavonoids) extracted from experimental media, roots and leaves with organic solvents and quantified by UV and the Folin colori metric reagent. 4 Plant model systems include crops (sunflower, corn, chickpea, thistle) and herbaceous wild species (Nerium oleander, Canna sativa, wild sunflower relatives, etc .) . Results Nine hundreds plants belonging to two hundred fifty six taxa (51 families, 156 genera) have been collected during periodical surveys. There is a broad diversity on plant growth and metal accumulation depending on the species, developmental stage, metal and area. Adventitious species are predominant. Different plant species, as Astragalus spp., Solanum melongena, Capsicum annuum and Lycopersicom esculentum, have been evaluated for their capacity to accumulate and tolerate arsenic. Astragalus baeticus accumulated the highest levels of arsenic in aerial parts although it did not tolerate high levels of arsenic, while the other plant species did. Fifty bacteria isolates have been obtained from roots and the surrounding soil in the contaminated Aznalcollar area. Four of these isolates have been selected on the basis of their tolerance to heavy metals. These populations when added to sterile and nat-

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March 2001

ALCÁNTARA

PHYTOREMEDIATION OF A METAL CONTAMINATED AREA IN SOUTHERN SPAIN

II.—Metals concentration in sunflower aerial part in experimental field plot located at Aznalcollar contaminated area on March 2000.

TABLE

DAM

Fe

Zn

Mn

Cu

Pb

Cd

pg g- 1 dry weight baerial part 41 769±91 22±2 14±2 2±0.4 335±73 254±59 63 965±90 358±49 302±43 30±2 15±1 4±0.3 8±2 901±114 348±54 212±27 25±3 2±0.2 96 Total extracted (pg in aerial part per plant) 6±1 111±31 10±2 1±0.3 41 313±51 166±58 63 3159±571 1204±250 1013±218 99±18 53±11 19±2 96 13604±2026 5604±1065 3207±504 383±52 131±30 28±5 a

) Days after sowing. b) Average±standard error (n=20).

ural soil improved plant growth, development and metal tolerance and accumulation. 5 By using sunflower and tobacco as experimental model systems we have observed a good correlation between the induced synthesis of phenolics and tolerance to and accumulation of heavy metals. 6 According to field and greenhouse experiments, sunflower seems to be a possible candidate to be used for metal phytoremediation because a good level of tolerance, biomass production and metal accumulation were obtained (Table II). It can be used in continuous phytoextraction protocols as well as induced ones by using chelates like EDTA (Table III). Other plant species also present promising results as referred to biomass production and tolerance (corn, thistle) although the level of total accumulation is not always elevated. In general, we have observed high accumulation in the root and much lower in the aerial part. This is one of the critical further steps in our objectives.

Discussion and conclusions Phytoremediation is a very new field. In order to realize its great potential, it is neccessary to build a

Vol. 13 - No. 1

III. —Metals concentration in sunflower aerial part after irrigation with EDTA (25 mM), 63 days after sowing, in experimental field plot located at Aznalcollar contaminated area on March 2000. Data obtained on plants harvested 96 days after sowin

TABLE

Para meters

Dry weight per plant (g)

Control EDTA

14±1 11±1

a

)

pg Fe

e

Zn

dry weight Mn

Cu

Pb

Cd

1131±262 a 309±38 109±32 29±4 11±2 1±0.2 1626±390 678±79 281±56 41±7 19±4 0.4±0.4 =

Average±standard error (n 5).

greater understanding of the many and varied processes involved. This requires a multidisciplinary approach as diverse as plant biology, agronomy, agricultural practices, soil science, microbiology and biochemistry. Screening of cultivated and wild species and genotypes for metal accumulation and tolerance will broaden the spectra of genetic material available for optimization and transfer. Optimizing agronomic practices such as irrigation, fertilization, planting and harvest time, and amendment application will increase the efficiency of the phytoremediation processes. References 1. Salt DE, Smith RD, Raskin I. Phytorremediation. Annu Rev Physiol Plant Mol Biol 1998;49:643-68. 2. Simón M, Ortíz I, García I, Fernández E, Fernández J, Dorronsoro C et al. El desastre ecológico de Doñana. Boletin de la Sociedad Española de las Ciencias del Suelo 1999;5:153-61. 3. Hoagland DR, Arnond DI. The water culture method for growing plants without soil. Calif Agric Exp Stat Circ 1950;N:1-347. 4. López-Valbuena R. Efectos del mildiu (Plasmopara halstedii) en el metabolismo de compuestos fenólicos de girasol. Ph. D. Thesis Dissertation. Universidad de Córdoba, Spain. 1980. 5. Shilev S, Benlloch M, Puig M, Sancho E. Rhizospheric bacteria promotes sunflower plant (Helianthus annuus) growth and tolerance to heavy metals. In: Perotto S, Benedetti A, Schwitzguebel JP, Gianinazzi S, Massacci A. editors. Intercost Workshop on Bioremediation, 2000:133. 6. Hernández M, Zapata J, Prats-Pérez E, Ojeda MA, Jorrín J. Toxic metals accumulation and total soluble phenolics in sunflower and tobacco plants. Minerva Biotecnol 2001;13(1):

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