UNIVERSIDAD AUTNOMA DE MADRID. Departamento de Qumica Agrcola

TESIS DOCTORAL

PLANT-BASED METHODS FOR REMEDIATING ARSENIC-POLLUTED MINE SOILS IN SPAIN.

RECUPERACIN DE SUELOS MINEROS CONTAMINADOS CON ARSNICO MEDIANTE FITOTECNOLOGAS.

EDUARDO MORENO JIMNEZ Madrid, 2010

Directores: Dr. Jess M. Pealosa Olivares y Dra. Elvira Esteban Fernndez

Facultad de Ciencias Departamento de Qumica Agrcola

PLANT-BASED METHODS FOR REMEDIATING ARSENIC-POLLUTED MINE SOILS IN SPAIN.

Memoria presentada por EDUARDO MORENO JIMNEZ para optar al grado de Doctor en Ciencias.

Firmado

Eduardo Moreno Jimnez

Los Directores

Dr. Jess M. Pealosa Olivares

Dra. Elvira Esteban Fernndez

Facultad de Ciencias Departamento de Qumica Agrcola

AGUSTN GARATE ORMAECHEA, CATEDRTICO Y DIRECTOR DEL DEPARTAMENTO DE QUMICA AGRCOLA, DE LA FACULTAD DE CIENCIAS DE LA UNIVERSIDAD AUTNOMA DE MADRID.

CERTIFICA: Que el trabajo de investigacin titulado: PLANT-BASED METHODS FOR REMEDIATING ARSENICPOLLUTED MINE SOILS IN SPAIN se ha realizado en este Departamento bajo la direccin del Dr. Jess M. Pealosa Olivares y la Dra. Elvira Esteban Fernndez, lo que ha dado lugar a la Memoria que presenta D Eduardo Moreno Jimnez para optar al Grado de Doctor.

Y para que conste a los efectos oportunos firmamos el presente certificado en Madrid, a 3 de Marzo de 2010.

Fdo: Agustn Grate Ormaechea

AbstractArsenic is a trace element which is naturally found in the environment, but human activities have increased its concentration in soils. Pyritic mining is a major source of As in the soil, but there are others, such as agriculture or coal burning. Arsenic speciation in soils is complex, with organic forms including methylated As (MMA, DMA), or As bound to sugars, whilst inorganic species include arsenite and arsenate, which are the most abundant. If flooded soils are excluded, the majority of As in aerated soils is H2AsO4- (acid soils) or HAsO42- (neutral and basic). The availability of As in soils is usually driven by many factors, such as pH, Feoxides, phosphorus, (co)precipitation in salts, organic matter, clays, rainfall, etc. The available fraction (which is the most mobile as well) can be potentially taken up by plant roots, although its concentration is usually low. As far as we know, arsenic has no biological function in plants. Once inside root cells, As(V) is quickly reduced to As(III), and complexed in many plant species. Plants cope with As using detoxification mechanisms (complexation, compartmentalization and cell wall retention), but phytotoxicity symptoms appear once those mechanisms are saturated. Phytotoxic effects include growth inhibition, chlorophyll and nutrient degradation or oxidative stress. Phosphorus nutrition will influence As(V) uptake and toxicity in plants. Phytoremediation has been recently reported as an emerging, promising technology for soil reclamation, and the soil-plant system has been intensively studied to achieve the success of this technology. Within this technology phytoextraction consists of the uptake of As and other elements and translocation to the harvestable parts of plants, after which they are removed; whilst phytostabilization and phytoimmobilisation are plant-aided stabilization techniques to reduce As mobility. In many cases, the use of native plants would be an advantage from the technical and ecological point of view. We selected two arsenopyritic mine soils as working scenarios: (i) An experimental plot in Aznalcllar (Seville, Spain). In 1998 one wall of the pond containing mine wastes was breached and large amounts of pyritic sludge and water flooded over a surface of more than 4000 ha. After cleaning activities (physical removal of the top layer), a vast proportion of land (55 km2) was included in an ecological restoration project (Green Corridor of the Guadiamar River), but the experimental plot remained unaltered for research. There is a lot of information on trace elements in this site. (ii) A mountain valley in Bustarviejo (Madrid, Spain), where an As-mine was active for longer than 300 years. The mine is now derelict but large amounts of pyritic wastes remain. There was no previous information about trace element dispersal to the adjacent soils.

The main objective of this Thesis is to study how Mediterranean native plants respond to elevated arsenic concentrations, the transfer of this element from the mine soils to plants and the identification of native plant species that may be useful for the phytoremediation of As-polluted mine soils. Dealing with this objective, the experimental part covered several scales of working: (i) hydroponics under controlled conditions to evaluate As resistance and As accumulation in Mediterranean shrubs, (ii) short-term pot trials with soil to study As phytoavailability, (iii) long-term pot experiments with soils to study As dynamics in the soil-plant system, the transfer of As to plants and the effects of plant culture on soil quality, (iv) field trials to evaluate the fate of As under real conditions, the factors affecting its mobility, transfer to plants, and the efficiency of re-vegetation procedures.

The conclusions derived from these experiments procedures are: 1) A valid methodology was developed to analyze As in the soil-plant system. Total element determination in both plants and soils after digestion with nitric acid and hydrogen peroxide showed good recoveries and a high reproducibility. Ammonium sulphate (0.1 M) was a good extractant for assessing As phytoavailability in our soils, both under laboratory and field conditions. 2) The selection of a phytotechnology for a particular As contaminated soil requires studies at different scales, from hydroponics to field conditions. Working in this way, we obtained complementary information: i) After a hydroponic experiment with different As doses to 6 plant species, Retama sphaerocarpa and Tamarix gallica were the most resistant species, and T. gallica accumulated the largest amount of As. ii) Four plant species selected in the previous hydroponic experiment were tested in a pot trial under semi-controlled conditions with a moderately As-contaminated soil, additionally polluted by pyritic sludge application in some treatments. Plant establishment induced a positive effect on soil quality, improving C, N and enzymatic activity in soils. The best effects were obtained for R. sphaerocarpa and Myrtus communis. Despite the promising results of T. gallica in hydroponics, this plant did not perform well in the pot trial. iii) In a field trial in the Green Corridor of the Guadiamar river (Aznalcllar, Spain) using the same plant species as in the pot trials, R. sphaerocarpa showed the highest survival rate, Rosmarinus officinalis survived efficiently in soils at pH>4, and T. gallica and M. communis showed a low survival rate. Soil to plant transfer of As was low.

The multi-scale approaches allowed us to select Retama sphaerocarpa plants as the best candidate for As phytostabilisation of mine soils between the plant species used for the revegetation of the Green Corridor of the Guadiamar river. 3) Twenty five plant species (ferns, herbs, and woody plants) and waters (surface waters and interstitial pore water) were collected in a field survey around a derelict As-mine in Bustarviejo (Madrid, Spain). All the plants showed low concentrations of As in shoots despite the high levels of As in some soils at this site. Salix atrocinerea and Cytisus scoparius plants showed high bioaccumulation factors ([As]shoot/[As]available) and had some interesting characteristics. For instance, S. atrocinerea was abundant in the riparian communities and has a quick growth rate, whilst C. scoparius is a leguminous species frequent in the drier soils. These properties make both species interesting candidates for phytoremediation, and further studies will be carried out to assess their potential. 4) Arsenic was generally retained in the roots of Mediterranean plants, with little translocation to shoots. This reduces the potential for As toxicity in shoots, even under hydroponics. Consequently, most of the studied plants had low As concentration in aboveground tissues. Arsenic supply in the hydroponic experiment promoted toxicity symptoms in plants: growth inhibition, oxidative stress, chlorophyll depletion, and induction of detoxification mechanisms (thiol synthesis). 5) The fraction of mobile/available As was always small in well aerated Mediterranean soils affected by arsenopyritic mining, with arsenic bound in insoluble fractions. Only