RICERCA ITALIANA     

BIOTIC AND ABIOTIC PROCESSES AFFECTING THE MOBILITY AND BIOAVAILABILITY OF TOXIC ELEMENTS IN THE SOIL-PLANT SYSTEM

Università degli Studi di Napoli "Federico II"

Abstract

We intend to carry out researches on abiotic and biotic processes which influence the mobility of As, Cu and Cr in soil environments and their bioavailability and toxicity for microorganisms and plants.
Heavy metals and metalloids easily interact with soil components (clay minerals, microorganisms, organic matter, Fe and Al oxides, enzymes and so on). However, abiotic and biotic components should not be considered as separate entities but rather as a united system constantly in close association and interactions with each other in terrestrial environment. It is essential that scientists interested in different disciplines related to environmental soil chemistry, soil biochemistry, soil microbiology, plant nutrition and plant biochemistry interact each other to seek understanding the mechanisms regarding the mobility and bioavailability of xenobiotics in the soil-plant system.
Chemical, physico-chimical and mineralogical analyses of soil samples of polluted sites will be carried out and then a multistep selective sequential extraction will be used to characterize different species such as exchangeable, bound to carbonates, metal-organic complex-bound, amorphous mineral colloid-bound, crystalline Fe oxide-bound, and residual. On these soils, the microbial biomass, some soil enzymatic activities, soil respiration, substrate-induced respiration as well as taxonomical and functional diversity of the bacterial communities will be performed. Furthermore, we will try to select plant growth promoting (PGP) bacterial strains, among the metal-resistant bacterial populations; preliminary tests (pack growth pouches, pot experiments) will be carried out with isolated model strains to verify PGP ability towards ethylene-sensitive vegetable species. Experiments will be carried out on the phytoavailability of toxic elements (mainly Cu) by growing seeds of durum wheat into plastic pots, containing polluted soils, on which different species were previously determinated, in order to correlate the amount of element present in the plants with the different fractions of a heavy metal in soils, which appears more phytoavailable and then more toxic for plants.
In order to evaluate the mobility and bioavailability of toxic elements studies will be carried out on the influence of biomolecules, soil organic soluble fractions (DOM), or phosphate (in order to simulate the influence of root exudates and fertilizers) on the adsorption/desorption of toxic elements on/from Al and Fe oxides and organo-mineral complexes at different pH values and Eh. The interactions between different organic fractions isolated from variously contaminated soils and different amounts of mixtures of different heavy metals will be studied in order to evaluate selective mechanisms and specific affinity.
In order to know the processes which regulate the transport and accumulation of heavy metals in the apoplastic system studies will be carried out on the interactions which may occur between different forms of toxic elements and the acid polysaccharidic components of the root mucilages in the absence or presence of some low molecular mass root exudates and/or hydrolytic species of Al and Fe.
We intend to study the effects of low molecular weight organic acids or DOM on Cu toxicity maize seedlings grown in hydroponic controlled environmental conditions by taking into account the physiological and biochemical parameters related to the oxidative status and antioxidative response of maize roots. Finally, field experiments will be performed on plants (principally maize) growing in high copper polluted fields in Piemonte and the polyamine (putrescine, spermine) content will be detected to understand if these polyamines could be used as markers of the oxidative stress in plants growing in polluted areas. <<<

Principal Investigator

Antonio VIOLANTE Università degli Studi di NAPOLI "Federico II"

Research Objectives

Researches on the factors which may influence the mobility and phytoavailability of Cu, As and Cr deserve particular attention because their presence in soils and other environments is of great concern. In fact, these elements are included in the U.S. Environmental Protection Agency's list of priority pollutants.
In order to know the potential toxicity of a trace element in soil environments it is necessary to study the factors which affect its mobility and bioavailability for plants and microorganisms.
The mere presence of a toxic element in soil may not adversely affect either a single organism or a community of organisms if that element is not in a biologically, chemically, or physically unavailable chemical form. It is accepted that total soil heavy metal content alone is not a good measure of bioavailability and not a very useful tool to determine the potential risks from soil contamination. The mobility and bioavailability, and then potential toxicity of a heavy metal in the soil depend on its concentration in soil solution, the nature of its association with other soluble species and the soil's ability to release the heavy metal from the solid phase to replenish that removed from the soil solution by the plants.
Heavy metals and metalloids easily interact with soil components (clay minerals, microorganisms, organic matter, Fe and Al oxides, enzymes and so on). However, abiotic and biotic components should not be considered as separate entities but rather as a united system constantly in close association and interactions with each other in terrestrial environment. Interactions of these components have enormous impact on terrestrial processes critical to environmental quality and ecosystem health.
Six scientific groups from the University of Naples, Milan, Viterbo, Sassari, Turin and Bari are jointly involved in this proposal in order to study the factors which influence the mobility of arsenic, chromium and cupper in soil environments and their bioavailability and toxicity for microorganisms and plants. This working group plans to study:

a) Speciation and phytoavailability of Cu and As in polluted soils and effects on soil biomass. Studies will be carried out on: i) fractionation of different species of Cu and As in polluted soils with a wide range of chemical and mineralogical properties; ii) to correlate the relative importance of the fractions of a heavy metal (Cu) in soils and soil solutions in influencing their phytoavailability and toxicity; iii) to study taxonomical and functional diversity of the bacterial communities living in polluted soils and determine soil enzymatic activities before and after cellular lysis.

b) Selection of plant growth promoting bacterial strains among the metal-resistant bacterial populations
Studies will be carried: i) to select plant growth promoting (PGP) bacterial strains among the Cu-resistant bacterial populations; preliminary tests (pack growth pouches, pot experiments) will be carried out with isolated model strains to verify PGP ability towards ethylene-sensitive vegetable species; ii) to study taxonomical and functional diversity of the bacterial communities living in soils polluted with arsenic (arsenate and arsenite); iii) to evaluate the dynamics correlated to the developing or decline status of the microbial biomass in the contaminated soils.

c) Evaluation of mobility and bioavailability of toxic elements using polluted soil and synthesis models.
Studies will be carried out on: i) the influence of biomolecules (root exudates), soil organic soluble fractions (DOM), or phosphate (fertilizers) on the adsorption of toxic elements on Al and Fe oxides and organo-mineral complexes at different pH values and Eh;ii) the desorption of Cu, As and/or Cr from polluted soils or sorbed on or coprecipitated into metal oxides or organo-mineral complexes by organic ligands and phosphate; iii) the interactions between different organic fractions isolated from variously contaminated soils and different amounts of mixtures of different heavy metals in order to evaluate selective mechanisms and specific affinity.

d) Evaluation of possible biotic and abiotic processes which affect the accumulation and transport of toxic elements at soil-root interface
Studies will be carried out: i) to evaluate the role of Fe and/or Al oxides, trapped inside the acid polysaccharidic components of the root mucilages, in the accumulation of selected toxic elements, and in the redox and ion complexation processes by the root exudates; ii) to evaluate the role of the exudates of metal resistant bacterial strains on the phenomena of mobilization of Cu and Cr ions accumulated in the Ca-polygalacturonate fibrils; iii) to define the mechanisms of reduction and complexation of caffeic acid and its derivates towards Cu, Cr and As in either free form or accumulated in the Ca-polygalacturonate fibrils; iv) to evaluate the role of Fe and/or Al oxides, oxyhydroxides or hydroxides, trapped inside the polysaccharidic matrix, in the accumulation of Cr, Cr, As, As e Cu, and in the redox and ion complexation processes by the root exudates (in collaboration with the UO of Torino and Napoli); v) to evaluate the role of the exudates of metal resistant bacterial strains on the phenomena of mobilization of Cu and Cr ions accumulated in the Ca-polygalacturonate fibrils.

e) Metabolic reaction of root maize seedlings to copper in the absence or presence of organic molecules with different chelating power

Studies will be conducted on i) the effects of low molecular weight organic acids or DOM on Cu toxicity maize seedlings grown in hydroponic controlled environmental conditions; ii) the physiological and biochemical parameters related to the oxidative status and antioxidative response of maize roots; iii) the influence of low molecular weight organic acids on Cu influx in roots

The objectives a) and b) may give useful information on the species of a selected trace element which appears more phytoavailable and then more toxic for plants and may permit to evaluate the dynamics correlated to the developing or decline status of the microbial biomass in the contaminated soils. The experiments which will be carried out on the adsorption/desorption of heavy metals in the presence of low molecular mass biomolecules, usually released by plant roots and microorganisms, and inorganic anions, phosphate and sulfate, added to soils as fertilizers (objective c) may give useful information on some of the most important processes which influence the mobility and the potential toxicity of heavy metals in agricultural soils and, in particular, in the rhizosphere. For example, application of phosphate fertilizers is a management practice that can have a direct effect on the mobility of As in soils and its presence in ground waters. Soil and ground water contamination by As is dramatic in many countries but particularly in many Asian countries, where the presence of As is due both to the parent materials and to indiscriminate use of arsenical pesticides, until the mid-1900s, and anthropogenic wastes.
The findings of the objective d) should allow to establish the action combined of the root mucilages, organic molecules of low molecular weight and of Fe and/or Al hydroxides in the processes of accumulation and mobilization of heavy metal ions at the soil-root interface. Furthermore, it will be possible to individuate eventual synergism and competition phenomena among the organic and inorganic components considered, which could affect the mechanisms of detoxification from heavy metals which occur in the rhizosphere. Finally, the findings of the objective e) may give information on the effect of root exudates or soluble organic compounds (DOM) on the toxicity of heavy metals for plants and on their translocation in plant tissue and could permit to understand if some polyamines could be used as markers of the oxidative stress in plants growing in polluted areas. <<<

First Results

- Speciation of different forms of Cu and As in polluted soils. Definition of efficient and reproducible analytical methodologies to measure trace metals of environmental concern.
- To correlate the relative importance of the fractions of a heavy meta <<<

Timescale

24 months

National and international background

Researches on the factors which may influence the mobility and phytoavailability of copper, arsenic and chromium deserve particular attention because their presence in soils and other environments is of great concern (Adriano, 1992). These elements are included in the U.S. Environmental Protection Agency's list of priority pollutants. They exist in more than one oxidation state [As(V), As(III) or Cu(I), Cu(II)] and some of them may be present as cations (Cr3+) and anions (chromate) (Sparks, 1995; Jackson, 1998). These toxic elements have deleterious effects on humans through contamination of the food chain or drinking waters, such as disorders of the heart and circulatory system, cancer, anemia, liver disfunction and so on. Arsenic pollution toxicity and increased appearance in the biosphere has triggered public and political concern (Huang et al., 1998; 2002; Frankerberg, 2002; Violante et al. 2002). As(III) is more mobile and about 200 times more toxic than As(V). The presence of As in the environment is attributable both to the parent materials and to anthropogenic waste. Arsenic may lead to contamination of both agricultural soils and surface waters and sediments in areas where mining and smelting are present (Smith et al., 1998; ). In many countries indiscriminate use of arsenical pesticides until the mid-1900 has led to extensive contamination of soil worldwide. Many countries (including Italy) are severely affected by the arsenic crisis, but the situation is especially alarming in south and southeast Asia . The oxidation of As (III) to As (V) can be considered a detoxification process (Tamaki & Frankenberger, 1992; Neff, 1997). Some of the arsenite-oxidising bacteria, that are good candidates for arsenic mitigation in heavily polluted sites (Weeger et al., 1999) exhibit high tolerance to As (III) and other heavy metals such as Cu, Cd, Zn (Bansal et al., 2001).
Copper represents an important plant nutrient, although at high concentrations becomes toxic, particularly at pH < 6.0, affecting negatively the growth and development of plants. This toxic action is noticeable on cellular membrane through the oxidation of sulfur bridges and the production of oxygen radicals which induces the peroxidation of polyunsaturated fatty acids (Weckx & Clijsters, 1996).
Copper application to agricultural land in quantities in excess of that required by crops occurs in the case of fertilizers, sewage sludge, agricultural or municipal wastes and in vineyard where copper salts are used as fungicides (Iskandar, 2001; Huang et al, 2002; Kabata-Pendias, 2002). Copper is more soluble and therefore toxic. Its solubility increases if complexed with organic ligands (e.g. root exudates). The distribution of Cu within plants is highly variable (Kabata-Pendias, 2002).
The large use of chromium in the industries including textile, lether tanning, electroplating and metal finishing has resulted in significant pollution of the environment and now most soils contain measurable level of chromium extractable by dilute HCl. The hexavalent form of chromium in soils is of the major concern due to its toxicity and high mobility in soils. Symptoms of Cr toxicity for plants appear as wilting of tops and root injury; also chlorosis in young leaves are typical features (Kabata-Pendias, 2001).

Toxic elements interacts with abiotic and biotic soil components, such as phyllosilicates, microorganisms, organic matter (humic and fulvic acids, polysaccharides, phenolic compounds, root exudates and so on), Al-, Fe- and Mn-oxides, extracellular enzymes. However, soil components should not be considered as separate entities but rather as a united system constantly in close association and interactions with each other in the terrestrial environment. Each of the soil components not only participates in the transformation of xenobiotics but also modifies the participation of the three other factors. Microorganisms degrade xenobiotics in addition to degrading many natural products, including humic materials and extracellular enzymes. They also contribute to the weathering of minerals. Extracellular enzymes are also influenced by pollutants but their activity could be affected by the colloids on which they are immobilized and the nature of pollutant. Humic acids are believed to catalyse certain transformation reactions, but their major impact lies in the ability to adsorb microorganisms, enzymes and chemicals and to be adsorbed on soil minerals. Minerals exhibit mixed functions as well.
Among soil processes, chemical and biogeochemical reactions occurring in soil environments play a vital role in governing mechanisms of transformations, speciation, bioavailability, toxicity, dynamics, and transport processes of pollutants of environmental concern.
Therefore, soil chemical and biogeochemical processes have a tremendous impact on soil quality and the stability and sustainability of the ecosystem. It is essential that scientists interested in different disciplines related to environmental soil chemistry, soil biochemistry, microbiology, plant nutrition and plant biochemistry interact each other to seek understanding of pertinent chemical and biogeochemical reactions pertaining to soil quality and the abiotic and biotic processes which control the mobility of toxic elements in the soil-plant system.
The interactions between heavy metals and soil components are particularly important in the rhizosphere for a greater amount of microorganisms and for the more intense release of biomolecules by plant roots (root exudates) and microorganisms (Marchner, 1995; Violante & Gianfreda, 2000; Huang et al., 2002). The presence of root exudates may greatly affect the bioavailability of heavy metals (Erricade & Campbell, 2000) by promoting complexation and desorption reactions. The most important high- molecular-weight root exudates are mucilages, polysaccharides, phenolic compounds, and proteins (enzymes), whereas the main low molecular weight root exudates are carbohydrates, organic acids, aminoacids, phenols and polyphenols. The main class of binding sites is made up of the carboxylate groups of acid polysaccharides, mainly polygalacturonic acid (PGA), which are deposited outside the root hairs forming a gelatinous envelope known as "mucigel" (Gessa & Deiana, 2001). Microorganisms, soil particles and organic acids of both high and low molecular weight, such as phenolics, organic acids, sugars and amminoacids, produced by the soil, plant and microbial activity, are commonly found inside the mucigel. Furthermore, the mucigel contains humo-similar organic compounds, formed upon oxidation of phenolics, clay minerals and inorganic precipitates, like iron, manganese and aluminium oxides and oxyhydroxides (11). PGA, which constitutes also an important fraction of the apoplastic system, has a high ability to complex metals, thus immobilizing them in the reticulation sites of the root free space.
The rhizosphere of plants is a zone of intense microbial activity and the rhizobacteria, exhibit active root colonization. Plant growth promoting rhizobacteria (PGPR) represent a wide variety of soil bacteria which when grown in association with a host plant, result in stimulation of growth of their host. PGPR may colonize the rhizosphere, the root surface, or superficial intercellular spaces. Not any soil bacterium can colonize these areas.
High concentrations of heavy metals have been shown to adversely affect the size, diversity, and activity of microbial population in soil, even if many microorganisms have evolved mechanisms of metal resistance (Andreoni et al., 2003; Mazzocchi et al., 2004; PRIN2002). Some mechanisms are specific for a particular metal; others are general, conferring resistance to a variety of metals.
When plants are exposed to conditions that threaten their ability to survive, they respond to a variety of different environmental stresses by synthesizing " stress" ethylene (Salisbury, 1994).
The toxicity of heavy metals in the apoplast strongly depends on the presence in rhizodepositions of organic molecules with chelating activity able to reduce their mobility and bioavailability. In particular, it has been reported that Cu or Al exposition can elict citric and malic acid exudation, making plant more tolerant to these metals (Zhu et al., 2003; Nian et al., 2002). Recently, Deiana et al. (2003a-c; PRIN2002) have demonstrated that the uptake of Cu, Fe or Al by the polysaccharide matrix is regulated by the affinity of the ion with the polyelectrolyte and the exudates at low molecular weight. In the presence of weak ligands, as D-galacturonic and pyruvic acids, the uronic polymer acts as Cu accumulator, while in the presence of strong ligands, as citric acid, the polymer does not accumulate. Further studies are necessary on the influence of increasing concentrations of different root exudates or soluble organic matter (DOM) on plant grown in hydroponic controlled environmental conditions containing different amounts of a toxic elements. Experiments on the oxidative stress induced by Cu in roots of maize seedlings have been recently carried out in the absence of biomolecules (Zacchini et al., 2003; PRIN2002).
The toxicity, bioavailability and mobility of heavy metals in the soil-root system and particularly in the root mucilages and in the apoplast are mainly determined by the system pH and redox potential (pe), by the presence of organic molecules with complexing and reducing activity produced by the microorganism and plant biological activity, as well as by the presence of Fe-, Mn- and Al-oxides and oxyhydroxides. The latter can affect the complexation and reduction reactions of toxic elements with different oxidation state (as As, Cu and Cr) by the root and microbial exudates being able to interact with both the metal ions and the exudates through adsorption phenomena.

The surfaces of Fe, Al and Mn oxides (present in the rhizosphere as well as in the root mucilages and in the apoplast) are particularly reactive for the presence of hydroxyl functional groups. Many trace elements in anionic form (e.g. arsenate) form strong inner-sphere surface complexes with these type of sites. When they do, they are not readily desorbed and, as such, are relatively unavailable for plants and microorganisms. In contrast, those metals that form weaker out-sphere surface complexes may be more readily desorbed and remain available when conditions that favor desorption in the soil solution result. Unfortunately, few studies have been carried out on the interaction between toxic elements and biomolecules (root exudates, DOM) or nutriens added as fertilizers (phosphate, sulfate), particularly in replacing heavy metals previously sorbed on the surfaces of soil colloids. Recent studies carried out for the previous proposal PRIN2002 concerning the effect of organics and fertilizers on the sorption of arsenate and Cu(II) have demonstrated that phosphate and organic ligands strongly affect heavy metal adsorption on soil components (Violante and Pigna, 2002; Violante et al., 2003; Violante et al., 2004). Till today it is not known if trace elements coprecipitated with Al and/or Fe into oxides or in the presence of organic ligands into organo-mineral complexes may be partially removed by phosphate or root exudates (Selim & Sparks, 2000; Huang et al., 2002). Furthermore, the knowledge of the role of abiotic and biotic components on the redox and chelation processes which determine the accumulation and mobilization of the Cr, As and Cu species at the soil-root interface and in the apoplast is still lacking.

The mere presence of a toxic element in soil may not adversely affect either a single organism, or a community of organisms if that element is not in a biologically, chemically, or physically unavailable chemical form. The ecotoxicological significance of the environmental impact of heavy metals in soils is determined by the specific form and reactivity of their association with particulate forms and solid-solution equilibria at the solid-water interface. It is accepted that total soil heavy metal content alone is not a good measure of bioavailability and not a very useful tool to determine the potential risks from soil contamination. The mobility and bioavailability, and then potential toxicity of a heavy metal in the soil depend on its concentration in soil solution, the nature of its association with other soluble species, and the soil's ability to release the heavy metal from the solid phase to replenish that removed from the soil solution by the plants. Solid-phase fractionation, however, is particularly important as it determines the rate of replenishment of heavy metal in the soil solution phase and its phytoavailability.
Metal cations may be soluble, readily exchangeable, complexed with organic matter or hydrous oxides or occluded in mineral structures. Delineating the fractionation of metals in soils is considered essential for understanding the mobility, bioavailability, and toxicity of the metals. Chemical sequential extractions are reported in the literature which permit to determine different fractions of heavy metals as exchangeable, specifically adsorbed, bound to metal-organic complexes, easily reducible metal oxides, organics, amorphous mineral colloids, crystalline Fe oxides, and bound to mineral lattices (Krishnamurti et al., 2000; 2002). The determination of different species of a selected heavy metal is particularly important when experiments are carried out on the amounts of the toxic element which may be uptaken by plants which are allowed to grow on the soils under investigations. In this case the phytoavailable toxic metal may be significantly correlated with some species of the metal present in the soil. Leita et al. (1999) showed significant correlations between the available fraction of heavy metals (DTPA extractable) and the amount and activity of biomass and demonstrated the selectivity of interactions between heavy metals and microorganisms from equilibrated, enriched and contaminated soils. Thus, it appears very important to consider, besides the chemico-physical factors, also the biological component to evaluate the availability of pollutants. Biotic and abiotic stresses can modify the soil microbial equilibriums compromising in different ways the natural nutrient cycles with important consequences for the soil-plant system.
In soil environments, particularly at soil-root interface, the associations among soil components (phyllosilicates, humic substances, oxides, organo-mineral complexes), microorganisms and plant roots are particularly important. Therefore, the reactions and processes in soil environments, mainly in the rhizosphere can only be interpreted satisfactorily with interdisciplinary approaches. It is difficult to separate the impacts on the chemistry of the contaminant of microbial activity from those of plant root activity. Further, the effect of the nature and properties of soil particles on plant-microorganism interactions and the impact on metal uptake remain to be established. The impacts of physico-chemical, biochemical, and biological processes in the rhizosphere on metal mobility, phytoavailability, toxicity, uptake, food chain contamination, and ecosystem health merit increasing attention. <<<