Session 2. Risk assessment / Evaluation du risque (Oral presentations) General introduction. What risks? Dispersal of contaminants in the environment (food chain, aquatic ecosystem, groundwater) before/during/after phytoremediation. E. Vindimian........................................................................................................................ Modelling trace element exposure and effects on plants. S. Sauvé ............................................................................................................................... What tools for defining the PNEC values ? M. Mench ............................................................................................................................ Tools to assess bioavailability: ex situ techniques and analytical challenges D. van der Lelie, S. Brown, M. Mench and J. Vangronsveld .............................................. Biosensor, biomonitor, phytoremediation of radionuclides. M. Dutton ............................................................................................................................

Banquet, short lecture. The Bordeaux mixture. J. Delas ................................................................................................................................

Session 2. (Poster presentations) Characterisation of mercury-thiol complexes in maize root. L. E. Hernández, L. A. Arroyo-Méndez, S. Vázquez, F. F. del Campo, R. O. Carpena-Ruiz ....................................................................................................................... Relationship between plant structure, physiological processes, uptake and accumulation of Cd. A. Lux, A. Šottníková, L. Lunácková, E. Masarovicová, D. Lišková, K. Králová, V. Streško, P. Capek ............................................................................................ Cadmium and Pb toxicity in sugar beet (Beta vulgaris L.) F. Morales, A. Larbi, A. Álvarez-Fernández, A.F. López-Millán, N. Molías, Y. Gogorcena, J.J. Lucena, A. Abadía and J. Abadía 1 ............................................................ Evaluation of the potential biotoxicity / essentiality of Zinc and Cadmium in suspended cells of Cynara cardunculus and Centaurea calcitrapa. M. A. G. Oliveira, S. Raposo and M. E. Lima-Costa..........................................................

Role of root exudates in metal tolerance: lessons from aluminium research. Ch. Poschenrieder and J. Barceló ........................................................................................ Zinc uptake by Buddleia davidii cultured in vitro. Effect on the growth and on the polyamine content analysed as stress markers. P. Schnekenburger, G. Charles, A. Hourmant and M. Branchard....................................... Why it may be critical to rely on Cattail for phytoremediation of organic contaminants. P. Schröder, B. Huber and C. Scheer ..................................................................................

Session 2. (Poster presentations) n° 12. Characterisation of mercury-thiol complexes in maize root. L. E. Hernández, L. A. Arroyo-Méndez, S. Vázquez, F. F. del Campo, R. O. Carpena-Ruiz .......................................................................................................................

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n° 13. Relationship between plant structure, physiological processes, uptake and accumulation of Cd. A. Lux, A. Šottníková, L. Lunácková, E. Masarovicová, D. Lišková, K. Králová, V. Streško, P. Capek ............................................................................................

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n° 14. Cadmium and Pb toxicity in sugar beet (Beta vulgaris L.) F. Morales, A. Larbi, A. Álvarez-Fernández, A.F. López-Millán, N. Molías, Y. Gogorcena, J.J. Lucena, A. Abadía and J. Abadía...............................................................

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n° 15. Evaluation of the potential biotoxicity / essentiality of Zinc and Cadmium in suspended cells of Cynara cardunculus and Centaurea calcitrapa. M. A. G. Oliveira, S. Raposo and M. E. Lima-Costa..........................................................

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n° 16. Role of root exudates in metal tolerance: lessons from aluminium research. Ch. Poschenrieder and J. Barceló ........................................................................................

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n° 17. Speciation of metals in plants: analytical challenges and strategies. D. Schaumlöffel, L. Ouerdane, S. Mounicou, J. Szpunar and R. Lobinski.........................

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n° 18. Zinc uptake by Buddleia davidii cultured in vitro. Effect on the growth and on the polyamine content analysed as stress markers. P. Schnekenburger, G. Charles, A. Hourmant and M. Branchard.......................................

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n° 19. Why it may be critical to rely on Cattail for phytoremediation of organic contaminants. P. Schröder, B. Huber and C. Scheer .................................................................................. Session 2.

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General introduction. What risks? Dispersal of contaminants in the environment before / during / after phytoremediation.

E. Vindimian INERIS, Departement Ecotoxicologie,Verneuil en Halatte, France.

Modelling trace element exposure and effects on plants

S. Sauvé Department of Chemistry, Université de Montréal, 2900 Édouard-Montpetit, Montréal, Québec, Canada, H3C 3J7. Introduction Modelling Chemical speciation drastically modulates the bioavailability of trace metals and needs to be incorporated into the assessment of soil quality. Analytical determinations of free metal ions in soil solutions are unlike routine analyses and are difficult to determine and validate. Regulatory agencies require some easier means of estimating the influence of chemical speciation upon bioavailability for integration into environmental guidelines. Some semi-empirical models to predict the soil solution speciation of divalent free metal can be combined with plant toxicity data to improve predictions of toxic effects. Because trace elements in soils have a very low solubility that does not conform with predictions using mineral solubility equilibrium, an alternative modelling approach is therefore required. A semi-empirical regression predicting soil solution free metal activities using total metal content and pH was derived by analogy to a competitive sorption of free metals and protons (McBride et al., 1997, Sauvé 2001). This model begins with the simple equilibrium sorption of a metal (Me) to a protonated surface (SurH):

Me + SurH y ⇔ MeSur + y ⋅ H + .

Equation 1

Transforming into an equilibrium constant and log-transforming yields: pMe 2+ = a + b ⋅ pH + c ⋅ log(Total Metal ) + d ⋅ log(Sur ) ,

Equation 2

Analysis of variance shows that often, the Sur term can be neglected, i.e., most of the variability is explained by pH and Total Metal, yielding: pMe 2+ = a + b ⋅ pH + c ⋅ log10 (Total Metal )

Equation 3

This form of the model (Equation 3) has been applied to the modelling of free Cu2+, Cd2+, Pb2+ and Zn2+. Materials and methods Soil Characteristics Contaminated soils were sampled from various origins (agricultural, urban, forest and industrial sites) and cover diverse contaminations sources (atmospheric emissions, smelting, pesticides, sewage sludge, industrial, etc.). Soil total metals were determined using digestion in concentrated nitric acid (i.e. total-recoverable metals). Soil solutions were obtained using 1:2 soil:solution extractions with 0.01 M dilute

salts (CaCl2 for Cu and KNO3 for Cd, Pb, and Zn). Slurries were shaken 2 hours and centrifuged at 10000 g. The supernatants were filtered through 0.45-µm cellulosic membranes and analysed for total dissolved metals by GF-AAS and electrochemical speciation by ion selective electrode for Cu and anodic stripping voltammetry for Cd, Pb, and Zn and has been supplemented with resin-based speciation data from Knight et al. (1998). Methodological details for the different datasets are available from the original publications (see details in Sauvé 2001).

Results and discussion The dataset for the chemical speciation of Cd2+ in soil solution is illustrated in Figure 1. This dataset includes a series a spiked and field-collected soils and surprisingly, it shows very little difference between both datasets. Equation 3 has been applied to the different datasets and yields semi-empirical regressions of free metal activities based on simply soil pH and total metal content (Sauvé 2001).

pCd 2 + = 5. 14 + 0. 61⋅ pH − 0.79 ⋅ log10 (Total Cd ) R 2 = 0. 696, n = 64, p < 0. 001 Equation 4 pCu 2 + = 3. 20 + 1. 47 ⋅ pH − 1.84 ⋅ log10 (Total Cu) R 2 = 0.921, n = 94, p < 0. 001 Equation 5 pPb 2+ = 6. 78 + 0. 62 ⋅ pH − 0. 84 ⋅ log10 (Total Pb) R 2 = 0. 643, n = 84, p < 0.001 Equation 6 pZn 2 + = 4.70 + 0. 95 ⋅ pH − 1. 71⋅ log10 (Total Zn) R 2 = 0.760, n = 30, p < 0.001 Equation 7 It is interesting to note that the coefficients for pH and Total Metal vary according to metals. Cu has the steepest slopes regarding both pH and total metal leve ls. On the other hand Cd2+ and Pb2+ seem to have the smoothest slope with regards to pH and total metal. The constants actually reflect the relative solubility of each metal, with Pb, which with no surprise is the least soluble one. Equations 4 to 7 highlight the importance of preserving some flexibility in how contamination is evaluated to better address the specificity of given environmental issues. References 1. McBride M.B., S. Sauvé, and W. Hendershot. 1997. Solubility control of Cu, Zn, Cd and Pb in contaminated soils. Europ. J. Soil Sci. 48, 337-346. 2. Sauvé, S. 2001. Chapter 2: Speciation of metals in soils, p. 7-58, In H. E. Allen, ed. Bioavailability of Metals in Terrestrial Ecosystems: Importance of Partitioning

for Bioavailability to Invertebrates, Microbes and Plants. Society for Environmental Toxicology and Chemistry. Pensacola, FL, USA.

What tools for defining the PNEC values?

M. Mench ER BGETA, Unité d’Agronomie, Centre INRA Bordeaux-Aquitaine, 71 avenue E. Bourlaux, BP81, F-33883 Villenave d’Ornon, France. Introduction. Early warning before the pollution of sites, reducing the contaminant exposure at polluted sites, and (bio)monitoring the remediated sites are nowadays 3 major aspects of ecotoxicology and plant biotechnologies. Risk assessment and biomonitoring must allow preserving the ecosystem functions. Knowing pollution sources and their potential effects, especially on primary producers such as plant species, in view to limit susceptible rejects is a prerequisite. Exposure, which exceeds levels giving rise to deleterious effects recorded at either short or long term, must be avoided. The exposure assessment aims to predict the probable concentration in the environment accounting for the physico-chemical properties of the contaminant, its behaviour in the environment, its use, the amount released, etc. that will condition its distribution in the environment compartments. This concentration is so-called PEC (Predicted Environmental Concentration). To assess the relationship dose (concentration)-effect aims to determine the concentration under which the contaminant would not have adverse effects on the environmental component considered. This concentration is socalled PNEC (Predicted No Effect Concentration). A risk situation at one site is characterized when the ratio PEC vs. PNEC exceeds 1. What tools are available for defining the PNEC values, especially in the case of plant species? In the past, germination and growth parameters were used. Genotoxic effects were neglected. Literature shows that trace elements affect the plant functioning before one observes adverse effects on their germination and their growth. Information can be obtained at several sub-cellular levels, i.e. metabolites, proteins, gene transcripts. Does this info rmation end to decrease PNEC values? We give an example using biochemical biomarkers in maize exposed to a series of soils contaminated by incinerator fly ashes. Materials and methods. Plant chronic exposure was carried out using 800 g potted soils. The standard OCDE soil was used. Incinerator fly ashes were added into the soil at 21 levels increasing from 0 to 2%. Soils were rehydrated with distilled water and a modified Hoagland n°2 nutrient solution was added. Five seeds (Zea mays L. cv. Volga) were sowed. Plants were grown for 2 weeks with 16h/8h day/night, 180 µmol m-2 s-1 , 75% RH, 25/20°C. Soil moisture was maintained at 60% of the water holding capacity. Fresh and dry weight of shoots, 3rd and 4th leaves were determined. Density of chlorophylleous pigments was measured in 3rd and 4th leaves. Root and leaf samples were frozen in liquid nitrogen. Total soluble protein concentration, glutathion reductase (GR) and ascorbate peroxydase (APx) activities, total thiol concentration and phytochelatins were quantified (Lagriffoul, 1998; Mench et al, 2001). Plant tissues were wet digested in HNO3 and H2 O2 , and trace elements were quantified using axial ICP-AES. Results and discussion. Maize seeds have germinated in all soils. Shoot yield reached 3 g FW plant -1 in the range 0 – 0,19% addition rate, and then started to decrease. Similar effects were obtained for the 3rd and 4th leaf FW yields. Among trace elements, only Cd

concentration in the 3rd-leaf was related (r = 0,93) with the fly ash application rate. Above the 0.19% level, APx activity in the 3rd leaf increased with a dose-effect relationship demonstrating the development of an oxidative stress. Cystein, (GluCys), (Glu-Cys-Glu) concentrations increased in roots in relation with the soil contamination, especially by Cd (Fig. 1). These changes occurred before a decrease in growth parameters. Among other thiol compounds, increased concentrations in roots were found for (Glu-Cys)3 and PC3 , and in a lesser extent for (Glu-Cys)4 , PC2 and PC4 , in relation with the addition rate. Effective concentrations (EC 10 i) were calculated for each parameter, and converted into toxic units (UTi). The scale of potential ecotoxic effect (BEEP) was calculated using the formula BEEP = Log10 [1+ n(Σ i=1 to n UTi)/N], with the number of tests giving a dose-effect relationship (n) and the total number of tests (N) (Table 1).

Fig.1. (Glu-Cys) concentration in the maize roots. Fig.2. Hill relationship between BEEP values and exposure to fly ash. Table 1. Effective concentrations leading to a 10% decrease (EC10) (expressed in % fly ash addition rate per kg soil air-dried weight), toxic units (UT), and BEEP values (Log10 UT). EC10 UT BEEP -------------------------------------------------Glu-Cys 0.0996 1004 1.927 PC2 0.190 526 2.408 PC4 0.207 483 2.702 4th-leaf FW yield 0.219 457 2.916 Glu-Cys-Glu 0.231 433 3.082 (Glu-Cys)3 0.245 408 3.219 (Glu-Cys)4 0.226 375 3.332 Shoot FW yield 0.272 368 3.431 3rd-leaf FW yield 0.274 365 3.520 Cys 0.329 304 3.595 PC3 0.350 286 3.662 APx 0.400 250 3.720

Root (Glu-Cys) concentration gave the lowest EC 10 . Using data from biochemical biomarkers and growth parameters, the BEEP value showed a Hill relationship with the rate of fly ash addition into the soil (Fig. 2). Its EC 10 and EC 50 values were 0.015% and 0.08%, and corresponded to 1.3 mg and 11.9 mg Cd kg-1 in the maize 3rdleaf. The BEEP EC 10 value could be proposed as a provisional PNEC value. Screening of Cd-responsive genes in Arabidopsis thaliana suggests that oxidative stress and protein denaturation are important components of Cd toxicity (Suzuki et al, 2001). Changes in transcript populations may help to propose new PNEC values. References. Lagriffoul A, 1998 Biomarqueurs métaboliques de toxicité du cadmium chez le maïs (Zea mays L.). Mécanismes de tolérance, relation dose-effet et précocité de la réponse. Thèse Doctorat, Ecotoxicologie, Bordeaux 1, Unité d’Agronomie INRA Centre Bordeaux-Aquitaine.

Mench M., Ruttens A., Girardi S., Vangronsveld J., Corbisier P., Van der Lelie D., 2001. Evaluation de l’écotoxicité d’un sol et d’un déchet de référence par les tests BIOMET et PLANTOX. Rapport intermédiaire, convention Ademe – INRA n°A846, Villenave d’Ornon cedex. Suzuki N., Koizumi N., Sano H. 2001. Screening of cadmium-responsive genes in Arabidopsis thaliana. Plant, Cell, and Environment 24, 1177-1188.

Tools to assess bioavailability: ex situ techniques and analytical challenges D. van der Lelie 1,2, S. Brown 3 , M. Mench 4 and J. Vangronsveld 5 1

Brookhaven National Laboratory (BNL), Biology Department, Building 463, Upton, NY 11973-5000, USA ([email protected]) 2 Vlaamse Instelling voor Technologisch Onderzoek (VITO), Environmental Technology, Boeretang 200, B-2400 Mol, Belgium 3 Ecosystem Sciences, University of Washington, Seattle, Washington 98195, USA 4 Agronomy Unit, INRA Bordeaux Aquitaine Research Center, BP 81, 33883 Villenave d’Ornon, France 5 Limburgs Universitair Centrum, Centre for Environmental Sciences, Universitaire Campus, B-3590 Diepenbeek, Belgium

Introduction A number of chemical analysis methods are currently used to determine the total metal concentrations in solid samples. However, such methods do not provide information about the biological available fraction of the detected pollutants in the samples, despite the fact that this parameter provides important information on the ecological impact of the pollution (both for heavy metals and organic xenobiotics). Since its provides a risk based classification and decision tool, the biological available fraction is of particular practical importance. In addition, it permits the evaluation of which point bioremediation targets can be achieved, and can be used to evaluate the efficiency and sustainability of immobilization based remediation methods. Therefore a need exists to develop and validate cost efficient and ecological relevant tests to determine the bioavailability and environmental impact of a pollutant. These tests can be enzymatic tests, tests based on specific organs, whole cells and organisms, or studies of complex ecosystems that include different trophic levels. With an increasing complexity of the test, the ecological significance of the test’s outcome might improve; however, the costs to perform the test will also increase. Therefore an optimum has to be defined between ecological relevance and the costs to perform the test. The challenge will be to design a test panel of complementary biological tests, this in addition to chemical analysis. Bacterial tests Bacterial tests have been developed to assess the impact of pollution. The pioneer system was the MicroTox test (Bulich and Isenberg, 1981), that is based on a constitutively light producing strain of Vibrio fischeri: in the presence of a toxic, bioavailable contaminant, the light production of this strain will decrease, allowing to determine the EC20 and EC50 toxicity values of the sample. This concept has been copied and improved by using more relevant bacterial strains, such as a genetically manipulated Pseudomonas strain, which is of more ecological relevance to study contaminated soil samples (Bundi et al, 2001). However, none of these bacterial biosensors is able to discriminate between different types of pollution. To overcome this limitation, bacterial biosensors were designed where the reporter system is placed under the control of a promoter-operator sequence whose transcription is specifically induced in the presence of an environmental insult. This concept can be applied for

the specific detection of heavy metals, organic xenobiotics or any compound that induces a specific form of genetic response. Bacterial biosensors, referred to as the BIOMET test, were developed that detect the presence of a specific heavy metal or a group of heavy metals (Corbisier et al, 1994 and 1996; van der Lelie et al, 2000). These sensors are based on gene fusions between heavy metal resistance operons e.g. of Ralstonia metallidurans CH34 and the luxCDABE operon of Vibrio fischeri. The presence of bioavailable heavy metals results in the specific and quantitative induction of light production. These sensors are presently available for the specific detection of bioavailable Zn and Cd, Cu, Pb, Ni, As and Cr(VI) (Corbisier et al, 1993 and 1999; Tibazarwa et al, 2000; van der Lelie et al, 2000) and have been successfully used to assess the bioavailability of heavy metals in different environmental samples including metal contaminated soils and river sediments. The BIOMET test shows a good correlation with the bioavailability data as predicted using sequential extraction (Tessier et al, 1979), phytotoxicity (Vangronsveld and Clijsters, 1992; Van Assche and Clijsters, 1990) and zootoxicity tests. Heavy metal bioavailability and microbial ecology Since heavy metal contamination might cause a selective pressure on the microbial community, addition of soil additives that results in a decrease in bioavailability of the heavy metals could have effects on the proportion of heavy metals resistant bacteria in the total bacterial population. If this should be the case, changes in the proportion of heavy metal resistant bacteria would reflect changes in bioavailability of heavy metals. This concept was recently demonstrated with soils from Leadville (USA) that received different treatments: changes in heavy metal bioavailability, as determined using the BIOMET test, resulted in a decrease in the bacterial community of the heavy metal resistant Ralstonia strain LV1. In situ monitoring using bacterial biosensors At present, few examples of on site monitoring are available. However, with the availability of new reporter systems, such as the green fluorescent protein (gfp), on site monitoring of the topsoil of large surfaces is theoretically feasible. This concept can be used to screen large surfaces for the presence of specific contaminants, or even mines. However, a general drawback of this technique is the necessity to deliberately release GMOs. References 1. Bulich, A. A.; Isenberg, D. L. ISA Trans. 1981, 20, 29-33. 2. Bundy, J. G.; Campbell, C. D.; Paton, G. I. J. Environ. Monit. 2001, 3, 404-410. 3. Corbisier, P.; Ji, G.; Nuyts, G.; Mergeay, M.; Silver, S. FEMS Microbiol. Lett. 1993, 110, 231-238. 4. Corbisier, P.; Thiry, E.; Masolijn, A.; Diels, L. In: Bioluminescence and Chemoluminescence: Fundamentals and Applied Aspects, Campbell A. K.; Cricka L. J.; Stanley P. E., Eds. John Wiley and Sons, Chichester, New York, Brisbane, Toronto, Singapore, 1994, 150-155. 5. Corbisier, P.; Thiry, E.; Diels. L. Environ Toxicol and Water Quality 1996, 11, 171-177. 6. Corbisier, P.; van der Lelie, D.; Borremans, B.; Provoost, A.; de Lorenzo, V.; Brown, L.B.; Lloyd, J. R.; Hobman, J.L.; Csöregi, E.; Johansson, G.; Mattiasson, B. Anal. Chimica Acta 1999, 387, 235-244. 7. Tessier, A; Campbell, P.G.C.; Bisson, M. Analytical Chemistry 1979, 51, 844-850.

8. Tibazarwa, C.; Wuertz, S.; Mergeay, M.; Wyns, L.; van der Lelie, D. J. Bacteriol. 2000, 182, 1339-1409. 9. Van Assche, F.; Clijsters, H. Environ. Pollut. 1990, 66, 157-172. 10. van der Lelie, D.; Verschaeve, L.; Regniers, L; Corbisier, P. In: New Microbiotests for routine toxicity screening and biomonitoring, Persoone, G.; Janssen, C.; De Coen. W., Eds. Kluwer Academic/Plenum Publishers, London, UK, 2000, 197-207. 11. Vangronsveld, J.; Clijsters, H. In: Metal compounds in environment and life, 4 (Interrelation between chemistry and biology); Merian, E.; Haerdi, W., Eds. Science Reviews Inc.: Wilmington, 1992, 117-125.

The Application of Short Rotation Coppice for Clean-up of Radionuclide Contaminated Sites M. Dutton.

THE BORDEAUX MIXTURE.

Jacques DELAS Directeur de Recherche honoraire INRA, Centre INRA Bordeaux-Aquitaine, F-33883 Villenave d’Ornon cedex, France. In the second half of the 19th Century, as maritime trades developed with the New World, several American parasites of the grapevine were successively introduced into the French vineyard where they caused considerable damage and threatened the future of viticulture. The most important of these were the fungal diseases powdery mildew (oïdium),downy mildew (mildiou), and black-rot, together with the insect parasite, Phylloxera. Efforts deployed to fight against these pests and plant diseases resulted in solutions still used today: sulphur against powdery mildew, grafting on rootstock- tolerant plants in the case of the phylloxera and the use of Bordeaux mixture against downy mildew. I plan to summarise the history of Bordeaux mixture. Downy mildew (mildiou), known in the USA as either grapevine mildew or wine pest, was first reported in 1878 near Bordeaux. Over several years, it spread across France many other parts of Europe. The causal agent is a fungus, Plasmopara viticola. Mildew causes considerable damage to both leaves and grapes. The early leaf senescence and defoliation that it provokes translates, in case of severe attack, into both a significant reduction in yield and an equally important decrease of both grape and wine quality. One spoke, at the time of the first attacks, about “mildiousés” wines. A. MILLARDET, Professor of Botany at the Science University of Bordeaux, was famous at this time due to his researches on phylloxera. He is also renowned as the discoverer of Bordeaux mixture. In late October 1882, when the whole Médoc region was strongly hit by the mildew, he observes " that all along the road, the grapevines have leaves while everywhere elsewhere they had been fallen for a long time " (due to the effect of the mildew attacks). These leaves present in end of the rows were covered "on the superior face with a slim layer, which sticks, a pulverulent substance of a bluish white". He learnt from E. DAVID, manager of the BEAUCAILLOU castle (vineyard) at SAINT JULIEN, that it is custom, in the Médoc, when grapes start to mature, to cover leaves with either green -grey (alkaline copper acetate) or copper sulphate mixed with lime. This " médocain mix" aimed to deter would-be grape thieves by making them fear poisoning by the blue vitriol. It was spread on the vine stocks the closest to roads with the help of heather brooms. MILLARDET was convinced by these observations that copper salts could ward off mildew attack on leaves by preventing the germination of spores (the antifungal capacity of copper, signalled in the preceding century, had been used against wheat smut). Therefore he decided to experiment with various formulations based on copper salts. He found that only those similar to the "médocain mix" appeared fully effective. He had also to convince detractors and wine growers of the efficacy of this fungicide treatment. On the 1st April 1885, he gave a lecture at the Gironde Agriculture Society where he presented the synthesis of his observations made with DAVID. He reported on the Bordeaux mixture formula, as well as its application method and 3 suitable periods for preventive treatments. The most efficient mixture in his tests was a 5.3% copper sulphate solution neutralized by addition of quick lime. Further work demonstrated the efficiency of lower concentrations of copper sulphate and that

it was not necessary to limit applications to 3 each year, but to adapt their number to prevailing climatic conditions. Another professor of the Bordeaux Science Faculty, U. GAYON, a disciple of PASTEUR, played an important role with MILLARDET in the formulation of Bordeaux mixture. He made two other important discoveries. Firstly, he demonstrated the reaction mechanism of copper in the Bordeaux mixture. No-one understood that copper, precipitated by lime, could have an action on the fungus. GAYON showed that the insoluble deposit formed by the mixture deposited on leaves transformed slowly, under the action of rainwater and dew which both contained dissolved carbon dioxide, into soluble copper hydroxide, releasing copper in an ionic form. Secondly, GAYON demonstrated that copper deposited onto the surface of grapes was precipitated during the fermentation and was not found in the wine. This replied to objections of some detractors of the Bordeaux mixture who put forward risks of human toxicity due to copper being concentrated in the wine. From 1885, birth date of the Bordeaux mixture, until the 1960’s when the first organic fungicides appeared, a period of 75 years, copper was the only treatment able to efficiently combat mildew. Due to its efficacy, it was used in all French vineyards with a frequency depending essentially on climatic conditions. For many years, Bordeaux mixture with a 2 % CuSO 4 , 5H2 O level was applied at a rate of 1 000 liters / ha. Therefore, each treatment results in an input of 5 kg Cu / ha and the number of applications ranged from 5 to 15 each year. As a result, one can estimate that the total amount of copper applied to French vineyard soils during this 75 year period represents between 200 to 2 000 mg Cu/kg of soil. Research at the end of 19th Century and the beginning of 20th Century focussed on the consequences that Cu from Bordeaux mixture could have on higher plants, independently of its toxicity to fungi. Experiments carried out at this time concluded there was an absence of Cu phytotoxicity at the levels used in viticulture. In fact, these reassuring results had been obtained with calcareous soils. One learns only 50 years later that Cu phytotoxicity occurred mainly in acidic soils! The first consequences of Cu accumulation in vineyard soils became apparent after 1950. The catastrophic frost of 1956 resulted in the death of many grapevines across France. This was followed by numerous replants. However, many of these displayed very serious damage, with either the death of young grapevines or annual crop plants. These were reported in many vineyard areas. Researches undertaken as a result of this emergency period took several years to find the cause of phytotoxicity and to develop remediation methods that have since been widely adopted in wine growing practice. These results can be briefly summarized: -

Phytotoxicity observed after the removal of old grapevine stocks and subsequent replanting is due to Cu accumulation in the soil as a result of the cumulative antifungic treatments. Cu toxicity does not lead to specific symptoms on the leaves but primarily reduces growth of the root system where the metal accumulates. Copper transfer coefficients from roots to aerial plant parts are low. Phytotoxicity is only evident in acidic soil (the acidity of pedogeochimic origin being worsened in some situations by the biological oxidation of the sulphur applied to combat powdery mildew of the grapevine); The more acid the soil, the more serious the Cu toxicity; occurrence of phytotoxicity is enhanced by a low CEC. The copper accumulated in the upper soil horizons and does not readily leach down the soil profile: this explains why Cu toxicity has generally never been demonstrated on grapevines where the root system is localised below the zone of Cu accumulation;

-

on the other hand, phytotoxicity occurs when a grapevine plantlet or an annual plant seedling is cultivated in the superficial accumulation zone; Liming (up to pH 6.5) before replanting (or the sowing) eliminated the risk of phytotoxicity. The use of organic amendments, which enhance the formation of organometallic Cu complexes, is an additional remediation treatment.

The increased production of either organometallic or organic fungicides in the 1960’s at the time where one discovered the toxicity of the copper accumulated in vineyard soils should have resulted in a total absence of Cu-fungicides in viticulture . This has not taken place. Whilst it is true to say that total Cu inputs have decreased, they are far from being null (the development of new delivery systems allows reduced doses for the same efficiency). The wine producers that still use copper for some of their treatments justify their choice by the fact that, contrary to organic fungicides, it does not induce resistance phenomena, or that it would be more efficient for late treatments during the season. Furthermore, copper is authorized in biological agriculture where it is used commonly. The absence of a substitute product for this type of agriculture would pose serious problems if the use of copper came to be forbidden. Bordeaux mixture has allowed French viticulture to overcome the very serious crisis provoked by the mildew invasion, and Plant pathology to become a fully-fledged scientific discipline. Well over 100 years after its development, it is still one of the best-investigated topics on consequences of trace element accumulation in agricultural soils where the inputs, speciation and consequences of soil Cu contamination are all clearly defined.

References DELAS J., 1963. La toxicité du cuivre accumulé dans les sols. Agrochimica, 7 ; 258-288. LAFON R., 1985. Le Médoc et la découverte de la bouillie bordelaise. MEDOC, Bulletin d’information du G.I.E. des vins du Médoc, 5-33. VIENNOT-BOURGIN G., LAFON R., 1985. La naissance de la Bouillie Bordelaise. Fungicides for Crop Protection. 100 years of progress. Monograph N° 31. Acknowledgements. Author is grateful to Dr. N. Lepp and Dr. M. Mench for helping in text editing.

Poster Session 2. n° 12. Characterisation of mercury-thiol complexes in maize root

L. E. Hernández*, L. A. Arroyo-Méndez*, S. Vázquez, F. F. del Campo*, R. O. Carpena-Ruiz *Laboratorio de Fisiología Vegetal, Departamento de Biología and Departamento de Química Agrícola, Universidad Autónoma de Madrid, Camp us de Cantoblanco, E28049 Madrid, E- mail: [email protected] Introduction. Heavy metals accumulation in the soil can lead to severe toxicity symptoms and inhibition of plant growth. Tolerant plants have developed several mechanisms to restrict the biological activity of the free toxic metal in the plant cell. One of them is their association to cell wall components, that decreases metal accessibility to the protoplast. Once the metal enters the cell, one of the proposed tolerance mechanisms is the chelation of metals to the thiol-containing peptides phytochelatins. This is supported by biochemical and genetical evidences showing that plants unable to synthesise phytochelatins were more sensitive to several heavy metals. Phytochelatins are non-ribosomally synthesised by phytochelatin synthase after activation of the enzyme in the presence of the metal from the ubiquitous peptide glutathione. Results and discussion. Mercury (Hg) is one of the most toxic pollutant heavy metals to plants. In Spain there are several areas contaminated with this metal, of which the Almaden mining facility is the most relevant. Previous field work in this area (1) showed important differences in the tolerance strategies of graminaceous and leguminous plants. Recently (2) we have studied some Hg-stress biomarkers in maize and pea plants, indicating that maize is less tolerant to Hg. Now we are studying the mechanisms of plant tolerance to Hg in maize grown hydroponically and exposed for 7 days to 30 µM Hg. Firstly, we have characterised the subcellular fractions of root and shoot where Hg accumulated. To analyse Hg accumulated, plant tissues were homogenised in a suitable buffer and the homogenate filtrated through nylon cloth. The residue consisted mainly of plant cell walls, and the filtrate was differentially centrifuged. The particulate and plant cell wall material were pooled together and considered as the insoluble fraction, and the supernatant recovered after centrifugation as the soluble fraction. The analysis of Hg accumulation in root fractions revealed that up to 20 % was associated to the soluble fraction. On the other hand, analysis of non-protein thiol-peptides indicated a significant increase as compared to the control (approx. from 250 to 1050 nmol/g FW). Furthermore, analysis of gluthatione revealed a modest increase in plants treated with Hg, indicating that other thiol-peptides might had been accumulated in response to the metal. To determine the possible association of Hg to thiol-peptides in the soluble fraction, this fraction was separated chromatographically by FPLC-DEAE. A peak of thiol-peptide co-eluted with Hg, from which we could recover up to 70% of the metal contained in the soluble fraction

sample loaded onto the DEAE column. These results suggest that most of the Hg found in the soluble fraction was associated with thiol-peptides. We are currently characterising the peptides found in the FPLC-DEAE fractions. Tentative identification by HPLC, after derivatisation with Ellman’s reagent, indicate that the thiol-peptides resemble some of those accumulated in maize after exposure to cadmium. References. 1. Lucena, J.J., Hernandez, L.E., Olmos, S., Carpena Ruiz, R. 1993. Micronutrient content in graminaceous and leguminous plants contaminated with mercury. In: Optimization of Plant Nutrition (M.A.C. Fragoso and M.L. Beusichem, eds.) pp.531-37. Kluwer Acad.Publs. Dordrecht. 2. S. Vázquez, L.E. Hernández, R.O. Carpena-Ruiz. 2001. Determinación de compuestos tiólicos como indicadores de contaminación de Hg en plantas de maíz y guisante. In: Nutrición mineral en una agricultura mediterránea sostenible (C.F. Alcaraz; M. Carvajal; V. Martínez eds.) pp. 357-363. CEBAS-CSIC, Consejería Agricultura, Agua, Medio Ambiente. Murcia, Spain

n° 13. Relationship between plant structure, physiological processes, uptake and accumulation of Cd

A. Lux, A. Šottníková, L. Lunácková, E. Masarovicová, D. Lišková1 , K. Králová, V. Streško, P. Capek1 Department of Plant Physiology, Institute of Chemistry and Geological Institute, Faculty of Natural Sciences, Comenius University, Mlynská dolina B2, SK-84215 Bratislava, Slovakia; 1 Institute of Chemistry, Slovak Academy of Sciences, Dúbravská cesta 9, SK-84238 Bratislava, Slovakia.

Introduction Cadmium is a rare element with important toxicological properties already in relatively low concentrations. The toxicity is related predominately to its similarity with zinc, which is an essential metal for living organisms. Unlike Zn, cadmium has no essential function in any living organism (Wagner, 1995), however, it can be transported by identical mechanisms as Zn and can substitute it in many organic compounds (Kalac and Tríska, 1998). For phytoremediation of soils contaminated by Cd several herbaceous species could be used due to their high accumulation abilities. On the other hand the deposition of metals in the trees is lower, but it can be advantageous because of the long- life span, deeply penetrating roots and favourable association with microorganisms. Relatively high sensitivity of trees to toxic metals together with the low extraction ability stimulate experiments aimed at physiology and structure of these species under influence of toxic metals as well as screening and selection of tolerant and hyperaccumulating species and clones. Materials and methods Various species and clones of the genus willow (Salix), and poplar (Populus) were used for experiments. Plants were cultivated in hydroponics and in vitro. Cd was applied in the form of Cd(NO3 )2 and CdCl2 , in concentrations 10-5 to 10-3 M. Growth characteristics of stem cuttings growing in hydroponics (number and cumulative length of roots and shoots, leaf area, specific leaf mass and biomass production of individual organs) were studied. Growth of organ cultures in vitro with and without Cd was evaluated. Accumulation of Cd in individual plant organs in vivo, in vitro and in callus cultures was determined by AAS Assimilation pigments (chlorophylls and carotenoids) were determined according to Lichtenthaler (1987). Structural analysis of roots was focused on the development of endodermis and exodermis and their relation with the development of vascular system. Methods of light, transmission electron microscopy and fluorescence microscopy (Brundrett et al. 1988) were used. For chemical analyses of plant cell walls gas and paper chromatography, mass spectrometry and NMR were used. Results and discussion

Studied willows and poplars belong to the fast growing species. Stem cuttings develop rapidly adventitious root from the existing root primordia and simultaneously new shoots are formed from the axial buds. The growth of these newly formed organs is influenced by the presence of Cd in cultivation media, the roots react most sensitively. The structure of adventitious roots in both genera is characterized by broad cortical layer with extensive intercellular spaces. Endodermis is formed close to the root apex in the distance of approximately 3 mm. Interspecific differences were found in formation of Casparian bands, in some cases preceding formation of vascular system. After Cd treatment Casparian bands were formed cons iderably closer to the apex. Exodemis with Casparian bands and suberin lamellae was also formed in all studied species, in variable distances from the root apex. The highest amounts of Cd were accumulated in roots of all species in hydroponics. However, significant interspecific differences of Cd content were found in stem cuttings and in newly formed shoots. In vitro cultures of several willow and poplar species were derived from the stem cuttings. The sensitivity to Cd treatment was documented also on rooted and non-rooted shoot cultures in vitro. The results confirmed the importance of the root in metal accumulation. The long-term poplar callus culture showed high tolerance to Cd. In this connection the effect of cadmium on callus cell walls was studied microscopically and by chemical analyses. Assimilation pigment concentration is considered to be decreased as a result of negative Cd effect (Barua and Jana 1986, Kummerová and Brandejsová 1994). The found results of assimilation pigment concentration showed chl a as the main target of Cd application. The values of chl a concentration significantly decreased in S. viminalis, S. alba and P. gigant. However, in P. robusta, S. purpurea and S. cinerea cadmium did not negatively affect concentration of this pigment. There were also no significant differences in determined values of carotenoids and chl b concentration. Acknowledgement The research was supported by COST Action 837 and Slovak Grant Agency VEGA grant No. 1/7258/20 References 1. Barua, B. and S. Jana. 1986. Effects of heavy metals on dark induced changes in Hill reaction activity, chlorophyll and protein contents, dry matter and tissue permeability in detached Spinacia oleracea L. leaves. Photosynthetica 20, 74-76. 2. Brundrett, M.C., D.E. Enstone and C.A. Peterson. 1988. A berberine - aniline blue fluorescent staining procedure for suberin, lignin and callose in plant tissue. Protoplasma 146, 133-142. 3. Kalac P. and J. Tríska. 1998. Chemistry of the Environment. University of South Bohemia, Ceské Budejovice, Czech Republic, p. 44. – in Czech 4. Kummerová, M.and R. Brandejsová. 1994. Project TOCOEN. The fate of selected pollutants in the environment. Part XIX. The phytotoxicity of organic and inorganic pollutants-cadmium. The effect of cadmium on the growth of germinating maize plants. Toxicol. Environ. Chem. 42, 115-122. 5. Lichtenthaler H.K. 1987. Chloroplylls and carotenoids: photosynthetic biomembranes. Methods Enzymol. 148, 350-382.

Pigments

of

6. Wagner G.J. 1995. Biochemical studies of heavy metal transport in plants. pp. 2122. In Current Topics in Plant Biochemistry, Physiology and Molecular Biology,

Randall D., Raskin I., Baker A., Blevis D., Smith R., eds. Univ. of Missouri, Columbia, Missouri.

n° 14. Cadmium and Pb toxicity in sugar beet (Beta vulgaris L.)

F. Morales1 , A. Larbi1 , A. Álvarez-Fernández1 , A.F. López-Millán1 , N. Molías1 , Y. Gogorcena 1 , J.J. Lucena 2 , A. Abadía1 and J. Abadía1 1

Departamento de Nutrición Vegetal, Estación Experimental de Aula Dei-CSIC, Apdo. 202, E-50080 Zaragoza, Spain. 2 Departamento de Química Agrícola, Geología y Geoquímica, Universidad Autónoma de Madrid, E-28049 Madrid, Spain.

Introduction Heavy metals constitute a heterogeneous group of elements, including both essential and non-essential ones. Essential elements are some of those known as micronutrients: Mn, Fe, Cu, Zn and Mo. Non-essential heavy metals, such as Pb, Cd and Al, can be phytotoxic even when present at relatively low concentrations. Cadmium and Pb are the most abundant non-essential heavy metals polluting the environment. Natural and anthropogenic sources of major heavy metals in soils have been reviewed recently (Baligar et al. 1998). Materials and methods Sugar beet plants were grown in growth chamber (Morales et al. 1990). Six-week old plants were transplanted to buckets containing half-Hoagland nutrient solution with 10 or 50 µM CdCl2 , 10 or 50 µM Cd-EDTA, or 2 mM Pb-EDTA. The global chemical speciation was estimated by using the MINTEQA2 software (Allison et al. 1991). The pH of the nutrient solutions was measured every 2-3 days and adjusted to 5.5 with diluted HCl. Iron reductase activity was measured in excised root tips following procedures described in Gogorcena et al. (2001). Photosynthetic pigments were quantified by HPLC as described in Gogorcena et al. (2001). Gas exchange measurements were made with a portable system (CIRAS-1, PP Systems, Herts, U.K.). Continuous Chl fluorescence measurements were made with the experimental set-up (Morales et al. 1990) and protocol (Belkhodja et al. 1998) described elsewhere. Modulated Chl fluorescence measurements were made as described in Belkhodja et al. (1998). Samples for heavy metal and nutrient analyses were analysed using standard procedures (Abadía et al. 1985; Lozano-Rodríguez et al. 1995). Results and discussion Cadmium and Pb toxicity have been investigated in sugar beet grown in hydroponics under growth chamber-controlled conditions. Chemical speciation was used to investigate the chemical species in equilibrium. Cadmium decreased root and shoot fresh and dry weight, and increased root/shoot ratio. Cadmium- treated plants developed few brownish roots with short laterals. Plants treated with Cd developed chlorosis. In leaves, Cd decreased N, P, Mg, K, Mn, Cu and Zn concentrations but decreased Ca concentrations. All Cd treatments increased Fe(III)-chelate reductase activities in root tips, although Fe concentrations in shoots were similar to those of controls. The changes in photosynthetic parameters observed with Cd in sugar beet

resembled those observed in Fe-deficient sugar beet plants (Morales et al. 1990, 1998; Belkhodja et al., 1998). These included increases of the lutein/Chl and VAZ pigments/Chl molar ratios, changes in the dark-adapted Chl fluorescence induction curve -reversible by far-red pre- illumination- and changes in the Chl fluorescence quenching parameters. All these data suggest the existence of a Cd-induced Fe chlorosis, resembling to that occurring in leaves from field-grown, Fe-chlorotic trees (Abadía et al. 1985). Lead chelated with EDTA increased root fresh and dry weight with no changes in the shoot mass, therefore increasing the root/shoot ratio. Changes in nutrient concentrations were much less marked with Pb than those found with Cd. It should be mentioned, however, that shoot Cu levels were close to deficiency critical levels. Leaves of Pb-treated plants remained green or showed a slight leaf pale green colour. In some cases, Pb-treated leaves rolled their edges inwards. Root tips from Pbtreated plants also had increased Fe(III)-chelate reductase activity. Lead had much less effects than Cd on all photosynthetic parameters measured. Photosynthesis and gas exchange measurements were similar in control and Pb-treated plants. The actual photosystem II efficiency was only slightly affected by Pb, resulting from both slight decreases in intrinsic photosystem II efficiency and slight decreases in photochemical quenching. The only photosynthetic parameter affected by Pb was non-photochemical quenching, that increased up to 2.6-fold in response to Pb. References 1. Abadía J., J.N. Nishio, E. Monge, L. Montañés, and L. Heras. 1985. Mineral composition of peach tree leaves affected by iron chlorosis. J. Plant Nutr. 8, 697708 2. Allison J.D., D.S. Brown, and K.J. Novo-Gradak. 1991. A geochemical assessment model for environmental systems v. 3.0. Washintong DC, Environ. Res. Lab., US Environ. Protection Agency 3. Baligar V.C., N.K. Fageria, and M.A. Elrashidi. 1998. Toxicity and nutrient constraints on root growth. HortSci. 33, 960-965. 4. Belkhodja R., F. Morales, R. Quílez, A.F. López-Millán, A. Abadía, and J. Abadía. 1998. Iron deficiency causes changes in chlorophyll fluorescence due to the reduction in the dark of the photosystem II acceptor side. Photosynth. Res. 56, 265-276. 5. Gogorcena Y., N. Molías, A. Larbi, J. Abadía, and A. Abadía. 2001. Characterization of the responses of cork oak (Quercus suber) to iron deficiency. Tree Physiol. 21, 1335-1340. 6. Lozano-Rodríguez E, M. Luguera, J.J. Lucena, and R.O. Carpena-Ruiz. 1995. Evaluation of two different acid digestion methods in closed systems for trace elements determinations in plants. Química Analítica 14, 27-30. 7. Morales F., A. Abadía, and J. Abadía. 1990. Characterization of the xanthophyll cycle and other photosynthetic pigment changes induced by iron deficiency in sugar beet (Beta vulgaris L.). Plant Physiol. 94, 607-613.

8. Morales F., A. Abadía, and J. Abadía. 1998. Photosynthesis, quenching of chlorophyll fluorescence and thermal energy dissipation in iron-deficient sugar beet leaves. Aust. J. Plant Physiol. 25, 403-412.

n°15. Evaluation of the potential biotoxicity / essentiality of Zinc and Cadmium in suspended cells of Cynara cardunculus and Centaurea calcitrapa

M. A. G. Oliveira, S. Raposo and M. E. Lima-Costa Faculty of Engineering of Natural Resources, University of Algarve, Campus de Gambelas, 8000-117 Faro, Portugal

Introduction From general biological, as well from plant physiological point of view, essential and non-essential heavy metals can be distinguished (Clijsters et al., 1999). There are three criteria for establishing whether or not a trace element is essential for the normal growth of plants: (i) the organism can neither grow nor complete its life cycle without it; (ii) the element cannot be wholly replaced by any other element; (iii) the element has a direct influence on the organism and is involved in its metabolism (Mas and Azcue, 1993). Heavy metal such as Cu2+, Zn2+, Mn2+, Fe2+, Ni2+ and Co2+ are essential micronutrients for plant metabolism, but when present in excess, these, and nonessential metals, such as, Cd2+, Hg2+, Ag2+ and Pb2+, can become extremely toxic (Williams et al., 2000). In this work we aimed to study the effect of increasing amounts, of an essential metal, Zn (kno wn enzyme activator), and a non-essential metal, Cd (normally considered toxic), on the growth of suspended cells of Cynara cardunculus and Centaurea calcitrapa. Materials and methods C. cardunculus and C. calcitrapa cells suspension culture were grown, respectively, on B5 (Gamborg et al., 1968) and SH (Schenk and Hildbrandt, 1972) nutrient medium. The Zn assays (with ZnSO4 ,7H2 O) were performed by addition of 20 and 200 mg/L Zn salt (CY1), and 200 and 600 mg/L Zn salt (CC1) respectively for C. cardunculus and C. calcitrapa suspended cells. The Cadmium assays (with CdSO4 ,8/3H2 O) were performed by addition of 1, 20 and 50 mg/L Cd salt (CC2), and 1, 10 and 20 mg/L Cd salt (CY2), respectively for C. calcitrapa and C. cardunculus suspended cells. In both Zn and Cd assays we performed, as a control, an assay with 2 (in CY1) and 1 mg/L Zn salt (in CC1), and an assay without Cd for both CY2 and CC2. The suspended cells were kept in Erlenmeyer flasks (500 mL), at a temperature of 25ºC with constant agitation (120 rpm). Fresh weight (FW) and dry weight (DW) were determined. Soluble proteins were determinated according to Bradford (1976) and phenol content according to Anselmo et al. (1985). Experiments were carried out in triplicates for each concentration. Results and discussion The specific growth rate, µg, obtained in CY1 cultures became smaller when the Zn salt concentration, in the culture medium, increased. The profile of biomass accumulation in both assays with excess Zn has also shown growth inhibition. The presence of 20 mg/L Zn salt yielded the best result in terms of soluble protein in CY1.

With 200 mg/L Zn salt the production of phenols was triggered sooner, in comparison to the other concentration assays, suggesting that a stress metabolism has been induced in C. cardunculus cells in the presence of this amount of Zn. With CC1 cultures, it was obtained an increase in terms of µg, with 200 mg/L Zn salt, being the best result in those conditions. It was obtained a similar µ g in the presence of 1 and 600 mg/L Zn salt, but the biomass accumulation and soluble protein content showed the highest values in the presence of 600 mg/L Zn salt. The phenols production was higher for the assay with 200 mg/L Zn salt. In both CY2 and CC2 cultures, it was obtained growth inhibition for the assays performed in the presence of Cd higher than 1mg/L Cd salt. However, with 1 mg/L Cd salt, both cell suspension cultures manifest different behaviours. In CC2 it was obtained a higher µg value and higher soluble protein increase, in comparison to the value obtained in the control assay, but with CY2 this result was not confirmed. The biomass accumulation was severely affected, either CC2 or CY2 growth conditions cultures. The profile of phenolic compounds production, in CC2, usually associated to plant stress metabolism, in the presence of 1 mg/L Cd salt, suggested that there was no additional stress in these cells, in comparison to the control culture. An opposite result was obtained with CY2 cell culture. The heavy metal Cd cannot be considered an essential element because it inhibits the growth of both suspended cells culture, in a higher proportion for C. cardunculus cells. The low biotoxicity of Zinc element was clearly demonstrated, except for higher Zn concentration, as referred by Williams et al., 2000. Another conclusion was that C. cardunculus suspended cells are more sensitive to the excess of Zn and to the presence of Cd, in the culture medium, than C. calcitrapa cell suspension. References 1. Anselmo A. M., J. M. S. Cabral, J. M. Novais. 1985. Degradation of phenol by immobilized cells of Fusarium flociferum. Biotech. Lett. 7 (12): 889-894 2. Bradford, M. M. 1976. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the princip le of protein dye binding. Anal. Biotech. 72: 248-254. 3. Clijsters H., A. Cuypers , J. Vangronsveld. 1999. Physiological responses to heavy metals in higher plants; defence against oxidative stress. Z. Naturforsch. 54c, 730-734 4 Gamborg O. L., A. Miller, K. Ojima. 1968. Nutrient requirements of suspension cultures of soybean root cells. Exp. Cell Res. 50: 151-154 5. Mas A., J. M. Azcue. 1993. Metals en Sistemas Biológicos. 1e d. Promociones y Publicationes Universitarias, S.A. 6. Skenk, R., A. Hildbrandt. 1972. Medium and techniques of induction and growth of monocotyledonous and dycotyledonous plant cell cultures. Can. J. Bot. 50: 199-204. 7. Williams L. E., J. J. Pittman, J. L. Hall. 2000. Emerging mechanisms for heavy metal transport in plants. Biochim. Biophys. Acta 1465, 104-126.

n° 16. Role of root exudates in metal tolerance: lessons from aluminium research

Ch. Poschenrieder and J. Barceló Laboratorio de Fisiología Vegetal, Facultad de Ciencias, Universidad Autónoma de Barcelona, E-08193 Bellaterra, Spain. E- mail: [email protected]

Introduction The importance of root exudates containing organic acids, phenolic substances, or phytosiderophores in the plant's capacity to acquire essential nutrients from sparingly soluble forms in soils is well established. Segregation of citrate from cluster roots enhances mobilization of P. Iron deficiency- induced exudation of citrate in dicots (strategy I) and of phytosiderophores in grasses (strategy II) favors Fe mobilization and uptake. Exudation of phytosiderophores also increases the rhizosphere solubility and mobility of other metallic micronutrients, especially Zn and Cu. Exudation of phenolics can be implied in Mn mobilization (Marschner, 1995). The use of synthetic chelators for metal mobilization is gaining increasing importance in both fertilization of crops and phytoremediation technologies. However, not in all cases root exudation of potential metal-chelators leads to enhanced metal availability, to more metal uptake, and, if present in excess in the soil, to increased danger of phytotoxicity. On the contrary, there is growing experimental evidence that root exudation of chelating substances may also play a major role in detoxification and exclusion from roots of certain potentially toxic metals such as Al and Pb (Ryan et al., 2001). During the last years fast advances have been made in the understanding of the role of root exudates in the mechanisms of Al resistance. Although there is no use to try to reduce Al contents in soils by phytoextractio n technologies, the lessons learnt from Al-research may be useful for improve experimental approaches of the role of root exudates in metal mobilization, metal resistance and metal uptake of species used for phytostabilization and phytoextraction technolo gies in heavy metal contaminated soils. Keys to understand Al toxicity and resistance Among the most remarkable points for the progress in understanding the mechanisms of Al toxicity and resistance in crop plants are the following (Barceló & Poschenrieder, 2002): 1) Identification of Al3+ as the main phytotoxic Al species. 2) Identification of root tips as the most Al susceptible site (effects on cytoskeleton, root cell elongation and root cell division) 3) Use of short-term root growth curves for distinguishing different response patterns a) threshold for toxicity (importance of Al speciation in substrate and internal effect concentration in tips) b) hormesis (e.g. by alleviation of proton toxicity)

4) 5)

6)

7) 8)

c) threshold for resistance (possible need for activation of resistance mechanisms) Localization at the tips of root exudate production specifically induced by Al 3+ Chemical characterization of different Al chelators in exudates of different species a) organic acids: malate, citrate, oxalate, b) flavonoid-type phenolics: catechin, quercetin Distinction between exudation patterns a) Pattern 1: immediate exudate release by activation of anion channels b) Pattern 2: induction of exudate release after a several hours lag-time (probable need for gene activation) Other Al exclusion mechanisms: mucilage, border cells… Root exudates, Al uptake, ligand exchange, and compartmentation in Al hyperaccumulators (e.g. tea and species of the Melastomataceae family)

Conclusions Organic acids (malate, citrate, oxalate) and/or flavonoid-type phenolics (catechin, quercetin) are very common components of root exudates of plants under different environmental conditions. The specific role of such compounds in the protection against Al toxicity in Al resistant genotypes resides in the ion-specific induction (Al3+) of these exudates at the precise region where the primary toxicity effects occur (root tip). The apoplastic accumulation of these chelators prevents both Al3+ toxicity in the cell wall - plasma membrane region and uptake of Al into the symplasm. Differences in either the distribution of anion efflux channel that are activated specifically by Al 3+ (pattern 1 of exudate release) or the genes responsible for exudate production upon specific activation (pattern 2 of release) seem to account for varietal differences in Al resistance by exclusion. Similar experimental approaches addressing the ion specificity of exudate induction, the site of exudate production, the chemical composition of the exudates and their time pattern of release using metals and plant species that are relevant in phytoremediation would contribute to a better understanding of the rhizosphere processes implied in these technologies. Acknowledgements Part of the author’s work cited in this paper was supported by the European Union (ICA4-CT-2000-30017) and by the Spanish Government (DGICYT, BFI2001-2475CO2-01)

References 1. Barceló, J., Poschenrieder, Ch., 2002 Fast root growth responses, root exudates, and internal detoxification as clues to the mechanisms of aluminium toxicity and resistance: a review. Env. Exp. Bot. (submitted) 2. Marschner, H., 1995. Mineral Nutrition of Higher Plants. 2nd ed. Academic Press, London.

3. Ryan PR, Delhaize E., Jones, DL., 2001 Function and mechanism of organic anion exudation from plant roots. Annu. Rev. Plant Physiol. Plant Mol. Biol. 52, 527-560.

n° 17. Speciation of metals in plants: analytical challenges and strategies

D. Schaumlöffel, L. Ouerdane, S. Mounicou, J. Szpunar and R. Lobinski CNRS, UMR 5034, Group of Bio- inorganic Analytical Chemistry, Hélioparc, 64053 Pau, France

Challenge The understanding of the uptake, homeostasis, storage or detoxification of metals in plants is hampered by the lack of information on the molecular level concerning the identity and structures of metallospecies and bioligands involved. From the point of view of analytical chemistry, speciation analysis of metals in plant tissues is a difficult task due to the complexity of the matrix and low concentration of chemical species, many of which have not yet been identified. Analytical strategy Multidimensional hyphenated techniques based on the combination of separation methods with sensitive element- and molecule specific detectors are proposed for characterization of metal species in plant tissues [1]. Different modes of high performance liquid chromatography (HPLC) or capillary zone electrophoresis (CZE) are used for separation of the metallocompounds present in a sample. On-line coupling of these separation methods to inductively coupled plasma mass spectrometry (ICP MS) as one of the most sensitive instruments in trace element analysis allows to detect the presence of metal compounds in plant samples on very low concentration levels. The use of electrospray tandem mass spectrometry (ESI MS/MS) enables the structural elucidation of the metal species. Fig. 1 gives a schematical overview on the possible combinations of the different analytical methods to a multidimensional strategy.

Fig. 1: Coupling techniques for biochemical speciation Results and discussion The analytical approach discussed in the presentation allowed for successful identification of phytochelatins (PC) isoforms in rice (Oryza sativa) exposed to Cd stress and of nickel species in hyperaccumulating plant Sebertia accuminata. A water extract of the Oryza sativa roots, preconcentrated by lyophilization, was characterized by preparative reversed phase chromatography followed by CZEESI MS showing the presence of a number of PC isoforms. PCs eluted from CZE in the range of migration times between 20 and 25 min and were detected by ESI MS and MS/MS allowing the identification of the bioligands. The direct analysis by CZEESI MS/MS without any purification or derivatisation of the analytes enabled the detection of three PC families: standard PCs, iso-PCs (Ser) and desGly-PCs [2]. A water extract from the latex of Sebertia accuminata, a Ni-hyperaccumulator from New-Caledonia, was investigated by size-exclusion (SEC) and anion exchange chromatography (AIC) coupled to ICP MS. Purified and preconcentrated fractions from AIC and preparative SEC were analysed by ESI MS/MS. Preliminary results showed the occurrence of at least five Ni- species, two of which were identified as Nicitrate (the storage form of nickel in Sebertia accuminata) and Ni-nicotianamine, which is probably responsible for the nickel transport.

References 1. Lobinski R. and Szpunar J. 1999. Biochemical speciation analysis by hyphena ted techniques. Analytica Chimica Acta 400, 321-332. 2. Mounicou S., Vacchina V., Szpunar J., Potin- Gautier M. and Lobinski R. 2001. Determination of phytochelatins by capillary zone electrophoresis with electrospray tandem mass spectrometry detection (CZE-ES MS/MS). Analyst 126, 624-632.

n° 18. Zinc uptake by Buddleia davidii cultured in vitro. Effect on the growth and on the polyamine content analysed as stress markers.

P. Schnekenburger, G. Charles, A. Hourmant and M. Branchard Plant Biotechnology and Physiology, ISAMOR-UBO, Technopôle Brest-Iroise, 29280 PLOUZANE, France. Introduction In order to clean up town refuse tips, whose lixiviates may disseminate trace elements, the main plant species growing on such sites under "natural" selective pressure are currently analysed for their ability to accumulate heavy metals (Zn2+ particularly). Among seven candidate species, Buddleia davidii was retained for its large and fast development. Furthermore, its wide geographical representation indicates a good tolerance for various climates. In order to study and to optimise the uptake of Zn by B. davidii, it was first acclimatised in vitro. By this mean, the genotypical effects have been suppressed, and the clones were grown under controlled conditions. In preliminary studies, various concentrations of Zn were added to the culture medium to analyse their impact both on the fresh weight and on the Zn content of the different plant organs. The effect of this trace metal was also evaluated on the content in polyamines, known as stress markers. Materials and methods Young nodal explants of B. davidii, growing spontaneously on a polluted site, were decontaminated and propagated in test tubes on MS medium (Murashige and Skoog, 1962) supplemented with 20 g.L-1 sucrose and 8 g.L-1 agar. The plants, placed under a 16 h photoperiod at 40 µmol.m-2 .s-1 and 22 °C, were subcultured every 4 weeks. Zn (0 to 1000 µM) was added as sulphate salt and dissolved in MES buffer (1 mM, pH = 5.7) and 10 ml of this solution were added to the culture medium. The different organs (roots, stems, leaves) were analysed separately 4 weeks after subculture. The fresh and dry weights were measured. The Zn content was determined by ICP-ES after digestion of dried plant material in concentrated HNO3 /HClO 4 mixture (3/1, v/v). The polyamines (putrescine, acetylspermine, spermidine and spermine) were extracted and analysed by HPLC, according to Flores and Galston (1982) slightly modified (Le Guen - Le Saos and Hourmant, 2002). Results and discussion The nodal explants rooted normally, and axillary buds developed and produced 6 nodes every 4 weeks. Addition of MES alone to the culture medium increased the fresh weight of the different organs. Zn (100 µM) further increased the fresh weight whereas a Zn concentration higher than 500 µM inhibited the growth. Whatever the Zn concentration, the number of nodes remained unchanged, but the leaves showed signs of necrosis over a 750 µM Zn concentration. The supply of MES or Zn did not change the water percentage of the different organs. The plant Zn content increased with the Zn addition. The amounts of Zn were higher in roots than in stems and

leaves, which represent respectively 12.5 %, 16.3 % and 71.2 % of the plant fresh weight. When the culture medium was supplemented with 1000 µM Zn, roots, stems and leaves accumulated respectively 7.8, 6.9 and 4.9 mg Zn.g-1 DW. With increasing levels of Zn in the medium, a decrease in the putrescine and spermidine content was observed in the leaves. By contrast, the polyamine amounts increased in stems and roots, where Zn accumulated preferentially. This latter finding agrees with that of Choudhary and Singh (2000) using Cd. Our results are consistent with the idea that polyamines protect against heavy metal stress. Work is now in progress with another species, Brassica juncea, frequently used in phytoremediation programs, in order to establish comparisons with our candidate species, Buddleia davidii. References 1. Choudhary A. and R. P. Singh. 2000. Cadmium- induced changes in diamine oxidase activity and polyamine levels in Vigna radiata wilczek seedlings. J. Plant Physiol. 156, 704-710. 2. Flores H. E. and A. W. Galston. 1982. Analysis of polyamines in higher plants by high performance liquide chromatography. Plant Physiol. 69, 701-706. 3. Le Guen – Le Saos F. and A. Hourmant. 2002. Stimulation of putrescine biosynthesis via ornithine decarboxylase pathway by gibberellic acid in the in vitro rooting of globe artichoke (Cynara scolymus). Plant Growth Regul. (in press). 4. Murashige T. and F. Skoog. 1962. A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol. Plant. 15, 473-479.

n° 19. Why it may be critical to rely on Cattail for phytoremediation of organic contaminants

P. Schröder, B. Huber and C. Scheer GSF National Research Center for Environment and Health, Neuherberg, D-85758 Oberschleissheim Introduction Cattail (Typhaspec) is a freshwater monocot species with Europe wide distribution. They are found abundantly in creeks, along swampy river banks and in drainages of agricultural areas. In the latter case, Typha is found very effective in removing nitrogen and phosphorus from the drainage water. Due to the strengthening of the water directive of the European Community the need to improve and ma intain high quality standards for sewage treatment effluents during the next years is of importance. Plant-based treatment systems may offer an adequate supplement to existing technologies. In order to test Typha´s suitability for the removal and metabolism of organic xenobiotics especially from sewage water Thypha latifolia (L.) and Typha angustifolia (L.) were investigated for a) the general detoxification capacity of organic xenobiotics; and b) the fate and specific breakdown of two persistent substances: bis (2-ethylhexyl)-phthalate (DEHP) (a plasticizer) and Lamotrigine (an antiepilepticum). These are substances of high environmental concern. Results and discussion Preliminary results indicate that Typha plants possess peroxidase activity and glutathione S-transferase (GST) activity for the conjugation of several xenobiotic model substrates (i.e. CDNB, DCNB etc.) in leaves, rhizomes and roots. Total GST activity seems to be shared by several GST- isoforms. Whereas the activity of some Glutathione S-transferase remains unaffected following the application of xenobiotics in induction experiments, other are induces by both chemicals and medicaments. In studies with Typha roots and rhizomes, the removal DEHP and Lamotrigine from water was observed. This disappearance seems to be connected to the activity of GSTs or Glucosyl transferases. However, the amount of bound residue formation seems to be low. Analysis of Typha rhizome indicates that the amount of sclerenchymateous tissue and lignification in this plant is rather low compared to other species. It is assumed that detoxified xenobiotics will only be bound to hemicelluloses and pectin of the cell wall. Both substances are easily degradable by soil bacteria and fungi. The significance of this effect for the utilisation of Typha in the treatment of sewage polluted with organic xenobiotics is discussed.

Modelling trace element exposure and effects on plants

S. Sauvé Department of Chemistry, Université de Montréal, 2900 Édouard-Montpetit, Montréal, Québec, Canada, H3C 3J7. Introduction Modelling Chemical speciation drastically modulates the bioavailability of trace metals and needs to be incorporated into the assessment of soil quality. Analytical determinations of free metal ions in soil solutions are unlike routine analyses and are difficult to determine and validate. Regulatory agencies require some easier means of estimating the influence of chemical speciation upon bioavailability for integration into environmental guidelines. Some semi-empirical models to predict the soil solution speciation of divalent free metal can be combined with plant toxicity data to improve predictions of toxic effects. Because trace elements in soils have a very low solubility that does not conform with predictions using mineral solubility equilibrium, an alternative modelling approach is therefore required. A semi-empirical regression predicting soil solution free metal activities using total metal content and pH was derived by analogy to a competitive sorption of free metals and protons (McBride et al., 1997, Sauvé 2001). This model begins with the simple equilibrium sorption of a metal (Me) to a protonated surface (SurH): Me + SurH y ⇔ MeSur + y ⋅ H + . Equation 1 Transforming into an equilibrium constant and log-transforming yields: pMe 2+ = a + b ⋅ pH + c ⋅ log(Total Metal ) + d ⋅ log(Sur ) , Equation 2 Analysis of variance shows that often, the Sur term can be neglected, i.e., most of the variability is explained by pH and Total Metal, yielding: pMe 2+ = a + b ⋅ pH + c ⋅ log10 (Total Metal ) Equation 3 This form of the model (Equation 3) has been applied to the modelling of free Cu2+, Cd2+, Pb2+ and Zn2+. Materials and methods Soil Cha racteristics Contaminated soils were sampled from various origins (agricultural, urban, forest and industrial sites) and cover diverse contaminations sources (atmospheric emissions, smelting, pesticides, sewage sludge, industrial, etc.). Soil total metals were determined using digestion in concentrated nitric acid (i.e. total-recoverable metals). Soil solutions were obtained using 1:2 soil:solution extractions with 0.01 M dilute salts (CaCl2 for Cu and KNO3 for Cd, Pb, and Zn). Slurries were shaken 2 hours and centrifuged at 10000 g. The supernatants were filtered through 0.45-µm cellulosic membranes and analysed for total dissolved metals by GF-AAS and electrochemical speciation by ion selective electrode for Cu and anodic stripping voltammetry for Cd, Pb, and Zn and has been supplemented with resin-based speciation data from Knight et al. (1998). Methodological details for the different datasets are available from the original publications (see details in Sauvé 2001).

Results and discussion The dataset for the chemical speciation of Cd2+ in soil solution is illustrated in Figure 1. This dataset includes a series a spiked and field-collected soils and surprisingly, it shows very little difference between both datasets. Equation 3 has been applied to the different datasets and yields semi-empirical regressions of free metal activities based on simply soil pH and total metal content (Sauvé 2001).

pCd 2 + = 5. 14 + 0. 61⋅ pH − 0.79 ⋅ log10 (Total Cd ) R 2 = 0. 696, n = 64, p < 0. 001 Equation 4 2+ pCu = 3. 20 + 1. 47 ⋅ pH − 1.84 ⋅ log10 (Total Cu) R 2 = 0.921, n = 94, p < 0. 001 Equation 5 2+ pPb = 6. 78 + 0. 62 ⋅ pH − 0. 84 ⋅ log10 (Total Pb) R 2 = 0. 643, n = 84, p < 0.001 Equation 6 2+ pZn = 4.70 + 0. 95 ⋅ pH − 1. 71⋅ log10 (Total Zn) R 2 = 0.760, n = 30, p < 0.001 Equation 7 It is interesting to note that the coefficients for pH and Total Metal vary according to metals. Cu has the steepest slopes regarding both pH and total metal levels. On the other hand Cd2+ and Pb2+ seem to have the smoothest slope with regards to pH and total metal. The constants actually reflect the relative solubility of each metal, with Pb, which with no surprise is the least soluble one. Equations 4 to 7 highlight the importance of preserving some flexibility in how contamination is evaluated to better address the specificity of given environmental issues. References 1. McBride M.B., S. Sauvé, and W. Hendershot. 1997. Solubility control of Cu, Zn, Cd and Pb in contaminated soils. Europ. J. Soil Sci. 48, 337-346. 2. Sauvé, S. 2001. Chapter 2: Speciation of metals in soils, p. 7-58, In H. E. Allen, ed. Bioavailability of Metals in Terrestrial Ecosystems: Importance of Partitioning for Bioavailability to Invertebrates, Microbes and Plants. Society for Environmental Toxicology and Chemistry. Pensacola, FL, USA.

Tools to assess bioavailability: ex situ techniques and analytical challenges Daniël van der Lelie 1,2, Sally Brown 3 , Michel Mench 4 and Jaco Vangronsveld 5 1

Brookhaven National Laboratory (BNL), Biology Department, Building 463, Upton, NY 119735000, USA ([email protected]) 2 Vlaamse Instelling voor Technologisch Onderzoek (VITO), Environmental Technology, Boeretang 200, B-2400 Mol, Belgium 3 Ecosystem Sciences, University of Washington, Seattle, Washington 98195, USA 4 Agronomy Unit, INRA Bordeaux Aquitaine Research Center, BP 81, 33883 Villenave d’Ornon, France 5 Limburgs Universitair Centrum, Centre for Environmental Sciences, Universitaire Campus, B-3590 Diepenbeek, Belgium

Introduction A number of chemical analysis methods are currently used to determine the total metal concentrations in solid samples. However, such methods do not provide information about the biological available fraction of the detected pollutants in the samples, despite the fact that this parameter provides important information on the ecological impact of the pollution (both for heavy metals and organic xenobiotics). Since its provides a risk based classification and decision tool, the biological available fraction is of particular practical importance. In addition, it permits the evaluation of which point bioremediation targets can be achieved, and can be used to evaluate the efficiency and sustainability of immobilization based remediation methods. Therefore a need exists to develop and validate cost efficient and ecological relevant tests to determine the bioavailability and environmental impact of a pollutant. These tests can be enzymatic tests, tests based on specific organs, whole cells and organisms, or studies of complex ecosystems that include different trophic levels. With an increasing complexity of the test, the ecological significance of the test’s outcome might improve; however, the costs to perform the test will also increase. Therefore an optimum has to be defined between ecological relevance and the costs to perform the test. The challenge will be to design a test panel of complementary biological tests, this in addition to chemical analysis. Bacterial tests Bacterial tests have been developed to assess the impact of pollution. The pioneer system was the MicroTox test (Bulich and Isenberg, 1981), that is based on a constitutively light producing strain of Vibrio fischeri: in the presence of a toxic, bioavailable contaminant, the light production of this strain will decrease, allowing to determine the EC20 and EC50 toxicity values of the sample. This concept has been copied and improved by using more relevant bacterial strains, such as a genetically manipulated Pseudomonas strain, which is of more ecological relevance to study contaminated soil samples (Bundi et al, 2001). However, none of these bacterial biosensors is able to discriminate between different types of pollution. To overcome this limitation, bacterial biosensors were designed where the reporter system is placed under the control of a promoter-operator sequence whose transcription is specifically induced in the presence of an environmental insult. This concept can be applied for the specific detection of heavy metals, organic xenobiotics or any compound that

induces a specific form of genetic response. Bacterial biosensors, referred to as the BIOMET test, were developed that detect the presence of a specific heavy metal or a group of heavy metals (Corbisier et al, 1994 and 1996; van der Lelie et al, 2000). These sensors are based on gene fusions between heavy metal resistance operons e.g. of Ralstonia metallidurans CH34 and the luxCDABE operon of Vibrio fischeri. The presence of bioavailable heavy metals results in the specific and quantitative induction of light production. These sensors are presently available for the specific detection of bioavailable Zn and Cd, Cu, Pb, Ni, As and Cr(VI) (Corbisier et al, 1993 and 1999; Tibazarwa et al, 2000; van der Lelie et al, 2000) and have been successfully used to assess the bioavailability of heavy metals in different environmental samples including metal contaminated soils and river sediments. The BIOMET test shows a good correlation with the bioavailability data as predicted using sequential extraction (Tessier et al, 1979), phytotoxicity (Vangronsveld and Clijsters, 1992; Van Assche and Clijsters, 1990) and zootoxicity tests. Heavy metal bioavailability and microbial ecology Since heavy metal contamination might cause a selective pressure on the microbial community, addition of soil additives that results in a decrease in bioavailability of the heavy metals could have effects on the proportion of heavy metals resistant bacteria in the total bacterial population. If this should be the case, changes in the proportion of heavy metal resistant bacteria would reflect changes in bioavailability of heavy metals. This concept was recently demonstrated with soils from Leadville (USA) that received different treatments: changes in heavy metal bioavailability, as determined using the BIOMET test, resulted in a decrease in the bacterial community of the heavy metal resistant Ralstonia strain LV1. In situ monitoring using bacterial biosensors At present, few examples of on site monitoring are available. However, with the availability of new reporter systems, such as the green fluorescent protein (gfp), on site monitoring of the topsoil of large surfaces is theoretically feasible. This concept can be used to screen large surfaces for the presence of specific contaminants, or even mines. However, a general drawback of this technique is the necessity to deliberately release GMOs. References 1. Bulich, A. A.; Isenberg, D. L. ISA Trans. 1981, 20, 29-33. 2. Bundy, J. G.; Campbell, C. D.; Paton, G. I. J. Environ. Monit. 2001, 3, 404-410. 3. Corbisier, P.; Ji, G.; Nuyts, G.; Mergeay, M.; Silver, S. FEMS Microbiol. Lett. 1993, 110, 231-238. 4. Corbisier, P.; Thiry, E.; Masolijn, A.; Diels, L. In: Bioluminescence and Chemoluminescence: Fundamentals and Applied Aspects, Campbell A. K.; Cricka L. J.; Stanley P. E., Eds. John Wiley and Sons, Chichester, New York, Brisbane, Toronto, Singapore, 1994, 150-155. 5. Corbisier, P.; Thiry, E.; Diels. L. Environ Toxicol and Water Quality 1996, 11, 171-177. 6. Corbisier, P.; van der Lelie, D.; Borremans, B.; Provoost, A.; de Lorenzo, V.; Brown, L.B.; Lloyd, J. R.; Hobman, J.L.; Csöregi, E.; Johansson, G.; Mattiasson, B. Anal. Chimica Acta 1999, 387, 235-244. 7. Tessier, A; Campbell, P.G.C.; Bisson, M. Analytical Chemistry 1979, 51, 844-850. 8. Tibazarwa, C.; Wuertz, S.; Mergeay, M.; Wyns, L.; van der Lelie, D. J. Bacteriol. 2000, 182, 1339-1409.

9. Van Assche, F.; Clijsters, H. Environ. Pollut. 1990, 66, 157-172. 10. van der Lelie, D.; Verschaeve, L.; Regniers, L; Corbisier, P. In: New Microbiotests for routine toxicity screening and biomonitoring, Persoone, G.; Janssen, C.; De Coen. W., Eds. Kluwer Academic/Plenum Publishers, London, UK, 2000, 197-207. 10. Vangronsveld, J.; Clijsters, H. In: Metal compounds in environment and life, 4 (Interrelation between chemistry and biology); Merian, E.; Haerdi, W., Eds. Science Reviews Inc.: Wilmington, 1992, 117-125.

Modelling trace element exposure and effects on plants

S. Sauvé Department of Chemistry, Université de Montréal, 2900 Édouard-Montpetit, Montréal, Québec, Canada, H3C 3J7. Introduction Modelling Chemical speciation drastically modulates the bioavailability of trace metals and needs to be incorporated into the assessment of soil quality. Analytical determinations of free metal ions in soil solutions are unlike routine analyses and are difficult to determine and validate. Regulatory agencies require some easier means of estimating the influence of chemical speciation upon bioavailability for integration into environmental guidelines. Some semi-empirical models to predict the soil solution speciation of divalent free metal can be combined with plant toxicity data to improve predictions of toxic effects. Because trace elements in soils have a very low solubility that does not conform with predictions using mineral solubility equilibrium, an alternative modelling approach is therefore required. A semi-empirical regression predicting soil solution free metal activities using total metal content and pH was derived by analogy to a competitive sorption of free metals and protons (McBride et al., 1997, Sauvé 2001). This model begins with the simple equilibrium sorption of a metal (Me) to a protonated surface (SurH):

Me + SurH y ⇔ MeSur + y ⋅ H + .

Equation 1

Transforming into an equilibrium constant and log-transforming yields: pMe 2+ = a + b ⋅ pH + c ⋅ log(Total Metal ) + d ⋅ log(Sur ) ,

Equation 2

Analysis of variance shows that often, the Sur term can be neglected, i.e., most of the variability is explained by pH and Total Metal, yielding: pMe 2+ = a + b ⋅ pH + c ⋅ log10 (Total Metal )

Equation 3

This form of the model (Equation 3) has been applied to the modelling of free Cu2+, Cd2+, Pb2+ and Zn2+. Materials and methods Soil Characteristics Contaminated soils were sampled from various origins (agricultural, urban, forest and industrial sites) and cover diverse contaminations sources (atmospheric emissions, smelting, pesticides, sewage sludge, industrial, etc.). Soil total metals were determined using digestion in concentrated nitric acid (i.e. total-recoverable metals). Soil solutions were obtained using 1:2 soil:solution extractions with 0.01 M dilute

salts (CaCl2 for Cu and KNO3 for Cd, Pb, and Zn). Slurries were shaken 2 hours and centrifuged at 10000 g. The supernatants were filtered through 0.45-µm cellulosic membranes and analysed for total dissolved metals by GF-AAS and electrochemical speciation by ion selective electrode for Cu and anodic stripping voltammetry for Cd, Pb, and Zn and has been supplemented with resin-based speciation data from Knight et al. (1998). Methodological details for the different datasets are available from the original publications (see details in Sauvé 2001).

Results and discussion The dataset for the chemical speciation of Cd2+ in soil solution is illustrated in Figure 1. This dataset includes a series a spiked and field-collected soils and surprisingly, it shows very little difference between both datasets. Equation 3 has been applied to the different datasets and yields semi-empirical regressions of free metal activities based on simply soil pH and total metal content (Sauvé 2001).

pCd 2 + = 5. 14 + 0. 61⋅ pH − 0.79 ⋅ log10 (Total Cd ) R 2 = 0. 696, n = 64, p < 0. 001 Equation 4 pCu 2 + = 3. 20 + 1. 47 ⋅ pH − 1.84 ⋅ log10 (Total Cu) R 2 = 0.921, n = 94, p < 0. 001 Equation 5 pPb 2+ = 6. 78 + 0. 62 ⋅ pH − 0. 84 ⋅ log10 (Total Pb) R 2 = 0. 643, n = 84, p < 0.001 Equation 6 pZn 2 + = 4.70 + 0. 95 ⋅ pH − 1. 71⋅ log10 (Total Zn) R 2 = 0.760, n = 30, p < 0.001 Equation 7 It is interesting to note that the coefficients for pH and Total Metal vary according to metals. Cu has the steepest slopes regarding both pH and total metal leve ls. On the other hand Cd2+ and Pb2+ seem to have the smoothest slope with regards to pH and total metal. The constants actually reflect the relative solubility of each metal, with Pb, which with no surprise is the least soluble one. Equations 4 to 7 highlight the importance of preserving some flexibility in how contamination is evaluated to better address the specificity of given environmental issues. References 1. McBride M.B., S. Sauvé, and W. Hendershot. 1997. Solubility control of Cu, Zn, Cd and Pb in contaminated soils. Europ. J. Soil Sci. 48, 337-346. 2. Sauvé, S. 2001. Chapter 2: Speciation of metals in soils, p. 7-58, In H. E. Allen, ed. Bioavailability of Metals in Terrestrial Ecosystems: Importance of Partitioning

for Bioavailability to Invertebrates, Microbes and Plants. Society for Environmental Toxicology and Chemistry. Pensacola, FL, USA.

What tools for defining the PNEC values?

M. Mench ER BGETA, Unité d’Agronomie, Centre INRA Bordeaux-Aquitaine, 71 avenue E. Bourlaux, BP81, F-33883 Villenave d’Ornon, France. Introduction. Early warning before the pollution of sites, reducing the contaminant exposure at polluted sites, and (bio)monitoring the remediated sites are nowadays 3 major aspects of ecotoxicology and plant biotechnologies. Risk assessment and biomonitoring must allow preserving the ecosystem functions. Knowing pollution sources and their potential effects, especially on primary producers such as plant species, in view to limit susceptible rejects is a prerequisite. Exposure, which exceeds levels giving rise to deleterious effects recorded at either short or long term, must be avoided. The exposure assessment aims to predict the probable concentration in the environment accounting for the physico-chemical properties of the contaminant, its behaviour in the environment, its use, the amount released, etc. that will condition its distribution in the environment compartments. This concentration is so-called PEC (Predicted Environmental Concentration). To assess the relationship dose (concentration)-effect aims to determine the concentration under which the contaminant would not have adverse effects on the environmental component considered. This concentration is socalled PNEC (Predicted No Effect Concentration). A risk situation at one site is characterized when the ratio PEC vs. PNEC exceeds 1. What tools are available for defining the PNEC values, especially in the case of plant species? In the past, germination and growth parameters were used. Genotoxic effects were neglected. Literature shows that trace elements affect the plant functioning before one observes adverse effects on their germination and their growth. Information can be obtained at several sub-cellular levels, i.e. metabolites, proteins, gene transcripts. Does this info rmation end to decrease PNEC values? We give an example using biochemical biomarkers in maize exposed to a series of soils contaminated by incinerator fly ashes. Materials and methods. Plant chronic exposure was carried out using 800 g potted soils. The standard OCDE soil was used. Incinerator fly ashes were added into the soil at 21 levels increasing from 0 to 2%. Soils were rehydrated with distilled water and a modified Hoagland n°2 nutrient solution was added. Five seeds (Zea mays L. cv. Volga) were sowed. Plants were grown for 2 weeks with 16h/8h day/night, 180 µmol m-2 s-1 , 75% RH, 25/20°C. Soil moisture was maintained at 60% of the water holding capacity. Fresh and dry weight of shoots, 3rd and 4th leaves were determined. Density of chlorophylleous pigments was measured in 3rd and 4th leaves. Root and leaf samples were frozen in liquid nitrogen. Total soluble protein concentration, glutathion reductase (GR) and ascorbate peroxydase (APx) activities, total thiol concentration and phytochelatins were quantified (Lagriffoul, 1998; Mench et al, 2001). Plant tissues were wet digested in HNO3 and H2 O2 , and trace elements were quantified using axial ICP-AES. Results and discussion. Maize seeds have germinated in all soils. Shoot yield reached 3 g FW plant -1 in the range 0 – 0,19% addition rate, and then started to decrease. Similar effects were obtained for the 3rd and 4th leaf FW yields. Among trace elements, only Cd

concentration in the 3rd-leaf was related (r = 0,93) with the fly ash application rate. Above the 0.19% level, APx activity in the 3rd leaf increased with a dose-effect relationship demonstrating the development of an oxidative stress. Cystein, (GluCys), (Glu-Cys-Glu) concentrations increased in roots in relation with the soil contamination, especially by Cd (Fig. 1). These changes occurred before a decrease in growth parameters. Among other thiol compounds, increased concentrations in roots were found for (Glu-Cys)3 and PC3 , and in a lesser extent for (Glu-Cys)4 , PC2 and PC4 , in relation with the addition rate. Effective concentrations (EC 10 i) were calculated for each parameter, and converted into toxic units (UTi). The scale of potential ecotoxic effect (BEEP) was calculated using the formula BEEP = Log10 [1+ n(Σ i=1 to n UTi)/N], with the number of tests giving a dose-effect relationship (n) and the total number of tests (N) (Table 1).

Fig.1. (Glu-Cys) concentration in the maize roots. Fig.2. Hill relationship between BEEP values and exposure to fly ash. Table 1. Effective concentrations leading to a 10% decrease (EC10) (expressed in % fly ash addition rate per kg soil air-dried weight), toxic units (UT), and BEEP values (Log10 UT). EC10 UT BEEP -------------------------------------------------Glu-Cys 0.0996 1004 1.927 PC2 0.190 526 2.408 PC4 0.207 483 2.702 4th-leaf FW yield 0.219 457 2.916 Glu-Cys-Glu 0.231 433 3.082 (Glu-Cys)3 0.245 408 3.219 (Glu-Cys)4 0.226 375 3.332 Shoot FW yield 0.272 368 3.431 3rd-leaf FW yield 0.274 365 3.520 Cys 0.329 304 3.595 PC3 0.350 286 3.662 APx 0.400 250 3.720

Root (Glu-Cys) concentration gave the lowest EC 10 . Using data from biochemical biomarkers and growth parameters, the BEEP value showed a Hill relationship with the rate of fly ash addition into the soil (Fig. 2). Its EC 10 and EC 50 values were 0.015% and 0.08%, and corresponded to 1.3 mg and 11.9 mg Cd kg-1 in the maize 3rdleaf. The BEEP EC 10 value could be proposed as a provisional PNEC value. Screening of Cd-responsive genes in Arabidopsis thaliana suggests that oxidative stress and protein denaturation are important components of Cd toxicity (Suzuki et al, 2001). Changes in transcript populations may help to propose new PNEC values. References. Lagriffoul A, 1998 Biomarqueurs métaboliques de toxicité du cadmium chez le maïs (Zea mays L.). Mécanismes de tolérance, relation dose-effet et précocité de la réponse. Thèse Doctorat, Ecotoxicologie, Bordeaux 1, Unité d’Agronomie INRA Centre Bordeaux-Aquitaine.

Mench M., Ruttens A., Girardi S., Vangronsveld J., Corbisier P., Van der Lelie D., 2001. Evaluation de l’écotoxicité d’un sol et d’un déchet de référence par les tests BIOMET et PLANTOX. Rapport intermédiaire, convention Ademe – INRA n°A846, Villenave d’Ornon cedex. Suzuki N., Koizumi N., Sano H. 2001. Screening of cadmium-responsive genes in Arabidopsis thaliana. Plant, Cell, and Environment 24, 1177-1188.

Tools to assess bioavailability: ex situ techniques and analytical challenges D. van der Lelie 1,2, S. Brown 3 , M. Mench 4 and J. Vangronsveld 5 1

Brookhaven National Laboratory (BNL), Biology Department, Building 463, Upton, NY 11973-5000, USA ([email protected]) 2 Vlaamse Instelling voor Technologisch Onderzoek (VITO), Environmental Technology, Boeretang 200, B-2400 Mol, Belgium 3 Ecosystem Sciences, University of Washington, Seattle, Washington 98195, USA 4 Agronomy Unit, INRA Bordeaux Aquitaine Research Center, BP 81, 33883 Villenave d’Ornon, France 5 Limburgs Universitair Centrum, Centre for Environmental Sciences, Universitaire Campus, B-3590 Diepenbeek, Belgium

Introduction A number of chemical analysis methods are currently used to determine the total metal concentrations in solid samples. However, such methods do not provide information about the biological available fraction of the detected pollutants in the samples, despite the fact that this parameter provides important information on the ecological impact of the pollution (both for heavy metals and organic xenobiotics). Since its provides a risk based classification and decision tool, the biological available fraction is of particular practical importance. In addition, it permits the evaluation of which point bioremediation targets can be achieved, and can be used to evaluate the efficiency and sustainability of immobilization based remediation methods. Therefore a need exists to develop and validate cost efficient and ecological relevant tests to determine the bioavailability and environmental impact of a pollutant. These tests can be enzymatic tests, tests based on specific organs, whole cells and organisms, or studies of complex ecosystems that include different trophic levels. With an increasing complexity of the test, the ecological significance of the test’s outcome might improve; however, the costs to perform the test will also increase. Therefore an optimum has to be defined between ecological relevance and the costs to perform the test. The challenge will be to design a test panel of complementary biological tests, this in addition to chemical analysis. Bacterial tests Bacterial tests have been developed to assess the impact of pollution. The pioneer system was the MicroTox test (Bulich and Isenberg, 1981), that is based on a constitutively light producing strain of Vibrio fischeri: in the presence of a toxic, bioavailable contaminant, the light production of this strain will decrease, allowing to determine the EC20 and EC50 toxicity values of the sample. This concept has been copied and improved by using more relevant bacterial strains, such as a genetically manipulated Pseudomonas strain, which is of more ecological relevance to study contaminated soil samples (Bundi et al, 2001). However, none of these bacterial biosensors is able to discriminate between different types of pollution. To overcome this limitation, bacterial biosensors were designed where the reporter system is placed under the control of a promoter-operator sequence whose transcription is specifically induced in the presence of an environmental insult. This concept can be applied for

the specific detection of heavy metals, organic xenobiotics or any compound that induces a specific form of genetic response. Bacterial biosensors, referred to as the BIOMET test, were developed that detect the presence of a specific heavy metal or a group of heavy metals (Corbisier et al, 1994 and 1996; van der Lelie et al, 2000). These sensors are based on gene fusions between heavy metal resistance operons e.g. of Ralstonia metallidurans CH34 and the luxCDABE operon of Vibrio fischeri. The presence of bioavailable heavy metals results in the specific and quantitative induction of light production. These sensors are presently available for the specific detection of bioavailable Zn and Cd, Cu, Pb, Ni, As and Cr(VI) (Corbisier et al, 1993 and 1999; Tibazarwa et al, 2000; van der Lelie et al, 2000) and have been successfully used to assess the bioavailability of heavy metals in different environmental samples including metal contaminated soils and river sediments. The BIOMET test shows a good correlation with the bioavailability data as predicted using sequential extraction (Tessier et al, 1979), phytotoxicity (Vangronsveld and Clijsters, 1992; Van Assche and Clijsters, 1990) and zootoxicity tests. Heavy metal bioavailability and microbial ecology Since heavy metal contamination might cause a selective pressure on the microbial community, addition of soil additives that results in a decrease in bioavailability of the heavy metals could have effects on the proportion of heavy metals resistant bacteria in the total bacterial population. If this should be the case, changes in the proportion of heavy metal resistant bacteria would reflect changes in bioavailability of heavy metals. This concept was recently demonstrated with soils from Leadville (USA) that received different treatments: changes in heavy metal bioavailability, as determined using the BIOMET test, resulted in a decrease in the bacterial community of the heavy metal resistant Ralstonia strain LV1. In situ monitoring using bacterial biosensors At present, few examples of on site monitoring are available. However, with the availability of new reporter systems, such as the green fluorescent protein (gfp), on site monitoring of the topsoil of large surfaces is theoretically feasible. This concept can be used to screen large surfaces for the presence of specific contaminants, or even mines. However, a general drawback of this technique is the necessity to deliberately release GMOs. References 1. Bulich, A. A.; Isenberg, D. L. ISA Trans. 1981, 20, 29-33. 2. Bundy, J. G.; Campbell, C. D.; Paton, G. I. J. Environ. Monit. 2001, 3, 404-410. 3. Corbisier, P.; Ji, G.; Nuyts, G.; Mergeay, M.; Silver, S. FEMS Microbiol. Lett. 1993, 110, 231-238. 4. Corbisier, P.; Thiry, E.; Masolijn, A.; Diels, L. In: Bioluminescence and Chemoluminescence: Fundamentals and Applied Aspects, Campbell A. K.; Cricka L. J.; Stanley P. E., Eds. John Wiley and Sons, Chichester, New York, Brisbane, Toronto, Singapore, 1994, 150-155. 5. Corbisier, P.; Thiry, E.; Diels. L. Environ Toxicol and Water Quality 1996, 11, 171-177. 6. Corbisier, P.; van der Lelie, D.; Borremans, B.; Provoost, A.; de Lorenzo, V.; Brown, L.B.; Lloyd, J. R.; Hobman, J.L.; Csöregi, E.; Johansson, G.; Mattiasson, B. Anal. Chimica Acta 1999, 387, 235-244. 7. Tessier, A; Campbell, P.G.C.; Bisson, M. Analytical Chemistry 1979, 51, 844-850.

8. Tibazarwa, C.; Wuertz, S.; Mergeay, M.; Wyns, L.; van der Lelie, D. J. Bacteriol. 2000, 182, 1339-1409. 9. Van Assche, F.; Clijsters, H. Environ. Pollut. 1990, 66, 157-172. 10. van der Lelie, D.; Verschaeve, L.; Regniers, L; Corbisier, P. In: New Microbiotests for routine toxicity screening and biomonitoring, Persoone, G.; Janssen, C.; De Coen. W., Eds. Kluwer Academic/Plenum Publishers, London, UK, 2000, 197-207. 11. Vangronsveld, J.; Clijsters, H. In: Metal compounds in environment and life, 4 (Interrelation between chemistry and biology); Merian, E.; Haerdi, W., Eds. Science Reviews Inc.: Wilmington, 1992, 117-125.

The Application of Short Rotation Coppice for Clean-up of Radionuclide Contaminated Sites M. Dutton.

THE BORDEAUX MIXTURE.

Jacques DELAS Directeur de Recherche honoraire INRA, Centre INRA Bordeaux-Aquitaine, F-33883 Villenave d’Ornon cedex, France. In the second half of the 19th Century, as maritime trades developed with the New World, several American parasites of the grapevine were successively introduced into the French vineyard where they caused considerable damage and threatened the future of viticulture. The most important of these were the fungal diseases powdery mildew (oïdium),downy mildew (mildiou), and black-rot, together with the insect parasite, Phylloxera. Efforts deployed to fight against these pests and plant diseases resulted in solutions still used today: sulphur against powdery mildew, grafting on rootstock- tolerant plants in the case of the phylloxera and the use of Bordeaux mixture against downy mildew. I plan to summarise the history of Bordeaux mixture. Downy mildew (mildiou), known in the USA as either grapevine mildew or wine pest, was first reported in 1878 near Bordeaux. Over several years, it spread across France many other parts of Europe. The causal agent is a fungus, Plasmopara viticola. Mildew causes considerable damage to both leaves and grapes. The early leaf senescence and defoliation that it provokes translates, in case of severe attack, into both a significant reduction in yield and an equally important decrease of both grape and wine quality. One spoke, at the time of the first attacks, about “mildiousés” wines. A. MILLARDET, Professor of Botany at the Science University of Bordeaux, was famous at this time due to his researches on phylloxera. He is also renowned as the discoverer of Bordeaux mixture. In late October 1882, when the whole Médoc region was strongly hit by the mildew, he observes " that all along the road, the grapevines have leaves while everywhere elsewhere they had been fallen for a long time " (due to the effect of the mildew attacks). These leaves present in end of the rows were covered "on the superior face with a slim layer, which sticks, a pulverulent substance of a bluish white". He learnt from E. DAVID, manager of the BEAUCAILLOU castle (vineyard) at SAINT JULIEN, that it is custom, in the Médoc, when grapes start to mature, to cover leaves with either green -grey (alkaline copper acetate) or copper sulphate mixed with lime. This " médocain mix" aimed to deter would-be grape thieves by making them fear poisoning by the blue vitriol. It was spread on the vine stocks the closest to roads with the help of heather brooms. MILLARDET was convinced by these observations that copper salts could ward off mildew attack on leaves by preventing the germination of spores (the antifungal capacity of copper, signalled in the preceding century, had been used against wheat smut). Therefore he decided to experiment with various formulations based on copper salts. He found that only those similar to the "médocain mix" appeared fully effective. He had also to convince detractors and wine growers of the efficacy of this fungicide treatment. On the 1st April 1885, he gave a lecture at the Gironde Agriculture Society where he presented the synthesis of his observations made with DAVID. He reported on the Bordeaux mixture formula, as well as its application method and 3 suitable periods for preventive treatments. The most efficient mixture in his tests was a 5.3% copper sulphate solution neutralized by addition of quick lime. Further work demonstrated the efficiency of lower concentrations of copper sulphate and that

it was not necessary to limit applications to 3 each year, but to adapt their number to prevailing climatic conditions. Another professor of the Bordeaux Science Faculty, U. GAYON, a disciple of PASTEUR, played an important role with MILLARDET in the formulation of Bordeaux mixture. He made two other important discoveries. Firstly, he demonstrated the reaction mechanism of copper in the Bordeaux mixture. No-one understood that copper, precipitated by lime, could have an action on the fungus. GAYON showed that the insoluble deposit formed by the mixture deposited on leaves transformed slowly, under the action of rainwater and dew which both contained dissolved carbon dioxide, into soluble copper hydroxide, releasing copper in an ionic form. Secondly, GAYON demonstrated that copper deposited onto the surface of grapes was precipitated during the fermentation and was not found in the wine. This replied to objections of some detractors of the Bordeaux mixture who put forward risks of human toxicity due to copper being concentrated in the wine. From 1885, birth date of the Bordeaux mixture, until the 1960’s when the first organic fungicides appeared, a period of 75 years, copper was the only treatment able to efficiently combat mildew. Due to its efficacy, it was used in all French vineyards with a frequency depending essentially on climatic conditions. For many years, Bordeaux mixture with a 2 % CuSO 4 , 5H2 O level was applied at a rate of 1 000 liters / ha. Therefore, each treatment results in an input of 5 kg Cu / ha and the number of applications ranged from 5 to 15 each year. As a result, one can estimate that the total amount of copper applied to French vineyard soils during this 75 year period represents between 200 to 2 000 mg Cu/kg of soil. Research at the end of 19th Century and the beginning of 20th Century focussed on the consequences that Cu from Bordeaux mixture could have on higher plants, independently of its toxicity to fungi. Experiments carried out at this time concluded there was an absence of Cu phytotoxicity at the levels used in viticulture. In fact, these reassuring results had been obtained with calcareous soils. One learns only 50 years later that Cu phytotoxicity occurred mainly in acidic soils! The first consequences of Cu accumulation in vineyard soils became apparent after 1950. The catastrophic frost of 1956 resulted in the death of many grapevines across France. This was followed by numerous replants. However, many of these displayed very serious damage, with either the death of young grapevines or annual crop plants. These were reported in many vineyard areas. Researches undertaken as a result of this emergency period took several years to find the cause of phytotoxicity and to develop remediation methods that have since been widely adopted in wine growing practice. These results can be briefly summarized: -

Phytotoxicity observed after the removal of old grapevine stocks and subsequent replanting is due to Cu accumulation in the soil as a result of the cumulative antifungic treatments. Cu toxicity does not lead to specific symptoms on the leaves but primarily reduces growth of the root system where the metal accumulates. Copper transfer coefficients from roots to aerial plant parts are low. Phytotoxicity is only evident in acidic soil (the acidity of pedogeochimic origin being worsened in some situations by the biological oxidation of the sulphur applied to combat powdery mildew of the grapevine); The more acid the soil, the more serious the Cu toxicity; occurrence of phytotoxicity is enhanced by a low CEC. The copper accumulated in the upper soil horizons and does not readily leach down the soil profile: this explains why Cu toxicity has generally never been demonstrated on grapevines where the root system is localised below the zone of Cu accumulation;

-

on the other hand, phytotoxicity occurs when a grapevine plantlet or an annual plant seedling is cultivated in the superficial accumulation zone; Liming (up to pH 6.5) before replanting (or the sowing) eliminated the risk of phytotoxicity. The use of organic amendments, which enhance the formation of organometallic Cu complexes, is an additional remediation treatment.

The increased production of either organometallic or organic fungicides in the 1960’s at the time where one discovered the toxicity of the copper accumulated in vineyard soils should have resulted in a total absence of Cu-fungicides in viticulture . This has not taken place. Whilst it is true to say that total Cu inputs have decreased, they are far from being null (the development of new delivery systems allows reduced doses for the same efficiency). The wine producers that still use copper for some of their treatments justify their choice by the fact that, contrary to organic fungicides, it does not induce resistance phenomena, or that it would be more efficient for late treatments during the season. Furthermore, copper is authorized in biological agriculture where it is used commonly. The absence of a substitute product for this type of agriculture would pose serious problems if the use of copper came to be forbidden. Bordeaux mixture has allowed French viticulture to overcome the very serious crisis provoked by the mildew invasion, and Plant pathology to become a fully-fledged scientific discipline. Well over 100 years after its development, it is still one of the best-investigated topics on consequences of trace element accumulation in agricultural soils where the inputs, speciation and consequences of soil Cu contamination are all clearly defined.

References DELAS J., 1963. La toxicité du cuivre accumulé dans les sols. Agrochimica, 7 ; 258-288. LAFON R., 1985. Le Médoc et la découverte de la bouillie bordelaise. MEDOC, Bulletin d’information du G.I.E. des vins du Médoc, 5-33. VIENNOT-BOURGIN G., LAFON R., 1985. La naissance de la Bouillie Bordelaise. Fungicides for Crop Protection. 100 years of progress. Monograph N° 31. Acknowledgements. Author is grateful to Dr. N. Lepp and Dr. M. Mench for helping in text editing.

Poster session 2 . n° 12. Characterisation of mercury-thiol complexes in maize root

L. E. Hernández*, L. A. Arroyo-Méndez*, S. Vázquez, F. F. del Campo*, R. O. Carpena-Ruiz *Laboratorio de Fisiología Vegetal, Departamento de Biología and Departamento de Química Agrícola, Universidad Autónoma de Madrid, Camp us de Cantoblanco, E28049 Madrid, E- mail: [email protected] Introduction. Heavy metals accumulation in the soil can lead to severe toxicity symptoms and inhibition of plant growth. Tolerant plants have developed several mechanisms to restrict the biological activity of the free toxic metal in the plant cell. One of them is their association to cell wall components, that decreases metal accessibility to the protoplast. Once the metal enters the cell, one of the proposed tolerance mechanisms is the chelation of metals to the thiol-containing peptides phytochelatins. This is supported by biochemical and genetical evidences showing that plants unable to synthesise phytochelatins were more sensitive to several heavy metals. Phytochelatins are non-ribosomally synthesised by phytochelatin synthase after activation of the enzyme in the presence of the metal from the ubiquitous peptide glutathione. Results and discussion. Mercury (Hg) is one of the most toxic pollutant heavy metals to plants. In Spain there are several areas contaminated with this metal, of which the Almaden mining facility is the most relevant. Previous field work in this area (1) showed important differences in the tolerance strategies of graminaceous and leguminous plants. Recently (2) we have studied some Hg-stress biomarkers in maize and pea plants, indicating that maize is less tolerant to Hg. Now we are studying the mechanisms of plant tolerance to Hg in maize grown hydroponically and exposed for 7 days to 30 µM Hg. Firstly, we have characterised the subcellular fractions of root and shoot where Hg accumulated. To analyse Hg accumulated, plant tissues were homogenised in a suitable buffer and the homogenate filtrated through nylon cloth. The residue consisted mainly of plant cell walls, and the filtrate was differentially centrifuged. The particulate and plant cell wall material were pooled together and considered as the insoluble fraction, and the supernatant recovered after centrifugation as the soluble fraction. The analysis of Hg accumulation in root fractions revealed that up to 20 % was associated to the soluble fraction. On the other hand, analysis of non-protein thiol-peptides indicated a significant increase as compared to the control (approx. from 250 to 1050 nmol/g FW). Furthermore, analysis of gluthatione revealed a modest increase in plants treated with Hg, indicating that other thiol-peptides might had been accumulated in response to the metal. To determine the possible association of Hg to thiol-peptides in the soluble fraction, this fraction was separated chromatographically by FPLC-DEAE. A peak of thiol-peptide co-eluted with Hg, from which we could recover up to 70% of the metal contained in the soluble fraction

sample loaded onto the DEAE column. These results suggest that most of the Hg found in the soluble fraction was associated with thiol-peptides. We are currently characterising the peptides found in the FPLC-DEAE fractions. Tentative identification by HPLC, after derivatisation with Ellman’s reagent, indicate that the thiol-peptides resemble some of those accumulated in maize after exposure to cadmium. References. 1. Lucena, J.J., Hernandez, L.E., Olmos, S., Carpena Ruiz, R. 1993. Micronutrient content in graminaceous and leguminous plants contaminated with mercury. In: Optimization of Plant Nutrition (M.A.C. Fragoso and M.L. Beusichem, eds.) pp.531-37. Kluwer Acad.Publs. Dordrecht. 2. S. Vázquez, L.E. Hernández, R.O. Carpena-Ruiz. 2001. Determinación de compuestos tiólicos como indicadores de contaminación de Hg en plantas de maíz y guisante. In: Nutrición mineral en una agricultura mediterránea sostenible (C.F. Alcaraz; M. Carvajal; V. Martínez eds.) pp. 357-363. CEBAS-CSIC, Consejería Agricultura, Agua, Medio Ambiente. Murcia, Spain

n° 13. Relationship between plant structure, physiological processes, uptake and accumulation of Cd

A. Lux, A. Šottníková, L. Lunácková, E. Masarovicová, D. Lišková1 , K. Králová, V. Streško, P. Capek1 Department of Plant Physiology, Institute of Chemistry and Geological Institute, Faculty of Natural Sciences, Comenius University, Mlynská dolina B2, SK-84215 Bratislava, Slovakia; 1 Institute of Chemistry, Slovak Academy of Sciences, Dúbravská cesta 9, SK-84238 Bratislava, Slovakia.

Introduction Cadmium is a rare element with important toxicological properties already in relatively low concentrations. The toxicity is related predominately to its similarity with zinc, which is an essential metal for living organisms. Unlike Zn, cadmium has no essential function in any living organism (Wagner, 1995), however, it can be transported by identical mechanisms as Zn and can substitute it in many organic compounds (Kalac and Tríska, 1998). For phytoremediation of soils contaminated by Cd several herbaceous species could be used due to their high accumulation abilities. On the other hand the deposition of metals in the trees is lower, but it can be advantageous because of the long- life span, deeply penetrating roots and favourable association with microorganisms. Relatively high sensitivity of trees to toxic metals together with the low extraction ability stimulate experiments aimed at physiology and structure of these species under influence of toxic metals as well as screening and selection of tolerant and hyperaccumulating species and clones. Materials and methods Various species and clones of the genus willow (Salix), and poplar (Populus) were used for experiments. Plants were cultivated in hydroponics and in vitro. Cd was applied in the form of Cd(NO3 )2 and CdCl2 , in concentrations 10-5 to 10-3 M. Growth characteristics of stem cuttings growing in hydroponics (number and cumulative length of roots and shoots, leaf area, specific leaf mass and biomass production of individual organs) were studied. Growth of organ cultures in vitro with and without Cd was evaluated. Accumulation of Cd in individual plant organs in vivo, in vitro and in callus cultures was determined by AAS Assimilation pigments (chlorophylls and carotenoids) were determined according to Lichtenthaler (1987). Structural analysis of roots was focused on the development of endodermis and exodermis and their relation with the development of vascular system. Methods of light, transmission electron microscopy and fluorescence microscopy (Brundrett et al. 1988) were used. For chemical analyses of plant cell walls gas and paper chromatography, mass spectrometry and NMR were used. Results and discussion

Studied willows and poplars belong to the fast growing species. Stem cuttings develop rapidly adventitious root from the existing root primordia and simultaneously new shoots are formed from the axial buds. The growth of these newly formed organs is influenced by the presence of Cd in cultivation media, the roots react most sensitively. The structure of adventitious roots in both genera is characterized by broad cortical layer with extensive intercellular spaces. Endodermis is formed close to the root apex in the distance of approximately 3 mm. Interspecific differences were found in formation of Casparian bands, in some cases preceding formation of vascular system. After Cd treatment Casparian bands were formed cons iderably closer to the apex. Exodemis with Casparian bands and suberin lamellae was also formed in all studied species, in variable distances from the root apex. The highest amounts of Cd were accumulated in roots of all species in hydroponics. However, significant interspecific differences of Cd content were found in stem cuttings and in newly formed shoots. In vitro cultures of several willow and poplar species were derived from the stem cuttings. The sensitivity to Cd treatment was documented also on rooted and non-rooted shoot cultures in vitro. The results confirmed the importance of the root in metal accumulation. The long-term poplar callus culture showed high tolerance to Cd. In this connection the effect of cadmium on callus cell walls was studied microscopically and by chemical analyses. Assimilation pigment concentration is considered to be decreased as a result of negative Cd effect (Barua and Jana 1986, Kummerová and Brandejsová 1994). The found results of assimilation pigment concentration showed chl a as the main target of Cd application. The values of chl a concentration significantly decreased in S. viminalis, S. alba and P. gigant. However, in P. robusta, S. purpurea and S. cinerea cadmium did not negatively affect concentration of this pigment. There were also no significant differences in determined values of carotenoids and chl b concentration. Acknowledgement The research was supported by COST Action 837 and Slovak Grant Agency VEGA grant No. 1/7258/20 References 8. Barua, B. and S. Jana. 1986. Effects of heavy metals on dark induced changes in Hill reaction activity, chlorophyll and protein contents, dry matter and tissue permeability in detached Spinacia oleracea L. leaves. Photosynthetica 20, 74-76. 9. Brundrett, M.C., D.E. Enstone and C.A. Peterson. 1988. A berberine - aniline blue fluorescent staining procedure for suberin, lignin and callose in plant tissue. Protoplasma 146, 133-142. 10. Kalac P. and J. Tríska. 1998. Chemistry of the Environment. University of South Bohemia, Ceské Budejovice, Czech Republic, p. 44. – in Czech 11. Kummerová, M.and R. Brandejsová. 1994. Project TOCOEN. The fate of selected pollutants in the environment. Part XIX. The phytotoxicity of organic and inorganic pollutants-cadmium. The effect of cadmium on the growth of germinating maize plants. Toxicol. Environ. Chem. 42, 115-122. 12.Lichtenthaler H.K. 1987. Chloroplylls and carotenoids: photosynthetic biomembranes. Methods Enzymol. 148, 350-382.

Pigments

of

13.Wagner G.J. 1995. Biochemical studies of heavy metal transport in plants. pp. 2122. In Current Topics in Plant Biochemistry, Physiology and Molecular Biology,

Randall D., Raskin I., Baker A., Blevis D., Smith R., eds. Univ. of Missouri, Columbia, Missouri.

n° 14. Cadmium and Pb toxicity in sugar beet (Beta vulgaris L.)

F. Morales1 , A. Larbi1 , A. Álvarez-Fernández1 , A.F. López-Millán1 , N. Molías1 , Y. Gogorcena 1 , J.J. Lucena 2 , A. Abadía1 and J. Abadía1 1

Departamento de Nutrición Vegetal, Estación Experimental de Aula Dei-CSIC, Apdo. 202, E-50080 Zaragoza, Spain. 2 Departamento de Química Agrícola, Geología y Geoquímica, Universidad Autónoma de Madrid, E-28049 Madrid, Spain.

Introduction Heavy metals constitute a heterogeneous group of elements, including both essential and non-essential ones. Essential elements are some of those known as micronutrients: Mn, Fe, Cu, Zn and Mo. Non-essential heavy metals, such as Pb, Cd and Al, can be phytotoxic even when present at relatively low concentrations. Cadmium and Pb are the most abundant non-essential heavy metals polluting the environment. Natural and anthropogenic sources of major heavy metals in soils have been reviewed recently (Baligar et al. 1998). Materials and methods Sugar beet plants were grown in growth chamber (Morales et al. 1990). Six-week old plants were transplanted to buckets containing half-Hoagland nutrient solution with 10 or 50 µM CdCl2 , 10 or 50 µM Cd-EDTA, or 2 mM Pb-EDTA. The global chemical speciation was estimated by using the MINTEQA2 software (Allison et al. 1991). The pH of the nutrient solutions was measured every 2-3 days and adjusted to 5.5 with diluted HCl. Iron reductase activity was measured in excised root tips following procedures described in Gogorcena et al. (2001). Photosynthetic pigments were quantified by HPLC as described in Gogorcena et al. (2001). Gas exchange measurements were made with a portable system (CIRAS-1, PP Systems, Herts, U.K.). Continuous Chl fluorescence measurements were made with the experimental set-up (Morales et al. 1990) and protocol (Belkhodja et al. 1998) described elsewhere. Modulated Chl fluorescence measurements were made as described in Belkhodja et al. (1998). Samples for heavy metal and nutrient analyses were analysed using standard procedures (Abadía et al. 1985; Lozano-Rodríguez et al. 1995). Results and discussion Cadmium and Pb toxicity have been investigated in sugar beet grown in hydroponics under growth chamber-controlled conditions. Chemical speciation was used to investigate the chemical species in equilibrium. Cadmium decreased root and shoot fresh and dry weight, and increased root/shoot ratio. Cadmium- treated plants developed few brownish roots with short laterals. Plants treated with Cd developed chlorosis. In leaves, Cd decreased N, P, Mg, K, Mn, Cu and Zn concentrations but decreased Ca concentrations. All Cd treatments increased Fe(III)-chelate reductase activities in root tips, although Fe concentrations in shoots were similar to those of controls. The changes in photosynthetic parameters observed with Cd in sugar beet

resembled those observed in Fe-deficient sugar beet plants (Morales et al. 1990, 1998; Belkhodja et al., 1998). These included increases of the lutein/Chl and VAZ pigments/Chl molar ratios, changes in the dark-adapted Chl fluorescence induction curve -reversible by far-red pre- illumination- and changes in the Chl fluorescence quenching parameters. All these data suggest the existence of a Cd-induced Fe chlorosis, resembling to that occurring in leaves from field-grown, Fe-chlorotic trees (Abadía et al. 1985). Lead chelated with EDTA increased root fresh and dry weight with no changes in the shoot mass, therefore increasing the root/shoot ratio. Changes in nutrient concentrations were much less marked with Pb than those found with Cd. It should be mentioned, however, that shoot Cu levels were close to deficiency critical levels. Leaves of Pb-treated plants remained green or showed a slight leaf pale green colour. In some cases, Pb-treated leaves rolled their edges inwards. Root tips from Pbtreated plants also had increased Fe(III)-chelate reductase activity. Lead had much less effects than Cd on all photosynthetic parameters measured. Photosynthesis and gas exchange measurements were similar in control and Pb-treated plants. The actual photosystem II efficiency was only slightly affected by Pb, resulting from both slight decreases in intrinsic photosystem II efficiency and slight decreases in photochemical quenching. The only photosynthetic parameter affected by Pb was non-photochemical quenching, that increased up to 2.6-fold in response to Pb. References 1. Abadía J., J.N. Nishio, E. Monge, L. Montañés, and L. Heras. 1985. Mineral composition of peach tree leaves affected by iron chlorosis. J. Plant Nutr. 8, 697708 2. Allison J.D., D.S. Brown, and K.J. Novo-Gradak. 1991. A geochemical assessment model for environmental systems v. 3.0. Washintong DC, Environ. Res. Lab., US Environ. Protection Agency 3. Baligar V.C., N.K. Fageria, and M.A. Elrashidi. 1998. Toxicity and nutrient constraints on root growth. HortSci. 33, 960-965. 4. Belkhodja R., F. Morales, R. Quílez, A.F. López-Millán, A. Abadía, and J. Abadía. 1998. Iron deficiency causes changes in chlorophyll fluorescence due to the reduction in the dark of the photosystem II acceptor side. Photosynth. Res. 56, 265-276. 5. Gogorcena Y., N. Molías, A. Larbi, J. Abadía, and A. Abadía. 2001. Characterization of the responses of cork oak (Quercus suber) to iron deficiency. Tree Physiol. 21, 1335-1340. 6. Lozano-Rodríguez E, M. Luguera, J.J. Lucena, and R.O. Carpena-Ruiz. 1995. Evaluation of two different acid digestion methods in closed systems for trace elements determinations in plants. Química Analítica 14, 27-30. 7. Morales F., A. Abadía, and J. Abadía. 1990. Characterization of the xanthophyll cycle and other photosynthetic pigment changes induced by iron deficiency in sugar beet (Beta vulgaris L.). Plant Physiol. 94, 607-613.

8. Morales F., A. Abadía, and J. Abadía. 1998. Photosynthesis, quenching of chlorophyll fluorescence and thermal energy dissipation in iron-deficient sugar beet leaves. Aust. J. Plant Physiol. 25, 403-412.

n°15. Evaluation of the potential biotoxicity / essentiality of Zinc and Cadmium in suspended cells of Cynara cardunculus and Centaurea calcitrapa

M. A. G. Oliveira, S. Raposo and M. E. Lima-Costa Faculty of Engineering of Natural Resources, University of Algarve, Campus de Gambelas, 8000-117 Faro, Portugal

Introduction From general biological, as well from plant physiological point of view, essential and non-essential heavy metals can be distinguished (Clijsters et al., 1999). There are three criteria for establishing whether or not a trace element is essential for the normal growth of plants: (i) the organism can neither grow nor complete its life cycle without it; (ii) the element cannot be wholly replaced by any other element; (iii) the element has a direct influence on the organism and is involved in its metabolism (Mas and Azcue, 1993). Heavy metal such as Cu2+, Zn2+, Mn2+, Fe2+, Ni2+ and Co2+ are essential micronutrients for plant metabolism, but when present in excess, these, and nonessential metals, such as, Cd2+, Hg2+, Ag2+ and Pb2+, can become extremely toxic (Williams et al., 2000). In this work we aimed to study the effect of increasing amounts, of an essential metal, Zn (kno wn enzyme activator), and a non-essential metal, Cd (normally considered toxic), on the growth of suspended cells of Cynara cardunculus and Centaurea calcitrapa. Materials and methods C. cardunculus and C. calcitrapa cells suspension culture were grown, respectively, on B5 (Gamborg et al., 1968) and SH (Schenk and Hildbrandt, 1972) nutrient medium. The Zn assays (with ZnSO4 ,7H2 O) were performed by addition of 20 and 200 mg/L Zn salt (CY1), and 200 and 600 mg/L Zn salt (CC1) respectively for C. cardunculus and C. calcitrapa suspended cells. The Cadmium assays (with CdSO4 ,8/3H2 O) were performed by addition of 1, 20 and 50 mg/L Cd salt (CC2), and 1, 10 and 20 mg/L Cd salt (CY2), respectively for C. calcitrapa and C. cardunculus suspended cells. In both Zn and Cd assays we performed, as a control, an assay with 2 (in CY1) and 1 mg/L Zn salt (in CC1), and an assay without Cd for both CY2 and CC2. The suspended cells were kept in Erlenmeyer flasks (500 mL), at a temperature of 25ºC with constant agitation (120 rpm). Fresh weight (FW) and dry weight (DW) were determined. Soluble proteins were determinated according to Bradford (1976) and phenol content according to Anselmo et al. (1985). Experiments were carried out in triplicates for each concentration. Results and discussion The specific growth rate, µg, obtained in CY1 cultures became smaller when the Zn salt concentration, in the culture medium, increased. The profile of biomass accumulation in both assays with excess Zn has also shown growth inhibition. The presence of 20 mg/L Zn salt yielded the best result in terms of soluble protein in CY1.

With 200 mg/L Zn salt the production of phenols was triggered sooner, in comparison to the other concentration assays, suggesting that a stress metabolism has been induced in C. cardunculus cells in the presence of this amount of Zn. With CC1 cultures, it was obtained an increase in terms of µg, with 200 mg/L Zn salt, being the best result in those conditions. It was obtained a similar µ g in the presence of 1 and 600 mg/L Zn salt, but the biomass accumulation and soluble protein content showed the highest values in the presence of 600 mg/L Zn salt. The phenols production was higher for the assay with 200 mg/L Zn salt. In both CY2 and CC2 cultures, it was obtained growth inhibition for the assays performed in the presence of Cd higher than 1mg/L Cd salt. However, with 1 mg/L Cd salt, both cell suspension cultures manifest different behaviours. In CC2 it was obtained a higher µg value and higher soluble protein increase, in comparison to the value obtained in the control assay, but with CY2 this result was not confirmed. The biomass accumulation was severely affected, either CC2 or CY2 growth conditions cultures. The profile of phenolic compounds production, in CC2, usually associated to plant stress metabolism, in the presence of 1 mg/L Cd salt, suggested that there was no additional stress in these cells, in comparison to the control culture. An opposite result was obtained with CY2 cell culture. The heavy metal Cd cannot be considered an essential element because it inhibits the growth of both suspended cells culture, in a higher proportion for C. cardunculus cells. The low biotoxicity of Zinc element was clearly demonstrated, except for higher Zn concentration, as referred by Williams et al., 2000. Another conclusion was that C. cardunculus suspended cells are more sensitive to the excess of Zn and to the presence of Cd, in the culture medium, than C. calcitrapa cell suspension. References 1. Anselmo A. M., J. M. S. Cabral, J. M. Novais. 1985. Degradation of phenol by immobilized cells of Fusarium flociferum. Biotech. Lett. 7 (12): 889-894 2. Bradford, M. M. 1976. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the princip le of protein dye binding. Anal. Biotech. 72: 248-254. 3. Clijsters H., A. Cuypers , J. Vangronsveld. 1999. Physiological responses to heavy metals in higher plants; defence against oxidative stress. Z. Naturforsch. 54c, 730-734 4 Gamborg O. L., A. Miller, K. Ojima. 1968. Nutrient requirements of suspension cultures of soybean root cells. Exp. Cell Res. 50: 151-154 5. Mas A., J. M. Azcue. 1993. Metals en Sistemas Biológicos. 1e d. Promociones y Publicationes Universitarias, S.A. 6. Skenk, R., A. Hildbrandt. 1972. Medium and techniques of induction and growth of monocotyledonous and dycotyledonous plant cell cultures. Can. J. Bot. 50: 199-204. 14. Williams L. E., J. J. Pittman, J. L. Hall. 2000. Emerging mechanisms for heavy metal transport in plants. Biochim. Biophys. Acta 1465, 104-126.

n° 16. Role of root exudates in metal tolerance: lessons from aluminium research

Ch. Poschenrieder and J. Barceló Laboratorio de Fisiología Vegetal, Facultad de Ciencias, Universidad Autónoma de Barcelona, E-08193 Bellaterra, Spain. E- mail: [email protected]

Introduction The importance of root exudates containing organic acids, phenolic substances, or phytosiderophores in the plant's capacity to acquire essential nutrients from sparingly soluble forms in soils is well established. Segregation of citrate from cluster roots enhances mobilization of P. Iron deficiency- induced exudation of citrate in dicots (strategy I) and of phytosiderophores in grasses (strategy II) favors Fe mobilization and uptake. Exudation of phytosiderophores also increases the rhizosphere solubility and mobility of other metallic micronutrients, especially Zn and Cu. Exudation of phenolics can be implied in Mn mobilization (Marschner, 1995). The use of synthetic chelators for metal mobilization is gaining increasing importance in both fertilization of crops and phytoremediation technologies. However, not in all cases root exudation of potential metal-chelators leads to enhanced metal availability, to more metal uptake, and, if present in excess in the soil, to increased danger of phytotoxicity. On the contrary, there is growing experimental evidence that root exudation of chelating substances may also play a major role in detoxification and exclusion from roots of certain potentially toxic metals such as Al and Pb (Ryan et al., 2001). During the last years fast advances have been made in the understanding of the role of root exudates in the mechanisms of Al resistance. Although there is no use to try to reduce Al contents in soils by phytoextractio n technologies, the lessons learnt from Al-research may be useful for improve experimental approaches of the role of root exudates in metal mobilization, metal resistance and metal uptake of species used for phytostabilization and phytoextraction technolo gies in heavy metal contaminated soils. Keys to understand Al toxicity and resistance Among the most remarkable points for the progress in understanding the mechanisms of Al toxicity and resistance in crop plants are the following (Barceló & Poschenrieder, 2002): 9) Identification of Al3+ as the main phytotoxic Al species. 10) Identification of root tips as the most Al susceptible site (effects on cytoskeleton, root cell elongation and root cell division) 11) Use of short-term root growth curves for distinguishing different response patterns a) threshold for toxicity (importance of Al speciation in substrate and internal effect concentration in tips) b) hormesis (e.g. by alleviation of proton toxicity)

c) threshold for resistance (possible need for activation of resistance mechanisms) 12) Localization at the tips of root exudate production specifically induced by Al 3+ 13) Chemical characterization of different Al chelators in exudates of different species a) organic acids: malate, citrate, oxalate, b) flavonoid-type phenolics: catechin, quercetin 14) Distinction between exudation patterns a) Pattern 1: immediate exudate release by activation of anion channels b) Pattern 2: induction of exudate release after a several hours lag-time (probable need for gene activation) 15) Other Al exclusion mechanisms: mucilage, border cells… 16) Root exudates, Al uptake, ligand exchange, and compartmentation in Al hyperaccumulators (e.g. tea and species of the Melastomataceae family) Conclusions Organic acids (malate, citrate, oxalate) and/or flavonoid-type phenolics (catechin, quercetin) are very common components of root exudates of plants under different environmental conditions. The specific role of such compounds in the protection against Al toxicity in Al resistant genotypes resides in the ion-specific induction (Al3+) of these exudates at the precise region where the primary toxicity effects occur (root tip). The apoplastic accumulation of these chelators prevents both Al3+ toxicity in the cell wall - plasma membrane region and uptake of Al into the symplasm. Differences in either the distribution of anion efflux channel that are activated specifically by Al 3+ (pattern 1 of exudate release) or the genes responsible for exudate production upon specific activation (pattern 2 of release) seem to account for varietal differences in Al resistance by exclusion. Similar experimental approaches addressing the ion specificity of exudate induction, the site of exudate production, the chemical composition of the exudates and their time pattern of release using metals and plant species that are relevant in phytoremediation would contribute to a better understanding of the rhizosphere processes implied in these technologies. Acknowledgements Part of the author’s work cited in this paper was supported by the European Union (ICA4-CT-2000-30017) and by the Spanish Government (DGICYT, BFI2001-2475CO2-01)

References 1. Barceló, J., Poschenrieder, Ch., 2002 Fast root growth responses, root exudates, and internal detoxification as clues to the mechanisms of aluminium toxicity and resistance: a review. Env. Exp. Bot. (submitted) 2. Marschner, H., 1995. Mineral Nutrition of Higher Plants. 2nd ed. Academic Press, London.

3. Ryan PR, Delhaize E., Jones, DL., 2001 Function and mechanism of organic anion exudation from plant roots. Annu. Rev. Plant Physiol. Plant Mol. Biol. 52, 527-560.

n° 17. Speciation of metals in plants: analytical challenges and strategies

D. Schaumlöffel, L. Ouerdane, S. Mounicou, J. Szpunar and R. Lobinski CNRS, UMR 5034, Group of Bio- inorganic Analytical Chemistry, Hélioparc, 64053 Pau, France

Challenge The understanding of the uptake, homeostasis, storage or detoxification of metals in plants is hampered by the lack of information on the molecular level concerning the identity and structures of metallospecies and bioligands involved. From the point of view of analytical chemistry, speciation analysis of metals in plant tissues is a difficult task due to the complexity of the matrix and low concentration of chemical species, many of which have not yet been identified. Analytical strategy Multidimensional hyphenated techniques based on the combination of separation methods with sensitive element- and molecule specific detectors are proposed for characterization of metal species in plant tissues [1]. Different modes of high performance liquid chromatography (HPLC) or capillary zone electrophoresis (CZE) are used for separation of the metallocompounds present in a sample. On-line coupling of these separation methods to inductively coupled plasma mass spectrometry (ICP MS) as one of the most sensitive instruments in trace element analysis allows to detect the presence of metal compounds in plant samples on very low concentration levels. The use of electrospray tandem mass spectrometry (ESI MS/MS) enables the structural elucidation of the metal species. Fig. 1 gives a schematical overview on the possible combinations of the different analytical methods to a multidimensional strategy.

Fig. 1: Coupling techniques for biochemical speciation Results and discussion The analytical approach discussed in the presentation allowed for successful identification of phytochelatins (PC) isoforms in rice (Oryza sativa) exposed to Cd stress and of nickel species in hyperaccumulating plant Sebertia accuminata. A water extract of the Oryza sativa roots, preconcentrated by lyophilization, was characterized by preparative reversed phase chromatography followed by CZEESI MS showing the presence of a number of PC isoforms. PCs eluted from CZE in the range of migration times between 20 and 25 min and were detected by ESI MS and MS/MS allowing the identification of the bioligands. The direct analysis by CZEESI MS/MS without any purification or derivatisation of the analytes enabled the detection of three PC families: standard PCs, iso-PCs (Ser) and desGly-PCs [2]. A water extract from the latex of Sebertia accuminata, a Ni-hyperaccumulator from New-Caledonia, was investigated by size-exclusion (SEC) and anion exchange chromatography (AIC) coupled to ICP MS. Purified and preconcentrated fractions from AIC and preparative SEC were analysed by ESI MS/MS. Preliminary results showed the occurrence of at least five Ni- species, two of which were identified as Nicitrate (the storage form of nickel in Sebertia accuminata) and Ni-nicotianamine, which is probably responsible for the nickel transport.

References 1. Lobinski R. and Szpunar J. 1999. Biochemical speciation analysis by hyphena ted techniques. Analytica Chimica Acta 400, 321-332. 2. Mounicou S., Vacchina V., Szpunar J., Potin- Gautier M. and Lobinski R. 2001. Determination of phytochelatins by capillary zone electrophoresis with electrospray tandem mass spectrometry detection (CZE-ES MS/MS). Analyst 126, 624-632.

n° 18. Zinc uptake by Buddleia davidii cultured in vitro. Effect on the growth and on the polyamine content analysed as stress markers.

P. Schnekenburger, G. Charles, A. Hourmant and M. Branchard Plant Biotechnology and Physiology, ISAMOR-UBO, Technopôle Brest-Iroise, 29280 PLOUZANE, France. Introduction In order to clean up town refuse tips, whose lixiviates may disseminate trace elements, the main plant species growing on such sites under "natural" selective pressure are currently analysed for their ability to accumulate heavy metals (Zn2+ particularly). Among seven candidate species, Buddleia davidii was retained for its large and fast development. Furthermore, its wide geographical representation indicates a good tolerance for various climates. In order to study and to optimise the uptake of Zn by B. davidii, it was first acclimatised in vitro. By this mean, the genotypical effects have been suppressed, and the clones were grown under controlled conditions. In preliminary studies, various concentrations of Zn were added to the culture medium to analyse their impact both on the fresh weight and on the Zn content of the different plant organs. The effect of this trace metal was also evaluated on the content in polyamines, known as stress markers. Materials and methods Young nodal explants of B. davidii, growing spontaneously on a polluted site, were decontaminated and propagated in test tubes on MS medium (Murashige and Skoog, 1962) supplemented with 20 g.L-1 sucrose and 8 g.L-1 agar. The plants, placed under a 16 h photoperiod at 40 µmol.m-2 .s-1 and 22 °C, were subcultured every 4 weeks. Zn (0 to 1000 µM) was added as sulphate salt and dissolved in MES buffer (1 mM, pH = 5.7) and 10 ml of this solution were added to the culture medium. The different organs (roots, stems, leaves) were analysed separately 4 weeks after subculture. The fresh and dry weights were measured. The Zn content was determined by ICP-ES after digestion of dried plant material in concentrated HNO3 /HClO 4 mixture (3/1, v/v). The polyamines (putrescine, acetylspermine, spermidine and spermine) were extracted and analysed by HPLC, according to Flores and Galston (1982) slightly modified (Le Guen - Le Saos and Hourmant, 2002). Results and discussion The nodal explants rooted normally, and axillary buds developed and produced 6 nodes every 4 weeks. Addition of MES alone to the culture medium increased the fresh weight of the different organs. Zn (100 µM) further increased the fresh weight whereas a Zn concentration higher than 500 µM inhibited the growth. Whatever the Zn concentration, the number of nodes remained unchanged, but the leaves showed signs of necrosis over a 750 µM Zn concentration. The supply of MES or Zn did not change the water percentage of the different organs. The plant Zn content increased with the Zn addition. The amounts of Zn were higher in roots than in stems and

leaves, which represent respectively 12.5 %, 16.3 % and 71.2 % of the plant fresh weight. When the culture medium was supplemented with 1000 µM Zn, roots, stems and leaves accumulated respectively 7.8, 6.9 and 4.9 mg Zn.g-1 DW. With increasing levels of Zn in the medium, a decrease in the putrescine and spermidine content was observed in the leaves. By contrast, the polyamine amounts increased in stems and roots, where Zn accumulated preferentially. This latter finding agrees with that of Choudhary and Singh (2000) using Cd. Our results are consistent with the idea that polyamines protect against heavy metal stress. Work is now in progress with another species, Brassica juncea, frequently used in phytoremediation programs, in order to establish comparisons with our candidate species, Buddleia davidii. References 5. Choudhary A. and R. P. Singh. 2000. Cadmium- induced changes in diamine oxidase activity and polyamine levels in Vigna radiata wilczek seedlings. J. Plant Physiol. 156, 704-710. 6. Flores H. E. and A. W. Galston. 1982. Analysis of polyamines in higher plants by high performance liquide chromatography. Plant Physiol. 69, 701-706. 7. Le Guen – Le Saos F. and A. Hourmant. 2002. Stimulation of putrescine biosynthesis via ornithine decarboxylase pathway by gibberellic acid in the in vitro rooting of globe artichoke (Cynara scolymus). Plant Growth Regul. (in press). 8. Murashige T. and F. Skoog. 1962. A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol. Plant. 15, 473-479.

n° 19. Why it may be critical to rely on Cattail for phytoremediation of organic contaminants

P. Schröder, B. Huber and C. Scheer GSF National Research Center for Environment and Health, Neuherberg, D-85758 Oberschleissheim Introduction Cattail (Typhaspec) is a freshwater monocot species with Europe wide distribution. They are found abundantly in creeks, along swampy river banks and in drainages of agricultural areas. In the latter case, Typha is found very effective in removing nitrogen and phosphorus from the drainage water. Due to the strengthening of the water directive of the European Community the need to improve and ma intain high quality standards for sewage treatment effluents during the next years is of importance. Plant-based treatment systems may offer an adequate supplement to existing technologies. In order to test Typha´s suitability for the removal and metabolism of organic xenobiotics especially from sewage water Thypha latifolia (L.) and Typha angustifolia (L.) were investigated for a) the general detoxification capacity of organic xenobiotics; and b) the fate and specific breakdown of two persistent substances: bis (2-ethylhexyl)-phthalate (DEHP) (a plasticizer) and Lamotrigine (an antiepilepticum). These are substances of high environmental concern. Results and discussion Preliminary results indicate that Typha plants possess peroxidase activity and glutathione S-transferase (GST) activity for the conjugation of several xenobiotic model substrates (i.e. CDNB, DCNB etc.) in leaves, rhizomes and roots. Total GST activity seems to be shared by several GST- isoforms. Whereas the activity of some Glutathione S-transferase remains unaffected following the application of xenobiotics in induction experiments, other are induces by both chemicals and medicaments. In studies with Typha roots and rhizomes, the removal DEHP and Lamotrigine from water was observed. This disappearance seems to be connected to the activity of GSTs or Glucosyl transferases. However, the amount of bound residue formation seems to be low. Analysis of Typha rhizome indicates that the amount of sclerenchymateous tissue and lignification in this plant is rather low compared to other species. It is assumed that detoxified xenobiotics will only be bound to hemicelluloses and pectin of the cell wall. Both substances are easily degradable by soil bacteria and fungi. The significance of this effect for the utilisation of Typha in the treatment of sewage polluted with organic xenobiotics is discussed.