Tesis sobre el tema "Copper accumulation"

Our knowledge of aquatic Cu toxicity has increased greatly over the past several years culminating with the incorporation of a model (the Biotic Ligand Model (BLM)) into the regulatory framework which allows for the site specific adjustment of water quality criteria based on water chemistry. However, our understanding of Cu toxicity in the aquatic environment is limited mostly to freshwater (FW). Because of this limited knowledge, this dissertation set out to examine the affect of salinity on Cu toxicity and accumulation across salinities from FW to sea water (SW). First, this work examined tissue specific Cu accumulation in five salinities (FW, 5 ppt, 11 ppt, 20 ppt, 28 ppt) from waterborne Cu exposure at two [Cu] (30 and 150 µg Cu L-1) in the euryhaline killifish, Fundulus heteroclitus. Branchial and hepatic accumulation followed a pattern that would be expected based on speciation and competition from cations. [Cu] were high in FW and decreased as salinity increased. However, in the intestine, [Cu] were highest at 5 ppt and were also elevated in the higher salinities. The elevation at the higher salinities was most likely due to drinking by the fish which increases as salinity increases above the isoosmotic point of the fish (~10 ppt) for osmoregulatory purposes and showed a trend toward increasing [Cu] with increasing salinity as would be expected. Secondly, the mechanism of Cu toxicity in FW and SW was examined in killifish. The mechanism of Cu toxicity in the killifish in FW was the same as had been seen for other FW fish. Cu exposure caused a decrease in Na+ / K+ ATPase activity which led to a decrease in whole body [Na+] which is the likely cause of death. In SW, surprisingly no ionoregulatory disturbances were observed. The only measured parameter that was changed in SW was net ammonia which showed a substantial decrease. Therefore, the mechanism of acute copper toxicity in FW and SW differed suggesting that physiology may need to be considered in future development of a BLM for SW. Next, the effect of salinity on Cu accumulation from a naturally incorporated dietary source was examined in FW and SW in Fundulus heteroclitus. Cu accumulation was not seen to differ in the two salinities in spite of differences in gut fluid chemistry that would lead to an ~11 fold difference in free Cu ion between FW and SW. This indicated that Cu accumulation from a dietary source was not as the free ion but most likely as organically (amino acid) bound Cu. In addition to this, subcellular fractionation of the diet revealed that Cu available to the next trophic level did not change with increasing waterborne [Cu], increasing tissue [Cu], or time in oysters which has implications for Cu accumulation in the environment. Finally, the molecular mechanisms behind effects and acclimation to waterborne and dietary Cu exposure in FW and SW were examined using suppression subtraction hybridization in the killifish intestine. Exposure to Cu induced a stress response which could be responsible for upregulation of genes involved in protein synthesis, proteolysis and ATP production. At the tissue level, two main responses were observed. First, genes necessary for muscle function were upregulated potentially in response to previously observed decreased intestinal motility. Secondly, apoptotic genes were upregulated corresponding to increased rates of apoptosis in intestines during dietary exposures. At the cellular level, metal chelators and oxidative stress genes were upregulated in response to increased free Cu in the cell and the subsequent free metal induce free radical formation. This work has advanced our understanding of Cu toxicology in saline environments and emphasized the importance of considering both chemistry and physiology in analyzing and interpreting Cu toxicology especially in saline environments. Hopefully, it will contribute to the future development of Cu water quality criteria.

Thesis (Ph. D.)--University of California, San Diego, 2009.
Title from first page of PDF file (viewed Jan. 19, 2010). Available via ProQuest Digital Dissertations. Vita. Includes bibliographical references (p. 132-148).

Thesis (Ph. D.)--University of California, San Diego, 2010.
Title from first page of PDF file (viewed June 3, 2010). Available via ProQuest Digital Dissertations. Vita. Includes bibliographical references (leaves 108-118).

Environmental contamination with heavy metals, especially of soils, has been a continuousproblem worldwide since the beginning of the industrial revolution. Heavy metal emissionshave increased continuously since 1900 and the metals accumulate in the environment. Pulpand paper mill factories, and factories which produce sulphuric acid emit heavy metals,among others lead (Pb), copper (Cu) and zinc (Zn). Results from previous studies showedthat the soils near factories had higher concentrations of these pollutants than the controlplace. In this study I want to determine the effects of industrial heavy metal emissions of onehistoric and one present industry on nearby soil heavy metal concentrations.Samples were taken near one historically heavily contaminated site, the old sulphite factoryin Rydöbruk (1897-1944) and near the present Stora Enso paper mill in Hyltebruk. As acontrol site, I compared these with Rocknen nature reserve area.At each site I took 6 soil samples, representing the top 15-20 cm of soil and organic material.Samples were analysed with an Atomic Absorption Spectrometer and the concentrations oflead, copper and zinc were determined per ashed dry material.I analyzed the samples at the laboratory using the guideline from the Swedish StandardInstitute for water analysis.The concentrations of the soil samples showed no significant difference between the naturereserve area and the industrial sites (One-way ANOVA). This was due to very variableorganic content within and between the sites.Organic content of the samples has a significantly effect on the metal concentrations for Pband Cu (Linear Regression test). In conclusion, metals can accumulate in the vegetation, andin case of Pb and Cu there is a correlation between their concentration and the amount oforganic matter. Therefore in future studies it is best if soil with very high organic content orvegetation is used to determine if forest soil near industries are affected by past or presentindustrial emissions of heavy metals.

L'application de fongicides à base de cuivre pour lutter contre le mildiou dans les vignobles a entraîné une accumulation de cuivre dans les sols, en particulier dans les grandes régions viticoles telles que la Nouvelle-Aquitaine. Cette contamination pose des problèmes environnementaux majeurs, affectant la qualité des sols et la biodiversité. Bien que la réduction de l'utilisation de cuivre soit amorcée, son usage reste essentiel pour lutter contre les maladies fongiques des vignes, avec un risque d'impact sur les écosystèmes viticoles.Ce travail vise à analyser la biodisponibilité et la spéciation du cuivre dans deux sols viticoles de la région Nouvelle-Aquitaine (un sol alcalin et un sol acide) et à évaluer la réponse de deux espèces de plantes spontanées (Amaranthus retroflexus et Chenopodium album) aux niveaux de cuivre présents naturellement dans ces sols, ainsi qu'enrichis en laboratoire. L'hypothèse est que ces plantes peuvent développer des mécanismes de tolérance au cuivre, offrant des perspectives pour la gestion des sols contaminés.La spéciation et la biodisponibilité du cuivre dans les sols ont été étudiées à l'aide de méthodes d'extractions chimiques simples, d’électrochimie et de modélisations géochimiques. Une expérience en pots a ensuite été menée sur ces sols enrichis ou pas en cuivre, pour examiner les mécanismes d'accumulation du métal au sein des plantes. Ces données ont ensuite été comparées à l'accumulation des plantes ayant poussé naturellement sur site viticole.Les résultats ont montré que, dans des conditions contrôlées, les deux plantes ont mis en œuvre une stratégie d'exclusion du cuivre dans le sol alcalin, en accumulant le cuivre dans les racines, associé à une faible translocation vers les parties aériennes. En revanche, aucune croissance n’a été observée sur le sol acide, indiquant une toxicité excessive du cuivre dans ce contexte. La spectroscopie d'absorption des rayons X (XANES et EXAFS) et l'imagerie par microfluorescence X sur rayonnement synchrotron (µXRF) couplée au µXANES, ont montré que le cuivre s'accumulait dans les racines sous forme de complexes avec des ligands carboxyle et hydroxyle, principalement au niveau du rhizoderme.L' étude menée sur Amaranthus retroflexus ayant poussé en conditions réelles sur le sol alcalin a révélé des concentrations importantes de cuivre dans les feuilles, notamment au niveau de la cuticule, suggérant une pénétration foliaire du fongicide. La présence spontanée d'Amaranthus retroflexus pourrait ainsi constituer une surface tampon lors des traitements phytosanitaires, réduisant ainsi la contamination directe des sols par ces produits.Ces résultats ouvrent la voie à une meilleure compréhension du rôle écologique de ces plantes dans la gestion des sols contaminés. À l'avenir, leur potentiel pour limiter l'impact du cuivre sur les sols pourrait être exploité dans des stratégies de gestion durable des agroécosystèmes viticoles
The application of copper-based fungicides to fight downy mildew in vineyards has led to copper accumulation in soils, particularly in major wine-growing regions such as Nouvelle-Aquitaine. This contamination raises serious environmental concerns, affecting soil quality and biodiversity. Despite recent efforts to reduce copper use, its application remains essential to struggle against fungal diseases in vines, with potential impact on the ecosystems.This work aims to analyze copper bioavailability and speciation in two vineyard soils from the Nouvelle-Aquitaine region (one alkaline and one acidic) and to assess the response of two spontaneous plant species (Amaranthus retroflexus and Chenopodium album) to copper levels naturally present in the vineyard sites, as well as those enriched in the laboratory. The hypothesis is that these plants may develop copper tolerance mechanisms, offering potential alternatives for vineyard soil management.Copper speciation and bioavailability in these soils were studied using simple chemical extraction methods, electrochemical analyses, and geochemical modeling. A pot experiment was then conducted on these native and copper-enriched soils to examine the mechanisms of metal accumulation developed by the plants. These data were then compared to copper accumulation of plants naturally grown on the site.The results showed that, under controlled conditions, the two plants developed a copper exclusion strategy in the alkaline vineyard soil, accumulating copper in their roots and preventing its translocation to aerial parts. In contrast, no growth was observed in the acidic soil, indicating excessive copper toxicity. X-ray absorption spectroscopy (XANES and EXAFS) and synchrotron µ X-ray fluorescence (µXRF) imaging coupled with µXANES showed that copper was accumulated in the roots as complexes with carboxyl and hydroxyl ligands, mainly located at the rhizodermis level.The study of Amaranthus retroflexus grown on the alkaline vineyard revealed high concentrations of copper in the leaves, mainly in the cuticle, suggesting a foliar uptake of fungicides. The spontaneous presence of Amaranthus retroflexus could thus be used as a buffer surface during phytosanitary treatments, thereby reducing the direct contamination of soils by these products.These results pave the way for a better understanding of the ecological role of these plants in managing contaminated soils. In the future, their potential to limit the impact of copper on vineyard soils could be harnessed in sustainable management strategies for vineyard agroecosystems

Discrepancies in ecotoxicity effects data derived from soils amended with dissolved metals in the laboratory and soils from historically aged metal-contaminated sites pose a challenge in deriving environmentally relevant soil quality criteria. In this thesis, the chemical artefacts produced by dissolved metal additions and the subsequent effects on metal bioavailability, toxicity and bioaccumulation are examined. The overall goal of this research is to advance the understanding of soil trace metal bioavailability mechanisms and improve current methods for ecotoxicity testing and bioavailability modeling.
The first objective was to develop a leaching method which would minimize the "salt-effect" commonly observed in freshly-spiked soil samples. This was achieved by adapting a column leaching procedure in order to spike and leach larger volumes of test soil as a preparatory step prior to ecotoxicity assays. An experiment was set up to observe changes to leachate chemistry resulting from the dissolved Cu additions and subsequent leaching with two weak electrolyte solutions. Results showed that leaching removed the excess dissolved Cu as well as the excess acidity and base cations that were solubilized during the Cu additions. Differences between control and spiked samples of some key soil solution parameters (e.g. pH, DOC and total dissolved Ca, Mg, Al, Fe) were reduced thereby resulting in a set of spiked samples more conducive to deriving causal dose-response relationships.
A second experiment compared soil and soil solution properties of samples which underwent the spike/leach procedure to those of freshly spiked samples. Lower soil pH (up to 0.81 pH units) and DOC concentrations were observed in non-leached samples as well as up to 35-fold and 55-fold increases of dissolved Ca and Mg concentrations, typical of the salt-effect. It was estimated that the non-leached samples contained up to 100-fold greater Cu2+ and 50-fold greater Al3+ activities which, I hypothesized, could result in greater phytotoxicity of non-leached samples. Bioassays were conducted wherein Hordeum vulgare (barley) seedlings were exposed to the leached and non-leached Cu-spiked soil samples for 14 days. The leached samples were less toxic to barley and showed significant increases (up to 1.7-fold) in median inhibitory concentrations (i.e. IC50) for root elongation in two of the three test soils. The Cu2+ fraction was able to explain much of the variability in toxicity between leached and non-leached samples, as well as among the different test soils. One exception was the most acidic test soil for which plants in the leached samples showed up to 10-fold greater toxicity than plants in the non-leached samples, when exposed to similar Cu2+ activities. Soil speciation and bioaccumulation data for Cu, Al and Ca were used to deduce that Ca deficiency and possibly Al toxicity contributed to the toxicity observed in this sample set.
In a modeling exercise, the concepts of the terrestrial biotic ligand model (TBLM), which take into account ion competition at the soil solution/root interface, to describe toxic response in our samples. Results showed that the additive effects of Cu and Al toxicity estimated from the Cu-root ligand complex (Cu-BL+) and Al-root ligand complex (Al-BL2+ ) accounted for more of the variability in toxic response data than did the Cu2+ fraction or the Cu-BL+ fraction alone. The model however, could not account for the effects of the Cu-induced Ca deficiency in the most acidic test soil, thus highlighting one of the limitations of the TBLM. This thesis has contributed to the understanding of metal bioavailability mechanisms affected by soil spiking procedures and highlights the benefits of soil leaching in preparing soil samples for ecotoxicity assays which should improve trace metal risk assessment in the future.

THESIS ORGANISATION This thesis is organised into nine chapters that include seven international and national publications (six accepted and one submitted for publication). The initial overview chapter outlines the justification and direction for this thesis. With the exception of chapter 8 (accepted for publication on the 1st May 2005); all chapters are exact duplicates of published articles in international and national refereed journals (chapters 2 to 7). The initial chapters (2 and 3) presents research findings using a marine fish species, mullet (Mugil cephalus), to measure trace metal bioavailability in Lake Macquarie, NSW Australia. While subsequent chapters (4 to 8) are presenting research under taken to improve the understanding of arsenic cycling in marine and estuarine environments. The final chapter (chapter 9) is a synopsis of the major findings presented in this thesis. Due to the publication nature of this thesis, an unavoidable degree of replication exists within chapters (publications).

Klövsjukdomen digital dermatit hos kor ger upphov till smärta hos djuren samt störningar i produktionen. För att förebygga och behandla klövsjukdomen används olika preparat, däribland CuSO4-lösning. Syftet med studien är att undersöka om och i så fall i vilken omfattning spridande av kopparförorenad gödsel påverkar Cu-halten i jordbruksmark. Studien genomfördes med provtagning av två oberoende jordbruksmarker där ett av områdena gödslas med kopparförorenad gödsel. Resultaten visar att jordbruksmark där förbrukad CuSO4 spridits med stallgödseln ger upphov till en signifikant ökad Cu-halt i markens översta lager, 0-25 cm.
The hoof disease digital dermatitis rise pain with the cows as well as production disruption. To prevent and treat the hoof disease different substances are used, including CuSO4 solutions. The purpose of this study is to examine if, and in that case in which incidence, the spread of copper contaminated manure affects the copper content in agricultural land. The study was performed sampling two independent agricultural areas. One of the areas was fertilized with copper contaminated manure. The results show that agricultural land where CuSO4 has been spread with the manure has a significant higher content of Cu in the upper level of the soil, 0-25 cm.

Introduction The work carried on in the triennium 2007-09 put itself in the field of research known as phytoremediation. This term identifies several techniques (phytoextraction, phytostabilization, phytovolatilization) based on using different plant species able to survive on substrates contaminated by many pollutants derived from human activities and to accumulate them in own tissues or volatilize by evapotranspiration or avoiding their leakage troughout the groundwater . This ability varies strongly trough species and inside a species between genetic pools. About the ability in heavy metals removing , researchers have identified two classes of plant species : Hyperaccumulators : species tipical of metalliferous soils, able to carry on their life cycle without showing symptoms of stress as heavy metals high concentration. (Baker et al., 2000). To be classified as hyperaccumulator a plant species must accumulate a minimum of : 100 mg/Kg d.w. of Cd, 1000 mg/Kg d.w. of Pb, 10000 mg/Kg d.w. of Zn ; ranges for As are not yet determined. (Baker and Brooks 1989, Baker et al., 2000). Some of most rapresentative hyperaccumulator species are Thlaspi caerulescens, Arabidopsis halleri and Viola calamarina, Astragalus spp and several other species of following families : Cariophyllaceae, Fabaceae, Poaceae, Chenopodiaceae, Brassicaceae e Cyperaceae. About As the best accumulator is Pteris vittata. The main limit of hyperaccumulators is little biomass production. Accumulators: in this group there are herbaceous and woody species able to couple a good accumulation with a good biomass production. Between them we can find Brassica juncea, end the genus Salix. This kind of accumulators are the best solution for pollutants removal programs in short and medium period. An high biomass production allows to plan a recycle of it in energy production and to recover heavy metals from ashes. ((Kabata-Pendias and Pendias, 1992 ; Pullford and Dickinson 2004 ; Paulson et al 2003; French et al 2005; Ebbs et al 1997 ; Ebbs et al 1998). This work, divided in several steps, as described below, wants to increase the knowledge on some of previous species, from a physiological point of view and evaluating performances of accumulation in order to replace, when it’s feasible, invading and expensive remediation techniques with phytoremediation that is, in its different applications, more respectful of landscape and more cost-effective. Trials to evaluate phytoextraction performances of four species of willow (Salix spp) , Phragmites australis and Brassica juncea grown on polluted sludge dredged from Venice Lagoon. In collaboration with Department I.M.A.G.E. of Faculty of Engenering of University of Padova, in the field of PRIN 2005 (National Significant Project) “Remediation of sea, lagoon and river sediments” experimental trials were planned in order to understand remediation and biomass production potential of Salix matsudana, S. babylonica, S. alba, S. fragilis, Brassica juncea and Phragmites australis growing on salty polluted sludge , dredged from Venice Lagoon (closest to Marghera harbor) and to think about a phytoextraction installation. Sludge samples were kept in four sites (North industrial channel, West industrial channel-two samples-, and Evolution Basin), then labeled and characterized. To dry, homogenize and ameliorate their drainage capability of sludge, samples were mixed with river sand in a 1:1 ratio. Trial on 50 L tanks : After preliminary trials that underlined impossibility to grow up in field conditions Salix babylonica, fragilis , alba, , Thypha latifolia and Zea mais, seven 50 L tanks were filled up with a bottom layer of expanded clay and sludge up to 35 L (2 with Phragmites, 2 with Salix Matsudana, 2 with Brassica juncea and one without plants ) each one with a tube coupled with a filter, linked in the bottom to collect water samples and favorite drainage. Willow cuttings and Indian mustard seedlings were planted with a universal soil bulk to attenuate transplanting shock and negative effect on young root system of direct contact with polluted substrate, Phragmites rhizomes instead, after an initial steady state, showed to be able to grow on direct contact with sludge. After two months from start of trial, EDTA was added to one of Phragmites tanks to increase phytoextraction performances. Hydroponic trials : hydroponic livestocks of S. Matsudana, S. Alba, S. Fragilis, s. Babylonica and Brassica juncea were prepared mixing Hoagland nutrient solution with same Ni, Cd, Zn, Cu and Pb concentrations detected in first water sample collected from tanks. For each willow species, were prepared one becker as control with Hoagland solution only, one with Hoagland plus metals and one with Hoagland, metals plus NaCl, supplied at increasing concentrations (from 0.05 M to 0.2 M). About brassica, five beckers with 40 seedlings for each one were prepared. One becker as control with Hoagland solution, two beckers with Hoagland plus metals in the same concentration as described above, and two beckers with metals plus raising up Na concentration as described above. At the end of experimental period, cuttings and seedlings were harvested, weighted and metals and Na contents were detected. Twenty L tanks trials : five 20 L tanks with a tap on the bottom were filled up with a layer of fine gravel, a layer of sludge and a top layer of universal soil. They were placed in growth chamber. Each tank hosted three cuttings (one species for each tank) and eight Paspalum vaginatum plants, the fifth one hosted a second series of S. Babylonica cuttings and eight Paspalum vaginatum plants. Trials were 50 days long. A week after trial start, in fifth tank EDDS chelant was added, in order to increase metals bioavailability. After plant harvesting, biometric and metal content measurement were done. Willow root deepening evaluation: to see possible differences in sediment exploration by root systems of willow species twenty four 2 L plastic bottles were prepared. Sixteen of that filled up with same 20 L tank content, and eight (two for species) with grave’, universal soil on the top and soil kept from Experimental farm “L.Toniolo”. Each bottle hosted a cutting (six bottles for species). At the end of trial, willow shoots were removed, then bottles were frozen and sawed in three sections corresponding to universal soil layer and two part of sludge layer corrispondenti alla sezione contenente il terriccio e a due parti conteneti il sedimento. All root systems were extracted, washed up, weighted, scanned and analyzed to determine total length and diameter. Results : Phragmites : we can see an EDTA effective action on Cd, Ni, Pb Zn and Na accumulation in shoots while in rhizomes, there are significative differences only about Pb and Na. No differences in term of biomass production. EDTA became more available Cu and Zn in soil solution; nevertheless metals concentrations remain pretty low. Willows in growth chamber : no differences in leaves accumulation between species while Babylonica EDDS treated became the best performer in Cu, Ni, Pb and Zn accumulation, while it diminuish Na concentration. In stems babylonica appears the best, with and without EDDS for Cu, Zn and Na. About roots Babylonica is again one of best performer in Na, Zn and Cd accumulation. No differences between species or treatment in Ni accumulation. Comparing growth chamber willows with S. matsudana grown in field conditions, this one shows best performaces in leaves accumulation for Na (followed by “growth chamber matsudana”, fact that confirms the ability of the species in Na translocation), and for Cd and Ni with babylonica EDDS treated. In stems, “field matsudana” is the best accumulator of Na, Pb and Ni and it remains the best performer for root system except for Na. hydroponic trials confirm in leaves the strong competition of Na versus accumulation of other anions, particularly versus Cd, Ni, Pb and Zn ; competition versus Cu appears lighter instead. In stems le migliori performances di S. babylonica is the best accumulator of Cd, Cu, Zn and Ni, nevertheless it appears at the same time the best Na accumulator. In root system too babylonica is the best accumulator of Cd, Ni Zn and Na. The roots deepening evaluation trial shows in comparison with control of each species that babylonica root system should be the less suffering (according to accumulation data), while between four species grown on sludge Salix alba shows the deepest root system coupled with smallest diameters. This two characteristics should be a signal of less stress as found by Vamerali et al. 2008. Brassica juncea hydroponic trial show clearly interference of Na in metals accumulation. Anyway accumulation performances in hydroponic are clearly better than that ones of brassica grown in field conditions, also in presence of Na. It should mean that : Ahydroponic trial in this case isn’t reliable in a comparison with field conditions B) probably other limiting factors, as nutrients could affect metal accumulation and biomass production that is pretty low also because the quick flowering, another symptom of stress. Interaction of selenate and molybdate with the transport and assimilation of sulfate in Brassica juncea Recently MOT1, a transporter with an high affinity for Mo was identified in Arabidopsis thaliana (Tomatsu et al., 2007), but it’s common opinion plants could uptake Mo also using S transporters because the two anions are analogs and could compete in binding of active site of the same transporters (Dudev e Lim 2004; Alhendawi et al 2005). Once inside the cell, Mo can interfere with S methabolism becoming substrate for reactions catalyzed by ATP sulfurilase (APS), as is selenate, but in contrast to selenate no Mo containing products are formed in the reaction (Reuveny 1977). Given the significant impact that S fertilizers application may have on Mo and Se distribution and accumulation in plants (Shinmachi et al., 2010), this work studied interactions of sulfur nutrition with molybdenum and selenium in Brassica juncea. To this aim Brassica juncea seedlings were grown with different combinations of S and Se or S and Mo. Experiments were carried out in a short time (24h) as selenate and molybdate were given to plants at a very high concentration (200 µM), which was the same of sulfate in the S-sufficient condition. Chemical and physiological analyses were performed, including quantification of elements and plant growth. Tolerance index (% of fresh weight relative to the control) and root/shoot ratio show a greater stress for plants grown in S deficiency and in presence of Se and Mo. Se and Mo were more absorbed in minus S condition. Both for total S and sulfate Se and Mo competition becomes clear only between 6 and 24 h of treatment, while within an hour there are not significant changes in S uptake. Study on potential effects of Se accumulation on drought stress in Brassica juncea e Stanleya pinnata . Some Se accumulator species (Brassica spp, Stanleya spp, Astragalus spp.) uses Se accumulated as a weapon against herbivors (Freeman et al., 2007; Galeas et al. 2007 ; ) Brassica juncea , differently from other toxic or potentially toxic trace elements, accumulates Se also in flowers and seeds giving value to the hypothesis of an happening evolutionary in the use of Se against parassites and herbivors adaptation (Quinn C. et al., 2008). This two species were grown up in gravel and in hydroponic in presence (20 µM) and in absence of Sodium selenate. The water deficit was experimented at first on gravel by irrigation stopping for 8 days, then in hydroponic by adding polietilenglicol (PEG 10000), to modify Ψw of nutrient solution. In two hydroponic trials’ Ψw was brought to -1.3 / -1.6 Mpa. Measures of photosintetic efficency, biomass and water and osmotic potential show that there are no evidences of a positve effect of Se on drought tolerance except for the non stress condition or light stress (-0.5/-0.6 Mpa). This action, actually not very strong, increase biomass production in plants treated with selenate according to trials on Triticum aestivum (Yao Xiaoqin et al. ; 2009). Study of potentials of Se-accumulator Astragalus racemosus and non accumulator Astragalus convallarius as Ni, Mo, Cr, V, W, Te, Se and As accumulators. The genus Astragalus is composed by many species distributed in northern america, some of that adapted to live in seleniferous soils, so able to develop an high tolerance versus Selenium (Cowgill, 1990). Concentration of Arsenic, Nichel Chromium, Vanadium, Tellurium, Tungsten and Molibdenum raised up in the environment because human activities ( Bamhart 1997 ; Zarchinas et al 2004 ; Zoller et al. 1973 ; Gott and McCarthy 1966 ; ) . the aim of this work is to understand if adaptation to Se accumulation is effective also versus other elements, some of them analogs of Se. These two species of Astragalus germinated in Petri dishes then grown up in growth chamber for 3 months in Murashige & Skooge addicted agargel plus salts of elements mentioned above , mixed in different concentrations based on their degree of toxicity, in order to avoid a too quick death. Results showed that Se hyperaccumulator is not always more tolerant than the non accumulator (A. Convallarius showed more tolerance in V accumulation). Effects of Cu deficiency on Cu-proteins activity and on expression of laccases in leaves and stem of Populus trichocarpa Cu has a role of cofactor in function of several dismutases (enzymes catalyzing formation of H2O2 from R.O.S.) and in laccases enzyme (Pilon et al. 2006). Many clones of P. Trichocarpa were multiplyed and grown up in MS addicted agargel with two different Cu concentrations (0.1 µM e 5 µM) to verify trough quantification of Cu-enzymes activity (western blot and native gel) if and how much these two Cu concentrations (the first closest to the limit of deficiency and the second pretty high but far from toxicity ), could affect Cu-enzymes activity. Once quantified activity of Cu-SODdepending on Cu concentration, a preliminary investigation on Laccases regulation was done. Because Laccases needs four Cu ions and because in a condition closest to Cu deficiency, cell inhibits all Cu requiring enzymes except that necessary to make photosintesis like plastocianin, we wanted verify if Laccases faith was exactly the same. Primers were drawn (RNA short sequences neded to make a Polimerase Chain Reaction in vitro ) to amplify the target sequence of a miRNA (RNA short sequences that, coupled with RISC complex, cut down mRNA inactivating it) (Yamasaky et al. 2007; Abdel Ghani et al 2008) and to obtain after an electrophoretic run, bands of different intensity, depending on the down regulation action made by cell. Preliminary results show different levels of expression for several kinds of laccase in stem and leaves.
Introduzione generale L’attività di ricerca svolta nel triennio 2007-2009 si colloca nell’ambito della fitodepurazione. Con questo termine si identifica una serie di tecniche (fitoestrazione, fitostabilizzazione, fitovolatilizzazione) basate sull’impiego di specie vegetali diverse (l’elenco delle quali è in continua evoluzione) ma accomunate dalla capacità di sopravvivere su substrati contaminati da diversi inquinanti prodotti dall’attività umana e di interagire con il substrato in modo tale, a seconda dei casi, da depauperarne il contenuto di inquinanti accumulandoli nei propri organi e/o liberandoli nell’atmosfera attraverso l’evapotraspirazione fogliare, oppure impedendone la lisciviazione. L’attitudine ad un’azione di fitorimedio varia fortemente da specie a specie e all’interno della stessa specie in base al corredo genetico . Relativamente alla capacità di rimozione di metalli pesanti, sono state individuate due classi di specie accumulatrici: Iperaccumulatrici : sono specie tipiche di suoli metalliferi, in grado di portare a termine il proprio ciclo vitale senza manifestare sintomi di deperimento dovuti all’alta concentrazione di metalli (Baker et al., 2000). Per essere definita iperaccumulatrice, una specie deve rientrare nei seguenti limiti inferiori di bioaccumulo: 100 mg/Kg s.s. di Cd, 1000 mg/Kg s.s di Pb, 10000 mg/Kg s.s. di Zn ; i ranges per quanto riguarda l’arsenico non sono ancora stati ben definiti (Baker and Brooks 1989, Baker et al., 2000). Tra le specie iperaccumulatrici le più rappresentative sono quelle appartenenti al genere Thlaspi, Arabidopsis halleri e Viola calamarina, Astragalus spp oltre a diverse specie appartenenti alle famiglie delle Cariophyllaceae, Fabaceae, Poaceae, Chenopodiaceae, Brassicaceae e Cyperaceae. Per quanto riguarda l’iperaccumulo di arsenico spicca Pteris vittata. Il limite delle specie iperaccumulatrici è la scarsa produzione di biomassa. Accumulatrici: a questo gruppo appartengono specie, ad habitus sia erbaceo che arboreo, in grado di ottenere buone concentrazioni di metalli e metalloidi nei propri organi, pur non arrivando a livello delle iperaccumulatrici, e una buona produzione di biomassa. Fra esse sono state individuate Brassica juncea, e il genere Salix. Le caratteristiche di queste accumulatrici le rendono idonee a programmi di disinquinamento a breve e medio termine, soprattutto per suoli poco o mediamente inquinati. La possibilità di avere una considerevole produzione di biomassa permette di pensare, come seconda fase, al reimpiego di questa per la produzione di energia ed il recupero dei metalli pesanti di interesse industriale ((Kabata-Pendias and Pendias, 1992 ; Pullford and Dickinson 2004 ; Paulson et al 2003; French et al 2005; Ebbs et al 1997 ; Ebbs et al 1998). Il lavoro triennale, suddiviso in diverse fasi, come descritto in seguito, si propone di approfondire la conoscenza su alcune delle specie suddette , sia dal punto di vista della risposta fisiologica sia , con un taglio maggiormente applicativo, dal punto di vista della valutazione di performaces di bioaccumulo al fine di sostituire, ove possibile, tecniche di bonifica invasive e costose con la fitodepurazione che, nelle sue diverse applicazioni, si dimostra piu’ rispettosa del territorio, anche dal punto di vista estetico e considerevolmente piu’ vantaggiosa economicamente. Prove di valutazione della capacita’ di accumulare metalli pesanti da parte di quattro specie di Salice (Salix spp) , di Phragmites australis e Brassica juncea. Date queste premesse, in collaborazione col dipartimento I.M.A.G.E. della Facoltà di ingegneria dell'Università di Padova, nell’ambito del PRIN 2005 ““Risanamento di sedimenti marini, lagunari e fluviali” sono state messe a punto delle prove sperimentali finalizzate a comprendere le potenzialità di Salix spp, di Brassica juncea e Phragmites australis nel mettere in atto un'azione di fitorimedio su sedimenti provenienti dal dragaggio della laguna di Venezia nella zona di porto Marghera. Il campionamento dei fanghi è stato effettuato in quattro siti (canale industriale nord, due punti del canale industriale ovest, bacino dell'Evoluzione), quindi i campioni sono stati catalogati e caratterizzati. In vista dell’allestimento di possibili impianti finalizzati al fitorisanamento dei fanghi dragati dall’area di Porto Marghera, sono state allestite delle prove sperimentali con l’intento di definire le potenzialità fitoestrattive di Salix matsudana, S. babylonica, S. alba e S. fragilis, Phragmites australis e Brassica juncea e valutarne quindi l’idoneità ad essere messe a dimora su questi sedimenti, anche in base alla capacità di sopravvivenza ed alla produzione di biomassa. I campioni provenienti dai 4 siti, sono stati miscelati tra loro ed in rapporto 1:1 con della sabbia di fiume, sia per ottenere un substrato con valori omogenei relativamente alle concentrazioni di inquinanti, sia per aumentare la capacità drenante di un substrato altrimenti totalmente asfittico. Prova su mesocosmo : Dopo test preliminari che hanno evidenziato l’impossibilita’ di condurre prove, ceteris paribus, anche su Salix Babylonica, Fragilis ed Alba, su Thypha latifolia e su Zea Mais, sono state allestite 7 vasche (2 dedicate a Phragmites australis, 2 a Salix Matsudana e 2 a Brassica juncea ed una priva di vegetazione, della capienza di 50 L (riempite fino a 35 L) ciascuna, munite di un tubo per il drenaggio del percolato alla base. Sul fondo delle vasche è stato depositato uno strato di argilla espansa e l’ingresso del tubo è stato protetto dall’intasamento con un filtro Le talee di salice ed i semenzali di brassica sono state messe a dimora con un piccolo pane di terriccio universale al fine di attutire gli effetti dello stress da trapianto e di permettere all’apparato radicale di accrescersi a sufficienza prima di esplorare il sedimento contaminato, mentre i rizomi di Phragmites hanno attecchito a diretto contatto col substrato oggetto di analisi. Dopo due mesi dall’inizio della prova, ad una delle vasche ospitanti la Phragmites e’ stato addizionato il chelante EDTA. Prove in idroponica : prendendo come riferimento le concentrazioni di 5 tra metalli pesanti riscontrati nell’analisi dei campioni di percolato (Ni,Cd, Zn, Cu, Pb,) e la concentrazione media del Na (intorno a 0,2 M) sono state allestite degli allevamenti in coltura idroponica con talee, oltre che di S. matsudana, anche di S. alba, S. babylonica , S. fragilis e di Brassica juncea. Per quanto riguarda i salici, la soluzione contenente inizialmente una soluzione nutritiva di tipo hoagland, è stata arricchita dai suddetti 5 metalli, in concentrazione pari a quella riscontrata nelle acque di percolazione. Per ogni specie di salice è stato allestito un beacker di controllo con sola soluzione nutritiva, un becker con soluzione nutritiva, più i metalli ed uno con le caratteristiche precedenti più NaCl a concentrazioni crescenti (da 0,05 M fino a 0,2 M). Per le brassiche sono stati allestiti 5 becker con 40 piantine ciascuno. Un beacker di controllo con soluzione nutritiva, due con concetrazione di metalli pari a quella del percolato, due con concentrazione di metalli pari al percolato più NaCl 0,05 M (aumentato fino a 0,2 M nel corso di 3 settimane). Alla fine del periodo di trattamento sono state separate e pesate parte aerea ed apparato radicale dei semenzali e ne sono stati analizzati i contenuti in metalli e Na. Prove su microcosmi : sono state allestite 5 taniche da 20 L ciascuna, dotate di rubinetto alla base e sistemate in cella climatica, tutte dotate di uno strato drenante di ghiaino alla base, un orizzonte costituito solo da sedimento ed uno, quello più superficiale, di terriccio universale. Quattro taniche hanno ospitato ciascuna tre talee (una specie per ogni tanica) e otto piantine di Paspalum vaginatum, la quinta tre talee di S. Babylonica e otto piantine di Paspalum. L’insediamento delle talee e della graminacea è durato 50 giorni. Nella settimana successiva alla messa a dimora dei S. Babylonica nella quinta tanica è stato aggiunto l’ EDDS, un chelante che ha lo scopo di rendere più biodisponibili i metalli pesanti presenti nel substrato. Studio dell’approfondimento radicale dei salici : per verificare la capacità delle quattro specie di salice, di esplorare il sedimento contaminato sono state allestite 24 bottiglie in pet da 2 L, di cui 16 riempite con lo stesso contenuto delle taniche e 8 (due per specie) riempite con del terreno proveniente dall’Azienda Sperimentale “L. Toniolo”. In ogni bottiglia è stata messa a dimora una talea di salice (6 bottiglie per specie). Al termine dei 50 giorni di prova condotti in camera di crescita, è stata asportata la parte aerea della talea, le bottiglie sono state congelate e poi segate in tre parti corrispondenti alla sezione contenente il terriccio e a due parti conteneti il sedimento. Tutte le radici presenti sono state estratte , lavate e pesate e sottoposte a scansione digitale. Risultati : Phragmites : azione efficace del chelante EDTA per quanto riguarda l’accumulo di Cd, Ni, Pb Zn e Na nei fusti di Phragmites, mentre differenze significative nei rizomi sono state riscontrate solo per Pb e Na. Nessuna differenza in termini di biomassa prodotta. Il chelante ha reso maggiormente disponibile nella soluzione circolante Cu e Zn anche se le concentrazioni restano molto basse. Salici nelle taniche in cella climatica : gli accumuli a livello fogliare non mostrano differenze significative tra le quattro specie non trattate. Mentre l’EDDS incrementa le prestazioni del babylonica rispetto a se stesso ed alle altre specie per quanto riguarda Cu, Ni, Pb e Zn mentre riduce la concentrazione di Na. A livello di fusti la suddetta differenza si esplica nelle concentrazioni di Cu, Zn e Na; questi ultimi risultano maggiormente accumulati dal babylonica sia con che senza chelante. A livello radicale il babylonica risulta ancora uno dei migliori accumulatori del lotto per quanto riguarda Na, Zn e Cd. Nessuna differenza ne tra specie ne tra presenza/ assenza di chelante per quanto riguarda il Ni. In un confronto tra questi salici ed il matsudana cresciuto piu’ a lungo e in condizioni di T , umidita’ e fotoperiodo variabili, a livello fogliare il salice cresciuto in mesocosmo presenta valori di Na significativamente piu’ alti degli altri salici, seguito dal matsudana cresciuto in cella, a conferma dell’atitudine di questa specie a traslocare piu’ facilmente il Na nella chioma. Anche riguardo Cd e Ni il matsudana di campo denuncia tra i valori piu’ elevati assieme al babylonica trattato con EDDS. Nei fusti, per Na ,Pb e Ni ci sono differenze significative in favore del S. matsudana di campo. A livello radicale il S. matsudana cresciuto in mesocosmo presenta valori significativamente piu’ alti rispetto alle prove in cella climatica ad eccezione del Na. Le prove in idroponica confermano, a livello fogliare che l’accumulo di Na compete fortemente con l’uptake dei metalli, in particolare nei confronti di Cd, Ni, Pb e Zn ; piu’ sfumata invece l’azione competitiva verso il Cu. Da rilevare, a livello di fusti le migliori performances di S. babylonica per Cd, Cu, Zn e Ni, nonostante si rilevi nel contenpo il primato nell’accumulo di Na. Anche a livello radicale il babylonica accumula piu’ delle altre specie per quanto riguarda Cd, Ni Zn e Na. La prova sullo sviluppo radicale mostra, nella comparazione col rispettivo controllo, che l’apparato radicale di S. babylonica risulta il meno sofferente tra le quattro specie (in accordo quindi con i dati di accumulo), mentre comparando le quattro specie cresciute sul sedimento, il Salix alba risulta avere l’apparato piu’ sviluppato in profondita’ e con i diametri minori , segno questo che potrebbe essere considerato di minor sofferenza, come riscontrato anche da Vamerali et al. 2008. L’allevamento in idroponica di Brassica juncea evidenzia differenze significative tra l’accumulo di metalli in presenza di Na rispetto all’accumulo in assenza di quest’ultimo. Le performances di accumulo rispetto alle brassiche cresciute su fango sono nettamente migliori, anche in presenza di Na , a testimonianza del fatto che A) l’idroponica non offre termini di paragone affidabili rispetto alle condizioni di campo B) probabilmente intervengono altri fattori, come la carenza di nutrienti, a condizionare l’accumulo di metalli e la produzione di biomassa che risulta bassa anche per la precocita’ della fioritura, sintomo della condizione di stress. Interazione del selenato e del molibdato con il trasporto e l’assimilazione di solfato in Brassica juncea Recentemente e’ stato individuato in Arabidopsis un trasportare (MOT1) con elevata affinita’ per il Mo (Tomatsu et al., 2007), ma si ritiene che le piante assorbano Mo anche attraverso i trasportatori del S poiche’ i due anioni hanno caratteristiche simili e potrebbero competere nell’occupare il sito attivo degli stessi trasportatori (Dudev e Lim 2004; Alhendawi et al 2005). Una volta all’interno della cellula, Mo puo’ interferire col metabolismo dello S divenendo substrato per le reazioni catalizzate dall’ ATP sulfurilasi (APS), come accade per il Se ma, a differenza di quest’ultimo non si riscontra la formazione di prodotti contenenti Mo derivanti da questa reazione (Reuveny, 1977). A causa del significativo impatto che l’applicazione di fertilizzanti a base di S puo’ avere sulla distribuzione di Se e Mo e sul loro accumulo nelle piante (Shinmachi et al., 2010), si e’ cercato di capire, in questo lavoro,quale sia l’interazione tra l’uptake di S e il Se e Mo in Brassica juncea. A questo scopo semenzali di Brassica juncea sono stati allevati con differenti combinazioni di S e Se o S e Mo. Gli esperimenti sono stati condotti nel breve periodo (24h) poiche’ selenato e molibdato sono stati somministrati ad alte concentrazioni (200 µM), le stesse del solfato in condizioni di normale apporto di S. Sono state condotte analisi relative all’accumulo di S, Se e Mo e ai paramentri di crescita. L’indice di tolleranza (% del peso fresco sul controllo) e il rapporto radici/fusti denuncia un maggiore stress per le piante cresciute in carenza di zolfo e in presenza di Se e Mo. Se e Mo sono risultati maggiormente assorbiti in carenza di S. Sia per quanto riguarda lo S totale , sia riguardo al ssolfato, la competizione con Se e Mo si nota solo tra le 6 e le 24 h di trattamento, mentre nel brevissimo periodo (entro 1 h) non si apprezzano cambiamenti significativi nell’assorbimento di S. Studio dei potenziali effetti del Selenio sulla tolleranza allo stress idrico da parte di Brassica juncea e Stanleya pinnata . Talune specie accumulatrici di Se (Brassica spp, Stanleya spp, Astragalus spp.) usano questa peculiarita’ per difendersi dai predatori (Freeman et al., 2007; Galeas et al. 2007 ; ) e recentemente si e’ visto che Brassica juncea , a differenza di quello che succede per altri elementi tossici o potenzialmente tossici, accumula selenio in grandi quantita’ anche nei fiori e nei semi, avvalorando l’ipotesi, per certe specie o sottospecie o addirittura per alcune popolazioni in via di speciazione, di un adattamento evoluzionistico nell’uso del selenio contro parassiti e predatori erbivori (Quinn C. et al., dati non ancora pubblicati).Su queste due specie sono state condotte prove su substrato ghiaioso e in idroponica in presenza (20 µM) e in assenza di Selenato di sodio. Il deficit idrico e’ stato applicato prima su substrato ghiaioso attraverso la mancata irrigazione, poi in idroponica attraverso l’aggiunta di polietilenglicol (PEG 10000), un composto organico che altera il Ψ idrico della soluzione nutritiva. Nei due esperimenti condotti in idroponica il potenziale e’ stato portato fino a -1.3 / 1.6 Mpa. Dalla misurazione della capacita’ fotosintetica sotto stress idrico, della biomassa e del Ψ idrico fogliare nei diversi esperimenti, si e’ arrivati alla conclusione che non vi e’ una evidente azione positiva del Se nei riguardi della tolleranza allo stress idrico se non per quanto riguarda la condizione di controllo (non stress) e di stress lieve (-0.5/0.6 Mpa). Questa azione, pur non marcata, si esplica nell’aumento di biomassa superiore in piante trattate con selenato rispetto a quelle non trattate in accordo con quanto riscontrato per semenzali di Triticum aestivum (Yao Xiaoqin et al. ; 2009). Studio delle potenzialita’ di Astragalus racemosus (iperaccumulatore di Selenio) e Astragalus convallarius (non accumulatore) nel ruolo di accumulatori di Nichel, Molibdeno, Cromo, Vanadio, Tungsteno, Arsenico, Tellurio e Selenio. Il genere Astragalus comprende diverse specie distribuite in america centro settentrionale, alcune delle quali adattate a vivere su suoli seleniferi e quindi in grado di sviluppare un’elevata tolleranza al Se, divenendo cosi’ iperaccumulatrici (Cowgill, 1990). Arsenico, Nichel Cromo, Vanadio, Tellurio, Tungsteno e Molibdeno sono tutti elementi la cui concentrazione nell’ambiente e’ aumentata a causa dell’attivita’ umana ( Bamhart 1997 ; Zarchinas et al 2004 ; Zoller et al. 1973 ; Gott and McCarthy 1966 ; ) . e l’obiettivo di questo studio e’ capire se gli adattamenti fisiologici nei confronti dell’accumulo di Se siano efficaci anche nei confronti di altri metalli pesanti e metalloidi, in particolare Cromo e Tellurio che presentano caratteristiche steriche simili al Selenio. Queste due specie di Astragalus sono state fatte germinare in piastra petri e quindi fatte crescere in camera di crescita per 3 mesi su substrato di agargel piu’ Murashige & Skooge e addizionato con sali degli elementi sopra indicati dosati in concetrazioni differenti in base al loro grado di tossicita’ (affinche’ le piante potessero crescere ed accumulare metalli senza patirne la tossicita’ fino a morirne troppo precocemente). I risultati hanno dimostrato che non sempre la specie iperaccumulatrice di Selenio ha le caratteristiche per tollerare l’accumulo anche di altri elementi (A. Convallarius si e’ dimostrato piu’ tollerante nell’accumulo di Vanadio). Studio degli effetti del Rame nella fisiologia di Populus trichocarpa ed in particolare sull’attivita’ della proteina Laccasi, coinvolta nella formazione della parete cellulare. Il Rame e’ coinvolto come cofattore, nel funzionamento di diverse dismutasi (proteine che trasformano i ROS in H2O2) oltre che essere cofattore per il funzionamento delle laccasi (Pilon et al. 2006). Allevando in agargel e moltiplicando diversi cloni di Populus trichocarpa generati a partire dallo stesso callo, con due diverse concentrazioni di Cu (0.1 µM e 5 µM) si e’ voluto verificare attraverso analisi dell’attivita’ proteica (western blot e native gel) se e quanto due concentrazioni di rame (una prossima al limite inferiore che ne decreta la carenza ed una piuttosto elevata ma ben entro i limiti di tossicita’), influenzino l’omeostasi cellulare. Una volta verificato la diversa intensita’ dell’attivita’ delle proteine in questione (MnSOD, Cu/ZnSOD, FeSOD, CCS ecc. In base alla concentrazione di rame fornita, si e’ passato ad uno studio preliminare dell’espressione della Laccasi in base alle due diverse concentrazioni di rame. Poiche’ la laccasi necessita anch’essa del Cu come cofattore (4 ioni Cu ) e poiche’ in uno stato di carenza o prossimo alla carenza di rame, la cellula inibisce l’espressione di tutte le proteine che richiedono rame e che non sono strettamente necessarie alla fotosintesi come invece lo e’ la plastocianina, si e’ voluto verificare se la laccasi subisse esattamente questo destino. Sono stati quindi disegnati dei primers (sequenze innesco per amplificare col metodo PCR il DNA retrotrascritto da RNA) che contemplassero l’amplificazione della sequenza bersaglio dei microRNA (sequenze brevi di RNA che in coppia col complesso proteico RISC tagliano e quindi inattivano l’RNA messaggero) (Yamasaky et al. 2007; Abdel Ghani et al 2008) in modo da avere, dopo la corsa elettroforetica su gel di agarosio, bande di intensita’ diversa in base all’azione di downregulation operata o meno dalla pianta. I risultati preliminari mostrano diversi livelli di espressione per diversi tipi di laccasi analizzati presenti nel fusto e nelle foglie,

L’analyse des données de biosurveillance (RNO/ROCCH, IFREMER, 2003-2014) des concentrations en argent (Ag) et cuivre (Cu) dans les huîtres sauvages de 13 sites de la côte atlantique française a montré des variations spatio-temporelles reflétant les pressions locales. De fortes relations entre Ag et Cu semblent en partie dues à des facteurs physiologiques impliqués dans leur bioaccumulation chez les huîtres. Le rapport élémentaire Cu/Ag a montré un potentiel intéressant pour détecter des sources de Ag et Cu, invisibles à travers les concentrations absolues qui sont caractérisées par une très forte variabilité inter-individuelle. Les mesures chimiques et les observations histologiques dans les huîtres sauvages de la Gironde, ont montré une accumulation préférentielle de Ag et Cu dans la glande digestive, accompagnée d’importantes dégradations cellulaires. La méthode innovante du dopage isotopique est très sensible et précise pour observer les cinétiques rapides d’accumulation in vivo de Ag et Cu par voie directe à des niveaux d’exposition réalistes. Les signaux isotopiques ont permis de quantifier l’accumulation rapide des ETM dans les différents organes et d’observer des phénomènes de co-régulation entre Ag et Cu. De plus, elle représente une réelle avancée pour des expérimentations écotoxicologiques permettant d’analyser l’état de contamination initial et après exposition dans le même individu. L’étude de la voie trophique suggère une accumulation de Ag supérieure à celle de Cu chez les algues. La fraction de Ag et Cu particulaire potentiellement biodisponible des particules estuariennes a été estimée à 60% et 82%, respectivement. Les résultats ont montré que cette estimation dépend du temps d’exposition et de la concentration en sédiment, pouvant entraîner d’importantes sous-estimations
The data-base analyses of the French biomonitoring program (RNO/ROCCH, IFREMER, 2003-2014) in terms of silver (Ag) and copper (Cu) concentrations accumulation in oysters from 13 sites along the French Atlantic Coast showed spatial and time-dependent variations reflecting local pressures. The high Ag and Cu relations seems to be partly due to physiological processes involved in their bioaccumulation by oysters. The Cu/Ag elementary ratios showed an interesting potential for Ag and Cu sources detection, which were invisible through the use of only absolute concentrations characterized by a high individual variability. Chemical measurements and histological observations performed in wild oysters from Gironde Estuary showed a preferential Ag and Cu accumulation in the digestive gland with important cellular damages. The innovative isotope spiking method is highly sensitive and precise to observe rapid in vivo Ag and Cu accumulation kinetics through direct pathway using close-to-real exposure concentrations. The isotopic signals allowed to quantify the rapid MTE accumulation in the different organs and to observe the Ag and Cu co-regulation. Furthermore, it represents a real step forward in ecotoxicological experiments allowing to obtain the initial and final contamination state inside the same organism. The study of the trophic pathway suggested a higher Ag accumulation in algae than for Cu. The Ag and Cu potentially bioavailable fraction in estuarine particles was estimated respectively at 60% and 82%. The results showed that this estimation depends on the exposure time and sediment concentration, which can lead to important under-estimations

L'activitat humana és una de les majors causes d'elevades concentracions de nutrients i substàncies tòxiques en els ecosistemes fluvials. Entre la gran varietat de factors que alteren aquests ecosistemes, l'eutrofització i la contaminació per metalls pesants són dos dels principals problemes ambientals en països desenvolupats. Els biofilms fluvials (també anomenats comunitats perifítiques) representen una eina valuosa per avaluar els efectes dels contaminants (ex. nutrients i metalls) en els ecosistemes aquàtics. Aquest treball pretén investigar el destí i els efectes del Cu en els ecosistemes fluvials centrant-se en les comunitats perifítiques. Diferents metodologies han estat desenvolupades i/o adaptades per investigar específicament la dinàmica del Cu, la seva toxicitat i bioacumulació en comunitats perifítiques naturals, i la interacció entre l'eutrofització i la toxicitat del Cu en aquests ecosistemes.
Human activity is one of the major causes of elevated concentrations of nutrients and toxic substances in fluvial ecosystems. Among the many ecosystem stressors, eutrophication and metal pollution are two of the major environmental problems in many developed countries. Fluvial biofilms (periphyton communities) are a useful tool for monitoring the effects of pollutants (e.g. nutrients and metals) into aquatic ecosytems. The present study aims to investigate the fate and effects of Cu in fluvial ecosystems focusing on periphyton communities. Different methodologies have been developed and/or adapted to specifically investigate the dynamics of Cu, its toxicity and bioaccumulation on natural periphyton communities, and the interaction between eutrophication and Cu toxicity in these ecosystems.

Zinc, copper and cadmium accumulation was measured in two herbivorous gastropod molluscs Austrocochlea constricta and Bembicium auratum from Lake Macquarie NSW an area with a history of trace metal pollution. The investigation consisted of three main parts. The first part examined the influence of organism mass and location within the Lake on whole body tissue metal concentrations. This part of the study also compared the distributions of tissue metal concentrations of populations from Lake Macquarie, a known polluted environment, with those of populations from Jervis Bay NSW, an unpolluted environment, to establish whether either species is a net accumulator of zinc copper or cadmium. The second part of the investigation examined a range of factors which may influence whole body metal concentrations. One location in Lake Macquarie was sampled monthly from August 1995 to July 1996. The factors examined were temporal variation, gender, breeding cycle, and tissue distribution. The final part of the investigation examined the detoxification and storage of excess metals in the gastropods from Lake Macquarie. The mechanisms studied were metallothioneins and granules. The tissue metal concentrations of both species were found to be independent of mass. Location within Lake Macquarie did not significantly influence tissue metal concentrations. Variation between individuals was the most significant contribution to overall variation, resulting in a positive skewing of sample trace metal distributions. B. auratum populations from Lake Macquarie had significantly higher copper and cadmium tissue concentrations and A. constricta populations had significantly higher zinc, copper and cadmium tissue concentrations than the populations from Jervis Bay. This suggests that regulation of these metals is not occurring. A. constricta may therefore be considered a net accumulator of zinc, copper and cadmium and B. auratum of copper and cadmium. Tissue metal concentrations did not vary significantly over time. It is suggested that the organisms are in equilibrium with their environment. B. auratum has higher natural equilibrium concentrations than A. constricta particularly for copper and cadmium, suggesting different routes of exposure, uptake or accumulation for the two species. Gender and breeding cycle did not significantly influence tissue metal concentrations. Most of the variability in total copper and cadmium concentrations of both species was explained by variability in gonad tissue metal concentration, while variability in the gonad and somatic tissues zinc concentration explained about an equal amount of the variability in total zinc concentration. A. constricta and B. auratum were both found to induce a cadmium binding protein which has some features in common with metallothionein. A protein of around 10 000 Da which binds approximately 60% of the soluble cadmium was isolated using gel filtration. This protein was further separated into two isoforms using anion exchange. The first isoform eluted at the same time as MT I and the second at the same time as MT II rabbit liver standard. Large cells containing granular material which stained positive for calcium were observed interspersed among the connective tissue immediately behind the columnar epithelial cells lining the gut wall in both species under a light microscope. Calcium positive granular particles were also observed within the columnar epithelial cells of B. auratum. These species have been shown to be net accumulators of the trace metals investigated, with the exception of zinc in B. auratum. It has also been established that organism mass, gender and reproductive state, the partitioning of metals between tissues, and temporal effects are not confounding factors for the purposes of comparing trace metal concentrations between populations. They should therefore be effective biomonitors of the trace metals investigated, with the exception of zinc in B. auratum.

On etudie l'indice cuprifere de santa blandina (bresil) provenant de l'alteration d'un skarn porteur d'une mineralisation sulfuree a chalcopyrite et bornite. Les produits argilomorphes provoquent l'epigenie des mineraux du skarn et des solutions percolantes provoquent l'evolution mineralogique de ces produits. Les analyses mineralogiques et cristallochimiques determinent la nature exacte de ces phases argilomorphes et montrent la difficulte de l'incorporation du cuivre dans un reseau regulier d'argile

碩士
國立臺灣海洋大學
水產養殖學系
106
In this study, the usage of cages made of AB65 copper alloys and hybrid copper alloys is evaluated in aquaculture. The released copper from copper alloys (0.10 ~ 1.00 ppm) were measured by atomic absorption spectrophotometer and the concentration were effective in inhibiting the growth of two common Vibrio sp. (V. alginolyticus and V. parahaemolyticus). In the three-month fouling test, the weight gain of groups without copper alloy was significantly higher (46.62 ~ 417.33 %) than those copper alloys (-15.99 ~ 17.02 %) (p < 0.05), indicating the copper alloy have better anti-fouling effect than commercial materials. The safety tests were further conducted in a three-month long period that hybrid grouper (Epinephelus fuscoguttatus × Epinephelus lanceolatus) were cultured in seawater with different cage materials. The concentration of released copper in copper alloys group (0.14 ppm) did not lead to over accumulation of copper in tissues, including gill (0.99~1.27 ppm), liver (25.29~25.52 ppm) and muscle (0.21~0.22 ppm), which were found no significant differences between control and treatment groups (p > 0.05). Additionally, the measurements of the weight gain, specific growth rate and survival rate were all shown not significant different between control and copper alloys groups. Hence, no adverse effects were found.

碩士
國立海洋大學
水產養殖學系
84
Abstract The experiment was to simulate the effects of acid rain on the accumulation of heavy metals in muscle of tilapia (Oreochromis niloticus, L). Tilapia averaged 10.5 g were reared for 8 weeks in water containing 4 metals with 3 concentrations (Cu: 3.5, 7.0 and 14.0 ppm; Cd: 0.4, 0.8 and 1.6 ppm; Pb: 7.0, 14.0 and 28.0 ppm; Zn: 13.0, 26.0 and 52.0 ppm) under 2 pH conditions (5.0 and 7.0). Each treatment combination had 2 replicates. Control group ( pH = 7, no metals added) had 4 replicates. Metals concentration in muscle was analyzed every 2 weeks. The results showed that Cu concentration in muscle of fish reared under pH 7.0 (8.91 ppm) was significantly(p ≦ 0.05) higher then that under pH 5.0 group(5.46 ppm). It may be attributed to the increase of Cu precipitation under high pH and the reaction towards the formation of copper sulfate which was easier absorbed by fish. Cu concentration in muscle increasd as Cu in water increased. Up to 6 weeks, Cu concentration in muscle increased with elapsed time. However, Cu concentration in muscle decreased from 6 to 8 weeks. Cd concentration in muscle of fish reared under pH 5.0 (0.41 ppm) was not significantly (p ＞ 0.05) different from that under pH 7.0 (0.39 ppm). Cd concentration in muscle increased as Cd in water increased. Cd concentration in muscle increased with elapsed time until 6 weeks and then decreased. Pb concentration in muscle of fish reared under pH 5.0 (1.98 ppm) environment was significantly higher then that under pH 7.0 (0.57 ppm). The higher Pb concentration in water, the higher Pb concentration in muscle. Pb concentration in muscle increased with elapsed time. Pb concentration in muscle of fish at 8 weeks was found significantly higher than that at 4 weeks, but there was no significant differences in Pb concentration in muscle of fish for 2, 4 and 6 weeks. Zn concentration in muscle of fish reared under pH 5.0 (131.49 ppm) was not significantly different from that under pH 7.0 (147.71 ppm). Zn concentration in muscle increased with increasing Zn concentration in water and elapsed time. There was no significantly difference in growth of fish among various pH and concentration of metals.

碩士
國立中央大學
環境工程研究所碩士在職專班
94
Abstract The objective of this study was to investigate potential sources of oyster contaminated by copper and arsenic in Sien-San area, Hsin-Chu City. Because most contaminants were regarded as coming from the Keya River, monitoring contaminant data from the river and the content of copper and arsenic in marine and sediment collected from web site and reports, which could indicate the fate of the selected metal in environment. Despite the data mentioned above, in situ sampling and analysis for selected metal in various matrixes including marine, sediment, and oyster were carried out in order to obtain the bioconcentration factor (BCF) and uptake rate constant of the selected metals with regard to oyster. By examining the BCF and uptake rate constant, the accumulative amounts of the metals in oyster can be predicted. The results of heavy metal analysis in various mediums and collected data from literatures indicate that there are no significant relation between contamination of Keya River and wastewater discharge from Science Park in Hsin-Chu City. The copper concentration in marine increases with increasing copper concentration in Keya River and decreases with increasing SS concentration in Keya River. Accordingly, the copper ions in marine and sediment were thought to result from plants near Keya River, but arsenic ions were not. As for the BCF, the ranges of BCF for copper and arsenic were set as104~106 and 102~104, respectively. The obtained BCF values in this study are slight higher than those in literature. The uptake rate constants (1/day) estimated for copper and arsenic are about 446~1066 and 3.21~4.63. Based on the predicted uptake rate constants, the accumulative amounts of heavy metal in oyster within one year can be calculated with which copper is about 512~1201 mg/kg and arsenic is about 2.21~3.97 mg/kg.

碩士
國立臺灣大學
農業化學研究所
93
Conventional cleanup technology is generally too costly, and often harmful to desirable soil properties (i.e., texture, organic matter) for the restoration of contaminated sites. More recently, increasing attention has been given to the development of a plant-based technology (phytoremediation) to remediate heavy metal contaminated soils without extensive excavation, disposal costs, and loss of topsoil associated with traditional remediation practices.The success of a phytoremediation process is dependent on adequate plant yield and high metal concentrations in plant shoots. The largest numbers of hyperaccumulating species in the world belong to Brassicaceae. The optimum plant for phytoextraction would be able to both tolerate and accumulate high levels of heavy metals and also grow with a high biomass yield. But there are many different species of Indian mustard in the world, and the growth and uptake of those Indian mustard are not clear. Therefore, the object of this study is to compare the growth and heavy metal accumulation of three Indian mustard (Brassica juncea) grown in soils contaminated by Copper, Zinc, Cadmium and Lead. The study site was located at the green house of National Taiwan University. The investigation was conducted from July 2004 to April 2005. Four salt solution were added to the air-dried soil to control the total concentration of four metals at 200, 400 mg Cu kg-1, 100, 200 mg Zn kg-1, 25, 50 mg Cd -1kg, 500, 1000 mg Pb kg-1. Three kinds of Indian mustard (Brassica juncea) grown in soils contaminated by zinc, cadmium, lead or coppe. The soil moisture content was maintained at 60% of the water holding capacity, by weighing and adding deionized water. Soil solution were collected directly by RSMS after seeding 0 , 7, 14, 21, 28, 35 day. Test plants were harvested after seeding 35 days, then harvested plant were digested by the H2SO4/H2O2 digestion method. The concentration of Cu, Zn , Cd, and Pb in soil solution and plant digested solution were determinated by inductively coupled plasma optical emission spectrometry (ICP-OES)(Perkin-Elmer 2000 DV). The total and available concentration of Cu, Zn , Cd, and Pb in soil were digested by aqua regia and EDTA, DTPA extractable methods , then determinated by atomic absorption spectrometry (Hitachi 180-30 type). The results indicated that the soil limiting concentrations of Cu and Zn of the three different species of Indian mustard were lower than 200 and 100 mg kg-1. The soil limiting concentration of Pb of the three different species of Indian mustard was 1000 mg kg-1, and the maximum Pb accumulation of the three Indian mustards was 200 mg kg-1, which was much lower than the accumulation of the Pb hyperaccumulator . The soil limiting concentration of Cd of the three different species of Indian mustard was 25 mg kg-1, and the maximum Cd accumulation of the three Indian mustards was 200 mg kg-1. It reaches the standard level of the Cd hyperaccumulator .The biomass of India and Pakistan Indian mustard were significantly higher than Afghanistan specie (p<0.05). There were no significant different of Cd accumulation between the three Indian mustard species. The Pb accumulation of the three Indian mustard species, the species of Afghanistan of Indian mustard was significantly higher than the species of Indian and Pakistan (p<0.05). The total Cd removal , the species of India and Pakistan were significantly higher than that of Afghanistan species (p<0.05). There were no significant different between the Pb removal of the three Indian mustard species. Harvestd at 7th day after applying EDTA, the Cd concentration of Indian mustard were increased form 200 to 330 mg kg-1, and the total removal of Cd were increased form 97 to 157 μg pot-1. Harvestd at 7th day after applying EDTA, the Pb concentration of Indian mustard were increased form 80 to 700 mg kg-1, and the total removal of Cd were increased form 38 to 250 μg pot-1. In conclusion, the ability of phytoremediation of the three species of Indian mustard are not different, and adding EDTA solution can significantly increase the uptake of heavy metal in contaminated soil.

Concentrations of selenium (Se), manganese (Mn), zinc (Zn), cadmium (Cd), lead (Pb) and arsenic (As) were measured in the water column, sediment and biota, in conjunction with selected physicochemical data, from representative wetland types at a mining site within Salt Lake County, Utah, USA. The selected field sites included Oolitic Pond (lentic) and Lee Creek (lotic), which are moderately contaminated brackish, alkaline aquatic wetlands managed by a copper mining industry. These fishless wetlands are located in a geographic region that poses risk to migratory shorebird populations from dietary Se. A spatial sampling study demonstrated the extent of variation in total Se concentration within the wetlands. With the exception of the sediment compartment, Se concentration did not differ significantly along the 2-mile length of Lee Creek or within the Pond. The differences in sediment total Se concentrations between the Creek East and West segments characterize lower Lee Creek as having two segments distinguished by unique processes that influence the sediment Se accumulation profiles. Se accumulation trends were observed temporally over 3-years (2000 to 2002) and over two seasons (spring and autumn). Total Se body burden in benthic invertebrates was more clearly associated with sediment/detritus Se concentrations than with surface water concentrations. Three invertebrate groups dominated the aquatic invertebrates assemblage in the lotic and lentic benthos; primary consumers (Chironomidae, Diptera), generalist feeders (Hemiptera) and predators (Odonata). The chironomid larvae accumulated 1.3 to 39 times the trace metal concentrations of the Hemiptera or odonate taxa, independent of trace metal type (essential or non-essential) or wetland occupation. Organism-specific factors, such as habitat selection and preferential feeding habits, were proposed to influence benthic invertebrate accumulation profiles by modifying trace metal exposure. Mixed diets, trophic omnivory and the complexity of wetland biogeochemistry limit the power of stable nitrogen fractionation signatures to define benthic food web relationships. Wetland site-specific processes impacted Se accumulating efficiency, with trace metal concentrations from 4 to 7 times greater within the lentic benthic system than the lotic. The fractionation of the natural abundant stable carbon isotopes revealed the importance of sedimentary and detrital organic carbon as dietary sources for the benthic food web. Sediment organic content was not significantly associated with sediment, or invertebrate, Se accumulation profiles. Ecological risk assessments based on sound understanding of metal chemistry and the interactions between the sediment matrix and benthic organisms are necessary to provide tools for environmental management.
Graduation date: 2005

The role of metal accumulation in the pathophysiology of neurodegenerative diseases has been a hot topic in recent years due to the possibility of its treatment by chelating agents. Although the mechanisms of neurodegeneration are well known, the role of metal accumulation is still unclear. The main limitation are unsatisfactory methods for in vivo metal imaging; the most widely used technique is magnetic resonance imaging (MRI). Our aim was to assess the possibility of using transcranial sonography (TCS) in differential diagnosis of neurodegenerative diseases and to further explore the underlying factors of echogenicity. In the first study, using TCS fusion with MRI, we focused on location verification of the commonly assessed structures (substantia nigra and nucleus lentiformis) and exclusion of possible focal structural changes affecting the echogenicity in WD and PD patients. Moreover, obtained MRI were used for semi-quantitative comparison with TCS images. Although TCS has been confirmed to be highly beneficial in differential diagnosis of Wilson's disease and it should be recommended as a screening method for extrapyramidal patients with atypical course of the disease, the direct relationship between TCS and metal deposits could not be proven. The obtained results from the ultrasound fusion...