Bioavailability of trace metals to aquatic microorganisms: importance of chemical, biological and physical processes on biouptake

An important challenge in environmental biogeochemistry is the determination of the bioavailability of toxic and essential trace compounds in natural media. For trace metals, it is now clear that chemical speciation must be taken into account when predicting bioavailability. Over the past 20 years, equilibrium models (free ion activity model (FIAM), biotic ligand model (BLM)) have been increasingly developed to describe metal bioavailability in environmental systems, despite the fact that environmental systems are always dynamic and rarely at equilibrium. In these simple (relatively successful) models, any reduction in the available, reactive species of the metal due to competition, complexation or other reactions will reduce metal bioaccumulation and thus biological effects. Recently, it has become clear that biological, physical and chemical reactions occurring in the immediate proximity of the biological surface also play an important role in controlling trace metal bioavailability through shifts in the limiting biouptake fluxes. Indeed, for microorganisms, examples of biological (transport across membrane), chemical (dissociation kinetics of metal complexes) and physical (diffusion) limitation can be demonstrated. Furthermore, the organism can employ a number of biological internalization strategies to get around limitations that are imposed on it by the physicochemistry of the medium. The use of a single transport site by several metals or the use of several transport sites by a single metal further complicates the prediction of uptake or effects using the simple chemical models. Finally, once inside the microorganism the cell is able to employ a large number of strategies including complexation, compartmentalization, efflux or the production of extracellular ligands to minimize or optimize the reactivity of the metal. The prediction of trace metal bioavailability will thus require multidisciplinary advances in our understanding of the reactions occurring at and near the biological interface. By taking into account medium constraints and biological adaptability, future bioavailability modeling will certainly become more robust.     

Favouring the bioavailability of Zn and Cu to enhance the production of lignin-modifying enzymes in Trametes versicolor cultures

Metal effect on the enzyme secretion in fungi is usually related to total concentrations but not to bioavailable metal species. In this work, we aimed at enhancing the secretion of lignin-modifying oxidoreductases in Trametes versicolor by favouring the bioavailability of essential metals. For this purpose, the fungus was exposed to Cu or Zn in liquid culture media exhibiting different complexation levels. Metal speciation was determined experimentally or theoretically to quantify free metal species, supposed to be the most bioavailable, and species complexed to ligands. Although Zn(2+) contents were high in media, Zn had no effect on the oxidoreductase production. Conversely, Cu highly induced the manganese peroxidase and laccase productions until 40 and 310 times when compared to unexposed controls. This inductive potential was highly correlated to Cu(2+) contents in media. Furthermore, in poorly complexing media, the response threshold of oxidoreductases to Cu greatly decreased and an unexpected production of lignin peroxidase occurred.     

Comparative metal content profiling of parasitic helminths by electron paramagnetic resonance spectrometry: significance for metalloprotein content

Variation in co-ordination geometries of metal ions bound to proteins imposes electronic states different from free (hydrated) ions in solution. Electron paramagnetic resonance spectroscopy has been used to analyse a selection of parasitic helminths for metal content as an initial step to determination of metallo-enzymes in their ES products under immune stress conditions. Characteristic paramagnetic resonance spectroscopy spectra show clear evidence for the presence of iron, copper, and manganese centres and in the selected parasites. The metals ions are identified as protein-bound as distinct from free metal ions present in aqueous solution, and distinguishable from parasite dietary components derived from host sources. Indication is given that superoxide dismutases may, in part, account for the metal ions observed. The use of electron paramagnetic resonance spectroscopy to identify specific protein-bound metals without prior isolation of the suspected protein is here applied.     

Research Advances on the Mechanisms of Heavy Metal Tolerance in Plants

Heavy metals impact on the cytoplasmic function in a number of different ways, principally by their binding to protein sulflhdryl groups, by producing a deficiency of essential ions and, eventually, by substituting the essemial ions. Other modes of toxicity are possible, including disruption of cell transport processes and oxidative damage by free radicals generated by metal redox cycling. Plants have developed a variety of biochemical defense strategies to prevent heavy metal poisoning. The possible defense mechanism in plant may involve: metal binding to cell walls, avoidance of uptake these toxic metal ions, reduction of heavy metal transport across the cell membrane, active efflux, compartmentalization and metal chelation. Phytochelatins that can tightly bind and sequester metals may play an important role in the accumulation of heavy metals and preventing them from entering the cell metabolic pathway, the rates of high molecular weight (HMW) metal phytochelatin complexes (Cd-Sa-complex) formation may be an important determinant of the plant tolerance. In addition, plants possess several antioxidant defense systems to protect themselves from the oxidative stress by heavy metals.     

Importance of Clean Techniques and Speciation in Assessing Water Quality for Metals

Trace metals in aquatic and soil systems exist in a number of different soluble and particulate forms that impact the effect of the metals on these ecosystems. Appropriate methods of sampling and analysis are required to accurately determine the low concentrations present. Although assessment of metals in many regulatory programs is based on data for total metal concentrations, such values rarely correlate with effects. Consequently, other means are needed for the prediction of risk. Bioavailability of metals depends on their speciation, whose importance was first established for copper in aquatic systems where the toxicity of metals is related to the activity of the free metal ion. Small concentrations of natural organic matter strongly complex metals ameliorating toxicity. Several electroanalytical techniques are available that allow the assessment of metal species. Recently, a modeling approach, the Biotic Ligand Model (BLM), has been applied to the prediction of acute toxicity. The model accounts for the effects of natural organic matter, pH, and hardness and is able to predict toxicity over several orders of magnitude of soluble metal concentration using only easily determined site parameters. Total metal concentrations in sediment cover several orders of magnitude with no distinction of sediments that cause effects and those that do not except at low total metal concentrations. Relating the metal concentration to the concentrations of sulfide and organic matter binding sites enables the sediments containing higher concentrations of metals to be divided into those that do and those that do not have adverse effects. It is essential that metal speciation be considered to realistically evaluate the potential of metals to pose risk.     

Metal oxidoreduction by microbial cells

Summary For many organisms, some heavy metals in external media are essential at low concentrations but are toxic at high concentrations. Strongly toxic heavy metals are toxic even at low concentrations. Recently, it was proven that changes of valencies of Fe, Cu and Mn were necessary for these metals to be utilized by organisms, especially microorganisms. The valencies of Hg and Cr are changed by reducing systems of cells in the process of detoxifying them. Thus, the processes of oxidoreduction of these metals are important for biological systems of metal-autoregulation and metal-mediated regulation. Metal ion-specific reducing enzyme systems function in the cell surface layer of microorganisms. These enzymes require NADH or NADPH as an electron donor and FMN or FAD as an electron carrier component. Electron transport may be operated by transplamsa-membrane redox systems. Metal ion reductases are also found in the cytoplasm. The affinities of metal ions to ligand residues change with the valence of the metal elements and mutual interactions of various metal ions are important for regulation of oxidoreduction states. Microorganisms can utilize essential metal elements and detoxify excess metals by respective reducing enzyme systems and by regulating movement of heavy metal ions.     

植物耐重金属机理研究进展

由于工业“三废”和机动车尾气的排放、污水灌溉及农药、除草剂和化肥的使用,严重地污染了土壤、水质和大气,其中土壤中的重金属（Ｈｇ、Ｃｄ、Ａｓ、Ｃｕ和Ａｌ）污染更为严重［１］。重金属在植物根、茎、叶及籽粒中的大量累积,不仅严重地影响植物的生长和发育［１～...     

Effects of metal speciation on growth of phytoplankton with special reference to iron

This paper is a compilation of studies concerning the effects of metal speciation on the growth of phytoplankton. Special attention is paid to the speciation and availability of iron in lake Tjeukemeer, The Netherlands. Under laboratory conditions the free ionic metal species generally appear to be most effective in determining metal availability and toxicity. A variety of factors controlling solubility, ion-exchange, complexation or chelation, sorption and electrostatical attraction of metal ions affect the metal speciation, mostly resulting in reduced availabilities. However, some organic metal chelates such as citrates, nitrillotriacetates and the specifically iron chelating siderophores, are sometimes more available than the corresponding free ions. The presence of other metals also influences the availability of a given metal by competing for the same binding sites on the algal cell. This competition leads to antagonism betweene.g. iron nutrition and cadmium toxicity in marine diatoms. In the eutrophic, alkaline, hard and humic lake Tjeukemeer, the free Fe³⁺ concentration is below measurable levels and does not match the iron requirement of the phytoplankton. So most of the algal iron must have been provided by the predominant inorganic iron colloids and particles bye.g. dissolution or photo-degradation (reduction). If the provision rates of available iron are slow in relation to that of iron uptake, the growth of some phytoplankton species may become iron-limited. Continuous culture work indicated that the iron fraction <0.2 m from Tjeukemeer,i.e., the soluble fraction, is about one third as much available as iron from NH₄Fe(SO₄)₂.12H₂O. Different phytoplankton species vary widely in their metal requirements and tolerances. Therefore, metal speciation and availability may affect species composition and succession within phytoplankton communities. So far the assessment of metal availability in natural waters has been complicated by the complex metal chemistry and by methodological limits.     

A spectrophotometric method for the determination of zinc, copper, and cobalt ions in metalloproteins using Zincon

Zincon (2-carboxy-2′-hydroxy-5′-sulfoformazylbenzene) has long been known as an excellent colorimetric reagent for the detection of zinc and copper ions in aqueous solution. To extend the chelator’s versatility to the quantification of metal ions in metalloproteins, the spectral properties of Zincon and its complexes with Zn²⁺, Cu²⁺, and Co²⁺ were investigated in the presence of guanidine hydrochloride and urea, two common denaturants used to labilize metal ions in proteins. These studies revealed the detection of metals to be generally more sensitive with urea. In addition, pH profiles recorded for these metals indicated the optimal pH for complex formation and stability to be 9.0. As a consequence, an optimized method that allows the facile determination of Zn²⁺, Cu²⁺, and Co²⁺ with detection limits in the high nanomolar range is presented. Furthermore, a simple two-step procedure for the quantification of both Zn²⁺ and Cu²⁺ within the same sample is described. Using the prototypical Cu²⁺/Zn²⁺-protein superoxide dismutase as an example, the effectiveness of this method of dual metal quantification in metalloproteins is demonstrated. Thus, the spectrophotometric determination of metal ions with Zincon can be exploited as a rapid and inexpensive means of assessing the metal contents of zinc-, copper-, cobalt-, and zinc/copper-containing proteins.     

Highlighting the roles of transition metals and speciation in chemical biology

Transition metal ions play key structural and functional roles, affecting structures of biomolecules and enzyme function. The importance of transition metal ions in chemical biology is, thus, undisputed. However, the aqueous chemistry of metal ions is complicated because they form species in several protonation and redox states. In the presence of metabolites, metal ions can also form coordination complexes. The existence of several species is relevant because enzymes and membrane receptors can distinguish between species even when they are rapidly equilibrating. Thus, metal ions, enzyme cofactors, and therapeutic agents are sensitive to the metal ion speciation chemistry because it affects their interaction with enzymes and other biomolecules. Speciation is also crucial for metal-containing bioorthogonal reactions, since water and metabolites stabilize active catalysts, affect chemoselectivity and reaction yields.     

PHYCOLOGY AND HEAVY-METAL POLLUTION

1. All heavy metals, including those that are essential micronutrients (e.g. copper, zinc, etc.), are toxic to algae at high concentrations. 2. One characteristic feature of heavy-metal toxicity is the poisoning and inactivation of enzyme systems. Many of the physiological and biochemical processes, viz., photosynthesis, respiration, protein synthesis and chlorophyll synthesis, etc., are severely affected at high metal concentrations. 3. Some algae inhabit waters chronically polluted with heavy-metal-laden wastes from mining and smelting operations; Nodularia sp., Oscillatoria sp., Cladophora sp., Hormidium sp., Fucus sp. and Laminaria sp., etc., occur in metal-rich waters. These algal forms are probably more capable of combating the toxic levels of heavy metals and this attribute is a result of physiological and/or genetic adaptations. The sensitivity or tolerance to heavy metals varies amongst different algae. The phenomena of multiple tolerance and co-tolerance may be exhibited by some algae. 4. Heavy-metal pollution causes reduction in species diversity leading to the dominance of a few tolerant algal forms. The primary productivity also decreases after metal supplementation. 5. The uptake and accumulation of heavy metals can be active (energy-dependent), passive (energy-independent), or both. 6. Heavy metals can be safely stored as intranuclear complexes by some algae. Notwithstanding this, some changes in the cell wall can enable the algae to tolerate heavy metals by checking the entry of the metals (exclusion mechanism). 7. The metal content of algae growing in a waterbody may yield valuable information for simulating heavy metal pollution: several species of Cladophora and Fucus have been extensively used for this purpose. 8. Several factors affect and determine toxicity of heavy metals to algae. At low pH, the availability of heavy metals to algae is greatly increased, as a consequence of which pronounced toxicity is evident. Hard waters decrease metal toxicity. Some ions, e.g., calcium, magnesium and phosphorus, can alleviate toxicity of metals. 9. The presence of other metals can influence toxicity of a heavy metal through simple additive effect or by synergistic and antagonistic interactions. Similarly, other pollutants can influence heavy-metal toxicity. 10. The toxicity of heavy metals depends upon their chemical speciation. Various ionic forms of a metal characterized by different valency states, may be differentially toxic to a test alga. 11. Amino acids, organic matter, humic acids, fulvic acid, EDTA, NTA, etc. can complex with heavy metals and render them unavailable. This may eventually lead to less toxicity. 12. Heavy-metal toxicity largely depends upon algal population density: the denser the population the more numerous the cellular sites available, leading to decreased toxicity.     

The biotic ligand model and a cellular approach to class B metal aquatic toxicity

The biotic ligand model (BLM) and a cellular molecular mechanism approach represent two approaches to the correlation of metal speciation with observed toxicity to aquatic organisms. The two approaches are examined in some detail with particular reference to class B, or soft metals. Kinetic arguments are presented to suggest situations that can arise where the BLM criterion of equilibrium between all metal species in the bulk solution and the biotic ligand may not be satisfied and what might the consequences be to BLM predictive capability. Molecular mechanisms of toxicity are discussed in terms of how a class B metal might enter a cell, how it is distributed in a cell, and how the cell might respond to the unwanted metal. Specific examples are given for copper as an organism trace essential metal, which is toxic in excess, and for silver, a non-essential metal. As class B metals all bind strongly to sulfur, regulation of these metals requires that all S(II-) species be accounted for in aquatic systems, even under oxic conditions.     

Mechanism of heavy metal ion activation of phytochelatin (PC) synthase: blocked thiols are sufficient for PC synthase-catalyzed transpeptidation of glutathione and related thiol peptides

The dependence of phytochelatin synthase (gamma-glutamylcysteine dipeptidyltranspeptidase (PCS), EC ) on heavy metals for activity has invariably been interpreted in terms of direct metal binding to the enzyme. Here we show, through analyses of immunopurified, recombinant PCS1 from Arabidopsis thaliana (AtPCS1), that free metal ions are not essential for catalysis. Although AtPCS1 appears to be primarily activated posttranslationally in the intact plant and purified AtPCS1 is able to bind heavy metals directly, metal binding per se is not responsible for catalytic activation. As exemplified by Cd(2+)- and Zn(2+)-dependent AtPCS1-mediated catalysis, the kinetics of PC synthesis approximate a substituted enzyme mechanism in which micromolar heavy metal glutathione thiolate (e.g. Cd.GS(2) or Zn.GS(2)) and free glutathione act as gamma-Glu-Cys acceptor and donor. Further, as demonstrated by the facility of AtPCS1 for the net synthesis of S-alkyl-PCs from S-alkylglutathiones with biphasic kinetics, consistent with the sufficiency of S-alkylglutathiones as both gamma-Glu-Cys donors and acceptors in media devoid of metals, even heavy metal thiolates are dispensable. It is concluded that the dependence of AtPCS1 on the provision of heavy metal ions for activity in media containing glutathione and other thiol peptides is a reflection of this enzyme's requirement for glutathione-like peptides containing blocked thiol groups for activity.     

Addressing speciation in the effect factor for characterisation of freshwater ecotoxicity—the case of copper

Purpose  

Determination of the ecotoxicity effect factor (EF) in life cycle impact assessment (LCIA) is based on test data reporting the total dissolved concentration of a substance. In spite of the recognised influence of chemical speciation and physico-chemical characteristics of the aquatic systems on toxicity of dissolved metals, these properties are not considered when calculating characterization factors (CFs) for metals. It is hypothesised that the main cause of the variation in reported EC50 values of Cu among published test results lies in different speciation patterns for Cu in the test media, and that the toxicity of Cu is predominantly caused by the free Cu²⁺ ion. Hence, the free Cu²⁺ ion concentration should substitute the total dissolved metal concentration when determining the EF.     

Bioreduction of precious and heavy metals by Candida species under oxidative stress conditions

The aim of the present work was to evaluate whether Candida species can reduce both precious and toxic pure metals from the respective molecular ions. From these results, the nanoparticles formed were studied using scanning electron microscopy with energy-dispersive spectroscopy, Raman spectroscopy, X-ray fluorescence spectroscopy and synchrotron radiation. Our results showed that the metal ions were reduced to their corresponding metallic nanoconglomerate or nanoparticles by Candida species. This is the first report on how yeasts of this genus are capable of achieving homeostasis (resilience) in the presence of metal ions of both precious and toxic metals by reducing them to a metallic state.     

Environmental and biochemical trace-metal speciation studies by radiotracers and neutron activation analysis

Although knowledge of the total concentrations of trace metals in the environment and living organisms is still the essential starting point for any toxicological evaluation, it is, however, not sufficient to explain the mechanisms responsible for retention and toxic effect of trace metals. Differentiation between their chemical and biochemical forms is necessary. The need to resolve the total concentration of trace metals into single chemical species poses great experimental difficulties and imposes the use of very sensitive analytical techniques for trace metal determinations after specific preseparation procedures of the different chemical species. Thus, once the chemical or biochemical metal species have been selectively isolated, the speciation becomes merely an analytical problem, which requires high sensitivity and accuracy, as well as the evaluation of matrix effects, blanks, contamination, loss, and sampling. This paper describes the use of nuclear and radiochemical techniques related to chemical and biochemical speciation problems at the Joint Research Centre, Ispra, with carrier-free radiotracers and radiochemical neutron activation analysis.     

X-ray absorption spectroscopy (XAS) of toxic metal mineral transformations by fungi

Fungi can be highly efficient biogeochemical agents and accumulators of soluble and particulate forms of metals. This work aims to understand some of the physico-chemical mechanisms involved in toxic metal transformations focusing on the speciation of metals accumulated by fungi and mycorrhizal associations. The amorphous state or poor crystallinity of metal complexes within biomass and relatively low metal concentrations make the determination of metal speciation in biological systems a challenging problem but this can be overcome by using synchrotron-based element-specific X-ray absorption spectroscopy (XAS) techniques. In this research, we have exposed fungi and ectomycorrhizas to a variety of copper-, zinc- and lead-containing minerals. X-ray absorption spectroscopy studies revealed that oxygen ligands (phosphate, carboxylate) played a major role in toxic metal coordination within the fungal and ectomycorrhizal biomass during the accumulation of mobilized toxic metals. Coordination of toxic metals within biomass depended on the fungal species, initial mineral composition, the nitrogen source, and the physiological state/age of the fungal mycelium.     

A high-performance liquid chromatography method for determining transition metal content in proteins

Transition metals are common components of cellular proteins and the detailed study of metalloproteins necessitates the identification and quantification of bound metal ions. Screening for metals is also an informative step in the initial characterization of the numerous unknown and unclassified proteins now coming through the proteomic pipeline. We have developed a high-performance liquid chromatography method for the quantitative determination of the most prevalent biological transition metals: manganese, iron, cobalt, nickel, copper, and zinc. The method is accurate and simple and can be adapted for automated high-throughput studies. The metal analysis involves acid hydrolysis to release the metal ions into solution, followed by ion separation on a mixed-bead ion-exchange column and absorbance detection after postcolumn derivatization with the metallochromic indicator 4-(2-pyridylazo)resorcinol. The potential interferences by common components of protein solutions were investigated. The metal content of a variety of metalloproteins was analyzed and the data were compared to data obtained from inductively coupled plasma-atomic emission spectroscopy. The sensitivity of the assay allows for the detection of 0.1-0.8 nmol, depending on the metal. The amount of protein required is governed by the size of the protein and the fraction of protein with metal bound. For routine analysis 50 microg was used but for many proteins 10 microg would be sufficient. The advantages, disadvantages, and possible applications of this method are discussed.     

In the absence of catalytic metals ascorbate does not autoxidize at pH 7: ascorbate as a test for catalytic metals

Trace amounts of adventitious transition metals in buffer solutions can serve as catalysts for many oxidative processes. To fully understand what role these metals may play it is necessary that buffer solutions be 'catalytic metal free'. We demonstrate here that ascorbate can be used in a quick and easy test to determine if near-neutral buffer solutions are indeed 'catalytic metal free'. In buffers which have been rendered free of catalytic metals we have found that ascorbate is quite stable, even at pH 7. The first-order rate constant for the loss of ascorbate in an air-saturated catalytic metal free solution is less than 6 X 10(-7) s-1 at pH 7.0. This upper limit appears to be set by the inability to completely eliminate catalytic metal contamination of solutions and glassware. We conclude that in the absence of catalytic metals, ascorbate is stable at pH 7.     

城市地表灰尘中重金属的来源、暴露特征及其环境效应

地表灰尘是城市环境重金属的“源”、“汇”载体,与环境质量和人类健康有着密切关系.分析了城市地表灰尘重金属污染研究的意义,综述了国内外城市地表灰尘重金属地球化学行为研究进展,包括地表灰尘重金属来源、暴露特征及其影响因素分析、环境效应等方面.提出应加强时间序列及小尺度微观研究;地理因素对地表灰尘中重金属暴露特征、环境效应的影响机理研究.