Speciation of heavy metals in environmental water by ion chromatography coupled to ICP-MS.

Ammann AA.

Swiss Federal Institute for Environmental Science and Technology (EAWAG), Dubendorf. [email protected]

Biogenic (e.g. phytochelatins, porphyrins, DOM) as well as anthropogenic (e.g. NTA, EDTA, phosphonates) chelators affect the mobility and cycling of heavy metals in environmental waters. Since such chelators can form strongly bound anionic heavy metal complexes that are stable and highly mobile, anion-exchange chromatography coupled to ICP-MS was investigated. A narrow bore HPLC system was connected to a micro concentric nebuliser for in-line sample introduction. A new chromatographic procedure based on a synthetic hydrophilic quaternary ammonium anion exchanger in combination with nitrate as a strong eluent anion, and gradient elution, provided high separation selectivity and a large analytical window. Low detection limits (nmol L(-1)) were achieved by on-column matrix removal and sample preconcentration. This allowed the method to be successfully applied to different environmental research areas. In ecotoxicological studies of heavy metal effects on algae low concentrations of metal EDTA complexes were determined in nutrient solutions without interference from high (buffer) salt concentrations. In groundwater, infiltrated by a polluted river, mobile metal EDTA species were observed. In river water of different pollution levels beside CuEDTA other anionic Cu-complexes were found in nmol L(-1) concentrations.

PMID: 11939532 [PubMed - in process]

Phytochelatins.

Rauser WE.

Department of Botany, University of Guelph, Ontario, Canada.

Publication Types:

Review

Review, Academic

PMID: 2197985 [PubMed - indexed for MEDLINE]

responses of the diatom Ditylum brightwellii with a gradual increase in copper.

Rijstenbil JW, Gerringa LJ.

NIOO-CEMO, Netherlands Institute of Ecology, Centre for Estuarine and Coastal Ecology, P.O. Box 140, NL-4400 AC NL, Yerseke, Netherlands. [email protected]

A continuous culture experiment was conducted to study interactions between copper-binding ligands released by light-limited Ditylum brightwellii, and toxic effects of Cu on this diatom. Over 6 months, the Cu concentration in the medium has been increased in seven steps (3-173 nM). At each Cu addition, Cu speciation, characteristics of Cu sorption to cellular binding sites, and cell characteristics were determined. Physiological effects of Cu were studied, using indicators for metal detoxification (thiols) and lipid peroxidation (malondialdehyde). Minor amounts of Cu (<1.4%) were chelated by a minimum amount of EDTA (57 nM), required to maintain a stable long-term continuous culture. The responses of D. brightwellii to Cu were monitored. (1) From 3 to 47 nM added Cu, decreasing pools of glutathione, increasing malondialdehyde contents, an increased release of lipophilic ligands, and cell lysis indicated the enhancement of lipid peroxidation. (2) From 47 to 94 nM Cu, a 16-fold increase in high-affinity (strong) hydrophilic ligands was measured (conditional stability constants K' approximately 10(12)) that complexed most Cu (maximum 97%); sexual reproduction was stimulated and cell volumes increased. (3) From 126 nM Cu, glutathione pools increased again, whereas cell division rates decreased slightly. (4) At 142 nM Cu, the number of lysed cells reached a maximum, as did the production of lipophilic compounds that complexed approximately 2% Cu. As the binding sites of the strong ligands became Cu-saturated above 142 nM Cu, larger amounts of Cu were bound to low-affinity (weak) dissolved ligands (3-30%) and cellular binding sites (0.2-2.5%). Probably due to saturation of organic complexes at 142 nM Cu, the MINEQL-calculated Cu2+ concentrations increased markedly; pCu values decreased from >11 to approximately 10; division rates were further inhibited; gamma-glutamylcysteine (phytochelatin precursor) was produced. (5) At 157 nM Cu, phytochelatin synthesis started, and Cu-sorption capacities (cell walls and internal binding sites) increased. (6) At 173 nM Cu, the phytochelatin pool sizes and the number of cellular Cu-binding sites increased further. These results suggest that ligands released by a dense bloom of D. brightwellii, either by active excretion or lysis, would have lower affinities for Cu (K' approximately 10(9)-10(12)) and moderate the availability of Cu less effectively than ligands in natural environments (10(13)-10(14)). In this diatom, the concurring release of ligands, enhanced malondialdehyde production, increasing numbers of presexual cells and cell enlargement may serve as early-warning signals for Cu toxicity, rather than metal-specific phytochelatins that appeared at a stage when cell division was already clearly inhibited.

PMID: 11755700 [PubMed - indexed for MEDLINE]

Phytochelatin production in freshwater algae Stigeoclonium in response to heavy metals contained in mining water; effects of some environmental factors.

Pawlik-Skowronska B.

Institute of Ecology, Polish Academy of Sciences, Experimental Station, Niecala 18/3, 20-080, Lublin, Poland. [email protected]

Production of phytochelatins (PC) in two freshwater, filamentous green algae of the genus Stigeoclonium, in response to heavy metals contained in mining water was studied. Stigeoclonium sp. grown abundantly in ditches with the mining water (southern Poland) accumulated high amounts of heavy metals. The other studied alga Stigeoclonium tenue Kutz. was isolated from unpolluted lake water in the Netherlands. Both algae exposed to the heavy metal mixture (17 microM; mainly zinc) contained in the hard, alkaline (pH 8.2) mining water produced similar amounts of phytochelatins (PC2 and PC3): 500-600 nmol SH g(-1) dry weight. After water acidification to pH 6.8, a > 2-fold increase of the total phytochelatin level as well as the appearance of longer chain peptide PC4 in the cells of both algae was observed. The concentration of labile forms of zinc in the acidified mining water was four times as high as that in the alkaline water. The heavy metal mixture (17 microM) in non-complexing solution of pH 6.8 caused a comparable phytochelatin production in both Stigeoclonium strains as the same mixture present in the acidified mining water. However, in the non-complexing solution of pH 8.2, the metal mixture induced in algae more PCs than at pH 6.8. A positive effect of bicarbonate enrichment in the solution on the PC production in S. tenue was also observed. Stigeoclonium sp. exposed to high concentrations (10 microM) of individual metals (Zn, Pb and Cd available as free cations) synthesised much higher amounts of phytochelatins (PC2-PC4) than in response to the metal mixture contained in the mining water. The order of PC induction by the studied metals in the Stigeoclonium sp. was Cd > Pb > Zn. Addition of suspended solid matter to the heavy metal solution resulted in essential quantitative changes in phytochelatins in algal cells; a considerable decrease of PC2, PC3 and PC4 levels was observed. The PC production in algae of the genus Stigeoclonium exposed to the heavy metal mixture is discussed in the context of various metal bioavailability and the algal adaptation to complex aquatic environment.

PMID: 11239685 [PubMed - indexed for MEDLINE]

Relationships between acid-soluble thiol peptides and accumulated Pb in the green alga Stichococcus bacillaris.

Pawlik-Skowro&nacute;ska B.

Institute of Ecology, Polish Academy of Sciences, Experimental Station, Niecala 18/3, 20-080, Lublin, Poland

Stichococcus bacillaris, an ubiquitous green microalga accumulated inorganic lead (Pb) from aqueous solutions extra- and intracellularly. In response to Pb uptake acid-soluble thiol peptides (glutathione - GSH and phytochelatins - PC) were synthesized. The proportion of the intracellular Pb uptake by algal cells was low and comprised only 3-6% of the total metal sorption. The intracellular uptake was dependent on external Pb concentration, time of metal exposure and cell metabolism. Pb accumulation in alga was determined by means of 210Pb radiometry. Reduced GSH and PC were determined in algal cells using HPLC with the post-column derivatization with Ellman's reagent. Within the studied concentration range 0.1-20 microM, inorganic lead caused a significant production of induced thiol peptides: PC (n=2-4) and some other unidentified oligopeptides, probably (GluCys)n. The time of appearance and the concentration of individual oligomers of phytochelatins were dependent on the external Pb concentration and time of metal exposure. In algal cells exposed to Pb, significant changes in the GSH level accompanying the formation of the induced thiol peptides were also observed. The GSH level decreased in the cells exposed to the lower (up to 10 microM) studied Pb concentrations or increased in the cells treated with higher (20 microM) Pb concentrations. The thiol groups originated from induced peptides (mainly phytochelatins) followed a stoichiometric relationship 2:1 to the intracellular Pb amounts, however, only at the lowest studied external concentration (0.1 microM). At higher concentrations (up to 2.5 microM), intracellular Pb concentration was equal or even exceeded (at Pb>2.5 microM) two to three times the level of induced thiols. S. bacillaris accumulated intracellularly by 46% more Pb in light than in dark and the level of induced thiol peptides was significantly higher in the cells exposed to Pb under illumination. The rapid formation of these peptides in S. bacillaris in response to Pb, and their elimination (by about 90%) when algae were placed into the Pb-free solution reveal a tight regulation of GSH and phytochelatin pools in the algal cells exposed to toxic metals. The obtained results suggest that both PCs and GSH are the primary line of defence against the Pb toxicity. Additionally, the induced thiol peptides in S. bacillaris could be a good indicator of intracellular Pb availability and stress at the metal concentrations found in polluted fresh waters.

PMID: 10958956 [PubMed - as supplied by publisher]

Determination of cadmium partitioning in microalgae and oysters: contribution to the assessment of trophic transfer.

Ettajani H, Berthet B, Amiard JC, Chevolot L.

CNRS, ISOMer, SMAB, Service d'Ecotoxicologie, Nantes, France.

Alternative methodologies have been applied to the study of cadmium transfer in a food chain: water, microalgae (Skeletonema costatum and Tetraselmis suecica), oysters (Crassostrea gigas). The potential bioavailability of Cd in organisms was assessed through partitioning at the cell or tissue levels, and the predictive value of this method was evaluated by determining directly the metal transfer in an experimental food chain model. Cd concentrations were lower in S. costatum than T. suecica, in controls as well as in contaminated algae. In both algal species, Cd was firmly bound to the cell wall or had entered the cell. Cytosolic Cd was bound to intracellular ligands, the biochemical characteristics of which were not consistent with the hypothesis of detoxification via phytochelatins. In both algal species, Cd was predominantly present in the insoluble fraction, but at pHs such as those existing in the digestive tract of bivalves, it was easily extracted from the cells. Thus, exposure to Cd through phytoplanktonic food induced a significant uptake of this metal in soft tissues of bivalves. Due to the difference in Cd accumulation in algae, Cd doses associated with S. costatum were lower than those bound to T. suecica. Moreover, oysters retained a lower percentage of the metal associated with S. costatum compared to T. suecica (9 and 20%, respectively, after 21 days of exposure). Cd doses potentially available to oysters exposed directly in sea water were considerably higher, and direct uptake induced the highest levels of Cd incorporation but only 2% of dissolved Cd was actually retained by oysters over 21 days of exposure. In the soft tissues of oysters, Cd was distributed equally between soluble and insoluble fractions. Cytosolic Cd was present predominantly in the heat-stable fraction and mainly bound to compounds of molecular weight equal to 13.5 kDa. Moreover, a positive correlation was observed between metallothionein-like protein (MTLP) levels and gross concentrations of Cd in the soft tissues of oysters. These data are consistent with the hypothesis of an important role of metallothioneins in Cd metabolism in oysters and suggest a potential availability of MT-bound fraction of Cd to the consumers. These data are in agreement with the response of oysters exposed to Cd in the field.

PMID: 11243323 [PubMed - indexed for MEDLINE]

X-ray absorption spectroscopy of cadmium phytochelatin and model systems.

Pickering IJ, Prince RC, George GN, Rauser WE, Wickramasinghe WA, Watson AA, Dameron CT, Dance IG, Fairlie DP, Salt DE.

Stanford Synchrotron Radiation Laboratory, Stanford University, CA 94309, USA. [email protected]

Higher plants, algae and some yeasts respond to potentially toxic heavy metals such as cadmium by synthesizing phytochelatins and related cysteine-rich polypeptides. We have used X-ray absorption spectroscopy to study the nature of cadmium binding in such peptides isolated from maize (Zea mays) exposed to low levels of cadmium, and in two synthetic cadmium-peptide complexes, Cd-(gamma-Glu-Cys)3Gly and Cd-(alpha-Glu-Cys)3Gly. We have used the synthetic ions [Cd(SPh)4]2-, [Cd4(SPh)10]2- and [S4Cd10(SPh)16]4-as crystallographically defined models for the cadmium site. The Cd K-edge extended X-ray absorption fine structure (EXAFS) data, together with the Cd K, LI, LII and LIII near-edge spectra, reveal a predominantly tetrahedral coordination of cadmium by sulfur in both the phytochelatin and synthetic peptide complexes. In particular, the Cd LIII-edge lacks a peak at 3534.9 e V which was found to be prominent for oxygen- or nitrogen-coordinated species. The Cd-S distance in the phytochelatin complex is 2.54 A. The Cd K-edge EXAFS does not show any isolated, well-defined Cd-Cd interactions; however, contrary to the conclusion of previous work, their absence is not necessarily indicative of isolated cadmium-thiolate ligation. Evidence from other studies suggests that high static disorder, combined with a large vibrational component, serve to effectively wash out this contribution to the EXAFS. The sulfur K-edge, moreover, shows a low-energy feature both in the phytochelatin and in the synthetic cadmium-peptide complexes which is consistent with sulfide bound in a cluster with cadmium as found for [S4Cd10(SPh)16]4-. This feature strongly suggests the presence of a polynuclear cadmium cluster in maize phytochelatin.

PMID: 9989220 [PubMed - indexed for MEDLINE]

Production of phytochelatins in the marine diatom Phaeodactylum tricornutum in response to copper and cadmium exposure.

Morelli E, Pratesi E.

Istituto di Biofisica, Via S. Lorenzo, 26, 56127 Pisa, Italy.

PMID: 9307434 [PubMed - indexed for MEDLINE]

Interaction of toxic trace metals and mechanisms of detoxification in the planktonic diatoms Ditylum brightwellii and Thalassiosira pseudonana.

Rijstenbil JW, Sandee A, Van Drie J, Wijnholds JA.

Netherlands Institute of Ecology, Centre for Estuarine and Coastal Ecology, Yerseke, The Netherlands.

Effects of cadmium (10 nM), copper (80 nM) and zinc (150 nM) additions were studied in the marine diatom Ditylum brightwellii and the riverine diatom Thalassiosira pseudonana. Defense against oxidative stress via cellular thiol (SH) pools and superoxide dismutase (SOD) activation, detoxification via phytochelatins and cell damage were monitored in metal-exposed exponential-phase cells and controls, grown in estuarine medium. Total SH and reduced+oxidized glutathione (GSH+GSSG) in T. pseudonana were much higher than in D. brightwellii. In T. pseudonana, total SH and GSH decreased at 322 nM Zn, and GSH increased at 80 nM Cu but decreased at 119 nM Cu. GSH:GSSG ratios were low, while phytochelatins were not detectable in metal-exposed D. brightwellii. Cd-exposed T. pseudonana made more phytochelatins than Cu-exposed cells, and in different proportions. At 322 nM Zn, SOD activity decreased in T. pseudonana. Zn caused a major, and Cu a minor increase of SOD activity in D. brightwellii; inhibition of photosynthesis was observed in Cu-exposed D. brightwellii, probably due to oxidative damage. The C:N ratios were higher and protein contents lower in Cu-exposed cells of both species, which might indicate excretion due to a loss of cell membrane integrity. From these results, it is hypothesized that T. pseudonana has evolved an effective detoxification mechanism as a result of a more severe exposure to toxic metals in rivers and estuaries. In contrast, D. brightwellii, a marine-estuarine species, cannot adjust well to metal exposure. Its poor defense against metal toxicity was marked by low SH-contents.

PMID: 7917426 [PubMed - indexed for MEDLINE]

Satofuka H, Fukui T, Takagi M, Atomi H, Imanaka T.

Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Yoshida-Honmachi, Sakyo-ku, Kyoto 606-8501, Japan.

Phytochelatins (PCs, (gamma Glu-Cys)(n)-Gly, n=2-11) are produced by higher plants, algae and some fungi in order to detoxify Cd(2+) by sequestration to form Cd-PCs complexes. In order to investigate what chemical structures of PCs are responsible for their metal-binding ability, various cysteine-rich peptides ((X-Cys)(7)-Gly, X=Glu, Asp, Lys, Gly, Ser and Gln) were chemically synthesized. Water-solubility, metal-binding property, and detoxification effect toward Cd(2+) were analyzed and compared with those of (gamma EC)(7)G. (SC)(7)G and (QC)(7)G were insoluble at pH below 10, and (GC)(7)G was not soluble at any pH between 1 and 12, indicating that charged side chains were at least required for the molecules to be solubilized in aqueous solution. By spectroscopic analyses using DTNB method and UV method, we found that (EC)(7)G and (DC)(7)G had almost equivalent abilities of Cd(2+)-binding as PC ((gamma EC)(7)G), indicating that the distance between each thiol group was not a major factor for the binding to Cd(2+). (beta DC)(7)G and (KC)(7)G interacted to Cd(2+) with fourth coordination as in the case of other soluble PC-related peptides. However, compared to (gamma EC)(7)G, (beta DC)(7)G displayed a slightly weaker binding to Cd(2+), and (KC)(7)G showed a drastic decrease in binding ability. The affinities of PC-related peptides toward Cd(2+) were evaluated as below; (gamma EC)(7)G=(EC)(7)G=(DC)(7)G>(beta DC)(7)G>>(KC)(7)G=weak binding. The results of Cd(2+)-detoxification assays were consistent with the affinity between Cd(2+) and the peptides. We concluded that the structure consisting of thiol and carboxyl groups were essential for the formation of a tight Cd-peptides complex such as Cd-PCs.

PMID: 11566332 [PubMed - indexed for MEDLINE]

Synthesis and stability of phytochelatins induced by cadmium and lead in the marine diatom Phaeodactylum tricornutum.

Morelli E, Scarano G.

Consiglio Nazionale delle Ricerche, Istituto di Biofisica, Pisa, Italy. [email protected]

The synthesis of phytochelatins (PC), intracellular metal-binding polypeptides characterized by a repeating sequence of gamma-glutamic acid- cysteine (gamma-Glu-Cys) pairs, has been studied in laboratory cultures of the marine diatom Phaeodactylum tricornutum exposed to Cd, Pb or Zn. Cd and Pb were able to induce PC of different degree of polymerization. The accumulation of the peptides follows a direct relationship with the metal exposure. No PC induction was observed in Zn-treated cultures, although the intracellular concentration of Zn increased during exposure. Both in short-term (7 h exposure, 10 microM Cd or Pb) and 3-day experiments (metal concentration less than 0.5 microM), the major fraction of total PC gamma-Glu-Cys subunits synthesized was polymerized as PC2 when cells were exposed to Pb, but as PC4 when cells were exposed to Cd. In short-term experiments about 50% of the gamma-Glu-Cys residues of the cellular pool of glutathione was quickly and almost quantitatively converted into PC. Recovery experiments, in which metal-stressed cells are suspended in a metal-free medium, showed a decrease of the PC pool and a concomitant increase of glutathione, suggesting a mechanism of degradation and release of metal-phytochelatin complexes.

PMID: 11695656 [PubMed - indexed for MEDLINE]

Phytochelatin production by marine phytoplankton at low free metal ion concentrations: laboratory studies and field data from Massachusetts Bay.

Ahner BA, Price NM, Morel FM.

R. M. Parsons Laboratory, Massachusetts Institute of Technology, Cambridge 02139.

Phytochelatins are small metal-binding polypeptides synthesized by algae in response to high metal concentrations. Using a very sensitive HPLC method, we have quantified phytochelatins from phytoplankton in laboratory cultures at environmentally relevant metal concentrations and in marine field samples. Intracellular concentrations of phytochelatin, in the diatom Thalassiosira weissflogii, exhibit a distinct dose-response relation with free Cd2+ concentration in the medium--not with total Cd(2+)--and are detectable even when the free Cd2+ concentration is less than 1 pM. In Massachusetts Bay, phytochelatin levels (normalized to chlorophyll a) in the particulate fraction are similar to those measured in laboratory cultures exposed to picomolar free Cd2+ concentrations and exhibit a decreasing seaward trend. Incubations of natural samples with added Cd2+ confirmed the induction of the peptides by this metal. Ambient phytochelatin concentrations thus appear to provide a measure of the metal stress resulting from the complex mixture of trace metals and chelators in natural waters.

PMID: 8078899 [PubMed - indexed for MEDLINE]

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