M. Fernanda C. Leal

Evidence for Strong Copper(I) Complexation by Organic Ligands in Seawater

Thiols like glutathione and cysteine form such stable complexes with copper(I) that they preclude the presence of copper(II). Conventionally seawater is titrated with copper(II) whilst monitoring the labile or reactive copper concentration by voltammetry or with other techniques, to determine the concentration of copper(II) binding complexing ligands in seawater. Titrations of seawater to which copper(I) binding ligands have been added reveal that the copper(I) binding ligands are detected when seawater is titrated with copper(II). The copper(II) in seawater is reduced to copper(I) within 2 to 40 minutes depending on the nature of the copper(I) binding ligand. The titrations of seawater with copper(II) thus give a response to the presence of copper(I) binding ligands indiscernible from that for copper(II) binding ligands. The stoichiometry of the detected apparent ligand concentrations for given concentrations of glutathione and cysteine suggest that 2 : 1 (thiol : Cu) complexes are formed. This was confirmed using voltammetry of free glutathione. Values of 21.2 and 22.2 were found for log β″CuL for glutathione and cysteine respectively (for the reaction of Cu′ + 2L′ ↔ CuL2). The complex stability is similar to that of natural organic species in the oceanic water column. The high stability of the copper(I) complexes was apparent from values of 32.1 and 32.6 for log β″Cu(I)L2 (for the reaction Cu+ + 2L″ ↔ CuL2) for the copper(I) complexes with glutathione and cysteine in seawater. Glutathione and other thiols are common in the marine system including the water column. It is therefore possible that at least some of the ligands detected in seawater, and previously assumed to be copper(II) binding ligands, are in fact strongly complexed as copper(I). The copper(I) oxidation state may thus be stabilised in seawater.

Influence of the nature of the exudates released by different marine algae on the growth, trace metal uptake and exudation of Emiliania huxleyi in natural seawater

Marine phytoplankton are known to release metal complexing ligands but little is known about the effect of exudates on the biological behaviour of the different microorganisms, including their toxicity and influence on trace metal availability. In this study,cultures ofEmiliania huxleyigrownin filtered seawater,enriched withnitrate andphosphateaswellasits ownexudates and those ofPhaeodactylum tricornutum,Porphyraspp. andEnteromorphaspp.,were used to investigate the effectsofalgal exudates on algal growth, uptake (extracellular adsorption plus intracellular uptake) of Cu, Pb, Cd, Zn, Fe, Mn, Ni and Co, and extent of exudation. Cathodic and anodic stripping voltammetry (CSVand ASV) were used to determine metals, both in the medium and taken up by the algae, and total complexing organic ligands in the medium. Among these ligands, thiol compounds (cysteine-like and glutathione-like) were quantified in the exudates of different origins and during the growth of E. huxleyi in media enriched with them. An improvement of the final cell yield of E. huxleyi was caused by the addition of Enteromorpha exudates (the richest inglutathione-likecompounds),andgrowthinhibition(adecreaseoffinalcellyieldandgrowthrate)wascausedbytheadditionof P. tricornutum exudates (the richest in cysteine-like compounds). The nature and concentration of the organic compounds present in the culture medium also influenced trace metal uptake and the concentration and composition of the exudates produced by E. huxleyi. Therefore, it can be speculated that a bloom of a species of algae that produces large amounts of specific exudates may favour or inhibit the local growth of other algal species and, in an extreme situation, change the biodiversity. D 2002 Elsevier Science B.V. All rights reserved.

Speciation of Cu, Pb, Cd and Hg in waters of the Oporto coast in Portugal, using pre-concentration in a Chelamine resin column 1☆

Abstract Sea water was collected monthly between January and March 1996 from the shore of the Oporto coast at three different representative sampling points and speciation of Cu, Pb, Cd and Hg were carried out. For the purpose, total particulate (retained on a 0.45 μm pore-size filter), particulate sorbed to inorganic matter, total dissolved, and dissolved and operationally labile metal was discriminated. The filters were digested by microwaves. The filtered water was pre-concentrated on a microcolumn of Chelamine, eluted with 5 ml of 2 mol l −1 HNO 3 and analysed by flame atomic absorption spectrometry (F-AAS) or mercury cold vapour (MCV-AAS) for Hg. Spike recovery percentages did not differ significantly ( P >0.05) from 100% for any metal studied. The organic matter (OM) destruction was performed by UV photolysis. For quality control of the results, determination of Cu, Pb and Cd total dissolved concentration by differential pulse anodic stripping voltammetry was also performed; for Hg only the pre-concentration in the column was changed to partial evaporation of the sea water, followed by MCV-AAS. No statistically significant difference was observed between the results provided by the comparative methods. Online pre-concentration with Chelamine showed to be expeditious and effective for extraction of the heavy metals in coastal waters. The distribution of each metal in the different fractions, including ascertainment of significant correlation between pairs of metal fractions and the respective partition coefficients, is discussed.

Exudates of different marine algae promote growth and mediate trace metal binding in Phaeodactylum tricornutum

Phaeodactylum tricornutum was grown in filtered natural seawater enriched with nitrate, phosphate, and silicate only (control) or with exudates from itself, from Emiliania huxleyi (a coccolithophore micro-alga), Porphyra spp. (a red macro-alga) or Enteromorpha spp. (a green macro-alga). Cathodic (and anodic) stripping voltammetry (C(A)SV) were used to determine the concentrations of trace metals, both in the medium and in the algae, as well as total Cu-complexing organic ligands in the medium and, among these, some thiols (compounds identified as cysteine- or as glutathione by CSV). Exudates of different marine micro- and macro-algae could cause allelopathic effects in P. tricornutum cultures. Cell yield of P. tricornutum was increasingly promoted by exudates of E. huxleyi >Porphyra >Enteromorpha. Although exudates strongly complex Cu (and probably other metals), their presence promoted Cu uptake. Significant changes of Ni, Cd, Fe, Zn and Mn uptake by P. tricornutum were also observed in the presence of exudates of different algal species. In addition, both intensity of production and nature of exudates released by P. tricornutum were markedly influenced by the presence of exudates of other algae, the allelopathic effects being very specific (variable from one species to another). Allelopathy will probably also occur in the aquatic environment, although to a lesser extent than in cultures, particularly during bloom events and may have effects on both chemical speciation and bioavailability of chemicals to phytoplanktonic species. Such changes might cause the predominance of some species over other species. Therefore, in future in vitro culture studies with the purpose of using them as models of the real environment, more attention should be paid to the role of algal exudates, in order to improve the environmental relevance and significance of the results.

Antagonistic interactions of Pb and Cd on Cu uptake, growth inhibition and chelator release in the marine algae Emiliania huxleyi

Abstract Some trace metals are known to act synergistically or antagonistically to influence phytoplankton growth limitation or toxicity. However, little is known about the effects of Pb and/or Cd on Cu uptake and liberation of exudates by marine micro-algae. The work presented here was undertaken using cultures of Emiliania huxleyi in coastal seawater enriched only with nitrate and phosphate, and in which the background total dissolved metal concentrations and Cu-, Pb- and Cd-complexing ligands had been previously determined. The concentrations of Cu, Pb and Cd, and the concentrations of released organic compounds and their ligand concentrations, in the cultures at different Pb and Cd levels, were monitored by cathodic and anodic stripping voltammetry (CSV and ASV). Compounds that behaved in CSV like cysteine and glutathione were also determined by an independent method. Addition of 10 nM Pb (64 pM ionic Pb ([Pb 2+ ])) reduced the growth rate but did not promote the liberation of organic ligands. The cellular levels of Cu decreased or did not change, depending on the age of the cultures, suggesting that Pb slightly antagonised Cu uptake ([Cu 2+ ]=0.32 pM, [Cu] d =29 nM). Similar effects were observed for 310 pM initial [Pb 2+ ] (25 nM [Pb] d ); 120 pM Initial [Cd 2+ ] (10 nM [Cd] d ) as well as 400 pM initial [Cd 2+ ] (25 nM [Cd] d ) reduced the rate of algal growth for the first 7 days, although the rate of cell division subsequently increased. It was also noted that 120 pM initial [Cd 2+ ] but not 400 pM initial [Cd 2+ ] slightly antagonised Cu uptake, whilst Cd strongly promoted the liberation of exudates, particularly those identified by CSV as glutathione. The effects of simultaneous additions of Pb and Cd (10 nM Pb and 10 nM Cd) could not be interpreted as simple additive effects.

Influence of the ratio copper(II) to ligand concentrations and the nature of entering and leaving ligands on the lability of copper(II) complexes

Abstract The influence of the ratio Cu : L on the lability of copper(II) complexes with simple ligands of different thermodynamic stabilities (L=EDTA, NTA, cysteine, proline or glycine) was investigated in the range 4:1–1:3. Chelex-100 and chitin were used parallel and batch wise, to cause the dissociation of the complexes, and the results were compared. A few experiments were also performed with the yeast, Saccharomyces cerevisiae . The effect of the exposure time, between 5 and 30 min, and mass (or number of sites) of the particulate matter (PM), always in large excess, were also studied. For all systems, but Cucysteine with chitin, the lability of CuL showed no marked dependence on the mass of PM: when the mass of PM was duplicated, an increase in lability by ≤ 10% was found. For cysteine and proline with Chelex-100, and cysteine and glycine with chitin, the lability of CuL complexes markedly increased with the time of exposure, which indicated that the kinetics of the substitution reactions were relatively slow. These results indicate the role of both the leaving and entering ligands in the kinetics of the ligand substitution reactions. The effect of Cu : L on the complex dissociation was more marked with chitin than with Chelex-100. When the leaving ligand is in excess ( Cu : L Cu : L > 1, the lability decreased with the increase of the excess of metal for cysteine or glycine with Chelex-100, and for all ligands in presence of chitin. The heterogenous behaviour of the PM may be partially responsible for these results. A maximum lability was observed for 1:1 ratio, particularly for the complexes that are thermodynamically more stable. In the presence of yeast cells, the tendencies mentioned above were confirmed. The present results reinforce the operational character of the speciation studies based on lability data, and that relationship between lability and bioavailability must be analysed for each individual case and should not be extrapolated to different PM or experimental conditions.

Cathodic voltammetric detection of diltiazem at a bismuth film electrode: application to human urine and pharmaceuticals

In this work it was developed a novel application of bismuth film electrodes (BiFEs) for the determination of the antihypertensive and coronary vasodilator diltiazem by square wave cathodic voltammetry. The bismuth film was deposited ex situ on a glassy carbon electrode for 90 s at -1.4 V vs. Ag/AgCl, from an acetate buffer (pH 4.5; 0.10 mol L-1) containing 5 or 30 mg L-1 Bi. Diltiazem analytical signal was obtained in phosphate buffer (pH 7.4; 0.25 mol L-1) where reduction takes place at -1.5 V vs. Ag/AgCl. The proposed methodology was applied to the quantification of diltiazem in pharmaceutical samples (dynamic linear range comprised between 90 and 900 µg L-1) and in human urine (dynamic linear range comprised between 45 and 270 µg L-1, and detection limit of 12 µg L-1).