M. Fernanda C. Leal

M. Fernanda C. LealUniversidade Fernando Pessoa | UFP · FP-ENAS, CEBIMED

· Ph.D., M.Sc.
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    Universidade Fernando Pessoa | UFP
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    • Associate Professor
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    Research Items (22)
    Metals may be released from toys via saliva during mouthing, via sweat during dermal contact, or via gastric and intestinal fluids after partial or whole ingestion. In this study it was determined the lead migration from toys bought on the Portuguese market, for children below 3 years of age. The lead migration was performed according to the European Committee on standards EN 71-3 that proposes a 2 h migration test which simulates human gastric conditions. The voltammetric determination of migrated lead was performed by anodic stripping voltammetry (ASV) at a bismuth film electrode (BiFE). For all the analysed toys, the values of migrated lead did not exceed the limits imposed by the European Committee on standards EN 71-3 (90 mg kg(-1)) and by the EU Directive 2009/48/EC (13.5 mg kg(-1)) on the safety of toys.
    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 mu g L-1) and in human urine (dynamic linear range comprised between 45 and 270 mu g L-1, and detection limit of 12 mu g L-1).
    An automatic system was developed to determine ofloxacin in biological fluids and pharmaceutical formulations. Drug detection was carried out by a potentiometric membrane sensor based on [bis(trifluoromethyl)phenyl]borate as molecular-recognition material. The tubular shaped detector system was solidly attached to the manifold, creating a high-throughput stable setup (50 samples per hour) appropriate for routine antibiotic assessment. Under the optimized flow conditions, the sensor displayed a mean detection limit of 1 × 10(-5) M, a linear response over the concentrations of 2 × 10(-5) to 5 × 10(-3) M (slope of 57.4 mV decade(-1)) and a wide working pH range (2.1 - 6.6). The procedure was successfully applied to ofloxacin analysis in pharmaceuticals (relative deviation lower than 6%) and biological fluids at levels usually found after drug administration of clinical doses (recoveries between 91 and 106%). No significant interference from common excipients found in commercial formulations and inorganic ions usually present in biological fluids was noticed.
    Metals such as copper and zinc are essential for the development and maintenance of numerous enzymatic activities, mitochondrial functions, neurotransmission, and also for memorization and learning. However, disruption in their homeostasis can cause neurodegenerative disorders such as the Alzheimer and Parkinson diseases. In this work, the speciation of copper and zinc in urine samples was carried out. To this end, free and total metal concentrations were determined by square wave anodic stripping voltammetry using a glassy carbon electrode coated with bismuth film. The digestion of the samples was performed in a microwave with the addition of oxidant reagents.
    An ofloxacin-selective electrode was developed for application in pharmaceutical preparations, serum and urine analysis. The electrode is based on tetrakis[3,5-bis(trifluoromethyl)phenyl]borate as ion exchanger in a poly(vinyl chloride) membrane. The sensor displayed a Nernstian response for ofloxacin over a wide concentration range (3×10−6–1×10−2 mol L−1) with a slope of 55±1 mV per decade of concentration and a practical detection limit of 1×10−6 mol L−1. The electrode was successfully applied to the determination of ofloxacin in samples with no pretreatment steps, adequate accuracy and precision and recoveries within the intervals of 94–107 %.
    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.
    The yield of cultures of Emiliania huxleyi, a coccolithophore marine micro-alga species, in natural seawater and in seawater previously exposed to elutriates of ZEBEN-06 and ZESTEC-56, two zeolitic-nature products (ZNPs) (supernatant of 2.0 g l−1 of ZNP in natural seawater, stirred for 30 min) was evaluated. Total concentrations of dissolved trace metals and organic ligands (and respective conditional stability constants) in the initial media and at 7th day cultures were determined, by anodic and cathodic striping voltammetry. The changes introduced by the ZNPs in the media significantly enhanced E. huxleyi growth yield, the effect being more extent in the ZESTEC-56 elutriate. It was observed that both the ZNPs enriched the seawater with trace and not toxic quantities of Mn. In addiction, ZEBEN-06 removed small fractions of Cu and Zn from the media. The ZEBEN-06 elutriate stimulated the cell exudation but that of ZESTEC-56 inhibited exudation. Algae were also incubated in a culture medium with 0.05 g l−1 ZESTEC-56 in situ, and the growth yield was similar to that of the control culture. ZEBEN-06 was not studied in situ because it was hard to distinguish its particles from the cells, during cells counting. The ZESTEC-56 in situ enriched the medium in Mn, Cu, Pb and Cd, but impoverished it in Zn. The cell exudation was about four times higher in the presence of the ZNP in situ. Liberation/adsorption of micro-nutrients at the surface of the zeolites seems to be the cause of the observed changes in the biological response of the algae. The yield of the algal growth has economic relevance in aquaculture. On the other hand, ZNP are cheap, only small amounts (few mg l−1) are required and the addition of some nicro-nutrients may be omitted. Therefore, the inclusion of zeolites in algal cultures in aquaculture may be economically advantageous. However, it is recommendable an previous investigation, in order to select the zeolitic characteristics and concentration that will maximise the algal yield in each particular case (alga nature and seawater trace metal contents).
    Growth of the marine alga, Emiliania huxleyi, and chemical changes in the culture medium were assessed in response to the addition of differing concentrations of ZESTEC-56, a product of zeolitic nature (PZN). E. huxleyi was inoculated in natural seawater containing 0.025, 0.050 and 0.10 g l−1 PZN. In addition, algae were also inoculated in a PZN elutriate (1.0 g l−1 PZN in seawater stirred for 30 min). Total concentrations of dissolved trace metals and organic ligands (and respective conditional stability constants) were determined, in all media including the controls, by anodic and cathodic striping voltammetry in the initial medium and after 7-day algal growth. When compared with the control culture, 0.025 and 0.050 g l−1 PZN in situ with E. huxleyi did not significantly change the micro-algae growth yield. The highest PZN amount (0.10 g l−1) caused a decrease both in growth rate and yield. In the PZN elutriate culture cell yield was significantly greater than the controls. The PZN caused changes in the total concentrations of dissolved metallic micro-nutrients, adsorbing some (e.g. Zn) and releasing others (e.g. Mn). The ion-exchange processes seem to be relatively slow, as the extent of changes in the elutriate culture, where the contact between PZN and medium was only 30 min, was much lower than that caused by in situ PZN for 7 days. In addition, the amount of exudates released per cell was also influenced by the PZN probably due to the changes in the available trace elements. This work indicated that the changes caused by zeolites in a culture medium, in terms of micro-nutrient available levels can influence the micro-algae yield in natural seawater cultures. Hence, it follows that the characteristics and concentration of the zeolite should be considered when one wishes to use it in a given aquacultural medium, for instance for stimulation of the bacterium-mediated decomposition of non-consumed fish feed at reduced levels of ammonia. Depending on the seawater trace element composition, characteristics and concentration of the zeolite and algal species, the presence of a suitable zeolite may improve the yield of the cultures, reducing the production costs. Otherwise, an inhibition of growth may occur, which is economically disadvantageous.
    The elutriate sediment toxicity test (ESTT) provides a measure of the amount of a substance that is exchanged between the sediment and the aqueous phase during resuspension processes such as floods or dredging operations. This study used ESTT with two complementary aims: a comparison of the elutriates of two estuarine sediments (anaerobic muddy [A] and aerobic sandy [B]) in terms of toxicity and a comparison of the response of three different microalgae (Emiliania huxleyi (coccolithophore), Dunaliella minuta (green alga), and Phaeodactylum tricornutum (diatom)) to each elutriate in terms of growth, heavy metals uptake, and organic ligands release or uptake in order to find eventual differences of sensitivity. The interpretation of the results was based on chemical speciation in the culture media. Both elutriates, particularly A, were much richer than seawater (control medium) in some heavy metals and organic ligands able to bind strongly heavy metals. Elutriate A slightly inhibited P. tricornutum growth but stimulated growth of E. huxleyi and D. minuta. Elutriate B stimulated the growth of the three algal species. Therefore, the diatom behaved differently from both the coccolithophore and the green alga. Strong complexation of trace metals by organic ligands could be the cause of absence of the metallic toxicity of the elutriates. Growth inhibition of P. tricornutum in elutriate A could be caused by ammonia-N and/or organic compounds. The concentration of the organic ligands decreased markedly (about 75%) in both elutriates after 10 d of incubation in contrast to the control culture, where their concentration increased about 50% because of exudation. This phenomenon was interpreted to result from ligand uptake by the algae, free or as metal complexes. This work demonstrated that beside the evaluation of toxicity of free heavy metals to alga species, the organic ligands must not be ignored. Depending on the amount of ligand present, the toxicity can be reduced (sequestration) or enhanced (better availability through uptake of metal-ligand complexes). Since the applied ESTT is a standard procedure (U.S. Environmental Protection Agency) for the evaluation of dredged material proposed for ocean disposal, it is necessary to discuss results obtained during toxicity tests with such elutriates in detail.
    The N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) is extensively used as pH buffer in culture media for testing chemicals. However, this study demonstrates that 0.01 M HEPES significantly reduces the rate of Cu, Pb, and Cd binding to Porphyra spp. and Enteromorpha spp. marine macroalgae. The HEPES also decreased the accumulation of Cu, Pb, and Cd but not Hg by these macroalgae. Both the extracellular adsorption and the intracellular uptake of the metals were influenced by HEPES to a similar extent. The HEPES also promoted the release of exudates by the algae, and these exudates form very stable complexes with Cu (and probably with other trace metal ions). The HEPES interference varied with the nature of the metal, the macroalga, and the season. The presence of 0.01 M HEPES in seawater cultures of the Emiliania huxleyi (a microalga) also interfered with E. huxleyi growth, liberation of Cu-complexing organic ligands, and Cu uptake. The HEPES, which displays surface activity, may facilitate the binding of metals to the algae for an initial exposure period. The metal taken up appears to stimulate the liberation of exudates that subsequently control the bioavailability of the metals and therefore metal uptake. Because HEPES can control the uptake of trace metals by algae and the production of organic ligands, the results obtained in cultures containing the HEPES pH buffer can be influenced by this component of the media.
    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 of Emiliania huxleyi grown in filtered seawater, enriched with nitrate and phosphate as well as its own exudates and those of Phaeodactylum tricornutum, Porphyra spp. and Enteromorpha spp., were used to investigate the effects of algal 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 (CSV and 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 in glutathione-like compounds), and growth inhibition (a decrease of final cell yield and growth rate) was caused by the addition of 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.
    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 ([Pb2+])) 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 ([Cu2+]=0.32 pM, [Cu]d=29 nM). Similar effects were observed for 310 pM initial [Pb2+] (25 nM [Pb]d); 120 pM Initial [Cd2+] (10 nM [Cd]d) as well as 400 pM initial [Cd2+] (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 [Cd2+] but not 400 pM initial [Cd2+] 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.
    Here it is demonstrated that both Porphyra spp. and Enteromorpha spp. of macro-algae display similar and very marked seasonal variations in their concentration factor (CF) of Cu, Pb, Cd and Hg in field conditions. The CF variations are specific for each metal and reproducible over several years. The way variations in the biological activity affect the equilibrium and kinetics of the interaction between trace metals and live algae was studied in vitro. Natural seawater was used as the culture medium. Voltammetry was used for the determination of natural organic ligands and trace metals except Hg, which was determined by mercury cold vapour after on-line pre-concentration. Titrations with the relevant metal demonstrated that the maximum binding capacity of the algae was not significantly dependent on the season for Pb (ca. 100 μmol gdry algae−1), Cd (ca. 50 μmol g−1) and Hg (80–100 μmol g−1). Marked seasonal variations were observed for Cu (ca. 40 μmol g−1 in January; 70 μmol g−1 in May; and 100 μmol g−1 in August). The conditional stability constants of metal–algae complexation sites were seasonally independent and similar for both algae: logKMS′=8.5±0.3 (Cu), 5.6±0.2 (Pb), 5.3±0.2 (Cd) and 18.0±0.3 (Hg). Exudates with a strong Cu complexing capacity (logK′CuL=12.47±0.06) were determined in cultures with added Cu, Pb or Cd concentrations, and identified by cathodic stripping voltammetry (CSV) as cysteine or glutathione. All the tested metals promoted the liberation of exudates, both cysteine- and glutathione-like ligands were exuded in the presence of Cu, only cysteine-like ligands in the presence of Pb, and only glutathione-like ligands in the presence of Cd, the rise depending of the season of the year, particularly for Cu. Highest levels were produced in the presence of added Pb. When exposed to either 1- or 100-μM total dissolved metal concentrations, the metal uptake, and its rate, varied with the season and the algae.
    Short-term kinetic experiments, carried out in natural coastal seawater (with predetermined background levels of trace metals and organic ligands, L) enriched with nitrate and phosphate, demonstrated that Emiliania huxleyi was able to uptake Cu very quickly. After 10 min of exposure (background Cu level in the inoculated cells: [Cu]total cellular = 9.3 x 10(-17) mol cell-1, [Cu]intracellular = 8.4 x 10(-17) mol cell-1, and [Cu]extracellular = 1.0 x 10(-17) mol cell-1) to a natural seawater which contained 29 nM total initial dissolved Cu concentration ([Cu]d) (29 nM [CuL] and 3.2 x 10(-13) M free Cu concentration, [Cu2+]) the intracellular Cu was already 28 x 10(-17) mol cell-1. This value corresponded to 85% of the intracellular metal observed in pseudoequilibrium conditions (after 24 h of exposure) and to 88% of the total metal sorption (adsorption plus uptake) after 10 min. In contrast, the external adsorption after 10 min was only 3.0 x 10(-17) mol cell-1 which corresponded to 60% of the extracellular metal in pseudoequilibrium conditions. Simultaneously occurred a very fast release of organic ligands (L) by E. huxleyi, the majority being identified by cathodic stripping voltammetry as glutathione. The production of exudates increased with both Cu concentration and exposure time. After 10 min of exposure, the production of exudates in a medium with 129 nM [Cu]d (72 nM [CuL] and 7.9 x 10(-13) M [Cu2+]) was 51 nM, about 42% of that observed in pseudoequilibrium. As the Cu complexes with the organic ligands present in the medium were very stable (logarithm of the conditional stability constant: 12.18 +/- 0.06) and the ligand concentration in the medium was relatively high (e.g. 123 nM CL in the medium with 129 nM initial [Cu]d after 10 min of exposure) most of the metal was organically bound in the medium.
    Marine microalgae (Emiliania huxleyi) were grown in seawater enriched only with nitrogen and phosphorus, without control of free metal concentrations using synthetic chelators. Complexing ligands and thiol compounds were determined by cathodic stripping volt-ammetry. Copper was added to these cultures, and ligands were produced in response to the copper addition. Parallel measurements of thiols showed that glutathione and other unidentified thiols (electrochemically similar to thioacetamide) were produced by the algae at rates and concentrations similar to those of the complexing ligands. Smaller amounts of thiols were produced when ligands including thiols were added to the culture. The results indicate that thiols can account for a major part or most of the copper-complexing ligands produced by these algae. Furthermore, a feedback mechanism exists in which the production of thiol-type complexing ligands is controlled by the free copper concentration, production already being stimulated by an increase of [Cu2+] from 0.4 to 1.5 pM. Incubations with added exudates, thiols, and salicylaldoxime (SA) showed much reduced copper toxicity even though copper uptake was increased by the exudates and the SA.
    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 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 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.
    The concentrations of dissolved Cd, Cu, Hg and Pb in seawater and algae (Enteromorpha spp. and Porphyra spp.) were determined in parallel on three beaches located in the Oporto coast (Portugal), during 8 successive months, comprising periods of low and high biological productivity. The concentrations of dissolved metals in seawater varied markedly during the sampling period and exhibited seasonality. The concentrations of Cd were high in autumn and winter, but low in spring. The concentrations of Cu and Hg were high in autumn, low in winter and peaked in May. The concentrations of Pb were low in autumn and winter, and also peaked in May. It is likely that biological activity was, at least partially, responsible for the observed seasonal changes. The mean concentrations of dissolved metals in seawater were similar to those reported for polluted and industrialized european coastal areas. Metal concentration factors, calculated month-to-month for each alga, metal and site, varied markedly during the sampling period, indicating that concentration factors based on single or few determinations could be misleading. On the contrary, mean concentration factors (calculated by dividing the mean metal concentration in the algae by the mean metal concentration in seawater) were relatively constant at all three sites, indicating that in the Oporto coast, Enteromorpha spp. can be used to estimate the mean concentration of dissolved Cd, Cu and Hg in seawater, and Porphyra spp., the mean concentration of Cd, Cu and Pb. However, considering that future estimates of the concentrations of these metals in seawater should be based on determinations of the metal contents in Enteromorpha spp. and Porphyra spp. carried out on several occasions spanning a considerable period of time, the use of these algae as monitors of heavy metal pollution can have limited practical advantages over the direct assay of the metals in seawater.
    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 HNO3 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.
    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 Cu-cysteine with chitin, the lability of Cut showed no marked dependence on the mass of PM: when the mass of PM was duplicated, an increase in lability by less than or equal to 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<1) the magnitude of that excess did not affect markedly the lability. But, when 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.
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