Acute toxicology to an estuarine teleost of mixtures of cadmium, copper and zinc salts

Effects of mixtures of chloride salts of cadmium, copper and zinc on survival, whole body residues, and histopathology of mummichog, Fundulus heteroclitus (L.), were investigated in synthetic sea water at 20‰ salinity and 20°C. Mixtures of Cu²⁺ and Zn²⁺ as indicated by 96 h bioassay studies produced more deaths than expected on the basis of toxicities of individual components. Concentrations of Cd²⁺ not ordinarily lethal exerted a negative effect on survival of fish intoxicated by salts of copper, zinc, or both.
Atomic absorption determinations of Cd, Cu, and Zn residues in mummichog which survived 96 h exposures to each of these toxicants provided useful indices of total body burdens for these metals. Residues from survivors held in mixtures, especially Cd²⁺ and Zn²⁺ mixtures, did not conform to patterns observed for single elements. Whole body aggregates of Cd, Cu, and Zn from dead mummichogs were of limited worth owing to possible accumulation of these metals from the medium after death.
Renal and lateral line canal lesions were noted in all fish subjected to copper concentrations of 1 mg/1 and higher. Renal lesions observed in fish immersed in mixtures of Cu²⁺ and Cd²⁺ assumed a damage pattern characteristic of Cd²⁺; with mixtures of Cu²⁺ and Zn²⁺, lesion were typical of Cu²⁺-induced damage. Lesions induced in lateral line epithelium by Cu²⁺ were not affected by either Cd²⁺ or Zn²⁺. Epithelia lining the oral cavity were necrotized by the caustic action of high levels of Zn²⁺ (60 mg/1) and of Cu²⁺ (8 mg/1).     

Decreased cupric ion uptake as the mechanism for cupric ion resistance in Escherichia coli

Abstract Plasmid-encoded copper (Cu²⁺) resistance in Escherichia coli was due to decreased uptake of Cu²⁺. The Cu²⁺-resistant E. coli Rtsl strain contained a 60 MDa plasmid which is known to encode for both Cu²⁺ and kanamycin resistance. A plasmid-free derivative of the same organism exhibited a greater uptake of Cu²⁺, and sensitivity to Cu²⁺ in both respiration and growth studies than the E. coli Rtsl strain.     

The regulation of Mn2+ and Cu2+ uptake in cells of the yeast Candida utilis grown in continuous culture

Abstract Transport of Mn²⁺ was repressed in Candida utilis cells grown in continuous culture in high-Mn²⁺ (100 μM Mn²⁺) medium as compared to cells grown in basic (0.45 μM Mn²⁺) and low-Mn²⁺ (< 0.05 μM Mn²⁺) media. In contrast, no repression of Cu²⁺ uptake occurred in high-Cu²⁺-grown (25 μM Cu²⁺) cells as compared to cells grown in basic medium (0.54 μM Cu²⁺). Cu²⁺-limited cells did not hyperaccumulate Cu²⁺ and there was not significant difference in initial uptake rates for all 3 Cu²⁺ conditions. Mn²⁺ uptake appears to be regulated by a mechanism sensitive to the external Mn²⁺ concentration, whereas Cu²⁺ transport is not governed in this way by the external Cu²⁺.     

Potentiation of 45Ca Uptake and Acute Toxicity Mediated by the N-Methyl-D-Aspartate Receptor: The Effect of Metal Binding Agents and Transition Metal Ions

Abstract: The activities mediated by the N -methyl-D-aspartate (NMDA) receptor were studied in cultured rat cerebellar granule cells. Micromolar concentrations of the metal binding compounds, EDTA, cysteine, and histidine, as well as serum albumin strongly potentiated receptor activity in the presence of millimolar concentrations of Ca²⁺ and Mg²⁺. The findings indicated that these agents remove an endogenous metal, probably Zn²⁺, which attenuates NMDA receptor-mediated ⁴⁵Ca uptake and toxicity. Several added metal ions were therefore tested at low micromolar concentrations. Zn²⁺ was found to be the most potent inhibitor of NMDA-induced ⁴⁵Ca uptake, followed by Cu²⁺ and Fe²⁺. Co²⁺, Cd²⁺, Fe³⁺, and AI³⁺ had no significant effect, whereas Ni²⁺ potentiated the ⁴⁵Ca uptake but inhibited at much higher concentrations. The potentiating agents that remove the endogenous metal had a particularly dramatic effect in the presence of Mg²⁺, the voltage-dependent suppressor of the NMDA receptor. Mg²⁺ also played an important role in the inhibitory effect of added Zn²⁺. Much lower concentrations of Zn²⁺ were needed to achieve inhibition of NMDA-induced ⁴⁵Ca uptake in the presence of Mg²⁺. Under a variety of conditions, a very good correlation was found between NMDA receptor-mediated ⁴⁵Ca uptake and the magnitude of acute neurotoxicity.     

Cupric ions induce both an efflux of potassium and low molecular mass metabolites in Pseudomonas syringae

Abstract Cu²⁺ induced an efflux of potassium, inorganic phosphate, 260 nm-absorbing materials and ribose-containing molecules in Pseudomonas syringae . No detectable amounts of aspartic and glutamic acids leaked from the cells. It is proposed that the release of inorganic phosphate and other low molecular mass anionic metabolites probably played a role in re-equilibrating the internal charge balance after the exit of K⁺ ions.     

Mechanism of copper-enhanced photoinhibition in thylakoid membranes

The effect of copper on photoinhibition of photosystem II (PSII) in vitro was studied in bean ( Phaseolus vulgaris L. cv. Dufrix) and pumpkin ( Cucurbita pepo L.) thylakoids. The thylakoids were illuminated at 200–2 000 μmol photons m⁻² s⁻¹ in the presence of 70–1 830 added Cu²⁺ ions per PSII. Three lines of evidence show that the irreversible damage of PSII caused by illumination of thylakoids in the presence of Cu²⁺ was mainly due to donor-side photoinhibition resulting from inhibition of the PSII donor side by Cu²⁺. First, addition of an artificial electron donor partially restored PSII activity of thylakoids that had been illuminated in the presence of Cu²⁺. Second, already moderate light was enough to cause rapid inhibition of PSII, and the inhibition could be saturated by light. Third, the extrinsic polypeptides of the oxygen-evolving complex were found to become oxidized by the combined effect of Cu²⁺ and light. The presence of oxygen was not necessary for the copper-induced enhancement of photoinhibition of PSII. When the illumination was prolonged, copper caused a gradual collapse of the thylakoid structure by increasing degradation of thylakoid proteins.     

Chromosomally-determined induced tolerance to copper in Escherichia coli

Pre-exposure of Echerichia coli to sub-lethal concentrations of cupric sulphate induced copper tolerance: pre-exposed (habituated) organisms were essentially unaffected by concentrations of Cu²⁺ which completely prevented colony formation by non-habituated ones. The observed copper tolerance was not dependent on the selection of copper-resistant mutants but resulted from a phenotypic change in the organisms during the pre-exposure period.     

Photosynthetic and respiratory electron transport in Cu2+-deficient Dunaliella

Growth inhibition of the green alga Dunalietla parva Lerche has been observed during cultivation in low Cu²⁺ media. A minimum endogenous Cu concentration for unrestricted growth of 100 to 200 nmol ml⁻¹ packed cell volume was estimated. At lower concentrations, Cu deficiency causes a decrease in photosynthesis and respiration. Assay of photosynthetic electron transport rates as well as the determination of several redox components showed that the target of Cu deprivation in the photosynthetic apparatus is the synthesis of Cu-containing plastocyanin. Consequently, inhibited formation of plastocyanin resulted in low activities of photosynthetic electron transport. A secondary, indirect effect of Cu deficiency is the reduction of thylakoid formation resulting in an additional decrease of photosynthesis compared to cultures with sufficient Cu²⁺.
The inhibitory influence of low Cu²⁺ on respiration was located at the site of cytochrome oxidase. In contrast to blue-green algae, a strong coordination of the biosynthesis of the cytochrome oxidase complex was evident. During restricted Cu²⁺ supply the formation of cytochiome aa₃ , another component besides Cu, was stalled. The resulting low activities of cytochrome oxidase are responsible for decreased respiratory electron transfer activity from NADPH to oxygen. At Cu²⁺ concentrations which exert only moderate effects on Dunalietla , the cytochrome oxidase reaction was more strongly affected than the photosystem I reaction.     

Ascorbate-Induced Oxidative Inactivation of Zn2+-Glycerophosphocholine Cholinephosphodiesterase

Abstract: Zn²⁺-glycerophosphocholine cholinephosphodiesterase, responsible for the conversion of glycerophosphocholine into glycerol and phosphocholine, was inactivated during incubation with ascorbic acid at 38°C. The inclusion of copper ions or Fe²⁺ accelerated the ascorbate-induced inactivation, with Cu²⁺ or Cu⁺ being much more effective than Fe²⁺, suggestive of ascorbate-mediated oxidation. Dehydroascorbic acid had no effect on the phosphodiesterase, but H₂O₂ inactivated the enzyme in a concentration-dependent manner. Also, the enzyme was inactivated partially by a superoxide anion-generating system but not an HOCl generator. In support of involvement of H₂O₂ in the ascorbate action, catalase and superoxide dismutase expressed a complete and a partial protection, respectively. However, hydroxy radical scavengers such as mannitol, benzoate, or dimethyl sulfoxide were incapable of preventing the ascorbate action, excluding the participation of extraneous ^•OH. Although p -nitrophenylphosphocholine exhibited a modest protection against the ascorbate action, a remarkable protection was expressed by amino acids, especially by histidine. In addition, imidazole, an electron donor, showed a partial protection. Separately, when Cu²⁺-induced inactivation of the phosphodiesterase was compared with the ascorbate-mediated one, the protection and pH studies indicate that the mechanism for the ascorbate action is different from that for the Cu²⁺ action. Here, it is proposed that Zn²⁺-glycerophosphocholine cholinephosphodiesterase is one of brain membrane proteins susceptible to oxidative inactivation.     

THE RELEASE OF COPPER-COMPLEXING LIGANDS BY THE BROWN ALGA FUCUS VESICULOSUS (PHAEOPHYCEAE) IN RESPONSE TO INCREASING TOTAL COPPER LEVELS

The growth of Fucus vesiculosus L. germlings in chemically defined culture media containing a range of Cu concentrations (20–1000 nM) was monitored simultaneously with measurement of the Cu speciation in the media by competitive equilibrium-adsorptive cathodic stripping voltammetry. Fucus vesiculosus germlings were found to exude Cu-complexing ligands with conditional stability constants of the order of 1.6 × 10¹¹. Ligand concentrations increased with increasing total dissolved Cu concentrations (Cu_T) until a concentration of 500–800 neq Cu·L⁻¹ was reached. Concentrations of the ligand exceeded Cu_T in treatments containing 20 and 100 nM Cu, were similar to Cu_T in the 500-nM Cu treatment, but were less than Cu_T in the 1000-nM treatment. Therefore, [Cu²⁺] were calculated to be at concentrations of 10⁻¹¹− 10⁻¹⁰ M in the 20- and 100-nM treatments, 10⁻⁹ M in the 500-nM treatment, and 10⁻⁷ M in the 1000-nM treatment. Growth rates were lowest at Cu²⁺ concentration > 10⁻⁹. These results are discussed within the context of the potential roles for exuded copper-complexing ligands.     

Bactericidal activity of catechin-copper (II) complexes on Escherichia coli ATCC11775 in the absence of hydrogen peroxide

Washed Escherichia coli ATCC11775 cells were killed by (–)-epigallocatechin (EGC) in the presence of a non- lethal concentration of Cu²⁺ (1 μmol l⁻¹) without additional H₂O₂, but not by (–)-epicatechin (EC). EGC alone (< 0·1 mmol l⁻¹) did not reduce the viability of the cells. The survival curve obtained in the presence of EGC and Cu²⁺ was similar to that obtained in the presence of (–)-adrenaline (EN) and Cu²⁺.     

GRAPPLING WITH CONFLICT AMONG INFORMATION SOURCES IN RECONSTRUCTION OF THE EARLY EVOLUTION OF LAND PLANTS

In an assay addressing the toxicity of copper to Dictyosphaerium pulchellum , a 48 hours' exposure of axenic cultures of the alga to metal concentrations ranging from 0 to 300 mg L⁻¹ caused a significant decrease in the growth rate and cell chlorophyll content. Extracellular mucilage remotion by controlled sonication before exposure to copper emphasized chlorophyll content reduction, but not cellular density diminution. Because mucilage acts as a cation interchanger, these results suggested that, during at least the first 48 hours, division rates might be affected by lower Cu⁺² concentrations than chlorophyll content. Ultrastructural observations showed that the normal organization of the chloroplasts was altered; they presented dilated lamellae, irregularly arranged with practically no stacking. Copper precipitates were mainly observed outside the plasmalemma or within the extracellular mucilage. Electron probe microanalysis confirmed these observations, showing only minor precipitates inside the cells.     

Removal of heavy metals using a brewer's yeast strain of Saccharomyces cerevisiae: advantages of using dead biomass

Aim:  The capacities of live and heat-killed cells of Saccharomyces cerevisiae at 45°C for the removal of copper, nickel and zinc from the solution were compared.
Methods and Results:  Kinetic studies have shown a maximum accumulation of Ni²⁺ and Zn²⁺ after 10 min for both types of cells, while for Cu²⁺ this was attained after 30 and 60 min for dead and live cells, respectively. Equilibrium studies have shown that inactivated biomass displayed a greater Zn²⁺ and Ni²⁺ accumulation than live yeasts. For Cu²⁺, live and dead cells showed similar accumulation. Fluorescence, scanning electron microscopy and infrared spectroscopy studies have shown that no appreciable structural or molecular changes occurred in the cells during the killing process. The increased metal uptake observed in dead cells can be most likely explained by the loss of membrane integrity, which allows the exposition of further metal-binding sites present inside the cells.
Conclusions:  Heat-killed cells showed a higher degree of heavy metal removal than live cells, being more suitable for further bioremediation works.
Significance and Impact of the Study:  Dead flocculent cells can be used in a low cost technology for detoxifying metal-bearing effluents as this approach combines an efficient metal removal with the ease of cell separation.     

sse of photosynthetic parameters for the diagnosis of copper deficiency in Pinus radiata seedlings

Six-month-old water cultures of Pinus radiataI D. Don seedlings showed optimal growth, and the highest CO₂ assimilation and photosystem I-dependent ascorbate/dichlorophenolindophenol → NADP⁺ electron flow, at 3.0 uM Cu²⁺ (excess) in the hydroponic media. In the nine-month-old water cultures, when the early Cu deprivation has been overcome, the optimum for plant growth and CO₂ fixation shifts to 0.3 u M Cu²⁺ (normal); at that time, the 3.0 uM Cu²⁺ water cultures showed toxic symptoms of foliar chlorosis. Under Cu²⁺ deficient levels (0.03 uM) a clear decrease in the photosystem I-linked electron transport and CO₂ assimilation rates, as well as in the whole plant development, could be observed. Both six- and nine-month-old water cultures showed a close relationship between the Cu²⁺ concentration of the media and the foliar Cu content. However, leaf chlorophyll and the Cu content of thylakoid lamellae showed such a correlation only in the Cu²⁺ deficient and Cu²⁺ normal water cultures. The conclusion from these results is that the electron transport rate ascorbate/dicblorophenolindophenol → NADP⁺, and the Cu content of the photosynthetic membranes, can be used to diagnose a Cu deficiency in Pinus radiata plants.     

Activity of methanotrophic bacteria in Green Bay sediments

Abstract Sediment pore water samples obtained from a 19 m station in Green Bay in Lake Michigan were examined for levels of ambient dissolved methane and copper, and for the potential for in situ methane oxidation by methanotrophs found within surface sediments. The in situ methane concentration in the upper oxic sediment layer ranged from 20–150 μmol · 1⁻¹ at this station. The activity of methanotrophs and the kinetics of methane oxidation in these sediments were demonstrated by the uptake of radiolabeled methane. K_s values varied between 4.1–9.6 nmol · cm³ of sediment slurry. High V_max values (12.7–35.2 nmol · cm⁻³ · h⁻¹) suggest a large population of methanotrophs in the sediments. An average methane flux to the oxic sediments of 0.24 mol · m⁻² · year⁻¹ was calculated from the pore water methane gradients. Pore water concentrations of copper in the upper sediment layer ranged from 10–120 nmol · 1⁻¹. Based upon the copper concentration, other measured parameters, and equilibrium conditions defined by WATEQF4, an estimate for dissolved free Cu²⁺ concentration of 5–38 nmol · 1⁻¹ pore water was obtained. Several factors control the rate of methane oxidation, including oxygen, methane, and the bioavailability of free Cu²⁺.     

The effect of hydrogen peroxide on spores of Clostridium perfringens

Dithiothreitol (DTT)-treated spores of Clostridium perfringens were much more sensitive to lysis by H₂O₂ in the presence of Cu²⁺ than untreated spores. Lysis was greatly inhibited by hydroxyl radical (.OH) scavengers such as thiourea, dimethylthiourea and dimethylsulfoxide, suggesting that lysis of spores by H₂O₂ involves formation of OH by Cu²⁺-catalysed decomposition of the peroxide. DTT-treated spores took up Cu²⁺ at almost the same rate and extent as did isolated cortical fragments. Hydrogen peroxide caused both the decrease in optical density and the hexosamine solubilization of cortical fragments which bound Cu²⁺.     

THE UBIQUITOUS PRESENCE OF CHLOROPHYLL D IN RED ALGAE FROM THE MONTEREY PENINSULA

Domoic acid (DA) is a neurotoxic amino acid produced by several members of the diatom genus Pseudo-nitzschia. Trophic transfer of DA up the food chain has been implicated in the deaths of 100's of marine birds and marine mammals along the central California Coast. The physiological function of DA in Pseudo-nitzschia spp. has not been defined, although some evidence indicates that elevated metal concentrations can induce DA accumulation (Subba RAO et al. , 1998, P.S.Z.N. Mar. Ecol. 19:31). Although California coastal waters have experienced a decline in several heavy metals from 1977–1990, copper concentrations have increased by as much as 25% (Stephenson, M. D. & Leonard, G. H., 1994, Mar. Poll. Bull. 28:148). Many algae produce chelators, including amino acids, in response to toxic [Cu²+] (Wu et al. 1998, J. Phycol. 34: 113). Domoic acid, a tricarboxylic acid, has 4 functional groups that may readily form chelation complexes with transition metals like copper. Copper enrichment experiments indicate that while Cu²+ is toxic to Pseudo-nitzschia multiseries at total [Cu] greater than 16.1μM (pCu 6.0), intracellular DA accumulation increases up to this point with no decline in growth rates relative to cultures grown in standard enriched seawater. These data suggest that DA may be accumulated by P. multiseries to mitigate the toxicity of elevated [Cu²+]. Chemiluminescence will be used to quantify the binding affinity (expressed as conditional stability constants, Kc) of DA for Cu²+. Defining the Cu-DA dose response relationship in Pseudo-nitzschia can facilitate prediction of future toxic bloom events.     

Effect of calcium and other metal ions on pectic activities from autolysed cultures of Alternaria alternata

Abstract The effect of increasing concentrations of Ca²⁺, Mg²⁺, Cu²⁺, Zn²⁺, Na⁺ and EDTA on the pectic enzymic activities (polymethylgalacturonase, endopectinase and pectin-lyase) present in the autolytic complex from Alternaria alternata has been studied. In all cases the divalent metal ions and EDTA produced an increased inhibition correlated with increasing concentration of each ion. An opposite effect was shown by the Na⁺ ion, which produced an increase in pectic enzymic activities, principally at low concentrations.     

Effect of disk rotational speed on heavy metal accumulation by rotating biological contactor (RBC) biofilms

The efficiency of a rotating biological contactor (RBC) in the treatment of waters contaminated with cadmium, copper and zinc, in particular the effect of rotational speed, namely 3, 15 and 25 rev min⁻¹, on heavy metal reclamation was evaluated. Atomic absorption spectrophotometric analysis indicated > 92% and < 20% removal of copper (Cu²⁺) and zinc (Zn²⁺), respectively. Cadmium (Cd²⁺) levels, however, remained constant. Similar results were obtained at all three rotation speeds tested.     

Hydrolysis of glucuronide-based substrates mediated by tungsten, Cu2+, Fe2+, and Zn2+

A variety of metal microprojectiles are currently used for carrying foreign DNA into living cells via particle-acceleration techniques. While developing a microprojectile-mediated protocol for transforming cells of sugarbeet ( Beta vulgaris L.), formation of a blue precipitate was observed with the indigoqenic substrate 5-bromo-4-chloro-3-indolyl-β-D-glucuronic acid (X-gluc) in the absence of gusA DNA encoding β-D-glucuronidase (GUS). Tungsten microcarriers, but not gold or silicon carbide, proved capable of catalyzing the cleavage of the glucuronide residue from three histochemical substrates evaluated: X-gluc, salmon X-gluc and magenta X-gluc. Indigo-stained sugarbeet cells were observed following bombardment with tungsten in the absence of DNA. Addition of oxidative catalysts to tungsten microcarriers during substrate incubation had no apparent effect on the metal-mediated catalysis. Treatment of microcarriers with Proteinase K and heat ruled out the presence of enzymes. Microbiological evaluation indicated the absence of contaminating microbes. Similarly, metal-catalyzed hydrolysis of the fluorogenic substrate 4-methylumbelliferyl-β-D-glucuronic acid (4-MUG) was observed in the presence of tungsten spheres but not with gold or silicon carbide particles. With this substrate, hydrolysis also occurred with millimolar concentrations of Cu²⁺, Fe²⁺ and Zn²⁺ ions. Consequently, careful monitoring of DNA-minus controls and avoidance of millimolar concentrations of Cu²⁺, Fe²⁺ and Zn²⁺ ions are recommended in microprojectile bombardment experiments where transient assays for gusA expression are performed.