Cobalt toxicity in anaerobic granular sludge: influence of chemical speciation

The influence of cobalt speciation on the toxicity of cobalt to methylotrophic methanogenesis in anaerobic granular sludge was investigated. The cobalt speciation was studied with three different media that contained varying concentrations of complexing ligands [carbonates, phosphates and ethylenediaminetetraacetic acid (EDTA)]. Three fractions (nominal added, dissolved and free) of cobalt were determined in the liquid media and were correlated with data from batch toxicity experiments. The average concentration of cobalt that was required for 50% inhibition of methanogenic activity (IC₅₀) for free Co²⁺ in the three sets of measurements was 13 μmol/L with a standard deviation of 22% and a similarity of 72% between the data obtained in the three different media for the range of cobalt concentrations investigated. The standard deviation of the IC₅₀ for the other two fractions was much higher, i.e. 85 and 144% for the added cobalt and dissolved cobalt, respectively, and the similarity was almost 0% for both fractions. Complexation (and precipitation) with EDTA, phosphates and carbonates was shown to decrease the toxicity of cobalt on methylotrophic methanogenesis. The free cobalt concentration is proposed to be the key parameter to correlate with cobalt toxicity. Thus, the toxicity of cobalt to granular sludge can be estimated based on the equilibrium-free cobalt concentration.     

Cobalt stimulates carotid body chemoreceptors

Because cobalt administration is known to elicit erythropoietin response, it is a reasonable hypothesis that cobalt would also stimulate the O2-sensing process in the peripheral chemoreceptors. We tested this hypothesis by measuring the effects of cobalt chloride on carotid chemosensory fibers in pentobarbital-anesthetized cats that were paralyzed and artificially ventilated. Responses of carotid chemoreceptor afferents to graded doses of cobalt given by intra-arterial injections (0.08-2.10 mumols) were measured at constant blood gases. Responses of the same chemoreceptor afferents to hypoxia, before and after a saturation dose of cobalt, were measured. In two experiments carotid body tissue PO2 was also simultaneously measured. The chemosensory fibers showed prolonged excitation after a brief period of inhibition subsequent to cobalt administration. The stimulatory effect showed a dose-dependent saturation response. Cobalt augmented rather than blocked carotid chemoreceptor response to hypoxia. The effect of cobalt was not mediated by tissue PO2. These results are consistent with the hypothesis that cobalt stimulates the O2-sensing mechanism, although a direct effect of cobalt on the excitability of the chemosensory terminal remains a possibility.     

The diverse chemical forms of heavy metals in tissue extracts of some metallophytes from Shaba Province,Zaïre

Copper and cobalt were determined in tissue extracts of five metallophytes from Shaba Province, Zaïre. About 4O% of the cobalt and copper was extractable into deionized water and a further 4O% was extractable in 0.2M hydrochloric acid. It was concluded that copper and cobalt are bound to several different ligands instead of a single ligand as in the case of nickel hyperaccumulators. Proton microprobe studies on the cobalt accumulator Haumaniastrum robertii showed a strong inverse correlation between the spatial distributions of cobalt and potassium, as well as a direct relationship between cobalt and calcium. It is suggested that some of the cobalt may be immobilized with calcium in oxalate crystals.     

Effects of Cobalt Nanoparticles on Human T Cells In Vitro

Limited information is available on the potential risk of degradation products of metal-on-metal bearings in joint arthroplasty. The aim of this study was to investigate the cytotoxicity and genotoxicity of orthopedic-related cobalt nanoparticles on human T cells in vitro. T cells were collected using magnetic CD3 microbeads and exposed to different concentrations of cobalt nanoparticles and cobalt chloride. Cytotoxicity was evaluated by methyl thiazolyl tetrazolium and lactate dehydrogenase release assay. Cobalt nanoparticles dissolution in culture medium was determined by inductively coupled plasma-mass spectrometry. To study the probable mechanism of cobalt nanoparticles effects on T cells, superoxide dismutase, catalase, and glutathione peroxidase level was measured. Cobalt nanoparticles and cobalt ions could inhibit cell viability and enhance lactate dehydrogenase release in a concentration- and time-dependent manner (P < 0.05). The levels of cobalt ion released from cobalt nanoparticles in the culture medium were less than 40% and increased with cobalt nanoparticles concentration. Cobalt nanoparticles could induce primary DNA damage in a concentration-dependent manner, and the DNA damage caused by cobalt nanoparticles was heavier than that caused by cobalt ions. Cobalt nanoparticles exposure could significantly decrease superoxide dismutase, catalase, and glutathione peroxidase activities at subtoxic concentrations (6 μM, <CC₅₀). These findings suggested that cobalt nanoparticles could generate potential risks to the T cells of patients suffer from metal-on-metal total hip arthroplasty, and the inhibition of antioxidant capacity may play important role in cobalt nanoparticles effects on T cells.     

An evaluation of cobalt chloride as an O2-sensitive chemoreceptor stimulant in channel catfish

The effects of cobalt chloride on heart rate, blood pressure, ventilatory frequency and opercular pressure amplitude in channel catfish, Ictalurus punctatus were measured to evaluate the potential of cobalt as a histochemical probe to study mechanisms of oxygen chemoreception, as well as assess the general effects of cobalt on the cardioventilatory physiology of fishes. Cobalt, like cyanide, has been previously used to stimulate oxygen chemoreceptors and hypoxic reflexes in mammals but there is little information on the cardioventilatory effects of cobalt on fish. Catfish were exposed to increasing concentrations (1-20 mg/kg) of cobalt in the water (external) or injections into the dorsal aorta (internal) and the cardioventilatory effects recorded. Mean arterial pressure showed a significant, dose-dependent increase in response to cobalt injections. Heart rate increased slowly, but significantly after cobalt injections but the magnitude of change was not dose-dependent. There was a small increase in ventilatory rate but no effect on amplitude. External cobalt had similar effects but the responses were weaker. Although cobalt stimulated some cardioventilatory reflexes the pattern and magnitude of the responses were noticeably different from those of cyanide and hypoxia. The results suggest that the cardioventilatory reflexes stimulated by cobalt were not mediated by O(2)-sensitive chemoreceptors and that cobalt is not an effective O(2) receptor stimulant in fishes.     

Cobalt-substituted hemoglobin Zürich (alpha 2 beta 263His leads Arg). Oxygen equilibria and EPR spectra.

Cobalt hemoglobin Zürich (alpha 2 beta 263His leads to Arg) has been successfully reconstituted from the apohemoglobin Zürich and cobaltous protoporphyrin IX. The oxygen affinity of cobalt hemoglobin Zurich, as well as that of iron hemoglobin Zürich, were measured in the absence and presence of organic phosphate and Cl-. The overall oxygen affinity of cobalt hemoglobin Zürich was found to be higher and the cooperativity as measured by the n value was smaller than those of cobalt hemoglobin A. Organic phosphate and Cl- affect the oxygen equilibrium properties of cobalt hemoglobin Zürich in a manner similar to that of cobalt hemoglobin A, but to a lesser extant than cobalt hemoglobin A. The EPR spectrum of oxy cobalt hemoglobin Zürich is less sensitive to the replacement of the buffer system from H2O to 2H2O, indicating that the hydrogen bond between the distal amino acid residue and the bound oxygen is not formed in the abnormal beta subunits. The deoxy EPR spectrum of cobalt hemoglobin Zürich is similar to that of deoxy cobalt hemoglobin A, suggesting that the deoxy cobalt hemoglobin Zürich is predominantly in the deoxy quaternary structure (T state).     

Increase of free cysteine and citric acid in plant cells exposed to cobalt ions

Cobalt complexation was investigated in a suspension cell culture of the cobalt hyperaccumulator Crotalaria cobalticola. C. cobalticola cells were more tolerant towards cobalt ions than the suspension cells of the non-accumulators Rauvolfia serpentina and Silene cucubalus. While the concentration of various compounds increased in cells of C. cobalticola challenged with cobalt ions, phytochelatin biosynthesis was not induced. Instead, the exposure to cobalt ions resulted in the increase of citrate and cysteine in cells. Size exclusion chromatography demonstrated the co-elution of cobalt and cysteine in C. cobalticola cell extracts. A significant increase in cysteine was observed also in cells of R. serpentina and S. cucubalus when they were exposed to cobalt ions. These results suggest that free cysteine is involved in cobalt ion complexation in plant cells.     

Mutagenicity, carcinogenicity and teratogenicity of cobalt metal and cobalt compounds

Cobalt metal and cobalt compounds are extensively used for the production of high-temperature alloys, diamond tools, cemented carbides and hard metals, for the production of various salts used in electroplating and as catalysts, drying agents in paints, additives in animal feeds and pigments. Cobalt oxides are used not only in the enameling industry and for pigments, but also in catalytic applications. There is no indication that cobalt metal and cobalt compounds constitute a health risk for the general population. Allergic reactions (asthma, contact dermatitis) can be induced by certain cobalt compounds. Interstitial fibrosis has also been observed in workers exposed to high concentrations of dust containing cobalt, tungsten, iron, etc., mainly in the cemented carbides and the diamond-polishing industries. Several experiments have demonstrated that single or repeated injections of cobalt metal powder or some forms of cobalt salt and cobalt oxide may give rise to injection site sarcoma in rats and in rabbits but the human health significance of such data is questionable. Intratracheal administration of a high dose of one type of cobalt oxide induces lung tumors in rats but not in hamsters. In the latter long-term inhalation of cobalt oxide (10 mg/m3) did not increase the incidence of lung cancer. The human data are too limited to assess the potential carcinogenic risk for workers. Co2+ interacts with protein and nucleic acid synthesis and displays only weak mutagenic activity in microorganisms. Some cobalt salts have been reported to enhance morphological transformation of Syrian hamster embryo cells. Cobalt chloride displays some limited mutagenic activity in yeast and some cobalt compounds are able to produce numerical and structural chromosome aberrations in plant cells. Cobalt and its salts appear to be devoid of mutagenic and clastogenic activity in mammalian cells. Cobaltous acetate and cobaltous chloride have not been found to be teratogenic in hamsters and rats respectively.     

Stimulation of methanol degradation in UASB reactors: in situ versus pre-loading cobalt on anaerobic granular sludge

The effect of pre-loading and in situ loading of cobalt onto a cobalt-limited granular sludge on the performance of methanol fed bioreactors was investigated. One upflow anaerobic sludge bed (UASB) reactor was inoculated with cobalt pre-loaded sludge (24h; 30 degrees C; 1 mM CoCl2) and a second UASB with unloaded sludge. The UASB reactors (30 degrees C; pH 7) were operated for 77 days at 8 h hydraulic retention time and organic loading rates ranging from 5 to 20 g COD.L reactor(-1).d(-1). Cobalt pre-loading clearly stimulated the methanogenic activity of the sludge with methanol as the substrate, e.g., after 30 days of reactor operation this activity was 5.8 times higher than that of the cobalt unloaded sludge. During the experiment, part of the cobalt leached from the pre-loaded sludge, i.e., 54% of the cobalt content was lost during the 77 days of reactor operation. Sequential metal extraction showed that losses mainly occurred from the exchangeable and carbonate fraction and in the sludge remaining cobalt was mainly present in the organic/sulfide fraction of the sludge. In situ loading of cobalt in the unloaded UASB reactor on day 57 by adding 31 microM cobalt to the influent for a 24-h period (16% of the cobalt present in the loaded sludge at day 11) resulted in a 4 time increase of the methanogenic activity of the sludge with methanol as the substrate at the end of the reactor experiment, while the accumulated amount of cobalt in the sludge only amounted to 6% of the cobalt accumulated in the loaded sludge (on day 11). This study showed that both pre-loading sludge and in situ loading are adequate for achieving an increased reactor performance of methanol fed UASB reactors operating under cobalt limitation. However, the in situ dosing procedure needs substantially lower amounts of cobalt, while it also gives significantly smaller losses of cobalt with the effluent.     

Importance of cobalt for individual trophic groups in an anaerobic methanol-degrading consortium.

Methanol is an important anaerobic substrate in industrial wastewater treatment and the natural environment. Previous studies indicate that cobalt greatly stimulates methane formation during anaerobic treatment of methanolic wastewaters. To evaluate the effect of cobalt in a mixed culture, a sludge with low background levels of cobalt was cultivated in an upflow anaerobic sludge blanket reactor. Specific inhibitors in batch assays were then utilized to study the effect of cobalt on the growth rate and activity of different microorganisms involved in the anaerobic degradation of methanol. Only methylotrophic methanogens and acetogens were stimulated by cobalt additions, while the other trophic groups utilizing downstream intermediates, H2-CO2 or acetate, were largely unaffected. The optimal concentration of cobalt for the growth and activity of methanol-utilizing methanogens and acetogens was 0.05 mg liter-1. The higher requirement of cobalt is presumably due to the previously reported production of unique corrinoid-containing enzymes (or coenzymes) by direct utilizers of methanol. This distinctly high requirement of cobalt by methylotrophs should be considered during methanolic wastewater treatment. Methylotroph methanogens presented a 60-fold-higher affinity for methanol than acetogens. This result in combination with the fact that acetogens grow slightly faster than methanogens under optimal cobalt conditions indicates that acetogens can outcompete methanogens only when reactor methanol and cobalt concentrations are high, provided enough inorganic carbon is available.     

Effects of acid water exposure on plasma cortisol, ion balance, and immune functions in the "cobalt" variant of rainbow trout

This study was undertaken to examine physiological responses to acidification of environmental water in the "cobalt" variant of rainbow trout (Oncorhynchus mykiss), which exhibits malformation of the pituitary, by following changes in plasma levels of cortisol and electrolytes, blood pH, gill Na(+), K(+)-ATPase activity, and immune functions after exposure to acid water (pH 4.5). Resting levels of plasma cortisol and lysozyme were significantly lower in the cobalt variant than in the normal trout, whereas plasma ceruloplasmin was significantly higher in the cobalt variant, suggesting that some endocrine factors, lacking or deficient in the cobalt variant, are important for the regulation of its immune functions. Blood pH was slightly but significantly lower in the cobalt variant at rest. After exposure to acid water for 24 h, both the normal trout and cobalt variant showed a significant elevation in plasma cortisol, although the increased level in the cobalt variant was still lower than that in the normal trout transferred to neutral water. No differences were seen in blood pH, plasma electrolytes, and gill Na(+), K(+)-ATPase activity between the normal trout and the cobalt variant, indicating that the cobalt variant regulates ion balance when exposed to acid water, despite malformation of the pituitary. Although the normal trout showed a reduction in plasma lysozyme level after acid exposure, there was no significant change in the cobalt trout. Adverse effects of pituitary malformation on ion balance and immune functions may be compensated by extrapituitary factors in the cobalt variant when it is exposed to acid water.     

Endogenous excretion and true absorption of cobalt as affected by the oral supply of cobalt

At the end of a 49-d experiment with 32 growing male rats, a period of 8 d was used to determine endogenous excretion and true absorption as well as apparent absorption and retention of cobalt with the aid of the isotope dilution technique. For this purpose, a single im dose of⁵⁸Co was applied at d 35 of the experiment. After that, urine and feces were collected separately from d 8 to 15 after injection of the isotope. The specific cobalt activity of the liver was used as an endogenous reference source. The basal diet provided 5.9 ppb cobalt, the different treatment groups were obtained by supplementing the diet with 0, 10, 50, 250, or 1250 ppb cobalt. The different diets were offered from the beginning of the experiment. In the balance period, apparent and true absorption as well as fecal excretion behaved similar to cobalt intake, whereas urinary excretion increased more rapidly with increasing cobalt supply. Endogenous fecal excretion accounted for 3.5 ng Co/d in the groups fed the diets without and with 10 ppb cobalt. An increase was not observed until supplementing the diet with 50 ppb cobalt. This increase between 250 and 1250 ppb cobalt was higher than the corresponding increase in the dietary cobalt supply. This indicates that endogenous fecal excretion might be more important for homeostatic regulation at a higher dietary cobalt concentration. Endogenous renal excretion as calculated from the results of the isotope dilution technique showed a similar kind of response to increasing cobalt supply as endogenous fecal loss. Nevertheless, the elimination of excessive cobalt mainly took place by adjusting urinary excretion, whereas the variations in true absorption and endogenous fecal excretion had no quantitative importance. Apparent and true absorption were on average 28.0 and 29.8%, respectively, of the cobalt intake. In the case of retention, a marked decline was observed from 19% in the depletion group to 3% with 1250 ppb cobalt, again demonstrating the importance of urinary excretion for controlling the cobalt content of the organism.     

Formation of bile pigments by coupled oxidation of cobalt-substituted haemoglobin and myoglobin.

Treatment of cobalt-substituted haemoglobin and myoglobin with ascorbate and molecular O2 (coupled oxidation) resulted in biliverdin formation from the cobalt(II) derivatives but not from the cobalt(III) derivatives. This was apparently due to the inability of ascorbate to reduce cobalt(III) haemoproteins. Isomer analysis of the biliverdins produced from coupled oxidation of cobalt(II) oxyhaemoglobin suggested that the orientation of the cobalt protoporphyrin IX in the haem pocket differed slightly from that of the haem in native haemoglobin.     

Cobalt activates potassium conductance in the plasma membrane of cultured renal epithelioid (MDCK)-cells

Cobalt has been shown to stimulate sodium transport across the distal nephron of the newt kidney. The mechanism of this action remained elusive. The present study has been performed to test for effects of cobalt on electrical properties of cultured subconfluent kidney (MDCK)-cells: cobalt (10 microM) leads to a rapid, sustained and reversible hyperpolarization of the cell membrane, paralleled by an increase of the potassium selectivity and a decrease of the resistance. Thus, cobalt increases the potassium conductance of the cell membrane. The half-maximal effect is elicited by approx. 1 microM. At extracellular calcium concentration reduced to less than 0.1 microM, cobalt (10 microM) leads to a transient hyperpolarization, which can be elicited only once. Thus, cobalt enhances the potassium conductance in a calcium dependent way. At higher concentrations (100 microM) cobalt hyperpolarizes the cell membrane only transiently even in the presence of extracellular calcium. Furthermore 100 microM cobalt interferes with ATP-induced hyperpolarization, which is known to result from calcium mediated activation of K+ channels. Thus, 100 microM cobalt may inhibit ATP-stimulated calcium entry into the cell.     

Genetic analysis of oxidative and endoplasmic reticulum stress responses induced by cobalt toxicity in budding yeast

BackgroundCobalt is an important metal cofactor of many living cells. However, excessive cobalt is toxic and can cause cell death and even several diseases in humans. Saccharomyces cerevisiae is a useful tool for studying metal homeostasis and many of the genes and pathways are highly conserved in higher eukaryotes including humans.MethodsThe intracellular cobalt and reactive oxygen species (ROS) levels were measured by an atomic absorption spectrometer and DHE staining method, respectively. The expression of genes involved in scavenging oxidative stress was tested by qPCR method, while the expression of UPRE-lacZ report gene was analyzed via β-galactosidase activity assay.ResultsUsing a genome-scale genetic screen, 153 cobalt-sensitive and 37 cobalt-tolerant gene deletion mutants were identified from Saccharomyces cerevisiae. We showed that 101 of the cobalt-sensitive mutants accumulated higher intracellular cobalt compared to wild-type. The intracellular ROS levels in 112 of the mutants were induced by cobalt, which might be caused by the decreased expression of genes involved in scavenging oxidative stress in response to cobalt. Moreover, more than one-third of the cobalt-sensitive mutants were also sensitive to tunicamycin, and cobalt stress might induce the unfolded protein response (UPR) through serine/threonine kinase and endoribonuclease Ire1.ConclusionsThis study reinforced the fact that cobalt toxicity might be due to the high intracellular cobalt and ROS levels, and the endoplasmic reticulum stress responses induced by cobalt.General significanceElucidating the toxicity mechanisms of cobalt stress response will help reveal new routes for the treatment of the diseases induced by cobalt.     

Cobalt-resistance in wall-less mutant (fz; sg; os-1) of Neurospora crassa

A cobalt-resistant wall-less mutant (slime) of Neurospora crassa was obtained by repeated sub-culturing of the sensitive wall-less mutant (W-sl) on agar medium containing toxic concentrations of cobalt. Resistance was stable on culturing Cor-sl on cobalt-free medium up to 15 weekly subcultures. Cor-sl is 10-fold more resistant to cobalt when compared to W-sl. It is also cross-resistant to Cu (10-fold) and Ni (3-fold). Cobalt accumulated by Cor-sl during growth and in short-term uptake experiments was lower when compared to W-sl. Cells previously loaded with cobalt was released into medium in both mutants, while in case of Cor-sl most of cobalt taken up (>80%), was released back into the medium when compared to W-sl. Metabolic inhibitor (Sodium azide) and magnesium ions inhibited cobalt uptake in both the mutants. Fractionation of cell-free extracts showed that most of the cobalt (70%) taken up by Cor-sl was bound to an inducible protein fraction which bound to DEAE-Cellulose, while in W-sl only 20% of cobalt was associated with this fraction. Subcellular localization of cobalt in W-sl indicated most of it to be cytoplasmic (70%) while nuclei and mitochondria had 10% and 5% respectively. In case of Cor-sl, mitochondrial cobalt accounted for only 2% while no significant differences were noted for other fractions. Our data implicate both transport block and intracellular sequestration of cobalt to play a major role in resistance.     

Effect of cobalt on the anaerobic degradation of methanol

The effect of trace elements on the methanogenesis from methanol and acetate was studied utilizing granular sludge obtained from an anaerobic wastewater treatment plant. The methanogenic activity from methanol was dramatically stimulated by the addition of a cocktail of trace elements in the basal medium. When trace elements were supplied individually, cobalt greatly stimulated methanogenesis which equalled the stimulation observed with the complete trace element mixture. No remarkable influence of any trace element was observed when acetate was used as the substrate. Two Upflow Anaerobic Sludge Blanket (UASB) reactors were operated with and without supplementation of cobalt. Cobalt greatly stimulated both acetogenesis in the initial operational phase and later methanogenesis. The cobalt sufficient column provided almost 3 times the methane productivity compared to the cobalt deprived column. At an organic loading rate of 8 g COD/l·d, 87% of the COD was converted to methane in the cobalt sufficient column. Under low cobalt concentration, methanogens compete better for cobalt than acetogens.     

Effect of long-term cobalt deprivation on methanol degradation in a methanogenic granular sludge bioreactor

The effect of the trace metal cobalt on the conversion of methanol in an upflow anaerobic sludge bed (UASB) reactor was investigated by studying the effect of cobalt deprivation from the influent on the reactor efficiency and the sludge characteristics. A UASB reactor (30 degrees C; pH 7) was operated for 261 days at a 12-h hydraulic retention time (HRT). The loading rate was increased stepwise from 2.6 g chemical oxygen demand (COD) x L reactor(-1) x d(-1) to 7.8 g COD x L reactor(-1) x d(-1). Cobalt deprivation had a strong impact on the methanogenic activity of the sludge. In batch tests, the methanogenic activity of the sludge with methanol as the substrate increased 5.3 (day 28) and 2.1 (day 257) times by addition of 840 nM of cobalt. The sludge had an apparent K(m) for cobalt of 948 nM after 28 days of operation and 442 nM at the end of the run. Cobalt deprivation during 54 days of operation led to a methanol conversion efficiency of only 55%. Continuous addition of cobalt (330 nM) for 33 days improved the methanol removal efficiency to 100%. In this period of cobalt dosing, the cobalt concentration in the sludge increased 2.7 times up to 32 microg x g TSS(-1). Upon omission of the cobalt addition, cobalt washed-out at a stable rate of 0.1 microg x g VSS(-1) x d(-1). At the end of the run, the cobalt concentration of the sludge was similar to that of the seed sludge.