Biomonitoring of chromium(VI) deposited in pulmonary tissues: Pilot studies of a magnetic resonance imaging technique in a post-mortem rodent model

The biomonitoring of individuals exposed to chromium(VI) by inhalation is often based on determinations of chromium in body fluids such as blood, plasma or urine, or on assessments of DNA damage in non-lung surrogate tissues such as peripheral blood lymphocytes. These techniques are of some use as biomarkers of internal exposure or biological effect, mainly in the case of soluble chromium(VI) compounds, but they provide at best only indirect information about chromium(VI) concentrations in the main target organ of interest - the lung. An urgent need exists for a non-invasive technique to permit the visualization and quantification of chromium(VI) in the lung of exposed humans. This study details the development of a lung imaging technique based on the detection of paramagnetic chromium using magnetic resonance imaging (MRI). The intracellular reductive conversion of chromium(VI) is a crucial bioactivation step in its carcinogenicity, and the MRI method described here relies on the conversion of non-paramagnetic (MRI 'silent') chromium(VI) to detectable paramagnetic species such as chromium(III). Initial studies with chromium(III) revealed that a range of 2.5-5 μg chromium(III) instilled in rat lung is considered to be the lower limit of detection of this method. It was possible to demonstrate the presence of 30 μg chromium(VI) in our post-mortem rat model. The ultimate objective of this work is to determine whether this technique has applicability to the biomonitoring of chromium(VI) inhalation exposures that result in internalized lung doses in human subjects.     

Role of physiological antioxidants in chromium(VI)-induced cellular injury.

Chromium(VI) compounds are well known to be potent toxic and carcinogenic agents. Because chromium(VI) is easily taken up by cells and is subsequently reduced to the trivalent form, the formation of chromium(III) or other intermediate oxidation states such as chromium(V) and (IV) is believed to play a role in the adverse biological effects of chromium(VI) compounds. Recent in vitro studies have shown that this reduction process generates free radical species such as active oxygen radicals. Furthermore, physiological antioxidants are reported to modify the genotoxic and toxic effects of chromate. This article reviewed the recent in vitro and in vivo studies of the effects of antioxidants including active oxygen scavengers; glutathione; vitamins B2, E, and C, on chromate-induced injury such as DNA lesions; lipid peroxidation; enzyme inhibition; cytotoxicity; mutation; and so on. In addition, the mechanism of action of these antioxidants was discussed with respect to the formation of active oxygen radicals and paramagnetic chromium such as chromium(V) and (III). Such studies may help elucidate the mechanism of chromium(VI) toxicity as well as the mechanism of protection.     

In vitro metabolism and permeation studies in rat jejunum: organic chromium compared to inorganic chromium.

The purpose of these studies was to compare the in vitro absorption of two inorganic chromium(III) compounds: chromium chloride and chromium nitrate, with organic chromium(III)-picolinate; and to investigate if any in vitro metabolism of chromium(VI) takes place. The in vitro metabolism studies showed that chromium (VI) was reduced by artificial gastric juice. The reduction followed first order kinetics with a half-life of 23 min. The studies also showed that the chromium picolinate complex was stable in artificial gastric juice for 4 hours. By the rat everted gut sac technique, chromium chloride, chromium nitrate and chromium picolinate penetrated the rat jejunum with 165 +/- 59, 160 +/- 26 and 127 +/- 36 ng chromium per g rat jejunum, respectively, whereas the permeability coefficients (Papp) were 0.7 +/- 0.3, 1.0 +/- 0.4, and 9.6 +/- 2.2 microns/min, respectively. Absorption studies on pig intestine in Ussing chambers showed a nearly total adsorption of chromium(III) by the chambers, resulting in unreliable data.     

Problems in the biological monitoring of chromium(VI) exposed individuals

The usefulness of currently available techniques for the biological monitoring of chromium(VI) exposed individuals is reviewed. Chromium levels in body fluids, such as urine and blood plasma, are reliable markers of exposure to chromium in oxidation states (VI) and (III) and provide a measure of the internalized dose of chromium. These markers are sufficiently sensitive to be useful in most occupational settings encountered today. In contrast, the majority of cytogenetic surveillance studies among chromium platers, ferrochromium workers and stainless steel welders using the manual metal arc (MMA) method have yielded negative or inconclusive results. As a marker for genotoxicity, the number of sister chromatid exchanges in blood lymphocytes proved to be relatively insensitive towards exposure to chromium(VI). There were however significant increases in rare chromosome aberrations among MMA stainless steel welders, although the reported levels of all aberrations combined were similar to those observed among control groups of many other studies. The relative lack of success of cytogenetic surveillance studies using blood lymphocytes is surprising in view of the strong genotoxicity of chromium(VI). A possible explanation comes from recent studies which showed that the differences in chromium lymphocyte levels between exposed and controls were disproportionately small. Another factor which complicates attempts to correlate genotoxic effects in lymphocytes with the processes giving rise to cancers of the respiratory system is the toxicokinetics of inhaled chromium(VI). Only small fractions of the total inhaled dose are distributed in the body while the bulk of chromium(VI) deposited in the lungs remains there for very long periods of time. The vast majority of lymphocytes will therefore come into contact with chromium(VI) not while travelling through the supporting tissues of the lungs but during their migration through the blood. There they take up chromium(VI) that has leached from the lungs. Blood lymphocytes therefore seem to be inappropriate for the monitoring of the biologically effective dose, and of early biological effects arising from exposure to chromium(VI). Thus there is an urgent need to develop techniques which would allow the non-invasive monitoring of internalized doses of chromium in the lung.     

Problems in the biological monitoring of chromium(VI) exposed individuals

The usefulness of currently available techniques for the biological monitoring of chromium(VI) exposed individuals is reviewed. Chromium levels in body fluids, such as urine and blood plasma, are reliable markers of exposure to chromium in oxidation states (VI) and (III) and provide a measure of the internalized dose of chromium. These markers are sufficiently sensitive to be useful in most occupational settings encountered today. In contrast, the majority of cytogenetic surveillance studies among chromium platers, ferrochromium workers and stainless steel welders using the manual metal arc (MMA) method have yielded negative or inconclusive results. As a marker for genotoxicity, the number of sister chromatid exchanges in blood lymphocytes proved to be relatively insensitive towards exposure to chromium(VI). There were however significant increases in rare chromosome aberrations among MMA stainless steel welders, although the reported levels of all aberrations combined were similar to those observed among control groups of many other studies. The relative lack of success of cytogenetic surveillance studies using blood lymphocytes is surprising in view of the strong genotoxicity of chromium(VI). A possible explanation comes from recent studies which showed that the differences in chromium lymphocyte levels between exposed and controls were disproportionately small. Another factor which complicates attempts to correlate genotoxic effects in lymphocytes with the processes giving rise to cancers of the respiratory system is the toxicokinetics of inhaled chromium(VI). Only small fractions of the total inhaled dose are distributed in the body while the bulk of chromium(VI) deposited in the lungs remains there for very long periods of time. The vast majority of lymphocytes will therefore come into contact with chromium(VI) not while travelling through the supporting tissues of the lungs but during their migration through the blood. There they take up chromium(VI) that has leached from the lungs. Blood lymphocytes therefore seem to be inappropriate for the monitoring of the biologically effective dose, and of early biological effects arising from exposure to chromium(VI). Thus there is an urgent need to develop techniques which would allow the non-invasive monitoring of internalized doses of chromium in the lung.     

Intracellular chromium reduction

Two steps are involved in the uptake of Cr(VI): (1) the diffusion of the anion CrO4(2-) through a facilitated transport system, presumably the non-specific anion carrier and (2) the intracellular reduction of Cr(VI) to Cr(III). The intracellular reduction of Cr(VI), keeping the cytoplasmic concentration of Cr(VI) low, facilitates accumulation of chromate from extracellular medium into the cell. In the present paper, a direct demonstration of intracellular chromium reduction is provided by means of electron paramagnetic (spin) resonance (EPR) spectroscopy. Incubation of metabolically active rat thymocytes with chromate originates a signal which can be attributed to a paramagnetic species of chromium, Cr(V) or Cr(III). The EPR signal is originated by intracellular reduction of chromium since: (1) it is observed only when cells are incubated with chromate, (2) it is present even after extensive washings of the cells in a chromium-free medium; (3) it is abolished when cells are incubated with drugs able to reduce the glutathione pool, i.e., diethylmaleate or phorone; and (4) it is abolished when cells are incubated in the presence of a specific inhibitor of the anion carrier, 4-acetamido-4'-isothiocyanatostilbene-2-2'-disulfonic acid.     

Effect of Rhamnolipids on Chromium-Contaminated Kaolinite

Hexavalent chromium Cr(VI) is a common environmental pollutant that is treated by its reduction to the trivalent form Cr(III). The latter can be re-oxidized to the toxic form, Cr(VI), under specific conditions. A study was conducted on the removal of Cr(III) to eliminate the hazard imposed by its presence in soil as there has been some evidence that organic compounds can decrease its sorption. The effect of addition of negatively-charged biosurfactants (rhamnolipids) on chromium contaminated kaolinite was studied. Results showed that the rhamnolipids have the capability of extracting 25% portion of the stable form of chromium, Cr(III), from the kaolinite, under optimal conditions. The removal of hexavalent chromium was also enhanced compared to water by a factor of 2 using a solution of rhamnolipids. Results from the sequential extraction procedure showed that rhamnolipids remove Cr(III) mainly from the carbonate and oxide/hydroxide portions of the kaolinite. The rhamnolipids had also the capability of reducing close to 100% of the extracted Cr(VI) to Cr(III) over a period of 24 days. This study indicated that rhamnolipids could be beneficial for the removal or long–term conversion of chromium Cr(VI) to Cr(III).     

Distribution and speciation of chromium accumulated in Gynura pseudochina (L.) DC.

Soil and water contamination with chromium is an issue of recent concern in Thailand due to increases in industrial activity. Gynura pseudochina (L.) DC., a chromium tolerance plant, could be employed to address this problem via phytoremediation. To understand the tolerance mechanism, this study investigated the speciation and distribution of chromium accumulated in G. pseudochina (L.) DC. using AAS, XAFS, μ-XANES, μ-XRF imaging and EPR. The plants were separately treated with K₂Cr₂O₇ and Cr₂(SO₄)₃ in a hydroponic system. μ-XRF imaging clarified the distributions of Cr, Fe, Zn, Ca, Cl, K and S within the samples. In G. pseudochina (L.) DC. treated with Cr(VI) solution, the Cr was mainly distributed in the vascular bundle and periderm of the tuber, the stem xylem, the vein and the epidermis, including the trichome of the leaf tissues. This Cr distribution corresponded to those of Cu, Fe and Zn. In G. pseudochina (L.) DC. treated with Cr(III) solution, the Cr was distributed in the periderm of the tuber, the stem cortex, and the epidermis and parenchyma of the leaf tissues. μ-XANES and XAFS indicated that highly toxic Cr(VI) was reduced to the intermediate Cr(V) and accumulated as less toxic Cr(III), and EXAFS spectra showed that the reduced Cr(III) was bound to oxygen ligands. The coordination number (N) and the interatomic distance (R) to the first shell were approximately 3–4 (N) and 2 Å (R), respectively. EPR spectra of the plant samples treated with Cr(VI) revealed the presence of Cr(V) and Cr(III). Thus, Cr(III) and Cr(VI) were taken up into the vascular system and transported from the roots to the leaves. Cr(III) was distributed via the symplast system to the ground tissue and accumulated mainly in the stem cortex. Cr(VI) was transported to the xylem via the apoplast system, and the adsorption of Cr(VI) and its reduction to Cr(V) and Cr(III) occurred on oxygen ligands in the lignocellulosic structure of the xylem and vein.     

Chromium-induced production of reactive oxygen species,DNA single-strand breaks,nitric oxide production,and lactate dehydrogenase leakage in J774A.1 cell cultures

The involvement of oxidative stress in the toxicity of chromium (VI) and chromium (III) has been proposed. We have therefore examined the effects of these cations on the production of superoxide anion, nitric oxide (NO), and DNA single strand breaks (SSB) in J774A.1 macrophage cells in culture as well as the effects on lactate dehydrogenase (LDH) leakage and cell viability. Following a 48 hour incubation, over twofold increases in superoxide anion and NO production were observed at concentrations of approximately 0.30 and 50 μM for Cr (VI) and Cr (III), respectively. The patterns of cell viability and LDH leakage paralleled superoxide anion and NO production for Cr (VI) and Cr (III). A 50% decrease in viability was observed at approximately the concentrations that produced a twofold increase in superoxide and NO production. Concentration-dependent increases in DNA-SSB were observed after incubation with Cr (III) with maximum increases occurring at a concentration of approximately 60 μM. Cr (VI) had no effect on the incidence of DNA-SSB at any of the tested concentrations. The results indicate that Cr (VI) and Cr (III) are toxic to the J774A.1 cell line, and the toxicity may be due at least in part to an oxidative stress induced by the production of reactive oxygen species. © 1996 John Wiley & Sons, Inc.     

Effects of chromium(VI) and chromium(III) on energy charge and oxygen consumption in rat thymocytes

The cytotoxic effects of chromium compounds in two oxidation states have been studied in rat thymocytes. endogenous nucleotide levels and oxygen consumption were examined as relevant parameters of the physiological state of the cell. Incubation of rat thymocytes with Cr(VI) produced a marked unbalance of endogenous purine nucleotide pool and a parallel decrease in oxygen consumption. A close correlation between the reduction of oxygen consumption and ATP level in rat thymocytes treated with increasing concentrations of Cr(VI) has been found. In rat thymocytes permeabilized with digitonin and in isolated rat liver mitochondria both Cr(VI) and Cr(III) showed, at different range of concentrations, a marked inhibition of maximal oxygen consumption rate (uncoupled respiration). The effects observed were depending on chromium oxidation state and on different mitochondrial sites of substrate oxidation.     

Simultaneous speciation of chromium by spectrophotometry and multicomponent analysis

Abstract

A simple, fast and sensitive spectrophotometric method for the simultaneous determination of Cr(III) and Cr(VI) in effluents and contaminated waters using a UV-visible spectrophotometer, which operates with an advanced software for multicomponent analysis, is proposed. The method consists in the complexation of Cr (III) with EDTA and reaction of Cr(VI) with diphenylcarbazide (DPC). Variables, such as pH and colour stability time, were studied. The effect of concomitant ions on the simultaneous Cr(III) and Cr(VI) determination was also investigated. The sums of the chromium species concentrations obtained by the proposed method were compared with the total chromium concentrations found by electrothermal atomic absorption spectrometry. Recoveries of the chromium species between 75 and 136% were obtained for spiked samples. The linear working range for Cr(III) was 0.5-30 mg L^?1, while for Cr(VI) was 0.005-0.30 mg L^?1. The detection limits were 0.3 mg L^?1 for Cr(III) and 0.003 mg L^?1 for Cr(VI) while the quantification limits were 1.0 mg L^?1 for Cr(III) and 0.01 mg L^?1 for Cr(VI).     

EPR detection of paramagnetic chromium in liver of fish (Anguilla anguilla) treated with dichromate(VI) and associated oxidative stress responses—Contribution to elucidation of toxicity mechanisms

The impact of chromium (Cr) on fish health has been the subject of numerous investigations, establishing a wide spectrum of toxicity, attributed particularly to the hexavalent form [Cr(VI)]. However, reports on the simultaneous assessment of Cr toxicity in fish and its toxico-kinetics, namely involving metal speciation, are scarce. Therefore, keeping in view the understanding of the mechanisms of Cr(VI) toxicity, this work intended to detect the formation of paramagnetic Cr species in liver of Anguilla anguilla following short-term dichromate(VI) intraperitoneal treatment (up to 180 min), assessing simultaneously the pro-oxidant properties. The formation of Cr(V) and Cr(III) was examined by electron paramagnetic resonance (EPR), as an innovative approach in the context of fish toxicology, and related with the levels of total Cr. Cr(V) was successfully detected and quantified by EPR spectrometry, showing a transient occurrence, mostly between 15 and 90 min post-injection, with a peak at 30 min. The limitations of EPR methodology towards the detection and quantification of Cr(III) were confirmed. Although Cr(VI) exposure induced the antioxidant system in the eel's liver, the oxidative deterioration of lipids was not prevented. Overall, the results suggested that Cr(V), as a short-lived species, did not appear to be directly and primarily responsible for the cellular damaging effects observed, since stress responses persisted up to the end of exposure regardless Cr(V) drastic decay. Though further research is needed, ROS mediated pathways (suggested by superoxide dismutase and catalase activity induction) and formation of Cr(III) complexes emerged as the most plausible mechanisms involved in Cr(VI) toxicity.     

Synthesis and characterization of new porphyrazine-Gd(III) conjugates as multimodal MR contrast agents

Magnetic resonance imaging (MRI) has long been used clinically and experimentally as a diagnostic tool to obtain three-dimensional, high-resolution images of deep tissues. These images are enhanced by the administration of contrast agents such as paramagnetic Gd(III) complexes. Herein, we describe the preparation of a series of multimodal imaging agents in which paramagnetic Gd(III) complexes are conjugated to a fluorescent tetrapyrrole, namely, a porphyrazine (pz). Zinc metalated pzs conjugated to one, four, or eight paramagnetic Gd(III) complexes are reported. Among these conjugates, Zn-Pz-8Gd(III) exhibits an ionic relaxivity four times that of the monomeric Gd(III) agent, presumably because of increased molecular weight and a molecular relaxivity that is approximately thirty times larger, while retaining the intense electronic absorption and emission of the unmodified pz. Unlike current clinical MR agents, Zn-Pz-1Gd(III) is taken up by cells. This probe demonstrates intracellular fluorescence by confocal microscopy and provides significant contrast enhancement in MR images, as well as marked phototoxicity in assays of cellular viability. These results suggest that pz agents possess a new potential for use in cancer imaging by both MRI and near-infrared (NIR) fluorescence, while acting as a platform for photodynamic therapy.     

Hexavalent chromium removal in vitro and from industrial wastes, using chromate-resistant strains of filamentous fungi indigenous to contaminated wastes

Two chromate-resistant filamentous fungi, strains H13 and Ed8, were selected from seven independent fungal isolates indigenous to Cr(VI)-contaminated soil because of their ability to decrease hexavalent chromium levels in the growth medium. Morphophysiological studies identified strain H13 as a Penicillium sp. isolate and Ed8 as an Aspergillus sp. isolate. When incubated in minimal medium with glucose as a carbon source and in the presence of 50 microg/mL Cr(VI), these strains caused complete disappearance of Cr(VI) in the growth medium after about 72 h of incubation. Total chromium concentration in growth medium was constant during culture growth, and no accumulation of chromium in fungal biomass was observed. Quantitative determinations of oxidized and reduced chromium species during the reduction process revealed stoichiometric conversion of Cr(VI) to Cr(III). A decrease in Cr(VI) levels from industrial wastes was also induced by Ed8 or H13 biomass. These results indicate that chromate-resistant filamentous fungi with Cr(VI)-reducing capability could be useful for the removal of Cr(VI) contamination.     

Generation of PM2 DNA breaks in the course of reduction of chromium(VI) by glutathione

The carcinogen chromate is efficiently taken up and reduced to chromium(III) compounds by various biological systems. To test the possible DNA damage induced in the course of chromium(VI) reduction, we used a combination of chromate with the reductant glutathione (GSH) as well as a green complex of chromium(V), which is formed in the reaction of chromate with GSH. The combination of chromate and glutathione was found to cause single-strand breaks in supercoiled circular DNA of the bacteriophage PM2. The green chromium(V) complex Na4(GSH)4Cr(V).8H2O, prepared from chromate and glutathione, also cleaved supercoiled PM2 DNA. No DNA-degrading effects were observed with either chromate or the final product of the reaction with GSH, a purple anionic chromium(III) GSH complex. The nature of the buffering agents revealed a strong influence on the extent of DNA strand breaks produced by chromate and GSH. A variation of the GSH concentration in the reaction with chromate and PM2 DNA, performed in sodium phosphate-buffered solutions showed an initial increase in the number of strand breaks at GSH concentrations up to 1 mM followed by a decline at higher GSH concentrations. Since neither chromate, when administered individually, nor the final product of chromium(VI) reduction, the purple chromium(III) GSH complex, produced any detectable DNA cleavage, the critical steps leading to DNA strand breaks occur in the course of the conversion of chromium(VI) to chromium(III) by GSH, the most abundant intracellular low molecular thiol. Moreover, the demonstration that DNA cleavage is induced in the presence of the chromium(V) complex identifies chromium(V) as the oxidation state of the metal, which is involved in the steps leading to DNA-damaging effects of chromate.     

Efficiency of Penicillium chrysogenum PTCC 5037 in reducing low concentration of chromium hexavalent in a chromium electroplating plant wastewater

The effectiveness of Penicillium chrysogenum was evaluated for reducing Cr(VI) from the wastewater of a chromium electroplating plant. Statistically-based experimental designs were applied to optimize the condition for reducing Cr(VI) to Cr(III). By applying Plackett-Burman factorial design and central composite design as the optimization step, attempts were made to identify optimal values of the three factors that bringing about maximum microorganism activity and therefore maximum hexavalent chromium(VI) bioreduction. It was found that each gram of P. chrysogenum of dry biomass condition could reduce 66 mg of Cr(VI) to Cr(III) in the wastewater of the chromium electroplating plant.     

DNA-protein crosslinks in peripheral lymphocytes of individuals exposed to hexavalent chromium compounds

Abstract

DNA-protein crosslinks were measured in peripheral blood lymphocytes of chrome-platers and controls from Bulgaria in order to evaluate a genotoxic effect of human exposure to carcinogenic Cr(VI) compounds. Chrome-platers and most of the unexposed controls were from the industrial city of Jambol; some additional controls were recruited from the seaside town of Burgas. The chrome-platers had significantly elevated levels of chromium in pre- and post-shift urine, erythrocytes and lymphocytes compared with the control subjects. The largest differences between the two groups were found in erythrocyte chromium concentrations which are considered to be indicative of Cr(VI) exposure. Despite the significant differences in internal chromium doses, levels of DNA-protein crosslinks were not significantly different between the combined controls and exposed workers. Individual DNA-protein crosslinks, however, correlated strongly with chromium in erythrocytes at low and moderate doses but at high exposures, such as among the majority of chrome-platers, these DNA adducts were saturated at maximum levels. The saturation of DNA-protein crosslinks seems to occur at 7–8 μg I-¹ chromium in erythrocytes whereas a mean erythrocyte chromium among the chrome platers was as high as 22.8 μg l^?1. Occupationally unexposed subjects exhibited a significant variability with respect to the erythrocyte chromium concentration, however erythrocyte chromium levels correlated closely with DNA-protein crosslinks in lymphocytes. The controls from Jambol had higher chromium concentrations in erythrocytes and elevated levels of DNA-protein crosslinks compared with Burgas controls. Occupational exposure to formaldehyde among furniture factory workers did not change levels of DNA-protein crosslinks in peripheral lymphocytes. DNA-protein crosslink measurements showed a low intraindividual variability and their levels among both controls and exposed indivduals were not affected by smoking, age or weight.     

Oligomerization of paramagnetic substrates result in signal amplification and can be used for MR imaging of molecular targets

Magnetic resonance imaging (MRI) has evolved into a sophisticated, noninvasive imaging modality capable of high-resolution anatomical and functional characterization of transgenic animals. To expand the capabilities MRI, we have developed a novel MR signal amplification (MRamp) strategy based on enzyme-mediated polymerization of paramagnetic substrates into oligomers of higher magnetic relaxtivity. The substrates consist of chelated gadolinium covalently bound to phenols, which then serve as electron donors during enzymatic hydrogen peroxide reduction by peroxidase. The converted monomers undergo rapid condensation into paramagnetic oligomers leading to a threefold increase in atomic relaxtivity (R1/Gd). The observed relaxtivity changes are largely due to an increase in the rotational correlation time tau r of the lanthanide. Three applications of the developed system are demonstrated: (1) imaging of nanomolar amounts of an oxidoreductase (peroxidase); (2) detection of a model ligand using an enzyme-linked immunoadsorbent assay format; and (3) imaging of E-selectin on the surface of endothelial cells probed for with an anti-E-selectin-peroxidase conjugate. The development of "enzyme sensing" probes is expected to have utility for a number of applications including in vivo detection of specific molecular targets. One particular advantage of the MRamp technique is that the same paramagnetic substrate can be potentially used to identify different molecular targets by attaching enzymes to various antibodies or other target-seeking molecules.     

Bacterial removal of chromium (VI) and (III) in a continuous system

The capacity of Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans to reduce different concentrations of hexavalent chromium in shake flask cultures has been investigated. A. ferrooxidans reduces 100% of chromium (VI) at concentrations of 1, 2.5 and 5 ppm, but in the presence of 10 ppm only 42.9% of chromium (VI) was reduced after 11 days of incubation. A. thiooxidans showed a lower capacity to reduce this ion and total reduction of chromium (VI) was only obtained for concentrations of 1 and 2.5 ppm, whereas 64.7% and 30.5% was reached for 5 and 10 ppm, respectively, after 11 days. A continuous flow mode system was subsequently investigated, in which A. thiooxidans was immobilized on elemental sulphur and the acidic medium obtained was employed to solubilize chromium (III) and to reduce chromium (VI) present in a real electroplating waste [30% of chromium (III) and 0.1% of chromium (VI)]. The system enabled the reduction of 92.7% of hexavalent chromium and represents a promising way to treat this type of waste in the industry.