Effects of Sulfide Input on Arsenate Bioreduction and Its Reduction Product Formation in Sulfidic Groundwater.

Microbes have important impacts on the mobilization of arsenic in groundwater. To study the effects of sulfide on As(V) bioreduction in sulfidic groundwater, Citrobacter sp. JH012-1 isolated from sediments in the Jianghan Plain was used in a microcosm experiment. The results showed that sulfide significantly enhanced As(V) bioreduction as an additional electron donor. The reduction rates of As(V) were 21.8%, 34.5%, 73.6% and 85.9% under 0, 15, 75 and 150 µM sulfide inputting, respectively. The main products of As(V) bioreduction were thioarsenite and orpiment and the concentration of thioarsenite reached to 5.5 and 7.1 µM in the solution with the initial 75 and 150 µM sulfide, respectively. However, under 0 and 15 µM sulfide inputting, the dominant product was arsenite with no thioarsenite accumulation. The decrease in pH enhanced the bioreduction of As(V) and promoted the formation of thioarsenite and orpiment. In addition, the percentage of thioarsenite in total arsenic decreased with the decrease in the ratio of sulfur to arsenic, indicating that the formation of thioarsenite was limited by the concentration of initial sulfide. Therefore, the presence of sulfide had a significant effect on the transformation of arsenic in groundwater. This study provides new insights into the bioreduction of As(V) and the formation of thioarsenite in sulfidic groundwater.

Thioarsenite Detection and Implications for Arsenic Transport in Groundwater.

Arsenic toxicity and mobility in groundwater depend on its aqueous speciation. Uncertainty about the methods used for measuring arsenic speciation in sulfate-reducing environments hampers transport and fate analyses and the development of in situ remediation approaches for treating impacted aquifers. New anion-exchange chromatography methods linked to inductively coupled plasma mass spectrometry (ICP-MS) are presented that allow for sample/eluent pH matching. Sample/eluent pH matching is advantageous to prevent thioarsenic species transformation during chromatographic separation because species protonation states remain unaffected, hydroxyl-for-bisulfide ligand substitution is avoided, and oxidation of reduced arsenic species is minimized. We characterized model and natural solutions containing mixtures of arsenic oxyanions with dissolved sulfide and solutions derived from the dissolution of thioarsenite and thioarsenate solids. In sulfidic solutions containing arsenite, two thioarsenic species with S/As ratios of 2:1 and 3:1 were important over the pH range from 5.5 to 8.5. The 3:1 thioarsenic species dominated when disordered As2S3 dissolved into sulfide-containing solution at pH 5.4. Together with the preferential formation of arsenite following sample dilution, these data provide evidence for the formation and detection of thioarsenite species. This study helps resolve inconsistencies between spectroscopic and chromatographic evidence regarding the nature of arsenic in sulfidic waters.

Adsorption and transformation of thioarsenite at hematite/water interface under anaerobic condition in the presence of sulfide.

The adsorption behavior of thioarsenite (TAsIII) on the surface of hematite (α-Fe2O3) is unknown at present. In the present study, we have investigated the transformation and reactions of TAsIII [monothioarsenite (MTAsIII) and dithioarsneite (DTAsIII)] on the surface of α-Fe2O3 in the presence of sulfide at S/As = 1 and 3 by X-ray absorption spectroscopy (XAS) and Raman spectroscopy. The adsorption envelopes reveal that the adsorption of TAsIII on α-Fe2O3 is significantly less than that of arsenite (AsIII) in the pH range from 7 to 11 with the initial As concentration of 25 mg L-1. However, at the initial As concentration of 135 mg L-1, the uptake of TAsIII by α-Fe2O3 is higher at pH 7 but lower at pH 8-11 than that of AsIII. The adsorption isotherms show that the adsorption of As on α-Fe2O3 is largely inhibited by the presence of aqueous sulfide at pH 7 with low As equilibrium concentration (<40 mg L-1). Whereas the uptake of As by α-Fe2O3 is highly elevated compared with the value predicted by Langmuir model at pH 7 with high As equilibrium concentration (>40 mg L-1), implying the formation of As-bearing (surface) precipitate. The As and S K-edge XAS as well as Raman spectroscopy confirm the formation of As sulfide precipitate on the surface of α-Fe2O3 in MTAsIII system. It is worth to note that the oxidation of (thio)AsIII occurs on the surface of α-Fe2O3 in DTAsIII system under strictly anaerobic conditions. These results shed new light on the understanding of the interfacial behavior of As and point to the potential implication in immobilization and removal of arsenic in sulfidic environment.

Arsenite removal without thioarsenite formation in a sulfidogenic system driven by sulfur reducing bacteria under acidic conditions.

Sulfidogenic process using sulfate-reducing bacteria (SRB) has been used to remove arsenite from the arsenic-contaminated waters through the precipitation of arsenite with sulfide. However, excessive sulfide production and significant pH increase induced by sulfate reduction result in the formation of the mobile thioarsenite by-products and the inefficiency and instability of arsenite removal, especially when the arsenite level fluctuates. In this study, we proposed a novel sulfidogenic process driven by sulfur reducing bacteria (S0RB) for the arsenite removal under acidic conditions. In a long term experiment, efficient sulfide production (0.42 ±â€¯0.2 kg S/m3-d) was achieved without changing the acidic condition (pH around 4.3) in a sulfur reduction bio-reactor. With the acidic sulfide-containing effluents from the bio-reactor, over 99% of arsenite (10 mg As/L) in the arsenic-contaminated water was precipitated without the formation of soluble thioarsenite by-products, even in the presence of excessive sulfide. Maintaining the acidic condition (pH around 4.3) of the sulfide-containing effluent was essential to ensure the efficient arsenite precipitation and minimize the formation of thioarsenite by-products when the arsenite to sulfide molar ratios ranged from 0.1 to 0.46. An acid-tolerant S0RB, Desulfurella, was found to be responsible for the efficient dissimilatory sulfur reduction under acidic conditions without changing the solution pH significantly. The microbial sulfur reduction may proceed through the direct electron transfer between the S0RB and sulfur particles, and also through the indirect electron transport mediated by electron carriers. The findings of this study demonstrate that the proposed sulfidogenic process driven by S0RB working under acidic conditions can be a promising alternative to the SRB-based process for arsenite removal from the arsenic-contaminated waters.

Determination of arsenic speciation in sulfidic waters by Ion Chromatography Hydride-Generation Atomic Fluorescence Spectrometry (IC-HG-AFS).

A method for the analysis of arsenic species in aqueous sulfide samples is presented. The method uses an ion chromatography system connected with a Hydride-Generation Atomic Fluorescence Spectrometer (IC-HG-AFS). With this method inorganic As(III) and As(V) species in water samples can be analyzed, including arsenite (HnAs(III)O3(n-3)), thioarsenite (HnAs(III)S3(n-3)), arsenate (HnAs(V)O4(n-3)), monothioarsenate (HnAs(V)SO3(n-3)), dithioarsenate (HnAs(V)S2O2(n-3)), trithioarsenate (HnAs(V)S3O(n-3)) and tetrathioarsenate (HnAs(V)S4(n-3)). The peak identification and retention times were determined based on standard analysis of the various arsenic compounds. The analytical detection limit was ~1-3 µg L(-1) (LOD), depending on the quality of the baseline. This low detection limit makes this method also applicable to discriminate between waters meeting the drinking water standard of max. 10 µg L(-1) As, and waters that do not meet this standard. The new method was successfully applied for on-site determination of arsenic species in natural sulfidic waters, in which seven species were unambiguously identified.

Arsenic speciation in sulfidic waters: reconciling contradictory spectroscopic and chromatographic evidence.

In recent years, analytical methods have been developed that have demonstrated that soluble arsenic-sulfur species constitute a major fraction of dissolved arsenic in sulfidic waters. However, an intense debate is going on about the exact chemical nature of these compounds, since X-ray absorption spectroscopy (XAS) data generated at higher (mmol/L) concentrations suggest the presence of (oxy)thioarsenites in such waters, while ion chromatographic (IC) and mass spectroscopic data at lower (µmol/L to nmol/L) concentrations indicate the presence of (oxy)thioarsenates. In this contribution, we connect and explain these two apparently different types of results. We show by XAS that thioarsenites are the primary reaction products of arsenite and sulfide in geochemical model experiments in the complete absence of oxygen. However, thioarsenites are extremely unstable toward oxidation, and convert rapidly into thioarsenates when exposed to atmospheric oxygen, e.g., while waiting for analysis on the chromatographic autosampler. This problem can only be eliminated when the entire chromatographic process is conducted inside a glovebox. We also show that thioarsenites are unstable toward sample dilution, which is commonly employed prior to chromatographic analysis when ultrasensitive detectors like ICP-MS are used. This instability has two main reasons: if pH changes during dilution, then equilibria between individual arsenic-sulfur species rearrange rapidly due to their different stability regions within the pH range, and if pH is kept constant during dilution, then this changes the ratio between OH(-) and SH(-) in solution, which in turn shifts the underlying speciation equilibria. This problem is avoided by analyzing samples undiluted. Our studies show that thioarsenites appear as thioarsenates in IC analyses if oxygen is not excluded completely, and as arsenite if samples are diluted in alkaline anoxic medium. This also points out that thioarsenites are necessary intermediates in the formation of thioarsenates.

A critical investigation of hydride generation-based arsenic speciation in sulfidic waters.

In sulfidic environments, hydride generation-based approaches are not suitable for arsenic determination because thioarsenates which can constitute the predominant arsenic species under these conditions (> 80% of total arsenic) are completely ignored. Sample acidification for preservation or during hydride generation leads to loss of total inorganic arsenic due to precipitation of arsenic-sulfur phases. Total concentrations can be determined correctly using 1% potassium iodide as prereducing agent while with L-cysteine (0.16 mol L(-1)), transformation of tetra-, tri-, and dithioarsenate to arsenite remains incomplete. By decreasing the original sample pH, hydride generation destroys thioarsenate species distribution because only monothioarsenate is stable over the whole pH range. Dithioarsenate transforms to arsenite below pH 4. Tetrathioarsenate transforms to trithioarsenate (pH 11.9) which subsequently transforms to arsenite (pH 5.6), followed by precipitation of arsenic-sulfur phases below pH 5. It is thus impossible to determine thioarsenates by hydride generation. The "As(III)"--fraction contains tetra-, tri-, and some dithioarsenate as well as arsenite, while monothioarsenate is determined with arsenate as "As(V)". Different analytical setups have substantial impact on thioarsenate hydride-generation behavior, thus provide little comparability and render reinterpretation of existing arsenic hydride-generation speciation data from sulfidic environments impossible. In natural geothermal water samples from Yellowstone National Park, total arsenic concentrations determined by ICP-MS and by HG-AFS with prereductant agreed well (< 6% relative difference). Speciation results deviated from the behavior predicted for thioarsenates from laboratory experiments, probably due to matrix effects.

Keratin mutation predisposes to mouse liver fibrosis and unmasks differential effects of the carbon tetrachloride and thioacetamide models.

BACKGROUND & AIMS: Keratins 8 and 18 (K8/K18) are important hepatoprotective proteins. Animals expressing K8/K18 mutants show a marked susceptibility to acute/subacute liver injury. K8/K18 variants predispose to human end-stage liver disease and associate with fibrosis progression during chronic hepatitis C infection. We sought direct evidence for a keratin mutation-related predisposition to liver fibrosis using transgenic mouse models because the relationship between keratin mutations and cirrhosis is based primarily on human association studies. METHODS: Mouse hepatofibrosis was induced by carbon tetrachloride (CCl(4)) or thioacetamide. Nontransgenic mice, or mice that over express either human Arg89-to-Cys (R89C mice) or wild-type K18 (WT mice) were used. The extent of fibrosis was evaluated by quantitative real-time reverse-transcription polymerase chain reaction of fibrosis-related genes, liver hydroxyproline measurement, and Picro-Sirius red staining and collagen immunofluorescence staining. RESULTS: Compared with control animals, CCl(4) led to similar liver fibrosis but increased injury in K18 R89C mice. In contrast, thioacetamide caused more severe liver injury and fibrosis in K18 R89C as compared with WT and nontransgenic mice and resulted in increased messenger RNA levels of collagen, tissue inhibitor of metalloproteinase 1, matrix metalloproteinase 2, and matrix metalloproteinase 13. Analysis in nontransgenic mice showed that thioacetamide and CCl(4) have dramatically different molecular expression responses involving cytoskeletal and chaperone proteins. CONCLUSIONS: Over expression of K18 R89C predisposes transgenic mice to thioacetamide- but not CCl(4)-induced liver fibrosis. Differences in the keratin mutation-associated fibrosis response among the 2 models raise the hypothesis that keratin variants may preferentially predispose to fibrosis in unique human liver diseases. Findings herein highlight distinct differences in the 2 widely used fibrosis models.

Pharmacological evaluation of contractile activity of the dog heartworm Dirofilaria immitis.

Effects of a variety of compounds on spontaneous contractile activity of whole, intact, adult canine heartworms (HW), which had been maintained in culture, were evaluated to improve understanding of the pharmacological sensitivities of this parasitic nematode. Acetylcholine, pilocarpine, imidazole, levamisole, and DL-tetramisole caused spastic paralysis. Gamma-aminobutyrate (GABA), the GABA-mimetic muscimol, the GABA amino transferase inhibitor 3-mercaptopropionic acid, fenthion, ketamine, levodopa, and salinomycin caused flaccid paralysis. Atropine and monensin had inhibitory effects. Neostigmine, the neuromuscular blocking agents decamethonium, succinylcholine, and D-tubocurarine, and the aminergic agents epinephrine, norepinephrine, and serotonin had little or no effect on contractile activity. Thiacetarsamide had a nonreversible, slow onset, inhibitory effect on contractile activity. Occurrence of spastic or flaccid paralysis was not correlated with gender or culture age and was never associated with the same compound. Submaximal stimulatory or inhibitory responses paralleled the type of maximal responses (spastic or flaccid paralysis) for most compounds. Concentration variations producing maximal effects suggested considerable variation in individual preparation sensitivity, which did not appear to involve cuticle defects or time in culture. Difference in gender sensitivity was noted only for levamisole, which caused greater stimulation of contractile activity in males than in females.

Complementary molecular and elemental detection of speciated thioarsenicals using ESI-MS in combination with a xenon-based collision-cell ICP-MS with application to fortified NIST freeze-dried urine.

The simultaneous detection of arsenic and sulfur in thioarsenicals was achieved using xenon-based collision-cell inductively coupled plasma (ICP) mass spectrometry (MS) in combination with high-performance liquid chromatography. In an attempt to minimize the (16)O(16)O(+) interference at m/z 32, both sample introduction and collision-cell experimental parameters were optimized. Low flow rates (0.25 mL/min) and a high methanol concentration (8%) in the mobile phase produced a fourfold decrease in the m/z 32 background. A plasma sampling depth change from 3 to 7 mm produced a twofold decrease in background at m/z 32, with a corresponding fourfold increase in the signal associated with a high ionization surrogate for sulfur. The quadrupole bias and the octopole bias were used as a kinetic energy discriminator between background and analyte ions, but a variety of tuning conditions produced similar (less than twofold change) detection limits for sulfur ((32)S). A 34-fold improvement in the (32)S detection limit was achieved using xenon instead of helium as a collision gas. The optimized xenon-based collision cell ICP mass spectrometer was then used with electrospray ionization MS to provide elemental and molecular-based information for the analysis of a fortified sample of NIST freeze-dried urine. The 3sigma detection limits, based on peak height for dimethylthioarsinic acid (DMTA) and trimethylarsine sulfide (TMAS), were 15 and 12 ng/g, respectively. Finally, the peak area reproducibilities (percentage relative standard deviation) of a 5-ppm fortified sample of NIST freeze dried urine for DMTA and TMAS were 7.4 and 5.4%, respectively.

Influence of sulfide (S2-) on preservation and speciation of inorganic arsenic in drinking water.

Generally, H2SO4, HNO3, HCl or the combination of ethylenediaminetetraacetate with acetic acid (EDTA-HAc) have been used to preserve arsenite and arsenate species prior to analysis. When these acidic preservatives are added in sulfidic water, instantaneous precipitation of poorly crystalline orpiment, As2S3(am), occurs, thereby lowering the total arsenic, As(Tot), analysis. A new method for the determination of As(Tot) was developed in which acid-preserved sulfidic water samples were oxidized with NaOCl, converting As2S3(am) and thioarsenic species to arsenate. A new method was also developed for the separation of uncharged arsenite and charged thioarsenic species in fresh, unpreserved sulfidic water by adsorbing the charged thioarsenic species while allowing uncharged arsenite to pass through a strong-base resin unhindered. The adsorbed thioarsenic species could be eluted efficiently with 0.16 M NaOCl solution.

Novel thioarsenic metabolites in human urine after ingestion of an arsenosugar, 2',3'-dihydroxypropyl 5-deoxy-5-dimethylarsinoyl-beta-D-riboside.

The presence of arsenic-containing carbohydrates, arsenosugars, in many seafoods raises questions of human health concerning the ingestion and metabolism of these compounds. A previous study investigating the metabolites in human urine after the ingestion of a common arsenosugar 2',3'-dihydroxypropyl 5-deoxy-5-dimethylarsinoyl-beta-d-riboside (oxo-arsenosugar) showed that the arsenic was rapidly excreted in the urine and was present as at least 12 metabolites, only three of which could be identified. In this repeat study with oxo-arsenosugar and using high-performance liquid chromatography/inductively coupled plasma mass spectrometry, we report the identification of seven arsenic metabolites, which together accounted for 88% of the total urinary arsenic collected over 61 h. The metabolites included previously reported human urinary arsenicals dimethylarsinate (DMA), oxo-dimethylarsenoethanol (oxo-DMAE), and trimethylarsine oxide, in addition to new human metabolites oxo-dimethylarsenoacetate (oxo-DMAA), thio-dimethlyarsenoacetate (thio-DMAA), thio-dimethylarsenoethanol (thio-DMAE), and the thio-arsenosugar. Cytotoxicity testing of the major metabolites DMA, oxo-DMAE, thio-DMAE, oxo-DMAA, and thio-DMAA showed that they were nontoxic even at 10 mM, except for DMA, which showed some toxic effects at 1 mM.

Effect of thioarsenite formation on arsenic(III) toxicity.

Soluble arsenic(III)-sulfide complexes (thioarsenites) play a significant role in the chemistry of arsenic in reducing, sulfidic environments at circumneutral pH. Chemical equilibrium calculations using thioarsenite thermodynamic data from the literature indicate that the formation of a dithioarsenite complex, AsS(OH)(SH)(-1), reduces the concentration of the uncomplexed inorganic As(III) species present (defined sigma H3AsO3, where sigma H3AsO3 = AsO3(-3) + HAsO3(-2) + H2AsO3(-1) + H3AsO3). With enough sulfide present, soluble As(III) is dominated by this complex. Therefore, it is of interest to examine the effect of dithioarsenite formation on As(III) toxicity. The Microtox acute toxicity test was used for this purpose. Tests performed on solutions with varying S:As ratios indicate that As(III) toxicity is a function of the uncomplexed As(III) concentration rather than the total As(III) concentration. This suggests that the dithioarsenite species is not bioavailable and that its formation reduces As(III) toxicity. Chemical equilibrium calculations and sediment pore-water field data from various sources indicate that, in many sediments, dithioarsenite formation can reduce toxicity.

Immunological anomalies and thrombocytopenia in 117 dogs and cats diagnosed with chronic fatigue syndrome (CFS).

Retrospective analysis of immune dysfunctions found in 55 dogs and 62 cats diagnosed with Chronic Fatigue Syndrome (CFS), revealed leukopenia in 11% of dogs (n = 6) and 22.5% of cats (n = 14), lymphopenia in 14.5% of dogs (n = 8) and 10% of cats (n = 6), hypogammaglobulinaemia in 9% of dogs (n = 5) and 13% of cats (n = 8) and thrombocytopenia in 20% of dogs (n = 11) and 68% of cats (n = 42). All patients had creatine kinase enzyme levels above the normal range (CK = 5-100 IU/L) and carried micrococcus-like organisms on erythrocytes. Blood cultures proved positive for Staphylococcus spp. in 16 cases. After low-dosage arsenic-based therapy (thiacetarsamide sodium) all animals experienced complete clinical remission. Subsequent controls demonstrated immune restoration in 4 representative FIV-FeLV negative cats, previously diagnosed with CFS associated with leukopenia, lymphopenia, hypogammaglobulinaemia and thrombocytopenia. The main conclusion is that a CFS-like disease in dogs and cats, characterised by the common hallmarks of high CK levels, absence of known causes of chronic fatigue in animals and presence of micrococcus-like organisms in the blood, can be associated with humoral and/or cellular immune deficiencies in 9-22.5% of cases and with thrombocytopenia in 20-68% of cases. Considerations are made on the possible role of micrococci in the aetiology of the condition and on the similarities with CFS in humans.

Chronic Fatigue Syndrome (CFS) in 15 dogs and cats with specific biochemical and microbiological anomalies.

A great deal of controversy and speculation surrounds the etiology of Chronic Fatigue Syndrome (CFS) in human patients and the existence of a similar illness in animals. To evaluate the association with a presumptive staphylococcal infection and bacteremia, seven dogs and eight cats diagnosed with CFS (two meeting the CDC working case definition) were submitted to rapid blood cultures and fresh blood smears investigations. Nine out of 15 blood cultures proved Staph-positive and four isolates were specified as S. xilosus (3) and S. intermedius (1). The presence of micrococci-like organisms in the blood was of common observation among these subjects, in association with fatigue/pain-related symptoms and biochemical abnormalities suggestive of a myopathy. Following treatment with a low dosage arsenical drug (thiacetarsamide sodium, Caparsolate, i.v., 0.1 ml/kg/day) all patients experienced complete remission. Micrococci disappeared from the blood at post-treatment controls made 10-30 days later. The outcomes were compared with those of five healthy controls and five 'sick with other illness' patients showing significant difference.

Chronic fatigue and immune dysfunction syndrome associated with Staphylococcus spp. bacteraemia responsive to thiacetarsamide sodium in eight birds of prey.

Chronic fatigue and immune dysfunction syndrome (CFIDS) is a recognized human illness with zoonotic implications that is rarely described in animals. Eight birds of prey examined between 1992 and 1995 and sharing common symptoms (asthenia, inability to fly, poor appetite and emaciation) underwent laboratory tests revealing immunodeficiency, anaemia, high creatine kinase levels and low serum magnesium levels. Diagnosis of CFIDS was based upon these features. The effectiveness of an arsenic-based medication, thiacetarsamide sodium, administered intravenously for 2-3 days at low dosages (0.1 ml/kg/day) has been demonstrated by checks carried out 10, 20 and 30 days after therapy. The symptoms and the immune and haematological dysfunctions disappeared within 2-4 weeks of treatment. In all patients, micrococcus-like organisms found adhering to the outer surface of many red blood cells, had disappeared at post-treatment controls. Two of five blood cultures were positive for Staphylococcus spp. (S. intermedius and S. xilosus). Consideration is given to the pharmacological activity of an arsenic-based drug in animal illnesses resembling CFIDS.

Chronic fatigue syndrome in horses: diagnosis and treatment of 4 cases.

A report from England has suggested that Chronic Fatigue Syndrome exists in equines and constitutes an emerging veterinary problem. Preliminary epidemiological studies seem to confirm the zoonotic implications of CFS. An arsenical drug, sodium thiacetarsamide, was administered to four horses with a diagnosis of Chronic Fatigue Syndrome (CFS), already treated unsuccessfully with different medications. The CFS-like lethargy, with accompanying symptoms and signs, of the four animals obtained a complete remission after intravenous treatment with this drug at low dosage (0.1 mg/kg/day). No adverse side effects were ever noticed. This clinical response was associated with recovery from anaemia and decrease of muscular enzyme values in two of the four horses. In all patients, micrococci-like bacteria found before treatment adhering to the outer surface of many red blood cells, disappeared at post-treatment controls. Considerations are made on the possible action of an arsenical drug, used in isolation, in the treatment of CFS.