Optimization and validation of a methodology to determine total arsenic, As(III) and As(V), in water samples, through graphite furnace atomic absorption spectrometry

The Graphite furnace atomic absorption spectrometry (GF AAS) was the technique chosen by the inorganic contamination laboratory (INCQ/ FIOCRUZ) to be validated and applied in routine analysis for arsenic detection and quantification. The selectivity, linearity, sensibility, detection, and quantification limits besides accuracy and precision parameters were studied and optimized under Stabilized Temperature Platform Furnace (STPF) conditions. The limit of detection obtained was 0.13 µg.L-1 and the limit of quantification was 1.04 µg.L-1, with an average precision, for total arsenic, less than 15% and an accuracy of 96%. To quantify the chemical species As(III) and As(V), an ion-exchange resin (Dowex 1X8, Cl- form) was used and the physical-chemical parameters were optimized resulting in a recuperation of 98% of As(III) and of 90% of As(V). The method was applied to groundwater, mineral water, and hemodialysis purified water samples. All results obtained were lower than the maximum limit values established by the legal Brazilian regulations, in effect, 50, 10, and 5 µg.L-1 para As total, As(III) e As(V), respectively. All results were statistically evaluated.

Effect of pH, dextrose and yeast extract on cadmium toxicity on Saccharomyces cerevisiae PE-2

The aim of this study was to evaluate the effects of pH, dextrose and yeast extract on the cadmium toxicity on Saccharomyces cerevisiae PE-2. In the first assay, the YED mediums with different pH (2, 3, 4, 5, 6, 7, and 8) containing 0.0 and 0.05 mmol Cd L-1 were inoculated with yeast suspension and incubated at 30 °C for 18 hours. During the anaerobic growth, the biomass concentration was determined. The yeast trehalose content, cell viability, and the growth rate were assessed at the beginning and at the end of the growth stages. In the second assay the YED mediums were diluted to the total, ½, and ¼ content of dextrose and yeast and 0.0 and 0.05 mmol Cd L-1 were added. The pH of the mediums was adjusted to 5. The culture mediums were inoculated and incubated at 30 °C for 18 hours. The yeast growth was not affected by cadmium at high pH, but at low pH the yeast becomes more sensitive to the toxic effect. The yeast susceptibility to cadmium was enhanced by the decrease of yeast extract strength and the increase of dextrose strength.

Arsenic and germanium determination by hydride generation in the D.C. plasma optical emission spectrometer

Modifications to the commercial hydride generator, manufactured by Spectrametrics, resulted in improved operating procedure and enhancement of the arsenic and germanium signals. Experiments with arsenic(III) and arsenic(V) showed that identical reiults could be produced from both oxidation states. However, since arsenic(V) is reduced more slowly than arsenic(III), peak areas and not peak heights must be measured when the arsine is immediately stripped from the system (approximately 5 seconds reaction). When the reduction is allowed to proceed for 20 seconds before the arsine is stripped, peak heights may be used. For a 200 ng/mL solution, the relative standard deviation is 2.8% for As(III) and 3.8% for As(V). The detection limit for arsenic using the modified system is 0.50 ng/mL. Studies performed on As(V) standards show that the interferences from 1000 mg/L of nickel(II), cobalt(II), iron(III), copper(II), cadmium(II), and zinc(II) can be eliminated with the aid of 5 M Hel and 3% L-cystine. Conditions for the reduction of germanium to the corresponding hydride were investigated. The effect of different concentrations of HCl on the reduction of germanium to the covalent hydride in aqueous media by means of NaBH 4 solutions was assessed. Results show that the best response is accomplished at a pH of 1.7. The use of buffer solutions was similarly characterized. In both cases, results showed that the element is best reduced when the final pH of the solution after reaction is almost neutral. In addition, a more sensitive method, which includes the use of (NH4)2S208' has been developed. A 20% increase in the germanium signal is registered when compared to the signal achieved with Hel alone. Moreover, under these conditions, reduction of germanium could be accomplished, even when the solution's pH is neutral. For a 100 ng/mL germanium standard the rsd is 3%. The detection limit for germanium in 0.05 M Hel medium (pH 1.7) is 0.10 ng/mL and 0.09 ng/mL when ammonium persulphate is used in conjunction with Hel. Interferences from 1000 mg/L of iron(III), copper(II), cobalt(II), nickel(II), cadmium(II), lead(II), mercury(II), aluminum(III), tin(IV), arsenic(III), arsenic(V) and zinc(II) were studied and characterized. In this regard, the use of (NH4)ZS20S and Hel at a pH of 1.7 proved to be a successful mixture in the sbppression of the interferences caused by iron, copper, aluminum, tin, lead, and arsenic. The method was applied to the determination of germanium in cherts and iron ores. In addition, experiments with tin(IV) showed that a 15% increase in the tin signal can be accomplished in the presence of 1 mL of (NH4)2S20S 10% (m/V).

Arsenic speciation by ion-exchange chromatography with on- line determination by hydride generation: electronic modification of the D.C. plasma by photon counting

A method using L-cysteine for the determination of arsenous acid (As(III)), arsenic acid (As(V)), monomethylarsonic acid (MMAA), and dimethylarsinic acid (DMAA) by hydride generation was demonstrated. The instrument used was a d.c. plasma atomic emission spectrometer (OCP-AES). Complete recovery was reported for As(III), As(V), and DMAA while 86% recovery was reported for MMAA. Detection limits were determined, as arsenic for the species listed previously, to be 1.2, 0.8, 1.1, and 1.0 ngemL-l, respectively. Precision values, at 50 ngemL-1 arsenic concentration, were f.80/0, 2.50/0, 2.6% and 2.6% relative standard deviation, respectively. The L-cysteine reagent was compared directly with the conventional hydride generation technique which uses a potassium iodide-hydrochloric acid medium. Recoveries using L-cysteine when compared with the conventional method provided the following results: similar recoveries were obtained for As(III), slightly better recoveries were obtained for As(V) and MMAA, and significantly better recoveries for DMAA. In addition, tall and sharp peak shapes were observed for all four species when using L-cysteine. The arsenic speciation method involved separation by ion exchange .. high perfonnance liquid chromatography (HPLC) with on-line hydride generation using the L.. cysteine reagent and measurement byOCP-AES. Total analysis time per sample was 12 min while the time between the start of subsequent runs was approximately 20 min. A binary . gradient elution program, which incorporated the following two eluents: 0.01 and 0.5 mM tri.. sodium citrate both containing 5% methanol (v/v) and both at a pH of approximately 9, was used during the separation by HPLC. Recoveries of the four species which were measured as peak area, and were normalized against As(III), were 880/0, 290/0, and 40% for DMAA, MMAA and As(V), respectively. Resolution factors between adjacent analyte peaks of As(III) and DMAA was 1.1; DMAA and MMAA was 1.3; and MMAA and As(V) was 8.6. During the arsenic speciation study, signals from the d.c. plasma optical system were measured using a new photon-signal integrating device. The_new photon integrator developed and built in this laboratory was based on a previously published design which was further modified to reflect current available hardware. This photon integrator was interfaced to a personal computer through an AID convertor. The .photon integrator has adjustable threshold settings and an adjustable post-gain device.

Continuous hydride generation in the presence of L- cysteine for the determination of arsenic, bismuth, antimony and tin in steels by inductively coupled plasma atomic emission spectrometry

Arsenic, bismuth, germanium, antimony and tin were simultaneously determined by continuous hydride generation and inductively coupled plasma-atomic emission spectrometry . I Hydrides were introduced into four different types of gas-liquid separators. Two of the gas-liquid separators were available in-house. A third was developed for this project and a fourth was based on a design used by CET AC. The best signal intensity was achieved by the type II frit-based gas-liquid separator, but the modified Cetac design gave promise for the future, due to low relative standard deviation. A method was developed for the determination of arsenic, bismuth, antimony and tin in low-alloy steels. Four standard reference materials from NIST were dissolved in 10 mL aqua regia without heat. Good agreement was obtained between experimental values and certified values for arsenic, bismuth, antimony and tin. The method was developed to provide the analyst with the opportunity to determine the analytes by using simple aqueous standards to prepare calibration lines. Within the limits of the samples analyzed, the method developed is independent of matrix.

Investigation into the determination of arsenic by direct current plasma atomic emission spectrometer

Improvements have been made on the currently available hydride generator system manufactured by SpectraMetrics Incorporated, because the system was found to be unsatisfactory with respect to the following: 1. the drying agent, anhydrous calcium chloride, 2. the special sample tube, 3. the direction of argon flow through the Buchner funnel when it came to dealing with real sample, that is, with reference only to aqueous extracts of soil samples. Changes that were made on the system included the replacement of anhydrous calcium chloride with anhydrous calcium sulphate and the replacement of the special sample tube with a modified one made from silica. Re-directing the flow of argon through the top of the Buchner funnel appeared to make the system compatible with aqueous extracts of soil samples. The interferences from 1000 ~g/mL of nickel(II) , cobalt(II), iron(III), copper(II) have been eliminated with the aid of 1.4 M hydrochloric acid and 1% (weight/volume) L-cystine. Greater than 90% recovery of 0.3 ~g/mL arsenic signal was achieved in each case. Furthermore, 103% of arsenic signal was accomplished in the presence of 1000 ~g/mL cadmium with 5 M Hel. tVhen each of the interferents was present in solution at 1000 ppm, a recovery of 85% was achieved by using 5 M hydrochloric acid and 3% (weight/volume) L-cystine. Without L-cystine and when 1.4 M hydrochloric acid was used, the recoveries were 0% (Ni), 0% (Co), 88% (Fe), 15% (Cu), 18% (Cd). Similarly, a solution containing 1000 ppm of each interferent gave a zero percent recovery of arsenic. The reduction of trivalent and pentavalent arsenic at a pH less than one has also been investigated and shown to be quantitative if peak areas are measured. The reproducibility determination of a 0.3 Vg/mL standard arsenic solution by hydride generation shows a relative standard deviation of 3.4%. The detection limits with and without Porapak Q have been found to be 0.6 ng/mL and 1.0 ng/mL, respectively.

The Na+/H+ exchanger Nhx1 of Saccharomyces cerevisiae is essential to limit drug toxicity

Nhx1 est un antiport vacuolaire de Na+/H+ chez la levure Saccharomyces cerevisiae. Nhx1 joue un rôle important dans le maintien de l’homéostasie ionique du cytoplasme de la cellule. En effet, la mutation du gène NHX1 chez la levure nhx1Δ entraîne une perte de l’homéostasie cellulaire quand les cellules sont cultivées dans un milieu de faible osmolarité. Ce travail rapporte pour la première fois, et contrairement à la cellule parentale, que la mutation du gène NHX1 a pour effet une sensibilité du mutant nhx1Δ à une variété des drogues et des agents cationiques et anioniques lorsque les cellules sont cultivées dans un milieu riche. En outre, dans ces conditions de culture, aucune sensibilité n’a été observée chez le mutant nhx1Δ quand les cellules sont traitées avec différentes concentrations de sel. Nous avons aussi démontré que la sensibilité du mutant nhx1Δ aux différents agents ainsi que la sécrétion de l’enzyme carboxypeptidase Y observé chez ce mutant n’ont pas été restauré lorsque les cellules sont cultivées dans des milieux avec différents pH ou avec différentes concentrations de sel. Enfin, une analyse génétique a révélé que le mutant nhx1Δ montre un phénotype distinct d’autres mutants qui ont un défaut dans le trafic entre le compartiment pré-vacuolaire et l’appareil de Golgi quand ces cellules sont traitées avec différents agents. Cette analyse prouve que la sensibilité de nhx1Δ aux différents agents n’est pas liée au trafic entre le compartiment pré-vacuolaire et l’appareil de Golgi.

Semisynthetic aminoglycoside antibiotics : toward biomimetic synthesis, evasion of bacterial resistance and reduced toxicity

Les antibiotiques aminoglycosidiques sont des agents bactéricides de grande valeur et d’efficacité à large spectre contre les pathogènes Gram-positifs et Gram-négatifs, dont plusieurs membres naturels et semisynthétiques sont importants dans l’histoire clinique depuis 1950. Des travaux crystallographiques sur le ribosome, récompensés par le prix Nobel, ont démontré comment leurs diverses structures polyaminées sont adaptées pour cibler une hélice d’ARN dans le centre de codage de la sous-unité 30S du ribosome bactérien. Leur interférence avec l’affinité et la cinétique des étapes de sélection et vérification des tARN induit la synthèse de protéines à basse fidélité, et l’inhibition de la translocation, établissant un cercle vicieux d’accumulation d’antibiotique et de stress sur la membrane. En réponse à ces pressions, les pathogènes bactériens ont évolué et disséminé une panoplie de mécanismes de résistance enzymatiques et d’expulsion : tels que les N acétyltransférases, les O phosphotransférases et les O nucleotidyltransférases qui ciblent les groupements hydroxyle et amino sur le coeur des aminoglycosides; des méthyl-transférases, qui ciblent le site de liaison ribosomale; et des pompes d’expulsion actives pour l’élimination sélective des aminoglycosides, qui sont utilisés par les souches Gram-négatives. Les pathogènes les plus problématiques, qui présentent aujourd’hui une forte résilience envers la majorité des classes d’antibiotiques sur le bord de la pan-résistance ont été nommés des bactéries ESKAPE, une mnémonique pour Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa et Enterobacteriaceae. La distribution globale des souches avec des mécanismes de résistance envers les standards cliniques aminoglycosides, tels que la tobramycine, l’amikacine et la gentamicine, est comprise entre 20 et 60% des isolées cliniques. Ainsi, les aminoglycosides du type 4,6-disubstitués-2-deoxystreptamine sont inadéquats comme thérapies anti-infectieuses à large spectre. Cependant, la famille des aminoglycosides 4,5-disubstitués, incluant la butirosine, la neomycine et la paromomycine, dont la structure plus complexe, pourrait constituter une alternative. Des collègues dans le groupe Hanessian et collaborateurs d’Achaogen Inc. ont démontré que certains analogues de la paraomomycine et neomycine, modifiés par désoxygénation sur les positions 3’ et 4’, et par substitution avec la chaîne N1-α-hydroxy-γ-aminobutyramide (HABA) provenant de la butirosine, pourrait produire des antibiotiques très prometteurs. Le Chapitre 4 de cette dissertation présente la conception et le développement d’une stratégie semi-synthétique pour produire des nouveaux aminoglycosides améliorés du type 4,5 disubstitués, inspiré par des modifications biosynthétiques de la sisomicine, qui frustrent les mécanismes de résistance bactérienne distribuées globalement. Cette voie de synthèse dépend d’une réaction d’hydrogénolyse de type Tsuji catalysée par palladium, d’abord développée sur des modèles monosaccharides puis subséquemment appliquée pour générer un ensemble d’aminoglycosides hybrides entre la neomycine et la sisomicine. Les études structure-activité des divers analogues de cette nouvelle classe ont été évaluées sur une gamme de 26 souches bactériennes exprimant des mécanismes de résistance enzymatique et d’expulsion qui englobe l’ensemble des pathogènes ESKAPE. Deux des antibiotiques hybrides ont une couverture antibacterienne excellente, et cette étude a mis en évidence des candidats prometteurs pour le développement préclinique. La thérapie avec les antibiotiques aminoglycosidiques est toujours associée à une probabilité de complications néphrotoxiques. Le potentiel de toxicité de chaque aminoglycoside peut être largement corrélé avec le nombre de groupements amino et de désoxygénations. Une hypothèse de longue date dans le domaine indique que les interactions principales sont effectuées par des sels des groupements ammonium, donc l’ajustement des paramètres de pKa pourrait provoquer une dissociation plus rapide avec leurs cibles, une clairance plus efficace et globalement des analogues moins néphrotoxiques. Le Chapitre 5 de cette dissertation présente la conception et la synthèse asymétrique de chaînes N1 HABA β substitutées par mono- et bis-fluoration. Des chaînes qui possèdent des γ-N pKa dans l’intervalle entre 10 et 7.5 ont été appliquées sur une neomycine tétra-désoxygénée pour produire des antibiotiques avancés. Malgré la réduction considérable du γ N pKa, le large spectre bactéricide n’a pas été significativement affecté pour les analogues fluorés isosteriques. De plus, des études structure-toxicité évaluées avec une analyse d’apoptose propriétaire d’Achaogen ont démontré que la nouvelle chaîne β,β difluoro-N1-HABA est moins nocive sur un modèle de cellules de rein humain HK2 et elle est prometteuse pour le développement d’antibiotiques du type neomycine avec des propriétés thérapeutiques améliorées. Le chapitre final de cette dissertation présente la proposition et validation d’une synthèse biomimétique par assemblage spontané du aminoglycoside 66-40C, un dimère C2 symétrique bis-imine macrocyclique à 16 membres. La structure proposée du macrocycle a été affinée par spectroscopie nucléaire à un système trans,trans-bis-azadiène anti-parallèle. Des calculs indiquent que l’effet anomérique de la liaison α glycosidique entre les anneaux A et B fournit la pré-organisation pour le monomère 6’ aldéhydo sisomicine et favorise le produit macrocyclique observé. L’assemblage spontané dans l’eau a été étudié par la dimérisation de trois divers analogues et par des expériences d’entre croisement qui ont démontré la généralité et la stabilité du motif macrocyclique de l'aminoglycoside 66-40C.

Toxicity of Effluent from a Titanium Dioxide Factory on some Marine Animals

Toxicity of effluent from a titanium dioxide factory containing sulphuric acid residue with soluble iron metallic salts and insoluble material such as silica, etc. on fishes, decapods and molluscs was studied. The effluent caused changes in pH and oxygen depletion of the sea water. Sublethal effects of the precipitate of ferrous salts were also studied. Dilutions of effluent up to 1:150 were LC100 for all organisms used while 1:200 dilution was LC50 for fishes at 36 hr and for other organisms at 48 hr. But death of organisms at this concentration was caused by pH changes and oxygen depletion and did not account for the effects of the precipitate. Below this level precipitation started soon after mixing with sea water causing death of organisms by choking their gills and siphons. Dilutions,< 1:1000 were 96 hr LCO.

HEAVY METAL TOXICITY IN BIVALVE- HISTOLOGICAL AND HISTOCHEMICAL ENQUIRY

Division of Marine Biology, Microbiology and Biochemistry, School of Marine Sciences, Cochin University of Science and Technology