Critical mercury concentration in tropical soils: Impact on plants and soil biological attributes.

Mercury is a toxic element that becomes a problem when present at high concentrations in soils. Mercury toxicity in soils varies depending on chemical species, concentration, exposure routes, and organism vulnerability. There is little information regarding the toxicity of Hg in tropical soils, especially for establishing safe levels of this pollutant. The purpose of this study was to investigate Hg concentrations in two tropical soils and their effect on oats and common beans, as well as on soil biological attributes. The experiment was carried out in a greenhouse, following ISO 11.269-2 and OECD-208 guidelines. Oat and common bean were cultivated in a Typic Hapludox (TyHpx) and Rhodic Acrudox (RhAcx) contaminated with HgCl2 at the following concentrations: 0, 2.5, 5.0, 10.0, 20.0, 40.0, and 80.0 mg of Hg kg-1 of dry soil. The biological variables analyzed were seedling emergence, vegetative growth, chlorophyll content (SPAD index), gas exchange (photosynthetic rate, internal CO2 concentration, transpiration rate, and stomatal conductance), and Hg concentration and accumulation in shoot dry matter. Microbial biomass carbon, soil basal respiration, and metabolic quotient (qCO2) were also analyzed. Due to the sorptive characteristics of TyHpx, it had higher Hg concentrations than RhAcx. Mercury showed toxic effects on both oat and common bean species. However, common bean was affected only at concentrations higher than 20 mg kg-1. The microbial community showed high sensitivity to soil Hg concentrations, but external factors, such as the plant species cultivated, influenced the sensitivity of the community. The microbiota was most sensitive in pots with common bean, and this effect was more pronounced at low clay and low organic matter contents (TyHpx). In this study, the concentration of 0.36 mg kg-1 was critical for Hg in these soils, based on its deleterious effects on oat and common bean and on biological soil attributes.

Selenium and mercury in Brazilian Cerrado soils and their relationships with physical and chemical soil characteristics.

The objective of this study was to determine the natural concentrations of Hg and Se in 45 representative soil profiles from the Cerrado biome in central Brazil, and to correlate their concentrations with soil chemical and physical characteristics. The study area was composed of three sub-regions: Goiás, Northwest of Minas Gerais, and Minas Gerais Triangle. Selenium and Hg concentrations were determined by acid digestion and atomic absorption spectroscopy. Data were subjected to analysis of variance on the means of the Hg and Se variables within each soil class at two depths, followed by multivariate statistical methods. The Hg concentrations ranged from 15 to 182 µg kg-1 and the Se concentrations ranged from 22 to 72 µg kg-1. The soil characteristics that most contributed to Hg concentrations in the soils, according to principal component analysis, were Fe2O3, FeO, TiO2, pH, P2O5, and effective CEC. In general, the soils of the Cerrado biome have deficient Se concentrations. The Humic Rhodic Acrustoxes have Hg concentrations above the prevention reference value for soils of Minas Gerais.

Two New Species of the Spittlebug Genus Ocoaxo Fennah (Hemiptera: Cercopidae) from Mexico, and Keys for the Groups, Group Three, and First Subgroup.

Insects of the family Cercopidae are known as spittlebugs or froghoppers and are represented by 62 genera in the Neotropical region. One of these genera is Ocoaxo Fennah, 1968 with 30 species. The most recent species to be accepted into this genus, Ocoaxo costaricanus, was described by Nast (Ann Zool 33:93-101, 1975). Herein, two new species of Ocoaxo from Mexico are described. One of these new species forms a complex together with Ocoaxo assimilis (Walker) and Ocoaxo varians (Stål). The complex has economic importance in the mountainous areas of the states of Puebla and Oaxaca because it attacks Pinus spp. and causes a disorder called "pine decline." Additionally, dichotomous keys were designed to identify the Ocoaxo Fennah groups and also the species of the subgroup bivittus.

Natural variation of selenium in Brazil nuts and soils from the Amazon region.

Brazil nut tree (Bertholletia excelsa) is native of the Amazon rainforest. Brazil nuts are consumed worldwide and are known as the richest food source of selenium (Se). Yet, the reasoning for such Se contents is not well stablished. We evaluated the variation in Se concentration of Brazil nuts from Brazilian Amazon basin, as well as soil properties, including total Se concentration, of the soils sampled directly underneath the trees crown, aiming to investigate which soil properties influence Se accumulation in the nuts. The median Se concentration in Brazil nuts varied from 2.07 mg kg-1 (in Mato Grosso state) to 68.15 mg kg-1 (in Amazonas state). Therefore, depending on its origin, a single Brazil nut could provide from 11% (in the Mato Grosso state) up to 288% (in the Amazonas state) of the daily Se requirement for an adult man (70 µg). The total Se concentration in the soil also varied considerably, ranging from <65.76 to 625.91 µg kg-1, with highest Se concentrations being observed in soil samples from the state of Amazonas. Se accumulation in Brazil nuts generally increased in soils with higher total Se content, but decreased under acidic conditions in the soil. This indicates that, besides total soil Se concentration, soil acidity plays a major role in Se uptake by Brazil nut trees, possibly due to the importance of this soil property to Se retention in the soil.

Phosphorus improves arsenic phytoremediation by Anadenanthera peregrina by alleviating induced oxidative stress.

Due to similarities in their chemical behaviors, studies examining interactions between arsenic (As)--in special arsenate--and phosphorus (P) are important for better understanding arsenate uptake, toxicity, and accumulation in plants. We evaluated the effects of phosphate addition on plant biomass and on arsenate and phosphate uptake by Anadenanthera peregrina, an important Brazilian savanna legume. Plants were grown for 35 days in substrates that received combinations of 0, 10, 50, and 100 mg kg(-1) arsenate and 0, 200, and 400 mg kg(-1) phosphate. The addition of P increased the arsenic-phytoremediation capacity of A. peregrina by increasing As accumulation, while also alleviating As-induced oxidative stress. Arsenate phytotoxicity in A. peregrina is due to lipid peroxidation, but not hydrogen peroxide accumulation. Added P also increased the activity of important reactive oxygen species-scavenging enzymes (catalase and ascorbate peroxidase) that help prevent lipid peroxidation in leaves. Our findings suggest that applying P represents a feasible strategy for more efficient As phytoremediation using A. peregrina.

Genetic variability of Mahanarva sp (Hemiptera: Cercopidae) collected from different sites in Brazil.

Spittlebugs are the leading cause of damage to tall grasses. Annual losses are estimated to reach 2.1 billion dollars in sugarcane crops and grazing land throughout the world. Correct identification of these species is difficult due to similarities in color, body size and male genitalia. Molecular markers have been useful in the identification and assessment of genetic diversity of many species. We investigated the genetic diversity of the spittlebug species Mahanarva fimbriolata, M. spectabilis and M. liturata and looked for markers that could aid in their identification. DNA from 34 spittlebug specimens, collected from six different regions of Brazil (Brasília, Campo Grande, Valença, Presidente Prudente, Juiz de Fora, and Porto Alegre), was analyzed with 29 RAPD primers, generating 501 polymorphic markers. High genetic variability was found among individuals M. fimbriolata (0.37), M. spectabilis (0.18) and M. liturata (0.69). Species-specific molecular RAPD markers were identified for each of the three species; these could be used as auxiliary tools for their correct identification.

Distribution patterns of Neotropical primates (Platyrrhini) based on Parsimony Analysis of Endemicity.

The Parsimony Analysis of Endemicity (PAE) is a method of historical biogeography that is used for detecting and connecting areas of endemism. Based on data on the distribution of Neotropical primates, we constructed matrices using quadrats, interfluvial regions and pre-determinated areas of endemism described for avians as Operative Geographic Units (OGUs). We codified the absence of a species from an OGU as 0 (zero) and its presence as 1 (one). A hypothetical area with a complete absence of primate species was used as outgroup to root the trees. All three analyses resulted in similar groupings of areas of endemism, which match the distribution of biomes in the Neotropical region. One area includes Central America and the extreme Northwest of South America, other the Amazon basin, and another the Atlantic Forest, Caatinga, Cerrado and Chaco.

Distribution patterns of Neotropical primates (Platyrrhini) based on Parsimony Analysis of Endemicity

A Análise de Parcimônia de Endemismo (PAE) é um método da biogeografia histórica que é usado para detectar e conectar áreas de endemismo. Baseando-se em dados de distribuição de primatas Neotropicais, construíram-se matrizes de dados utilizando-se quadrículas, regiões entre rios e áreas de endemismo pré-determinadas para aves como Unidades Geográficas Operacionais (OGUs). Codificou-se a ausência da espécie na OGU como 0 (zero) e a presença como 1 (um). Uma área hipotética com ausência total de espécies de primatas foi usada como grupo externo para polarização. Todas as três análises resultaram em grupos similares de áreas de endemismo, coincidindo com a distribuição de biomas na região Neotropical: uma área incluindo a América Central e o extremo Noroeste da América do Sul; outra, a Bacia Amazônica; e, uma terceira, a Mata Atlântica, Caatinga, Cerrado e Chaco.

In vitro osteoblastic differentiation of human bone marrow cells in the presence of metal ions.

For periods up to 21 days human bone marrow was cultured in control conditions that favor the proliferation and differentiation of osteoblastic cells. The effect of AISI 316L corrosion products and the corresponding major separate metal ions (Fe, Cr, and Ni) were studied in three different phases of the culture period in order to investigate the effects of metal ions in cell populations representative of osteoblastic cells in different stages of differentiation. Toxicity consequences of the presence of metal ions in bone marrow cultures were evaluated by biochemical parameters (enzymatic reduction of MTT, alkaline phosphatase activity, and total protein content), histochemical assays (identification of ALP-positive cells and Ca and phosphates deposits), and observation of the cultures by light and scanning electron microscopy. Culture media were analyzed for total and ionized Ca and P and also for metal ions (Fe, Cr, and Ni). The presence of AISI 316L corrosion products and Ni salt in bone marrow cultures during the first and second weeks of culture significantly disturbs the normal behavior of these cultures, interfering in the lag phase and exponential phase of cell growth and ALP expression. However, the presence of these species during the third week of culture, when expression of osteoblastic functions occurs (mineralization process), did not result in any detectable effect. Fe salt also disturbs the behavior of bone marrow cell cultures when present during the lag phase and proliferation phase, and a somewhat compromised response between the normal pattern (control cultures) and intense inhibition (AISI 316L corrosion products and Ni salt-added cultures) was observed. Fe did not affect the progression of the mineralization phase. Osteogenic cultures exposed to Cr salt (Cr3+) presented a pattern similar to the controls, indicating that this element does not interfere, in the concentration studied, in the osteoblastic differentiation of bone marrow cells. Quantification of metal ions in the culture media showed that Cr (originated from AISI 316L corrosion products but from not Cr3+ salt) and Ni (originated from AISI 316L corrosion products and Ni salt) appear to be retained by the bone marrow cultures.

Miniaturization and application of the MTT assay to evaluate metabolic activity of protozoa in the presence of toxicants.

This paper describes a critical evaluation of a miniaturised colorimetric assay, using MTT (3-[4,5-dimethyl-thiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) reduction, applied to protozoan viability testing. The toxic substances used were copper, zinc, Triton X-100 (a membrane surfactant) and cycloheximide (an inhibitor of the protein synthesis). The viability assay of the ciliate protozoan Tetrahymena pyriformis was optimised in terms of MTT concentration and incubation time. Since protozoa are non adherent cells the MTT assay was modified in order to maintain the medium in the well. MTT proved to be effective in the measurement of Tetrahymena pyriformis viability. Four hours of MTT incubation followed by 30 minutes of incubation with DMSO were found to be the best incubation times for optical density reading. Furthermore, 10 mg/ml of MTT solution was the concentration that gave higher values of optical densities with minor medium interference.

Decreased consumption of Ca and P during in vitro biomineralization and biologically induced deposition of Ni and Cr in presence of stainless steel corrosion products.

The purpose of this study was to investigate the effects of 316L stainless steel (SS) corrosion products on the in vitro biomineralization process, because tissue necrosis, bone loss, impaired bone mineralization, and loosening of orthopedic implants are associated with ions and debris resulting from biodegradation. Rat bone marrow cells were cultured in experimental conditions that favored the proliferation and differentiation of osteoblastic cells and were exposed to SS corrosion products obtained by electrochemical means for periods ranging from 1 to 21 days. Quantification of total and ionized Ca and P, as well as Fe, Cr, and Ni, ions in the culture media of control and metal added cultures during the incubation period was performed to study the influence of corrosion products on the Ca and P consumption that occurs during the mineralization process. Control cultures and metal effects on cultures were evaluated concerning DNA content, enzymatic reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and alkaline phosphatase (ALP) activity. Histochemical detection of ALP, Ca, and phosphate deposition, and examination of the cultures by scanning and transmission electron microscopy (SEM and TEM) were also performed. The presence of SS corrosion products resulted in impairment of the normal behavior of rat bone marrow cultures. Levels of Cr and Ni in the medium of cultures exposed to 316L SS corrosion products decreased throughout the incubation period, suggesting a regular deposition of these species; these results were supported by TEM observation of the cultures. Cultures exposed to the corrosion products presented lower DNA content, MTT reduction, and ALP activity and failed to form mineralized areas. These cultures showed negative staining on histochemical reactions for the identification of calcium and phosphate deposition and SEM and TEM examination did not show mineral globular structures or mineralization foci, respectively, which is characteristic of cultures grown in control conditions. These results suggest that metal ions associated with 316L SS are toxic to osteogenic cells, affecting their proliferation and differentiation.

Chromium determination in osteoblast-like cell culture medium by catalytic cathodic stripping voltammetry with a mercury microelectrode.

A catalytic cathodic stripping voltammetric procedure for the determination of total chromium in osteoblast-like cell culture medium using a mercury film microelectrode (MFM) was optimised. The method is based on the pre-concentration of the Cr(III)-diethylenetriaminepentaacetic acid (DTPA) complex by adsorption at the potential of-1.00 V (vs. Ag/AgC1) in the presence of 10 x 10(-3) mol/L DTPA, 0.70 mol/L sodium nitrate, 0.04 mol/L sodium acetate and 1.0 x 10(-3) mol/L potassium permanganate at pH 5.9-6.0. The limit of detection obtained for a 40 s collection time was 2.80 x 10(-10) mol/L of chromium. The results achieved by stripping voltammetry using the MFM were compared to those obtained by atomic absorption spectrometry (AAS) to ensure the reliability of the electrochemical method. This procedure proved to be an alternative to AAS and valuable in biocompatibility studies performed in vitro using osteoblast-like cells.

Effects of AISI 316L corrosion products in in vitro bone formation.

Rat bone marrow cells were cultured in experimental conditions that favour the proliferation and differentiation of osteoblastic cells (i.e., 2.52 x 10(-4) mol l(-1) ascorbic acid, 10(-2) mol l(-1) beta-glycerophosphate and 10(-8) mol l(-1) dexamethasone) in the absence and in the presence of stainless-steel corrosion products, for a period of 18 days. An AISI 316L stainless-steel slurry (SS) was obtained by electrochemical means and the concentrations of the major metal ions, determined by atomic absorption spectrometry, were 8.78 x 10(-3) mol l(-1) of Fe, 4.31 x 10(-3) mol l(-1) of Cr and 2.56 x 10(-3) mol l(-1) of Ni. Bone marrow cells were exposed to 0.01, 0.1 and 1% of the SS and at the end of the incubation period, control and treated cultures were evaluated by histochemical assays for the identification of the presence of alkaline phosphatase and also calcium and phosphate deposition. Cultures were further observed by scanning electron microscopy. Levels of total and ionised calcium and phosphorus in the culture media collected from control and metal exposed cell cultures were also quantified. Histochemical staining showed that control cultures presented a strong reaction for the presence of alkaline phosphatase and exhibited formation of calcium and phosphates deposits. The presence of 0.01% SS caused no detectable biological effects in these cultures, 0.1% SS impaired osteoblastic behaviour and, 1% SS resulted in cell death. In the absence of bone cells, levels of total and ionised calcium and phosphorus in the control and metal added culture medium were similar throughout the incubation period. A significant decrease in the levels of ionised calcium and phosphorus were observed in the culture medium of control cultures and also in cultures exposed to 0.01% SS after two weeks of incubation, an event related with the formation of mineral calcium phosphate deposits in these cultures. In cultures grown in the presence of 0.1 and 1% SS corrosion products, levels of calcium and phosphorus were similar to those observed in the absence of cells. Results showed that stainless-steel corrosion products above certain concentrations may disturb the normal behaviour of osteoblast-like rat bone marrow cell cultures.

In vitro biomineralization by osteoblast-like cells. I. Retardation of tissue mineralization by metal salts.

The cytocompatibility of stainless steel 316L (SS 316L) corrosion products was investigated with particular focus on the dose- and time-effect of electrochemically dissolved SS and the corresponding separate metal ions on osteogenic bone marrow derived cells. Type AISI 316L stainless steel (Fe 63.9%, Cr 18.0%, Ni 12.5%, Mo 2.8%, Si 1.2%, Mn 1.6% and C 0.025%, weight for weight) was anodically dissolved in Hank's Balanced Salt Solution (HBSS) and diluted to the following concentrations: 500 microg ml(-1) of Fe, 122 microg ml(-1) of Cr and 101 microg ml(-1) of Ni, as estimated by atomic absorption spectrometry. Similarly, salt solutions containing 50 microg ml(-1) of Fe (FeCl3 x 6H2O), 122 microg ml(-1) of Cr (CrCl3 x 6H2O) or 101 microg ml(-1) of Ni (NiNO3) were prepared. All solutions were diluted 1:10(3), 1:10(4) and 1:10(5) and their effects on cell proliferation and function of rabbit bone marrow cells were studied up to 28 days of culture. Bone marrow cells (second subculture) were cultured in alpha-Minimal Essential Medium (alpha-MEM) supplemented with 10% fetal bovine serum 10(-8) mol l(-1) dexamethasone, 2.52 x 10(-4) mol l(-1) ascorbic acid and 10(-2) mol l(-1) beta-glycerophosphate. The osteoblast response to the presence of metal ions was evaluated by biochemical assays (enzymatic reduction of MTT for evaluation of cell viability/proliferation, and estimation of alkaline phosphatase (ALP) activity) and histochemical assays (identification of ALP positive cells and calcium and phosphates deposits). Results suggest a decrease in the expression of the osteoblast phenotype in the presence of ion and alloy solutions. Stainless steel corrosion products elicited slight effects but the corresponding metal ions produced pronounced effects on the osteoblast phenotype, namely an alteration in the levels and temporal expression of ALP and lower and retarded tissue mineralization ability.

In vitro biomineralization by osteoblast-like cells. II. Characterization of cellular culture supernatants.

The quantification of total calcium, phosphorus, iron, chromium and nickel in cell culture medium by electrochemical or spectroscopic means may require digestion of samples. Nevertheless, when pH adjustment is performed for values higher than about 6.5, the formation of two phases occurs: a white precipitate and a clear solution. Analysing both phases using microelectrodes, atomic absorption spectrometry (AAS), diffuse reflectance infrared Fourier transform (DRIFT) spectroscopy, X-ray dispersive (XRD) analysis, scanning electron microscopy (SEM) and energy dispersive spectroscopic (EDS) analysis, it was observed that iron, chromium and nickel are not co-precipitating with the white solid phase. If quantification of calcium, phosphorus and magnesium is intended, a ten-fold dilution at least, must be performed to avoid most of these elements going into the precipitate. This knowledge is crucial if a mineralization study is going to be made.

The use of rat, rabbit or human bone marrow derived cells for cytocompatibility evaluation of metallic elements.

Rat, rabbit and human bone marrow cells were cultured according to the method previously reported for cells of rat origin [1] and were exposed, or not (control), to corrosion products of a Co-Cr orthopaedic alloy as well as to metal salts containing Co2+, Cr3+ and Cr6+. Cells were cultured for 21 days and analysed for the following biochemical parameters: intracellular MTT reduction (i.e. cell viability/proliferation), alkaline phosphatase (ALP) activity and protein production. Morphological observations included both histochemistry (detection of ALP-positive cells, calcium and phosphate deposits) and scanning electron microscopy (SEM). Control cultures of rat and rabbit cells showed higher proliferation rates than human cells at the start of culture, but they all reached similar values on day 21. Protein production was parallel to cell proliferation. In contrast, ALP activity of rat cultures was much stronger than rabbit or human cultures. All cell types were able to develop the osteogenic phenotype in vitro.Co-Cr extract caused inhibitory effects on cell viability, on ALP activity and, to a lower extent, on protein production of all rat, rabbit and human cell cultures. Compared to rat and rabbit cultures, human cultures were the most sensitive to metal ions exposure.

Mineralization in serially passaged human alveolar bone cells.

Well-characterized human bone cell cultures have been regarded as a useful tool to study bone control mechanisms and also to analyse bone/biomaterials interactions. In the present study, human alveolar bone cells were cultured in alpha-minimal essential medium (alpha-MEM) containing 10% foetal bovine serum (FBS), 50 microg/ml ascorbic acid, 10 mM sodium beta-glycerophosphate and either in the presence or in the absence of 10 nM dexamethasone (Dexa). Cultures were characterized concerning cell viability/proliferation, alkaline phosphatase (ALP), acid phosphatase (ACP) and tartraric acid resistant phosphatase (TRAP) activities, and formation of mineralized areas. Cell proliferation increased gradually for approximately 20 days. In the presence of Dexa, cells formed isolated or interconnected multilayered clusters that increased with culture time. Histochemical assays revealed strong positive reactions for ALP and calcium and phosphates deposits, mainly in relation t! o ce lls associated with the clusters. High levels of ALP activity (biochemical determination) were observed. Cells cultured in the absence of Dexa showed significantly lower ALP activity and no calcium and phosphates deposits were present. Serially passaged cells kept the proliferation rate constant but a decrease in ALP activity was observed either in the presence or in the absence of Dexa. The ability to form mineralized areas (cultures fed with Dexa) also decreased on serial subculture.

Use of microelectrodes as electrochemical sensors of metal ions released from biomaterials.

In vitro studies to determine Fe3+ levels in mice liver samples were performed using platinum microelectrodes and atomic absorption spectrophotometry. Microelectrodes have been shown to be useful for quantitative analysis of metal ions released during the biodegradation process that occurs on implanted metallic biomaterials.

Toxic effects caused by stainless steel corrosion products on mouse seminiferous cells.

Stainless steel type AISI 316L is widely used in orthopaedic surgery. The toxic effects of its corrosion products on the morphology of mouse seminiferous cells were investigated. Chemical elements from stainless steel were released into a physiological medium using an electrochemical method. This metallic solution was injected subcutaneously into male Charles River mice at 72 h intervals for 10 days. Electron microscopic observations of seminiferous tubule thin sections showed that the metallic suspension caused tissue vacuolation, cell degeneration, and multinucleated cell formation. This apparent tissue toxicity induced by stainless steel corrosion products suggests that long term implantation of such biomaterials may impair spermatogenesis.

Stimulation of human T lymphocytes via CD2 and CD3 molecules: establishment of the method and characterisation of proliferating cells.

The in vitro exposure of human lymphocytes to degradation products (Fe, Ni or Co) of metallic biomaterials causes a significant reduction of lymphocytes expressing the molecules involved in T lymphocyte activation, CD2 and CD3. A method is described which was developed for the stimulation of lymphocytes in which both CD2 and CD3 molecules were triggered simultaneously. For this purpose an anti-CD3 monoclonal antibody (mAb) was chemically bound to sheep (SE) or human (HE) erythrocytes, forming SE alpha CD3 or HE alpha CD3 conjugates, respectively, which were used for lymphocyte stimulation. Stimulation via CD2 and CD3 was compared with classical phytohaemagglutinin stimulation as well as with soluble alpha CD3 mAb stimulation. Phenotypical characterisation of DNA synthesising lymphocytes was assessed by autoradiography of 3H-thymidine pulsed cultures combined with immunocytochemical tests. Results indicate that this method of T lymphocyte stimulation via CD2 and CD3 is reliable and consistent, and it seems to be very convenient for the evaluation of immunocytotoxicity of metal ions derived from metallic biomaterials.