Chemical characterization and surface properties of a new bioemulsifier produced by Pedobacter sp. strain MCC-Z.

A novel biopolymer was described in the form of an extracellular polysaccharide (EPS) by Pedobacter sp. strain MCC-Z, a member of a bacterial genus not previously described as an emulsifier producer. The new biomolecule was extracted, purified and characterized, and its surface and emulsifying properties were evaluated. The purified bioemulsifier, named Pdb-Z, showed high emulsifying activity (E24% = 64%) and reduced the surface tension of water up to 41 mN/m with a critical micelle concentration value of 2.6 mg/mL. The chemical characterization of Pdb-Z was performed using (1)H NMR, FT-IR, HPLC/MS/MS and GC/MS. Pdb-Z was found to contain 67% of carbohydrates, consisting mainly of galactose and minor quantities of talose, 30% of lipids, being pentadecanoic acid the major lipidic constituent, and 3% of proteins. The bioemulsifier was a glycolipids-protein complex with an estimated molecular mass of 10(6)Da. Furthermore, Pdb-Z emulsified pure aliphatic and aromatic hydrocarbons as well as diesel more efficiently than commercial synthetic surfactants, used for comparison. Our results suggest Pdb-Z has interesting properties for applications in remediation of hydrocarbon-contaminated environments and bioremediation processes.

Plant growth promotion by inoculation with selected bacterial strains versus mineral soil supplements.

In the process of remediation of mine sites, the establishment of a vegetation cover is one of the most important tasks. This study tests two different approaches to manipulate soil properties in order to facilitate plant growth. Mine waste from Ingurtosu, Sardinia, Italy rich in silt, clay, and heavy metals like Cd, Cu, and Zn was used in a series of greenhouse experiments. Bacteria with putative beneficial properties for plant growth were isolated from this substrate, propagated and consortia of ten strains were used to inoculate the substrate. Alternatively, sand and volcanic clay were added. On these treated and untreated soils, seeds of Helianthus annuus, of the native Euphorbia pithyusa, and of the grasses Agrostis capillaris, Deschampsia flexuosa and Festuca rubra were germinated, and the growth of the seedlings was monitored. The added bacteria established well under all experimental conditions and reduced the extractability of most metals. In association with H. annuus, E. pithyusa and D. flexuosa bacteria improved microbial activity and functional diversity of the original soil. Their effect on plant growth, however, was ambiguous and usually negative. The addition of sand and volcanic clay, on the other hand, had a positive effect on all plant species except E. pithyusa. Especially the grasses experienced a significant benefit. The effects of a double treatment with both bacteria and sand and volcanic clay were rather negative. It is concluded that the addition of mechanical support has great potential to boost revegetation of mining sites though it is comparatively expensive. The possibilities offered by the inoculation of bacteria, on the other hand, appear rather limited.

Predictability of copper, irgarol, and diuron combined effects on sea urchin Paracentrotus lividus.

The aim of this work was to investigate the mixture toxicity of Irgarol (2-methylthio-4-t-butylamino-6-cyclopropylamino-s-triazine), Diuron (3-(3,4-dichlorophenyl)-1,1-dimethylurea), and copper upon the sea urchin Paracentrotus lividus and to compare the observed data with the predictions derived from approaches of Concentration Addition (CA) and Independent Action (IA). Copper spermiotoxicity was more sensitive (EC50 = 0.018 mg/L) than embryotoxicity (EC50 = 0.046 mg/L). The offspring malformations were mainly P1 type (skeletal alterations) in both cases, probably because copper competes to fix Ca2+. Irgarol and Diuron toxicity has been previously investigated. EC50 mixture embryotoxicity showed an EC50 of 1.79 mg/L, whereas spermiotoxicity mixture effects were lower than 11%. Both CA and IA modeling approaches failed to predict accurately mixture toxicity. For embryotoxicity, the IA model overestimated the mixture toxicity at effect levels of <80%. CA does not represent the worst-case approach showing values lower than IA (embryotoxicity) or similar (spermiotoxicity).

Toxic effects of irgarol and diuron on sea urchin Paracentrotus lividus early development, fertilization, and offspring quality.

Irgarol and Diuron are the most representative "organic booster biocides" that replaced organotin compounds in antifouling paints. It cannot be assumed beforehand that their use will have no environmental impact: more ecotoxicological data and a significant environmental monitoring are required. Spermio and embryotoxicities of the biocides Irgarol and Diuron were investigated on Paracentrotus lividus, the dominant echinoid species of the Mediterranean Sea. Spermiotoxicity was studied by assessing the effects of sperm exposure on fertilization rate as well as on the induction of transmissible damages to the offspring. Embryotoxicity was studied by assessing the developmental defects in the exposed larvae. The experimental results show a Diuron EC50 of 2.39 (+/- 0.21) mg/L with a NOEL of 0.25 mg/L for embryos, and of 5.09 (+/- 0.45) mg/L with a NOEL of 0.5 mg/L for sperms, respectively. Data obtained from the embryotoxicity test on Irgarol [EC50 0.99 (+/- 0.69) mg/L] are of the same order of magnitude as the literature data about Japanese urchins. Spermiotoxicity tests show an Irgarol EC50 of 9.04 (+/- 0.45) mg/L with a NOEL of 0.1 mg/L. These data show the different sensitivities of the two tests: embryos are more sensitive than sperms for both the tested chemicals and Diuron seems to be the less toxic one. Moreover, as a major output of the experimental work, tested herbicides exert transmissible damage to spermatozoa evidenced by larval malformations in the offspring, mainly P1 type (skeletal alterations). The comparison of the endpoints results offers an interesting indication of a probable different mode of action (Irgarol seems to interact with calcium homeostasis) of the two biocides.

Copper and other trace elements strongly bound to humic acids along sediment cores in the Ross Sea, Antarctica.

Marine sediment cores sampled in three different areas, during the 1997/98 Italian Antarctic Expedition, were studied. The cores (40 cm) were immediately subdivided into 13 or 14 layers (2-4 cm) in order to obtain a high-depth resolution in the metal content and humic substance analyses. The results obtained for the amount of metals strongly bound to humic acids showed the following order: Cu >> Zn >> Co > As >or= Mn. The fraction of Cu, Zn and As bound to humic acid (microg g(-1) of HA) increases in the first 10-12 cm of cores, then a decrease is observed. This observation could be related both to the presence of a mobile fraction of metals in sediments successively bound to humic acids and to different structural features showed by humic acids along the cores, due to transformation processes. For the other metals a variable pattern along the cores was found.

A kinetic study of trace element leachability from abandoned-mine-polluted soil treated with SS-MSW compost and red mud. Comparison with results from sequential extraction.

The effect of adding treated red mud, a by-product of alumina production, to soil polluted by an abandoned mine and characterised by high concentrations of heavy metals, relatively low reaction grade, and low organic carbon content, was investigated. Also studied was addition of both red mud and compost (produced from source-separated municipal solid waste)--the synergistic action of red mud and compost could be exploited to achieve both metal trapping and an increase in organic carbon content. Leaching batch tests were performed on four different systems: soil, soil and treated red mud, soil and compost, soil and compost plus treated red mud. Dilute sulfuric acid and EDTA solution (liquid/solid ratio 10:1) were used in the tests--sulfuric acid to "mimic" acid rain and EDTA in accordance with general methods for estimating "plant-available" metals. Sequential extraction was also applied to the same samples. The use of relatively non-specific extractant reagents in the leaching tests led to a kinetic approach (already proposed in literature), because measurements of trace elements extracted at equilibrium cannot be related to their speciation. Comparison of information obtainable by the kinetic approach to evaluation of data from leaching tests with results from sequential extraction enabled evaluation whether the "kinetic fractionation method", a relatively rapid and simple procedure, furnishes adequate information about the mobility and bioavailability of trace elements. Especially interesting results were obtained for Mn, Zn, and Ni, present in large amounts in the soil studied--their leachability was significantly reduced by addition of red mud and compost, suggesting interesting perspectives in soil-remediation activity.

Uranium daily intake and urinary excretion: a preliminary study in Italy.

An estimate was made of daily uranium intake in Italy through human diet and drinking water. The uranium concentration was determined in food, drinking water, and other beverages. Uranium concentration was also determined in a "total diet" sampled in 1999. The total daily dietary intake was estimated to be in the range 2.9-4.8 microg d(-1). The contribution of drinking water to the total uranium intake is sometimes significant as the uranium concentration range in 29 analyzed tap and bottled "mineral" waters is 0.04-5.86 microg L(-1) and the drinking water consumption about 1 L d(-1) or more. Data obtained in this study were compared with data from literature on the uranium daily intake in other countries. Urine samples were collected from 24 male and 14 female occupationally unexposed subjects, 20-50 y of age, living and working in the district of Rome (Italy). The mean concentration found was 10 +/- 7.0 ng L(-1); the inter-subject variability and the daily variability was evaluated. Analyses were performed using ICP-MS.

Evaluation of performance of time-saving extraction devices in the BCR three-step sequential extraction procedure.

Two alternative extraction methods--a routine ultrasonic bath and a microwave oven--were developed and optimized for their eventual exploitation in the three-stage sequential extraction procedure proposed by the European Standards, Measurements and Testing (S, M & T) Programme, formerly Bureau Communitaire de Reference (BCR), for the operationally defined speciation of Cd, Cr, Cu, Ni and Zn. The conventional BCR three-stage sequential extraction procedure was modified at each stage, applying ultrasonic or microwave devices in order to shorten the required 16 h of shaking in all three steps. The experimental tests and the optimization of the operating parameters were carried out on a highly homogenized estuarine sediment reference material (RM S7) prepared by the Joint Research Centre (JRC) of ISPRA. Extractable metal contents were measured by inductively coupled plasma-mass spectrometry (ICP-MS) and the results obtained in each fraction by all three methods were statistically compared for all the studied elements. The conventional and newly developed alternative extraction methods were finally compared by the analysis of BCR 601, which is certified for the three-step BCR sequential extraction procedure. According to the statistical evaluation of the results, the proposed accelerated sequential extraction methods are valid alternatives to conventional shaking, with much shorter extraction times.

Platinum levels in natural and urban soils from Rome and Latium (Italy): significance for pollution by automobile catalytic converter.

Platinum concentrations in topsoil samples collected in 1992 (48) and in 2001 (16) from the urban area of Rome have been determined by ICP-MS. Concentrations in 47 soil samples collected in 1992 from natural sites of Latium (an area around Rome) have been determined for a first assessment of natural background levels. The Pt concentrations in Rome urban soils collected in 1992 range from 0.8 to 6.3 ng/g (mean = 3.8 +/- 1.0) overlapping the concentration range of natural soils from Latium (mean = 3.1 +/- 2.1 ng/g). No significant correlation has generally been found between Pt contents in the 'natural' soils and related bedrock or major pedogenetic parameters. These results suggest that there is no evidence of Pt pollution in Rome urban soils at that time, because the massive use of the automobile catalytic converter has only just started. Higher (up to six times more) Pt concentrations, than those measured in the 1992 samples, have been measured, in some cases, in Rome urban soils collected in 2001, suggesting a possible start of Pt accumulation because of the large-scale use in the last decade of automobile catalytic converters. At the same time, a clear decrease of lead levels in Rome urban soils with respect to the levels measured in 1992 has been observed, paralleling the decreasing number of lead gasoline-fuelled cars. Here we present one of the first systematic studies for defining background levels of Pt in Italian natural soils, thus allowing for monitoring, in the future, should any possible Pt pollution caused by the use of automobile catalytic converter, especially in urban soils, occur.

Determination of PAH in atmospheric particulates in the area of the Italian base in Antarctica: report on monitoring activities during the last three scientific expeditions.

Concentrations of polycyclic aromatic hydrocarbons (PAH) in atmospheric particulates have been determined in the frame of the control activities for environmental protection during the VI (1990-1991), VII (1991-1992) and VIII (1992-1993) Italian expeditions in Antarctica. Samples were collected at the four cardinal points at about 200 m from the central area of the Italian scientific base. Data show low levels of the individual PAH (95% in the range 1-50 pg m(-3)) confirming the absence, up to now, of significant contamination from the base. These results could be used as a reference for the assessment of the air pollution due to combustion sources present in the base. Any possible variation in the base's activities in the future should be analysed and evaluated with respect to these findings.

Determination of 2-ethyl-1-hexanol as contaminant in drinking water.

An analytical procedure for the determination of 2-ethyl-1-hexanol (2-EH) in drinking water is presented. The method is based on volatile-compound stripping, adsorption on activated-charcoal-filled tubes, solvent elution, identification by gas chromatography/mass spectrometry, and determination by gas chromatography with flame ionization detection. Bottled samples with undesirable organoleptic characteristics were analyzed to determine a possible correlation with the presence of 2-EH. The presence of 2-EH at 2-10 micrograms/L was confirmed in several samples. The presence of di-2-ethylhexyl phthalate (D2EHP) was also checked in all samples. This compound was always found at 2-30 micrograms/L. Hydrolysis of D2EHP was carried out for 2 weeks to evaluate its possible contribution to water contamination by 2-EH. Tests did not show measurable amounts of the alcohol. Nonetheless, the hydrolysis of phthalates in the weakly acidic conditions of the examined waters would not justify the presence of 2-EH at the observed levels, and so it is reasonable to hypothesize a direct contamination from packaging materials containing 2-EH as residue from D2EHP synthesis.

Amperometric lysine bioprobes analysis in feeds.

Amperometric enzyme electrode probes have been constructed for the specific determination of L-lysine and used in batch and flow analysis. The enzyme lysine oxidase was immobilized on a preactivated polymer support which was placed on a platinum electrode. Additional blocking membranes conferred high stability, reproducibility and avoided electrochemical and enzyme interferences. Parameters including pH, temperature, storage and operational times were optimized. Lysine was determined in the range 10(-6)-2.10(-3)M with a detection limit of 5 x 10(-7)M. The Michaelis constant was 2 x 10(-3)M. This value was approximately two order of magnitudes higher than that reported in literature for the free enzyme. The response time of the probe was about 2 min in batch and flow analysis and 30 sec in flow injection analysis (FIA). The resulting probes were stable for more than three months with more than 300 analyses performed. The determination of lysine was carried out by both flow-through analysis and FIA. Analysis in feeds was carried out by acid hydrolysis to liberate lysine; then the solution was analyzed by the bioprobe and HPLC procedures. Results by the two methods correlated well.

Anticholinesterase activity measurement by a choline biosensor: application in water analysis.

A choline amperometric biosensor was assembled and used to measure the anticholinesterase activity due to compounds (which have the property to inhibit cholinesterase enzymes) present in water samples. This parameter can be used as a 'toxicological index', defined as the amount of compound which causes a certain percentage of cholinesterase inhibition equivalent to a known amount of a reference compound causing the same percentage inhibition. The organophosphorus insecticide Paraoxon, which has proved to be a strong inhibitor of cholinesterase enzymes, was chosen as the reference compound. The analysis was carried out by monitoring the decrease of cholinesterase activity in the presence of a pesticide and a substrate specific for the enzyme whose reaction produces choline. The decrease in choline production was measured by the choline sensor and correlated to the concentration of anticholinesterase compound present in the solution. Parameters such as buffer, pH, temperature and incubation time were optimized. The rate constant Ki was calculated experimentally for Paraoxon and used in the anticholinesterase activity measurements at different fixed incubation times. The probe was calibrated with different standard solutions of Paraoxon. The effect of Paraoxon and heavy metals on the choline probe was evaluated. This probe was then used for the determination of anticholinesterase activity of some organophosphorus pesticides, and heavy metals in spiked waters. Samples were also analysed by liquid/liquid extraction and GC determination. Results seem to correlate with acute toxicity expressed as LD50 (oral, rat). Analysis of water samples from different sources in central Italy were analysed for total anticholinesterase activity (TAA) and compared with a reference procedure.