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Rapid-cooling and storage of plant nematodes at -140 degrees C.

Low temperatures can assure the long-term or even indefinite preservation of important biological specimens. Nematode cryopreservation allows for the availability of large numbers of living nematodes at any one time, especially for experimental purposes. New isolates of Bursaphelenchus have recently been collected, including Bursaphelenchus eremus (Rühm) Goodey. This species was identified in north-central Italy on dying oak trees and from the bark beetle Scolytus intricatus Ratzeburg as dauer larvae. We therefore, sought to develop a cryopreservation technique for the long-term storage of all available Bursaphelenchus spp. The technique consists of a rapid-cooling protocol involving immersion in a liquid nitrogen bath before storage of the frozen samples in a mechanical freezer at -140 degrees C. The survival of nematodes subjected to this rapid-cooling protocol was higher than previously reported using slow-cooling methods and is suitable for several species of Bursaphelenchus and other phytoparasitic nematodes.

Animals↗

Ecotoxicological evaluation of cork-boiling wastewaters.

Toxicity tests with Vibrio fischeri, Daphnia magna, and Lemna minor were used to evaluate acute and chronic toxicity of cork-boiling wastewaters and correlation analysis was performed with physicochemical parameters. Acute toxicity values (EC(50)) ranged from 2.3% to 24.2% in the Microtox test and from 4.4% to 29.5% in the Daphnia test. According to these values, 78% of the samples were classified as acutely toxic to the most sensitive species (V. fischeri). Significant correlation was obtained between EC(50) and chemical oxygen demand, biochemical oxygen demand after 5 days, total organic carbon, tannin and lignin and total solids. No significant correlation was found between the two acute tests. The D. magna chronic test showed significant differences between all tested concentrations and the control, with an EC(50) of 0.32%. An EC(50) value of 26.0% was calculated for L. minor. For the analysis of cork-boiling wastewater toxicity and routine biomonitoring, the use of the Microtox test is suggested, to afford greater environmental protection.

Aliivibrio fischeri↗

Detecting changes in insect herbivore communities along a pollution gradient.

The forests surrounding the urban areas of the Los Angeles basin are impacted by ozone and nitrogen pollutants arising from urban areas. We examined changes in the herbivore communities of three prominent plant species (ponderosa pine, California black oak and bracken fern) at six sites along an air pollution gradient. Insects were extracted from foliage samples collected in spring, as foliage reached full expansion. Community differences were evaluated using total herbivore abundance, richness, Shannon-Weiner diversity, and discriminant function analysis. Even without conspicuous changes in total numbers, diversity or richness of herbivores, herbivore groups showed patterns of change that followed the air pollution gradient that were apparent through discriminant function analysis. For bracken fern and oak, chewing insects were more dominant at high pollution sites. Oak herbivore communities showed the strongest effect. These changes in herbivore communities may affect nutrient cycling in forest systems.

Air Pollution↗

Lichen biomonitoring of ammonia emission and nitrogen deposition around a pig stockfarm.

Effects of high ammonia emissions and nitrogen deposition were investigated on lichens around a pig stockfarm (ca. 7,000 animals) in central Italy. Four sites were selected along a transect at 200, 400, 1000 and 2500 m from the stockfarm, the diversity of epiphytic lichens was measured and transplanted thalli of Xanthoria parietina and Flavoparmelia caperata exposed, together with passive NH3 (diffusion tubes) samplers. Ammonia dramatically decreased from the centre of the stockfarm to the sampled sites, where it was correlated with bark pH. Total lichen diversity was not associated with either NH3 concentrations or bark pH, but the diversity of strictly nitrophytic species was highly correlated with both parameters. Physconia grisea was the best indicator species for NH3 pollution. Total N accumulated in X. parietina and F. caperata was correlated with NH3 concentrations.

Agriculture↗

Lichen and bryophyte distribution on oak in London in relation to air pollution and bark acidity.

Epiphytic lichen and bryophyte distribution and frequency were investigated on the trunks of 145 young oak trees throughout London and surrounding counties, and compared with pollution levels and bark pH. Sixty-four lichen and four bryophyte species were recorded. Three major zones were identified: (i) two central regions with a few lichens, bryophytes absent; (ii) a surrounding region with a more diverse flora including a high cover of nitrophyte lichens; and (iii) an outer region, characterised by species absent from central London, including acidophytes. Nineteen species were correlated with nitrogen oxides and 16 with bark pH, suggesting that transport-related pollution and bark acidity influence lichen and bryophyte distribution in London today. Lichens and bryophytes are responding to factors that influence human and environmental health in London. Biomonitoring therefore has a practical role to assess the effects of measures to improve London's air quality.

Air Pollution↗

Comparison of calculated and measured foliar O3 flux in crop and forest species.

We designed a new gas exchange system that concurrently measures foliar H2O, O3, and CO2 flux (HOC flux system) while delivering known O3 concentrations. Stomatal responses of three species were tested: snapbean, and seedlings of California black oak (deciduous broadleaf) and blue oak (evergreen broadleaf). Acute O3 exposure (120-250 ppb over an hour) was applied under moderate light and low vapor pressure deficits during near steady state conditions. The rate of stomatal closure was measured when the whole plant was placed in the dark. An adjacent leaf on each plant was also concurrently measured in an O3-free cuvette. Under some conditions, direct measurements and calculated foliar O3 flux were within the same order of magnitude; however, endogenously low gs or O3 exposure-induced depression of gs resulted in an overestimation of calculated O3 fluxes compared with measured O3 fluxes. Sluggish stomata in response to light extinction with concurrent O3 exposure, and incomplete stomatal closure likewise underestimated measured O3 flux.

Carbon Dioxide↗

Polyphasic identification of yeasts isolated from bark of cork oak during the manufacturing process of cork stoppers.

A two-step protocol was used for the identification of 52 yeasts isolated from bark of cork oak at initial stages of the manufacturing process of cork stoppers. The first step in the identification was the separation of the isolates into groups by their physiological properties and RFLPs of the ITS-5.8S rRNA gene. The second step was the sequencing of the D1/D2 domains of the 26S rRNA gene of selected isolates representing the different groups. The results revealed a predominance of basidiomycetous yeasts (11 species), while only two species represented the ascomycetous yeasts. Among the basidiomycetous yeasts, members representing the species Rhodosporidium kratochvilovae and Rhodotorula nothofagi, that have been previously isolated from plant material, were the most abundant. Yeasts pertaining to the species Debaryomyces hansenii var. fabryii, Rhodotorula mucilaginosa and Trichosporon mucoides were isolated in small numbers.

Base Sequence↗

Fungal strains isolated from cork stoppers and the formation of 2,4,6-trichloroanisole involved in the cork taint of wine.

Cork taint is mainly due to 2,4,6-trichloroanisole (TCA) produced through the activity of undesirable fungal strains. We observed that CFU mould number in TCA-containing stoppers was not quantitatively different to that of the stoppers not containing TCA (ca. 10(5)CFU/g). In contrast more fungi diversity was observed in TCA-containing stoppers. Penicillium spp (Penicillium chrysogenum, Penicillium glabrum), Aspergillus spp (Aspergillus niger and Aspergillus oryzae), Chrysonilia sitophila, Mucor racemosus, Paecilomyces sp. and Trichoderma viride were found in TCA-containing stoppers, while C. sitophila and Penicillium sp. were the main fungi in the stoppers devoid of TCA. Conidia were numerous close to the lenticels and present from the lateral surface through to the centre of the stoppers. Strains of Aspergillus, Mucor, Paecilomyces, Penicillium and Trichoderma isolated from TCA-containing stoppers were able to convert 2,4,6-trichlorophenol (TCP) in TCA in resting cell or growing conditions. The best yields of conversion were obtained by green fungi Paecilomyces sp. and P. chrysogenum, 17% and 20%, respectively. Chysonilia sitophila and Penicillium sp. did not produce TCA from TCP in our conditions.

Anisoles↗

Overall characterization of cork dust explosion.

Explosibility and ignitability studies of air/cork dust mixtures were conducted in a near-spherical 22.7 L explosibility test chamber using pyrotechnic ignitors and in a furnace of 1.23 L. The suspension dust burned as air-dispersed dust clouds and the uniformity of the dispersion inside the chamber was evaluated through optical dust probes. The range of tested particle sizes went from a mass median diameter of 47.4 to 438.3 microm and the covered dust cloud concentration was up to 700-800 g/m(3). Measured explosion parameters included minimum explosible concentration, maximum explosion pressure, maximum rate of pressure rise and minimum autoignition temperature. The effect of dust particle size on flammability was evaluated and it was found that the minimum explosible concentration is around 40 g/m(3) and it is relatively independent of particle size below 180 microm. Maximum explosion pressure of 7.2 bar and maximum rate of pressure rise of 179 bar/s were detected for the smallest tested sizes. The limitations on the rates of devolatilization of the solid particles became rate controlling at high burning velocities, at high dust loadings and for large particle sizes. The effect of initial pressure on the characteristic parameters of the explosion was studied by varying the initial absolute pressure from 0.9 bar to 2.2 bar, and it was found that as initial pressure increases, there is a proportional increase of minimum explosion limit, maximum explosion pressure, and maximum rate of pressure rise. The influence of the intensity of the ignition energy on the development of the explosion was evaluated using ignition energies of 1000 J, 2500 J and 5000 J, and the experimental data showed that the value of 2500 J is the most convenient to use in the determination of minimum explosion concentration. The behavior of the cork dust explosion in hybrid methane air mixtures was studied for atmospheres with 2% and 3.5% (v/v) of methane. The effect of methane content on the characteristic parameters of the explosions was evaluated. The conclusion is that, the hazard and explosion danger rise with the increase of methane concentration, characterized by the reduction of the minimum dust explosion concentration. The minimum autoignition temperature obtained with the thermal ignition tests was 540 degrees C and the results have shown that this value is independent of particle size, for particles with mass median diameters below 80 microm.

Dust↗

A protocol for direct sequencing of multiple gene specific PCR products from Discula umbrinella, a fungal endophyte, utilizing bufferless precast electrophoresis.

A protocol for direct sequencing of polymerase chain reaction (PCR) products from mycelia of Discula umbrinella, a fungal endophyte, using bufferless electrophoresis is described. This improved method allows researchers to conduct high-capacity screening of multiple gene regions for fungal endophytes applicable to microbial ecology and population genetic studies.

Chitin Synthase↗

Biomonitoring of trace elements in the leaves and fruits of wild olive and holm oak trees.

Biomonitoring of trace elements is essential to assess ecosystem health, in particular in landscapes influenced by human activity. The Guadiamar Valley (SW Spain) was polluted in 1998 by a spill from an open-pit pyrite mine affecting about 55 km2. In this paper, we used two common species of tree, namely wild olive and holm oak, to biomonitor the concentration of nine trace elements-As, Cd, Cu, Fe, Mn, Ni, Pb, Tl and Zn-in this spill-affected area over the 3-year period 1999-2001. We analysed the leaves and fruits of trees growing in the spill-affected terraces, and compared them with adjacent trees in the non-affected upper terraces. The main trace elements polluting the soil were Zn, As, Pb and Cu. In general, the oak leaves were richer in trace elements than the olive leaves, reaching phytotoxic levels for As and Pb, while the olive fruits (pulp) were more polluted than the oak seeds (protected inside a hard pericarp), reaching toxic values for Cd and Pb. The concentration of trace elements in the leaves and fruits decreased with time and, in consequence, the toxicity risk to the food web diminished.

Accidents, Occupational↗

Analysis of short-term influences of ambient aeroallergens on pediatric asthma hospital visits.

The objective of our study was to investigate the association between daily pediatric asthma hospital visits and daily concentrations of aeroallergens and their specific species. Records of daily asthma visits in Cincinnati area were retrieved from Cincinnati Children's Hospital Medical Center and aeroallergen sampling was performed by the Button Inhalable Sampler. The Poisson generalized linear model was carried out in which the log of the number of asthma hospital visits was related to aeroallergen level, treated as a continuous variable with adjustment for seasonal time trend, day of the week, ozone and PM(2.5) concentrations, temperature and humidity. The aeroallergens having a significant impact on asthma hospital visits were ragweed, oak/maple and Pinaceae pollen. Their relative risks on asthma hospital visits with respect to a 100 counts/m(3) increase in concentration were in the range of 1.23 to 1.54. The effects in causing the asthma exacerbation were delayed by 3 or 5 days.

Acer↗

REE characteristics and Pb, Sr and Nd isotopic compositions of steel plant emissions.

A comprehensive Pb-Sr-Nd isotope and REE tracer study of atmospheric trace metal pollution by a steel plant situated to the north of the urban communities of Strasbourg (France) and Kehl (Germany) has been performed using tree barks as biomonitors. The 206Pb/207Pb and 208Pb/207Pb isotopic ratios of the steel plant's filter dust are similar to values found in dust of waste incinerators. The 87Sr/86Sr ratio is similar to present-day ratios of Phanerozoic or Precambrian granitic rocks. The 143Nd/144Nd isotopic composition is very low and corresponds to an (Nd) value of -17.5. Such a low value is characteristic of old Precambrian granitic rocks and banded iron formations. Thus, this low (Nd) value might point to the origin of the iron necessary for the steel production. The fact, that this isotopic composition does not occur in crustal rocks of Western Central Europe makes the Nd isotope ratio a powerful tool to trace steel plants atmospheric emissions. The rare earth element (REE) distribution pattern of the steel plant's filter dust shows very specific fractionations like La and Nd enrichments which are traceable in tree barks over a distance of 4 km. The Pb, Sr and Nd isotope ratios not only enable the steel plant's emissions to be traced in a north-easterly direction, along the principal wind pathway but also enables the interference of this emission at 4 km NE from the steel plant with another atmospheric component originating from the Strasbourg Rhine harbour to be identified.

Air Pollutants↗

Characterization of oak and birch dust-induced expression of cytokines and chemokines in mouse macrophage RAW 264.7 cells.

Occupational exposure to wood dust is related to several respiratory diseases, such as allergic rhinitis, chronic bronchitis, and asthma. However, virtually nothing is known about molecular mechanisms behind wood dust-induced pulmonary inflammation. To elucidate the effects of wood dust exposure on cytokine and chemokine expression in murine macrophage cell line cells, mouse RAW 264.7 cells were exposed to two selected hardwood dusts, oak and birch. TiO2 and LPS were used as controls. Expression patterns of several cytokines, chemokines, and chemokine receptors were assessed by real-time quantitative PCR system and by ELISA. Exposure to birch dust caused a major increase in TNF-alpha and IL-6 protein levels whereas a weaker induction of TNF-alpha protein was found after exposure to oak dust. Inorganic TiO2 dust did not induce significant cytokine expression. With respect to the chemokines, a dose-dependent, about 10-fold induction of CCL2 mRNA and protein was found after exposure to birch dust. Oak dust induced weakly CCL2 protein. Similarly, birch dust induced a strong expression of CCL3, CCL4, and CXCL2/3 mRNA whereas only moderate levels of these chemokine mRNAs were detected after oak dust exposure. In contrast, expression of CCL24 mRNA was inhibited by more than 40-fold by both oak and birch dusts. TiO2 dust induced about five-fold expression of CCL3 and CCL4 mRNA but did not affect significantly other chemokines. These results suggest that exposure to birch or oak dusts may influence the development of the inflammatory process in the airways by modulating the expression of macrophage-derived cytokines and chemokines.

Animals↗

Efficient production and capture of 8-prenylnaringenin and leachianone G-biosynthetic intermediates of sophoraflavanone G--by the addition of cork tissue to cell suspension cultures of Sophora flavescens.

It has previously been demonstrated that the addition of cork tissue to cell suspension cultures of Sophora flavescens stimulates the production of sophoraflavanone G, most of which has been recovered from the added cork tissue. In the present study, it was found that two precursors of sophoraflavanone G, 8-prenylnaringenin (sophoraflavanone B) and leachianone G, both of which have never been detected either in cultured cells or in the original plants, also accumulated in the added cork tissue. Thirteen minor flavonoids including three prenylated flavonoids, in addition to 8-prenylnaringenin and leachianone G, were isolated from the cork tissue co-incubated with S. flavescens cells. The new compounds flavescenones A, B and C, were determined to be (3R)-5, 7, 2'-trihydroxy-6-gamma, gamma-dimethylallyl-4', 5'-methylenedioxyisoflavanone; 5, 7, 2'-trihydroxy-6-gamma, gamma-dimethylallyl-4', 5'-methylenedioxyisoflavone and 2-[2',4'-dihydroxy-3'-(gamma-hydroxymethyl-gamma-methylallyl)phenyl]-5,6-methylenedioxybenzofuran, respectively, by means of spectroscopic analyses that included 2D-NMR techniques.

Acetates↗

Measurement and characterization of cellulase activity in sclerophyllous forest litter.

Cellulases are enzymatic proteins which hydrolyze cellulose polymers to smaller oligosaccharides, cellobiose and glucose. They consist in three major types of enzymes: endoglucanases (EC 3.2.1.4), cellobiohydrolases (EC 3.2.1.91) and beta-glucosidases (EC 3.2.1.21) which play an essential role in carbon turnover of forest ecosystem. The aim of this study was firstly to determine the parameters (i.e. buffer type, pH, temperature, quantity of litter, incubation time and reagent type) which affect the measurement of cellulase activity in a sclerophyllous forest litter, and secondly to compare two methods for measuring cellulase activity: a direct method and an extraction method. In the direct method, the litter was directly incubated with a buffered solution containing the enzyme substrate, whereas in the extraction method, the cellulases were firstly extracted before measuring their activity. The results were compared with other studies about soil cellulase activity, and it appeared that several parameters (buffer type, pH, temperature and sample quantity) which influence the measurement of cellulase activity differ according to whether a soil or a litter is considered. Concerning the procedure used for the measurement of cellulase activity, results showed that the activity values were higher when using an extraction procedure than when using a direct procedure. The extraction procedure, combined with a concentration stage of the extract, also allowed electrophoretic analysis (PAGE) of the cellulases extracted from the litter. The electrophoretic pattern revealed two cellulase isoenzymes which may be related to the occurrence of two pH-activity peaks of these enzymes when citrate buffer was used for the measurement of cellulase activity in the litter.

Buffers↗

Determination of organically bound tritium background level in biological samples from a wide area in the south-west of France.

In this paper, the authors describe a sensitive method for low-level non-exchangeable OBT determination. This methodology combines suitable sample treatment, a combustion apparatus for large-sized samples and low-background liquid scintillation spectrometry, along with precautions that substantially reduce the risks of sample contamination. Great care must be taken in the measurement of non-exchangeable OBT at environmental levels. Many authors have discussed the opportunities for cross-contamination between samples and contamination by exchange with the laboratory atmosphere. The authors also describe an application of the methodology to a large-scale sampling and measurement campaign, aimed at the determination of the environmental non-exchangeable OBT background level in tree leaves and ferns collected on the site and in the vicinity of a research centre located in the south-west of France, not far from Bordeaux. This study constitutes a "zero level" for the non-exchangeable OBT activity, as, to our knowledge, there is no tritium source within or in the surroundings of the sampled area capable of producing non-exchangeable OBT activities above the natural levels. Our analyses showed that non-exchangeable OBT activities in the collected samples were very low, ranging from below the detection limit (ca 0.7 Bq kg(-1) of dry material) to ca 2 Bq kg(-1) of dry material. These values are similar to the natural tritium background measured in water samples. No discrepancies can be shown between fern samples and oak tree leaf samples or between samples collected inside and outside the research site.

Environmental Monitoring↗

Sampling optimization, at site scale, in contamination monitoring with moss, pine and oak.

With the aim of optimizing protocols for sampling moss, pine and oak for biomonitoring of atmospheric contamination and also for inclusion in an Environmental Specimen Bank, 50 sampling units of each species were collected from the study area for individual analysis. Levels of Ca, Cu, Fe, Hg, Ni, and Zn in the plants were determined and the distributions of the concentrations studied. In moss samples, the concentrations of Cu, Ni and Zn, considered to be trace pollutants in this species, showed highly variable long-normal distributions; in pine and oak samples only Ni concentrations were log-normally distributed. In addition to analytical error, the two main source of error found to be associated with making a collective sample were: (1) not carrying out measurements on individual sampling units; and (2) the number of sampling units collected and the corresponding sources of variation (microspatial, age and interindividual). We recommend that a minimum of 30 sampling units are collected when contamination is suspected.

Air Pollutants↗