Evaluation of interference to conventional and real-time PCR for detection and quantification of fungi in dust

Advances in polymerase chain reaction (PCR) have permitted accurate, rapid and quantitative identification of microorganisms in pure cultures regardless of viability or culturability. In this study, a simple sample processing method was investigated for rapid identification and quantification of fungal spores from dust samples using both conventional and real-time PCR. The proposed method was evaluated for susceptibility to interference from environmental dust samples. Stachybotrys chartarum and Aspergillus fumigatus were used as test organisms. The sensitivity of detection in pure culture was 0.1 spore DNA equivalents per PCR reaction corresponding to 20 spores ml(-1) in the sample. However, 1 spore DNA equivalent per PCR reaction corresponding to 200 spores ml(-1) in the sample was the lowest amount of spores tested without interference in dust samples spiked with spores of either fungal species. The extent of inhibition was calculated using conventional and real-time PCR reactions containing fungal spores, specific primers, specific probes (for real-time PCR) and various amounts of dust. The results indicate that the extent of inhibition by dust on PCR varies with the type and amount of dust, and number of spores. No interference in the analysis of spiked samples was detected from 0.2 mg ml(-1) of four real-life dust samples at p-value >0.05 using 2 x 10(4) spores for conventional PCR and 2 x 10(5) spores for real-time PCR. However, samples containing >0.2 mg ml(-1) real-life dust compromised the PCR assay. These results suggest the potential usefulness of a simple sample processing method in conjunction with PCR for monitoring the fungal content of aerosols collected from indoor environments.     

Pool of Dust Particles over the Asian Continent: Balloon-borne Optical Particle Counter and Ground-based Lidar Measurements at Dunhuang, China

Measurements of aerosols were made in 2001 and 2002 at Dunhuang (40 degrees 00'N, 94 degrees 30'E), China to understand the nature of atmospheric particles over the desert areas in the Asian continent. Balloon-borne measurements with an optical particle counter suggested that particle size and concentration had noticeable peaks in super micron size range not only in the boundary mixing layer but also in the free troposphere. Super-micron particle concentration largely decreased in the mid tropopause (from 5 to 10 km; above sea level, a.s.l.). Lidar measurements made during August 2002 at Dunhuang suggested the possibility that mixing of dust particles occurred from near the ground to about 6 km even under calm weather conditions, and a large depolarization ratio of particulate matter was found in the aerosol layer. The top of the aerosol layer was found at heights of nearly 6 km (a.s.l.). It is strongly suggested that nonspherical dust particles (Kosa particles) frequently diffused in the free atmosphere over the Taklamakan desert through small-scale turbulences and are possible sources of dust particles of weak Kosa events that have been identified in the free troposphere not only in spring but also in summer over Japanese archipelago. Electron microscopic experiments of the particles collected in the free troposphere confirmed that coarse and nonspherical particles observed by the mineral particle were major components of coarse mode (diameter larger than 1 microm) below about 5 km over Dunhuang, China.     

Sediment quality in the Douro river estuary based on trace metal contents, macrobenthic community and elutriate sediment toxicity test (ESTT)

The aim of this study was to evaluate the sediment quality in the mesotidal Douro River estuarine environment, in order to identify areas where sediment contamination could cause ecosystem degradation. Samples were obtained in five locations and sediment characterised for grain size, total organic matter, total-recoverable metals (Al, Fe, Cu, Pb, Cr, Ni, Cd, Zn and Mn), as well as acid volatile sulfide (AVS) and simultaneously extracted metals (SEM). In situ effects were evaluated by examining the macrobenthic community structure. An elutriate sediment toxicity test (ESTT) was used to estimate the amount of metals and nutrients that could be exchanged with the water column through resuspension, and its positive or negative effects on the growth of the micro-alga Emiliania huxleyi in a 10 day test. Anthropogenic metal contamination was identified at the north bank of the Douro estuary, with deleterious effects on the macrobenthic community, namely decrease in number of species and diversity. This contamination could possibly also be toxic for water column organisms, in case of resuspension, as shown by the ESTT. Sediments from the salt marsh at the south bank showed an impoverished macrobenthic community and elutriate toxicity, which appeared to be due to anaerobic conditions. This study clearly shows the usefulness of the ESST approach to assess the biological effect of resuspension of estuarine sediments.     

Assessment of Metal Contamination in Doñana National Park (Spain) using Crayfish (Procamburus Clarkii)

Water quality assessment in the Aznalcollar area was attempted using multivariate methods based on heavy metal concentrations in red swamp crayfish (Procamburus clarkii). Trace levels of four heavy metals, copper (Cu), zinc (Zn), cadmium (Cd) and lead (Pb), were detected in crayfish from eleven different stations. Principal component analysis (PCA) highlighted a gradient of contamination between the sampling stations. Cluster analysis (CA) distinguished three groups of stations. Discriminant analysis also differentiated three groups. The group centroids of the first discriminant function were used to devise an index that varies according to the source of the crayfish. These standardized values are proposed for use as a water quality index. The ability of this index to successfully predict environmental quality was proved with random samples.     

Total versus inhalable sampler comparison study for the determination of asphalt fume exposures within the road paving industry

Exposure to asphalt fumes has a threshold limit value (TLV of 0.5 mg m(-3) (benzene extractable inhalable particulate) as recommended by the American Conference of Governmental Industrial Hygienists (ACGIH). This reflects a recent change (2000) whereby two variables are different from the previous recommendation. First is a 10-fold reduction in quantity from 5 mg m(-3) to 0.5 mg m(-3). Secondly, the new TLV specifies the "inhalable" fraction as compared to what is presumed to be total particulate. To assess the impact of these changes, this study compares the differences between measurements of paving asphalt fume exposure in the field using an "inhalable" instrument versus the historically used 'total' sampler. Particle size is also examined to assist in the understanding of the aerodynamic collection differences as related to asphalt fumes and confounders. Results show that when exposures are limited to asphalt fumes, a 1:1 relationship exists between samplers, showing no statistically significant differences in benzene soluble matter (BSM). This means that for the asphalt fume ACGIH TLV, the 'total' 37-mm sampler is an equivalent method to the "inhalable" method, referred to as IOM (Institute of Occupational Medicine), and should be acceptable for use against the TLV. However, the study found that when confounders (dust or old asphalt millings) are present in the workplace, there can be significant differences between the two samplers' reported exposure. The ratio of IOM/Total was 1.37 for milling asphalt sites, 1.41 for asphalt paving over granular base, and 1.02 for asphalt over asphalt pavements.     

A study of the elemental leachability and retention capability of compost

In this work a comparison is made between the different approaches that can be taken to evaluate the mobility of elements in compost. The practical consequences of the results obtained are also discussed in terms of methods for cleaning up compost and using compost in environmental remediation. The mobility of potentially toxic elements in compost is evaluated by leaching with four selected eluents, i.e. diluted sulfuric acid, oxalate, citrate and EDTA. In contrast to the chelating agents, diluted sulfuric acid was found to generally have a low leaching capability for removal of heavy metals from compost. This implies that the risk of heavy metal leaching caused by natural rainfall is likely to be low. The results obtained in the leaching experiment were compared with previous results obtained from sequential fractionation. This comparison confirmed that both methods gave similar results for predicting the lability of elements in compost. A non-linear regression analysis of the leaching curves was also conducted. The leaching curves for elements with high lability could be fitted with a two components model. The labile components identified by the kinetic model are approximately in accordance with the fractions obtained from the first step of the sequential extraction method. The kinetic speciation method is shown to be a relatively rapid and simple procedure for compost which gives more information about element lability than simple leaching experiments. The leaching reagents used in this work were not effective enough to be used for cleaning up compost with a high metal content. Compost was however shown to have a high affinity for heavy metals, with the order of affinity of metal for the compost being very similar to that seen for humic acid. Compost may therefore prove to be a good remediation material for metal contaminated waste.     

monitoring bioaccumulation of contaminants in the belted kingfisher (ceryle alcyon)

The belted kingfisher (Ceryle alcyon), acommon piscivore in the eastern United States, hasbecome a common endpoint in ecological riskassessments (ERA) because of their high consumptionof potentially contaminated aquatic prey. Whilebioaccumulation data and biosurveys may be used tosupport conclusions of ERAs for kingfishers, thereare currently no published data on contaminantconcentrations in kingfishers. Additionally,methods available for collecting biological samples(e.g., feathers, eggs, food debris, etc.) fromkingfisher burrows can be detrimental to thereproductive success of the birds. We present amethod for obtaining samples from burrows during orfollowing the nesting season. The method wasapplied to kingfisher burrows on the Oak RidgeReservation (ORR) in eastern Tennessee. Feathers,eggshells, and nestlings were collected from burrows and analyzed. In addition, carcasses ofthree adult kingfishers found dead on the ORR wereanalyzed. Metals and radionuclides were accumulatedby both juvenile and adult birds. Body burdens ofcadmium, lead, and cesium-137 in adult birds were belowlevels associated with toxicity. Concentrations of selenium and mercury were observed at potentiallytoxic levels. Contaminants in eggshells andnestling feathers indicate exposure, however, thereis insufficient information to evaluate thetoxicological significance of this contamination.National Laboratory Oak Ridge National Laboratory is managed for the U.S. Department of Energy by Lockheed Martin Energy Research Corp. under contract DE-AC05-96OR22464     

Methods for constructing and balancing ecosystem flux charts: new techniques and software

We describe a general computer program which employs novel techniques to analyze carbon (energy) and nutrient flows in complex food webs. The program uses estimates of metabolic/physiological parameters and ecosystem fluxes to simultaneously calculate the most likely balanced flux charts for carbon and a nutrient. We give an example of how the model can explore the theoretical issues involved with nutrient limitation and recycling.     

Fractionation of butyltin species during sample extraction and preparation for analysis

Solvent extraction and evaporative concentration steps are often used in procedures for the measurement of butyltins in environmental samples. As part of a larger study utilizing radiolabeled butyltins, the loss and fractionation of butyltins during sample preparation was investigated. TBT, DBT, and MBT were extracted from acidified seawater by hexane with efficiencies of about 95–99, 50–60 and 11% respectively. In addition, losses of about 70% of DBT were found during evaporative concentration of hexane. A variety of sediment extraction procedures were tested and none were found to be highly efficient for total butyltin extraction.     

Within-and among-clutch variation of organochlorine residues in eggs of black-crowned night-herons

Within-clutch variability of DDE and PCB residues in eggs from 62 clutches of black-crowned night-herons (Nycticorax nycticorax) was small (12% and 17%) compared to among-clutch variability (88% and 83%). Significant correlations between concentrations of DDE (median r=0.8885) and of PCBs (median r=0.8244) occurred when 501 correlations were run on two randomly selected eggs from within the same clutch; no significant correlation occurred for either concentrations of DDE (median r=0.0353) or PCBs (median r=–0.0843) when eggs were not restricted to the same clutch but were restricted to the same colony. The probability of finding infrequently detected organochlorine contaminants (e.g., DDT, cis-chlordane) in eggs from the same clutch varied from 43–96% and increased as the chemical became more prevalent and the number of eggs per clutch became smaller. These results further support one of the basic assumptions of the sample egg technique, that the chemical residues in one egg in a clutch accurately reflect residues in the remaining eggs of the clutch.