Comparison of Endotoxin Exposure Assessment by Bioaerosol Impinger and Filter-Sampling Methods

Environmental assessment data collected in two prior occupational hygiene studies of swine barns and sawmills allowed the comparison of concurrent, triplicate, side-by-side endotoxin measurements using air sampling filters and bioaerosol impingers. Endotoxin concentrations in impinger solutions and filter eluates were assayed using the Limulus amebocyte lysate assay. In sawmills, impinger sampling yielded significantly higher endotoxin concentration measurements and lower variances than filter sampling with IOM inhalable dust samplers. Analysis of variance for repeated measures showed that this association remained after controlling for other factors such as replicate, sawmill, sawmill operation, wood type, and interaction terms. Endotoxin concentrations in the swine barns were 10-fold higher on average than in sawmills. These samples demonstrated comparable endotoxin concentration estimates for impinger and filter methods although the variability was lower using the impinger method. In both occupational settings, side-by-side replicates were more uniform for the impinger samples than for the filter samples. This study demonstrates that impinger sampling is an acceptable method for quantitation of area endotoxin concentrations. Further, when sampling is performed with impingers for airborne microorganism quantitation, these same impinger solutions can yield valid endotoxin exposure estimates, negating the need for additional filter sampling.     

The measurement of aerosolized endotoxin from land application of Class B biosolids in Southeast Arizona

The purpose of this study was to determine aerosolized endotoxin concentrations downwind of a biosolids land application site. Aerosol samples were collected from biosolids land application sites, tractor operation, and an aeration basin located within an open-air wastewater treatment plant. Aerosolized endotoxin above background concentrations was detected from all sites, at levels ranging from below detection up to 1800 EU m-3 of air. Biosolids loading operations resulted in the greatest concentrations of endotoxin (mean 344 EU m-3). As downwind (perpendicular to wind vector) distance increased from sources (2-200 m), levels of endotoxin decreased to near background (without biosolids application) concentrations. Overall, the detected levels of aerosolized endotoxin were within past proposed aerosolized endotoxin limits (250-2000 EU m-3) by other occupational exposure studies. Occasionally, peak concentrations were found to be above these limits. Sites in which soil was being aerosolized resulted in greater concentrations of endotoxin with or without biosolids, which suggested that the majority of endotoxin may in fact be of soil origin. This study evaluated the presence of aerosolized endotoxin from the land application of biosolids and showed that these levels were within ranges for concern suggested by other studies and that this area of research needs further investigation.     

Comparison of endotoxin exposure assessment by bioaerosol impinger and filter-sampling methods

Environmental assessment data collected in two prior occupational hygiene studies of swine barns and sawmills allowed the comparison of concurrent, triplicate, side-by-side endotoxin measurements using air sampling filters and bioaerosol impingers. Endotoxin concentrations in impinger solutions and filter eluates were assayed using the Limulus amebocyte lysate assay. In sawmills, impinger sampling yielded significantly higher endotoxin concentration measurements and lower variances than filter sampling with IOM inhalable dust samplers. Analysis of variance for repeated measures showed that this association remained after controlling for other factors such as replicate, sawmill, sawmill operation, wood type, and interaction terms. Endotoxin concentrations in the swine barns were 10-fold higher on average than in sawmills. These samples demonstrated comparable endotoxin concentration estimates for impinger and filter methods although the variability was lower using the impinger method. In both occupational settings, side-by-side replicates were more uniform for the impinger samples than for the filter samples. This study demonstrates that impinger sampling is an acceptable method for quantitation of area endotoxin concentrations. Further, when sampling is performed with impingers for airborne microorganism quantitation, these same impinger solutions can yield valid endotoxin exposure estimates, negating the need for additional filter sampling.     

Year-long assessment of airborne endotoxin at a concentrated dairy operation

In this study, we monitored total airborne endotoxins at upwind and downwind sites at a large open-lot dairy each month for a year. At the upwind site, the average airborne concentration was 28.5 endotoxin units (EU) m⁻³, while at the downwind edge of the lot and 200 m from the lot edge, the average concentrations were 169 and 72 EU m⁻³, respectively. At the downwind edge of the lot, there was a significant correlation between the airborne endotoxin concentration and wind speed or air temperature. A comparison between total and inhalable airborne endotoxin concentrations, near the end of the study, revealed no significant differences between the two endotoxin collection methods. Our data suggest that endotoxin exposure can be reduced as one increases their distance from the open-lot dairy.     

Comparison of the Rotorod to other air samplers for the determination of Ambrosia artemisiifolia pollen concentrations conducted in the Environmental Exposure Unit

The Environmental Exposure Unit (EEU) is a 924 m³ facility (Kingston General Hospital, Ontario) in which uniform concentrations of various pollens in HEPA-filtered air at known rates of laminar airflow can be maintained. This facility provided a unique opportunity to compare several air samplers without the environmental variation inherent in outdoor comparisons. The purpose of this study was to conduct a quantitative comparison of pollen measurements using the Rotorod, Burkard™ Personal Volumetric Air Sampler, Air-O-Cell™ and a 37 mm open-faced filter cassette with a microporous filter in the EEU. Pollen samples were taken during clinical trials being conducted in the Unit. Raw pollen counts/m³ obtained using the different methods were corrected using published particle collection efficiencies for the particle size (∼ ∼20 μm) and airflow. Data were analyzed by ANOVA/Tukey HSD. No statistically significant differences were found between pollen concentrations determined by Rotorod, Air-O-Cell and filter cassette. Pollen levels determined by the Burkard were up to 2 times higher than the other sampling methods. Relative standard deviations were similar for the Rotorod, Burkard, and filter cassette and higher for the Air-O-Cell. This study demonstrated that, under our conditions, the Rotorod sampler provides consistent and reliable measurements of ragweed pollen concentrations.     

The abundance of urban endotoxins as measured with an impinger-based sampling strategy

Endotoxins are components of Gram-negative bacteria with inherently high pro-inflammatory potential. In an urban environment, airborne endotoxins may associate with pollutants such as particulate matter, increasing the severity of the immune response by acting as a natural adjuvant to augment inflammatory respiratory disease development. Here, we present a closer look at outdoor urban endotoxins by applying a microbial-targeted collection strategy. Results from 87 samples distributed throughout the city of Antwerp ranged from 0.45 to 93.71 EU/m³, with a geometric mean of 4.49 EU/m³ and 95% confidence interval of 3.53–5.71 EU/m³. Sample collection was also coupled with the use of a Coulter counter, for which the particle count (2.5–10 μm/m³) showed a significant correlation with endotoxin concentration (R²?=?0.24; p?<?0.0001; n?=?64). In addition, the analysis of the cultivable bacterial colony-forming units on Reasoner’s 2A agar (expressed CFU/m³) showed to be a good indicator for airborne endotoxins (R²?=?0.57; p?<?0.0001; n?=?58). Moreover, identification of dominant bacterial colonies on these culture plates gave some indications on potential sources of these urban outdoor bacteria and endotoxins.     

Evaluation of four sampling devices for Burkholderia pseudomallei laboratory aerosol studies

Previous field and laboratory studies investigating airborne Burkholderia pseudomallei have used a variety of different aerosol samplers to detect and quantify concentrations of the bacteria in aerosols. However, the performance of aerosol samplers can vary in their ability to preserve the viability of collected microorganisms, depending on the resistance of the organisms to impaction, desiccation, or other stresses associated with the sampling process. Consequently, sampler selection is critical to maximizing the probability of detecting viable microorganisms in collected air samples in field studies and for accurate determination of aerosol concentrations in laboratory studies. To inform such decisions, the present study assessed the performance of four laboratory aerosol samplers, specifically the all-glass impinger (AGI), gelatin filter, midget impinger, and Mercer cascade impactor, for collecting aerosols containing B. pseudomallei generated from suspensions in two types of culture media. The results suggest that the relative performance of the sampling devices is dependent on the suspension medium utilized for aerosolization. Performance across the four samplers was similar for aerosols generated from suspensions supplemented with 4% glycerol. However, for aerosols generated from suspensions without glycerol, use of the filter sampler or an impactor resulted in significantly lower estimates of the viable aerosol concentration than those obtained with either the AGI or midget impinger. These results demonstrate that sampler selection has the potential to affect estimation of doses in inhalational animal models of melioidosis, as well as the likelihood of detection of viable B. pseudomallei in the environment, and will be useful to inform design of future laboratory and field studies.     

Evaluation of Four Aerobiological Sampling Methods for the Retrieval of Aerosolized Pseudomonas syringae

The Andersen six-stage impactor, the SAS (Surface Air System) impactor, the AGI-30 impinger, and gravity plates were evaluated for the retrieval of aerosol-released Pseudomonas syringae. The upper limits of the impactor samplers were exceeded at a spray concentration of 10⁷ CFU/ml, indicating that these samplers are not appropriate for monitoring high airborne concentrations. Decreased cell concentrations were retrieved with increased sampling time for the Andersen and AGI samplers, indicating that a minimum sampling time is preferable for monitoring aerosolized vegetative cells.     

Dispersal of Pseudocercosporella herpotrichoides Spores from Infected Wheat Straw

Evidence from spore samples collected amongst infected straw spread on fallow ground supported the conclusion that spores of Pseudocercosporella herpotrichoides are dispersed mostly by rainsplash. Most spores travelled a short distance in the larger ballistic splash droplets, although some may have travelled further in smaller airborne droplets. Weekly spore counts from microscope slides under rainshields, a funnel and an impinger, evaluated as samplers for spores of P. herpotrichoides, showed a similar seasonal pattern. The funnel, as the largest sampler, generally collected most spores, but the impinger collected more spores per unit area of sampling surface. Slides sometimes collected spores when none was recovered from other samplers. Young wheat plants, exposed with the samplers and changed weekly, subsequently developed eyespot symptoms for most of the season.     

Soil CO2 efflux in a beech forest: comparison of two closed dynamic systems

The aim of this study was to understand why two closed dynamic systems with a very similar design gave large differences in soil CO₂ efflux measurements (PP systems and LI-COR). Both in the field (forest beech stand) and in the laboratory, the PPsystems gave higher estimations of soil CO₂ efflux than the LI-COR system (ranging from 30% to 50%). The difference in wind speed occurring within the soil respiration chambers (0.9 m s⁻¹ within the SRC-1 and 0.4 m s⁻¹ within the LI-6000-09 chambers) may account for the discrepancy between the two systems. An excessive air movement inside the respiration chamber is thought to disrupt the high laminar boundary layer over the forest floor. This would promote an exhaust of the CO₂ accumulated into the upper soil layers into the chamber and a lateral diffusion of CO₂ in the soil towards the respiration chamber. The discrepancy between the two systems was reduced (i) by decreasing fan speed within the SRC-1, (ii) by increasing wind speed over the soil surface outside the respiration chamber, or (iii) by using an artificial soil design without high CO₂ concentration in soil pores. We show that wind speed is an important component of soil CO₂ diffusion which must be taken into account when measuring soil CO₂ efflux, even on very fine textured soil like silt-loam soil. Proper measurement can be achieved by maintaining wind speed inside the chamber below 0.4 m s⁻¹ since low wind speed conditions predominate under forest canopies. However, more accurate measurements will be obtained by regulating wind speeds within the chamber at a velocity representative of the wind speed recorded simultaneously at the floor surface. This revised version was published online in June 2006 with corrections to the Cover Date.     

Exposure to airborne microorganisms,hyphal fragments,and pollen in a field of organically grown strawberries

Many working environments are predisposed for larger than average amounts of fungi and other microorganisms often due to organic material being handled. From 2003 to 2007, the area used for strawberry production in Denmark increased by 62%. The purpose of this study was to determine the levels of exposure to microorganisms, endotoxin, (1→3)-β-d-glucan (β-glucan), and pollen in a field of strawberries. The study was carried out in eastern Denmark from the middle of June to the beginning of August 2008. The strawberries were grown organically, and microbiological pest control agents (MPCAs) were applied during this and former growth seasons. In order to measure exposure to inhalable bioaerosol components, we used stationary filter samplers. Bioaerosol sampling was performed during 4 working days, and a total of 57 samplings were performed. The filters were analysed for contents of fungi, MPCAs, endotoxin, β-glucan, and pollen. The mean exposure was 6,154 CFU Cladosporium sp. m⁻³, 1.0 × 10⁵ fungal spores m⁻³, 4.1 × 10⁴ hyphal fragments m⁻³, 5.8 × 10³ pollen m⁻³, 57.3 ng β-glucan m⁻³, and 8.9 endotoxin units (EU) m⁻³. A significant and positive correlation was found between β-glucan and fungal spores and between CFU of Cladosporium sp. and CFU of fungi. We selected specifically for Metarhizium anisopliae, Beauveria bassiana, and the applied MPCAs Trichoderma harzianum, T. polysporum, and Bacillus thuringiensis but found none of these species. In conclusion, our study shows that berry pickers in this organic strawberry field were potentially subjected to higher levels of fungal spores, Cladosporium sp., hyphal fragments, pollen, and thus also β-glucan than is usually seen in outdoor air. Exposure to MPCAs was not seen. The exposure to endotoxin was only slightly higher than e.g. in a town.     

Removal of Endotoxin during Purification of Poly(3-Hydroxybutyrate) from Gram-Negative Bacteria

Poly(3-hydroxybutyrate) (PHB) was produced by cultivating several gram-negative bacteria, including Ralstonia eutropha, Alcaligenes latus, and recombinant Escherichia coli. PHB was recovered from these bacteria by two different methods, and the endotoxin levels were determined. When PHB was recovered by the chloroform extraction method, the endotoxin level was less than 10 endotoxin units (EU) per g of PHB irrespective of the bacterial strains employed and the PHB content in the cell. The NaOH digestion method, which was particularly effective for the recovery of PHB from recombinant E. coli, was also examined for endotoxin removal. The endotoxin level present in PHB recovered by 0.2 N NaOH digestion for 1 h at 30°C was higher than 10⁴ EU/g of PHB. Increasing the digestion time or NaOH concentration reduced the endotoxin level to less than 1 EU/g of PHB. It was concluded that PHB with a low endotoxin level, which can be used for various biomedical applications, could be produced by chloroform extraction. Furthermore, PHB with a much lower endotoxin level could be produced from recombinant E. coli by simple NaOH digestion.     

Optimization of the Endotoxin Removal Performance of Solid-Phase Conjugated S3E3 Antimicrobial Peptide Using Response Surface Methodology

S3E3 is a new variant of S3 antimicrobial peptide (AMP) derived from factor C of horseshoe hemolymph and features a high binding affinity for endotoxin. In this research, site-specific conjugated S3E3 AMP onto Sepharose 6% solid phase support (S3E3-S-Sepharose) was applied for endotoxin removal from protein solutions. The bovine serum albumin (BSA) was chosen as a protein model due to its acidic-sticky nature interfering with the endotoxin removal process. The batch process parameters including, endotoxin concentration, pH, and ionic strength of the sample were optimized by response surface methodology to reach maximum endotoxin binding capacity and protein recovery. The predicted optimal conditions for enhanced endotoxin removal performance were as follows: pH 4.5, 25 mM NaCl, and 68,500 EU/ml endotoxin leading to a maximum endotoxin binding capacity of 3.114?×?10⁺⁶ EU/ml of resin and a 95.89% protein recovery. S3E3-S-Sepharose could be applied as an efficient endotoxin removal affinity chromatography matrix at downstream processes of recombinant therapeutics due to its high capacity and protein recovery.

     

A rain tower and wind tunnel for studying the dispersal of plant pathogens by rain and wind

The rain tower/wind tunnel complex at Rothamsted consists of a rain tower (height 11 m, cross-section 1 m²) linked to the upwind end of a wind tunnel (length 12 m, cross-section 1 m²), which may be operated in either an open or a closed configuration. At the top of the rain tower, water drops with diameters of 2.5 to 5 mm are produced by a drop generator, which can be fitted with different nozzles. Simulated rain with a drop diameter of 1 to 3 mm is produced at a rate of 8 to 12 mm h^-1 by a rain generator with an area of 52 × 67 cm. The rain tower may be operated in conjunction with the wind tunnel in an open configuration. The windspeed can be decreased from a maximum of 8 m s^-1 by decreasing the speed of the fan. The wind tunnel has its own internal lighting. When the wind tunnel is in a closed configuration, temperature and humidity can be controlled in the range 12 ^oC (62–80% r.h.) to 35 ^oC (22–50% r.h.). Data presented illustrate the use of this rain tower/wind tunnel complex to study dispersal of plant pathogen spores by rain-splash or wind.     

Using SPMDs to Assess Natural Recovery of PCB-contaminated Sediments in Lake Hartwell,SC: I. A Field Test of New In-Situ Deployment Methods

Results from the field testing of some innovative sampling methods developed to evaluate risk management strategies for polychlorinated biphenyl (PCB) contaminated sediments are presented. Semipermeable membrane devices (SPMDs) were combined with novel deployment methods to quantify the availability of PCBs at the Sangamo-Weston Lake Hartwell Superfund Site in SC. Three locations in Lake Hartwell were examined: a background site (BKG) with little detectable contamination and two contaminated sites (T-M/N and T-O). PCB availability was quantified using sediment surface samplers designed to hold SPMDs in contact with surface sediments, benthic dome samplers designed to enclose and suspend SPMDs at the sediment-water interface, and commercially obtained SPMD cages suspended in the water column. A two-way analysis of variance showed significant effects by sampler type (P < 0.0001) and site (P < 0.0001) for mean time-weighted average (TWA) total PCBs (t-PCBs). Regardless of the SPMD sampler used, mean TWA t-PCBs were directly proportional to the level of contamination present (BKG < T-M/N < T-O). Water column and surface sediment sampler t-PCB uptake patterns were described by a significant linear fit of the data (r² = 0.9625, p < 0.0001, and r² = 0.8188, p < 0.0001, respectively). Dome and sediment surface SPMD samples had a higher percentage of higher chlorinated PCBs compared to water column samples.     

Cyanobacteria and cyanotoxins in the source water from Lake Chivero,Harare, Zimbabwe,and the presence of cyanotoxins in drinking water

The phytoplankton community and cyanotoxins in Lake Chivero (formerly Lake McIlwaine) and the presence of cyanotoxins in treated drinking water were investigated between 2003 and 2004. A typical seasonal succession of Cyanobacteria species occurred from January to April, Bacillariophyta from May to July, and Cryptophyta and Chlorophyta from August to December. Microcystis aeruginosa and M. wesenbergii, known producers of the toxin microcystin, and the non-toxic cyanobacterium M. novacekii dominated during summer. The highest concentrations of microcystins and lipopolysaccharide endotoxins occurred when cyanobacterial biomass was highest. Lipopolysaccharide endotoxin concentrations in the lake ranged between 8 and 3 200 Endotoxin Units (EU) ml^?1. Microcystin concentrations in treated water were below the recommended safe limit for drinking water. Lipopolysaccharide endotoxin concentrations in treated water ranged from 0.15 to 11 EU ml^?1. The phytoplankton community comprised non-microcystin-producing species for the greater part of the study period.     

Vegetative and reproductive phenology of the mangrove Bruguiera gymnorrhiza (L.) Lam. on Okinawa Island, Japan

Vegetative and reproductive phenology of the subtropical mangrove Bruguiera gymnorrhiza was investigated at Manko Wetland, Okinawa Island, Japan. Phenology was assessed using litterfall data over 4 years. Leaf and stipule litterfall occurred throughout the year, with distinct seasonal patterns. Kendall’s coefficient of concordance, W, revealed that the monthly changes in leaf and stipule litterfall were strongly and significantly concordant among years. Leaf litterfall was linked to monthly day length and maximum wind speed, and stipule litterfall was linked to monthly mean air temperature, monthly rainfall, and maximum wind speed. Branch litterfall showed no clear monthly pattern, but tended to vary with monthly maximum wind speed. Mean leaf longevity was 19 months. Mean total litterfall was 10.1 Mg ha^?1 year^?1, with the largest component being vegetative organs (58 %). Flower and propagule litterfall were highest in autumn and summer, respectively, and lowest in winter. The W values revealed that, of the reproductive organs, only flower litterfall had a significant monthly trend. Flower litterfall was correlated with monthly mean air temperature and relative humidity. The average development periods from flower buds to flowers and from flowers to mature propagules were approximately 1 and 8 months, respectively. It took 9 months to produce mature propagules from flower buds. Except for branches, all vegetative and reproductive components of litterfall had clear annual cycles.     

Effects of wind speed and direction on monthly fluctuations of <Emphasis Type="Italic">Cladosporium</Emphasis> conidia concentration in the air

This study determined the relationship between airborne concentration of Cladosporium spp. spores and wind speed and direction using real data (local wind measured by weather station) and modelled data (air mass flow computed with the aid of HYbrid Single Particle Lagrangian Trajectory model). Air samples containing fungal conidia were taken at an urban site (Worcester, UK) for a period of five consecutive years using a spore trap of the Hirst design. A threshold of ≥6000 s m^?3 (double the clinical value) was applied in order to select high spore concentration days, when airborne transport of conidia at a regional scale was more likely to occur. Collected data were then examined using geospatial and statistical tools, including circular statistics. Obtained results showed that the greatest numbers of spore concentrations were detected in July and August, when C. herbarum, C. cladosporioides and C. macrocarpum sporulate. The circular correlation test was found to be more sensitive than Spearman’s rank test. The dominance of either local wind or the air mass on Cladosporium spore distributions varied between examined months. Source areas of this pathogen had an origin within the UK territory. Very high daily mean concentrations of Cladosporium spores were observed when daily mean local wind speed was v _s ≤ 2.5 m s^?1 indicating warm days with a light breeze.     

Phenological traits of the mangrove Rhizophora stylosa Griff. at the northern limit of its biogeographical distribution

The vegetative and reproductive phenology of the subtropical mangrove species Rhizophora stylosa was investigated at Manko Wetland, Okinawa Island, Japan. We assessed phenology using litterfall data over four years. Leaf and stipule litterfall occurred throughout the year, with distinct seasonal patterns. Kendall’s coefficient of concordance, W, revealed that the monthly changes in leaf and stipule litterfall were strongly and significantly concordant among years. Leaf litterfall was significantly correlated with monthly maximum wind speed and monthly day length, and stipule litterfall was significantly correlated with monthly mean air temperature and relative humidity. Branch litterfall showed no clear monthly pattern and correlated well with monthly maximum wind speed. Mean total litterfall was 11.2 Mg ha^?1 year^?1, with the largest component being vegetative organs (78.7 %). Flower, fruit, and propagule litterfall were all highest in summer and lowest in winter. The W values revealed that most reproductive organs in litterfall had significant monthly trends. Flower and fruit litterfall were significantly correlated with monthly day length and monthly mean air temperature, respectively. The average development periods from flower buds to flowers, fruits, and mature propagules were approximately 2–3 months, 4–5 months, and 11–12 months, respectively. Except for branches, all vegetative and reproductive components of litterfall had approximately one year cycles.     

Peculiarities of Lycopodium spores sampling from the ambient atmosphere: physical effects and problems of representativeness

The process of aspiration of monodisperse Lycopodium spores (d = 32 μm) from the weakly disturbed atmosphere and the calm air into aerosol samplers of various designs was experimentally investigated. Besides, some qualitative and quantitative characteristics of the interaction of spores with inner surfaces of sampling lines were studied, three experimental installations being employed.

Thin- and thick-walled cylindrical probes, their inner diameters being 0.3–1.5 cm and their length being 10–50 cm, as well as probes of other geometries were used in the experiments. The flow rate varied from 1 to 1000 cm³ s^?1; the mean wind speed value varied from 0 to 2 m s^?1.

Some regularities of sampling of air-spores were obtained. The aerosol concentration distortions caused by particle inertia and rebound as well as the wall loss effect were determined. The analysis is provided with a number of practical recommendations for sampling air spores into probes of aerosol measurement devices.