Anne Straumfors

Work Tasks as Determinants of Grain Dust and Microbial Exposure in the Norwegian Grain and Compound Feed Industry

OBJECTIVES The grain and compound feed industry entails inevitable risks of exposure to grain dust and its microbial content. The objective of this study was therefore to investigate task-dependent exposure differences in order to create knowledge basis for awareness and exposure reducing measures in the Norwegian grain and compound feed industry. METHODS A total of 166 samples of airborne dust were collected by full-shift personal sampling during work in 20 grain elevators and compound feed mills during one autumn season and two winter seasons. The personal exposure to grain dust, endotoxins, β-1→3-glucans, bacteria, and fungal spores was quantified and used as individual outcomes in mixed models with worker nested in company as random effect and different departments and tasks as fixed effects. RESULTS The exposure levels were highest in grain elevator departments. Exposure to endotoxins was particularly high. Tasks that represented the highest and lowest exposures varied depending on the bioaerosol component. The most important determinants for elevated dust exposure were cleaning and process controlling. Cleaning increased the dust exposure level by a factor of 2.44 of the reference, from 0.65 to 1.58mg m(-3), whereas process controlling increased the dust exposure level by a factor of 2.97, from 0.65 to 1.93mg m(-3). Process controlling was associated with significantly less grain dust exposure in compound feed mills and the combined grain elevators and compound feed mills, than in grain elevators. The exposure was reduced by a factor of 0.18 and 0.22, from 1.93 to 0.34mg m(-3) and to 0.42mg m(-3), respectively, compared with the grain elevators. Inspection/maintenance, cleaning, and grain rotation and emptying were determinants of higher exposure to both endotoxin and β-1→3-glucans. Seed winnowing was in addition a strong determinant for endotoxin, whereas mixing of animal feed implied higher β-1→3-glucan exposure. Cleaning was the only task that contributed significantly to higher exposure to bacteria and fungal spores. CONCLUSION Cleaning in all companies and process controlling in grain elevators were the strongest determinants for overall exposure, whereas seed winnowing was a particular strong determinant of endotoxin exposure. Exposure reduction by technical intervention or personal protective equipment should therefore be considered at work places with identified high exposure tasks.

Mycotoxins and other fungal metabolites in grain dust from Norwegian grain elevators and compound feed mills

Employees at grain elevators and compound feed mills are exposed to large amounts of grain dust during work, frequently leading to airway symptoms and asthma. Although the exposure to grain dust, microorganisms, β-1→3-glucans and endotoxins has been extensively studied, the focus on the mycotoxin content of grain dust has previously been limited to one or few mycotoxins. Our objective was therefore to screen settled grain dust from grain elevators and compound feed mills for fungal metabolites by LC/MS-MS and explore differences between work places, seasons and climatic zones. Seventy fungal metabolites and two bacterial metabolites were detected. Trichothecenes, depsipeptides, ergot alkaloids, and other metabolites from Fusarium, Claviceps, Alternaria, Penicillium, Aspergillus, and other fungi were represented. The prevalence of individual metabolites was highly variable, and the concentration of each metabolite varied considerably between samples. The prevalence and concentration of most metabolites wer...

Profile and Morphology of Fungal Aerosols Characterized by Field Emission Scanning Electron Microscopy (FESEM)

Fungal aerosols consist of spores and fragments with diverse array of morphologies; however, the size, shape, and origin of the constituents require further characterization. In this study, we characterize the profile of aerosols generated from Aspergillus fumigatus, A. versicolor, and Penicillium chrysogenum grown for 8 weeks on gypsum boards. Fungal particles were aerosolized at 12 and 20 L min−1 using the Fungal Spore Source Strength Tester (FSSST) and the Stami particle generator (SPG). Collected particles were analyzed with field emission scanning electron microscopy (FESEM). We observed spore particle fraction consisting of single spores and spore aggregates in four size categories, and a fragment fraction that contained submicronic fragments and three size categories of larger fragments. Single spores dominated the aerosols from A. fumigatus (median: 53%), while the submicronic fragment fraction was the highest in the aerosols collected from A. versicolor (median: 34%) and P. chrysogenum (median: 31%). Morphological characteristics showed near spherical particles that were only single spores, oblong particles that comprise some spore aggregates and fragments (<3.5 μm), and fiber-like particles that regroup chained spore aggregates and fragments (>3.5 μm). Further, the near spherical particles dominated the aerosols from A. fumigatus (median: 53%), while oblong particles were dominant in the aerosols from A. versicolor (68%) and P. chrysogenum (55%). Fiber-like particles represented 21% and 24% of the aerosols from A. versicolor and P. chrysogenum, respectively. This study shows that fungal particles of various size, shape, and origin are aerosolized, and supports the need to include a broader range of particle types in fungal exposure assessment.

Submicronic Fungal Bioaerosols: High-Resolution Microscopic Characterization and Quantification

ABSTRACT Submicronic particles released from fungal cultures have been suggested to be additional sources of personal exposure in mold-contaminated buildings. In vitro generation of these particles has been studied with particle counters, eventually supplemented by autofluorescence, that recognize fragments by size and discriminate biotic from abiotic particles. However, the fungal origin of submicronic particles remains unclear. In this study, submicronic fungal particles derived from Aspergillus fumigatus, A. versicolor, and Penicillium chrysogenum cultures grown on agar and gypsum board were aerosolized and enumerated using field emission scanning electron microscopy (FESEM). A novel bioaerosol generator and a fungal spores source strength tester were compared at 12 and 20 liters min−1 airflow. The overall median numbers of aerosolized submicronic particles were 2 × 105 cm−2, 2.6 × 103 cm−2, and 0.9 × 103 cm−2 for A. fumigatus, A. versicolor, and P. chrysogenum, respectively. A. fumigatus released significantly (P < 0.001) more particles than A. versicolor and P. chrysogenum. The ratios of submicronic fragments to larger particles, regardless of media type, were 1:3, 5:1, and 1:2 for A. fumigatus, A. versicolor, and P. chrysogenum, respectively. Spore fragments identified by the presence of rodlets amounted to 13%, 2%, and 0% of the submicronic particles released from A. fumigatus, A. versicolor, and P. chrysogenum, respectively. Submicronic particles with and without rodlets were also aerosolized from cultures grown on cellophane-covered media, indirectly confirming their fungal origin. Both hyphae and conidia could fragment into submicronic particles and aerosolize in vitro. These findings further highlight the potential contribution of fungal fragments to personal fungal exposure.

Cross-shift study of exposure–response relationships between bioaerosol exposure and respiratory effects in the Norwegian grain and animal feed production industry

Objective We have studied cross-shift respiratory responses of several individual bioaerosol components of the dust in the grain and feed industry in Norway. Methods Cross-shift changes in lung function and nasal congestion, as well as in respiratory and systemic symptoms of 56 exposed workers and 36 referents, were recorded on the same day as full-shift exposure to the inhalable aerosol fraction was assessed. Exposure–response associations were investigated by regression analysis. Results The workers were exposed on average to 1.0 mg/m3 of grain dust, 440 EU/m3 of endotoxin, 6 µg/m3 of β-1,3-glucans, 17×104/m3 of bacteria and 4×104/m3 of fungal spores during work. The exposure was associated with higher prevalence of self-reported eye and airway symptoms, which were related to the individual microbial components in a complex manner. Fatigue and nose symptoms were strongest associated with fungal spores, cough with or without phlegm was associated with grain dust and fungal spores equally strong and wheeze/tight chest/dyspnoea was strongest associated with grain dust. Bioaerosol exposure did not lead to cross-shift lung function decline, but several microbial components had influence on nose congestion. Conclusions Exposure to fungal spores and dust showed stronger associations with respiratory symptoms and fatigue than endotoxin exposure. The associations with dust suggest that there are other components in dust than the ones studied that induce these effects.

Indirect Immunodetection of Fungal Fragments by Field Emission Scanning Electron Microscopy.

ABSTRACT Submicronic fungal fragments have been observed in in vitro aerosolization experiments. The occurrence of these particles has therefore been suggested to contribute to respiratory health problems observed in mold-contaminated indoor environments. However, the role of submicronic fragments in exacerbating adverse health effects has remained unclear due to limitations associated with detection methods. In the present study, we report the development of an indirect immunodetection assay that utilizes chicken polyclonal antibodies developed against spores from Aspergillus versicolor and high-resolution field emission scanning electron microscopy (FESEM). Immunolabeling was performed with A. versicolor fragments immobilized and fixed onto poly-l-lysine-coated polycarbonate filters. Ninety percent of submicronic fragments and 1- to 2-μm fragments, compared to 100% of >2-μm fragments generated from pure freeze-dried mycelial fragments of A. versicolor, were positively labeled. In proof-of-concept experiments, air samples collected from moldy indoor environments were evaluated using the immunolabeling technique. Our results indicated that 13% of the total collected particles were derived from fungi. This fraction comprises 79% of the fragments that were detected by immunolabeling and 21% of the spore particles that were morphologically identified. The methods reported in this study enable the enumeration of fungal particles, including submicronic fragments, in a complex heterogeneous environmental sample.

Fungal Fragments and Fungal Aerosol Composition in Sawmills

Abstract Assessment of exposure to fungi has commonly been limited to fungal spore measurements that have shown associations between fungi and development or exacerbation of different airway diseases. Because large numbers of submicronic fragments can be aerosolized from fungal cultures under laboratory conditions, it has been suggested that fungal exposure is more complex and higher than that commonly revealed by spore measurements. However, the assessment of fungal fragments in complex environmental matrix remain limited due to methodological challenges. With a recently developed immunolabeling method for field emission scanning electron microscope (FESEM), we could assess the complex composition of fungal aerosols present in personal thoracic samples collected from two Norwegian sawmills. We found that large fungal fragments (length >1 µm) dominated the fungal aerosols indicating that the traditional monitoring approach of spores severely underestimate fungal exposure. The composition of fungal aerosols comprised in average 9% submicronic fragments, 62% large fragments, and 29% spores. The average concentrations of large and submicronic fragments (0.2–1 µm) were 3 × 105 and 0.6 × 105 particles m−3, respectively, and correlated weakly with spores (1.4 × 105 particles m−3). The levels of fragments were 2.6 times higher than the average spore concentration that was close to the proposed hazardous level of 105 spores per m3. The season influenced significantly the fungal aerosol concentrations but not the composition. Furthermore, the ratio of spores in the heterogeneous fungal aerosol composition was significantly higher in saw departments as compared to sorting of green timber departments where the fungal fragments were most prevalent. Being the dominating particles of fungal aerosols in sawmills, fungal fragments should be included in exposure-response studies to elucidate their importance for health impairments. Likewise, the use of fungal aerosol composition in such studies should be considered.

Profiling of 3-hydroxy fatty acids as environmental markers of endotoxin using liquid chromatography coupled to tandem mass spectrometry.

3-Hydroxy acids are constituents of the lipid A part of lipopolysaccharides and may potentially be used as chemical markers of endotoxin. While commercial enzymatic assays, such as the widely used Limulus amebocyte lysate (LAL) assay, commonly detect merely the water-soluble fraction of the bioactive endotoxin, the chemical approach aims to estimate the total amount of endotoxin present in a sample. Our objective was to develop a simple method for quantitative profiling of 3-hydroxy fatty acids in occupational and environmental samples based on detection with HPLC-MS/MS. We included eleven 3-hydroxy fatty acids (3-hydroxyoctanoic acid to 3-hydroxyoctadecanoic acid) in the HPLC-MS/MS based method, which involved base hydrolysis of filter samples using 1M sodium hydroxide and removal of the base as well as concentration of the fatty acids using solid-phase extraction on a functionalized polystyrene-divinylbenzene polymer. Recovery trials from spiked glass fiber filters, using threo-9,10-dihydroxyhexadecanoic acid as internal standard, gave an overall recovery of 54-86% for 3-hydroxy fatty acids of medium chain length (3-hydroxynonanoic to 3-hydroxypentadecanoic acid). 3-Hydroxyoctanoic acid and the longer chain fatty acids were more problematic yielding overall spike recoveries of 11-39%. While the 3-hydroxy fatty acid profile of pure lipopolysaccharides was dominated by 3-hydroxydecanoic, 3-hydroxydodecanoic and 3-hydroxytetradecanoic acid the aqueous phase from drilling mud contained in addition relatively high amounts of 3-hydroxyoctanoic and 3-hydroxynonanoic acid. Endotoxin activity as measured by the LAL assay was reasonably correlated (R(2)=0.54) to the sum of 3-hydroxydecanoic acid, 3-hydroxydodecanoic acid and 3-hydroxytetradecanoic acid in these samples.

Predictors for Increased and Reduced Rat and Mouse Allergen Exposure in Laboratory Animal Facilities

Abstract Introduction Exposure to rat and mouse allergens during work in laboratory animal facilities represents a risk for being sensitized and developing allergic diseases, and it is important to keep the exposure level as low as possible. The objective of this study was to characterize the personal Mus m 1 and Rat n 1 exposure during work in laboratory animal facilities, and to investigate the effect of identified predictors of increased and reduced exposure. Methods Mus m 1 and Rat n 1 were analysed in whole day or task-based personal air samples by enhanced sensitivity sandwich enzyme-linked immunosorbent assay. Information about cage-and-rack systems, tasks, and other conditions known to influence the allergen exposure was registered. Predictors for allergen exposure were identified by multiple linear regression analyses. Results The median allergen exposure was 3.0 ng m−3 Mus m 1 and 0.5 ng m−3 Rat n 1, with large task-dependent variations among the samples. The highest exposed job group were animal technicians. Cage emptying and cage washing in the cage washroom represented the highest exposure, whereas animal experiments in the lab/operation room represented the lowest exposure, with laminar airflow bench being an exposure-reducing determinant. Cage changing was the highest exposed task in the animal room, where individually ventilated cages (IVCs) were predictors of reduced exposure for both Mus m 1 and Rat n 1, whereas cage-rack systems with open shelves and sliding doors were predictors of increased Rat n 1 exposure. Cages of IVC type with positive air pressure (IVC+) as well as open shelves and sliding doors were strong predictors of increased exposure during cage emptying and cage washing. Conclusions Significant different exposure levels depending on type of work and task imply different risks of sensitization and allergy development. The fact that IVC+ cages have opposite impact on Mus m 1 and Rat n 1 exposure during different tasks may have positive clinical implications when taken into account.