Airborne endotoxin associated with particles of different sizes and affected by water content in handled straw

International Journal of Hygiene and Environmental Health 213 (2010) 278–284

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International Journal of Hygiene and Environmental Health journal homepage: www.elsevier.de/ijheh

Airborne endotoxin associated with particles of different sizes and affected by water content in handled straw A.M. Madsen ∗ , S.H. Nielsen The National Research Centre for the Working Environment, Lersø Parkallé 105, 2100 Copenhagen Ø, Denmark

a r t i c l e

i n f o

Article history: Received 8 September 2009 Received in revised form 8 February 2010 Accepted 3 March 2010 Keywords: Bioaerosol Endotoxin Exposure Inflammation Respirable endotoxin Straw Thoracic endotoxin PM10

a b s t r a c t High exposures to endotoxin are observed in environments where organic materials are handled and lower exposures are found in e.g. indoor air. Inhaled endotoxin contributes significantly to the induction of airway inflammation and dysfunction. The size of an inhaled particle influences the deposition in the airways and the following health symptoms. The objective is to characterise the distribution of endotoxin on airborne particles of different sizes in straw storage halls with high exposure and in other environments with lower exposure levels to endotoxin. Furthermore we have studied the influence of water content of handled straw on the size distribution of endotoxin containing particles. Total, inhalable, thoracic and respirable endotoxin and particles have each been quantified in aerosols from boiler rooms and straw storage halls at 24 power plants, including 21 biofuel plants. Inhalable, thoracic and respirable endotoxin have been quantified in aerosols from offices and outdoor air. The endotoxin concentration was higher in airborne thoracic dust than in airborne ‘total dust’. The median respirable fraction in the straw storage halls, boiler rooms at biofuel plants, boiler rooms at conventional plants, offices and outdoors was respectively 42%, 9%, 19%, 24% and 34%. Thoracic endotoxin per number of thoracic particles was higher than respirable endotoxin per number of respirable particles at the biofuel plants. In straw storage halls the fraction of endotoxin of respirable size was highest on the days with lowest water content in the received straw. Furthermore the exposures to all endotoxin fractions were highest on days with the lowest water content in the received straw. In conclusion the highest exposures and concentrations of endotoxin occur or tend to occur from thoracic dust. A high variation in endotoxin concentrations and in fractions of respirable or thoracic size is found in the different working areas. This is important in the risk assessment and makes attempts to influence the endotoxin exposure a possibility. Water content in straw affected the concentration, exposure level and size distribution of airborne endotoxin. © 2010 Elsevier GmbH. All rights reserved.

Background Endotoxin is a cell wall component from Gram-negative bacteria, and it is primarily composed of lipopolysaccharides (LPS), with secondary constituents of protein and phospholipids. High exposures to endotoxin are observed in environments where organic materials are handled (Madsen, 2006b; Madsen et al., 2009; Martens et al., 2001). Inhaled endotoxin contributes significantly to the induction of airway inflammation and dysfunction (Keman et al., 1998; Pirie et al., 2003) and several occupational studies have shown positive associations between endotoxin exposure and respiratory disorders, including asthma-like syndrome, chronic airway obstruction, organic dust toxic syndrome, byssinosis, bronchitis, and increased airway responsiveness (Alwis et al., 1999; Douwes et al., 2003; Smit et al., 2005; Williams et al., 2005). For

∗ Corresponding author. Tel.: +45 39165242; fax: +45 39165201. E-mail address: [email protected] (A.M. Madsen). 1438-4639/$ – see front matter © 2010 Elsevier GmbH. All rights reserved. doi:10.1016/j.ijheh.2010.03.001

example a study of joinery workers showed significant associations between personal exposures to inhalable and respirable endotoxin and chronic bronchitis. In the same study, sawmill and chipmill workers showed significant relationships for personal exposures to respirable endotoxin, but not to inhalable endotoxin and chronic bronchitis (Alwis et al., 1999). On the other hand, recent studies suggest that environmental exposure to endotoxin in early childhood may protect against the development of allergy diseases later in life (Williams et al., 2005). Respiratory disorders caused by microorganisms can be dependent on the exposure levels (Eduard et al., 2001; Rylander et al., 1985) and with respect to particles, the size of particles has also been observed as significant (Brunekreef and Forsberg, 2005; Oberdorster, 2001). Thus it is well known that both the damage to and treatment of the pulmonary system has direct connection to particle deposition in the human lung. Therefore, assessing the risk of exposure to a given pollution such as endotoxin, and estimating the distribution of particle deposition throughout the lung is significant. Threshold concentrations for both total and respirable

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endotoxin have been suggested based on exposure-response for livestock workers and poultry workers (Donham and Cumro, 1999). These suggested threshold concentrations for total and respirable endotoxin were 614 EU/m3 and 0.35 EU/m3 respectively. Lower occupational exposure limits for total or inhalable endotoxin have also been suggested (Dutch Expert Committee on Occupational Standards, 1998). The objective of this study is to characterise the distribution of endotoxin on airborne particles of different sizes. We have sampled airborne particles of different sizes in four kinds of working areas and in outdoor air. These working areas included straw storage halls at biofuel plants. Another type of working area selected was boiler rooms at biofuel plants where straw is converted to energy. Previous studies have shown that dust from straw storage halls has a higher concentration of endotoxin than dust from boiler rooms (Madsen, 2006b; Madsen et al., 2008). Furthermore we have included boiler rooms at conventional power plants, where we expect a lower exposure to endotoxin. Finally we have included measurements at offices and outdoors, where low exposures to endotoxin are usually found (Hines et al., 2000; Madsen, 2006a). We have measured endotoxin concentrations as a function of particle size. Furthermore we have studied whether the water content in the straw handled in straw receptions influences the size of the fraction of endotoxin which is of respirable or thoracic size. Methods The power plants The study included 21 biofuel plants situated all over Denmark. In this paper the plants are called plant 4 to 24. The plants generated energy using straw as the fuel. We sampled airborne dust in working areas in combined straw receiving and storage halls, which in the following are called straw storage halls. Furthermore, at plants 4, 5 and 6, airborne dust was sampled in combined straw feeding and boiler rooms situated at higher levels in the buildings, and in the following these areas are called boiler rooms. In addition airborne dust was sampled in boiler rooms at three conventional power plants mainly generating energy from coal. The plants are called plant 1, 2 and 3. At 10 of the biofuel plants straw was received on one of the days of dust sampling and at 11 of the plants straw was received on both days of sampling; up to 36 trucks arrived per day with straw. On receipt, the straw was unloaded in the straw storage hall. On receipt, the water content in the received straw was measured by the people working at the plants as between 10.2% and 15.2% by weight. The relative humidity of the air in the straw storage halls was between 63.3 and 86.7 (av. = 78.0%) and the temperature between 4.2 and 12.4 ◦ C (av. = 8.8 ◦ C). In the boiler room at the biofuel plants the average relative humidity of the air was 33.6% and the average temperature 23.1 ◦ C. In the boiler room at the conventional plants average relative humidity of the air was 38.1% and the average temperature 21.9 ◦ C. Sampling of airborne dust at the power plants Measurements were performed in the late autumn, winter and early spring season in 2004–2006 and at 20 of the plants during two successive working days. At plant 15, sampling was performed in three periods over two years; in the last period sampling was only performed during one day. At plants 2, 5, 8 and 19, sampling was only performed during one day. The sampling and measurement of numbers and aerodynamic diameters (dae ) of particles were performed 1.5 m above floor level. The samplings using different kinds of samplers were done in parallel.

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Size-fractionated dust sampling was performed by a three-stage virtual impactor called a Respicon (TSI Incorporated, MN, USA). Thus airborne dust was sampled using a Respicon at boiler rooms at biofuel plants (n = 5), conventional plants (n = 5), at straw storage halls at biofuel plants (n = 40), in offices (n = 5) and outdoors (n = 5). The people working at the plants usually spend more working hours in the straw storage hall than in the boiler room. The people in the storage hall moved around and thus were at different distances from the stationary measurement. Our stationary measurements were placed 8–12 m away from where unloading of straw commenced. The Respicon has been shown to meet the ACGIH/ISO/CEN particle size selective sampling criteria (Koch et al., 2002; Tatum et al., 2002). Stage 1 of the device collects respirable particles and stage 2 collects tracheobronchial particles. Extra-thoracic particles are collected on stage 3 of the Respicon. Therefore, particles collected with the Respicon sampler are categorised by appropriate combination of the results from the three stages; thus, thoracic = respirable (stage 1) + tracheobronchial (stage 2), and inhalable = thoracic + extrathoracic (stage 3). Thoracic particles can also be called PM10 and respirable particles PM2.5 . The Respicon was fitted with Teflon filters and used at a flow rate of 3.1 l/min. On average the Respicones were sampling for 6 h and 7 min per day. Airborne particles were also sampled using a Triplex cyclone (BGI, MA) with a flow rate of 3.5 l/min. The Triplex cyclone has a well defined sharp penetration curve at a flow rate of 3.5 l/min with a cut point of 1 ␮m (D50 ) and only about 1% of particles with dae between 1.7 and 2.0 ␮m penetrate the cyclone (Gussman and Kenny, 2000). The Triplex cyclone was fitted with a polycarbonate filter with a pore size of 1.0 ␮m. PM1 dust was sampled at boiler rooms from conventional plants (n = 3) and biofuel plants (n = 3) and at straw storage halls (n = 10). On average the Triplex cyclones were sampling for 6 h and 58 min per day. ‘Total dust’ has been defined as the dust collected by a sampler with an entry velocity of 1.25 m/s (Kenny and Ogden, 2000); we sampled ‘total dust’ using 25 mm closed-face cassettes (Millipore holder; Millipore, Bedford, MA, USA, with a 5.6 mm inlet and a flow of 1.9 l/min corresponding to an inlet velocity of 1.25 m/s) in the areas where the Respicon sampled simultaneously. ‘Total dust’ can also be called TSP (total suspended dust). A total of 40 samples were taken in straw storage halls and five in boiler rooms at biofuel plants and five in boiler rooms at conventional plants. The samplers were fitted with Teflon filters (pore size 1.0 ␮m). On average the Millipore samplers were sampling for 6 h and 9 min per day. After sampling, the filters were transported carefully to the laboratory. Sampling in offices and outdoors Size-fractionated dust sampling was performed by Respicons at five offices (n = 5) at the same company and outdoors (n = 5). The offices were located at a company not related to any production plant and they had no humidity problems. The outdoor measurements were performed in the city outside these offices. The sampling was performed in May and June 2009. On average the Respicones were sampling for 7 h and 22 min per day. Gravimetric analysis The mass of dust components collected on the Teflon filters with Millipore samplers was determined by weighing the filters before and after sampling. Before weighing, the filters were equilibrated at constant air temperature (22 ◦ C) and humidity (50%) for 20–24 h. The detection limit was 0.037 mg dust/m3 for the filters from the Millipore samplers, and all concentrations were above the detection limit. The detection limit was calculated as three-times the

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standard deviation of 10 blanks and divided by the mean sampled volume.

Results Endotoxin per mg airborne dust

Dust extraction The filters were carefully removed from the samplers 20–24 h after sampling. The dust on the Teflon filters from the Millipore sampler and Respicons was extracted in respectively 6.0 ml and 5.0 ml pyrogen-free water with 0.05% Tween 20 by orbital shaking (300 rpm) at room temperature for 60 min. The dust on the polycarbonate filters was extracted in 5.0 ml sterile 0.05% Tween 80 and 0.85% NaCl aqueous solution by shaking for a 15 min period (500 rpm) at room temperature. The dust suspensions were centrifuged (1000 × g) for 15 min. The supernatants were stored at −80 ◦ C until they were used to determine the endotoxin content. Endotoxin quantification The supernatant was analysed (in duplicate) for endotoxin using the kinetic Limulus Amboecyte Lysate test (Kinetic-QCL endotoxin kit, BioWhittaker, Walkersville, Maryland, USA). A standard curve obtained from an Escherichia coli O55:B5 reference endotoxin was used to determine the concentrations in terms of endotoxin units (EU) (12.0 EU ≈ 1.0 ng). The range of the standard curve was 0.05–50 EU/ml. The limits of detection were between 0.2 and 0.3 EU/m3 and all concentrations were above the detection limit. The endotoxin measurements are presented as EU/mg dust, fraction of respirable endotoxin (%) calculated from data of e.g. respirable endotoxin (EU) per m3 air, EU/number of particles, and as EU/m3 /number of trucks arriving with straw.

Number and size distribution of airborne particles An APS (APS-3321; TSI Inc., USA) measured the number concentration of particles from 0.542 to 19.81 ␮m (aerodynamic diameter) over 1-min intervals at each sample station at the power plants. The theoretical aspiration of the APS is near 100% for particles as large as 20 ␮m (Peters et al., 2006). On average the APS was measuring for 6 h and 7 min per day. From these measurements, the numbers and weights of thoracic, respirable and PM1 size were calculated (EN 481, 1993).

The ‘total’ endotoxin concentration (EU/mg dust) was higher in straw storage halls than in boiler rooms, but the concentrations were also very different in the straw storage halls at the different plants (Fig. 1a). The endotoxin concentration was higher in thoracic dust than in ‘total dust’ at the power plants. Only a low endotoxin concentration was found in PM1 dust (Table 1). Respirable endotoxin per cubic metre air The median concentration of respirable endotoxin in the straw storage halls was 141 EU/m3 , in the boiler rooms at the biofuel plants was 16.1 EU/m3 , in the boiler rooms at the conventional plants was 2.6 EU/m3 , in the offices was 0.33 EU/m3 and in the outdoor air was 0.40 EU/m3 . Fraction of endotoxin in thoracic, respirable and PM1 dust The fractions of inhalable endotoxin that were of respirable size were of different sizes, ranging from 3% to 90% (Fig. 1b). At some plants the variation in the respirable fraction from day to day was only small (e.g. plant 3 and 10). The median exposures to airborne respirable endotoxin (EU/m3 ) in the straw storage halls, boiler rooms at biofuel plants, and boiler rooms at conventional plants were 42%, 9% and 19% of the inhalable endotoxin respectively (Table 2). Thus significant differences were seen in straw storage halls compared with boiler rooms. The measurements in offices showed 24% respirable endotoxin and the outdoor measurements 34% respirable endotoxin (median values). Only a small fraction of the endotoxin exposure occurred from PM1 dust when considered per m3 air (Table 1). Endotoxin per number of particles The amount of thoracic endotoxin per number of thoracic particles was higher than respirable and PM1 endotoxin per number of particles of the same size categories in both straw storage halls and boiler rooms at the biofuel plants (Table 3). Endotoxin amount in dust from the straw storage halls was lower for respirable than for thoracic particles (Table 3). Endotoxin as affected by water content in handled straw

Treatment of data The dust and endotoxin exposures were log-transformed. At plants where straw was received on both days of sampling, the effect of the water content in the handled straw on the size distribution of endotoxin and on endotoxin concentration (EU/mg) was calculated using Proc Mixed in SAS 9.1. with the biofuel plants as the random effect. The concentrations of endotoxin in different fractions and areas were compared using Proc Anova and the standard deviation (s*) is calculated according to (Limpert et al., 2001). Different numbers of trucks with straw were arriving and unloading straw at the straw storage halls over the two days of sampling at each plant. To be able to compare the exposure level at two days of sampling at the same plant, we balanced the exposure level with the number of trucks arriving with straw. Subsequently, the effect of water content in the handled straw on the endotoxin levels was calculated on the log-transformed data also using Proc Mixed with the biofuel plants as the random effect. The dust and endotoxin concentrations were log-transformed and the Pearson correlation coefficients between endotoxin concentrations and fractions were calculated in SAS 9.1.

At some plants (measurements from 24 days at 11 plants) straw was received on all days of aerosol sampling. At these plants the sizes of the respirable (%) and thoracic fraction (%) was compared on the two days of sampling. The size of the fraction of respirable size depended on the average water content in the received straw (Fig. 2a) (p = 0.029). Consequently the respirable fraction was higher on the day where water content in the received straw at the same plant was lower. This was also seen for 75% of the measurements of the thoracic endotoxin fraction (Fig. 2b). However, because 25% of the measurements behaved differently, the trend was not significant (p = 0.16). The concentration (EU/mg dust) of endotoxin also depended on the average water content in the received straw. Consequently the respirable endotoxin concentration (respirable endotoxin/mg respirable dust) was higher on the day where water content in the received straw at the same plant was lower (p = 0.035). This was also seen for the measurements of the thoracic endotoxin fraction (p = 0.042) but not for total endotoxin per mg total dust (p = 0.23). The exposure to ‘total’, inhalable, thoracic and respirable endotoxin per number of trucks with straw arriving in the straw

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Fig. 1. Airborne endotoxin in boiler rooms at conventional (plants 1, 2 and 3) and biofuel (plants 4, 5 and 6) plants and at straw storage halls (plants 6–24). Endotoxin (EU) per mg ‘total dust’ (a) and respirable (black part of bars) and thoracic (grey part of bars) endotoxin fraction of inhalable endotoxin (EU/m3 ) (b). Table 1 Median endotoxin (EU) concentration in total, thoracic, respirable and PM1 dust per mg dust particles of the same size category.

Straw storage hall Boiler room biofuel Boiler room conventional a b

‘Total’ dusta , b

s*

n

Thoracic dust

s*

n

Respirable dust

s*

n

PM1 dust

s*

n

558b 88c 25d

1.9 1.5 1.7

40 5 5

1804a 1733a 110c

1.4 1.2 1.3

40 5 5

1289a 815b 69cd

1.2 1.2 1.3

40 5 5

160c 104c 52d

1.3 1.2 1.4

9 3 3

Numbers followed by the same letter or letters are not statistically significantly different. Total dust was measured by weighting while amounts of thoracic, respirable and PM1 dust was calculated from APS-data.

Table 2 Median fraction (%) of endotoxin (EU/m3 ) present in thoracic, respirable and PM1 dust.

Straw storage hall Boiler room biofuel Boiler room conventional Office Out door air a

Thoracic dusta

s*

n

Respirable dust

s*

n

PM1 dust

s*

n

81a 48b 36bc 73a 58b

1.3 1.3 1.2 1.1 1.2

40 5 5 5 5

42b 9d 19c 24cd 34c

1.4 1.6 1.2 1.5 1.5

40 5 5 5 5

2.6e 0.5e 0.7e nm nm

2.1 2.4 1.7

9 3 3 0 0

n

PM1 dust

s*

n

1.4 1.9 1.7

9 3 3

Numbers followed by the same letter or letters are not statistically significantly different.

Table 3 Median (EU) concentration in thoracic, respirable and PM1 dust per numbers of thoracic, respirable and PM1 particles. Thoracic dusta ) Straw storage hall Boiler room biofuel Boiler room conventional a

−6

4.35 × 10 a 1.70 × 10−6 bc 1.49 × 10−7 d

s* 1.3 1.5 1.5

n 40 5 5

Respirable dust −6

2.03 × 10 b 9.05 × 10−7 c 9.25 × 10−8 d

Numbers followed by the same letter or letters are not statistically significantly different.

s* 1.4 1.6 1.5

40 5 5

−8

2.60 × 10 e 9.94 × 10−9 f 7.19 × 10−9 f

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Fig. 2. Respirable (r) (a) and thoracic (t) (b) endotoxin fraction (%) as a function of water content (%) in the straw received during the two days of aerosol sampling at straw storage halls at plants 7, 9, 11, 12, 15, 4, 20, 21, 23, 6 and 24.

Fig. 3. Examples of exposure to airborne respirable (r), thoracic (t) (a), inhalable (i) and ‘total’ (tot) (b) endotoxin/m3 /number of trucks with straw as a function of water content in received straw during the two days of aerosol sampling at straw storage halls at plants 9, 15, 4, 20, 21 and 23.

Discussion storage hall as affected by water content in the received straw was calculated. The exposure to respirable (p = 0.0017) and thoracic (p = 0.0103) (examples are given in Fig. 3a) and ‘total’ (p = 0.0176) and inhalable (p = 0.0318) (examples are given in Fig. 3b) endotoxin was significantly lower on the days where the water content of the received straw was highest.

Correlations between exposures The exposure (EU/m3 ) to respirable endotoxin correlated significantly with the exposure to thoracic (r = 0.93, p < 0.0001) and inhalable (r = 0.87, p < 0.0001) endotoxin. The exposure to thoracic endotoxin correlated significantly with the exposure to inhalable endotoxin (r = 0.97, p < 0.0001). The respirable endotoxin fraction (%) correlated positively and significantly with exposure to ‘total dust’ (r = 0.61, p < 0.0001). There were no significant correlations between exposure to numbers of PM1 , respirable and thoracic particles and exposure to endotoxin in the same size categories.

A fraction of 42%, 9% and 19% (median values) of the airborne endotoxin (EU/m3 ) in respectively the straw storage halls, boiler rooms at biofuel plants, and boiler rooms at conventional plants were present on respirable particles. The 9% and 19% found in boiler rooms corresponds to what is found in different wood-working sites. Thus Alwis et al. (1999) have found geometric mean values between 0.13 and 2.03 ng respirable endotoxin/m3 and between 0.74 and 21.08 ng inhalable endotoxin/m3 in wood-working sites, corresponding to around 10–18% respirable endotoxin. In a study in sheds, a median exposure to endotoxin was found to be 36 inhalable EU/m3 and 2 respirable EU/m3 ; this corresponds to around 6% respirable endotoxin (Heinrich et al., 2003). In different agricultural environments Nieuwenhuijsen et al. (1999) found that about 3% of the endotoxin was of respirable size during cow milking and about 28% during pruning of trees. In relation to these three studies and to this study, the respirable fraction of endotoxin in straw storage halls can be considered as high. On the other hand, in the straw storage halls a large variation in the respirable fraction from plant to plant was found (Fig. 1b). In sheds with different animals, a large variation in the fraction of respirable size is also found, and around 0.3–32% airborne endotoxin was of respirable size (Schierl et al., 2007).

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High airborne concentrations of respirable endotoxin (median = 141 EU/m3 ) were found in straw storage halls. The concentration of airborne respirable endotoxin in boiler rooms at biofuel plants was at the level of what is earlier found at saw mills; the level in boiler rooms at conventional plants is at the level of what is earlier found at logging sites (Alwis et al., 1999). The airborne concentrations found at the biofuel plants were lower than what is earlier found in a single biofuel plant where sampling was performed both during night and day time (Madsen et al., 2008). As expected, lower airborne concentrations were found outdoor and in offices. From data in studies of exposure to endotoxin during night and day in different animal sheds, the exposure to endotoxin was often highest during day time (Schierl et al., 2007; Seedorf et al., 1998) and the respirable fraction in most animal sheds was lowest when the highest exposure was measured. In our study in the straw storage halls we found a positive association between size of the respirable fraction (%) and dust exposure level, but not between the respirable fraction and endotoxin exposure. The exposure to respirable endotoxin correlated significantly with the exposure to thoracic and inhalable endotoxin. In wood dust a positive significant correlation was also found between inhalable and respirable endotoxin (Alwis et al., 1999). At some power plants the variation in the respirable fraction from day to day was only small (e.g. plant 10 and 12), even though the endotoxin concentrations (EU/mg dust) were very different from day to day. This indicates that the equipment used at the plants influences the endotoxin particle size distribution and this makes attempts to influence the endotoxin particle distribution a possibility. Plant 15 was visited five times, in two periods of two successive days in spring and one day in autumn. The respirable fraction had almost the same size on two successive days but not during the three periods. From this study we cannot explain this difference but it might be because of the fact that different vacuum cleaners were used during unloading of straw in the different periods or because measurements were performed during different seasons. In this study only five outdoor measurements were performed. These measurements showed that 58% of the airborne endotoxin was of thoracic size and it was about 1.5 times higher in thoracic dust than in respirable. In outdoor air in towns endotoxin exposures 10-times higher have been found from thoracic dust than from the PM2.5 particle fraction independently, irrespective of whether the concentrations were expressed per mg sampled mass or per m3 air (Heinrich et al., 2003). Consequently, a considerable part of the endotoxin exposure outdoors seems to occur from thoracic–endotoxin-containing particles and it is relevant to study whether this is generally seen for outdoor air. In offices and straw storage halls in this study respectively 73% and 81% (median value) of the airborne endotoxin was present in thoracic dust. Increased level of thoracic dust has been related to adverse health effects including increased use of asthma medication, attacks of asthma in pre-existing asthmatics and attacks of Chronic Obstructive Pulmonary Disease (Donaldson and Macnee, 2001). The concentration of endotoxin (EU/mg dust) was very different in the studied environments. In airborne ‘total dust’ from the conventional boiler rooms we found a median endotoxin concentration of 25 EU/mg dust. This is at the level of the median concentration on 15–20 EU/mg settled floor dust found in other studies (Allermann et al., 2006; Bouillard et al., 2005) and at the level of what is found in mattress dust (Braun-Fahrlander et al., 2002). In one study a concentration of total endotoxin larger than 100 EU/mg family room house dust is considered as elevated. The elevated concentrations was associated with increased risk of any wheeze and of repeated wheeze in the first year of life in a cohort of children genetically predisposed to asthma and allergy (Park et al., 2001). In straw storage halls we found a much higher con-

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centration of endotoxin in ‘total dust’ (median = 558 EU/mg), and this concentration is at a similar level to what is found in settled dust from stables (Mutius et al., 2000) and in airborne ‘total’ dust in cucumber nurseries (Madsen et al., 2009). The concentration of endotoxin in respirable dust from the 19 straw storages halls (median = 1289 EU/mg) was significantly higher than in respirable dust from boiler rooms (815 EU/mg). Using a Gravikon for sampling respirable dust we have at a single biofuel plant found a endotoxin concentration of 1100 EU/mg dust in March and 480 EU/mg in August (Madsen and Sharma, 2008). Previous studies have shown that respirable dust from a straw storage hall can induce a stronger inflammatory response in mice than dust from a boiler room at the same biofuel plant (Madsen et al., 2008). The dusts used in the mice exposure study had lower concentrations of respirable endotoxin/mg dust than the median values found in this study. The water contents in the received straw were between 10.2% and 15.2%. Microorganisms have a limiting water activity (aw ) level below which they will not grow. The water activity (aw ) level that limits the growth of the vast majority of bacteria is below 0.90aw and for molds below 0.70aw . Thus the effect of water content in straw on the exposure to endotoxin is not expected to be caused by different degrees of growth of Gram-negative bacteria in a period close to the time of handling. In contrast the effect of water content in the straw on the exposure to endotoxin is expected to be caused by the effect on particle release. The respirable endotoxin fractions increased as the water content in the received straw decreased. Thus only small differences in water content in the handled straw affect how much of the endotoxin was of respirable size. Consequently this will also affect how long the endotoxin particles remain airborne, their ability to be inhaled and penetrate through the upper respiratory tract into lung airways and the following deposition. From plant to plant there was no association between water content in straw and size (%) of the respirable fraction. This is probably caused by factors like different straw handling processes at different plants and different distances to the endotoxin sources (straw handling). The endotoxin exposure per amount of received straw (EU/m3 /number of trucks arriving with straw) was also affected by the water content in the received straw. Small increases in water content in the straw caused a lower exposure to endotoxin. We have found no other work place studies of how release of endotoxin from mechanically handled organic material is affected by the humidity of the material. However studies using rotating drums have been performed. Using a rotating drum, wood chips with a high watercontent were less dusty and had a lower particle generation rate than chips with lower water content, and the straw with the lowest water content released most particles, etc. (Madsen et al., 2004). Also studies of dustiness of coal using a rotating drum show that increasing water content causes decreasing dust release (Hjemsted and Schneider, 1996). In some working environments, attempts to reduce dust exposure using e.g. oil or lignosulfonate or other suspensions have been made (Bhattacharyya and Roe, 1983; Breum et al., 1999; Takai et al., 1995). We have seen no such attempts concerning reducing dust exposure at biofuel plants. In conclusion the highest concentrations of endotoxin both when considered per mg dust and per number of particles occur, or tend to occur, from thoracic dust. A high variation in endotoxin concentrations and in fractions of respirable or thoracic size was seen. A higher fraction of the endotoxin was present as thoracic and respirable endotoxin in the straw storage halls than in boiler rooms and in outdoor air. To be able to reduce exposure identification of factors affecting the size distribution of endotoxin containing particles released from straw will be helpful. In this study we found that the factor water content in handled straw affected both the concentration in dust and air, and size distribution of endotoxincontaining particles.

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