Farmers’ exposure to dusts and gases in modern Finnish cubicle cow houses Kyösti Louhelainen, Juhani Kangas, Marjut Reiman Kuopio Regional Institute ofOccupational Health, PO Box 93, FIN-70701 Kuopio, Finland, e-mail: kyosli. louhelainen @ occuphealth.fi Pentti Kalliokoski University ofKuopio, Department ofEnvironmental Sciences, PO Box 1627, FIN-70211 Kuopio, Finland The occurrence of airborne dust, gases, microbes, endotoxin and bovine epithelial antigens (BEA, BDA2O) was studied in 26 modern, mainly cubicle, cow houses. Air samples of total dust, total spores, endotoxin and bovine epithelial allergens were collected on membrane filters with portable or piston pumps and analyzed with appropriate methods. Concentrations of gases (ammonia, carbon dioxide, hydrogen sulfide) were measured with diffusion tubes. Airborne viable spores were collected with a cascade impactor on five selective culture media for the identification of xerophilic, mesophilic and thermotolerant fungi and thermophilic actinomycetes. The geometric mean concentrations of total dust, BEA and BDA2O were 0.2-1.9 mg/m 3 , 5.2- 9.7 pg/m 3 and 50-260 ng/m 3 , respectively. The mean concentrations of ammonia and carbon dioxide were between 2.8-15 ppm and 2200-3200 ppm, respectively. The geometric mean of endotoxins was 19 ng/m 3 and the concentrations of fungi were at the lO'-IO3 cfu/m 3 level. In general, the variation in concentrations of total dust, viable fungi and endotoxin was large. The concentrations of total dust and fungi were lower than in earlier studies. Thus new cubicle houses provide a better working environment with regard to airborne hazards than the traditional cow houses. Key words: agriculture, airborne health hazards, allergens, micro-organisms - - V, 'Z'Z o ftS)f Z M Introduction (Malmberg 1990,Terho et al. 1987,Tammilehto et al. 1994,Donham 1993). Symptoms ofchronic bronchitis and farmer’s lung are still the most common respiratory complaints among dairy farmers (Tammilehto et al. 1994). In Finland, during recent years the dominant etiologic fac- tor for occupational respiratory diseases among Farm workers are exposed to a variety of agents that can cause injury by inhalation. Epidemio- logical studies indicate a high prevalence of res- piratory symptoms or diseases among farmers © Agricultural and Food Science inFinland Manuscript received June 1997 207 Vol. 6(1997): 207-217. AGRICULTURAL AND FOOD SCIENCE IN FINLAND https://www.c-info.fi/en/info/?token=n6weQyAhFrKX8W-P.xQoYkrePRms1dZyxNR4Lsg.0YSpRgrY_-Jj5oIbS7JasK0AznMZo2_cKXE-M3Q-u6teyaMiUt62rVmrhKFOAmP97sk7dbkBIaQRnuK09DFTmT4w6H9sJA2WarSSz4eTFu3e8shh3dE-H1cFiypIVbwwFj5dn1fg0s79ja6dQ5ZpIbNZahaYjKcgG4G2b4zhw5JwmJMVkNFQZvFhuHvcqpcPVvz8LfZyzsaQk7lGIc-mr5nWB4SJcxFOzoessLciLh3_jJ2qkyC2O8u-zyU1HIAIKokbGn-EIjetd62z6Ju5lRuiCZeB4WdZSB4MSYJBG5oMgGbAFFrTGGwNt9Zsp3pp-jFSJ7Jnjiq1ZPM_iuWU-w Louhelainen, K. et al. Farmers ’ exposure to dusts and gases in cubicles farmers has been cow dander, but microbial ex- posure also has caused hundreds of cases of farmer’s lung (Karjalainen et al. 1996, 1997, Kauppinen et ai. 1994, 1995). The mean levels of total dust in cow houses have been low (range from 0.7 to 1.5 mg/m3 ) (Louhelainen et ai. 1987a, Virtanen et al. 1986, Virtanen et ai. 1988) related to swineries (Louhelainen et ai. 1987b, Gustafsson 1988, Donham et ai. 1995). Thus other factors which are constituents of dust explain the etiology of respiratory diseases in the farming population. The airborne concentrations and serological studies of bovine epithelial antigen (BEA) and 20 kD bovine dander antigen (BDA2O) have been reported mainly by Finnish researchers (Virtanen et al. 1986, Virtanen et ai. 1988. Virtanen et ai. 1992, Ylönen et ai. 1990, Ylönen et ai. 1994). Several studies have concluded that the main sources of microbial dust in agriculture are hay, straw and grain (Kotimaa et al. 1987, Kotimaa 1990, Kotimaa et ai. 1991). Straw samples lib- erate significantly larger numbers of spores than hay and grain do (Kotimaa 1990). Thermoactin- omyces vulgaris has been the predominant mi- crobe from straw and Aspergillus umbrosus from hay and grain. On farms where fodder was dried artificially the concentration of thermophilic actinomycetes was lower than on farms with a traditional storage system (Dalphin et al. 1991). Malmberg and coworkers concluded that aller- gic alveolitis was associated with high levels of exposure to mold spores on most weekdays for weeks at a time and that ODTS (organic dust toxic syndrome) was associated with extreme exposure on a single day (Malmberg et al. 1993). In agriculture, endotoxins can be found in vari- ous materials and environments (Olenchock et al. 1990, Liesivuori et al. 1994). Ammonia is the most harmful of the gases causing respiratory symptoms normally found in animal houses (Kangas et al. 1987, Donham 1991, Donham et al. 1995). In cow houses, the lowest concentrations of airborne ammonia have been found in facilities equipped with a low ex- haust system of ventilation through the manure channels (Gustafsson 1988, Linnainmaa et al. 1993). The objective of this study was to investigate airborne health hazards during the working pe- riod in 26 modern, mainly cubicle, cow houses. The factors measured were total dust, bovine epithelial and dander allergens, microbial dust, endotoxins, ammonia, carbon dioxide and hydro- gen sulfide. Material and methods A total of 26 farms were included in this study. They were chosen among about 80 farms pro- vided by agricultural extension services. The main criteria for including a farm in the study were that the cow houses had to be new, the number of milking cows high (20-30) and it had to be a family farm. The cow houses were new, less than five years old; the mean year of con- struction or complete renovation was 1991,range 1989-93. Each farm was run by two persons and the mean number of milking cows was 22 (range 17-40). Twenty-three farms had cubicle houses (loose-housing barns), two had traditional tie stall barns and one had a cold cubicle barn. The cold cubicle (cold loose-housing barn) is the lat- est method of barn construction, but not yet com- mon in Finland. The cow department is uninsu- latedand equipped with wooden walls and roof. The ventilation is natural with openings in the roof. The milking parlor is a separate warm and insulated room beside the cold barn. All warm barns had a mechanical system of ventilation, but only one farm had a detailed design for ven- tilation. The farms were situated in middle and southern Finland. All except two of the barns had an automatic feeding system for milking cows, but the heifers had to be fed manually. The normal working tasks included cleaning the feeding table anu feeding the cows with forage, fodder grain and dry hay. Hay for fodder was used on 23 of the 208 AGRICULTURAL AND FOOD SCIENCE IN FINLAND 26 farms; straw was used for bedding on 6 farms, wood shavings on 13 farms; and seven of the farms did not use bedding at all. Measurements were made during winter and early autumn between 1994-1995 in each cow house. The indoor temperatures and relative hu- midity measured in the barn varied from 7 to 17°C (mean 12.7°C) and 55 to 95% (mean 87%), respectively. At the time of measurement the tem- perature in the cold loose barn was -6°C. The outdoor temperature was from +2 to -20°C dur- ing farm visits. The total dust was sampled gravimetrically according to the Finnish standard method (SFS 3860). For results below the limit of detection (0.1 mg/m 1 for area sampling and 0.5 mg/m5 for breathing zone sampling according to the stand- ard) half of the limit of detection has been used for interpreting the results (CEN 1995). Dust samples were collected on membrane filters (Millipore, USA) mounted in open-faced three- piece cassettes (Millipore, USA). Personal sam- pling was done at air flow rates of 2-2.5 L/min (SKC, USA) and stationary sampling at flow rates of 20-25 L/min (Piston pump, Finland). Samples were taken during the whole working period, and therefore the sampling time varied from 1.5 to 3.5 hours. The sampling sites for sta- tionary sampling in cubicle houses were located in the milking parlor and in the feeding aisle, and in tied barns they were located in the feed- ing and manure aisles. The purpose of the sta- tionary samples was to evaluate spreading of the dust. For personal sampling 1-2 samples were taken. The pumps were weekly calibrated with a calibrator (Gilian, USA) and the pumps were checked visually during the measurements. Samples for bovine epithelial antigen and specific bovine dander antigen were collected in the same way as samples for total dust. After sampling the filters were frozen at -20°C until analyzed. The samples were analyzed for BEA by a immunochemical method described by Vir- tanen et al. (1992) and for BDA2O with two-site immunometric assay described in detail by Ylönen et al. (1994). Viable fungal spores were sampled with a six- stage Andersen impactor (Model 10-800, An- dersen Inc., USA). Sampling volume was 50 to 100 liters. The culture media and incubation tem- peratures and timesfor analysis of viable spores were; xerophilic fungi on NaCl malt extract agar or on Dichloran Glycerol 18 (DG18) agar at +2O°C for 7 days, mesophilic fungi on Hagem agar at +2O°C for 7 days, thermotolerant fungi on Hagem agar at +4O°C for 3-5 days, ther- mophilic actinomycetes on half-strength nutri- ent agar at +55°C for 3 days. Xerophilic fungi are low-moisturefungi found for example in hay. The optimum temperature range for mesophilic fungi is 20-25°C, whereas thermotolerant fungi are heat-tolerant up to 45°C and thermophilic fungi can grow at temperatures up to 60°C (Dix & Webster 1995). Viable spore counts were cal- culated as colony forming units per cubic meter of air (cfu/m3), and the fungal or bacterial group was identified with a light microscope. Three types of samples were taken, one background sample one hour before work, one during milk- ing when hay, forage and fodder grain were also delivered to cows and heifers, and the third one two hours after the work was finished. The concen- trations of total spores were determinedby the CAM- NEA method describedby Palmgren et al. (1986). Endotoxin sampling and analysis is described in detailby Laitinen et al. (1994). One nanogram of endotoxin standard from Esherichia coli 01 11 :B4 corresponds to 12 endotoxin units. Concentrationsof airborne ammonia, carbon dioxide, and occasionally also hydrogen sulfide were measured withpassive diffusion tubes (am- monia 20/a-D, carbon dioxide 500/a-D, hydro- gen sulfide 10/a-D, Draeger, Germany) from feeding aisles or in the case in tied barns in ma- nure aisle and in milking parlor. Sampling time for gases was the whole working period of the farmers, 1.5 to 3.5 h. When the results were interpreted, the milk- ing parlors were divided into 1) open-parlors open to the barn, separated only by a fence about 2 m high and 4-5 m long and 2) closed parlors totally separated from the rest of the barn. 209 Vol. 6 (1997): 207-217. AGRICULTURAL AND FOOD SCIENCE IN FINLAND Louhelainen, K. et al. Farmers’ exposure to dusts and gases in cubicles Data analysis Most of the statistical analyses were performed with log-transformed data, because the data was not normally distributed. When the GM (geomet- ric mean) values were calculated for fungi and actinomycetes, 1 was added to each result be- cause the results included zero values. The sta- tistical significance of differences between mi- crobial concentrations measured in three consec- utive periods (before work, during milking and 2 h after milking) were testedby repeated meas- ures analysis of variance (ANOVA) for normal distribution and Friedmanrank ANOVA for non- normal distribution. The differences in the con- centrations of total dust, endotoxin, bovine epi- thelial antigens, and gases between the milking parlors and stationary and personal sampling sites were examined with the t-test for normal distribution or Mann-Whitney U-test for non- normal distribution. The correlations between the variables were tested with Pearson product mo- ment correlation coefficient or with Spearman production moment regression. Results The geometric mean concentrations oftotal dust did not exceed the OFF (Occupational Exposure Level) of 5 mg/m3 for organic dust (Työminis- teriö 1996). Seven samples from a total 52 sam- ples in area measurements and 15 samples from a total 45 personal samples were under the re- spective detection limits. There were no statisti- cal differences in the total dust concentrations between sampling sites or between persons do- ing feeding and milking work (Table 1). The personal concentrations oftotal dust were 2 to 3 times higher than the area concentrations (t-test, p<0.001). The highest personal total dust con- centration (14.5 mg/m3 ) was measured during feed delivery in a barn where the farmer had to take feed to the calves from a silo with a showel. The GM concentrations of BEA were at the same level in area sites and breathing zones in both tied houses and cubicle houses (Table 2). Concentrations of BDA2O antigen were 2 to 3 times higher in the breathing zone samples than in the area site samples in cubicles (t-test, p<0.001) (Table 2). However, no correlations were found between concentrations of total dust and BEA or BDA2O in open- or closed-type par- lors either in the breathing zone samples or at stationary site samples. In all 26 barns the GM of endotoxin concen- tration was 19 ng/m3 . The endotoxin concentra- tion was 5.5 ng /m3 in the cold cow barn, and in other barns the GMs varied from 16 to 27 ng/m3 . The concentrations of viable fungi varied strongly during different sampling periods (Ta- ble 3). For every group of microorganisms, in the closed parlors, the concentrations were 2-3 times higher during milking than during other measurement periods, but in the open parlors, in only with thermophilic actinomycetes. In addi- tion, the concentrations of microorganisms in the cold barn parlor were at about the same level as in other parlors. Statistical differences (ANO- VA or Friedman rank ANOVA, p<0.05) between measuring periods were found with both xerophilic (NaCl-Malt and DG18), mesophilic fungi and thermophilic actinomycetes in closed -type parlors, but not with any group of fungi or actinomycetes in open-type parlors. In the tied barns during milking, the geometric means of all microorganisms except thermophilic actino- mycetes were about ten times higher than con- centrations measured from all other cow hous- es. This difference was not noticed for before work or after work measurements. The mean total spore count during milking was 1.2 x I05 spores/m3 (Table 3). The viable fungal spores of xerophilic, mesophilic, thermo- tolerant fungi and thermophilic actinomycetes consisted of about 1-77% of the total spores measured during milking. There was no correla- tion between the concentration of viable spores from any groupof fungi and the total spore count. The prevalence of viable fungal groups or gen- 210 AGRICULTURAL AND FOOD SCIENCE IN FINLAND Table 1. Concentrations of total dust during indoor farm work, mg/m3 . (n = number of samples, AM = arithmetic mean, GM = geometric mean, s = stationary sampling site, bz = breathing zone sampling) Feeding aisle, s Milking parlor, s Milking, bz Feeding, bz nAM GM range nAM GM range nAM GM range nAM GM range cold bam I 0.7 I 0.1 I 0.3 1 1.8 tied bam 2 0.2 0.2 0.2 - 0.3 2 0.2 0.2 0.2 -0.3 2 1.0 0.8 0.25-1.8 2 0.3 0.3 0.25-0.3 cubicle 23 0.8 0.3 0.05-11.7 23 0.3 0.2 0.05-1.2 17 1.1 0.6 0.25-4.5 23 1.9 0.8 0.25^1.5 closed 12 1.2 0.3 0.05-11.7 12 0.4 0.2 0.05-1.2 9 1.3 0.7 0.25-1.5 12 2.5 0.9 0.25^1.5 open 11 0.4 0.2 0.05- 1.3 11 0.2 0.1 0.05-0.4 8 0.8 0.5 ll 0.8 0.7 0.25-2.2 Table 2. Concentrations of BEA (bovine epithelial antigen) and BDA2O (bovine dander antigen) in cow houses, (n = number of samples, AM = arithmetic mean, GM = geometric mean, s = stationary sampling site, bz = breathing zone sampling) Feeding aisle, s Milking parlor, s Milking, bz Feeding, bz nAM GM range nAM GM range nAM GM range nAM GM range HEA. Hg/m3 cold bam I 2.8 1 1.3 1 0.9 1 10.0 tied bam 2 10.6 7.3 2.9-18.3 2 5.4 5.2 3.8- 7.0 2 6.5 4.6 1.1-18.4 223.4 9.7 2.1-44.8 cubicle 23 9.2 6.1 1.3-35.5 22 8.0 5.3 1.4-25.8 18 8.6 5.6 1.4-28.4 21 12.9 8.6 1.7-43.2 BDA2O. ng/m’ cold bam I 40 1 44 1 90 I 310 tied bam 2 50 50 43-62 2 25 25 24-26 2 210 200 140-270 2 210 200 150-270 cubicle 23 124 85 12-320 22 99 76 21-450 18 240 200 63-550 21 320 260 60-700 era is presented in Table 4. The most prevalent fungus was Aspergillus, followed by Cladospo- rium, Paecilomyces, Penicillium, and Wallemia sebi. The most abundant species of thermoac- tinomycetes were Thermoactinomyces Candidas and T vulgaris. In addition, many yeasts were found on mesophilic media and on both types of xerophilic culture media. Hydrogen sulfide was not detected in any of the cow houses. In two tied barns, the mean con- centration of ammonia was 7 ppm in the feeding aisle and 11 ppm in the manure aisle. In the cold loose barn, the concentration of ammonia was under the limit of detection at both sampling sites. The concentrations of ammonia measured in all 26 cow houses were significantly lower in milking parlors or aisles than in feeding aisles (Mann-Whitney U-test, p