Environ. Sci. Technol. 2008, 42, 8849–8857
Investigation of Bioaerosols Released from Swine Farms using Conventional and Alternative Waste Treatment and Management Technologies G W A N G P Y O K O , * ,† O T T O D . S I M M O N S I I I , ‡ C H R I S T I N A A . L I K I R D O P U L O S , ‡,| LYNN WORLEY-DAVIS,§ MIKE WILLIAMS,§ AND MARK D. SOBSEY‡ Department of Environmental Health and Institute of Health and Environment, Department of Biological Sciences and Institute of Microbiology, Seoul National University, Seoul, Korea, Department of Environmental Sciences and Engineering, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, and Animal and Poultry Waste Management Center, North Carolina State University, Raleigh, North Carolina 27695
Received April 21, 2008. Revised manuscript received July 24, 2008. Accepted August 22, 2008.
Microbial air pollution from concentrated animal feeding operations (CAFOs) has raised concerns about potential public health and environmental impacts. We investigated the levels of bioaerosols released from two swine farms using conventional lagoon-sprayfield technology and ten farms using alternative waste treatment and management technologies in the United States. In total, 424 microbial air samples taken at the 12 CAFOs were analyzed for several indicator and pathogenic microorganisms, including culturable bacteria and fungi, fecal coliform, Escherichia coli, Clostridium perfringens, bacteriophage, and Salmonella. At all of the investigated farms, bacterial concentrations at the downwind boundary were higher than those at the upwind boundary, suggesting that the farms are sources of microbial air contamination. In addition, fecal indicator microorganisms were found more frequently near barns and treatment technology sites than upwind or downwind of the farms. Approximately 4.5% (19/424), 1.2% (5/424), 22.2% (94/424), and 12.3% (53/424) of samples were positive for fecal coliform, E. coli, Clostridium, and total coliphage, respectively. Based on statistical comparison of airborne fecal indicator concentrations at alternative treatment technology farms compared to control farms with conventional technology, three alternative waste treatment technologies appear to perform better at reducing the airborne release of fecal indicator microorganisms during on-farm treatment and management processes. These results demonstrate that airborne microbial contaminants are released from swine farms and pose possible exposure risks to farm workers and nearby neighbors.
* Corresponding author e-mail:
[email protected]. † Seoul National University. ‡ University of North Carolina at Chapel Hill. § North Carolina State University. | Current Address: U.S. Geological Survey, Columbus, OH 43229. 10.1021/es801091t CCC: $40.75
Published on Web 10/08/2008
2008 American Chemical Society
However, the release of airborne microorganisms appears to decrease significantly through the use of certain alternative waste management and treatment technologies.
Introduction The rapid industrialization of livestock production in the 1990s generated numerous concentrated animal feeding operations (CAFOs) in the United States, with more than 1 million animals on the largest farms (1-3). The number of CAFOs increased by 51% from 1982 to 1992 (1). According to the U.S. Department of Agriculture (USDA) and Environmental Protection Agency (EPA), CAFOs generate about 500 million tons of manure annually, which is more than three times the amount of human sanitary wastes. Large concentrations of animal waste are often stored in lagoons, piled on farm property, or sprayed over agricultural land. Due to the enormous amount of animal wastes produced on these CAFOs, potentially high concentrations of zoonotic pathogens in the waste, and only marginally effective management processes, airborne contaminants, including microbial pathogens, can be released from animal feeding operations and may effect human and animal health through many different routes of exposure. Airborne releases from CAFOs may be an important pathway for pathogen movement off farms with high animal density and improper waste treatment. Bioaerosols released from CAFOs can possibly cause adverse health effects in animals on the farms, in farm workers, and in nearby neighbors, as well as be responsible for environmental contamination (2-5). Increased levels of respiratory illnesses, including infectious diseases, allergy, and toxicosis, have been reported among farmers and neighbors (6). Epidemiological studies have shown increases in the incidence rates of enteric diseases during farm irrigation seasons, possibly due to exposure to bioaerosols (7, 8). Exposure to bioaerosols released from wastewater has also been associated with gastrointestinal infections among wastewater workers (9). New manure treatment technologies to better manage animal wastes have been developed amid growing concerns over human and animal health risks due to animal wastes released from animal operation facilities (10-12). However, few studies have examined airborne microorganisms released from these alternative animal waste treatment processes. Thus, to determine the health effects and environmental contamination associated with these technologies, the concentrations of airborne microbial contaminants released from these facilities must be assessed. We investigated the release of microorganism from two farms with conventional animal waste lagoons and ten farms with alternative manure treatment technologies. Our objectives were to quantitatively investigate the amount of airborne microorganisms released from swine CAFOs, determine the types of airborne fecal indicator microorganisms on farm properties, evaluate manure treatment and management technologies that minimize the release of airborne fecal indicator microorganisms from farms, and determine the effect of environmental conditions on the levels of airborne microorganisms associated with each technology. We also characterized the effectiveness of various waste treatment technologies in reducing the airborne release of fecal contamination.
Materials and Methods Farm Site Sampling. Microbiological air samples were collected and analyzed from 12 farms using various waste VOL. 42, NO. 23, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
9
8849
TABLE 1. Summary of Investigated Farms with Different Manure Treatment Technologies farm
treatment technology
1 2 3
number of animals and type of farm
types of treatment on farm
treatment system open to the environment?
conventional lagoon (control farm 1) conventional lagoon (control farm 2) solids separation-constructed wetlands system ambient temperature anaerobic digester
4,874-head finishing farm 7,200-head finishing farm 3,520-head finishing farm
biological biological physical-biological
entire system open entire system open entire system open
4,000 head farrow-to-weana
biological
5 6
solids separation-reciprocating wetland solids separation-liquid biofiltration using upflow aerated biological filters
1,600-head finishing farm 4,000-head finishing farm
physical-biological physical-biological
7
solids separation-nitrification-denitrification/ soluble phosphorus removal/solids processing system solids separation-filtramat separator solids separation-screw-press separator permeable anaerobic lagoon with cover, aerated nitrification pond, denitrification/ irrigation storage pond sequencing batch reactor aerobic blanket-aerated nitrification pond and denitfication/irrigation storage pond
4,360-head finishing farm
physical-biologicalchemical
digester closed, holding ponds open entire system open solids separation open, upflow biological filters closed entire system open
4,048-head finishing farm 3,320-head finishing farm 2,448-head finishing farm
physical-biological physical-biological biological
2,700-head finishing farm 6,480-head finishing and 1,067-head sow farm
biological biological
4
8 9 10
11 12 a
This type of farm houses sows and newborns up to 3 weeks old.
management technologies located in central and eastern North Carolina. We included two control farms with conventional technology (lagoon-sprayfield) and ten farms using alternative technologies. Table 1 briefly summarizes the farm characteristics, with in-depth descriptions previously outlined (13). Microbiological air samples were collected at key sites on the farms over 68 sampling dates from April 2002 to August 2004. To cover seasonal variations, we sampled each farm at least once in each of the four seasons and collected duplicate samples at barn exhaust fans, downwind lagoon edges, wastewater treatment unit processes, and the upper (upwind) and lower (downwind) boundaries of the facility. Due to variations in the positions of facilities on the farms and to maintain consistency in evaluating each technology, “property boundary” data were collected approximately 150 m upwind and downwind of facilities. This distance was chosen to represent a “worst-case” scenario for key sites (e.g., barns, manure holding structures, waste management technology) located close to a farm boundary, given a particular wind direction. We measured airborne microbial concentrations for culturable bacteria, culturable fungi, spores of Cl. perfringens, fecal coliforms, E. coli, bacteriophage, and Salmonella. Microbiological Air Sampling and Analysis. Microbiological air samples were collected using AGI-30 impingers (Ace Glass, Vineland, NJ) with 1% peptone-distilled water (DW) supplemented with 0.01% Tween 80 and 0.005% antifoam A (14). Environmental air was sampled at 12.5 LPM for 30 min. During the course of microbiological air sampling, the inlet of the sampler (AGI-30) was oriented into the wind. Airborne microorganism concentrations were measured by culturing bacteria and fungi, Cl. perfringens spores, fecal coliform, E. coli, and Salmonella. Culturable bacteria and fungi were quantified by 10-fold serial dilution and spread plating in duplicate on R2A and malt extract agar (MEA), respectively, and then cultured 5-7 days at room temperature (15). We used the most-probable number (MPN) assay system to detect and quantify fecal coliform and E. coli that were assayed and enumerated using Defined Substrate Technology (Colilert; IDEXX, Portland, ME) and IDEXX Quanti-Tray. The samples were incubated at 37 °C for 4 h and then at 44.5 °C for 18 h to resuscitate any injured bacteria (16). For positive wells, samples were aseptically recovered, and E. coli was further cultured on E. coli 4-methylumbelliferyl-R-D-glucu8850
entire system open entire system open anaerobic lagoon covered, nitrification and storage pond open entire system open entire system open
9
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 42, NO. 23, 2008
ronide (EC-MUG) at 37 °C overnight, with isolated colonies archived for further biochemical characterization. Cl. perfringens was quantified by a three-dilution, five-tube MPN using the iron milk media method (IMM) with incubation for 24 h at 42 °C (17). Total bacteriophages were measured using U.S. EPA Method 1601, a two-step enrichment method using E. coli c3000 as the host bacteria (18). Salmonella was measured using a MPN method, with pre-enrichment in buffered peptone water and Rappaport-Vassiliadis broth, followed by streaking on Salmonella-Shigella agar for distinctive colonies (19). Environmental Data. Environmental conditions were recorded simultaneously at the sampling points where air samples were collected at 1.5 m above ground level. Temperature and relative humidity (RH) were measured with a temperature and RH sensor (model 8720; TSI Inc., Shoreview, MN), solar irradiation was measured with a radiometer (model 1400A; International Light Inc., Newburyport, MA), and wind velocity and direction were measured with a vane thermo-anemometer (Extech Inc., Waltham, MA). Data Analysis. Airborne microbial concentrations were estimated by dividing the number of colony forming units (CFUs) detected in the impinger liquid by the product of the sampling airflow rate and duration. Statistical analysis was performed using STATA software (College Station, TX). We used a t test to compare the concentrations of total culturable bacteria and fungi and a χ2 test to compare the percentage of positive fecal indicator microorganisms at different sampling sites on the farms. Nonparametric statistical analyses [Wilcoxon’s rank-sum (Mann-Whitney) test] were performed to compare concentrations of airborne fecal indicators at the alternative technology sites with those at the conventional technology farms (Farm 1 and 2). Spearman’s rank correlation test was performed on microbial concentrations to evaluate differences under various environmental conditions.
Results Total Culturable Bacteria and Fungi in Air Samples. Culturable bacteria and fungi were detected in all air samples collected at all farm sites. The results are summarized in Table 2 and Figure 1. Concentrations of airborne culturable bacteria generally ranged from 102 to 105 CFU/m3. Bacterial concentrations at the lower (downwind) boundary (t test; p
TABLE 2. Average Concentrations (CFU/m3) of Total Culturable Aerobic Bacteria and Fungi in Bioaerosol Samples at Control and Alternative-Technology Farms farm
upper boundarya
lower boundaryb
1 2 3 4 5 6 7 8 9 10 11 12 overall
1.1 × 104 1.4 × 103 1.0 × 105 2.9 × 103 1.8 × 103 1.1 × 104 1.0 × 103 3.7 × 102 9.5 × 102 4.1 × 102 5.5 × 10 6.6 × 102 1.0 × 104
total culturable bacteria 6.9 × 103 3.2 × 104 8.6 × 103 1.7 × 105 1.7 × 105 7.6 × 105 1.2 × 105 3.3 × 104 4.6 × 103 2.7 × 103 1.6 × 104 1.5 × 105 4.4 × 103 4.7 × 103 1.2 × 103 3.4 × 103 6.0 × 103 4.1 × 103 2.7 × 104 1.1 × 104 1.3 × 102 1.4 × 103 1.4 × 103 6.1 × 103 2.5 × 104 1.0 × 105
1 2 3 4 5 6 7 8 9 10 11 12 overall
2.9 × 103 9.6 × 102 3.1 × 103 8.3 × 103 1.2 × 103 2.2 × 103 3.8 × 102 2.7 × 102 4.1 × 102 2.3 × 102 7.3 × 10 3.8 × 102 1.6 × 103
4.5 × 103 4.4 × 103 5.0 × 103 6.3 × 103 3.6 × 103 3.5 × 103 3.6 × 102 8.0 × 102 4.2 × 102 5.1 × 102 3.0 × 10 5.1 × 102 2.6 × 103
barn
total culturable fungi 3.5 × 103 7.1 × 103 4.0 × 103 3.7 × 103 1.3 × 103 7.0 × 103 2.5 × 102 5.8 × 102 1.0 × 103 4.1 × 102 5.7 × 10 3.1 × 102 3.0 × 103
a Upper boundary: approximately 150 m upwind of facility. facility. c n/a: not applicable.
) 0.03) and near barn (t test; p < 0.01) sites were higher than those at the upper (upwind) boundary on all of the sampled farms. Except for Farm 5, which had a solids separationreciprocating wetland, bacterial aerosol concentrations were highest at barn exhaust fans. Overall, our results indicated higher airborne bacteria levels on the farms than at the upwind boundaries. The difference in overall airborne bacterial concentrations between the control and alternative farms was not statistically different, except for Farm 11 with a sequencing batch reactor having significantly lower concentrations. Airborne culturable fungal concentrations generally ranged from 10 to 103 CFU/m3 at all of the study sites. Like bacterial aerosols, concentrations of total culturable fungi were significantly higher at the lower (downwind) than at the upper (upwind) farm boundaries (t test; p ) 0.03). However, total culturable fungal concentrations were not significantly higher near barns (t test; p ) 0.07) than at the upper (upwind) boundary. Overall, airborne fungal concentrations on the new waste-technology farms were generally similar to those on the control farms (Farms 1 and 2). The difference in overall airborne fungal concentrations between the control and alternative farms was not statistically significant, except for Farm 11 with a sequencing batch reactor having significantly lower concentration. Airborne Fecal Indicators and Pathogenic Bacteria (Salmonella). The data for airborne microorganisms associated with fecal contamination (fecal coliform, E. coli, Cl. perfringens spores, coliphages, and Salmonella), summarized in Table 3, show the percentage of samples testing positive for any fecal indicator microorganism, along with the mean and median concentrations of airborne microorganisms of fecal origin in positive samples. Overall, 4.5% (19/424), 1.2% (5/424), 21.7% (92/424), and 12.5% (53/424) of the samples were positive for fecal coliform, E. coli, Clostridium, and coliphage, respectively. Salmonella was not detected at any
b
lagoon
technology
overall
2.0 × 104 2.4 × 104 2.5 × 105 1.5 × 105 2.8 × 104 1.7 × 104 n/a 5.8 × 102 1.7 × 103 n/a 1.3 × 102 1.2 × 103 3.6 × 104
n/ac n/a 2.1 × 105 9.2 × 104 1.3 × 104 2.3 × 104 1.4 × 103 1.8 × 103 1.1 × 103 1.0 × 104 2.9 × 102 n/a 3.7 × 104
1.6 × 104 5.5 × 104 3.0 × 105 7.8 × 104 1.0 × 104 4.3 × 104 2.6 × 103 1.5 × 103 2.8 × 103 1.2 × 104 4.0 × 102 2.3 × 103
3.0 × 103 2.7 × 103 1.9 × 103 1.2 × 103 3.2 × 103 5.3 × 103 n/a 4.1 × 102 5.7 × 102 n/a 2.7 × 10 6.8 × 102 2.1 × 103
n/ac n/a 3.5 × 103 5.8 × 103 1.3 × 103 4.2 × 103 2.1 × 102 4.0 × 102 5.5 × 102 4.6 × 102 4.0 × 102 n/a 1.6 × 103
3.3 × 103 3.8 × 103 3.4 × 103 5.1 × 103 2.1 × 103 4.4 × 103 2.9 × 102 4.9 × 102 6.0 × 102 4.0 × 102 1.2 × 102 4.7 × 102
Lower boundary: approximately 150 m downwind of
farm. Fecal coliform was detected in up to 7.5% of the samples at the control farms and up to 15.4% at alternative-technology farms. Airborne E. coli was not detected at the control farms but was found in up to 10.3% of the alternative-technology farm samples. Cl. perfringens was the most frequently found fecal indicator microorganism, in about 12.5-25% of samples at the control farms and up to 54.2% of samples at alternativetechnology farms. Coliphage was detected in 2.5-20.3% of control-farm samples and up to 43.3% of alternativetechnology farm samples. The frequencies at which air samples were positive for fecal indicator microorganisms were lowest (below detection limits) at the farm utilizing a solids separation-reciprocating wetland (Farm 5) compared to the frequencies at the other farms (38 of 416 samples, or 9%). On the farms using the conventional management technique (lagoons), up to 25% of samples were positive for fecal microorganisms. On the alternative-technology farms, positive sample rates ranged from below detection limits to 54.2%, varying with the type of alternative management system. For all of the microbial indicators, the rate of positive samples was highest at or near barns and next highest around lagoons or other holding structures (Table 4). Thus, barns and most waste management technologies contributed similar measurable fecal indicator bacteria to the air on the farms (χ2test; p < 0.001) As indicated in Table 4, the concentrations of fecal indicators at the lower boundary (downwind) were consistently higher than those at the upwind boundary (χ2 test; p ) 0.005). Approximately 2.6% (10/388), 7.2% (25/347), 21% (82/388), 8.5% (26/304), and 9.3% (26/280) of samples were positive at the upper boundary, lower boundary, near the barn, lagoon, and at the wastemanagement site, respectively. We used Wilcoxon’s rank-sum (Mann-Whitney) test for statistical comparisons of airborne fecal indicator levels among alternative-technology farms (Farms 3-12) compared VOL. 42, NO. 23, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
9
8851
FIGURE 1. Concentrations (CFU/m3) of airborne total culturable bacteria (a) and fungi (b) at control farms and farms with alternative waste treatment technologies. The box plots show medians: 10th, 25th, 75th, and 90th percentiles, and outliers. to conventional-technology farms (Farms 1 and 2; Table 5). Among farms with alternative technologies, Farms 5, 7, and 11 performed better than those using conventional management practices; Farms 8, 9, and 10 performed equivalently to them; and Farms 3, 4, and 12 performed lower than the conventional control farms. Environmental Conditions. Figure 3 summarizes environmental conditions on the farms at the times of air sample collections. Temperatures varied somewhat because of seasonal differences among sampling dates. The mean relative humidity, wind velocity, and solar irradiation were similar on each of the farms tested. Air temperatures ranged from -12 to 37.2 °C, with a mean of 19.1 °C (SD ) 11.7); RH ranged from 15.8 to 100%, with a mean of 52.3% (SD ) 22.6); solar irradiation ranged from 0 to 12.8 mW/cm (2), with a mean of 5.5 mW/cm (2) (SD ) 3.4); and wind velocity ranged from 0 to 8.1 m/s, with a mean of 1.8 m/s (SD ) 1.3). Temperature was positively associated with airborne fungi and negatively associated with airborne bacteriophage and Cl. perfringens (Table 5). The concentrations of airborne 8852
9
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 42, NO. 23, 2008
bacteria, fungi, fecal coliform, and Cl. perfringens were positively associated with increased relative humidity. All of the measured airborne microorganisms were negatively associated with solar irradiation. Wind velocity was negatively associated with airborne culturable bacteria and fungi.
Discussion To our knowledge, this is the most extensive investigation (424 samples over 2 years at 12 different swine CAFOs) of the microbiological air quality related to CAFOs. In addition, this is the first study to evaluate microbial releases from CAFOs with conventional (lagoon) and ten different alternative wastewater treatment technologies. Our study indicated that swine CAFOs may be an important source of airborne transmission of fecally associated microorganisms to animals and humans in communities close to the farm boundaries. Airborne microorganisms and associated components released from farm waste management sites may pose health risks to exposed humans and animals both on and off the
FIGURE 2. Concentrations (CFU/m3) of airborne culturable bacteria (a) and fungi (b) at farm locations. The box plots show medians: 10th, 25th, 75th, and 90th percentiles, and outliers. (N ) 424). farms. The concentrations of culturable bacteria and various fecal indicator microorganisms sampled downwind of the farm sites were consistently higher than those at the upwind farm boundaries, suggesting that waste management systems on the farms are the sources of the airborne fecal contamination. Concentrations of culturable bacteria were highest near barn air exhausts and at certain waste treatment operations (solids separation, aeration processes, and waste treatment unit processes), suggesting these as sources of the bacteria. Fecal indicator microorganisms, such as fecal coliform, Clostridium, and bacteriophage, were more frequently detected in air near barns and wastewater treatment technologies. These results strongly suggest that airborne microorganisms released from farm waste management
systems are present at and potentially beyond farm boundaries. Three types of microorganisms were analyzed in this study: heterotrophic airborne microorganisms (total culturable bacteria and fungi), fecal indicator organisms (fecal coliform, E. coli, Clostridium, and bacteriophage), and a human bacterial pathogen (Salmonella). The concentrations of total culturable bacteria and fungi in this study are within the ranges found in previous studies of swine CAFOs (20-23). Total culturable bacteria and fungi, which are common in air and can arise from a variety of sources, are not specific to fecal contamination. Our main goal in sampling for these ubiquitous airborne microorganisms was to characterize the general microbiological air quality on and between different VOL. 42, NO. 23, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
9
8853
TABLE 3. Percentage of Positive Samples of Fecal Coliform, E. coli, Clostridium perfringens Spores, and Total Coliphage at the Sampled Farms (No Salmonella was Detected in Any Microbiological Air Sample) farm
fecal coliform a
1 2 3 4 5 6 7 8 9 10 11 12 Total a
7.5% (3/40) 0% (0/64) 15.4% (6/39) 14.3% (5/35) 0% (0/40) 3.3% (1/30) 0% (0/40) 0% (0/20) 0% (0/20) 9.4% (3/32) 0% (0/40) 4.2% (1/24) 4.5% (19/424)
E. coli
Clostridium perfringens spores
total coliphage
Salmonella
0% (0/40) 0% (0/64) 10.3% (4/39) 2.9% (1/35) 0% (0/40) 0% (0/30) 0% (0/40) 0% (0/20) 0% (0/20) 0% (0/32) 0% (0/40) 0% (0/24) 1.2% (5/424)
12.5% (5/40) 25.0% (16/64) 46.2% (18/39) 31.4% (11/35) 0% (0/40) 50.0% (15/30) 2.5% (1/40) 5.0% (1/20) 25.0% (5/20) 21.9% (7/32) 0% (0/40) 54.2% (13/24) 21.7% (92/424)
2.5% (1/40) 20.3% (13/64) 23.1% (9/39) 5.7% (2/35) 0% (0/40) 43.3% (13/30) 2.5% (1/40) 10.0% (2/20) 5.0% (1/20) 3.1% (1/32) 12.5% (5/40) 20.8% (5/24) 12.5% (53/424)
0.0% (0/40) 0.0% (0/64) 0.0% (0/39) 0.0% (0/35) 0.0% (0/40) 0.0% (0/30) 0.0% (0/40) 0.0% (0/20) 0.0% (0/20) 0.0% (0/32) 0.0% (0/40) 0.0% (0/24) 0.0% (0/424)
Percentage of positive samples (the number of positive samples/the total number of analyzed samples).
TABLE 4. Percentage of Positive Samples of Fecal Coliform, E. coli, Clostridium perfringens Spores, and Total Coliphage at Different Sampling Sites on the Farms (The Numbers Indicate Percent Positive Samples of Tested Microorganisms in 0.375 m3 of Sampled Air; No Salmonella was Detected in Any Microbiological Air Sample) location
fecal coliform
E. coli
Clostridium perfringens spores
total coliphage
overall (%) positive
P-valuea
upper boundary lower boundary barn lagoon waste management sites total
1.0% (1/97)b 5.7% (5/87) 7.4% (7/94) 2.6% (2/76) 2.9% (2/70) 4.0% (17/424)
0% (0/97) 0% (0/87) 4.3% (4/94) 0% (0/76) 1.4% (1/70) 1.2% (5/424)
4.1% (4/97) 18.4% (16/87) 50.0% (47/94) 18.4% (14/76) 18.6% (13/70) 22.2% (94/424)
5.2% (5/97) 3.5% (4/86) 25.5% (24/94) 13.2% (10/76) 14.3% (10/70) 12.3% (53/423)
2.6% (10/388) 7.2% (25/347) 21.1% (82/388) 8.5% (26/304) 9.3% (26/280) 9.9% (169/1707)
0.005
