Estimation of bioaerosol risk of infection to residents adjacent to a land applied biosolids site using an empirically derived transport model

AIM: The purpose of this study was to develop an empirically derived transport model, which could be used to predict downwind concentrations of viruses and bacteria during land application of liquid biosolids and subsequently assess microbial risk associated with this practice. METHODS AND RESULTS: To develop the model, coliphage MS-2 and Escherichia coli were aerosolized after addition to water within a biosolids spray application truck, and bioaerosols were collected at discrete downwind distances ranging from 2 to 70 m. Although coliphage were routinely detected, E. coli did not frequently survive aerosolization. Data on aerosolized coliphage was then used to generate a virus transport model. Risks of infection were calculated for various ranges of human virus concentrations that could be found in biosolids. CONCLUSIONS: A conservative estimate at 30.5 m (assumed to be nearest adjacent residences) downwind, resulted in risks of infection of 1 : 100,000, to the more realistic 1 : 10,000,000 per exposure. Conservative annual risks were calculated to be no more than 7 : 100,000 where as a more realistic risk was no greater than 7 : 10,000,000. Overall, the viral risk to residences adjacent to land application sites appears to be low, both for one time and annual probabilities of infection. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrated a simple approach towards modelling viral pathogens aerosolized from land applied liquid biosolids, and offers insight into the associated viral risk.     

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

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

Diversity of aerosolized bacteria during land application of biosolids

AIMS: The purpose of this study was to determine the diversity of bacterial communities associated with bioaerosols generated during land application of biosolids using 16S ribosomal RNA (16S rRNA) PCR. METHODS AND RESULTS: Anaerobically digested Class B biosolids were land applied to an agricultural site located in South Central Arizona. Aerosol samples were collected downwind of the biosolids operations and were collected via the use of SKC Biosamplers and subsequently extracted for the presence of bacterial community DNA. All DNA was amplified using 16S rRNA primers, cloned and sequenced. All sequences were aligned and phylogenetic trees were developed to generate community profiles. The majority of aerosolized bacterial clone sequences belonged to the Actinobacteria and alpha- and beta-proteobacterial taxa. Aerosol samples collected downwind of soil aerosolization produced similar profiles. These profiles differed from upwind and background samples. CONCLUSIONS: No one clone sequence isolated from the aerosol samples could be solely attributed to biosolids; on the contrary, the majority appeared to have arisen from soil. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates that in dry, arid climates the majority of aerosols associated with biosolids land application appear to be associated with the onsite soil.     

Field studies measuring the aerosolization of endotoxin during the land application of Class B biosolids

Endotoxins are a component of Gram-negative bacteria cell walls and are known to be present in biosolids. Endotoxins have been shown to be potent stimulators of the innate immune response causing airway irritation and shortness of breath. Class B biosolids are routinely applied to agricultural lands to enhance soil properties and can be used as an alternative to chemical fertilizers. This study investigated the aerosolized endotoxin dispersed during the land application of Class B biosolids on agricultural land and a concrete surface at two sites in Colorado, USA. Aerosolized endotoxin was captured using HiVol samplers fitted with glass fiber filters, polycarbonate filter cassettes (both open and closed) and BioSampler impinger air samplers. Endotoxins were also measured in the biosolids to allow for correlating bulk biosolids concentrations with aerosol emission rates. Endotoxin concentrations in biosolids, impinger solutions and filter extracts were determined using the kinetic Limulus amebocyte lysate assay. Aerosolized endotoxin concentration was detected from all sites with levels ranging from 0.5 to 642 EU/m³. The four types of sampling apparatus were compared, and the HiVol and open-faced cassette samplers produced higher time-weighted average (TWA) measurements (EU/m³) than the impinger and closed cassette samplers. Ambient wind speed was found to be the variable best describing the observed results with optimal wind speed for highest deposition estimated at 5 m s^?1. It is argued that HiVol air samplers are a particularly reliable approach and subsequent analyses relating TWA measurements to wind speed and biosolids characteristics were based on the measurements collected with those samplers.     

Survey of wastewater indicators and human pathogen genomes in biosolids produced by class a and class B stabilization treatments

Accurate modeling of the infectious aerosol risk associated with the land application of biosolids requires an in-depth knowledge of the magnitudes and changes in pathogen concentrations for a variety of class A and class B stabilization methods. The following survey used quantitative PCR (qPCR) and culture assays to detect environmentally resistant bacterial and viral pathogens and biosolid indicator organisms for 36 biosolid grab samples. Biosolids were collected from 14 U.S. states and included 16 class B mesophilic anaerobic digestion (MAD) samples and 20 class A biosolid samples from temperature-phased anaerobic digestion (TPAD), MAD plus composting (COM), and MAD plus heat pelletization processes. The indicator concentrations of fecal coliforms and male-specific coliphages as well as pathogen genome concentrations for human adenovirus species, Legionella pneumophila, Staphylococcus aureus, and Clostridium difficile were significantly lower in the class A samples, and a multivariate analysis of variance ranked the stabilization processes from the lowest pathogen/indicator load to the highest as (i) class A COM, (ii) class A TPAD, and (iii) class B MAD. Human adenovirus genomes were found in 88% of the class B samples and 70 to 100% of the class A samples. L. pneumophila, S. aureus, and C. difficile genomes were detected at the qPCR assay detection limits in 19 to 50% of the class B and class A anaerobic digestion samples, while L. pneumophila was detected in 50% of the class A compost samples. When considering all the stabilization methods, both the fecal coliform and the male-specific coliphage concentrations show a significant linear correlation with the pathogen genome concentrations. This survey provides the necessary pathogen concentrations to add to biosolid aerosol risk and pathogen exposure analyses and clarifies the effectiveness of class A stabilization methods with the pathogen and indicator loads in biosolids.     

Source tracking aerosols released from land-applied class B biosolids during high-wind events

DNA-based microbial source tracking (MST) methods were developed and used to specifically and sensitively track the unintended aerosolization of land-applied, anaerobically digested sewage sludge (biosolids) during high-wind events. Culture and phylogenetic analyses of bulk biosolids provided a basis for the development of three different MST methods. They included (i) culture- and 16S rRNA gene-based identification of Clostridium bifermentans, (ii) direct PCR amplification and sequencing of the 16S rRNA gene for an uncultured bacterium of the class Chloroflexi that is commonly present in anaerobically digested biosolids, and (iii) direct PCR amplification of a 16S rRNA gene of the phylum Euryarchaeota coupled with terminal restriction fragment length polymorphism to distinguish terminal fragments that are unique to biosolid-specific microorganisms. Each method was first validated with a broad group of bulk biosolids and soil samples to confirm the target's exclusive presence in biosolids and absence in soils. Positive responses were observed in 100% of bulk biosolid samples and in less than 11% of the bulk soils tested. Next, a sampling campaign was conducted in which all three methods were applied to aerosol samples taken upwind and downwind of fields that had recently been land applied with biosolids. When average wind speeds were greater than 5 m/s, source tracking results confirmed the presence of biosolids in 56% of the downwind samples versus 3% of the upwind samples. During these high-wind events, the biosolid concentration in downwind aerosols was between 0.1 and 2 microg/m3. The application of DNA-based source tracking to aerosol samples has confirmed that wind is a possible mechanism for the aerosolization and off-site transport of land-applied biosolids.     

Comparison of coliphage and bacterial aerosols at a wastewater spray irrigation site.

Microbiological aerosols were measured on a spray irrigation site at Fort Huachuca, Ariz. Indigenous bacteria and tracer bacteriophage were sampled from sprays of chlorinated and unchlorinated secondary-treatment wastewaters during day and night periods. Aerosol dispersal and downwind migration were determined. Bacterial and coliphage f2 aerosols were sampled by using Andersen viable type stacked-sieve and high-volume electrostatic precipitator samplers. Bacterial standard plate counts averaged 2.4 x 10(5) colony-forming units per ml in unchlorinated effluents. Bacterial aerosols reached 500 bacteria per m3 at 152 m downwind and 10,500 bacteria per m3 at 46m. Seeded coliphage f2 averaged 4.0 x 10(5) plaque-forming units per ml in the effluent and were detected 563 m downwind. Downwind microbial aerosol levels were somewhat enhanced by nighttime conditions. The median aerodynamic particle size of the microbial aerosols was approximately 5.0 micrometer. Chlorination reduced wastewater bacterial levels 99.97% and reduced aerosol concentrations to near background levels; coliphage f2 was reduced only 95.4% in the chlorinated effluent and was readily measured 137 m downwind. Microbiological source strength an meteorological data were used in conjunction with a dispersion model to generate mathematical predictions of aerosol strength at various sampler locations. The mean calculated survival of aerosolized bacteria (standard plate count) in the range 46 to 76 m downwind was 5.2%, and that of coliphage f2 was 4.3 %.     

Effects of Environmental Factors on the Survival of Airborne T-3 Coliphage

Experiments were conducted to determine the effects of storage temperatures, relative humidity, and additives on the survival of aerosolized Escherichia coli phage T-3. The aerosol stability of the coliphage, calculated as per cent recovery, was not affected by storage at 10 or -70 C for up to 4 months. However, an increase in aerosol decay rate of coliphage stored at 10 C was observed. The effect of humidities ranging from 20 to 90% relative humidity was studied, and it was observed that humidities lower than 70% relative humidity significantly reduce the survival of airborne coliphage. The effect of various compounds on the aerosol decay rate of T-3 coliphage was studied at 50 and 85% relative humidity. Addition of dextrose in 0.1 M concentrations to the disseminating fluid significantly reduced aerosol decay rate at 50% relative humidity without affecting the decay at 85%. Addition of spermine, spermidine-phosphate, thiourea, galacturonic acid, and glucosaminic acid, individually or in combination, had no effect on aerosol decay rates. The use of deuterium oxide as the suspending fluid for dissemination had no effect on aerosol stability of the coliphage.     

Influence of long‐term land application of Class B biosolids on soil bacterial diversity

Aim: To evaluate the effect of long‐term annual land applications of Class B biosolids on soil bacterial diversity at University of Arizona Marana Agricultural Field Center, Tucson, Arizona. Methods and Results: Following the final of 20 consecutive years of application of Class B biosolids in March 2005, followed by cotton growth from April to November 2005 surface soil samples (0–30 cm) were collected from control (unamended) and biosolid‐amended plots. Total bacterial community DNA was extracted, amplified using 16S rRNA primers, cloned, and sequenced. All 16S rRNA sequences were identified by 16S rRNA sequence analysis and comparison to known sequences in GenBank (NCBI Blast N and Ribosomal Database Project II, RDP). Results showed that the number of known genera (identifiable > 96%) increased in the high rate biosolid plots compared to control plots. Biosolids‐amended soils had a broad phylogenetic diversity comprising more than four major phyla: Proteobacteria (32%), Acidobacteria (21%), Actinobacteria (16%), Firmicutes (7%), and Bacteroidetes (6%) which were typical to bacterial diversity found in the unamended arid southwestern soils. Conclusion: Bacterial diversity was either enhanced or was not negatively impacted following 20 years of land application of Class B biosolids. Significance and Impact of the Study: This study illustrates that long‐term land application of biosolids to arid southwestern desert soils has no deleterious effect on soil microbial diversity.     

Occurrence of microbial indicators and Clostridium perfringens in wastewater, water column samples, sediments, drinking water, and Weddell seal feces collected at McMurdo Station, Antarctica

McMurdo Station, Antarctica, has discharged untreated sewage into McMurdo Sound for decades. Previous studies delineated the impacted area, which included the drinking water intake, by using total coliform and Clostridium perfringens concentrations. The estimation of risk to humans in contact with the impacted and potable waters may be greater than presumed, as these microbial indicators may not be the most appropriate for this environment. To address these concerns, concentrations of these and additional indicators (fecal coliforms, Escherichia coli, enterococci, coliphage, and enteroviruses) in the untreated wastewater, water column, and sediments of the impacted area and drinking water treatment facility and distribution system at McMurdo Station were determined. Fecal samples from Weddell seals in this area were also collected and analyzed for indicators. All drinking water samples were negative for indicators except for a single total coliform-positive sample. Total coliforms were present in water column samples at higher concentrations than other indicators. Fecal coliform and enterococcus concentrations were similar to each other and greater than those of other indicators in sediment samples closer to the discharge site. C. perfringens concentrations were higher in sediments at greater distances from the discharge site. Seal fecal samples contained concentrations of fecal coliforms, E. coli, enterococci, and C. perfringens similar to those found in untreated sewage. All samples were negative for enteroviruses. A wastewater treatment facility at McMurdo Station has started operation, and these data provide a baseline data set for monitoring the recovery of the impacted area. The contribution of seal feces to indicator concentrations in this area should be considered.     

Nutrient and Microbial Dynamics in Biosolids Amended Soils Following Rainfall Simulation

Municipal waste treatment plants are mandated by U.S. EPA to treat domestic wastewater prior to releasing it to receiving streams. The dewatering and high temperature drying processes at the plant are considered effective in reducing microbial contaminants in the waste. The resulting solid material (biosolid) is rich in nutrients that may serve as a value-added product for plant growth. In this study, we examined the nutrient value of biosolids, their potential biological and chemical risks that could result from surface application to two Mid-Atlantic soils: Bojac (coarse-loamy, mixed, thermic Typic Hapludult) and Cullen (clayey, mixed, thermic Typic Hapludult). Soils were placed on tilt beds and packed to their respective bulk density. Biosolids were added at a rate of 2.24 Mg/ha equivalent and mixed with the top 5 cm of the soil bed. Simulated rain was applied at a rate of 65 mm h^?1 for 45 minutes. Surface runoff and percolation water were collected and analyzed for elemental content, Escherichia coli (E. coli) and total coliform bacteria. Among the nutrient elements of concern (P, Zn, Mn, and Cu) in biosolids, none were found to be higher than the specified EPA limits. The concentration of P was highest in runoff and percolation water from beds packed with Bojac and biosolids. The combined effects of high clay (35%), Al (1.14%), and Fe (5.11%) in Cullen increased its P-adsorbing capacity. Low levels of E. coli and other coliform bacteria were present in samples from biosolids-treated beds packed with Cullen. Microbial counts in runoff and percolation samples varied with soil type; in some instances they were ten-fold higher in Bojac than in Cullen. The results obtained in this study suggest that surface runoff from land applications of biosolids might contribute to microbial contamination of receiving waters near agricultural fields.     

The assessment of airborne bacterial contamination in three composting plants revealed site-related biological hazard and seasonal variations

AIMS: The purpose of this study was to evaluate the degree of bacterial contamination generated by three Italian composting plants (1, 2 and 3) in two different seasons and to assess the health risk for the employees. METHODS AND RESULTS: Aerosols samples were collected with an agar impact sampler. Several plant sites and external upwind and downwind controls were examined. Total colony-forming counts of mesophilic and thermophilic bacteria, actinomycetes and streptomycetes, Gram-negatives, coliforms and sulfite-reducers were determined. Selective media were used in order to isolate pathogenic bacteria. The levels of total mesophilic and thermophilic micro-organisms ranged between 33 and >40,000 CFU m(-3) in plant 1, 39 and 18,700 CFU m(-3) in plant 2 and 261 and 6278 CFU m(-3) in plant 3. Strains of Escherichia coli, Staphylococcus aureus and Clostridium perfringens were also found. CONCLUSIONS: The plants monitored in this study have proved to be sources of aerosolized bacteria. The activities involving mechanical movement of the composting mass and the indoor activities were of greatest potential risk. In all the studied plants, a statistically significant dependence was found between the bacterial contamination and the season for some or almost all the analysed parameters, but a clear seasonal trend could not be observed. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides broad evidence of bacterial aerosol dispersion and site-related biological hazards that may be useful to the regional government to implement regulations on worker safety in composting plants.     

Bacterial Indicator Occurrence and the Use of an F+ Specific RNA Coliphage Assay to Identify Fecal Sources in Homosassa Springs, Florida

Abstract A microbiological water quality study of Homosassa Springs State Wildlife Park (HSSWP) and surrounding areas was undertaken. Samples were collected in November of 1997 (seven sites) and again in November of 1998 (nine sites). Fecal bacterial concentrations (total and fecal coliforms, Clostridium perfringens, and enterococci) were measured as relative indicators of fecal contamination. F⁺-specific coliphage genotyping was performed to determine the source of fecal contamination at the study sites. Bacterial levels were considerably higher at most sites in the 1997 sampling compared to the 1998 sampling, probably because of the greater rainfall that year. In November of 1997, 2 of the 7 sites were in violation of all indicator standards and guidance levels. In November of 1998, 1 of 9 sites was in violation of all indicator standard and guidance levels. The highest concentrations of all fecal indicators were found at a station downstream of the animal holding pens in HSSWP. The lowest levels of indicators were found at the Homosassa Main Spring vent. Levels of fecal indicators downstream of HSSWP (near the point of confluence with the river) were equivalent to those found in the Southeastern Fork and areas upstream of the park influences. F⁺ specific RNA coliphage analysis indicated that fecal contamination at all sites that tested positive was from animal sources (mammals and birds). These results suggest that animal (indigenous and those in HSSWP) and not human sources influenced microbial water quality in the area of Homosassa River covered by this study. Received: 12 May 1999; Accepted: 11 August 1999; Online Publication: 2 March 2000     

Evaluation of F+ RNA and DNA coliphages as source-specific indicators of fecal contamination in surface waters

Male-specific (F+) coliphages have been investigated as viral indicators of fecal contamination that may provide source-specific information for impacted environmental waters. This study examined the presence and proportions of the different subgroups of F+ coliphages in a variety of fecal wastes and surface waters with well-defined potential waste impacts. Municipal wastewater samples had high proportions of F+ DNA and group II and III F+ RNA coliphages. Bovine wastewaters also contained a high proportion of F+ DNA coliphages, but group I and IV F+ RNA coliphages predominated. Swine wastewaters contained approximately equal proportions of F+ DNA and RNA coliphages, and group I and III F+ RNA coliphages were most common. Waterfowl (gull and goose) feces contained almost exclusively F+ RNA coliphages of groups I and IV. No F+ coliphages were isolated from the feces of the other species examined. F+ coliphage recovery from surface waters was influenced by precipitation events and animal or human land use. There were no significant differences in coliphage density among land use categories. Significant seasonal variation was observed in the proportions of F+ DNA and RNA coliphages. Group I F+ RNA coliphages were the vast majority (90%) of those recovered from surface waters. The percentage of group I F+ RNA coliphages detected was greatest at background sites, and the percentage of group II F+ RNA coliphages was highest at human-impacted sites. Monitoring of F+ coliphage groups can indicate the presence and major sources of microbial inputs to surface waters, but environmental effects on the relative occurrence of different groups need to be considered.     

Leaching of phage from Class B biosolids and potential transport through soil

The objective of this study was to investigate leaching and transport of viruses, specifically those of an indigenous coliphage host specific to Escherichia coli ATTC 15597 (i.e., MS-2), from a biosolid-soil matrix. Serial extractions of 2% and 7% (solids) class B biosolid matrices were performed to determine the number of phage present in the biosolids and to evaluate their general leaching potential. Significant concentrations of coliphage were removed from the biosolids for each sequential extraction, indicating that many phage remained associated with the solid phase. The fact that phage was associated with or attached to solid particles appeared to influence the potential for release and subsequent transport of phage under saturated-flow conditions, which was examined in a series of column experiments. The results indicated that less than 8% of the indigenous coliphage initially present in the biosolids leached out of the biosolid-soil matrix. A fraction of this was subsequently transported through the sandy porous medium with minimal retention. The minimal retention observed for the indigenous phage, once released from the biosolids, was consistent with the results of control experiments conducted to examine MS-2 transport through the porous medium.     

F+ RNA coliphage typing for microbial source tracking in surface waters

AIMS: The utility of coliphages to detect and track faecal pollution was evaluated using South Carolina surface waters that exceeded State faecal coliform standards. METHODS AND RESULTS: Coliphages were isolated from 117 surface water samples by single agar layer (SAL) and enrichment presence/absence (EP/A) methods. Confirmed F+ RNA coliphages were typed for microbial source tracking using a library-independent approach. Concentrations of somatic coliphages using 37 and 44.5 degrees C incubation temperatures were found to be significantly different and the higher temperature may be more specific for faecal contamination. The EP/A technique detected coliphages infecting Escherichia coli Famp in 38 (66%) of the 58 surface water samples negative for F+ coliphages by the SAL method. However, coliphages isolated by EP/A were found to be less representative of coliphage diversity within a sample. Among the 2939 coliphage isolates tested from surface water and known source samples, 813 (28%) were found to be F+ RNA. The majority (94%) of surface water F+ RNA coliphage isolates typed as group I. Group II and/or III viruses were identified from 14 surface water stations, the majority of which were downstream of wastewater discharges. These sites were likely contaminated by human-source faecal pollution. CONCLUSIONS: The results suggest that faecal contamination in surface waters can be detected and source identifications aided by coliphage analyses. SIGNIFICANCE AND IMPACT OF THE STUDY: This study supports the premise that coliphage typing can provide useful, but not absolute, information to distinguish human from animal sources of faecal pollution. Furthermore, the comparison of coliphage isolation methods detailed in this study should provide valuable information to those wishing to incorporate coliphage detection into water quality assessments.     

Occurrence of antibiotic-resistant bacteria and endotoxin associated with the land application of biosolids

The purpose of this study was to determine the prevalence of antibiotic-resistant bacteria and endotoxin in soil after land application of biosolids. Soil was collected over a 15 month period following land application of biosolids, and antibiotic resistance was ascertained using clinically relevant antibiotic concentrations. Ampicillin, cephalothin, ciprofloxacin, and tetracycline resistance were all monitored separately for any changes throughout the 15 month period. Endotoxin soil concentrations were monitored using commercially available endotoxin analysis reagents. Overall, land application of biosolids did not increase the percentage of antibiotic-resistant culturable bacteria above background soil levels. Likewise, land application of biosolids did not significantly increase the concentration of endotoxin in soil. This study determined and established a baseline understanding of the overall effect that land application of biosolids had on the land-applied field with respect to antibiotic-resistant bacterial and endotoxin soil densities.     

Human Exposure Pathways of Heavy Metals in a Lead-Zinc Mining Area,Jiangsu Province,China

Heavy metal pollution is becoming a serious issue in developing countries such as China, and the public is increasingly aware of its adverse health impacts in recent years. We assessed the potential health risks in a lead-zinc mining area and attempted to identify the key exposure pathways. We evaluated the spatial distributions of personal exposure using indigenous exposure factors and field monitoring results of water, soil, food, and indoor and outdoor air samples. The risks posed by 10 metals and the contribution of inhalation, ingestion and dermal contact pathways to these risks were estimated. Human hair samples were also analyzed to indicate the exposure level in the human body. Our results show that heavy metal pollution may pose high potential health risks to local residents, especially in the village closest to the mine (V1), mainly due to Pb, Cd and Hg. Correspondingly, the residents in V1 had higher Pb (8.14 mg/kg) levels in hair than those in the other two villages. Most of the estimated risks came from soil, the intake of self-produced vegetables and indoor air inhalation. This study highlights the importance of site-specific multipathway health risk assessments in studying heavy-metal exposures in China.     

Survival of T3 Coliphage in Varied Extracellular Environments. I. Viability of the Coliphage During Storage and in Aerosols

The objective of this study was to determine the feasibility of using airborne T3 coliphage as a viral tracer in microbial aerosols. Although T3 coliphage was relatively stable when stored either at temperatures ranging from 21 to 37 C or in the frozen state at -20 C, there was a 2-log loss in infectivity when stored for 72 days at 4 C. Either agitation of stored coliphage suspensions held at 31 C or wide fluctuations in storage temperature produced an increased loss of infectivity. In the airborne state, freshly prepared coliphage and stored coliphage behaved similarly, with survival diminishing as the relative humidity (RH) was lowered. The greatest loss occurred during the first five min following aerosolization. The results showed that only under certain conditions of temperature and relative humidity can T3 coliphage be used as a satisfactory aerosol tracer.