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ASAE Journal Article

Technical Note: Effect of Sprinkler Pressure and Spray Plate on Culturable Microorganism Concentrations During Simulated Irrigation of Dairy Wastewater

R. S. Dungan, D. L. Bjorneberg, A. B. Leytem


Published in Transactions of the ASABE Vol. 54(5): 1669-1673 ( 2011 American Society of Agricultural and Biological Engineers ).

Submitted for review in March 2011 as manuscript number SW 9116; approved for publication as a Technical Note by the Soil & Water Division of ASABE in August 2011.

The authors are Robert S. Dungan, Research Microbiologist, David L. Bjorneberg, ASABE Member, Supervisory Agricultural Engineer, and April B. Leytem, Research Soil Scientist, USDA-ARS Northwest Irrigation and Soils Research Laboratory, Kimberly, Idaho. Corresponding author: Robert S. Dungan, USDA-ARS Northwest Irrigation and Soils Research Laboratory, 3793 North 3600 East, Kimberly, ID 83341; phone: 208-423-6553; fax: 208-423-6555; e-mail: robert.dungan@ars.usda.gov.


Abstract. In this study, we conducted simulated spray irrigation events of dairy wastewater to assess the impact of pressure and sprinkler type on post-sprinkler culturable microorganism concentrations. Dairy wastewater was sampled before and after it was pumped through sprinklers typically used on center-pivot irrigation systems. Three different sprinklers types were used at three different operating pressures to give a range of water drop sizes. The microorganisms quantified in this study were total coliforms, Escherichia coli , Clostridium perfringens , heterotrophic bacteria, and coliphages. In most cases, the pre- and post-sprinkler concentrations were determined to be statistically similar, suggesting that culturable viability was not affected when wastewater flowed through these sprinklers. When an impact was found to occur, there was usually an increase in the post-sprinkler microorganism concentration. While this increase can be attributed to the disruption of microbial aggregates during the spraying process, there was no apparent relationship with pressure setting or spray plate. Understanding impacts at the sprinkler level should be considered an integral part of the dispersion modeling process, as it may influence the number of viable microorganisms that become aerosolized during pressurized irrigation events.

Keywords. Culturable, Dairy, Irrigation, Microorganism, Sprinkler, Wastewater.

Concentrated animal feeding operations (CAFOs) generate large quantities of feces and urine that are typically stored at the facility until they are either treated or beneficially used in some manner (Rice et al., 2006). The animal wastes are frequently applied to land as a cost-effective means for disposal, although additional benefits are an increase in the soil nutrient status (Sims et al., 2005). While manure solids are often applied using a box-type spreader, liquid wastes are typically broadcast or directly injected using a tank-type applicator or spread using sprinkler irrigation (Pfost and Fulhage, 2001). Use of these land application techniques enhances the aerosolization of zoonotic pathogens and microbial byproducts in the untreated manures, thus increasing the risk of exposure to farm workers and individuals in the downwind environment (Dungan, 2010).

Although there is a move to directly inject liquid wastes into soil to mitigate ammonia emissions (Morken and Sakshaug, 1998; Hansen et al., 2003; Lovanh et al., 2010), it is also common practice to pump liquid waste from storage tanks and ponds to pressurized irrigation systems (Sharpe and Harper, 1997). Investigations into the transport of aerosolized microorganisms during the spray application of animal wastes have shown a decrease in the airborne concentration with distance from the irrigation source, suggesting a low respiratory hazard for nearby individuals (Boutin et al., 1988; Hutchison et al., 2008). Murayama et al. (2010) used a PCR-based assay to identify culturable airborne microorganisms during the aerial spraying of a swine slurry and found that none of the organisms were described previously as having an inhalation route of infection. Decreases in the airborne microorganism concentration with downwind distance were also observed during spray irrigation events of municipal wastewaters (Katzenelson and Teltch, 1976; Bausum et al., 1982; Camann et al., 1988).

To assess the transport and diffusion of airborne microorganisms during wastewater irrigation events, models based on Pasquill's (1961) particle dispersion model have been utilized (Parker et al., 1977; Teltsch et al., 1980). Since the Pasquill model was based on inert particles, a biological decay term ( ? ) was added to account for the death of aerosolized microorganisms (Lighthart and Frish, 1976). In addition to ? , a dispersion model later used by the U.S. EPA (1982) included a microorganism impact factor ( I ), which was added to represent the initial shock associated with the aerosolization process (e.g., rapid evaporation of water, turbulent forces). To date, however, researchers have not defined the suspected individual components of I . The objective of this study was to assess post-sprinkler impacts on culturable microorganisms by pumping dairy wastewater through center-pivot sprinklers with a rotating or fixed (flat or grooved) spray plate under various pressures. The premise for this work was based on the hypothesis that increases in sprinkler pressure would cause reductions in microorganism survivability. This is the first assessment of I since the U.S. EPA (1982) work, with a specific focus on physical stresses prior to aerosolization of microorganisms.

Materials and Methods

Wastewater Storage Pond

Wastewater from a storage pond at an open-freestall dairy with 10,000 Holstein cows was used for this study. Manure in the freestall barns was flushed daily from the alleys and then sent to a solids separator. Some of the liquid waste was utilized in an anaerobic digester system for biogas production. Undigested liquid waste, as well as digester effluent, was then stored in a series of ponds. The dairy commonly pumps the wastewater through center pivots to irrigate nearby fields.

Spray Chamber and Sprinkler

A spray chamber was used to contain the wastewater application from the sprinkler. The chamber was constructed from a 132 L opaque plastic drum (47 cm dia. × 76 cm high) by cutting a window (28 cm wide × 33 cm high) into the side of the drum. Galvanized steel piping (1.9 cm dia.) was installed through the top of the drum to allow a sprinkler to be mounted inside. The drum was mounted on a stand (55 cm high), and the piping was connected to a 0.56 kW centrifugal pump. After the centrifugal pump, a valve was installed so samples could be collected prior to the sprinkler. Pressure regulators were used to maintain water pressures of 138, 207, or 276 kPa at the sprinkler. A pressure gauge was installed before the pressure regulator to ensure that the water pressure was adequate for the test. Sprinklers (3000 Series, Nelson Irrigation Corp., Walla Walla, Wash.) were used with either a brown rotating plate (Part No. 10681), fixed flat smooth plate (Part No. 9591), or fixed concave grooved plate (Part No. 10721). A Nelson 3TN No. 21 nozzle (4.2 mm orifice dia.) was used, which produces flow rates of 12.7, 15.6, and 18 L min -1 at pressures of 138, 207, and 276 kPa, respectively.

Experimental Design

The spray chamber was placed near the edge of the wastewater pond, so the wastewater was sprayed back into the pond. A gas-powered generator was used to supply electricity to the centrifugal pump. Using a flotation device, the inlet end of the hose was suspended on the surface of the pond to avoid the intake of settled solids into the pump and clogging of the nozzle. Wastewater was pumped through the system for 5 min before collecting samples. A series of 10 pre- and post-sprinkler samples were then collected with approximately 2 min intervals between each collection. The samples were collected in sterile 500 mL plastic bottles and immediately placed into a cooler with ice packs. Enumeration of culturable microorganisms was conducted on the same day, usually within 8 h of sample collection.

Wastewater Analyses

The pH and temperature of the wastewater was determined in situ using a portable meter (model 260A, Thermo Scientific Orion, Beverly, Mass.). Total solids, dissolved solids, and total suspended solids were determined according to Standard Methods 2540b, 2540c, and 2540d, respectively (Eaton et al., 2005).

Enumeration of Microorganisms

The concentrations of total coliform, Escherichia coli , Clostridium perfringens , heterotrophic bacteria, and coliphages in the pre- and post-sprinkler samples were determined via cultivation. Prior to cultivation, 10-fold serial dilutions of the samples were prepared in phosphate-buffered saline. Total coliform and E. coli were assayed by filtering 100 mL aliquots through membrane filters (47 mm, 0.45 µ m pore size) and incubating them at 35°C for 1 d on MI media (U.S. EPA, 2002). Vegetative cells and spores of C. perfringens were assayed using membrane filtration onto mCP media (Neogen, Lansing, Mich.) with mCP selective Supplement I (Sigma-Aldrich, St. Louis, Mo.), as described by Armon and Payment (1988). For the enumeration of clostridia spores, dilutions were heat-shocked at 80°C for 5 min prior to membrane filtration. The mCP plates were anaerobically incubated at 44.5°C for 1 d, with exposure to ammonia hydroxide vapors afterwards to quantify presumptive C. perfringens colonies. Heterotrophic bacteria were cultivated by spreading 0.1 mL aliquots on tryptic soy agar (TSA) media and incubating at 25°C for 5 d. Coliphages were assayed using the pour plate method with overnight incubation at 35°C (Eaton et al., 2005). Positive controls consisted of E. coli (ATCC 13706), C. perfringens (ATCC 13124), and coliphages (ATCC 13706-B1). All plates were manually counted, with bacterial and coliphage counts reported as colony forming units (CFU) and plaque forming units (PFU) per mL, respectively.

Statistical Analysis

Two-sample paired t -tests for the means were performed using SAS (2008). Statements of statistical significance were based on p < 0.05 unless otherwise stated.

Results and Discussion

During the sprinkler studies, the ambient air temperature ranged from 9.2°C to 25°C with a mean of 18.5°C. The relative humidity ranged from 19.3% to 84.1% with a mean of 42.8%. While the ambient weather data are not presented, select properties of the wastewater are presented in table 1. The mean pH and electrical conductivity were 7.7 and 12.3 mS m -1 , respectively. The wastewater temperature ranged from 13.7°C to 21.1°C with a mean of 18.2°C. The mean total solids, suspended solids, and total dissolved solids contents were 14,589, 7,055, and 8,000 mg L -1 , respectively.

Table 1. Properties of the dairy wastewater during the sprinkler study.

Sprinkler

Pressure

(kPa)

pH

EC

(mS m -1 )

Temp.

(°C)

Total

Solids

Suspended

Solids

(mg L -1 )

Total

Dissolved

Solids

Rotating plate

138

7.7

12.4

16.5

15,640

7,906

7,716

207

7.6

11.6

20.1

12,330

4,938

7,478

276

7.7

12.7

21.1

13,810

8,894

9,654

Fixed flat plate

138

7.6

12.1

19.2

13,185

5,282

7,336

207

7.8

12.2

18.2

16,300

9,906

7,376

276

7.8

12.8

19.3

15,810

5,764

8,530

Fixed grooved plate

138

7.8

12.6

13.7

17,485

10,672

6,934

207

7.9

12.3

18.5

14,260

5,428

9,280

276

7.7

11.9

17.4

12,480

4,702

7,692

The pre- and post-sprinkler microorganism concentrations under pressure settings of 138 and 207 kPa with rotating, flat, or grooved spray plates are presented in table 2. Due to the consistency of the non-diluted wastewater, during the 276 kPa runs it was often necessary to remove the nozzle to maintain proper pressure. As a result, these data are presented separately in table 3. In general, the pre- and post-sprinkler microorganism concentrations were similar and within the same order of magnitude. A statistical analysis of the data, however, revealed that the post-sprinkler concentration was statistically greater than the pre-sprinkler concentration in several instances. For example, at a pressure of 207 kPa with a rotating spray plate, the post-sprinkler C. perfringens spore concentration was about 2-fold greater (p < 0.01) than the pre-sprinkler concentration (table 2). Similarly, under the same test conditions, post-sprinkler E. coli and coliphage concentrations were found to be greater (p < 0.01). At a pressure of 138 kPa with use of a grooved spray plate, both the C. perfringens spore and coliphage concentrations were statistically greater (p < 0.05) in the post-sprinkler discharge (table 2). When the sprinkler pressure was increased to 276 kPa, total coliform and coliphage concentrations were found to be greater (p < 0.01). None of the post-sprinkler concentrations were found to be statistically greater at each of the pressures tested when a flat spray plate was used (table 3).

Table 2. Mean culturable microorganism concentrations (CFU or PFU mL -1 ) in pre- and post-sprinkler samples at pressure settings of 138 and 207 kPa. [a]

Microorganism

138 kPa

207 kPa

Pre-Sprinkler

Post-Sprinkler

Pre-Sprinkler

Post-Sprinkler

Rotating plate

E. coli

433 (20)

408 (21)

1016 (28)

1154 (31)**

Total coliforms

3020 (271)

3220 (259)

3650 (212)

3360 (310)

C. perfringens (spore)

1185 (126)

1325 (53)

170 (26)

335 (51)**

C. perfringens (vegetative)

1990 (117)

1805 (106)

645 (75)

580 (80)

Heterotrophic bacteria

11,130,000 (801,395)

12,060,000 (1,293,505)

19,405,000 (1,596,427)

21,420,000 (1,357,985)

Coliphage

260 (62)

290 (64)

524 (26)

798 (69)**

Fixed flat plate

E. coli

2260 (163)

2540 (139)

3930 (715)

3890 (288)

Total coliforms

4250 (143)

4430 (173)

5160 (468)

6130 (318)

C. perfringens (spore)

245 (34)

40 (16)***

975 (96)

1055 (86)

C. perfringens (vegetative)

590 (69)

635 (53)

1680 (86)

1775 (67)

Heterotrophic bacteria

34,230,000 (4,148,709)

30,005,000 (1,721,021)

8,970,000 (695,390)

10,570,000 (822,874)

Coliphage

1350 (133)

1640 (125)

1720 (339)

1550 (147)

Fixed grooved plate

E. coli

148 (14)

141 (8)

1865 (119)

1975 (96)

Total coliforms

2110 (177)

1720 (278)

4500 (226)

4340 (434)

C. perfringens (spore)

1425 (94)

1885 (126)*

681 (52)

562 (42)**

C. perfringens (vegetative)

2595 (136)

3050 (333)

885 (111)

800 (44)

Heterotrophic bacteria

8,470,000 (1,024,700)

6,550,000 (1,148,840)

2,370,000 (168,689)

2,290,000 (287,306)

Coliphage

212 (18)

250 (17)*

1865 (216)

1737 (273)

[a] Values in parentheses are SEM ( n = 10). Asterisks (*, **, and ***) indicate significant differences at the 0.05, 0.01, and 0.001 probability levels, respectively.

Table 3. Mean culturable microorganism concentrations (CFU or PFU mL -1 ) in pre- and post-sprinkler samples at a pressure setting of 276 kPa without a nozzle. [a]

Microorganism

Pre-Sprinkler

Post-Sprinkler

Rotating plate

E. coli

47 (8)

46 (8)

Total coliforms

950 (118)

1040 (91)

C. perfringens (spore)

480 (39)

475 (56)

C. perfringens (vegetative)

725 (37)

798 (70)

Heterotrophic bacteria

3,550,000 (229,129)

3,560,000 (581,416)

Coliphage

1445 (127)

1540 (127)

Fixed flat plate

E. coli

33 (6)

34 (7)

Total coliforms

1720 (181)

2330 (310)

C. perfringens (spore)

482 (37)

366 (29)

C. perfringens (vegetative)

1050 (125)

1130 (131)

Heterotrophic bacteria

4,290,000 (276,265)

4,030,000 (401,400)

Coliphage

1410 (92)

1890 (238)

Fixed grooved plate

E. coli

1608 (69)

1716 (65)

Total coliforms

3510 (181)

4810 (249)***

C. perfringens (spore)

191 (25)

218 (10)

C. perfringens (vegetative)

533 (28)

520 (32)

Heterotrophic bacteria

14,080,000 (831,939)

16,884,167 (1,171,621)

Coliphage

136 (20)

358 (81)**

[a] Values in parentheses are SEM ( n = 10). Asterisks (*, **, and ***) indicate significant difference at the 0.05, 0.01, and 0.001 probability levels, respectively.

In only two instances were the post-sprinkler concentrations found to be statistically lower than the pre-sprinkler concentrations (table 2). At a sprinkler pressure of 138 kPa with a flat spray plate, the mean pre- and post-sprinkler C. perfringens spore concentrations were 245 and 40 CFU mL -1 , respectively. When a grooved spray plate was used at 207 kPa, the respective mean pre- and post-sprinkler C. perfringens spore concentrations were 681 and 562 CFU mL -1 . These results were unexpected, as unlike vegetative cells, clostridia spores are resistant to high pressures and other physical and chemical treatments (Paredes-Sabja et al., 2007). With respect to the rotating spray plate, no statistically significant post-sprinkler decreases occurred (tables 2 and 3).

In this study, we used fecal indicator organisms to investigate the effect of pressure and spray plate on post-sprinkler culturable concentrations. Indicator organisms are generally used in water quality studies and investigations into bioaerosol formation and transport since they are more abundant than target pathogens and are easily identified (Teltsch and Katzenelson, 1978; Brenner et al., 1988; Pianetti et al., 2004). While the use of indicator organisms provides a good approximation of sprinkler effects on microbial populations, effects on pathogens cannot be directly assessed from this data set, as they may respond differently to environmental stresses (Moriñigo et al., 1990; Harwood et al., 2005). Although there was no clear trend between the post-sprinkler microorganism concentrations and the sprinkler configuration, the results suggest that spray irrigation influences viable microorganism concentrations in wastewater in some cases.

In cases where the post-sprinkler concentrations were found to be lower ( Clostridium spores only), it is likely that the integrity of the cellular membranes was disrupted due to the physical force of the microorganisms hitting the spray plate or by the sudden decrease in pressure as they were discharged from the nozzle. Shear stress in particular has been shown to be responsible for the inactivation of viral particles (Segura et al., 2006; Peixoto et al., 2007), although larger viral particles have been shown to be relatively unaffected (Michalsky et al., 2008). One must also consider the fact that these conditions could have transformed the microorganisms into a viable non-culturable condition (Bogosian and Bourneuf, 2001). When culture-based techniques are used to quantify microorganisms, those in a viable but non-culturable condition can cause underestimation of the actual risk of exposure and infection (Jones et al., 1991; Colwell et al., 1996). Use of alternative techniques (e.g., molecular-based techniques) can be employed to enhance the detection of non-culturable microorganisms, although some of these techniques cannot differentiate between viable and non-viable organisms (Dungan and Leytem, 2009).

Increases in the post-sprinkler concentration of vegetative cells, spores, and coliphages may be attributable to the disruption of microbial aggregates (e.g., via shear stress) during release of wastewater from the nozzle and impaction on the spray plate. While disruption of aggregates does not increase the total number of microorganisms per unit volume of wastewater, it increases the number of individual particles. This is an important consideration when using a dispersion model to predict downwind concentrations, as the actual number of microorganisms available for aerosolization could be substantially greater than the values determined in the wastewater prior to spray irrigation. During wastewater spray irrigation studies conducted by the U.S. EPA (1982), I was developed to account for the loss of microorganism viability during the spray process. In some cases, I was greater than 1, suggesting that wastewater aggregates were being broken apart during the aerosolization process. Since I was used as a catch-all for all undefined processes that influence the viable microorganism source strength (i.e., organisms s -1 ), no efforts were made to define separate values for individual component of I . Our results indicate that the physical stresses associated with the spraying process can also influence microorganism viability prior to aerosolization, which should be accounted for in dispersion models.

Conclusions

The results from this study, while not producing any clear trends, indicate that spraying dairy wastewaters can result in both increases and decreases in post-sprinkler microorganism concentrations. However, the data do not support our hypothesis that increases in sprinkler pressure result in decreases in microorganism survivability. In most cases where a statistical difference was noted between pre- and post-sprinkler concentrations, an increase most often occurred. The concentration increase can be attributed to the disruption of microbial aggregates during the spraying process, whereas concentration decreases were likely a result of microorganism death. It should be noted that both disruption and inactivation processes may be a result of the same shear stresses and may occur simultaneously.

Acknowledgements

The authors would like to thank Sheryl VerWey, Myles Miller, and Susie Hansen for performing the work in this study.

REFERENCES

Armon, R., and P. Payment. 1988. A modification of m-CP medium for enumeration Clostridium perfringens from water samples. Canadian J. Microbiol. 34(1): 78-79.

Bausum, H. T., S. A. Schaub, K. F. Kenyon, and M. J. Small. 1982. Comparison of coliphage and bacterial aerosols at a wastewater spray irrigation site. Appl. Environ. Microbiol. 43(1): 28-38.

Bogosian, G., and E. V. Bourneuf. 2001. A matter of bacterial life and death. EMBO Reports 2(9): 770-774.

Boutin, P., M. Torre, R. Serceau, and P.-J. Rideau. 1988. Atmospheric bacterial contamination from landspreading of animal wastes: Evaluation of the respiratory risk for people nearby. J. Agric. Res. 39(3): 149-160.

Brenner, K. P., P. V. Scarpino, and C. S. Clark. 1988. Animal viruses, coliphages, and bacteria in aerosols and wastewater at a spray irrigation site. Appl. Environ. Microbiol. 54(2): 409-415.

Camann, D. E., B. E. Moore, H. J. Harding, and C. A. Sorber. 1988. Microorganism levels in air near spray irrigation of municipal wastewater: The Lubbock infection surveillance study. J. Water Pollut. Control Fed. 60(11): 1960-1970.

Colwell, R. R., P. Brayton, D. Herrington, B. Tall, A. Huq, and M. M. Levine. 1996. Viable but non-culturable Vibrio cholerae O1 revert to a cultivable state in the human intestine. World J. Microbiol. Biotech . 12(1): 28-31.

Dungan, R. S. 2010. Board-invited review: Fate and transport of bioaerosols associated with livestock operations and manure. J. Animal Sci. 88(11): 3693-3706.

Dungan, R. S., and A. B. Leytem. 2009. A concise review of methodologies used to collect and characterize bioaerosols and their application at concentrated animal feeding operations. World J. Microbiol. Biotech. 25(9): 1505-1518.

Eaton, A. D., L. S. Clesceri, E. W. Rice, and A. E. Greenberg. 2005. Standard Methods for the Examination of Water and Wastewater. 21st ed. Washington, D.C.: American Public Heath Association.

Hansen, M. N. S. G. Sommer, and N. P. Madsen. 2003. Reduction of ammonia emission by shallow slurry injection: Injection efficiency and additional energy demand. J. Environ. Qual. 32(3): 1099-1104.

Harwood, V. J., A. D. Levine, T. M. Scott, V. Chivukula, J. Lukasik, S. R. Farrah, and J. B. Rose. 2005. Validity of the indicator organism paradigm for pathogen reduction in reclaimed water and public health protection. Appl. Environ. Microbiol. 70(6): 3163-3170.

Hutchison, M. L., S. M. Avery, and J. M. Monaghan. 2008. The airborne distribution of zoonotic agents from livestock waste spreading and microbiological risk to fresh produce from contaminated irrigation sources. J. Appl. Microbiol. 105(3): 848-857.

Jones, D. M., E. M. Sutcliffe, and A. Curry. 1991. Recovery of viable but non-culturable Campylobacter jejuni . J. Gen. Microbiol. 137(10): 2477-2482.

Katzenelson, E., and B. Teltch. 1976. Dispersion of enteric bacteria by spray irrigation. J. Water Pollut. Con. Fed. 48(4): 710-716.

Lighthart, B., and A. S. Frisch. 1976. Estimation of viable airborne microbes downwind from a point source. Appl. Environ. Microbiol. 31(5): 700-704.

Lovanh, N., J. Warren, and K. Sistani. 2010. Determination of ammonia and greenhouse gas emissions from land application of swine slurry: A comparison of three application methods. Biores. Tech. 101(6): 1662-1667.

Michalsky, R., P. H. Pfromm, P. Czermak, C. M. Soresen, and A. L. Passarelli. 2008. Effects of temperature and shear force on infectivity of the baculovirus Autographa californica M nucleopolyhedrovirus. J. Virol. Meth . 153(2): 90-96.

Moriñigo, M. A., R. Córnax, M. A. Muñoz, and P. Romero. 1990. Relationships between Salmonella spp. and indicator microorganisms in polluted natural waters. Water Res . 24(1): 117-120.

Morken, J., and S. Sakshaug. 1998. Direct ground injection of livestock waste slurry to avoid ammonia emission. Nutrient Cycl. Agroecosyst. 51(1): 59-63.

Murayama, M., Y. Kakinuma, Y. Maeda, J. R. Rao, M. Matsuda, J. Xu, P. J. Moore, B. C. Millar, P. J. Rooney, C. E. Goldsmith, A. Loughrey, M. Ann, S. McMahon, D. A. McDowell, and J. E. Moore. 2010. Molecular identification of airborne bacteria associated with aerial spraying of bovine slurry waste employing 16S rRNA gene PCR and gene sequencing techniques. Ecotoxicol. Environ. Safety 73(3): 443-447.

Paredes-Sabja, D., M. Gonzalez, M. R. Sarker, and J. A. Torres. 2007. Combined effects of hydrostatic pressure, temperature, and pH on the inactivation of spore of Clostridium perfringens Type A and Clostridium sporogenes in buffer solutions. J. Food Sci. 72(6): M202-M206.

Parker, D. T., J. C. Spendlove, J. A. Bondurant, and J. H. Smith. 1977. Microbial aerosols from food-processing waste spray fields. J. Water Pollut. Control Fed. 49(12): 2359-2365.

Pasquill, F. 1961. The estimation of the dispersion of windborne material. Meteorol. Mag . 90(1063): 33-49.

Peixoto, C., M. F. Q. Sousa, A. C. Silva, M. J. T. Carrondo, and P. M. Alves. 2007. Downstream processing of triple-layered rotavirus-like particles. J. Biotech . 127(3): 452-461.

Pianetti, A., L. Sabatini, F. Bruscolini, F. Chiaverini, and G. Cecchetti. 2004. Faecal contamination indicators Salmonella, Vibrio, and Aeromonas in water used for the irrigation of agricultural products. Epidemiol. Infect. 132(2): 213-238.

Pfost, D. L., and C. D. Fulhage. 2001. Land application equipment for livestock and poultry manure management. EQ383. Columbia, Mo.: University of Missouri Extension.

Rice, J. M., D. F. Caldwell, and F. J. Humenik. 2006. Animal Agriculture and the Environment: National Center for Manure and Animal Waste Management White Papers . St. Joseph, Mich.: ASABE.

SAS. 2008. SAS/STAT 9.2 User's Guide. Cary, N.C.: SAS Institute, Inc.

Segura, M. M., A. Kamen, and A. Garnier. 2006. Downstream processing of oncoretroviral and lentiviral gene therapy vectors. Biotech. Adv . 24(3): 321-337.

Sharpe, R. R., and L. A. Harper. 1997. Ammonia and nitrous oxide emissions from sprinkler irrigation applications of swine effluent. J. Environ. Qual. 26(6): 1703-1706.

Sims, J. T., L. Bergström, B. T. Bowman, and O. Oenema. 2005. Nutrient management for intensive animal agriculture: Policies and practices for sustainability. Soil Use Mgmt. 21(supp. 1): 141-151.

Teltsch, B., and E. Katzenelson. 1978. Airborne enteric bacteria and viruses from spray irrigation with wastewater. Appl. Environ. Microbiol. 35(2): 290-296.

Teltsch, B., H. I. Shuval, and J. Tadmor. 1980. Die-away kinetics of aerosolized bacteria from sprinkler application of wastewater. Appl. Environ. Microbiol. 39(6): 1191-1197.

U.S. EPA. 1982. Estimating microorganism densities in aerosols from spray irrigation of wastewater. EPA-600/9-82-003. Cincinnati, Ohio: U.S. EPA.

U.S. EPA. 2002. Method 1604: Total coliforms and Escherichia coli in water by membrane filtration using a simultaneous detection technique (MI medium). EPA 821-R-02-024. Washington, D.C.: U.S. EPA, Office of Water.