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Risk of infection from Legionella associated with spray irrigation of reclaimed water
Water Research 139 (2018) 101e107
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Risk of infection from Legionella associated with spray irrigation of reclaimed water Ian L. Pepper, Charles P. Gerba* Department of Soil, Water and Environmental Science, University of Arizona, 2959 W. Calle Agua Nueva, Tucson, AZ, 85745, USA
a r t i c l e i n f o
a b s t r a c t
Article history: Received 13 April 2017 Received in revised form 16 March 2018 Accepted 1 April 2018 Available online 2 April 2018
Legionella pneumophila has been detected in reclaimed water used for spray irrigation of turfgrass in public parks and golf courses. This study determined the risks of infection from exposure to various levels of Legionella in reclaimed waters considering: the method of spray application; and the duration and frequency of exposure. Evaluation of these factors resulted in a risk of infection greater than 1:10,000 for several scenarios when the number of Legionella in the reclaimed water exceeded 1000 colonyforming units (CFU) per ml. Most current guidelines for control of Legionella in distribution systems recommend that increased monitoring or remedial action be taken when Legionella levels exceed 1000 to 10,000 CFU/ml. Based upon our risk assessment, these guidelines seem appropriate for reclaimed water systems where spray irrigation is practiced. © 2018 Elsevier Ltd. All rights reserved.
Keywords: Legionella Reclaimed water Risk assessment
1. Introduction Legionella is a water based opportunistic pathogen found naturally in freshwater environments including rivers and lakes. In addition, it can be found in municipal water supply distribution systems and reclaimed waters (Leoni et al., 2005; Ajibode et al., 2013). Legionellosis, commonly known as Legionnaires'’ disease, frequently results from infection with the bacterium Legionella pneumophila. The disease includes a serious sometimes-lethal form of pneumonia, and the less serious Pontiac fever that results in selflimiting flu-like illness. Despite the fact that many cases are likely unreported or undiagnosed, recent estimates are that Legionnaires disease accounts for between 3% and 6% of community-acquired pneumonia that occur in the United Kingdom and United States each year (Moore et al., 2015). The disease typically occurs following inhalation of droplets or aerosols containing Legionella bacteria that are deposited in the lungs. Droplets range in size from five to 100 mm, which normally only travel a few feet in the air (Osterholm et al., 2015). In contrast, true aerosols are <5 mm and can travel greater distances through the air. Thus aerosols released from water sources such as spas or sprayed mists used for cooling are potential routes of exposure to Legionella. Water based pathogens including Legionella are
* Corresponding author. E-mail address: [email protected] (C.P. Gerba). https://doi.org/10.1016/j.watres.2018.04.001 0043-1354/© 2018 Elsevier Ltd. All rights reserved.
frequently found in reclaimed waters. In a recent study Ajibode et al. (2013), detected Legionella spp. in 40% (of 432 samples) of all samples collected from a reclaimed water distribution system from two utilities in the Southwest USA over a one-year period. In some samples, Legionella concentrations reached 228,000/100 ml. In arid regions, reclaimed wastewaters are commonly used for landscape irrigation of parks, golf courses, playgrounds and home lawns (Metcalf and Eddy, Inc. 2003). Spray or sprinkler irrigation being the method of application of choice for grass lawns. This creates an opportunity for the aerosolization of Legionella and subsequent exposure by inhalation. To assess this potential route of transmission, we applied quantitative microbial risk assessment (Haas et al., 2014). 2. Materials and methods 2.1. Quantitative microbial risk assessment The risk assessment paradigm involves four basic steps: hazard identification; exposure assessment; dose response; and risk characterization. However, there are uncertainties associated with each of these steps when assessing the public health risk from Legionella (Whiley et al., 2014). This is particularly true for assessing the exposure to Legionella via reclaimed water, since exposure via spray irrigation will not only depend on the concentration of Legionella in the reclaimed water, but also the size of the droplets or aerosols produced during irrigation, the distance from the spray
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source, and the duration of exposure.
D a R¼1 1þ
2.1.1. Dose response To date, there have been no studies conducted with humans to develop a dose response model for Legionella, however, there have been studies done on animals that provide surrogate dose response data. Most of the animal studies conducted to evaluate the effects of inhalation exposure of Legionella have used the guinea pig (Cavia porcellus) as the common testing animal model, to predict the effects on humans. Based on comparative in vitro macrophage studies and cell-mediated immunology on multiple animal species, guinea pigs were found to be the most suitable animal model for human risk assessment of Legionella pneumophila (Kliment, 1973). Deposition of aerosols 5 mm in the alveolar macrophages spaces of both guinea pigs and humans resulted in the same response to Legionella pneumophila. Thus the guinea pig, which shares with humans a susceptibility to lung infection with L. pneumophila, is an excellent animal model for the study of Legionnaires' disease (Kliment, 1973; Berendt et al., 1980; Davis et al., 1982; Breiman and Horwitz, 1987). Thus, data from these studies have been used to assess the infectivity of various doses of Legionella (Armstrong and Haas, 2007, 2008). For exposure in our study, we considered a number of factors, including the potential concentrations of Legionella within reclaimed water, which we obtained from a previous study conducted in our laboratory (Ajibode et al., 2013). When guinea pigs are exposed to aerosols of L. pneumophila they develop a pneumonic illness characterized by fever, weight loss and labored respirations that sometimes results in death. This syndrome resembles Legionnaires disease in humans (Berendt et al., 1980; Baskerville et al., 1981; Breiman and Horwitz, 1987). Berendt et al. (1980) carried out experiments to determine the infectivity and lethality of exposure to L. pneumophila, using guinea pigs. Muller et al. (1983) repeated the dose response experiments except that they anesthetized the guinea pigs after exposure to L. pneumophila, and tested their lungs and spines for infection. Breiman and Horwitz (1987) exposed guinea pigs to aerosols generated by a pump that projected a suspension of L. pneumophila into the air for 30 min. By examining the lungs of the animals after sacrifice, the number of L. pneumophila inhaled and retained by the animals was determined, allowing for the effects of specific numbers of inhaled Legionella on the animals to be determined. Based on an analysis of these studies, we used the Muller et al. (1983) experimental dataset in this current study to develop the dose response model, since their experiment predicted infections when the guinea pigs were sacrificed before mortality, and hence these data better represented the infection dose rather than mortality. This is the same dataset that was used by Armstrong and Haas (2007). Maximum Likelihood Estimates (MLE) were used to fit the measured data to exponential and beta Poisson statistical models. The exponential model is expressed as equation (1):
R ¼ 1 EXPðk DÞ
(2)
b
For both models, the Solver routine in Microsoft Excel program (Microsoft Inc. Redmond, WA) was used to optimize the models by varying the k value in equation (1); and a and b in equation (2). Here the objective was to maximize likelihood given the observed and the expected probability of infection. A likelihood ratio test was used to assess model fit. The null hypothesis test states that there is no statistically significant difference between the observed and the expected probability values for the respective dose. The research hypothesis assumes that there is a significant statistical difference in at least one observed and expected probability value for the respective dose. The test statistic used is the c2. The rejection region of the null hypothesis is defined based on rejection area of 0.05, and a degree of freedom ¼ dose group minus1 and the exponential and the dose group minus 2. The test statistics for both models were equal as shown in Fig. 1, which means that equation (1) with k ¼ 0.058836 or equation (2) with a ¼ 1.13 107 and b ¼ 1.98 108 can be used. Thus both the exponential and Beta-Poison does response models predict the same probality of infection for the same dose. Fig. 2 shows the dose response model for inhaled L. pneumophila (CFU). Data represent both the exponential and the Beta-Poison dose response models as shown in equations (3) and (4). Both equations give the same probability of infection for similar doses. Armstrong and Haas (2007) concluded that these two models are appropriate for low doses exposures for mechanistic reason for L. pneumophila. Our assessment confirmed their results and we used the exponential model in our study.
R ¼ 1 EXPð0:058836 DÞ R¼1 1þ
D 1:98 108
1:13107 (4)
2.2. Exposure assessment 2.2.1. Droplet size of sprinkler irrigation systems During sprinkler (spray) irrigation, irrigation water is segregated into spray particles or droplets of variable size. The size of water droplets is critical for determining the distance that the droplets
(1)
where: R: is the probability of risk of infection, Response, (decimal); k: is the infectivity per viable L. pneumophila concentration inhaled and retained CFU; D: is the Dose of viable L. pneumophila that is inhaled and retained (CFU). The Beta-Poisson model can be estimated by equation (2), in which a and b are constants and R is again the probability of risk of infection.
(3)
Fig. 1. Fitted exponential and beta Poisson models".
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Fig. 2. Dose Response Model for inhaled L. pneumophila (CFU).
will be transported through the air, and as predicted by Stoke's Law, the smaller the droplet size, the further the droplets will be transported. In addition, as the size of spray droplets decrease, their numbers increase, and the potential for drift also increases. Overall, the characteristics of sprinkler nozzle such as: type, fan angle, orifice size, and sprinkler operational pressure all influence droplet size. Kinkaid et al. (1996) measured droplet size for different sprinkler nozzles with different configurations and different operational pressures using a laser-optical method. They derived an exponential function with parameters that are measurable to define droplet size distribution. The function is shown as equation (5) below:
2.2.2. Drift distance due to wind Wolf et al. (2013) sprayed water at 206.8 Kilo Pascal (30 pounds per square inch) pressure, and measured water droplet drift distances as a function of droplet size with relative humidity of 75%; temperature of 23.9 C (75 F); and wind speed of 2.235 m per second (5 mile per hour). Humidity and temperature will affect droplet size over distance depending on the distant traveled. Ordinary linear regression was performed on the data in Table 1 and yielded a goodness of fit of 0.9994 for a fourth order polynomial function shown as equation (6), in which Drift is the drift distance in feet and S is the droplet size in mm.
Drift ¼
Pv ¼ 100%
d n 1 e EXP e0:693 d50
2:0 109 S4 4:0 107 S3 þ 2:7 103 S2 ð0:7375 SÞ þ 75:783
(6)
(5)
where: d ¼ droplet diameter (mm) Pv ¼ percent (%) of total discharge in drops smaller than d d50 ¼ volume mean drop diameter (mm) n ¼ dimensionless exponent Using equation (5) to determine percent of total discharge by 75 different sprinkler types and configurations in drops less than d ¼ 10 mm, and using the parameters provided by Kinkaid et al. (1996), yields a list of 75 values that can be used to determine the percent of total discharge by the sprinkler system of drops smaller than 10 mm. The Bootstrapping routine in the R-Language (R Core Team, 2013) with 100,000 iterations was used to determine the statistical distribution of the aerosol particles generated (Fig. 3). The average distribution of total discharge by the sprinkler system with drops smaller than 10 mm was 0.01572%, and the 95% Confidence Interval (CI) was [0.0116%, 0.0220%].
The ordinary linear regression equation was used to extrapolate the data to droplet sizes below 50 mm as shown in Fig. 4. Fig. 4 shows that droplet size of 10 mm or less has the potential to drift 70e75 feet away from spraying sprinklers.
2.2.3. Inhalation volume of recycled wastewater droplets The model presented in equation (7) was developed to determine inhalation water droplets with size smaller than 10 mm generated from a sprinkler irrigation system operating at 30 psi (206.8 Kilo Pascal) pressure, air relative humidity of 75%, air temperature of 75 F (23.9 C), and wind speed of 5 mile per hour (2.235 m per second)
Vt ¼
Exp X t¼1
where:
Hrate Hvol Cdroplets < 10 dt
(7)
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equation (8).
Cdroplet < 10 ¼
Q Pv < 10 t
p r2 H ð100%Þ
(8)
where: Q: is discharge rate from the sprinkler (ml/minute) Pv < 10: % of droplets generated with size less than 10 mm (%) r: is drift distance for a sprinkler (mm) H: is maximum height to which sprinkler is generating droplets (mm); Assumed to be 2000 mm (2.0 m). 2.3. Risk of infection 2.3.1. One-time event In order to calculate the risk of infection, the exposure was calculated taking into account: the concentration of Legionella in the reclaimed water; the distance from the spray source; the droplet size and the duration of exposure. Here we assume the distance from the source to be 72.5 feet (22 m) (assumed distance of a front house lawn) from an operational sprinkler that is spraying 30 gallons (113 L) per minute of recycled wastewater at operational pressure of 30 psi (206.8 Kilo Pascal). From Fig. 3 and bootstrapping results presented earlier, it was determined that the average of the statistical distribution was 0.01572% of droplets generated with size less than 10 mm and the 95% Confidence Interval (CI) was [0.0116%, 0.0220%]. Conservatively, we assumed the 95% upper limit of the confidence interval of Pv<10 to be 0.0220%. According to Kliment (1973) an average adult who weighs about 80 kg breathes 7.5 breaths per minute with an air breathing volume of 800 cm3/min. 3. Results Fig. 3. Statistical distribution of droplet size. Percent of total discharge with droplets size less than 10 mm obtained for 75 irrigation sprinkler types, configurations and pressures (t* represents % of total discharge with droplets' size smaller than 10 mm).
Table 1 Droplet size and drift distance. Droplet Size Descriptiona
Dropletsa size (micron)
Drift Distance Feet
meter
Extremely Coarse Very Coarse Coarse Medium Fine Very Fine Ultra Fine
600 500 400 300 200 100 50
0.20 0.30 0.50 1.30 5.00 25.00 45.00
0.06 0.09 0.15 0.40 1.52 7.62 13.72
a
American Society of Agricultural Engineers.
Vt: total volume of droplets with size < 10 mm, inhaled during exposure time (ml) Hrate: inhalation rate for humans (breaths/minute) Hvol: inhalation volume (ml/breath) Cdroplets<10: Concentration of droplets with size less than 10 mm in the air at any time (ml droplets/ml air) dt: incremental time in minutes: 1, 2, 3, … Exp: total exposure time (minutes) Concentration of droplets with size less than 10 mm in the air at any time (ml droplets/ml air), Cdroplets<10 can be calculated from
Fig. 4 shows the risk of infection due to inhalation of recycled wastewater containing L. pneumophila for different exposure times in minutes based on L. pneumophila concentrations obtained from a previous study (Ajibode et al., 2013) for a onetime event. Table 2 shows the risk of infection for different concentrations of L. pneumophila in recycled sprayed irrigation water with different exposure times for a one-time exposure event. 3.1. One-time exposure event per week over summer months Table 3 shows the risk of infection of legionnaire's disease as a function of L. pneumophila concentration in recycled sprayed irrigation water, with different exposure times for a one-time exposure event per week for 12 weeks (summer months). 3.2. One-time exposure event per week for a year Table 4 shows the risk of infection of Legionnaire's disease as a function of L. pneumophila concentration in reclaimed water with different exposure times for a one-time exposure event per week for an entire year. Table 5 shows the risk of infection of legionnaire's disease for the average L. pneumophila concentration found in recycled water with different exposure times and exposure events. 4. Discussion The recent detection of Legionella in reclaimed water distribution systems (Whiley et al., 2015; Ajibode et al., 2013) indicates that
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Fig. 4. Risk of infection of legionnaire's disease for different exposure times.
Table 2 Risk of infection of legionnaire's disease as a function of L. pneumophila concentration in recycled sprayed irrigation water and with different exposure times for a one-time event. L. pneumophilia in Recycled Wastewater (CFU/100 ml)
1 10 100 500 1000 5000 10,000 50,000 100,000 500,000 1,000,000
Exposure Time (Minutes) 1
5
10
20
30
60
4.9E-10 4.9E-09 4.9E-08 2.4E-07 4.9E-07 2.4E-06 4.9E-06 2.4E-05 4.9E-05 2.4E-04 4.9E-04
2.4E-09 2.4E-08 2.4E-07 1.2E-06 2.4E-06 1.2E-05 2.4E-05 1.2E-04 2.4E-04 1.2E-03 2.4E-03
4.9E-09 4.9E-08 4.9E-07 2.4E-06 4.9E-06 2.4E-05 4.9E-05 2.4E-04 4.9E-04 2.4E-03 4.9E-03
9.8E-09 9.8E-08 9.8E-07 4.9E-06 9.8E-06 4.9E-05 9.8E-05 4.9E-04 9.8E-04 4.9E-03 9.8E-03
1.5E-08 1.5E-07 1.5E-06 7.3E-06 1.5E-05 7.3E-05 1.5E04 7.3E-04 1.5E-03 7.3E-03 1.5E-02
2.9E-08 2.9E-07 2.9E-06 1.5E-05 2.9E-05 1.5E-04 2.9E-04 1.5E-03 2.9E-03 1.5E-02 2.9E-02
Table 3 Risk of infection of Legionnaire's disease as a function of L. pneumophila concentration in recycled sprayed irrigation water, with different exposure times for one exposure event per week for 12 weeks. L. pneumophila in Recycled Wastewater (CFU/100 ml)
1 10 100 500 1000 5000 10,000 50,000 100,000 500,000 1,000,000
Exposure Time (Minutes) Once a Week for 12 Weeks - Summer Time 1
5
10
20
30
60
0.0Eþ00 2.5E-09 3.3E-08 1.7E-07 3.3E-07 1.7E-06 3.4E-06 1.7E-05 3.4E-05 1.7E-04 3.4E-04
2.5E-09 1.8E-08 1.7E-07 8.4E-07 1.7E-06 8.4E-06 1.7E-05 8.4E-05 1.7E-04 8.4E-04 1.7E-03
2.5E-09 3.3E-08 3.3E-07 1.7E-06 3.4E-06 1.7E-05 3.4E-05 1.7E-04 3.4E-04 1.7E-03 3.3E-03
7.6E-09 6.8E-08 6.7E-07 3.4E-06 6.7E-06 3.4E-05 6.7E-05 3.4E-04 6.7E-04 3.3E-03 6.7E-03
1.0E-08 1.0E-07 1.0E-06 5.0E-06 1.0E-05 5.0E-05 1.0E-04 5.0E-04 1.0E-03 5.0E-03 1.0E-02
2.0E-08 2.0E-07 2.0E-06 1.0E-05 2.0E-05 1.0E-04 2.0E-04 1.0E-03 2.0E-03 1.0E-02 2.0E-02
a potential risk exists if the organisms are aerosolized during spray irrigation. Spray irrigation of reclaimed for water irrigation of house lawns, parks, playgrounds and other public areas is practiced in the United States resulting in exposure of susceptible individuals. No outbreaks have been reported associated with reclaimed water, but
outbreaks by unknown sources occur and most cases are not diagnosed or reported (Beer et al., 2015). We determined that a one-time event resulted in a risk of infection of 1:10,000 from a 60-min exposure to spray irrigated reclaimed water with a concentration of 5000 CFU/100 ml at a
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Table 4 Risk of infection of legionnaire's disease as a function of L. pneumophila concentration in reclaimed water with different exposure times for one exposure event per week for a full year. L. pneumophila in Recycled Wastewater (CFU/100 ml)
Exposure Time (Minutes) Once a Week for 50 Weeks - One Year
1 10 100 500 1000 5000 10,000 50,000 100,000 500,000 1,000,000
1
5
10
20
30
60
2.5E-09 1.5E-08 1.4E-07 7.0E-07 1.4E-06 7.0E-06 1.4E-05 7.0E-05 1.4E-04 7.0E-04 1.4E-03
7.6E-09 7.0E-08 7.0E-07 3.5E-06 7.0E-06 3.5E-05 7.0E-05 3.5E-04 7.0E-04 3.5E-03 7.0E-03
1.5E-08 1.4E-07 1.4E-06 7.0E-06 1.4E-05 7.0E-05 1.4E-04 7.0E-04 1.4E-03 7.0E-03 1.4E-02
2.8E-08 2.8E-07 2.8E-06 1.4E-05 2.8E-05 1.4E-04 2.8E-04 1.4E-03 2.8E-03 1.4E-02 2.8E-02
4.3E-08 4.2E-07 4.2E-06 2.1E-05 4.2E-05 2.1E-04 4.2E-04 2.1E-03 4.2E-03 2.1E-02 4.1E-02
8.3E-08 8.4E-07 8.4E-06 4.2E-05 8.4E-05 4.2E-04 8.4E-04 4.2E-03 8.3E-03 4.1E-02 8.0E-02
Table 5 Probability of Infection based on the average L. pneumophila measured in the distribution system (4971 CFU/100 ml) (Ajibode et al., 2013). Number of Events
One Event Once a Week for 12 Weeks: Summer Months Once a Week for 50 Weeks: One Year
Exposure Time (Minutes) 1
5
10
20
30
60
1.4E-07 1.7E-06 6.9E-06
6.9E-07 8.3E-06 3.5E-05
1.4E-06 1.7E-05 6.9E-05
2.8E-06 3.3E-05 1.4E-04
4.2E-06 5.0E-05 2.1E-04
8.3E-06 1.0E-04 4.2E-04
distance of 72.5 feet from the spray source. Using the data from Ajibode et al. (2013), we found this value was exceeded in 12% (432 samples) of all the reclaimed water samples analyzed. In contrast, Bouwknegt et al. (2013) conducted a quantitative risk assessment for L. pneumophila following exposure via whirlpool use. They conducted an in-depth calculation of exposure via air bubbles taking into account the number of Legionella inhaled via water droplets. Using a dose-response model for guinea pigs to represent humans, infection risks for active whirlpool use with 100 CFU/L of water for 15 min were 0.29 (x 0.11e0.48) for susceptible males, and 0.22 (x 0.06e0.42) for susceptible females. In addition, risk was shown to be dependent on both the concentration of Legionella and duration of exposure. Specifically exposure to 1000 CFU/L almost always resulted in infection. Likewise a 2 h exposure to 100 CFU/L also resulted in a very high risk of infection (0.86e0.97). A factor that would underestimate the risk includes the use of analytical methods for the detection of Legionella that are not 100% efficient. Factors that under-estimate or over-estimate risk would include: 1) data for dose response in animals that is not reflective of dose response in humans; 2) variable concentrations of viable Legionella in the water during exposure or through the course of a year; 3) wind speed, 4) movement of individuals in and out of the aerosol plume. Detection of Legionella at low levels in distribution systems and cooling towers is not uncommon and infections are increasing (Parr et al., 2015; Sikora et al., 2015; Jjemba et al., 2015). Most guidelines recommend an action level for control of Legionella when levels of Legionella exceed 1000 to 10,000 CFU/per liter (Parr et al., 2015). Recommended actions include: increased levels of disinfection; pro-active control of biofilms; reduced nutrient levels; increased water temperatures in heated systems; flushing the distribution systems; and increased monitoring. In the United States, the U. S. Environmental Protection Agency has set guidance for drinking water treatment to reduce the risk of infection for one year to 1:10,000 per year (USEPA, 2002). This requires a risk of daily infection of 106 to 107 (Signor and Ashbolt, 2009). The degree of risk is highly dependent upon the duration of exposure, the size of droplets generated and the numbers of
Legionella in the reclaimed water. Recommended USEPA guidelines for risks for drinking water are approximately 106 for a one-time exposure. This level would be reached when Legionella concentrations in water in water are >1000 CFU/ml. In the current study, at least one reclaimed water sample contained Legionella concentrations greater than 200,000 CFU/100 ml. In addition, 12% of all samples contained Legionella at concentrations that posed significant risk. These data suggest that there is a potential for infection from sprayed irrigation water and based on this, routine monitoring of reclaimed systems would be prudent. This concurs with another recent study with a similar recommendation (Parr et al., 2015). 5. Conclusions The water based pathogen Legionella is frequently found in reclaimed water When concentrations exceed 1000 per ml there is a significant risk of infection In this study, 12% of all reclaimed water samples exceeded this value thereby imposing significant risk Based on this, routine monitoring of reclaimed water spray irrigation systems would be prudent Acknowledgements We would like to thank Akrum Tamimi for helping with statistical analyses. This research was funded by National Science Foundation Grant #1361505. References Ajibode, O.M., Rock, C., Bright, K., McLain, E.T., Gerba, C.P., Pepper, I.L., 2013. Influence of residence time of reclaimed water within distribution systems on water quality. J. Desalination Water Reuse 33, 185e196. Armstrong, T.W., Haas, C.N., 2007. A quantitative microbial risk assessment model for Legionnaires' disease: animal model selection and dose-response modeling. Risk Anal. 27, 1581e1596. Armstrong, T.W., Haas, C.N., 2008. Legionnaires' disease: evaluation of a quantitative microbial risk assessment model. J. Water Health 6, 149e166. Baskerville, A., Fitzgeorge, R.B., Broster, M., Hambleton, P., Dennis, P.J., 1981. Experimental transmission of Legionnaires' disease by exposure to aerosols of
I.L. Pepper, C.P. Gerba / Water Research 139 (2018) 101e107 Legionella pneumophila. Lancet 1389 ii. Beer, K.D., Gargano, J.W., Roberts, A.S., Reese, H.E., Hill, V.R., Garrison, L.E., Kutty, P.R., Hilborn, E.D., Wade, T.J., Fullerton, K.E., Yoder, J.S., 2015. Outbreaks associated with environmental and undetermined water exposures e United States. Morb. Mortal. Wkly. Rep. 64, 849e851. Berendt, R.F., Young, H.W., Allen, R.G., Knutsen, G.L., 1980. Dose-response of Guinea pigs experimentally infected with aerosols of Legionella pneumophila. J. Infect. Dis. 141 (2). Bouwknegt, M., Schijven, J.F., Schalk, J.A.C., de Roda Husman, A.M., 2013. Quantitative risk estimation for a Legionella pneumophila infection due to whirlpool use. Risk Anal. 33, 1228e1236. Breiman, R.F., Horwitz, M.A., 1987. Guinea pigs sublethally infected with aerosolized Legionella pneumophila develop humoral and cell-mediated immune responses and are protected against lethal aerosol challenge: a model for studying host defense against lung Infections caused by intracellular pathogens. J. Exp. Med. 3, 799e811. R Core Team, 2013. R: a Language and Environment for Statistical Computing. R Foundation for Statistical Computing. Vienna, Austria. http://www.R-project. org/. Davis, G.S., Winn Jr., W.C., Gump, D.W., Crayhead, J.E., Beaty, H.N., 1982. Legionnaires' pneumonia after aerosol exposure in Guinea pigs and rats. Am. Rev. Respir. Dis. 126, 1050. Haas, C.N., Rose, J.B., Gerba, C.P., 2014. Quantitative Microbial Risk Assessment. John Wiley, NY. Jjemba, P.S., Johnson, W., Bukhari, Z., LeChevallier, M.W., 2015. Occurrence and control of Legionella in recycled water systems. Pathogens 4, 470e502. Kinkaid, D.C., Solomon, K.H., Oliphant, J.C., 1996. Drop size distribution for irrigation sprinklers. Trans. ASAE (Am. Soc. Agric. Eng.) 39 (3), 839e845. Kliment, V., 1973. Similarity and Dimensional Analysis, Evaluation of Aerosol Deposition in the Lungs of Laboratory Animals and Man, vol. 21, pp. 59e64. Issue 1. Leoni, E., DeLuca, G., Legnami, P.P., Sacchetti, R., Stampi, S., Zanetti, F., 2005. Legionella waterline colonization: detection of Legionella species in domestic, hotel and hospital hot water systems. J. Appl. Microbiol. 98, 373e379. Metcalf and Eddy, Inc, 2003. Wastewater engineering: treatment and reuse. In:
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Tchobanoglou, G., Burton, F.L., Stensel, H.D. (Eds.), McGraw-Hill Series in Civil and Environmental Engineering, fourth ed. Moore, G., Hewitt, M., Stevenson, D., Walker, J.T., Bennett, A.M., 2015. Aerosolization of respirable droplets from a domestic spa pool and the use of MS-2 coliphage and Pseudomonas aeruginosa as markers for Legionella pneumophila. Appl. Environ. Microbiol. 81, 555e561. Muller, D., Edwards, M.L., Smith, D.W., 1983. Changes in iron and transferrin levels and body temperature in experimental airborne Legionellosis. J. Infect. Dis. 147 (2). Osterholm, M.T., Moore, K.A., Kelley, N.S., Brosseau, L.M., Wong, G., Murphy, F.A., Peters, C.J., LeDuc, J.W., Russell, P.K., Van Herp, M., et al., 2015. Transmission of Ebola viruses: what we know and what we do not know. mBio 6 (2) e00137e15. doi:10.1128. Parr, A., Whitney, E.A., Berkelman, R.L., 2015. Legionellosis on the rise: a review of guidelines for prevention in the United States. J. Publ. Health Manag. Pract. 21, E17eE26. Signor, R.S., Ashbolt, N.J., 2009. Comparing probabilistic microbial risk assessment for drinking water against daily rather than annualized infection probability targets. J. Water Health 7, 535e543. Sikora, A., Wojtowicz-Bobin, A., Koziol-Montewka, M., Magrys, A., Gladysz, I., 2015. Prevalence of Legionella pneumophila in water distribution systems in hospitals and public buildings of the Lublin region of eastern Poland. Ann. Agric. Environ. Med. 22, 195e201. USEPA. United States Environmental Protection Agency, 2002. Long Term Enhanced Surface Treatment Rule Manual. EPA 815-F-02-001 (Washington, DC). Whiley, H., Keegan, A., Fallowfield, H., Ross, K., 2014. Uncertainties associated with assessing the public health risk from Legionella. Front. Microbiol. 5, 501. Whiley, H., Keegan, A., Fallowfield, H., Bentham, R., 2015. The presence of opportunistic pathogens, Legionella spp., L. pneumophila and Mycobacterium avium complex, in South Australian reuse water distribution pipelines. J. Water Health 13, 553e561. Wolf, B., Grisso, B., Hipkins, P., Reed, T., 2013. Spray Droplet Size and Drift. Accessed December 13, 2013. http://offices.ext.vt.edu/carroll/programs/anr/cc_pesticide_ training_files/droplets_v6.pdf.
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Risk of infection from Legionella associated with spray irrigation of reclaimed water Ian L. Pepper, Charles P. Gerba* Department of Soil, Water and Environmental Science, University of Arizona, 2959 W. Calle Agua Nueva, Tucson, AZ, 85745, USA
a r t i c l e i n f o
a b s t r a c t
Article history: Received 13 April 2017 Received in revised form 16 March 2018 Accepted 1 April 2018 Available online 2 April 2018
Legionella pneumophila has been detected in reclaimed water used for spray irrigation of turfgrass in public parks and golf courses. This study determined the risks of infection from exposure to various levels of Legionella in reclaimed waters considering: the method of spray application; and the duration and frequency of exposure. Evaluation of these factors resulted in a risk of infection greater than 1:10,000 for several scenarios when the number of Legionella in the reclaimed water exceeded 1000 colonyforming units (CFU) per ml. Most current guidelines for control of Legionella in distribution systems recommend that increased monitoring or remedial action be taken when Legionella levels exceed 1000 to 10,000 CFU/ml. Based upon our risk assessment, these guidelines seem appropriate for reclaimed water systems where spray irrigation is practiced. © 2018 Elsevier Ltd. All rights reserved.
Keywords: Legionella Reclaimed water Risk assessment
1. Introduction Legionella is a water based opportunistic pathogen found naturally in freshwater environments including rivers and lakes. In addition, it can be found in municipal water supply distribution systems and reclaimed waters (Leoni et al., 2005; Ajibode et al., 2013). Legionellosis, commonly known as Legionnaires'’ disease, frequently results from infection with the bacterium Legionella pneumophila. The disease includes a serious sometimes-lethal form of pneumonia, and the less serious Pontiac fever that results in selflimiting flu-like illness. Despite the fact that many cases are likely unreported or undiagnosed, recent estimates are that Legionnaires disease accounts for between 3% and 6% of community-acquired pneumonia that occur in the United Kingdom and United States each year (Moore et al., 2015). The disease typically occurs following inhalation of droplets or aerosols containing Legionella bacteria that are deposited in the lungs. Droplets range in size from five to 100 mm, which normally only travel a few feet in the air (Osterholm et al., 2015). In contrast, true aerosols are <5 mm and can travel greater distances through the air. Thus aerosols released from water sources such as spas or sprayed mists used for cooling are potential routes of exposure to Legionella. Water based pathogens including Legionella are
* Corresponding author. E-mail address: [email protected] (C.P. Gerba). https://doi.org/10.1016/j.watres.2018.04.001 0043-1354/© 2018 Elsevier Ltd. All rights reserved.
frequently found in reclaimed waters. In a recent study Ajibode et al. (2013), detected Legionella spp. in 40% (of 432 samples) of all samples collected from a reclaimed water distribution system from two utilities in the Southwest USA over a one-year period. In some samples, Legionella concentrations reached 228,000/100 ml. In arid regions, reclaimed wastewaters are commonly used for landscape irrigation of parks, golf courses, playgrounds and home lawns (Metcalf and Eddy, Inc. 2003). Spray or sprinkler irrigation being the method of application of choice for grass lawns. This creates an opportunity for the aerosolization of Legionella and subsequent exposure by inhalation. To assess this potential route of transmission, we applied quantitative microbial risk assessment (Haas et al., 2014). 2. Materials and methods 2.1. Quantitative microbial risk assessment The risk assessment paradigm involves four basic steps: hazard identification; exposure assessment; dose response; and risk characterization. However, there are uncertainties associated with each of these steps when assessing the public health risk from Legionella (Whiley et al., 2014). This is particularly true for assessing the exposure to Legionella via reclaimed water, since exposure via spray irrigation will not only depend on the concentration of Legionella in the reclaimed water, but also the size of the droplets or aerosols produced during irrigation, the distance from the spray
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source, and the duration of exposure.
D a R¼1 1þ
2.1.1. Dose response To date, there have been no studies conducted with humans to develop a dose response model for Legionella, however, there have been studies done on animals that provide surrogate dose response data. Most of the animal studies conducted to evaluate the effects of inhalation exposure of Legionella have used the guinea pig (Cavia porcellus) as the common testing animal model, to predict the effects on humans. Based on comparative in vitro macrophage studies and cell-mediated immunology on multiple animal species, guinea pigs were found to be the most suitable animal model for human risk assessment of Legionella pneumophila (Kliment, 1973). Deposition of aerosols 5 mm in the alveolar macrophages spaces of both guinea pigs and humans resulted in the same response to Legionella pneumophila. Thus the guinea pig, which shares with humans a susceptibility to lung infection with L. pneumophila, is an excellent animal model for the study of Legionnaires' disease (Kliment, 1973; Berendt et al., 1980; Davis et al., 1982; Breiman and Horwitz, 1987). Thus, data from these studies have been used to assess the infectivity of various doses of Legionella (Armstrong and Haas, 2007, 2008). For exposure in our study, we considered a number of factors, including the potential concentrations of Legionella within reclaimed water, which we obtained from a previous study conducted in our laboratory (Ajibode et al., 2013). When guinea pigs are exposed to aerosols of L. pneumophila they develop a pneumonic illness characterized by fever, weight loss and labored respirations that sometimes results in death. This syndrome resembles Legionnaires disease in humans (Berendt et al., 1980; Baskerville et al., 1981; Breiman and Horwitz, 1987). Berendt et al. (1980) carried out experiments to determine the infectivity and lethality of exposure to L. pneumophila, using guinea pigs. Muller et al. (1983) repeated the dose response experiments except that they anesthetized the guinea pigs after exposure to L. pneumophila, and tested their lungs and spines for infection. Breiman and Horwitz (1987) exposed guinea pigs to aerosols generated by a pump that projected a suspension of L. pneumophila into the air for 30 min. By examining the lungs of the animals after sacrifice, the number of L. pneumophila inhaled and retained by the animals was determined, allowing for the effects of specific numbers of inhaled Legionella on the animals to be determined. Based on an analysis of these studies, we used the Muller et al. (1983) experimental dataset in this current study to develop the dose response model, since their experiment predicted infections when the guinea pigs were sacrificed before mortality, and hence these data better represented the infection dose rather than mortality. This is the same dataset that was used by Armstrong and Haas (2007). Maximum Likelihood Estimates (MLE) were used to fit the measured data to exponential and beta Poisson statistical models. The exponential model is expressed as equation (1):
R ¼ 1 EXPðk DÞ
(2)
b
For both models, the Solver routine in Microsoft Excel program (Microsoft Inc. Redmond, WA) was used to optimize the models by varying the k value in equation (1); and a and b in equation (2). Here the objective was to maximize likelihood given the observed and the expected probability of infection. A likelihood ratio test was used to assess model fit. The null hypothesis test states that there is no statistically significant difference between the observed and the expected probability values for the respective dose. The research hypothesis assumes that there is a significant statistical difference in at least one observed and expected probability value for the respective dose. The test statistic used is the c2. The rejection region of the null hypothesis is defined based on rejection area of 0.05, and a degree of freedom ¼ dose group minus1 and the exponential and the dose group minus 2. The test statistics for both models were equal as shown in Fig. 1, which means that equation (1) with k ¼ 0.058836 or equation (2) with a ¼ 1.13 107 and b ¼ 1.98 108 can be used. Thus both the exponential and Beta-Poison does response models predict the same probality of infection for the same dose. Fig. 2 shows the dose response model for inhaled L. pneumophila (CFU). Data represent both the exponential and the Beta-Poison dose response models as shown in equations (3) and (4). Both equations give the same probability of infection for similar doses. Armstrong and Haas (2007) concluded that these two models are appropriate for low doses exposures for mechanistic reason for L. pneumophila. Our assessment confirmed their results and we used the exponential model in our study.
R ¼ 1 EXPð0:058836 DÞ R¼1 1þ
D 1:98 108
1:13107 (4)
2.2. Exposure assessment 2.2.1. Droplet size of sprinkler irrigation systems During sprinkler (spray) irrigation, irrigation water is segregated into spray particles or droplets of variable size. The size of water droplets is critical for determining the distance that the droplets
(1)
where: R: is the probability of risk of infection, Response, (decimal); k: is the infectivity per viable L. pneumophila concentration inhaled and retained CFU; D: is the Dose of viable L. pneumophila that is inhaled and retained (CFU). The Beta-Poisson model can be estimated by equation (2), in which a and b are constants and R is again the probability of risk of infection.
(3)
Fig. 1. Fitted exponential and beta Poisson models".
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Fig. 2. Dose Response Model for inhaled L. pneumophila (CFU).
will be transported through the air, and as predicted by Stoke's Law, the smaller the droplet size, the further the droplets will be transported. In addition, as the size of spray droplets decrease, their numbers increase, and the potential for drift also increases. Overall, the characteristics of sprinkler nozzle such as: type, fan angle, orifice size, and sprinkler operational pressure all influence droplet size. Kinkaid et al. (1996) measured droplet size for different sprinkler nozzles with different configurations and different operational pressures using a laser-optical method. They derived an exponential function with parameters that are measurable to define droplet size distribution. The function is shown as equation (5) below:
2.2.2. Drift distance due to wind Wolf et al. (2013) sprayed water at 206.8 Kilo Pascal (30 pounds per square inch) pressure, and measured water droplet drift distances as a function of droplet size with relative humidity of 75%; temperature of 23.9 C (75 F); and wind speed of 2.235 m per second (5 mile per hour). Humidity and temperature will affect droplet size over distance depending on the distant traveled. Ordinary linear regression was performed on the data in Table 1 and yielded a goodness of fit of 0.9994 for a fourth order polynomial function shown as equation (6), in which Drift is the drift distance in feet and S is the droplet size in mm.
Drift ¼
Pv ¼ 100%
d n 1 e EXP e0:693 d50
2:0 109 S4 4:0 107 S3 þ 2:7 103 S2 ð0:7375 SÞ þ 75:783
(6)
(5)
where: d ¼ droplet diameter (mm) Pv ¼ percent (%) of total discharge in drops smaller than d d50 ¼ volume mean drop diameter (mm) n ¼ dimensionless exponent Using equation (5) to determine percent of total discharge by 75 different sprinkler types and configurations in drops less than d ¼ 10 mm, and using the parameters provided by Kinkaid et al. (1996), yields a list of 75 values that can be used to determine the percent of total discharge by the sprinkler system of drops smaller than 10 mm. The Bootstrapping routine in the R-Language (R Core Team, 2013) with 100,000 iterations was used to determine the statistical distribution of the aerosol particles generated (Fig. 3). The average distribution of total discharge by the sprinkler system with drops smaller than 10 mm was 0.01572%, and the 95% Confidence Interval (CI) was [0.0116%, 0.0220%].
The ordinary linear regression equation was used to extrapolate the data to droplet sizes below 50 mm as shown in Fig. 4. Fig. 4 shows that droplet size of 10 mm or less has the potential to drift 70e75 feet away from spraying sprinklers.
2.2.3. Inhalation volume of recycled wastewater droplets The model presented in equation (7) was developed to determine inhalation water droplets with size smaller than 10 mm generated from a sprinkler irrigation system operating at 30 psi (206.8 Kilo Pascal) pressure, air relative humidity of 75%, air temperature of 75 F (23.9 C), and wind speed of 5 mile per hour (2.235 m per second)
Vt ¼
Exp X t¼1
where:
Hrate Hvol Cdroplets < 10 dt
(7)
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equation (8).
Cdroplet < 10 ¼
Q Pv < 10 t
p r2 H ð100%Þ
(8)
where: Q: is discharge rate from the sprinkler (ml/minute) Pv < 10: % of droplets generated with size less than 10 mm (%) r: is drift distance for a sprinkler (mm) H: is maximum height to which sprinkler is generating droplets (mm); Assumed to be 2000 mm (2.0 m). 2.3. Risk of infection 2.3.1. One-time event In order to calculate the risk of infection, the exposure was calculated taking into account: the concentration of Legionella in the reclaimed water; the distance from the spray source; the droplet size and the duration of exposure. Here we assume the distance from the source to be 72.5 feet (22 m) (assumed distance of a front house lawn) from an operational sprinkler that is spraying 30 gallons (113 L) per minute of recycled wastewater at operational pressure of 30 psi (206.8 Kilo Pascal). From Fig. 3 and bootstrapping results presented earlier, it was determined that the average of the statistical distribution was 0.01572% of droplets generated with size less than 10 mm and the 95% Confidence Interval (CI) was [0.0116%, 0.0220%]. Conservatively, we assumed the 95% upper limit of the confidence interval of Pv<10 to be 0.0220%. According to Kliment (1973) an average adult who weighs about 80 kg breathes 7.5 breaths per minute with an air breathing volume of 800 cm3/min. 3. Results Fig. 3. Statistical distribution of droplet size. Percent of total discharge with droplets size less than 10 mm obtained for 75 irrigation sprinkler types, configurations and pressures (t* represents % of total discharge with droplets' size smaller than 10 mm).
Table 1 Droplet size and drift distance. Droplet Size Descriptiona
Dropletsa size (micron)
Drift Distance Feet
meter
Extremely Coarse Very Coarse Coarse Medium Fine Very Fine Ultra Fine
600 500 400 300 200 100 50
0.20 0.30 0.50 1.30 5.00 25.00 45.00
0.06 0.09 0.15 0.40 1.52 7.62 13.72
a
American Society of Agricultural Engineers.
Vt: total volume of droplets with size < 10 mm, inhaled during exposure time (ml) Hrate: inhalation rate for humans (breaths/minute) Hvol: inhalation volume (ml/breath) Cdroplets<10: Concentration of droplets with size less than 10 mm in the air at any time (ml droplets/ml air) dt: incremental time in minutes: 1, 2, 3, … Exp: total exposure time (minutes) Concentration of droplets with size less than 10 mm in the air at any time (ml droplets/ml air), Cdroplets<10 can be calculated from
Fig. 4 shows the risk of infection due to inhalation of recycled wastewater containing L. pneumophila for different exposure times in minutes based on L. pneumophila concentrations obtained from a previous study (Ajibode et al., 2013) for a onetime event. Table 2 shows the risk of infection for different concentrations of L. pneumophila in recycled sprayed irrigation water with different exposure times for a one-time exposure event. 3.1. One-time exposure event per week over summer months Table 3 shows the risk of infection of legionnaire's disease as a function of L. pneumophila concentration in recycled sprayed irrigation water, with different exposure times for a one-time exposure event per week for 12 weeks (summer months). 3.2. One-time exposure event per week for a year Table 4 shows the risk of infection of Legionnaire's disease as a function of L. pneumophila concentration in reclaimed water with different exposure times for a one-time exposure event per week for an entire year. Table 5 shows the risk of infection of legionnaire's disease for the average L. pneumophila concentration found in recycled water with different exposure times and exposure events. 4. Discussion The recent detection of Legionella in reclaimed water distribution systems (Whiley et al., 2015; Ajibode et al., 2013) indicates that
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Fig. 4. Risk of infection of legionnaire's disease for different exposure times.
Table 2 Risk of infection of legionnaire's disease as a function of L. pneumophila concentration in recycled sprayed irrigation water and with different exposure times for a one-time event. L. pneumophilia in Recycled Wastewater (CFU/100 ml)
1 10 100 500 1000 5000 10,000 50,000 100,000 500,000 1,000,000
Exposure Time (Minutes) 1
5
10
20
30
60
4.9E-10 4.9E-09 4.9E-08 2.4E-07 4.9E-07 2.4E-06 4.9E-06 2.4E-05 4.9E-05 2.4E-04 4.9E-04
2.4E-09 2.4E-08 2.4E-07 1.2E-06 2.4E-06 1.2E-05 2.4E-05 1.2E-04 2.4E-04 1.2E-03 2.4E-03
4.9E-09 4.9E-08 4.9E-07 2.4E-06 4.9E-06 2.4E-05 4.9E-05 2.4E-04 4.9E-04 2.4E-03 4.9E-03
9.8E-09 9.8E-08 9.8E-07 4.9E-06 9.8E-06 4.9E-05 9.8E-05 4.9E-04 9.8E-04 4.9E-03 9.8E-03
1.5E-08 1.5E-07 1.5E-06 7.3E-06 1.5E-05 7.3E-05 1.5E04 7.3E-04 1.5E-03 7.3E-03 1.5E-02
2.9E-08 2.9E-07 2.9E-06 1.5E-05 2.9E-05 1.5E-04 2.9E-04 1.5E-03 2.9E-03 1.5E-02 2.9E-02
Table 3 Risk of infection of Legionnaire's disease as a function of L. pneumophila concentration in recycled sprayed irrigation water, with different exposure times for one exposure event per week for 12 weeks. L. pneumophila in Recycled Wastewater (CFU/100 ml)
1 10 100 500 1000 5000 10,000 50,000 100,000 500,000 1,000,000
Exposure Time (Minutes) Once a Week for 12 Weeks - Summer Time 1
5
10
20
30
60
0.0Eþ00 2.5E-09 3.3E-08 1.7E-07 3.3E-07 1.7E-06 3.4E-06 1.7E-05 3.4E-05 1.7E-04 3.4E-04
2.5E-09 1.8E-08 1.7E-07 8.4E-07 1.7E-06 8.4E-06 1.7E-05 8.4E-05 1.7E-04 8.4E-04 1.7E-03
2.5E-09 3.3E-08 3.3E-07 1.7E-06 3.4E-06 1.7E-05 3.4E-05 1.7E-04 3.4E-04 1.7E-03 3.3E-03
7.6E-09 6.8E-08 6.7E-07 3.4E-06 6.7E-06 3.4E-05 6.7E-05 3.4E-04 6.7E-04 3.3E-03 6.7E-03
1.0E-08 1.0E-07 1.0E-06 5.0E-06 1.0E-05 5.0E-05 1.0E-04 5.0E-04 1.0E-03 5.0E-03 1.0E-02
2.0E-08 2.0E-07 2.0E-06 1.0E-05 2.0E-05 1.0E-04 2.0E-04 1.0E-03 2.0E-03 1.0E-02 2.0E-02
a potential risk exists if the organisms are aerosolized during spray irrigation. Spray irrigation of reclaimed for water irrigation of house lawns, parks, playgrounds and other public areas is practiced in the United States resulting in exposure of susceptible individuals. No outbreaks have been reported associated with reclaimed water, but
outbreaks by unknown sources occur and most cases are not diagnosed or reported (Beer et al., 2015). We determined that a one-time event resulted in a risk of infection of 1:10,000 from a 60-min exposure to spray irrigated reclaimed water with a concentration of 5000 CFU/100 ml at a
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Table 4 Risk of infection of legionnaire's disease as a function of L. pneumophila concentration in reclaimed water with different exposure times for one exposure event per week for a full year. L. pneumophila in Recycled Wastewater (CFU/100 ml)
Exposure Time (Minutes) Once a Week for 50 Weeks - One Year
1 10 100 500 1000 5000 10,000 50,000 100,000 500,000 1,000,000
1
5
10
20
30
60
2.5E-09 1.5E-08 1.4E-07 7.0E-07 1.4E-06 7.0E-06 1.4E-05 7.0E-05 1.4E-04 7.0E-04 1.4E-03
7.6E-09 7.0E-08 7.0E-07 3.5E-06 7.0E-06 3.5E-05 7.0E-05 3.5E-04 7.0E-04 3.5E-03 7.0E-03
1.5E-08 1.4E-07 1.4E-06 7.0E-06 1.4E-05 7.0E-05 1.4E-04 7.0E-04 1.4E-03 7.0E-03 1.4E-02
2.8E-08 2.8E-07 2.8E-06 1.4E-05 2.8E-05 1.4E-04 2.8E-04 1.4E-03 2.8E-03 1.4E-02 2.8E-02
4.3E-08 4.2E-07 4.2E-06 2.1E-05 4.2E-05 2.1E-04 4.2E-04 2.1E-03 4.2E-03 2.1E-02 4.1E-02
8.3E-08 8.4E-07 8.4E-06 4.2E-05 8.4E-05 4.2E-04 8.4E-04 4.2E-03 8.3E-03 4.1E-02 8.0E-02
Table 5 Probability of Infection based on the average L. pneumophila measured in the distribution system (4971 CFU/100 ml) (Ajibode et al., 2013). Number of Events
One Event Once a Week for 12 Weeks: Summer Months Once a Week for 50 Weeks: One Year
Exposure Time (Minutes) 1
5
10
20
30
60
1.4E-07 1.7E-06 6.9E-06
6.9E-07 8.3E-06 3.5E-05
1.4E-06 1.7E-05 6.9E-05
2.8E-06 3.3E-05 1.4E-04
4.2E-06 5.0E-05 2.1E-04
8.3E-06 1.0E-04 4.2E-04
distance of 72.5 feet from the spray source. Using the data from Ajibode et al. (2013), we found this value was exceeded in 12% (432 samples) of all the reclaimed water samples analyzed. In contrast, Bouwknegt et al. (2013) conducted a quantitative risk assessment for L. pneumophila following exposure via whirlpool use. They conducted an in-depth calculation of exposure via air bubbles taking into account the number of Legionella inhaled via water droplets. Using a dose-response model for guinea pigs to represent humans, infection risks for active whirlpool use with 100 CFU/L of water for 15 min were 0.29 (x 0.11e0.48) for susceptible males, and 0.22 (x 0.06e0.42) for susceptible females. In addition, risk was shown to be dependent on both the concentration of Legionella and duration of exposure. Specifically exposure to 1000 CFU/L almost always resulted in infection. Likewise a 2 h exposure to 100 CFU/L also resulted in a very high risk of infection (0.86e0.97). A factor that would underestimate the risk includes the use of analytical methods for the detection of Legionella that are not 100% efficient. Factors that under-estimate or over-estimate risk would include: 1) data for dose response in animals that is not reflective of dose response in humans; 2) variable concentrations of viable Legionella in the water during exposure or through the course of a year; 3) wind speed, 4) movement of individuals in and out of the aerosol plume. Detection of Legionella at low levels in distribution systems and cooling towers is not uncommon and infections are increasing (Parr et al., 2015; Sikora et al., 2015; Jjemba et al., 2015). Most guidelines recommend an action level for control of Legionella when levels of Legionella exceed 1000 to 10,000 CFU/per liter (Parr et al., 2015). Recommended actions include: increased levels of disinfection; pro-active control of biofilms; reduced nutrient levels; increased water temperatures in heated systems; flushing the distribution systems; and increased monitoring. In the United States, the U. S. Environmental Protection Agency has set guidance for drinking water treatment to reduce the risk of infection for one year to 1:10,000 per year (USEPA, 2002). This requires a risk of daily infection of 106 to 107 (Signor and Ashbolt, 2009). The degree of risk is highly dependent upon the duration of exposure, the size of droplets generated and the numbers of
Legionella in the reclaimed water. Recommended USEPA guidelines for risks for drinking water are approximately 106 for a one-time exposure. This level would be reached when Legionella concentrations in water in water are >1000 CFU/ml. In the current study, at least one reclaimed water sample contained Legionella concentrations greater than 200,000 CFU/100 ml. In addition, 12% of all samples contained Legionella at concentrations that posed significant risk. These data suggest that there is a potential for infection from sprayed irrigation water and based on this, routine monitoring of reclaimed systems would be prudent. This concurs with another recent study with a similar recommendation (Parr et al., 2015). 5. Conclusions The water based pathogen Legionella is frequently found in reclaimed water When concentrations exceed 1000 per ml there is a significant risk of infection In this study, 12% of all reclaimed water samples exceeded this value thereby imposing significant risk Based on this, routine monitoring of reclaimed water spray irrigation systems would be prudent Acknowledgements We would like to thank Akrum Tamimi for helping with statistical analyses. This research was funded by National Science Foundation Grant #1361505. References Ajibode, O.M., Rock, C., Bright, K., McLain, E.T., Gerba, C.P., Pepper, I.L., 2013. Influence of residence time of reclaimed water within distribution systems on water quality. J. Desalination Water Reuse 33, 185e196. Armstrong, T.W., Haas, C.N., 2007. A quantitative microbial risk assessment model for Legionnaires' disease: animal model selection and dose-response modeling. Risk Anal. 27, 1581e1596. Armstrong, T.W., Haas, C.N., 2008. Legionnaires' disease: evaluation of a quantitative microbial risk assessment model. J. Water Health 6, 149e166. Baskerville, A., Fitzgeorge, R.B., Broster, M., Hambleton, P., Dennis, P.J., 1981. Experimental transmission of Legionnaires' disease by exposure to aerosols of
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