Supplementary Information: Free Chlorine and Monochloramine Inactivation Kinetics of Aspergillus and Penicillium in Drinking Water

Xiao Ma1, Kyle Bibby1, 2*

1Department of Civil and Environmental Engineering, University of Pittsburgh, Pittsburgh, PA 15261, 2Department of Computational and Systems Biology, University of Pittsburgh Medical School, Pittsburgh, PA 15261, USA *Corresponding author

*Corresponding Author: Kyle Bibby, 709 Benedum Hall, Pittsburgh, PA 15261 BibbyKJ@pitt.edu, 412-624-9207

Keywords:Disinfection, fungi, Aspergillus, Penicillium, opportunistic pathogen, premise plumbing

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WinBUGS code for delayed Chick-Watson model fitting

This code was derived from previous study (Sivaganesan et al. 2003)

Model

{

for (i in 1:N) {

Y[i] ~ dnorm(mu[i], tau)

mu[i] <- beta * (CT[i] - CTlag) * step(CT[i] - CTlag)

}

#non_informative priors (variance sigma2 = 1/tau, CTobs is the CT value corresponding to 10% of lowest Ln(N/N0))

tau ~ dgamma(0.001, 0.001)

beta ~ dnorm(0.0, 1.0E-6)

CTlag ~ dunif(0, CTobs)

sigma2 <- 1/tau

}

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0

0.2

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0.6

0.8

1

1.2

0 20 40 60 80

Free

chl

orin

e co

ncen

trat

ion

(mg/

L)

Time (min)

A. fumigatus type strain, 1 mg/L experiment

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4

0 10 20 30 40

Free

chl

orin

e co

ncen

trat

ion

(mg/

L)

Time (min)

A. fumigatus type strain, 4 mg/L experiment

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0.2

0.4

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0.8

1

0 20 40 60 80Free

chl

orin

e co

ncen

trat

ion

(mg/

L)

Time (min)

A. fumigatus clinical strain, 1 mg/L exeperiment

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0.4

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0.8

1

0 20 40 60 80Free

chl

orin

e co

ncen

trat

ion

(mg/

L)

Time (min)

A. versicolor, 1 mg/L exeperiment

0

0.2

0.4

0.6

0.8

1

0 20 40 60 80Free

chl

orin

e co

ncen

trat

ion

(mg/

L)

Time (min)

P. purpurogenum, 1 mg/L exeperiment

00.5

11.5

22.5

33.5

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0 10 20 30 40Free

chl

orin

e co

ncen

trat

ion

(mg/

L)

Time (min)

A. fumigatus clinical strain, 4 mg/L experiment

SI-Figure 1. Measured free chlorine residual concentration (mg/L)

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1.2

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Free

chl

orin

e co

ncen

trat

ion

(mg/

L)

Time (min)

A. fumigatus type strain, 1 mg/L experiment

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4

0 10 20 30 40

Free

chl

orin

e co

ncen

trat

ion

(mg/

L)

Time (min)

A. fumigatus type strain, 4 mg/L experiment

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0.2

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0.6

0.8

1

1.2

0 20 40 60 80Free

chl

orin

e co

ncen

trat

ion

(mg/

L)

Time (min)

A. fumigatus clinical strain, 1 mg/L exeperiment

00.5

11.5

22.5

33.5

4

0 10 20 30 40Free

chl

orin

e co

ncen

trat

ion

(mg/

L)

Time (min)

A. fumigatus clinical strain, 4 mg/L experiment

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0.2

0.4

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0.8

1

0 20 40 60 80Free

chl

orin

e co

ncen

trat

ion

(mg/

L)

Time (min)

A. versicolor, 1 mg/L exeperiment

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1.2

0 20 40 60 80Free

chl

orin

e co

ncen

trat

ion

(mg/

L)

Time (min)

P. purpurogenum, 1 mg/L exeperiment

SI-Figure 2. Measured monochloramine residual concentration (mg/L)

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-1

-0.5

0

0.5

1

0 20 40 60

Ln(N

/N0)

Time (min)

Free chlorine control experiment

A. fumgiatus typestrain

A. fumigatus clinicalstrain

Aspergillus spp.

Penicillium spp.

-1

-0.5

0

0.5

1

0 20 40 60

Ln(N

/N0)

Time (min)

Monochloramine control experiment

A. fumigatus typestrain

A. fumigatus clinicalstrain

A. versicolor tapwater isolate

P. purpurogenumtap water isolate

A. fumigatus type strain

A. fumigatus clinical strain

A. versicolor

P. purpurogenum

SI-Figure 3. Measured mean Ln(N/N0) for free chlorine and monochloramine control experiments, which no disinfectant was used;

Further F-test results (conducted using Prism, GraphPad Software) suggested that the measured Ln(N/N0) of all tested strains did not significantly deviated from 0

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y = 0.97xR² = 0.95

-8

-6

-4

-2

0-8 -6 -4 -2 0

Pred

icte

d Ln

(N/N

0)

Measured Ln(N/N0)

y = 0.87xR² = 0.75

-12

-10

-8

-6

-4

-2

0-12 -10 -8 -6 -4 -2 0

Pred

icte

d Ln

(N/N

0)

Measured Ln(N/N0)

y = 0.93xR² = 0.87

-3

-2

-1

0-3 -2 -1 0

Pred

icte

d Ln

(N/N

0)

Measured Ln(N/N0)

y = 0.96xR² = 0.93

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-4

-2

0-6 -4 -2 0

Pred

icte

d Ln

(N/N

0)

Measured Ln(N/N0)

y = 0.91xR² = 0.80

-8

-6

-4

-2

0-8 -6 -4 -2 0

Pred

icte

d Ln

(N/N

0)

Measured Ln(N/N0)

y = 0.81xR² = 0.56

-12

-10

-8

-6

-4

-2

0-12 -10 -8 -6 -4 -2 0

Pred

icte

d Ln

(N/N

0)

Measured Ln(N/N0)

y = 0.93xR² = 0.86

-3

-2

-1

0-3 -2 -1 0

Pred

icte

d Ln

(N/N

0)

Measured Ln(N/N0)

y = 0.96xR² = 0.91

-6

-5

-4

-3

-2

-1

0-6 -4 -2 0

Pred

icte

d Ln

(N/N

0)

Measured Ln(N/N0)

Classical Chick-Watson Model

Delayed Chick-Watson Model

A. fumigatus type strain

A. fumigatus clinical strain

A. versicolortap water isolate

P. purpurogenumtap water isolate

y = 0.97xR² = 0.95

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-6

-4

-2

0-10 -5 0

Pred

icte

d …

Measured …

1 mg/L assay

4 mg/L assay

SI-Figure 4. Comparison of model predicted Ln(N/N0) against measured Ln(N/N0) for both delayed Chick-Watson model and classical Chick-Watson model, free chlorine disinfection

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A. fumigatus clinical strain

y = 0.99xR² = 0.97

-5

-3

-1

-6 -4 -2 0

Mea

sure

d Ln

(N/N

0)

Measured Ln(N/N0)

y = 0.94xR² = 0.87

-8

-6

-4

-2

0-8 -6 -4 -2 0

Mea

sure

d Ln

(N/N

0)

Measured Ln(N/N0)

y = 0.98xR² = 0.97

-3

-2

-1

0-3 -2 -1 0

Mea

sure

d Ln

(N/N

0)

Measured Ln(N/N0)

y = 0.87xR² = 0.75

-1

-0.5

0-1 -0.5 0

Pred

icte

d Ln

(N/N

0)

Measured Ln(N/N0)

y = 0.98xR² = 0.95

-6

-4

-2

0-6 -4 -2 0

Estim

ated

Ln(

N/N

0)

Measured Ln(N/N0)

y = 0.94xR² = 0.88

-6

-4

-2

0-6 -4 -2 0

Estim

ated

Ln(

N/N

0)

Measured Ln(N/N0)

y = 0.93xR² = 0.86

-3

-2

-1

0-3 -2 -1 0

Estim

ated

Ln(

N/N

0)

Measured Ln(N/N0)

y = 0.88xR² = 0.75

-1

-0.5

0-1 -0.5 0

Estim

ated

Ln(

N/N

0)

Measured Ln(N/N0)

Classical Chick-Watson Model

Delayed Chick-Watson Model

A. fumigatus type strain

A. versicolortap water

isolate

P. purpurogenumtap water isolate

y = 0.99xR² = 0.97

-5

-3

-1-6 -4 -2 0

Mea

sure

d …

Measured …

1 mg/L assay

4 mg/L assay

SI-Figure 5. Comparison of model predicted Ln(N/N0) against measured Ln(N/N0) for both delayed Chick-Watson model and classical Chick-Watson model, monochloramine disinfection

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Table SI-1. Comparison of estimated 3-log inactivation Ct values of the tested fungal strains to previously tested virus and bacteria, by free chlorine

Ct (mg min/L) References(Previously tested virus and bacteria, 3-log inactivation)Escherichia coli (pH 7.4) 0.032 ±0.009 (Helbling and

VanBriesen 2007)Staphylococcus epidermidis (pH 7.4) 0.221 ±0.08 (Helbling and

VanBriesen 2007)Klebsiella pneumoniae (22.5 ºC, pH 7.2) 13.06 ±0.91 (Goel and Bouwer

2004)Mycobacterium avium strain 5502 (23 ºC, pH 7) 51 ±10 (Taylor et al. 2000)Mycobacterium avium strain 1508 (23 ºC, pH 7) 164 ±28 (Taylor et al. 2000)

Mycobacterium aurum (pH 7.4) 42.9 ±2.71 (Helbling and VanBriesen 2007)

Legionella pneumophila (25 ºC, pH 7) 365.3 (Jacangelo 2002)

EPA required Ct for 3-log inactivation of Giardia (20 - 25 ºC, pH 7)

37 (Crittenden et al. 2005)

(Fungal strains in the current study, 3-log inactivation)Aspergillus fumigatus type strain (22.5 ºC, pH 7) 61.42 ±4.74Aspergillus fumigatus clinical strain (22.5 ºC, pH 7) 48.99 ±11.36Aspergillus versicolor (22.5 ºC, pH 7) 84.72 ±6.85Penicillium purpurogenum (22.5 ºC, pH 7) 194.7 ±21.6

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Table SI-2. Comparison of estimated 3-log inactivation Ct values of the tested fungal strains to previously tested virus and bacteria, by monochloramine

Ct (mg min/L)

References

(Previously tested virus and bacteria, 3-log inactivation)Adenovirus (19 ºC, pH 7) 1.24 (Jacangelo 2002)E. coli (25 - 35 ºC, pH 7.5 to 8.5) 10.3 ±1.67 (Jakubek et al. 2013)Corynebacterium glutamicum (25 - 35 ºC, pH 7.5 to 8.5)

16.84 ±1.18 (Jakubek et al. 2013)

Pseudomonas fluorescens (25 - 35 ºC, pH 7.5 to 8.5) 22.19 ±3.04 (Jakubek et al. 2013)Klebsiella pneumoniae (22.5 ºC, pH 7.2 to 7.5) 39.57 ±1.27 (Goel and Bouwer

2004)Mycobacterium avium strain 5502 (23 ºC, pH 7) 91 ±34 (Taylor et al. 2000)Mycobacterium avium strain 5002 (23 ºC, pH 7) 1710 ±814 (Taylor et al. 2000)Legionella pneumophila environmental strain 1 (25 - 35 ºC, pH 7.5 to 8.5)

16.14 ±3.07 (Jakubek et al. 2013)

Legionella pneumophila environmental strain 2 (25 - 35 ºC, pH 7.5 to 8.5)

64.88 ±19.07 (Jakubek et al. 2013)

EPA required Ct for 3-log inactivation of Giardia (20 ºC, pH 6-9)

1100 (Crittenden et al. 2005)

(Fungal strains in the current study, 3-log inactivation)Aspergillus fumigatus type strain (22.5 ºC, pH 8) 103.9 ±6.64Aspergillus fumigatus clinical strain (22.5 ºC, pH 8) 90.33 ±7.17Aspergillus versicolor (22.5 ºC, pH 8) 531.3 ±87.11Penicillium purpurogenum (22.5 ºC, pH 8) 153.2 ±9.07

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Reference:Crittenden, J.C., Trussell, R.R., Hand, D.W., Howe, K.J. and Tchobanoglous, G. (2005) MWH's Water Treatment: Principles and Design, Second Edition, pp. 1914-1917, John Wiley & Sons, Inc.Goel, S. and Bouwer, E.J. (2004) Factors influencing inactivation of Klebsiella pneumoniae by chlorine and chloramine. Water Research 38(2), 301-308.Helbling, D.E. and VanBriesen, J.M. (2007) Free chlorine demand and cell survival of microbial suspensions. Water Research 41(19), 4424-4434.Jacangelo, J.G. (2002) Inactivation of waterborne emerging pathogens by selected disinfectants, American Water Works Association.Jakubek, D., Guillaume, C., Binet, M., Leblon, G., DuBow, M. and Le Brun, M. (2013) Susceptibility of Legionella Strains to the Chlorinated Biocide, Monochloramine. Microbes and Environments 28(3), 336-345.Sivaganesan, M., Rice, E.W. and Mariñas, B.J. (2003) A Bayesian method of estimating kinetic parameters for the inactivation of Cryptosporidium parvum oocysts with chlorine dioxide and ozone. Water Research 37(18), 4533-4543.Taylor, R.H., Falkinham, J.O., Norton, C.D. and LeChevallier, M.W. (2000) Chlorine, chloramine, chlorine dioxide, and ozone susceptibility of Mycobacterium avium. Applied and Environmental Microbiology 66(4), 1702-1705.

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