technical    BRIEF
    Decontamination of Drinking Water Infrastructure
             Contaminated with Bacillus Spores:
               Iron and Cement-mortar Surfaces
INTRODUCTION

This study examines the effectiveness of decontaminating corroded iron and cement-mortar
coupons that have been contaminated with spores of Bacillus atrophaeus subsp. globigii (B.
globigii), which is often used as a surrogate for pathogenic B. anthracis (anthrax) in disinfection
studies.

Bacillus spores are persistent on common drinking water material surfaces like corroded iron,
requiring physical or chemical methods to decontaminate the infrastructure. In the United
States, free chlorine and monochloramine are the primary chemical disinfectants used by the
drinking water industry to inactivate microorganisms.
Flushing is also a common, easily implemented practice in drinking water distribution systems,
although large volumes of contaminated water needing treatment could be generated.

Identifying readily available alternative disinfectant formulations for infrastructure
decontamination give water utilities options for responding to specific types of contamination
events.
In addition to presenting data on flushing alone, which demonstrated the  persistence of spores
on water infrastructure in the absence of high levels of disinfectants, data on acidified nitrite,
chlorine dioxide, free chlorine, monochloramine, ozone, peracetic acid, and followed by flushing
are provided. [2]

DISINFECTANT USE IN U.S. WATER UTILITIES

In the most recent American Water Works Association survey of disinfectant use [1], 63% of
U.S. water utilities reported using chlorine gas; 30% used chloramine; 8% used chlorine dioxide;
and 9% reported using ozone. Some utilities  used multiple forms of chlorine or combinations of
chemical and physical disinfection practices.
In addition to investigating chlorine and chloramine, U.S. EPA's Homeland Security Research
Program chose ozone and chlorine dioxide, used at water treatment plants for disinfection and
taste and odor control.  Because they are strong oxidants, their reactivity may limit their
application to small areas in a distribution system.

Two other disinfectants, not reported in use by water utilities, were used to see if they were
candidates for efficacious spore removal. Peracetic acid (PAA) is used in the food and beverage
industry, as well as the medical device industry for cleaning equipment. It is gaining acceptance
as an effective decontaminating agent for drinking water. Acidified nitrite, which can be
formulated from common reagents, is less reactive on infrastructure than oxidant disinfectants
and could  potentially be effective against spores in water.
EPA/600/S-15/168 October 2015

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INVESTIGATION [2]

Decontamination of two common drinking water infrastructure surfaces contaminated with 6.
globigii spores was evaluated using six disinfectants, plus flushing with water.

The study was conducted in a drinking water distribution system simulator (DSS). The DSS
consisted of 23 m (approximately 75 ft) of 15 cm (approx. 6 in) diameter polyvinyl chloride
(PVC)  pipe connected in a rectangular shape to an in-line recirculation tank. Total DSS volume
was 832 L (approximately 220 gal). A 3780 L (approximately 1000 gal) feed tank supplied tap
water from the Greater Cincinnati Water Works (GCWW) to the DSS. For details, see [3].
Two types of coupons (6.5 cm2) represented the infrastructure surfaces: corroded iron cut from
a water main and cement-mortar.1 The coupons were conditioned and allowed to form biofilm in
the DSS for one month prior to contamination with spores.
For all  coupons, unless adjusted, baseline conditions were: pH's ranging from 8.4 to 8.6 and
free chlorine levels2 during all disinfectant and flushing treatments ranging from 0.9 to 1.1 mg/L.
Water temperature fluctuated between 25 ° and 30 °C. Water was kept stagnant for the
disinfectant tests.3 All disinfectant treatments were followed by flushing at 0.3 m/sec (1 ft/sec).
Table 1 lists disinfectants and conditions.
Table 1. Disinfectants Tested: Concentrations, pH Levels, and Flushing (25 ° to 30 °C)
             0.3 m/sec is 1 ft/sec
+ flow of 314 L/min during disinfectant testing
1 Made using ANSI/AWWA method C104/A21.4-08. Standards for Cement-Mortar Lining for Ductile-Iron
Pipe and Fittings. Effective September 23, 2008, AWWA. Denver: American Waterworks Association.
2 Free chlorine was also tested at two pH levels and two concentrations.
3 During the ozone treatment (2 mg/L), decontamination occurred for 12 hours in the presence of flow at
314 L/min before flushing.
EPA/600/S-15/168  October 2015

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RESULTS

In the current study, flushing was tested at 0.5 m/sec (1.7 ft/sec) without disinfectants. This
reduced adhered spores by 0.5 log™ from iron and 2.0 log™ from cement-mortar.

More of the disinfectant treatments on contaminated cement-mortar coupons reduced spores to
undetectable levels than on iron. However, after 6 hours of treatment, chlorine dioxide (25 mg/L)
did reduce spores to undetectable levels on iron coupons.

Acidified nitrite (pH 2, 0.1 mol/L) was the only tested disinfectant that performed better on iron
coupons than on cement-mortar. There were undetectable spore levels on the iron surfaces
during the flushing phase (at 0.3 m/sec (0.1 ft/sec)) that followed the disinfection treatment.

Overall, chlorine dioxide was the best performing disinfectant on both surfaces with >3.0 log™
removal from cement-mortar at 5 and 25 mg/L at 2 hours of treatment. For acidified nitrite and
peracetic acid,  there were no test conditions under which spores were reduced to undetectable
levels on  cement-mortar coupons.


Table 2 summarizes the effective disinfectant concentrations and conditions.
Table 2. Disinfectants Were Observed to Reduce the Number of Spores to Undetectable
Levels on Cement-mortar Coupons (25° to 30°C) under these Time and Conditions
Conditions
Concentration (mg/L)
Time (hours)
Chlorine Dioxide
5
25
2
Disinfects
Free Chlorine (pH 7)
25
18
nts
Monochloramine
25
18
Ozone
2
1
Tables 3 (cement-mortar) and 4 (iron) present all the experimental results for disinfectant and
flushing treatments.
EPA/600/S-15/168 October 2015

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 Table 3. Surface Concentrations of Surviving Bacillus globigii Spores (cfu/cm2) with Decontaminant Effectiveness (log
 reduction) in Parenthesis for Various Decontamination and Flushing Durations on Cement-mortar Coupons (6.5 cm2)(25c
 to 30°C)
Experimental
Phase
c
a> o
§_ & Decontamination with Disinfectant
<"jp
Flushing at
0.3 m/sec
(1 ft/sec)
Time (hr)
0 (initial)
1
2
3
4
6
18
22
24
26
44
Disinfectant wi
Free
Chlorine
pH8,
5 mg/L
8.5x1 04
(0.0)
7.0x103
(1.1)
1.3x104
(0.8)
7.8x103
(1.0)
7.0x103
(1.1)
6.8x103
(1.1)
2.0x103
(1.6)
1.5x103
(1.8)
1.4x103
(1.8)
2.5x103
(1.5)
5.0x102
(2.2)
pH8,
25 mg/L
4.1x104
(0.0)
4.6x1 03
(1.0)
2.0x103
(1.3)
2.5x1 03
(1.2)
3.3x1 03
(1.1)
1.5x103
(1.4)
5.0x102
(1.9)
2.5x1 02
(2.2)
5.0x102
(1.9)
0.0
(>2.9)
0.0
(>2.9)
pH7,
25 mg/L
7.9x104
(0.0)
7.5x102
(2.0)
1.5x103
(1.7)
2.5x102
(2.5)
1.5x103
(1.7)
3.3x103
(1.4)
0.0
(>3.2)
0.0
(>3.2)
3.8x102
(2.3)
0.0
(>3.2)
0.0
(>3.2)
;h water pH (when
Chlorine
Dioxide I
5 mg/L
5.5x104
(0.0)
5.0x102
(2.0)
0.0
(>3.0)
0.0
(>3.0)
0.0
(>3.0)
0.0
(>3.0)
0.0
(>3.0)
0.0
(>3.0)
0.0
(>3.0)
0.0
(>3.0)
0.0
(>3.0)
25 mg/L
5.2x104
(0.0)
2.5x102
(2.3)
0.0
(>3.0)
0.0
(>3.0)
0.0
(>3.0)
0.0
(>3.0)
0.0
(>3.0)
0.0
(>3.0)
0.0
(>3.0)
0.0
(>3.0)
0.0
(>3.0)
adjusted) an
Monochlor-
amine 1
25 mg/L
4.4x1 04
(0.0)
3.5x1 04
(0.1)
2.0x104
(0.3)
1.2x104
(0.6)
1.1x104
(0.6)
2.3x1 03
(1.3)
0.0
(>2.9)
0.0
(>2.9)
5.0x101
(2.9)
0.0
(>2.9)
0.0
(>2.9)
d concentration
Acidified
Nitrite
pH2,
0.1 mol/L,
1.9x104
(0.0)
2.8x104
(0.0)
2.5x104
(0.0)
1.8x104
(0.0)
1.2x104
(0.2)
1.5x104
(0.1)
2.0x103
(1.0)
4.3x103
(0.6)
0.0
(>2.6)
5.0x102
(1.6)
1.5x103
(1.1)
pH3,
0.1 mol/L
1.2x105
(0.0)
1.1X1Q5
(0.1)
6.1x104
(0.3)
4.0x104
(0.5)
5.5x104
(0.3)
4.6x104
(0.4)
4.6x104
(0.4)
7.9x104
(0.2)
6.0x104
(0.3)
3.2x104
(0.6)
5.4x103
(1.4)
Ozone *
2 mg/L
8.0x104
(0.0)
0.0
(>3.2)
0.0
(>3.2)
0.0
(>3.2)
0.0
(>3.2)
0.0
(>3.2)
0.0
(>3.2) +
No Data
0.0
(>3.2)
0.0
(>3.2)
0.0
(>3.2)
PAAJ
25 mg/L
2.6x104
(1.7)
7.3x103
(0.6)
4.8x1 03
(0.7)
3.3x1 03
(0.9)
3.3x1 03
(0.9)
1.5x103
(1.2)
3.5x1 03
(0.9)
2.8x103
(1.0)
1.9x103
(1.1)
5x102
(1.7)
5x102
(1.7)
 tpH ranging from 8.4 to 8.6; free chlorine ranging from 0.9 toll mg/L.    * Flow 314 L/min before flushing.
• Data is from 12 hr from start of treatment.
EPA/600/S-15/168  October 2015

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 Table 4. Surface Concentrations of Surviving Bacillus globigii Spores (cfu/cm2) with Decontaminant Effectiveness (log
 reduction) in Parenthesis for Various Decontamination and Flushing Durations on Corroded Iron Coupons (6.5 cm2) (25°
 to 30°C)
Experimental
Phase
Spore
Injection
Decontamination with Disinfectant
Flushing at
0.3 m/sec
(1 ft/sec)
Time (hr)
0( initial)
1
2
3
4
6
18
22
24
26
44
Disinfe
Free
Chlorine
pH8,
5 mg/L
2.2x105
(0.0)
1.8x105
(0.1)
1.5x105
(0.2)
1.3x105
(0.2)
2.3x105
(0.0)
1.4x105
(0.2)
1.6x105
(0.1)
1.3x105
(0.2)
9.8x104
(0.3)
7.3x104
(0.5)
8.1x104
(0.4)
pH8,
25 mg/L
2.5x1 05
(0.0)
4.2x1 04
(0.8)
3.8x1 04
(0.8)
4.2x1 04
(0.8)
4.1x104
(0.8)
2.8x1 04
(1.0)
2.5x1 04
(1.0)
1.6x104
(1.2)
1.4x104
(1.3)
1.2x104
(1.3)
7.3x1 03
(1.5)
pH7,
25 mg/L
1.7x105
(0.0)
1.5x104
(1.1)
1.5x104
(1.1)
1.7x104
(1.0)
2.0x104
(0.9)
1.7x104
(1.0)
1.8x104
(1.0)
1.2x104
(1.2)
9.8x103
(1.2)
1.3x104
(1.1)
7.3x103
(1.4)
ctant with water pH (w
Chlorine
Dioxide t
5 mg/L
2.2x105
(0.0)
6.3x104
(0.5)
5.6x104
(0.6)
4.9x104
(0.7)
3.5x104
(0.8)
2.7x104
(0.9)
2.5x104
(0.9)
2.7x104
(0.9)
2.0x104
(1.0)
1.6x104
(1.1)
1.5x104
(1.2)
25 mg/L
2.0x105
(0.0)
2.5x103
(1.9)
1.5x104
(1.1)
2.8x103
(1.9)
4.5x103
(1.6)
0.0
(>3.6)
0.0
(>3.6)
0.0
(>3.6)
2.5x102
(2.9)
2.5x102
(2.9)
5.0x102
(2.6)
ien adjuste
Monochlor-
amine t
25 mg/L
1.9x105
(0.0)
2.0x105
(0.0)
2.0x105
(0.0)
1.0x105
(0.3)
1.1X105
(0.2)
9.8x104
(0.3)
2.5x1 04
(0.9)
1.7x104
(1.0)
1.9x104
(1.0)
1.7x104
(1.1)
1.3x104
(1.2)
J) and concentration
Acidified
Nitrite
pH2,
0.1 mol/L
2.2x105
(0.0)
7.0x103
(1.5)
4.9x103
(1.6)
4.0x103
(1.7)
2.4x103
(2.0)
5.2x103
(1.6)
5.0x102
(2.6)
1.0x103
(2.3)
0.0
(>3.6)
2.5x102
(2.9)
0.0
(>3.6)
pH3,
0.1 mol/L
7.1x105
(0.0)
9.7x104
(0.9)
1.5x105
(0.7)
9.9x104
(0.9)
1.8x105
(0.6)
1.1x105
(0.8)
2.7x104
(1.4)
1.5x105
(0.7)
1.6x104
(1.6)
1.9x104
(1.6)
6.6x103
(2.0)
Ozone
2 mg/L
2.0x105
(0.0)
2.5x104
(0.9)
1.5x104
(1.1)
2.0x104
(1.0)
1.9x104
(1.0)
1.2x104
(1.2)
1.4x104
(1.2)+
No Data
4.5x103
(1.6)
1.8x103
(2.1)
2.0x103
(2.0)
PAA±
25 mg/L
1.5x105
(0.0)
8.6x104
(0.2)
7.0x104
(0.3)
8.5x1 04
(0.2)
4.7x104
(0.5)
4.5x1 04
(0.5)
2.3x104
(0.8)
1.9x104
(0.9)
1.6x104
(0.9)
2.1x104
(0.8)
1.6x104
(1.0)
   pH ranging from 8.4 to 8.6; free chlorine ranging from 0.9 to 1.1 mg/L.    * Flow 314 L/min before flushing.     + Data is from 12 hr from start of treatment.
EPA/600/S-15/168  October 2015

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The study concludes: "These data will help individuals such as incident commanders and
drinking water utility personnel make informed decisions about how to decontaminate a drinking
water distribution system after a biological contamination incident. Ultimately, decontamination
performance along with the cost and effort of disseminating disinfectants over a sufficient area
of the distribution system will dictate their use."


REFERENCES


[1] AWWA Disinfection Systems Committee. 2008. Committee Report: Disinfection Survey, Part
1 - Recent changes, current practices, and water quality. Journal AWWA, 100(10):76-90.

[2] Szabo, J. G., Meiners, G., Heckman, L, Rice,  E. W, and Hall, J. 2015. Decontamination of
Bacillus spores  adhered to iron and cement-mortar drinking water infrastructure in a model
system using disinfectants.


[3] U.S.  EPA. 2008. Pilot-scale Tests and Systems Evaluation for the Containment, Treatment,
and Decontamination of Selected Materials from T&E Building Pipe Loop Equipment.
Washington, D.C.:  U.S. Environmental Protection Agency.  EPA/600/R08/016.


CONTACT INFORMATION

For more information, visit the EPA Web site: http://www2.epa.gov/homeland-securitv-research.

Technical Contact: Jeff Szabo (szabo.jeff@epa.gov)

General Feedback/Questions: Kathy Nickel (nickel.kathv@epa.gov)

If you have difficulty accessing this PDF document, please contact Kathy Nickel
(nickel.kathy@epa.gov) or Amelia McCall (mccall.amelia@epa.gov) for assistance.
  U.S. EPA's Homeland Security Research Program (HSRP) develops products based on scientific
  research and technology evaluations. Our products and expertise are widely used in preventing,
  preparing for, and recovering from public health and environmental emergencies that arise from terrorist
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  contaminants that could affect indoor areas, outdoor areas, or water infrastructure. HSRP provides these
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  National Response Framework, statutory requirements, and Homeland Security Presidential Directives.
EPA/600/S-15/168  October 2015

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