United States
Environmental Protection
Agency
Water Engineering Research
Laboratory
Cincinnati OH 45268
Research and Development
EPA/600/S2-87/045 Sept. 1987
&EPA Project Summary
Influence of Phosphate
Corrosion Control
Compounds on Bacterial
Growth
William D. Rosenzweig
The influence of two phosphate
corrosion control compounds on the
growth and survival of coliform and
other heterotrophic bacteria was inves-
tigated. The compounds studied
included Shan-No-Corr* (a sodium,
zinc-metaphosphate) and Virchem 932
(a zinc-orthophosphate).
The investigation was conducted in
three parts:
A. Growth of Citrobacter freundii.
Enterobacter cloacae, and Kleb-
siella pneumonias was studied in
pure culture (laboratory) investi-
gations in the presence of various
concentrations of Shan-No-Corr
(0.1 to 2.0 mg/L of water, as
product) and in the presence of
Virchem 932 (0.01 to 1.0 mg/L
of water, as zinc). In some exper-
iments Fe2O3 (100 ijg/L). an iron
corrosion product, was also
added.
B. Field investigations were con-
ducted in three water distribution
systems. Two interconnected
systems received Shan-'No-Gorr
and the third Virchem 932. Total
coliform, total heterotrophic bac-
teria, and 16 different physico-
chemical parameters of the water
were measured, twice weekly,
before and after the addition of
the corrosion control com-
pounds. Statistical correlations
were sought among observed
changes in bacterial counts and
'Mention of trade names or commercial products
does not constitute endorsement or recommenda-
tion for use
the various physicochemical
water parameters.
C. Model system studies were con-
ducted by adding coliform (C.
freundii) and other heterotrophic
bacteria to a model water distri-
bution system, establishing a
steady-state bacterial population,
adding the phosphate corrosion
control compounds, and then
comparing the growth of the
bacteria before the addition of the
phosphate compounds to growth
after the additon.
Results presented no evidence that
the corrosion control compounds stim-
ulated coliform growth and some
evidence that they might inhibit
growth. Growth of the other heterotro-
phic bacteria might be stimulated by
the compounds, although the evidence
is not conclusive.
This Project Summary was devel-
oped by EPA's Water Engineering
Research Laboratory, Cincinnati, OH,
to announce key findings of the
research project that is fully docu-
mented in a separate report of the same
title (see Project Report ordering
information at back}.
Introduction
One major problem facing water utility
companies is the control of pipe corro-
sion. Each year, hundreds of millions of
dollars are spent by utility companies to
maintain and replace pipes damaged by
corrosion.
Different treatment methods exist to
prevent pipe corrosion in water distribu-
tion systems. The most common treat-
-------
ments are those that rely on the addition
of various inorganic compounds that will
form a protective film on the walls of the
pipes. Included in this group are car-
bonates, silicates, and phosphates
Phosphate corrosion inhibitors are
quite popular and have been used for
over 30 years. They come in three
principal forms orthophosphates, bime-
tallic phosphates, and molecularly dehy-
drated polyphosphates
Objectives of the Project
Due to a lack of information on the
influence of phosphate addition (for
corrosion control) on microbial growth
and the potential public health signifi-
cance, the objective of this project was
to determine the influence of phosphate
addition for corrosion control in water
distribution systems on the survival and
growth of cohform and other heterotro-
phic bacteria. Correlations between
bacterial growth, phosphate addition,
and various physicochemical parameters
(i e., pH, temperature, chlorine levels) of
the water were also investigated
Materials and Methods
The influence of phosphate corrosion
control compounds on the growth of
cohforms and other heterotrophic bacte-
ria was determined by comparing bac-
terial growth in the presence and
absence of the compounds Investiga-
tions were performed on three levels:
A Pure culture investigations in the
laboratory.
B Field investigations in three, small,
public groundwater distribution
systems.
C Model system investigations in the
laboratory
This investigation utilized two com-
mercially available phosphate corrosion
control compounds. Shan-No-Corr, a
sodium, zinc-metaphosphate manufac-
tured by the Shannon Chemical Com-
pany, Malvern, PA, and Virchem 932, a
zmc-orthophosphate manufactured by
Technical Products Corporation, Ports-
mouth, VA
Pure Culture Investigations
Experiments were performed in the
laboratory to determine the influence of
various concentraitons of Shan-No-Corr
and Virchem 932 on the growth of pure
cultures of C. freundii (ATCC 8090), E.
cloacae (ATCC 13047), and K. pneumo-
niae (ATCC 13883).
Cohforms for the studies were cultured
for 24 hr in tryptic-soy broth (Difco).
Serial dilutions of 1 mL of this culture
were done in sterile saline (0.85% w/v,
pH 7.0) to obtain a final dilution of 10~6.
A 1 -mL dose of this suspension (approx-
imately 1,000 to 6,000 cells/mL) was
then added to 99 ml of sterile, dechlo-
rinated tap water (in a 250 ml flask)
amended with a phosphate corrosion
control compound. The tap water was
batch dechlormated with activated car-
bon and sterilized by membrane filtra-
tion. Shan-No-Corr concentrations of
01,02, 0.3, 0.5, 1.0, 1.5, and 2.0 mg/L
(as product) were tested. In the case of
Virchem 932, the concentrations tested
were 001, 0.1, 0.3, 0.5, 0.75, and 1.0
mg/L (as zinc). In both studies, tap water
without the phosphate corrosion control
compound was used as a control. Various
chemical characteristics of each batch of
tap water (total and free chlorine, pH,
alkalinity, turbidity, sparged and non-
sparged total organic carbon, total,
f iltrable, and dissolved solids, nitrogen as
N02-N03, zinc, orthophosphate, and total
phosphate) were determined, prior to
dechlormation, according to Standard
Methods (1985) The flasks were incu-
bated at 25°C and the coliforms were
counted after 24, 48, and 168 hr by the
spread plate procedure using one-tenth-
strength tryptone-glucose-yeast agar as
the plating medium, an incubation time
of 48 hr for C. freundii and E. cloacae
and 168 hr for K. pneumoniae, and an
incubation temperature of 35°C.
Since the phosphate corrosion control
compounds interact with iron corrosion
products, it was of value to determine
if there were any synergistic effects
between the iron corrosion products and
the phosphate compounds on coliform
growth. To test this, pure culture exper-
iments, as described above, were con-
ducted with the addition of 100 //g of iron
(as FesOsl/L of water. An additional
control, consisting of tap water, FeaOa,
and no phosphate compound was added.
Field Investigations
A field study was undertaken to
deter mine the influence of Shan-No-Corr
and Virchem 932 on the growth of
coliforms and other heterotrophic bacte-
ria in operating water distribution
systems.
Three, small, public, water distribution
systems m Chester County, PA, were
used in the field investigations:
A. Bradford Glen: supplies ground-
water chlorinated at a dose of 1.0
mg/L. The system consists of
100% ductile iron pipes and
approximately 250 services. The
system was started in 1975 and
portions are under construction as
houses are being built.
B. Spring Run: supplies groundwater
chlorinated at a dose of 1.0 mg/L.
The system consists of 30% ductile
iron/70% asbestos/cement pipes
and approximately 220 services.
The system was installed during
the period 1972-1975.
C Federal Drive: supplies ground-
water chlorinated at a dose of 1.0
mg/L. The system consists of
100% ductile iron pipes and 67
services. The system was installed
during the period 1972-1 975.
Sampling
The systems were sampled, at least
twice a week, at or near the source water,
at system midpoint, and at the end. At
each sampling site, four 900-mL serial
water samples were collected in clean,
sterile polypropylene bottles containing
0.2 mL of a 20% (w/v) sodium thiosulfate
solution added before autoclaving. The
sodium thiosulfate was added to neutral-
ize any residual chlorine present in the
water. All samples were protected from
sunlight and transported with non-
contaminating artificial coolants to the
laboratory for immediate processing. A
pre-additive, 1985 (prior to addition of
either Shan-No-Corr or Virchem 932)
sampling phase of 2 months for Bradford
Glen and Spring Run and 3 months for
Federal Drive was performed to deter-
mine the background bacteriological and
chemical profile of the systems. After the
pre-additive sampling phase, the phos-
phate corrosion control compounds were
added and sampling (1985 post-additive
phase) continued until the winter. Sam-
pling was stopped during the winter,
although the phosphate compounds
were added throughout the winter.
Sampling resumed the following spring
(1986 post-additive phase).
Addition of Phosphate Corrosion
Control Compounds
-------
After the pre-additive, 1985 sampling
phase, Shan-No-Corr was added to
Bradford Glen and Spring Run (treated
as one system because of an intercon-
nect) water at a rate of 3.0 mg/L (as
product) for 4 months (for passivation)
and then reduced to 1.0 to 1 5 mg/L (for
maintenance). Virchem 932 was added
to Federal Drive water at a rate of 1.0
mg/L (as zinc) for 1 month (for passi-
vation) and then reduced to 0.3 mg/L (for
maintenance). The pH of all systems was
adjusted to pH 6.5 to 7.0, according to
manufacturer recommendations, with
commercial caustic soda (50% NaOH)
Bacteriological Analysis
The following bacteriological tests
were performed on the samples'
A. Portions of each sample (100 mL)
were analyzed for total cohforms by
the membrane filter procedure
(Standard Methods, 1985) using
mENDO LES medium (Difco) and
24 hr incubation at 35°C. Typical
coliform colonies were verified by
growth and gas production in lauryl
tryptose broth (Difco) and brilliant
green bile broth (Difco). Approxi-
mately 23% of verified isolates
were identified by the API Rapid E
system (Analytab Products).
B. Heterotrophic plate counts were
performed on 5 to 20 mL portions
of each sample by the membrane
filter procedure (Standard
Methods, 1985) using R2A
medium and 168 hr incubation at
28°C. After incubation, the colo-
nies were counted and categorized
by color (red, orange, yellow, and
other). For comparative purposes,
every fifth sample was analyzed
(total counts only) by the spread
plate procedure (Standard
Methods, 1985) (1 mL of sample,
R2A medium, and 168 hr incuba-
tion at 28°C) and by the standard
pour plate procedure (Standard
Methods, 1985).
Chemical Analysis
Various physicochemical parameters
of the water were determined (Standard
Methods, 1985). Free and total chlorine
and temperature were measured in the
field while turbidity; pH; alkalinity; total,
filtrable, and dissolved solids; orthophos-
phate, total phosphate (acid-
hydrolyzable); non-sparged and sparged
total organic carbon; nitrite-nitrate
nitrogen; zinc; and calcium were mea-
sured in the laboratory.
Model System Investigations
A model water distribution system was
constructed as the third component of
this research
The model system was a re-circulating
system (Figure 1) and consisted of three
asbestos/cement and three ductile iron
pipe segments (4-inch diameter, 3 feet
in length). Each pipe segment was
supplied with Bradford Glen well water
from a 10-gallon reservoir. Polyvinyl
chloride tubing connected each pipe
section to its own individual reservoir.
For each pipe type, one segment served
as a control, one received Shan-No-Corr,
and the third received Virchem 932. The
system was inoculated with heterotro-
phic bacteria to establish a steady-state
population of approximately 104 to 10s
cells/mL. The heterotrophic population
consisted of coliform and non-cohform
bacteria isolated from the distribution
systems They were added at approxi-
mately the same ratio (colifornrrnon-
coliform) observed in the distribution
systems The model system was run at
25°C and at a flow rate (0.3 ft/sec)
sufficient to induce turbulent flow and
insure complete mixing of the water.
At the conclusion of the last experi-
ment, various parts of the model system
were sampled for cohforms Areas
sampled included the inner wall surface
of the ductile iron and asbestos-cement
pipes, reservoirs, and the polyvinyl
chloride tubing. Sampling was accom-
plished by swabbing each area with two
sterile cotton swabs andthen placing one
swab into a tube of lauryl tryptose broth
(Difco) and the other into tryptic-soy broth
(Difco). In the case of the pipes, a sterile
stainless steel spatula was used to
scrape the pipe wall and the collected
material was placed into the broths. Use
of the spatula was necessitated by the
rough surface of the pipe walls. Swab-
bing with a cotton swab would have
resulted in loss of the cotton from the
swab. Tubes demonstrating growth after
48 hr were examined for cohforms by
standard procedures (Standard Methods,
1985).
The model system was generally
sampled daily following the same pro-
tocol used in the field investigations. The
influence of the following physicochem-
ical parameters on the growth of the
bacteria were tested:
A. Phosphate corrosion control
compounds.
B. As in A but with the pH increased
from approximately 6.2 (natural pH
of the water) to 8.5.
C. Phosphate corrosion control com-
pounds along with an assimilable
carbon source (50 /^/g of total
organic carbon as glucose/L of
water).
Each experiment was run until a
steady-state (or predictable rate of
growth) heterotrophic population (coli-
form and non-coliform) was attained.
Prior to the beginning of each new
experiment, the heterotrophic population
within the system was adjusted (if
necessary) to the starting point of 103
to 104cells/mL.
Reagent/Culture
Addition
10-gal
Reservoir
Pump, Mag Drive, Polypropylene
Head and Fittings
Sampling
rjaiii/jiiny
r— " —|
Figure 1. Diagram of model system.
-------
Analysis of Data
Analyses were performed using the
BMDP Statistical Software available on
Prime computer systems, and no data
were treated as outliers.
Pure Culture Investigations
The equivalence of repeated runs with
the same compound - organism set was
tested by one-way Analysis of Variance
(ANOVA), grouped by run. At the same
time the Levene test for equal variances
was performed The significance of
grouped means was tested over the
range of concentrations by multiple
linear regression, and against the control
by Student's t-test.
Field Investigations
Field data were tested by both para-
metric and non-parametric methods.
Correlations of bacteriological and phys-
icochemical parameters over the test
period were sought using multiple linear
regression for all systems combined or
simple linear regression for individual
systems. Spearman and Kendall rank
correlations were also performed for the
same data sets. The significance of
correlations was tested using, where
appropriate, tables of critical values for
the appropriate r- or t-transforms
Equivalence of parameters for the pre-
additive and post-additive periods was
tested by a one-way ANOVA grouped by
period and the significance of differences
further tested by a separate-variance t-
test. In addition, simple linear regres-
sions on the two data sets were also
performed and tested by appropriate
methods. Both direct and LOdo bacterial
counts were used in these analyses.
Results and Discussion
Pure Culture Investigations
Growth of C. freundii, E. cloacae, and
K. pneumonias was followed in the
presence of various concentrations of
Shan-No-Corr and Virchem 932 in
dechlorinated, filter-sterilized tap water.
In the presence of Shan-No-Corr there
was no significant (Student's t-test and
multiple linear regression analysis)
pattern of growth inhibition or stimula-
tion when compared to the control after
24,48, 168 hr of incubation The addition
of 100 /ug/L of iron (as Fe203) did not
change the results.
In the presence of Virchem 932 growth
of E. cloacae was not significantly
(Student's t-test) different from the
control. C. freundii showed a significant
(Student's t-test) decrease in growth
(Table 1) after 48 and 168 hr of incu-
bation in the presence of 0.3 mg/L and
above of Virchem 932. At concentrations
less than 0.3 mg/L or after 24 hr of
incubation, there was no significant
difference in growth. K. pneumoniae
showed a significant (Student's t-test)
decrease in growth (Table 2) after all
three incubation times and generally at
Virchem 932 concentrations of 0.3 mg/
L and above. The addition of 100 //g/L
of iron to the tap water enhanced the
inhibitory action of the Virchem 932 with
all three coliforms significantly (Stu-
dent's t-test) inhibited.
Chemical analysis of the tap water
used in the pure culture investigations
indicated that the different batches of tap
water used were uniform and did not vary
greatly in their chemical properties.
Field Investigations
Investigations were performed in three
water distribution systems comparing
coliform and heterotrophic bacterial
counts prior to the addition of phosphate
corrosion control compounds to counts
obtained after the addition of the com-
pounds. In Bradford Glen (BG), Spring
Run (SR), and Federal Drive (FD) the
mean total coliform counts per 100 ml_
sample for the period prior to the addition
of the corrosion control compounds (pre-
additive 1985) were 2.14,1 19, and 1 88,
respectively (Figure 2). Mean heterotro-
phic bacterial counts per mL were 6.43
for BG, 7 29 for SR, and 5 33 for FD
(Figure 3) After the addition of Shan-No-
Corr to BG and SR and Virchem 932 to
FD (post-additive 1985), there was no
significant change (Student's t-test) in
the mean total coliform counts (Figure
2). However, the mean heterotrophic
counts increased significantly (Student's
t-test) in BG and SR (Figure 3). There was
no significant change in FD. The corro-
sion control compounds were added over
the 1985-1986 winter and sampling
resumed in the spring of 1986 (post-
additive 1986) Mean total coliform
counts obtained in 1986 were signifi-
cantly lovver (Student's t-test) m BG and
FD when compared to the pre-additive
1985 counts (Figure 2). Although lower,
the count in SR was not significantly
different from the 1985 count. Mean
heterotrophic counts obtained in 1986
were significantly (Student's t-test)
increased in all three systems when
compared to the pre-additive 1985
counts (Figure 3).
Statistical analyses revealed little
evidence that the corrosion inhibitors
stimulate coliform growth in water
Table 1.
Growth ol'Citrobacter Freundii in the Presence of Various Concentrations of Virchem
932
Time Cone
(hi {mg/L}
24 O.O
0.01
0.1
0.3
0.5
0.75
1.0
48 00
0.01
0.1
0.3
0.5
0.75
1.0
168 0.0
0.01
0.1
0.3
0.5
0.75
10
Mean Cell Count
+ SD (per ml}*
4O4O ±
180.6 +
252 7 +
28.6 ±
77±
7.3 +
4.3 +
12.3 +
31.9 ±
8.2 ±
02±
57 ±
345 ±
4.3 ±
66 +
65±
7.0 ±
39 +
1.8 +
2.9 ±
05 +
691 2O
275.38
442 10
1932
10.46
7.20
4.14
19. 38 £4
56 34 E4
14.98E4
028 E4
1003 E2
3541 E2
3.60E2
1 61 E5
1.40 E5
257E5
3 14 E5
2.61 E5
3.69 E5
054E5
Level of
Significance^
/VS
/VS
/VS
/VS
/VS
NS
/VS
/VS
<0.02
<0.05
<005
<005
NS
/VS
<002
<0001
<0001
<0001
"Mean of 4 runs. 3 plates/run
^Student's t-test; NS, not significant (p >0.05J
-------
Table 2.
Time
Growth of Klebsiella Pneumomae in the Presence of Various Concentrations of
Virchem 932
Cone
(mg/L)
Mean Cell Count
+ S.D. (per ml)*
Level of
Significance^
24
48
00
0.01
0 1
03
05
075
1 0
00
0.01
0 1
03
05
0.75
1 0
96 1 ± 71.03 E3
1055 +81 13E3
19.4± 6.47E3
6.0 ± 3.39 E3
57 ± 793E3
1 2 ±91 OOE3
1.1 ± 036 E3
93.8 + 20.38 E4
131 0 ±5806E4
970 + 1728E4
382 ±3801 E4
6.7 + 4.72 E4
1 9± 1 69E4
29.2 ± 42 85 E4
NS
<0.01
<0.002
<0002
<0.05
<0.002
/VS
/VS
<0.002
<0.002
<0.001
<0.001
168
00
001
0.1
03
05
0 75
1 0
85 ± 1 06 E5
126+ 321E5
8 1 ± 1 15 E5
5.9 ± 1.96E5
6.6 ± 361 E5
54 + 344E5
55+ 277E5
--
<0005
NS
<0002
/VS
<005
<0.01
'Mean of 3 runs, 3 plates/run
UStudent's t-test. NS. not significant (p >0 05)
25 •
2.0 •
Mean Cell ; 5 .
Count per
WO mL of
Sample 7 0 .
05 •
n n .
WT
1"'
\<£
I
&*y
?
t
§1
sC"
n
^
1
Is
i
Z
/j
^
/i
\
iaj Pre-Corrosion
Control Additive
(1985)
Q Post-Corrosion
Control Ad/t/ve
(1985)
l~l Post-Corrosion
Control Aditive
11986)
BG SR
System
Figure 2. Mean total coliform counts
distribution systems. In addition, the
observed increase in heterotrophic
counts, after the addition of the inhib-
itors, could not be conclusively correlated
to the phosphate compounds While
strong, consistent correlations do exist
between the heterotrophic counts and
indicators of the presence of the mhib-
FD
itors (e.g , total phosphates and zinc),
they are tempered by other strong
correlations in uncontrolled water
parameters such as nitrogen levels
The API Rapid E system identified 23%
of the isolated coliforms. C. freundii, E.
cloacae, and K. pneumoniae accounted
for 94% of the identified isolates prior
to the addition of the phosphate corrosion
control compounds and 81% of the
isolates after the addition.
The spread plate procedure using R2A
medium and 28°C for 168 hr proved far
superior to the standard pour plate
procedure in recovering heterotrophic
bacteria. Counts were 10 to 13 times
higher with the spread plate procedure
than with the pour plate method.
Model System Investigations
Six experiments were conducted with
the model system and consisted of the
following treatments of the water:
A. Passivation dosages of Shan-No-
Corr and Virchem 932.
B. Maintenance dosages of Shan-No-
Corr and Virchem 932.
C. Same as B.
D. Maintenance dosages of Shan-No-
Corr and Virchem 932; pH of water
adjusted to 8.0
E. Maintenance dosages of Shan-No-
Corr and Virchem 932 plus 50 ug
of carbon as glucose per L of water.
F. Same as E.
The model system was inoculated at
the beginning of each experiment (if
necessary) with non-coliform hetero-
trophs and C. freundii to achieve an
approximate total population of 104 to
105/ml_ (C. freundii population approx-
imately 101 to 102/100 mL). All exper-
iments were run for 2 to 5 weeks.
Experiment A, which ran for 5 weeks,
was re-inoculated with C. freundii (to a
level of 104/100 mL) after approximately
2 weeks.
In* all experiments we were able to
establish the desired non-coliform het-
erotrophic population, however, we were
unable to establish a coliform population
that was detectable by the standard
membrane filtration procedure. The
various treatments did not appear to have
any influence on the survival and growth
of the established heterotrophic
population.
Swabbing various parts of the model
system (pipes, tubing, reservoirs) isolated
bacteria. However, none of the isolated
bacteria were coliforms
Conclusions and
Recommendations
1 There is little evidence that either
Shan-No-Corr or Virchem 932
-------
Log Mean
Cell Count
per mL of
Sample
1.8-,
1.4.
1.2-
10-
0.8-
0.6-
0.4-
0.2 •
I
vwr
\
I
—
BG SR FD
System
LJ Pre-Corrosion
Control Additive
I1985J
T7\ Post-Corrosion
Control Aditive
{1985}
CD Post-Corrosion
Control Aditive
(1986)
Figure 3. Log mean total heterotrophic plate counts
3.
stimulate coliform growth or sur-
vival in water distribution systems.
However, the pure culture inves-
tigations suggest that coliform
growth is inhibited by Virchem 932.
While field studies with Shan-No-
Corr and Virchem 932 resulted in
increased heterotrophic counts,
this increase could not be conclu-
sively correlated to the presence of
the phosphate corrosion control
compounds. Strong, consistent
correlations do exist but are tem-
pered by other strong correlations
in uncontrolled physicochemical
parameters such as nitrogen.
The field portion of this investiga-
tion was probably not of sufficient
duration to get a good picture of
the relationship between the phos-
phate corrosion control compounds
and bacterial growth. Yearly sam-
pling over a 5-year (minimum)
period would probably be more
reasonable.
4. It might be of value to analyze
sampling data of water companies
that add phosphate corrosion con-
trol compounds to their water.
Many companies have been adding
these compounds for decades. A
comparison of pre- and post-
additive data from such companies
might reveal a relationshp between
bacterial growth and the presence
of these compounds.
The full report was submitted in
fulfillment of Cooperative Agreement No.
CR-811613-01-0 by Drexel University
under the sponsorship of the U.S.
Environmental Protection Agency.
William D. Rosenzweig is with Drexel University, Philadelphia, PA. 19104.
Donald J. Reasoner is the EPA Project Officer (see below).
The complete report entitled "Influence of Phosphate Corrosion Control
Compounds on Bacterial Growth."(Order No. PB 87-198 297/AS; Cost: $13.95,
subject to change)
will be available only from:
National Technical Information Service
5285 Port Royal Road
Springfield. VA 22161
Telephone: 703-487-4650
The EPA Project Officer can be contacted at:
Water Engineering Research Laboratory
U.S. Environmental Protection Agency
Cincinnati, OH 45268
-------
United States
Environmental Protection
Agency
Center for Environmental Research
Information
Cincinnati OH 45268
BULK RATE
POSTAGE & FEES P/
EPA
PERMIT No G-35
Official Business
Penalty for Private Use $300
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0000329 PS
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CHICAGO
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