EPA-BOO/2-76-244
November 1976
Environmental Protection Technology Series
|U
PROCEEDINGS OF WORKSHOP ON
MICROORGANISMS IN URBAN STORMWATER
~*
:
- V
'ii;X. ' i -i.v.y-''j>'
jJ*-- " "•: -„ %
.
* • v ? * > '->>-V<^-
' •; •- l-;a>-- '^
^.w*ii
I
£s>1-'-itvt'l-^lfc'Xk/.':--»V-. -* ' •£*
-------
EPA-600/2-76-244
November 1976
PROCEEDINGS OF WORKSHOP ON
MICROORGANISMS IN URBAN STORMWATER
Edison, New Jersey
March 24, 1975
by
Richard Field
Municipal Environmental Research Laboratory (Cincinnati)
Edison, New Jersey 08817
Vincent P. Olivieri
The Johns Hopkins University
Baltimore, Maryland 21205
Ernst M. Davis
University of Texas at Houston
Houston, Texas 77025
James E. Smith
Syracuse University
Syracuse, New York 13210
Edwin C. Tifft, Jr.
O'Brien & Gere Engineers, Inc.
Syracuse, New York 13201
Based on Project Nos. S802433, R802709, S802400
Project Officer
Richard Field
Storm and Combined Sewer Section
Wastewater Research Division
Municipal Environmental Research Laboratory (Cincinnati)
Ediaon, New Jersey 08817
MUNICIPAL ENVIRONMENTAL RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
-------
DISCLAIMER
This report has been reviewed by the Municipal Environmental Research
Laboratory, U.S. Environmental Protection Agency, and approved for publi-
cation. Mention of trade names or commercial products does not constitute
endorsement or recommendation for use.
ii
-------
FOREWORD
The Environmental Protection Agency was created because of increasing
public and government concern about the dangers of pollution to the health
and welfare of the American people. Noxious air, foul water, and spoiled
land are tragic testimony to the deterioration of our natural environment.
The complexity of that environment and the interplay between its components
require a concentrated and integrated attack on the problem.
Research and development is that necessary first step in problem
solution and it involves defining the problem, measuring its impact, and
searching for solutions. The Municipal Environmental Research Laboratory
develops new and improved technology and systems for the prevention,
treatment, and management of wastewater and solid and hazardous waste
pollutant discharges from municipal and community sources, for the
preservation and treatment of public drinking water supplies, and to
minimize the adverse economic, social, health, and aesthetic effects of
pollution. This publication is one of the products of that research; a
most vital communications link between the researcher and the user
community.
It is with these objectives in mind that the results of this workshop
on microorganisms in urban stormwater are published.
Francis T. Mayo
Director
Municipal Environmental Research
Laboratory
111
-------
. , ABSTRACT
was held--oft- March 29, 1975 at Edison, New Jersey.
the aim was to -exchange Information obtained from USEPA Office of Research
and Development\ Stbofm and .Combined Sewer Program sponsored projects so as
to foster -a better understanding of mierodrganisms in urban storm runoff
and combined sewer overflow.' ".
Workshop emphasis was placed on the following aspects:
a. Procedures for pathogenic microorganism assays
b. Relationship between pathogenic and coliform group microorganisms
c- Disinfection and aftergrowth of microorganisms
d. Viruses in stomswater
IV-
-------
CONTENTS
Foreword lii
Abstract iv
List of Workshop Participants vl
Acknowledgments vill
Microorganisms In Urban Stormwater—A U.S. Environmental
Protection Agency Program Overview
Richard Field 1
Experience On The Assay Of Microorganisms In Urban Runoff
Vincent P. Olivier! and Stephen C. Rigglo 8
Public Health Aspects Of Surface Waters In The Woodlands
E. M. Davis, J. D. Moore, and D. Casserly 52
Experiences With Recovery of Viruses From Storm Water
J. E. Smith 88
The Enhancement Of High-Rate Disinfection By The Sequential
Addition Of Chlorine And Chlorine Dioxide
Edwin C. Tifft, Peter E. Moffa, Steven L. Richardson, and
Richard field 96
-------
LIST OF WORKSHOP PARTICIPANTS
LECTURERS
Ernst M. Davis, Ph.D., Associate Professor, University of Texas at Houston,
Health Science Center, School of Public Health, Houston, Texas 77025
Richard Field, WORKSHOP DIRECTOR, Chief, Storm & Combined Sewer Section,
Wastewater Research Division, Municipal Environmental Research Laboratory,
Office of Research and Development, U.S. Environmental Protection
Agency, Edison, New Jersey 08817
Vincent P. Olivieri, Sc.D., Assistant Professor, The Johns Hopkins University,
School of Hygiene and Public Health, Department of Environmental Health,
615 N. Wolfe Street, Baltimore, Maryland 21205
James E. Smith, Ph.D., Associate Professor of Microbiology, Department of
Biology, Syracuse University, 130 College Place, Syracuse, New York 13210
Edwin C. Tifft, Jr., Ph.D., Laboratory Supervisor, O'Brien & Gere Engineers,
Inc., 1304 Buckley Road, Syracuse, New York 13201
PARTICIPANTS -
Felipe C. Alfonso
School of Hygiene and Public Health
The Johns Hopkins University
615 N. Wolfe Street
Baltimore, Maryland 21205
Gerald Berg
Biological Methods Branch
Environmental Monitoring and
Support Laboratory
U.S. Environmental Protection Agency
Cincinnati, Ohio 45268
Fran Brezenski
Chief, Technical Support Branch
Surveillance & Analysis Division
Region II
U.S. Environmental Protection Agency
Edison, New Jersey 08817
David J. Cesareo
Storm & Combined Sewer Section
Municipal Environmental Research
Laboratory-Cincinnati
U.S. Environmental Protection Agency
Edison, New Jersey 08817
Cecil W. Chambers
Treatment Process Development Branch
Wastewater Research Division
Municipal Environmental Research
Laboratory
U.S. Environmental Protection Agency
Cincinnati, Ohio 45268
Carl 0. A. Charles
Storm & Combined Sewer Section
Municipal Environmental Research
Laboratory-Cincinnati
U.S. Environmental Protection Agency
Edison, New Jersey 08817
vi
-------
Francis Condon
Transport & Treatment Systems Branch
Municipal Pollution Control Division
U.S. Environmental Protection Agency
Washington, B.C. 20460
Fred Ellerbush
Industrial Waste Treatment Research
Laboratory
U.S. Environmental Protection Agency
Edison, New Jersey 08817
Chi-Yuan Fan
WORKSHOP CO-DIRECTOR
Storm & Combined Sewer Section
U.S. Environmental Protection Agency
Edison, New Jersey 08817
Edwin E. Geldreich
Microbiological Treatment Branch
Water Supply Research Division
Municipal Environmental Research
Laboratory
U.S. Environmental Protection Agency
Cincinnati, Ohio 45268
Steven Lawrence Goldstein
School of Hygiene and Public Health
The Johns Hopkins University
615 N. Wolfe Street
Baltimore, Maryland 21205
Irwin Katz
Microbiology Section
Technical Support Branch
Surveillance & Analysis Division
Region II
U.S. Environmental Protection Agency
Edison, New Jersey 08817
Cornelius W. Kruse
Department Chairman
Department of Environmental Health
School of Hygiene and Public Health
The Johns Hopkins University
615 N. Wolfe Street
Baltimore, Maryland 21205
Jan Alan Markowitz
School of Hygiene and Public Health
The Johns Hopkins University
615 N. Wolfe Street
Baltimore, Maryland 21205
Hugh E. Masters
Storm & Combined Sewer Section
Municipal Environmental Research
Laboratory-Cincinnati
U.S. Environmental Protection Agency
Edison, New Jersey 08817
Peter E. Moffa
O'Brien & Gere Engineers, Inc.
1304 Buckley Road
Syracuse, New York 13201
Clive C. Rutherford
Onondaga County
Department of Public Works
25 Elwood Davis Road
North Syracuse, New York 13201
Anthony N. Tafuri
Storm & Combined Sewer Section
Municipal Environmental Research
Laboratory-Cincinnati
U.S. Environmental Protection Agency
Edison, New Jersey 08817
vii
-------
ACKNOWLEDGMENTS
Mr. Chi-Yuan Fan, previously staff engineer of the Storm and Combined
Sewer Section and now managing engineer with Envirosphere, Inc.j New York,
NY deserves special recognition for devoted effort in helping to organize
the workshop.
Aside from the lecturers, the other workshop participants (listed on
pages vi and vii) are thanked for their contributions in discussions and
their reviews of various facets o£ the projects involved with the workshop.
These efforts have subsequently helped to better direct the EPA Stormwater
Program.
Sincere thanks are given to Mrs. Elizabeth H. Mjohary for her most
valuable assistance in organizing these proceedings and in typing.
Richard Field
Workshop Director
vii!
-------
MICROORGANISMS IN URBAN STORMWATER --
A U.S. ENVIRONMENTAL PROTECTION AGENCY PROGRAM OVERVIEW
by
Richard Field
Urban storm discharges whether they be combined with municipal sewage or
separate stormwater occur on an intermittent and random basis. After rain-
fall, these flows exhibit highly varying patterns in both pollutant and
microorganism quality and hydraulic quantity over short periods of time. A
sewer or channel can flow from completely dry to a 1000 times the nearly
steady-state flow conditions of sanitary wastewater. Temperature fluctuation
is also much greater for stormwater than for sanitary sewers which points to
the possible importance of temperature in addition to the usual time and
dosage disinfection control parameters. Consequently, it has been difficult
to adapt existing analytical and disinfection methods to microorganisms in
storm flow.
Three basic needs have ariain for the control of microorganisms in storm
flow:
First, to know its microorganism pathogenetic quality and the pathogens'
relationships to other indicator microorganisms. The pathogen indicators, i.e.
total collform and sometimes fecal coliform and fecal streptococcus, are con-
ventionally used in stormwater studies. These parameters have been adopted
almost blindly out of their routine use for sanitary sewage analysis into a
new consideration — urban stormwater. Resulting analytical data can mis-
-------
represent the actual harmful potential or pathogenic microorganism contents
of the storm flow dealt with. Extremely high coliform counts can come from
sources such as soils and animals, other than humans. Unnecessary and
costly disinfection facilities could result or possibly underdesigned
facilities could also result;
The second need is to develop high-rate disinfection systems to save on
large tankage or dosage requirements for the high storm flow rates en-
countered; and
Third, to develop disinfection facility design and operation techniques
for the highly varying qualitative and quantitative character of the storm-
generated inflows.
I do not want to give the impression that methods for a truer analysis
of pathogen quality, and more rapid and better operation of disinfection
facilities are no longer needed for domestic waste flows — for we all know
that they are. What I am implying is that the need is more apparent for
storm flows because the dry-weather municipal waste flows are nearly steady-
state, more historical data is known for them, and they are less intense as
compared to wet-weather flows.
Over the past seven or eight years our urban stormwater research,
development, and demonstration program has instituted and implemented a
number of projects wholly or partially dedicated to the determination or
disinfection of microorganisms in urban stormwater and combined sewer
overflows; and some impressive results have been obtained. Some of these
results will be discussed here today by others.
-------
Previous stormwater program ventures in the area of microorganisms
and disinfection included:
First: Thecharacterization of indicator microorganisms, where it was
found that coliform counts ranged from the order of magnitude found in
domestic wastewater to a few orders less; however, in almost all cases,
disinfection would be required based on the present standards recommended.
Second: On-site disinfectant generation by raw materials' batching and
2
electrolysis in. order to provide the large and intermittantly unpredict-
able quantities of disinfectant required economically; and
Third: High-rate disinfection with the help of more rapid oxidants (e.g.
345 3
chlorine dioxide and ozone ' ), two stage "booster" disinfectant dosing,
4-6
and by imparting greater turbulence by static and mechanical means. We
have made a special point not to just arbitrarily increase dosages without
regard for the potential residual toxlclty problems. In this regard,
chlorine dioxide which does not form chloramines, and ozone with its re-
latively short half-life, may also be the better choice.
In our study In Philadelphia, it was found that by using principles
from the field of particle flocculation for potable water treatment, a dis-
infection tank with closely spaced corrugated baffles could be designed to
provide adequate coliform kills in under two minutes contact time with less
than three mg/1 chlorine. The closely spaced baffle arrangement also
insures plug flow for optimum utilization of the tank volume. Four mg/1 of
ozone with a contact time of 3.3 minutes was also shown to be effective in a
side-by-slde comparison. "
-------
At present, ozone does not appear as suitable as chlorine due to com-
parative costs. This is most likely caused by the power requirements for
on-site ozone generation. However, the use of ozone should not be over-
looked since the cost gap may be closing and there may be an economically de-
fined need for on-site chlorine generation when it comes to storm flow appli-
cation (as previously discussed). Other advantages of ozone are that it —
(a) is a more powerful oxidant reacting more vigorously with organics, (b) de-
structs phenols rather than forming obnoxious chlorophenols, (c) may be a
better viriclde, and (d) by the method of air application increases effluent
dissolved oxygen.
Again, some of the ongoing research that will be discussed today covers
the areas of primary need which Include characterization, high-rate disinfec-
tion, and adequate operational control.
In 1962, the ASCE committee on Public Health Activities examined the
relationship between collform concentration in bathing waters and the
occurrence of enteric diseases among bathers. It was stated that "there is
a remarkable lack of evidence that high concentrations of colifonn organisms
in recreational waters are accompanied by enteric disease cases." In a
recent EPA statement on proposed wastewater disinfection policy mention was
made to eliminate coliform limitations from secondary treatment require-
ments and to establish disinfection requirements case-by-case according to
water quality and public health needs. The benefits from microorganism kill
should be weighed against environmental risks from toxic residuals.
-------
The evidence points to a strong need for more effective analysis and'
disinfection of microorganisms. Perhaps we. all agree that the only con-
ceivable way to go about this is by the interrelation with carefully planned
epidemiological studies; however, we may also agree tha.t in the interim the
development of more meaningful relationships between, levels of the customary
indicators to pathogenic microorganisms, and direct pathogen examination
could lead us in the right direction towards an -ultimate cure.
The principal purpose of today's workshop is for an exchange of project
information and data, and to establish, liasons between principal investigators
on three EPA sponsored research projects for the mutual benefit of their
respective investigations. The first speaker will be Dr. Vincent Olivieri
from the Johns Hopkins' School of Public Health and Hygiene. His project,
"Microorganisms in Stormwater»" began in May 1974 and is-slated for comple-
tion in December 1975. The objective of Dr,. Olivier!'s study (EPA Research
Grant No. R802709) is to provide basie information for the conduct of 'more
scientific evaluations on the health hazard potential from-bacteria in urban
storm flows. . - • • '•
f-' • '
The next speaker will be Dr. Ernst Davis from the University of Texas'
School of Public Health. His study is under an EPA project - dealing with
total water resources for a planned community near Houston (EPA Research
Grant No. S802433). Dr. Davis will present data from his study to deter-
mine disinfection requirements for the community. Indicator and pathogen
analyses have been conducted on stream and runoff samples. Eventually he
hopes to demonstrate how urbanization influences bacterial quality as the
-------
community develops. The project began during the summer of 1973 and is due
to be completed the summer of 1976. I would like to mention that a colleague
of Dr. Davis' (also involved in the project) is performing fish assays to
analyze the after-effects of chlorine and ozone.
Our third speaker will be Dr. James Smith of Syracuse University who
will discuss viral recovery and inactivation as a result of his experiences
from two stormwater program projects, the previously mentioned Johns Hopkins
study in Baltimore and a demonstration project with Onondaga County in
Syracuse, New York (EPA Demonstration Grant No. S802400).
Lastly, Dr. Ted Tifft of O'Brien & Gere, Consulting Engineers, will
present interesting results from his work on disinfection of combined sewer
overflows with chlorine and chlorine dioxide from the Onondaga study. The
project started in July 1972 and is due to be completed the end of 1976.
Each speaker will have 45 minutes for his discussion, which includes
30 minutes for presentation and 15 minutes for questions and answers. A de-
tailed discussion is scheduled after all presentations are completed. With
the assistance of Frank Condon, who has a thorough familiarity of stormwater
applications, and Cecil Chambers, Ed Geldrlch, Gerald Berg, Irwin Katz,
Cornelius Kruse and Fran Brezinski, who are world renowned experts in the
field of waterborne microorganisms and disinfection, I am hopeful that the
discussion periods will be worthwhile.
-------
REFERENCES
1. Pontius, U. R., et al. Hypochlorination of Polluted Stonnwater Pumpage
at New Orleans. EPA-670/2-73-067. U.S. Environmental Protection
Agency, 1973. 189 pp.
2. Ionics, Incorporated. Hypochlorite Generator for Treatment of Combined
Sewer Overflows. 11023 DM 03/72, U.S. Environmental Protection
Agency, 1972. 90 pp.
3. Moffa, P. E., et^ al_. Bench-Scale High-Rate Disinfection of Combined
Sewer Overflows. EPA-670/2-75-021, U.S. Environmental Protection
Agency, 1975. 181 pp.
4. Cochrane Division, Crane Co. Microstraining and Disinfection of
Combined Sewer Overflows. 11023 EVO 06/70, U.S. Environmental Protec-
tion Agency, 1970. 78 pp.
5. Glover, G. E., and G. R. Herbert. Microstraining-and Disinfection of
Combined Sewer Overflows - Phase II. EPA-R2-73-124, U.S. Environmental
Protection Agency, 1973. 117 pp.
6. Maher, M. B. Microstraining and Disinfection of Combined Sewer Overflows
Phase III. EPA-670/2-74-049, U.S. Environmental Protection Agency, 1974.
82 pp.
-------
EXPERIENCE ON THE ASSAY OF MICROORGANISMS IN URBAN RUNOFF
Workshop on Microorganisms In Stormwater
by
Vincent P. Ollvierl
and
Stephen C. Riggio
The Johns Hopkins University
School of Hygi-ene and Public Health
615 N. Wolfe Street
Baltimore, Maryland 21205
-------
INTRODUCTION
The high levels In urban runoff of microorganisms indicative of fecal
contamination has been well documented in the literature '(Weibei et al> (1),
Geldreich et al. (2), Evans et at. (3), and Burm &t al* (4)). However, little
information is available on the level of pathogenic microorganisms in storm-
water runoff in urban areas.. The specific objectives of this project are
to obtain information on the levels of pathogenic microorganisms in storm-
water and evaluate the relationships between indicator microorganisms and
pathogens.
The evaluation of methods of detection and enumeration is a fundamental
prerequisite for obtaining reliable results. Techniques and procedures
employed in the laboratory for a given set of samples may yield poor results
in another laboratory for a different sample. The microbial flora can vary
significantly in water samples depending on environmental conditions and
sources of contamination. The types and levels of interfering microorganisms
become an important factor and will vary with the procedure employed.
Techniques, methods and culture media developed for microbial assays of
clinical specimens nay yield poor results when applied to water samples
where different interfering microorganisms are present. No one method for
the detection and enumeration of a particular group of microorganism can
be universally employed.
The early phase of the current project Involved the evaluation of
methods of detection and enumeration of the microorganisms to be assayed.
Techniques and culture procedures described in Standard Methods for the
Analysis of Water and Wastewater (5) and in recent literature were evaluated.
Where similar results were obtained, the cultural procedures in Standard
Methods were chosen. "Where the procedures in Standard Methods were found
lacking, alternative methods were developed. The final selection of techniques,
methods and procedures was based on simultaneous analysis of water samples
in our laboratory and information in the literature. Multiple tube dilution
procedures which permitted a calculation of a most probable number (MFN)
were generally favored because of the wide variability in the chemical and
physical characteristics (particularly solids) expected in the samples from
the urban water courses.
Grab samples were collected in the Baltimore metropolitan area and
assayed for the levels of total coliform, fecal coliform, fecal streptococci,
Salmonella, Shigellas Pseudomonaa aerugi-noaa* Staphlocoaaua oupeus, and
animal viruses. Viral assays were performed by Dr. James Smith of Syracuse
University. Two sets of samples were taken In the study. Background samples
were collected from three urban streams and raw sewage on a routine schedule
regardless of rainfall. Recently an upland water source has been added.
These samples are designated background samples. Six samples were collected
during storms at outfalls within the Baltimore metropolitan area to provide
information on the levels of microorganisms in runoff. Both combined and
separate sewers were sampled. Sites were selected to provide a cross section
of drainage areas found in an urban environment.
-------
METHODS
DIFFERENTIAL TESTS
The following tests were employed to provide information for the tenta-
tive identification of isolates obtained from the microbial assays. A spot
inoculation procedure on differential agar plates was employed where possible.
Isolates were transferred with sterile toothpicks to a 35 or 50 place grid
pattern on 100 mm Petri dishes containing the appropriate agar medium.
Phenylalanine deaminase
Isolates were spotted on phenylalanine agar (Difco) and incubated for
24 hours at 37°C. Phenylalanine deaminase activity was indicated by a green
zone around the colony after flooding the plate with a 0.5M ferric chloride
solution (6).
Oxidase
Oxidase production was determined for isolates spotted on tech agar
(Baltimore Biological Laboratory) after incubation at 37°C for 18 to 24 hours.
The tech agar plate was flooded with a 1% p-amlno dimethylaniline taonohydro-
chloride. Oxidase positive colonies turn pink within one minute.
Triple Sugar Iron Agar (TSI)
TSI slants were prepared in 13 x 100 mm culture tubes. Stab-streaks
were prepared for each isolate and incubated at 37°C. pH changes in the
butt and slant were observed after 18 to 24 hours. Hydrogen sulfide pro-
duction was observed after 48 hours incubation.
Lysine Iron Agar (LIA)
LIA slants were prepared as above for TSI and used in conjunction with
TSI as recommended by Edwards and Ewing (7).
Malonate Utilization
Malonate utilization was determined in malonate broth in 13 x 100 mm
culture tubes according to the procedures described in Edwards and Ewing (7).
The change in indicator from green to prussian blue indicated malonate
utilization.
Lysine Decarboxylase
Lysine decarboxylase activity was determined by the method of Moeller
described in Edwards and Ewing (7). Decarboxylase base medium with and
without lysine was inoculated from fresh agar slant cultures, overlaid with
sterile mineral oil and incubated at 37°C. The cultures were examined daily
for four days. Lysine decarboxylase activity was indicated by the change
10
-------
from yellow to violet in the tubes containing lysine. Control tubes without
lysine remained yellow.
Salmonella Polyvalent Antisera
Isolates that yielded typical or suspicious Salmonella reactions were
submitted to preliminary serological examination, with Salmonella 0 antiaerum
Poly A-I including Vi (Difco) by slide agglutination. Positive tests are
indicated by rapid complete agglutination of the bacterial cells.
Urease
Urease activity was determined by the spot inoculation procedure on
plates prepared with Christensen's urea agar (7). Urease activity was indi-
cated by the formation of a red zone around the colony after 2 to 6 hours of
incubation. Delayed reactions could not be observed by this procedure.
Citrate Utilization
Citrate utilization was determined by the spot inoculation procedure
on plates containing Simmons citrate agar. Colony formation and color change
from green to blue in the medium indicated citrate utilization (7).
Coagulase
The elaboration of coagulase was determined by the plate method described
by Esber and Faulconer (8). Isolates were transferred to coagulase agar
(Difco) and incubated overnight. Staph. aureua stock cultures were included
for each fresh batch of media. Coagulase positive and mannitol positive
strains yield a yellow opaque zone around the colony. The plate procedure
was evaluated by comparing the elaboration of coagulase by the conventional
tube procedure for 150 isolates. 100% agreement was observed for the two
procedures.
DNase
DNase production was performed by the method of Streitfeld et al. (9).
Colonies were transferred to DNase test agar (Baltimore Biological Labor-
atories) and incubated overnight. After incubation the plate was flooded
with 0.1% toluidine blue. DNase production is indicated by a rose pink
zone around the colonies.
Lipovitellenin-Lipase
Lipovitellenin-Lipase was determined on Lipovitellenin-salt-mannitol
agar (LSM) (10). Opaque- zones around the colonies indicated lipovitellenin-
lipase activity.
Mannitol Fermentation
Mannitol fermentation was observed on LSM and coagulase agar. Yellow
zone around the colonies was taken as positive mannitol fermentation.
11
-------
Anaerobic Glucose Fermentation
Representative'isolates that yielded typical Staph. aureus reactions
were further tested for the ability to ferment glucose anaerobically by the
method described by Evans and Kloos (11) to separate any possible soil
micrococci. An overnight culture on brain heart infusion (BHI) broth at
37 °C was used to Inoculate tubes of Brewer's fluid thioglycolate medium
(BTM) containing 0.35% agar. Growth throughout the tube after 24 hours was
indicative o£ anaerobic glucose fermentation.
Gram Stain
Gram stains were prepared according to the procedures described in
Standard Methods (5).
Gelatin Liquefaction
Gelatin liquefaction was determined on BHI containing 120 g/llter of
gelatin. Isolates were spot inoculated on plates and incubated at 10°C
for five days. Liquid zones around colonies indicated gelatin liquefaction,
Mannitol and Arabinose Fermentation
Mannitol and arabinose fermentation were determined separately on
plates of phenol red carbohydrate fermentation medium containing 10 g/liter
of each sugar and 2% agar. Plates were incubated at 37°C for 18 hours.
Yellow zones around the colonies indicated fermentation.
Growth in Bile
Growth in 40% bile and 6.5% NaCl was determined on separate BHI agar
plates containing 40 g/liter oxgall and 65 g/liter NaCl. Colony formation
at the point of inoculation after 48 hours incubation at 35°C was considered
an indication of growth.
Growth at 45°C and 10°C
Growth at 45 °C and 10°C was determined on BHI agar after incubation
for 48 hours and five days, respectively. Colony formation at the point
of inoculation was considered an indication of growth.
Catalase
Catalase activity was determined for isolates from the fecal streptococci
assay be adding a drop of 3% hydrogen peroxide to each colony on the BHI
agar replicate control plate after Incubation at 37°C for 24 hours. Gas
bubbles indicated catalase activity. Catalase activity for Stccphyloooooi
isolates was determined by transferring a portion of the colony to 3% hy-
drogen peroxide. Evolution of gas indicated a positive catalase.
12
-------
Starch Hydrolysis
Starch hydrolysis was determined on nutrient agar containing 10 g/liter
of soluble starch and 8 g/liter NaCl. After incubation at 37°C for 48
hours each plate was flooded with Gram's iodine. Clear zones around the
colony indicated starch hydrolysis.
Casein Hydrolysis
Casein hydrolysis was determined on plates of skim milk agar (10 g/liter
skim milk and 15 g/liter agar) after incubation for three to five days at
37°C (12). Hydrolysis of casein was indicated by the formation of clear
zones around the colony. Paeudomonaa isolates were observed for fluorescence
when exposed to ultra-violet light.
Growth on Acetamide
Growth on acetamide • was rechecked by spotting isolates on acetamide
agar (5) and incubated for 48 hours. Pink to red colonies with blue fluor-
escence when exposed to ultra-violet light were considered positive.
Growth at 42°C
Acetamide and oxidase positive isolates were transferee! to Drake's medium
#10 for evaluation of growth at 42°C (13) after 48 hours incubation. Growth
was indicated by turbidity with blue green fluorescence.
13
-------
CULTURE EVALUATION AND PROCEDURES
SALMONELLA sp.
A multiple concentration and enrichment procedure was employed to
permit the calculation of a MPN Salmonella for each sample. The procedure
was similar to that described by Kenner and Clark (14) except that diatomaceous
earth was used for concentration. The multiple concentration and enrich-
ment procedure is shown in schematic form in Figure 1. A three replicate
MPN plus three seeded controls were employed to permit an evaluation of the
recovery procedures for each sample. A laboratory strain, Salmonella typh-
imurivm SB558, resistant to 1000 yg/ml of streptomycin was used as the seed
Salmonella, The Salmonella seed was prepared from cultures stored at -40°C
in 23% glycerol. The low temperature glycerol storage provided a readily
available test organism at a known density. The three seeded controls eval-
uated sample toxicity, diatomaceous earth concentration and the overall
recovery procedure. A 100~fold dilution of the Salmonella seed stock was
prepared fo.r each sample to evaluate sample toxicity. The mixture was
maintained at room temperature for the duration of the sample processing
period and plated on brain heart infusion (BHI) agar containing 1000 ug/ml
streptomycin. Samples were considered toxic when more than 90% inactivation
was observed. The diatomaceous earth concentration was evaluated by the
addition of 5 to 50 Salmonella to a replicate of each sample filtered. The
diatomaceous earth plug was transferred to enrichment medium containing
1000 ug/ml of streptomycin. The recovery of streptomycin-resistant Sal-
monella was considered positive concentration on the diatomaceous earth. The
overall concentration and culture procedure was evaluated with an additional
replicate of each sample prepared as above. The streptomycin, however, was
omitted from the enrichment medium. Isolates obtained from the primary
plates were tested for streptomycin resistance. The isolation of streptomycin-
resistant Salmonella indicated a positive recovery.
During the initial portion of the study various combinations of enrich-
ment media, enrichment temperatures and primary plating media were evaluated
using 22 samples prepared with a multiple concentration on diatomaceous
earth and multiple enrichment. The results are given in Table 1. The
enrichment media and temperatures evaluated were selenite broth at 37°C,
selenite broth at 4l°C, GN broth at 37°C, tetrathionate broth at 37°C and
dulcitol selenite broth at 40°C. The primary plating media employed were
bismuth sulfite (BS), brilliant green agar (EGA), Salmonella Shigella agar
CSS) and xyiose lysine desoxycholate agar (XLD). A. total of 527 isolates
were tested during this phase of the investigation. The elevated temperature
enrichments consistently yielded higher numbers of Salmonella isolates for
primary plating media. The final choice was dulcitol selenite at 40°C
coupled with primary plating on XLD similar to the procedure reported by
Kenner and Clark (14). Identification of the members of the genus Salmonella
was performed according to the schematic given in Figure 2. Typical Salmonella
colonies on XLD (pink colonies with black centers or pink colonies) were
screened for phenylalanine deaminase and oxidase activities. Enrichment
cultures that did not yield typical Salmonella colonies were restreaked at
48 or 72 hours on XT.n and subsequent typical colonies were handled as above.
14
-------
Figure 1. Schematic - Multiple concentration and
enrichment for the MPN determination of Salmonella
Seeded Controls
Sample:
3 or 5 replicates
and seeded con-
trol.
r; r;
Concentration:
diatomaceous
earth.
Multiple Enrichment:
(1) 10 g filter
plug suspended in
1 x enrichment
medium to 50 ml.
Effective volume: ,
0.9 x sample. \
(2) 10-fold dilution:
5.0 ml of (1) to
5.0 ml 1 x enrich-
ment medium.
Effective volume:
0.09 x sample.
(3) 10-fold dilution:
1.0 ml of (2) to
9.0 ml 1 x enrich-
ment medium.
Effective volume:
0.009 x sample.
(4) Subsequent 10-
fold dilutions if
necessary. 1.0 ml
final dilution re-
moved to waste to
maintain serial
dilution.
(
* Seed - 10 to 50 organisma/sample vol. of streptomycin
resistant Salmonella typhiman-um SB558
15
-------
Table 1. COMPARISON OF ENRICHMENT AND PRIMARY
PLATING MEDIA FOR THE ISOLATION OF SAIMONELLA
Enrichment
Temp.
Primacy Pine lag
f Positive
1 Isolates
Primary Plata
f Positive
f T Dieted on
..ufichaent
Enrichment
f Positive
f Isolated on
Earicfment
f Positive
Total f Positive
Selenito F
37*C
BS BGA SS ILD
1 0 2 U
83 16 36 12
1 0 2 11
147 147 147 147
14
147
li
90
Belenita
41*C
BB BOA SS ILD
0 1 37
9 21 67
0 1 37
" 97 97 97
SB
97
38
90
CH
37*0
BS BOA SS XLD
- 0 0 1
- 16 59 45
001
120 120 120
1
120
1
90
Tetrathionate
37"C
BB BGA SS XLD
0000
2 14 32 33
0000
81 81 81 81
0
81
0
90
Dulcltol-Selenite
40'G
BS BOA SS XLD
1 B 28
9 32 41
- .1 8 28
02 82 82
37
82
37
90
Totals
90
527
Note (+) Data based on 22 eaoplea (7 row aewage and 15 urban atrcau) prepared with a Miltipla concentration on cellte
and wiltlple enrlctaent
-------
Figure 2. Schematic - Isolation and identification
of Salmonella sp.
xylose lysine desoxycholate agar
i i
typical Salmonella absence of typical
colonies Salmonella colonies
I
restreak
_
I
typical Salmonella
colonies
I
biochemical screen
phenylalanine oxidase mannitol
deamlnase fermentation
11 r
streak purity
I
triple sugar iron agar
lysine iron agar
typical Salmonella
reactions
i i i
lysine malonata Salmonella
decarb oxylose
t
polyvalent antlsera
I
Salmonella sp.
17
-------
Phenylalanine deaminase negative and oxldaae negative isolates were streak
purified again and transferred to triple sugar iron agar (TSI) and lysine
iron agar (LIA). Typical Salmonella was tested for malonate utilization and
lyaine decarboxylase activity and submitted to a serological examination with
polyvalent antisera A-I (including ?i).
Phenylalanine deaminase and oxidase were employed to eliminate the
most probable interferences from members of the genus Proteus^ Provideneia
and Pseudomonas and, thereby, minimize the number of isolates submitted
to further tests. Table 2 shows a comparison of the phenylalanine deaminase
and oxidase screen with the commonly used TSI-LIA screen for suspicious
Salmonella isolates. Typical Salmonella reactions on TSI-LIA were obtained
for 92.8% of the isolates from XLD that were phenylalanine deaminase and
oxidase negative. The spot inoculation procedure for phenylalanine deaminase
and oxidase compares favorably with the TSI-LIA screen for Salmonella and
provides a rapid, inexpensive and effective method to screen large numbers
of suspicious Salmonella isolates. Table 2 also indicated the relative
efficiency of XLD as a primary plating medium after dulcitol selenite enrich-
ment at 40°C. About 53 percent of the isolates obtained from XLD yielded
typical TSI-LIA reactions. A marked difference, however, can be seen for the
two types of colony morphology for suspicious Salmonella on XLD. 84.5%
of the black centered but only 4.3 percent of the red colonies were positive
through the screening procedure.
Table 3 shows a tentative identification of the genus of microorganisms
commonly encountered during the procedure for the detection and enumeration
of Salmonella. Each of the microorganisms yield suspicious Salmonella
colonies on XLD. The tentative grouping of isolates into a particular genus
was based on a limited number of biochemical tests. The predominant inter-
fering microorganisms were members of the genus Proteus^ Providenoia and
Peeudomonaa* Arizona sp, and CitTabactev sp. were about 3% each of the
isolates tested after the phenylalanine deaminase, oxidase and TSI-LIA
screens.
SEIGELIA sp.
A multiple concentration and enrichment procedure was used to permit
a calculation of a most probable number. Two liters to 3.79 liters were
initially filtered through celite for concentration. The entire plug of
celite was transfered to GN broth for enrichment. Ten-fold dilutions of the
celite suspension were prepared in GN broth. Three replicates were run at
10 to SO microorganisms to evaluate recovery. The multiple concentration
and enrichment procedure for Shigella was similar to that given for Salmon-
ella in Figure 1 except that Shigella sonnei was used for the seed control.
After incubation at 37°C each dilution was streaked on xylose lysine deoxy-
cholate agar and suspicious Shigella colonies (red) were tested biochemically
according to the protocol shown in Figure 3.
Eighteen samples of raw sewage and urban streams were assayed according
to the above procedure. It should be noted that each sample represents
nine attempts to isolate Shigella (3 replicates x 3 dilutions). More than
18
-------
Table 2. COMPARISON OF THE PHENYLALANINE DEAMINASE () AND OXIDASE (OX)
SCREEN WITH TSI AND LIA REACTIONS FOR THE DIFFERENTIATION OF SALMONELLA
Number Typical Salmonella Reactions (%)
Colony morphology
on XLD
Black centered
colony
Red colony
Total
Number of
isolates
6,258
3,457
9,715
<|> and OX
screen
5,286 (84.5)
288 (8.3)
5,589 (57.5)
$ and OX followed
by TSI-LIA
5,142 (82.2)
149 (4.3)
5,186 (53.4)
Table 3. GENUS OF MICROORGANISMS COMMONLY ENCOUNTERED
DURING THE ISOLATION OF SALMONELLA *
Proteus sp. and
Provictencia sp. Peeudomonas sp. Arizona sp. ' Citrobacter sp.
Number
isolated
Number
tested
1,250
8,995
13.9
2,809
8,995
31.6
108
2,967
3.6
96
2,967
3.2
* Tentative identification based on a limited number of biochemical tests
19
-------
Figure 3. Schematic-- Isolation and
identification of Shigella sp.
multiple enrichaent
GS broch 24 hr. at 37'C
priaary placing for Isolation
ZLD 24 hr. ae 37*C
r
typical Shigella. colonies
typical Shigalla
colonies absenc
1
reaCreak on 3CLD
typical Sftigalla colonies
biochemical scraea
urei
1
9« phanylalanin* c:
daaoinaaa
I
_
I
.crata casein oxif
hydrolysis
1
336
i
streak purify
cripla sugar iron agar
lysiaa iron agar
Shigalla
reaction
•cypical Szlxssmlla
reactioo
FP*
polyvalent aaciatra
and
biochenlcal casts
polyvalent ancisera
sad
biochemical tests
* FP indicates false positive
20
-------
1,100 suspicious isolates from XLD were submitted to further biochemical
tests. Shigella was not found in any of the samples tested nor was the
seeded Shigella ever recovered. Approximately 10% of the isolates were
negative in preliminary biochemical screen indicating possible Shigella.
One isolate yielded typical reactions for Shigella on TSI and L1A. but was
serotypically negative and hydrolyzed casein. Table 4 is a tentative identi-
fication of the major groups of microorganisms isolated. Pseudomonaa species,
Providenaia species and non-H2S producing members of the genus Proteus
yield red colonies on XLD agar indistinguishable from Shigella.
Levels of indicator microorganisms and Salmonella for the samples
negative for Shigella are given in Table 5. One would expect to isolate
Shigella from the raw sewage and streams containing relatively high levels
of total and fecal coliforms, fecal streptococci and Salmonella. The limit-
ation, appears to make the methodology poor.
The celite concentration procedure employed appears to function reason-
ably well. Table 6 shows the results of control experiments with low level
seeded Shigella in phosphate-buffered saline. The recovery of Shigella
on celite is well within.the 95% confidence limits for the MPN procedure.
The major difficulty appears to be in the enrichment procedures and methodology.
Shigella does not appear to compete and survive well in the presence of
other actively growing microorganisms. Hentges (15) reported the inhibition
of Shigella when grown with coliforms and Klebsiella. He attributed the
inactivation of Shigella to organic acids produced by the other microorganism
and that a pH below 7 enhanced the effect.
Rather than continue the unproductive Shigella assays, the effort was
directed at providing some information to explain the inability to1 isolate
Shigella. Older literature suggests that Shigella is a fragile microorganism
in the environment. This assumption of fragility is adequately dispelled
by the reports of the ability of laboratory cultures of Shigella to survive
when added to a variety of polluted and non-polluted waters. The absolute
survival times vary markedly from study to study dependent on environmental
conditions. Laboratory experiments indicate that at 20°C Shigella persisted
for 12 days in farm pond water (16). McFeters et al. (17) reported half
time die-off rates of 22.4 hours, 24.5 hours and 26.8 hours for Shigella
dyeenteriae, Shigella sonnei and Shigella flexneri at 9.5 to 12.5°C in well
water. Bartos et al. (18) reported recovery of added dysentery bacilli after
22 days in well water and 40 days in well water with coliforms added.
Dolivo-Dobrovol'skii and Rossovaskaia (19) reported Shigella survivals from
30 minutes 'to four days and indicated that aeration markedly reduced survival
time. Under conditions of extreme cold (-45°C) Shigella persisted 145 days
in feces, 135 days in soil and 47 days in frozen river water (20). Although
conditions vary, Shigella does not appear to be any more fragile than other
pathogens. In fact, where comparative studies were reported Shigella is
significantly more persistent than strains of Salmonella and Vibrio aholerae
(17).
Survival studies conducted in our laboratory also indicated that Shigella
will persist for reasonable lengths of time in aqueous systems. Shigella
flexneri was seeded into phosphate-buffered saline (pH 6.8), sterile storm
21
-------
Table 4. GENUS OF MICROORGANISMS FOUND
DURING ATTEMPTS TO ISOLATE SHIGELLA
10
A.
C.
D.
Site
Raw Sewage
Stream
Stream
PvoteuB
#/Total
221/720
26/106
81/225
sp.
% Total
31
24
36
Providenoia
it/Total %
173/720
17/106
14/225
ep.
Total
24
16
6
Pseudomonas
# /Total %
276/720
39/106
41/225
3p.
Total
38
37
18
Others
///Total %
50/720
24/106
89/225
Total
7
23
40
Total
328/1051
31
204/1051
19
356/1051
34
163/1051
16
-------
Table 5. LEVELS OF INDICATOR MICROORGANISMS AND
SALMONELLA IN SAMPLES NEGATIVE FOR SHIGELLA
Site Date
A. Raw Sewage 10/2
(9/30)
10/7
10/14
10/21
10/28
11/4
11/11
11/18
12/2
12/9
12/17
C. Stream 12/2
12/17
D. Stream 11/4
11/11
11/18
12/2
12/9
TC
MPN/lOOml
2.4
2.4
3.5
2.9
5.4
<2.6
3.5
1.7
2.4
7.0
1.6
2.4
9.2
4.6
2.2
1.7
3.3
3.5
x 107
x 107
x 107
x 106
x 107
x 106
x 107
x 107
x 106
x 106
x 107
x 10*
x 10*
x 102
x 103
x 103
x 10*
x 10*
EC
MPN/lOOml
2
4
4
4
1
<2
7
7
1
3
4
2
3
2
4
3
1
3
.4
.9
.6
.9
.1
.6
.9
.9
.3
.3
.6
.4
.5
.3
.9
.3
.3
.3
x
x
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
107
106
106
10 6
107
106
106
10 6
106
10 6
10 5
10*
10*
102
102'
102
10*
103
FS
tf/lOOml
4.
1.
1.
8.
4.
2.
1.
1.
3.
9.
3.
7.
4.
1.
2.
1.
1.
2.
1
1
2
3
5
0
1
1-
4
7
9
6
2
2
1
2
0
4
x 106
x 106
x 106
x 105
x 105
x 10*
x 106
-x 106
x 105
x 105
x 105
x 10*
x 10*
x 102
x 102
x 102
x 10s
x 10*
Salmonella
MPN/10 1
>2.9
1.2
4.8
5.1
1.7
2.8
5.1
4.8
1.0
2.6
1.3
2.7
4.4
1.3
2.2
7.0
7.0
x 103
x 103
x 102
x 103
x 103
-
x 102
x 102
x 101
x 102
x 101
x 102
x 101
x 10°
x 101
x 101
x 101
x 101
23
-------
CO
Table 6. RECOVERY OF SHIGELLA ON DIATOMACEOUS EARTH
PHOSPHATE-BUFFERED SALINE pH 7.2 TEMPERATURE 20-25°C
Shigella seeded
MPN Recovered
on Celite
95% Confidence
Limit for MPN
S.
# liter
9
13
2-70
eonnei
% Recovery
-
144
22-777
5.
# liter
13
7
1-38
flexneri
% Recovery
-
54
2-292
S.
it liter
10
7
1-38
fiexneri*
% Recovery
_
70
2-292
S. fleameri 3 strain 3-5 Op, resistant to 500 ug/ml streptomycin
-------
water, and stream water at a level of 3 x 106 Shigella/Tnl and stored at
40°C. Samples were removed for determination of the levels of Shigella
flexnevi. Figure 4 shows the survival of Shigella flesmeri. 'After eight
days 37%, 41% and 26% of the seeded organisms remained in the saline, sterile
stream water, and stream water, respectively. The stream water contained
7 x 102 total coliforms, 7 x 102 fecal coliforms, 5.1 x 103 fecal strep-
tococci, 3.3 x 102 Pseudomonaa aeruginosa and less than 1.8 Staphloaoaaus
aureus per 100 ml and 2.7 x 101 Salmonella/10 liters. The stream water pH
was 7.5.
The major difficulty at present appears to be the enrichment step.
Enrichment is necessary because of the low levels of Shigella present in
the aquatic environment. The antagonism between the normal intestinal
flora and enteric pathogens has received attention for quite some time.
Eentges (15) recently reported the ability of 22 strains of microorganisms
to suppress the growth of Shigella in the mouse intestine. All strains of
Eaoheirichia eoli and Enterobacter aevogenea and most strains of Proteus
vlugoris tested were antagonistic to Shigella. The bacteriostatic and
bactericidal effects of the coliform organisms were attributed to volatile
acids produced as metabolic end products. The effects were enhanced at
lower pH values.
The application of the findings of Hentges (15) to the development
of a suitable enrichment procedure has yielded some success. Enrichment
conditions were set up to minimize the production and effect of volatile
acids. Nutrient broth, a complex medium which contains low levels of car-
bohydrates, was adjusted to pH 8.0 and inocualted with 59 Shigella sormei
and Shigella flexnevi. One ml of stream water or 1 ml of raw sewage was
added as a source of interfering microorganisms, comparable cultures were
set up with GN broth. Replicate cultures were incubated under aerated and
stationary conditions. Shigella sarmei was recovered from the flasks seeded
with stream water with an initial pH of 8.0. The predominant interference
was members of the genus Paeudcmonas. One subsequent attempt to recover
low levels of Shigella (14 S. sannei, 28 S. flexneri and 9 5. flexneri)
did not produce any Shigella isolates. Of 148 suspicious colonies, 129
were tentatively identified as Pseudamonas. The minimal content of carbohy-
drate in the medium and the aerated conditions favored the growth of the
Pseudomonas. Experiments conducted in the laboratory indicate that the
growth of laboratory cultures of Pseudamonas aemginoBa was not antagonistic
to Shigella and the results are in agreement with the reports of Hentges
(15). PseudamonaB apparently masks the presence of Shigella.
STAPHYLOCOCCUS aupeus
Serious difficulties were encountered with the determination of levels
of StaphylooooauB aureuB. Membrane filter procedures using m-staphylococcus
broth and Vogel-Johnson medium (V-J) and plate counts on tellurite glycine
agar were evaluated with samples of sewage and urban streams. Each medium
yielded many auspicious Staph. anxeua colonies with typical morphology.
However, those colonies failed to yield typical biochemical reactions, Gram
stains and microscopic morphology. Table 7 shows the recovery of confirmed
Staph. aureus for 1,249 isolates from four procedures for 66 samples of raw
sewage and urban streams. High levels of interfering microorganisms were
found with m-staphylococcus broth, tellurite glycine and VJ medium. The
25
-------
Figure 4. Survival of SkigelTa flexnem. at 4°C
O
O
-J
^ -2
a
<
IT
U.
IT
3-3
-4
C 2
O
A
A SALINE pH 6.3
O STERILE STREAM
• STREAM
4 6
TIME IN DAYS
8
10
26
-------
Table 7. RECOVERY OF STAPH. AVREUS
Medium
Tellurite-glycine
Vogel-Johnaon
M-staphylococcus broth
M-staphylococcus broth +
Procedure
plate count
membrane filter
membrane filter
MPN
Number
of
samples
19
13
16
18
Number of
typical
Isolates
265
109
344
531
Number of
confirmed
Staph. aureue
7
1
30
507
%
confirmed
Staph. caa-eue
2.6
0.9
8.7
95.4
0.75mM azlde enrichment
and LSM primary plate
Totals
66
1,249
-------
predominant Interference on tellurite glyclne was Gram positive cocci that
were catalase negative, DNase negative and yielded pink to red colonies of
KF streptococcus agar. The typical colony morphology expected for Staph.
aureus on V-J medium is a black colony surrounded by a yellow halo which
indicates mannitol fermentation. Stock Staph. aupeus on V-J medium ia a
black colony surrounded by a yellow halo which indicates mannitol fermen-
tation. Stock Staph. at&eus cultures when collected on membrane filters
and grown on V-J medium did yield black colonies but the yellow halo was
obscured by the membrane filter and difficult to detect. The isolates
obtained from the membrane filters Incubated on m-staphylococcus broth were
predominantly Gram positive and Gram negative, rod-shaped bacteria. In
each case the presence of Staph. aureus was heavily masked by interfering
microorganisms that gave typical colony morphology. Simple enumeration of
suspicious colonies would yield a gross overestimate of the levels of Staph.
aureus for each sample.
Smuckler and Appleman (21) reported similar difficulties with Staphy-
lococcus medium 110 when attempting to enumerate Staph. aureus in meat pot
pies. Staphylococcus medium 110 was the medium from which m-staphylococcus
broth was developed for use with membrane filters. The former was prepared
as a solid medium with agar and contains gelatin. The latter is a broth
without gelatin. Similar to our observations with m-staphylococcus broth,
Smuckler and Appleman (21) observed high levels of interfering rod-shaped
bacteria that yield typical colonies on Staphylococcus medium 110. After
testing several inhibitors they recommended the addition of 0.75 mM sodium
azlde to the Staphylococcus medium 110 to inhibit the growth of the rod-shaped
bacteria.
Sodium azide was added to m-staphylococcua broth at a concentration
of 0.75 mM and the modified medium was employed as an enrichment broth for
Staph. aureus. A multiple tube dilution procedure was utilized to permit
the calculation of a MPN. Lipovitellenin-salt-mannitol agar (LSM) was employ-
ed as a primary Isolation medium from the modified m-staphylococcus broth.
Gunn et at. (10) used LSM to isolate and identify Staph. aureus. The produc-
tion of opaque yellow zones around the colonies was considered positive
evidence for lipovitellenln-lipase activity (opaque) and mannitol fermentation
(yellow). Lipovitellenin-lipase activity was found to correlate with coag-
ulase production for 94.7% of the Isolates tested by Gunn et al. (10). Table
7 shows the recovery of Staph. ai&eus for typical colonies on LSM after
enrichment in the modified m-staphyloccus broth with 0.75 mM sodium aside.
507 of 531 or 95.4% of the typical isolates after enrichment and plating
on LSM were confirmed Staph. aureus.
The correlation of biochemical characteristics with pathogeniclty of
Staphylococcus isolates has been the subject of numerous reports. Unfor-
tunately, no one criterion is adequate for the identification of Staph.
OL&eus. Even coagulase production, which has traditionally been used to
differentiate Staph. aureus from Staph. epidermis (formerly Staph. albus)
is not absolute (22). The enumeration must be based on several biochemical
characteristics that will enable a differentiation between not only Staph.
epidermis but other genera of microorganisms not normally encountered at
high levels in clinical samples.
28
-------
Enrichment tubes in the multiple dilutions procedure were considered
positive when isolates were recovered that were catalase negative, coagulase
positive, DNase positive, fermented mannitol, fermented glucose anaerobically,
yielded typical microscopic morphology and Gram positive. A schematic of
the culture procedure is given in Figure 5 .
PSEUDOMOHAS aeruginoaa
Culture media for the enumeration of Pseudomonas aeruginosa were eval-
uated with samples of raw sewage and urban streams. MPN procedures using
L-asparagine broth and confirmation on acetamide broth according to
Methods (5), Drake's asparagine broth #10 (23) confirmed on acetamide broth
and membrane filter procedures using MacConkey agar according to Nixon and
Brodsky (24) were tested. Table 8 shows the results of the initial culture
evaluation for 16 samples of raw sewage and urban streams, L-asparagine brotk
according to Standard Methods yielded consistently higher levels of confirmed
Pseudomonas aeruginosa than either of the other methods tested and was em-
ployed in the remainder of the study. Spot inoculation on acetamide agar
(acetamide broth + 1.5% agar) was compared to acetamide broth for a confirm-
atory procedure to permit the use of acetamide agar as a one step isolation
and confirmatory procedure and as confirmatory screening procedure before
streak isolation. Table 9 shows the comparative levels of P. aerugi.no so.
calculated from confirmation on acetamide broth and acetamide agar. Each
presumptive positive dilution tube was transferred for confirmation to ace-
tamide broth and spotted or streaked on acetamide agar. The levels of P.
aeruginosa. were similar for each confirmatory method except for one stream
sample.
The final procedure for the enumeration and identification of P.
ginosa is given in schematic form in Figure 6. Asparagine tubes showing
growth with fluorescence after incubation at 37 °C for 48 hours were streaked
on acetamide agar or spotted on acetamide agar and subsequently streaked
on PAP agar for isolation. Isolates were submitted to the tests indicated
for verification of P. aeruginoGa* Calculation of the MPN P. aeTuginosa
was based on the recovery of isolates that were acetamide and oxidase posi-
tive and grew at 42°C. Casein hydrolysis with fluorescence (25) was employ-
ed as a secondary characteristic.
COLIFOBMS
Total coliforms were determined by the multiple tube dilution procedure
with lactose broth as the presumptive medium according to Standard Methods
(5). Positive tubes were confirmed on brilliant green lactose broth with
2% bile.
Fecal coliforms were determined by confirmation of positive lactose
broth presumptive tubes on EC medium incubated at 44.5°C for 24 hours (5).
The effect of homogenization on the levels of microorganisms was eval-
uated. Stream samples were blended at room temperature for varying time
periods and the levels of total and fecal coliforms determined. The results
29
-------
Figure 5. Schematic - Isolation and identification of Staphylocoacus
aureus
m-staphylococcus broth
-H3.75 mM sodium azide
I
turbidity
I
streak for isolation
on LSM*
r
typical colonies//
absence of
typical colonies
I
restreak
typical colonies^
I
Gram
stain
catalase
anaerobic coagulase mannitol
glucose fermentation
fermentation
DNase
Staphyloaoccua ouxeus
* LSM - lipovitellenln salt mannitol agar
# typical colonies - colonies surrounded by opaque and/or yellow
zones
30
-------
Table 8. EVALUATION OF PRESUMPTIVE MEDIA FOR
THE ENUMERATION OF PSfflJDOMONAS ASRUaiNOSA
L-Asparagine
broth a
MPN/ 100ml
Raw Sewage 9.2 x 10s
3.5 x 10s
2.3 x 10s
1.7 x 106
Stream 1.7 x 102
1.7 x 103
1.6 x 10s
2.2 x 103
2.3 x 10*
2.4 x 10*
3.5 x 10s
1.1 x 103
1.7 x 10s
2.8 x 10*
7.0 x 10*
5.4 x 103
Drakes #10
broth a.
MPH/ 100ml
3.5 x 10s
2.3 x 10s
9.2 x 10s
4.6 x 10s
1.1 x 102
1.4 x 102
1.6 x 10s
1.1 x 102
2.2 x 10*
1.7 x 103
1.9 x 10*
3.1 x 102
3.3 x 10*
4,9 x 103
3.3 x 103
7.9 x 102
MacConkey
agar - mf a b
# /100ml
4.5 x 10s
m
4.5 x 10s
1.0 x 105
1.4 x 102
4.0 x 103
2.0 x 10*
ND
1.1 x 103
3.0 x 10*
2.0 x 10*
4.0 x 103
1.5 x 10*
1.5 x 10*
1.0 x 10*
1.5 x 103
a Confirmed in acetamide broth
ND No data
b Brodsky and Nixon (24)
31
-------
Table 9. LEVELS OP PSEUDOMONAS AEBUGINOSA CONFIRMED
OK ACETAMIDE BROTH AND ACETAMIDE AGAR
Ps&udomanae aeyuffinosa KPN/ 100ml
Ace t amide confirmation
Sample Broth Agar
Raw Sewage 2.3
1.7
-9.2
4.6
Stream 1.6
2.4
2.8
2.2
3.5
1.6
1.7
4.9
1.7
3.3
x IO6
x 10s
x 10s
x 10s
x 10s
x 10"
x 10"
x IO3
x 10s
x IO5
x IO3
x IO3
x 10"
x IO3
2.3
2.8
1.6
1.7
2.2
2.4
7.0
3.5
1.6
1.6
1.3
1.3
1.8
3.5
x IO6
x io6
x IO5
x 10s
x 10"
x 10"
x 10"
x IO3
x 10s
x 10s
x IO3
x 10"
x IO3
x IO3
32
-------
Figure 6. Schematic - Identification of Ps&udomonas
1-asparagine broth
!
turbidity with
fluorescence
ace tamide agar
confirmation
i
streak for
isolation
1
r
growth \
fluoresct
I
oxidaae
•
I
spot
inoculation
1
• i r i
*ith - growth with
snce • fluorescence
1
streak for isolation
Pseudomonas agar P
1
1 I
acetamide growth
. 42°C
1
at tech agar
i
1
casein
growth 4-
primary criteria
Pseudamonaa
growth with hydrolysis
fluorescence with
fluorescence
secondary criteria
33
-------
Figure 7. Effect of homogenization on
the levels of total and fecal coliforms
TOTAL COLIFORM LOG MPN/ 100 ml.
PO
01
Cl
ro
o
CD
r
m
z
o
•7
I
*
ffi
FECAL COLIFORM LOG MPN/ 100 ml.
ro
O)
-------
Figure 8. Identification of the fecal streptococci
KF streptococcus agar
pink-red colonies
catalase
growth in
40% bile
growth at
45°G
ENTEROGOCCI
I
starch hydrolysis
1
atypical
S. f 000011*3
growth at
10°C
f
growth at
6.5% NaCl
I
casein hydrolysis
starch hydrolysis
I
gelatin liquefaction
5. faecalie
var. liquefaciens
var. zymogensa
arabinose fermentation
. i
S, faeaiim
i
mannitol fermentation
r
•¥
S. faeoal-is
S. durans
35
-------
for five trials are shown in Figure 7. No consistent trends and little sig-
nificant differences were observed for the levels of total coliforms after
blending for time periods up to 180 seconds. Two trials showed an increase
in levels of fecal conform while three trials showed little significant
differences'with blending time. Again no consistent trends were observed
and no blending time necessary for optimum levels of coliforms could be
predicted. As a result, a preparative blending step for each sample before
the microbial assays would be of questionable value.
FECAL STREPTOCOCCAL GROUP
The determination of fecal streptococci has found increasing usage for
evaluating the microbial quality of surface waters. However, there is some
controversy concerning the sanitary significance of the microorganisms that
comprise this indicator group. Standard Methods (5) defines the fecal strep-
tococcal group to contain the followin species: S. faeoalis^ S. faeaalia
var. liqu-ifaaienSt S. faeaali,s var. zymogenes, S. dw?ans3 S. faea-Lum, S.
bovis and S. equinus and is considered synonymous with "Lancefield's Group
D. Streptococcus". The more restrictive term "enterococcus11 excludes S.
bovis and S. eqirinus. Geldreich (26) assigns limited sanitary significance
to the 5. faecal-is strain capable of starch hydrolysis (designated atypical)
and S. faecalis var. liquifaciens and provides evidence to suggest that the
subgroup of S. bovis and S. equinus may be indicative of non-human animal
pollution. The latter two streptococcal species were found in significant
levels of animal feces but not recovered from human feces.
The initial effort was directed toward evaluation of media for the enu-
meration of the fecal streptococcal group. Multiple tube dilution technique
with azide dextrose broth confirmed on ethyl violet azide (EVA) and/or enter-
ococci confirmatory agar, M-enterococcus plate counts and KF streptococcus
plate counts were evaluated with samples of raw sewage and urban streams.
KF streptococcus plate counts consistently yielded the highest recovery of
fecal streptococci. M-enterococcus agar consistently yielded low recovery.
Subsequent evaluation of the more recent selective enterococcus medium (Pfizer
Diagnostics) (PSE) yielded results similar to KF streptococcus agar with
the advantage of a shorter incubation period. Based on the results of the
preliminary culture evaluations and the similar findings of Pavlova (27),
Hartman et al. (28), and Kenner (29), KF streptococcus agar was employed for
the enumeration of the fecal streptococcal group.
Isolates obtained from KF plates were differentiated according to the
scheme given in Figure 8. Generally, 35 to 50 isolates were randomly picked
from the countable plates for further differentiation. This represents a
minimum of 5.7% to 58% of the available isolates on duplicate plates con-
taining 30 to 300 colonies. All colonies were picked when the number of
colonies were below 30 at the lower sensitivity limit of the assay. Two.
types of replicate plate procedures were employed to enable the differentiation
of a large number of isolates. Colonies were transferred with sterile tooth-
picks to a grid pattern on a master plate and incubated. The master plate
was then used to inoculate a velveteen pad and transferred in the grid pattern
to the appropriate differential agars. The second procedure employed the
preparation of a master plate as above but a multiple point inoculation
device utilizing toothpicks was used. Each isolate was picked from the
36
-------
master plate with a sterile toothpick and transferred to a plexiglass tooth-
pick holder in a similar grid pattern. The charged toothpicks were then
used to inoculate the appropriate differential agars. The last plate In
each replication, series was a BHI agar control to evaluate the transfer.
Isolates not replicated through the final control plate were not considered.
Each procedure would easily transfer microorganisms through 15 plates.
The velveteen and toothpick replicator were evaluated by comparing
results obtained in simultaneous determinations with conventional tube
methods. The results for 250 trials of isolates obtained from KF streptococcus
agar is given in Table 10. With the exception of arabinose fermentation
94.4% agreement or better was observed between the replication procedures
and standard tube methods. The relatively poor agreement on arabinose
fermentation appears to be a function of the initial pH of the medium. If
the initial pH of the medium is too low the fermentation reaction was dif-
ficult to read. Subsequent comparison with 50 isolates where the pH was
adjusted to 7.4 before autoclaving yielded 98% agreement. The overall agree-
ment on species identification by the replication procedures and the tube
methods was 93.0% for the velveteen technique and 94.5% for the toothpick
method. Many of 'the isolates that did not agree with the tube procedures
for arabinose fermentation did not require arabinose fermentation for the
determination of species. Considering the group identification of entero-
coccus, S. bowis and equinust the liquefac-iens and zymogenes variations of
S. faeaalisi atypical 5. faecal-is and false positives, the percent agreement
for the overall group identification was 97.0% for both procedures.
Both replication procedures were rapid, inexpensive and reasonably
accurate. The toothpick replicator yielded a more positive (small notice-
able holes in the agar at the time of replication) inoculation of the agar
and was less sensitive to plate moisture than the velveteen replication
technique. The major portion of the assays was conducted with the toothpick
replicator.
37
-------
Table 10. COMPARISON OF VELVETEEN AND TOOTHPICK REPLICATION PROCEDURES TO
CONVENTIONAL TUBE METHODS FOR THE DIFFERENTIATION OF FECAL STREPTOCOCCI
Velveteen
# of #
Test tests agree
Growth in 40% bile
Growth, in 6.5% NaCl
Growth at 45 °C
Growth at 10°C
Starch hydrolysis
Gelatin liquafaction
Casein hydrolysis a
Arabinose fermentation
Mannitol fermentation
Species identification b
Group identification
250 250
250 237
250 242
250 242
250
150 150
250 250
200 166
250 240
-
- —
%
agreement
100
94.8
96.8
96.8
-
100
100
83.0
96.0
93.0
97.0
Toothpick
# %
agree agreement
250 100
236 94.4
243 97.2
240 96.0
-
150 100
250 100
184 92.0
243 97.2
94.5
97.0
a Casein hydrolysis was compared to peptonization of litmus milk
° S. durans classified as a variation of 5. faeeiwn
38
-------
RESULTS
A comprehensive presentation and discussion of the information obtained
during the current investigation is beyond the scope of this report. Some
highlights and observed trends are given below.
The ranges in the levels of microorganisms found in the background
and stormwater samples are given in Table 11 and 12, respectively. The
minimum and maximum levels of indicator and pathogenic microorganisms observed
are reported as MPN or number per 100 ml with the exception of the data for
Salmonella sp. which is reported as MPN Salmonella/10 liters. The final
two columns are the minimum and maximum levels for the ratio of fecal coliform
to total coliform and fecal coliform to fecal streptococci. It is evident
from both tables that the ranges of the levels of the different microorganisms
and the magnitude of the ratios of indicator microorganisms varies widely
for each sample station. Differences between the minimum and maximum levels
of several orders of magnitude are observed in each case for each micro-
organism group. The wide variations are not unexpected. Each sample is a
grab sample and reflects the microbial quality at the time the sample was
taken. Large differences in. flow, quantity of rainfall, antecedent rainfall, and
portion of the stormwater sampled (first flush, mid-storm, tail) were observed
from sample to sample at each station. These wide variations emphasize the
need to obtain a large number of samples over a reasonable length of time
to provide valid information to evaluate trends and to observe any specific
correlation between the levels of different microorganisms.
Table 13 shows the frequency of detection of Salmonella at different
levels of fecal coliforms and a comparison of similar data reported in the
literature. The levels of fecal coliforms in raw sewage exceeded 2,000
per 100 ml in each sample, and Salmonella was isolated in every case. The
samples collected and assayed from an upland reservoir contained low levels
of fecal coliforms. Salmonella was isolated from one sample. The majority
of the samples collected from urban streams and stormwater contained greater
than 2,000 fecal coliforms per 100 ml. Salmon&lla was found routinely.
The exception was not isolating Salmonella from samples of urban stremas
and stormwater. For the overall total of 273 samples collected from the
urban aquatic environment, Salmonella was isolated in 27%, 89% and 96% of
the samples containing 0-200, 201-2,000 and greater than 2,000 fecal coliforms
per 100 ml, respectively. The frequency of Salmonella isolation compares
favorably with the fresh water data reported by Geldreich and Van Donsel (30)
and differs significantly from the estuary data reported by these authors
and Brezenski and Russomanno (31).
An important aspect of the current study has been in inclusion of
seeded Salmonella controls to evaluate the steps in the recovery of Salmon-
ella. Table 14 shows the frequency of recovery of the seeded Salmonella
for each sample station. Only two storm samples were found to cause more
than 90% inactivation of the seeded Salmonella* The test organism was con-
sistently recovered after exposure to the water samples from the different
sources and indicated that the water samples were not bactericidal to the
seeded Salmonella. After concentration on diatomaceous earth, the seed
Salmonella was recovered in 67% to 100% of the samples depending on sample
39
-------
Table 11. LEVELS OF MICROORGANISMS IN RAW SEWAGE AND URBAN STREAMS
Station
Raw Sewage
rain.
max.
Herring Run
Din.
max.
Jones Falls
min.
max.
Cwynns Falls
min.
max.
Loch Raven
min.
max.
Number
of
Samples
34
34
34
34
8
Total
Coliform
HPN/lOOnl
1.3 x 106
1.6 x 109
5.0 i 101
3.5 x 10s
1.1 x 1011
3.3 x 105
3.4 x 10Z
2.4 x 10s
<2.0 x 10°
4.0 x 102
Fecal
Coliform
MPN/lOOnl
3.3 x 10s
9.2 x 108
2.0 x 101
3.5 x 10s
1.1 x ID11
2.4 x 10s
8.0 x 101
2.4 x 106
<2.0 x 10°
8.0 x 101
Fecal
Streptococci
MPM/lOthnl
2.0 x 101*
3.3 x 106
1.9 x 102
4.0 x 10"
2.6 x 103
9.2 x 1011
<1.0 x 102
>1.0 x 105
<5.0 x 10°
2.0 x 102
PaeudomonaB
aerug-Lnosa
HPN/lOOnl
3.3 x 103
5.6 x 107
5.0 x 10°
1.6 x 105
1.1 x 102
3.5 x 10s
3.0 x 10°
1.7 x 10s
<2.0 x 10°
2.3 x 101
Staph.
OUF0UB
HPH/lOOml
4.3 x 101
4.6 x 103
<2.0 x 10°
7.0 x 10°
2.0 x 10°
1.8 x 102
<1.0 x 10°
9.3 x 10*
<3.0 x 10°
Salmonolla
sp.
HPN/10 liters
2.6 x 101
2.7 x 1011
<9.0 x 101
1.3 x 102
1.0 x 10°
3.2 x 102
4.1 x 10°
1.3 x 102
<8.8 x 10-1
8.8 x 10"1
FC/TC
.03
1.0
.08
1.0
.15
1.0
.03
1.0
0.5
1.0
FC/FS
1.2
25.0
< .1
21.9
.3
46.9
.13
37.7
.2
.4
144
-------
Table 12. LEVELS OF MICROORGANISMS IN STOEMWATER RUNOFF
rfx
H*
Station
Stoney Run
rain.
max.
Glen Ave.
mln.
max.
Howard Park,
win.
roox.
Jones Falls
mln.
max.
Dueh Street
tain.
max.
Northvood
rain.
max.
Number Total
of Collform
Samples HPN/lOCtal
17
5.4
1.6
17
7.9
1.6
17
4.9
2.8
17
3.3
>2.4
17
7.9
2.4
14
1.3
1.7
x 103
x 106
x 103
x 10s
x 103
x 107
x 101*
x 106
x ID3
x 106
x 103
x 105
Fecal
Collform
HPH/lOChnl
1.3 x 103
5.4 x 10*
1.4 x 10s
2.3 x 105
2.3 x 103
2.9 x 10s
5.0 x 103
>1.6 x 106
1.7 x 103
2.4 x 106
8.0 x ID1
7.9 x 10*
Fecal
Streptococci
MPH/lOOml
5.3
3.0
9.2
2.8
1^4
2.6
8.0
2.5
1.9
1.7
3.0
x 10Z
x 10s
x 103
x 106
x 103 .
x 106
x 103
x 10s
K 103
x 106
x 103
x 10s
Peeudcmpnac
aeruginooa
HPH/lOOal
2.3
2.4
1.3
2.6
7.9
5.4
9.4
1.6
1.1
7.5
1.7
9.2
x 102
x 105
x 102
x 105
x 10*
x 1011
x 102
x 106
x 102
x 1011
x 101
x 103
Staph.
aureuB
HPH/lOOwl
<3.0 x 10°
7.9 x 101
<3.0 x 10°
1.5 x 102
6.0 x 10°
9.2 x 102
4.0 x 10°
1.1 x 101
<3.0 x 10°
4.6 x 103
<3.0 x 10°
4 .6 x 10*
Salmonella
HPN/10 liters
2.9 x 10°
>1.3 x 103
1.7 x 10°
>1.1 x 10*
3.9 x 10°
>1.3 x 103
1.7 x 10
2.7 x 10
<1.7 x 10°
2.7 x 103
<1.7 x 10°
4.3 x 10'
FC/TC
< .07
1.0
.06
1.0
.02
1.0
.01
1.0
.05
1.0
.01
.65
FC/FS
.06
92.5
.02
5.9
.05
32.7
.05
4.4
.02
12.9
.01
4.6
99
-------
Table 13. COMPARISON OF THE FREQUENCY OF DETECTION
OF SAIMONELIA WITH THE LEVELS OF FECAL COLIFORMS
Fecal Coliform
Report
J.H.U.
Geldreich &
Van Donsel (30)
Brezenski &
Russomanno (31)
Sample
raw
sewage
urban
streams
upland
reservoir
storm
runoff
overall
total
fresh
water
estuary
estuary
Range
MPN/lOOml
0-200
201-2000
>2000
0-200
201-2000
>2000
0-200
201-2000
>2000
0-200
201-2000
>2000
0-200
201-2000
>2000
0-200
201-2000'
>2000
0-200
201-2000
>2000
0-200
201-2000
>2000
Number of
Samples In
Range
0
0
32
3
34
55
13
0
0
1
12
123
17
46
210
29
27
54
258
91
75
34
43
73
Salmonella.
Number of
Samples
Positive
32
3
31
53
1
1
10
117
5
41
202
19
53
33
33
40
45
6
13
43
Percent
Positive
100
100
91
96
8
100
83
95
29
89
96
27
70
98
13
44
60
18
30
59
42
-------
w
o
0 0
*»
en
UJ
O
_J
<
O)
o
o
. •
0
345
LOG TC/IOO ml.
8
Figure 10. Relationship between total coliform and Salmonella in background (solid point) and
stormwater (open point) samples.
-------
station. The Salmonella seed was recovered in 87% of all samples. This
suggests that the diatomaceous earth concentration procedure for the recovery
of low levels of Salmonella from large volumes of water was effective.
After enrichment and primary plating, the recovery of the seeded Salmonella
decreases markedly and was recovered in only 30% of the samples. The frequency
of recovery for the overall culture procedure varied with the sample site.
In general, the samples with the higher levels of microorganisms yielded
poorer recoveries, and samples with low levels of microorganisms yielded bet-
ter recoveries. The data suggest that the major difficulty with the procedures
for the detection and enumeration of Salmonella in water lies in the enrich-
ment step.
The relationship between the logarithm of the levels of Salmonella
sp. (MPN/10 liters) and the logarithm of the levels of total coliform (MPN/
100 ml), fecal coliform (MPN/100 ml) and fecal streptococci (#/100 ml) can
be seen in Figures 10, 11, and 12, respectively. In each case there is some
relationship between the pathogen and the indicator group. However, there
is a noticable difference between the least squares curve for the background
samples and the storm water samples. Table 15 summarizes the correlation
coefficients (r) for the levels of Salmonella and indicator microorganisms.
Good correlation ( r = ~ 0.7) was observed for total coliform, fecal coliforta,
fecal streptococci and enterococci for the background samples. For the
stormwater samples the correlation coefficient was far less significant part-
icularly for the fecal streptococci and enterococci. The correlation coef-
ficient between indicator microorganism and Salmonella when all samples are
considered was between 0.53 and 0.59, The data suggest that the levels of
Indicator microorganisms are less meaningful in stormwater runoff.
44
-------
Table 14. FREQUENCY OF RECOVERY OF SEEDED SALMONELLA AFTER
EXPOSURE TO THE SAMPLE,' CONCENTRATION ON DIATOMACEOUS
EARTH AND ENRICHMENT WITH PRIMARY PLATING
% of the samples positive
Enrichment with
Sample station Exposure a Concentration primary plating
Background
Raw sewage 100 100 0
Herring Run .100 88 67
Jones Falls 100 88 17
Gwynns Falls 100 82 33
Loch Raven 100 100 83
Storm drain
Stoney Run 100 79 0
Glen Ave. 100 93 25
Howard Park 95 100 0
Jones Falls 95 79 0
Bush Street 100 86 50
Northwood 100 67 25
All samples 99 87 30
a Recovery of the seeded Salmonella after exposure to the sample was
considered positive if less than 90% inactivation was observed.
43
-------
*-
O5
w
«5
3
_J
LU
Z
O
CO
o
• I » • • t *°
345
LOG FC/IOO ml.
B
Figure 11. Relationship between fecal coliform and Salmonella in background (solid point) and
stormwater (open point) samples.
-------
CO
Q>
_J
111
z
o
CO
C!>
O
45
LOG FS/IOO ml.
Figure 12. Relationship between fecal streptococci and Salmonella In background (solid point) and
stonnwater (open point) samples.
-------
Table 15. CORRELATION COEFFICIENTS FOR THE LEVEL OF SALMONELLA
AND THE LEVELS OF INDICATOR MICROORGANISMS
Correlation coefficient
Background Urban runoff ' All
samples samples samples
Total coliform 0.67 0.49 0.54
Fecal coliform 0.72 0.36 0.59
Fecal streptococci 0.71 0.19 0.53
Enterococci 0.73 0.18 0.54
-------
REFERENCES
1. Weibel, S.R., R.J. Anderson, and R.L. Woodware. Urban Land Runoff as
a Factor in Stream Pollution. J. Water Poll. Control Fed., 36:914-924,
1964.
2. Geldreich, E.E., L.C. Best, B.A. Kenner, and D.J. Van Donsel. The
Bacteriological Aspects of Stormwater Pollution. J. Water- Poll. Control
Fed., 40:1861-1872, 1968.
3. Evans, F.L., III, E.E. Geldreich, S.R. Weibel, and G.G. Robeck. Treat-
ment of Urban Stonnwater Runoff, J. Water Poll. Control Fed.3 40:162-
171, 1966.
4. Bunn, R.J. and R.D. Vaughan. Bacteriological Comparison Between Combined
and Separate Sewer Discharges in Southeastern Michigan. J. Water Poll.
Control Fed., 38:400-409, 1966.
5. Standard Methods for the Examination of Water and Wastewater. Thirteen-
th Edition. American Public Health Association Inc.
6. Ewing, W.H., B.R. Davis, and R.W. Reavis. Phenylalanine and Malonate
Media Avel Their Use in Enteric Bacteriology. Publ 'Health Lab., 15:
153-167, 1957.
7. Edwards, P.R. and W.H. Ewing. Identification of Enterobacteriaceae.
Burgess Publishing Co., Minneapolis, Minn., 1972.
8. Esber, R.J. and R.J. Faulconer. A Medium for Initial Visual Demonstr-
ation of Production of Coagulase and Fermentation of Mannitol by Patho-
genic Staphylococci. Technical Bull, of the Registry of Medi-cal Teeh.3
29(7):108-110, 1959.
9. Streitfeld, M.M., E.M. Hoffman, and H.M. Janklow. Evaluation of Extra-
cellular Deoxyribomiclease Actively in Pseudomonas. «/". Bact., 84:77-80,
1962.
10. Gunn, B.A., Capt., MSC, W.E. Dunkelberg, Jr., Major, MSC, and J.R.
Creitz, Col., MSC. Clinical Evaluation of 2% LSM Medium for Primary
Isolation and Identification of Staphylococci. Amef. J. Clin. "Path.,
57:236-240, 1972.
11. Evans, J.B. and W.E. Kloos. Use of Shake Cultures in a Semisolid Thio-
glycolate Medium for Differentiating Staphylococci from Micrococci.
Appl. Microbiol., 23, 1972.
12. Rhodes, M.E. The Characterization of Pseudomonas fluorescens. J. Gen.
Mierobiol., 21:221-263, 1959.
13. Brodsky, M.H. and M.C. Nixon. Membrane Filter Method for the Isolation
and Enumeration of Paeudomonas aeruginosa from Swimming Pools. Appl.
Microbiol., 938-943, 1973.
49
-------
14. Kenner, B.A. and H. Clark. Detection and Enumeration of Salmonella
and Ps&udomonas aeruginosa. J. Water Poll* Control Fed.3 46:2163, 1974.
15. Hentges, D.J. Inhibition of Shigella Fl&xneri by the Normal Intestinal
Flora. II Mechanism of Inhibition by Coliform Organisms. J. Bacti.j
97:513, 1969.
16. Andre, D.A., H.H. Weiser, and G.W. Malaney. Survival of Enteric Patho-
gens in Farm Pond Water. J. American Water Works Assoe.j, 59:503, 1967.
17. McFeeters, G.A., G.K. Bissonnette, J.J. Jezeski, C.A. Thomson, and D.G.
Stuart. Comparative Survival of Indicator Bacteria and Enteric Pathogens
in Well Water, Appl. Microbiol., 27:823, 1973.
18. Bartos, B., J. Bansagi, and K. Bakas. Survival Time of Enteric Patho-
genic Bacteria in Well Water. Zeita. Hyg. Infektkr., 127:347, 1947.
19. Dolivo-Dobrovol'skii, L.B. and V.S. Rossovskaia. Survival of Shigella
dysenteriae in Water Supply. Gigiena i San** 21:52, 1956.
20. Geldreich, E.E. Water-Borne Pathogens in Water Pollution Microbiology
edited by R. Mitchell, John Wiley and Sons, Inc., New York, 1972.
21. Smuckler, S.A. and M.D. Appleman. Improved Staphylococcus Medium No.
110. Appl. Microbiol., 12(4):355-359, 1964.
22. Morton, H.E. and J. Cohn. Coagulase and Desoxyribonuclease Activities
of Staphylococci Isolated from Clinical Sources. Appl. Mierobiol.s
23:725, 1972.
23. Drake, C.H. Evaluation of Culture Media for the Isolation and Enumer-
ation of Pseudomonas aeruginoaa. Health Lab* Sci.j 3:10, 1966.
24. Brodsky, M.H. and M.C. Nixon. Rapid Method for Detection of Pseudomonas
aernginosa on MacConkey Agar Under Ultraviolet Light. Appl. Miarobiol..,
26(2):219-220, 1973.
25. Levin, M.A. and V.J. Cabelli. Membrane Filter Technique for Enumeration
of Pseudomonas aeruginosa. Appl. Mierobiol., 24:864-870, 1972.
26. Geldreich, E.E. and B.A. Kenner. Concepts of Fecal Streptococci in
Stream Pollution. J. Water Poll. Control Fed.j 41:336-352, 1969.
27. Paulova, M.T., F.T. Brezenski, and Warrenlitsky. Evaluation of Various
Media for Isolation, Enumeration and Identification of Fecal Streptococci
from Natural Sources. Health Lab. Soi^ 9:289-297, 1972.
28. Hartman, P.A., G.W. Reinbold, and D.S. Saraswat. Media and Methods for
Isolation and Enumeration of the Enterococci. Advances in Applied
Microbiol.j edited by W.W. Umbreit, Academic Press, 8:253, New York,
1966.
50
-------
29. Kenner, B.A., H.F. Clark, and P.W. Kabler. Fecal Streptococci (1)
Cultivation and Enumeration of Streptococci in Surface Waters. Appl.
Mierobiol., 9:15, 1961.
30. Geldreich, E.E. and D.J. Van Donsel. Salmonsllae in Fresh Water Pol-
lution in Proc. National Specialty conference on Disinfection, American
Society of Civil Engineers, New York, 1970.
31. Brezenski, F.T. and R. Russomanno. The Detection and Use of Salmonellae
in Studying Polluted Tidal Estuaries. J. Water Poll. Control Fed.3
41:725, 1969.
51
-------
PUBLIC HEALTH ASPECTS OP SURFACE WATERS IN
THE WOODLANDS
E. M. Davis,
(2) (2)
J. D. Moore, D. Casserly
The bacteriological content of surface waters in The
Woodlands, Texas has been the subject of a multipurpose research
investigation which was initiated in late November, 1973.
Conceptually, that test site is a planned community covering
approximately 20,000 acres and is located 35 miles north of
Houston, Texas off Interstate Highway 45, and is being designed
to ultimately house about 120,000 residents. An outline of the
test area and the principal watercourses involved is included
as Figure 1. It appeared at the onset of this investigation
that the area would be ideal to study the effect on the bac-
teriological quality of stream waters by urbanization. None
of the lakes indicated in Figure 1 are natural lakes but have
been constructed for use as irrigation water sources or re-
creational waters. Since one of the objectives was to quantify
the indicator bacteria and some pathogenic bacterial groups or
species in the streams, the result of impounding the stream-
waters became equally as important an objective.
Associate Professor, UTSPH, Houston.
Research Stat. Aide, UTSPH, Houston.
52
-------
Determining stormwater runoff quality in Panther Branch
and Bear Branch was another purpose of this investigation; de-
pending on the viewpoint of the reader, perhaps the single most
important purpose. Several research subsets of this latter
purpose were developed. One parameter considered when judging
water quality from a public health viewpoint is a series of
indicator organism test results. Total coliform, fecal coli-
form, and fecal streptococci are currently in use for that rea-
son and the ratio of fecal coliform to fecal streptococci has
been given considerable research attention with regard to the
source of the polluting bacterial groups. Analyses of those
bacteria and their numerical ratios in stream waters in The
Woodlands have been conducted for yet another reason. Point
source wastewater discharges were believed to have been non-
existent in the test area. Therefore the data obtained from
such an undeveloped watershed would be useful for comparison
with other regions of the Country and evaluating the effective-
ness of the fecal coliform (FC) to fecal streptococci (FS)
ratio. Other tasks which were pursued included the quantifi-
cation of pathogenic bacterial species or groups during periods
of low flow in the streams as well as throughout storm event
hydrographs and the determination of stormwater disinfection
requirements employing chlorine and ozone. Three genera of
pathogens have been quantified and include Salmonella sp.,
Staphylocoecus sp., and Pseudomonas aeruginosa.
53
-------
Total coliform bacteria were enumerated on m-Endo Agar
(BBL) plates using Gelman membrane filters of 0.45p porosity.
Fecal coliform bacteria and fecal streptococci were plated on
m-FC Agar (Difco) and m-Enterococcus Agar (BBL) respectively,
also on membrane filters. Staphylococcus sp. colonies were
enumerated using Vogel-Johnson Agar followed by testing most
representative colonies for catalase positive reactions. Most
plates yielded well over 90% catalase positive staphylococci.
Pseudomonas aeruginosa was quantified on membrane filters using
m-PA medium, the formula for which was provided by Dr. E. E.
Geldreich, U.S.E.P.A., Cincinnati. Salmonella sp. quantifica-
tion required the use of several media. Water samples were
first plated onto Xylose Lysine Deoxycholate Agar (XLD) (BBL).
All positive colonies were then transferred to Triple Sugar
Iron Agar (TSI) (BBL) slants. From those, positives were
further tested and confirmed using API test strips which employ
screening via 20 biochemical reactions.
Initially, fifteen locations for low flow water quality
monitoring were established in the test area. Those stations
are indicated in Figure 2. Within the group, P-10*and P-30*
\
were considered to be of prime importance due to their location
above and below, respectively, the area in which construction
was started following the initiation of this investigation.
Data presented later in this treatise will refer to certain of
those sample locations by code number.
*See next page.
54
-------
*During the period of time that the data reported herein were developed some
of the Woodlands area above station P-30 was under construction. A compari-
sion of land use above station P-10 and above P-30 is as follows:
Station P-10 (Upstream) Station P-30 (Downstream)
Drainage Area, acres
% Impervious
Developed and/or under
construction
16,050
1%
1%
21,606
1%
10%
FM 2978
WOODLANDS
BOUNDARY
INTERSTATE
45
FIGURE 1
THE WOODLANDS AREA
MONTGOMERY COUNTY TEXAS
55
-------
en
Figure 2
Location of Low-Flow Water Quality
Sampling Sites on Panther and Bear
Branches
S-10
S-09
-------
One of the factors which had to be identified was the
variability in density of the aforecited bacteria groups and/or
species during low-flow with time. To this purpose a diurnal
sampling scheme was initiated at stations P-10 and P-30 in
September, 1974. The data developed from that project are
listed in Tables 1 through 4, and represent sampling over appro-
ximately an 18-hour period of time. For all practial purposes
the waters at both stations appeared to have been unfit for con-
tact or noncontact recreation throughout the entire sampling
period, if total coliform densities are the only parameter to
be considered. Attention is directed to the high variability
in concentrations in all organism counts. Those data suggest
the possibility that typical approaches normally taken in
judging water quality from a bacterial content viewpoint via
one grab sample per month, for example, are, at best, only
approximations. Mean values compared with standard deviations
for each parameter in those Tables further the meaning of that
suggestion. It is equally as important to take note of the
overall increase in the FC/FS ratios and the FC/FS mean ratio
between the two stations. The increase in densities of each
occurred with distance downstream. The densities of Staphylo-
coccus sp. and Pseudomonas aeruginosa definitely indicate a
quality of water which posed a threat to the public health
insofar as contact recreation was concerned.
During the course of this investigation, to date of
the workshop, seven storm events have been monitored. To the
57
-------
purpose of brevity however, data for one representative event
in the test site is included in this treatise. Naturally, all
detailed data has been made available to USEPA personnel through
monthly reports and year-end reports and may be obtained from
the author. Tables 5 through 8 contain the bacteriological data
for the storm event which was monitored from December 5 through
December 9, 1974. Differentiation of water quality between the
upper location's water quality and that at the downstream
station (P-30) by examining the data for total coliform bacteria
alone is obviously not possible. However, if one would compare
the other indicators and pathogen densities over time, a pattern
appears, and has appeared in other storm event data. An immedi-
ate increase in total coliform bacteria held, with highly vari-
able densities throughout the major portion of the measured
hydrograph. Fecal coliform and fecal streptococci, on the other
hand, increased by several orders of magnitude during the
initial phases of the storm event and decreased with time. They
also exhibited higher densities in the downstream waters (P-30;
Table 7) than at station P-10, upstream. FC/FS ratios were not
consistent within the waters at either station throughout the
entire storm event, i.e., if a basis is used for evaluating
FC/FS ratios as to 4 (human, pollution), to <1 (animal pollu-
tion) . On the other hand, Pseudomonas sp. concentrations were
higher at station P-10 than at P-30 during the storm. Staphylo-
cocci may warrant special attention due to their behavior which
is unlike that of any of the other groups. A general increase
58
-------
of from 700 or 800/100 ml to about 2,000/100 ml held throughout
the early part of the storm event at P-10 whereas erratic varia-
tions, yet high concentrations occurred at station P-30 through-
out the entire time of the storm event. A graphical represen-
tation of those parameters plus suspended solids and turbidity
is presented in Figures 3 through 11. Initial runoff carries
peak, loading and/or concentrations of certain bacteria groups
or species. Those graphs permit such a conclusion to be made
as it becomes obvious when comparing points within the first
twelve hours of the hydrograph. Among those factors which
peaked prior to the hydrograph maximum flow rate were suspended
solids, turbidity, and total coliform. Leaching from or wash-
out of soil and forest floor cover is presumed to have been
partially responsible for the peaks in bacterial concentrations
following the hydrograph peak.
Disinfection requirements of stormwaters vary between
times during the storm event as well as the concentration of
suspended solids and other oxidant demanding (or combining)
substances. Several experiments have been conducted to deter-
mine chlorine and ozone demand of varying qualities of water
during storm events and of the requirements for disinfection
to meet or exceed established or proposed standards. Tables 9
through 12 contain disinfectant data for preselected dosages
of chlorine and ozone of discharge waters from Lake A (The
Woodlands), the sample for which was taken at 0900 hrs. on
March 13, 1975 during the storm event of 3/12-14/75. Lower
59
-------
initial (time zero) counts than Are represented in earlier data
are suspected to have been the result of that small impoundment
(12.6 ac. surface} 90 ac-ft. vol.) acting as a settling basin.
Values represented for "less than" some numerical figure imply
a positive count but high turbidity impeded additional filtra-
tion, as has been the case in numerous instances. Ozonation
was effective but not nearly so much within the first 30-minutes
exposure as chlorine. S'taphylo coccus sp. demonstrated a marked
capability to regenerate an appreciable population following
ozonation. The total coliform group exhibited the greatest
capacity for aftergrowth two days following exposure even up to
*
16 mg/1 .chlorine.
Over thirty composited soil samples have been collected
from swales, forest floor, and hear each sampling station
(Figure 2) for the purpose of attempting to determine whether
a principal source of bacteria in storm runoff was indeed the
soil or whether unidentified point sources may have been in
existence in the test area. Table 13 contains a partial listing
of the sampled areas and bacteriological results from each.
The results suggest that sufficient numbers of those listed
bacteria groups or -species exist in high humus soil to be
carried out by rainfall leachate of high enough volume. To
date no point sources of wastewater discharge have been identi- .
fied in the upper reaches of the test site.
Currently, additional research is being directed to
critical evaluations of the meaningfulness of the indicator
60
-------
bacteria counts and their related water quality factors. To the
purpose of proposing the most useful relationship analyses have
been conducted weighing all factors listed earlier. With 231
data points in the first attempt sequence, it was determined
that the highest R value was obtained for fecal streptococci.
This was 0.82 and speaks for itself. Raw data did not produce
as high a value but the factors followed normal distribution
patterns and were mathematically sound when converted by log
transformation. A useful expression developed from these data
was as follows:
Log (FS/100 ml) = 0.72 Log (PC/100 ml) + 0.002 (discharge)
+ 0.003 (Turbidity) + 0.002 (Susp. Solids)
+ 0.612
One aspect of stormwater quality which was not dis-
cussed earlier is its geographical location. The test site is
located, obviously, in a semitropical climate with virtually
no frost-line and high humidity year-round. It is, in the
opinion of the principal investigator, highly conductive to the
growth of .indicator bacteria and perhaps even pathogens, given
enough organic nutrient in the water or soil. This should be
taken into consideration when comparing stormwater quality
between different locations of the Country.
This research was supported in Part under USEPA Prime
Contract R802433, Subgrant 733, Storm and Combined Sewer Tech-
nology Branch. Data collection of discharge, suspended solids,
and turbidity by Rice University faculty and staff is hereby
acknowledged.
61
-------
Figure 3. Volume vs Time; Storm Event of 12/5-9/74
Station P-10.
The Woodlands,
to
300 -
250 .
05
o 200 -
-------
o>
CO
125
100
CO
T3
-H
i-l
o
en
•a
0)
-a
c
0)
ft
en
3
Vt
75 -
50 -
25 -
12
24
36
60
72
84
Time (Hours)
Figure 4. Suspended Solids vs Time; Storm Event of 12/5-9/74
Woodlands, Station P-10.
The
-------
o>
100
90
80
70
60
50
3 40-1
E«
30 '
20
10
12
24
36
48
60
72
84
Time (Hours)
Figure 5. Turbidity vs Time; Storm Event of 12/5-9/74. The Woodlands,
Station P-10.
-------
Cn
c
.-I
X
C
o
w
6
M
O
VM
•rt
i-H
O
CJ
4J
0
Figure 6. Total Coliform vs Time; Storm Event of 12/5-9/74
The Woodlands, Station P-10.
200
ISO'
100 -
50
12
24
36
48
60
72
84
Time (Hours)
-------
125-1
05
O5
CN
o
100-
15"
c
o
I-H
\
U)
E
O
U-l
^ 50-
o
o
1— 1
to
D
Q)
A
A
/ \
r VJ
3 2
24
36 48
Time (Hours)
60
72
84
Figure 7. Fecal Coliform vs Time; Storm Event of 12/5-9/74. The
Woodlands, Station P-10.
-------
25-
O
a
cH
^
-H
O
o
O
u
0
4J
01
rt!
U
0)
[(4
20-
15-
10-
5-
12
Figure 8
24
36 4S
Time (Hours)
60
72
84
Pecal Streptococci vs Time; Storm Event of 12/5-9/74
The Woodlands, Station P-10.
-------
Figure 9. Pseudomonas aeruginosa vs Time; Storm Event of 12/5-9/74
The Woodlands, Station P-10-
oo
o
r-i
X
c
o
nj
in
o
c
M
HI
(fl
Uli
(0
c
0
E
o
OJ
(0
Time (Hours)
-------
O3
CM
C
o
o
01
i-i
O
o
C
o
o
ff.
a
to
*"
to
10
12
Figure 10
24
36 48
Time (Hours)
60
72
Staphylococcus aureus vs Time; Storm Event of 12/5-9/74.
The Woodlands, Station P-10.
-------
300-
o
c
rH
X.
OJ
c
o
(LI
C
o
e
•-H
US
Vi
200-
100-
12
Figure 11
24
36
48
60
72
84
Time (Hours)
Salmone 11 a- Arizona sp. vs Time;
The Woodlands, Station P-10.
Storm Event of 12/5-9/74.
-------
Table 1
MICROBIOLOGICAL ANALYSIS OF SURFACE WATERS IN THE WOODLANDS
SAMPLE DATE: 9/21/74 - 9/22/74 (No./lOO ml) DIURNAL STUDY.
STATION: P-10
Date & Total
Time Coliform,
9/2
5:
6:
7 :
8:
9:
10:
11:
9/2
12:
1:
2:
3:
4:
5:
6:
7:
8:
9:
10:
n =
1/74
45 PM
45
45
45
50
50
50
2/74
50 AM
50
50
50
50
55
55
55
55
55
55
18 2=1
y — »
a=
201,
60,
70,
51,
56,
78,
51,
65,
48,
62,
55,
61,
22,
39,
6,
24,
39,
31,
,019,
56,
40,
000
000
000
000
000
000
000
000
000
000
000
000
000
000
000
000
000
000
000
611
478
Fecal
Coliform,
150
130
210
310
220
200
190
150
180
70
80
80
10
15
50
200
580
130
2,955
164
127
Fecal
Streptococci ,
130
180
190
250
290
310
300
250
310
320
260
270
200
200
170
130
140
180
4,080
227
65
FC/FS
1.15
.72
1.11
1.24
.76
.65
.63
.60
.58
.22
. 31
.29
.05
.08
.29
1.54
4.14
.72
15.08
.84
.92
71
-------
Table 2
RESULTS OF PATHOGEN ANALYSES OP SURFACE WATERS IN THE WOODLANDS,
SAMPLE DATE: 9/21/74 - 9/22/74 (NO./100 ml) DIURNAL STUDY.
STATION: P-10
Date &
Time
9/21/74
5:45 PM
6:45
7:45
8:45
9:50
10:50
11:50
9/22/74
12:50 AM
1:50
2:50
3:50
4:50
5:55
6:55
7:55
8:55
9:55
10:55
n = 18
Staphylococcus
sp.
300
510
250
200
310
210
400
450
200
350
400
90
200
150
50
200
350
650
Z=5,170
X= 287
0= 195
Pseudomonas
aeruginosa
30
<10
880
40
40
130
250
450
150
750
650
950
80
50
10
50
60
60
4,640
258
325
Salmonella
sp.
200
100
400
200
600
<100
300
500
100
1,500
400
1,600
100
<100
1,200
<100
<100
<100
7,200
(n = 13)
554
532
72
-------
Table 3
MICROBIOLOGICAL ANALYSIS OP SURFACE WATERS? THE WOODLANDS.
SAMPLE DATE: 9/21/74 - 9/22/74 {No./lOO ml) DIURNAL STUDY.
STATION: P-30
Date fi
Time
9/21/74
4:50 PM
5:50
6:50
7:50
8:50
10:20
11:20
9/22/74
12:50 AM
1:55
2 :55
3:55
5:30
6:30
7:30
8:30
9:30
10:30
11:30
12:30 PM
1:30
2s 30
3:30
4:30
n = 23 Z=
X=
cr=
Total
Colif orm
81,000
78,000
83,000
69,000
83,000
50,000
112 ,000
96,000
100,000
90,000
101,000
75,000
79,000
51,000
48,000
76,000
115,000
55,000
79,000
71,000
80,000
122, 000
62,000
1,856,000
80,696
20,379
Fecal
Coliform
ISO
230
270
160
220
200
290
280
160
100
150
100
160
350
420
230
220
200
180
180
250
200
160
4,890
213
66
Fecal
Streptococci
210
210
270
150
350
230
240
250
180
260
180
190
100
70
90
140
130
220
110
300
430
560
450
5,320
213
237
FC/PS
.86
1.10
1.00
1.07
.63
.87
1.21
1.12
.89
.38
.83
.53
1.60
5.00
4.67
1.64
1.69
.91
1.64
.60
.58
.36
.36
29.54
1.28
1.19
73
-------
Table 4
RESULTS OF PATHOGEN ANALYSES OP SURFACE WATERS IN THE WOODLANDS
SAMPLE DATE: 9/21/74 - 9/22/74 (No./lOO ml) DIRUNAL STUDY.
STATION: P-30
Date S
Time
9/21/74
4 50 PM
5 50
6 50
7 50
8 50
10 20
11 20
9/22/74
12
1
2
3
5
6
7
8
9
10
11
12
1
2
3
50 AM
55
55
55
30
30
30
30
30
30
30
$0 PM
30
30
30
4 30
n = 23
Staphylococcus
sp.
650
550
1, 100
500
2 ,000
950
1,500
750
250
250
150
650
100
700
610
1,000
400
2,500
800
650
500
400
1,250
2=18,210
X= 792
0= 582
Pseudomonas
aeruginosa
1-80
260
340
110
190
190
90
170
180
150
110
90
100
60
50
160
120
110
130
150
370
540
400
4,250
185
123
Salmonella
sp.
100
600
700
100
<100
200
200
400
300
200
100
400
<100
<100
<100
<100
<100
<100
<100
100
<100
300
200
3,900
(n = 14)
279
188
74
-------
MICROBIOLOGICAL ANALYSIS
STORM EVENT OF 12/5/74 -
Table 5
OF SURFACE WATERS IN THE WOODLANDS;
12/9/74, STATION P-10.
(No./lOO ml)
Date &
Time
12/5/74
2130
2200
2300
2400
12/6/75
0100
0200
0300
0400
0500
0600
0730
0830
1015
1115
1215
13-15
1415
1515
1615
1715
1815
1919
2015
2115
2145
2245
Total
Colif orm
3xl04
BOxlO4
23xl04
61xl04
69xl04
210xl04
174xl04
7xl04
7xl04
161xl04
llxlO4
32xl04
ISxlO4
66x10
68xl04
llxlO4
19xl04
52xl04
ISxlO4
4
8x10
6xl04
7xl04
30xl04
ISxlO4
63xl04
42xl04
Fecal
Colif orm
100
1,100
1,900
3,100
3,500
4,100
3,400
2 ,600
2,800
2,700
7,100
7,400
5,900
7,900
8,300
11,500
7,800
5,000
6,800
8,200
5,900
6,600
7,300
5,000
1,500
1,900
Fecal
Streptococci
100
200
500
600
900
600
300
500
400
700
1,800
2,300
1,500
2,900
1,700
1,900
1,900
2,700
2,200
2,600
1,600
1,900
2,100
1,300
200
200
FC/FS
1.0
5.5
3.8
5.2
3.9
6.8
11. 3
5.2
7.0
3.8
3.9
3.2
3.9
2.7
4.9
6.0
4.1
1.8
3.1
3.1
3.7
3.5
3.5
3.8
7.5
9.5
75
-------
Table 5 (Cont'd)
MICROBIOLOGICAL ANALYSIS OF SURFACE WATERS IN THE WOODLANDS:
STORM" EVENT OF 12/5/74 - 12/9/74. STATION P-10.
(No./lOO ml)
Date S
Time
2345
12/7/74
0045
0145
0245
0345
0445
0545
0645
0715
0815
0915
1045
1245
1445
1645
1845
2045
2245
12/8/74
0045
0245
0445
0545
1115
1315
1515
1715
Total
. Coliform
lllxlO4
5xl04
7xl04
llxlO4
9xl04
7xl04
lOlxlO4
27xl04
39xl04
9xl04
ISxlO4
21xl04
14xl04
31xl04
41xl04
5xl04
3xl04
IxlO4
4xl04
IxlO4
16xl04
5xl04
2xl04
IxlO4
6xl04
23xl04
Fecal
Coliform
1,600
1,100
1,500
1,600
1,900
1,700
1,600
1,900
1,400
1,500
1,500
1,200
1,900
900
1,100
900
600
200
200
800
100
500
800
800
300
300
Fecal
Streptococci
700
400
400
500
400
700
300
400
100
400
400
500
500
200
100
500
300
100
100
100
200
200
100
100
100
200
FC/FS
2,3
2.7
3.7
3.2
4.7
. 2.4
5.3
4.7
14.0
3. 7
3.7
2.4
3.8
4.5
11.0
1.8
2.0
2.0
2.0
8,0
0.5
2.5
8.0
8.0
3.0
1.5
76
-------
Table 5 (Cont'd)
MICROBIOLOGICAL ANALYSIS OF SURFACE WATERS IN THE WOODLANDS:
STORM EVENT OF 12/5/74 - 12/9/74. STATION P-10.
(NO./100 ml)
Date S, Total Fecal Fecal
Time Colif orm Coliform Streptococci FC/FS
1915
2115
2315
3xl04
' 4xl04
5xl04
500
200
300
500
300
200
5.0
0.7
1.5
12/9/74
0115 4xl04 600 200 3.0
0315 IxlO4 500 100 5.0
77
-------
Table 6
PATHOGEN ANALYSIS OP SURFACE WATERS IN THE WOODLANDS:
STORM EVENT OP 12/5/74 - 12/9/74. STATION P-10.
(No./lOO ml)
Date &
Time
12/5/74
2130
2200
2300
2400
12/6/74
0100
0200
0300
0400
0500
0600
0730
0830
1015
1115
1215
1315
1415
1515
1615
1715
1815
1915
2015
2115
2145
2245
Pseudomonas
aeruginosa
100
10
10
20
40
10
10
60
60
20
200
100,
200
100
200
100
200
100
500
200
200
100
100
200
100
300
Staphy loco ecus
sp.
700
800
1,300
2 ,000
1,400
1,400
1,500
600
900
700
2,800
5,400
1,800
2,100 '
1,600
1,400
3,000
2,700
3,200
2,700
2,400
3,500
2,000
2,800
600
400
Salmonella
sp.
33
<33
<33
33
66
<33
<33
<33
<33
99
33
132
33
66
66
33
165
297
297
330
165
i
198
198
33
<33
<33
78
-------
Table 6 (Cont'd)
PATHOGEN ANALYSIS OF SURFACE WATERS IN THE WOODLANDS:
STORM EVENT OF 12/5/74 - 12/9/74. STATION P-10.
(No./lOO ml)
Date S
Time
2345
12/7/74
0045
0145
0245
0345
0445
0545
0645
0715
0815
0915
1045
1245
1445
1645
1845
2045
2245
12/8/74
0045
0245
0445
0545
1115
1315
1515
1715
Pseudoraonas
aeruginosa
600
300
200
600
200
300
10, 100
300
200
100
200
700
200
200
300
100
100
100
100
100
200
500
600
700
200
100
Staphy loco ecus
sp .
300
100
200
300
400
1,164
400
100
400
800
500
600
400
200
300
300
400
200
400
200
300
<10
10
30
140
<10
Salmone lla
sp.
<33
33
33
<33
33
<33
165
<33
<33
33
<33
33
<33
198
132
66
99
<33
66
231
<33
<25
<25
75
<25
100
79
-------
Table 6 (Cont'd)
PATHOGEN ANALYSIS OP SURFACE WATERS IN THE WOODLANDS:
STORM EVENT OF 12/5/74 - 12/9/74. STATION P-10.
(No./lOO ml)
Date S Pseudomonas Staphylococcus Salmonella
Time aeruginosa sp. sp.
1915 100 <10 25
2115 100 <10 50
2315 200 100 <25
12/9/74
0115 100 100 <25
0315 100 200 <25
80
-------
Table 7
MICROBIOLOGICAL ANALYSIS OF SURFACE WATERS IN THE WOODLANDS:
STORM EVENT OF 12/5/74 - 12/9/74. STATION P-30.
(No./lOO ml)
Sample
No.
1
3
5
7
9
10
11
12
13
14
15
16
17
19
20
22
24
Total
Coliform
3x10
15x10
104x10
5x10
55x10
34x10
5x10
73x10
26x10
27x10
4x10
110x10
112x10
57x10
311x10
9x10
3x10
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
Fecal
Coliforra
5,
6,
12,
18,
11,
14,
1,
13,
7,
14,
9,
16,
8,
34,
10,
400
200
900
800
900
100
800
200
700
700
600
200
300
300
000
400
800
Fecal
Streptococci
2
3
3
17
1
14
3
5
2
13
2
1
6
3
3
100
500
,200
,200
,600
,500
,800
,200
,100
,200
,700
,000
,800
,900
,100
,800
,900
PC/FS
4.
10.
3.
4.
5.
0.
8.
0.
4.
1.
5.
0.
5.
4.
5.
2.
0.
0
4
1
0
2
6
2
1
4
5
4
7
8
4
6
7
2
81
-------
Table 8
PATHOGEN ANALYSIS OF SURFACE WATERS IK THE WOODLANDS:
STORM EVENT OF 12/5/74 - 12/9/74. STATION P-30.
(No./lOO ml)
Sample
No.
1
3
5
7
9
10
11
12
13
14
15
16
17
19
20
22
24
Pseudomonas Staphy lococcus
aeruginosa
100
800
500
400
300
1,000
200
1,600
100
11,200
100
8,500
100
100
5,100
2 ,500
1,000
13
1
3
2
1
6
1
11
1
5
1
2
7
3
3
sp.
,700
,500
,500
800
900
,500
,500
,300
,500
,600
,400
,800
,800
,100
,900
,300
,100
Salmonella
sp-
33
33
66
264
198
33
66
66
165
231
132
33
66
33
66
231
429
82
-------
Table 9
DISINFECTION REQUIREMENTS OF STORM WATER IN THE WOODLANDS.
SAMPLE TAKEN DURING STORM EVENT OF 3/12/75 - 3/14/75.
STATION: LAKE A; 0900 3/13/75 (No./lOO Ml).
Chlorination (mg/1)
Time
t=0 hrs.
30 min .
Control
2
4
8
16
24 hrs.
Control
2
4
a
16
43 hrs.
Control
2
4
8
16
96 hrs.
Control
2
4
8
16
8 Days
Control
2
4
8
16
Total Fecal Fecal
Coliform Coliform Streptococci
160xl03 100 4,000
3
157x10 <10 4,000
200 <10 3,980
<100 <10 <10
<100 <10 <10
<100 <10 <10
3
8x10 10 3,500
<100 <10 10
<100 <10 <10
<100 <10 <10
<100 <10 <10
<100 <10 1,490
<100 <10 <10
<100 <10 <10
<100 <10 <10
<100 <10 <10
3
26.2x10 <10 520
600 . <10 • <10
380 xlO <10 <10
60 xlO_ <10 <10
113 xlO <10 <10
24 XlO* <10 50
8 X10e <10 <10
19 X10 <10 <10
36 XlO* <10 <10
5 xlO <10 <10
83
-------
Table 10
DISINFECTION REQUIREMENTS OF STORM WATER IN THE WOODLANDS.
SAMPLE TAKEN DURING STORM EVENT OP 3/12/75 - 3/14/75.
STATION: LAKE A; 0900 3/13/75 (No./lOO nil) .
Chlorination (mg/1)
Time
t=0 hrs.
30 min.
Control
2
4
8
16
24 hrs.
Control
2
4
8
16
48 hrs.
Control
2
4
8
16
96 hrs.
Control
2
4
8
16
8 Days
Control
2
4
8
16
S taphylococcus Pseudomorias
aureus aeruginosa
180 40
160 10
60 10
10 10
<10 <10
<10 10
80 <10
50 <10
10 <10
10 <10
<10 <10
460 10
120 <10
<10 <10
160 <10
40 <10
10 <10
300 <10
1,280 <1Q
20 <10
<10 <10
80 <10
110 <10
150 <10
50 <10
<10 <10
Salmonella
sp.
<25
<25
<25
<25
<25
<25
<25
<25
<25
<25
<25
<25
<25
<25
<25
<25 '
<25
<25
<25
<25
<25
<25
<25
<25
<25
<25
84
-------
Table 11
DISINFECTION REQUIREMENTS OF STORM WATER IN THE WOODLANDS.
SAMPLE TAKEN DURING STORM EVENT OF 3/12/75 - 3/14/75.
STATION: LAKE A; 0900 3/13/75 (No./lOO ml) .
Ozonation (mg/1)
Time
t=0 hrs.
30 min.
Control
2
4
8
16
24 hrs.
Control
2
4
8
16
48 hrs.
Control
2
4
8
16
96 hrs.
Control
2
4
8
16
8 Days
Control
2
4
8
16
Total
Colif orm
219xl02
157xl02
102x10
647xl02
114X102
26xl02
IxlO2
<100
<100
<100
<100
153xl02
232xl02
57xl02
400xl02
575xl02
146xl02
124xl02
126x102
217xl03
400x103
17xl04
5x104
IxlO4
SxlO4
83xl04
Fecal
Colif orm
160
130
100
40
20
90
20
20
40
110
90
<10
<10
<10
<10
<10
' 10
<10
20
10
10
<10
<10
<10
<10
<10
Fecal
Streptococci
3,500
3,100
100
<10
100
<10
600
900
910
1,070
60
200
100 "
<100
<100
<100
400
200
600
400
<100
<100
<100 ,
<100
<100
<100
85
-------
Table 12
DISINFECTION REQUIREMENTS OF STORM WATER IN THE WOODLANDS.
SAMPLE TAKEN DURING STORM EVENT OF 3/12/75 - 3/14/75.
STATION: LAKE A? 0900 HRS. 3/13/75 (No./lOO ml).
PATHOGEN ANALYSIS.
Time
t=0 hrs .
30 min.
Control
2
4
8
16
24 hrs.
Control
2
4
8
16
48 hrs.
Control
2
4
8
16
96 hrs.
Control
2
4
8
16
8 Days
Control
2
4
8
16
Ozonation
Staphy loco ecus
aureus
160
110
290
90
110
60
220
250
110
180
160
540
840
790
740
50
1,350
1,100
740
1,090
1,500
60
150
90
170
50
(mg/1)
Pseudomonas
ajaruginosa
50
40
30
<10
60
40
40
50
50
20
20
100
150
<10
100
110
<10
<10
<10
<10
10
<10
<10
<10
<10
<10
Salmonella
sp .
<25
<25
<25
<25
<25
<25
<25
<25
<25
<25
<25
<25
<25
<25
<25
<25
<25
<25
<25
<25
<25
<25
<25
<25
<25
<25
86
-------
Table 13
INDICATOR AND PATHOGEN CONCENTRATIONS IN SOILS AT FIVE LOCATIONS
IN THE WOODLANDS .
SAMPLE DATE: 10/20/74
(No./gram)
Location
Woods
Woods
at Lake B
at Lake A
Woods South of
Conference Center
Ditch
Blvd.
Swale
on Woodlands
Entering
Total Fecal Fecal
Coliform Coliform Streptococci FC/PS
124
68
58
IS
9
,000
,000
,000
,600
,400
35
250
250
90
210
100
240
100
10
390
0
1
2
9
0,
. 3
.0
.5
.0
.5
Lake B
Location
Woods at Lake B
Woods at Lake A
Woods South of
Pseudomonas
sp.
300
90
100
Staphylo coccus
sp.
50
160
150
Salmonella
sp .
<30
<30
30
Conference Center
Ditch of Woodlands
Blvd.
Swale Entering
Lake B
230
120
600
250
<30
30
87
-------
EXPERIENCES WITH RECOVERY OF VIRUSES FROM STORM WATER
J. E. SMITH, Biology Department, Syracuse University
ABSTRACT
A study was made to determine the feasibility of using the Aquella
virus concentrator (Carborundum Co.) to detect live animal viruses in real
and simulated combined storm overflows. Storms in two locations are being
investigated presently—Syracuse, N. Y. and Baltimore, Md. In Syracuse
55 gallon samples are collected, concentrated and stored; Baltimore samples
of 5 to 20 gallons are shipped by bus to Syracuse for analysis. Analysis
of virus survival subsequent to shipping (Table 1) showed that little in-
activation occurred as a result of thermal shock since viruses in TPB or
sewage plant influent were not appreciably reduced in titer. However,
Gwynns Palls creek water seeded with laboratory viruses commonly showed re-
ductions of 50 to 60 per cent. This is not a progressive inactivation since
seeded creek water maintained its titer for nine days when stored at 4C
(Table 2). Other work showed that the creek water inactivated seed virus
within a few minutes after the virus was diluted in it and before any
thermal inactivation could take place.
Two concentration techniques have been employed to adsorb viruses
preferentially to Cox epoxy fiberglass filters (Figure 1). Picornaviruses
can tolerate a combination of low pH for adsorption (pH 3.5) and high pH
for elution (pH 11.5). This effectively eliminates adenoviruses and reo-
viruses. To preserve them, adsorption is at pH 4.5 and elution, at pH 9.0.
Picornaviruses are also adsorbed under these conditions and they are
eliminated routinely by the addition of antisera. More than 60 percent of
the isolates in combined storm overflow are polioviruses—probably vaccine
strains—and it is necessary to suppress them since they quickly overgrow
most other viruses. Benzlmidazoles suppress many single stranded RMA viruses
but have little effect on DNA adenoviruses or double-strand SNA reoviruses.
The latter two are easily differentiated on the basis of CPE and cytopathic
inclusions.
Isolations have been made principally on HEp-2, Wl-38 and pig kidney
PK15 cells. More recently BGM cells and FLOW Labs #2000 fetal lung cells
have been studied for comparative plating efficiency. It is anticipated
that BGM cells will be the cell line of choice for most water-borne viruses
of human origin. Pig kidney PK15 cells did not prove to be a broad adeno-
virus indicator line as we had anticipated and were decidedly inferior to
HEK cells.
88
-------
General operating experience with the Aquella virus concentrator
suggests that recovery of snail numbers of viruses from large volumes of
storm overflow is possible but the efficiency is more in the range of 30
per cent, particularly when a combination of heavy silt and high organic
content is encountered. The number of 50 gallon samples which can be
processes completely in one working session is limited to about two/day. As
a consequence we either store 50 gallon samples at 4C or else concentrate
the sample to the first elution step and maintain the sample while we
process other large samples. Enteroviruses survive the holding processes
well but the data concerning longevity of adenoviruses and reoviruses are
still incomplete.
Table 3 summarizes the qualitative aspects of storm samples which have
been studies to date. Viruses have been found in nearly all sewage samples
in both the raw sewage and the chlorinated effluents. Poliovirus is the most
frequent but other viruses are usually in the same sample of fecal polluted
water. (The values are based on assays which directly examined 20 per cent
of the whole concentrated sample). Despite the obvious difference in sample
sizes, Syracuse storm overflow and Baltimore storm overflow do not differ
greatly in the detection frequency. This probably is fortuitous, but
suggests that the Aquella concentrator might be redesigned to handle more
samples at lower expense.
A limited number of Syracuse storms have been analyzed at the Maltbie
Street treatment facility. Viruses were detected in storm overflows with
one sample being particularly marked (7/3/74). Experience with the chlorine
dioxide treatment is still too limited to make any real conclusions con-
cerning its effectiveness against naturally occurring viruses. Some diffi-
culties in logistics were experienced when samples were first being acquired
and these samples tended to be collected at widely spaced intervals in the
storm. Samples collected later in the season were pumped into drums and are
more representative of water for any given period in the storm.
TI
Although the project is still in its early stage, viruses have been
recovered from both dry flow of urban Baltimore creeks and from storm over-
flows (Table 5). The viruses are barely at the detectable level and the
identification of the serotypes of the viruses is still in progress. As
might be anticipated, dry season flows tend to show higher titers than
storm flows. Those areas with no intentional sanitary discharge such as
Northwood have proved to be negative so far. Those areas with overflow dis-
charge, Howard Park, Jones Falls Storm Outlet, and Bush Street, reflect
fecal discharge.
89
-------
Pig. 1. Identification and quantification of vi-
ruses In storm waters after concentrating them with
the Aquella instrument. Cell culturej HEp-2 and
BGM cells.
Adsorption to Cox
epoxyflberglasa
filter, pH 3.5.
0.0005 M A1C13
PICORNAVISUSE3
Elutlon pU 11.5»
0.05 M glyoina
20 ml
Neutralize
«x\
STORAGE
-65C
PBS or TPB
plus 2%
. PH 7.
serum
2
Adsorption to Coz
epoxyflborglaos
filter, pH 4.5.
0.05 M HgCl2
HEOVIRU3B3
Elutionwith ADENOVIHU3E3
boef extrao
pH 9.0 plus
aonioatloa,
20 ml
Neutralize
Anti-polio serum
and/or
•Ati-ooxsaokia
Three 10-fold
dilutions
PLAQUES
DIBECT COUNTS,
KPH ESTIMATES
Sterile toothpick
transfer method
MICHOTITEH SUBCULTURE
TPB
HEPEBENCS CJULTOBE
\ t
SPECIFIC
SEEOTXPING
HICROTITEH SUBCULTURE PLUS SERA AGAINST
(a) polio 1,2,3
(b) polio 1,2,3 &*& ooxsaokie B (pooled goat antioerc)
^IDENTIFIED VIRUS WHICH BREAKS THBOUGB (a) and (b)
1) pooled antlaera against eohoviruses
sensitivity to toenzlmidazolea and guanidlne HC1
aeridine orange staining
CPE with Giemsa staining
2)
I
90
-------
Table 1. Relative survival of enteroviruses during shipment.
Following collection the water samples were maintained
at ambient temperatures for 10 hours and at AC for 12
to 15 hours.
Sample
&
Number Virus
7/29 Polio- I
8/5 Pol!o-l
9/1? ' Pollo-1
9/24 Pol to- 1
10/29 Polio- I
1/14 Potlo-l
(#27)
1/20 Poll o-l
(130)
Per cent survivors after dilution
and shipment in:
Creek Water Sewage
Gwynns Falls Back River
TP8 (Site D) Treatment Plant
(Site A)
100 20 80
pH 7.3 pH 7.0 pH 6.0
5
P» 6.8
«<100 13 #100
P« 7-3 pH 6.9
36
pH 6.9
160 16
pH 6.9
69 50
pH 7.3 pH 7.3
100
pH 7.3
110 70 81
pH 7.3 pH 7.25 pH 6.9
9/24 Coxsackte 83
1/20 Coxsacfde 83
(#30)
9/24 Echo-7
66 33
pH 7.3 pH 6.9
90 82 95
PH 7.3 pH 7.25
68 54
pH 7-3 pH 6.9
1/20 Echo-7
99 77 69
pH 7.3 P« 7-25 pH 6.9
91
-------
Table 2- Survival of pollovirus 1 supplied as a tissue culture
supernatant, diluted 1:10 in Gwynns Falls Creek water
and shipped to Syracuse, N.Y. The tissue culture
maintenance medium contained Eagle's minimum essential
medium (E-MEM) with 5 per cent fetal calf serum.
Vi rus
treatment
Hours
Relative
titers
(PFU/ml)
Per cent
survival
TC vi rus, thawed
and titered
TC vi rus , diluted in
creek water and
shipped to Syracuse
TC virus diluted In
creek water, shipped
and stored at 4C
10
216
6 x 10
7.9 x 10
8.3 x 105
100
13
92
-------
Table 3. Summary of natural oocurronoe of viruses in untreated water
sources. Estimates are based on MPN values for 0 tubes inoculated with
each of three suacosslvo 10-fold dilutions.
Total no.
rfater Source No. positive
Samples samples
No. positive
samples Avernsre
after add'n sample
anti-Polio serum (liters)
Sewage
Sewage
Storm
Storm
Creelcb
Syracuse0
Baltimore*'0
Syracuse
Baltimore6
Baltlmorea
11
l*
1*
16
10
11
k
6
7
6
6
k
it
5
0
4 to 260
19
111 to 193
19
19
^Through
sDry weather flow
'Treatment plant influent
93
-------
Table
—
1
0
0
0
0
0
0
15
0
0
Other
0
0
9
21
0
1
0
0
0
0
0
0
0
0
lchlorlne dioxide
-------
Table 5. Recovery of viruses from Baltimore storm overflows.
co
en
Water Place
A Back Hiver Treatment Plant 375
I Dry Herring Bun tt
C Flow Jones Falls 70
D Gwynns Falls 3
F Stoney Bun 0 Oj, 1
G Western Bun 0 2x10 0
H Stona Howard Park i 20
K Overflow Jones Falls (Storm outlet) 0 4-30
L Bush Street 5 11
M Korthwood 0 0
-------
THE ENHANCEMENT OF HIGH-RATE DISINFECTION
BY THE SEQUENTIAL ADDITION OF CHLORINE
AND CHLORINE DIOXIDE
Edwin C. Tifft, Peter E. Moffa'and Steven L. Richardson
O'Brien & Gere Engineers, Inc.
1304 Buckley Road
Syracuse, New York 13201, U.S.A.
and
Richard Field
U.S. Environmental Protection Agency
Storm and Combined Sewer Section
Edison, New Jersey, 08817, U.S.A.
ABSTRACT
The magnitude of combined sewer overflows (CSO) that occur within urban
areas limits the use of conventional disinfection techniques that necessitate
large facilities. Point-source treatment of CSO by high-rate application of
25 mg/1 chlorine (C^) or 12 rag/1 chlorine dioxide (CIO,) reduced indicator
bacterial and viral counts to levels acceptable for discharge to recreational
waters. When added sequentially at 15 to 30 second intervals, 8 mg/1 Cl2
followed by 2 mg/1 C102 achieved lower bacterial counts after two minutes
contact time than would be predicted from corresponding single-stage disinfec-
tion results. This effect may be due to an interaction between the two disin-
fectants rather than a reduction in demand for C102 caused by the prior addi-
tion of Cl2« It is hypothesized that the more potent disinfectant, CK^, is
regenerated by the reaction of Cl2 with chlorite (C102), the decomposition
product of C102.
96
-------
Introduction
Combined sewer overflows (CSO) remain one of the most
neglected sources of microbial contamination of surface waters.
Although intermittent in nature, the volumes of these overflows
are sufficient to raise the indicator bacterial counts in
receiving waters to levels that preclude contact recreation.
The occurrence of most CSO within highly developed urban areas,
where land is often unavailable or expensive, restricts the
use of conventional treatment techniques that require long
detention times and large facilities. Several methods of
high-rate point-source treatment of CSO were studied in an
effort to circumvent this restriction. The facilities, which
are described in detail elsewhere (Moffa et al. , 1975),
include fine-mesh screening or swirl concentration to reduce
suspended solids followed by disinfection with chlorine (Clg)
or chlorine dioxide (C102) to reduce microbial organisms.
Clg has been found to give satisfactory disinfection in
similar applications (Glover and Herbert, 1973). However,
concern about the generation of possible carcinogens through
chlorination (Dowty et al., 1975), and the cost and availability
of C~\2 would indicate the desirability of an alternate disin-
fectant. ClOg nas snown potential in previous limited appli-
cations and was investigated as an alternative. Although many
reactions for the manufacture of C102 use Cl2 as a starting
material, at least one method of generation (Callerame, 1973)
requires only other common chemicals.
97
-------
The evaluation was conducted in two parts. First a series
of bench-scale studies was performed to determine the approxi-
mate dosages of Cl2 and C102 that would reduce microbial
counts to acceptable levels. The second phase consisted of
verification of these findings by operation of two full-scale
prototype treatment facilities for CSO.
Methods
The bench-scale studies were conducted according to the
flow charts given in Figures 1 and 2. The disinfectants, Cl2
and C102» were applied individually and sequentially (single-
stage and two-stage studies, respectively) to portions of a
simulated combined sewer overflow (SCSO). The SCSO was a
mixture of equal parts distilled deionized water and influent
to the Onondaga County Metropolitan Treatment Plant and was
chosen for ease and consistency of comparisons to be carried
out over several months. Samples for bacterial and viral
analysis were collected in bottles containing an amount of
sodium thiosulfate sufficient to halt the disinfection by
either Cl2 or C102. Bench-scale screening was performed by
passing the samples through a screen with a 23 micron pore
diameter.
The site plans for the prototype treatment facilities are
shown in Figures 3 and 4 for Maltbie Street and Figure 5 for
West Newell Street. The Maltbie Street facility was designed
around the concept of high-rate, fine-mesh screening followed
by high-rate disinfection. Three parallel screening units,
98
-------
a Crane Microstrainer (Crane Co., King of Prussia, Pa.) with
a screen aperture of 23 microns, a Zurn Micro-Matic (Zurn In-
dustries, Inc., Erie, Pa.) with a screen aperture of 71 microns
and a Sweco Wastewater Concentrator (Southwestern Engineering
Co., Massilon, Ohio) with a screen aperture of 105 microns,
can each receive equal flow up to a maximum of 219 I/sec
(5 mgd) flow. The effluent from each unit can be disinfected
with either Cl£ or C102 for a contact time of up to two min-
utes. A proportioning weir was constructed so the contact
time could be held constant with different flows.
The West Newell Street facility was designed around the
swirl concentrator conceived in England (Smisson, 1967) and
further developed by the U. S. Environmental Protection Agency
(Field, 1973). The influent to the swirl concentrator may
reach a maximum of 400 I/sec (8.9 mgd) and any flow in excess
of 66 I/sec (1.5 mgd) will overflow the regulating weir. The
outfall pipe for this overflow serves as the contact chamber
for disinfection so the contact time will vary with flow up
to one minute.
Samples were collected at the designated locations in
Figures 4 and 5 with refrigerated sequential sampler (Sigma-
motor, Inc., Middleport, N.Y.). For disinfected samples,
sodium thiosulfate was added to each of the bottles before
sample collection. The standard bacterial indicators of
water quality, total coliforms (TC), fecal coliforms (FC) and
fecal streptococci (FS) were enumerated using the membrane
99
-------
filter (MF) technique (Standard Methods, 1971) with three
replicates for each sample. Although there is some question
surrounding the validity of the MF technique in chlorinated
waste-waters, periodic correlation with the most probable
number (MPN) technique established the reliability of the MF
technique in chlorinated CSO for this study. All samples were
blended for approximately six seconds at approximately 3000'
rpm in order to enumerate any bacteria that may have been har-
bored within suspended matter and grease particles. The
effects of disinfection were also measured by enumeration of
two bacterial viruses, f2 and 0X174 and one enteric virus,
Poliovirus Sabin Type 1 (Polio-1). Due to the'relatively low
natural levels of virus in wastewater, the samples were seeded
with a known concentration of test virus prior to disinfection.
The viruses were preconcentrated on a membrane filter before
enumeration by the plaque assay method (Dulbecco and Vost,'*
1954).
Cl2 was obtained as a five percent solution of sodium
hypochlorite 1n water. Standardization of stock solutions and
determinations of residual Cl2 were accomplished using the
thiosulfate and DPD methods (Standard Methods, 1971). C102
was generated on a bench-scale by acidification of a solution
of sodium chlorite (Standard Methods, 1971) and on a full-scale
by the nitrosyl chloride method (Callerame, 1973). The
measurement of C102 concentrations was performed by the DPD
technique and the use of electron spin resonance (esr) which
100
-------
has been advocated as a primary standard method for the analy-
sis of C102 (Murphy and Tifft, 1973).
Results and Discussion
The results of single-stage disinfection of bacteria in
SCSO with Cl2 and C102 are shown in Tables 1 through 4. Since
each trial was run on a separate batch of SCSO, the initial
bacterial counts varied by an order of magnitude or more.
Consequently, the results were converted to fraction survivors
in order to average the values. Two of the trials were run
on screened SCSO (23 micron aperture) to determine the effects
of screening, but the variations in duplicate trials were of
the same magnitude as the difference between screened and
unscreened trials. The variations in fraction survivors from
the reported averages ranged from 10 to 50 percent. The re-
peated measurement of bacterial populations in CSO shows an
approximate average maximum value of 5,000,000 TC/100 ml
(Moffa et al.} 1975). The general guidelines for maximum
bacteria levels in the discharge are 1,000 TC/100 ml and a
fraction survivors of 0.0002 would correspond to the desired
effluent population under adverse conditions. The results in-
dicate that these reductions can be obtained with 25 mg/1 Cl2
or 12 mg/1 C102 within 120 seconds. The results also show that
FS may be more resistant to disinfection and perhaps a more
reliable bacterial indicator.
The results of single-stage high-rate bench-scale disin-
fection of viruses with C12 and C102 are summarized in Table 5.
101
-------
Table 1
H>
O
to
Contact Tim
(seconds )
0
30
60
120
180
300
S
ingl
e
ft
1.00
0.
1.
1 .
1.
1.
86
10
28
00
06
e-Stage
Col iform
Disinfection of
in SCSO
wi
th Cl
Cl? Dosage mg/
4
1 .00
0.32
0.63
0.22
0.88
0.44
8
1 .00
0."83
0.26
0.19
0.18
0.079
1.
0.
0.
0.
0.
0.
12
00
10
049
043
013
0052
Total
?a
1
1.
0.
0.
0.
0.
0.
ie
00
13
037
034
00039
00059
1
2
0
0
0
0
20
.00
.11
.0020
.0011
.00012
.000057
25
1 .00
0.0067
0.0030
0.00021
0.00093
0.00087
aValues in fraction survivors
-------
Table 2
o
co
Contact Time
(seconds)
0
30
60
120
180
300
Si
0
1 .00
0.99
1.14
0.89
0.93
0.98
ngl
1
0
0
0
0
0
e-Stage
Strep 1
i
.00
.77
.77
.68
.58
.44
Disinfect
n SCSO
8.
1 .00
0.78
0.47
0.23
0.22
0.08
ion of
Fecal
With Cl?a
1
1.
0.
0.
0.
0.
0.
£
00
44
30
18
10
05
1
1.
0.
0.
0.
0.
0.
6_
00
27
26
11
06
03
20.
1 .00
0.17
0.10
0.02
0.02
0.01
25.
1.00
0.056
0.009
0.006
0.001
0.002
aValues in fraction survivors
-------
Table 3
Single-Stage
on tact Time
(seconds)
0
30
60
120
180
300
0
1.00
0.92
0.80
1.07
0.74
0.90
Col iform
4
1.00
0.199
0.120
0.088
0.024
0.127
Disinfection of Total
in SCSO
CIO?
8
1.00
0.018
0.014
0.010
0.001
0.012
With C10?a
Disage (mg/1 )
'12 16
1.00 1.00
0.0060 0.0049
0.003 0.0017
0.0008 0.0012
0.0002 0.0001
0.0009 0.0012
20. 25.
1.00 1.00
0.0121 0.0017
0.0007 0.0009
0.0005 0.0005
0.0001 0.0001
0.0002 0.0003
aValues in fraction survivors
-------
Table 4
o
en
Contact Time
(seconds )
0
30
60
120
180
300
Si
1
0
0
0
0
0
ngle-Stage Di
S
0
.00
.98
.97
.83
.88
.95
trep in
1.
0.
0.
0.
0.
0.
4
00
30
50
21
13
25
sinfection
of
Fecal
SCSO With C10?a
1
0
0
0
0
0
CIO?
8_
.00
.064
.056
.041
.0016
.0054
Dosage (mg/1 )
1
0
0
0
0
0
11
.00
.020
.01
.01
7
6
.0010
.0016
1
1 .
0.
0.
0.
0.
0.
6
00
010
0026
0016
0002
0009
20 25.
1.00 1.00
0.0026 0.0034
0.0022 0.0022
0.0010 0.0006
0.0001 0.0001
0.0002 0.0001
aValues in fraction survivors
-------
Table 5
Disinfection of Viruses
Contact
Dosage Time Log
Virus Disinfectant mg/1 (seconds) Red
0X174 C12 20 120 4.7
0X174 Cl£ 25 120 5.0
0X174 C102 12 30 5.4
Polio-1 CIO? 8 30 2.2
Pol1o-l C102 12 120 4.0
Polio-1 C102 16 120 5.0
f2 C12 20 120 3.0
f2 C102 4 120 2.8
f2 C102 7 120 3.6
f2 C102 9 120 4.5
106
-------
The values are reported in terms of logarithm reductions for
the following reasons. Natural virus levels in sewage and
overflows are generally unknown, but it is assumed that the
values would rarely exceed 1 X 105 pfu (plaque forming unit)/
ml. Thus, a reduction of five logarithms would bring the
virus levels to 1 X 10° pfu/ml or less. This target is com-
pletely arbitrary and should not be taken as a viral standard
because the ingestion of only one virus particle may be an in-
fectious dose. Present methods of viral enumeration do not
permit the rapid detection of counts below 1 X 10^ pfu/ml in
wastewaters. Therefore^ in order to facilitate the experi-
ments, the samples were seeded to give initial levels of 1 X
10° pfu/ml. It was assumed that a reduction from 10^ to 10^
pfu/ml can be accomplished under the same disinfectant condi-
tions as a reduction from 10 to 10° pfu/ml. This is based on
the fact that in these ranges the number of disinfectant-deman-
ding organisms is negligible compared to the number of disin-
fectant molecules. Therefore, the rate of kill is a function
of disinfectant concentration and contact time but not viral
population. These results indicate that 25 mg/1 Cl2 and 12 mg/1
C102 would give satisfactory virus kills in 120 seconds. The
virus studies are discussed in greater detail elsewhere (Smith
and McVea, 1975).
The next step involved verification of these findings in
the operation of the full-scale prototype treatment facilities.
Table 6 summarizes the work that was completed in 1974. For
107
-------
Table 6
Disinfection of CSO
o
oo
Facility
Maltbie Sweco
Newell Swirl
Maltbie Zurn
Overfl
Date
6/10/74
6/10/74
6/14/74
6/14/74
7/29/74
7/29/74
7/29/74
9/3/74
9/3/74
9/3/74
9/21/74
ow
Time
20:00
20:20
11 :30
10:30
18:10
18:50
19:30
9:10
11:00
11:30
17:30
Flow Rate
(mgd)
1.88
1.88
0.75
0.62
3.40
3.40
3.40
0.50
0.50
0.50
1 .00
Disin-
fectant
C102
C102
C102
C102
C102
C102
C102
C102
C102
C102
C102
Dosage
(mg/1)
14.6
14.6
7.5
8.5
7.0
7.0
7.0
8.0
8.0
8.0
12.2
Bacterial Reduction
Fraction Survivors
TC
0.32
0.02
0.0027
0.010
-_
--
--
0.00084
0.030
0.0004
0.097
FC FS
0.30
__
0.012 0.017
0.024 0.015
0.011
0.012
0.012
0.0093
0.0067
0.028
__
-------
all trials the contact time was approximately one minute. The
data shows that the target disinfection, as defined previously
in this paper, is possible.
The effect of one disinfectant (C12) followed by a second
(C102) after an interval of 15 to 30 seconds (two-stage disin-
fection) is presented in Table 7.
Table 7
Two-Stage Disinfection of Bacteria in SCSO
Fraction Survivors at
Cl2 Dosage C102 Dosage Interval 120 Seconds
(mg/1) (mg/1) (Seconds) J_£ £S FT
4 2 15 0.015 0.039
4 2 30 0.034 0.120
4 4 15 0.032 0.055
4 4 30 0.0050 0.031
8 2 15 0.00059 -- 0.00074
8 2 30 0.00045 -- 0.0011
8 415 0.00015 0.00099 0.000030
8 4 30 0.00010 0.00059 0.000040
Varying the interval between the addition of each disinfectant
from 15 to 30 seconds did not affect the rate of kill, presum-
ably an effect of the rate of mixing. In order to determine
1f disinfection had been enhanced beyond the additive Effects
of the different agents, the log reductions were compared.
Table 8 includes the log reductions in TC accomplished by 4
mg/1 and 8 mg/1 C12» and 2 mg/1 and 4 mg/1 C102 as determined
in the single-stage studies (from Tables 1 and 3, respective-
109
-------
1y). The sums of the reductions were compared to the observed
log reductions in the two-stage studies for the corresponding
dosages in Table 9.
Table 8
Average TC Reductions for
Log
Disinfectant (mg/1 ) (seconds) Reduction
C12 4 120 0.66
Single-Stage
Dosage
(mg/1)
4
8
2
4
Disinfection
Time
(seconds)
120
120
90
90
C12 8 120 0.72
C102 2 90 0.503
C102 4 90 0.99a
alnterpolated Values
Table 9
Average TC Reductions
Two-Stage Disl
First
Disinfectant
C12
C12
C12
C12
Dosage
(mg/1)
4
4
8
8
Second Dis-
infectant
C102
C102
C102
C102
for
infection
Dosage
(mg/1)
2
4
2
4
Log Reduction
at 120 Seconds
Predicted Observed
1.16 1.60
1.65 1.80
1.22 3.28
1.71 3.92
At low reductions there were small differences between
observed and predicted values, most likely because of diffi-
culties in measuring small differences in large numbers. As
HO
-------
the reliability in reported differences increased, the en-
hancement of disinfection became more easily discernible.
It was first thought that C12 might precondition the SCSO
by reducing the demand for C102. To test this hypothesis, a
sample of SCSO was split into two portions for determination
of Cl2 and C102 demands. The sample for which the Clg d.emand
had been satisfied was then dechlorinated with thiosulfate.
The C102 demand of this sample was found to be about 80% of
the C102 demand of the original sample. Thus, some other
mechanism must be responsible for the enhanced disinfection.
It is possible that the two disinfectants could Interact
through the following mechanism. One.of the reactions for
the preparation
-------
8.5 to 1. Current figures show that the cost of one pound of
C102 is three to four times the cost of one pound of C^.
Thus, two-stage application of Cl2 and C102 may result in sig-
nificant savings.
CONCLUSIONS
The economic and environmental objections to the use of
chlorine for terminal wastewater disinfection have led to the
investigation of alternative disinfectants. Twelve mg/1 of
chlorine dioxide has given the same bacterial and viral kills
as twenty-five mg/1 chlorine in two minutes contact time.
This is especially pertinent to disinfection of combined sewer
overflows. The effects of chlorite ion, the by-product of
disinfection with C102» remain to be investigated. The possi-
bility of using both disinfectants in combination (8 mg/1 Cl£
and 2 mg/1 C102) may be a way to Increase efficiency and reduce
operating expenses.
112
-------
ACKNOWLEDGEMENTS
The virus studies were conducted by James E. Smith of
the Biological Research Laboratories of Syracuse University.
The assistance of John M, Karanik, Project Officer for the
Division of Sanitation and Drainage, Department of Public
Works, County of Onondaga, New York, is acknowledged. This
work was co-sponsored by the County of Onondaga and the U. S.
Environmental Protection Agency, Grant No. S-802400.
REFERENCES
Callerame, J. Nitrosyl Chloride Generation of Chlorine Dioxide.
U. S. Patent No. 375079, 1973.
Dowty, B., Carlisle, D., Laseter, J.L. and Storer, J.
Halogenated Hydrocarbons in New Orleans Drinking Water and
Blood Plasma. Science 187, 75-77, 1975.
Dulbecco, R. and Vogt, M. Plaque Formation and Isolation of
Pure Lines of Poliomyelitis viruses. J. Exp. Med. 99, 167-182,
1954.
Field, R. Dual Function Swirl Combined Sewer Overflow Regulator/
Concentrator. EPA-670/2-73-059, U.S. Environmental Protection
Agency, 1973. 49 pp.
Glover, G.E., and G.R. Herbert. Hicrostraining and Disinfection
of Combined Sewer Overflows - Phase II. EPA-R2-73-124,
U.S. Environmental Protection Agency, 1973. 116 pp.
Moffa, P.E., et al. Bench-Scale High-Rate Disinfection of Com-
bined Sewer Overflows with Chlorine and Chlorine Dioxide.
EPA-670/2-75-021, U.S. Environmental Protection Agency, 1975.
181 pp.
113
-------
Murphy, C.B. and E.G. Tifft. A Novel Application of Electron
Spin Resonance; The Analysis of Chlorine Dioxide in Wastewater.
Presented at the 5th Northeast Regional Meeting of the American
Chemical Society, Rochester, N.Y. 1973.
Smisson, B.S. Construction and Performance of Vortex Overflow.
Proceedings of the Symposium on Storm Sewage Overflows,
Institute of Civil Engineers, 1967. pp. 99.
Smith, J.E. and J.L. McVea. Virus Inactivation by Chlorine
Dioxide and Its Application to Stormwater Overflow. Proceedings
of the 166th American Chemical Society Meeting, Chicago, 111.
1973.
Standard Methods for the Examination of Water and Wastewater,
13 ed. American Public Health Association, New York. 1971.
114
-------
2.6
gol«
3 gait
RAW SEWAGE
DISTILLED WATER
SIMULATED OVERFLOW
23 MICRON SCREEN
0«
NO SCREEN
Y T T V T V TT DISINFECTANT
4 mg/l 8 mg/l B mg/i 2 mg/l- 2 mj/l 4 mg/l 4mg/l DOSAGE
15 QR 30
SECONDS MIXING
I'1 STAQE
-JOR
i
i
1
1
Cl,
I
1
1
1
T T T T T T T T DISINFECTANT
2 mg/l 4 mg/t 2 mg/l 4 mg/l 2 mg/l 4 mg/t 2 mg/l 4 mg/l DOSAGE
11111111
llOj
t
CONTINUOS
MIXING
2nd STASE
SAMPLES TAKEN AT
0,15,30,60,120,180,300 SECONDS
FIGURE 2. OUTLINE FOR TWO-STAGE DISINFECTION
116
-------
2.9
RAW SEWASE
I.I DISTILLED WATER
SIMULATED OVERFLOW
I
23 MICRON SCREEN
OR
NO SCREEN
DISINFECTANT
0 mg/I 4 mo/I 8mg/l I2mg/| temg/l 20mg/l 23mg/l DOSAGE
IS SECONDS
MIXING
I LITER
BEAKERS
1
1
1
1
JL
i
1
1
J,
1
JL
|
i
|
SAMPLES TAKEN AT
0,15,30,50,120,180,300 SECONDS
FIGURE 1. OUTLINE FOR SINGLE-STAGE DISINFECTION
115
-------
ONONOAGA
CHEEK
DISINFECTION CONTACT
TANKS
TOTAL DISINFECTED
EFFLUENT
OVERFLOW REGULATOR
-W/ LEAPING WEIR
&
COLLECTOR
TRUNK
TRANSPORT
TRUNK
SOLIDS CONCENTRATE-
LINE
SIPHON TO MAIN
INTERCEPTOR
FIGURE 3. MALTBIE STREET SITE PLAN
-------
SWECO
WASTE -
WATER
CONCENT-
RATOR
SCREENING BUILDING AND DISINFECTION TANKS
KEY
M-FLOW METER
X - SAMPLING LOCATION
— -FLOW DIRECTION
PUMPIN9 STATION
FIGURE 4. MALTBIE STREET PROCESS ORIENTATION
118
-------
EXISTING
5.
WEST
SITE
119
-------
TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
, REPORT NO.
EPA-600/2-76-244
3. RECIPIENT'S ACCESSION-NO.
4. TITLE AND SUBTITLE
PROCEEDINGS OF WORKSHOP ON
MICROORGANISMS IN URBAN STORMWATER
5. REPORT DATE
November 1976 (Issuing Date)
8. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
Richard Field,
James E. Smith,
Vincent P. Olivieri,
Edwin C. Tifft, Jr.
Ernst M. Davis,
B, PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Storm and Combined Sewer Section
Wastewater Research Division
Municipal Environmental Research Laboratory (Cincinnati)
Edison, New Jersey 08817
10. PROGRAM ELEMENT NO.
1BC611
11. CONTRACT/GRANT NO.
12. SPONSORING AGENCY NAMi AND ADDRESS
Municipal Environmental Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, Ohio 45268
13. TYPE OF REPORT AND PERIOD COVERED
Workshop Proceedings
£&_
ING
14. SPONSORING AGENCY CODE
EPA-ORD
16. SUPPLEMENTARY NOTES
Workshop Proceedings based on SPA Grants S802433, R802709 and S802400.
P.O. Richard Field (201) 548-3347 X503 (8-342-7503)
16. ABSTRACT
This workshop was held on March 29, 1975 at the USEPA Office, Edison,
New Jersey. The aim was to exchange information obtained from USEPA Office of
Research and Development, Storm and Combined Sewer Program sponsored projects
so as to foster a better understanding of microorganisms in urban storm runoff
and combined sewer overflow.
Workshop emphasis was placed on the following aspects:
a. Procedures for pathogenic microorganism assays
b. Relationship between pathogenic and coliform group microorganisms
c. Disinfection and aftergrowth of microorganisms
d. Viruses in stormwater
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.lDENTIFIERS/OPEN ENDED TERMS
c. COSATI Field/Group
Waste water, Sewage, Microorganisms,
Viruses, Coliform bacteria, Disinfection,
Contaminants, Waste treatment, Water
pollution, Water treatment, Bacteria,
Salmonella, Water quality, Shigella,
Streptococcus, Storm sewers, Pseudomonas,
Surface water runoff, Runoff
Pathogenic microorganisms
/coliform bacteria
relationship, Combined
sewer overflows, Waste-
water treatment, Water
pollution sources,
Storm runoff
13B
18. DISTRIBUTION STATEMENT
RELEASE TO PUBLIC
19. SECURITY CLASS (ThisReport)
UNCLASSIFIED
21. NO. OF PAGES
128
20. SECURITY CLASS (Thtspage}
UNCLASSIFIED
22. PRICE
EPA Form 2220-1 (9-73)
120 &U.$.GOMBNMEHrrtlimNG OFFICE: 1979-657-060/1671 Region No. 5-11
------- |