Environmental Protection Technology Series

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                                          EPA-600/2-77-087
                                          July 1977
          MICROORGANISMS IN URBAN STORMWATER
                         by
                  Vincent P. Olivier!
                  Cornelius W. Kruse
                   Kazuyoshi Kawata
             The Johns Hopkins University
           School of Hygiene & Public Health
          Department of Environmental Health
              Baltimore, Maryland  21205
                         and

                    James E. Smith
                  Syracuse University
               Syracuse, New York  13210
                   Grant No. R802709
                    Project Officer

                     Richard Field
           Storm and Combined Sewer Section
             Wastewater Research Division
Municipal Environmental Research Laboratory (Cincinnati)
               Edison, New Jersey  08817
       MUNICIPAL ENVIRONMENTAL RESEARCH LABORATORY
           OFFICE OF RESEARCH AND DEVELOPMENT
          U.S. ENVIRONMENTAL PROTECTION AGENCY
                CINCINNATI, OHIO  45268
                                                                   "*••
                   EERU-TIX
RECEIVED"
 APR 18 1989
 EERU-TIX

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                                    DISCLAIMER
         This report has been reviewed by the Municipal Environmental Research
    Laboratory,  U.S. Environmental Protection Agency,  and approved for publica-
    tion.  Approval does not signify that the contents necessarily reflect the
    views and policies of the U.S. Environmental Protection Agency,  nor does
    mention of trade names or commercial products constitute endorsement or
    recommendation for use.
C 3 VI ;:*:„•
                                        ii

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\
                                        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,  treat-
       ment, 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 publica-
       tion is one of  the products of  that research; a most vital  communications
       link between  the researcher and the user community.

            This report documents microbiological  quantitative assays of  Baltimore
       City urban  runoff to show  the relationships to several factors such as
       separate or combined sewer flow, urban characteristics of drainage area,
       rainfall, and quantity of  flow  during and between rain storms.  In general,
       there was a consistently high recovery of both pathogenic and indicator
       microorganisms  throughout  the study.
                                             iii

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                             ABSTRACT
     Microbiological quantitative assays of Baltimore City urban runoff
were conducted throughout a 12 month period to show the relationships
to several factors such as separate or combined sewer flow, urban
characteristics of drainage area, rainfall, and quantity of flow during
and between rain storms.  In general, there was a consistently high
recovery of both pathogenic and indicator organisms throughout the
study except for Shigella sp. which is believed to have been present
but could not be isolated due to interferences during the culture pro-
cedure.  There appeared to be little relationship between pathogen
recovery and season of the year, amount of rainfall, period of the
antecedent rainfall, and stream flow.  The most concentrated pathogens
were Pseudomonas aevug-inosa and Staphylooocaus auveus at levels ranging
from 103 to 105 and from 10° to 103/100ml, respectively.  Salmonella
and enteroviruses, though frequently isolated, were found at levels
of only 10° to lOvlO 1 of urban runoff. The background samples (sewage,
urban streams and reservoirs) between storms gave good positive corre-
lation between indicators and pathogens at a 95 to 99% level of con-
fidence, whereas, the stormwater had no or poor correlation.  The
ratios of indicators, such as FC/FS, gave some indications of pollution
by human sewage, but it was the presence of enteroviruses that definitely
showed the mixing of sewage with rain water, whether in a storm sewer
or in the combined sewer overflow.  The logical solution would point
to the removal of sanitary sewage overflows rather than the disinfection
of all urban runoff for removing the health hazard and improving the
quality of urban runoff.

     This report was submitted in fulfillment of grant no. R802709
by the Johns Hopkins University under the sponsorship of the U.S.
Environmental Protection Agency.  This report covers a period from
July 1, 1974 to December 31, 1976 and work was completed as of
August 1975.
                                    iv

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                               TABLE OP CONTENTS
                                                                            Page
Abstract 	•	:••   iv
List of Figures 	   vi
List of Tables	*	    x
Acknowledgement 	•	  xii
  1.  Introduction 	    1
  2.  Conclusions	•	    5
  3.  Recommendations	•	    6
  4.  Study Site	•	    8
        The Sampling Sites  .,	    8
  5.  Method^ and Materials  	•	   30
        Sample collection	•	   30
        Estimation of  flows  	   30
        Physical measurements  	   31
        Microbiological procedures  	   32
  6.  Results	••   65
        Rainfall	* • • • • ^ •   65
        Occurrence and levels  of microorganisms	   65
        Relationship between pathogens  and  indicators  	   88
        Indicator ratios  	   94
  7.  Discussion	•	  H8
        Sampling 	• •  H8
        Enumeration of pathogenic microorganisms	  118
        Distribution of fecal streptococci	  130
        Relationship between indicator  and  pathogenic  microorganisms .....  133
        Quality of urban  surface waters	  135
        Health Hazard	•••	  135
 References	  140
 Appendices
   A.   Daily precipitation in inches	  146
   B.   Levels of Bacteria	•	•. •  153
   C.   Levels of enteric viruses	,	  164
   D.   Distribution of  fecal streptococci	,..  167
   E.   Physical and chemical characteristics	  177
   F.   Frequency of Detection of Salmonella and Animal Virus 	  180
 Glossary	  "181
                                         v

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                                 LIST OF FIGURES
 Number
                                                                            Page
   1      The four major drainage  areas  of  the  Baltimore metropolitan
         area	    9

   2      Open areas  and stream valley park land  in Baltimore City  	,...   10

   3      Baltimore City storm sewers	«	   11

   4      Location of the sampling stations for urban streams and storm
         water outfalls studied in Baltimore City	   12
  5     Characteristics of the Herring Run drainage area, sample site
        B	rf


  6     Characteristics of the Jones Falls  drainage area, sample site
        Characteristics of the Gwynns Falls drainage area, sample site
        D
  8


  9

 10


 11


 12


 13


 14


 15


 16


17

18
 Characteristics of the Stoney Run drainage area, sample site
 F ................................................
 Characteristics of the Glen Avenue drainage area, sample site G

 Characteristics of the Howard Park combined sewer drainage
 area, sample site H ..,	 	
 Characteristics of the drainage area of the Jones Falls storm
 drain, sample site K • »	
 Characteristics of the Bush Street drainage area,  sample site
 L

 Characteristics of the Bush Street drainage area,  sample site
 L

 Characteristics  of the Northwood  drainage  area, sample site
 Schematic - Multiple  concentration and enrichment for the MPN de-
 termination of Salmonella  ..........................
Schematic - Isolation and identification of Salmonella sp.,


Schematic - Isolation and identification of Sh-Lgella sp.

Survival of Shigella flesmeri, at 4°c
 14


 16



 18



 20


 22


 23



 25



 26



.27



 29


 36

 38


 41


 46
                                        vi

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Number

  19    Schematic - Isolation and identification of Staphylocoocus
        aureus 	•       50

  20    Schematic - Identification of Pseudomonas aevuginosa 	       54

  21    Effect of hombgenization on the levels of total and fecal
        cpliforms	       55

  22    Schematic - Identification of the fecal streptococci	       57

  23    Presumptive assay for virus concentrates (Micro Test 11
        plate)	       61

  24a   Levels of indicator microorganisms in Herring Run sample site
        B, during the sampling period	       73

  24b   Levels of pathogenic microorganisms in Herring Run, sample
        site B, during the sampling period	       74

  25a   Levels of indicator microorganisms in Jones Falls, sample
        site C, during the sampling period	       75

  25b   Levels of pathogenic microorganisms in Jones Falls, sample
        site C during the sampling period	.*	       76

  26a   Levels of indicator microorganisms in Gwynns Falls, sample
        site D, during the sampling period	       77

  26b   Levels of pathogenic microorganisms in Gwynns Falls, sample
        site '.D, during the sampling period	       78

  27    Effect of stream flow on the levels of fecal coliform in
        Herring Run, sample site B	     80

  28    Effect of stream flow on the levels of fecal coliform in Gwynns
        Falls, sample site D	     81

  29    The effect of period in days since last rain storm on the fecal
        coliform density measured in the background samples	     82

  30    The relationship of stream flow at the time of sampling on the
        fecal coliform density in stormwater 	     87

  31    The effect of number of days since the last rain storm on the
        fecal coliform density of stormwater	... • •     89

  32a   Relationship between total coliform and Salmonella in background
        and stormwater samples 	     90

  32b   Relationship between fecal coliform and Salmonella in background
        and stormwater samples	,	     91
                                     vii

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Number
                                                                            Page
  32c   Relationship between fecal streptococci and Salmonella in back-
        ground and stormwater samples 	     92

  32d   Relationship between enterococci and Salmonella in background
        and stormwater samples 	     93

  33a   Relationship between total coliform and Pseudomonas aevuginosa
        in background and stormwater samples 	     95

  33b   Relationship between fecal coliform and Pseudomonas aevuginosa
        in background and stormwater samples <.	     96

  33c   Relationship between fecal streptococci and Pseudomonas aeru.-
        ginosa in background and stormwater samples 	     97

  33d   Relationship between enterococci and Pseudomonas  aerug-inosa in
        background and stormwater samples	     98

  34a   Relationship between total coliform and Staphylocoecus  aureus
        in background and stormwater samples 	    99

  34b   Relationship between fecal colifrom and Staphylocoecus  aureus
        in background and stormwater samples 	 	   100

  34c   Relationship between fecal streptococci and Staphylocoecus  aureus
        in background and stormwater samples	   101

  34d   Relationship between enterococci and Staphylococous aureus  in
        background and stormwater samples  	   102

  35a   Relationship between total coliform and enterovirus in back-
        ground and stormwater samples	   103

  35b    Relationship  between fecal coliform and enterovirus in back-
        ground and stormwater samples	   104

  35c    Relationship between fecal  streptococci  and enterovirus in back-
        ground and  stormwater samples  	   105

  35d    Relationship between  enterococci and enterovirus in background
        and stormwater samples 	   10g

 36a   Back.River raw sewage, site A.  Ratio of fecal coliform to total
        coliform, fecal streptococci, and enterococci 	   108

 36b   Herring Run, site B.  Ratio of fecal coliform to total coliform,
       fecal streptococci and enterococci  	  109

 36c   Jones Falls, site C.  Ratio of fecal coliform to total coliform,
       fecal streptococci, and enterococci  	  110

 36d   Gwynns Falls, site D.  Ratio of fecal coliform to  total coliform,
       fecal streptococci, and enterococci	  HI

                                    viii

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Number

  37a   Stoney Run stormwater, site F.  Ratio of fecal coliform to
        total coliform, fecal streptococci and enterococci	<
  37b
  37c
  37d
   37f
   38
Glen Avenue stormwater, site G.  Ratio' of fecal coliform to
total coliform, fecal streptococci, and enterococci	
Howard Park combined sewer, site H.  Ratio of fecal coliform
to total coliform, fecal streptococci, and enterococci	
Jones Falls stormwater, site K.  Ratio of fecal coliform to
total coliform, fecal streptococci, and enterococci	
  37e   Bush Street stormwater, site L.  Ratio of fecal coliform to
        total coliform, fecal streptococci and enterococci	
Northwood stormwater, site M.  Ratio of fecal coliform to
total coliform, fecal streptococci, and enterococci	
Comparison of the levels of Salmonella in sewage with the in-
cidence of salmonellosis in Baltimore City  	
                                                                          Page
                                                                  112
                                                                          113
                                                                          114
                                                                          115
                                                                  116
                                                                          117
                                                                           125
                                                                  r-fr, Teg
                                       IX

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                                LIST OF TABLES
Number

   1

   2


   3


   4
   8

   9


  10


  11
 13

 14

 15


 16

 17

 18
                                                                    Page

 Characteristics of Urban Stormwater Sampling Sites 	   19

 Comparison of Enrichment and Primary Plating Media for the Iso-
 lation of Salmonella	   37

 Comparison of the Phenylalanine Deaminase and Oxidase Screen with
 TSI and LIA Reactions for the Differentiation of Salmonella	   40

 Genus of Microorganisms Commonly Encountered During the Isolation
 of Salmonella	,	   40
        Genus of Microorganisms Found During Attempts to Isolate Shi-
        gella 	<....
                                                                    43
 Levels  of Indicator Microorganisms  and Salmonella in Samples
 Negative for Shigella 	  44

 Recovery of  Sh-igella on Diatomaceous  Earth,  Phosphate Buffered
 Saline, pH 7.2  Temperature  20-25°D  	;	  45

 Recovery of  Staphyloaocous  aureus  	,	,	  43

 Evaluation of Presumptive Media for the Enumeration  of Pseudomonas
 aevuginosa	  52

 Levels  of Pseudomonas  aevuginosa Confirmed on Acetamide Broth and
 Acetamide Agar	  53

 Comparison of Velveteen and Toothpick Replication Procedures to
 Conventional Tube Methods for the Differentiation of  Fecal Strepto-
       cocci
 12    Diagnostic Pattern  for Presumptive Identification of Enteroviruses
       in Tissue Culture
                                                                           59
                                                                           62
Control Samples for Losses Due to Shipping 	•.....  54

Number of Bacterial Isolates Tested During the Project Period ....  66

Monthly Precipitation in cm (inches) During the Stormwater Study
Period at Three Gauging Stations of Baltimore City	  57

Occurence of Selected Pathogenic Bacteria in Background Samples ..  69

Occurence of Viruses in Background Samples 	,...  69

Distribution of Fecal Streptococci in Background Samples 	  70

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Humber

  19


  20

  21

  22

  23


  24


  25
   27



   28


   29

   30
                                                                 Page

Geometric Mean Density of Selected Pathogens and Indicator Micro-
organisms in Background Samples  	  72

Occurence of Selected Pathogenic Bacteria in Stormwater Samples   83

Occurence of Selected Viruses in Stormwater Samples 	   83

Distribution of Fecai Streptococci in Stormwater Samples .......   84

Geometric Mean Density of Selected Pathogens and Indicator Micro-
organisms in Stormwater	
                                                                  86
Levels of Total and Fecal Coliforms at Various Sites Within a
Drainage Area 	

Comparison of the Frequency of Detection of Salmonella with the
Levels of Fecal Coliforms	
                                                                 119
   26     Comparison of  the Levels  of Salmonella Found  in Surface Water,
         and  Sewage
                                                                          122
Frequency of Recovery, of Seeded Salmonella After Exposure to the
Sample, Concentration on Diatomaceous Earth, and Enrichment and
Primary Plating	

Recovery and Level of Enteric Virus with Respect to  the Mean
Levels of Total  Coliforms	••	fjaujeavtH •   -*-29

Frequency of Occurence of  the FC/FS Ratio at Each  Sample Station  132

Overall Ratio of Pathogens to Indicator Microorganisms	   134
                                       XI

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                          ACKNOWLEDGEMENTS
      The  cooperation of  the  Bureau of  Engineering  of  Baltimore  City
 is  gratefully  acknowledged.  We  are particularly indebted to Mr. John
 Trussing  and Mr.  Earl Hartman, principal  engineers, Division of Waste-
 water,  for providing maps  and drawings of the Baltimore City storm
 sewer system and  local rainfall  data.

      The  United States Department  of Commerce, Weather Bureau provided
 the hourly precipitation data taken at the Custom  House in Baltimore,
 Maryland.

      The  cooperation of Mr.  Jerry Valcik,  plant superintendent, Montebello
 Water Treatment Plant is gratefully acknowledged.

      The  following individuals contributed to the  project:  Glorestine
 Toles,  Steve C. Riggio, Jan A. Markowitz,  Charles  I. Noss, Felipe C.
Alfonso,  Steven L. Goldstein and Philip Driscoll.  John Hasegawa and
Michael C. Snead  assisted in the data  analysis.
                                  xii

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                           INTRODUCTION
     Little is known of the early urban sewerage systems in the United
States.  Often they were constructed by individuals or the small district
at their own expense and with little or no engineering or public super-
vision.  The oldest sewers date from 1805 to 1810.  It should be noted
that these sewers were intended only for the removal of stormwater,
and excreta were excluded.  However, the runoff from the growing towns
and cities containing all kinds of matter washed from unpaved streets,
livery stables, dead animals, overflowing cesspools and privy vaults
contributed to the rise of the great urban epidemics.  These diseases
were then known as "pythogenic" (born of putridity) and although scienti-
fically unsound, started the sanitary movement for better town drainage.
Col. George E. Waring, Jr. (1) believed in town drainage but promoted
the "dry-earth system of sewerage".  In a paper on sanitary drainage
in 1875 he wrote:

     "I should never recommend water (carriage) sewerage; yet
     the dry earth in the present states of its development, is
     so inapplicable to a large majority of cases, or so dis-
     tasteful to a mass of persons whose necessities demand
     immediate relief, that, without in any way receding from
     its advocacy, I freely acknowledge that the practical good
     which is to come from water sewerage is evident."

     Already in Boston in 1833 house connections and cesspool overflows
were being made to storm sewers.  The quantities of domestic and indus-
trial waste collections were comparatively small to stormwater flow
so  that the inclusion of sewage did not require an appreciably larger
conduit.  The practice of introducing sanitary sewage into the nearest
storm  drain became wide spread with disasterous results.  There had
been a tendency to employ needlessly large dimensions for stormwater
sewers and coupled with flat grade resulted in accumulation of decom-
posing sludge  during dry weather and sluicing of  solids  into receiving
streams during rainstorms.  The first application of engineering skill
in  sewer design was in Brooklyn, New York in 1857 followed in Chicago
in  1858.  The  combined sewer system design with provision for self-
cleaning dry weather  flow became established and by 1875, of the 67
cities so  sewered having  a population >^ 100,000 not one  was treating
its waste but  merely  discharging to the nearest water course.  The
increasingly  foul  runoff  into  the  neighborhood  from these sewers was
exceedingly disagreeable  and a health hazard.   In many  cities  the  streams
had to be  covered  to  minimize  the  nuisance  and  boat  docks near polluted
waters were often  relocated  so as  not  to offend passengers.  The solution
for the  pollution  crises  was expensive  and  involved  extensive  inter-
ception  of  the dry weather  flows  from hundreds  of combined  sewer outlets
 for conveyance or  pumping to plants  for treatment prior to  discharge.

      The first system of  separate  sanitary  sewers for the  collection
 of  domestic wastes was built  in  1880 by no  other  than Col.  Waring  in
Memphis,  Tennessee.   Although  the  system was  a comparative  failure,

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 it did much to establish design principles and benefits of the system
 over the combined sewer.  These benefits were apparent in small com-
 munities where the receiving streams provided insufficient dilution
 for combined sewer overflow and the treatment was necessary.  Where
 stormwater was conveyed by naturally occurring open drains, the separate
 system permitted the best sanitary solution with least cost and delay.
 Nevertheless, there still remained mistrust regarding the principle
 of separation and the U.S. Board of Health in 1880 sent an engineer,
 Rudolph Herring, to Europe to observe the extensive experiences they
 had with both combined and separate systems.  In his report Mr. Herring
 (2) concluded:

      "The principle of separation, although often ostensively
      preferred on sanitary grounds, does not necessarily give
      the system in this respect any decided advantage over the
      combined, except under certain definite conditions."

 Thus,  the construction of combined systems was continued and was extended
 on economic grounds.   Most of the major cities in the United States:
 New York,  Boston, Philadelphia,  St. Louis, Chicago,  Cleveland, Portland,
 and Washington,  B.C.,  to mention a few, are still largely served with
 combined sewer systems.  Only Baltimore, Maryland at the turn of the
 century elected to provide the separate system of sewers.

     As the cities expanded and became more congested, the sewage and
 storm  flow increased.   Combined interceptors could no longer contain
 even the most  modest  rain and frequent flooding of streets and basements
 was common.   Interceptor overflows were used to divert the stormwater
 commingled with  sewage to surface waterways away from direct  contact
 with the citizens.  Treatment was bypassed and the general sanitary
 quality of water  fronts and urban streams began to deteriorate.   As
 early  as 1900  many state regulatory agencies would not permit  further
 construction of  combined sewers.

     Even where separate .systems  were  built,  the  problems  of urban
 runoff  were  not completely  resolved.   Sewer mains, interceptors,  pumping
 stations and treatment plants  did  not  grow but  the demand  for  sewer
 service did.  The  post-World War  II boom for  sewer service has  yet to
 be  satisfied with  the  extension of  sewer lines  into  fast growing  suburban
 fringe  areas, often with high  infiltration rates  and many  illegal  rain
 water connections which overtax the entire system.   In order to limit
 the backing up of  raw  sewage into basements, hundreds  of bleeders  are
 provided which discharge sewage into storm sewers.  The net effect is
 that sewage  is not reaching the treatment  plant.   In a way the problems
 of the  combined and separate systems are becoming more similar.  The
 engineering solution of parallel interceptors and  increased pumping and
 treatment capacities is much more economical for the separate system
 than for abating the combined sewerage problem.

     As in the past, today the problem of urban runoff involves a
variety of pollutants as well as the sewage component.  Urban stormwater
 is consistently found to contribute pollutants proportioned to popu-
 lation density, new construction, street and vegetation litter, motor
vehicle contaminants,  fertilizer, and animal manure as well as intentional
 and unintentional sewage discharge.  Many studies and conferences have

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"been directed to the urban runoff problem.  More research Is indicated
in several areas including:  street materials such as salt, grit, oil
and garbage; non-street materials including animal wastes, fertilizers,
and pesticides; construction activity; sanitary sewage; airborne parti-
culates; commercial and industrial wastes, and unrecorded and undetected
discharges to storm drains.  The cost of correcting the problem is
apt to be enormous depending upon the degree, and the strategy of abate-
ment is deemed necessary.  It has been estimated that the runoff in
the urbanized area portion of the Standard Metropolitan Statistical
Areas of the United States in 1973 is 85 million m3/day (22.5 billion
gallons/day).  The strategy of treatment depending upon the abatement
levels required may have a capital cost of $134 to $350 billion for
simple screening to advance waste treatment.  The operation and main-
tenance might vary from $93 to $2,700 million/year (3).

     The fact that stormwater runoff from urban areas contains large
concentrations of microorganisms has been well established.  It has
been generally supposed that the great occurrence of microorganisms
is discharged with the first flush of the combined storm and sanitary
sewer which acts as a settling basin during dry weather flow.  Also,
it has been assumed that the bacterial concentration will be lowered
by the dilution of rain water after the entrained pollution within the
sewer has been purged.  What concentration of microorganisms might be
expected in urban runoff from areas with well functioning sewer systems
had not been clearly defined.  Studies of the bacterial quality of such
runoff have been limited to the determination of the densities of
indicator microorganisms.  The major  limitation to the total coliform
or  fecal coliform index  of stormwater quality has been the uncertain
correlation to the occurrence of pathogenic bacteria and viruses.
                                                             ,-H. erf-t fj.7
     While  fragments  of  such data  are available, more  is needed on
what health hazard potential actually exists keeping in mind the possible
priority of modes of  pathogen entry.  Entry by ingestion has high
priority in the  case  of  eating raw shellfish but low  in the case of
accidental  drinking of surface water.  Entry by contact may be a major
factor  in ear, nose and  throat  infections among those  engaged in swim-
ming but a  low priority  for pathogen  penetration of the mucous membrane
or  through  cuts.  The latter represents water-borne epidemiological
curiosities such as central nervous system  amoebiasis  caused by Naegleoria
gruber-i, or leptospirosis, or Myaobaateri-um balnei  skin ulcers.

      It is  known that rural  stormwater  runoff  from  pastures,  farms and
barnyards have fecal  coliform discharges  that  may be  equal  to or exceed
that  of municipal  sewage.  While  the  problem of nutrients  and BOD  of
cattle waste exists,  the potential health threat  of  the microorganism
 remains to  be  demonstrated.  Literature  is  replete  with references
 to  high densities  of  total coliforms,  fecal coliforms,  and  fecal  strep-
 tococci in stormwater samples  from forested (4,  5),  agricultural,  and
 rural (4,  5,  6), urban and suburban  (4,  5,  6,  7,  8)  drainage areas.
 Typhoid fever  is not  an indigenous health problem in the  U.S.  today.
 However, with enormous numbers  of people travelling abroad and  returning
 to  metropolitan areas the health threat  from exotic enteric diseases
 cannot be  dismissed.   This is  especially true in view of  the deficiencies

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 known to exist in the wastewater collection,  transmission and disposal
 systems  and the tremendous  opportunities for  adults  and children to
 contact  the waters receiving the urban runoff.

      .The large urban waterway and its numerous small urban streams
 (flood ways) are very much  in demand for recreational and aesthetic
 parks serving the city population.   Unfortunately, the quality of these
 waters is greatly impaired  even when separate sewers are provided for
 stormwater and sanitary sewage.   Whitman in 1968 (9) surveyed the
 sources  of pollution  in the urban streams in  Baltimore (separate sewers)
 and Washington,  B.C.  (combined sewers)  and revealed  that the largest
 cause of poor water quality in both cities was  sewer malfunction.
 Although the contribution of indicator and pathogenic microorganisms
 from  the feces of pets,  birds and rodents cannot be  ignored,  the pollution
 of urban waterways with untreated sewage presents a  greater  potential
 health threat.   Many  regulatory  agencies post signs  printed  "Danger,
 Polluted  Water,  Keep  Out!"   Obviously,  this conflicts with the  fullest
 use and  enjoyment  of  the waterway.

      The National Commission on Water  Quality study  (3) could  find no
 information  on any runoff sampling  studies conducted specifically for
 human pathogens.   The main  objective of  this report  is to provide the
 needed information on the presence  and  concentrations of selected
 pathogenic bacteria and viruses  in  the urban runoff from areas served
by combined and  separate sewers.

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                            CONCLUSIONS
     1.  High densities of indicator microorganisms were found in the
urban streams.  Only three samples of 92 were less than the National
Technical Advisory Council suggested standard for recreational waters.
The mean concentration was 30 times the 200 fecal coliform/100 ml desired.
Pathogenic microorganisms were consistently recovered.  P. ae-^uginosa
were the most  numerous,  Staphylocoocus awceus was present at low levels,
and Salmonella and enteric viruses required concentration for enumeration.

     2.  High densities of indicator microorganisms were also found
in urban storm runoff.  Only one sample of 136 was less than the 200
fecal  coliform/100 ml standard.  Pathogens were consistently recovered
at approximately 10-fold higher densities than in the stream samples.
The same order of occurrence was observed.

     3.  There was no marked   seasonal  variation in the  levels of micro-
organisms in  the urban streams.  There was a  slight elevation of con-
centration  in the density of P. aeTuginosa in the early fall when
rainfall was  minimal and water temperature was the highest.

     4.  The  density of fecal  coliform was independent of flow and
antecedent  rainfall in the urban streams.

     5.  In the  routinely examined background samples  (sewage, urban
streams  and reservoirs),  there was a strong positive  correlation between
the levels  of total coliform,  fecal coliform, fecal streptococci and
enterococci and  the levels  of  pathogenic bacteria.  Only  the  levels  of
total  coliform and  fecal  coliform correlated  well with  the levels  of
enteric  viruses.

      6.   In the  stormwater  samples the densities of  indicators and
 pathogenic bacteria were  for the most part independent  with no sig-
 nificant positive or  negative correlation.   The only exceptions were
 for total coliform to Salmonella,  and total coliform and  fecal coliform^
 to P.  aeruginosa.   No correlation was observed  between indicator  bacteria
 and enteric viruses.

       7.   Although in the examination of samples,the recovery of Shigella
 sp. was negative, there is circumstantial evidence that the genus is
 present in urban surface water and could present a significant health
 hazard.

       8.  Members of the genus Salmonella were recovered from all the
 raw sewage,  91% of the urban  stream and 94%  of the stormwater samples.
 The parallel seeding experiments  suggested that the levels of Salmonella
 reported in  this study and probably in other studies were underestimates
 of the true  densities.  Methods for the enumeration of enteric viruses
 and pathogenic Staphylococc'L  also appear to  have inherent short comings.

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                               RECOMMENDATIONS
       1.   Although pathogenic microorganisms were consistently recovered
  from urban storm runoff in this study,  the densities  of enteric  pathogenic
  £S£rrt  f   K1^863  Tre f°Und t0 be relati^ly low.   It  is  highly unlikely
  tive stoS UrbaJ/°pulf lons wil1 c^sume the large quantities of  unattrac-
  takinf £?„£??? required to cause overt diseases.   In coastal  areas, the
  sewer? ?f f  K  S  I™* ^^ ** **  immediate vicinity  of  urban storm
  ™f     Bidden due to unsatisfactory sanitary survey, if not  for ex-
  cessive colxform or fecal coliform densities.   It is  acknowledged  that
  although  commercial harvesting  for direct marketing is  prohibited  in such
  SIS; r"? r?"ea^°nal takin§ of shellfish presents a more  plausible
  threat to  health since  pathogens  are  concentrated and shellfish are often
  further diJ^ / ?? f °™ter  contaminants  reaching recreational water are
  further diluted.  If beaches  are  close  by, more often than not they are closed
 wSn^ °f**c&ed^Z°f  Prevailing coliform  standards.  The threat to those
 who persist in swimming  is small  since prodigious swallowing of water would
 be required to increase  enteric disease risk.  Pathogenic organisms not
 requring ingestion, namely, P. aerug-inosa and Staphylocoocus auveus are
 eff infS- SbUndant'  f fc th* eviden^ ^ not available that skin, eye,  or
 JaL ?5eCtlons arise from the °rganism in the recreational water.  Thus,
 with the exception of the members of the genus Shigella, there is little
 waX™ ?OW? ^ ?r extensive ?ublic  health concern in recreational
 waters receiving urban storm runoff.  Furthermore, the urban storm runoff
 and many of the receiving bodies of water are aesthetically unattractive
 during and immediately following storm events and thereby receive little
 use,  if at all,  when  the levels of microorganisms are  the highest.   Given
 these conditions existing today, there is little justification based on
 of*! ^~     /°nuiderati°nS t0 warrant a Program of disinfection  of billions
 fhnS If8 ?  U    n -St0rm runoff'   On the other hand,  more  serious  effort
 should be  given  to increasing the  carrying capacities  of sanitary sewers  and
 to  maintaining them so as to minimize overflows of raw sewage into urban
 waterways .
*11v                2* Pf hogenic microorganisms observed in the intention-
ally constructed combined sewer in  this study  (Howard Park, site H) were
generally 10-fold higher than the levels in the urban sto™ runoff!  Even
at these higher levels, the enteric health hazard is believed to be limited
unless there is the likelihood of the direct consumption of combined waste-
water or the water in the stream receiving combined wastewater.  Combined
where ?hev ^1-* 1S° plaCS added loads on downstream water treatment works,
ha^rH   y I    ;«  f WatSr C°ntaCt recreati°n areas exist downstream, the
hazard may be a little more real because of the nature of the escape of the

2£.?V   ?      ^ th£ St°rm Water flow'   In the absence of any cost-
effective and practical method of separating storm water from the sanitary

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sewage, disinfection of the combined wastewater to reduce the mlcrobial
load for subsequent water treatment and to minimize the hazard to down-
stream users of direct water contact recreational areas may be indicated.

     3.  Better methods and procedures should be developed for the
enumeration of pathogenic microorganisms.  Although the techniques for
the quantitative determination of Salmonella, sp. and enteric viruses have
improved dramatically in the last ten years, there is considerable room
for advancement.  Particular attention should be directed toward the
improvement of assay methods for these microorganisms for moderately to
heavily contaminated samples.  Superior media and methods are lacking for
the rapid, selective enumeration of Staph, aureus in water.  Reliable
qualitative recovery procedures for Shigella sp. are not available.

     4.  Since there is little danger of direct consumption of urban
storm runoff, information on survival of the natural pathogen popula-
tions would be useful to better evaluate the impact of the storm runoff
on receiving bodies of water.  This would be of particular importance for
recreational waters and shellfish harvesting waters in the coastal regions.

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                             STUDY SITE
      The investigations on microorganisms in urban stormwater were
 carried out in Baltimore City, Maryland.  Baltimore is unique in being
 the first large city in the United States to construct a comprehensive
 system of separate sanitary and stormwater sewers.  The sanitary sewerage
 plan was conceived in 1907, completed construction in 1915,  including
 secondary treatment, and 90% of the population of the city was connected
 by 1920.  Today the sewer system extends into the metropolitan areas
 of Baltimore County.  The Baltimore metropolitan area, as shown in
 Figure 1, is intersected by four separate drainage areas which dis-
 charge into the tidal estuary of the Chesapeake Bay.   The main arm of
 the Baltimore harbor is the Patapsco River which drains the  area south
 and west of Baltimore and does not directly effect the drainage of the
 city.   The principal drainage areas are the Gwynns Falls, Jones Falls
 and Herring Run.   These stream valleys at one time had numerous mill
 dams by virtue of an average stream slope of 0.73% with certain locations
 through the fall zone with slopes as high as 1.5%.   The topography of
 the area greatly facilitated the drainage scheme.   Much of the area
 is served by sanitary sewers which flow by gravity to the Back River
 Wastewater Treatment Plant,  the largest plant located some 9.26 km (5.75
 miles)  east of the city.   A second but a much smaller plant  on the Patapsco
 River  in the south harbor will be enlarged to provide relief for the
 wastewater from the Gwynns Falls interceptor in the near future.   The
 valleys fan out into wide flatlands along the coastal plain.   Here the
 collected sewage  requires considerable pump lift of some 21.3  m (70
 feet)  to discharge into  the main outfall sewer to  Back River.

     The stormwater drainage was developed separately.   In 1902  a
 provision was  made to set aside the stream valleys  for floodway  parks
 under public ownership.   This action was a forerunner of" the modern
 concept of flood  plain management.   The  railroads ran down the valleys
 to  reach the harbor.   This,  along with the old water  power mills  attract-
 ed  some industry  which still exists in the lower Jones  and Gwynns  Falls
 reaches.   The  extent  of open areas and valley  park  is shown  for  the
 three drainage areas  in Figure  2.

 THE SAMPLING SITES

     Although  separate systems  of  storm  and  sanitary  sewers have been
provided  it  is  still  possible to  find  a  few  small old  subdivision
drainage  areas which  have  combined  sewers.  All have been  intercepted
and during heavy  rain storms  discharge into the nearest water course.
Figure  3  shows  the system  of  stormwater  sewers for Baltimore City and
Figure  4  shows  the location of background  stream and  stormwater sampling
sites.  The  stream valleys are an  integral part of the storm system.
The channels drain a  completely urban  and  suburban city-county popu-
lation  and the water  samples from which present an integrated picture
of the microbiological quality of runoff from a major metropolitan
area.  Background samples were taken from each stream throughout the
study period as well as samples from storm drains and combined sewers

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                     RURAL-SUBURBAN
                ^
          SCALE !N MILES
    101  23436769
Figure 1.  The four major  drainage areas of the Baltimore
           metropolitan area;  Patapsco River, Gwynns
           Falls, Jones Falls  and  Herring Run.

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      I  "\WESTERN RUN
             PARK
                                                   MOORES
                                                    RUN
                                                    PAflK
       \PARK
        x INDUSTRIAL
    ORUID HILL *
         ,PARK
H1LLSOALE
  PARK
              GWYNNS FALLS \
                  PARK     V\
                            \
           EXISTING PARK AND RECREATIONAL1
           AREAS ALONG STREAMS
            SCALE  IN MILES
           0    I     '2     3
Figure  2.  Open areas  and  stream valley park land in
             Baltimore City.
                                10

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Figure 3.  Baltimore City storm sewers.  The solid lines repre-
           sent underground conduits, the thickness of which
           are proportional to area of cross section.  The
           lightly shaded lines are natural channels.
                              11

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 •if  ROUTINE SAMPLING SITE

 *  STORM WATER SAMPLING SITE

 •  RAIN  GAGE

 o  SEWAGE OVERFLOW LOCATION
Figure 4.  Location of the sampling stations  for urban streams and storm-
           water  outfalls studied in Baltimore  City.   The shaded areas
           indicate the catchment for the  stormwater  outfall.
                                    12

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during heavy rains.  With the exception of the unsewered country-estate
zone indicated as the Green Spring Valley the entire catchment is
indistinguishable on either side of the city limits with regard to typical
suburban development.  It is estimated (1972) that 905,000 people live
in the drainage area under study, with 805,000 being within the city.
The population density within the city averages 45.7 persons/ha. (18.5/
acre) and 37.3/ha. (15.I/acre) in the county.

Background Samples

     For the integrated urban surface water background samples, the
following waters were collected routinely regardless of the rainfall:

     (A)  Raw sewage taken at the Back River Wastewater Treatment Plant.
     (B)  Herring Run at the Mount Pleasant golf course.
     (C)  The Jones Falls at the Baltimore Street Car Museum.
     (D)  Gwynns Falls at the Dickeyville Dam.
     (E)  Loch Raven reservoir (Montebello Filter Plant raw water).

The detail location of these and other samples are shown in Figure 4.
A brief description of these drainage areas is given below.

Back River Sewage Treatment Plant—
     The Back Rivers Sewage Treatment Plant, started in 1911, is a com-
plete primary plus secondary plant, but its age and lack of maintenance
through the years requires a program of reconstruction and modernization
now underway.  The plant is serving an estimated 1.3 million population
in tKe Baltimore metropolitan region.  The average flow in 700,000 m3/
day (185 mgd) but with a peak at 1.2 million m3/day (350 mgd) during
heavy rains indicating the large component of infiltration and inflow
into the separate sewer system.  The raw sewage, sample A, was taken
at the head of the plant and had an average five-day BOD at 20°C of
209 mg/1 and a suspended solids content of 198 mg/1.

Herring Run—
     The Herring Run valley is the smallest of the drainage areas under
study having an area of 58.8 km2 (23.5 square miles).  The average slope
of the streams is 0.74%.  As seen in Figures 1 and 3 the tributaries
are the Chinquapin, the West Branch, East Branch and Moore's Run.
The average flow of Herring Run is 3.67 m3/min. (2.16 cfs) with a
minimum of zero and a normal maximum of 1020 m3/min. (600 cfs).  Hurri-
cane storm runoff flows are, of course, much higher.  There are no
heavy industrial wastes and the interceptor, since construction, is
functioning fairly well.  There are about 10 sewage bleeders, particularly
in the lower less attractive part of the drainage area where considerable
erosion has taken place requiring concrete lining (Figure 5a).  The
quality of the stormwater taken in the lower reaches was unusually poor
before Moore's Run interceptor was completed.  Records show a maximum
of 3,192 mg/1 of suspended solids, total coliform MPN of 31 million/
100 ml, and BOD of 110 mg/1.  Today, under normal flow conditions
the stream is clear and attractive as seen in Figure 5b.  Note the manhole
for the sewer interceptor along the stream.  The characteristic dwellings
are group row homes and apartments (Figure 5c).  The routine sample,
identified as B, is taken in the park section of the East Branch of
                                    13

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-------
Herring Run  (Figure 5d).  The spillway in the foreground conveniently
provided the means for  estimating the flow at the time of sampling by
use of the Francis formula modified for a flat crested weir.

Jones Falls—
     The Jones Falls and its tributaries, Western Run and Stoney Run,
encompass some 151 km2  (58.3 square miles) of drainage area.  The stream
at one time provided the water supply for the city from the Roland im-
pounding reservoir downstream from the rural Green Spring Valley.
The Northern Central (now the Pennsylvania Central) Railroad used the
gentle grades of the valley which attracted industries that still
exist.  The lower reaches of the Jones Falls run through the central
business district and are completely covered for 2.4 km (1.5 miles)
before discharging into the middle harbor.  In the city the natural
state of the Jones Falls cannot compete aesthetically with either the
Herring Run or Gwynns Falls due to the presence of railroads, expressways,
commercial institutions and industries (see Figure 6a).  The lower
portion of the Jones Falls drains typical Baltimore row housing as
shown in Figure 6b.  The routine sample site C is located on the edge
of the residential and  central business district where the stream is
not very attractive (Figure 6c).

     The quality of the runoff into the Jones Falls has markedly im-
proved with the completion of the Jones Falls interceptor in 1956 which
eliminated the sewage treatment plant at Towson.  Metropolitan sewerage
service is now provided for the entire drainage area excluding the
high-income, low density, country estates of the Green Spring Valley
district.  The growth of the suburban area in the Towson-Lutherville
corridor followed the sewer service.  When the Jones Falls interceptor
and pumping station were placed into operation in 1956, the capacities
provided were considered adequate until 1970.  However, the flow dramat-
ically exceeded expectations.  By 1962 the flow reached 35,400 m3/day
(9.33 mgd), a flow not  anticipated before 1970.  Peak flows could no
longer be handled and were discharged into the Jones Falls at the
pumping station.  Today this condition has been remedied through pro-
visions for gravity diversions, additional pump capacities and parallel
force mains.

     The heavy built-up character of the metropolitan areas results
in high flows in the Jones Falls in excess of 10,200 m3/min (6,000 cfs)
resulting in flood problems in the industrial areas.  The average flow
is 820 I/sec. (29 cfs)  and the minimum is 84 I/sec.  (3.1 cfs).  The
water quality in the lower reaches within the city is not good.  The
maximum suspended solids have reached 404 mg/1.  Considerable numbers
.of industrial waste connections and dozens of sewer bleeders contribute
to the poor water quality.  The Western Run portion is an early sub-
division area which although served by separate sewers has been provided
with bleeders.  The upper city section of the drainage area is quite
a contrast to the lower Jones Falls in terms of parks, housing type
and open areas as suggested by the typical homes on the Jones Falls
drainage area (Figure 6d).

Gwynns Falls—-
     Of the urban stream drainage basins the Gwynns Falls is the largest
having an area of 164.5 km2  (63.5 square miles).  This valley offers
                                    15

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the greatest recreational potential since it is still in a beautiful
natural state seldom found in a modern city.  The average stream flow
is 850 I/sec. (30.8 cfs) and the minimum is 110 I/sec. (3.6 cfs).
Flooding is common with flows up to 140 m3/sec. (5,000 cfs).  Hurricanes
have produced much higher discharges with considerable damage to bridges
and timber areas.  The stream is "flashy" with a rapid rise and fall,
is highly turbid, and has much driftwood and floatage.  During such
periods the BOD5 reaches a maximum of 46 mg/1; suspended solids, 341
mg/1; and total coliform MPN, 9 million/100 ml.  During hot weather
and low stream flow there is a sewage odor nuisance in the valley.
Fortunately, the numerous rapids quickly reaerate the water so that
with normal flow and in winter the stream is very attractive to children
and adults who play along the Falls in warm weather and skate on the
numerous pools in winter.  The upper valley is the fastest growing
portion of Baltimore County.  The Gwynns Falls and Dead Run sewer inter-
ceptors which extend into the new area are reaching design capacities
and surcharge into the Gwynns Falls at several points along the sewer
in the city and county during heavy rains.  Numerous temporary sanitary
bleeders have been installed to prevent sewage backing into basements
and a State Health Department moratorium has been ordered on new building
permits until a parallel interceptor is constructed.  Sampling site
D is below an old mill dam as seen in Figure 7a.  The spillway serves
as a flow gauging weir for measurement of the discharge at time of
sample collection.  The interceptor may be seen along the stream by
the manhole structure in Figures 7b and c.  Figure 7d shows one of
the many pools of Gwynns Falls which attract wild water birds such as
herons and ducks and provide fishing for perch, catfish and carp.

Loch Raven Reservoir—-
     Loch .Raven is an impoundment on the Gun Powder River with a 784.8
km2  (303 sq. miles) of protected catchment area in rural Baltimore
County.  It delivers to the Montebello Filter Plant 566 to 755 thousand
m3/day (150 to 200 mgd) of good quality raw water.  The average daily
total coliform MPN/100 ml is 118 with a range from 0 to 2,400.  Sample
(E) was taken at the raw water sampling tap at Montebello Water Treat-
ment Plant.

Storm Sites

     The sites selected for the study of the quality of urban runoff
during storm episodes are given in Figure 4 and indicated by the solid
star.  The drainage areas served by the outfalls are shown in dark
shade.  The physical characteristics of the stormwater sampling sites
are given in Table 1.

Stoney Run—
     The Stoney Run drainage area measures 567 ha.  (1,379 acres) in
the Jones Falls valley in the north-central section of Baltimore City.
The sampling point is identified as site F.  The drainage area is pri-
marily residential with a population density of 20.7 people/ha. (8.4/
acre).

     The dwellings in the area are often large houses on wooded lots
as shown in Figure 8a.  Although park land occupies only about four
                                    17

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acres along the lower open section of Stoney Run, several private
schools and religious institutions occupy sizable tracts of land with
a park-like nature.  Figure 8b shows a view of one of the small private
schools in the area.  No industry is located in the area and only about
five acres are occupied by commercial establishments.  The lower section
of Stoney Run is lined with concrete (Figure 8c).  The sample site was
located in this section of the stream and can be seen in Figure 8d.
The hash marks shown on the side of the invert in the lower left hand
corner were used to determine depth of flow.  Note the flow in the
drain during dry weather.

     The composition of the waters sampled at Stoney Run is believed
to be primarily storm runoff from an upper middle class residential
community.  Three known sanitary, sewage bleeders are located within
the drainage area.  The degree to which these sewage overflows function
during storms is not known.

Glen Avenue—
     The Glen Avenue storm drain is located on Western Run in the Jones
Falls valley in the northwest section of Baltimore City.  The sampling
point is identified as site G.  The drainage area measures 80.3 ha.
(198 acres) with a population density of 65 persons/ha. (26.2/acre).
The neighborhood consists of cottages and apartment complexes on a
rolling timbered terrain.  Figures 9a and b show the typical street
scenes with the small cottages that comprise the major portion of the
drainage area.  Fig\ire 9c; shows a small wooded park which occupies about
two acres in the lower section of the drainage area.  No industrial
and very little commercial land uses are found in this area.  Figure
9d shows the outfall where the runoff samples were taken and an apartment
complex in the background.  Note the flow in Western Run during dry
weather.  Western Run represents a major open storm drain in the north-
west section of Baltimore.  During storms the Glen Avenue outfall is
often surchargedi

     The composition of the waters from the Glen Avenue drain is believed
to be primarily storm runoff from a middle class residential community.
One known sanitary sewage bleeder is located in the drainage area.
The extent of sewage overflows during storms is not known.

Howard Park—
     The Howard Park drainage area is located in western Baltimore
in the Gwynns Falls drainage basin.  The sampling point is identified
as site H.  The .catchment area is 31.6 ha.  (780 acres) with a population
density of 738 persons/ha. (29.9/acre).  A golf course occupies approx-
imately 22% of the drainage area (Figure lOa).  The remainder of the
area is characterized by  small detached dwellings on rolling wooded
lots, as shown in Figures lOb and lOc, with some small commercial zones
and an apartment complex  interspersed.  The Howard Park site is unique
since it represents one of the few intentional combined sanitary and
stormwater sewer systems  in Baltimore City.  The sanitary sewage is
collected and transported to the Gwynns Falls interceptor in a conduit
which is also used for stormwater runoff.  The Gwynns Falls interceptor
runs parallel to the Gwynns Falls and is located under the spillway
shown in Figure lOd.  During dry weather, the sanitary sewage flows
down through a grate located just behind the outfall pictured in Figure
                                    21

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 lOd into the sanitary sewer.  When the grate is clogged with debris
 or when the interceptor is overloaded, the sanitary sewage from Howard
 Park or the Gwynns Falls interceptor flows to the spillway and into
 the Gwynns Falls.  The overflow of sanitary sewage into the Gwynns
 Falls has been a common occurrence at this outfall during dry weather.
 During periods of rainfall, the runoff and sanitary sewage flow by the
 grate to the spillway and into the Gwynns Falls.   In addition, sanitary
 sewage from the overloaded Gwynns Falls interceptor mixes with the runoff.
 Samples were collected at the spillway shown in Figure lOd.

      The composition of water collected at Howard Park is believed to
 represent combined sanitary and storm flow.  In addition four sanitary
 sewer overflows are located in the catchment area.

 Jones Falls Storm Drain—
      The Jones Falls storm drain is located in the central section
 of Baltimore City and occupies 253 ha.  (625 acres).   The sampling
 point is identified as site K.  The population density is approximately
 110 persons/ha.  (44.8/acre) and is characteristic of a downtown resi-
 dential-commercial district.   The typical dwellings  in this  area are
 the row homes pictured in Figure lla.   The characteristic small back-
 yards,  generally paved,  with a service alley are  shown in Figure lib.
 A small commercial district with some  light industry occupies about
 8.5 ha.  (21 acres)  in the drainage area.   Although not intentional,
 the sanitary sewage from a small area  comprising  a few square blocks
 is discharged directly to the storm drain.   Figure lie shows  the outfall
 where the samples were collected.   Note the dry weather flow.

      The character of the water collected at the  Jones Falls  storm
 drain is believed to be  representative  of combined sanitary sewage
 and stormwater runoff from an inner city  residential-commercial district.
 No other known sanitary  sewage overflows  are believed to  be located
 in the drainage  area.

 Bush Street—
      The Bush Street drainage area has  an area  of 440 ha.  (1,087 acres)
 in the central section of  Baltimore City  between  the  Jones Falls and
 Gwynns Falls  drainage basins.   The sampling  point is  identified as site
 L.   The  catchment  area is  independent of  the valley streams and discharges
 directly into  the  tidal  portion of the  Baltimore harbor.  The  Bush
 Street drainage  area contains  a large residential district characterized
by  row homes with both low and  middle income neighborhoods (Figures
 12a  and  12b) with a  population  density  of  228 persons/ha.  (92.5/acre).
Typical  inner  city commercial districts are  shown in  Figure 12c.  The
industrial  areas are  composed of warehouses, truck terminals and mis-
 cellaneous  light industries as  shown in Figure  13a.  A large park oc-
cupies approximately 16% of the  catchment  area  and is  located between
 the  industrial and residential  districts  (Figure 13b).  Samples were
collected at the point where  the storm drain empties  into the Baltimore
harbor.  The sampling point is  shown in Figure  13c.  The Bush Street
outfall was always surcharged and was influenced by the tides.  During
storms the flow was always observed in the direction of the harbor.
                                    24

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      The  samples  collected  at  Bush Street  are believed  to be represen-
 tative of separate  storm runoff  from inner city  residential, commercial
 and  industrial  districts.   No  known sewage overflows are believed to
 be located in this  drainage area.

 Northwood—
      The  Northwood  drainage area measures  20.2 ha.  (50  acres) in the
 Herring Run drainage basin  in  the  northeast section of  Baltimore City.
 The  sampling point  is identified as site M.  The population density
 is 65.6 persons/ha.  (26.6/acre).   About 60% of the catchment area is
 composed  of a residential area.  The typical group houses can be seen
 in Figures  14a  and  14b.  The remainder of  the catchment area (40%)
 is occupied by  a  shopping center shown in  Figure 14c.  The outfall is
 shown in  Figure 14d.  A  concrete Parshall  flume with a 12 foot throat
width was located immediately  down stream  from the outfall and was used
 to measure  the  storm flow from the Northwood area.  The Parshall flume
was  constructed in  1963  for  a  storm drainage research project of the
Johns  Hopkins University sponsored by Baltimore City, Baltimore County,
The  State of Maryland and the U.S.  Bureau  of Public Roads.

     The  samples collected at Northwood are believed to represent storm
runoff  only.  No sewage overflows  are known to be located in the North-
wood catchment  area.
                                  28

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                        METHODS AND MATERIALS
 SAMPLE COLLECTION
      All of the water samples in this study were analyzed for micro-
 organisms within four hours after collection.  The exceptions were
 those samples collected for virus assay which were specially handled
 and prepared for analysis at a later time.  The samples were collected
 in clean, heat sterilized 20 liter (5 gallon) plastic containers.

      The volume of water collected was 20 liters for each sample site
 for both the routine and storm stations.  Additional 20 liters were
 collected for virus samples.  Background samples were collected each
 week from the three urban streams,  Herring Run, Jones Falls and Gwynns
 Falls, from the raw water at the Montebello Water Treatment Plant, and
 the raw sewage at the Back River Wastewater Treatment Works.  After
 February 1975, background samples were collected biweekly.   Virus samples
 were collected every other week.

      The storm sewer discharges and combined sewer overflows were, of
 course,  sampled whenever rain storms occurred.   Since automatic sampling
 of the storms was deleted from the  study,  a manual "grab" sampling pro-
 cedure was used.   Samples were taken as early in the storm as possible.

      Each technician was responsible for the sample collection at out-
 falls near his residence or conveniently located on his way back to the
 laboratory.   Storm warning forecasts were  posted and when the probabilities
 for rain were high an alert was  posted in  the laboratory or the super-
 visor called each technician at  home before or  after work hours.   In this
 manner a sampling of a representative number of storms was  achieved.

 ESTIMATION OF FLOWS

      The flows  in the channel  or outfall sewer  were estimated by  the
 technicians  using relatively simple methods.  Three sample  sites  in-
 volved a weir  or  spillway  from which  simple measurement of  head,  H,
 in feet  of water  flowing over  the lip or crest  give the flow estimate,
 Q,  in cubic  feet/seconds,  (cfs).  The sites  and appropriate  discharge
 forumlae used were  as  follows:
              B.  Herring Run
              D.  Gwynns Falls
              H.  Howard Park
(Q = 125.5 x H1'5)
(Q = 343 x H1'5)
(Q = 134 x H1'5)
     For the remaining site the estimation of flow required the measure-
ments of the average velocities and the cross sectional areas of the
waterways during the time of sampling.  The flow velocity meters used
were manufactured by the Tsurumi Precision Instrument Company and
were of the propeller type identified as 313 TS Flowmeter calibrated
to give velocity in meters/seconds.  A 100 seconds timed by stop watch
made the velocity calculation easy.  .The flow meter was fixed on the
end of six foot staff in order that the traverse across the outfall
pipe could be made by the observer on top of the invert without dis-
turbing the flow pattern.  For other than pipes the velocity measurements
                                     30

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were made from bridges spanning open channels.  The areas of cross
sections in the channel were determined for each unit depth of flow
at the invert.  The flow in m3/sec. was calculated as the product of
the average velocity in m/sec. and the cross sectional area in m as
measured when samples were collected.  The following channels were so
estimated:
               F.  Stoney Run
Q = (6.36 x 1(T2 x d) + 0.54 x V .,
Q = discharge, m3/sec.
d = depth of water in channel, cm
V = average velocity, m/sec.
     Flow formulae for various shapes of outfalls including an ellipse,
a rectangular section with rounded invert, and Parshall flume were
derived.
               G.  Western Run
               K.   Jones  Falls
                          where
                L.   Bush Street
                M.   Northwood
                          where
(for depth in excess of 0.5 ft.)
Q = (6.18 x d) - 2.5 x V
Q = discharge, cfs
d = depth of flow in ft.
V = average velocity, ft./sec.

Q = 1.98 + 7 x (d-1) x V
Q = discharge, cfs
d = depth of water at the invert, ft.
V = average velocity, ft./sec.

Q = 25 x d x V
Q = discharge cfs
d = depth of water at the invert, ft.
V = average velocity, ft./sec.

Parshall flume available from previous
stormwater studies.

Q = 48.8 x d
Q = discharge is cfs
d = depth of water at entrance  section,  ft.
 The relationship of cross sectional areas and depth of water flow was
 plotted for each outfall so that the area could be readily obtained
 for any depth without calculation.  All calculations of flow in cfs
 were converted to metric units as follows:  cfs x 2.83 x 10~2 = m /
 sec. or 1000 I/sec.

 PHYSICAL MEASUREMENTS

      The only physical measurement made in the field was for temperature.
 However, the collector indicated certain organoleptic values as appro-
 priate with regard to appearance and odor.  In the laboratory the pH
 measurements were made with a Beckman Zeromatic meter.  The data for
 temperature, pH and flow is given in Appendix E.
                                      31

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 MICROBIOLOGICAL PROCEDURES

 Bacteriological Assays

 Differential Biochemical Tests—
      The following tests were employed to provide information for the
 tentative 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 appro-
 priate 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 0.5 M ferric chloride solution (10).

 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-amino
 dimethylaniline monohydrochloride.  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 production 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 (11).

 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  (11).  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 (11).   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 decar-
 boxylase activity was indicated by the change  from yellow to  violet
 in  the tubes  containing  lysine.  Control  tubes without lysine remained
 yellow.

 Urease—Urease  activity was determined by  the  spot inoculation procedure
 on  plated prepared with  Christensen's urea agar (11).  Urease activity
was  indicated 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 (11).
                                    32

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Coasulase—TKe elaboration of coagulase was determined by the plate
method described by Esber and Faulconer (12).  Isolates were transferred
to coagulase agar (Difco) and incubated overnight.  Staphyloaooffus
aureus 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.  (13).  Colonies were transferred to DNase test agar  (Baltimore
Biological Laboratories) 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  lipo-
vitellenin-salt-mannitol agar  (LSM)  (14).  Opaque  zones  around  the
colonies indicated lipovitellenin-lipase activity.

Mannitol fermentation—Mannitol fermentation was  observed  on LSM and
coagulase agar.Yellow  zones  around the colonies  were taken as positive
mannitol fermentation.

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 (15) to separate any possible soil micrococci.   An overnight
 culture on brain Heart infusion (BHI)  broth at 37°C was used to ino-
 culate tubes of Brewer's fluid thioglycolate medium (BTM)  contaxning
 0.35% agar.  Growth throughout the tube after 24 hours was indicative
 of anaerobic glucose fermentation.

 Gram stain—Gram stains were prepared according to the procedures
 described in Standard Methods (16).

 Gelatin liquefaction—Gelatin liquefaction was determined on BHI con-
 taining 120 g/1 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 fermen-
 tation  medium containing 10 g/1 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/1  oxgall and  65 xg/l 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.

                                     33

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  Catalase—-Catalase activity was determined for isolates from the fecal
  streptococci assay by 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 Staphyloeoooi, isolates was determined by transferring  a
  portion of  the colony to 3% hydrogen peroxide.   Evolution of  gas indicated
  a positive  catalase.

  Starch hydrolysis—Start* hydrolysis was determined on nutrient  agar
  containing  10 g/1 of  soluble starch and 8 g/1 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/1 skim milk and 15  g/1  agar) after  incubation  for three
  to five days at 37°C  (17).   Hydrolysis  of  casein was  indicated by the
  formation of clear zones  around the colony.  Pseudomonas  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  (16)  and  incubated for 48 hours.  Pink to
  red  colonies with blue  fluorescence when exposed to ultra-violet lieht
 were considered positive.

 Growth at 42°C—Acetamide and oxidase positive isolates were transferred
 to Drake s medium #10 for evaluation of growth at 42°C  (18) after 48
 hours incubation.   Growth was indicated by turbidity with blue-green
 fluorescence.

 Cultural Evaluation and Final Procedures—
      The evaluation of methods of detection and enumeration is a funda-
 mental 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 important factors  and will
 vary with the procedure employed.  Techniques, methods and culture media
 developed for microbial assays of clinical specimens may yield  poor
 results when applied to water samples where different interfering micro-
 organisms 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 (16) 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 the  laboratory and infor-
mation  in the  literature.  Multiple  tube dilution procedures which
 permitted a calculation  of a  most probable number (MPN) were generally
 favored because of  the wide variability in the chemical, microbiological

                                   34

-------
and physical characteristics (particularly solids) expected in the
samples from the urban water courses.

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 (19)^
except that diatomaceous earth was used for concentration.  The multiple
concentration and enrichment procedure is shown in schematic form in
Figure 15.  Three replicate tests plus three seeded controls were
employed to permit an evaluation of  the recovery procedures for each
sample.  A laboratory strain, Salmonella typhimurium SB558, resistant
to 1000 yg/ml of streptomycin was used as the seed Salmonella.  The
Salmonella seed was prepared from culture 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 evaluated sample
toxicity, diatomaceous earth concentration and the overall recovery
procedure.  A 100-fold dilution of the Salmonella seed stock was prepared
for 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% inacti-
vation 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   g/ml of streptomycin.  The recovery of strep-
tomycin-resistant Salmonella 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
 enrichment 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 2.   The enrichment media and temperatures evaluated were selenite
 broth at 37°C,  selenite broth at 41°C,  GN broth at 37°C,  tetrathionate
 broth at 37°C and dulcltol selenite broth at 40°C.   The primary plating
 media employed were bismuth sulfite (BS), brilliant  green agar (EGA),
 Salmonella Shigella agar (SS)  and xylose 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 (19).
 Identification of the members of the genus Salmonella was performed
 according to the schematic given in Figure 16.  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 XLD and subsequent typical colonies
 were  handled as above.  Phenylalanine deaminase negative and oxidase
 negative  isolates were  streak purified again and transferred to triple

                                     35

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                                                  Seeded Controls
 Sample:
   3 or 5 replicates
 and seeded control.
                                           71
 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
r1  (
   (2)  10-fold dilu-
 tion:
 5.0 ml of (1) to
 5.0 ml 1 x enrich-
 ment medium.
  •C
 Effective volume:
   0.09 x sample.
   (3) 10-fold dilu-
tion:
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 removed
to waste to maintain
serial dilution.
        * Seed - 10 to 50 organisms/sample vol. of streptomycin
              resistant Salmonella typhimurium SB558


              Figure 15.  Schematic - Multiple concen-
                          tration and enrichment for the
                          MPN determination  of Salmonella.
                                   36

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              xylose lysine desoxycholate agar
               I	
     typical Salmonella
          colonies
              absence of typical
              Salmonella colonies
                       I
                   restreak
              	  I
                           typical Salmonella
                                colonies
                                                       l
                     biochemical screen
                I	
          phenylalanine
            deaminase
                1
    oxidase
	1
   mannitol
 fermentation
       I
                       streak purify
                              I
                   triple sugar iron agar
                      lysine iron agar
                 r	
         typical Salmonella
             reactions
        I	—
     lysine
 decarboxylase
        I
malonate
	1
     Salmonella
 polyvalent  antisera
                   Salmonella  sp.

Figure 16.  Schematic - Isolation  and  identification of
            Salmonella sp.
                          38

-------
sugar iron agar (TSI) and lysine iron agar (LIA).  Typical Salmonella
was tested for malonate utilization and lysine decarboxylase activity
and submitted to a serological examination with polyvalent antisera
A-I (including Vi) (11).

     Phenylalanine deaminase and oxidase were employed to eliminate
the most probable interferences from members of the genus Proteus^
Providencia and Pseudomonas and, thereby, minimize the number of isolates
submitted to further tests.  Table 3 shows a comparison of the pheny-
lalanine 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  3 also indicated the relative  efficiency of XLD as a
primary plating medium  after dulcitol selenite  enrichment at 40°C.
About  53% 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% of  the red colonies were positive through
the screening procedure.

     Table  4  shows a tentative  identification  of the  genus  of  micro-
organisms  commonly  encountered  during the procedure  for the detection
 and enumeration of Salmonella.   Each of the microorganisms  yield sus-
 picious Salmonella colonies on  XLD.   The tentative grouping of isolates
 into a particular genus was based on a limited number of  biochemical
 tests.  The predominant interfering microorganisms were members of the
 genus Proteus,  Providencia and Pseudomonas.  Arizona sp.  and Citvobaoter
 sp. were about 3% each of the isolates tested after the phenylalanine
 deaminase,  oxidase and TSI-LIA screens.

 Sh-ictella 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 transferred to GN broth for enrichment.
 Ten-fold dilutions of the celite suspension were prepared in GN broth.
 Three  replicates were run at 10 to 50 microorganisms to evaluate recovery.
 The multiple concentration and enrichment procedure  for Shigella_was
 similar to that  given for Salmonella in Figure 15 except that^Sfogella
 sonnei was used  for the seed control.  After incubation at 37°C each
 dilution was streaked on xylose lysine deoxycholate  agar and  suspicious
 Shigella colonies (red) were tested biochemically according to- the
 protocol shown in Figure  17.

       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 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
                                     39

-------
 Table 3.  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  and OX
isolates screen
6,258 5,286 (84.5)
3,457 288 (8.3)
•
9,715 5,589 (57.5)
 and OX followed
by TSI-LIA
5,142 (82.2)
149 (4.3)
5,186 (53.4)
         Table 4.  GENUS  OF MICROORGANISMS  COMMONLY ENCOUNTERED
                   DURING THE ISOLATION OF  SALMONELLA  *
            Proteus  sp. and
            Providenoia sp.  Pseudomonas  sp.  Arizona  sp.  C-itrobaotev 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
                                    40

-------
                 multiple enrichment
               GN broth 24 hr. at 37 °C

            primary plating for isolation
                 XLD 24 hr. at 37°C .
                          r	
typical Shigella colonies
                    typical Shigella
                    colonies absent
                            I
                     restreak on XLD
                            I
                            typical Shigella colonies
                           	I
                          I
                 biochemical screen
 urease   phenylalanine  citrate    casein      oxidase
            Ideaminase      (      hydrolysis       I
               I           I           I           I
 Li	I	"	I	U
                          i     :[
                    streak purify
               triple sugar iron agar
                  lysine iron agar
               	I	
   typical Shigella
       reaction
          I
 polyvalent antisera
         and
  biochemical tests
 I                  I

    Shigella sp.
atypical Salmonella
     reaction
         I
polyvalent antisera
       and
.biochemical tests
  Salmonella sp.
 false
positive
Figure 17.  Schematic -  Isolation and  identification
               of Shigella  sp.
                                41

-------
 possible Shigella.   One  isolate yielded  typical  reactions for Shigella
 on TSI and LIA but was serotypically negative  and hydrolyzed casein.
 Table 5 is a tentative identification of the major groups of micro-
 organisms isolated.  Pseudomonas sp., Providencia sp. 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 6.  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 limitation appears to be the poor methodolody.

      The celite concentration procedure  employed appears to function
 reasonably well.  Table 7 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 (20)  reported the inhibition of Shigella when grown with coli-
 forms 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
 to 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  (21).   McFeters et  al.  (22)  reported  half time  die-off
 rates of  22.4 hours,  24.5 hours and 26.8  hours  for Shigella  dysenteviae,
 Shigella sonnei  and Shigella flexneri  at  9.5  to 12.5°C in well  water.
 Bartos  et al.  (23) reported recovery of added dysenteTy  bacilli after
 22 days in well water and 40 days in well water with  coliforms  added.
 Dolivo-Dobrovol'skii  and  Rossovaskaia  (24)  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 (25).  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 oholevae (22).

     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 stormwater, and stream water at a level of 3 x 106 Shigella/TtiL
and stored at 40°C.  Samples -were removed for determination of the
levels of Shigella flexnevi.  Figure 18 shows the survival of Shigella
flexnevi.  After eight days  37%, 41% and  26% of the seeded organisms

                                    42

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Table 6.  LEVELS OF INDICATOR MICROORGANISMS AND
   SALMONELLA IN SAMPLES NEGATIVE FOR SHIGELLA
Sample
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
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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
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
10?
107
107
It)6
107
10s
107
107
10s
105
107
10"
10"
102
103
103
10"
10"
FC
MPN/lOOml
2.4
4.9
4.6
4.9
1.1
<2.6
7.9
7.9
1.3
3.3
4.6
2.4
3.5
2.3
4.9
3.3
1.3
3.3
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
107
10s
10s
10 6
107
10s
10s
106
10 6
10s
105
10"
10"
102
102
102
10"
103
FS
#/100ml
4.1
1.1
1.2
8.3
4.5
2.0
1.1
X
X
X
X
X
X
X
1.1 -ac
3.4
9.7
3.9
7.6
4.2
1.2
2.1
1.2
1.0
2.4
X
X
X
X
X
X
X
X
X
X
10s
10s
106
10 5
10s
10"
10 6
10s
10s
10s
1QS
10"
10"
102
102
102
10s
10"
Salmoneila
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
                      44

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remained in the saline, sterile stream water, and stream water, respec-
tively.  The stream water contained 7 x 102 total coliforms, 7 x 102
fecal coliforms, 5.1 x 103 fecal streptococci, 3.3 x 102 Pseudomonas
aerug-inosa and less than 1.8 Staphloeoeaus awceus per 100 ml and 2.7
x 10* Salmonella/10 1.  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 Sh-igella present
in the aquatic environment.  The antagonism between the normal intestinal
flora and enteric pathogens has received attention for quite some time.
Hentges (20) recently reported the ability of 22 strains of microorganisms
to suppress the growth of Sh-igetta in the mouse intestine.  All strains
of Escheri.ohi.a ool-i and Enterobaeter aevogenes and most strains of
Proteus vulgar-is tested were antagonistic to Shigella.  The bacterio-
static and bactericidal effects of the coliform organisms were attri-
buted to volatile acids produced as metabolic end products.  The effects
were enhanced at lower pH values.

     The application of the findings of Hentges (20) to the development
of a suitable enrichment procedure has yielded some success.  Enrich-
ment conditions were set up to minimize the production and effect of
volatile acids.  Nutrient broth, a complex medium which contains :low
levels of carbohydrates, was adjusted to pH 8.0 and inoculated with
59 Shigella sonne-i 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.  Sh-igella
sonnei, was recovered from the flasks seeded with stream water with an
initial pH of 8.0.  The predominant interference was members of the
genus Pseudomonas.  One subsequent attempt to recover low levels of
Shigella (14 S. sonne-i, 28 S. flexner-i. and 9 S. flexnevi) did not
produce any Sh-igella isolates.  Of 148 suspicious colonies, 129 were
tentatively identified as Pseudomonas.  The minimal content of carbohydrate
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 Pseudomonas aevuginosa was not
antagonistic to Sh-igella and the results are in agreement with the
reports of Hentges  (20).  Pseudomonas apparently masks the presence of
Sh-igella.

StaphyloQOQQUS aureus—Serious difficulties were encountered with the
determination of levels of Staph. aureus.  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 suspicious Staph. auveus
colonies with typical morphology.  However, those colonies failed to
yield typical biochemical reactions.  Gram stains and microscopic
morphology.  Table  8 shows the recovery of confirmed Staph. auveus
for 1,249 isolates  in  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 V-J medium.  The
predominant interference on tellurite glycine 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

                                     47

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by a yellow halo which indicates mannitol fermentation.  Stock Staph.
duveus on V-J medium is a black colony surrounded by a yellow halo which
indicates mannitol fermentation.  Stock Staph. awfeus 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 nagative, rod-shaped bacteria.  In each case the presence
of Staph. aia>eus 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  (26) reported similar difficulties with
staphylococcus medium 110 when attempting to enumerate Staph. awceus
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 these observations
with m-staphylococcus broth, Smuckler and Appleman (26) 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 azide to the staphylococcus
medium 110 to inhibit the growth of the rod-shaped bacteria.

     Sodium azide was added to m-staphylococcus 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 employed as a primary isolation medium from the modified
m-staphylococcus broth.  Gunn et ul.  (14) used LSM to isolate and identify
Staph. aureus.  The production of opaque yellow zones around the colonies
was considered positive evidence for lipovitellenin-lipase activity
 (opaque) and mannitol fermentation  (yellow).  Lipovitellenin-lipase
activity was found to correlate with coagulase production for 94.7%
of the isolates tested by Gunn et al.  (14).  Table 8 shows the recovery
of Staph. auveus for typical colonies .on LSM  after enrichment in the
modified m-staphylococcus broth with 0.75 mM  sodium azide.  507 of 531
or 95.4% of the typical isolates after enrichment and plating on LSM
were confirmed Staph. awceus.

     The correlation of biochemical characteristics with pathogenicity
of staphylococcus  isolates  has been the subject of numerous reports.
Unfortunately, no  one criterion is adequate for the identification
 of Staph. aureus.  Even coagulase production, which has traditionally
been used to.differentiate  Staph. aureus  from Staph. epidermis  (formerly
 Staph. albus)  is  not absolute  (27).   The  enumeration must be based on
 several  biochemical  characteristics that will enable a differentiation
 from not only  Staph. epidermis but  other  genera of microorganisms not
 normally encountered at high  levels in clinical samples.

     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  glu-
 cose  anaerobically,  yielded typical microscopic morphology  and  Gram
 positive.   A schematic  of the  culture procedure is  given in Figure 19.
                                    49

-------
                     m-staphylococcus broth
                      +0.75 mM sodium azide
                                I
                            turbidity
                    	I	
          streak for isolation
                 on LSM*
         	I	
  typical colonies//
                                   absence of
                                 typical colonies

                                    restreak
                      typical colonies)1/
Gram     catalase    anaerobic   coagulase   mannitol
stain        I         glucose             fermentation
  I           I      fermentation
                                                         DNase >
                     Staphyloaoaaus aureus


*  LSM - lipovitellenin salt mannitol agar

t  typical colonies - colonies surrounded by opaque and/or yellow
   zones
Figure  19.   Schematic  - Isolation  and  identification
               of  Staphyloaoocus  au&eus.
                              50

-------
Pseudomonas aeruginosa—Culture media for the enumeration of Pseudomonas
aevug-lnosa were evaluated with samples of raw sewage and urban streams.
MPN procedures using L-asparagine broth and confirmation on acetamide
broth according to Standard Methods (16), Drake's asparagine broth #10
(28) confirmed on acetamide broth and membrane filter procedures using
MacConkey agar according to Nixon and Brodsky (29) were tested.  Table
9 shows the results of the initial culture evaluation for 16 samples
of raw sewage and urban streams.  L-asparagine broth according to
Standard Methods yielded consistently higher levels of confirmed Pseu-
domonas aevug'Lnosa 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 confirmatory 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 10 shows the comparative
levels of P. aevuginosa calculated from confirmation on acetamide broth
and acetamide agar.  The contents of each presumptive positive dilution
tube were transferred for confirmation to acetamide broth and spotted
or streaked on acetamide agar.  The levels of P. aevuginosa were similar
for each confirmatory method except for one stream sample.

     The final procedure for the enumeration and identification of
P. a&YU.g-inosa is given in schematic form in Figure 20.  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. aeruginosa.
Calculation of the MPN P. aeruginosa was based on the recovery of
isolates that were acetamide and oxidase positive and grew at 42°C.
Casein hydrolysis with fluorescence  (30) was employed as a secondary
characteristic.

Coliform group—Total coliforms were determined by the multiple tube
dilution procedure with lactose broth as the presumptive medium according
to Standard 'Methods  (16).  Positive tubes were confirmed on brilliant
green lactose broth with 2% bile at 35°C.

     Fecal coliforms were determined by confirmation of positive lactose
broth presumptive tubes on EC medium incubated at 44.5°C for 24 hours
(16).

     The effect of homogenization on the levels of microorganisms was
evaluated.  Stream samples were blended at room temperature for varying
time periods and the levels of total and fecal coliforms determined.
The results for five trials are shown in Figure 21.  No consistent
trends and little significant 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 coliform 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.
                                    51

-------
                Table  9.  EVALUATION  OF PRESUMPTIVE MEDIA FOR
                  THE  ENUMERATION  OF  PSEUDOMONAS AERUGINOSA
L-Asparagine
broth a
MPN/lOOtnl
Raw Sewage 9.2 x 10 5
3.5 x 105
2.3 x 106
1.7 x 106
Stream 1.7 x 102
1.7 x 103
1.6 x 105
2.2 x 103
2.3 x 10k
2.4 x 10^
3.5 x 105
1.1 x 103
1.7 x 10s
2.8 x 101*
7.0 x 10^
5.4 x 103
Drakes #10
broth a
MPN/lOOml
3.5 x 105
2.3 x 10s
9.2 x 105
4.6 x 105
1.1 x 102
1.4 x 102
1.6 x 105
1.1 x 102
2.2 x 101*
1.7 x 103
1.9 x 10*
3.1 x 102
3.3 x 101*
4.9 x 103
3.3 x 103
7.9 x 102
MacConkey
agar - mf a b
#/100ml
4.5 x 105
ND
4.5 x 106
1.0 x 106
1.4 x 102
4.0 x 103
2.0 x 101*
ND
1.1 x 103
3.0 x 104
2.0 x 104
4.0 x 103
1.5 x 10^
1.5 x 101*
1.0 x 10^
1.5 x 103
a  Confirmed in acetamide broth

ND No data

b  Brodsky and Nixon (29)
                                    52

-------
         Table 10.  LEVELS OF PSEUDOMONAS AERUGINOSA CONFIRMED
                 ON ACETAMIDE BROTH AND ACETAMIDE AGAR
 Sample
                                Pseudomonas  qevuginosa MPN/100ml
                                     Acetamide confirmation
  Broth
  Agar
Raw Sewage
2.3 x 10s

1.7 x 10s

9.2 x 105

4.6 x 10s
2.3 x 10s

2.8 x lO6

1.6 x 10s

1.7 x 105
Stream
1.6 x 105

2.4 x 101*

2.8 x 101*

2.2 x 103

1.5 x 105

1.6 x 10s

1.7 x 103

4.9 x 103

1.7 x lO1*

3.3 x 103
2.2 x

2.4 x

7.0 x

3.5 x 103

1.6 x 10s

1.6 x 105

1.3 x 103

1.3 x lO1*

1.8 x 103

3.5 x 103
                                 53

-------
                     1-asparagine broth

                       turbidity with
                       fluorescence
                     	I	
                    -t-
                    I
              acetand.de agar
               confirmation
          streak for
           isolation
   growth with
  fluorescence
                  spot
               inoculation
        growth with
       fluorescence
             I
   streak for isolation
    Pseudomonas agar P
 oxidase
             acetamide
              growth
    I
growth at
  42°C
    I
    -f-
      \            I
  tech agar     casein

      I            I
 growth with  hydrolysis
fluorescence     with
            fluorescence
        primary criteria                secondary criteria

                       ?seudonana.a aevuginosa


Figure 20.   Schematic -  Identification  of Pseu-
               domonas aevuginosa.
                             54

-------
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                                                     4-1
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-------
 Fecal streptococcal group—There has been an increasing usage of fecal
 streptococci 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 (16) defines the fecal streptococcal group to contain the following
 species:   S.  faecaUs,  S. faeoalis var.  liquifaoiens3  S. faeoal-ls
 var.  zymogenes,  S.  duvans,  S.  faecium, S.  bovis  and S.  equinus and is
 considered synonymous  with  "Lancefield's Group D.  Streptococcus".
 The more  restrictive term "enterococcus" excludes S.  bovis and S.  equinus.
 Geldreich (31)  assigns limited sanitary  significance to the S.  faeoal-Ls
 strain^capable of .starch hydrolysis (designated  atypical)  and S.  faecalis
 var.  t-iquifaoi-ens and  provides evidence  to suggest that the subgroup
 of  S.  bovis and S.  equinus  may be indicative of  non-human  animal pollu-
 tion.   The latter two  streptococcal species were found in  significant
 levels of animal feces but  not recovered from human feces.

      An initial effort in this study was directed toward evaluation of
 media for the enumeration of the fecal streptococcal group.   Multiple
 tube  dilution technique with azide dextrose broth confirmed on ethyl
 violet azide (EVA)  and/or enterococci confirmatory agar, M-enterococcus
 plate counts and KF streptococcus plate  counts were evaluated with
 samples of raw sewage  and water from urban streams.   KF streptococcus
 plate counts consistently yielded the highest recovery of  fecal strep-
 tococci.   M-enterococcus agar  consistently yielded low recovery.   Sub-
 sequent 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 et al.  (32),  Hartman et al. (33),  and Kenner et at.  (34),
 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  22.   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 containing 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  toothpicks 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  master plate
with  a sterile toothpick and transferred to a  plexiglass toothpick
 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.
                                    56

-------
              KF streptococcus agar
                pink-red colonies
             	I	
catalase      growth in     growth at
             40% bile       45°C
              ENTEROCOCCI
                   I
            starch, hydrolysis
               growth at
                 10"C
               	I	
                                      growth at
                                      6.5% tlaCl
                                          I
                                             starch hydrolysis
                      casein hydrolysis
                     	I	
           gelatin liquefaction
          	    I	
    S. faecalis
  var. liquefaaiens
   var. zymogenes
arabinose fermentation
                        S. fieai-wn
                                       mannitol fermentation
                                  S. fa.eadli.8
                                                      S.
Figure  22.   Schematic  - Identification of the fecal
               streptococci.
                              57

-------
      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 strep-
 tococcus agar is given in Table 11.  With the exception of arabinose
 fermentation 94.4% agreement or better was observed between the repli-
 cation 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 was too low
 the fermentation reaction was difficult to read.  Subsequent comparison
 with 50 isolates where the pH was adjusted to 7.4 before autoclaving
 yielded 98% agreement.  The overall agreement 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.   With respect to the group identification of enterococcus,
 S.  bovis and equinuSj  the l-iquefaoiens and zymogenes variations of S.
 faecaHsj  atypical S.  faeoal-ls and false positives,  the percent agreement
 overall was  97.0% for both procedures.

      Both replication procedures were rapid, inexpensive and reasonably
 accurate.  The toothpick replicator yielded a more positive (small
 noticeable 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.

 Animal Virus Assays

      Animal  virus assays were conducted by Dr.  James E.  Smith at the
 Syracuse University Biological  Research Laboratories in  Syracuse,  New
 York.

 Tissue Cultures'—
      BGM cells were grown in a  1:1  mixture of MEM medium (Hanks  basal
 salts)  and L-15  medium (Leibovitz)  using 10% fetal calf  serum and  1%
 of  a 7.5% NaHCOa stock (35).  Human HEp-2  cells  were grown  in H-MEM
 above plus 5% fetal calf  serum.   Human  fetal embryonic lung  (HEL)  cells
 were obtained from Flow Laboratories (as Flow 4000)  and  grown for
 4 to 8 passages  in E-MEM  (Earles  basal  salts) plus 10% fetal  calf  serum.
 Addition of  gentamicin  (5 yg/ml)  to the growth media for BGM  and HEL
 cells markedly improved the  growth  and  plaquing  capabilities  of  these
 cell lines.

 Plaque  Formation—
     BGM and  Hep-2  cells were used  for  plaque assays  as  described by
Dahling &t al.  (36) using an agar overlay  containing  per 100 ml:  H-
MEM, L-glutamine, MEM nonessential  amino acids,  2% fetal calf  serum,
 3.0 ml  7.5% NaHCOs, 1.0 ml 1% MgCl2  • H20  solution,  1.5  ml 0.1%  neutral
red  solution, 200 units penicillin,  100  yg streptomycin  and 50 pg
mycostatin.  HEL cells were overlaid with  E-MEM, 5% fetal bovine serum,
pH 7.4.  All agar overlays contained either  0.8% ion  agar No.  2  (Oxoid)
or 0.8-1.0% Noble agar  (Bacto).  Plaque  formation was observed in 60 mm
polystyrene plates and in Multiwell  (Falcon) plates  (37, 38).
                                   58

-------
Table 11.  COMPARISON OF VELVETEEN AND TOOTHPICK REPLICATION PROCEDURES TO
CONVENTIONAL TUBE METHODS FOR THE DIFFERENTIATION OF FECAL STREPTOCOCCI
                                   Velveteen
Toothnick
# of
Test tests
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
250
250
150
250
200
250
-
_
#
agree
250
237
242
242
-
150
250
166
240
-
—
%
agreement
100
94.8
96.8
96.8
-
100
100
83.0
96.0
93.0
97.0
#
agree
250
236
243
240
-
150
250
184
243
-
—
%
agreement
100
94.4
97.2
96.0
-
100
100
92.0
97.2
94.5
97.0
 a Casein hydrolysis was compared to peptonization of litmus milk

 k S. durans  classified as a variation of 5. faecivm
                                    59

-------
 Microtiter TCIDso Endpoint Assays  and CPE—
      Techniques described by Conrath  (39) were used for viral neutrali-
 zation and metabolic inhibition  tests of virus concentrates eluted from
 sewage solids.  Microtest II plates were employed and the cell monolayers
 were stained with Giemsa reagent according to the following schedule
 (40).  After 2 to 10 days the Microtest II culture plates were washed
 by immersion in PBS and fixed by immersion in methanol 5 minutes.  The
 monolayers were washed 15 minutes  in dilute.Giemsa solution (stock
 Giemsa diluted 1:15) and rinsed 20-40 seconds in tap water and distilled
 water.  The plates were inverted and air-dryed.  (Stock Giemsa:  Ig
 Giemsa powder was dissolved in 66 ml glycerol, 55 C, 1 hr. and 66 ml
 methanol was added.)

 Methods of Identification—
      A two-step scheme was employed to make quantitative estimates
 of the number of viruses in each of five groups:   polio-, Coxsackie B-,
 echo-, adeno- and reoviruses.   A presumptive test of the virus concen-
 trates was made first in Microtest II plates to determine which groups
 were represented in the sample and what was their approximate TCID5Q
 titer.   The dilution sequences,  the challenge with eight neutralizing
 units  of antipoliovirus IgG (rabbit)  and the cell sensitivity spectrum
 are illustrated in Figure 23.   The interpretation of the patterns obtained
 by this procedure are shown in Table  12.   Generally it was possible
 to differentiate the virus groups even when they  occurred in mixtures
 by suppressing one or two groups  with antiserum or a nonpemissive host.
 In those samples where a fourth  or an unrecognized component was  present
 the samples were plated and  the  individual plaques were  picked with
 sterile toothpicks and a confirmed test was applied.   For this test
 the unknown plaques  were subcultured  in microtiter wells,  the  resulting
 virus was passed back through  the screen  (Figure  23)  and/or  the cytopathology
 of the  infected cells was  examined  by  staining with May-Grunwald  Giemsa
 stains.   If adeno- or reoviruses  were  suspected,  the  plaques were grown
 in 16-chamber Lab-Tek well slides and  stained  with  acridine  orange as
 well as MGG  (41).

 Virus Concentration—
     Five gallon  stormwater samples were decanted and viruses  in  the
 supernatant were isolated by selective adsorption  to membrane  filters.
 The Aquella Virus Concentrator (Carborundum Co.) was used  to remove
 the remaining solids  and to adsorb  the viruses to "sandwich" of three
 cellulose  acetate membrane filters  - 5 ym and  0.47 ym porosities.
 Orion (10  and 1 ym porosities) and  cellulose acetate  (0.8 ym) wound
 textile filters  ("Fulflo") were used ahead of the membrane filters to
 remove suspended solids which would clog the membrane filters  (42, 43).
Virus samples were adjusted to pH 4.5 with HC1 and MgCl2  (0.05 M) was
added after the prefiltration step.  The adsorbed viruses were eluted
from the membrane with 0.05 M glycine, pH 11.5 or 3% beef extract,
pH 9.5.  Eluates were collected in cold buffer, pH 7.2 to prevent
damage to the virions (individual virus particles) and sterilized with
chloroform.  Two percent fetal calf serum and 1% MgCl2 were added to
the preparations which were then frozen and stored at -40°C.

Sampling and Shipping—
     Twenty liter samples were collected in polystyrene containers and
shipped by express bus from Baltimore  to Syracuse, N.Y.  During shipment

                                    60

-------
      BGM
      HEp-2
               HEL
 o
co —
0



io-i



10~2



10-3



10-
o o
0 -- 1

— o

£o

oooo
oooo
oooo
oooo
oooo
oooo
oooc
oooo
oooo
oooo
oooo
oooo
oooo
ooo
oooo
oooo
           oooo
           oooo
           oooo
           oooo
           oooo
          oo
            ooo
           oooo
           oooo
  Figure 23. Presumptive assay for virus concentrates (Micro Test

     11 plate).
         61

-------
  Table 12.  DIAGNOSTIC PATTERN FOR PRESUMPTIVE IDENTIFICATION
              OF ENTEROVIRUSES IN TISSUE CULTURE
  Virus
  Group
Treatment
  with
anti-polio
   IgG1
  Cytopathic effect on;

BGM        HEp-2       HEL
Polioviruses
   No
   Yes
Coxsackie B
viruses
Echoviruses
   No
   Yes
   No
   Yes
                       -or+
                       -or+
                                   +or-
                                   +or-
1 Rabbit sera fractionated by precipitation with ammonium sulfate
  and cross-absorbed with packed cells of the three indicator
  cultures.
                               62

-------
the samples were maintained at ambient temperatures for 9 to 12 hours;
on arrival they were refrigerated overnight at 4°C.  Thermal controls
were included in each grab sample.  One ml aliquots of commercial polio-
virus vaccine which had been diluted 1:25 in tryptose phosphate broth
(TPB) were mixed with 9 ml of each water sample in tightly capped tubes.
These tubes .plus one with virus in sterile TPB were placed in the
water containers during shipment.  Upon receipt they were sterilized
by chloroform extraction, aerated to remove the chloroform and; frozen,
-40°C, after the addition of 2% calf serum and MgCl2 to 0.05 M.The
plaque titers of the surviving viruses were compared to reference
stocks of the original vaccine maintained at -65°C.        ;-   a  ; .

     Shipment of waste water samples by bus was found to be an	adequate
procedure for the holding time involved in these experiments -^eight
hours at ambient temperatures.  When temperatures  of newly arrived^
samples were measured, they were not more than one or two degrees  C
above external  temperatures, suggesting that the cargo hold? remained
cool throughout the trip.  Trivalent oral polio vaccine was chosen for
the thermal control since there are some differences in temperature
sensitivity among the polio serotypes.

     Average recoveries  of PFU are shown in Table  13, ranging from 84
to 94%  of  the  expected values.  The average values for recoveries of
virus diluted  in water samples were consistently lower than the  Values
for viruses diluted in TPB.  Although  those differences were not signi-
ficant  enough  to  affect  the method of  handling samples,  it did  suggest
that  the viruses  may be  osmotically shocked when diluted  in stream
water or that  TPB may protect  them against  thermal damage in  some way.

      An earlier progress report  appeared to  show marked  losses  and
 irregular  recoveries  of  enteroviruses  when their plaque  recoveries were
measured.   However,  this problem was  corrected  later when it was demon-
 strated that  the chloroform-preserved thermal control samples were not
 being adequately aerated.   Traces of  chloroform in some  samples reduced
 the plating efficiency and the apparent recovery of  virus.
                                    63

-------
       Table 13.   CONTROL SAMPLES FOR LOSSES  DUE TO  SHIPPING
Percent survival compared to
frozen polio vaccine
Sample
no.
36
38
39
46
48
52
55
57
60
61
63
Average
a
A
88
79
103
90
87
96
85
91
88
73
81
87.4
12.2
Sampling site
BCD
91
61
100
79
100
89
93
105
ND
86
97
90.1
12.1
90
80
101
99
112
82
87
114
96
90
78
93.5
11.4
76
95
91
78
87
80
67
93
92
ND
99'
83.7
10.0
TPB
104
120
99
91
111
86
ND
99
ND
88
100
99.7
10. 6
ND - No Data
                                 64

-------
                              RESULTS
     Samples were collected for microbial analysis from August 1974
through September 1975.  The samples collected in August and September
of 1974 were used to evaluate culture methods and set logistical pro-
cedures for the routine microbial assays of subsequent background and
storm samples.  The data presented cover 13 months, from September
1, 1974 through September 30, 1975.  During this period, 35 sets of
background samples and samples from 24 storm events were collected
and assayed.'  This represents a total of 298 samples, 154 background
and 144 storm samples.  Table 14 gives some idea of the magnitude of
effort to obtain information on the levels of pathogenic and indicator
microorganisms.  A total of 34,511 isolates were tested.  Each isolate
was processed through a series of biochemical tests to enable a tentative
identification of pathogenic and indicator microorganisms.

RAINFALL

     Rainfall data were obtained at three rainfall gauges in Baltimore
City.  The three locations are indicated on Figure 4 by filled circles.
The Customs House station was operated by the U.S. Weather Bureau and
located close to the downtown business district.  The Woodborne and
Ashburton gauges were  operated by  the City of Baltimore and located
in the northeastern and northwestern sections of the city.  Rainfall
data during gauge malfunction at Woodborne and Ashburton were estimated
front the measurements  taken  at the Customs House.  The monthly pre-
cipitation data  from  September 1974 to August 1975 at the three gauging
stations in Baltimore  City are"given in Table 15.  The 50 year normal
precipitation data are given in  the column on the  right for comparison.
The Customs House station  in downtown Baltimore  recorded considerably
more rainfall than the 50 year normal in  September and December in
1974 and March,  May and July in  1975.  The annual  rainfall  at the
Customs House exceeded the 50 year normal by more  than 40 cm.  The
Woodborne  gauge  recorded higher  rainfalls  in  September and  December
1974 and March,  May,  June  and July 1975.   The annual  total  at this
station was more than 17 cm higher than  the  50 year normal.   The Ash-
burton gauge  in  the northwestern section recorded  almost  8  cm more
rain than  the 50 year normal.   The amount  of  precipitation  measured
at three different  locations in Baltimore City varied considerably.
The daily  rainfall  at each gauging station can be seen in Appendix A.

OCCURRENCE AND LEVELS OF MICROORGANISMS

     The  raw data for the  levels of bacteria and animal viruses  for
 each  sample are  given in Appendix B and Appendix C,  respectively.
 The data are presented for each sample station.   The levels of  indicator
microorganisms,  P.  aerugi-nosa and Staph.  aureus  are reported as MPN
 or number/100 ml.  The Salmonella sp.  and animal virus are reported
 as MPN/10 1 and PFU/10 1,  respectively.   It is  important  to recognize
 that  the denominator for the Salmonella and animal virus  data is  100
 fold greater than the denominator for the indicator and other micro-
                                     65

-------
        Table 14.  NUMBER OF BACTERIAL ISOLATES
           TESTED DURING THE PROJECT PERIOD
Bacterial Assay
Number of Isolates
Salmonella sp.

Shigella sp.

Pseudamonas aemuginosa

Staph. aweeus

Fecal streptococci
                         Total
      10,242

       1,051

       7,389

       7,587

       8.242

      34,511
                           66

-------
       Table 15.  MONTHLY PRECIPITATION IN cm (INCHES) DURING
            THE STORMWATER STUDY PERIOD AT THREE GAUGING
                     STATIONS OF BALTIMORE CITY
Period
1974
September
October
November
December
1975
January
February
March
April
May
June
July
August
12 month
total
Woodborne

13.59
2.79
4.70
15.37

8.76
5.72
12.07
5.21
11.94
11.05
27.94
6.60

125.73


(5.35)
(1.10)
a,
,85)
(6.05)

(3
(2
(4
(2
(4
(4
(11
(2

(49

.45)
.25)
.75)
.05)
.70)a
.35)a
.00)a
.60)

.50)a
Ashburton

10.67
2.41
4.32
15.62

4.95
3.18
10.67
6.73
10.29
8.51
27.94
10.92

116.21

(4.
(0.

20)
95)
(1.70)
(6.15)a

(1.
(1,
(4,

.95)
.25)
.20)
(2.65)
(4
(3
(11
(4

(45
.05)
.35)a
.00)a
.30)

.75)a
Custom

15.27
3.40
4.50
20.47

9.14
6.35
15.01
7.72
20.52
9.40
29.67
9.50

150.95
House


(6.01)
(1.34)
(1.77)
(8.06)

(3
(2
(5
(3
(8
(3
(11
(3

(59

.60)
.50)
.91)
.04)
.08)
.70)
.68)
.74)

.43)
50 year
normal

9.22
10.24
9.84
11.80

12.09
12.09
13.58
12.20
12.52
14.05
16.92
16.49

108.41

(3.63)
(2.82)
(2.71)
(3.25)

(3.33)
(3.33)
(3.74)
(3.36
(3.45)
(3.87)
(4.66)
(4.53)

(42.68)
a - estimated
                                     67

-------
  organisms.  The levels  of viruses  are  reported as  TCIDsn/10 1 for the
  presumptive test and PFU/10 1.  All  calculations involving enteric
  viruses were made using the PFU/10 1 data.

  Background Samples

       The occurrence of selected pathogenic bacteria is summarized in
  Table 16 for the background samples  (sewage, urban streams and reservoirs)
  Salmonella sp. were recovered from all the raw sewage samples, 84%
  of the samples from Herring Run, 94% of the samples from the Jones Falls
  and 100% of the samples from Gwynns Falls.  Only 7% (1 of 14) of the
  samples from Loch Raven reservoir were found to contain Salmonella.
 P. aevugvnoaa, the most abundant pathogen, was found in all the samples
  of raw sewage, Herring  Run, and Jones Falls, and 97% of the samples
  from Gwynns Falls.   P.  aeruginosa was isolated in 62% of the Loch Raven
 reservoir samples.   Staph.  aureus was found in 93%, 57%, 93% and 59% of
 the raw sewage, Herring Run, Jones Falls, and Gwynns Falls samples,
 respectively.   No Staph. auveus was recovered from Loch Raven reservoir.
 It should be noted that the lower sensitivity limit for the Staph.
 aureus assay was an MPN of 2/100 ml.

 The occurrence of viruses in the background samples is shown in
 Table 17.   The percent  occurrence was based on the presumptive test
 of the virus  concentrates with neutralizing antisera and cell sensitivity
 spectrum for  five virus groups;  poliovirus,  Coxsackie B virus,  echo-
 virus,  adenovirus and reovirus.   Animal-viruses were isolated in 93%,  82%,
 75% and 79% of the  raw  sewage,  Herring Run,  Jones  Falls,  and  Gwynns Falls'
 samples,  respectively.   A surprisingly high 71%  (5  of 7 samples)  of the
 samples from Loch Raven reservoir contained viruses.   Poliovirus  was the
 predominant virus recovered  in  Gwynns Falls  and Loch Raven reservoir.
 Similar frequencies  of  recovery of  poliovirus  and  Coxsackie B virus were
 observed  for faw sewage and  Herring Run.   Coxsackie B virus was the pre-
 dominant  group  found in the  Jones Falls.   Echovirus was recovered from  8%
 to 29%  of the background samples.   Adenovirus was recovered from  raw
 sewage  and  Gwynns Falls, and reovirus was  found in  Jdnes Falls and Gwynns
 Falls.

      The fecal streptococcal group contains several  species and  strains
 that have limited sanitary significance.   Table 18  shows the distribution
 of fecal streptococci in the background samples.  The occurrence  is
 presented as the average percent of the isolates and the percent  samples
 positive for each subgroup of fecal streptococci.   The average percentage
 of enterococci  isolates  varied from 5.0.5%  to 56.5%  for the raw sewage
 and urban streams with  typical S. faeoalis and S.  faeo-ivm representing
 43.4% to 48.4%.  S.  faeoalis var. liquefaoiens and  S. faeoalis var.
 zymogenes were  found in  58% to 73%  of the background samples and  repre-
 sented a mean of 6.7% to 9.8% of the  isolates.  Atypical S. faeoalis3
 capable of hydrolyzing starch, was  recovered from 24%, 32%, 9% and 10%
 of the samples of raw sewage, Herring Run, Jones Falls and Gwynns Falls,
 respectively.   The mean  percent of  the isolates for this strain of
 S. faecaUs was 0.5% to  1.4%.  The  liquefaoiens and zymogenes and atypical
 strains of S.  faeoalis were not found to predominate in any of the
background samples.   S.  bovis and  S.  equinus similarly, were observed
at mean levels of 7.5% to 9.9% of the isolates tested.  The frequency
of positive samples for S. bovis and S.  equinus were 88.2%, 50.0%,
71.9% and 48.4% for raw sewage,  Herring Run, Jones Falls and Gwynns

                                    68

-------
         Table  16.  OCCURRENCE OF  SELECTED PATHOGENIC

           BACTERIA IN BACKGROUND  SAMPLES, PERCENT
Sample site Salmonella sp.
A
B
C
D
E

Raw sewage
Herring Run
Jones Falls.
Gwynns Falls
Loch Raven
reservoir
100
84
94
100
7

P. aevug-lnosa
100
100
100
97
62

Staph. aureus
93
57
93
59
0

                Table 17-  OCCURRENCE OF VIRUSES

                     IN BACKGROUND  SAMPLES
                                   	 Occurrence*,  %	  '	
                Number   	——       ~~
                   of     Animal             Coxsackie
                samples  Virus   Poliovir.us    virus B    Echovirus  Other
Sample site
A
B
C
D
E

Raw sewage
Herring Run
Jones Falls
Gwynns Falls
Loch Raven
reservoxr
15
11
12
14
7

93
82
75
, 79
71

53
36
33
57
43

53
36
58
50
29

20
27
8
29
14

13a
0
8b
21a, 1
14C

* - Occurrence based on presumptive test of virus concentrates


a - Adenovirus


b - Reovirus


c - Not identified
                                    69

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Falls, respectively.  False positive non-fecal streptococci were found
in all the background samples and represented a mean of 34.4% to 37%
of the isolates tested.

     The geometric mean densities for the microorganisms assayed for
each background sample station are given in Table 19.  The raw sewage
and Loch Raven reservoir provide information on the microbial water
quality that can be expected under the worst and best conditions,
respectively, in an urbanized area.  Each of the urban streams contain
high levels of each of the indicator microorganisms and would be judged
contaminated regardless of the indicator of fecal contamination employed.
The relative order of levels of pathogens in the urban streams was
P. aeruginosa >'Staph. aureus > Enterovirus > Salmonella sp.  It should
be stressed that the levels of enterovirus and Salmonella sp. are
reported with a denominator 100-fold higher than the other microorganisms
and represent a 100-fold more sensitive assay.

     The relative levels of indicator and pathogenic microorganisms
in the urban streams during the sampling period are shown graphically
in the following three figures.  Figure 24a shows the log; density of
each  of the indicator  groups of microorganisms by date for Herring
Run  (sample site B).   Consistently high levels of total coliform, fecal
coliform and fecal  streptococci were observed throughout the year.
Although variation  can be  seen in the data, there does not appear to
be any marked variation with season.  The levels of  fecal coliform
in Herring Run  exceeded  the recreational standard of 200 fecal  coli-
forms/100  ml in 30  of  33 or 91%  of  the  samples during  the 13 month  sampling
period.  The levels of pathogenic microorganisms in  Herring  Run can be
seen in Figure  24b.  Again, noticeable  variation is  shown but there
is no apparent  dependence  upon season  of the year.   Staph.  awceus
was  found  at low levels  in each  sample.  Salmonella  sp. were consistently
found in Herring Run and isolates were recovered in  27 of  32 or; 84%
of  the samples.                                                 \

      The  levels of indicator microorganisms in the Jones  Falls  iare
 shown in  Figure 25a.  The total coliform,  fecal coliform and fecal
 streptococci densities were somewhat higher than in  Herring Run and
 no apparent seasonal variation was observed.   All  samples collected
 from the Jones Falls exceeded the fecal coliform recreational water
 standard by several orders of magnitude.   The levels of -P.  aertig-inosa,
 Staph.  aureus,  and Salmonella sp.  (Figure 25b) are also considerably
 higher than in Herring Run.  P.  aeruginosa was the predominant pathogen
 followed by Staph.  auveus.  Salmonella was recovered from 29 of 31 or
 94% of the samples collected from the Jones Falls.

      Similar results are shown for Gwynns Falls in Figures  26a and 26b.
 High levels of indicators were observed with no apparent seasonal
 variation.  The fecal coliform recreational water standard was met
 in only one sample during the sample period. The levels of  pathogens
 follow the same order as in the previous urban streams.  Seasonal
 variation again does not appear.  A possible peak was observed for
 P. aeruglnosa for the Gwynns Falls in September 1974 but was not observed
 in September of 1975.  Salmonella was recovered from all samples col-
 lected in the Gwynns Falls.
                                      71

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     The effect of stream flow on the levels of fecal coliforms in
Herring Run and Gwynns Falls is shown in Figures 27 and 28, respectively.
Stream flow was determined at the time of sample collection.  The minimum
stream flow that could be estimated at Herring Run was 28 I/sec. (0.95 cfs)
The highest recorded flow was 608 I/sec. (21.5 cfs) and is not recorded
on the graph.  During periods of low flow the levels of fecal coliform
varied from just more than 10 to greater than 105 MPN/100 ml.  There
was no apparent relationship between stream flow and fecal coliform
densities for Herring Run.  The stream flow in the larger Gwynns Falls
varied from 110 to 8,490 I/sec. (3.9 to 300 cfs).  As in Herring Run,
there appears to be little correlation between stream flow and levels
of fecal coliforms.

     The effect of previous rainfall prior to the time of sampling on
the microbial quality was evaluated for the background samples.  The
levels of fecal coliform was compared to the antecedent or number of
days since the last rainfall.  Antecedent rainfall, in days, .appears
to have little effect on the fecal coliform density in raw sewage and
urban streams (Figure 29).

Storm Samples

     The occurrence of selected pathogenic bacteria in storm runoff
is shown in Table 20. Salmonella sp. were recovered from all the sam-
ples collected at Stoney Run, Glen Avenue, and Howard Park, 96% of the
samples collected at Jones Falls storm drain and Bush Street, and
52% of the Northwood samples.  P. aevugi-nosa was found in all the storm
samples at levels approaching those found for the indicator microorganisms.
Staph. aureus was isolated from 83%, 71%, 95%, 100%, 96%, and 82% of
the samples from Stoney Run, Glen Avenue, Howard Park, Jones Falls
storm drain, Bush Street and Northwood, respectively.  The lower relative
recovery of Staph. aiweus was due to the higher sensitivity limit for
the Staph. auveus assay.  No concentration procedure was employed and
the maximum sample volume assayed was 10 ml.

     Animal viruses were recovered at a high frequency in the storm
runoff samples.  Table 21 shows the occurrence of selected animal
viruses for these samples.  Animal viruses were recovered from all
the samples at Stoney Run and Howard Park, 92% of the Glen Avenue,
83% of the Jones Falls and Northwood and 75% of the Bush Street samples.
The predominant virus groups found were polioviruses and Coxsackie B
viruses.  Echovirus was observed at a lower frequency.  Adenovirus was
found in 1 of 12 samples at Bush Street and reoviruses were found at
a  similar, frequency at Glen  Avenue and Howard Park.

     The distribution of fecal streptococci in the storm runoff samples
is given in Table 22.  The mean percent of isolates that were found
for each component of the fecal streptococci for each storm sample
site is shown.  The number in parenthesis was the frequency with which
the member microorganisms were observed.  Enterococci were found in
all samples and were 39.6% to 51.5% of  the isolates tested.  The major
portion of the enterococcal group were  typical strains of S. faecalls
and S. faeoum.  A small percentage  (1.3% - 7.1%) of the enterococci
isolates were the  liquefao-iens - zymogenes varieties of S. faeoal-is
                                     79

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             4      8       12      16

            ANTECEDENT    DAYS
                                   20
Figure 29.   The effect of period in days since last
           rain storm on the fecal coliform density
measured in the background samples, A,  raw sewage
(o), B, Herring Run (*), C, Jones Falls (a),
D, Gwynns Falls (A), and E,  Loch Raven (•).
                      82

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           Table 20.  OCCURRENCE OF SELECTED PATHOGENIC
                  BACTERIA IN STORMWATER SAMPLES

Sample site
F Stoney Run
G Glen Avenue
H Howard Park

Salmonella sp.
100
1QO
100
Occurrence, %
P. aevug'inosq
100
100
100

Staph. awceus
83
71
95
K  Jones Falls
   storm drain

L  Bush Street

M  Northwood
96

96

52
100

100

100
100

 96

 82
             Table 21.  OCCURRENCE OF SELECTED VIRUSES
                       IN STORMWATER SAMPLES
Occurrence, %
Animal
Sample site virus Poliovirus
F
G
H
K
L
M
a
b
c
Stoney Run 100 73
Glen Avenue 92 75
Howard Park 100 42
Jones Falls 83 67
storm drain
Bush Street 75 25
Northwood 83 42
- Adenpvirus
- Reovirus
- Not identified
Coxsackie
virus B Echovirus Other
73 27 9C
42 17 8b
58 8 8b
50 33 8C
42 . 25 8a
50 33 8C



                                    83

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and were found in 10% to 24% of the samples.  S. bovis and S. equinus
were recovered in 65% to 91% of the storm samples and represent 10.5
to 17.5% of the isolates.  The false positive and non-fecal streptococci
were found in all the storm samples and were 38.8% to 48.1% of the
isolates.

     The geometric mean densities of the selected pathogens and indicator
microorganisms for each storm sample site are given in Table 23 to pro-
vide an indication of the relative microbial quality of the storm runoff
at each location.  The levels of indicators, P. aerugi-nosa-and Staph,
ccureus are reported in the conventional units of MPN or number/100 ml
while the levels of enterovirus and Salmonella sp. are reported as
PFU/10 1 and MPN/10 1, respectively.  The levels of total coliform,
fecal coliform, fecal streptococci and enterococci suggest that the
runoff at each location was heavily contaminated and from a microbio-
logical standpoint was of poor quality.  The densities of indicator
microorganisms found in storm runoff were generally about 10-fold higher
than that found in urban streams and approached the indicator densities
of raw sewage.  The levels of indicators in storm runoff was several
orders of magnitude above that found in the reservoir samples.  Regard-
less of the indicator employed, the runoff from each of the sample
locations would be considered heavily contaminated.  With the exception
of Howard Park (H) and Northwood (M) the mean levels of indicators
are surprisingly similar.  The Howard Park sample, representative of
a true combined system, contained consistently higher levels of indicators
than the other stormwaters.  Northwood, a small drainage area of storm
runoff only, contained consistently lower levels of indicators than
from the remaining runoff sites.  The Jones Falls storm drain is known
to receive some raw sewage but the ratio of raw sewage to runoff is
believed to be small.  The Stoney Run (F) and Glen Avenue (G) drainage
areas contain bleeders from the sanitary sewers which occasionally
operate during heavy rain storms.  The Bush Street, drain (L), although
believed to carry only storm runoff, has a large low lying catchment
area with many possibilities for sewage contamination.

     The stormwater levels of enteroviruses, Salmonella sp., P. aerugi-nosa
and Staph. aureus were several fold higher than that found in the urban
streams but significantly lower than the levels in raw sewage.  P.
a&Tugi/nosa, was the most predominant pathogen in stormwater followed
by Staph. aureus, enteroviruses, and Salmonella sp.  Howard Park samples
from the combined sewer yielded considerably higher levels of pathogenic
microorganisms than the other sampling locations.

     Fairly reliable flow estimations were obtained at the time of
sampling for three stations; Stoney Run (F), the Jones Falls storm
drain (K), and the Northwood storm drain (M).  Flow estimation at the
remaining three stations were unreliable due to surcharging, loss of
a velocity meter and washouts.  Figure 30 shows the relationship of
instantaneous flows to the levels of fecal coliforms determined at
the time of sample collection for Stoney Run, the Jones Falls storm
drain and Northwood.  At low flows the range of levels of fecal coli-
forms was between 102 to almost 106/100 ml.  A similar range of fecal
coliform density was observed at moderate and high flows.  There appeared
to be little correlation between instantaneous discharge and the levels
of microorganisms.
                                    85

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     The effect of the number of days since the last storm on the
levels of fecal coliform is shown in Figure 31.  Similar to that observed
for the background samples, the levels of fecal coliform observed in
the storm runoff appears to be independent of the time between storms.

RELATIONSHIP BETWEEN PATHOGENS AND INDICATORS

     The relationship between indicator and pathogenic microorganisms
was evaluated in the following Figures 32 through 35.  The logarithm
of the density of the pathogen was compared to the logarithm of the
density of the indicator group of microorganisms for all samples.
The lower sensitivity limit of the pathogen determination is a function
of the volume of sample assayed.  In cases where no pathogen was recovered
the lower sensitivity limit was about one microorganism per unit volume.
For purposes of graphic representation and statistical analysis the
value of one was assigned  to these samples.  Since the logarithm of
one is zero, the minimum detectable concentration points were plotted
as zero logs in the graphs.  Lines of best fit for background and
storm samples were calculated by the method of }.east squares and presented
on each graph.  The correlation coefficients, r, were calculated for
four groups of samples; all, background, storm and stream.  These are
presented in the upper left hand corner  of each graph.  In addition
the 99% confidence intervals for each value of r are shown.  The linear
relationship between the levels of indicator and pathogenic microorganisms
was tested by  evaluating the null hypothesis for the correlation coef-
ficient  (r - 0).  The significance of r  was tested using the lower
limit of the 99%  confidence interval  for r.  Conclusions indicating
a linear relationship are  affected by the number of pairs of samples
and the magnitude of r.  The number  of pairs for sample groups  varied
slightly for each comparison of pathogens and  indicators.  The  numbers
of the pairings of pathogenic bacteria and various  indicators in the
analysis for all, background, storm  and  stream samples were  in  the
ranges 218  to  266, 119  to  136,  112 to 130 and  70 to  93, respectively.

     The relationships  between  the levels of Salmonella sp.  and total
coliform,  fecal coliform,  fecal streptococci and enterococci are shown
in Figures  32a, b,  c,  and  d,  respectively.  The levels  of  Salmonella
are presented  as  MPN/10 1  and the levels of  indicators  are presented
as MPN or  number/100  ml.   Thus,  it  should be recognized that the levels
of Salmonella *are 100-fold lower than the levels of indicator micro-
organisms.   The data show considerable variation.   However,  the positive
linear relationships  between indicators  and  Salmonella for all  the  samples
are  unmistakable.  The correlation coefficients for total coliform,
fecal coliform, fecal streptococci and enterococci were 0.54,  0.59,
 0.53 and 0.54, respectively,  and are significant  at the 1%^level.
There appears to be a reasonably good correlation between indicator
 and Salmonella for the overall data.  Although fecal coliforms  yielded
 the highest correlation coefficient, each of the indicator groups  tested
 gave good linear relationships to the levels of Salmonella.   The cor-
 relation coefficients for the background samples:   raw sewage,  urban
 streams and reservoir, showed somewhat higher correlation coefficients.
 The r values of 0.67 for total coliform, 0.72 for fecal coliform,  0.71
 for fecal streptococci and 0.73 for enterococci were also significant
 at the 1% level.  Again, little difference was observed for the linear
 relationships between the indicator groups and Salmonella.  However,

                                     88

-------
   10
    8

                                            6
               •-•^-a-
              48       12      16

              ANTECEDENT    DAYS
20
 Figure 31. The effect  of number of  days since the
           last rainstorm on the fecal coliform
density of stormwater at F, Stoney Run (A), G,
Western Run (a), H,  Howard Park (•), K, Jones
Tails (A), L, Bust St.  (o), and M, Northwood (•),
                       89

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 the  correlation between Salmonella  and  indicators  in the stormwater
 runoff was not so good.  The  low values of r were  0.49, 0.36, 0.19
 and  0.18 for total coliform,  fecal  coliform, fecal streptococci and
 enterococci, respectively.  Only the correlation between total coliform
 and  Salmonella was significant at the 1% level.  The remaining correlation
 coefficients were not significant at the 5% level  in the storm samples.
 The  samples collected from the three urban streams showed no correlation
 between the levels of Salmonella and indicator microorganisms.

      The relationship between the levels of P. aeruginosa and the
 levels of total coliform, fecal coliform, fecal streptococci and entero-
 cocci are shown in Figures 33a, b,  c and d, respectively.  The densities
 of P. aerugvnesa and indicator groups were reported in comparable units.
 Although P. aevuginosa is not an enteric pathogen it is associated
 with and commonly found in feces.  The r values for "all" and background
 samples are highly significant at the 1% level and were the highest
 observed for the data.  The total and fecal coliform appeared to cor-
 relate slightly better to the levels of P.  aerug-inosa than the strep-
 tococcal indicators.   For the storm samples,  the significant correlation
 was found at the 5% level for total and fecal coliforms.   No correlation
 was observed for fecal streptococci and enterococci.   P.  aeruginosa
 was found to correlate at the 99% significance level only to fecal coli-
 forms for the stream samples.

      The relationships between Staph.  aureus  and total coliform,  fecal
 coliform,  fecal streptococci and  enterococci  are shown in Figures  34a,
 b,  c, and d,  respectively.   The levels of both pathogen and indicators
 are given in comparable units.  Similar to  the data presented for
 Salmonella and  P.  aerug-Lnosa correlations were found  at the 1% signi-
 ficance  level for  "all" and  background samples.   The  values for  the
 correlation coefficient were significantly  higher for the latter samples.
 No  significant  correlation was observed  between Staph.  auveus and  the
 indicator microorganisms  for the  storm and  stream samples.

      Figures  35a,  b,  c,  and  d  show  the  correlation  between  the levels
 of  enteric  virus and  the  levels of  the total coliform,  fecal coliform,
 fecal streptococci and enterococci,  respectively.   The  number of samples
 assayed  for animal viruses was considerably lower than  those assayed
 for the bacterial  pathogens.   The numbers of sample pairs in the analysis
 in  each  sample  grouping ranged as follows:  94  to 123 for "all" samples,
 46  to 57 for background samples, 54  to 66 for  storm samples  and 28  to
 35  for stream samples.  The  95% confidence intervals were calculated
 for correlation coefficients.  In general, the  correlations  were
 poor  and significant  correlations at the 5% level were  only  found in
 the background  sample group for total coliform and  fecal coliform.
 Otherwise, no significant correlation between the levels of  enteric
 viruses and indicators was observed.

 INDICATOR RATIOS

      The relationship between the levels of the indicator groups of
microorganisms at each sample location is shown in  the following series
 of graphs.  The levels of total coliform, fecal streptococci and entero-
 cocci were compared to the levels of fecal coliforms.  Theoretical
                                    94

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ratios are indicated by solid and dashed lines on each plot.  The
scatter of the data relative to the theoretical ratios can be seen.
The ratios of total coliform to fecal colifonn (FC/TC) and fecal coli-
form to fecal streptococci (FC/FS) are often used to evaluate the pos-
sible source of contamination;  A FC/TC ratio of greater than 0.1 is
believed to be indicative of sewage.  A FC/FS ratio of 4.0 or greater
is believed to be indicative of human feces and a ratio of 1.0 or less
is believed to be indicative of animal feces.

     Figure 36a shows the relative levels of total coliform, fecal
streptococci and enterococci compared to the levels of fecal coliform
for raw sewage taken at Back River Wastewater Treatment Plant, sample
site A.  The raw sewage provides a point of comparison for the data
obtained for urban streams and storm runoff.  FC/TC ratios in raw
sewage lie between 0.01 to 1.0 with the. large majority of samples having
ratios of 0.1 to 1.0.  The FC/FS ratios observed for raw sewage lie
between 0.1 to 100 with 61% of the samples having ratios of 4.0 or
greater.  Similar relationships can be seen between enterococci to fecal
coliform (FC/Ent).  It should be noted that even in raw sewage a sizable
variation between the ratios of indicators was observed.

     The relationship between the levels of fecal coliforms and the
other bacterial indicators in the urban streams:  Herring Run, Jones
Falls and Gwynns Falls is shown in Figures 36b, c and d, respectively.
FC/TC ratios lie predominantly between 0.1 and 1.0 for Herring Run and
Jones Falls.  In the Gwynns Falls similar ratios predominate but more
samples were found to have FC/TC ratios less than 0.1.  Significant
variability in FC/FS ratios was observed for the urban streams.  About
20%, 42% and 22% of the samples had FC/FS ratios of 4.0 or greater in
Herring Run, Jones Falls and Gwynns Falls, respectively.  The frequency
of samples with FC/FS ratios of 1 or less was 46% in Herring Run, 27%
in Jones Falls and 33% in Gwynns Falls.  A large portion of the samples
in each of the urban streams had FC/FS ratios in the intermediate range
of between 1.0 and 4.0.  Similar results were obtained with enterococci
levels when compared to fecal coliform data.

     The relationships between the indicator groups of microorganisms
for the storm samples are shown by site in Figure 37a through 37f.
FC/TC ratios in the large majority of the samples collected at each
of the storm locations lie between 0.1 to 1.0.  FC/FS ratios found
in the storm samples are markedly different from those observed for
the background samples.  More than 90% of the samples collected at
Stoney Run  (Figure 37a), Glen Avenue  (Figure 37b), Bush Street  (Figure
37e) and Northwood Figure 37f) had FC/FS ratios less than 4.0.  Greater
than 80% of the samples from these stations had FC/FS ratios of 1.0
or less.  Only 18% and 12% of the samples collected at Howard Park
(Figure 37c) and the Jones Falls storm drain  (Figure 37d) had FC/FS
ratios of 4.0 or greater.  The frequency of samples of these stations
with FC/FS ratios of 1.0 or less was  41% for Howard Park and 76% for
the Jones Falls storm drain.  Ratios  of FC/Ent. appeared to shift up
slightly for the storm runoff samples compared to the FC/FS.  In each
case a larger percentage of samples had FC/Ent. ratios greater than
1.0.  Unfortunately, the significance of FC/Ent. ratios have not been
evaluated.
                                   107

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                     456789
                       LOG  FC/ 100 ml.

Figure 36a.  Back River raw  sewage,  site A.  Ratio of fecal coliform
             to total coliform, fecal streptococci, and enterococci.
                                108

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                1234567


                      LOG  FC/ 100 ml.



Figure 36b.  Herring Run, site B.  Ratio of fecal coliform to total

             coliform, fecal streptococci and enterococci.
                                109

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Figure 36c.
     I    234567


           LOG  FC/ 100 ml.




Jones Falls,  site C.   Ratio of fecal coliform to

total coliform,  fecal streptococci,  and  enterococci,
                              110

-------
Figure 36d.
             45678
           LOG  FC/IOOml.

Gwynns Falls, site D.   Ratio  of  fecal  coliform to total
coliform, fecal streptococci, and  enterococci.
                                Ill

-------
                  345678

                    LOG  FC/ 100 ml.


Figure 37a.  Stoney Run stormwater ,  site  F.   Ratio  of  fecal
             coliform to total coliform, fecal streptococci and
             enterococci.
                              112

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                 234567
                        LOG  FC/ 100 ml.

Figure 37b.   Glen Avenue  stormwater, site G.  Ratio of fecal coli-
             form to  total  coliform, fecal streptococci, and
             enterococci.
                               113

-------
                     345678

                        LOG  FC/ 100 ml.


Figure 37c.  Howard Park combined sewer,  site H.  Ratio of fecal
             coliform to total coliform,  fecal  streptococci, and
             enterococci.
                              114

-------
                      345678

                        LOG FC/IOO ml.
Figure 37d.  Jones Falls storrawater,  site K.   Ratio  of fecal coli-
             form to total coliform,  fecal streptococci, and
             enterococci.
                               115

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Figure 37e.  Bush Street storrawater, site L.  Ratio of fecal coli-
             form to total coliform, fecal streptococci and
             enterococci.
                               116

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Figure 37f.  Northwood stormwater,  site M.   Ratio  of  fecal  coliform

             to total coliform,  fecal streptococci, and  enterococci.
                                117

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                            DISCUSSION
 SAMPLING

      It  is  evident  from the  tables  and  graphs presented in the Results
 section  that  there  was  a wide variation in the levels of each of the
 selected microorganisms for  each of the samples collected at each of
 the  sampling  locations.  Differences of several orders of magnitude
 between  the minimum and maximum densities of each microbial group
 were observed.  This wide variation was not unexpected particularly
 in storm runoff.  Each  sample was a "grab" sample and reflects the
 microbial quality of the water at the time the sample was taken.
 Betson and  Buckingham (4) observed  differences of several orders of
 magnitude in  the levels of indicator bacteria over a span of two minutes.
 Factors  that  will influence  the levels  of microorganisms in each of
 the  water samples are numerous and  the  relationship between each of
 these factors are complex.   The magnitude and intensity of the rainfall
 within each catchment area and the  time, magnitude and intensity of
 the  antecedent storm are parameters associated with each storm event
 that will influence the levels of microorganisms.  The flow and the
 temporal discharge  characteristics  for  a given storm drain will vary
 with each storm event and be affected by many factors including the
 topography, land use, relative impervious areas, vegetation and type
 of soil  in  the drainage area.  In addition, the flow time to a given
 observation point,  even in a very small catchment, will influence
 the  magnitude of flow.   The  source  and  magnitude of the microbial
 flora in the  drainage area along with the relative volume of water
 conveyed will dramatically influence the levels of microorganisms found.
 The  drainage  area characteristics mentioned above along with the density
 of animal populations (including man) and general sanitary conditions
 in the area will influence the microbial quality of the runoff.   The
mean levels of total and fecal coliforms observed by Geldreich et al.
 (5)  in rainwater was less than 1/100 ml.  Betson and Buckingham (4)
 reported the  levels of  total and fecal  coliforms for various sampling
points within a well maintained residential area with sanitary sewers.
A portion of  their data  is reproduced in Table 24.  The rainwater
becomes  contaminated at  the earth's  surface and the degree of contam-
 ination varied dramatically with sampling location.   The waters col-
 lected from roof downspouts and foundations had lower levels of total
and  fecal coliforms than any of the  samples where the rainwater flowed
over  the land.  The wide variation  in the magnitude of the density of
microorganisms from various sources  coupled with the wide variation
 in the magnitude of flows from different types of drainage areas and
 the  infinite  permutations and combination of these factors provide some
insight  into  the nature of the wide variabilities in the densities
of microorganisms observed.

ENUMERATION OF PATHOGENIC MICROORGANISMS

     A major  objective of this investigation was to provide information
on the levels of selected pathogenic microorganisms in the urban aquatic
environment.  The recovery of pathogens from water has been recognized
to be heavily dependent on the methods,  techniques and procedures employed.

                                    118

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Table 24.  LEVELS OF TOTAL AND FECAL COLIFOSMS
   AT VARIOUS SITES WITHIN A DRAINAGE AREA.
   DATA TAKEN FROM BETSON AND BUCKINGHAM (4)
Sample site
Gage (Q = 0.3 cfs) (8.5 I/sec.)
Foundation and roof drain tile
Street gutter sample below litter pile
Roof downspout - no trees near house
Gage (Q = 1.4 cfs) (39.6 I/sec.)
Roof downspout - trees overhanging roof
Overland flow at curb
Overland flow and gutter flow
Gage (Q = 6.2 cfs) (175.4 I/sec.)
Gage (Q = 3.8 cfs) (107.5 I/sec.)
Gage (Q = 0.3 cfs) (8.5 I/sec.)
Culvert draining street
Roof downspout - trees overhanging
Foundation and roof drain tile
Street gutter near gage (West)
Gage (Q = 0.16 cfs) (4.5 I/sec.)
Time
1156
1207
1208
1224
1229
1234
1250
1258
1303
1323
1749
1753
1800
1802
1806
1807
MPN/100
Total
coliform
510,000
4,500
1,240,000
100
460,000
2,500
360,000
470,000
340,000
360,000
43,000
30,000
<10
1,800
26,000
58,000
ml
Fecal
coliform
7,300
<10
25,000
<10
27,000
740
21,400
58,000
44,000
26,000
8,400
800
<10
10
3,000
18,000
                          119

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 Salmonella

      Salmonella in water has been a major concern for the last 75
 years.  The majority of the information in the literature, however,
 has been qualitative in nature and data have been reported as frequency
 of isolation.  Table 25 shows the frequency of detection of Salmonella
 at different levels of fecal coliforms in the current study and a com-
 parison to similar data reported in the literature.   The levels of fecal
 coliforms in raw sewage exceeded 2,000/100 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 of 14 samples.   Only three samples taken
 from the urban streams had less than 200 fecal coliform/100 ml and
 Salmonella was found in each of these samples.  At fecal coliform den-
 sities of 201 to 2,000 and greater than 2,000/100 ml the frequency
 of isolation of Salmonella was 91 and 96%, respectively.  The storm
 runoff samples had only one sample with less than 200 fecal coliforms/
 100 ml and 12 samples with between 201 to 2,000  fecal coliforms/100 ml.
 Salmonella was recovered in the low fecal coliform level sample and in
 83% of the latter samples.   Storm samples with fecal coliform levels
 greater than 2,000/100 ml were 95% positive for  Salmonella.   The overall
 Salmonella isolation was 28%,  89% and 96% for the samples with 0-200,
 201-2,000 and greater than 2,000 fecal coliforms/100 ml, respectively.
 The frequency of Salmonella isolation for all the samples compares
 favorably with the fresh water data reported by  Geldreich and Van Donsel
 (44)  but differs significantly from the estuary  data reported by these
 authors and Brezenski and Russomanno (45).

      Recently,  limited data on the levels of Salmonella  in water have
 been reported.   Table 26 shows a comparison of the range in  the density
 of Salmonella reported for  various types  of surface  water to the infor-
 mation obtained in the Johns Hopkins study.   Each of the procedures
 for the enumeration of Salmonella was multiple dilution  technique
 with a MPN estimate of density.   All the  data, in Table 26 were corrected
 to MPN/10 1 for comparison.  Enrichment,  plating media and recovery
 techniques differed from laboratory to laboratory.   The  levels of
 Salmonella in sewage varied from less than 300 to 1,100,000  Salmonella!
 10 1.   The ranges  observed  in  the Baltimore sewage are much -lower
 than that reported by Cheng et al.  (46) but  similar  in orders  of  magnitude
 to the reports  of  Kampelmacher and van Noorle  Jansen (47), Phirke (48),
 and Kenner and  Clark (19).   The ranges  in  the  levels  of  Salmonella in
 Baltimore sewage significantly overlap  the  ranges reported by  the latter
workers.   The urban streams  in Baltimore appear  to contain lower  levels
 of Salmonella than the  river and  creek  samples collected  by  Kenner and
 Clark  (19).   The levels  of  Salmonella in storm runoff reported by
 these  authors were similar  to  the  levels observed in Baltimore..   Geldreich
 et al.  (5)  reported  4,500 Salmonella/'100 ml  (450,000/10  1) in  one  storm
 sample  from a business  district but was unable to isolate  Salmonella
 from other  storm samples.

     An important  aspect of the current study has been the inclusion
of  seeded  Salmonella controls  to evaluate the steps in the recovery
of  Salmonella.  Table 27 shows  the frequency of recovery of the seeded
Salmonella for each  sample station.  Three distinct controls were per-
formed to evaluate the recovery procedure.  A streptomycin resistant

                                    120

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          Table 25.  COMPARISON OF THE FREQUENCY OF DETECTION
           OF SALMONELLA WITH THE LEVELS OF FECAL COLIFORMS*
Fecal Coliform

Report
This study











Geldreich &
Van Dons el (44)


Brezenski &
Russomanno (45 )

Sample
raw
sewage
urban
streams

upland
reservoir

storm
runoff

overall
total

fresh
water
estuary

estuary

Range
MPN/ 100ml
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
14
0
0
1
12
123
18
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
7

100
83
95
28
89
96
27
70
98
13
44
60
18
30
59
*The frequency of detection of Salmonella with  alternative fecal coliform
 ranges can be seen in Appendix F.
                                     121

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         Table 26.  COMPARISON  OF THE LEVELS  OF SALMONELLA
                  FOUND  IN  SURFACE WATER AND  SEWAGE
# of
Sample samples
Sewage
Sewage
Sewage
Sewage
Sewage
River
Creek
Urban streams
3 C
3
8
17
15
34
8
2
94
Range of
Salmonella
MPN/10 liters
20,000
Reference

Kampelmacher and Van Noorle
Jansen (47), 1970
11,000 to 1,100,000 Cheng &t al. (46), 1971
700 to 25,000
<300 to 150,000
3 to >27,000
150 to >30,000
450 to 1,200
<0.9 to 320
Phirke (48), 1974
Kenner and Clark (19),
This study
Kenner and Clark (19),
Kenner and Clark (19) ,
This study

1974

1974
1974

Storm runoff     2

Storm runoff   140
20 to 150        Kenner and Clark (19), 1974

<0.9 to >11,000  This study
                                  122

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  Table 27.   FREQUENCY OF RECOVERY OF .SEEDED SALMONELLA AFTER
     EXPOSURE TO THE SAMPLE, CONCENTRATION ON DIATOMACEOUS
          EARTH,  AND .ENRICHMENT'AND  PRIMARY PLATING
% of the samples positive
Enrichment with
Sample station Exposure a Concentration primary plating
Background
Raw sewage
Herring Run
Jones Falls
Gwynns Falls
Loch Raven
Storm drain
Stoney Run
Glen Ave.
Howard Park
Jones Falls
Bush Street
Northwood
All samples

100
100
100
100
100
100
100
95
95
100
100
99

100
88
88
82
100
79
93
100
79
86
67
87

0
67
17
33
83
'& 5 £ *
0
= '•'?".. I 5 j 1
25
0
0
50
25
30
a Recovery of the seeded Salmonella, after exposure to the sample was
  considered positive if less than 90% inactivation was observed.
                                123

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  strain of  Salmonella typhimur-iim was  used as  the  test  organism.   Sample
  toxicity to  the  seeded  Salmonella was evaluated by  exposure  of the
  Salmonella seed  to  each sample  for  the length of  time  necessary to
  process the  sample  in the  laboratory.   The recovery of the seeded Sal-
  monella after exposure  to  the sample  was  considered positive if less
  than 90% inactivation was  observed.   The  concentration with  diatomaceous
  earth  for  low levels  of seeded  Salmonella was  evaluated by the recovery
  of streptomycin  resistant  Salmonella  from seeded  replicates  of each
  sample after filtration.   Streptomycin was included in the enrichment
  medium to  minimize  the  effect of  other microorganisms  and favor growth
  of the seeded Salmonella.  The  overall recovery of  seeded Salmonella
 was evaluated by incorporating  an additional seeded replicate and testing
  the Salmonella isolated  after enrichment  and primary plating (no strep-
  tomycin) for streptomycin  resistance.  Recovery of  streptomycin resistant
 batmoneL-la was considered  positive.

      Only  two storm samples were  found to  cause more than 90% inacti-
 vation of  the seeded Salmonella.  The  test organism was consistently
 recovered after exposure to the water  samples from the different sources
 and indicated that the water samples were not bactericidal to the seeded
 Saimonella.  After concentration on diatomaceous earth, the seed Sal-
 monella was recovered in 67% to 100% of the samples  depending on sample
 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 decreased 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  better  recoveries.

     The Salmonella  seeding experiments conducted  simultaneously with
 replicates  of the background and storm runoff  samples provided useful
 information to evaluate  the Salmonella data presented and  to  point out
 problem areas in  the recovery procedures.   Essentially  no  acute bacteri-
 cidal effect  was  observed for the aquatic  samples.   The concentration
 of low  levels of  Salmonella on diatomaceous earth  appears  to  be a viable
 method.   The  culturing methods,  once the bacteria  are concentrated,
 however,  have certain  limitations.   As a result, the levels of Salmonella
 reported in this  and probably other  studies, are under  estimates of the
 actual  levels.  This underscores the need  for  the  development of more
 efficient enrichment media.

     In sewage there was  a  noticeable  seasonal variation in the levels
 of Salmonella with a peak density  of 27,000/10 1 in  the late  summer.
 The comparison of the  levels  of  Salmonella in sewage with the incidence
 of salmonellosis  in  Baltimore City is  shown in Figure 38.  Note the parallel
 in peaks in late  summer  for Salmonella  density and incidence of the
disease.

Enteric Viruses

     Samples for the assay  of enteric viruses were shipped by bus from
Baltimore, Maryland,  generally immediately after collection, to  Syracuse,

                                    124

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  5





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 IO
 L06 SALMONELLA/10 liters
 NO. SALMONELLOSIS CASES IN  WEEK
    I.
                                                         5S=SS?g^^i
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 New York.   Concentration procedures were initiated as soon as possible.
 Trivalent  oral poliovaccine thermal controls were included in each
 sample to  evaluate the eight to ten hour transit time.   Irregular re-
 coveries and noticeable losses were observed in some of the early samples.
 The initial control losses were due to inadequate aeration of the control
 samples to remove the chloroform included as a preservative.   Traces
 of chloroform noticeably reduced the plating efficiency.   After correcting
 the problem, average recoveries of 84% to 96% of the expected  values  were
 observed and suggested that the shipping procedure had  a minimal effect
 on the viruses.

      In a  large  sampling study such as this, unusual observations occur
 which need further study or some special attention.   For example, samples
 D8 and D15 (Appendix C.   Station D,  Gwynns Falls,  run no.  9 and no.  15)
 appeared to have extraordinarily high titers and a broad spectrum of viruses.
 It is possible that samples were obtained when "hot samples were in  the
 pipe" or that solids with adsorbed viruses had settled  near the sampling
 point and  were leaking viruses.   Nevertheless, the distribution of viruses
 among nearest neighbors  showed that the titers were most probably due
 to condensation  on lids  of the Microtest II plates which spread the  inoculum
 from low dilutions to high ones.   The above two samples were  omitted
 from the subsequent analysis.

      This  condensation problem was eliminated by carrying  out  the inoc-
 ulations in a laminar flow hood,  placing the lidless plates on sterile
 cafeteria  trays  and covering the  first tray with an inverted  second
 tray.   The plates  were then incubated in a humidified,  five percent
 C02 atmosphere.

      The level of  viruses in raw  sewage provides  a reasonable  means
 of comparing  the virus data to other  reports.   Although the physical,
 chemical and  biological  characteristics of  raw sewage are  quite variable,
 the raw sewage samples were more  similar than were stream  and  storm
 samples  which were highly dependent upon local conditions.  Sigh  levels
 of viruses were  obtained in most  sewage samples.   These were principally
 poliovirus and Coxsackievirus  B.   There were no clear trends toward  a
 seasonal productivity of either type  of enteroviruses.  Roughly equal
 numbers  of Coxsackie and polio isolations were made.  It is not clear
 from these data  whether  it Was the output of a generally infected popu-
 lation or  the yield from a small  number of very productive  shedders.
 The levels of enteroviruses observed  here were of  the same  order  of
 magnitude  of  sewage enteroviruses as  reported  by Dahling et al.  (36)
 and by Kalter and Millstein (49) .  Metcalf eb  ail.  (50)  calculated the
 numbers  of enteric  viruses  discharged  from two activated sludge type
 waste treatment  plants in Houston.  The largest number of virus isolates
were polioviruses.   In the  Houston sewage effluents, however, echoviruses
were nearly as frequent  as  polioviruses  and  Coxsackievirus B isolations
were very much in the minority.   These  ratios  undoubtedly fluctuate nor-
mally and  individual  types  rise and decline  frequently.   Sabin polio
 strains are artificially maintained by  continuing immuinzation programs.

     The use  of  the presumptive analysis allowed rapid identification
 of  about two-thirds of the  samples; the other  third  (42/130) required
 a  confirmed test for single plaques from positive isolates.  The value
of  the confirmed test is shown by  the fact that of the 42 presumptive

                                     126

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guesses, only 12 were correct.  The other 30 guesses failed to predict
some types or indicated others which could not be verified.

     In the presumptive test polioviruses are suppressed with IgG and
Coxsackie B viruses with the nonpermissive HEL cells.  Ostensibly this
should increase the number of adenovirus isolations.  However, only five
samples were found to have DNA viruses with typical adenovirus morphology.
Certainly this must be low and one can conclude that the screening method
and/or the concentrating techniques have failed to identify the wild
adenoviruses.  A partial explanation of this failure may be the pH at
which the adsorption-filtration was conducted (pH 4.5).  Fields and
Metcalf (43) reported that at pH 4.7, 31% of adenovirus 5 was inactivated,
and at pH 3.5, 39% was lost.  The three reovirus isolates probably in-
dicate that the wild reoviruses also underwent a similar degradation
at pH 4.5 (50).

     Plaque forming units were almost always lower than predicted by
TCID5Q titrations in presumptive tests.  This was not unexpected since
the TCID50 was based on four wells per dilution and thus provides a
large degree of error.  The presence of agar and neutral red appears
to suppress plaque formation during primary isolations of many groups
other than enteroviruses.                                  .-£•:£-.».*,•'.•:  >.>

     Virus isolations were made from the majority of samples :ffom''the
urban streams  (Appendix C) during dry weather flows.  The TClDsQ • titers
were at minimal levels for detection by this method.  Since the 'samples
could not be diluted very far, difficulty with toxic residues was'''exper-
ienced in many samples and this may, in part, explain the instances
where no viruses were recovered.  The distribution of virus types re-
sembled the pattern seen with sewage from the Back River Treatment
Plant.

     Six stormwater flows were examined.  Viruses were isolated from
all of them.  For the most part there were few perceptible differences
from the urban streams in the titers, the distribution of types, or in
the number of  isolates without viruses.  The principal difference between
them is in the fact that the high "bursts" of stormwater viruses were
more frequent  than in urban streams and greater in amplitude.  It suggests
that storms may have actually flushed sewage solids without large
dilution effects.

     Loch Raven reservoir, site E, was included as background infor-
mation  on a relatively uncontaminated natural water.  Enteroviruses
were found in  three of seven  samples.  An echovirus  of unknown serotype
and poliovirus I with vaccine strain genetic markers  (d~T~) were identi-
fied.  However, the titers were low and the possibility of a  laboratory
contamination  could explain the occurrence.  However, the  finding of
virus in the raw water for Baltimore City is not unusual.  For example,
Foliguet et al.  (51) detected virus in 81% of the raw water in France.
Chang  (52) suggested that 30 PFU/1 could be expected in moderately
polluted raw water sources.   In this regard, the highest virus concen-
tration in the Loch Raven reservoir was 13 PFU/1 with a mean  of all
samples throughout the study  of only 6 PFU/1.
                                    127

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      Since  the  examination of  samples  for enterovirus was less exten-
 sive  than for other pathogens,  it must be acknowledged that the presence
 of  trace levels of virus  in  the clean  waters of the Loch Raven reser-
 voir  deserve some discussion.   The question arises how was it possible
 to  isolate  only one confirmed  Salmonella throughout the study, but
 on  three separate occassions enteroviruses were found?  While it is
 possible for raw sewage to enter the reservoir through the failure of
 a sewage pumping station  on  the metropolitan sanitary district, no such
 failure was known to have occurred.  Furthermore, the normal indicator
 of  pollution gave no suggestion of an  unusual entrance of sewage.  The
 mean  total  coliform concentration of Loch Raven during the study was
 a MPN 26/100 ml.

      Rather than to postulate  an explanation such as differential decay
 rates between viruses and bacteria in  several months of storage in the
 reservoir,  it would be useful  to look  more closely at the virus recovery
 techniques  for  opportunities for false negatives and positives.  This
 is  supported by the fact  that  cleaner  waters produced almost as many
 positive virus  isolations as the sewage polluted waters.  Table 28
 summarizes  the  recovery of viruses for dirty to clean waters.

      To .suggest that the  Loch  Raven waters contain one virus particle/
 1-771 does  not  seem reasonable in view of the fact that only one
 Salmonella/130  1 was isolated.  The ratio of virus to Salmonella in
 raw sewage  was  2 to 1 or  1 virus/10 ml and 1 Salmonella I'19 ml which
 seems more  reasonable than the 70 to 1 in Loch Raven.  It is also difficult
 to  accept the findings that 100% of the samples contained viruses at
 Northwood compared to only 87%  in raw  sewage.  Any conclusions reached
 from  these  data should be tentative and be reserved for future confir-
 mation.

Pseudomonas aeyuginosa

     No serious  difficulties were encountered in the enumeration of
P.  aemginosa.   It should be emphasized that the calculations of the
MPN were based  on a series of biochemical characteristics and not solely
 on  the growth in asparagine broth and  confirmation on acetamide.  The
 levels of P. aeruginosa observed in sewage and streams were of the same
 order of magnitude as those reported by Levin and Cabelli (30) for
 sewage and  Cabelli et al.  (53)  in fresh water.  The densities in storm-
water were  generally 10-fold higher than the urban streams and often
 approached and sometimes exceeded the levels of indicator microorganisms.
P. aeruginosa was the most predominant and ubiquitous pathogen observed
 in  this study.   It was found in all sewage and storm samples, in nearly
all stream  samples, and in 62%  of the  samples from Loch Raven reservoir.

Staphylococcus aweus

      Serious problems were observed in the enumeration of Staph.  aureus.
The conventional media recommended for this pathogen do indeed support
its growth with a particular colony morphology but are not sufficiently
selective for use in water.  Many microorganisms not commonly encountered
at relatively high densities in clinical samples will mask the presence
of Staph.  aureus in water samples.
                                    128

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Table 28.  RECOVERY AND LEVEL OF ENTERIC VIRUSES WITH
   RESPECT TO THE MEAN LEVELS OF TOTAL COLIFORMS
Virus isolation
Site frequency, %
Raw sewage
Howard Park
(stormwater)
Jones Falls Drain
(stormwater)
Bush Street
(stormwater)
Northwood"--— ---— >*«—
(stormwater)

Western Run
(stormwater)
Jones Falls
(stream)
Gwynns Falls
(stream)
Stoney Run
(stormwater)
Herring Run
(stream)
Loch Raven
(reservoir)
87

100

75

42
..wr.-™*^^*..^^.™^,-, .„,-*-——-<.
100


82

84

67

91
73

43
Virus density
Coliform index
per 100 ml TC/virus ml/virus
2.3 x 107 2.2 x 106 10

1.1 x 106 7.1 x 105 64

4.3 x 105 1.3 x 106 300

3.5 x 105 1.0 x 106 285
„ 	 ,-„.-, .. ,,,.,,--- »«..---~~r— ^— ^— ~ 	 —- • • »-. -* '"•— "•' '
3.4 x 105 6.6 x'105--^itl95
- - - '• i~ * • ; i -
,;.,'. , . :
1.6 x 106 8.0 x 105'. -.-• ;;50^
•'•"••-.;.:. .•;•:•; ; ; •.
4.0 x I0k 5.5 x Wk 138
I C " .*
4.0 x 10^ 2.9 x 105 726
1 It
3.1 x 104 8.5 x 10^ 273
4.9 x 103 1.7 x lO4 345

2.6 x 101 4.6 x 102 1,770
                            129

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      The levels of Staph. am>eus presented in this study were MPN members
 estimates based on the demonstration of coagulase positive staphylococci.
 Staph. aureus is not found in large numbers in water except in swimming
 pools (54).  The highest levels of Staph. auveus were observed in raw
 sewage presumably from the human intestinal tracts, and the bath and
 wash waters.  Staph. aureus was also observed in the majority of stream
 samples at low levels.  However, the storm samples contained significantly
 higher levels of Staph. auveus than the urban streams.  With the exception
 of the Northwood station (site M), this pathogen was recovered from 96%
 or more of the storm runoff samples.

 DISTRIBUTION OF FECAL STREPTOCOCCI

      Considerable effort was directed toward enumerating the members
 of the fecal streptococcal group to provide an insight into the use-
 fulness of this indicator in storm runoff.  According to Standard Methods
 (16)  the fecal streptococci are considered synonymous'with "Lancefield's
 Group D Streptococcus" and include S.  faecalis,  S.  faeaium, S.  durans
 and their varieties or biotypes, S.  bovis and S.  equinus.   S.  durans
 in this study was considered a variety of S.  faeaium.   The more restrictive
 term enterococcus refers to all of the above species except S.  bovis
 and S.  equinus.   These two species of streptococci  are believed to be
 indicative of animal feces and dieaway rapidly in the aquatic  environment.
 The l-utuefaeiens and zymogenes biotypes of S.  faecalis and atypical
 S.  faecalis are believed to be ubiquitous and have  little  sanitary
 significance (31).   In the heavily contaminated  urban aquatic  environment
 S.  faecalis var.  liquefaciens and zymogenes were found .in  a significant
 percentage (30%  to  73.5%)  of thesamples and support the contention
 of  their relative ubiquity.   However,  these varieties  were "never  observed
 to  be predominant fecal streptococci in either background  or routine
 samples.   Only an average  of 1.3 to  9.8% of the  isolates tested belonged
 to  this  subgroup.   Atypical S.  faecalis was observed as  less than 1.6%
 of  the isolates  in  from 9%  to 23% of the samples.

      The percentage of  isolates and  the frequency of isolation  of S.
 bovis and S.  equinus differed from stream and  storm runoff samples.
 Approximately 8%  of the isolates obtained  from the  urban streams  were
 S.  bovis and  S. equinus and were found  in 48 to  72% of the samples.
 The isolates  from four  of the six storm sites  had 15 to  17% S.  bovis
 and S. equinus in 66  to 91%  of  the samples.  The  remaining two  sites
 were  just slightly  higher in  both percent  of isolates  and  frequency
 of  isolation  than the stream  samples.   The higher levels of S.  bovis
 and S. equinus in the storm  sample suggests a  stronger influence  of
 animal feces  on the microbial quality.

      Enterococci were the predominant group in the  fecal streptococci
 isolates.  However, high levels  of false positive, not fecal streptococci
 isolates, were observed in all  the samples.  Slightly more  than 35% of
 the isolates  from background  samples belonged to this group.  A noticeable
 increase  (35% to 42%) in the number of  false positive non-fecal strep-
 tococci were  found in the storm samples.

      The  occurrence of false positive non-fecal streptococci on KF
medium has been reported.  Pavlova et al.  (32) found 18.6% of the isolates
                                    130

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obtained from food, sewage, and feces to be non-fecal streptococci.
Facklam and Moody (55) indicated that many kinds of streptococci can
grow and give the appropriate reactions on KF medium.  Mossel (56)
showed that KF support the growth of Staph. aweeus and Raibaud et al.
(57) reported overgrowth from Lacto'baG'i'LI'i.

     The ratio of indicator microorganisms has been employed to provide
some insight into the source of microbial contamination.  A firm FC/TC
ratio has been difficult to establish  (58).  The FC/FS ratio, however,
has been utilized more frequently (31, 59, 60, 61) to determine whether
the pollution was of human or animal origin.  The basis for the FC/FS
ratio can be found in the early literature  (62, 63, 64).  More recent
studies demonstrated that the fecal streptococci were present in
greater numbers than coliform bacteria in the feces of animals  (5, 65,
66, 67, 68, 69).   In human feces, however, fecal coliforms were found
in  greater numbers than fecal streptococci.  Geldreich and Kenner  (31)
reported that FC/FS ratios for human feces and wastewater were  greater
than 4.0 and for animal feces, separate  stormwater systems and  farm
drainage are less  than 0.7.  The ratios  between 0.7 and 4.0 are diffi-
cult to interpret.  The authors also suggested that  the FC/FS ratios
should be  applied  carefully  and that the ratios are most meaningful
when the microbial density data are collected at 'outfalls  into  the stream.
Upon entering  the  stream  the levels of each of the microorganisms may,
be  affected by numerous environmental  factors and differentialmicrpbial
die-away.   They also  concluded  that the value of  the FC/FS ratio  for:
stream samples would  only be useful during the  initial 24  hours;df ; ;;
downstream travel  from the point  of discharge.   Table 29  shows  the :
 frequency of  occurrence of FC/FS  ratios at the  sample stations  in^
 Baltimore.  The majority  of the samples from raw sewage had a FC/FS
ratio  of  greater than 4.0.  However,  12% of the samples had a FC/FS
 ratio of  less than 1.0.   The distribution of the FC/FS ratio in the
 urban streams was difficult to interpret.   A large percentage of samples
 lie in the grey area between animal and human contamination.  If FC/*b
 ratio above 1.0 is considered to be of human origin, then in 54%, 734,
 and 77% of the samples for each of the streams there was a suggestion
 of the presence of significant levels of contamination with human fecal
 material.  The FC/FS ratios in the large majority of the storm samples
 were less than 1.0.  At the stations  representative of combined sanitary
 sewage and storm runoff, only 18% and 12% of the samples had FC/FS
 ratios greater than 4.0.  In fact, 41%  and 76% of these samples indicated
 animal contamination.  In a large portion of storm samples the presence
 of human contamination was masked.

      The data obtained in the stream  and  storm samples emphasize  the
 difficulties with the application  of  FC/FS ratio.  The flow times from
 the points of contamination to the points of observation  for the urban_
 streams in this study are well within the  24 hours  suggested by  Geldreich
 and Kenner (31).  This was  also the case  for the storm drains, although
 it was impossible to  evaluate the  time between the deposition  of animal
 feces and their incorporation into the storm runoff.   Interpretation
 of the FC/FS  ratio less  than  4.0  leave much to be desired.  Too  many
 factors will  influence the  densities  of fecal coliform and  fecal strep-
 tococci.   The magnitude  of  these  densities along with the volume of water
 which carries the contamination,  combined with  the  numerous  environmental
  factors  that  will affect the  levels of these microorganisms, make the

                                     131

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               Table 29.  FREQUENCY OF OCCURRENCE OF THE
                  FC/FS RATIO AT EACH SAMPLE STATION
     Sample station
Less than
   1.0
                                           Ratio FC/FS
1.0 to 4.0
Greater than
    4.0
     Background Samples

 A  Raw sewage                 12

 B  Herring Run
      (stream)                 46

 C  Jones  Falls
      (stream)                 27

 D  Gwynns Falls
      (stream)                 33

 E  Loch Raven reservoir       ID
                  27


                  34


                  31


                  45

                  ID
                    61


                    20


                    42


                    22

                    ID
       Storm Samples

F   Stoney Run
      (storm runoff)            82

G   Glen Avenue
      (storm runoff)            89

H   Howard Park
      (combined sewage)         41

K   Jones Falls storm drain
      (combined sewage)         76

L   Bush Street
      (storm runoff)            81

M  Northwood
      (storm runoff)            92
                  12
                  41
                  12
                  13
                    18
                   12
                                  132

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useful application of the FC/FS ratio  difficult in the urban environment.
The determination of these microorganisms and the calculation of the
FC/FS ratio for a storm outfall or a stream must be recognized to be
the net result of an innumerable number of contamination events of
variable sources and magnitudes and the effects of many different localized
environmental conditions that alter the microbial populations.  Effectively,
the presence of human contamination may be obscured.  The FC/FS ratio
was not intended nor should it be employed as a "magic number" to
evaluate the source of contamination in a complex system.

RELATIONSHIP BETWEEN INDICATOR AND PATHOGENIC MICROORGANISMS

     The evaluation of the naturally occurring relationships between
indicator and pathogenic microorganisms in the urban aquatic environ-
ment was an important objective of the present study.  The correlations
for the levels of total coliform, fecal coliform, fecal  streptococci
and enterococci and bacterial pathogens were highly significant at
the 1% level when all of the samples and the background  samples were
considered.  However, little or no correlations were found between
indicator and pathogenic bacteria in the storm and stream samples.
In each case where significant correlations were observed, large numbers
of samples, raw sewage and reservoir samples were considered. — -The
raw sewage samples contained relatively high levels and  the reservoir
samples contained very low levels of indicator and pathogenic  bacteria.
The latter two samples play a large role in defining the regression
line.  Despite the variability observed, the levels of microorganisms
at a  given sample station were surprisingly similar.  A  large  majority
of the samples were  found in a one to  two  log range.   It should be
stressed  that most  of  the microbial  assays were MPN estimates  of  the
bacterial densities.   The 95%  confidence  interval  for  a  five  place
multiple  tube  procedure  span almost  a  factor of ten around the MPN.
Whether the  lack  of  correlation  between indicator  and  pathogen simply
reflects  the poor precision  for  the MPN or that  the indicators are
 less  meaningful  in  these samples remains  to be determined.

      Significant  correlations  at the 95% level were observed  between
 the levels  of  enteric viruses  and total coliform and  fecal  coliform
 in the background samples.   No significant correlation was  observed
when all  the samples,  or the storm and stream samples  were  considered
 separately.   Negative correlations,  although not .significant, were
 often observed for the storm samples.   The uniformly  poor bacterial
 indicator-virus correlation may be explained by 1)  the smaller number
 of samples assayed,  2)   the different response between virus and bacteria
 to the environment,  3)   the difference in survival,  and  4)   the
 difficulties associated with concentration,  recovery and assay of low
 levels of virus.

      Despite the poor correlations between indicator and pathogens
 observed for several sample categories, the overall ratio of pathogen
 to indicator provides a useful tool to evaluate the relative order of
 magnitude of the presence of these microorganisms.  The ratio of pathogen
 to indicator varied considerably from sewage to clean water.  The  overall
 ratios obtained in this study are given in Table 30.   P. aeruginosa
 was  so abundant, either in fact or through multiplication in the field,
                                     133

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Table 30.  OVERALL RATIO OF PATHOGENS
     TO INDICATOR MICROORGANISMS
Microorganisms
P. a&ruginosa
P. aerug-i-nosa
P. a&puginosa
P. a&Tuginosa
Staph. awceus
Staph. aweus
Staph. awceus
Staph. aureus
Salmonella
Salmonella
Salmonella
Salmonella
Enteric virus
Enteric virus
Enteric virus
Enteric virus
to TC
to FC
to FS
to ENT.
to TC
to FC
to FS
to ENT.
to TC
to FC
to FS
to ENT.
to TC
to FC
to FS
to ENT.
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
Ratio
: 45
: 14
: 18
: 5
: 4,780
: 1,410
: 2,000
: 630
: 141,000
: 105,000
: 147,000
: 45,500
: 151,000
: 50,000
: 85,500
: 40,700
                134

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that the levels of common indicators of pollution were only an order of
magnitude greater than this pathogen.  Staph. aweeus* however, was less
abundant and Salmonella sp. and enteric virus almost rare compared to
the indicator microorganism.

QUALITY OF URBAN SURFACE WATERS

     It has been shown from results of the study that the water quality
of the urban streams based on detailed examination of the levels of
pathogens and indicators of pollution was uniformly poor.  The recovery
of pathogenic bacteria and viruses was accomplished in almost every
sample examined throughout the period of study.  Only three samples out
of 92 taken from the urban streams met the 200 fecal coliform MPN/100 ml
for recreational water use  (National Technical Advisory Council Standard,
NTAC) (70).  The mean fecal coliform density was, in fact, in the order
of 6,000/100 ml with a range of 200 to 2.4 million MPN/100 ml, and this
certainly is not an acceptable quality for water contact recreation.
The levels of microorganisms in the urban streams was independent of
season, flow and the number of days since the last rainfall.  This
apparent independence, relative to factors that have been repeatedly
demonstrated to have an effect on the microbial quality, was not surprising.
It has been long recognized that the seaspnal variation and effect of
rainfall on the bacterial  quality of surface waters  are dependent on
the overall condition of the stream.  Kisskalt  (71)  in 1906 compared
the seasonal fluctuations  in the levels of bacteria  in a good,,quality
and a highly polluted stream.  In the clean  stream the levels "of bacteria
were highest during periods of rain  or high water.   In the highly polluted
stream the levels  of bacteria_were hJLgher^ durmg per^dj_^f^J:p^,fj£W.	
Frost ^nd''street^r"T7"2) "'in 1924 reported little seasonal fluctuations
in the level of bacteria in the Ohio River below Cincinnati.  They
concluded that the normal  fluctuations in the bacterial  count were masked
by the effect  of  the pollution from  the city.  The consistently high
levels of indicator microorganisms,  routine  recovery of  pathogenic
bacteria  and enteric viruses, and the  absence  of normal  fluctuation
levels of microorganisms  suggest a high level  of continuous pollution.
Although  the identification of the  sources  of  contamination within an
urban environment were  well beyond  the scope of  the  present investigation,
 it is difficult  to conceive of any  other  source but  raw  sewage  that
would possess  the necessary magnitude  of microbial  content and  continuous
presence  to yield the  observed data  for  the urban  streams.

 HEALTH HAZARD

      It  has  been hypothesized that  an urban area supplied with separate
 sewer systems  for stormwater  and for sanitary  sewage will have cleaner,
 less hazardous,  urban streams than those with  combined systems.   There
 is no doubt  that the hypothesis  is  supported where properly constructed
 separate sewer systems are provided in new towns and subdivisions where
 none of the defects of age, overloading and poor maintenance has appeared.

      The area for study is believed to represent a rather typical old,
 central city with a growing suburban belt,  a situation shared by metro-
 politan areas throughout the United States.  The study area,  unlike
 many along the Eastern seaboard, is primarily one of separate sewers
                                     135

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 and  open channels  for  the  convenience  of  storm runoff  through existing
 and  proposed  recreational  areas.   The  population's contact with the urban
 streams  is heavy and will  increase as  playgrounds and  parks are extended
 in the future.  An important  aspect of assessing the public health
 threat from contact with these urban streams  is the density of pathogens
 recovered as  was summarized in Tables  19  and  23.

     Pseudomonas aerug-Lnosa was the most  abundant pathogen in all the
 streams.  The mean MPN/100 ml was  1 x  103 with a range from 3 x 10°
 to 3.5 x 10 .  This secondary pathogen is of  interest  because of its
 association with eye and ear  infections,  its  resistance to antibiotics
 and  proclivity for invading individuals in debilitated states.  Infections
 with P.  aevug-inosa are the most dreaded,  second only to antibiotic
 resistant strains  of streptococcus.  The  organism is rather ubiquitous
 and  is known  to be discharged in the feces.   Among other organisms
 proposed as possible indicators of recreational water  quality, P.
 aeruginosa has the advantage  of allegedly being of human rather than
 of animal origin (73).  The city sewage had relatively large numbers
 of P. aevuginosa averaging 2.2 x 105/100 ml as compared to an average
 of 7.0 x 106  fecal coliform/100 ml (1:32).  However, there were numerous
 samples  which gave a P. qeTug-Lnosa MPN equal  to or in  excess of the
 fecal coliform MPN.  This  had be'en found  in other studies and were
 attributed to possible multiplication  under natural conditions.  This
 may  be an explanation  for  the peak numbers of P. aevugi-nosa at all
 study sites during the late summer months when water temperatures were
 in the 18-26?C range and stream flows  were low.  Should this, in fact,
 be the characteristics of  this pathogen,  its  usefullness as a recreational
 water quality indicator is limited.  The least square  fit of fresh and
 estuarine water data reported by Cabelli et at. (53) gave 12 P. aevu-
 ginosa MPN/100 ml  at the National  Technical Advisory Council suggested
 standard of 200 FC/100 ml, whereas,  the stormwater in  this study gave
 63/100 ml.  In both instances the  data were quite variable.  The inability
 of past  studies to  show any relationship between levels of P. aevugi-nosa
 in bathing waters  and  ear  infections weakens  the public health concern
 for  the  abundance  of the organism  in urban streams.  It must be cautioned,
 however,  that the  urban runoff studied had concentrations two orders
 of magnitude higher than values observed in bathing beach studies.

     The skin which is exposed to  the  external environment provides
 an environment for  a variety  of microorganisms.  Staph. aureus, the
 coagulase-positive  organism,  is an important  human pathogen and is
 responsible for a wide spectrum of clinical diseases.  Usually boils,
 carbuncles, abscesses, and impetigo  are the common skin leasions seen.
 Obviously, staphlococcal infections may develop anywhere since the
 organism is indigenous on  the skin.  Direct contact with infected in-
 dividuals are the most important sources and  asymptomatic staphylococcal
 carriers  and air play very minor roles.  However, prolonged contact
with water carrying concentrations  of  a wide  variety of Staph.  auveus
 strains,  some antibiotic resistant  or  highly  virulent, could be an impor-
 tant factor in the  infection  of cuts and abrasions acquired while playing
 in the urban streams.  The presence of Stapkyloooooi, in raw sewage is
believed  to be primarily contributed by the bath and laundry waters
and had a mean concentration  of 820 MPN/100 ml with a maximum of 4,600
 to a minimum of 42/100 ml.  In the urban streams the concentrations
of StaphyloooGci. were not impressively high.  The mean concentration
                                    136

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was 5 MPN/100 ml with a minimum of less than 1 and a maximum 93/100 ml.
At the NTAC suggested standard of 200 FC/100 ml the calculated concen-
tration of Staph. aux>eus was 2.25/100 ml.  Unfortunately, little infor-
mation is available to correlate the degree of risk associated with the
levels of Staph. aureus in the water.

     The primary human enteric disease-producing bacterial agents
associated with the ingestion of water are:  Salmonella typhi  (typhoid
fever), Salmonella pavatyphi-A  (paratyphoid fever), Salmonella species
(salmonellosis), Skigella species  (bacillary dysentery) and cholera.
Today all of these organisms but Salmonella and Shigella  are epidemic-
logical curiosities having been brought under control by  environmental
sanitation practices and maintenance.

     The assessment of the potential health hazard from the Salmonella-
Shigella organisms in the urban waterways will depend upon the numbers
of organisms ingested, the virulence of the bacterial strain,  the
susceptability  of  genetically heterogenous human populations,  the  age
and physiological  state of individuals, the multiplicity  of factors
affecting the immunity of the host, and the interaction of the pathogen
with the microbial flora and food  in the  gastroin^Xi^3^^§J^^,l!P=Tr=-~-^^-

     MacKenzie  and Livingstone  (74) indicated  that  the Salmonella- ^...  .
infecting dose  vafies with susceptibility, and is smaller for  infants
and  the  aged.   Species and strain differences  gave  different-level!:?
of organisms  to produce clinical illness.  In  healthy adult.volunteers
fed  experimental dosages,  the minimal  numbers  causing symptoms.varied
enormously  from 10s  to 109 cells.   Comparable  data  for  typhoid fever
 (75) showed that a dose of 103  organisms  produced no disease,  whereas,
105  typhoid bacteria resulted  in illness  of  28% of  persons exposed.
The  estimated typhoid LD50  (76)  was placed at  107  organisms.   In/general
it was stated:

           "There are so many gaps  in our understanding of the
           infectivity of  Salmonellae that it  is not possible
           to  give any reliable figure for the infecting dose
           in man".

 In any case,  the number of Salmonella required to be ingested is re-
 latively large compared to the evidence regarding Sh-igella as shown
 below:

                 Infective Dose of Enteric Pathogens

                 Shigella	  . . 101 to 10 2
                 Salmonella	10
                 Escherisdh-La coli	10
                        cholefae	108
      In the urban streams the mean Salmonella density was low with a
 MPN of 8.7/10 1  (8.7 x 10~2 MPN/100 ml).  The minimum was less  than
 1/10 1 and a maximum of  320/10 1.  At  the NTAC  suggested standard of
 200 FC/100 ml the most probable number of Salmonella was 5.8/10 1.
 If this water is consumed at the maximum possible  intake per day of
 2 I/capita, the number of Salmonella ingested at the worst  condition

                                   137

-------
  (32/1) would be only 64 organisms.  Thus, the salmonellosis health hazard
 in water contact with urban streams is believed to be small.  'The density
 of Salmonella in stormwater exceeded 10,000/10 1 in only one case.  If
 we use a value of 10,000 Salmonella /10 1 for a similar calculation, the
 number of Salmonella ingested per day would be 2,000 organisms and is
 still more than a factor of 10 lower than the infective dose listed above.
 This coupled with the highly unlikely event of consuming 2 liters of
 stormwater make the health hazard also small.

      Shigellosis,  on the other hand, may present a problem because for
 reasons already discussed in the section under analytical methods.  There
 is every reason to believe that Shigella sp. are consistently present in
 the sewage and in the urban runoff.   The reported, cases of shigellosis in
 the city peak at the same late summer period as salmonellosis but are only
 0.7 of the reported cases of the latter.   The degree of health hazard cannot
 be verified until  methods for the isolation and enumeration of Shigella
 under varying environmental conditions have been accomplished.   The Dubuque,
 Iowa episode (77)  where the transmission of shigellosis by swimming in a
 contaminated river supports this concern.   The study revealed a mean FC
 MPN/100 ml, of 17,500 in samples of water in the swimming area.   This is
 greatly in excess  of the NTAC suggested standard of 200 FC/100 ml.  S.
 sonne-i was isolated from a sample of the river water containing 400 FC/100
 ml.   While the density of Shigella organisms per unit volume was not
 determined,  the role of this pathogen could be a hazard in view of the
 evidence of  fecal  contamination.

      The exact quantity of jmteroviruses which must be ingested to produce
 injurious  infections is not Known. Poliovirus infection by the  oral route
 has been studied and Sabin (78)  reported that non-human primates and man
 did not have comparable susceptibility.  He reported that  if  fewer than
 10 tissue  culture  doses  of vaccine poliovirus were  ingested by  a human,
 the virus would  bypass  the pharynx but  infect the intestines.   If only  one
 poliovirus particle  ordinarily  infected a  cell,  thousands  of  virus units
 must  be defective.   The  literature is  contradictory in this regard since
 some  claim that  a much  lower  concentration can infect  children, while
 others  feel  that more than 104  tissue  culture doses  of vaccine  are needed
 to infect infants  (79).  Despite  this, the authors  of  several reviews of
 the problem  of viral minimal  infective dose  (MID) (80,81,82,83) have
 generally arrived at the conclusion  that one  tissue  culture infective dose
 (TCID50) correlates  well with one MID for  a broad spectrum  of viruses.
 This  principle applies to both water and airborne infections  (80)  and is
based not only on work with experimental animals but administration of
viruses to humans as well.  It is particularly germane to this discussion
 that  these observations on MID included human viruses such  as polioviruses
1 and 3  (84,81), coxsackievirus A21  (85),  coxsackievirus B4 (86),  rhino-
virus (80) and adenovirus  (80).
                                    138

-------
     Admittedly the viruses achieved their high degree of efficiency
after careful instillation of the inoculum under optimal conditions with
minimum interference from environmental factors and host resistance
factors.  Nevertheless the potential for establishing the infection
warrants concern.  It should be noted that infection does not always lead
to overt disease.

     Whether or not this potential will be realized is largely a matter
of probability and frequency of contact.  If enough of a polluted water
supply is consumed, infections are inevitable.  It probably makes no
difference whether it is small amounts consumed by numerous individuals
or larger quantities consumed by fewer persons.  One simply cannot destroy
viral infectivity by diluting the viruses.

     In contrast to this, a rather large number of viable cells of bacterial
pathogens must be consumed by a single host to establish an infection.   In
a very dilute suspension it is impossible to consume enough quantities  to
establish the infective dose.  A comparison of the bacterial  and viral
health hazards involve unknown factors such as the influence  of particle
aggregations.  Thus, even though the infective agents  are diluted  to  low
levels, the  occurrence of clusters of virions or microbial  cells may  defeat
protection offered by the average low concentration.        -£, osi £J-'T  = --.
                                                           •• - o. r. fj rl^ -^ i i" i^lt    '
      Goldfield in ,1976 (87)  reviewed the epidemiological evidence for ^the
 transmission of virus diseases by the water route.   He concluded , ^similar ,
 to Mosely in 1965 (88),  that the demonstrated health hazard bf -.viruses 'in
 water has been limited to an occasional outbreak of infectious he^atitus
 associated with the direct consumption of contaminated water and 'raw ^ "
 shellfish, a rare occurrence of poliomyelitis, and adenovirus infection
 associated with swimming pools.  At present, the etiology of acute infec-
 tious non-bacterial (viral)  gastroenteritis remains unclear.  Thus, even
 though viruses are detected at low levels in urban waterways and storm
 runoff, and the minimum infective dose may be small, the epidemiological
 information at present indicates that the threat to health is low.
                                      139

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87.
Goldfield, M.  Epidemiological Indicators For Transmission of Viruses
by Water.  In Viruses in Water3 Edited by G. Berg, H.L. Bodily, E.H.
Lennette, J.L. Melnick and T.G. Metcalf.  American Public Health
Association Inc., 1976.
88.
Mosley, J.W., Transmission of Viral Diseases by Drinking Water.
Transmission of Viruses by the Water Route.  Edited by G. Berg.
Interscience, N.Y., p. 5-23, 1967.
In
                                      145

-------
                               APPENDICES
APPENDIX A.  Daily Precipitation in Inches  at  the  Customs House  (CH),
Woodbourne  (W)  and Ashburton (A)  in Baltimore  City from September  1,
1974  to September  30,  1975.

                   Daily Precipitation  (inches)

      September 1974	                 October 1974

Day
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30

W
0
0
0.55
0
.0
1.40
0.60
0
, _o
;o
0
0
0.75
0.10
0
0
0
0
0
0
0.45
0
0
0
0
0
0
1.50
0
0
Site
A
0
0
0
0
0
1.65
0
0
0
0
0
0
0.45
0.20
0
0
0
0
0
0
0.25
0
0
0
0
0
0
1.65
0
0

CH
0.04
0.08
1.04
0.02
0
1.53
0.50
0
0
0
0.03
0
0.52
0.16
0
0
0
0
0
0
0.36
0
0
0
0
0
0
1.73
0
0

Day
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16*
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31

W
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0.45
0.65
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Site
A
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0.40
0.55
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0

CH
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0.58
0.76
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Total  5.35
4.20
6.01
                                            1.10
                                     0.95
1.34
    Storms sampled
                                146

-------
APPENDIX A.  Daily Precipitation in Inches at the Customs House  (CH),
Woodbourne  (W) and Ashburton  (A) in Baltimore City from September 1,
1974 to September 30, 1975.

                  Daily Precipitation (inches)

	    November 1974                        December 1974

Day
1
2
3
4
5*
6
7
8
9
10
11
12*
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30

W
0
0
0
0
0.65
0
0
0
0
0
0
0.55
0
0.15
0.05
0
0
0
0
0.15
0
0
0
0
0.30
0
0
0
0
0
Site
A
0
0
0
0
0.40
0
0
0
0
0
0
0.75
0
0.05
0.10
0
0
0
0
0.15
0
0
0
0
0.25
0
0
0
0
0


CH
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0




.58






.65

.06
.12




.10




.26






Day
1
2
3
4 '
5
6
7
8
9
10
11
12
13
14
15
16*
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31

W
2.25
0.05
0
0
0
0
0.35
1.00
0
0
0
0
0.05
0.10
0.05
2.20
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0.40
Site
A
2.50**
0
0
0
0
0
0.20
1.15
0
0
0
0 1:
0.05
0.15
0
1.70
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0.40

CH
3.87
0.08
0
0
0
0
0.15
1.28
0
0
:o
.' 0
<0
:o.i5
:o.os
2.09
0
0
0
0
0.03
0
0
0
0
0
0
0
0
0
0.36
Total  1.85
1.70
1.77
6.05
6.15**  8.06
*   Storms sampled

**  Estimates based on Customs House data
                                 147

-------
APPENDIX A.  Daily Precipitation in Inches at the Customs House  (CH),
Woodbourne  (W) and Ashburton  (A) in Baltimore City from September 1,
1974 to September 30, 1975.
                  Daily Precipitation (inches)
        January 1975

Day
1
2
3
4
5
6*
7
8
9
10
11*
12
13*
14
15
16
17
18
19
20*
21
22
23
24
25
26
27
28
29
30
31

W
0
0
0
0
0
0.45
0
0.40
0.15
a
0.15
d
0.45
0
0
0
0
0.70
0.20
0.30
0
0
0
0.10
0.35
0
0
0
0
0
0.20
Site
A
0
0
0
0
0
0
0
0
0
0
0
0
0.50
0
0
0
0
0.50
0.45
0.10
0
0
0
0.25
0
0
0
0
0
0
0.15

CH
0
0
0
0.01
0
0.44
0
0.22
0.38
0
0.16
0
0.58
0
0
0
0
0.55
0.38
0.20
0
0
0
0.02
0.42
0
0
0
0
0
0.24
                               February 1975

Day
1
2
3
4
5*
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23*
24
25
26
27
28

W
0
0.05
0
0.15
0.50
0.05
0
0
0
0
0
0.45
0
0
0
0
0.15
0
0
0
0
0
0.50
0.40
0
0
0
0
Site
A
0
0
0
0.40
0.25
0
6
0
0
0
0
0.40
0
0
0
0
0
0
0
0
0
0
0.20
0
0
0
0
0

CH
0
0.06
0
0.41
0.34
0.05
0
0
0
0
0
0.43
0
0
0
0
0.15
0
0
0
0
0
0.65
0.36
0
0
0
0
Total  3.45
1.95
3.60
2.25
1.25
2.50
     Storms  sampled
                                 148

-------
APPENDIX A.  Daily Precipitation in Inches at the Customs House  (CH),
Woodbourne (W) and Ashburton  (A) in Baltimore City from September 1,
1974 to September 30, 1975.
                  Daily Precipitation (inches)
        March 1975

Day
1
2
3
4
5
6
7
8
9
10
11
12*
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31

¥
0
0
0
0
0
0
0
0
0
0.10
0
0.45
0.10
0.95
0
0.05
0.20
0
1.85
0
0
0
0
0.55
0
0
0
0
0.10
0.40
0
Site
A
0
0
0
0
0
0
0
0
0
0
0
0.55
0
0.70
0
0
0
0
1.85
0
0
0
0
0.60
0
0
0
0
0.10
0.40
0

CH
0
0
0
0
0
0
0.05
0
0
0.09
0
0.43
0.03
1.10
0
0.09
0.29
0
2.34
0
0
0
0
0.88
0
0
0
0
0.08
0.53
0


Day
1
2
3*
4
5
6
7
8
9
10
11
12
13
14
15*
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
April

W
0
0
0.10
0
0
0
0
0
0
0
0
0
0
0
0.30
0
0
0
0
0
0
0
0.05
0.45
1.05
0
0
0
0.10
0
1975
Site
A
0
0
0.15
0
0
0
0.10
0
0
0
0
0
0
0
0.35
0.05
0
0
0
0
0
0
0
0.75
0.90
0.05
0
0.05
0.25
0


CH
0
0
0.30
0
0
0
0
0
0
o,
:I.Q
0
»i,0
0;
0}.42
0
0
0.02
0.14
0
0
0
0.05
0.37
1.47
0
0
0.02
0.25
0
Total   4.75
4.20
5.91
2.05
2.65
3.04
     Storms  sampled
                                 149

-------
APPENDIX A.  Daily Precipitation in Inches at the Customs House (CH),
Woodbourne (W) and Ashburton  (A) in Baltimore City from September 1,
1974 to September 30, 1975.

                   Daily Precipitation (inches)

         May 1975                              June 1975

Day
1*
2
3
4
5
6*
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31

W
0.85
0
0.20
1.65
0
0.20
0
0
, 0
0
jO
:<3.35
j. ;0.20
, , 0
*f 0.10
0.25
0
0
0
0
0
0
0
0
0
0
0
0
0
.1**
.8**
Site
A
0.45
0
0.10
1.55
0
0.30
0
0
0
0
0
0.65
0.30
0
0
0
0
0
0
0
0
0.40
0
0.30
0
0
0
0
0
0
0

CH
0.71
0
0.14
1.95
0.02
0.48
0
0
0
0
0
0.89
0.08
0
0.14
0.21
0
0
0
0
0
0.58
0
1.77
0
0
0
0
0
0.15
0.86

Day
1
2
3
4
5
6
7
8
9
10
11*
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30*

W
.5**
o**
o**
o**
1.1**
o**
0
0
0
0
0.30
0.50
0.75
0
0
0
0
0
0
0
0
0
0
0
0
0.05
0
0.95
0.20
0
Site
A
.5**
o**
o**
o**
1.1**
o**
0
0
0
0
0
0.55
0,30
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0.80
0.10
0

CH
0.49
0
0
0
1.15
0.15
0
0
0
0
0.26
0.39
0.15
0
0
0.05
0
0
0
0
0
0
0
0
0.04
0.16
0.11
0.55
0.20
0
Total  4.70**  4.05
8.08
4.35**  3.35**  3.70
*   Storms sampled

**  Estimates based on Customs House data
                                150

-------
APPENDIX A.  Daily Precipitation in Inches at the Customs House  (CH) ,
Woodbourne (W) and Ashburton (A) in Baltimore City from September 1,
1974 to September 30, 1975.

                  Daily Precipitation (inches)

        July 1975	           	August 1975

Day
1
2
3
4
5
6
7
8
9
10*
11
12
13 -
14*
15
16
17
18
19
26
21
22
23
24
25
26
27*
28
29
30
31

W
0
0
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
"ND
ND
ND
ND
ND
0
0.70
0
0
0
0.15
0
0
0
0
0
0
0
Site
A
0
0
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
0
0.45
0
0
0
0
0
0
0
0
0
0
0

CH
0
0
0.50
0
0
0
0
0
0
4.66
0
0
3.85
1.66
0.02
0
0
0
0
0.83
0
0
0
0.19
0
0
0
0.02
0
0
0

Day
1
2
3
4
5
6*
7
8
9
10
11
12
13*
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31

W
0
0
0
0.50
0.10
0.15
0
0
0
0
0
0
0.60
1.05
0
0.20
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Site
A
0
0
0
0.40
0.35
0
0
0
0
0
0
0
0.454 -
1.45
0 -J
0.25"
0.05
0
0
0
0
0
0
0
0
0
0
0
d
0
1.35

CH
0
0
0
0.73
0.07
0.23
0
0
0
to
'•• 0
-0
-0.69
'1.31
"0
0.17
0.04
0
0
0
0
0
0
0
0
0
0
0
0
0
0.50
Total  11.0**   11.0**   11.68
2.60
4.30
3.74
*    Storms  sampled

**   Estimates based  on Customs House  data

ND   No Data
                                 151

-------
APPENDIX A.  Daily Precipitation in Inches at the Customs House  (CH),
Woodbourne  (W) and Ashburton  (A) in Baltimore City from September 1,
1974 to September 30, 1975.

                  Daily Precipitation (inches)

      September 1975

Day
1
2
3
4
5
6
7
8
9
10
11
12*
13
14
15
16
17
18*
19
20
21
22
23
24
25
26
27
28
29
30

W
0.25
0
0
0
0
0
0
0
0
0
0
0.65
0
0
0
0
0
0.45
0.05
0
0
0.60
2.30
1.00
1.70
1.15
0
0
0
0
Site
A
0
0
0
0
0
0
0
0
0
0
0
0.45
0
0
0
0
0
0.45
0.15
0
0
0
1.15
1.55
1.40
1.40
0
0
0
0

CH
0.25
0
0
0
0
0.7
0
0
0
0
0.02
0.55
0
0
0 ,
0
0
0.68
0.10
0
0
0.67
2.40
1.15
1.55
1.35
0
0
0
0
Total  8.15
6.55
8.79
    Storms sampled
                                 152

-------
  APPENDIX B.  Levels of Bacteria,  Station A - Raw Sewage
Date
07/17/74
07/23/74
07/30/74
08/05/74
08/12/74
09/09/74
09/16/74
09/23/74
09/30/74
10/07/74
10/14/74
10/21/74
10/28/74
11/04/74
11/11/74
11/18/74
12/02/74
12/09/74
12/17/74
12/24/74
01/06/75
01/20/75
01/27/75
02/03/75
02/17/75
03/03/75
03/17/75
03/31/75
04/14/75
04/28/75
05/12/75
05/19/75
06/10/75
06/24/75
07/07/75
07/21/75
08/04/75
08/18/75
09/02/75
09/16/75
Geom. Mean
Run
Number
1
2
3
4
5
7
8
9
10
11
12
14
15
16
18
20
21
22
24
25
26
30
31
32
34
36
38
39
41
43
46
47
48
50
52
55
57
60
61
63

Total
Coliform
MPN/lOOml
>2.9 x 10s
5.4 x 108
2.8 x 108
1.3 x 108
3.3 x 107
3.3 x 107
1.7 x 107
3.5 x 107
2.4 x 107
2.4 x 107
3.5 x 107
2.9 x 106
5.4 x 107
2.6 x 10s
3.5 x 107
1.7 x 107
2.4 x 106
7.0 x 106
1.6 x 107
1.6 x 108
3.5 x 107
3.5 x 10s
>2.4 x 107
1.7 x 107
2.8 x 107
1.3 x 107
1.6 x 107
4.6 x 107
1.72 x 1§
>1.6 x 109
3.5 x 107
5.4 x 107
2.2 x 10s
1.3 x 10s
9.2 x 107
3.4 x 10s
4.9 x 107
9.2 x 107
5.4 x 107
5.4 x 107
2.2 x 107
Fecal
Coliform
MPN/lOOml
3.5 x 107
1.7 x 108
7.9 x 107
8.0 x 105
2.3 x 107
3.3 x 107
4.9 x 106
3.5 x 107
2.4 x 107
4.9 x 106
4.6 x 10s
4.9 x 106
1.1 x 107
>2.6 x 10s
7.9 x 106
7.9 x 10s
1.3 x 10s
3.3 x 10s
4.6 x 105
1.7 x 107
7.9 x 106
3.3 x 105
5.4 x 106
3.3 x 10s
3.3 x 106
3.3 x 105
3.3 x 106
1.3 x 107
4.9 x 107
9.2 x 108
3.5 x 107
7.9 x 106
1.7 x 106
3.3 x 105
4.9 x 106
4.9 x 10s
1.1 x 107
1.4 x 107
1.3 x 107
3.5 x 107
6.3 x 106
Fecal
Streptococci
no. /100ml
3.5 x 106
ND
2.4.x 106
5.4 x 10s
ND
5.2 x 10s
9.8 x 105
1.2 x 107
4.1 x 106
1.1 x 106
1.2 x 106
8.3 x 105
4.4 x 105
2.0 x 101*
1.1 x 106
1.1 x 10s
3.4 x 10s
9.7 x 105
3.9 x 105
1.1 x 10s
9.5 x 10s
5.4 x 105
4.5 x 107
1.1 x 10s
1.8 x 106
1.4 x 106
2.7 x 106
9.4 x 10s
1.7 x 106
1.6 x 106
2.4 x 106
3.0 x 106
3.1 x 10s
3.8 x 105
3.3 x 106
9.9 x 105
8.0 x 105
1.3 x 10s
1.4 x 106
9.5 x 105
1.4 x 10s
Pseudomonas
aeruginosa
MPN/lOOml
7.0 x 103
ND
7.9 x 101*
ND
3.5 x 10s
9.2 x 10s
3.5 x 105
2.3 x 106
1.7 x 106
1.1 x 106
3.5 x 105
7.9 x 10s
1.7 x 105
3.3 x 103
9.2 x 106
9.2 x 10s
1.3 x 10s
7.0 x 104
2.2 x 10s
3.5 x 10s
3.5 x 105
7.0 x 101*
3.3 x 105
3.3 x 105
1.4 x 105
1.4 x 105
7.9 x 101*
9.2 x 105
>1.6 x 10s
7.9 x 106
5.4 x 107 .
5.0 x 103
1.4 x 10s
2.3 x 103
2.3 x 101*
3.5 x 105
7.8 x lO1*
1.7 x 101*
2.7 x 101*
2.8 x 105
2.3 x 105
Staph.
aureus
MPN/lOOml
ND
ND
ND
ND
ND
ND
ND
ND'
ND
ND
ND
ND
3.6
2.4 x 102
4.5 x 101
9.4
4.6 x 102
5.8 x 102
2.4 x 103
1.7 x 103
2.0 x 101
3.9 x 102
4.6 x 103
9.3 x 102
2.4 x 103
2.1 x 103
2.4 x 103
2.4 x 103
4.6 x 103
2.9 x 102
4.6 x 103
2.4 x 103
1.5 x 102
6.1 x 101
4.5 x 101
2.6 x 102
7.8 x 101
LSL
3.3 x 102
LSL
2.6 x 102
Salmonella
MPN/10 liters
LSL
LSL
LSL
1.4 x 103
ND
5.6 x 102
5.3 x 101
2.6 x 102
>2.9 x 103
1.2 x 103
4.8 x 102
5.1 x 103
1.7 x 103
ND
2.8 x 102
5.1 x 102
4.8 x 101
1.0 x 102
2.6 x 101
>2.7 x 103
>2.7 x 103
5.1 x 102
5.1 x 102
1.2 x 103
2.7 x 102
4.9 x 102
4.9 x 102
2.2 x 102
1.7 x 102
4.3 x 102
8.3 x 102
2.2 x 102
2.7 x 104
5.1 x 103
6.1 x 102
1.2 x 10**
1.2 x 101*
2.7 x 101*
2.7 x 103
2.7 x 103
5.0 x 102
ND - No Data
LSL - Lower sensitivity limit of the assay.  No microorganisms recovered.
                                        153

-------
 APPENDIX  B.  Levels  of Bacteria, Station  B - Herring Run


Date
07/17/74
07/23/74
07/30/74
08/05/74
08/12/74
09/09/74
09/16/74
09/23/74
09/30/74
10/07/74
10/14/74
10/21/74
10/28/74
11/04/74
11/11/74
11/18/74
12/02/74
12/09/74
12/17/74
12/24/74
01/06/75
01/20/75
01/27/75
02/03/75
02/17/75
03/03/75
03/17/75
03/31/75
04/14/75
04/28/75
05/12/75
05/19/75
06/10/75
06/24/75
07/07/75
07/21/75
08/04/75
08/18/75
09/02/75
09/16/75

Run
Number
1
2
3
4
5
7
8
9
10
11
12
14
15
16
18
20
21
22
24
25
26
30
31
32
34
36
38
39
41
43
46
47
48
50
52
55
57
60
61
63
Total
Coliform
MPN/lOOml
1.1 x 10"
3.3 x 103
5.4 x 101*
3.3 x 10"
2.3 x 102
9.2 x 103
2.8 x 103
3.5 x 10s
1.3 x 103
5.4 x 103
5.0 x 101
5.4 x 102
1.6 x 103
3.5 x 102
1.7 x 10"
9.2 x 102
2.4 x 10s
>2.4 x 10s
2.4 x 10"
1.4 x 10"
3.5 x 103
1.7 x 103
2.4 x 103
2.2 x 103
3.5 x 103
3.3 x 103
1.4 x 10"
1.09 x 10"
1.09 x 10"
1.1 x 103
1.3 x 103
1.7 x 103
1.7 x 10"
2.4 x 103
5.4 x 103
1.6 x 103
3.3 x 103
9.2 x 103
1.1 x 10"
5.4 x 103
Fecal
Coliform
MPN/lOOml
2.3 x 103
1.7 x 103
2.4 x 10"
7.9 x 103
1.3 x 102
2.2 x 103
ND
3.5 x 10s
7.9 x 102
1.3 x 103
LSL .
5.4 x 102
5.4 x 102
1.1 x 102
1.4 x 103
5.4 x 102
2.4 x 10s
ND
1.3 x 10"
1.4 x 10"
7.9 x 102
2.1 x 102
1.3 x 103
2.2 x 103
3.5 x 103
2.4 x 103
3.3 x 103
7.9 x 103
7.0 x 103
4.9 x 102
7.9 x 102
1.7 x 103
1.3 x 103
1.3 x 103
1.3 x 103
2.8 x 102
2.0 x 102
1.4 x 103
3.3 x 103
7.9 x 102
Fecal
Streptococci
no ./100ml
2.2 x 103
2.2 x 103
1.7 x 10"
7.9 x 103
8.0 x 102
1.3 x 103
8.0 x 102
1.6 x 10"
1.0 x 103
4.5 x 102
7.5 x 102
4.5 x 102
3.0 x 102
1.9 x 102
2.0 x 102
5.3 x 102
4.0 x 10"
2.4 x 103
3.8 x 103
1.9 x 103
5.6 x 102
1.3 x 10"
3.9 x 103
2.3 x 103
1.1 x 10"
2.8 x 103
2.0 x 10"
4.1 x 102
4.2 x 102
3.1 x 102
2.3 x 102
3.9 x 102
8.0 x 103
2.1 x 103
2.7 x 103
1.4 x 10"
2.4 x 102
3.5 x 103
2.5 x 103
2.7 x 103
Paeudomonas
aeruginosa
MPN/lOOml
ND
ND
2.2 x 103
ND
3.4 x 103
1.7 x 102
1.7 x 103
1.6 x 10s
2.2 x 103
2.4 x 103
5.4 x 10"
3.3 x lO1
1.7 x 102
5.4 x 10"
4.9 x 102
5.4 x 103
1.3 x 103
9.4 x 101
1.4 x 102
7.0 x 101
4.6 x 101
2.3 x 102
1.1 x 102
1.7 x 102
3.4 x 101
4.9 x 101
4.9 x 101
2.78 x 102
2.78 x 103
5.2 x 102
3.3 x 101
3.3 x 101
7.9 x 102
1.1 x 101
4
1.7 x 103
ND
1.7 x 102
1.7 x 103
4.0 x 101
Staph.
aweeus
MPN/lOOml
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
Nl5
ND
LSL
LSL
LSL
6.8 x 101
2.0
ND
4.0
1.3

3.6
7.3
3.6
3.6
LSL
3.6
LSL
LSL
LSL
LSL
LSL
3.6 x 101
LSL
2.0
7.4
LSL
1.1 x 101
4.8
2.0
Salmonella

MPN/10 liters
LSL
LSL
LSL
LSL
ND
2.0
ND
ND
ND
4.1
1.2
2.7 x Ifl1
4.4
6.7
6.7
1.3 x 101
1.3 x 102
2.6
6.7
LSL
2.6
4.38 x 101
1.17
2.63
7.01 x 101
2.04
6.72
1.12
LSL
1.3 x 101
LSL
LSL
4.4 x 101
1.3 x 101
1.2
5.8
LSL
1.2
1.1 x 101
2.63
Geora. Mean
4.Sx 103   l.lxlO3    1.5 x 103
ND  - No Data
LSL - Lower sensitivity limit of the assay.
2.9 x 102   3.2
                      No microorganisms recovered.
                                                                             4.6
                                          154

-------
APPENDIX B.  Levels  of Bacteria, Station  C - Jones  Falls


Date
07/17/74
07/23/74
07/30/74
08/05/74
08/12/74
09/09/74
09/16/74
09/23/74
09/30/74
10/07/74
10/14/74
10/21/74
10/28/74
11/04/74
11/11/74
11/18/74
12/02/74
12/09/74
12/17/74
12/24/74
01/06/75
01/20/75
01/27/75
02/03/75
02/17/75
03/03/75
03/17/75
03/31/75
04/14/75
04/28/75
05/12/75
05/19/75
06/10/75
06/24/75
07/07/75
07/21/75
08/04/75
08/18/75
09/02/75
09/16/75

Run
Number
1
2
3
4
5
7
8
9
10
11
12
14
15
16
18
20
21
22
24
25
26
30
31
32
34
36
38
39
41
43
46
47
48
50
52
55
57
60
61
63
Total
Coliform
MPN/lOOml
7.9 x 10"
1.1 x 10"
3.3 x 105L'
1.3 x 10s
1.7 x 10s
3.3 x 105
7.9 x 10"
2.3 x 105'
1.6 x 10s
1.4 x 105
9.2 x 10"
9.2 x 10"
1.7 x 105
1.7 x 105
3,3 x 10"
4.6 x 10"
2.4 x 10"
1.6 x 10s
9.2 x 10"
1.7 x 105
1.3 x 105
3.3 x 10"
3.5 x 10"
5.4 x 10"
3.5 x 10"
1.3 x 10"
5.4 x 10"
3.4 x 10s
1.3 x 10s
7.9 x 10"
1.1 x 10"
3.5 x 10"
1.7 x 10s
3.3 x 10"
,1.7 x 105
2.2 x 10"
1.7 x 10s
5.4 x 10"
ND
9.2 x 10"
Fecal
Coliform
MPN/lOOtnl
4.9 x 10"
1.1 x 10"
1.7 x 10s
4.9 x 10"
1.3 x 10s
2.3 x 10s
4.9 x 10"
2.3 x 10s
1.6 x 10s
9.4 x 10"
1.4 x 10"
3.5 x 10"
1.7 x 10s
9.2 x 10"
3.5 x 10"
2.1 x 10"
2.4 x 10"
2.4 x 10"
3.5 x 10"
3.5 x 10"
4.9 x 10"
7.9 x 103
3.5 x 10"
3.5 x 10"
3.5 x 10"
7.9 x 103
1.3 x 10"
2.4 x 105
4.9 x 10"
7.9 x 10"
4.9 x 103
2.4 x 10"
4.9 x 10"
2.3 x 10"
1.3 x 10s
1.4 x 10"
2.2 x 10"
7.9 x 103
ND .
4.7 x 103
Fecal
Streptococci
no. /100ml
1.1 x 10"
1.7 x 10"
4.6 x 10"
9.2 x 10"
4.9 x 103
2.3 x 10"
1.5 x 10"
3.2 x 10"
5.3 x 10"
1.3 x 10"
1.4 x 10"
5.9 x 10"
2.3 x 10"
3.2 x 10"
3.6 x 10"
2.6 x 103
7.6 x 10"
2.6 x 10"
4.2 x 10"
7.1 x 103
4.0 x 103
1.1 x 10"
2.4 x 103
3.3 x 103
3.1 x 10"
4.2 x 103
7.3 x 10"
3.3 x 10"
4.6 x 103
3.5 x 10"
6.1 x 103
4.7 x 103
1.8 x 10"
8.5 x 103
5.1 x 10"
1.3 x 10"
2.0 x 103
6.7 x 103
7.3 x 103
4.9 x 103
Pseudomonas
aeruginosa
MPN/lOOml
9.2 x 102
2.4 x 102
5.4 x 103
ND
2.3 x 10"
5.4 x 103
2.3 x 10"
2.4 x 10"
3.5 x 10s
9.2 x 103
1.7 x 103
9.2 x 102
1.1 x 103
9.2 x 103
9.4 x 103
1.4 x 10"
2.7 x 103
1.7 x 103
1.7 x 103
>2.4 x 10"
3.5 x 103
1.1 x 103
1.1 x 103
7.9 x 102
5.4 x' 102
4.9 x 102
1.3 x 102
5.42 x 103
1.72 x 10"
1.6 x 10"
2.2 x 102
1.1 x 102
1.3 x 103
2.2 x 10*
8.4 x 101
2.0 x 102
7.9 x 102
2.4 x 103
9.2 x 102
9.2 x 102
Staph.
aureus
MPN/lOOml
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
2.0
4.6 x 101
1.8 x 102
1.1 x 101
2.0
LSL
1.7 x 101
1.2 x 101
2.0
4.3 x 101
7.3
4.3 x 101
2.3 x 101
9.1
9.1
3.6
9.1
4.3 x 101
4.3 x 101
3.6
9.3 x 101
4.0
2.0
1.1 x 102
4.0
LSL
2.0
1.8
Salmonella

MPN/10 liters
LSL
LSL
LSL
2.0
ND
ND
ND
ND
ND
2.6
2.0
1.2
1.2
1.2
1.3 x 101
4.4 x 101
1.3 x 102
7.0 x 101
2.7 x 101
-•; 8.2 ;TV-
2.0
4.38 x 101
2.63
7.88
2.72 x 101
6.72
3.21 x 102
2.04
1.17
3.2 x 102
2.2 x 101
4.4 x 10l
- : 1.1 x 101
.88
3.2 x 102
4.4
1.2
.88
6.13
7.0 x 101
 Geom. Mean
4.0 x 10"   1.5 x 10"    1.5x 10"     2.1 x 103    977
                                                                             9.1
 ND - No Data
 LSL - Lower sensitivity limit of the assay.  No microorganisms recovered.
                                          155

-------
APPENDIX B.   Levels of  Bacteria,  Station D -  Gwynns Falls

t y f A, f v
Date
07/17/74
07/23/74
07/30/74
08/05/74
08/12/74
09/09/74
09/16/74
09/23/74
09/30/74
10/07/74
10/14/74
10/21/74
10/28/74
11/04/74
11/11/74
11/18/74
12/02/74
12/09/74
12/17/74
12/24/74
01/06/75
01/20/75
01/27/75
02/03/75
02/17/75
03/03/75
03/17/75
03/31/75
04/14/75
04/28/75
05/12/75
05/19/75
06/10/75
06/24/75
07/07/75
07/21/75
08/04/75
08/18/75
09/02/75
09/16/75

Number
1
2
3
4
5
7
8
9
10
11
12
14
15
16
18
20
21
22
23
25
26
30
31
32
34
36
38
39
41
43
46
47
48
50
52
55
57
60
61
63
Total
Coliform
MPN/lOOml
2.2 x 10*
7.9 x 103
3.5 x 10s
3.3 x 10"
2.2 x 103
1.7 x 10"
5.4 x 10s
1.3 x 10*
2.4 x 10s
3.5 x 10*
1.3 x 10*
9.2 x 10*
3.4 x 102
4.6 x 102
2.2 x 103
1.7 x 10s
3.3 x 10*
3.5 x 10*
3.5 x 10s
1.1 x 10*
7.0 x 102
7.0 x 10s
2.1 x 10*.
1.3 x 103
7.0 x 103
4.9 x 103
2.4 x 10"
>2.4 x 106
3.3 x 103
3.3 x 10*
1.1 x 10*
3.5 x 103
1.7 x 10*
5.4 x 103
1.6 x 10*
1.3 x 10*
3.3 x 103
3.5 x 10*
ND
3.5 x 10*
Fecal
Coliform
MPN/lOOml
2.7 x 103
5.0 x 102
1.1 x 10s
1.1 x 10*
8.0 x 102
7.0 x 103
4.9 x 10s
1.3 x 10*
1.6 x 10*
1.7 x 10*
5.0 x 102
4.6 x 103
8.0 x 101
2.3 x 102
4.9 x 102
3.3 x 102
1.3 x 10*
3.3 x 103
1.3 x 10"
1.3 x 103
7.0 x 102
1.1 x 10s
3.3 x 103
7.9 x 102
3.3 x 103
7.0 x 102
5.4 x 103
>2.4 x 106
1.3 x 103
4.0 x 103
1.1 x 103
4.9 x 102
3.3 x 103
7.0 x 102
4.9 x 102
3.3 x 103
7.0 x 102
1.3 x 103
ND
1.1 x 10"
Fecal
Streptococci
no . /100ml
ND
2.0 x 102
3.5 x 10"
1.3 x 103
4.6 x 102
1.7 x 103
2.3 x 10"
5.5 x 103
8.5 x 103
3.8 x 103
3.0 x 102
1.7 x 103
LSL
1.2 x 102
2.1 x 102
1.2 x 102
>1.0 x 10s
2.4 x 10"
1.0 x 105
6.0 x 102
5.1 x 103
4.5 x 10*
7.0 x 102
LSL
2.2 x 103
2.4 x 103
1.9 x 10*
3.4 x 10*
5.5 x 102
6.0 x 102
3.0 x 102
1.0 x 102
3.6 x 103
2.1 x 103
6.7 x 102
2.8 x 10*
4.5 x 101
8.3 x 102
ND
3.3 x 103
Pseudomonas
aevuginosa
MPN/lOOml
2.2 x 102
2.8 x 102
2.8 x 103
ND
1.6 x 105
1.1 x 103
1.7 x 10s
2.8 x 10*
7.0 x 10*
1.6 x 10*
9.4 x 102
7.0 x 102
7.0 x 101
1.3 x 102
8.0 x 10l
4.6 x 102
1.1 x 10*
1.1 x 103
7.9 x 102
2.1 x 102
3.3 x 102
4.6 x 102
2.2 x 101
1.1 x 102
2.2 x 102
4.9 x 102
9.4 x 101
2.21 x 103
2.21 x 103
2.8 x 103
7.9 x 10l
1.7 x 10l
2.2 x 102
1.09 x 102
1.41 x 102
3.5 x 103
ND
1.7 x 102
ND
2.3 x 102
Staph.
aureus
MPN/lOOml
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
3.6 x 101
3.6 x 101
ND
1.7
2.6 x 101
4.0
6.8
1.4 x 101
LSL
LSL
LSL
9.1
LSL
LSL
7.3
9.3 x 101
LSL
3.0
7.3 '
LSL
2.1 x 101
6.8
LSL
4.0
4.0
LSL
ND
LSL
Salmonella
MPN/10 liters
LSL
LSL
LSL
6.1
ND
4.1
ND
ND
ND
2
1.3 x 10Z
1.3 x 10l
6.1
6.7
4.4
1.3 x 101
2.2 x 101
1
7.0 x 101
7.0 x 101
1
2.7 x 101
2.7 x 10l
27.2
4.38 x 101
2.19 x 101
2.04
1.34 x 102
6.72
2.72 x 101
1.26 x 101
6.7
2. .7 x 101
8.2
5.8
6.1 x 10*
•
1.2 x 10*
*
2.7 x 101
2.0
2.0
4.4
ND
i
2.7 x 101
 Geom. Mean
                   4.0 x 10*   5.9 x 103    1.7 x 103      4.7 x 102   4.5
1.5 x 101
 ND - No Data
 LSL - Lower sensitivity limit of the assay.  No microorganisms  recovered.
                                          156

-------
APPENDIX B.   Levels of Bacteria,  Station E - Loch Raven Reservoir


Date
03/17/75
03/31/75
04/14/75
04/28/75
05/12/75
05/19/75
Q6/10/75
06/24/75
07/07/75
07/21/75
08/04/75
08/18/75
09/02/75
09/16/75
Geom. Mean

Run
Number
38
39
41
43
46
47
48
50
52
55
57
60
61
63

Total
Coliform
MPN/lOOml
LSL
LSL
LSL
1.7 x 101
5.0
2.0
4.0 x 102
1.7 x 102
4.9 x 101
3.4 x 102
4.5
ND
3.3 x 101
7.9 x 101
2.6 x 101
Fecal
Coliform
MPN/lOOml
LSL
LSL
LSL
'1.7 x 101
5.0
LSL
7.0 x 101
8.0 x 10l
2.3 x 101
1.7 x 102
2.0
ND
1.3 x 101
4.9 x 101
1.5 x 101
Fecal
Streptococci
no . /100ml
LSL
5.0
LSL
2.0 x 102
2.0 x 10l
LSL
5.0
4.0 x 101
<5.0
ND
<5.0
ND
<5.0
ND
1.0 x 101
Pseudomonas
aerug-inosa
MPN/lOOml

2.3 x 101
LSL
2.0
LSL
-LSL
0
0
2.3 x 10l
4.5
ND
ND
2.0
4.5
3.1
Staph.
aureua
MPN/lOOml
LSL
LSL
LSL
LSL
LSL
LSL
LSL
LSL
LSL
LSL
LSL
ND
LSL
LSL
<2.5
Salmonella

r,MPN/10 liters
LSL *
LSL
LSL
0.88
LSL
LSL
LSL
LSL
LSL
LSL
LSL
ND
LSL
LSL
0
 ND  - No Data
 LSL - Lower sensitivity limit of the assay.
No microorganisms recovered.
                                         157

-------
APPENDIX B.   Levels of  Bacteria,  Station F -  Stoney Run


Date
10/16/74
11/05/74
11/12/74
12/16/74
01/06/75
01/11/75
01/13/75
01/20/75
02/05/75
02/12/75
03/12/75
04/03/75
04/15/75
05/01/75
05/06/75
06/11/75
06/30/75
07/10/75
07/14/75
07/27/75
08/06/75
08/13/75
09/12/75
09/18/75

Run
Number
13
17
19
23
27
28
29
30
33
35
37
40
42
44
45
49
51
53
54
56
58
59
62
64
Total
Coliform
MPN/lOOml
3.5 x 10s
>2.4 x 10s
1.3 x 104
5.4 x 103
1.1 x 10"
1.7 x 104
3.5 x 104
7.0 x 103
2.6 x 104
4.9 x 103
3.3 x 104
7.9 x 103
2.4 x 10"
3.3 x 104
5.4 x 103
3.5 x 103
1.6 x 10s
1.3 x 10s
1.7 x 10s
4.9 x 10"
7.0 x 103
9.2 x 104
7.9 x 10s
5.6 x 104
Fecal
Coliform
MPN/lOOml
4.9 x 104
1.7 x 10"
4.9 x 103
'1.7 x 103
1.7 x 104
3.3 x 103
2.4 x 104
3.1 x 103
1.7 x 10"
3.3 x 103
1.4 x 103
7.9 x 103
1.3 x 104
1.7 x 104
3.3 x 103
1.3 x 103
5.4 x 105
7.9 x 104
1.7 x 105
4.9 x 104
2.3 x 103
1.7 x 104
1.1 x 105
5.4 x 10s
Fecal
Streptococci
no. /100ml
5.3 x 104
9.8 x 104
8.4 x 103
2.4 x 10s
1.1 x 10s
1.9 x 105
3.1 x 104
2.5 x 104
1.7 x 104
8.0 x 103
2.4 x 104
2.3 x 104
5.6 x 104
6.5 x 104
8.0 x 104
3.1 x 104
ND
3.0 x 105
1.9 x 105
4.2 x 10s
LSL
1.7 x 105
1.2 x 10s
3.7 x 10s
Pseudamonas
aeruginosa
MPN/lOOml
2.4 x 10s
3.3 x 102
2.3 x 102
1.1 x 103
5.4 x 102
1.7 x 103
1.8 x 103
>2.4 x 103
1.8 x 104
2.2 x 102
1.4 x 103
1.41 x 103
4.0 x 102
1.7 x 103
4.8 x 102
7.0 x 102
4.9 x 103
1.48 x 10a
2.3 x 103
1.7 x 103
1.3 x 103
3.2 x 102
4.9 x 103
1.7 x 103
Staph.
aureus
MPN/lOOml
ND
2.0
4.9 x 101
2.7 x 101
2.3 x 101
4.3 x 101
4.3 x 101
2.3 x 101
2.8 x 101
9.0
7.3
2.0 x 101
LSL
2.3 x 101
2.3 x 101
1.8
2.2 x 101
4.5
2.2 x 101
1.7 x 101
LSL
LSL
1.0 x 101
LSL
Salmonella

MPN/10 liters
6.1 x 102
2.9
5.0
5.17 x 101
6.12 x 102
2.56 x 102
>1.33 x 103
2.56 x 102
1.33 x 102
3.89
3.89
2.11 x 101
>1.3 x 103
2.7 x 102
2.6 x 102
3.5
6.1 x 102
4.2 x 101
6.7 x 101
2.2
1.2 x 101
6.1
2.5 x 101
1.67
 Goom. Mean
                  4.8 x 104   1.9 x 104    4.1 x 104
             1.3 x 103    1.2 x 101
3.0 x 101
HD  - No Data
LSL - Lower sensitivity limit of the assay.
No microorganisms recovered.
                                          158

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APPENDIX B.   Levels of  Bacteria,  Station G -  Glen Avenue


Date
10/16/74
11/05/74
11/12/74
12/16/74
01/06/75
01/11/75
01/13/75
01/20/75
02/05/75
02/23/75
03/12/75
04/03/75
04/15/75
05/01/75
Q5/Q6/75
06/10/75 .
06/30/75
07/10/75
07/14/75
07/27/75
08/06/75
08/13/75
09/12/75
09/18/75

Run
Number
13
17
19
23
27
28
29
30
33
35
37
40
42
44
45
49
51
53
54
56
58
59
62
64
Total
Coliform
MPN/lOOml
2.4 x 10s
2.4 x 105
1.7 x 105
1.3 x 10"
2.4 x 10s
2.8 x 105
3.5 x 10"
7.9 x 103
5.4 x 10"
7.9 x 10"
3.3 x 10"
1.3 x 105
2.4 x 105
1.3 x 105
1.4 x 10s
4.6 x 10"
1.6 x 10s
ND '
7.9 x 10s
9.4 x 105
>1.6 x 10s
1.4 x 106
>1.6 x 10s
1.7 x 10s
Fecal
Coliform
MPN/lOOml
4.9 x 10"
5.4 x 10"
1.7 x 10s
7.9 x 103
4.9 x 10"
4.0 x 10"
2.4 x 10"
1.4 x 103
3.3 x 103
7.9 x 10"
1.7 x 10"
3.3 x 10"
1.3 x 105
2.2 x 10"
2.3 x 105
1.7 x 10"
1.7 x 10s
ND
2.3 x 105
2.3 x 10s
5.4 x 10"
2.2 x 105
1.6 x 106
4.9 x 10s
Fecal
Streptococci
no ./100ml
1.6 x 106
9.2 x 10"
5.2 x 10s
4.3 x 10s
8.4 x 10"
2.4 x 10s
1.7 x 10s
3.8 x 10"
3.4 x 10"
8.3 x 105
2.3 x 105
1.2 x 106
9.2 x 10s
6.8 x 10s
2.8 x 10s
3.7 x 105
ND
ND
9.8 x 105
5.2 x 106
7.3 x 105
4.6 x 106
4.8 x 105
9.3 x 10s
Pseudomonas
aeruginosa
MPN/lOOml
2.6 x 10s
1.4 x 10"
7.0 x 103
7.9 x 102
2.4 x 10"
1.7 x 103
1.4 x 103
1.3 x 102
ND
1.1 x 103
3.4 x 102
2.78 x 103
9.2 x 103
1.7 x 103
3.5 x 103
3.5 x 102
7.9 x 103
ND
3.3 x 103
1.6 x 10"
3.5 x 103
5.6 x 103
2.1 x 10"
1.4 x 103
Staph.
aureus
MPN/lOOml
ND
ND
1.3 x 101
7.9 x 101
7.8
1.5 x 102
1.3 x 102
1.2 x 102
9.3 x 101
1.1 x 103
LSL
3.6
LSL
LSL
LSL
5.6
2.6 x 101
ND
1.1 x 101
6.1
ND
LSL
9.3
LSL
Salmonella

MPN/10 liters
6V2 x 102
>1.1 x 10"
2.6 x 103
1.33 x 102
2.45 x 101
7.78
1.28 x 101
1.28 x 101
2.39 x 10J
6.12 x 102
2.39 x 101
LSL
7.8
2.6 x 101
1.7 x 101
1.1 x 101
1.2 x 101
ND
5.0
5.0
2.1 x 101
1.1 x 101
5.2 x 101
LSL
 Geom. Mean
                  2.4 x 105   8.1 x 10"    6.6 x 10s
             3.3 x 103    1.4 x 101
2.4 x 101
ND  - No Data
LSL - Lower sensitivity limit of the assay.
No microorganisms recovered.
                                          159

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APPENDIX B.  Levels  of Bacteria,  Station H - Howard Park
Date
10/16/74
11/05/74
11/12/74
12/16/74
01/06/75
01/11/75
01/13/75
01/20/75
02/05/75
02/23/75
03/12/75
04/03/75
04/15/75
05/01/75
05/06/75
06/11/75
06/30/76
07/10/75
07/14/75
07/27/75
08/06/75
08/13/75
09/12/75
09/18/75
Gcom. Mean
Run
Number
13
17
19
23
27
28
29
30
33
35
37
40
42
44
45
49
51
53
54
56
58
59
62
64

Total
Collform
MPN/lOOml
1.7 x 10s
3.5 x 10s
4.9 x 103
3.5 x 106
3.5 x 10s
5.4 x 10s
2.4 x 10s
7.9 x 106
ND
1.7 x 10s
7.9 x 10s
2.8 x 107
2.4 x 106
5.4 x 10s
2.2 x 106
ND
3.5 x 106
7.9 x 106
3.3 x 10s
2.8 x 106
3.5 x 10s
7.0 x 10s
3.5 x 106
5.6 x 101*
1.2 x 106
Fecal
Coliform
MPN/lOOml
4.9 x 101*
7.0 x 101*
2.3 x 103
1.1 x 10s
7.9 x 10s
4.9 x 101*
2.4 x 106
2.3 x 105
ND
7.9 x 10s
2.2 x 10s
2.9 x 106
2.4 x 106
2.4 x 10s
4.0 x 101*
ND
2.4 x 10s
8.0 x 10s
2.3 x 105
1.7 x 10s
4.1 x 101*
1.3 x 10s
7.0 x 105
3.5 x 105
4.5 x 10s
Fecal
Streptococci
no. /100ml
1.5 x 10s
3.2 x 10s
1.0 x 103
7.2 x 105
1.4 x 10s
1.7 x 10s
3.5 x 10s
3.7 x 105
3.0 x 103
8.7 x 105
2.4 x 10s
1.4 x 106
7.0 x 10s
5.1 x 10s
8.1 x 10s
ND
ND
6.7 x 105
1.7 x 10s
1.2 x 10s
4.9 x 10s
1.7 x 105
7.1 x 105
6.3 x 101*
2.4 x 105
Pseudomonas
aeim.gi.no8a
MPN/lOOml
5.4 x 10s
1.7 x 101*
1.7 x 104
7.0 x 103
1.8 x 103
1.1 x 101*
2.8 x 101*
2.4 x 101*
1.8 x 103
1.3 x 101*
4.6 x 103
>1.6 x 101*
4.0 x 103
2.4 x 103
3.5 x 103
ND
1.41 x 101*
1.41 x 102
3.5 x 102
8.0 x 101
1.7 x 103
3.7 x 103
9.2 x 101*
3.1 x 103
5.2 x 103
Staph.
aupeus
MPN/lOOml
ND
9.2 x 102
1.7 x 101
1.3 x 102
3.5 x 102
4.6 x 102
1.5 x 102
1.6 x 101
9.1 .
2.1 x 102
4.6 x 102
9.3
1.1 x 101
9.3 x 101
4.6 x 102
ND
8.2
LSL
2.2 x 101
1.7 x 101
1.8
1.8
1.3 X 101
5.5
3.6 x 101
Salmonella
MPN/10 liters
6.2 x 102
1.4 x 103
3.5 x 101
1.50 x 101
1.33 x 101
3.89
>1.33 x 103
6.12 x 102
6.12 x 102
1.72 x 101
2.56 x 102
>1.334 x 103
1.2 x 103
2.7 x 102
1.2 x 103
ND
1.3 x 103
1.1 x 101
1.3 x 102
1.3 x 103
8.3 x 101
1.3 x 103
6.1 x 102
5.0
1.4 x 102
ND - No Data
LSL - Lower sensitivity limit of the assay.
No microorganisms recovered.
                                        160

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APPENDIX B.  Levels  of Bacteria,  Station R - Jones Falls Storm Drain


Date
10/16/74
11/05/74
11/12/74
12/16/74
01/06/75
01/11/75
01/13/75
01/20/75
02/05/75
02/23/75
03/12/75
04/03/75
04/15/75
05/01/75
05/06/75
06/11/75
06/30/75
07/10/75
07/14/75
07/27/75
08/06/75
08/13/75
09/12/75
09/18/75

Run
Number
13
17
19
23
27
28
29
30
33
35
37
40
42
44
45
49
51
53
54
56
58
59
62
64
Total
Coliform
MPN/lOOml
>1.6 x 106
>2.4 x 106
5.4 x 106
1.7 x 105
3.3 x 101*
1.6 x 10s
9.2 x 104
3.3 x 105
1.7 x 10s
1.7 x 106
1.4 x 10s
1.3 x 106
1.3 x 106
1.1 x 106
7.9 x 104
7.9 x 104
1.6 x 10s
3.3 x 10s
1.3 x 106
1.1 x 10s
9.2 x 104
2.2 x 105
9.2 x 10s
ND
Fecal
Coliform
MPN/lOOml
>1.6 x 10s
1.3 x 104
3.5 x 10s
4.9 x 101*
3.3 x 104
1.1 x 105
9.2 x 10"
1.7 x 10s
5.0 x 103
7.0 x 10s
9.4 x 10k
4.9 x 105
1.3 x 106
1.3 x 105
3.3 x 104
3.5 x 101*
3.5 x 103
3.3 x 105
4.9 x 10s
4.9 x lO4
2.4 x 104
4.9 x 104
5.4 x 105
ND
Fecal
Streptococci
no ./100ml
2.7 x 10s
4.6 x 105
8.0 x 105
3.4 x 10s
1.6 x 105
7.9 x 10s
2.5 x 10s
6.9 x 104
1.4 x 10s
7.5 x 105
1.3 x 10s
2.4 x 105
1.8 x 105
3.4 x 105
2.5 x 10s
5.5 x 10s
ND
3.7 x 10s
8.3 x 105
7.2 x 101*
5.5. x 10s
4.7 x 104
2.9 x 105
7.6 x 105
Pseudomonas
aevuginosa
MPN/lOOml
7.0 x 104
1.7 x 103
1.6 x 105
3.3 x 103
9.4 x 102
5.4 x 103
1.1 x 101*
9.2 x 103
1.6 x 10s
5.4 x 103
1.1 x 103
4.6 x 103
1.4 x 103
2.4 x 103
9.2 x 103
2.8 x 103
1.09 x 104
2.6 x 103
3.1 x 103
1.8 x 103
3.3 x 103
6.4 x 103
1.1 x 104
2.1 x 104
Staph.
aureus
MPN/lOOml
ND
2.7 x 102
1.6 x 103
1.7 x 102
7.0 x 101
1.5 x 101
1.4 x 101
1.5 x 102
4.6 x 102
2.4 x 102
9.3 x 101
4.6 x 102
1.6 x 101
1.5 x 102
9.3 x 101
3.7
2.1 x 101
6.8
3.4 x 101
1.0 x 101
1.8
1.8
1.1 x 101
1.8
Salmonella

MPN/10 liters
3.3
9.4 x 101
1.3 x 102
5.00
6.12
2.39 x 101
1.33 x 102
1.33 x 103
1.67
LSL
2.22
1.17 x 101
2.6 x 101
4.1 x 101
1.0 x 102
ND
1.3 x 103
6.1 x 102
2.1 x 101
3.9
1.7
: 1.3 x 102
6.1 x 102
1.7 x 101
 Geom. Mean
                  2.9 x 105   1.2 x 105    2.8 x 105
             6.6 x 103    4.0 x 101
2.5 x 101
 ND  - No Data
 LSL - Lower sensitivity limit of the assay.
No microorganisms recovered.
                                          161

-------
APPENDIX B.   Levels of Bacteria,  Station L  - Bush Street
Date
10/16/74
11/05/74
11/12/74
12/16/74
01/06/74
01/11/75
01/13/75
01/20/75
02/05/75
02/23/75
03/12/75
04/03/75
04/15/75
05/01/75
05/06/75
06/10/75
06/30/75
07/10/75
07/14/75
07/27/75
08/06/75
08/13/75
09/12/75
09/18/75
Run
Number
13
17
19
23
27
28
29
30
33
35
37
40
42
44
45
49
51
53
54
56
58
59
62
64
Total
Colifonn
MPN/lOOml
2.4 x 10s
3.5 x 101*
5.4 x 10"
1.1 x 10s
1.1 x 10s
9.2 x 10s
1.1 x 106
>2.4 x 106
5.4 x 106
7.9 x 103
5.4 x 105
9.4 x 10s
2.21 x 10s
3.3 x 10s
3.5 x 10s
7.9 x 101*
2.4 x 10s
7.9 x 10s
1.1 x 10s
3.5 x 10s
1.6 x 10s
>1.6 x 10s
5.4 x 10s
1.7 x 106
Fecal
Coliform
MPN/lOOml
4.9 x 101*
1.7 x 103
7.9 x 103
7.0 x 101*
4.9 x 10"
1.1 x 104
2.6 x 10s
>2.4 x 10s
1.7 x 101*
7.9 x 103
5.4 x 101*
7.0 x 10s
1.41 x 10s
8.0 x 1011
2.3 x 101*
9.4 x 103
1.6 x 106
4.9 x 105
3.1 x 105
7.0 x 104
1.4 x 10"
1.7 x 105
3.5 x 10s
3.3 x 10s
Fecal
Streptococci
no. /100ml
3.8 x 10s
5.0 x 103
4.3 x 103
1.2 x 10s
6.5 x 10s
1.4 x 105
3.5 x 105
7.6 x 10s
2.4 x 105
2.5 x 103
9.4 x 10s
1.2 x 106
3.6 x 10s
8.5 x 10s
1.9 x 10s
5.3 x 10s
ND
8.4 x 10s
5.2 x 105
3.8 x 106
4.1 x 105
7.2 x 105
1.1 x 106
1.4 x 106
Pseudomonas
aeruginosa
MPN/lOOml
7.5 x 104
1.1 x 102
1.7 x 103
2.3 x 103
4.9 x 102
2.2 x 103
1.3 x 10"
3.3 x 102
ND
1.7 x 102
1.3 x 103
9.2 x 103
1.10 x id"
5.4 x 102
3.5 x 103
3.5 x 103
2.21 x 101*
3.45 x 103
3.8 x 101
2.2 x 103
4.9 x 103
4.5 x 103
2.2 x 10"
7.8 x 102
Staph.
OUTBU8
MPN/lOOml
ND
7.9 x 101
7.0 x 101
2.3 x 101
9.3
7.3
4.3 x 101
4.6 x 102
4.3 x 102
LSL
9.3 x 101
LSL
1.5 x 101
3.6
2.4 x 102
2.9 x 101
4.0
ND
9.2
5.5
1.8
LSL
1.4 x 101
LSL
Salmonella
MPN/10 liters
1.8 x 101
6.7
8.3
8.34
1.56 x 101
3.89
1.33 x 102
1.50 x 101
5.00
LSL
2.56 x 102
2.06 x 101
5.1 x 102
5.1 x 102
5.1 x 102
8.3
1.3 x 103
3.9
5.0
LSL
1.8 x 101
1.3 x 103
1.3 x 103
1.2 x 102
Geom. Mean
3.8 x 10s   8.3 x 10"    5.6 x 10s
ND  - No Data
LSL - Lower sensitivity limit of the assay.
2.0 x 103   1.2 x 102
                      No microorganisms recovered.
                                                                             3.0 x 101
                                         162

-------
 APPENDIX B.   Levels  of Bacteria,  Station M - Northwood
Date
12/16/74
01/06/75
01/11/75
01/13/75
01/20/75
02/05/75
02/23/75
03/12/75
04/03/75
04/15/75
05/01/75
05/06/75
06/11/75
06/30/75
07/10/75
07/14/75
07/27/75
08/06/75
08/13/75
09/12/75
09/18/75
Geom. Mean
Run
Number
23
27
28
29
30
33
35
37
40
42
44
45
49
51
53
54
56
58
59
62
64

Total
Coliform
MPN/lOOml
2.4 x 10"
3.3 x 10"
2.4 x 103
1.7 x 10s
4.6 x 103
1.7 x 10"
5.4 x 10"
1.3 x 103
3.48 x lb"
4.6 x 10"
1.7 x 10"
1.3 x 10"
5.4 x 10"
1.1 x 10s
ND
1.4 x 10E
7.0 x 10"
3.5 x 10"
3.5 x 10"
1.4 x 105
3.5 x 10s
3.8 x 10"
Fecal
Coliform
MPN/lOOml
1.3 x 10"
7.9 x 103
8.0 x 101
7.9 x 10"
4.0 x 102
<2.0 x 102
1.3 x 10"
2.0 x 102
1.4 x 103
2.0 x 103
1.1 x 10"
2.0 x 102
1.3 x 102
4.0 x 10"
ND
3.3 x 10"
2.3 x 10"
2.4 x 10"
3.5 x 10"
9.2 x 10"
2.4 x 10s
6.9 x 103
Fecal
Streptococci
no. /100ml
1.0 x 10s
1.7 x 103
1.8 x 10"
ND
4.4 x 10"
6.0 x 103
3.6 x 10"
1.7 x 10"
3.1 x 10"
1.2 x 10"
4.1 x 10"
1.7 x 10s
3.0 x 10s
ND
ND
1.1 x 10s
3.7 x 10s
1.5 x 10s
1.1 x 10"
1.1 x 10s
2.0 x 10s
5.0 x 10"
Pseudomanas
aeruginosa
MPN/lOOml
1.8 x 102
4.9 x 102 .
1.7 x 103
3.5 x 103
2.3 x 102
1.1 x 103
3.5 x 102
9.2 x 103
7.0 x 102
2.8 x 102
1.7 x 101
3.4 x 102
7.9 x 103
ND
<8.0
1.1 x 10"
2.3 x 103
1.7 x 10"
2.6 x 103
1.2 x 103
5.9 x 102
Staph.
aureus
MPN/lOOml
2.3 x 101
2. a
7.0
4.3 x 101
4.6 x 102
4.3 x 101
LSI,
9.3 x 101
LSL
1.5 x 101
4.0
2.4 x 102
2.4 x 102
4.0
ND
LSL
LSL
LSL
LSL
1.4 x 101
LSL
1.2 x 101
Salmonella
MPN/10 liters
LSL
LSL
5.00
LSL
LSL
7.78
LSL
2.22
LSL
7.8
4.3 x 101
LSL
LSL
1.7
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2.6 x 102
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5.2 x 101
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3.3
5.7
ND - No Data
LSL - Lower sensitivity limit of the assay. No microorganisms recovered.
                                        163

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                                     GLOSSARY


background samples:  Water samples collected on a routine basis, regardless of
     rainfall to obtain background information on the microbiol levels in the
     urban aquatic environment.  In this study, the background samples consisted
     of raw sewage, a reservoir and three urban streams.

bleeder:  Intentional sanitary sewage overflow from sewage interceptors.  The over-
     flows are diverted directly ot indirectly into the storm drainage system.

combined sewer:  A sewer intended to receive both wastewater and storm ar surface
     runoff.

dry weather flow:  The flow in storm or sanitary sewers that contains no stormwater.

enterococci:  Members of the fecal streptococcal group containing the species S
     facoal-Ls and s. faeoium.

F.C.:   fecal coliform

first  flush:  The initial  portion of a storm or combined  sewer  discharge.

F.S.:   fecal streptococci

grab sample:  A single sample  collected at  neither  a  set  time or  flow.

MPN:   Most  probable number - that number of  microorganisms per unit volume  that,
     in accordance with  stastical theory, would be  more  likely  than  any other
     number to  yield  the observed test  result.  The MPN  is generally computed from
     the number of positive  findings from a multiple  - portion  -  decimal dilution
     planting.

 stormwater:  The water  resulting from  a  precipitation  event which may  stay on the
     land surface, percolate into the ground,  runoff  into a body  of  water,  enter a
      storm-sewer or  enter a  combined sewer, infiltrate  a sanitary sewer or evaporate.

 stormwater  runoff:   The stormwater which  flows  overland.

 T.C:   Total coliform

 urban  stream:   A course  of running  water  flowing in a particular  direction in definite
      channel through an urban area  and  discharging into some  other  stream or body of
     water.

 urban runoff:   The stormwater  runoff which flow overland through urban areas.
                                           181

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                                   TECHNICAL REPORT DATA
                            (Please read Instructions on the reverse before completing)
 1, REPORT NO.
  EPA-600/2-77-087
                                    3. RECIPIENT'S ACCESSION-NO.
 4. TITLE AND SUBTITLE
 MICROORGANISMS IN URBAN STORMWATER
                                    5. REPORT DATE
                                      July 1977
(Issuing Date)
                                                           6. PERFORMING ORGANIZATION CODE
 7. AUTHOR(S)
  Vincent P.  Olivieri,
  Kazuyoshi Kawata
                                                           8. PERFORMING ORGANIZATION REPORT NO.
Cornelius W. Kruse,  and
9. PERFORMING ORGANIZATION NAME AND ADDRESS
  The Johns  Hopkins University
  School  of  Science & Public Health
  Department of Environmental Health
  615 N.  Wolfe Street, Baltimore. Maryland
                                    10. PROGRAM ELEMENT NO.

                                      1BC611
                                    11. CONTRACT/GRANT NO.

                                      R802709
                       21205
 12. SPONSORING AGENCY NAME AND ADDRESS
 Municipal Environmental Research Laboratory—Cin.,OH
 Office of Research and Development
 U.S. Environmental Protection Agency
 Cincinnati,  Ohio 45268 .	
                                    13. TYPE OF REPORT AND PERIOD COVERED
                                    14. SPONSORING AGENCY CODE

                                      EPA/600/14
 15. SUPPLEMENTARY NOTES

 Project Officer:   Richard Field, (201) 321-6674,  (8-340-6674)
 16. ABSTRACTMicrobiological quantitative assays  of  Baltimore City urban runoff were  con-
 ducted throughout a 12 month period to show the  relationships to several factors  such
 as separate  or combined sewer flow, urban  characteristics of drainage area, rainfall,
 and quantity of flow during and between rain  storms.   In general, there was a consis-
 tently high  recovery of both pathogenic and indicator organisms throughout the study '
 except for Shigella sp. which is believed  to  have  been present but could not be
 isolated due to interferences during the culture procedure.   There appeared to be
 little relationship between pathogen recovery and  season of  the year, amount of rain-
 fall, period of the antecedent rainfall, and  stream flow. The most concentrated
 pathogens were Pseudomonas aeruginosa and  Staphylococcus aureus at levels ranging from
 103 to 105 and from 10° to 103/100ml, respectively.   Salmonella and enteroviruses,
 though frequently isolated, were found at  levels of only W® to loVlO 1 of urban run-
 off.  The background samples (sewage, urban streams  and reservoirs) between storms
 gave good positive correlation between indicators  and pathogens at a 95 to 99% level
 of confidence,  whereas, the stormwater had no or poor correlation.  The ratios of
 indicators,  such as FC/FS, gave some indications of pollution by human sewage, but it
 was the presence of enteroviruses that definitely  showed the mixing of sewage with
 rain water,  whether in a storm sewer or in the combined sewer overflow.  The logical
 solution would point to the removal of sanitary  sewage overflows rather than the  dis-
 infection of all urban runoff for removing the health hazard and improving the quality
 7.
                               KEY WORDS AND DOCUMENT ANALYSIS
                  DESCRIPTORS
                                             b.lDENTIFIERS/OPEN ENDED TERMS  C. COS AT I Field/Group
 Microorganisms, Bacteria,  Viruses, Storm
 sewers, Streams,  Urban areas
                       Urban stormwater micro-
                       organisms, Pathogenic
                       microorganisms .enumera-
                       tion
     13  B
 8. DISTRIBUTION STATEMENT

 RELEASE TO PUBLIC
                       19. SECURITY CLASS (ThisReport/

                       UNCLASSIFIED	
 21. NO. OF PAGES
    194
                                             20. SECURITY CLASS (Thispage)
                                              UNCLASSIFIED
                                                                        22. PRICE
EPA Form 2220-1 (9-73)
                                           182
                                                    U. S. GOVERNMENT PRINTING OFFICE: 1977-757-056/6462 Region No. 5-11

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