Microbiological Water Quality in Relation 

to Water-Contact Recreation, Cuyahoga 

River, Cuyahoga Valley National Park, Ohio, 

2000 and 2002

By Rebecca N. Bushon and G.F. Koltun

In collaboration with the National Park Service, Cuyahoga Valley National Park

Water-Resources Investigations Report 03-4333

U.S. Department of the Interior

U.S. Geological Survey
U.S. Department of the Interior

Gale A. Norton, Secretary

U.S. Geological Survey

Charles G. Groat, Director

U.S. Geological Survey, Reston, Virginia: 2004

Box 25286, Denver Federal Center

Denver, CO 80225

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For sale by U.S. Geological Survey, Information Services
iii

Contents

Abstract.......................................................................................................................................................... 1

Introduction ................................................................................................................................................... 2

Purpose and Scope ............................................................................................................................. 2

Description of Study Area .................................................................................................................. 2

Previous Studies .................................................................................................................................. 4

Acknowledgments............................................................................................................................... 4

Overview of Microbiological Indicators and Pathogens ....................................................................... 4

Methods of Study.......................................................................................................................................... 7

Sample Collection................................................................................................................................ 8

Physical Characteristics and Chemical Constituents.................................................................... 8

Microbiological Methods ................................................................................................................... 8

Escherichia coli........................................................................................................................... 8

Salmonella ................................................................................................................................... 9

F-specific and somatic coliphage............................................................................................ 9

Enteric viruses............................................................................................................................. 9

Infectious enteroviruses by ICC-PCR ........................................................................... 10

Enteric viruses by RT-PCR.............................................................................................. 10

Clostridium perfringens ........................................................................................................... 10

Cryptosporidium and Giardia .................................................................................................. 10

Characteristics of Streamflows during Sampling Periods................................................................... 11

Analysis of Microbiological Data............................................................................................................. 11

General Trends and Patterns ........................................................................................................... 13

Relation of Escherichia coli Water-Quality Standards to Presence of Selected 

 Infectious Microorganisms................................................................................................ 14

Associations Between Bacteria, Viruses, and Protozoa ............................................................ 15

Relation of Streamflow and Water-Quality Measures to Bacteria, Viruses, and 

 Protozoa ................................................................................................................................ 19

Assessment of Between-Site Differences in Microbiological Characteristics ...................... 19

Summary and Conclusions........................................................................................................................ 21

References Cited ........................................................................................................................................ 23

Appendix A. Quality-control samples for the detection of enterovirus and hepatitis A 

 virus by means of the reverse transcriptase-polymerase chain reaction (RT-PCR) 

 method............................................................................................................................................... 26

Appendix B. Streamflow and microbiological data for Cuyahoga River samples and 

 Akron Water Pollution Control Station (WPCS) effluent samples collected in 2000

 and 2002............................................................................................................................................. 27
iv 

Figures

 1. Map showing the location of the study area in northeastern Ohio...................................... 3

 2–3. Boxplots showing:

 2. Distribution of instantaneous streamflows measured in conjunction 

 with samples collected from the Cuyahoga River at Old Portage, by 

 calendar year ....................................................................................................................... 11 

 3. Distribution of somatic coliphage, F-specific coliphage, infectious 

 enterovirus, and Clostridium perfringens concentrations as a 

 function of the presence of hepatitis A virus in Cuyahoga River samples ................ 17

 4. Scatterplots showing relation between streamflow and concentrations 

 of Escherichia coli and F-specific coliphage in Cuyahoga River samples ............... 18

 5–7. Boxplots showing:

 5. Distribution of pH as a function of site on the Cuyahoga River, 

 including the Akron Water Pollution Control Station..................................................... 19 

 6. Distribution of Escherichia coli concentrations as a function of 

 site on the Cuyahoga River, including the Akron Water Pollution 

 Control Station...................................................................................................................... 20

 7. Distribution of Clostridium perfringens concentrations as a function 

 of site on the Cuyahoga River, including the Akron Water Pollution 

 Control Station...................................................................................................................... 20

Tables

 1. Ohio water-quality standards for Escherichia coli in recreational waters......................... 5

 2. Microorganisms studied and methods used for analyses..................................................... 6

 3. Summary statistics for concentrations of Escherichia coli,

 Clostridium perfringens, F-specific and somatic coliphage, infectious 

 enterovirus, and Giardia at selected sites in the Cuyahoga River and 

 at the Akron Water Pollution Control Station......................................................................... 12

 4. Geometric mean Escherichia coli concentrations for samples collected 

 during May, July, and September 2000 at selected sites on the 

 Cuyahoga River........................................................................................................................... 13

 5. Numbers and percentages of time microorganisms were present 

 in Cuyahoga River samples when the indicated threshold 

 Escherichia coli concentrations were not exceeded........................................................... 14

 6. Numbers and percentages of time microorganisms were present 

 in Cuyahoga River samples when the indicated threshold 

 Escherichia coli concentrations were exceeded ................................................................. 15

 7. Correlation between microorganism concentrations in Cuyahoga 

 River samples and combined river and Akron Water Pollution Control 

 Station effluent samples............................................................................................................ 16
v

Multiply By To obtain

 Length

inch (in.) 25.4 millimeter (mm)

foot (ft) 0.3048 meter (m)

mile (mi) 1.609 kilometer (km)

 Area

square mile (mi2) 2.590 square kilometer (km2) 

 Volume

ounce, fluid (fl. oz) 0.02957 liter (L) 

gallon (gal) 3.785 liter (L) 

cubic inch (in3) 0.01639 liter (L)

 Flow rate

cubic foot per second (ft3/s) 0.02832 cubic meter per second (m3/s)

million gallons per day (Mgal/d) 0.04381 cubic meter per second (m3/s)

Temperature in degrees Celsius (°C) may be converted to degrees Fahrenheit (°F) as follows:

 °F = (1.8 × °C) + 32

Temperature in degrees Fahrenheit (°F) may be converted to degrees Celsius (°C) as follows:

 °C = (°F - 32) / 1.8

Specific conductance is given in microsiemens per centimeter at 25 degrees Celsius (μS/cm at 

25°C).

Turbidity is given in Nephelometric Turbidity Units (NTU).

Concentrations of chemical constituents in water are given either in milligrams per liter (mg/L) 

or micrograms per liter (μg/L).

Concentrations of bacteria in water are given in colonies per 100 milliliters (col/100 mL).

Concentrations of coliphage in water are given in plaques per 100 milliliters (plaques/100 mL).

Concentrations of infectious enteroviruses in water are given in most probable number per 100 

liters (MPN/100 L).

Concentrations of Cryptosporidium in water are given in oocysts per 10 liters (oocysts/10 L).

Concentrations of Giardia in water are given in cysts per 10 liters (cysts/10 L).

Conversion Factors and Abbreviated Water-Quality Units

Abstract

The microbiological water quality of a 23-mile segment 

of the Cuyahoga River within the Cuyahoga Valley National 

Park was examined in this study. This segment of the river 

receives discharges of contaminated water from stormwater, 

combined-sewer overflows, and incompletely disinfected 

wastewater. Frequent exceedances of Ohio microbiological 

water-quality standards result in a health risk to the public who 

use the river for water-contact recreation. 

Water samples were collected during the recreational 

season of May through October at four sites on the Cuyahoga 

River in 2000, at three sites on the river in 2002, and from the 

effluent of the Akron Water Pollution Control Station (WPCS) 

both years. The samples were collected over a similar range 

in streamflow in 2000 and 2002. Samples were analyzed for 

physical and chemical constituents, as well as the following 

microbiological indicators and pathogenic organisms: Escherichia coli (E. coli), Salmonella, F-specific and somatic coliphage, enterovirus, infectious enterovirus, hepatitis A virus, 

Clostridium perfringens (C. perfringens), Cryptosporidium, 

and Giardia. The relations of the microorganisms to each other 

and to selected water-quality measures were examined.

All microorganisms analyzed for, except Cryptosporidium, were detected at least once at each sampling site. 

Concentrations of E. coli exceeded the Ohio primary-contact 

recreational standard (298 colonies per 100 milliliters) in 

approximately 87 percent of the river samples and generally 

were higher in the river samples than in the effluent samples. 

C. perfringens concentrations were positively and significantly 

correlated with E. coli concentrations in the river samples and 

generally were higher in the effluent samples than in the river 

samples.

Several of the river samples that met the Ohio E. coli 

secondary-contact recreational standard (576 colonies per 100 

milliliters) had detections of enterovirus, infectious enterovirus, hepatitis A virus, and Salmonella, indicating that there are 

still risks even when the E. coli standard is not exceeded. River 

samples in which the secondary-contact recreational standard 

for E. coli was exceeded showed a higher percentage of the 

co-occurrence of pathogenic organisms than samples that met 

the standard. This indicates that in this study area, E. coli is a 

useful indicator of human health risk.

Detections of hepatitis A virus tended to be associated 

with higher median concentrations of somatic coliphage, 

F-specific coliphage, and infectious enterovirus. In addition, 

geometric mean C. perfringens concentrations tended to be 

higher in samples where hepatitis A virus was present than 

in samples where hepatitis A virus was absent. Hepatitis A 

virus was not detected in samples collected upstream from 

the Akron WPCS; all downstream detections had coincident 

detections in the Akron WPCS effluent, suggesting that Akron 

WPCS was a principal source of hepatitis A virus at the downstream sites.

Geometric mean concentrations of E. coli were calculated 

on the basis of analytical results from at least five samples 

collected at each river site during May, July, and September of 

2000. In each case, the Ohio geometric-mean primary-contact 

recreational standard of 126 col/100 mL was exceeded.

E. coli concentrations were significantly correlated with 

streamflow and increased with streamflow at sites upstream 

and downstream from the Akron WPCS. This indicates that 

E. coli loads from sources upstream from the Akron WPCS 

have the potential to appreciably influence the frequency of 

attainment of recreational water-quality standards at downstream locations.

Microbiological Water Quality in Relation to WaterContact Recreation, Cuyahoga River, Cuyahoga Valley 

National Park, Ohio, 2000 and 2002

By Rebecca N. Bushon and G.F. Koltun
2 Microbiological Water Quality, Cuyahoga River, Cuyahoga Valley National Park 

Introduction

A 23-mile segment of the Cuyahoga River that flows 

through the Cuyahoga Valley National Park (CVNP) receives 

discharges of stormwater, combined-sewer overflows, and 

incompletely disinfected wastewater from urban areas. These 

discharges can result in health risks to people who use the 

river for water-contact recreation. Escherichia coli (E. coli)

frequently is found in the Cuyahoga River at concentrations 

that exceed recommended maximum levels for water-contact 

recreation. When present in water, E. coli is an indicator of 

contamination from human and (or) animal feces. E. coli concentration has been shown to be a reliable factor in explaining rates of gastrointestinal illness in swimmers exposed to 

contaminated water (U.S. Environmental Protection Agency, 

1986) and is an indicator of the risk of exposure to other 

pathogenic organisms. Elevated levels of fecal-indicator organisms (such as E. coli) are cause for concern in any river but are 

of particular concern for the Cuyahoga River because of the 

CVNP and its goal of promoting water-contact recreation. 

A considerable amount of information is available on 

concentrations of E. coli and selected other indicator organisms in the Cuyahoga River within the CVNP; however, very 

little is known about the co-occurrence of waterborne pathogens and how they relate to concentrations of the indicator 

organisms. Information on the relation of indicator organisms 

and other environmental factors to the occurrence of waterborne pathogens within the Cuyahoga River is needed for 

a better understanding of the human-health risk associated 

with water-contact recreation; such information can aid park 

managers in formulating recommendations for recreational 

use of the Cuyahoga River. To meet these information needs, 

the U.S. Geological Survey (USGS), in collaboration with the 

National Park Service, conducted an in-depth study to investigate microbiological water quality of the Cuyahoga River 

within the CVNP. 

Purpose and Scope

This report describes the results of a study to (1) characterize the occurrence and distribution of selected microbiological pathogens and indicator organisms in the Cuyahoga River 

within the CVNP, (2) examine the relations of microorganisms 

to each other and to selected hydrologic and water-quality 

measures, and (3) provide general information on the occurrence of waterborne pathogens in relation to E. coli-based recreational-use water-quality standards. Data used in this study 

were collected during the May through October recreational 

seasons in 2000 and 2002 and consisted of analytical results 

for water samples collected from four sites in 2000 and three 

sites in 2002 on the Cuyahoga River within the CVNP and 

from Akron Water Pollution Control Station (WPCS) effluent. 

Samples collected during the 2000 recreational season were 

analyzed for concentrations and (or) the presence/absence of 

E. coli, Salmonella, F-specific coliphage, enterovirus, infectious enterovirus, hepatitis A virus, and selected chemical 

constituents. Samples collected during the 2002 recreational 

season were analyzed for concentrations and (or) the presence/

absence of E. coli, Salmonella, F-specific and somatic coliphage, enterovirus, infectious enterovirus, hepatitis A virus, 

Clostridium perfringens (C. perfringens), Cryptosporidium, 

Giardia, and selected chemical constituents.

Description of Study Area

The study area consists of a 23-mi segment of the 

Cuyahoga River, in northeastern Ohio, within the CVNP 

(fig. 1). The 51.6-mi2 CVNP, administered by the National 

Park Service (NPS), was established in 1974 to preserve and 

protect the natural and recreational values of the Cuyahoga 

River and adjacent lands (National Park Service, 2003a). 

According to the National Park Service (2003a), about 3.5 

million people visit the park annually; attendance peaks during 

the May–October recreational season.

The area surrounding the CVNP is predominantly urban 

and includes two nearby major metropolitan areas—Akron, 

which is upstream from the study area, and Cleveland, which 

is downstream from the study area. Most of the undeveloped 

land near the study area is confined to areas of steep terrain 

along the Cuyahoga River and tributaries draining into it 

(Schiefer, 2002). A detailed description of the Cuyahoga River 

and basin can be found in Myers and others (1998). 

The Ohio Environmental Protection Agency (Ohio EPA) 

has designated the segment of the Cuyahoga River within the 

CVNP as a warm-water habitat and assigned a recreational-use 

designation of primary-contact recreation. Primary-contact 

waters are classified as suitable for full-body contact such as 

swimming, canoeing, and scuba diving; however, because of 

poor water quality, these recreation activities are discouraged 

by the NPS, especially after periods of heavy precipitation 

(National Park Service, 2003b). 

Water quality of the Cuyahoga River within the CVNP is 

affected during wet weather by discharges of partially treated 

domestic sewage, intermittent discharges of storm water, and 

combined-sewer overflows (CSOs). Water quality also is 

affected by continuous discharge of treated municipal wastewater from the Akron WPCS and other upstream facilities. 

During the study period, the average daily effluent flow from 

the Akron WPCS was approximately 68 Mgal/d, and the maximum daily effluent flow was 207 Mgal/d (Donald Calvert, 

City of Akron, written commun., 2003). Effluent flows less 

than or equal to 110 Mgal/d receive secondary treatment, 

whereas wet-weather flows in excess of 110 Mgal/d receive 

only partial treatment (City of Akron, 2003). Peak effluent 

flow at the Akron WPCS can reach about 280 Mgal/d during 

wet weather (City of Akron, 2003). 
Introduction 3

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Figure 1. Location of the study area in northeastern Ohio.
4 Microbiological Water Quality, Cuyahoga River, Cuyahoga Valley National Park 

Previous Studies

The microbiological and chemical water quality and 

hydrology of the Cuyahoga River and tributaries have been 

described in previous reports (Childress 1984, 1985; Ohio 

Environmental Protection Agency, 1994, 1999; Francy and 

others, 1996; Myers and others, 1998). Concentrations of fecal 

bacteria that exceed Ohio’s bathing-water, primary-contact, 

and secondary-contact water-quality standards are well documented in the lower 50 mi of the Cuyahoga River (Shindel and 

others, 1992, 1993; Shindel and others, 1994; Ohio Environmental Protection Agency, 1994, 1999, 2003a). During dryweather periods investigated in 1992, the river typically met 

Ohio’s geometric-mean primary-contact recreational standard 

for fecal coliform bacteria (Ohio Environmental Protection 

Agency, 1994). In an earlier study, Childress (1985) documented elevated concentrations of fecal coliform bacteria in a 

reconnaissance during summer low-flow conditions in 1982. 

Fecal coliform concentrations ranged from 38 to 1,900,000 

col/100 mL.

The entire reach of the Cuyahoga river was assessed for 

water-quality impairment by the Ohio EPA as part of their 

statewide activities in 1991 (Ohio Environmental Protection 

Agency, 1994) and 1996 (Ohio Environmental Protection 

Agency, 1999). Improvements observed in microbiological water quality were attributed to improved sewage treatment and disinfection (Ohio Environmental Protection 

Agency, 1994). Biological and water-quality conditions in 

the Cuyahoga River reflected minimal change in 1996 (Ohio 

Environmental Protection Agency, 1999). The Ohio EPA identified the lower 50 mi. of the Cuyahoga River, which includes 

the study area, as a priority impaired water on the 1998 and 

2002 Section 303(d) lists required by the Clean Water Act. 

The Ohio EPA lists organic enrichment, nutrient enrichment, 

low instream dissolved oxygen, toxicity, sedimentation, and 

habitat degradation as the primary causes of impairments and 

lists point sources, nonpoint sources, and CSOs as sources of 

impairment (Ohio Environmental Protection Agency, 2003a).

During USGS field studies in 1991–93, the highest 

concentration of E. coli detected in the Cuyahoga River at the 

upstream boundary of the CVNP (Francy and others, 1993; 

Myers and others, 1998) was 2,400,000 col/100 mL. That 

sample concentration exceeded Ohio’s single-sample primarycontact recreational standard (298 col/100 mL) by a factor of 

more than 8,000. The Akron WPCS was identified as the largest source of fecal bacteria to the Cuyahoga River within the 

CVNP during runoff periods when large volumes of influent 

result in inadequately disinfected effluent (Myers and others, 

1998). The next largest source of fecal bacteria was uncontrolled combined and sanitary sewers that also discharged to 

the river upstream from the park. 

Acknowledgments

The authors thank Meg Plona, Garree Williamson, Brian 

McHugh, John Debo, and William Carroll of the National Park 

Service; Donald Calvert of the Akron Water Pollution Control Station; and Steven Davis, former Cuyahoga American 

Heritage River Navigator for their assistance during all data 

collection and report-review phases of the project. 

Overview of Microbiological 

Indicators and Pathogens

The presence of pathogenic microorganisms in recreational waters is of great concern. Monitoring the waters for 

specific pathogens would be ideal but, in practice, is unrealistic because of constraints of time, cost, available methods, laboratory capabilities, and the multitude of pathogens involved 

in waterborne-disease outbreaks. Instead, indicator organisms 

traditionally have been used to assess the microbiological 

quality of water and indicate the potential for pathogens to 

be present. Fecal-indicator organisms are excreted in large 

numbers by humans and warmblooded animals; therefore, the 

concentrations of indicator organisms in fecally contaminated 

water generally are much higher and more consistent than 

those of pathogens (Moe, 2002). The numbers of pathogens, 

which are excreted only by infected individuals, depend on the 

excretion level and the number of infected individuals in the 

area. Relative to methods for detecting pathogens, methods to 

detect indicator organisms are generally much easier and less 

costly to perform, are easily adaptable by most laboratories, 

and produce results that are available more quickly. This overview section of the report discusses indicator organisms and 

pathogens of interest to this study and makes brief mention of 

analytical approaches for each.

In 1986, the U.S. Environmental Protection Agency 

(USEPA) recommended the use of E. coli as a bacterial indicator organism for monitoring microbiological water quality in 

freshwater. This recommendation was based on epidemiological studies that showed a strong positive correlation between 

E. coli and the occurrence of swimming-associated gastrointestinal illness (U.S. Environmental Protection Agency, 1986). 

The Ohio water-quality standards for E. coli in recreational 

waters are listed in table 1 (Ohio Environmental Protection 
Overview of Microbiological Indicators and Pathogens 5

Agency, 2003b). Relative to many viral and protozoan pathogens, E. coli has a shorter survival time in water and a greater 

susceptibility to water-treatment processes. As a consequence, 

E. coli tends to be a poor indicator for viral and protozoan 

pathogens (Moe, 2002). Other microorganisms, including coliphages and C. perfringens, are being investigated as alternative 

indicators that may better model the survival and disinfection 

resistance of viruses and protozoa (Moe, 2002).

Coliphages currently are used as indicators of fecal 

contamination and of the microbiological quality of water. 

Coliphages are viruses that infect and replicate in coliform 

bacteria. They are similar to pathogenic human enteric viruses 

in size and shape, transport characteristics, and resistance to 

disinfection processes. F-specific and somatic coliphages are 

two main groups of coliphages used as indicator organisms. 

They differ in how they infect the coliform bacteria. F-specific 

coliphage infect the bacteria by attaching to hairlike projections called F pili, and somatic coliphages infect the bacteria 

by attaching to the outer cell wall. Both groups of coliphage 

are found in high numbers in sewage and are thought to be 

reliable indicators of sewage contamination of waters (International Association of Water Pollution Research and Control 

Study Group on Health Related Microbiology, 1991). Raw 

sewage typically contains F-specific and somatic coliphage 

concentrations of about 1,000 plaques per milliliter (Sobsey 

and others, 1995). A single-agar layer (SAL) direct plating 

method can be used to enumerate F-specific and somatic 

coliphage in water samples (Ijzerman and Hagedorn, 1992). 

Results from this method are available in 24 hours. 

C. perfringens has been proposed as an indicator of 

the presence and density of pathogenic viruses and other 

stress-resistant microorganisms, such as Cryptosporidium

and Giardia (U.S. Environmental Protection Agency, 1996). 

C. perfringens is a bacterium of fecal origin that is consistently associated with human waste and sewage (Bisson and 

Cabelli, 1980; Fujioka and Shizumura, 1985). It produces 

spores that are resistant to disinfection practices and environmental stresses; it is an indicator of present contamination, 

as well as a conservative tracer of past fecal contamination 

(U.S. Environmental Protection Agency, 1996). The method 

for detecting C. perfringens is a standard membrane filtration 

procedure that is similar to that for E. coli and produces results 

in 24 hours. The method requires overnight incubation in an 

anaerobic environment.

In addition to the organisms discussed previously, 

samples collected for this study were analyzed for selected 

pathogens that are of concern in recreational waters. Concentrations of infectious enterovirus, Cryptosporidium, and 

Giardia were determined, as was the presence or absence of 

Salmonella, enterovirus, and hepatitis A virus. The microorganisms analyzed for and the methods used in this study are 

summarized in table 2.

Salmonella continues to be one of the main causes of 

waterborne illness worldwide (Arvanitidou and others, 1995). 

Every year in the United States, there are approximately 

800,000 to 4 million cases of salmonellosis, which result in 

[Effective from May 1 through October 15. All values are in colonies per 100 milliliters; na, not applicable. Source: Ohio Environmental 

Protection Agency, 2003b]

Type of recreational water

Bathing watersa Primary contactb Secondary contactc

Geometric meand 126 126 na

Single samplee 235 298 576

a

 Bathing waters are suitable for swimming and other full-body-contact exposure where a lifeguard or bathhouse is present.

b

 Primary-contact waters are suitable for full-body contact, such as swimming, canoeing, and scuba diving.

c

 Secondary-contact waters are suitable for partial-body contact, such as wading. 

d

 The geometric mean is based on a minimum of five samples in a 30-day period.

e

 This value cannot be exceeded in more than 10 percent of the samples collected in a 30-day period.

Table 1. Ohio water-quality standards for Escherichia coli in recreational waters.

Type of standard
6 Microbiological Water Quality, Cuyahoga River, Cuyahoga Valley National Park 

about 500 deaths (Centers for Disease Control and Prevention, 

1999). In Ohio, occurrence of salmonellosis increases slightly 

during midsummer, with most cases being reported in children 

under 5 years, adults 20–39 years, and adults over 60 years; 

however, all ages are at risk (Ohio Department of Health, 

1993b). Humans can acquire Salmonella from the ingestion of 

contaminated food or water or by direct contact with infected 

animals. Symptoms of salmonellosis include acute gastrointestinal illness, characterized by headache, diarrhea, fever, 

and vomiting. Infection can progress from gastrointestinal 

illness to septicemia or a focal infection; for example, meningitis (Ohio Department of Health, 1993b). One method for 

determining the presence or absence of Salmonella involves 

concentration by membrane filtration, selective enrichment in 

a growth medium, screening for morphological and biochemical characteristics, and serotyping (American Public Health 

Association and others, 1998, p. 9–22 to 9–25). 

More than 100 types of human pathogenic enteric viruses 

may be present in fecal-contaminated waters (Havelaar and 

others, 1993). Viruses generally are more persistent in the 

environment than bacteria and are not removed completely by 

common treatment processes. The enteric viruses examined 

in this study were enterovirus and hepatitis A virus. Enteroviruses account for an estimated 10–15 million symptomatic 

infections in the United States each year (Centers for Disease 

Control and Prevention, 2000). These viruses can cause a 

variety of illnesses ranging from gastroenteritis to myocarditis 

and aseptic meningitis (Melnick, 1990). Enteroviruses in the 

environment pose a health risk because they are transmitted through the fecal-oral route through contaminated water, 

and low numbers are able to initiate an infection in humans 

(Abbaszadegan and others, 1993). 

Hepatitis A occurs worldwide with a higher prevalence 

in developing nations, where sanitary conditions are poor. In 

the United States, 17,147 cases of hepatitis A were reported 

to the Centers for Disease Control and Prevention in 1999 

(Centers for Disease Control and Prevention, 2003b). Ohio 

reports approximately 800 cases of the disease each year 

(Ohio Department of Health, 2000). The hepatitis A virus is 

transmitted by the fecal-oral route. Symptoms can include 

Analytical 

laboratory

Volume 

analyzed

 Escherichia coli Membrane 

 filtration on

 mTEC agar

 USGS ODML / 

 Akron WPCS

 0.1 to 30 mL Colonies per 

 100 mL

 USEPA, 1985

 Salmonella Enrichment USGS National

 Wildlife

 Health Center

 100 mL Presence or 

 absence per

 100 mL

 APHA and 

 others, 1998

 F-specific and

 somatic

 coliphage

 Single-agar

 layer

 USGS ODML 100 mL Plaques per 

 100 mL

 Ijzerman and

 Hagedorn, 

 1992

 Infectious 

enterovirus

 ICC-PCR EHL 100 L MPN per 100 L Reynolds and

 others, 1996

 Enterovirus RT-PCR USGS ODML 100 L Presence or

 absence per

 100 L

 Fout and others, 

 2003

 Hepatitis A virus RT-PCR USGS ODML 100 L Presence or

 absence per

 100 L

 Fout and others,

 2003

 Clostridium 

perfringens

 Membrane 

 filtration on

 mCP agar

 USGS ODML 0.1 to 30 mL Colonies per 

 100 mL

 USEPA, 1996

 Cryptosporidium USEPA Method

 1623

 EHL 10 L Oocysts per 

 10 L

 USEPA, 2001

 Giardia USEPA Method

 1623

 EHL 10 L Cysts per 10 L USEPA, 2001

[USGS, U.S. Geological Survey; ODML, Ohio District Microbiological Laboratory; WPCS, Water Pollution Control Station; mL, milliliters; 

USEPA, U.S. Environmental Protection Agency; APHA, American Public Health Association; ICC-PCR, integrated cell culture-polymerase chain 

reaction; EHL, Environmental Health Laboratories; MPN, most probable number; L, liters; RT-PCR, reverse transcriptase-polymerase chain reaction]

Table 2. Microorganisms studied and methods used for analyses.

Microorganism Method Reporting units Reference
Methods of Study 7

fever, malaise, anorexia, nausea, abdominal discomfort, and 

jaundice. 

Several methods are available for examining water for 

enteric virus contamination, all with limitations. Two of the 

most common methods are cell culture and reverse transcriptase-polymerase chain reaction (RT-PCR). The cell culture 

method provides information on the infectivity of the viruses, 

but it is expensive and time consuming, requiring several 

weeks for confirmed positive results. Not all enteric viruses 

of public-health concern can be detected by this method, and 

specific strains of viruses in the water samples cannot be 

determined. Use of this method can be further complicated by 

the presence of toxic substances in the sample, which could 

interfere with the analysis. The RT-PCR method is more rapid 

and less expensive than the cell culture method and allows 

for detection of the small numbers of viruses usually found in 

environmental samples. This method can determine specific 

virus strains and can detect viruses that are unable to grow in 

cell culture. The disadvantages to the RT-PCR method are the 

inability to determine the infectivity of the viruses (the viruses 

do not have to be intact to be detected) and the sensitivity to 

inhibitory compounds that may be present in the sample and 

would prevent completion of the analysis.

An integrated cell culture-PCR (ICC-PCR) method can 

be used to determine concentrations of infectious enteroviruses. The ICC-PCR method combines the cell culture and 

RT-PCR methods to reduce the effects of toxic compounds on 

cell culture and inhibitory compounds on PCR. This method 

increases the sample volume examined and the chance for a 

more rapid detection of infectious viruses compared to the cell 

culture method (Reynolds and others, 1996).

Protozoan pathogens are distributed widely in the aquatic 

environment and have been implicated in several outbreaks of 

waterborne disease (Lee and others, 2002; Rose and others, 

1997). The principal protozoan pathogens that affect the public 

health acceptability of waters in the United States are Cryptosporidium and Giardia. Both organisms produce environmentally resistant forms, which facilitate their extended survival in 

water. These pathogens are transmitted through the fecal-oral 

route. The infectious dose for both organisms is very small, at 

approximately 10 organisms.

Cryptosporidiosis and giardiasis occur worldwide. In the 

United States, 3,785 cases of cryptosporidiosis were reported 

to the Centers for Disease Control and Prevention in 2001 

(Centers for Disease Control and Prevention, 2003a). Symptoms of cryptosporidiosis include diarrhea, stomach cramps, 

and fever. In Ohio, there is a seasonal peak of giardiasis in late 

summer, with most cases occurring in children under 5 years 

and in adults 30–39 years; however, all ages are at risk (Ohio 

Department of Health, 1993a). Symptoms include chronic 

diarrhea, cramps, bloating, and weight loss. USEPA Method 

1623 is used to detect Cryptosporidium and Giardia in water 

by filtration, immunomagnetic separation (IMS), and immunofluorescence assay (FA) microscopy.

Methods of Study

Samples were collected at five sites in 2000: (1) 

Cuyahoga River upstream from the CVNP at Old Portage (site 

1 in fig. 1), (2) effluent from the Akron WPCS (site 2 in fig. 

1), (3) Cuyahoga River at the upstream boundary of the CVNP 

at Botzum (site 3 in fig. 1), (4) Cuyahoga River midway 

through the CVNP at Jaite (site 4 in fig. 1), and (5) Cuyahoga 

River at the downstream end of the CVNP at Independence 

(site 5 in fig. 1). An appreciable amount of streamflow and 

water-quality data is available for these sites because they 

were sampled as part of a previous study on the Cuyahoga 

River (Myers and others, 1998). 

Five samples were collected at each of the five sampling 

sites during May, July, and September of 2000 and were analyzed for E. coli. The presence or absence of Salmonella was 

determined in samples collected once in May and twice in July 

and September at each site. Water samples to determine waterquality characteristics and F-specific coliphage concentrations 

were collected once in May, July, and September at each site. 

Because of the high cost of analysis for enterovirus, infectious 

enterovirus, and hepatitis A virus, samples were collected at 

only three sites (Old Portage, the Akron WPCS, and Botzum) 

during the three sampling months. Samples were collected 

each month for suspended-sediment concentration and chemical water quality. Field measurements of temperature, pH, specific conductance, and dissolved oxygen were made each time 

samples were collected. Streamflow was obtained from USGS 

streamflow-gaging stations, where available. Where there were 

no daily streamflow gages, streamflow was determined from 

rating curves on the basis of water-level measurements made 

with a wire-weight gage. 

In 2002, samples were collected at four of the five 

sampling sites; Old Portage, the Akron WPCS, Botzum, and 

Jaite. (One site from 2000 was dropped becaue of fiscal and 

logistical constraints.) Water samples were collected during three sampling periods, one in May, June, and July 2002. 

During each period, samples were collected for determination 

of the following constituents: E. coli, Salmonella, F-specific 

and somatic coliphage, enterovirus, infectious enterovirus, 

hepatitis A virus, C. perfringens, Cryptosporidium, Giardia, 

suspended sediment, turbidity, and chemical water quality. 

Field measurements also were made, and streamflow data 

were obtained from USGS streamflow-gaging stations where 

available or determined from rating curves. 
8 Microbiological Water Quality, Cuyahoga River, Cuyahoga Valley National Park 

Sample Collection

Stream-water samples were collected by USGS personnel by means of the equal-width-increment method described 

by Edwards and Glysson (1999). Samples for the analysis of 

E. coli, Salmonella, and turbidity were collected in sterile 1-L 

polypropylene bottles. Samples for the analysis of coliphage 

and C. perfringens were collected in sterile 3-L polypropylene bottles. Stream water to be analyzed for Cryptosporidium

and Giardia was composited into a 10-L sterile cubitainer. 

Samples for the analysis of suspended sediment and chemical 

constituents were composited in a churn splitter (Wilde and 

Radtke, 1999).

Samples from the Akron WPCS effluent were collected in the center of the outfall using the hand-dip method 

described by Myers and Sylvester (1997). During the recreational season, the effluent from the Akron WPCS is dechlorinated before it is discharged; therefore, further steps to 

dechlorinate the samples before microbiological analysis were 

not necessary.

A portable virus-sampling apparatus designed by USGS 

engineers was used to sample for enteric viruses. The sampler 

design was based on the protocol described in the USEPA 

Information Collection Rule (U.S. Environmental Protection 

Agency, 1996). The apparatus contained a submersible pump, 

pressure regulator, cartridge housing containing a 1MDS filter 

(Cuno Inc., Meriden, Conn.), and discharge line. Samples analyzed for enteric viruses were collected by inserting a sterile 

1MDS filter into the cartridge housing of the virus sampler 

and pumping 100 L of water through the filter. 

Because of the complexity of the virus-sampling apparatus, stringent disinfection protocols were established. Before 

sampling and after each use, the sampling equipment was 

cleaned with a dilute, nonphosphate, laboratory-grade detergent and then sterilized with a 10 percent household bleach 

(0.5 percent sodium hypochlorite) solution. The sampler 

was rinsed with a 0.2 percent sodium thiosulfate solution to 

remove residual chlorine and then was rinsed with sterile 

deionized water. 

Physical Characteristics and Chemical 

Constituents

Water and air temperature, pH, specific conductance, and 

dissolved oxygen were measured at each site when samples 

were collected. Water-quality meters were calibrated daily 

in the field before use. Streamflow data were obtained from 

USGS streamflow-gaging stations where available or determined from rating curves. Instantaneous flow from the Akron 

WPCS (provided by the City of Akron) was based on a standard flume computation.

Turbidity was measured in the field by means of a Hach 

2100P portable turbidimeter (Hach Company, Loveland, 

Colo.). Measurements of each sample were made in duplicate until two consecutive measured values agreed within 

5 percent. Turbidimeter calibration was checked daily against 

turbidity standards before use in the field.

Suspended-sediment samples were sent to the Heidelberg 

College Water-Quality Laboratory in Tiffin, Ohio, for analysis 

by means of the filtration method described by Guy (1969). 

Nutrient and chloride samples were processed in the field 

and then sent overnight on ice to the USGS National WaterQuality Laboratory (NWQL) in Lakewood, Colo. Phosphorus 

samples were collected in polyethylene bottles and acidified with 1 mL of 4.5 N sulfuric acid. Nitrogen and chloride 

samples were filtered through a 0.45-μm filter and collected in 

polyethylene bottles. Quality-control samples for nutrients and 

chloride included one field blank and one replicate for each 

constituent. No problems were evident from the quality-control results.

Microbiological Methods

All field and laboratory personnel were trained in sample 

collection and analysis procedures. Quality-assurance and 

quality-control procedures (Francy and others, 1998; Myers 

and Sylvester, 1997) were followed for all phases of the project. Unless indicated otherwise, no problems were observed 

in quality-control samples analyzed for microorganisms 

described below. The microbiological methods used in this 

study are summarized in table 2.

Escherichia coli

Water samples were analyzed for concentrations of 

E. coli by membrane filtration using the USEPA-recommended mTEC agar method (U.S. Environmental Protection 

Agency, 1985). All samples were analyzed in the field within 

6 hours of collection to meet specified holding times. A range 

of volumes was plated to obtain at least one plate with an ideal 

count of 20 to 80 colonies. Plates were incubated at 35°C for 2 

hours (to resuscitate injured or stressed bacteria) then transferred to 44.5°C for 20–22 hours. After incubation, filters were 

transferred to a pad soaked with a urea-phenol red solution. 

Colonies that remained yellow after 15 minutes, indicating a 

negative test for the urease enzyme, were counted as E. coli. 

The results were calculated as described by Myers and Sylvester (1997) and reported as colonies of E. coli per 100 milliliters (col/100 mL).
Methods of Study 9

Quality-control samples included a field blank, filter 

blanks, procedure blanks, and replicates. One field blank 

was analyzed to assess field contamination of samples and to 

ensure that equipment cleaning and sterilization techniques 

were adequate. Filter blanks were analyzed with every water 

sample to measure the sterility of the equipment and supplies. Procedure blanks were analyzed with every fourth water 

sample to measure the effectiveness of the analyst’s rinsing 

technique. Seventeen percent of the water samples were analyzed as nested replicates to determine sampling and analytical 

variability. A nested replicate consists of two water samples 

collected in separate bottles, each bottle plated in duplicate for 

concentrations of E. coli.

Salmonella

For Salmonella analyses, water samples were filtered 

through 0.45-μm membrane filters (Advantec, MFS, Inc., 

Pleasanton, Calif.) in the field within 1 hour of sample collection. In 2000, 500 mL of water was filtered; however, in 2002, 

only 100 mL of water was filtered. Each filter was placed in a 

bottle of 100 mL chilled Rappaport-Vassiliadis medium (RV) 

for Salmonella screening. The bottles were shipped overnight 

on ice to the USGS National Wildlife Health Center in Madison, Wis., for analysis. The bottles were incubated at 42°C for 

16 to 18 hours. After incubation, an aliquot of the RV enrichment broth was transferred to XLT4 Agar (Difco Laboratories, 

Detroit, Mich.) and Brilliant Green Agar (Becton Dickinson, 

Cockeysville, Md.). Both media were incubated at 35–37°C 

for 18 to 24 hours. If no Salmonella isolates were found on 

the media, another aliquot of the original RV enrichment broth 

was added to new RV enrichment broth, and the incubation 

and plating processes were repeated to enhance recovery. All 

bacterial colonies were screened to identify Salmonella spp., 

and those matching morphological and biochemical characteristics were subcultured on 5 percent sheep blood agar (Becton 

Dickinson, Cockeysville, Md.). Suspected Salmonella isolates 

were biochemically characterized by either the API-20E or 

Vitek systems (bioMerieux, St. Louis, Mo.). Isolates yielding 

a Salmonella identification were screened using a polyvalent 

antisera for Salmonella (Becton Dickinson, Cockeysville, Md.) 

before being serotyped for confirmation at the U.S. Department of Agriculture National Veterinary Services Laboratory 

(Ames, Iowa).

Quality-control samples for Salmonella analyses included 

one field blank and one matrix spike. A matrix spike was analyzed in order to determine the effect of the matrix on method 

performance. The matrix spike was prepared by adding a 

known amount of Salmonella to a water sample in the field.

F-specific and somatic coliphage

Water samples collected for the analysis of F-specific and 

somatic coliphage were shipped overnight on ice to the USGS 

Ohio District Microbiology Laboratory (ODML) for analysis. 

All coliphage samples were analyzed within the 48-hour holding time. Samples collected in 2000 were analyzed for F-specific coliphage, and samples collected in 2002 were analyzed 

for F-specific and somatic coliphage. Samples were analyzed 

using a method described by Ijzerman and Hagedorn (1992), 

which is based on the induction of the β-galactosidase enzyme 

found in E. coli. Antibiotics are used in this method to prevent 

interference due to the growth of background bacteria in the 

sample. Antibiotic-resistant E. coli host cultures are used to 

test for the presence of coliphage. E. coli F-amp, which is 

resistant to the antibiotics ampicillin and streptomycin, was 

used as a host culture for the detection of F-specific coliphage. 

E. coli CN-13, which is resistant to the antibiotic nalidixic 

acid, was used as a host culture for somatic coliphage. Sample 

volumes of 100 mL were mixed with the host culture, antibiotics, molten agar, and chemicals that react with the β-galactosidase enzyme: isopropyl-β-D-thiogalactoside (IPTG) acts as an 

inducer of β-galactosidase, and 5-bromo-4-chloro-3-indolylβ-D-galactopyranoside (X-gal) is a substrate for the enzyme. 

The mixtures were poured into four 150- x 15-mm sterile 

petri dishes and incubated for 24 hours at 35°C. If coliphage 

were present in the samples, the E. coli cells were lysed and a 

stable, dark blue indolyl product was released. Coliphage were 

easily identified and enumerated by the distinct blue circle 

within the viral plaque. Results were reported in plaques per 

100 milliliters (plaques/100 mL).

Quality-control samples for the analysis of coliphage 

included positive controls, negative controls, and replicates. A 

positive control of dilute sewage and a negative control of sterile water were analyzed each day water samples were analyzed 

to ensure that the method was performing adequately, that 

the coliphages were correctly identified, and that there was 

no laboratory contamination. Nine percent of the F-specific 

coliphage samples and 17 percent of the somatic coliphage 

samples were analyzed as replicates (one sample plated in 

duplicate). 

Enteric viruses

Cartridge housings containing the 1MDS filters were 

shipped overnight on ice to the ODML for initial processing. 

Once received, viruses that were attached to the 1MDS filter 

via charge interactions were eluted from the 1MDS filter using 

a beef extract solution at pH 9.5. The eluate was concentrated on a filter using celite at pH 4.0 and then eluted from 

the filter with sodium phosphate at pH 9.5. The 1MDS filter 

was left in beef extract solution overnight and reconcentrated 

the following day. The final concentrate was neutralized and 

frozen at -70°C to await further processing. An aliquot of each 

sample was shipped overnight on dry ice to Environmental 

Health Laboratories (EHL) (South Bend, Ind.) to be analyzed 

for infectious enteroviruses by the ICC-PCR method, and an 

aliquot was saved for the ODML to be analyzed for enteric 

viruses by the RT-PCR method. 
10 Microbiological Water Quality, Cuyahoga River, Cuyahoga Valley National Park 

Infectious enteroviruses by ICC-PCR

Water samples were analyzed for infectious enteroviruses 

by EHL by means of a method described by Reynolds and 

others (1996). For each concentrated sample, a volume of 10 

mL was analyzed using 10 cell-culture flasks of Buffalo Green 

Monkey Kidney (BGMK) monolayer cells. The cells were 

incubated for 14 days at 36.5°C. Each flask was then treated 

as an individual sample and was subjected to RNA extraction 

and then RT-PCR. After RT-PCR, samples were confirmed 

for enterovirus by oligonucleotide probe hybridization. There 

was a total of 10 RT-PCR reactions per sample. The MPNV 

computer program (U.S. Environmental Protection Agency, 

2003) was used to compute the most probable number (MPN). 

The inoculation volume, number of flasks, number of positive 

RT-PCR reactions, and dilution factors were the parameters for 

calculation of the MPN, which is reported as MPN/100 L. 

Quality-control samples for the analysis of enteroviruses 

consisted of two equipment blanks, one field blank, and positive and negative controls for both the cell-culture and RT-PCR 

steps of the method. The equipment blanks were produced in a 

laboratory and were analyzed to determine potential contamination from the equipment cleaning and sterilization process. 

The field blank was analyzed to assess field contamination of 

samples and to ensure that field-equipment cleaning and sterilization techniques were adequate. For the cell-culture step, 

the positive control consisted of 20–200 plaques of poliovirus 

inoculated into one flask, and the negative control consisted 

of 1 mL of sterile sodium phosphate buffer inoculated into 

one flask. Both were processed the same as water samples. 

For the RT-PCR step, 1 mL of cell suspension from a positivecontrol cell-culture flask and viral RNA from polioviruses was 

used as a positive control, and 1 mL of cell suspension from a 

negative-control cell-culture flask and 10 μL of sterile buffer was used as a negative control. With the exception of one 

equipment blank, the quality-control samples for infectious 

enteroviruses by ICC-PCR were acceptable. A detectable level 

of infectious enteroviruses was observed in the first equipment 

blank collected in May 2000. As a result, the water samples 

collected in May 2000 that did not exceed the concentration of 

viruses measured in the equipment blank were omitted from 

the data set.

Enteric viruses by RT-PCR

Water samples were analyzed for enteric viruses by the 

ODML by means of a method described by Fout and others 

(2003). Instead of using the multiplex RT-PCR as described in 

the method (where multiple viruses are analyzed for in a single 

reaction), each virus was analyzed for in a separate reaction. 

Viruses in the eluate were concentrated by ultracentrifugation 

through a sucrose gradient. The concentrates were treated with 

a solvent mixture that was designed to remove inhibitors, such 

as humic substances, which can interfere with the activity of 

the enzymes used in the RT-PCR step. All of the target viruses 

are RNA viruses; therefore, the enzyme reverse transcriptase 

(RT) was used to transcribe the RNA into DNA. The DNA 

was then amplified by means of PCR. All PCR products were 

confirmed with nucleic acid hybridization. Initially, tests were 

done for five viruses; however, the results were inconclusive 

for reovirus, rotavirus, and Norwalk virus, so results for those 

viruses are not reported.

Quality-control samples for enteric virus analysis by 

RT-PCR included two equipment blanks, one field blank, 

and laboratory quality controls for the method. Appendix A 

contains a list and descriptions of quality-control samples for 

enteric virus analysis by RT-PCR. 

Clostridium perfringens

Water samples to be analyzed for C. perfringens were 

shipped overnight on ice to the ODML. Concentrations of 

C. perfringens were determined by means of membrane filtration on mCP agar with some modifications (U.S. Environmental Protection Agency, 1996). The procedure for the inactivation of vegetative cells as described in the method was not 

followed for the analysis of these samples. All samples were 

analyzed within a 48-hour holding time. A range of volumes 

was plated to obtain at least one plate with an ideal count 

of 20 to 80 colonies. Plates were incubated in an anaerobic 

environment for 24 hours at 42°C. All straw-colored colonies 

that turned dark pink to magenta after exposure to ammonium 

hydroxide were counted as C. perfringens, and results were 

recorded as col/100 mL.

Quality control for the analysis of C. perfringens

included positive controls, filter and procedure blanks, and 

replicates. A positive control of diluted sewage was analyzed 

each day that water samples were analyzed to ensure that the 

analytical method was performed adequately and that the 

C. perfringens colonies were correctly identified. Filter blanks 

were analyzed with each water sample. Procedure blanks were 

analyzed with every fourth water sample. Seventeen percent 

of the water samples were analyzed as replicates (one sample 

plated in duplicate).

Cryptosporidium and Giardia

Water samples were shipped overnight on ice to EHL. 

Filtration of the bulk 10-L samples was initiated within 48 

hours of sample collection. Samples were analyzed for concentrations of Cryptosporidium oocysts and Giardia cysts by 

means of USEPA Method 1623 (U.S. Environmental Protection Agency, 2001), a performance-based method that requires 

filtration, immunomagnetic separation of the oocysts/cysts 

from the material captured, and an immunofluorescence assay 

for determination of oocyst/cyst concentrations, with confirmation through vital dye staining and differential interference 

contrast microscopy. The Filta-Max filtration system (IDEXX, 

Westbrook, Maine) was used in this study. The filter is made 

of open-cell foam discs that have been compressed to give a 

nominal porosity of 1 μm. After filtering, the foam is decompressed, enabling the captured oocysts and cysts to be recovered. 
Analysis of Microbiological Data 11

Matrix-spike samples were collected during each 

sampling trip in 2002 at the Botzum site. The matrix-spike 

samples consisted of a second 10-L sample that was spiked 

with a known amount of oocysts and cysts in the laboratory. 

These samples were analyzed with the regular water sample 

to determine the effect of the water matrix on the method’s 

recovery.

Characteristics of Streamflows during 

Sampling Periods

Streamflow was measured by means of standard USGS 

techniques (Rantz and others, 1982) at the times when nearly 

all microbiological samples were collected. More than 80 

percent of the samples (and streamflow measurements) were 

obtained during the 2000 recreational season, the remainder being obtained during the 2002 recreational season. To 

facilitate comparison of streamflow conditions between sampling years, the following discussion focuses on streamflows 

measured at the Old Portage site, which has operated as a 

continuous-record streamflow-gaging station from September 

1921 to December 1935 and from October 1939 to the present 

(November 2003). Instantaneous streamflows measured when 

microbiological samples were collected from Cuyahoga River 

sites in 2000 ranged from 111 to 799 ft3/s; median streamflow 

was 167 ft3/s (fig. 2). In comparison, instantaneous streamflows measured when microbiological samples were collected 

in 2002 ranged from 92 to 960 ft3/s; median streamflow was 

485 ft3/s (fig. 2). As a point of reference, the median of daily 

mean streamflows for the period of record at the Old Portage 

streamflow-gaging station through water year 2002 is 266 

ft3/s (Shindel and others, 2003). Ten percent of the daily mean 

streamflows exceeded 1,020 ft3/s during the same period, and 

90 percent of the daily-mean streamflows exceeded 78 ft3/s 

(Shindel and others, 2003). Although instantaneous streamflows are not strictly comparable to daily mean streamflows, 

these statistics indicate that the samples were not collected at 

extremes of streamflow. It also is notable that samples were 

collected in 2000 and 2002 over similar ranges of streamflow.

Analysis of Microbiological Data

Microbiological data used in this study came from analyses of samples collected in 2000 and 2002 from two sources; 

selected sites on the Cuyahoga River and effluent from the 

Akron WPCS. Data from the two sources were not combined 

for analysis except when assessing between-site differences 

and in selected instances where a combined analysis seemed 

appropriate. Appendix B lists the microbiological data from 

samples collected in 2000 and 2002.

The presence of censored observations (“less-than” 

values) in the data set required special handling. Analytical results for F-specific coliphage, infectious enterovirus, 

Giardia, and Cryptosporidium each included some censored 

values. Of the four microorganisms, only infectious enterovirus and F-specific coliphage had sufficiently small percentages of censored values (7 and 14 percent, respectively) to 

consider statistical analyses by means of parametric tests. In 

all applicable parametric tests involving microorganisms with 

censored values, half of the censoring level was substituted for 

the censored value.

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Figure 2. Distribution of instantaneous streamflows measured 

in conjunction with samples collected from the Cuyahoga River at 

Old Portage, by calendar year.
12 Microbiological Water Quality, Cuyahoga River, Cuyahoga Valley National Park 

Because of a lack of general public agreement on methods for handling censored values, data for microorganisms 

with censored values were also rank transformed. Applicable 

tests involving infectious enterovirus and F-specific coliphage 

were done on the both ranks and the base numerical values. 

Tests involving Giardia always were done using the ranktransformed concentrations.

Microbiological data collected for this study were 

analyzed qualitatively and quantitatively to identify relevant 

associations between microorganisms and to assess spatial 

trends in microorganism concentrations. Qualitative analyses were directed at identifying general trends and patterns. 

Quantitative analyses were directed at identifying correlations 

between concentrations of organisms and comparing differences in central values (mean concentration, for example) of 

one organism as a function of some characteristic of a second 

organism, as a function of location, or as a function of some 

other environmental measure. 

Correlation analyses were done by computing Spearman’s rho. Spearman’s rho was chosen over other measures of 

correlation because it can be used to identify correlations that 

are not necessarily linear and because it can be used with censored data sets (as long at their ordination can be determined). 

Comparison tests were done by means of analysis of variance (ANOVA) coupled (where appropriate) with a post hoc 

Tukey-Kramer multiple-comparison test. The Tukey-Kramer 

multiple-comparison test is an extension of Tukey’s test that 

facilitates comparisons of classes with unequal sample sizes. 

Site name Statistic

Clostridium 

perfringens 

(col/

100 mL)

F-specific 

coliphage 

(plaques/

100 mL)

Somatic 

coliphage 

(plaques/

100 mL)

Infectious 

enterovirus 

(MPN/

100 L)

Old Portage geometric mean 1,320 115 na 195 na na

 median 1,350 120 18 130 35 1

 maximum 18,000 190 160 790 190 3

 minimum 64 67 <1 72 <16 <1

 N 18 3 6 3 5 3

Akron WPCS geometric mean 122 1,140 43 157 112 na

 median 77 1,100 32 170 110 8

 maximum 2,900 1,700 180 290 190 10

 minimum 8 800 12 79 73 <1

 N 19 3 6 3 5 3

Botzum geometric mean 1,380 353 11 112 na na

 median 1,250 350 8 63 111 <1

 maximum 12,000 370 63 470 190 16

 minimum 170 340 3 47 8 <1

 N 18 3 6 3 6 3

Jaite geometric mean 898 227 na 76 106 na

 median 710 270 5 78 109 <1

 maximum 22,000 360 18 170 190 6

 minimum 160 120 <1 33 56 <1

 N 19 3 6 3 3 3

Independence geometric mean 975 nd 6 nd nd nd

 median 620 nd 5 nd nd nd

 maximum 25,000 nd 11 nd nd nd

 minimum 210 nd 3 nd nd nd

 N 15 0 3 0 0 0

Table 3. Summary statistics for concentrations of Escherichia coli, Clostridium perfringens, F-specific and somatic 

coliphage, infectious enterovirus, and Giardia at selected sites in the Cuyahoga River and at the Akron Water Pollution Control Station (WPCS), Ohio, 2000 and 2002.

[col/100 mL, colonies per 100 milliliters; mL, milliliters; MPN/100 L, most probable number per 100 liters; L, liters; N, sample size; na, not applicable; nd, not determined]

Escherichia 

coli

(col/

100 mL)

Giardia 

(cysts/

10 L)
Analysis of Microbiological Data 13

The Tukey-Kramer multiple-comparison test is an exact alphalevel test if the sample sizes are the same, and it is slightly 

conservative for unequal sample sizes (Rafter and others, 

2002). An alpha level of 0.05 was used to judge significance 

of all tests discussed in this report.

The Tukey-Kramer test is based on the assumption that 

the data are normally distributed, random, and independent 

and that they have common variance. Quantile-quantile plots 

and the Shapiro-Wilk test were used to assess normality of 

the microorganism concentration data. On the basis of these 

tests, it was determined that logarithmic transformations of 

concentrations of E. coli, F-specific and somatic coliphage, 

and C. perfringens were necessary to meet the normality 

assumption. No transformation was required for concentrations of infectious enterovirus, and none of the other microorganism data included sufficient percentages of uncensored 

values to warrant parametric analysis. Bartlett’s test (Snedecor 

and Cochran, 1980) was used to test for departures from the 

assumption of equal variance (no significant departures were 

detected) and the data were assumed to be both random and 

independent based on the sampling design.

General Trends and Patterns

E. coli concentrations in samples collected for this study 

ranged from 64 to 25,000 col/100 mL in Cuyahoga River samples and from 8 to 2,900 col/100 mL in Akron WPCS effluent 

samples (table 3). E. coli concentrations exceeded 298 col/

100 mL (Ohio’s single-sample primary-contact recreational 

standard) 87 percent of the time in Cuyahoga River samples as 

compared to 26 percent of the time in Akron WPCS effluent 

samples. Similarly, E. coli concentrations exceeded 576 col/

100 mL (Ohio’s single-sample secondary-contact recreational 

standard) 64 percent of the time in Cuyahoga River samples 

as compared to 26 percent of the time in Akron WPCS effluent samples. Both threshold concentrations were exceeded 

multiple times at all sites. The lower threshold-exceedance 

rates for the Akron WPCS effluent samples presumably are 

due to chlorination of the effluent.Those samples that did not 

exceed the standards generally were associated with the lower 

range of streamflows for the respective sites. For the range 

of effluent flow rates sampled in this study, Myers and others 

(1998) reported E. coli concentrations in the Akron WPCS 

effluent that were lower than concentrations in Cuyahoga 

River samples at equivalent flow rates. At effluent flow rates 

larger than observed in this study, the study by Myers and others (1998) indicated that the Akron WPCS effluent tended to 

have appreciably higher concentrations of E. coli than did river 

sites at equivalent flow rates. This trend in E. coli concentration occurs because capacity of the Akron WPCS to provide 

full secondary treatment of the effluent stream is exceeded at 

an effluent flow rate of about 110 Mgal/d.

At least five E. coli samples were collected at the 

Old Portage, Botzum, Jaite, and Independence sites on the 

Cuyahoga River during May, July, and September of calendar 

year 2000. Geometric mean E. coli concentrations calculated 

from analytical results from each site and month ranged from 

466 to 2,070 col/100 mL (table 4) and in each case exceeded 

Ohio’s geometric-mean primary-contact recreational standard 

of 126 col/100 mL

Salmonella was detected in 15 of 29 river samples (52 

percent) and 3 of 7 effluent samples (43 percent) (Appendix 

B); all but one of the detections were found in 2000, and 

multiple detections were noted at each sampling location. 

Salmonella was never detected in combination with hepatitis 

A virus; however, the majority of their respective detections 

were in different years.

There are over 2,300 known serotypes of Salmonella. In 

Ohio, the Salmonella serotypes Typhimurium and Enteritidis 

account for more than half of all human isolates (Ohio Department of Health, 1993b). Salmonella serotypes Braenderup, 

Hartford, Java (also called paratyphi B variant java), Manhattan, Miami, Muenster, Newport, Oranienburg, and Senftenberg 

were identified in samples collected in 2000. Braenderup, 

Java, and Newport were the most commonly found Salmonella

serotypes. Salmonella Arizona was identified in the one detection (at Jaite) in 2002. 

F-specific coliphage samples were collected in 2000 and 

2002; however, somatic coliphage, C. perfringens, Cryptosporidium, and Giardia samples were collected in 2002 only. No 

samples were collected from the Independence site in 2002. 

Concentrations of somatic coliphage and F-specific coliphage 

Geometric mean Escherichia coli concentration (top number), in col/100 mL, and number of 

observations on which it was based (bottom number), at indicated site

Month Old Portage Botzum Jaite Independence

 May 1,880

 5

 1,020

 5

 697

 6

 961

 5

 July 2,060

 5

 1,960

 5

 1,270

 5

 466

 5

 September 841

 5

 1,510

 5

 1,070

 5

 2,070

 5

Table 4. Geometric mean Escherichia coli concentrations for samples collected during May, July, and 

September 2000 at selected sites on the Cuyahoga River, Ohio.

[col/100 mL; colonies per 100 milliliters]
14 Microbiological Water Quality, Cuyahoga River, Cuyahoga Valley National Park 

ranged from 79 to 290 plaques/100 mL and 12 to 180 plaques/

100 mL, respectively, in the Akron WPCS effluent samples 

(table 3). Concentrations of somatic coliphage ranged from 33 

to 790 plaques/100 mL in the nine river samples collected in 

2002. F-specific coliphage concentrations ranged from <1 to 

160 plaques/100 mL in 21 river samples collected in 2000 and 

2002 and were less than detection in two (about 10 percent) of 

the river samples (table 3).

Hepatitis A virus was detected only in samples collected 

in 2002. Of 21 samples analyzed for hepatitis A virus, 6 tested 

positive (contained hepatitis A virus), with 3 of the 6 samples 

coming from the Akron WPCS effluent (Appendix B). The 

Centers for Disease Control and Prevention (2003b) states that 

hepatitis A virus is killed by adequate chlorination of water; 

however, hepatitis A virus was found in half of the Akron 

WPCS effluent samples analyzed. The Akron WPCS effluent is chlorinated (with a contact time of approximately 30 

minutes) and treated with sodium bisulfite (to reduce chlorine 

residual) just prior to discharge (City of Akron, 2003).

Hepatitis A virus was not detected in any of the six 

samples from the Old Portage site ( upstream from the Akron 

WPCS). Hepatitis A virus was detected in samples from 

Akron WPCS, Botzum, and Jaite collected May 21–22, 2002; 

in samples from the Akron WPCS and Botzum collected July 

15–16, 2002; and in a sample from the WPCS only on June 

17, 2002. In no case was hepatitis A virus detected in samples 

from a site other than the Akron WPCS without a coincident 

detection of hepatitis A virus in the Akron WPCS sample. The 

fact that hepatitis A virus was never detected upstream from 

the Akron WPCS, coupled with the fact that all downstream 

detections of hepatitis A virus had coincident detections of 

hepatitis A virus in the Akron WPCS effluent, suggests that 

the Akron WPCS was a principal source of hepatitis A virus at 

the downstream sites.

Enterovirus (as determined by the RT-PCR method) was 

detected when tested for in most samples (19 out of 21) and in 

all samples from the Akron WPCS and Botzum site (Appendix 

B). Infectious enterovirus concentrations (as determined by the 

ICC-PCR method) ranged from <16 to 190 MPN per 100 L in 

river samples and from 73 to 190 MPN per 100 L in samples 

from the Akron WPCS effluent (table 3). 

Concentrations of C. perfringens in samples collected 

from river and effluent sources exceeded 100 col/100 mL 

with only one exception (at Old Portage). Concentrations 

of C. perfringens in samples collected from the Cuyahoga 

River ranged from 67 to 370 col/100 mL, whereas concentrations in samples from the Akron WPCS effluent ranged from 

800 to 1,700 col/100 mL (table 3). The mean concentration 

of C. perfringens in Akron WPCS effluent samples (1,200 

col/100 mL) was more than 4 times the mean concentration in 

river samples (243 col/100 mL). This result is not unexpected 

because C. perfringens is present in large numbers in human 

and animals wastes, and its spores are resistant to disinfection 

(Francy and others, 2000).

Cryptosporidium concentrations greater than or equal to 

1 oocyst/10 L were not found in any sample. Giardia concentrations were greater than 1 cyst/10 L in four of the nine (44 

percent) Cuyahoga River samples and two of the three (67 percent) Akron WPCS effluent samples analyzed for Giardia and 

Cryptosporidium. The highest measured Giardia concentration 

(15.8 cysts/10 L) was from a sample collected at the Botzum 

site. The second and third highest concentrations (9.5 and 8.2 

cysts/10 L, respectively) were from samples collected from the 

Akron WPCS effluent. Giardia was detected at least once at 

each site sampled in 2002.

Relation of Escherichia coli Water-Quality 

Standards to Presence of Selected Infectious 

Microorganisms

Ohio’s water-quality standards for E. coli vary as a 

function of designated recreational use (table 1). Ohio has 

established standards for three categories of recreational 

uses: bathing waters, primary contact, and secondary contact. 

Bathing-water standards are applicable to designated swimming areas and therefore are the most stringent of the three. 

In contrast, primary-contact standards are applicable to waters 

that are not designated as swimming areas but are capable of 

Ohio contact 

recreation 

standard

Threshold 

Eschericha 

coli concentration (col/100 

mL)

Number of 

river samples 

not exceeding 

threshold

Number of current analyses 

for enterovirus, 

infectious 

enterovirus, and 

hepatitis A virus

Number of 

concurrent 

analyses for 

Salmonella

Number of times (percentage of time) indicated 

organism was present

Enterovirus 

and infectious 

enterovirus

Hepatitis A 

virus

Salmonella

 Bathing 235 5 0 0 NA (NA) NA (NA) NA (NA)

 Primary 298 9 1 1 1 (100%) 0 (0%) 1 (100%)

 Secondary 576 25 7 10 5 (71%) 1 (14%) 3 (30%)

Table 5. Numbers and percentages of time microorganisms were present in Cuyahoga River samples when the indicated 

threshold Escherichia coli concentrations were not exceeded, Ohio, 2000 and 2002.

[Total number of river samples analyzed for Escherichia coli = 70; NA, not applicable; col/100 mL, colonies per 100 milliliters]
Analysis of Microbiological Data 15

supporting full-body-contact recreation (such as swimming 

or canoeing). Secondary-contact standards are applicable to 

waters that can support only partial-body-contact recreation 

(such as wading). 

The E. coli water-quality standards were based in part 

on results of a USEPA study in which rates of swimmingassociated gastrointestinal illness were found to be related 

to concentrations of E. coli in the water (Dufour, 1984). An 

equation developed by Dufour (1984) relating illness rate to 

E. coli concentration shows that exposure to waters that meet 

E. coli water-quality standards is not necessarily without risk. 

For example, bathing waters with a geometric-mean E. coli

concentration of 126 col/100 mL (based on a minimum of 

five samples in a 30-day period) have an associated risk of 

8 gastrointestinal illnesses per 1,000 bathers (according to 

Dufour’s equation). Given that some gastrointestinal illnesses 

can be expected even when E. coli standards are not exceeded, 

an analysis was done to examine the frequency of occurrence 

of pathogenic organisms when selected recreational standards 

were not exceeded. 

As listed in table 5, only 5 out of the 70 river samples 

analyzed had E. coli concentrations less than 235 col/100 mL 

(Ohio’s single-sample bathing-water recreational standard). 

Unfortunately, no other bacterial or viral analyses were done 

for those samples. 

One of nine river samples with E. coli concentrations 

less than 298 col/100 mL was analyzed for other bacteria 

and viruses. Both infectious enterovirus and Salmonella were 

detected in the sample; however, hepatitis A was not detected 

(table 5). 

Seven of the 25 river samples with E. coli concentrations 

less than 576 col/100 mL were analyzed for enterovirus, infectious enterovirus, and hepatitis A virus. Of those seven samples, five (71 percent) were found to contain enterovirus and 

infectious enterovirus at concentrations above detection, and 

one (14 percent) was found to contain hepatitis A virus. Ten of 

the 25 river samples with E. coli concentrations less than 576 

col/100 mL were analyzed for Salmonella, which was found 

in three (30 percent) of the samples (table 5). By comparison, 

E. coli concentrations were greater than 576 col/100 mL in 45 

river samples (table 6). Eight of those 45 samples were analyzed for enterovirus, infectious enterovirus, and hepatitis A 

virus with rates of detection of 100 percent, 75 percent, and 25 

percent, respectively. Nineteen of the 45 samples with E. coli

concentrations greater than the single-sample secondary-contact recreational standard were analyzed for Salmonella, which 

was detected in approximately 63 percent of those samples. In 

all cases, percentages of pathogenic-organism detections were 

larger in samples where the single-sample secondary-contact 

recreational standard for E. coli was exceeded than in samples 

where the standard was not exceeded. A comparable analysis 

cannot be made with respect to the single-sample primarycontact recreational standard because only one sample that did 

not exceed that standard was analyzed for other pathogenic 

organisms. 

Associations Between Bacteria, Viruses, and 

Protozoa

Much information has been published on the relation of 

E. coli concentrations to fecal coliform concentrations in the 

Cuyahoga River and other selected rivers in Ohio (Francy and 

others, 1993; Myers and others, 1998); however, little information is available about the relation between concentrations of 

E. coli and other microorganisms or between combinations of 

other microorganisms. 

Ohio contact 

recreation 

standard

Threshold 

Eschericha 

coli concentration (col/100 

mL)

Number of 

river samples 

exceeding 

threshold

Number of current analyses 

for enterovirus, 

infectious 

enterovirus, and 

hepatitis A virus

Number of 

concurrent 

analyses for 

Salmonella

Number of times (percentage of time) indicated 

organism was present

Enterovirus 

and infectious 

enterovirus

Hepatitis A 

virus

Salmonella

 Bathing 235 65 15 29 13a (87%)

11b (73%)

 3 (20%) 15 (52%)

 Primary 298 61 14 28 12a (86%)

10b (71%)

 3 (21%) 14 (50%)

 Secondary 576 45 8 19 8a (100%) 

 6b (75%)

 2 (25%) 12 (63%)

Table 6. Numbers and percentages of time microorganisms were present in Cuyahoga River samples when the indicated 

threshold Escherichia coli concentrations were exceeded, Ohio, 2000 and 2002.

[Total number of river samples analyzed for Escherichia coli = 70; col/100 mL, colonies per 100 milliliters]

a

Value is for enterovirus.

b

Value is for infectious enterovirus.
16 Microbiological Water Quality, Cuyahoga River, Cuyahoga Valley National Park 

A matrix of correlation information for samples collected 

for this study is shown in table 7. The shaded cells in table 7 

list correlation information for concentration data for samples 

collected from Cuyahoga River sites only. The unshaded 

cells show correlation information for concentration data for 

samples collected from river sites combined with concentration data for samples collected from the Akron WPCS effluent 

(hereafter referred to as the “combined data set”). 

Absolute values of correlation coefficients were greater 

than 0.5 for only three microorganism pairs (table 7) and 

only two of those pairs had coefficients that were statistically 

significant. C. perfringens concentrations were positively 

(rho = 0.68) and significantly correlated with concentrations 

of E. coli in river samples. The correlation between these 

two microorganisms was also positive in the combined data 

set; however, the correlation was weaker (rho = 0.21) and not 

statistically significant. The poorer correlation in the combined 

data set is attributed to the markedly different characteristics 

of C. perfringens and E. coli concentrations in the effluent as 

compared to river samples.

Somatic coliphage concentrations and F-specific coliphage concentrations were positively and significantly correlated (rho = 0.63) in the combined data set. The correlation 

between this same pair of microorganisms was also positive 

(rho = 0.54) in samples collected from river sites only; however, the number of observations was smaller and the correlation was not statistically significant (p = 0.13). 

Infectious enterovirus concentration was negatively correlated with somatic coliphage concentrations in river samples 

(rho = -0.55) and in the combined data set (rho = -0.35). The 

magnitudes and signs of these correlation coefficients, however, were influenced strongly by one observation, and neither 

correlation was statistically significant. As a matter of note, 

Gantzer and others (1998) found a significant positive correlation between the concentration of somatic coliphage and the 

presence of infectious enterovirus in treated wastewater. 

ANOVAs on data for the Cuyahoga River sites indicated 

that neither geometric-mean somatic coliphage concentration nor geometric-mean F-specific coliphage concentration 

differed significantly as a function of the presence/absence 

of enterovirus, infectious enterovirus, or hepatitis A virus. 

Similarly, an ANOVA on data for the Cuyahoga River sites 

indicated that mean infectious enterovirus concentrations did 

not differ significantly as a function of the presence/absence of 

hepatitis A virus. In spite of the lack of significant differences, 

there were observable tendencies for the presence of hepatitis 

A virus to be associated with higher median concentrations 

of somatic coliphage, F-specific coliphage, and infectious 

enterovirus (fig. 3). When considering the significance of 

these results, it is important that the reader be aware that they 

are based on a small sample: only 9 observations for somatic 

coliphage, 15 observations for F-specific coliphage, and 14 

observations for infectious enterovirus.

Hepatitis A virus was not detected in samples when 

infectious enterovirus concentrations were less than 80 MPN/

100 L. There were instances, however, when infectious enterovirus was detected (in some cases at concentrations greater 

than 80 MPN/100 L) and hepatitis A virus was not detected.

Table 7. Correlation between microorganism concentrations in Cuyahoga River samples and combined river and Akron Water 

Pollution Control Station (WPCS) effluent samples, Ohio, 2000 and 2002.

[Shaded cells designate river samples only, unshaded cells designate combined river and effluent samples]

Spearman’s rho (top number), p value (middle number), and number of observations (bottom number) for 

indicated microorganism pairs

Escherichia coli F-specific coliphage Somatic coliphage Infectious 

enterovirus

Clostridium 

perfringens

Escherichia coli 0.09

 0.652

 27

 0.37

 0.240

 12

 -0.04

 0.661

 19

 0.21

 0.505

 12

F-specific 

coliphage

 0.33

 0.145

 21

 0.63

 0.027

 12

 0.17

 0.490

 19

 0.13

 0.687

 12

Somatic coliphage 0.37

 0.332

 9

 0.54

 0.130

 9

 -0.35

 0.264

 12

 0.11

 0.737

 12

Infectious 

enterovirus

 -0.25

 0.383

 14

 -0.22

 0.445

 14

 -0.55

 0.125

 9

 -0.01

 0.966

 12

Clostridium 

perfringens

 0.68

 0.045

 9

 0.06

 0.881

 9

 -0.11

 0.781

 9

 0.03

 0.949

 9
Analysis of Microbiological Data 17

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Figure 3. Distribution of (A) somatic coliphage, (B) F-specific coliphage, (C) infectious enterovius, and 

(D) Clostridium perfringens concentrations as a function of the presence of hepatitis A virus in Cuyahoga River 

samples, Ohio, 2000 and 2002.
18 Microbiological Water Quality, Cuyahoga River, Cuyahoga Valley National Park 

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Figure 4. Relation between streamflow and concentrations of (A) Escherichia coli and (B) F-specific 

coliphage in Cuyahoga River samples, Ohio, 2000 and 2002.
Analysis of Microbiological Data 19

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Figure 5. Distribution of pH as a function of site on the Cuyahoga River, including the Akron 

Water Pollution Control Station (WPCS), Ohio, 2000 and 2002.

An ANOVA followed by a Tukey-Kramer test on the 

combined data set indicated that geometric-mean C. perfringens concentration was significantly higher in samples where 

hepatitis A virus was present (detected) than in samples where 

hepatitis A virus was absent (not detected). Figure 3 (D) 

shows that a similar tendency was present for data from the 

Cuyahoga River sites alone; however, the difference in geometric-mean concentration was not statistically significant.

Relation of Streamflow and Water-Quality 

Measures to Bacteria, Viruses, and Protozoa

Scatterplots and correlation analyses were used to 

assess the relation of streamflow to concentrations of E. coli, 

F-specific coliphage, somatic coliphage, infectious enterovirus, C. perfringens, and Giardia at the Cuyahoga River sites. 

E. coli had a weak (rho = 0.35) but significant positive correlation with streamflow. F-specific coliphage concentration 

also was positively correlated (rho = 0.42) with streamflow; 

however, the p value for this correlation (p = 0.06) was slightly 

larger than alpha, and so the relation was not statistically significant. The strength of these relations are illustrated further 

in figure 4 which shows scatterplots of E. coli and F-specific 

coliphage concentrations against streamflow. No other microorganisms exhibited a notable trend with streamflow.

E. coli concentration tended to increase with increasing streamflow at Cuyahoga River sites both upstream and 

downstream from the Akron WPCS, an indication that E. coli

loading is an increasing function of rainfall /runoff processes 

and that E. coli sources upstream from the 

Akron WPCS may have appreciable influence on concentrations in the river downstream from the Akron WPCS. These 

results are consistent with the findings of 

Myers and others (1998).

A variety of water-quality characteristics, measured in selected samples, 

may be related to observed concentrations 

of E. coli. Because many water-quality 

characteristics are highly correlated with 

streamflow, apparent correlations between 

these measures and E. coli concentrations may be due solely to the influence 

of streamflow. As a consequence, it is 

necessary to account for the influence of 

streamflow on concentrations of E. coli

before assessing its correlation with 

water-quality characteristics. This was 

done by regressing logarithms of E. coli

concentration on logarithms of streamflow and using the residuals (the differences between the observed and predicted 

values of E. coli) for further analyses. 

The residuals represent the distribution 

of log-transformed E. coli concentrations with the variability 

attributable to streamflow removed.

Residuals from the regression of logarithms of E. coli

concentration on logarithms of streamflow (for Cuyahoga 

River sites) were analyzed for correlation with the water-quality characteristics. Fluoride, pH, specific conductance, and 

total phosphorus were the only water-quality characteristics 

with significant correlation to the residuals. Site-by-site 

investigations of these correlations suggest that the observed 

correlation resulted from the association of these parameters 

with spatial trends in concentration as opposed to within-site 

associations of the parameters with E. coli concentrations. 

To better illustrate how some water-quality parameters 

can exhibit spatial trends, figure 5 shows boxplots of pH 

measured in Cuyahoga River samples and in Akron WPCS 

effluent samples. An ANOVA followed by a Tukey-Kramer 

test indicates that mean pH in the Akron WPCS effluent 

samples was statistically lower than in samples from any of 

the river sites. Further, the mean pH in samples from Botzum, 

the site immediately downstream from the Akron WPCS, was 

statistically lower than in samples from the Old Portage and 

Jaite sites.

Assessment of Between-Site Differences in 

Microbiological Characteristics

Between-site differences in central values of concentrations of microorganisms were assessed by means of a combi-
20 Microbiological Water Quality, Cuyahoga River, Cuyahoga Valley National Park 

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Figure 7. Distribution of Clostridium perfringens concentrations as a function of 

site on the Cuyahoga River, including the Akron Water Pollution Control Station 

(WPCS), Ohio, 2000 and 2002.

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Figure 6. Distribution of Escherichia coli concentrations as a function of site on the 

Cuyahoga River, including the Akron Water Pollution Control Station (WPCS), Ohio, 

2000 and 2002.
Summary and Conclusions 21

nation of ANOVA and (where appropriate) the Tukey-Kramer 

test. Two basic types of assessments were done. 

The first type of assessment focused on identifying differences in concentrations between samples collected from 

all combinations of the Cuyahoga River sites and the Akron 

WPCS site. Based on an ANOVA coupled with the TukeyKramer test, geometric-mean E. coli and C. perfringens

concentrations were found to be statistically lower and higher, 

respectively, in the Akron WPCS effluent than in water 

samples from the Cuyahoga River sites. These relations are 

evident in figures 6 and 7. A comparable analysis on ranks 

of F-specific coliphage concentration indicated that the mean 

rank in samples from the Akron WPCS was statistically higher 

than in samples from Jaite; significant differences in ranks of 

F-specific coliphage concentration were not found between 

other sites. ANOVAs on the logarithms of somatic coliphage 

concentrations, logarithms and ranks of infectious enterovirus 

concentrations, and on ranks of Giardia concentrations did 

not show any significant between-site differences for these 

microorganisms.

The second type of assessment was done to determine 

whether significant differences existed between central values 

of concentration or rank considering only those samples from 

Cuyahoga River sites immediately upstream (Old Portage) 

and downstream (Botzum) from the Akron WPCS. Because 

the minimum significant difference between mean concentrations determined in the Tukey-Kramer test is a function of the 

weighted average variance (Sokal and Rohlf, 1981), it was felt 

that inclusion of the Akron WPCS results (as was done in the 

first type of assessment) potentially could increase or decrease 

the weighted average variance in a way that could obscure 

differences between central values observed for the river 

sites. Based on ANOVAs and a post hoc Tukey-Kramer test, 

C. perfringens was the only microorganism to exhibit significant between-site differences (in this case for geometric-mean 

values). Elevated concentrations of C. perfringens in Akron 

WPCS effluent (fig. 6) discharged to the Cuyahoga River 

likely explain why geometric-mean concentrations at Botzum 

were significantly higher than at Old Portage.

Summary and Conclusions

Discharges of stormwater, combined-sewer overflows, 

and incompletely disinfected wastewater to the Cuyahoga 

River cause frequent exceedances of the bacteriological 

water-quality standards for body-contact recreation. People 

who come in contact with the contaminated water are at risk 

for gastrointestinal and (or) other illnesses. Elevated levels 

of fecal-indicator organisms would be cause for concern in 

any river but are of particular concern for the Cuyahoga River 

because of the CVNP and its goal of promoting water-contact 

recreation. In response to this concern, the U.S. Geological 

Survey, in collaboration with the National Park Service, conducted an in-depth study to investigate microbiological water 

quality of the Cuyahoga River within the CVNP. 

Water samples were collected from the Cuyahoga River 

and from the effluent of the Akron Water Pollution Control 

Station (WPCS) in 2000 and 2002. Four river sites were sampled in 2000, but only three river sites were sampled in 2002. 

The samples were collected over a similar range in streamflow 

in 2000 and 2002. In 2000, water samples were analyzed for 

E. coli, Salmonella, F-specific coliphage, enterovirus, infectious enterovirus, hepatitis A virus, and physical and chemical 

characteristics. In 2002, water samples were analyzed for the 

same constituents as in 2000, as well as for somatic coliphage, 

C. perfringens, Cryptosporidium, and Giardia. 

With the exception of Cryptosporidium, all microorganisms analyzed for were detected one or more times at each 

location sampled. E. coli concentrations in river samples 

exceeded the single-sample primary-contact recreational standard in approximately 87 percent of the samples and tended 

to be higher than in the effluent samples (presumably because 

of chlorination of the effluent). In contrast, concentrations of 

C. perfringens, a spore-forming bacterium present in large 

numbers in human and animal waste, tended to be higher in 

effluent samples than in river samples.

Detections of Salmonella were, with one exception, 

limited to samples collected in 2000; however, detections of 

hepatitis A virus were limited exclusively to samples collected 

in 2002. It is unknown why the detection of hepatitis A virus 

was limited to one year; however, the reduction in detections 

of Salmonella in 2002 may be due to analysis of smaller 

sample volumes. 

Hepatitis A virus was not detected in samples collected 

upstream from the Akron WPCS. All downstream detections 

of hepatitis A virus were coincident with detections in the 

Akron WPCS effluent. The pattern of detections and the presence of hepatitis A virus in the Akron WPCS effluent suggests 

that the Akron WPCS was a principal source of hepatitis A 

virus at the downstream sites.

Geometric-mean concentrations of E. coli were calculated 

on the basis of analytical results from at least five samples collected at each river site during May, July, and September 2000. 

In each case, the geometric-mean primary-contact recreational 

standard of 126 col/100 mL was exceeded.

Microbiological data for river samples in which E. coli

concentrations were less than the single-sample secondarycontact recreational standard (576 col/100 mL) were examined 

to assess the co-occurrence of other microorganisms. Seven of 

the 25 river samples with E. coli concentrations less than 576 

col/100 mL were analyzed for enterovirus, infectious enterovirus, and hepatitis A virus. Of those seven samples, five (71 

percent) contained enterovirus and infectious enterovirus at 

concentrations above detection, and one contained hepatitis A 

virus. Ten of the 25 river samples with E. coli concentrations 

less than 576 col/100 mL were analyzed for Salmonella, which 

was detected in three (30 percent) of the samples. An analysis 

of the co-occurence of pathogenic microorganisms in samples 

in which the single-sample secondary-contact recreational 
22 Microbiological Water Quality, Cuyahoga River, Cuyahoga Valley National Park 

standard was exceeded consistently showed higher percentages 

of co-occurrence than in samples in which the single-sample 

secondary-contact recreational standard was not exceeded.

C. perfringens concentrations were positively and 

significantly correlated with concentrations of E. coli in river 

samples. The correlation between these two microorganisms 

was also positive in samples collected from Cuyahoga River 

sites combined with samples from the Akron WPCS effluent 

(the combined data set); however, the correlation was weaker 

and not statistically significant. 

Somatic coliphage concentrations and F-specific coliphage concentrations were positively and significantly 

correlated in the combined data set. The correlation between 

this same microorganism pair also was positive in samples collected from river sites alone; however, the number of observations was smaller and the correlation was not statistically 

significant.

ANOVA results indicated that geometric-mean concentrations of somatic coliphage and F-specific coliphage 

in river samples did not differ significantly as a function of 

the presence/absence of enterovirus, infectious enterovirus, 

or hepatitis A virus. Similarly, mean infectious enterovirus 

concentrations did not differ significantly as a function of the 

presence/absence of hepatitis A virus. In spite of the lack of 

statistically significant differences, there was an observable 

tendency for detections of hepatitis A virus to be associated 

with higher median concentrations of somatic coliphage, 

F-specific coliphage, and infectious enterovirus. There also 

appears to be a relation between C. perfringens and hepatitis A 

virus wherein geometric mean C. perfringens concentrations 

were significantly higher in combined dataset samples where 

hepatitis A virus was present than in samples where hepatitis 

A virus was absent. A similar tendency was present for data 

from the Cuyahoga River sites alone; however, the difference 

in geometric-mean concentration was not statistically significant.

E. coli concentrations exhibited a weak but significant 

positive correlation with streamflow. The tendency for E. coli

concentration to increase with streamflow was observed for 

Cuyahoga River sites upstream and downstream from the 

Akron WPCS, indicating that E. coli loading is an increasing 

function of rainfall /runoff processes and that E. coli sources 

upstream from the Akron WPCS may have appreciable influence on concentrations in the river downstream from the 

Akron WPCS. 

F-specific coliphage concentration also was positively 

correlated with streamflow; however, the p value for that correlation was slightly larger than alpha, so the relation was not 

statistically significant. Correlations between streamflow and 

concentrations (or ranks of concentrations) of other microorganisms were not significant, nor were there significant sitespecific correlations between physical or chemical water-quality measures and concentrations of microorganisms. 

ANOVA and the Tukey-Kramer test were used to assess 

between-site differences in central values of concentrations 

(or ranks of concentrations) of microorganisms. E. coli and 

C. perfringens concentrations were statistically lower and 

higher, respectively, in the Akron WPCS effluent than in water 

samples from the other Cuyahoga River sites. The mean rank 

of F-specific coliphage concentrations in samples from the 

Akron WPCS was statistically higher than in samples from 

the Jaite site; however, no significant differences in ranks of 

F-specific coliphage concentration were found between other 

sites. No significant between-site differences in central values 

of concentrations (or ranks of concentrations) were detected 

for other microorganisms. 

Between-site differences in central values of concentrations (or ranks of concentrations) also were assessed on the 

basis of samples collected at the Old Portage and Botzum 

sites only. That assessment was aimed at identifying differences that likely can be attributed to the Akron WPCS, which 

discharges its effluent to the Cuyahoga River between the two 

sites. C. perfringens was the only microorganism that exhibited significant between-site differences, probably because 

of the elevated concentrations of C. perfringens in the Akron 

WPCS effluent.

Results from this study demonstrate that pathogenic 

microorganisms can be present in the Cuyahoga River even 

when E. coli concentrations do not exceed the single-sample 

primary- or secondary-contact recreational standards. However, the fact that the percentage of detections of pathogenic 

organisms consistently was greater in samples in which the 

single-sample secondary-contact recreation standard was 

exceeded demonstrates that E. coli is a useful indicator of 

human health risk in the study area.
References Cited 23

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26 Microbiological Water Quality, Cuyahoga River, Cuyahoga Valley National Park 

Appendix A. Quality-control samples for the detection of enterovirus and hepatitis A virus by means of the reverse transcriptase-polymerase chain reaction (RT-PCR) method.

[PCR, polymerase chain reaction]

Sample type Description Purpose

Negative-process control Sterile water analyzed with each batch of 

five samples, beginning at the inhibitor 

removal step.

To determine whether cross-contamination 

of samples occurs during the inhibitor removal, RT-PCR, and hybridization steps.

Seeded negative-process control Positive-control viruses that are added to a 

duplicate negative-process control with 

each batch of five samples before the 

RT- PCR step.

To verify the proper completion of the RTPCR reaction.

Matrix spike Positive-control viruses added to duplicate 

environmental samples before the RTPCR step.

To indicate whether inhibitors are present in 

the environmental samples.

PCR positive control Positive-control viruses added to sterile 

water before the RT-PCR step (also 

serves as the hybridization positive 

control).

To verify the proper completion of the RTPCR and hybridization steps.

PCR negative control Sterile water analyzed at the RT-PCR step. To determine whether cross-contamination 

of samples occurs during the RT-PCR 

step.

Hybridization negative control Sterile water analyzed at the hybridization 

step.

To determine whether cross-contamination 

of samples occurs during the 

hybridization step.

Appendix A
Appendix B 27

Old Portage

05/03/00 290 780 -- -- -- -- -- -- -- -- -- --05/10/00 290 12,000 -- -- -- -- -- -- -- -- -- --05/11/00 253 1,000 -- -- -- -- -- -- -- -- -- --05/17/00 152 350 -- -- -- -- -- -- -- -- -- --05/23/00 799 7,200 present S. java 18 -- 33a present absent -- -- --07/06/00 185 1,400 -- -- -- -- -- -- -- -- -- --07/12/00 113 2,400 absent -- -- -- -- -- -- -- -- --07/18/00 290 4,600 present S. braenderup 18 -- 82 present absent -- -- --07/25/00 117 1,600 -- -- -- -- -- -- -- -- -- --07/26/00 113 1,500 -- -- -- -- -- -- -- -- -- --09/05/00 115 520 -- -- -- -- -- -- -- -- -- --09/06/00 111 64 -- -- -- -- -- -- -- -- -- --09/11/00 167 18,000 present S. hartford -- -- -- -- -- -- -- --09/19/00 125 540 absent -- 2 -- <16 present absent -- -- --09/26/00 350 1,300 -- -- -- -- -- -- -- -- -- --05/21/02 960 3,000 absent -- 34 790 35 present absent 190 <1 <106/17/02 485 370 absent -- 160 130 <16 absent absent 67 <1 307/15/02 92 350 absent -- <1 72 190 present absent 120 <1 1

Akron WPCS

05/23/00 143 2,500 -- -- -- -- -- -- -- -- -- --05/23/00 143 2,400 present S. miami 180 -- 33a present absent -- -- --05/24/00 112 110 -- -- -- -- -- -- -- -- -- --06/01/00 118 240 -- -- -- -- -- -- -- -- -- --

Appendix B. Streamflow and microbiological data for Cuyahoga River samples and Akron Water Pollution Control Station (WPCS) effluent samples collected in 2000 and 2002.[ft3/s, cubic feet per second; col/100 mL, colonies per 100 milliliters; plaques/100 mL, plaques per 100 milliliters; MPN/100 L, most probable number per 100 liters; oocysts/10 L, oocysts per 10 liters; cysts/10 L, cysts per 10 liters;--, no data; bold, results based on colony count outside the ideal range of 20-80 colonies per plate; <, less than]

Streamflow (ft3/s) Salmonella serotype

Hepatitis A

virus

(presence/

absence)

Giardia

(cysts/10 L)

Appendix B

Cryptosporidium

(oocysts/10 L)

Date

Escherichia

coli

(col/100 mL)

Salmonella

(presence/

absence)

F-specific

coliphage

(plaques/100 mL)

Somatic

coliphage

(plaques/100 mL)

Infectious

enterovirus

(MPN/100 L)

Enterovirus

(presence/

absence)

Clostridium

perfringens

(col/100 mL)
28 Microbiological Water Quality, Cuyahoga River, Cuyahoga Valley National Park 

Appendix B. Streamflow and microbiological data for Cuyahoga River samples and Akron Water Pollution Control Station (WPCS) effluent samples collected in 2000 and 2002.—Continued [ft3/s, cubic feet per second; col/100 mL, colonies per 100 milliliters; plaques/100 mL, plaques per 100 milliliters; MPN/100 L, most probable number per 100 liters; oocysts/10 L, oocysts per 10 liters; cysts/10 L, cysts per 10 liters;--, no data; bold, results based on colony count outside the ideal range of 20-80 colonies per plate; <, less than] Stream- flow (ft3/s) Salmonella serotype Hepatitis A virus (presence/ absence) Giardia (cysts/10 L) 06/05/00 196 30 -- -- -- -- -- -- -- -- -- -- 06/08/00 114 77 -- -- -- -- -- -- -- -- -- -- 06/12/00 192 32 -- -- -- -- -- -- -- -- -- -- 07/11/00 102 2,200 present S. braenderup -- -- -- -- -- -- -- -- 07/18/00 109 2,900 present S. manhattan 28 -- 190 present absent -- -- -- 09/19/00 86 30 -- -- -- -- -- -- -- -- -- -- 09/19/00 86 110 absent -- 110 -- 73 present absent -- -- -- 09/26/00 93 38 -- -- -- -- -- -- -- -- -- -- 09/28/00 89 32 -- -- -- -- -- -- -- -- -- -- 09/29/00 87 8 -- -- -- -- -- -- -- -- -- -- 10/02/00 90 14 -- -- -- -- -- -- -- -- -- -- 10/03/00 88 18 -- -- -- -- -- -- -- -- -- -- 10/16/00 86 38 -- -- -- -- -- -- -- -- -- -- 10/23/00 84 55 -- -- -- -- -- -- -- -- -- -- 05/21/02 119 210 absent -- 29 79 81 present present 1,700 <1 <1 06/17/02 108 970 absent -- 34 290 110 present present 800 <1 10 07/15/02 84 1,300 absent -- 12 170 150 present present 1,100 <1 8 Botzum 05/03/00 -- 580 -- -- -- -- -- -- -- -- -- -- 05/10/00 474 8,900 -- -- -- -- -- -- -- -- -- -- 05/17/00 283 170 -- -- -- -- -- -- -- -- -- -- 05/24/00 987 1,700 present S. muenster 7 -- 120 present absent -- -- -- 05/25/00 811 730 -- -- -- -- -- -- -- -- -- -- 07/06/00 317 2,600 -- -- -- -- -- -- -- -- -- -- 07/12/00 217 2,400 absent -- -- -- -- -- -- -- -- -- 07/18/00 486 3,200 present S. braenderup 6 -- 73 present absent -- -- -- 07/19/00 511 900 -- -- -- -- -- -- -- -- -- -- 07/26/00 226 1,600 -- -- -- -- -- -- -- -- -- -- 09/05/00 224 490 -- -- -- -- -- -- -- -- -- -- Date Escherichia coli (col/100 mL) Salmonella (presence/ absence) F-specific coliphage (plaques/100 mL) Somatic coliphage (plaques/100 mL) Infectious enterovirus (MPN/100 L) Enterovirus (presence/ absence) Clostridium perfringens (col/100 mL) Cryptospo- ridium (oocysts/10 L)
Appendix B 29

Appendix B. Streamflow and microbiological data for Cuyahoga River samples and Akron Water Pollution Control Station (WPCS) effluent samples collected in 2000 and 2002.—Continued[ft3/s, cubic feet per second; col/100 mL, colonies per 100 milliliters; plaques/100 mL, plaques per 100 milliliters; MPN/100 L, most probable number per 100 liters; oocysts/10 L, oocysts per 10 liters; cysts/10 L, cysts per 10 liters;--, no data; bold, results based on colony count outside the ideal range of 20-80 colonies per plate; <, less than]

Streamflow (ft3/s) Salmonella serotype

Hepatitis A

virus

(presence/

absence)

Giardia

(cysts/10 L)

09/11/00 314 12,000 -- -- -- -- -- -- -- -- -- --09/12/00 226 2,600 present S. java -- -- -- -- -- -- -- --09/19/00 228 700 present S. oranienburg 3 -- <16 present absent -- -- --09/26/00 521 730 -- -- -- -- -- -- -- -- -- --05/22/02 992 7,300 absent -- 24 63 110 present present 370 <1 <106/18/02 413 440 absent -- 8 47 110 present absent 350 <1 1607/16/02 176 410 absent -- 63 470 190 present present 340 <1 <1

Jaite

05/03/00 -- 900 -- -- -- -- -- -- -- -- -- --05/11/00 429 430 -- -- -- -- -- -- -- -- -- --05/17/00 300 160 -- -- -- -- -- -- -- -- -- --05/18/00 320 730 -- -- -- -- -- -- -- -- -- --05/24/00 1,090 2,300 absent -- 18 -- -- -- -- -- -- --05/25/00 896 1,100 -- -- -- -- -- -- -- -- -- --07/06/00 309 710 -- -- -- -- -- -- -- -- -- --07/07/00 331 680 -- -- -- -- -- -- -- -- -- --07/11/00 356 5,500 present S. seftenberg -- -- -- -- -- -- -- --07/17/00 488 4,600 present S. newport 4 -- -- -- -- -- -- --07/25/00 248 270 -- -- -- -- -- -- -- -- -- --09/06/00 243 250 -- -- -- -- -- -- -- -- -- --09/11/00 396 22,000 present S. newport -- -- -- -- -- -- -- --09/18/00 240 350 present unknown 10 -- -- -- -- -- -- --09/20/00 253 480 -- -- -- -- -- -- -- -- -- --09/26/00 524 1,500 -- -- -- -- -- -- -- -- -- --05/22/02 1,040 2,200 absent -- 4 170 110 present present 270 <1 <106/18/02 472 470 absent -- 6 78 56 absent absent 360 <1 607/16/02 183 240 present S. arizona <1 33 190 present absent 120 <1 <1

Date

Escherichia

coli

(col/100 mL)

Salmonella

(presence/

absence)

F-specific

coliphage

(plaques/100 mL)

Somatic

coliphage

(plaques/100 mL)

Infectious

enterovirus

(MPN/100 L)

Enterovirus

(presence/

absence)

Clostridium

perfringens

(col/100 mL)

Cryptosporidium

(oocysts/10 L)
30 Microbiological Water Quality, Cuyahoga River, Cuyahoga Valley National Park Independence 05/03/00 676 1,800 -- -- -- -- -- -- -- -- -- -- 05/04/00 550 620 -- -- -- -- -- -- -- -- -- -- 05/11/00 454 1,200 -- -- -- -- -- -- -- -- -- -- 05/18/00 361 470 -- -- -- -- -- -- -- -- -- -- 05/24/00 1,070 1,300 present S. java 11 -- -- -- -- -- -- -- 07/07/00 385 260 -- -- -- -- -- -- -- -- -- -- 07/11/00 390 320 present S. newport -- -- -- -- -- -- -- -- 07/12/00 356 210 -- -- -- -- -- -- -- -- -- -- 07/17/00 532 5,700 absent -- 5 -- -- -- -- -- -- -- 07/25/00 315 220 -- -- -- -- -- -- -- -- -- -- 09/06/00 284 400 -- -- -- -- -- -- -- -- -- -- 09/11/00 592 25,000 present multiple -- -- -- -- -- -- -- -- 09/18/00 340 320 absent -- 3 -- -- -- -- -- -- -- 09/25/00 854 4,600 -- -- -- -- -- -- -- -- -- -- 09/26/00 614 2,600 -- -- -- -- -- -- -- -- -- --

aResults were omitted from dataset because concentrations did not exceed levels measured in the equipment blank.

Appendix B. Streamflow and microbiological data for Cuyahoga River samples and Akron Water Pollution Control Station (WPCS) effluent samples collected in 2000 and 2002.—Continued[ft3/s, cubic feet per second; col/100 mL, colonies per 100 milliliters; plaques/100 mL, plaques per 100 milliliters; MPN/100 L, most probable number per 100 liters; oocysts/10 L, oocysts per 10 liters; cysts/10 L, cysts per 10 liters;--, no data; bold, results based on colony count outside the ideal range of 20-80 colonies per plate; <, less than]

Streamflow (ft3/s) Salmonella serotype

Hepatitis A

virus

(presence/

absence)

Giardia

(cysts/10 L) Date

Escherichia

coli

(col/100 mL)

Salmonella

(presence/

absence)

F-specific

coliphage

(plaques/100 mL)

Somatic

coliphage

(plaques/100 

mL)

Infectious

enterovirus

(MPN/100 L)

Enterovirus

(presence/

absence)

Clostridium

perfringens

(col/100 mL)

Cryptosporidium

(oocysts/10 L)