Effects Of Receiving-Water Quality And 

Wastewater Treatment On Injury, Survival, 

And Regrowth Of Fecal-Indicator Bacteria 

And Implications For Assessment Of 

Recreational Water Quality

By Donna S. Francy, Teresa L. Hart, and Cathy M. Virosteck

U.S. Geological Survey

Water-Resources Investigations Report 96-4199

Prepared in cooperation with 

OHIO WATER DEVELOPMENT AUTHORITY, 

NORTHEAST OHIO REGIONAL SEWER DISTRICT, 

SUMMIT COUNTY

Columbus, Ohio 

1996
U.S. DEPARTMENT OF THE INTERIOR 

BRUCE BABBITT, Secretary

U.S. GEOLOGICAL SURVEY 

Gordon P. Eaton, Director

For additional information 

write to:

District Chief 

U.S. Geological Survey 

975 West Third Avenue 

Columbus, Ohio 43212-3192

Copies of this report can be 

purchased from:

USGS Branch of Information Services

Box 25286

Denver, Colorado 80225
CONTENTS

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

Introduction ...............................................................................................................................................................^ 2

Purpose and scope ...................................................................................................................................................... 2

Previous studies.......................................................................................................................................................... 2

Methods of study.................................................................................................................................................................. 3

Analysis of microbiological properties....................................................................................................................... 3

Field studies......................................................................................................................................^ 5

Statistical methods..................................................................................................................................................^ 7

Quality assurance and quality control........................................................................................................................ 9

Evaluation of enhanced-recovery methods for use in field studies...................................................................................... 10

Effects of receiving-water quality and wastewater treatment on injury, survival, and regrowth of fecal-indicator

bacteria.................................................................................................................................................................................. 12

Receiving-water quality.............................................................................................................................................. 12

Concentrations of fecal-indicator bacteria.................................................................................................................. 13

Percent injury of fecal-indicator bacteria................................................................................................................... 20

Percent survival of fecal-indicator bacteria................................................................................................................ 26

Implications for assessment of recreational water quality.................................................................................................... 27

Relation of enhanced-recovery method concentrations to standard-method concentrations..................................... 27

Relation of enhanced-recovery and standard-method concentrations to Ohio recreational water-quality

standards....................................................................................................^^ 34

Sunimary and conclusions........................................................................^^ 38

References cited.................................................................................................................................................................... 41

FIGURES

1. Diagram showing incubation chamber used in field studies................................................................................... 5

2. Map showing collection locations and field-study sites.......................................................................................... 6

3-7. Graphs showing mean fecal-coliform concentrations determined by use of standard and enhanced-recovery 

methods in:

3. Wastewater effluent during May 1994 and August 1994 field studies at Lake Erie, Cleveland, Ohio:

unchlorinated, chlorinated, and dechlorinated samples................................................................................. 16

4. Unchlorinated wastewater effluent during June 1994, August 1995, and September 1995 field

studies at Cuyahoga River sites, Ohio........................................................................................................... 17

5. Chlorinated wastewater effluent during June 1994, August 1995, and September 1995 field studies

at Cuyahoga River sites, Ohio....................................................................................................................... 18

6. Dechlorinated wastewater effluent during June 1994, August 1995, and September 1995 field

studies at Cuyahoga River sites, Ohio........................................................................................................... 19

7. Combined-sewer-overflow effluent during May and June 1995 field studies at Cuyahoga River at

Independence, Ohio: unchlorinated, chlorinated, and dechlorinated samples .............................................. 21

Contents I
8-10. Boxplots showing percent injury of fecal coliforms in relati on to treatment type in:

8. Wastewater effluent collected from Easterly Wastewater Treatment Plant and incubated at Lake Erie

field site, Cleveland, Ohio: May 1994 and August 1994.............................................................................. 23

9. Wastewater effluent collected from Southerly Wastewater Treatment Plant and incubated at Cuyahoga

River at Peninsula and West Third field sites, Cleveland area, Ohio: June 1994 and August 1995 ............ 24

10. Combined-sewer-overflow effluent collected from Mill Creek and incubated at Cuyahoga River at

Independence, Cleveland area, Ohio: May 1995 and June 1995 .................................................................. 25

11-14. Graphs showing:

11. Regression relation between enhanced-recovery and standard-method concentrations of fecal coliforms 

in seven field studies of wastewater and combined-sewer-overflow effluent collected in the Cleveland 

area, Ohio...................................................................................................................................................... 28

12. Residuals from regression relation between enhanced-recovery and standard-method concentrations of 

fecal coliforms in seven field studies against predicted enhanced-recovery concentrations, elapsed 

time, and treatment type................................................................................................................................ 30

13. Regression relation between enhanced-recovery and j tandard-method concentrations of fecal coliforms 

and associated residuals against predicted enhanced-rscovery concentrations for data grouped by 

magnitude of standard-method concentrations: <5,00() colonies per 100 milliliters and >5,000 

colonies per 100 milliliters............................................................................................................................ 32

14. Regression relation between enhanced-recovery and standard-method concentrations of fecal coliforms 

and associated residuals against predicted enhanced-recovery concentrations for data grouped by 

treatment: unchlorinated, chlorinated, and dechlorinated............................................................................. 33

15-17. Bar graphs showing percentage of samples, by treatment type, in which fecal-coliform concentrations 

determined by standard and enhanced-recovery methods:

15. Met and exceeded Ohio's bathing-water single-sample standard: (A) wastewater effluent, low 

concentrations of bacteria, (B) wastewater effluent, high concentrations of bacteria, and 

(C) combined-sewer-overflow effluent......................................................................................................... 35

16. Met and exceeded Ohio's primary-contact single-sample standard: (A) wastewater effluent, low 

concentrations of bacteria, (B) wastewater effluent, high concentrations of bacteria, and 

(C) combined-sewer-overflow effluent........................!................................................................................. 36

17. Met and exceeded Ohio's secondary-contact single-sample standard: (A) wastewater effluent, low 

concentrations of bacteria, (B) wastewater effluent, high concentrations of bacteria, and 

(C) combined-sewer-overflow effluent.......................................................................................................... 37

TABLES

1. Differences in mean rank of concentrations of fecal coliforntis obtained by use of enhanced-recovery 

methods and concentrations of fecal coliforms obtained by use of standard method in unchlorinated, 

chlorinated, and dechlorinated wastewater effluent................................................................................................ 11

2. Dates, sites, and durations of field studies done with wastewater or combined-sewer-overflow effluent

from three collection locations, 1994-1995............................................................................................................ 12

3. Mean values of water-quality field measurements and constituent concentrations for wastewater and

combined-sewer-overflow effluent field studies...................J................................................................................. 14

4. Percent verifications of fecal coliforms in wastewater or combined-sewer-overflow effluent by use of

standard or enhanced-recovery method.................................................................................................................. 15

5. Mean percent injury of fecal coliforms in unchlorinated, chlorinated, and dechlorinated wastewater or

combined-sewer-overflow effluent field studies..................................................................................................... 22

6. Mean percent survival of fecal coliforms in unchlorinated, chlorinated, and dechlorinated wastewater or

combined-sewer-overflow effluent in field studies................................................................................................. 26

7. Regression statistics for enhanced-recovery and standard-method concentrations of fecal coliforms in

wastewater and combined-sewer-overflow effluent collected in the Cleveland area, Ohio ................................... 29

II Contents
CONVERSION FACTORS AND ABBREVIATED WATER-QUALITY UNITS

Multiply

liter (L)

milliliter (mL)

milligram per liter (mg/L)

colonies per 100 milliliters

(coI/lOOmL)

micron (|xm)

millimeter (mm)

By

0.03531

0.0607

0.00006243

283.2

0.00003937

0.03937

To obtain

cubic foot

cubic inch

pound per square inch

colonies per cubic foot

inch

inch

Water and air temperatures in degrees Celsius (°C) can be converted to degrees Fahrenheit (°F) by the 

following equation: °F = 1.8 (°C) + 32.

Abbreviated water-quality units used in this report: Chemical concentrations are given in metric units. 

Chemical concentration is given in milligrams per liter (mg/L). Milligrams per liter is a unit expressing the 

concentration of chemical constituents in solution as weight (milligrams) of solute per unit volume (liter) 

of water.

Specific conductance of water is expressed in microsiemens per centimeter at 25 degrees Celsius (|xS/cm).

U.S. Geological Survey scientist retrieving chambers in the river.

Contents III
Effects of Receiving-Water Quality and Wastewater 

Treatment on Injury, Survival, and Regrowth of FecalIndicator Bacteria and Implications for Assessment of 

Recreational Water Quality

By Donna S. Francy, Teresa L. Hart, andCathy M. Virosteck

Abstract

Bacterial injury, survival, and regrowth 

were investigated by use of replicate flow-through 

incubation chambers placed in the Cuyahoga 

River or Lake Erie in the greater Cleveland metropolitan area during seven 4-day field studies. 

The chambers contained wastewater or combined-sewer-overflow (CSO) effluents treated 

three ways unchlorinated, chlorinated, and 

dechlorinated. At timestep intervals, the chamber 

contents were analyzed for concentrations of 

injured and healthy fecal coliforms by use of standard selective and enhanced-recovery membranefiltration methods.

Mean percent injuries and survivals were 

calculated from the fecal-coliform concentration 

data for each field study. The results of analysis of 

variance (ANOVA) indicated that treatment 

affected mean percent injury and survival, 

whereas site did not. In the warm-weather Lake 

Erie field study, but not in the warm-weather 

Cuyahoga River studies, the results of ANOVA 

indicated that dechlorination enhanced the repair 

of injuries and regrowth of chlorine-injured fecal 

coliforms on culture media over chlorination 

alone. The results of ANOVA on the percent 

injury from CSO effluent field studies indicated 

that dechlorination reduced the ability of organisms to recover and regrow on culture media over 

chlorination alone. However, because of atypical 

patterns of concentration increases and decreases 

in some CSO effluent samples, more work needs

to be done before the effect of dechlorination and 

chlorination on reducing fecal-coliform concentrations in CSO effluents can be confirmed. The 

results of ANOVA on percent survivals found statistically significant differences among the three 

treatment methods for all but one study. Dechlorination was found to be less effective than chlorination alone in reducing the survival of fecal 

coliforms in wastewater effluent, but not in CSO 

effluent.

If the concentration of fecal coliforms 

determined by use of the enhanced-recovery 

method can be predicted accurately from the concentration found by use of the standard method, 

then increased monitoring and expense to detect 

chlorine-injured organisms would be unnecessary. 

The results of linear regression analysis, however, 

indicated that the relation between enhancedrecovery and standard-method concentrations was 

best represented when the data were grouped by 

treatment. The model generated from linear 

regression of the unchlorinated data set provided 

an accurate estimate of enhanced-recovery concentrations from standard-method concentrations, whereas the models generated from the 

chlorinated and dechlorinated data sets did not. In 

addition, evaluation of fecal-coliform concentrations found in field studies in terms of Ohio recreational water-quality standards showed that 

concentrations obtained by standard and 

enhanced-recovery methods were not comparable. Sample treatment and analysis methods were

Abstract 1
found to affect the percentage of samples meeting 

and exceeding Ohio's bathing-water, primarycontact, and secondary-contact standards. Therefore, determining the health risk of swimming in 

receiving waters was often difficult without information on enhanced-recovery method concentrations and was especially difficult in waters 

receiving high proportions of chlorinated or 

dechlorinated effluents.

Introduction

The U.S. Environmental Protection Agency 

(USEPA) required States to develop new chlorine 

water-quality standards for protection of aquatic life, 

such that chlorine concentrations in receiving waters 

must be substantially reduced (U.S. Environmental 

Protection Agency, 1985). To meet these new chlorine 

standards, many wastewater treatment plants in Ohio 

and elsewhere that chlorinate for disinfection must 

now dechlorinate effluents before discharging them to 

inland waterways. This process results in a reduction 

in chlorine-contact time for fecal-indicator bacteria 

and pathogenic microorganisms whose concentrations 

water-resource managers seek to control. However, 

little information exists on the effect of dechlorination 

on the repair and survival of chlorine-injured fecalindicator bacteria and the possible increased risk of 

swimming in waters with an undetected population of 

injured bacteria.

To augment the literature on recovery of injured 

fecal-indicator bacteria and to explore the implications 

of bacteria recovery for recreational water-quality 

standards, the U.S. Geological Survey (USGS), in 

cooperation with the Northeast Ohio Regional Sewer 

District (NEORSD), the Ohio Water Development 

Authority, and Summit County, Department of Environmental Services, studied the effects of receivingwater quality and wastewater-plant chlorination practices on fecal-coliform injury and survival in receiving 

waters and on fecal-coliform regrowth on growth 

medium.

Purpose and Scope

This report describes seven field studies done 

during the recreational seasons of 1994 and 1995 at 

one Lake Erie location and three Cuyahoga River 

locations in the greater Cleveland, Ohio, metropolitan

area. For each field study, bacterial injury, survival, 

and regrowth were investigated by use of replicate 

flow-through incubation chambers placed in the water 

column of the receiving stream or lake. The chambers 

contained wastewater or combined-sewer-overflow 

(CSO) effluents treated three ways: unchlorinated, 

chlorinated, and dechlorinated. At timestep intervals, 

the Chamber contents were analyzed for concentrations of injured and healthy fecal coliforms by use of 

standard selective and enhanced-recovery membranefiltration (MF) methods. Physical and chemical waterquality characteristics also were monitored at timestep 

intervals. Percent injury and survival were calculated

sach timestep. The effects of treatment and ambiwater-quality characteristics on percent injury andfor 

ent

survival were determined by use of analysis of variance (ANOVA). The relation between concentrations 

of fecal coliforms by use of standard and enhancedrecovery methods was determined by use of linear 

regression analysis. Concentrations of fecal coliforms 

were compared to Ohio water-quality standards to 

determine the importance of bacteria injury, survival, 

and regrowth on growth medium in the assessment of 

baci:erial water quality This report provides waterresource managers with information on the use of 

enhanced-recovery methods to assess recreational 

water quality and on the effectiveness of different 

chlorination practices in reducing fecal-coliform concentrations.

Previous Studies

Considerable information is available from laboratory studies showing that fecal-indicator bacteria 

surviving chlorination are able to repair injuries and 

survive in the aquatic environment. Investigators 

determined that the proportion of bacteria surviving 

chlorination increased when chlorinated wastewater 

was diluted with streamwater or distilled water (Heukelekian, 1951), as chlorine-contact time decreased 

(Braswell and Hoadley, 1974), or as residual chlorine 

concentration decreased (Shuval and others, 1973). In 

another study (Kinney and others, 1978), investigators 

found that the concentration of coliform bacteria in 

uncnlorinated wastewater effluent, including those in 

the fecal-coliform group, steadily declined during a 5- 

day study, whereas the concentration of coliforms in 

the same effluent increased after chlorination. Because 

of me natural die-off pattern noted in the unchlorinated wastewater, the authors attributed the increase in

2 Effects of Receiving-Water Quality and Wastewater Treatment on Injury, Survival, and Regrowth of Fecal-Indicator Bacteria
bacteria concentration in chlorinated wastewater to 

repair of injured organisms rather than growth of 

healthy organisms. It was also suggested that standard 

selective MF methods for enumerating fecal-indicator 

bacteria do not support the growth of chlorine-injured 

bacteria (Kinney and others, 1978). Standard methods, 

defined as those methods believed to represent the best 

current practice of American water analysts, are generally applicable to ordinary problems of sanitary investigations (American Public Health Association and 

others, 1992). Selective methods are defined as those 

methods that inhibit growth of nontarget bacteria 

groups in order to encourage growth of target organisms.

The most probable number (MPN) method, containing a nonselective enrichment step, was superior to 

the standard selective MF method in parallel tests for 

enumerating chlorine-injured fecal coliforms (Lin, 

1973) and in parallel tests for enumerating unchlorinated and chlorine-injured coliforms (Mowat, 1976). 

The USEPA (U.S. Environmental Protection Agency, 

1978) recommends that the standard selective MF 

method should not be used with chlorinated wastewaters and that any decision to use this test requires parallel MF/MPN evaluations to determine applicability 

of the MF method. However, because the MPN 

method is time-consuming, cumbersome, and an estimate of the "most probable number," many investigators sought improvements to the standard-selective 

MF method that would enhance growth of chlorineinjured bacteria (Camper and McFeters, 1979). Many 

enhanced-recovery MF methods have a pre-enrichment step on nonselective media, a temperature acclimation time, and (or) an alternate media devoid of 

inhibitory chemicals.

Several investigators used standard-selective 

MF methods and (or) MPN methods to investigate survival of chlorine-injured bacteria in the field. Silvey 

and others (1974) determined concentrations of total 

coliforms and fecal coliforms by use of MPN methods 

in the Trinity River in Texas, downstream from four 

wastewater treatment plants. The investigators suggested that nonfecal-coliform strains exhibited significant regrowth following chlorination, whereas fecalcoliform strains failed to regrow in the river. In a study 

in the Chicago area (Haas and others, 1988), investigators used standard selective MF methods to compare 

concentrations of fecal coliforms in receiving waters 

before and after a wastewater treatment plant discontinued chlorination. They suggested that, beyond a

certain affected zone, chlorination of an effluent may 

not improve microbiological water quality in the 

receiving stream. However, these studies were not 

done by use of a controlled group of fecal-indicator 

bacteria; and contributing influences from surfacewater runoff and combined-sewer overflows could not 

be ruled out.

Bissonnette and others (1975) investigated the 

influence of environmental stress on Escherichia coli 

(E. coli) injury and survival by use of membrane-filter 

chambers containing a controlled population of bacteria. E. coli is a major species in the fecal-coliform 

group and a natural inhabitant of the intestinal tract of 

warm-blooded animals. The investigators observed 

that, upon exposure to the aquatic environment, concentrations of E. coli obtained by use of a selective 

method were less than those obtained by use of an 

enhanced-recovery method. They also observed substantial variation in percentage of injury and survival 

of E. coli in various stream environments.

Information regarding the effects of ambient 

water-quality conditions and wastewater-plant chlorination practices on injury and survival of fecal-indicator bacteria in receiving waters is lacking. However, 

the survival of chlorine-injured fecal-indicator bacteria may have implications from a public-health and 

water-quality perspective. Fecal-indicator bacteria that 

survive but are injured from chlorination may be able 

to repair their injuries and regrow, provided that a suitable growth medium is available. That growth 

medium may be enhanced-recovery medium or the 

gastrointestinal tract of an unsuspecting swimmer. 

Standard selective methods may not detect injured 

organisms; therefore, the enumeration of fecal-indicator bacteria and the pathogenic bacteria whose presence they indicate may be underestimated.

Methods of Study

Analysis of Microbiological Properties

Sterile techniques were used throughout all 

phases of collecting and processing samples for microbiological analysis. All samples for microbiological 

determinations were analyzed by MF techniques and 

were filtered onto 0.45-|am-pore-size membrane filters 

within 6 hours of collection and treatment. Several 

volumes of sample were plated in order to obtain 

countable plates within the ideal range of 20-60

Methods of Study 3
colonies per plate (Britton and Greeson, 1989). Dilutions of more concentrated samples were made in 

buffered dilution water with peptone (Britton and 

Greeson, 1989).

Enumeration of fecal-indicator bacteria.

Samples were analyzed for concentrations of fecal 

coliforms by use of two MF methods: (1) the standard 

selective method (Britton and Greeson, 1989) and (2) 

an enhanced-recovery method designed to support 

growth of chlorine-injured organisms. Several 

enhanced-recovery methods for fecal coliforms have 

been developed by other researchers and are listed in 

"Standard Methods for the Examination of Water and 

Wastewater" (American Public Health Association, 

1992).

Standard selective methods for enumeration of 

fecal coliforms are widely used for monitoring recreational waters and Wastewater effluents. The fecal 

coliform MF method (mFC) involves incubation of the 

filtered water sample on mFC agar (Difco Laboratories, Detroit, Mich. 1 ) for 22 to 24 hours at 44.5°C. 

After incubation, all blue colonies are counted as fecal 

coliforms. The mFC agar contains growth substances, salts, and several compounds to inhibit 

growth of nontarget organisms.

Several enhanced-recovery methods were tested 

for use in field studies. One method tested was a twolayer method in which a nonselective, enriched overlay of agar-lactose broth (Difco) was added to plates 

containing mFC agar less than 1 hour prior to use 

(Rose and others, 1975). The plates were incubated for 

2 hours at 35°C which allowed for repair and resuscitation of damaged cells, after which the temperature 

was increased to 44.5°C for 22 hours to attain the necessary selectivity. Investigators also tested a method 

that used bovine liver catalase (Sigma Chemical Co., 

St. Louis, Mo.) or sodium pyruvate (Sigma) with mFC 

agar and a 24-hour incubation at 44.5°C (Calabrese 

and Bissonnette, 1990). The developers of this method 

hypothesized that chlorine reduces activity of the 

enzyme catalase, causing the accumulation of toxic 

hydrogen peroxide. Pyruvate is a hydrogen peroxidedegrading compound. Another modification of mFC 

agar that was tested in this investigation was the elimination of rosolic acid (Difco) from the media with or 

without a resuscitation time at a lower temperature.

'Use of trade, brand, or firm names in this report is for 

identification purposes only and does not constitute endorsement 

by the U.S. Geological Survey.

Presswood and Strong (1978) showed that the elimination of rosolic acid from mFC agar resulted in 

improved recovery of chlorine-injured cells. Other 

investigators found that a 5-hour (Green and others, 

1977) or 4-hour (LeChevallier and others, 1984) 

resuscitation at 35°C followed by an 18- or 19-hour

o

incubation at 44.5 C yielded the highest recovery of 

fecall coliforms from chlorinated effluents, without 

excessive background growth. Finally, the investigators tested a new enhanced-recovery medium, mT7 

agai, developed by LeChevallier and others (1983) for 

recovery of chlorine-injured fecal coliforms. This agar 

contains growth substances, a surfactant (Tergitol-7), 

and penicillin G. After an 8-hour resuscitation at 

35°C, and a 12-hour incubation at 44.5°C, all yellow 

colonies on mT7 agar were counted as fecal coliforms.

Treatment of cell suspensions. Secondarytreated wastewater (hereinafter called wastewater) or 

CSO effluent was collected, kept on ice, and transportsd to the laboratory for treatment. The wastewater

or CSO effluent (hereinafter called cell suspension) 

was split into two containers. In one container, one 

part cell suspension was diluted with two parts autoclaved receiving water and refrigerated (the "unchlorinatejd" sample). A chlorine stock solution, prepared 

daity from dilution of commercially purchased bleach 

(5.2}> percent sodium hypochlorite), was added to the 

other cell suspension to achieve a total-residual chlorine concentration of 0.3 to 0.5 mg/L. Total residual 

chlorine was measured by use of a residual chlorine 

electrode. The chlorinated cell suspension was mixed 

intermittently for contact times from 8 to 30 minutes 

(during testing of enhanced-recovery methods before 

field studies) or 25 minutes (during field studies). The 

chlorinated cell suspension was then split in half. 

One-half of the cell suspension was diluted with two 

parts sterile receiving water to one part cell suspension; this is the "chlorinated" sample. The other half 

of trie cell suspension was dechlorinated by adding 0.1 

mL bf a sterile, 10 percent sodium thiosulfate solution 

per 100 mL cell suspension (American Public Health 

Association, 1992, Section 9060 A) and then diluted 

as above; this is the "dechlorinated" sample. After 

treatment, the cell suspensions were filtered and plated 

on various media formulations by use of standard and 

enh£.need-recovery methods.

4 Effects of Receiving-Water Quality and Wastewater Treatment on In ury, Survival, and Regrowth of Fecal-Indicator Bacteria
Field Studies

Injury, survival, and regrowth were investigated 

by use of replicate incubation chambers submerged in 

the water column of the receiving stream or lake. Incubation chambers were constructed of autoclavablepolycarbonate cylinders with 142-mm diameter, 0.45- 

(nm-pore-size sterile membrane filters placed at the 

two open ends (fig. 1). Similar incubation chambers 

and their use are described by Bissonnette and others 

(1975), McFeters and Stuart (1972), Hazen and Esch 

(1983), and Carillo and others (1985). The chambers 

were laboratory and field tested for another USGS 

project (D.N. Myers, U.S. Geological Survey, oral 

comm., 1994). The membranes were kept in place by 

securing screened endplates with stainless steel hardware. Outer endplates made of polycarbonate were 

used during transport or filling of the chambers and 

were removed while chambers were submerged in 

river or lake water. Each chamber has a capacity of 

approximately 600 mL. The incubation chambers 

retain bacteria while maintaining diffusion of water 

and nutrients, and they do not restrict the transmission 

of ultraviolet light.

RAIN PLUG

AUTOCLAVABLE-POLYCARBONATE 

CYLINDER

MEMBRANE FILTER

SCREENED ENDPLATE 

OUTER ENDPLATE

Figure 1. Incubation chamber used in field studies.

Site selection and sampling frequency. Field 

studies were done during recreational seasons (MayOctober) in 1994-95. The field studies were done by 

use of chambers containing cell suspensions from 

three collection locations: (1) a CSO that discharges 

untreated sewage combined with storm water into Mill 

Creek, Garfield Heights, Ohio, (2) the Cleveland 

Southerly Wastewater Treatment Plant that discharges 

tertiary-treated effluent into the Cuyahoga River, and 

(3) the Cleveland Easterly Wastewater Treatment plant 

that discharges secondary-treated effluent into Lake

Erie (fig. 2). The Cleveland Easterly and Southerly 

plants receive the same proportions of domestic and 

industrial inputs in their influent waste streams; however, because the Easterly plant pumps activated 

sludge to Southerly for treatment, concentrations of 

metals and other constituents may be higher in the 

activated sludge process at Southerly than at Easterly.

Because bacteria survival has been shown to be 

affected by temperature (Flint, 1987), field studies 

were done at some locations when water temperatures 

were between (1) 10°C and 19.5°C and (2) 20°C and 

30°C. To determine the effect of receiving-water quality on injury, survival, and regrowth, some river studies were done at two field sites simultaneously. Seven 

field studies were done for this investigation, each one 

lasting approximately 4 days.

Field study sites consisted of three sites in the 

Cuyahoga River and a site in Lake Erie (fig. 2). At the 

Cuyahoga River at Peninsula, Ohio (Peninsula), chambers were placed in a pooled reach of the river within 

the Cuyahoga Valley National Recreation Area, downstream from the city of Akron. The water quality at 

Peninsula is affected by CSO's and treated wastewater 

upstream, and to a lesser extent, by industrial sources. 

The Cuyahoga River at Independence (Independence) 

is 16.5 river miles downstream from Peninsula in a 

residential and light industrial area. The Cuyahoga 

River at West Third Street, Cleveland, Ohio (West 

Third) is in a highly industrialized section of the Cuyahoga River in the Cleveland Ship Channel. At this site, 

water temperatures vary the least of all the field study 

sites and are elevated because of thermal effluents 

from industrial processes. The Lake Erie site is on a 

breakwall on the property of the Westerly Wastewater 

Treatment Plant, less than a mile from a bathing beach 

at Edgewater Park. All sites are in areas that support 

recreational uses such as boating and canoeing.

Measurement of fecal-indicator injury, survival, and regrowth. Fecal-indicator injury, survival, 

and regrowth were measured in cell suspensions of 

wastewater or CSO effluent. Wastewater-effluent samples were collected at both treatment plants from a 

final clarifier before chlorination. CSO effluent was 

collected by use of an automatic sampler having the 

intake and flow-triggering sensor in the CSO pipe. The 

sampler was set to collect the first flush of CSO effluent into a sterile 10-L sample bottle. A technician 

checked the sampler after a heavy rain, and if sufficient water was collected, removed the CSO effluent. 

All cell suspensions were kept at 4°C during transport

Methods of Study
41°30'

GARFIELDJHEIGHTS

INDEPENDENCE

CUYAHOGA COUNTY 

SUMMIT COUNTY

Cuyah

Valley \-. 

\ National 

' . Recreation ''-

CUYAHOGA COUNTY 

MEDINA COUNTY

Base modified from U.S. Geological Survey 

Cleveland, 1:250,000, 1956, revised 1972

Study Area

OHIO

10 MILES

10 KILOMETERS

Figure 2. Collection locations and field study sites.

EXPLANATION

FIELD STUDY SITES

1 Cuyahoga River at Peninsula

2 Cuyahoga River at Independence

3 Cuyahoga River at West Third Street

4 Lake Erie at Cleveland 

COLLECTION LOCATIONS

1 Combined-sewer overflow effluent

2 Cleveland Southerly Wastewater Treatment Plant

3 Cleveland Easterly Wastewater Treatment Plant

6 Effects of Receiving-Water Quality and Wastewater Treatment on Injury, Survival, and Regrowth of Fecal-indicator Bacteria
and storage. At a local laboratory (the Fishcreek 

Wastewater Treatment Plant in Stow, Ohio, or the 

Cuyahoga County Sanitary Engineers laboratory in 

Valley View, Ohio), the cell suspensions were treated 

and analyzed by USGS employees within 2 hours for 

wastewater effluent or 24 hours for CSO effluent.

Cell suspensions were treated three ways: 

unchlorinated, chlorinated, or dechlorinated. (Details 

of the treatment scheme were described previously.) 

An initial sample of each type of cell suspension was 

plated in triplicate for fecal coliforms by use of standard selective (standard) and enhanced-recovery 

methods. Six to eight replicate chambers were filled 

with each type of treated cell suspension, kept on ice, 

and transported to the site within 4 hours after treatment. At the Peninsula and West Third sites, chambers 

were suspended in crates in the water column, approximately 18 in. below the water surface. The crates 

were held in place by tethers tied to cement blocks. At 

the Lake Erie and Independence sites, chambers were 

suspended in a plastic swimming pool through which 

the lake or river water was constantly circulated by a 

progressive-cavity pump. The pool (8 ft diameter and 

4 ft deep) was lined with a green plastic sheet to mimic 

the color and reflectiveness of the receiving water.

One type of each replicate chamber (unchlorinated, chlorinated, and dechlorinated) was retrieved at 

approximately 8,16,24, 36,48, and 72 hours. The 

retrieved chambers were then placed on ice and transported to the laboratory for microbiological analysis 

within 6 hours after removal from the stream or lake. 

In the laboratory, the entire chamber contents were 

emptied into sterile bottles, and fecal-coliform concentrations were determined by use of standard and 

enhanced-recovery methods in the same manner as the 

initial samples.

Analysis of receiving-water quality. Field 

measurements of dissolved-oxygen concentration, 

specific conductance, temperature, and pH were measured in the lake, river, and pool at the time of chamber removal by use of a four-parameter water-quality 

meter. The meter was calibrated according to manufacturer's guidelines each time it was used. At sites 

where chambers were incubated in the river, field measurements were made near the chambers about 18 in. 

below the water surface. At sites where chambers 

were placed in the pool, field measurements were 

made at the pump intake and at the center and outflow 

of the pool. The average of the three pool measurements was used in this report. Field measurements

were also made in the river or lake about 3 ft from the 

pump intake line.

Water samples for chemical analyses were collected daily in river and lake field studies according to 

USGS sampling methods (Ward and Harr, 1990). At 

river sites, a single-vertical sample was collected near 

the chambers by use of the US-DH81 depth-integrating sampler. At lake sites, a single weighted-bottle 

sampler was submerged 2 ft below the lake surface 

about 3 ft off the breakwall. In field studies in which 

chambers were placed in the pool, three vertically 

integrated subsamples from the pool were collected 

into a single bottle or composited in a chum splitter 

and then poured into a sample bottle. Water-quality 

samples for determination of nutrients and total nonfilterable residue were collected into clean polypropylene sample bottles, kept in ice chests, and transported 

to the NEORSD laboratory in Cuyahoga Heights, 

Ohio, within 24 hours after sample collection. Samples for determination of total-organic carbon concentration were collected into 125-mL amber glass 

bottles, kept in ice chests or refrigerated, and sent to 

the USGS National Water Quality Laboratory in 

Arvada, Colo. within 3 days after sample collection.

All determinations of inorganic constituents 

were made by use of methods recommended by the 

USEPA for analysis of water and wastes (U.S. Environmental Protection Agency, 1979a). Samples were 

analyzed for total nonfilterable residue (103-105°C) 

(EPA 160.3), nitrate- plus nitrite-nitrogen 

(EPA 350.4), ammonia as nitrogen (EPA 350.1), total 

kjeldahl nitrogen (EPA 351.2), and total phosphorus 

(EPA 365.2). Samples were analyzed for total organic 

carbon by use of the method described by Wershaw 

and others (1987).

Statistical Methods

Analysis of variance (ANOVA) was used 

throughout this investigation to compare more than 

two groups of data. Groups of data were defined by 

one or more factors, a factor being a variable that 

influences the magnitude of the data. ANOVA is based 

on the assumption that all groups of data are normally 

distributed and have equal variances (Helsel and Hirsch, 1992). Therefore, the groups were tested for normality by use of the probability plot correlation 

coefficient (PPCC) test (Looney and Gutledge, 1985) 

and for equal variances by use of Hartley's test (Ott, 

1993). If any of the groups did not pass either test at

Methods of Study 7
a=0.05, the nonparametric rank transformation test 

was used (Conover and Iman, 1981) instead of the 

parametric ANOVA. The a-value, or significance 

level, is the probability of rejecting the null hypothesis 

when it is in fact true. In the rank transformation test, 

all data are combined and ranked from lowest to highest value, and an ANOVA is computed on the ranks.

The parametric and nonparametric ANOVA 

determines whether the mean or mean rank, respectively, differs between groups. The null hypothesis is 

that each group mean or mean rank is the same; the 

alternative is that at least one is different. In a two-way 

ANOVA, the factor effect explains whether or not 

deviations from the overall mean or mean rank are due 

to one or more factors. In a two-way ANOVA, an 

interaction effect is present if the effects of each factor 

on deviations from the overall mean or mean rank are 

not independent from each other.

Tukey's multiple comparison test was used to 

compare pairs of means or mean ranks (Helsel and 

Hirsch, 1992). This test computes a least significant 

range (LSR), the distance between any two means or 

mean ranks that must be exceeded in order for the two 

groups to be considered statistically different at a = 

0.05. The two groups can be considered different if the 

difference between the two group means or mean 

ranks is greater than the LSR.

Evaluation of enhanced-recovery methods. 

Because enhanced-recovery methods have not been 

widely used, several of these methods were evaluated 

before field studies to determine recovery enhancement of chlorine-injured fecal coliforms and suitability for use in large-scale field studies. Recovery was 

measured by determining concentrations of fecal 

coliforms in unchlorinated, chlorinated, and dechlorinated cell suspensions. In initial experiments, qualitative examinations of the fecal-coliform concentrations 

and enhanced-recovery protocols were done. The 

enhanced-recovery methods that were relatively easy 

to use and had higher recoveries of chlorine-injured 

cells over the standard method were tested further and 

evaluated statistically.

The statistical analysis of recoveries was done 

by use of two-way ANOVA and Tukey's multiple 

comparison test. The effects of method type and treatment type (unchlorinated, chlorinated, and dechlorinated) on recovery of fecal coliforms were 

determined. Use of a suitable enhanced-recovery 

method should result in higher concentrations of chlorine-injured bacteria compared with the standard

method. At the same time, the difference between the 

standard and enhanced-recovery methods in recovery 

of bacteria in unchlorinated samples should be statistically insignificant.

Analysis of data collected during field studies, 'ercent injury was calculated for each replicate 

plating at each timestep. The standard method support; the growth of healthy organisms, whereas the

enhanced-recovery method supports the growth of 

both healthy and injured organisms. Therefore, the 

percent injury at each timestep for each replicate plating was computed as follows (Bissonnette and others, 

1973):

ERMJ

where Pi is percent injury,

SSMis concentration of fecal coliforms by use 

of standard method, and

ERMis concentration of fecal coliforms by use 

of enhanced-recovery method.

The percent survival at each timestep for each 

replicate plating was computed as follows:

Ps =

f ERMt 

\ERMun x 100

where Ps is percent survival,

ERMt is concentration of fecal coliforms at 

time "t" by use of enhanced-recovery method, and

ERMuriQ is mean concentration of fecal 

coliforms at initial time in unchlorinated sample by 

use of enhanced-recovery method.

Regrowth, or cell division and multiplication, 

was not measured directly but was inferred from 

examining changes in standard- and enhanced-recovery-method concentrations. In this investigation, 

regrowth refers to the ability of the bacterial cells to be 

resuscitated on enhanced-recovery medium and does 

not refer to the ability of the bacterial cells to regrow 

in receiving waters.

The statistical relation between standard- and 

enhalnced-recovery-method concentrations of fecal 

coliforms was determined by linear regression analysis of base 10 logarithmically transformed data. 

Regression diagnostics were done to test five assumptions that must be met to provide the best, unbiased 

estimator of concentrations obtained by use of the 

enhanced-recovery method from concentrations 

obtained by use of the standard method: (l)y (the 

dependent variable) is linearly related to x (the

8 Effects of Receiving-Water Quality and Wastewater Treatment on Injury, Survival, and Regrowth of Fecal-Indicator Bacteria
independent variable), (2) the data used to fit the 

model are representative of the data of interest, (3) the 

variance of the regression residuals is constant (it does 

not depend on the independent variable or other factors), (4) the regression residuals are independent, and 

(5) regression residuals are normally distributed 

(Helsel and Hirsch, 1992, p. 224-225).

Log transformations of fecal-coliform concentrations resulted in a linear fit of the data and satisfied 

the first assumption. The second assumption was met 

because the samples were analyzed by the best methods available and cell suspensions were incubated in 

waters used for recreation. To evaluate assumptions 3 

and 4, the investigators plotted regression residuals by 

predicted values, elapsed time, and treatment to determine whether residuals were similar in range and 

evenly distributed above and below the zero line 

throughout the entire range of observations. To evaluate assumption 5, the regression residuals were tested 

to determine whether they were normally distributed 

by use of the Probability Plot Correlation Coefficient 

test (PPCC) (Looney and Gutledge, 1985).

Analysis of covariance (ANCOVA) was used to 

determine if the regression model would be more 

accurately described by grouping the data by some 

categorical variable. ANCOVA incorporates the use of 

dummy variables, blending regression and analysis of 

variance into one analysis (Helsel and Hirsch, 1992). 

The results of the ANCOVA determined the best way 

to group the data; data sets having statistically different slopes or y-intercepts would not be best represented in a model of the entire data set.

To determine whether concentrations obtained 

by standard and enhanced-recovery methods were 

comparable as indicators of recreational-water quality 

in the seven field studies, the percentage of samples by 

treatment type meeting and exceeding Ohio's bathingwater, primary-contact, and secondary-contact singlesample standards were determined.

Quality Assurance and Quality Control

Quality-assurance and quality-control (QA/QC) 

practices were done for all phases of data collection 

and analysis in the field and laboratory, and during 

data validation and assessment.

Field and laboratory quality assurance/quality control. Field and laboratory protocols were written and distributed to ensure that procedures were

done according to established methods and in a uniform manner by all personnel.

In the field, 5 percent of the water-quality samples collected were field blanks samples of inorganic-free water passed through the sampling 

equipment at the field site to detect contamination in 

sample collection, handling, and shipping. The integrity of the incubation chambers was tested by filling 

5 percent of the chambers with sterile river or buffer 

water and plating the contents of the chambers after a 

48-hour incubation in the stream or pool. Reproducibility of chamber bacterial contents was determined 

by enumerating the contents of at least 10 percent of 

the chambers placed in receiving waters as timestep 

replicates. In pool studies, field measurements were 

made hourly, when possible, to ensure that pool measurements were similar to river or lake measurements. 

If a large deviation in a field measurement was found, 

the pump rate on the pool was adjusted. In addition, 

concurrent water samples for chemical analyses were 

collected from the pool and the lake or river to compare the concentrations of chemical constituents.

In the laboratory, equipment and supplies were 

regularly checked to ensure proper performance. The 

incubators were monitored throughout experiments 

and field studies to ensure temperatures were 35°C + 

0.5°C or 44.5°C ± 0.2°C. The sterility of the buffer 

water and media were tested by use of blanks aliquots of buffer water filtered before each sample and 

after each third sample. Performance of media and 

buffer water was tested by use of lyophilized qualitycontrol bacterial isolates of E. coli obtained from the 

U.S. Environmental Protection Agency Quality Assurance Laboratory in Cincinnati, Ohio. Sample results 

were rejected if incubator temperatures were outside 

acceptable ranges or quality-control testing showed 

contaminated blanks or values outside the acceptable 

range for recovery of E. coli. The autoclave operating 

temperature and pressure were checked for each run 

and heat-indicating tape was used to identify supplies 

that had been sterilized. Other good laboratory practices cleanliness, safety practices, procedures for 

media preparation, specifications for reagent water 

quality were adopted by USGS employees as set 

forth by American Public Health Association (1992) 

and Britton and Greeson (1989).

The NEORSD laboratory's QA/QC program 

includes daily duplicates, standards, spiked samples, 

and check standards. Analysts update precision and 

accuracy control charts daily. The NEORSD

Methods of Study 9
laboratory participated each year in the USGS Standard Reference Water Sample Program and the 

USEPA Performance Evaluation Program. In addition, 

at least 15 percent of the samples in the field studies 

were QA/QC samples submitted to the NEORSD laboratory. These included duplicate samples, reference 

samples, and split samples analyzed by NEORSD and 

the USGS National Water Quality Laboratory.

Management and validation of fecal-indicator data. Bacteriological data were checked for errors 

and monitored for precision. All data obtained from 

plates having less than 8 colonies were deleted from 

the data set. Data from colony counts outside the ideal 

range of 20-60 colonies per plate were flagged as 

"nonideal count," and data from colony counts with 

high numbers of nontarget colony types were flagged 

as "high background." Precision control limits were 

established on the replicate analyses done during the 

first field study according to the Shewhart method 

(described in detail in U.S. Environmental Protection 

Agency, 1979b). The differences between three log 

transformed replicate observations were calculated, 

and the average difference was determined. The Upper 

Warning Limit (UWL), corresponding to the 95-percent confidence level, was calculated. In the first field 

study, and in later studies, replicate samples with differences greater than the UWL were examined. An 

observation was omitted from the data set if (1) the 

observation differed from the other two replicate 

observations by more than the UWL, or (2) the observation differed from at least one other replicate observation by more than the UWL and was either (a) 

flagged as "nonideal" or "high background" or (b) 

looked suspicious for any other reason. Plots of 

elapsed time by bacteria concentrations were used to 

ensure the data were representative in a historical 

sense, and deviant observations were examined for 

error. Before statistical analysis, all data not meeting 

quality-control requirements were deleted.

Percent verifications were determined for colonies isolated from cell suspensions during evaluation 

of enhanced-recovery methods and during field studies. A verified membrane-filter test establishes the 

validity of colony differentiation and is recommended 

when new procedures are being used (U.S. Environmental Protection Agency, 1978). Tests were run to 

compare the number of false positive fecal coliforms 

from the standard method to the number of false positives from the enhanced-recovery method.

For data collected during field studies, percent 

verifications were determined on colonies from plates 

of standard and enhanced-recovery methods. To 

obtain a good cross-section of colonies isolated during 

each field study, plates were selected from initial and 

24-mour samples for each treatment type (unchlorinated, chlorinated, and dechlorinated). Eight to twelve 

well-isolated colonies were randomly selected and 

verified from each plate. The colonies were transferred 

to lauryl tryptose broth (Difco Laboratories) and tryptic soy broth (Difco Laboratories). Tryptic soy broth 

was used to resuscitate any injured organisms unable 

to grow in lauryl tryptose broth and to detect nonviable cultures. Colonies failing to grow in either lauryl

tryptose broth or tryptic soy broth within 48 hours 

wers considered nonviable and were not included in 

the : inal count. Lauryl tryptose broth tubes having no 

groAvth after a 24-hour incubation at 35°C were reinocu ated from growth-positive tryptic soy broth test 

tubts. Gas-positive lauryl tryptose broth test tubes 

wer; transferred to EC broth (Difco Laboratories) and 

incubated at 44.5 °C. Cultures that produced gas in 

EC broth after a 24-hour incubation were interpreted 

as verified fecal coliforms (U.S. Environmental Protection Agency, 1978). A percent verification for each 

method was determined as follows:

Percent verification =

Number of positive verifications ^ . 

Number of verification tests performed )

Contingency-table analysis was done (Helsel 

and Hirsch, 1992) to measure the association between

metiod and number of positive verifications. If 

method type was found to affect the number of positive verifications, then method-specific percent verifications would have to be applied to the data set.

Evaluation of Enhanced-Recovery 

Methods for Use in Field Studies

Several enhanced-recovery methods for fecal 

coliforms were evaluated qualitatively to determine 

whether they were suitable for use in field studies. The 

two-layer overlay method (Rose and others, 1975) was 

cumbersome and resulted in increased recoveries of 

chlorine-injured fecal coliforms that were only 1.4 

times that of the standard method (mFC). The addition 

of catalase to mFC (Calabrese and Bissonnette, 1990) 

was found to be too time-consuming for a large-scale

10 Effects of Receiving-Water Quality and Wastewater Treatment on I njury, Survival, and Regrowth of Fecal-Indicator Bacteria
project, and the addition of pyruvate, although simple, 

resulted in increased recoveries of fecal coliforms in 

chlorinated wastewater of only 1.27 to 1.45 times that 

of mFC. Two other modifications of mFC mFC with 

a 4-hour resuscitation at 35°C (mFC4) and mFC without rosolic acid and a 4-hour resuscitation at 35°C 

(mFC-R4) increased recoveries of fecal coliforms in 

chlorinated wastewater effluent 3.3 to 14 times that of 

mFC. At the same time, increased recoveries of fecal 

coliforms in unchlorinated wastewater were considerably lower only 1.4 to 1.8 times that of mFC. This is 

expected from a suitable enhanced-recovery method, 

as described previously. Recoveries from the mT7 

agar enhanced-recovery method (mT7) (LeChevallier 

and others, 1983) were 1.9 to 11.8 times those 

obtained from mFC in chlorinated samples; in unchlorinated samples, recovery ratios were 1.5 to 3.5 times 

those obtained from mFC. Therefore, three enhancedrecovery methods tested (mFC4, mFC-R4, and mT7) 

were suitable for recovery of chlorine-injured fecal 

coliforms from wastewater effluent in field studies. 

Recoveries of fecal coliforms by use of these 

three enhanced-recovery methods (mT7, mFC4, and 

mFC-R4) were evaluated statistically. Concentrations 

of fecal coliforms obtained by use of the enhancedrecovery methods and mFC in unchlorinated, chlorinated, and dechlorinated wastewater effluent on different days were combined and analyzed by use of twoway nonparametric ANOVA. The two factors (treatment and media type) were found to significantly 

affect concentrations of fecal coliforms (pO.OOOl); an 

interaction effect also was found between the two factors (p=0.0192). Tukey's multiple comparison test was 

done to compare the differences in mean ranks of concentrations obtained by use of each enhanced-recovery 

method to the mean rank of concentrations obtained 

by use of the standard method (table 1). For unchlorinated wastewater, the differences in mean ranks 

between mT7, mFC-R4, and mFC4 as compared to the 

mean rank of mFC ranged from 5.5 to 9.8; these differences were statistically insignificant. For chlorinated wastewater, only the differences in mean ranks 

between mFC-R4 and mFC (difference=25.6) were 

statistically significant. However, for dechlorinated 

wastewater, the mean ranks of all three enhancedrecovery methods were statistically different than the 

mean rank of mFC, the differences ranging from 27.6 

to 43.3.

LeChevallier and others (1984) found that the 

mT7 method increased recoveries of chlorine-injured 

fecal coliforms and provided higher percent verifications when the fecal coliforms were resuscitated at 

37°C instead of 35°C. Using the 37°C resuscitation 

temperature, LeChevallier and others (1984) found 

83.2-percent verification of colonies from chlorinatedwastewater effluent. In this investigation, however, 

aluminum-block incubators could only be operated at 

35°C and 44.5°C. Using the 35°C temperature in this 

investigation, only 58 percent of colonies isolated 

from mT7 agar could be verified as fecal coliforms 

from chlorinated wastewater effluent. In addition, 

heavy background growth often made counting of 

fecal coliforms difficult and inaccurate, especially in 

samples from chambers that had been incubated in the 

receiving water for more than 24 hours. Therefore, 

although mT7 increased recovery of chlorine-injured 

organisms over mFC, the resulting low percent

Table 1. Differences in mean rank of concentrations of fecal coliforms obtained by use of enhanced-recovery methods (mT7, 

mFC-R4, and mFC4) and concentrations of fecal coliforms obtained by use of standard method (mFC) in unchlorinated, 

chlorinated, and dechlorinated wastewater effluent

[The difference in concentrations was determined by ranking the data, doing two-way analysis of variance on the ranks with treatment and method as 

factors, and comparing the mean ranks of the treatment groups by use of Tukey's multiple comparison test]

Treatment mT7a mFC-R4b mFC4c

Unchlorinated

Chlorinated

Dechlorinated

7.7 

22.7 

27.6d

9.8 

25.6d 

43.3d

5.5 

18.7 

33.2d

aM-Tergitol-7 media (mT7) with an 8-hour incubation at 35 C foifbwed by 12-hour incubation at 44.5°C.

bM-fecal coliform media (mFC) without rosolic acid (mFC-R4) with 4-hour incubation at 35°C followed by 20-hour incubation at 44.5°C. 

cM-fecal coliform media (mFC) with 4-hour incubation at 35°C followed by 20-hour incubation at 44.5°C.

A statistically significant difference exists between the mean ranks of concentrations of fecal coliforms by use of the enhanced-recovery methods as compared to the standard method at a = 0.05.

Evaluation of Enhanced-Recovery Methods for Use in Field Studies 11
verifications and high background growth limited its 

usefulness in field studies.

On the basis of statistical results of recovery 

enhancement and because of problems encountered 

with mT7 agar, mFC-R4 was selected as the best 

available method for increased recovery of chlorineinjured fecal coliforms; mFC-R4 was used as the 

enhanced-recovery method in all subsequent field 

studies.

Effects of Receiving-Water Quality and 

Wastewater Treatment on Injury, Survival, 

and Regrowth of Fecal-Indicator Bacteria

Receiving-Water Quality

Dates, sites, and durations of seven field studies 

done during the 1994-95 recreational seasons are 

listed in table 2. In two field studies, May and August 

1994, investigators used wastewater effluent from the 

Easterly Wastewater Treatment Plant and incubated 

chambers at the Lake Erie field site. The June 1994 

and August 1995 Cuyahoga River studies were done at 

two sites with wastewater effluent from the Southerly

Wastewater Treatment Plant; however, chamber loss 

and 1 >reakage from an increase in streamflow at the 

Peninsula site caused early termination of field studies 

at this site. A September 1995 Cuyahoga River field 

study was done at the Independence site in the incubation >ool to investigate injury, regrowth, and survival 

at cooler temperatures without risking chamber loss. 

In May and June 1995, investigators used CSO effluent from Mill Creek and incubated chambers at the 

Independence site in the pool.

Field measurements in the pool were similar to 

and showed the same fluctuations as field measurements in the lake or river (unpublished data on file at 

USGS, Columbus, Ohio). In all pool field studies, concentrations of nitrate- plus nitrite-nitrogen, ammonianitrogen, and total organic carbon in the incubation 

pool were similar to those concentrations found in the 

lake or river. However, concentrations of total nonfilteraMe residue and total phosphorus in the pool were 

often 2-3 times lower than those in the Cuyahoga 

River during the May and June 1995 CSO studies, 

especially when concentrations of total nonfilterable 

residue in the river were greater than 100 mg/L 

(unpublished data on file at USGS, Columbus, Ohio). 

These differences were likely due to the inability of

Table 2. Dates, sites, and durations of field studies done with wastewater or combined-sewer-overflow effluent from three 

collection locations, 1994-1995

Date Field site

Duration of study 

(hours)

Easterly Wastewater Treatment Plant effluent, Cleveland, Ohio

May 1994 

August 1994

June 1994 

August 1995 

September 1995

May 1995 

June 1995

Lake Erie at Westerly Wastewater Treatment Plant, Cleveland, Ohio 69.0 

Lake Erie at Westerly Wastewater Treatment Plant, Cleveland, Ohio 69.0

Southerly Wastewater Treatment Plant efflu

Cuyahoga River at Peninsula, Ohio 

Cuyahoga River at West Third Street, Cle 

Cuyahoga River at Peninsula, Ohio 

Cuyahoga River at West Third Street Brie 

Cuyahoga River at Independence, Ohio

Combined-sewer-overflow effluent, Mill Cn

Cuyahoga River at Independence, Ohio 

Cuyahoga River at Independence, Ohio

;nt, Cuyahoga Heights, Ohio

36.5 

^eland, Ohio 70.0 

48.0 

ge, Cleveland, Ohio 46.5 

66.0

sek, Garfield Heights, Ohio

62.0 

67.5

12 Effects of Receiving-Water Quality and Wastewater Treatment on Injury, Survival, and Regrowth of Fecal-Indicator Bacteria
the pump to circulate water fast enough in the pool to 

keep up with increasing total nonfilterable residue 

concentrations in the river.

Mean values of water-quality field measurements and constituent concentrations for each study 

are reported as pool values (Lake Erie and Independence sites) or river values (Peninsula and West Third 

sites) in table 3. At the Lake Erie field site, the largest 

differences in water quality between May and August 

1994 field studies were temperatures and dissolvedoxygen concentrations. In May, the mean temperature 

was 12.8°C compared to 22.6°C in August. Because 

solubility of oxygen is affected by temperature, mean 

dissolved-oxygen concentration was higher in May 

(8.5 mg/L) than in August (5.8 mg/L).

The major difference in water quality in June 

1994 and August 1995 Cuyahoga River field studies 

was mean dissolved-oxygen concentration between 

sites (table 3). At Peninsula, mean dissolved-oxygen 

concentrations were 7.3 and 7.7 mg/L, whereas at 

West Third concentrations were 5.2 and 4.3 mg/L. 

Less apparent were differences in temperature 

between field studies or sites. Large differences in 

mean concentrations of water-quality constituents 

were not apparent between field studies or sites in June 

1994 and August 1995 field studies. In 

September 1995 at Independence, the mean water temperature was lower and mean dissolved-oxygen concentration was higher than those at Peninsula and West 

Third in June 1994 and August 1995.

The major water-quality differences between the 

May 1995 and June 1995 CSO field studies at the 

Cuyahoga River at Independence were temperature 

and dissolved-oxygen concentrations (table 3). In 

May, the mean water temperature and dissolved-oxygen concentration were 15.6°C and 8.6 mg/L, whereas 

in June they were 23.1°C and 6.6 mg/L. In addition, 

the mean concentration of total nonfilterable residue 

was higher in samples collected in June (150 mg/L) 

than in May (50 mg/L).

Concentrations of Fecal-Indicator Bacteria

Percent verifications were determined on colonies isolated during each field study except for September 1995 (table 4). Generally, percent verifications 

were higher for colonies isolated from standardmethod plates than colonies isolated from enhancedrecovery plates. Results of contingency table tests, 

however, showed that differences of percent verifications between methods were not statistically significant (p>0.05) (unpublished data on file at USGS, 

Columbus, Ohio). Therefore, percent verifications 

were not applied to the data sets.

Concentrations of fecal coliforms during 

Lake Erie wastewater-effluent field studies. Mean 

fecal-coliform concentrations in wastewater effluent 

from the Easterly Wastewater Treatment Plant during 

May and August 1994 field studies at Lake Erie are 

shown in figure 3. In unchlorinated (fig. 3A), chlorinated (fig. 3B), and dechlorinated (fig. 3C) wastewater 

effluent, concentrations of fecal coliforms determined 

by use of the enhanced-recovery method were 1.1 to 

46 times greater than those concentrations determined 

by use of the standard method.

Seasonal differences of fecal-coliform concentrations can be found between the May 1994 and 

August 1994 field studies. Fecal-coliform concentrations were higher in August 1994 than in May 1994 

for all treatments. In addition, during the August 1994 

field study, concentrations of fecal coliforms in 

unchlorinated and dechlorinated samples increased 

during about the first 13 and 10 hours, respectively; 

however, in May 1994, concentrations of fecal 

coliforms remained fairly constant over the sampling 

period for all treatments. As temperature rises, chemical and enzymatic reactions in the cell proceed at 

more rapid rates and cell growth becomes faster 

(Brock and Madigan, 1988). High water temperatures 

in August, therefore, led to higher replication and 

decay rates of healthy organisms in unchlorinated and 

dechlorinated samples. In May 1994, concentrations 

in all samples remained fairly constant because of to 

lower water temperatures and slower bacteria metabolic rates.

In the chlorinated samples in August 1994 (fig. 

3B), the pattern of fecal-coliform concentration 

increases and decreases was strikingly different from 

that found in unchlorinated and dechlorinated samples 

in August 1994 and all treatments in May 1994. In 

chlorinated samples in August 1994, fecal coliform 

concentrations on enhanced-recovery media dropped 

off sharply during the first 7 hours and remained low 

throughout the remainder of the study. A large proportion of the organisms recovered from initial chlorinated samples were, therefore, injured organisms. This 

suggests that a large proportion of the chlorine-injured 

cells in the chlorinated samples were unable to repair 

injuries and regrow on enhanced-recovery media during the August 1994 study.

Effects of Receiving-Water Quality and Wastewater Treatment on Injury, Survival, and Regrowth of Fecal-Indicator Bacteria 13
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njury, Survival, and Regrowth of Fecal-Indicator Bacteria
Table 4. Percent verifications of fecal coliforms in wastewater or combined-sewer-overflow effluent by use of standard or 

enhanced-recovery method

[Total number of colonies tested is shown in parentheses]

Method

Study date Standard3 Enhanced-recoveryb

Easterly Wastewater Treatment Plant effluent, Cleveland, Ohio

May 1994 

Aug. 1994

80.0 (60) 

74.6 (59)

73.3 (60) 

72.4 (58)

Southerly Wastewater Treatment Plant effluent, Cuyahoga Heights, Ohio

June 1994 

Aug. 1995

77.3 (53) 

75.0 (60)

81.0 (47) 

60.0 (60)

Combined-sewer-overflow effluent, Mill Creek, Garfield Heights, Ohio

May 1995 

June 1995

79.7 (69) 

74.5 (51)

75.0 (72) 

68.6 (51)

aStandard method is m-fecal coliform media (mFC) incubated at 44.5°C for 22-24 hours.

bEnhanced-recovery method is mFC agar without rosolic acid incubated at 35°C for 4 hours and 44.5°C for 18-20 hours.

Concentrations of fecal coliforms during 

Cuyahoga River wastewater-effluent field studies.

Mean fecal-coliform concentrations in wastewater 

effluent from the Southerly Wastewater Treatment 

Plant during June 1994, August 1995, and September 

1995 field studies are shown in figures 4 through 6. 

Chambers were incubated at the Cuyahoga River at 

Peninsula and West Third sites during June 1994 and 

August 1995 field studies and at the Cuyahoga River 

at Independence during the September 1995 field 

study. Concentrations in unchlorinated (fig. 4), chlorinated (fig. 5), and dechlorinated (fig. 6) wastewater 

effluent ranged from 10 to 20 times higher in June 

1994 than in August 1995 or September 1995. In all 

studies, concentrations were highest in the unchlorinated samples, and concentrations in the dechlorinated 

samples were about an order of magnitude higher than 

those in the chlorinated samples. During all studies, 

concentrations of injured fecal coliforms varied considerably, indicating that the differences between 

enhanced-recovery and standard method concentrations were due not only to method differences, but also 

to population differences.

During the first half of the June 1994 study at 

Peninsula and West Third, fecal-coliform concentrations for all treatments generally increased slightly or 

remained the same. In the August 1995 unchlorinated 

samples at West Third and Peninsula, a pattern of 

decreasing fecal-coliform concentrations was found.

In the August 1995 chlorinated and dechlorinated 

samples, concentrations increased and decreased at 

West Third and remained about the same at Peninsula. 

Concentration increases in unchlorinated samples may 

be due to replication of healthy cells and (or) repair of 

nonchlorine-induced injures and regrowth on culture 

media. In dechlorinated and chlorinated samples, 

increases in concentrations may be due to these factors 

and one additional factor, the repair of chlorineinduced injuries and regrowth on culture media. A 

decrease in fecal-coliform concentrations in unchlorinated samples and a concomitant increase in fecalcoliform concentrations in dechlorinated and chlorinated samples at West Third indicates that the 

increases in chlorinated and dechlorinated samples 

were due in part to repair of chlorine-induced injuries 

and regrowth on culture media.

In September 1995, concentrations of fecal 

coliforms in unchlorinated samples fluctuated over 

time. The wide fluctuations may have been due to the 

fact that unchlorinated samples were plated only once 

during this field study (and not in triplicate as in other 

studies). Fecal-coliform concentrations in chlorinated 

and dechlorinated samples were very low throughout 

the September 1995 field study.

Concentrations of fecal coliforms during 

Cuyahoga River combined-sewer-overflow effluent 

field studies. Mean fecal-coliform concentrations in 

CSO effluent in May and June 1995 field studies at the

Effects of Receiving-Water Quality and Wastewater Treatment on Injury, Survival, and Regrowth of Fecal-Indicator Bacteria 15
A. UNCHLORINATED SAMPLES

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Figure 3. Mean fecal-coliform concentrations determined by use of standard and enhanced-recovery

methods in wastewater effluent during May 1994 and August 1994 field studies at Lake Erie, Cleveland,

Ohio in (A) unchlorinated, (B) chlorinated, and (C) dechlorinaled samples. (Different scales were used 

for different magnitudes of data to enhance visual interpretations.)

16 Effects of Receiving-Water Quality and Wastewater Treatment on Injury, Survival, and Regrowth of Fecal-Indicator Bacteria
CO

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Single plating '.

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ELAPSED TIME, IN HOURS

Figure 4. Mean fecal-coliform concentrations determined by use of standard and enhanced-recovery 

methods in unchlorinated wastewater effluent during June 1994, August 1995, and September 1995 

field studies at Cuyahoga River sites, Ohio. (Different scales were used for different magnitudes of 

data to enhance visual interpretation.)

Effects of Receiving-Water Quality and Wastewater Treatment on Injury, Survival, and Regrowth of Fecal-Indicator Bacteria 17
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120

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SEPTEMBER 1995

0 10 20 30 40 50 60 70

ELAPSED TIME, IN HOURS

Figure 5. Mean fecal-coliform concentrations determined by 

methods in chlorinated wastewater effluent during June 1994 

field studies at Cuyahoga River sites, Ohio. (Different scales 

data to visual interpretation.)

use of standard and enhanced-recovery 

, August 1995, and September 1995 

were used for different magnitudes of

18 Effects of Receiving-Water Quality and Wastewater Treatment on Injury, Survival, and Regrowth of Fecal-Indicator Bacteria
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Figure 6. Mean fecal-coliform concentrations determined by use of standard and enhanced-recovery 

methods in dechlorinated wastewater effluent during June 1994, August 1995, and September 1995 

field studies at Cuyahoga River sites, Ohio. (Different scales were used for different magnitudes of 

data to visual interpretation.)

Effects of Receiving-Water Quality and Wastewater Treatment on Injury, Survival, and Regrowth of Fecal-Indicator Bacteria 19
Independence site are shown in figure 7. In the unchlorinated (fig. 7A) and chlorinated (fig. 7B) samples, 

fecal-coliform concentrations were higher and 

increased more rapidly in June 1995 than in May 

1995, probably due to warmer water temperatures in 

June 1995. In the dechlorinated samples (fig. 7C), 

however, fecal-coliform concentrations were not 

higher in June 1995 than in May 1995.

During the first half of the May 1995 field study 

in the unchlorinated samples, the proportions of organisms obtained by use of the enhanced-recovery 

method remained about the same throughout the study. 

In dechlorinated and chlorinated samples in the May 

1995 field study, fecal-coliform concentrations determined by use of the enhanced-recovery method 

increased more rapidly from about 20-32 hours than 

those determined by use of the standard method. In the 

May 1995 field study in chlorinated and dechlorinated 

samples, therefore, repair of chlorine-induced injuries 

from about 20-32 hours may have enabled some previously undetected organisms to grow on enhancedrecovery media.

In the chlorinated samples in June 1995, fecalcoliform concentrations peaked at 14 hours and again 

at 48 hours. CSO effluents contain more solid materials than treated wastewater effluent because they are a 

mixture of untreated wastewater and stormwater. Bacteria attach to solid materials, which offer a nutrientrich environment and protection from chlorination. 

Perhaps the second peak in fecal-coliform concentrations at 48 hours was due to solids breaking apart and 

releasing a reservoir of bacteria and nutrients into the 

water.

Percent Injury of Fecal-Indicator Bacteria

Mean percent injury (table 5) was calculated for 

each wastewater-effluent and CSO field study by site 

for unchlorinated, chlorinated, and dechlorinated samples. The lowest mean percent injuries among treatments were found in unchlorinated samples, ranging 

from 34.8 percent at Peninsula in August 1995 to 54.8 

percent at West Third in June 1994. As expected, 

mean percent injuries in the chlorinated samples were 

higher than those in unchlorinated samples, ranging 

from 51 to 84.5 percent. Mean percent injuries for 

dechlorinated samples showed the largest spread, 

ranging from 46.3 to 90.3 percent. The two lowest 

mean percent injuries for dechlorinated samples were 

found in samples from the August 1994 (48.1 percent)

and September 1995 (46.3 percent); these values were 

similar to those found in the corresponding unchlorinated samples.

Percent injury of fecal coliforms during Lake 

Erie wastewater-effluent field studies. Relations 

among percent injuries and treatments in field studies 

of wastewater effluent from the Easterly Wastewater 

Treatment Plant incubated in Lake Erie in May and 

August 1994 were examined by use of ANOVA (fig. 

8). In the May field study (fig. 8A), percent injuries in 

the chlorinated and dechlorinated samples were statistically higher than percent injuries in the unchlorinated 

samples. In contrast, in the August 1994 field study 

(fig. 8B), percent injuries in the chlorinated samples 

were statistically higher than percent injuries in the 

uncnlorinated and dechlorinated samples. Therefore, 

in May 1994, when water temperatures were lower 

and bacteria metabolic rates were slower than those in 

August 1994, many fecal coliforms in chlorinated and 

dechlorinated samples were unable to repair chlorineinduced injuries and regrow on culture media. In 

August 1994, a large proportion of fecal coliforms in 

dechlorinated samples were able to repair injuries 

caused by chlorination and regrow on culture media; 

percent injuries equaled those found in the unchlorinated population. However, even in the warm August 

waters, fecal coliforms in chlorinated samples were 

una )le to repair their injuries and regrow on culture 

mec ia.

Percent injury of fecal coliforms during 

Cuyahoga River wastewater-effluent field studies.

Relations among percent injuries, treatments, and sites 

in field studies of wastewater effluent from Southerly 

Wastewater Treatment Plant were examined by use of 

a two-way ANOVA (fig. 9). Field studies were done in 

Juno 1994 and August 1995 at two Cuyahoga River 

sites, Peninsula and West Third.

In June 1994 and August 1995, treatment 

affected percent injury, site did not, and no interaction 

effects were found between treatment and site. In the 

June 1994 field study (fig. 9A), a statistically significant difference was found in percent injuries between 

unchlorinated and chlorinated samples only at the Peninsula site (p<0.01); all other paired comparisons were 

not statistically significant at o=0.05. In the June 

1994 field study, however, ^-values near o=0.05 were 

found between two other comparisons: (1) unchlorinated and dechlorinated samples at the Peninsula site 

(p=0.07) and (2) unchlorinated and chlorinated samples at the West Third site (p=0.07). In the August

20 Effects of Receiving-Water Quality and Wastewater Treatment on Injury, Survival, and Regrowth of Fecal-Indicator Bacteria
A. UNCHLORINATED SAMPLES

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Figure 7. Mean fecal-coliform concentrations by use of standard and enhanced-recovery methods in 

combined-sewer-overflow effluent during May and June 1995 field studies at Cuyahoga River at 

Independence, Ohio: (A) unchlorinated, (B) chlorinated, and (C) dechlorinated (Different scales were 

used for different magnitudes of data to enhance visual interpretation.)

Effects of Receiving-Water Quality and Wastewater Treatment on Injury, Survival, and Regrowth of Fecai-lndicator Bacteria 21
Table 5. Mean percent injury of fecal coliforms in unchlorinated, chlorinated, and dechlorinated wastewater or combinedsewer-overflow effluent field studies

[Percent injury is computed as [1-(SSM/ ERM)] x 100, where SSM is colonies per 100 milliliters recovered by standard-selective method and ERM is 

colonies per 100 milliliters recovered by enhanced-recovery method. ND, not determhed]

Wastewater,

Lake Erie at

Cleveland, Ohio

Wastewater, Cuyahoga River

Peninsula West Third

Combined-sewer

 overflow, Cuyahoga 

Independence River at Independence

Treatment

May 

1994

Aug. 

1994

June 

1994

Aug. 

1995

June 

1994

Aug. 

1995

Sept. 

1995

May 

1995

June 

1995

Unchlorinated 51.2 49.8 51.2 34.8 54.8

Chlorinated 72.8 76.6 78.2 55.9 72.1

Dechlorinated 80.1 48.1 71.7 62.6 68.6

40.5

51.0

56.2

51.5 

ND 

46.3

45.0

84.5

90.3

39.8

53.7

71.7

1995 field study (fig. 9B), the data sets were smaller 

and percent injury differences between groups were 

not as pronounced as those in the June data set. The 

only statistically significant difference was found 

between percent injuries in unchlorinated and dechlorinated samples at the Peninsula site (/?=0.03).

In both the June 1994 and August 1995 Cuyahoga River studies, no statistically significant differences were found in percent injuries between 

dechlorinated and chlorinated samples. Therefore, in 

the warm-weather Cuyahoga River studies, and unlike 

the warm-weather Lake Erie field study, dechlorination did not enhance the repair and regrowth of chlorine-injured fecal coliforms over chlorination alone. 

This bacterial response may be due to water-quality 

differences between Lake Erie and the Cuyahoga 

River. During field studies, concentrations of nutrients 

and total organic carbon were somewhat lower and 

concentrations of total nonfilterable residue were considerably lower in the lake than in the river (table 3). 

Because of lower suspended materials in the lake, 

transmission of ultraviolet light, which is bactericidal, 

would be enhanced in the lake and not in the river.

The different levels of treatment of wastewater 

effluents from the Easterly Plant (Lake Erie field studies) compared to the Southerly Plant (Cuyahoga River 

field studies) may have also contributed to different 

responses of the dechlorinated and chlorinated populations. Secondary-treated wastewater effluent was used 

in Lake Erie field studies, whereas tertiary-treated 

wastewater effluent was used in Cuyahoga River studies. The increased residence time of bacteria in tertiary-treated effluent over secondary-treated effluent 

may have resulted in different physiological states of

bacteria cells. In addition, activated sludge from the 

Easterly Plant is pumped to the Southerly plant for 

treatment; thus, the wastewater stream in the Southerly plant may have had higher concentrations of metals than the wastewater stream in the Easterly Plant. 

More work needs to be done to determine why dechlorination enhanced the repair and regrowth of chlorineinjured fecal coliforms over chlorination alone in the 

wanln-weather lake studies, but not in the river studies.

Percent injury of fecal coliforms during 

Cuyahoga River combined-sewer-overflow effluent 

field studies. Relations among percent injuries and 

treatments were examined in field studies of CSO 

effluent collected from Mill Creek and incubated at the 

Cuyahoga River at Independence in May and June 

1995 (fig. 10). Statistically significant differences 

were found in percent injuries between all possible 

pairs of treatment groups in each field study. In both 

studies, the lowest percent injuries were found in 

unchlorinated samples, and the highest percent injuries were found in dechlorinated samples. Contrary to 

what was found in wastewater-effluent field studies, 

the results from CSO studies indicated that dechlorination may reduce the ability of organisms to recover 

and regrow on culture media compared with chlorination alone. The presence of untreated waste and high 

concentrations of solids in CSO effluent may have 

contributed to this result. As explained earlier, solid 

materials in the chlorinated samples may have broken 

apart, releasing nutrients and bacteria into the chamber, 

pies

Why this did not happen in the dechlorinated samwas not determined. Because of the uncertain

nature of CSO effluents, however, more work needs to

22 Effects of Receiving-Water Quality and Wastewater Treatment on Injury, Survival, and Regrowth of Fecal-Indicator Bacteria
A. MAY 1994

160

140

120

100

80

60

40

20

£ 0

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£ 160 

a.

140 

120 

100

80 -

60 -

40

20 -

Analysis of variance on entire population p<0.000l 

p<0.01

p<0.01 pX).1

B. AUGUST 1994

i i 

Analysis of variance on entire population p=0.00063

p>0.1 

p<0.01 i | p<o.01 |;

11 : i

17

T

EXPLANATION

Sample number 

Upper whisker1

75th percentile

Median

25th percentile

Lower whisker1 

Outside values2

1 Upper whisker is defined as 

the largest data point less than 

or equal to the upper quartile 

plus 1.5 times the interquartile 

range. Lower whisker is minus 

1.5 times the interquartile range.

2An outside value is defined as 

greater than 1.5 and less than 

or equal to 3 interquartile 

ranges from the box.

UNCHLORINATED CHLORINATED DECHLORINATED

Figure 8.

treatment Plant and incubated at Lake Erie, Cleveland, Ohio field site in (A) 

May 1994 and (B) August 1994. (Dashed lines indicated results of paired Tukey's multiple 

comparison tests.)

Percent injury of fecal-col if orms in relation to treatment type in wastewater effluent from 

Easterly Wastewater Tr

Effects of Receiving-Water Quality and Wastewater Treatment on Injury, Survival, and Regrowth of Fecal-Indicator Bacteria 23
A. JUNE 1994

IOU

140

120

100 

80

60

40

20

OC Q 

i

i i

Two-way analysis of variance on ranks of entire populatic 

effect of site p=0.7293, effect of treatment p<0.0( 

interaction effect =0.4958

-

p=.07

P<.01 : : p>0.1

i 1

12

: r

-

-

L

2

|

p>0.1

p=.07 I ! p>0.1

'' 1

4

i I _L

n p<0.0001, - 

101,

f 1

-

7

I -

-

-

-

ILU 

O

S 160 

a.

140

120

100

80 

60

40

20

B. AUGUST 1995

Two-way analysis of variance on ranks of entire population p<0.0001, 

effect of site p=0.7367, effect of treatment p<0.0001, 

interaction effect =0.6370

-

p=.03

p>0.1 p>0.1 i

p>0.1

P>0.1 : P>0.1

' i I T ?

1

-

-

0 , I

10

- I '

'

-

-

(

f Y

)

L

-

L

EXPLANATION

12 Sample number 

Upper whisker1

75th percentile

Median

25th percentile

Lower whisker1 

Outside values2

1 Upper whisker is defined as 

the largest data point less than 

or equal to the upper quartile 

plus 1.5 times the interquartile 

range. Lower whisker is minus 

1.5 times the interquartile range.

2An outside value is defined as 

greater than 1.5 and less than 

or equal to 3 interquartile 

ranges from the box.

PENINSULA WEST THIRD

Figure 9. Percent injury of fecal-coliforms in relation to treatment type in wastewater effluent from 

Southerly Wastewater Treatment Plant and incubated at Cuyahoga River at Peninsula and West 

Third, Cleveland, Ohio field sites in (A) June 1994 and (B) August 1995. (Dashed lines indicated 

results of paired Tukey's multiple comparison test.)

24 Effects of Receiving-Water Quality and Wastewater Treatment on Injury, Survival, and Regrowth of Fecal-Indicator Bacteria
A. MAY 1995

160 

140 

120 

100

80

60

40 h

20

I °

Analysis of variance on ranks of entire population p<0.0001 

P<-01

P<-01 P=-02

23

20

14

1

EXPLANATION

iLJJ 

O

160

140

120

100

80

60

40

20

20

T

1

*

Sample number 

Upper whisker1

75th percentile 

Median 

25th percentile

Lower whisker1 

Outside values2

B. JUNE 1995

Analysis of variance on ranks of entire population p<0.0001

p<.01 

| i p=.02 | ! p<.0l i i

13

I

17

T

'Upper whisker is defined as 

the largest data point less than 

or equal to the upper quartile 

plus 1.5 times the interquartile 

range. Lower whisker is minus 

1.5 times the interquartile range.

2An outside value is defined as 

greater than 1.5 and less than 

or equal to 3 interquartile 

ranges from the box.

UNCHLORINATED CHLORINATED DECHLORINATED

Figure 10. Percent injury of fecal-coliforms in relation to treatment type in combined sewer overflow 

effluent collected from Mill Creek and incubated at Cuyahoga River at Independence, Ohio in the 

Cleveland area: (A) May 1995 and (B) June 1995. (Dashed lines indicated results of paired 

Tukey's multiple comparison test.)

Effects of Receiving-Water Quality and Wastewater Treatment on Injury, Survival, and Regrowth of Fecal-Indicator Bacteria 25
be done before the effect of dechlorination and chlorination on reducing fecal-coliform concentrations in 

CSO effluents can be confirmed.

Percent Survival of Fecal-Indicator Bacteria

Percent survival at each timestep was calculated as a percentage of the mean concentration from 

the enhanced-recovery method in the initial unchlorinated sample. Mean percent survival is a measure of 

the ability of fecal coliforms to survive in the aquatic 

environment after being discharged from the wastewater treatment plant or the CSO. For chlorinated and 

dechlorinated samples, mean percent survival is also a 

measure of the effectiveness of treatment in reducing 

fecal-coliform concentrations.

Mean percent survival was calculated for each 

field study by treatment and site (table 6). The highest 

percent survivals were found in unchlorinated samples, some values greater than 100 percent. (Mean 

percent survivals greater than 100 percent indicate that 

cells replicated after the initial timestep.) Mean percent survivals in unchlorinated CSO-effluent samples 

were generally less than those found in unchlorinated 

wastewater-effluent samples, an indication that fecal 

coliforms in CSO effluent were not able to survive in 

the aquatic environment as well as fecal coliforms 

from wastewater effluent. In all but one field study

(June 1995 CSO field study), mean percent survivals 

were greater in dechlorinated samples than in chlorinated samples. Therefore, chlorination alone was generally more effective than dechlorination in reducing 

fecal-coliform concentrations after treatment and (or) 

reducing survival of fecal coliforms in the aquatic 

environment.

ANOVA was used to examine relations among 

percjent survival, treatment, and site. For all studies, 

statistically significant differences were found in mean 

percent survivals among the three treatments, except 

in the June 1995 CSO field study where no statistical 

difference (p>0.10) was found in percent survivals 

between the chlorinated and dechlorinated populations. During this study, a second atypical peak in 

fecal-coliform concentrations was found in chlorinated samples (see fig. 7B).

In the June 1994 Cuyahoga River field study, no 

statistically significant differences were found in percent survivals between the Peninsula and West Third 

sites. In the August 1995 field study, however, percent 

survivals in the dechlorinated population at West 

Third (mean percent survival=18.2) were statistically 

higher (p<0.01) than those in the dechlorinated population at Peninsula (mean percent survival=10.6). During this field study, mean water temperature and total 

organic carbon concentrations were higher and mean 

dissolved-oxygen and total nonfilterable residue concentrations were lower at West Third than at Peninsula

Table 6. Mean percent survival of fecal coliforms in unchlorinated, chlorinated, and dechlorinated wastewater or combinedsewer-overflow effluent in field studies

[Survival is computed as [ERMt/(ERMuno>] x 100, where ERM is enhanced-recovery method, ERMt is colonies per 100 milliliters recovered at selected 

timesteps, and ERMuriQ is mean colonies per 100 milliliters recovered at timeo in unchlorinated samples; mean percent survival is the mean for all 

timesteps in each study]

Wastewater, Wastewater, Cuyahoga River Combined-sewer

tvcxn^ ki i^ cat

Cleveland, Ohio Peninsula V\

May Aug. June Aug. June 

Treatment 1994 1994 1994 1995 1994

UVBIIIUW, uuycuiuya

est Third Independence River at Independence

Aug. Sept. May June 

1995 1995 1995 1995

Unchlorinated 86.3 137.9 155.2 43.3 160.3 67.4 139.3 83.0 64.4

Chlorinated 3.3 1.6 3.2 1.4 3.5 1.5 .1 .4 .3b

Dechlorinated 23.2 47.0 26.2 10.6a 19.0 I8.2a .6 .9 .l b

aBy use of analysis of variance, a statistically significant difference was found in the August 1995 study between percent survivals of dechlorinated samples at Peninsula and West Third; no other statistically significant differenc; in percent survivals between sites was found.

bBy use of analysis of variance, a statistically significant difference was found between unchlorinated, chlorinated, and dechlorinated samples in 

all studies, but no statistically significant difference was found in the June 1995 combined-sewer-overflow effluent field study between percent survival of 

chlorinated and dechlorinated samples.

26 Effects of Receiving-Water Quality and Wastewater Treatment on Injury, Survival, and Regrowth of Fecal-Indicator Bacteria
(table 3). Whether or not these water-quality differences affected fecal-coliform survival is unknown; 

however, during these field studies, water-quality differences between Peninsula and West Third were less 

influential than treatment in affecting the survival of 

fecal coliforms.

Implications for Assessment of 

Recreational Water Quality

Relation of Enhanced-Recovery Method 

Concentrations to Standard-Method 

Concentrations

Currently, recreational water quality in Ohio is 

monitored by use of the standard method for fecal 

coliforms (Ohio Environmental Protection Agency, 

1990). Enhanced-recovery methods, however, were 

found to be superior to the standard method in recovering chlorine-injured organisms in other investigations (Rose and others, 1975; Green and others, 1977; 

LeChevallier and others, 1983, 1984) and in this 

investigation. If the concentration of fecal coliforms 

determined by use of the enhanced-recovery method 

can be predicted accurately from the concentration 

found by use of the standard method, then increased 

monitoring and expense to detect chlorine-injured 

organisms would be unnecessary. However, if the concentration of fecal coliforms found by use of the 

enhanced-recovery method cannot be predicted accurately from the concentration found by use of the standard method, water-resource managers could possibly 

misinterpret the health risk of swimming in some 

waters, especially those waters with a high proportion 

of chlorine-injured, undetected fecal coliforms.

Data from seven field studies (not grouped). 

The data from the seven field studies described in this 

report are shown on a scatterplot (fig. 11), and associated regression statistics are shown in table 7. Each 

data point on the scatterplot represents a pair of logtransformed standard and enhanced-recovery fecalcoliform concentrations from a single sample for all 

field studies. The concentration found by use of the 

standard method was highly correlated with the concentration found by use of the enhanced-recovery 

method (r= 0.962). The slope (0.906) is a measure of 

the rate of change in log standard method concentration with change in log enhanced-recovery method 

concentration. The y-intercept (0.795) is the value for

log enhanced-recovery method that corresponds to a 

zero value for log standard method. These values seem 

reasonable in sign and magnitude, and t- tests 

(a=0.05) indicate that the slope and y-intercept were 

statistically different than zero (unpublished data on 

file at USGS, Columbus, Ohio). The standard error of 

the regression (also known as the standard deviation of 

the residuals) measures the degree of deviation of 

observed values from the regression line and is an 

indicator of the level of uncertainty associated with a 

prediction, expressed as a percentage of the predicted 

mean. The standard error indicated that predicted 

enhanced-recovery concentrations can vary as much 

as 34.2 percent. The coefficient of determination, or R2 

(0.932), showed that a large amount of the variation in 

results from the enhanced-recovery method was 

explained by results from the standard method. An 

overall F-test of the regression indicated that the 

regression relation was statistically significant and that 

the relation between the two variables was not likely 

due to chance alone (unpublished data on file at 

USGS, Columbus, Ohio).

In spite of these statistics, however, regression 

diagnostics indicated that the regression model on the 

entire data set was inadequate. Regression residuals, 

the differences between observed and predicted values, for enhanced-recovery method concentrations 

were calculated. A PPCC plot of the residuals showed 

a large departure from normality with a probability of 

less than 0.01 (table 7). When the residuals were plotted against the predicted values (fig. 12A), heteroscedasticity (nonconstant variance) was strongly 

indicated. The variance was greater for smaller predicted concentrations and decreased as predicted concentrations increased. When the residuals were plotted 

against elapsed time (fig. 12B), there appeared to be a 

small decrease in variance with samples collected late 

in the field studies as compared to samples collected 

early in the field studies. When residuals were put into 

boxplots by treatment type (unchlorinated, chlorinated, and dechlorinated), greater residuals, wider 

interquartile ranges, and more outside values were 

found in chlorinated and dechlorinated sample pairs 

than in unchlorinated sample pairs (fig. 12C). In addition, 6 outliers with standard deviations greater than 2 

were found among the 382 observations in the data set 

(unpublished data on file at USGS, Columbus, Ohio). 

The outliers were all from chlorinated and dechlorinated sample pairs and the first 20 hours of incubation; 

however, because the data sets were large, the outliers

Implications for Assessment of Recreational Water Quality 27
log ERM = 0.906 (log SM) + 0.795

LLI

2 3 

STANDARD METHOD FECAL-COLIFORM CONCENTRATION, 

IN LOG COLONIES PER 100 MILLILITERS

Figure 11. Regression relation between enhancedconcentrations of fecal coliforms in seven field 

overflow effluent collected in the Cleveland area, Ohio

recovery (ERM) and standard-method (SM) 

studies of wastewater and combined-sewer28 Effects of Receiving-Water Quality and Wastewater Treatment on Injury, Survival, and Regrowth of Fecal-Indicator Bacteria
Table 7. Regression statistics for enhanced-recovery and standard-method concentrations of fecal coliforms in wastewater 

and combined-sewer-overflow effluent collected in the Cleveland area, Ohio

Data set

Sample 

size

S

(percent)

Slope 

(log units)

y-intercept 

(log units)

p-value for

normality of

residuals

All data:

SM concentration as explanatory 

variable

382 0.962 34.2 0.906 0.795

SM concentration and time as 382 .968 33.3 

expanatory variables

Data grouped by magnitude of standard method concentration:

0.906 (SM cone.) .901 

- .004 (time)

0.932

.936

0.01

SM concentraton <5,000

SM concentration >5,000

Data grouped by treatment type:

Unchlorinated

Chlorinated

Dechlorinated

256

117

131

125

126

0.897

.968

0.988

.904

.930

39.50

16.03

16.74

41.41

37.23

0.943

.924

0.984

.948

.930

0.722

.683

0.378

.746

.770

0.804

.936

0.976

.817

.866

O.01

.305

0.126

<01

<01

exhibited low leverage and influence on the regression 

relation.

A departure of residuals from normality results 

in hypothesis tests that have low power (slopes or 

explanatory variables will be falsely declared statistically insignificant), and results in a false estimate of 

confidence or prediction intervals (Helsel and Hirsch, 

1992, p.236). Heteroscedasticity of residuals results in 

the failure of the model to accurately predict 

enhanced-recovery concentrations for all standardmethod concentrations. Decreasing residuals with 

larger predicted values, decreasing residuals over 

time, and a larger spread of residuals for chlorinated 

and dechlorinated samples indicate that additional 

explanatory variables should be included in the model. 

Therefore, the regression model on the entire data set 

does not adequately explain the relation between standard-method concentrations and enhanced-recovery 

concentrations.

Time was included as an explanatory variable, 

in addition to log standard-method concentration, in 

the regression model. The addition of time as an 

explanatory variable resulted in an additional variable 

in the regression equation with a slope of-0.004 (table 

7); this slope was found to be statistically different 

than zero. It also resulted in a change in the y-intercept of the regression model from 0.795 to 0.901. The 

other regression statistics generated with two explanatory variables were similar to those resulting from one 

explanatory variable. Most important, the inclusion 

of time as an explanatory variable did not reduce heteroscedasticity of the residuals (unpublished data on

file at USGS, Columbus, Ohio). Therefore, the model 

that included time did not explain the variation in 

enhanced-recovery concentrations any better than the 

model containing log standard-method concentration 

alone as the explanatory variable.

Other factors that may influence the enhancedrecovery concentration are not appropriately 

expressed as continuous variables. The data were 

placed into groups based on two such factors: (1) magnitude of standard-method concentration and (2) treatment. These factors were individually tested to 

determine their importance as explanatory variables. 

ANCOVA was done to determine if the regression 

equations generated by each group were statistically 

different in terms of slopes and y-intercepts. An Fratio was tabulated from an analysis of variance of 

regression coefficients over groups. If the F-ratio tabulated was statistically significant (/?<0.05), then the 

regression equations generated by the grouped data 

(the complex model) improved the fit of the model 

compared to the equation generated by the entire data 

set (simple model). Diagnostics also were done on 

residuals generated from regressions of the grouped 

data.

Data grouped by magnitude of standardmethod concentration. Data were placed into two 

groups on the basis of magnitude of standard-method 

concentration. For Ohio, the single-sample secondarycontact recreational standard is 5,000 col/100 mL 

(Ohio Environmental Protection Agency, 1990). This 

is the highest recreational water-quality standard; for 

samples whose fecal-coliform concentration is less

Implications for Assessment of Recreational Water Quality 29
CO

<

Q

CO

LU 

CL

A. PRECIDTED ENHANCED-RECOVERY 

CONCENTRATIONS

-1

-2

PREDICTED ENHANCED RECOVERY CONCENTRATIONS, 

IN LOG COLONIES PER 100 MILLILITERS

-1

-2

B. ELAPSED TIME

10 20 30 40 50 60 

ELAPSED TIME, IN HOURS

70 80

i

i o

CO

LU 

DC

-2

C. TREATMENT TYPE

1 I

EXPLANATION

Upper whisker1 

75th percent! le 

Median 

25th percentile

Lower whisker1 

Outside value2

'Upper whisker is defined as 

the largest data point less than 

or equal to the upper quartile 

plus 1.5 times the interquartile 

range. Lower whisker is minus 

1.5 times the interquartile range.

2An outside value is defined as 

greater than 1.5 and less than 

or equal to 3 interquartile 

ranges from the box.

UNCHLORINATED CHLORINATED DECHLORINATED

Figure 12. Residuals from regression relation betwecm enhanced-recovery and standard-method 

concentrations of fecal conforms in seven field studies against: (A) predicted enhanced-recovery 

concentrations, (B) elapsed time, and (C) treatment lype. (Data for wastewater and combinedsewer-overflow effluent, Cleveland area, Ohio.)

30 Effects of Receiving-Water Quality and Wastewater Treatment on Injury, Survival, and Regrowth of Fecal-Indicator Bacteria
than this standard, it is important to accurately predict 

enhanced-recovery concentrations from standardmethod concentrations. Therefore, data were placed 

into two groups on the basis of magnitude of standardmethod concentration: (1) less than or equal to 5,000 

col/100 mL (<5,000) and (2) greater than 5,000 

col/100 mL (>5,000). The data representing the two 

groups are shown on scatterplots (fig. 13) and regression statistics are included in table 7.

The model generated from >5,000 data set (fig. 

13B) provided a better linear fit of the data than the 

model generated from <5,000 data set (fig. 13A). The 

correlation coefficient was higher for >5,000 data set 

(0.968) than <5,000 data set (0.897); the standard error 

of the regression was lower for >5,000 data set (16.03 

percent) than for <5,000 data set (39.5 percent); and 

the coefficient of variation was larger for >5,000 data 

set (0.936) than for <5,000 data set (0.804) (table 7). 

In addition, the residuals from the >5,000 data set 

were normally distributed, whereas the residuals generated from the <5,000 data set were not (table 7). The 

residuals generated from the model with data >5,000 

(fig. 13B) were smaller in magnitude and less heteroscedastic than residuals generated with <5,000 data 

(fig. 13A). However, the F-ratio calculated from analysis of variance of regression coefficients (Fratio=1.713) resulted in a/>-value of 0.179, indicating 

that the slopes or y-intercepts were not statistically different between the regression equations generated 

from the two groups. Thus, the relation between 

enhanced-recovery and standard-method concentrations are better represented by the simple model than 

by the complex model.

Data grouped by treatment. Data were placed 

in three groups based on type of treatment: (1) unchlorinated, (2) chlorinated, and (3) dechlorinated. For 

brevity, these data groups are referred to as "unchlorinated data," "chlorinated data," and "dechlorinated 

data." The data representing the three groups are 

shown on scatterplots (fig. 14), and regression statistics are included in table 7.

The model generated from unchlorinated data 

(fig. 14A) provided a better linear fit of the data than 

the models generated from the chlorinated (fig. 14B) 

and dechlorinated data (fig. 14C). For the unchlorinated data, the correlation coefficient was higher 

(0.988), the standard error of the regression was considerably lower (16.74 percent), and the coefficient of 

variation was larger (0.976) than values generated 

from chlorinated and dechlorinated data (table 7). The

F-ratio calculated from analysis of variance of regression coefficients among the three groups (F-ratio=5.2) 

resulted in ap-value of 0.005, an indication that the 

slopes or intercepts were statistically different among 

the regression equations generated from the three 

groups. Therefore, the data are best represented in 

regression relations generated from the complex 

model (data grouped by treatment type).

The residuals generated from the regression on 

unchlorinated data were normally distributed; those 

generated from chlorinated and dechlorinated data 

were not (table 7). The residuals generated from the 

regression on unchlorinated data (fig. 14A) were 

homoscedastic, satisfying an important assumption of 

linear regression; however, the residuals generated 

from the chlorinated data (fig. 14B) and the dechlorinated data (fig. 14C) showed considerable heteroscedasticity. Therefore, the regression equations 

generated from the unchlorinated samples met the 

assumptions of linear regression and provided the best 

estimate of enhanced-recovery concentrations from 

standard-method concentrations among the three treatment groups. For the regression equations generated 

from dechlorinated and chlorinated data, the normality 

of residual assumption and the equal variance assumption were not met. The assumption of a normal distribution is involved in testing hypotheses (Helsel and 

Hirsch, 1992) and, in this case, the hypothesis tests of 

whether the slope coefficient was statistically different 

than zero generated very high /-values of 23.56 (chlorinated) and 28.43 (dechlorinated); therefore, violation 

of this assumption was not a serious limitation. However, because of considerable heteroscedasticity of 

residuals, the regression equations generated from 

dechlorinated and chlorinated data did not provide a 

good estimate of enhanced-recovery concentrations 

from standard-method concentrations for all data.

Therefore, it appears that concentrations of fecal 

coliforms found by the enhanced-recovery method 

cannot be accurately predicted by standard-method 

concentrations for all receiving waters in this investigation. Accurate models were not generated for waters 

that contained high proportions of chlorinated or 

dechlorinated organisms or waters with low numbers 

of fecal coliforms. Presently, the standard method for 

fecal coliforms is the most widely used method to 

assess recreational water quality; enhanced-recovery 

methods are not routinely used. Therefore, the results 

of this investigation indicate that water-resource

Implications for Assessment of Recreational Water Quality 31
A. LESS THAN OR EQUAL TO 5,000 COLONIES PER 100 MILLILITERS

CO

cc 111 t

cc 111

0.

co

UJ

I

o o

o 

o

o

111 

o

o

o

cc

o

o

o

111

o

o

LLJ

cc

Q 

LU 

O

2 -

1 -

log ERM = 0.943 (log SM) + 0.722

, i , , , i i . , i , i , , i

CO

go

1 2 3

STANDARD-METHOD

FECAL-COLIFORM CONCENTRATION,

IN LOG COLONIES PER 100 MILLILITERS

B. GREATER THAN 5,000 COLONIES PER 100 MILLILITERS

-1 -

-2

012345 

PREDICTED ENHANCED-RECOVERY METHOD 

FECAL-COLIFORM CONCENTRATION, IN LOG 

COLONIES PER 100 MILLILITERS

log ERM = 0.924 (log SM) + 0.683

1 -

CO

CO 

UJ

cc

-1 -

-2

4567

STANDARD-METHOD

FECAL-COLIFORM CONCENTRATION,

IN LOG COLONIES PER 100 MILLILITERS

PREDICTED ENHANCED-RECOVERY METHOD

FECAL-COLIFORM CONCENTRATION, IN LOG

COLONIES PER 100 MILLILITERS

Figure 13. Regression relation between enhanced-recovery (ERM) and standard-method (SM) 

concentrations of fecal coliforms and associated residuals against predicted ERM concentrations 

for data grouped by magnitude of SM concentrations: (A) less than or equal to 5,000 

colonies per 100 milliliters and (B) greater than 5,000 colonies per 100 milliliters. (Data for 

wastewater and combined-sewer-overflow effluent, Cleveland area, Ohio.)

32 Effects of Receiving-Water Quality and Wastewater Treatment on Injury, Survival, and Regrowth of Fecal-Indicator Bacteria
A. UNCHLORINATED

7

6

5

4

3h

log ERM = 0.984 (log SM) + 0.378

CO 1

i o

co

LLJ 

DC

-1

-2

8

STANDARD-METHOD FECAL-COLIFORM CONCENTRATION, 

IN LOG COLONIES PER 100 MILLILITERS

PREDICTED ENHANCED-RECOVERY METHOD

FECAL-COLIFORM CONCENTRATION, IN LOG

COLONIES PER 100 MILLILITERS

B. CHLORINATED

log ERM = 0.948 (log SM) + 0.746

CO '

lo

CO 

LU 

DC

-2

STANDARD-METHOD FECAL-COLIFORM CONCENTRATION, 

IN LOG COLONIES PER 100 MILLILITERS

PREDICTED ENHANCED-RECOVERY METHOD

FECAL-COLIFORM CONCENTRATION, IN LOG

COLONIES PER 100 MILLILITERS

C. DECHLORINATED

5 -

log ERM = 0.930 (log SM) + 0.770

co 1

Q 0

-1

-2

.' » . -V . 

*« * . t

0

STANDARD-METHOD FECAL-COLIFORM CONCENTRATION, 

IN LOG COLONIES PER 100 MILLILITERS

PREDICTED ENHANCED-RECOVERY METHOD

FECAL-COLIFORM CONCENTRATION, IN LOG

COLONIES PER 100 MILLILITERS

Figure 14. Regression relation between enhanced-recovery (ERM) and standard-method (SM) 

concentrations of fecal coliforms and associated residuals against predicted enhanced-recovery 

concentrations for data grouped by treatment: (A) unchlorinated, (B) chlorinated, and (C) 

dechlorinated. (Data for wastewater and combined-sewer-overflow effluents, Cleveland 

area, Ohio.)

Implications for Assessment of Recreational Water Quality 33
managers may not have enough information about 

concentrations of injured fecal coliforms to correctly 

interpret the health risk of swimming in waters with 

high proportions of chlorinated or dechlorinated 

organisms.

Relation of Enhanced-Recovery and 

Standard-Method Concentrations to Ohio 

Recreational Water-Quality Standards

Fecal-coliform concentrations found in the 

seven field studies were evaluated in terms of Ohio 

water-quality standards (Ohio Environmental Protection Agency, 1990). The investigators calculated the 

percentage of samples in each field study by treatment 

type that met Ohio's single-sample bathing water (fig. 

15), primary-contact (fig. 16), or secondary-contact 

(fig 17) fecal-coliform recreational standards by use of 

the standard or enhanced-recovery methods.

Unchlorinated, chlorinated, and dechlorinated 

samples were placed in the following categories:

1. samples whose fecal-coliform concentration 

met recreational water-quality standards, regardless of 

method,

2. samples whose fecal-coliform concentration 

met recreational water-quality standards if the standard method was used but exceeded the standards if 

the enhanced-recovery method was used (referred to 

as mixed category), and

3. samples whose fecal-coliform concentration 

exceeded recreational water-quality standards, regardless of method.

Samples that fall into the mixed category are 

particularly troublesome. These samples may lead to 

misinterpretations of the health risk of swimming in 

contaminated waters if only results from standard 

methods are used to assess risk.

For all samples, patterns emerged that placed 

the field studies into three categories based on results 

of meeting or exceeding recreational-water quality 

standards. The observed patterns were the following:

1. field studies of wastewater effluents with low 

concentrations of fecal coliforms concentrations 

were < 10,000 col/100 mL throughout each field study 

(May 1994, August 1995, and September 1995);

2. field studies of wastewater effluent with high 

concentrations of fecal coliforms initial unchlorinated sample concentrations were > 10,000 col/100 

mL and increased at least tenfold throughout the field 

study (June 1994 and August 1994); and

3. CSO field studies (May and June 1995).

Single-sample bathing-water standard. Bathing waters are defined as those waters suitable for 

swimming and other full-body contact where a lifeguard or bathhouse is present (Ohio Environmental 

Protection Agency, 1990). Fecal-coliform concentrations in most of the unchlorinated samples exceeded 

the single-sample bathing-water standard of 400 

col/100 mL (fig. 15). All chlorinated samples from 

wastewater-effluent field studies with low concentrations of fecal coliforms (fig. 15 A), met the bathingwater standard regardless of the method used to determine those concentrations. However, of the dechlorinatod samples, 75 percent in May 1994 and 48 percent 

in August 1995 were in the mixed category. In contrast, all the dechlorinated samples from wastewatereffl aent field studies with high concentrations of fecal 

coliforms (fig. 15B) exceeded the bathing-water standard regardless of method; however, a considerable 

percentage of chlorinated samples fell into the mixed 

category. Dechlorinated and chlorinated samples from 

the May 1995 CSO field study (fig. 15C) either 

exceeded the standard regardless of method or were in 

the mixed category; thus, none of these samples met 

the bathing-water single-sample standard. In the June 

1995 CSO field study, dechlorinated and chlorinated 

samples were in all categories.

Generalizations on the health risk of swimming 

in receiving waters during this investigation based on 

the single-sample bathing-water standard can be made. 

The data indicate that the health risk of swimming in 

waters receiving unchlorinated effluent was high. 

Chlbrination of wastewater effluents that had low concentrations of fecal coliforms resulted in receiving 

waters meeting bathing-water single-sample standards, even if enhanced-recovery methods were used. 

However, risks associated with dechlorinated, lowfecal-coliform effluents were unclear because many of

dechlorinated samples were in the mixed category, 

wastewater-effluent field studies with high conthe 

For

cenirations of fecal coliforms in dechlorinated effluents, interpretation of the health risk of swimming in 

receiving waters was simpler because these samples 

did not meet the bathing-water standard by use of 

either method. However, for chlorinated, high-concentration effluents, the health risk of swimming in 

receiving waters was uncertain because many of these 

samples were in the mixed category. In this investigation, interpretation of the health risk of swimming in 

CSO-contaminated waters was generally difficult,

34 Effects of Receiving-Water Quality and Wastewater Treatment on Injury. Survival, and Regrowth of Fecal-Indicator Bacteria
120

100

80

60

40

20

0

A. WASTEWATER EFFLUENT, LOW CONCENTRATIONS OF BACTERIA

120 , , , 120

May 1994, Lake Erie

CL DE

August 1995, Cuyahoga River September 1995, Cuyahoga River

CL DE CL DE

B. WASTEWATER EFFLUENT, HIGH CONCENTRATIONS OF BACTERIA

110

120

100

80

60

40

20

0

UN CL DE UN CL DE

C. COMBINED-SEWER OVERFLOW EFFLUENT

120

May 1995, Cuyahoga River

CL DE UN 

TREATMENT

DE

EXPLANATION

Concentration met bathingwater standard

Concentration from 

standard method met 

bathing-water standard, 

and concentration from 

enhanced-recovery 

method exceeded 

bathing-water standard 

(mixea category)

Concentration exceeded 

bathing-water standard

Figure 15. Percentage of samples, by treatment type, in which fecal-coliform concentrations 

determined by standard and enhanced-recovery methods met and exceeded Ohio's bathingwater single-sample standard: (A) wastewater effluent, low concentrations of bacteria, (B) 

wastewater effluent, high concentrations of bacteria, and (C) combined-sewer-overflow effluent. 

(UN is unchlorinated; CL is chlorinated; and DE is dechlorinated.)

Implications for Assessment of Recreational Water Quality 35
o

CD

O

1

CO 

LLJ

LL 

O

O

cc

i r 

May 1994, Lake Erie

A. WASTEWATER EFFLUENT, LOW CONCENTRATIONS OF BACTERIA

120 120

100

80

60

40

20

0

i i i 

August 1995, Cuyahoga River

DE

100

80

60

40

20

0

September 1995, Cuyahoga River

CL

I

DE CL DE

B. WASTEWATER EFFLUENT, HIGH CONCENTRATIONS OF BACTERIA

120

100

80

60

40

20

0

T I I

August 1994, Lake Erie

I I T^

June 1994, Cuyahoga River

120

100

80

60

40

20

0

C. COMBINED-SEWER OVERFLOW EFFLUENT

120

EXPLANATION

Concentration met primarycontact standard

Concentration from standard 

method met primarycontact standard, and 

concentration from 

enhanced-recovery method 

exceeded primary-contact 

standard (mixed category)

Concentration exceeded 

primary-contact standard

UN DE UN 

TREATMENT

DE

Figure 16. Percentage of samples, by treatment type, in which fecal-coliform concentrations 

determined by standard and enhanced-recovery methods met and exceeded Ohio's primary-contact 

single-sample standard: (A) wastewater effluent, low concentrations of bacteria, (B) wastewater 

effluent, high concentrations of bacteria, and (C) combined-sewer overflow effluent. (UN is 

unchlorinated; CL is chlorinated; and DE is dechlorinated.)

36 Effects of Receiving-Water Quality and Wastewater Treatment on injury, Survival, and Regrowth of Fecal-Indicator Bacteria
September 1995, Cuyahoga River

UN

A. WASTEWATER EFFLUENT, LOW CONCENTRATIONS OF BACTERIA

120 i i i 120

August 1995, Cuyahoga River 

100 - - 100

80 - - 80

60 - - 60

40 - - 40

20 - - 20

o Hh o

\£\) 

100 

80

60

40

20 

0

i i 

May 1994, Lake Erie

-

-

-

-

i i

i

i

-

-

-

-

CL DE " UN CL DE UN CL 

B. WASTEWATER EFFLUENT, HIGH CONCENTRATIONS OF BACTERIA

DE

120

100

80

60

40

20

0

UN CL DE

C. COMBINED-SEWER-OVERFLOW EFFLUENT 

\ \ \ 120

May 1995, Cuyahoga River

CL

100

80

60

40

20

0 

DE UN

TREATMENT

i i r 

June 1995, Cuyahoga River

EXPLANATION

Concentration met 

secondary-contact 

standard

Concentration from standard 

method met secondarycontact standard, and 

concentration from 

enhanced-recovery 

method exceeded 

secondary-contact 

standard (mixed category)

Concentration exceeded 

secondary-contact 

standard

Figure 17. Percentage of samples, by treatment type, in which fecal-coliform concentrations 

determined by standard and enhanced-recovery methods met and exceeded Ohio's secondarycontact-single-sample standard: (A) wastewater effluent, low concentrations of bacteria, (B) 

wastewater effluent, high concentrations of bacteria, and (C) combined-sewer-overflow effli 

(UN is unchlorinated; CL is chlorinated; and DE is dechlorinated.)

Implications for Assessment of Recreational Water Quality 37
because many of the chlorinated and dechlorinated 

samples fell into the mixed category.

Single-sample primary-contact standard.

The Ohio Environmental Protection Agency (1990) 

defines primary-contact waters as those waters suitable for full-body contact, such as swimming, canoeing, and scuba diving. The single-sample primarycontact standard in Ohio for fecal coliforms is 2,000 

col/100 mL (Ohio Environmental Protection Agency, 

1990). For unchlorinated samples, the primary-contact standard was sometimes met in wastewater-effluent field studies where concentrations of fecal 

coliforms were low (fig. 16A); however, the standard 

was exceeded in unchlorinated samples from wastewater-effluent field studies where concentrations of 

fecal coliforms were high (fig. 16B) and in CSO field 

studies (fig. 16C). All chlorinated and dechlorinated 

samples from wastewater-effluent field studies where 

concentrations of fecal coliforms were low (fig. 16A) 

met the primary-contact standard by use of either the 

standard or enhanced-recovery method. In contrast, 

in wastewater-effluent field studies where concentrations of fecal coliforms were high (fig. 16B), 12-60 

percent of the chlorinated and dechlorinated samples 

were in the mixed category; the remaining chlorinated 

samples met the standard and the remaining dechlorinated samples exceeded the standard. Therefore, with 

regard to the single-sample primary contact standard 

and waters receiving wastewater with high concentrations of fecal coliforms, chlorination alone was more 

effective than chlorination followed by dechlorination 

in reducing fecal-coliform concentrations to acceptable levels in these field studies. No pattern emerged 

from the evaluation of the CSO effluents in terms of 

the primary-contact standard (fig. 16C), probably 

because of the uncertain composition of CSO materials. As with the bathing-water standards, many CSO 

effluent samples in this investigation fell within the 

mixed category, thus making the evaluation of the 

health risk of swimming in CSO-contaminated waters 

difficult.

Single-sample secondary-contact standard. 

The Ohio Environmental Protection Agency (1990) 

defines secondary-contact waters as those waters that 

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

The single-sample secondary-contact standard is 5,000 

col/100 mL (Ohio Environmental Protection Agency, 

1990). Most samples collected from wastewater-effluent field studies with low concentrations of fecal 

coliforms met this standard (fig. 17A). Wastewater

effluents containing low concentrations of fecal 

coliforms are often released during cool weather, 

when secondary contact is the common water-based 

recreational activity. For wastewater-effluent field 

studies where concentrations of fecal coliforms were 

high (fig. 17B), all the unchlorinated samples 

exceeded the secondary-contact standard, most of the 

chlorinated samples met the secondary-contact standard, and the dechlorinated samples were within all 

three categories. Similarly, CSO effluent samples were 

within all three categories (fig. 17C), again owing to 

the uncertain composition and unpredictability of CSO 

materials. In the June 1995 CSO field study, the 

dechlorinated samples met the secondary-contact standard more often than chlorinated samples did. 

i

Summary and Conclusions

To meet new chlorine water-quality standards 

required by the U.S. Environmental Protection 

Agency, many wastewater treatment plants in Ohio 

and elsewhere must now dechlorinate effluents before 

discharging them to inland waterways. However, there 

is little information on the effect of dechlorination on 

the repair of injury and survival of chlorine-injured 

fecal-indicator bacteria in receiving waters and on 

their regrowth on growth medium.

Seven field studies were done during the recreational seasons of 1994 and 1995 at one Lake Erie 

location and three Cuyahoga River locations in the 

Cleveland, Ohio metropolitan area to study fecalcoliform injury, survival, and regrowth. Incubation 

chambers, containing unchlorinated, chlorinated, or 

dechlorinated wastewater or combined-sewer overflow (CSO) effluents were placed in river or lake 

water. The contents of the chambers were analyzed for 

concentrations of injured and healthy fecal coliforms 

by use of standard and enhanced-recovery membranefiltration (MF) methods. Standard methods support the 

growth of healthy organisms, whereas enhancedrecovery methods support the growth of both healthy 

and injured organisms. The enhanced-recovery 

method selected for the field studies differed from the 

standard method in that rosalic acid was omitted from 

the medium and a 4-hour resuscitation at 35°C preceded incubation at 44.5°C.

' Field studies were done in May 1994 and in 

August 1994 at the Lake Erie site with wastewater 

effluent from the Easterly Wastewater Treatment 

Plant. In May, when mean water temperature was

38 Effects of Receiving-Water Quality and Wastewater Treatment on Injury, Survival, and Regrowth of Fecai-lndicator Bacteria
12.8°C, fecal-coliform concentrations in all samples 

remained fairly constant, probably because of slow 

bacteria metabolic rates. In August 1994, when mean 

water temperature was 22.6°C, concentrations in 

unchlorinated and dechlorinated samples increased 

during the first 10 to 13 hours. In contrast, in August

1994 in chlorinated samples, concentrations on 

enhanced-recovery media dropped off sharply during 

the first 7 hours, indicating that the chlorinated organisms were unable to repair injuries and regrow on 

enhanced-recovery media.

Field studies were done in June 1994 and 

August 1995 at two Cuyahoga River sites Peninsula 

and West Third with wastewater effluent from the 

Southerly Wastewater Treatment Plant. During both 

field studies, mean dissolved-oxygen concentrations 

were 2-3 mg/L higher at Peninsula than at West Third. 

In June 1994 at both sites, fecal-coliform concentrations generally increased slightly or remained the 

same after the initial timestep. However, in August

1995 at West Third, unchlorinated concentrations 

decreased, whereas chlorinated and dechlorinated concentrations increased and decreased, an indication that 

the increases in concentrations in chlorinated and 

dechlorinated samples were due in part to repair of 

chlorine-injured cells and regrowth on culture media.

Field studies were done in May 1995 and June 

1995 at the Cuyahoga River at Independence with 

CSO effluent collected from Mill Creek. In May 1995, 

mean water temperature was 15.6°C, whereas in June 

1995, mean water temperature was 23.1°C. In May 

1995, in dechlorinated and chlorinated samples but not 

in unchlorinated samples, fecal-coliform concentrations by use of the enhanced-recovery method 

increased more rapidly from about 20-32 hours than 

those by use of the standard method. This finding indicates that repair of chlorine-induced injuries may have 

enabled some previously undetected organisms in 

dechlorinated and chlorinated samples to grow on 

enhanced-recovery media. In the chlorinated samples 

in June 1995, fecal-coliform concentrations peaked at 

14 hours and again at 48 hours. CSO effluent samples 

contain more solid materials than treated wastewater 

effluent; perhaps the second, atypical fecal-coliform 

concentration peak in chlorinated samples was due to 

solids breaking apart and releasing a reservoir of bacteria and nutrients in the water.

The effects of wastewater treatment and site on 

percent injury of fecal coliforms were determined by 

use of analysis of variance (ANOVA). At the Lake

Erie site in May 1994, percent injuries in the dechlorinated and chlorinated samples were statistically higher 

than percent injuries in the unchlorinated samples. In 

contrast, in August 1994 at Lake Erie, percent injuries 

in the chlorinated samples were statistically higher 

than percent injuries in the unchlorinated and dechlorinated samples. Therefore, in May 1994, the rate of 

repair and regrowth on culture media was lower in 

chlorinated and dechlorinated samples than in unchlorinated samples. In August 1994, however, fecal 

coliforms in dechlorinated samples were able to repair 

injuries caused by chlorination and regrow on culture 

media, whereas those in chlorinated samples could 

not.

ANOVA on the data from two Cuyahoga River 

sites in June 1994 and August 1995 showed that treatment did affect percent injury, site did not affect percent injury, and there was no interaction effect 

between site and treatment. In both studies, statistically significant differences in percent injuries were 

found between some unchlorinated as compared to 

dechlorinated and chlorinated samples; however, no 

statistically significant differences were found 

between dechlorinated and chlorinated samples. 

Therefore, in the warm-weather Cuyahoga River field 

studies (unlike the warm-weather Lake Erie field 

study), dechlorination did not enhance the repair and 

regrowth of chlorine-injured fecal coliforms over 

chlorination alone. This result may be due to waterquality differences between the lake and river or due 

to the quality of the wastewater effluents used in lake 

and river field studies.

ANOVA on the data from CSO field studies in 

May and June 1995 revealed statistically significant 

differences in percent injuries between all possible 

pairs of treatment groups. The highest percent injuries 

were found in dechlorinated samples, contrary to what 

was found in wastewater-effluent field studies. 

Dechlorination, therefore, appeared to reduce the ability of organisms to recover and regrow on culture 

media over chlorination alone. The presence of 

untreated waste and high solid concentrations may 

have contributed to this result.

The effects of wastewater treatment and site on 

percent survival were determined by ANOVA. Mean 

percent survivals were highest in the unchlorinated 

population for all field studies. Mean percent survivals 

were higher in the dechlorinated than chlorinated populations, except in the June 1995 CSO field study. Statistically significant differences in mean percent

Summary and Conclusions 39
survivals were found between treatments for all field 

studies except in June 1995, wherein no statistically 

significant difference was found between chlorinated 

and dechlorinated samples. In the June 1994 Cuyahoga River wastewater-effluent field study, no statistically significant differences were found in percent 

survivals between Peninsula and West Third sites. In 

the August 1995 Cuyahoga River wastewater-effluent 

field study, however, percent survivals in the dechlorinated samples at the West Third site were statistically 

higher than those found in the dechlorinated samples 

at the Peninsula site. Water-quality differences, however, were not as influential a factor as treatment in 

affecting the survival of fecal coliforms.

Linear regression analysis of data from the 

seven field studies showed that standard-method and 

enhanced-recovery method concentrations were 

highly correlated and that the regression relationship 

was statistically significant; however, regression diagnostics indicated heteroscedasticity (nonconstant variance) of the residuals. Upon further analysis, it was 

found that the data were best represented in regression 

relations generated from data grouped by treatment 

type. The model generated from the unchlorinated 

data met the assumptions of linear regression and provided the best estimate of enhanced-recovery concentrations from standard-method concentrations. 

However, the residuals generated from regression of 

the chlorinated and dechlorinated data showed considerable heteroscedasticity. Therefore, concentrations of 

fecal coliforms determined by the enhanced-recovery 

method probably cannot be accurately predicted from 

standard-method concentrations for all receiving 

waters. The results of this investigation indicate that 

water-resource managers may not have enough information about concentrations of injured fecal coliforms 

from standard-method concentrations to correctly 

interpret the health risk of swimming in waters with 

high proportions of chlorinated or dechlorinated 

organisms.

The investigators determined whether concentrations obtained by standard and enhanced-recovery 

methods were comparable as indicators of recreationwater quality in the seven field studies. The percentage of samples by treatment types in which fecalcoliform concentrations met and exceeded Ohio's 

bathing-water, primary-contact, and secondary contact 

single-sample standards were classified in three categories: (1) samples whose fecal-coliform concentration met recreational water-quality standards

reigardless of the method used to determine that concentration, (2) samples whose fecal-coliform concentration met recreational water-quality standards if the 

standard method was used but exceeded the standards 

if the enhanced-recovery method was used (referred to 

as mixed category), and (3) samples whose fecalcoliform concentration exceeded recreational waterquality standards regardless of the method used. Samples in the mixed category may lead to misinterpretations of the health risk of swimming in contaminated 

waters if results from standard methods only are used 

tc assess water quality.

For all samples, patterns emerged that placed 

the field studies into three categories based on results 

of meeting or exceeding recreational-water quality 

standards: (1) field studies of wastewater effluents 

with low concentrations of fecal coliforms (May 1994, 

August 1995, and September 1995); (2) field studies 

of wastewater effluent with high concentrations of 

fecal coliforms (June 1994 and August 1994); and (3) 

CSO field studies (May and June 1995). In wastewater-effluent field studies where concentrations were 

lojw, chlorinated samples met the recreational-water 

quality standards regardless of the method used; howeter, dechlorinated and unchlorinated samples were 

placed in all three categories. In wastewater-effluent 

fiuld studies where concentrations were high, unchlorinated samples exceeded the standards regardless of the 

method used; however, many chlorinated samples and 

some dechlorinated samples were placed in the mixed 

category. In CSO effluent studies, although all unchloriiated samples exceeded the standards, no pattern 

emerged in terms of chlorinated and dechlorinated 

samples meeting and exceeding any of the standards, 

and many of the chlorinated and dechlorinated samples were placed in the mixed category. Therefore, 

evaluation of fecal-coliform concentrations found in 

fidd studies in terms of Ohio recreational water-quality standards showed that concentrations obtained by 

standard and enhanced-recovery methods were not 

always comparable.

Therefore, determining the health risk of swimming in receiving waters is difficult if information on 

enhanced-recovery method concentrations is not available. Wastewater treatment affects the injury, survival, 

and regrowth of fecal-indicator bacteria; receivingwater quality at the two Cuyahoga River sites had little 

effect on these processes. Dechlorination enhanced the 

repair of injuries and survival of chlorine-injured fecal 

coliforms over chlorination alone in the lake, but not

40 Effects of Receiving-Water Quality and Wastewater Treatment dn Injury, Survival, and Regrowth of Fecal-Indicator Bacteria
in the river. Whether this was because of water-quality 

differences between the lake and the river or because 

of the quality of the wastewater effluents used in the 

lake and river field studies was not determined in this 

investigation. Patterns of concentration increases and 

decreases in CSO effluents were atypical, so the 

effects of wastewater treatment on injury, survival, 

and regrowth in CSO effluents could not be determined.

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42 Effects of Receiving-Water Quality and Wastewater Treatment on Injury, Survival, and Regrowth of Fecal-Indicator Bacteria