Environmental Protection Technology Series
DISINFECTION EFFICIENCY AND
RESIDUAL TOXICITY OF
SEVERAL WASTEWATER DISINFECTANTS
Volume I - Grandville, Michigan
Municipal Environmental Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, Ohio 45268
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development, U.S. Environmental
Protection Agency, have been grouped into five series. These five broad
categories were established to facilitate further development and application of
environmental technology. Elimination of traditional grouping was consciously
planned to foster technology transfer and a maximum interface in related fields.
The five series are:
1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
4. Environmental Monitoring
5. Socioeconomic Environmental Studies
This report has been assigned to the ENVIRONMENTAL PROTECTION
TECHNOLOGY series. This series describes research performed to develop and
demonstrate instrumentation, equipment, and methodology to repair or prevent
environmental degradation from point and non-point sources of pollution. This
work provides the new or improved technology required for the control and
treatment of pollution sources to meet environmental quality standards.
This document is available to the public through the National Technical Informa-
tion Service, Springfield, Virginia 22161.
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EPA-600/2-76-156
October 1976
DISINFECTION EFFICIENCY AND RESIDUAL TOXICITY
OF SEVERAL WASTEWATER DISINFECTANTS
Volume I Grandville, Michigan
by
Ronald W. Ward
Department of Biology
Grand Valley State Colleges
Allendale, Michigan 49401
Randall D. Giffin
City of Wyomin-g, Michigan 49509
G. Michael DeGraeve
Department of Biology
Grand Valley State Colleges
Allendale, Michigan 49401
Richard A. Stone
City of Wyoming, Michigan 49509
Grant No. S-802292
(F 17060 HJB)
Project Officer
Cecil W. Chambers
Wastewater Research Division
Municipal Environmental Research Laboratory
Cincinnati, Ohio 45268
MUNICIPAL ENVIRONMENTAL RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
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DISCLAIMER
This report has been reviewed by the Municipal Environmental Research
Laboratory, U.S. Environmental Protection Agency, and approved for
publication. Approval does not signify that the contents necessarily
reflect the views and policies of the U.S. Environmental Protection
Agency, nor does mention of trade names or commercial products con-
stitute endorsement or recommendation for use.
ii
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FOREWORD
The Environmental Protection Agency was created because of
increasing public and government concern about the dangers of pollution
to the health and welfare of the American people. Noxious air, foul
water, and spoiled land are tragic testimony to the deterioration of
our natural environment. The complexity of that environment and the
interplay between its components require a concentrated and integrated
attack on the problem.
Research and development is that necessary first step in problem
solution and it involves defining the problem, measuring its impact,
and searching for solutions. The Municipal Environmental Research
Laboratory develops new and improved technology and systems for the
prevention, treatment, and management of wastewater and solid and
hazardous waste pollutant discharges from municipal and community
sources, for the preservation and treatment of public drinking water
supplies, and to minimize the adverse economic, social, health, and
aesthetic effects of pollution. This publication is one of the products
of that research; a most vital communications link between the researcher
and the user community.
This study was concerned with comparing the disinfection efficiency
of chlorine with and without dechlorination, ozone, and bromine chloride
on parallel wastewater streams and evaluating the potential toxicity of
those streams to aquatic life. Intimate knowledge of wastewater disin-
fection principles and the effects of wastewater disinfection practices
on man and his environment is vital to the proper control of disease
transmission and preservation of wildlife. This project has contributed
valuable information in the quest for these goals.
Francis T. Mayo, Director
Municipal Environmental Research
Laboratory
iii
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ABSTRACT
This study was conducted to determine the comparative effectiveness of chlo-
rine, bromine chloride, and ozone as wastewater disinfectants, and to deter-
mine any residual toxicity associated with wastewater disinfection with these
agents or with chlorinated wastewater which had been dechlorinated with
sulfur dioxide.
Streams of nondisinfected and chlorinated wastewater were pumped from the
Grandville, Michigan Wastewater Treatment Plant to the project laboratory.
Part of the chlorinated wastewater stream was delivered directly to the
toxicity laboratory for bioassay studies while the remainder of the chlor-
inated stream was dechlorinated with sulfur dioxide prior to its use in
bioassay tests. A portion of the nondisinfected wastewater stream was
delivered to the toxicity laboratory for use in bioassays while the remain-
ing portion was split to receive bromine chloride and ozone prior to use in
the bioassay studies.
Total and fecal coliform densities, suspended solids, volatile solids, COD,
ammonia nitrogen, phosphate, turbidity, color, and pH were measured in the
wastewater streams. Each of the five wastewater streams was used in acute
toxicity tests with several species of fishes and the freshwater macroinver-
tebrate Daphnia magna, and in a life cycle toxicity study with the fathead
minnow, Pimephales promelas, as the test subject.
Disinfection standards were met most frequently by chlorinated and dechlorinated
effluents and less frequently by chlorobrominated effluent. The only time
disinfection standards were met consistently by ozonated effluent was when
filtration preceded ozone injection.
Chlorine was found to be most toxic to aquatic life while sulfur dioxide
dechlorination completely eliminated the toxicological effects of chlorine.
Bromine chloride was less toxic than chlorine. Ozone was found to be neither
acutely nor chronically toxic to the aquatic animals tested.
iv
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CONTENTS
Page
Foreword iii
Abstract iv
List of Figures vi
List of Tables vii
Acknowledgements xi
I Introduction 1
II Conclusions 9
III The Wastewater Treatment Systems and 11
The Characteristics of the Wastewater Streams
IV Disinfection Studies 31
V Life Cycle Residual Toxicity Studies 59
VI Acute Toxicity Tests 113
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FIGUSES
No.
1 Flow of Effluent and Well (Dilution) Water 4
2 Monthly and Yearly Average Plant Flows at the Grandville 12
Wastewater Treatment Plant
3 Monthly and Yearly Average Suspended Solids at the Grand- 14
ville Wastewater Treatment Plant
4 Monthly and Yearly Average Biochemical Oxygen Demand at the 15
Grandville Treatment Plant
5 Monthly and Yearly Average Phosphorus Concentrations at the 16
Grandville Treatment Plant
6 Flow of the Various Wastewater Streams in the Grandville 20
Disinfection Study
7 Monthly Geometric Means of Total Coliform Densities (MF) 44
8 Monthly Geometric Means of Fecal Coliform Densities (MF) 45
9 Percent of Samples with Total Coliform Densities (MF) 46
Below 1000 per 100 ml
10 Percent of Samples with Fecal Coliform Densities (MF) 47
Below 200 per 100 ml
11 Schematic Drawing of the Modified Mount-Brungs Proportional 62
Diluter Used in This Study
vi
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TABLES
No. Page
1 Dates of Some Important Events During the Grandville Study 5
2 Physical-Chemical Characteristics of the Test Streams During 25
the Test Period - January 2, 1974 to November 30, 1974
3 A Summary of the pH Values Measured in the Various Waste- 27
water Streams
4 Reduction in Coliform Numbers by Chlorine, Chlorine Followed 34
by Dechlorination, Ozone, and Bromine Chloride During
January Through February 22, 1974
5 Reduction in Coliform Numbers by Chlorine, Chlorine Followed 35
by Dechlorination, Ozone, and Bromine Chloride During
February 25 Through April 15, 1974
6 Reduction in Coliform Numbers by Chlorine, Chlorine Followed 36
by Dechlorination, Ozone, and Bromine Chloride During
April 16 Through August 9, 1974
7 Reduction in Coliform Numbers by Chlorine, Chlorine Followed 37
by Dechlorination, Ozone, and Bromine Chloride During
August 12 Through September 30, 1974
8 Reduction in Coliform Numbers by Chlorine, Chlorine Followed 38
by Dechlorination, Ozone, and Bromine Chloride During
October 1 Through November 19, 1974
9 Reduction in Coliform Numbers by Chlorine, Chlorine Followed 39
by Dechlorination, Ozone, and Bromine Chloride During
November 19 Through November 27, 1974
10 Frequency That Daily Samples of Disinfected Effluents Achieved 40
Project Bacteriological Standards January Through November,
1974
11 Effectiveness of Ozone Disinfection on Filtered and Unfiltered 50
Effluent
12 Correlation and Regression of MPN vs MF Total Coliform Densi- 51
ties (Number/100 ml) January Through November, 1974
13 Characteristics of the Dilution Water 60
vii
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TABLES (continued)
No. Page
14 The Mean Residual Chemical Levels (mg/1), Sample Sizes, 66
and Standard Deviations Measured in Head Tanks and Adult
Test Chambers During the Life Cycle Tests
15 The Mean Residual Chemical Levels (mg/1), Sample Sizes, 67
and Standard Deviations Measured in Fry Test Chambers
During the Life Cycle Tests
16 The Mean Dissolved Oxygen Concentrations (mg/1) Measured 69
in Storage Tanks and Test Chambers During the Life Cycle
Tests
17 The Mean Water Chemistry Values Measured in Head Tanks 70
18 The Mean Water Chemistry Values Measured in the Highest 71
Effluent Concentration Adult Test Tanks Containing Live
Fish
19 Mean Water Temperatures (°C) Measured in Storage Tanks and 72
Adult Test Chambers During the Life Cycle Studies
20 Number of First Generation £. promelas Surviving in Non- 74
disinfected Effluent
21 Number of Second Generation £. promelas Surviving in 75
Nondisinfected Effluent
22 Number of First Generation J?. promelas Surviving in 77
Chlorinated Effluent
23 Number of Second Generation £. promelas Surviving in 78
Chlorinated Effluent
24 Number of First Generation JP. promelas Surviving in 80
Dechlorinated Effluent
25 Number of Second Generation ]?. promelas Surviving in 81
the Dechlorinated Effluent
26 Number of First Generation _P. promelas Surviving in 82
Chlorobrotninated Effluent
27 Number of Second Generation £. promelas Surviving in 84
the Chlorobrominated Effluent
viii
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TABLES (continued)
No. Page
28 Number of First Generation £. promelas Surviving in 85
Ozonated Effluent
29 Number of Second Generation P_. promelas Surviving in 86
the Ozonated Effluent
30 Mean Lengths (In mm) of First and Second Generation 89
P_. promelas Reared in Nondisinfected Effluent and in
Dilution Water
31 Mean Lengths (In mm) of First and Second Generation 91
—' Pamelas Reared in Chlorinated Effluent and in
Dilution Water
32 Mean Lengths (In mm) of First and Second Generation 92
P_. promelas Reared in Dechlorinated Effluent and in
Dilution Water
33 Mean Lengths (In mm) of First and Second Generation 93
P_. promelas Reared in Chlorobrominated Effluent and
in Dilution Water
34 Mean Lengths (In mm) of First and Second Generation 94
P_. promelas Reared in Ozonated Effluent and in
Dilution Water
35 Mean Lengths (In mm) of First Generation 7_. promelas 96
At Day 23 of the Life Cycle Test
36 Mean Lengths (In mm) of First Generation ]?. promelas 97
At Day 53 of the Life Cycle Test
37 Mean Lengths (In mm) of First Generation P_. promelas 98
At the Termination (Day 330) of the Life Cycle Test
38 Mean Lengths (In mm) of 30 Day Old Second Generation 99
P_. promelas in the Life Cycle Test
39 Mean Lengths (In mm) of 60 Day Old Second Generation 100
P_. promelas in the Life Cycle Test
40 Mean Weights (In grams) of First Generation _P. promelas 101
at the Termination (330 Days) of the Life Cycle Test
ix
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TABLES (continued)
No. Page
41 Mean Number of Viable Eggs Produced per Female and the 103
Mean Disinfectant Residual (mg/1) in Each Concentration
of Each Effluent Stream
42 Mean Number of Eggs per Spawning in the Various Concen- 105
trations of Each Effluent Stream
43 Percent Hatchability, Mean Disinfectant Residual (mg/1), 107
and Incubation Attempts in the Various Effluent Streams
44 Percent Hatchability of Eggs Incubated in Water Different 108
From That in which They Were Spawned
45 Results of Acute Toxicity Tests with Chlorinated Effluent 116
46 Results of Acute Toxicity Tests with Dechlorinated Effluent 119
47 Results of Acute Toxicity Tests with Chlorobrominated 122
Effluent
48 Results of Acute Toxicity Tests with Ozonated Effluent 125
49 Fathead Minnow Acclimation Test in Chlorinated Effluent 128
50 Lake Trout Fingerling Acclimation Tests in Chlorobrominated 130
Effluent
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ACKNOWLEDGEMENTS
We are indebted to numerous persons from the cities of Grandville and Wyoming,
Michigan, and from The Grand Valley State Colleges, for their assistance on
this project. The cooperation and assistance of Mr, Glenn Folk, Superinten-
dent of the Grandville Wastewater Treatment Plant, and all of his staff, was
greatly appreciated. Drs. Gary Griffiths and John Quiring, College of Arts
and Sciences, The Grand Valley State Colleges, provided assistance in the
processing and analysis of data. Dr. Stephen Clark, College of Arts and
Sciences, The Grand Valley State Colleges, participated in the early stages of
the project. Mr. William Stonebrook, Superintendent of the Wyoming Wastewater
Treatment Plant, and Ms. Grace Raven, Secretary, assisted the project in many
ways.
Mr. Paul Spelman was instrumental in the design and construction of the water
treatment facilities, and served as budget officer and on-site director of
the project.
Dr. William Brungs, Mr. Robert Andrew, and Mr. John Arthur, all of the Nation-
al Water Quality Laboratory in Duluth, Minnesota, and Drs. Quint Pickering and
Tim Neiheisel of the Newtown Fish Toxicology Laboratory, provided assistance
and advice for the bioassay portion of this project. We also acknowledge the
assistance of Dr. Jack Mills and Mr. Sidney Jackson of the Dow Chemical Com-
pany, Drs. Allen Filbey and Michael McCuen of Ethyl Corporation, and Dr.
Harvey Rosen and other employees of W. R. Grace and Company. The personnel
of several Federal and State of Michigan fish hatcheries were generous in
providing test animals for acute toxicity studies.
The assistance of our laboratory personnel, Mr. Terry Cruzan, Mr. Dale DeKaaker,
Mr. Irwin Jousma, Mr. Richard Lincoln, Ms. Patricia Matthews, Mr. Michael
Stifler, and Ms. Bonnie White, is gratefully acknowledged.
The suggestions, assistance and critical evaluations provided by Messrs. Cecil
W. Chambers and Albert D. Venosa, EPA Project Officers, were invaluable to the
completion of this project.
This report was submitted in fulfillment of Grant Number S802292 by the City
of Wyoming, Michigan, under the partial sponsorship of the Environmental
Protection Agency.
xi
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SECTION I
INTRODUCTION
AN OVERVIEW OF THE PROBLEM
Over the past seventy years chlorination has evolved as the commonly used
method of disinfecting water supplies, wastewater, and industrial wastes in
the United States. This extensive use of chlorine has been fostered by its
powerful disinfection capabilities, availability, ease of application, and
relatively low cost.
Even though the toxicity of chlorine has long been recognized, it is only
recently that much attention has been given to the possible toxic side effects
of chlorination. The principal exceptions to this were the hobbyists and
aquaculturists, who quickly recognized the toxicity of chlorinated water
supplies to aquatic organisms and tried to solve their toxicity problems as
early as 1930.1»2 However, over the past two decades, and especially since
Rachel Carson published her book, Silent Spring.3 increasing attention has
been given to the environmental impact of chlorinated compounds. The various
chlorinated hydrocarbon pesticides, such as DDT, elicited much of the early
concern, but the chlorine compounds found in chlorinated wastewater also
received attention.
An increasing number of laboratory and field investigations have documented
the toxic effects potentially associated with the chlorination of wastewater.
Principal among these were the studies of Arthur, et al.,5 Arthur and Eaton,
Esvelt, et al.,7 Tsai,8*9 and Zillich.10 The literature on residual chlorine
toxicity to aquatic life was reviewed by Brungs.H
More recently an interest has arisen in the carcinogenic effects of the ^
chlorinated organics which might be formed in chlorinated water supplies.
This concern for public health will undoubtedly lead to a broader under-
standing of the formation and the effects of chlorinated compounds.
One avenue that some investigators have taken to solve the potential problem
of residual toxicity of chlorinated effluent to aquatic life has been de-
chlorination. Thus, Arthur, et al., and Collins and Deener,13 have success-
fully dechlorinated with S02; Esvelt, et al.,7 with bisulfite; and Zillich10
with sodium thiosulfate, with no apparent adverse effects on their test
animals.
A second approach to the residual toxicity problem has been the substitution
of other means of disinfection for chlorination. Ozone has received con-
siderable attention over the past decade as an alternative to chlorine, and
during the past several years, a major effort has been initiated to determine
the feasibility of bromine chloride as an alternative to chlorine.
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Ozone is commonly used In Europe to disinfect water supplies ar
stronger and faster acting oxidizing agent than chlorine.1^> Various
reports have, appeared on the superiority of ozone over chlorine in killing
bacteria and viruses, 16-21 ancj on the ability of ozone to reduce the color,
odor, oxygen demand, and turbidity of wastewater.21-24 while some investi-
gators^' believe that the by—products of the ozonation of wastewater lack
the potential toxicity of the by-products of chlorination, some studies5>26,27
indicate that undesirable biological effects could possibly be associated
with the ozonation of wastewater. This point requires additional investiga-
tion before final conclusions may be drawn.
In addition to our increased knowledge of the biological characteristics of
ozone as a disinfectant of wastewater, technological advances have been made
in generating ozone. These advances have decreased the costs of treating
wastewater with ozone and, at least in some cases, have apparently made dis-
infection with ozone economically competitive with disinfection with chlorine
followed by dechlorinat;ion and reaeration.^ A general review of the chemical
reactivity and characteristics of ozone and its applicability to water and
wastewater treatment may be found in Evans' work.22
The modern-day interest in the disinfection capabilities of bromine began
with the investigations of Wood and Tiling29 and Beckwith and Moser30 during
the 1930's. During the same decade bromine was suggested as an agent f°r32
disinfecting water supplies as well as swimming pools.31 Johnson, et _al.
concluded that bromine had several advantages over chlorine in the disin-
fection of water, including the ability to kill both viruses and spores.
33
Kamlet advocated bromine chloride as a water and wastewater disinfectant
on the basis of its greater disinfection effectiveness and economy when
compared with either bromine or chlorine. More current estimates of the
economic advantages of wastewater disinfection with bromine chloride over
disinfection with either ozone or chlorination-dechlorination-rfeaeration
were presented by Wilson. in addition to the potential economic advantages
of bromine chloride for wastewater disinfection, Mills35,36 concluded that,
compared to chlorination, chlorobromination produces a better kill of viruses
and bacteria and a reduced residual toxicity.
OBJECTIVES OF PROJECT
This project was designed to investigate the problems mentioned above, i.e.,
the undesirable toxicity problem sometimes associated with the chlorination
of wastewater, the bactericidal efficacy of alternative wastewater disin-
fection processes in parallel on identical wastewater streams, as well as
any undesirable toxic effects associated with those alternative processes.
Two contrasting study sites were included in this project, one being the
Grandville, Michigan, Wastewater Treatment Plant, the other being the Wyoming,
Michigan, Wastewater Treatment Plant. The Grandville plant is an activated
sludge facility that treats wastewater derived almost totally from domestic
sources. The Wyoming plant has both activated sludge and trickling filter
treatment facilities, either of which might treat an influent composed of
35-45 percent industrial wastes. The design of this project called for the
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study of wastewater treated by the trickling filter process at the Wyoming
plant.
This report deals only with the Grandville portion of the project described
above. Utilizing a primarily domestic source wastewater receiving secondary
treatment by the activated sludge process, the specific objectives of this
portion of the project were to determine the disinfection efficiency of
chlorine (with and without sulfur dioxide dechlorination), ozone, and bromine
chloride in parallel on identical wastewater streams, and any residual
toxicity associated with these processes.
THE GRANDVILLE STUDY SITE
The Grandville Wastewater Treatment Plant, which receives primarily domestic
wastewater, is an activated sludge plant with chemical removal of phosphates.
The plant has a capacity of 12,000 cu m/d (3.2 mgd) and an average flow of
9800 cu m/d (2.6 mgd). The effluent is chlorinated with a manually controlled
feed system adjusted with the aid of a continuous residual chlorine analyzer
and recorder.
A laboratory building constructed at the plant provided space for the efflu-
ent treatment and toxicity studies. Approximately 2.40 liters/sec (37 gpm)
of chlorinated final effluent, which normally had a chlorine residual of 1.0
to 2.0 ing/1, was pumped from the chlorine contact chamber of the main plant
to the effluent treatment area of the laboratory (Figure 1). Part of this
chlorinated stream was pumped directly to the toxicity testing area, while
part was dechlorinated with sulfur dioxide and then pumped to the toxicity
testing area.
Nondislnfected effluent was pumped from the final settling tanks of the main
plant to the effluent treatment area where it was split into three streams,
one leading directly to the toxicity testing area, one flowing to the ozone
contact system and then to the toxicity testing area, and the third flowing
to the bromine chloride contacting system and then to the toxicity testing
area. Detailed information on the flow rates, dose rates and resulting
residuals, and other characteristics of the wastewater may be found in
Section III, while bacteriological data are discussed In Section IV.
After they were warmed to test temperature, the various wastewater streams
were each piped to a proportional diluter, which also received a supply of
warmed dilution water from a well. Undiluted wastewater and dilution water
and six concentrations of wastewater were then delivered to aquaria in which
test organisms were maintained. Several species of fishes and invertebrates
served as subjects for acute toxicity studies, while life-cycle studies were
conducted on fathead minnows (Pimephales promelas) in each wastewater stream.
A detailed account of the toxicity testing may be found in Sections V and VI.
IMPORTANT DATES
Table 1 shows some of the important dates during the Grandville study and
will serve as a useful reference while reading and interpreting the other
sections of this report.
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SOURCE OF WATER
BIOASSAY LABORATORY
END OF CHLORINE
CONTACT CHAMBER
FROM FINAL
SETTLING TANKS
INONDISINFECTED)
WATER WELL
(20O FT DEEP)
WATER CONDITIONING BUILDING
DECHLORINATION
(10 MIN. CONTACT TIME)
CL ANALYZER
\0.02 ,
CL ANALYZER
385 LITER
OZONATION (10 MIN
385 LITER
X0.02 ,
CONTACT TIME)
CHLOROBROMINATION
(10 MIN. CONTACT
TIME)
385 LITER
385 LITER
\OQ2_.
IRON REMOVAL FILTER
180 LITER
COLD EFFLUENT
HEAD TANK
CHRONIC TABLE
EACH ACUTE TABLE
CHRONIC TABLE
EACH ACUTE TABLE ]
CHRONIC TABLE
EACH ACUTE TABLE
CHRONIC TABLE
EACH ACUTE TABLE
CHRONIC TABLE
EACH ACUTE TABLE
EACH CHRONIC AND
ACUTE TABLE
EACH ACCLIMATION TANK
COLD ACUTE TABLE
EACH COLD ACCLIMATION
TANK
COLD ACUTE
TABLE
g. NUMBERS ON LINE INDICATE THE APPROXIMATE FLOW RATE IN LITERS PER SECOND
b. SOURCE OF EFFLUENT IS ONE OF THE EFFLUENT HEAD TANKS IN THE BIOASSAY LABORATORY.
Figure 1. FLOW OF EFFLUENT AND WELL (DILUTION) WATER
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Table 1. DATES OF SOME IMPORTANT EVENTS DURING THE GRANDVILLE STUDY
Start (End) of disinfection studies
Start (End) of life cycle bioassay studies
Last day of spring flooding (overloading of treatment plant)
Alum substituted for ferric chloride in the removal of
phosphate from the wastewater
Average chlorine feed lowered from 2.90 mg/1 to 2.73 mg/1
(Average chlorine residual reduced from 2.0 to 1.5 mg/1)
Average chlorine feed lowered from 2.73 mg/1 to 2.31 mg/1
(Average chlorine residual reduced from 1.5 to 1.0 mg/1)
Sulfur dioxide feed lowered from 7.0 mg/1 to 4.0 mg/1
(Measured mean residual sulfite reduced from 5.12 mg/1
to 2.88 mg/1)
Measured mean residual BrCl lowered from 3.6 mg/1 to 3.0 mg/1
Measured mean residual BrCl lowered from 3.0 mg/1 to 2.5 mg/1
Measured mean residual BrCl lowered from 2.5 mg/1 to 2.0 mg/1
Start of filtration of wastewater prior to ozonation
January 9
January 8
(November 27)
(December 5)
March 24
June 21
July 8
August 12
April 1
February 22
March 28
July 8
September 26
All dates are during the 1974 calendar year
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1. Hubbs, C. L. The High Toxicity of Nascent Oxygen. Physiol Zool.
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2. Coventry, F. L., V. E. Shelford, and L. F. Miller. The Condition-
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3. Carson, R. Silent Spring. Boston, Houghton Mifflin, 1962.
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16. Fetner, R. H., and R. S. Ingols. A Comparison of the Bactericidal
Activity of Ozone and Chlorine Against Escherichia coli at 1°.
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17. Buffle, J. P. Comparison of Bactericidal Action of Chlorine and Ozone
and Their Use for Disinfection of Water. Tech Sanit Munic. 45:74-82,
1950.
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18. Smith, W. W. and R. E. Bodkin. The Influence of Hydrogen Ion Concen-
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47:445, 1944.
19. Kessel, J. R., D. K. Allison, F. J. Moore, and M. Kaime. Comparison
of Chlorine and Ozone as Virucidal Agents of Poliomyelitis Virus. Proc.
Soc Ex Bio and Med. 53:71-73, 1943.
20. Carazzone, M. M., and G. C. Vanani. Experimental Studies on fht Effect
of Ozone on Viruses. I. Effect on Bacteriophage T. G Batt Virol
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21. Nebel, C., R. D. Gottschllng, R. L. Hutchinson, T. J. McBride, D. M.
Taylor, J. L. Pavoni, M. E. Tittlebaum, H. E. Spencer, and M. Fleisch-
man. Ozone Disinfection of Industrial-Municipal Secondary Effluents.
Jour Water Poll Cont Fed. 45(12):2493-2507, 1973.
22. Evans, F. L. III. Ozone in Water and Wastewater Treatment. Ann Arbor,
Ann Arbor Science Publishers, Inc., 1972. 185pp.
23. Greening, E. Feasibility of Ozone Disinfection of Secondary Effluent.
Chicago. Document No. 74-3. Illinois Inst. for Environmental Quality.
1974. 33pp.
24. Huibers, D. T. A., R. McNabney, and A. Halfron. Ozone Treatment of
Secondary Effluents from Wastewater Treatment Plants. Cincinnati.
Report No. TWRC-4. Robert A. Taft Water Research Center. 1969. 62pp.
25. Harr, Thomas. Residual Chlorine in Wastewater Effluents Resulting
from Disinfection. Albany. Tech Paper 38. New York State Dept of
Env Conservation. 1975. 202pp.
26. Maclean, S. A., A. C. Longwell, and W. J. Blogoslawski. Effects of
Ozone-Treated Seawater on the Spawned, Fertilized, Meiotic, and
Cleaving Eggs of the Commercial American Oyster. Mutation Res.
21:283-285, 1973.
27. Rosenlund, Bruce. Disinfection of Hatchery Water Supply by Ozonation
and the Effects of Ozonated Water on Rainbow Trout. Paper presented
at the International Ozone Institute Workshop on Aquatic Applications
of Ozone. Boston, Mass. 1974.
28. Rosen, H. M. Ozone Generation and its Relationship to the Economical
Application of Ozone in Wastewater Treatment. Ozone in Water and
Wastewater Treatment. Evans, F. L. Ced.). Ann Arbor, Ann Arbor Science
Publishers, 1972. P. 101-122.
29. Wood, D. R., and E. T. Illing. The sterilization of Sea Water by Means
of Chlorine. Analyst. 55:125-126, 1930.
30. Beckwith, T. D., and J. R. Moser. Germicidal Effectiveness of Chlorine,
Bromine, and Iodine. Jour Am Wat Works Assn. 25(3):367-374, 1933.
31. Hildesheim, H. The Bromination of Swimming Pool Water. Technische
Gemeindeblott. 39:36, 1936.
-------
32. Johnson, J. D., and R. Overby. Bromine and Bromamine Disinfection
Chemistry. Jour San Eng Div. Proc Am Soc Civil Eng.
97CSA5):617-628, 1971.
33. Kamlet, Jonas. 1953. U.S. Patent No. 2,662,855.
34. Wilson, H. 0. Costs for Alternate Methods of Wastewater Disinfection.
Presented at the Chlorine Residual Policy Seminar held at the Engineer-
ing Society of Baltimore, Maryland, on November 14, 1974.
35. Mills, J. F. The Disinfection of Sewage by Chlorobromination.
Presented before the Division of Water, Air and Waste Chemistry,
American Chemical Society Meeting. Dallas, Texas. April, 1973.
36. Mills, J. F. The Chemistry of Bromine Chloride in Wastewater Disin-
fection. Presented before the Division of Water, Air and Waste
Chemistry, American Chemical Society Meeting. Chicago, Illinois.
August, 1973.
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SECTION II
CONCLUSIONS
1. With the exception of the residual toxicity imparted to the effluent by
some of the disinfection processes, no change in wastewater quality that
might create an environmental problem under the more common conditions
of effluent release to waterways vas observed as the result of disinfec-
tion with chlorine, bromine chloride, or ozone, or dechlorination with
sulfur dioxide.
2. The frequency of disinfection by chlorine, bromine chloride, and ozone
was directly related to wastewater quality, as indicated by suspended
solids and biochemical oxygen demand.
3. Aftergrowth of microorganisms was considerably more apparent in the
dechlorinated effluent stream than in the chlorinated or chlorobromi-
nated stream. Less prominent aftergrowth was also observed in the
ozonated effluent stream.
4. The respective fecal and total coliform densities (MF) in the chlori-
nated, dechlorinated, and chlorobrominated effluents did not differ
significantly during the first four treatment intervals (January through
September). Coliform densities in chlorobrominated effluent were signif-
icantly higher (p>0.99) than those in chlorinated and dechlorinated
effluents during treatment intervals five and six (October 1 - November 27).
5. Fecal and total coliform densities (MF) in the ozonated effluent were
significantly higher (p>0.99) than those in the other disinfected efflu-
ents (during all intervals) when there was no multimedia pressure fil-
tration. But, when preceded by pressure filtration (October 1 - November
19), ozonated effluent displayed coliform densities which were not
significantly higher than those in the other disinfected effluents.
6. The fecal coliform standard (-£. 200/100 ml) was met more than 80 percent
of the time during all treatment intervals by the chlorinated and de-
chlorinated effluents, during all but one interval (November 19 - Novem-
ber 27) by the chlorobrominated effluent, and during only one interval
(October 1 - November 19, during pressure filtration) by the ozonated
effluent.
7. The total coliform standard (£ 1000/100 ml) was met more than 80 percent
of the time during the second through fifth intervals 0?ehruary 22 -
November 19) by the chlorinated and dechlorinated effluents, during the
second through fourth intervals (February 22 - September 30) by the
chlorobrominated effluent, and during no interval by the ozonated effluent.
-------
8. Examination of the disinfection capability of ozone was often limited by
an inadequate dosage resulting from design limitations, mechanical fail-
ures, and operator inexperience.
9. Since 100 percent nondisinfected effluent was lethal to fathead minnows
(Pimephales promelas) less than 60 days old, no conclusions could be
drawn on the toxicity of the 100 percent disinfected effluents.
10. Fourteen and twenty percent chlorinated effluent concentrations with mean
total chlorine residuals of 0.045 mg/1 or more caused growth retardation
and mortality of continuously exposed fathead minnows less than 60 days
old. The maximum mean residual chlorine concentrations which failed to
show such effects varied from 0.01 to 0.03 mg/1, depending upon the
quality of the effluent.
11. Dechlorination with sulfur dioxide eliminated the lethal and growth
inhibiting effects on fathead minnows reared in 14 and 20 percent chlori-
nated effluent concentrations. No effects were observed on the growth,
reproduction, or survival of fathead minnows continuously exposed to
dechlorinated effluent concentrations of 50 percent or less.
12. Continuous exposure to chlorobrominated effluent concentrations of 50 per-
cent or less containing mean residual bromine chloride levels of 0.043
mg/1 or less had no effect on the growth, reproduction, or survival of
fathead minnows.
13. No effects were observed on the survival or reproduction of fathead
minnows continuously exposed to ozonated effluent concentrations of 50
percent or less with mean residual ozone levels of 0.005 mg/1 or less.
Fathead minnows continuously exposed to 100 percent ozonated effluent
with a maximum mean ozone residual of 0.016 mg/1 exhibited greater mean
lengths at 30, 60, and 330 days of age than their counterparts in 100 per-
cent concentrations of nondisinfected, dechlorinated or chlorobrominated
effluent.
14. None of the disinfected effluent streams had any effect on the number of
eggs produced by those fathead minnows which survived to maturity or on
the probability of those eggs hatching.
15. Acute toxicity tests conducted on several species of fishes and the macro-
invertebrate Daphnia magna indicated that chlorine was the most toxic
disinfectant tested.
16. Fathead minnows exposed to gradually increased residual chlorine and
bromine chloride concentrations tolerated higher halogen residuals than
fish which lacked prior exposure to the halogens.
10
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SECTION III
THE WASTEWATER TREATMENT SYSTEMS AND
THE CHARACTERISTICS OF THE WASTEWATER STREAMS
INTRODUCTION
A variety of interactions and effects have been shown to occur when disin-
fectants are applied to wastewater. For instance, the organic load and the
pH of the wastewater often affect the bactericidal activity of a disinfec-
tant. Conversely, the disinfectant might affect wastewater parameters such
as dissolved oxygen, pH, or residual toxicity to aquatic organisms.
This section discusses the characteristics of the Grandville wastewater, and
outlines the treatment processes at the Grandville Wastewater Treatment Plant.
The design of the test treatment systems and their effects on the character-
istics of the wastewater are described.
THE GRANDVILLE WASTEWATER TREATMENT PLANT
The wastewater treatment plant at Grandville, Michigan, is a secondary
activated sludge system with chemical removal of phosphate. It has a
20,000 population equivalent1 of biochemical oxygen demand (BOD) and a
14,000 population equivalent of suspended solids. The wastewater is
primarily of domestic origin, with only three industrial inputs which
contribute an estimated 20 percent of the plant's BOD load.
Expansion of the plant from 6,000 cu m/day (1.6 mgd) to a design capacity
of 12,000 cu m/day (3.2 mgd) was completed just prior to the January 2,
1974 start of this project. Operational and equipment problems at the
enlarged plant resulted in highly variable effluent quality during the
first months of the project. Heavy precipitation and the subsequent
infiltration of Grand River flood waters into the plant's influent lines
occurred during the first two weeks of each of the first five months of
1974 and caused numerous treatment problems. The infiltration of flood
waters was the main cause of an estimated 568,000 cu m (150 mg) excess
annual plant flow over the previous year, and the subsequently lower than
average effluent quality.
The plant influent flow for 1974 averaged 9,600 cu m/day (2.54 mgd) with
daily flows varying from 6,000 to 19,300 cu m/day (1.60-5.09 mgd). The
influent pH ranged from 6.3 to 8.4. Figure 2 shows the average flows for
1974. The high flows occurred in the first five months. The poor removal
of solids, BOD, and phosphates in the first months of 1974 were largely
the result of the high daily plant flows and the numerous operational
problems experienced during this time.
11
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15£00 -
15,000
14,500
14,000
13,500
13,000
12,500
12,000
11,500
-,11,000
^10,600
EIQPOO
9,500
9,000
8,500
epoo
7,500
TJOOO
6,900
6,000
MONTHLY AVERAGE
YEARLY AVERAGE
JAN FEB MAR APR MAY JUN JUL AUG SEPT OCT NOV DEC
1974
Figure 2. MONTHLY AND YEARLY AVERAGE PLANT FLOWS AT THE GRANDVILLE WASTEWATER TREATMENT PLANT
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Sludge removal was also a problem during the first part of the project.
This problem was solved by wasting activated sludge to an aerobic digester
and primary sludge to an anaerobic digester. These sludges were not mixed
together and this type of sludge removal resulted in better effluent quality
and fewer problems with bulking.
Figure 3 shows the monthly average total suspended solids content in the
influent and final effluent for 1974. The average total suspended solids
for the year 1974 was 146 mg/1 for the influent and 17 mg/1 for the effluent.
The daily range of total suspended solids in the influent was 25 to 361 mg/1,
while the effluent range was 1 to 179 mg/1. The average removal of total
suspended solids was 88.0 percent.
The monthly average BOD concentrations, for the influent and effluent
streams (as determined by the Grandville Wastewater Treatment Plant labora-
tory) are given in Figure 4. The daily influent BOD ranged from 23 to .
760 mg/1 with an average of 158 mg/1, while the daily effluent BOD ranged
from 4 to 100 mg/1 with an average of 21 mg/1. The average BOD removal
over the year was 86.8 percent.
The use of ferric chloride and polymer to remove phosphate from the plant's
effluent was begun shortly before this project was started (December, 1973).
After extensive experimentation with application sites and feed rates of
polymer and ferric chloride, and many problems with the ferric chloride
feed system, alum was substituted for ferric chloride on June 21, 1974. In
June, alum and polymer were being split-fed 80 percent to the primary and
20 percent to the final settling basins. Feed rates were adjusted to obtain
alum concentrations of 150 mg/1 and polymer concentrations of 0.5 mg/1.
Beginning in October, both alum and polymer were fed at the end of the aer-
ation tanks, giving a retention time of 5 to 7 minutes before entering the
final settling tanks. Starting in October, the application of alum and
polymer was gradually reduced to achieve concentrations of 80 mg/1 and 0.4
mg/1, respectively.
The problems initially encountered with the phosphorus removal system are
clearly seen in Figure 5, which illustrates the monthly average total phos-
phorus in the influent and effluent streams. It was not until July that
the plant achieved a monthly average phosphorus removal greater than 75
percent. The influent stream averaged 8.1 mg/1 phosphorus with the extremes
being 3.6 and 16.8 mg/1. The effluent averaged 3.4 mg/1 with a range of
0.6 to 11.9 mg/1. The average total phosphorus removal for the entire year
was 57.6 percent.
DESIGN OF TEST WASTEWATER TREATMENT SYSTEMS
Because of the need for chlorine-free makeup water for the dilutions re-
quired in the bioassay laboratory, a water well was drilled at the project
location. Water from the well was passed through an iron removal filter
prior to its delivery to the bioassay laboratory (Figure 1).
After final settling, nondisinfected effluent was pumped to a treatment
13
-------
INFLUENT
YEARLY AV
MONTHLY AVERAGES
YEARLY AVERAGE
EFFLUENT
JAN FEB MAR APR MAY JUN JUL AUG SEPT OCT NOV DEC
1974
Figure 3. MONTHLY AND YEARLY AVERAGE SUSPENDED SOLIDS AT THE GRANDVILLE WASTEWATER TREATMENT PLANT
-------
240
230
220
210
200
190
180
170
160
ISO
. INFLUENT
YEARLY AVERAGE
130
120
_H 0
£5-100
E 90
80
70
60
50
40
30
20
10
0
ONTHLY AVERAGES
YEARLY AVERAGE
JAN FEB MAR
APR MAY JUN
1974
JUL AUG SEPT OCT NOV DEC
Figure 4. MONTHLY AND YEARLY AVERAGE BIOCHEMICAL OXYGEN DEMAND AT THE GRANDVILLE TREATMENT PLANT
-------
lOh
9
8
7
6
_ 5
E 4
3
2
I
0
INFLUENT
\
MONTHLY
AVERAGES
EFFLUENT
YEARLY AVERAGE
YEARLY AVERAGE
I I
J 1
JAN FEB MAR APR MAY JUN JUL AUG SEPT OCT NOV DEC
1974
Figure 5. MONTHLY AND YEARLY AVERAGE PHOSPHORUS CONCENTRATIONS AT THE GRANDVILLE TREATMENT PLANT
-------
building and divided into three streams; one was treated with bromine
chloride, one with ozone, and one was passed directly to the bioassay
laboratory (Figure l). Chlorinated effluent was pumped from the end of
the chlorine contact chamber of the main plant to the treatment building,
where a portion of this stream was dechlorinated with sulfur dioxide and
the remaining portion was carried directly into the bioassay laboratory.
The plant chlorinating system consisted of two Fisher-Porter chlorinators.
A Fisher-Porter Anachlor continuous titrator was installed in the line
between the end of the chlorine contact chamber of the main plant and the
bioassay laboratory. This unit measured the total chlorine residual and
provided information for the manual adjustment of the chlorine feed rate
in order to hold a constant chlorine residual. (Normally, adjustments of
the feed rate were made at intervals of 1-2 hours and the total chlorine
residual was maintained within ±0.3 mg/1.) The main problem with control-
ling the chlorine residual was that the time between the application of
chlorine and the measurement of the residual varied between 20 and 60
minutes, depending upon the plant flow.
The 30 minute residual chlorine concentration was maintained at 2.0 mg/1
from the beginning of the study until July 8th. During this period, the
chlorine feed averaged 2.90 mg/1 (24 Ib/mil gal). In order to achieve
minimum residual chlorine levels and still obtain adequate disinfection
(less than 1000 total coliforms per 100 ml and less than 200 fecal coliforms
per 100 ml), the residual concentration was lowered to 1.5 mg/1 and was
held there until August 12th. The chlorine feed during this period averaged
2.73 mg/1 (23 Ib/mil gal). Since the 1.5 mg/1 residual chlorine concen-
tration appeared to be more than adequate for disinfection, the residual
was lowered to 1.0 mg/1 on August 12th and was held there throughout the
remainder of the project. The feed rate of chlorine while the residual
was being held at 1.0 mg/1 averaged 2.31 mg/1 (19 Ib/mil gal).
A portion of the chlorinated stream (2.2 I/sec (35 gpm)) was treated with
sulfur dioxide (802). The S02 was fed into the chlorinated stream by an
aspirator and regulated by a Wallace and Tiernan Model 20-055 chlorinator.
The only problem associated with this system was the occasional inter-
ruption in liquid flow through the" aspirator as a result of high solids
levels in the wastewater. The dechlorinated stream flowed into a contact
tank having a 30-minute residence time at a flow rate of 2.2 I/sec (35 gpm).
The dimensions of the steel contact tank measured 3.66 m long by 1.22 m wide
by 0.91 m deep. Steel baffles were welded to the bottom at intervals of
1.22 and 2.44 m from the end, and wooden baffles were inserted from the top
at 0.61, 1.83, and 3.0 m from the end. This arrangement of baffles provided
an under-over-under flow configuration. The contact tank was constructed
with three outlets so that effluent could be pumped to the bioassay labora-
tory after 10-, 20-, or 30-minute contact tiaes.
Although S02 reacts with chlorine in a 1:1 ratio, the initial feed rate of
S02 was set at 7 mg/1 (58 Ib/mil gal) to protect the subjects of the bio-
assay tests from accidental exposure to residual chlorine. This application
rate was found to be higher than necessary, and was reduced on April 1, 1974
to 4 mg/1 (33 Ib/mil gal).
17
-------
The mean sulfite residual after 30 minutes of contact was 5.12 mg/1 when
the feed rate was 7.0 mg/1, and 2.88 mg/1 when the feed rate was 4.0 mg/1.
For the entire project the mean sulfite residual was 3.0 mg/1 (105 total
analyses).
To insure that no chlorine residual carried over to the bioassay laboratory,
another Fisher—Porter anachlor unit continuously monitored the dechlorinated
stream. This unit was designed so that the presence of any residual chlorine
tripped a switch which caused a signal to be sent to the control panel of the
bioassay laboratory. Upon receipt of this signal, the control panel stopped
the flow of treated effluent to the fish tanks and simultaneously triggered a
bell and light alarm system, thus insuring that fish in the dechlorinated
stream were not exposed to residual chlorine.
The components of the bromine chloride (BrCl) dosing system were similar to
the dechlorinated system except that the BrCl was vaporized and then injected
into the effluent stream. This was accomplished by use of a dip pipe whereby
the liquid was removed under its own pressure (2038 newtons/m2 ). The liquid
BrCl was then vaporized by heat and metered by a Wallace and Tiernan Model
20-055 chlorinator. It was necessary to heat the feeder and the piping to
the aspirator in order to keep the BrCl in a gaseous state.
Unlike the chlorination system which was regulated by residual control, the
chlorobromination system was regulated by dosage control. Originally the
BrCl feed rate was set at 3.6 mg/1. Because the immediately preceding BrCl
feed rates were determined to be greater than required for disinfection,
the dosage was lowered to 3.0 mg/1 on February 22, to 2.5 mg/1 on March 28,
and to 2.0 mg/1 on July 8, where it remained until the completion of the
project.
The problems encountered in keeping the BrCl feeder operating included
plugging of feeder and feed lines by the condensation of BrCl and by con-
taminating materials presumably originating in the BrCl tanks. As with
the SC>2 system, the aspirator tended to plug when the wastewater contained
high levels of solids.
The ozonating system consisted of an Ingersoil-Rand Model ESV-NL compressor,
a Pall-Trinity Model 35 HA1 dryer, a W. R. Grace Model LG-16 ozonator, a
W. R. Grace contactor, and a contact chamber identical to those used for the
S02 and BrCl systems. The compressor produced approximately 28.4 I/sec
(18 scfm) of air, which was dried to a -50°C dew point, and then passed on
to the ozone generator. The ozone-air mixture was introduced along with the
wastewater at the top of a 3.66 m column through a positive pressure injector.
The gas/liquid mixture flowed cocurrently from the injector down a 10.2 cm
diameter central pipe. The mixture reversed direction at the base of the
central pipe and flowed upwards through a concentric 30.5 cm diameter circu-
lar tank, which was open at the top and allowed the water to fall into a steel
contact chamber. Detention time was 40 seconds in the vertical contactor and
10 minutes in the steel contact chamber. Most of the ozone contacting appear-
ed to occur in the vertical contactor. A limited amount of analytical data
suggested that the ozone contacting system was very inefficient, and that most
of the ozone that was introduced was lost in the off-gasses from the contactor.
18
-------
Because of the limited success in disinfecting with ozone during the early
stages of the project, a Baker Model HRC-330D Hi-Rate filter system was
installed to filter the wastewater prior to ozonation. The support media
consisted of 64 mm pea gravel and 1.4 mm garnet, and the working media of
0.3 mm garnet and 0.6 mm anthracite. This filter was in operation from
September 26 until the completion of the project.
Ozone was applied at rates of 2.5 to 8.5 mg/1, calculated on the basis of the
amount of ozone in the gas stream and the wastewater flow to the contactor.
The variation in application rates was the result of the problems experienced
with the ozone system. Some of the problems were largely due to inexperience
with the system, while others were of a mechanical nature such as malfunction
of the compressor, ozone generator, and drier system.
In general, the performance of the chlorination and dechlorination systems
exceeded that of the ozonation and chlorobromination systems because the
former were less complex, simpler to operate, and better understood by the
operating personnel.
The average detention times in the various systems prior to sampling for
chemical analyses are shown in Figure 6. Point A represents the location of
the nondisinfected effluent pumps. Identical samples were taken from each
stream for the period January 9 to November 26, 1974, at a frequency of once
per day, five days per week, usually between the peak flow hours of 8:00 A.M.
and 12:00 noon. Thus, the chemical samples did not represent the same ef-
fluent due to the different detention times in the various systems. The
detention times given for the chlorinated and dechlorinated streams are the
average values over the entire project. The contact time for the chlorinated
stream varied from 20 minutes to more than 60 minutes, depending on the plant
flow. However, even though the different detention times resulted in the
sampling of potentially different test streams, this variable was apparently
of little significance to the final results, since the characteristics of
each treated stream over the entire study period were similar.
REACTIONS OF DISINFECTANTS
The extensive use of chlorine as a disinfectant has resulted in a thorough
understanding of the chemistry of chlorine in water.^ Elemental chlorine
hydrolyzes in water to form hypochlorous acid (equation 1). The hypochlorus
acid is a weak acid and it dissociates according to equation 2.
C12 -f H20 c > HOC1 + H+ + Cl~ (1)
HOC1 < >H+ + OC1~ (2)
Thus, free available chlorine is present as hypochlorous acid (HOC1), hypo-
chlorite ion (OC1~), and elemental chlorine (C^). The relative abundance
of these three species is temperature and pH dependent. The equilibrium
reaction of equation 1 lies far to the right at neutral pH, so that the pre-
dominant species at that pH are HOC1 and OC1~. HOC1 is a much more effective
disinfectant than OC1~. The ratio of^HOCl and OC1~ in aqueous solution is
19
-------
HOLDING
CHAMBER
40min.« I2,
FINAL
SETTLING
TANKS
AVERAGE DETENTION
TIMES FROM POINT A.
CHLORINATED
HOLDING
CHAMBER
30min ^2.2 IpS
NON -DISINFECTED
HOLDING
CHAMBER
30/ntn 6.6 Ips
47MINUTES
77 MINUTES
-JX\ » 1 MINUTE
3OMINUTES
1OMINUTES
INDICATES SAMPLING POINTS
FOR CHEMICAL ANALYSIS
Figure 6. FLOW OF THE VARIOUS WASTEWATER STREAMS IN THE GRANDVILLE DISINFECTION STUDY
-------
inversely proportional to pH. Thus, at 20 C the ratio of HOC1 to OC1~ at
pH 6, 7, 8, and 9 are approximately 32, 4, 0.39, and 0.04, respectively.
Ammonia is present to a significant degree in most wastewater and is of prime
importance in wastewater treatment plants using halogenation for disinfection.
The addition of ammonia to water results in the formation of ammonium and
hydroxide ions (equation 3).
NH, + H00 < > NH,+ + OH~ (3)
32 4
At pH 4.5-8.5 and 20°C, chlorine reacts with ammonia in wastewater to produce
monochloramine (NH^Cl) and dichloramine (NHC12) as in equations 4 and 5.
HOC1 + NH3 ). H20 + NH2C1 (4)
HOC1 + NH2C1 > H20 + NHC12 (5)
The ratio of monochloramine to dichloramine increases directly with pH. Only
dichloramine exists at pH 4.5, while only monochloramine exists above pH 8.5.
When the pH is less than 4.4, trichloramine (nitrogen trichloride or NC1-)
predominates (equation 6).
HOC1 + NHC12 > H20 + NC13 (6)
Complex organic chloramines may also be formed upon chlorination of waste-
water containing reactive organic amines.
The chloramines are considerably less microbiocidal than free chlorine.
Although discussion continues on the relative residual toxicity of "free"
and "combined" chlorine to aquatic organisms, the residual toxicity of both
forms has been conclusively demonstrated.^
Bromine chloride (BrCl) exists in equilibrium with bromine and chlorine in
both the gas and liquid phase (equation 7).
2BrCl ,, > Br2 + C12 (7)
In the vapor phase, BrCl is about 40 percent dissociated over a wide range of
temperature.5 The addition of BrCl vapor to water results in equilibrium
solutions represented by the following equations:
BrCl + H20 > HOBr + HC1 (8)
Br2 + HO ;, ) HOBr + HBr (9)
C12 + H20 •<• * HOC1 + HC1 (10)
BrCl hydrolizes exclusively to hypobromous acid (equation 8). Any HBr
formed by dissociation of elemental bromine would be quickly oxidized by
HOC1 to HOBr.5
HBr + HOC1 $ HOBr + HC1 (11)
21
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Hypobromous acid also dissociates to give hydrogen and hypobromite ions
(equation 12) .
HOBr ^ > H+ + OBr~ (12)
Because the hypohalous acids are more active disinfectants than the hypo-
halite ions, it is of interest to compare the equilibria of chlorine and
bromine chloride in wastewater. At pH 8.0, only 19 percent of the chlorine
exists as hypochlorous acid, while 90 percent of the dissociated bromine
chloride is hypobromous acid. 6 Bromine chloride combines with the ammonia
in wastewater to form bromamines (equations 13-15) . At the usual pH range
of typical wastewater effluent, mono- and di-bromamines are predominant.
NH3 + HOBr _ ^ NH2Br + H20 (13)
NH2Br + HOBr - > NHBr + H0 (14)
NHBr2 + HOBr
Mills reported that bromamines are unstable in wastewater and exhibit a half
life of less than 10 minutes in secondary wastewater effluent. Furthermore,
both the bactericidal and virucidal activity of bromamines have been reported
to be superior to those of chloramines in situations where the halogen demand
is low and the pH is high. Mills** also reported that wastewater disinfected
with bromine chloride exhibited a lower residual toxicity to aquatic organisms
than wastewater disinfected with chlorine.
Sulfur dioxide (S09) dissociates in aqueous solution in the following manner:
(16)
(17)
(18)
Equilibrium of the above reactions favors formation of sulfite (equation 18)
at pH >5, whereas at pH^5, bisulfite predominates (equation 17).
SO., reacts with hypochlorous acid or chloramines as follows:
S02 + HOC1 + H20 - > H2S04 + HC1 (19)
S02 + NH Cl + 2H20 - > NH^Cl + H SO, (20)
2S02 + NHC12 + 4H20 . - > NH^l + 2H2SO + HC1 (21)
3S02 + NC13 + 6H20 , - > NH4C1 + 3H2S04 + 2HC1 (22)
If the concentration of S02 exceeds the stoichiometric amount described in
equations 19 to 22, the resulting excess sulfite (equation 18) will react
with dissolved oxygen (DO) to form sulfate ion, thereby lowering the DO
content of the effluent by the amount of the excess S02.
22
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Ozone Is one of the most powerful oxidizing agents known. Its high oxida-
dion-reduction potential is thought to be due to the formation of the highly
reactive free radical 0' (equation 23).^
0 -- > 0 + 0- (23)
Recent work indicates that reactions of ozone are more dependent on the con-
centration of decomposition products than on the ozone concentration. 9,10
Hewes and Davison-*-0 have proposed that the free radicals and ions formed by
ozone decomposition are the chief reacting species. Their proposed mechanism
for aqueous decomposition of ozone is as follows:
03 + H20 - > H03+ 4- OH~ (24)
H03 + OH~ -- > 2H02 (25)
0 + H0 . - :> HO + 20 (26)
Venosa reports that the same free radicals are produced by irradiation of
the water, and that HO and H02 radicals contribute significantly to the
killing of bacteria by irradiation.
The reaction of ozone with ammonia is first-order with respect to ammonia
concentration, and the rate increases with increasing pH over the range 7-9,
and with increasing ozone partial pressure. In wastewater, the reaction is
only effective if the pH of the wastewater is maintained alkaline. Ozone
is a powerful oxidizing agent, and thus its rapid reactions with most compounds
in wastewater have limited investigations in this field.
There are many problems involved in identifying the composition of wastewater
and the products formed by chemical disinfectants. However, concern about
the environmental effects of reaction by-products formed by chemical disin-
fection is increasing. Studies on the chemical interactions of disinfectants
with organic compounds and wastewater components have been carried out by
Sawyer and McCarty, Mills, Hewes and Davison, ^ Bailey, ^ and others.
MATERIALS AND METHODS
Chemical tests performed during the project were those routinely used to
characterize wastewater. The analyses performed were: total and volatile
suspended solids, turbidity, color (apparent and true), pH, ammonia-nitrogen,
total phosphorus, dissolved oxygen, and chemical oxygen demand CCOD) . Bio-
chemical oxygen demand (BOD) was not run due to the uncertain nature of this
analysis on disinfected effluents even when neutralizing agents are used, and
the error introduced by the seed correction.
Suspended solids were extracted by passing water samples through Gooch
crucibles with glass fiber filters. ^ Color, determined by the platinum
cobalt method, and turbidity were measured on a Hach-DR colorimeter.
Phosphate was measured using the persulfate digestion-stannous chloride
method. Ammonia nitrogen was determined by direct Nesslerization after
clarification with zinc sulfate and alkali. ^ The colors developed in the
phosphate and ammonia tests were read on a Model 300 Turner spectrophoto-
23
-------
meter. Because of the rather high quality of the activated sludge effluent,
the COD test was modified by using 0.0625N dichromate solution instead of
the 0.25N solution prescribed in Standard Methods.1 Dissolved oxygen was
measured by use of a membrane probe (Yellow Springs Instrument).
The residual ozone in the ozonated effluent was measured by the iodometric
method.1 The residual chlorine and the residual bromine chloride in the
respective chlorinated and chlorobrominated effluent streams were determined
with a spectrophotoiodometric method of analysis which has been shown to
measure residual chlorine with approximately the same accuracy as the ampero-
metric titration method.14 Residual sulfite in the dechlorinated effluent
stream was measured by an amperometric titration method in which sensitivity
was increased through the use of a polarograph and a strip chart recorder
for end point determination.15
Statistical differences among respective mean test results in the various
wastewater streams were determined by subjecting the data to a two-tailed
t-test (P<0.05).
RESULTS AND DISCUSSION
Table 2 summarizes the physical-chemical characteristics of the test streams
during the test period. The unfiltered values represent samples taken from
January through September and two weeks in November. The filtered values
represent samples taken after filtration and before ozonation.
In all of the treated streams, the mean suspended solids levels were signif-
icantly (P<0.05) lower than the levels in the nondisinfected stream. Sedi-
mentation in the contact chambers accounted for a large part of the observed
decrease. Because the systems did not all have the same size contact chambers
and flow rates, the settling time for the different effluent streams varied,
thus making it difficult to determine if any system removed solids better
than another. Suspended solids reduction by an ozone-induced flotation pro-
cess has been reported by Nebel jit al. ,!*> and Greening. 17 However, Snider
and Porter9 reported no significant decrease in total or volatile solids when
the flotation process was not used, as was the case in this project.
All of the treated wastewater streams exhibited significantly lower turbidity
than was observed in the nondisinfected stream. At least part of this re-
duced turbidity was attributed to the detention times in the respective
contact chambers of the treated streams.
No significant difference in turbidity was observed among the four treated
test streams. This was of interest, since the settling times resulting from
the special treatment systems ranged from 30 minutes in the dechlorinated
and chlorobrominated systems to 10 minutes in the ozonated system. No data
were collected to determine if the reduced turbidity of the ozonated stream
resulted from the additional 10 minutes of settling time or if the ozone
per se acted to reduce turbidity.
All but the chlorinated stream showed significantly lower mean apparent and
24
-------
Table 2.
PHYSICAL-CHEMICAL CHARACTERISTICS OF THE TEST STREAMS
DURING THE TEST PERIOD - JANUARY 2, 1974 TO NOVEMBER 30, 1974*
Parameter
Total
Suspended Solids
(mg/1)
Volatile
Suspended Solids
(mg/1)
Turbidity
(J.T.U.)
Apparent Color
(Platinum
Cobalt units)
True Color
(Platinum
Cobalt units)
Chemical
Oxygen Demand
(mg/1)
Ammonia
Nitrogen (mg/1)
Dissolved
Oxygen (mg/1)
Total Phosphate
(mg/l)c
Nondis- ,
infected
19.9
(31.0)
14.2
(19.6)
23.4
(43.3)
56.0
(83.6)
11.9
(9.5)
38.3
(18.7)
7.58
(2.66)
2.70
(0.99)
0.63
(0.30)
Chlor-
inated
13.0
(12.1)
9.6
(8.8)
15.2
(10.5)
37.5
(30.8)
10.4
(10.9)
28.7
(14.6)
7.81
(2.60)
5.60
(0.86)
0.64
(0.35)
De chlor-
inated
11.0
(7.2)
8.0
(5.0)
12.6
(7.4)
28.9
(23.0)
8.1
(8.2)
34.1
(17.2)
8.10
(2.27)
4.88
(0.88)
0.63
(0.38)
Chloro-
brominated
11.8
(7.1)
8.8
(5.1)
12.8
(7.9)
29.6
(23.1)
7.6
(8.4)
33.2
(15.5)
7.74
(2.66)
2.82
(1.01)
0.57
(0.34)
Unfiltered Effluent
Nondis- .
infected
20.6
(33.5)
14.7
(21.1)
25.6
(46.7)
58.3
(90.8)
11.3
(9.8)
38.5
(19.6)
6.86
(2.74)
2.76
(1.03)
0.56
(0.34)
Ozonated
12.2
(8.2)
8.9
(5.6)
12.3
(8.0)
24.8
(22.7)
2.5
(4.8)
33.4
(19.4)
6.76
(2.80)
10.16
(0.86)
0.51
(0.42)
Filtered Effluent
Nondis- ,
infected
16.1
(7.9)
11.1
(6.6)
11.2
(5.5)
44.1
(14.4)
15.3
(7.1)
36.9
(13.4)
9.36
(1.34)
2.34
(0.62)
0.72
(0.21)
Ozonated
5.6
(4.3)
4.3
(3.2)
4.8
(3.2)
17.4
(13.4)
6.4
(3.6)
17.4
(6.8)
9.40
(1-38)
9.68
(0.87)
0.33
(0.22)
NJ
Ul
values depicted are means, while numbers in parentheses are standard deviations.
The mean values in the "Nondisinfected" columns vary because they represent different time periods.
The "Nondisinfected" column on the left was derived from data collected during the entire study period.
The second "Nondisinfected" column was derived from samples collected between January 2 and September
26 when no filtration was carried out. The third "Nondisinfected" column was derived from the data
gathered between September 26 and November 30 on the test stream before it was passed through pressure
filters prior to ozonation.
Pleasured only from August through November.
-------
true color levels than the nondisinfected stream, and the mean apparent and
true color levels were significantly lower in the ozonated stream than in
each of the other treated streams. The decolorizing properties of ozone
have been widely reported.9,12,16,17 on several occasions, usually when
effluent Was of exceptionally good quality or when the filters were in
operation, the ozonated effluents, exhibited a dilute permanganate color.
This was reflected in a higher true color when filtering than when not fil-
tering and was due to some unidentified compound in the effluent.
At the dosages and detention times in this project, chlorination signifi-
cantly reduced the chemical oxygen demand (COD) of the effluent, while
ozonation only reduced the COD of the filtered effluent. Dechlorination
and chlorobromination did not result in significantly different COD levels.
Several investigators 16-19 have reported COD reductions with ozone, but
such a reduction was not demonstrated in this project. The reduction of COD
in the filtered and ozonated stream appeared to be due to filtration. The
reason that only chlorination showed a significant reduction in COD is pre-
sumably due to the longer contact time than the other treatments.
None of the treatments significantly affected the ammonia nitrogen levels.
Total phosphorus concentrations were reduced only in the filtered ozonated
stream, presumably because of the physical removal of phosphates bound to
suspended solids.
The mean DO concentration was significantly greater in the chlorinated, de-
chlorinated, and ozonated streams than in the nondisinfected stream. The
ozonated stream, as expected, exhibited the highest level of dissolved oxygen,
because oxygen is the major decomposition product of ozonation. The mean DO
concentration in the dechlorinated stream was significantly higher than that
in the nondisinfected stream, but lower than that in the chlorinated stream.
The reason for the latter observation is that the excess sulfite is oxidized
by oxygen to sulfate.
The pH values of the five wastewater streams are summarized in Table 3.
Chlorination, dechlorination, and chlorobromination did not significantly
affect the pH of the effluent. However, ozonation caused an increase in
the recorded pH values. Nebel, et^ai^.^ and Greening!' have reported
similar increases in pH as a result of ozonation, and have attributed them
to the removal of carbon dioxide from the water.
CONCLUSIONS
Since the study of water quality improvement was not a major objective in
this project, the experimental design was not optimized for the comparison
of the physical and chemical changes of the various wastewater streams in-
duced by disinfection processes. Factors such as flow rates, contact times,
contactor designs, and methods of controlling the feed of the disinfectants
were not uniform in the various test streams. Nevertheless, some conclusions
pertaining to water quality may be drawn from the observations made in this
project.
26
-------
Table 3. A SUMMARY OF THE pH VALUES MEASURED IN THE VARIOUS WASTEWATER STREAMS
No. of samples
Range in pH
% of samples:
pH 7.0
pH 7.0-7.4
pH 7.5-7.9
pH interval with
highest percentage
of samples
Central range of pH
in which approx.
90% of samples fell
Stream
Nond is inf e c t ed
212
6.7-7.8
12.2
73.1
14.6
7.1
6.9-7.5
(90.5%)
Chlorinated
209
6.8-7.8
5.8
80.5
13.9
7.1
7.0-7.5
(90.1%)
Dechlorinated
204
6.5-7.7
7.4
88.7
4.0
7.1
7.0-7.5
(91.2%)
Chlorobrominated
202
6.8-8.0
4.0
87.0
8.5
7.1
7.0-7.5
(92.0%)
Ozonated
200
6.9-8.1
0.5
53.0
46.0
7.4
7.1-7.7
(91.5%)
-------
The application of chlorine, bromine chloride, and ozone for disinfection
and sulfur dioxide for dechlorination did not cause any adverse changes in
the physical and chemical characteristics of the Grandville effluent. The
dechlorination process did significantly lower the mean dissolved oxygen
level observed in the chlorinated stream. However, the magnitude of the
observed decrease in DO suggests that reaeration may not be necessary if
the dechlorination process is adequately controlled. Ozonation caused the
pH to rise in the treated effluent, but not enough to be detrimental.
The improvements in physical and chemical quality of the water that were
demonstrated in this project were a reduction in COD as the result of
chlorination, a reduction in apparent and true color as the result of
ozonation, and an increase in DO as the result of both chlorination and
ozonation.
28
-------
BIBLIOGRAPHY
1. Standard Methods for the Examination of Water and Wastewater. 13th ed.
American Public Health Association, New York, N.Y., 1971 874p.
2. Sawyer, C. N., and P. L. McCarty. Chemistry for Sanitary Engineers,
2nd ed. New York, McGraw-Hill Book Co., 1967.
3. Fair, Gordon M., John C. Geyer, and Daniel A. Okun. Elements of Water
Supply and Wastewater Disposal, 2nd ed. New York, John Wiley and Sons,
1972. 752p.
4. Brungs, W. A. Literature Review of the Effects of Residual Chlorine on
Aquatic Life. Jour Water Poll Cont Fed. 45:2180-2193, 1973.
5. Jackson, S. C. Chlorobromination of Secondary Sewage Effluent. Dow
Chemical Company. (Presented at Workshop on Disinfection of Wastewater
and Its Effect on Aquatic Life, Wyoming, Michigan. October 30-31, 1974.)
19p.
6. Mills, J. F. Disinfection of Sewage by Chlorobromination. Am Chem Soc.
Division of Water, Air and Wastes Chemistry. 13:1 (Presented at 165th
National Meeting, Dallas, Texas. April 8-13, 1973.)
7. Mills, Jack F. The Chemistry of Bromine Chloride in Wastewater Disin-
fection. Amer Chem Soc. Division of Water, Air and Wastes Chemistry.
(Presented at Chicago, Illinois. August, 1973.)
8. Layton, R. F. Analytical Methods for Ozone in Water and Wastewater
Applications. In: Ozone in Water and Wastewater Treatment, Evans, F. L.
(ed.), Ann Arbor, Ann Arbor Science Publishers Inc. 1972. p 15-28.
9. Snider, E. H., and J. J. Porter. Ozone Treatment of Dye Waste. Jour
Water Poll Cont Fed. 46:886-894, 1974.
10. Hewes, C., G., and R. P. Davison. Kinetics of Ozone Decomposition and
Reaction with Organics in Water. Am Inst Chem Engr Jour. 17:141, 1971.
11. Venosa, A. D. Ozone as a Water and Wastewater Disinfectant: A Liter-
ature Review. In: Ozone in Water and Wastewater Treatment, Evans, F. L.
(ed.). Ann Arbor, Ann Arbor Science Publishers Inc., 1972. p 83-100.
12. Singer, P. C., and W. B. Zilli. Ozonation of Ammonia in Wastewater.
Water Res. 9:127-134, 1975.
13. Bailey, P. S. Organic Groupings Reactive Toward Ozone Mechanisms in
Aqueous Media. In: Ozone in Water and Wastewater Treatment, Evans, F.L.
(ed.). Ann Arbor, Ann Arbor Science Publishers Inc., 1972. p 29-59.
14. Mills, J. F. A Spectrophotometric Method for Determining Microquantities
of Various Halogen Species. Draft Report, The Dow Chemical Company,
Midland, Michigan, 1971. 6p.
29
-------
15. Andrew, R. W., and G. E. Glass. Amperometrtc Tltration Methods for
Total Residual Chlorine, Ozone and Sulfite. Draft Report, National
Water Quality Laboratory, Duluth, Minn., 1974.
16. Nebel, C. R., D. Gottschling, R. L. Hutchinson, T. J. McBride, D. M.
Taylor, J. L. Pavoni, M. E. Tittlebaum, H. E. Spencer, and Mr. Fleisch-
man. Ozone Disinfection of Industrial-Municipal Secondary Effluents.
Jour Water Poll Cont Fed. 45:2493-2507, 1973.
17. Greening, E. Feasibility of Ozone Disinfection of Secondary Effluent.
Chicago. Document No. 74-3. Illinois Institute for Environmental
Quality, Project No. 20,028. January 1974. 39p.
18. Kinman, R. N. Ozone in Water Disinfection. In: Ozone in Water and
Wastewater Treatment, Evans F.L. (ed.). Ann Arbor, Ann Arbor Science
Publishers Inc., 1972. p 123-143.
19. Kirk, B. D., R. McNabney, and C. S. Wynn. Pilot Plant Studies of
Tertiary Wastewater Treatment with Ozone. In: Ozone in Water and
Wastewater Treatment, Evans, F. L. (ed.). Ann Arbor, Ann Arbor Science
Publishers Inc., 1972. p 61-82.
30
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SECTION IV
DISINFECTION STUDIES
INTRODUCTION
The role of disinfection in wastewater treatment is to destroy pathogenic
microorganisms in the wastewater and thereby provide a reasonable margin of
safety in controlling the spread of disease in natural waters. Total and
fecal coliform concentrations have been widely used as fecal pollution indi-
cators , and maximum allowable concentrations of these bacteria have been
assigned as public health standards.!
The objective of this part of the project was to evaluate the efficiency of
wastewater disinfection by chlorination (with and without dechlorination),
chlorobromination, and ozonation on parallel wastewater effluent streams.
The experimental site was the Grandville, Michigan activated sludge waste-
water treatment plant. This plant received primarily domestic wastewater
as raw influent and produced a good quality secondary effluent. For a
description of the Grandville study site and a detailed discussion of the
effectiveness and chemical reactivity of the disinfection processes, refer
to Section III.
MATERIALS AND METHODS
The standard membrane filtration (MF) technique was used to enumerate coli-
form bacteria in the five effluent streams. For isolating total coliforms,
samples were filtered through membrane filters (Gelman) having an average
pore size of 0.45 micrometers (pm). The filters were placed on pads satu-
rated with M-Endo broth (Difco), and incubated at 35±0.5°C for 24 hours
before counting. For isolating fecal coliforms, membranes were placed on
absorbent pads saturated with MFC broth (Difco) and incubated for 22 hours
at 44.5±0.2°C.
To check the accuracy of the MF technique, the multiple tube fermentation
method (MPN)^ was performed on every fifth sample in addition to membrane
filtration. A minimum of three sample dilutions with five tubes per dilu-
tion were tested, using Lauryl Tryptose broth (Difco) as the presumptive
medium2 and Brilliant Green Bile broth (Difco) as the confirmatory medium.
Samples from nondisinfected, chlorinated, chlorobrominated, and ozonated
streams were placed in sterile bottles containing sodium thiosulfate.
During the first 14 weeks, the dechlorinated stream was sampled only once
per week. Since coliform densities were found to deviate significantly
from those of the chlorinated stream, the dechlorinated stream was subse-
quently sampled on a daily basis.
31
-------
A number of tests were conducted to determine the best location and time of
day to sample the effluent. It was observed that the highest bacterial
density appeared in the late morning, while the lowest density occurred in
the early morning hours between 2 and 7 A.M. Bacteriological sampling at
specifLed Intervals along each stream was conducted to determine the minimum
detention time sufficient to achieve the desired disinfection efficiency with
a minimum of bacterial aftergrowth.
Based upon findings from the above study, a routine sampling program was
established. Each, weekday morning and periodically in the afternoon, samples
were collected at the. following locations:
(1) Nondisinfected wastewater - immediately prior to entering the
treatment systems.
(2) Chlorinated stream - at a site corresponding to a mean detention
time of 30 minutes (see Section III) in the chlorine contactor.
(3) Dechlorinated stream - at a site in the dosing unit immediately
following the SC»2 injection point, to minimize microbial after-
growth.
(4) Chlorobrominated stream - at the end of the BrCl contact chamber,
corresponding to a mean detention time of 30 minutes.
(5) Ozonated stream - at a site in the holding tank after 10 minutes
contact time.
Data were tabulated and statistically analyzed to determine the pertinent
relationships among the various wastewater characteristics.
The target coliform densities were arbitrarily established at 200 fecal coli-
forms per 100 ml and 1000 total colifonns per 100 ml, the former based on the
Environmental Protection Agency's 1973 Secondary Effluent Standards and the
latter on the onetime State of Michigan standard. Disinfection efficiency
was calculated by dividing the number of bacteriological samples which met
the above standards by the total number of samples taken from each effluent.
The central tendencies of coliform densities were calculated both as arithmetic
and geometric means. The geometric mean was used to minimize the effects of
extremely high and low values in a sampling period and because they were the
basis of the Federal standard at the time the project was conceived. Standard
deviations were calculated as measures of dispersion. Mean differences were
analyzed by t-test, analysis of variance, least significant difference (a
priori) and Tukey's procedure Ca posteriori). Linear regression analyses were
performed to determine the relationship between MF and MPN total coliform den-
sities and between suspended solids and coliform density.
32
-------
RESULTS
Observations of Accumulated Data
In order to determine and compare the relative bactericidal effects of each
disinfectant, coliform data were grouped into time segments during which no
changes in experimental design occurred. A new group was formed each time
a dose rate, residual, or other controllable system parameter was changed.
This resulted in nine groups of data consisting of four different BrCl
dosages (changed February 25, March 28, July 8), three different Cl2 residu-
als (changed July 8, August 12), four concentration ranges of 03 dosage
(changed April 16, August 12, October 28), and addition of a filtration step
prior to ozonation later in the project (i.e., pressure filtration added
October 1, removed again November 19). On one occasion, Cl2 and BrCl were
changed simultaneously (July 8) and, on another occasion, 03 and Cl£ were
changed simultaneously (August 12).
The above data groupings were subjected to analyses of variance followed by
Tukey's procedure to determine whether neighboring changes of a disinfectant
concentration resulted in significant differences in coliform survival. When
no significant difference was found between these neighboring groups, they
were combined into one larger group. This resulted in the final formation
of six data groups by aggregation of data involving two changes in BrCl
dosage (February 25 and March 8), two different concentrations of BrCl dosage
and Cl2 residual (changed July 8), and two changes in ozone application (with-
in the addition of filtration on October 1, a dosage reduction on October 28).
Tables 4 through 9 summarize the disinfectant levels, arithmetic and geometric
means of total and fecal coliform concentrations, and standard deviations of
the arithmetic means for the six intervals. Table 10 presents the frequency
that each disinfectant reduced total and fecal coliform densities to project
standards.
Mean total coliform densities (MF) per 100 ml nondisinfected wastewater
ranged from a high of 1.4 x 10° (4.0 x 10& arithmetic) in the first interval
(Table 4) to a low of 7.2 x 10* (8.5 x 10^ arithmetic) in the last interval
(Table 9). This corresponds in the same intervals to a high fecal coliform ,
density of 1.6 x 105 (6.6 x 105 arithmetic) and a low of 7.9 x 103 (9.9 x 10
arithmetic) organisms per 100 ml. Overall means for the entire test period
were 3.1 x 105 (1.2 x 106 arithmetic) total coliforms per 100 ml and 1.6 x 105
(4.3 x 105 arithmetic) fecal coliforms per 100 ml of nondisinfected wastewater.
Coliform densities in the disinfected streams were highest in the first time
segment (Table 4, when wastewater quality was low due to hydraulic overload)
and lowest in the third interval (Table 6, when biological treatment was more
uniform). Nevertheless, coliform density in the ozonated effluent was lowest
in the fifth interval (Table 8) when multimedia filtration preceded ozonation.
Variance in coliform data was greatest in the ozonated stream, probably due to
frequent breakdowns in the ozone generation equipment.
From the foregoing tables, it is possible to determine the relative ability
of each disinfectant to reduce coliform densities to acceptable levels under
33
-------
Table 4. REDUCTION IN COLIFORM NUMBERS BY CHLORINE, CHLORINE FOLLOWED BT DECHLORINATION, OZONE, AND BROMINE CHLORIDE
DURING JANUARY THROUGH FEBRUARY 22, 1974
Treatment
Nondisinfected
Chlorinated**
Dechlorinated
Chlorobromlnated
Ozonatedc
Total Collfonn Den«ity
(number/100 ml)
Membrane Filtration
No. of
Samples
34
35
13
34
36
Arith.
Mean
4.0xl06
5,900
4,600
1,900
18,000
S.D.
5.53xl06
9,860
5,780
3,790
24,900
Geom.
Mean
1.4xl06
2,000
1,700
1,100
8,900
Multii
No. of
Samples
2
2
2
2
2
)le Dilution Tube
Arith.
Mean
2.4xl05
2,500
3,600
200
5,200
S.D.
3.07xl05
3,430
4,790
42
2,600
Geom.
Mean
l.lxlO5
500
1,300
200
4,800
Fecal Coliform Density
(number/100 ml)
Membrane Filtration
No. of
Samples
33
34
12
33
34
Arith.
Mean
6.6xl05
65
33
210
1,700
S.D.
1.33xl06
86
34
700
2,360
Geom.
Mean
1.6xl05
35
19
57
860
aCl_ residual 2.0 mg/1 after 30 minutes contact
bBrCl dosage 3.6 mg/1
C0- dosage 5-8 mg/1
-------
Table 5. REDUCTION IN COLIFORM NUMBERS BY CHLORINE, CHLORINE FOLLOWED BY DECHLORINATION, OZONE, AND BROMINE CHLORIDE
DURING FEBRUARY 25 THROUGH APRIL 15, 1974
Treatment
Nondisinfected
Chlorinated a
De chlorinated
Chlorobrominated
Q
Ozonated
Total Coliform Density
(number/100 ml)
Membrane Filtration
No. of
Samples
37
33
12
35
30
Arith.
Mean
2.8xl05
480
1,000
47
4,100
S.D.
2.57xl05
971
1,510
64
4,480
Geom.
Mean
l.SxlO5
71
380
22
2,500
Multiple Dilution Tube
No. of
Samples
6
5
5
6
4
Arith.
Mean
3.9xl05
360
540
53
1,800
S.D.
3.09xl05
312
670
62
1,990
Geom.
Mean
1.9xl05
36
280
31
1,100
Fecal Coliform Density
(number /100 ml)
Membrane Filtration
No. of
Samples
32
27
9
32
28
Arith.
Mean
8.5xl04
64
7
8.6
1,200
S.D.
7.3x10*
269
7.8
13.1
1,750
Geom.
Mean
3.6xl04
4.5
3.6
3.8
400
aCl2 residual 2.0 mg/1 after 30 minutes contact
bBrCl dosage 3.0 mg/1, then 2.5 mg/1 (no significant differences between coliform densities at these two dosages)
C03 dosage 2.5 - 4 mg/1
-------
Table 6. REDUCTION IN COLIFORM NUMBERS BY CHLORINE, CHLORINE FOLLOWED BY DECHLORINATION, OZONE, AND BROMINE CHLORIDE
DURING APRIL 16 THROUGH AUGUST 9, 1974
u>
Treatment
Nondisinfected
Chlorinated
De chlorinated
Chlorobrominated
Ozonatedc
Total Collform Density
(number/100 ml)
Membrane Filtration
No. of
Samples
76
75
68
68
75
Arith.
Mean
6.7xl05
52
95
160
1,300
S.D.
l.OAxlO6
60
115
253
1,620
Geom.
Mean
2.0xl05
27
54
56
570
Multiple Dilution Tube
No. of
Samples
14
14
11
15
13
Arith.
Mean
5.6xl05
97
160
490
1,100
S.D.
7.32xl05
141
167
1,370
2,240
Geom.
Mean
2.0xl05
38
99
84
460
Fecal Coliform Density
(number/100 ml)
Membrane Filtration
No. of
Samples
77
72
66
66
74
Arith.
Mean
9.5xl04
3.7
7.5
23
180
S.D.
1.98xl06
7.0
206
33.0
257
Geom.
Mean
2.8xl04
1.9
2.4
8.5
80
Cl_ residual 2.0 mg/1 to July1 8, then 1.5 mg/1 (no significant differences between coliform densities at these two residuals)
BrCl dosage 2.5 ng/1 to July 8, then 2.0 mg/1 (no significant differences between coliform densities at these two dosages)
C03 dosage 5-8 ng/1
-------
Table 7. REDUCTION IN COLIFORM NUMBERS BY CHLORINE, CHLORINE FOLLOWED BY DECHLORINATION, OZONE, AND BROMINE CHLORIDE
DURING AUGUST 12 THROUGH SEPTEMBER 30, 1974
Treatment
Nondisinfected
Chlorinated
De chlorinated
Chlorobromlnated
Ozonated
Total Coliform Density
(number/ 100 ml)
Membrane Filtration
No. of
Samples
33
31
33
34
32
Arith.
Mean
1.2xl06
380
560
630
2,700
S.D.
1.82xl06
433
532
965
2,350
Geom.
Mean
4.2xl05
210
380
220
1,900
Multiple Dilution Tube
No. of
Samples
7
6
6
7
7
Arith.
Mean
S.OxlO5
540
1,000
270
2,500
S.D.
8.53xl05
328
1,260
162
3,000
Geom.
Mean
1.9xl05
410
600
280
1,700
Fecal Coliform Density
(number/100 ml)
Membrane Filtration
No. of
Samples
31
32
32
31
29
Arith.
Mean
2.2xl05
17
15
53
240
S.D.
3.61xl05
20.5
16.7
76.7
250
Geom.
Mean
4.7xl04
8.3
7.5
23
130
3C12 residual 1.0 mg/1
BrCl
2.0 mg/1
0. dosage started at 8 mg/1. Due to mechanical breakdown, dropped to 3 mg/1 August 28
(no significant differences between colif orm densities at these two dosages)
-------
Table 8. REDUCTION IN COLIFORM NUMBERS BY CHLORINE, CHLORINE FOLLOWED BY DECHLORINATION, OZONE, AND BROMINE CHLORIDE
DURING OCTOBER 1, THROUGH NOVEMBER 19, 1974
Treatment
Nondislnfected
Chlorinated3
De chlorinated
Chlorobromlnated
Ozonated0
Total Coliform Density
(number/100 ml)
Membrane Filtration
No. of
Samples
25
26
25
25
20
Arith.
Mean
7.8xl05
760
310
1,700
600
S.D.
9.18xl05
638
1,430
1,140
561
Geom.
Mean
3.4xl05
610
620
620
370
Multiple Dilution Tube
No. of
Samples
5
5
5
3
5
Arith.
Mean
1.6xl05
570
1,100
780
280
S.D.
2.16xl05
577
1,340
724
194
Geom.
Mean
8.5xl04
430
720
570
210
Fecal Coliform Density
(number/100 ml)
Membrane Filtration
No. of
Samples
20
22
21
20
14
Arith.
Mean
1.3xl05
20
26
120
58
S.D.
1.83xl05
17.2
22.6
85.5
67.2
Geom.
Mean
2.9x10*
13
16
82
28
oo
aC!2 residual 1.0 mg/1
bBrCl dosage 2.0 mg/1
CCK dosage 3 mg/1 to October 28, then 6 mg/1 (no significant differences between coliform densities at these two dosages)
Pressure filtration added October 1
-------
Table 9. REDUCTION IN COLIFORM NUMBERS BY CHLORINE, CHLORINE FOLLOWED BY DECHLORINATION, OZONE, AND BROMINE CHLORIDE
DURING NOVEMBER 19 THROUGH NOVEMBER 27, 1974
Treatment
Nondisinfected
Chlorinated
Dechlorlnated
Chlorobrominated
Ozonated
Total Collform Density
(number/ 100 ml)
Membrane Filtration
No. of
Samples
7
7
7
7
5
Arith.
Mean
8.5xl04
760
940
2,300
4,500
S.D.
5.2x10*
333
421
1,300
1,280
Geom.
Mean
7.2xl04
700
880
1,800
4,400
Multiple Dilution Tube
No. of
Samples
1
0
0
0
0
Arith.
Mean
2.2x10*
S.D.
0
Geom.
Mean
2.2xl04
Fecal Coliform Density
(number/100 ml)
Membrane Filtration
No. of
Samples
6
5
6
6
3
Arith.
Mean
9.9xl03
68
71
170
290
S.D.
8.3xl03
48
18
46
55
Geom.
Mean
7.9xl03
55
69
170
280
aC!2 residual 1.0 mg/1
bBrCl dosage 2.0 mg.l
C0 dosage 6 mg/1. Pressure filtration bypassed
-------
Table 10. FREQUENCY3 THAT DAILY SAMPLES OF DISINFECTED EFFLUENTS*5 ACHIEVED PROJECT BACTERIOLOGICAL STANDARDS
JANUARY THROUGH NOVEMBER, 1974
Dates
Jan. - Feb. 22
Feb. 22 - Apr. 15
Apr. 16 - Aug. 9
Aug. 12 - Sept. 30
Oct. 1 - Nov. 19
Nov. 19 - Nov. 27
Total Coliforms
% of Samples Below 1000 per 100 ml
Membrane Filtration
C12
31.4
(35)c
84.8
(33)
100
(75)
93.5
(31)
84.6
(26)
71.4
(7)
so2
46.2
(13)
83.3
(12)
100
(68)
93.9
(33)
84.0
(25)
71.4
(7)
BrCl
26.5
(34)
100
(35)
100
(68)
79.4
(34)
36.0
(25)
14.3
(7)
°3
0
(36)
20.7
(30)
64.5
(75)
21.9
(32)
75
(20)
0
(5)
Multiple Tube Dilution
C12
50
(2)
83.3
(5)
100
(14)
100
(6)
100
(5)
100
(1)
so2
50
(2)
80
(5)
100
(11)
83.3
(6)
100
(5)
(0)
BrCl
100
(2)
100
(6)
93.3
(15)
100
(7)
80
(3)
(0)
°3
0
(2)
50
(4)
75
(13)
14.3
(7)
92.9
(5)
(0)
Fecal Coliforms
% of Samples Below 200 $er 100 ml
Membrane Filtration
C12
93.9
(34)
96.3
(27)
100
(72)
100
(32)
100
(22)
100
(5)
so2
100
(12)
100
(9)
100
(66)
100
(32)
100
(21)
100
(6)
BrCl
90.9
(33)
100
(32)
100
(66)
93.5
(31)
80
(20)
66.7
(6)
°3
0
(34)
29.6
(28)
71.1
(74)
65.5
(29)
92.9
(14)
0
(3)
frequency calculated as number of samples that met coliform standards divided by total number of samples
times 100Z.
For disinfectant concentrations, refer to Tables 4-9.
cNumber of samples in parentheses.
-------
a particular set of operating conditions. A number of statistical tests were
performed to determine significant differences in disinfection efficiency
among the disinfection processes and effective differences among the various
disinfectant concentrations.
A two-way analysis of variance was computed on the total coliform and fecal
coliform data (MF). The two factors under consideration were treatment
effects (Cl2» S0£» BrCl, 0,) and time segment effects. A highly significant
interaction between treatment and time segment was demonstrated. This was
not unexpected and suggested that some of the changes in disinfectant dosage
or residual concentration significantly affected relative fecal and total
coliform densities in the disinfected streams. Consequently, each of the six
time segments was subjected individually to analyses of variance and Least
Significant Difference procedures to determine significant differences among
treatment means.
In the first time interval (January - February 22), mean coliform densities
were higher than in any other interval (Table 4). Poor water quality was
most probably responsible (see below) for these increased bacterial levels.
Both arithmetic and geometric means for all disinfection processes exceeded
the total coliform standard of 1000/100 ml. On the other hand, mean fecal
coliform levels from chlorinated, dechlorinated, and chlorobrominated efflu-
ents met or closely approached the project standard of 200/100 ml. Mean
coliform densities in the ozonated effluent were significantly higher (p?0.99)
than all other streams. Results from all disinfection processes except ozone
were not significantly different from one another.
Table 10 more clearly depicts these findings. All disinfection processes
were relatively ineffective in reducing total coliform levels (MF) to below
1000/100 ml more than 50 percent of the time. Nevertheless, fecal coliform
levels (MF) were reduced more than 90 percent of the time by all treatments
except ozone, which failed to meet either standard at any time during the
interval.
The second time interval (Table 5) was initiated by decreasing the BrCl dose
from 3.6 to 3.0 mg/1, and later to 2.5 mg/1. The geometric mean coliform
densities for chlorinated, dechlorinated, and chlorobrominated effluents were
below 1000 total coliforms and 200 fecal coliforms per 100 ml, but mean coli-
form densities for ozonated effluent were well above the project standards
as well as significantly above (p>0.99) mean fecal and total coliform densi-
ties for the other disinfected effluents.
Disinfection effectiveness (Table 10) was:> 80 percent for total coliforms
and>95 percent for fecal coliforms in the chlorinated, dechlorinated, and
chlorobrominated streams, but only 20 to 30 percent for the ozonated stream.
In the third time segment (Table 6), improvement in ozonation capacity was
accomplished by increasing dosage to a range of 5 to 8 mg/1. Concomitantly,
BrCl and Cl2 dose rates were lowered, but coliform densities were not signifi-
cantly changed. All systems functioned satisfactorily due to a considerable
improvement in wastewater quality (see Section III). Thus, during this time
segment, mean coliform densities were lowest in all effluents except the
-------
ozonated stream. The only other time coliform levels were lower In the
ozonated stream was in the fifth interval (.October 1 - November 19) when
a multimedia pressure filter was installed prior to ozone application.
Chlorine with and without dechlorination and bromine chloride achieved
project standards 100 percent of the time during this third period, while
ozone achieved the standards more than 64 percent of the time (Table 10).
Mean colif orm densities, however, were significantly higher (p>0.99) in
the ozonated stream than in the other disinfected streams.
The fourth interval (Table 7) was initiated by decreasing the chlorine resid-
ual from 1.5 to 1.0 mg/1 (the BrCl dosage remained the same). Although
coliform densities were higher in this interval, chlorine with and without
dechlorination and bromine chloride still achieved project standards satis-
factorily. Chlorine reduced total and fecal coliform densities to desired
levels more than 90 percent of the time. BrCl reduced total coliform densi-
ties to desired levels about 80 percent of the time, and fecal coliform den-
sities more than 90 percent of the time (Table 10). Although fecal coliform
standards were met by ozone almost as frequently as in the previous interval,
total coliform standards were met only about 22 percent of the time. While
chlorination, dechlorination, and chlorobromination produced fecal and total
coliform means which were not significantly different from each other, ozon-
ation still produced significantly higher (p> 0.99) coliform means.
In the fifth time segment, a multimedia filter was installed in front of the
ozonation system in an attempt to achieve better disinfection efficiency by
reducing the suspended solids level in the effluent. The other streams were
not filtered. This treatment was apparently successful, since total and fecal
coliform densities in the ozonated stream were lower than in any other interval
(Table 8). Total coliform standards were met 75 percent of the time and fecal
coliform standards 93 percent of the time (Table 10). Furthermore, mean total
coliform density in the ozonated stream was, for the first time, not signifi-
cantly higher than the levels produced by the other disinfection processes.
Disinfection efficiency in the BrCl system fell sharply in this interval, the
coliform levels being significantly higher (p>0.99) than the other systems.
The pressure filter was bypassed for the final, short interval (Table 9) to
reaffirm its effect on the ozonation process. In this interval, ozone failed
to meet project standards all of the time (Table 10). Coliform densities in
the ozonated effluent again increased to levels significantly higher (p>0.99)
than the other disinfection processes (Table 9). Coliform densities in the
chlorobrominated stream were also significantly higher (p>0.99) than the
chlorinated and dechlorinated effluents. Table 10 illustrates this marked
reduction in disinfection effectiveness of BrCl and also indicates a slight
reduction in disinfection effectiveness of Cl2« These effects were attributed
to a substantial decline in effluent quality due to seasonal changes in temper-
ature.
When the analyses of variance for all disinfectants were calculated using the
MPN data, it was found that ozone was the only disinfection process which
produced data quantitatively higher than the other processes, and this
was only significant (p>0.90) in the fourth interval (August 12-September 30).
42
-------
Because MPN's were performed only on every 5th sample, the above anomaly may
be a consequence of an insufficient number of samples. Thus, caution should
be exercised in forming conclusions from MPN data alone.
Observations of Monthly Data
Total and fecal coliform levels varied widely over the entire span of the
project. In order to analyze the temporal relationships of the various dis-
infection processes and to present them in a more typical fashion, mean coli-
form data are here presented in terms of monthly intervals. Additional in-
formation is presented in Appendices 1A-1E which show samples sizes, standard
deviations, and arithmetic and geometric means.
Figures 7 and 8 show the monthly geometric means of total coliform densities
(MF) and fecal coliform densities (MF), respectively, in all test streams.
All process flows exhibited a major peak in total and fecal coliform levels
early in the project. This was due to excessive hydraulic flows overloading
the final clarifiers, causing poor solids separation and reducing chlorine
residence time (see Section III). These high liquid flow rates in January,
trailing into February, were caused by heavy seasonal rain and snowfall,
flooded river conditions, and heavy infiltration which overloaded the plant's
treatment systems. Suspended solids levels and biochemical oxygen demand
(BOD) were high in both influent and effluent (see Section III, Figs. 3 and 4).
Coliform levels declined in spring and early summer as effluent quality
returned to normal.
Fecal and total coliform densities in nondisinfected wastewater remained
fairly constant for the remainder of the project period until a decline was
seen in the last month. As autumn approached (July-September), coliform
concentrations in the chlorinated, dechlorinated, and chlorobrominated streams
increased without a concomitant rise in suspended solids or BOD (the ozonated
stream was being filtered at this time). The rise in coliform levels was
partly due to a lowering of the halogen feed rates to determine the minimal
effective concentrations necessary to maintain the desired bacteriological
quality. Chlorine residual was decreased from 2.0 to 1.5 mg/1 on July 8, and
then to 1.0 mg/1 on August 12. Bromine chloride dosage was lowered from 2.5
to 2.0 mg/1 on July 8.
Figures 9 and 10 indicate the frequency that samples from each treatment met
project disinfection criteria. These data closely parallel those discussed
above. Disinfection was least effective during the first part of the project.
Then, as effluent quality improved, disinfection efficiency rose sharply.
Finally, at the end of the project, a decline in bacteriological quality was
again observed, partially because minimal concentrations of chlorine and
bromine chloride were employed, and partially because effluent quality decreas-
ed due to seasonal temperature changes.
Disinfection with ozone varied considerably throughout the project. Although
a liquid flow rate of 2.2 Ips C35 gpm) was the original project requirement,
a 6.3 Ips (100 gpm) contactor was provided. The ozone generator was theoret-
ically capable of dosing the wastewater flow to 20 mg/1, but in actuality
43
-------
1000000
CHI .OROBROMINATP 0
OZONATED
CI2 RESIDUAL (mg/l)
JAN. FEB. MAR. APR. MAY JUN. JLY AUG. SEP OCT. NOV.
2.0 | L5 i LQ
3.6 i 3.0 i
2.5
BrCI DOSAGE (mg/l)
03 DOSAGE (mg/l) i 6 » 2.5 I
PRESSURE FILTRATION
I
2.0
1
5-e
ON
Figure 7. MONTHLY GEOMETRIC MEANS OF TOTAL COLIFORM DENSITIES (MF)
-------
100000
10000
IOOO
E
0
0
£
OL
S 100
0
U.
8
2
UJ
CI2 RESIDUAL (mg/l)
BrCI DOSAGE (mg/l)
03 DOSAGE (mg/l)
PRESSURE FILTRATION
JAN. FEB. MAR. APR. MAY JUN. JLY. AUG. SEP OCT. NOV.
2.0 1.5 1.0
3.6 3.0
2.5
2.0
I
8
-2.5 |
5 — 8
OFF
3 i 6
ON (OFF
Figure 8. MONTHLY GEOMETRIC MEANS OF FECAL COLIFORM DENSITIES (MF)
45
-------
--
r
100
JAN.
CI2 RESIDUAL (mg/l) I—
BrCI DOSAGE (mg/l) h-
Oa DOSAGE (mg/l) i
FEB. MAR APR.
2.0
MAY
JUN.
JLY
1.5
AUG.
SEP
1.0
OCT
NOV.
—I
3.6
3.0
2.5
2.0
-*- 2.5
5-8
PRESSURE FILTRATION
OFF
ON
OFF1
Figure 9. PERCENT OF SAMPLES WITH TOTAL COLIFORM DENSITIES (MF) BELOW 1000 PER 100 ml
-------
100
-•
-.
E
O
o
•x
0
o
o
CM
3
LD
CD
10
U-l
a
uo
CI2 RESIDUAL (mg/l)
BrCI 00SAGE (mg/l) f
03 DOSAGE fmg/l) t
PRESSURE FILTRATION
3.6
3.0 .
8
2.5
5-8
NOV.
6
OFF
ON
OFF'
Figure 10. PERCENT OF SAMPLES WITH FECAL COLIFORM DENSITIES (MF) BELOW 200 PER 100 ml
-------
only a maximum of 8 mg/1 was ever achieved. This prevented determination
of the true minimal dosage required to achieve adequate disinfection with-
out filtration.
During the early weeks of the project, chlorine demand was approximately
1.4 mg/1 (see Section III). At this time, chlorine residual was regulated
at 2 mg/1 after about 30 minutes contact, although the detention time varied
with plant flow. In March, chlorine demand declined 50 percent and deten-
tion time increased slightly.
Disinfection with bromine chloride was regulated by dosage control rather
than residual control, because bromine chloride residual in wastewater
declines very rapidly and consequently is difficult to monitor accurately
at low concentrations. A dosage of 2.0 mg/1 was found to be adequate in
meeting project bacteriological standards only occasionally. The mean
30 minute residual at this dosage was calculated to be 0.5 mg/1, but ranged
from 0.1 to 1.5 mg/1.
Coliform levels in the ozonated stream fluctuated widely throughout the
project because of the difficulty in maintaining a constant dosage. This
was a result both of mechanical breakdowns of the ozone generator and in-
experience in handling ozonation equipment. At the beginning of the project
ozone dosage was 8 mg/1, but the gas-liquid contacting was inefficient. To
improve mass transfer, it was necessary to lower the gas flow rate. However,
the gas control valve was damaged by abrasives which had been introduced into
the ozone generator from the compressor and air dryer. A filter was installed
on March 8 to trap the abrasives, but little improvement resulted. It was
then found that excessive moisture was present in the air line, thus limiting
ozone production. New desiccant was placed in the dryer on April 3, and
still no improvement in ozone generation ensued. Upon further examination,
it was found that the dielectric cells had been damaged by the abrasives in
February. New cells were installed on April 16 and an immediate improvement
in ozone dosage and disinfection efficiency occurred.
The above mechanical upsets were not recognized rapidly by the relatively
inexperienced on-site personnel. The gas-to-liquid ratio necessary for
optimum mass transfer in the positive pressure injector contacting system
was about 0.025. But, because at the time there was no good method of
quantifying ozone concentration either in the liquid stream or in the exhaust
gas, the amount of ozone being lost in the exit gas could only be grossly
approximated.
On May 8, 1974, the Air Pollution Control division of the Michigan Department
of Natural Resources estimated ozone loss in the contacting unit by a series
of measurements with an Ecolyzer (Energetic Sciences, Inc.), a continuous
ozone monitor. Ozone concentrations were measured in the inlet and exhaust
gas streams, and from these data mass transfer efficiency was calculated.
Results indicated that 70 to 90 percent of the applied ozone was lost in the
off-gas, based on an assumed flow rate of 850 cfm through, the exhausting
stack (not a sealed system). Thus, mass transfer efficiency was extremely
low.
48
-------
In September, ozone production declined to 3 mg/1 due once again to excessive
moisture in the air and remained at this level until late October. New desi-
cant did not arrive until the second week in October.
Effect of Multimedia Filtration on Disinfection Efficiency of Ozone
Table 11 summarizes the effect of ozonation on filtered wastewater effluent
compared to a similar unfiltered wastewater effluent. Because the system
could not be run in parallel, filtered effluent data (October 1 - November 19)
were compared with unfiltered effluent data from the immediately proceeding
time interval (August 15 - September 17). The suspended solids levels in the
filtered effluent were significantly different (p>0.99) from the suspended
solids levels in the unfiltered effluent. Total coliform densities in the
filtered wastewater were significantly different (p? 0.999) from those in un-
filtered wastewater. It was concluded that filtration enhanced the effective-
ness of disinfection with ozone.
Correlation and linear regression analyses were performed between suspended
solids and total coliform densities (Table 11) before and/or after pressure
filtration. The results indicated no significant correlation before filtra-
tion but a significant (p>0.99) correlation after filtration. Since the
samples were not taken in parallel and did not contain a wide range of sus-
pended solids concentrations, caution should be exercised in forming con-
clusions. Nevertheless, it appears that "large" particles, which are removed
by pressure filtration, may have played an important role in limiting ozone
disinfection effectiveness. Further investigations need to be performed to
confirm this phenomenon.
Comparison of Membrane Filter and Multiple Tube Fermentation Procedures^
MPN total coliform data were compared with MF data statistically by way of
t-test and linear regression procedures. Table 12 gives the results of
those analyses. The MF means were not significantly different from MPN means
(p<0.50). Correlation between the two analytical procedures was linear in
all cases at the p> 0.99 level. The linear model chosen had an intercept
of zero. The results indicated a set of relationships very close to ideal
(slope of 1.0). From these tests, it was concluded that the MF data were
valid estimates of total coliform densities in all streams studied at Grand-
ville.
DISCUSSION AND CONCLUSIONS
All disinfection systems displayed a capability of achieving adequate disin-
fection efficiency when effluent quality was good and sufficient disinfectant
dosages were maintained. As effluent quality declined, the frequency of fail-
ures increased. Chlorine, with and without dechlorination, was least affected
by plant upsets.
State-of-the-art disinfection technology with bromine chloride and ozone was
not as advanced as chlorination technology, and consequently breakdowns were
more pervasive in these two systems. Bromine chloride application was regu-
lated by dosage control rather than residual control, and this was found to be
49
-------
Table 11. EFFECTIVENESS OF OZONE DISINFECTION ON FILTERED AND UNFILTERED EFFLUENT
Unfilterei
Effluent
Filtered
Effluent0
Oo Feed
(mg/1)
4.4
3.8
Total Coliform Density3
(number/ 100 ml)
No. of
Samples
30
20
Arith.
Mean
2700
600
S.D.
2400
560
Geom.
Mean
1800
370
Suspended Solids
(me/1)
No. of
Samples
27
19
Arith.
Mean
10.5
6.3
S.D.
6.9
5.1
Total Coliform Density vs Suspended Solids
No. of
Samples
27
19
Corr.
Coeff .(r)
0.036
0.678d
Slope (Coliforms/
Suspended Solids)
13
75
Intercept
Total Coli-
forms/100 ml
2700
144
Ol
o
Membrane filter determinations
Samples taken August 15 - September 27
cSamples taken October 1 - November 18
Correlation was linear at the p 7- 0.99 level
-------
Table 12. CORRELATION AND REGRESSION OF MPN vs MF TOTAL COLIFORM DENSITIES (NUMBER/100 ml)
JANUARY THROUGH NOVEMBER, 1974
Treatment
Nondlsinfected
Chlorinated
Dechlorinated
Chlorobrominated
Ozonated
Ho. of
Samples
32
30
23
30
29
Arithmetic Mean Coliform Densities
MPNa
3.8xl05
1000
760
230
1700
S.D.
5.3xl05
3160
1560
327
2260
MFa
4.3xl05
1600
1100
260
2200
S.D.
6.5xl05
1560
3490
404
2540
t-Statistic
for Means
0.2543b
0.5593b
0.3886b
0.2090b
0.5371b
Corr.
Coeff. (r)
0.7890°
0.9352C
0.9170°
0.8625°
0.9429°
Slope
(Intercept = 0)
1.02
1.55
1.93
1.08
1.13
aMFN data were independent variables, MF were dependent variables
b0.10£p*: 0.50
Correlation was linear at the p.? 0.99 level
-------
the primary cause of system failure. Another frequent repair item in the
BrCl dosing system was the evaporator. BrCl must be vaporized prior to
injection into the wastewater stream. The evaporator unit tended to accu-
mulate solids with time, thereby blocking flow. If an adequate means of
control by residual can be developed and refined, and if improvements in
the dosing system can be made, bromine chloride may be an effective waste-
water disinfectant.
Mechanical breakdowns in the ozonation system were more frequent and severe
and more difficult to diagnose and correct. Experience gained in this
project suggest that a fairly extensive shakedown period is needed by plant
operating personnel in learning to handle and control ozonation equipment
properly. Dosing of ozone appeared to be quite sensitive to shifts in demand
and changes in flow.
The duration of this project was 11 months. Data were grouped into six main
intervals or time segments, during which experimental conditions were fairly
stable in all systems. .Within these intervals, fecal and total coliform
levels in chlorinated, dechlorinated, and chlorobrominated effluents generally
were not significantly different. Only in the last two intervals were coli-
form densities in chlorobrominated effluent significantly higher than in the
chlorinated and dechlorinated streams.
Fecal and total coliform numbers in the ozonated stream were significantly
higher than in the other disinfected streams in all intervals but the fifth
(when filtration preceded ozonation). It appeared that particles larger
than about 10 um more readily interfered with the disinfection efficiency
of ozone than smaller particles.
Fecal coliform standards were met more frequently in each time interval and
for each treatment than corresponding total coliform standards. At times the
difference in frequency was 60 percent. This suggests that fecal coliforms
are more sensitive to disinfectants than those organisms comprising the
total coliform population. However, this conclusion may be somewhat question-
able, since recovery of fecal coliforms on membrane filters in some cases is
inferior to recoveries determined by the MPN technique.
Conclusions regarding the relative disinfection efficiencies of the various
disinfectants studied, based on the statistical analyses described in detail,
should be viewed from the perspective that dosage control of the various
disinfection systems was vastly different. For example, in order to maintain
a desired chlorine residual, dosages were changed in accordance with changes
in demand. Thus, disinfection efficiency of chlorine was fairly consistent.
However, the rate of addition of bromine chloride and ozone could not be
correlated with demand fluctuations, but rather was held constant at all times
during a specific interval. As a result, sudden changes in demand could easily
have caused concurrent changes in disinfection efficiency by BrCl and ozone,
and these changes would not have been accounted for in the statistical analysis.
52
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BIBLIOGRAPHY
1. Standard Methods for the Examination of Water and Wastewater, 13th ed.
American Public Health Association, New York, N.Y., 1971 874p.
2. The Federal Register, Vol. 38, No. 159. Fri., Aug. 17, 1973. p22298.
Title 40, Chapter I, Subchapter D, Part 133.102.
53
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APPENDIX A-l
COLIFORM DENSITIES OF NONDISINFECTED WASTEWATER
JANUARY THROUGH NOVEMBER, 1974
(number/100 ml)
January
February
March
April
May
June
July
August
September
October
November
Total Coliform Density
Membi;
No. of
Samples
18
Arith.
Mean
6.0xl06
20 ;1.3xl06
20
24
20
19
20
20
19
17
15
2.7xl05
2.7xl05
S.lxlO5
1.2xl06
3.3xl05
l.OxlO5
l.OxlO6
l.lxlO6
l.lxlO5
ane Filtration
S.D.
6.6xl06
2.4xl06
2 . 3xl05
3.8xl05
1.2x10
1.4xl06
4.9xl05
1.6xl06
l.SxlO6
9.8xl05
9 . 5xl04
Geom.
Mean
3.0xl06
4.4xl05
1.7xl05
1.2xl05
2.8xl05
3.3xl05
1.6xl05
4.3xl05
3.5xl05
5.9xl05
8.8xl04
Multi
No. of
Samples
2
4
4
3
4
4
4
4
6
pie Dilution Tube
Arith.
Mean
2.4xl05
3.8xl05
2.3xl05
9.8xl05
6.5xl05
5.0xl05
2.6xl05
6.6xl05
l.SxlO5
S.D.
3.1xl05
4.3xl05
l.SxlO5
1 . 3xl06
7.8xl05
4 . 8xl05
1.7xl05
1.2xl06
2.0xl05
Geom.
Mean
l.lxlO5
1.4xl05
9 . 2xl04
2.6xl05
2. 1x10 5
4.4xl05
2.2xl05
1 . 8xl05
6 . 8x10 4
Fecal Coliform Density
Membrane Filtration
No. of
Samples
17
20
18
23
21
19
20
19
18
15
11
Arith.
Mean
l.SxlO6
2 . 3xl05
5.2xl04
7.7xl04
l.lxlO5
2.0xl05
4.7x10^
2.8xl05
l.lxlO5
1.7xl05
9.5xl03
S.D.
9 . 8xl05
3.0xl05
7.0xl04
9 . IxlO4
1.6xl05
2 . 8xl05
5 . 8xl04
4.0xl05
2.4xl05
2.0xl05
7.3xl03
Geom.
Mean
3.0xl05
6.8xl04
2.4xl04
2.6xl04
4.0xl04
4.1xl04
2.8xl04
8.0x10
2.1xl04
4.8xl04
7.0xl03
-------
APPENDIX A-2
COLIFORM DENSITIES "OF CHLORINATED EFFLUENT
JANUARY THROUGH NOVEMBER, 1974
(number/100 al)
January
February
March
April
May
June
July
August
September
October
November
Total Coliform Density
Membrane Filtration
No. of
Samples
20
19
16
24
22
17
19
20
17
18
15
Arith.
Mean
5000
5700
300
460
27
72
66
95
580
700
830
S.D.
8900
1000
520
1200
41
78
48
69
500
700
410
Geom.
• Mean
2100
950
71
68
15
45
45
62
390
540
750
Multiple Dilution Tube
No. of
Samples
2
3
4
3
3
5
4
3
5
Arith.
Mean
2500
310
230
67
60
160
350
660
570
S.D.
3400
419
260
56
61
220
380
150
580
Ceom.
Mean
490
110
54
53
42
43
230
650
430
Fecal Coliform Density
Membrane Filtration
No. of
Samples
18
20
14
20
20
16
19
21
17
17
10
Arith.
Mean
69
59
5
82
2.2
7.1
4.7
6.2
24
15
54
S.D.
59
96
9.8
310
2.3
13
4.8
9.0
24
15
36
Geom.
Mean
35
22
2
4
2
2
3
3
14
10
46
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APPENDIX A-3
COLIFORM DENSITIES OF DECHLORINATED EFFLUENT
JANUARY THROUGH NOVEMBER, 1974
(number/100 ml)
January
February
March
April
May
June
July
August
September
October
November
Total Collform Density
Membrane Filtration
No. of
Samples
6
8
6
12
21
17
17
20
19
17
15
Arith.
Mean
4100
4400
280
870
54
140
120
210
770
940
850
S.D.
5500
6200
370
1600
57
190
89
210
600
1400
410
Geoin.
Mean
1500
1400
160
120
35
70
97
140
600
600
760
Multiple Dilution Tube
No. of
Samples
2
3
3
3
3
3
4
3
5
Arith.
Mean
3600
220
690
56
140
350
320
1700
1100
S.D.
4800
240
890
12
160
190
320
1600
1300
Geom.
Mean
1300
150
240
56
85
310
230
1200
720
Fecal Coliform Density^
Membrane Filtration
No. of
Samples
5
8
5
10
20
16
17
21
17
16
Arith.
Mean
52
17
1.6
8.0
4.5
17
6.2
7.4
19
S.D.
46
13
0.9
9.2
12
38
6.0
13
18
19 i 18
11 : 60
23
Geom.
Mean
30
11
1
3
2
3
4
3
11
12
55
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APPENDIX A-4
COLIFORM DENSITIES OF CHLOROBROMINATED EFFLUENT
JANUARY THROUGH NOVEMBER, 1974
(number/100 ml)
01
January
February
March
April
Mayo
June
July
August
September
October
November
Total Coliform Density
Membrane Filtration
No. of
Samples
IB
20
19
23
13
18
20
21
19
17
15
Arith.
Mean
3100
530
42
24
110
340
120
350
770
1800
1800
S.D.
5000
440
50
38
190
400
91
880
910
1300
1100
Geom.
Mean
1700
460
28
B
26
190
9
94
400
1400
1400
Multiple Dilution Tube
No. of
Samples
2
4
4
3
4
5
4
4
3
Arith.
Mean
200
70
10
91
110
1300
180
340
780
S.D.
42
71
10
74
76
2300
71
180
720
Geom.
Mean
200
17
6
68
91
350
210
290
570
Fecal Coliform Density
Membrane Filtration
No. of
Samples
17
20
17
21
12
18
20
20
17
15
11
Arith.
Mean
320
85
7.9
8.9
23
34
25
36
58
120
140
S.D.
950
220
12
16
33
47
25
76
68
97
62
Geom.
Mean
77
28
3
3
6
13
14
12
31
80
130
-------
APPENDIX A-5
COLIFORM DENSITIES OF OZONATED EFFLUENT
JANUARY THROUGH NOVEMBER, 1974
(number/100 ml)
oo
January
February
March
April
May
June
July
August
September
October
November
Total Coliform Density
Membrane Filtration
No. of Arith.
Samples Mean .. S.D.
20 23000 28000
16 12000 20000
19 5000 5200
22
21
19
20
1400
300
2700
1600
17 1600
19 j 3600
12
13
800
1900
2100
260
2000
1500
1100
2600
630
2300
Geom.
Mean
12000
5900
3100
420
190
1800
1200
1200
2800
540
680
Multiple Dilution Tube
No. of
Samples
2
3
3
•j
3
4
4
4
5
Arith.
Mean
S.D.
Geom.
Mean
5200 1 2600
2000
500
300
1900
1700
950
3500
280
2600
700
220
1500
2400
420
3800
4800
1000
200
200
1400
910
870
2400
190
210
Fecal Coliform Density
Membrane Filtration
No. of
Samples
18
16
17
22
21
19
20
17
16
9
8
Arith.
Mean S.D.
2000 2300
1300 2400
2100 ! 1300
510 840
85 97
380 410
180 120
190 240
260 ; 230
74 78
130 ' 140
Geom.
Mean
1100
630
350
110
38
180
150
100
150
37
49
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SECTION V. LIFE CYCLE RESIDUAL TOXICITY STUDIES
INTRODUCTION
The current concern for maintaining the quality of our environment requires
that chemical agents used in the treatment of wastewater not only function
as efficient disinfectants, but that they also show minimal potential to
exert a toxic effect on the aquatic life of receiving waters. This project
was, therefore, designed to simultaneously investigate the disinfection
effectiveness of several mlcrobiocidal agents and their potential residual
toxicity to aquatic life.
The toxicity of wastewater effluent disinfected with chlorine (Cl2) has
received attention in several recent studies-*-""' and the potential for un-
desirable toxic effects by such effluents is generally acknowledged. As a
result, research has progressed to the stage where successful elimination
of the acute residual toxicity of chlorinated effluents is possible through
the use of sulfur dioxide (802)^> bisulfite^ and sodium thiosulfate.6
On the other hand, both the disinfection effectiveness and the residual
toxic properties of bromine chloride (BrCl) and ozone (03) have received
much less study. Mills^' recently investigated wastewater disinfection
with BrCl and performed static tests to determine acute (96-hour) toxic
effects, if any, as a result of chlorobromination. No life cycle studies
with bromine chloride are reported in the literature. Venosa^O reviewed
the literature on water and wastewater disinfection with ozone and found it
to be confusing and contradictory. Investigations of the residual toxicity
of ozonated water under different conditions have produced varying results.
Arthur, et_ a_l., observed no acute or life-cycle toxic effects on aquatic
life exposed to ozonated effluent in which no measurable residual ozone was
present. The same investigators found that ozonated effluent containing a
measurable ozone residual was lethal to fathead minnows. Likewise, Rosen-
lundll reported that rainbow trout died soon after exposure to ozonated
lake water which contained residual ozone. Thus, it is clear that the poten-
tial for residual toxicity in both chlorobrominated and ozonated effluents
merits additional attention.
This residual toxicity study was designed to simultaneously test in parallel
the toxicity of a nondisinfected effluent stream; identical effluent streams
disinfected with chlorine, bromine chloride or ozone; and a chlorinated
stream dechlorinated with sulfur dioxide. Life cycle studies were run with
fathead minnows (Pimephales promelas) as test subjects while acute studies
were conducted with JP. promelas and other species of fish, and the freshwater
macroinvertebrate Daphnia magna.
59
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MATERIALS AND METHODS
Water Supplies
For a detailed description of the treatment site and flow schemes, refer to
Section III.
The dilution water used for diluting the treated effluent streams delivered
to the fish tanks was well water from which excess iron was removed by pas-
sage through an iron removal filter. This water was of high enough quality
to enable fathead minnows (Pimephales promelas) and Daphnia magna to grow
and reproduce satisfactorily. The chemical characteristics of the dilution
water are shown in Table 13. The pH was 7.6 and the conductivity was 859
mi cr omho s/cm.
Table 13. CHARACTERISTICS OF THE DILUTION WATER
Concentration
Analysis in mg/1
Hardness (as CaC03) 464
Calcium 160
Magnesium 13
Sulfate 270
Chloride 8
Iron 0.68
NH3-N 0.16
N02~-N 0.0
Alkalinity (as CaCO.,) 194
Acidity (as CaCOj) 15
Chemical Analyses
Residual chlorine, bromine chloride, ozone, and sulfite (residual sulfur
dioxide was measured as sulfite) were measured daily in the respective ef-
fluent storage tanks and in one aquarium containing the highest effluent
concentration. These same analyses were performed at least once per week
in the aquaria containing each lower effluent concentration. Standard
amperometric titration procedures^ were modified to improve the sensitivity
for determining the amperometric end point.13 ^he modification included a
polarograph (Heath EU-401 Series), a strip chart recorder (Heath model
EUW-20A), a synchronous motor electrode rotator, a platinum electrode, a
magnetic stirrer, and a microburet. An accuracy test using a volumetric
dilution of a known chlorine standard indicated that our procedure was
accurate to ±0.002 mg/1 for halogen determinations, and to ±0.08 mg/1 for
sulfite titrations.
A portable dissolved oxygen meter (Yellow Springs Instrument Model 54) was
used to measure oxygen concentrations daily in the highest effluent concen-
tration tanks, at least once per week in each of the other test chambers,
and three times per week in each of the effluent storage tanks. The temper-
60
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ature of the contents of one aquarium receiving effluent diluted 50 percent
was continuously monitored on each bioassay table. Also, the temperature
of each effluent storage tank was monitored twice per day.
Acidity, alkalinity, total ammonia, conductivity, hardness, and pH were
measured weekly in the effluent storage tanks, in the test aquaria contain-
ing the highest effluent concentration, and in the dilution water (.control)
test tanks. Acidity, alkalinity, and hardness samples were analyzed accord-
ing to procedures outlined in Standard Methods.12 Conductivity was measured
with a Hach conductivity meter (Model 2510), and pH was measured with an
Orion pH meter (Model 701). A modified Seligson-Seligson-^ method was used
in running total ammonia samples.
Bioassay Methods
The effluents and well water were heated to 25C for bioassays with warm water
species, or chilled to 13C for testing cold water species. Proportional di-
luters (Mount and Brungs15) with some refinements (Figure 1]) were used to
achieve the desired test concentrations and to mix effluents with dilution
water. Only PVC, silicone rubber, stainless steel, neoprene rubber and glass
materials were used in the construction of the diluters. Each diluter was
operated continuously on a four minute (±10 seconds) cycle time, with each adult
test chamber receiving 700 ml per cycle. With the exception of the chlori-
nated effluent diluter system, all diluters in the life cycle systems were
calibrated to deliver 100 percent effluent and 100 percent well water and
six intermediate concentrations. The six nominal intermediate concentrations
were 50.00, 25.00, 12.50, 6.25, 3.12, and 1.56 percent effluent. The chlori-
nated life cycle diluter was designed to deliver seven nominal dilutions of
chlorinated effluent, 20.00, 14.00, 9.80, 6.86, 4.80, 3.46, and 2.35 percent,
and 100 percent dilution water. Lower concentrations of chlorinated effluent
were used in the life cycle study because of the previously demonstrated
toxicity of chlorinated effluent to aquatic life.'
The calibration of all life cycle diluters was checked volumetrically each
week and adjusted, if necessary, to maintain the proper effluent concen-
tration and turnover time in each test chamber. Usually only minor adjust-
ments of the diluters were required to maintain proper calibration. Most of
the problems encountered with the diluters occurred during the first several
months of the project when effluent quality was low.
Test chambers were 60 x 29 x 30 cm (28.4 1) glass aquaria, which received
one complete volume change every 2.6 hours, or 9.2 tank volumes per 24-hour
period. Duplicate test chambers were randomly located on a 1.2 x 3.0 m
table for each type of treated effluent. The life cycle test tables were
isolated behind a black curtain to minimize the visual stimulation of the
test animals by laboratory traffic. This was particularly important during
the reproductive period when the fish were most sensitive to external stimuli.
On the life cycle test tables, fry chambers (30 x 30 x 30 cm (14.2 1)) were
located on a shelf below the spawning chambers and received the same concen-
trations of effluent at the same rate as the adult tanks. Those adult and
fry tanks receiving the four highest effluent concentrations (100, 50, 25,
and 12.5 percent) were continuously aerated with oil-free air to prevent
61
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DILUTION
WATER
CELL
102 MM
SLOPE OF W-AND C-CELL
IS 20MM PER 30.5CM
ie LEFT SIDE IS 38MM
HIGHER THAN RIGHT.
MICROSWITCH
o o-
o
,I8MM SLEEVES TO
ADULT CHAMBERS
-16MM SLEEVES TO
FRY CHAMBERS
NOTE-
DIMENSIONS OF CELLS
AND SPLITTER BOXES
ARE TO SCALE-GLASS
AND TYGON TUBING
NOT TO SCALE.
TO
DRAIN
Figure 11. SCHEMATIC DRAWING OF THE MODIFIED MOUNT-BRUNGS
PROPORTIONAL DILUTER USED IN THIS STUDY
62
-------
excessively low dissolved oxygen concentrations. This process was not neces-
sary on the chlorinated effluent life cycle table because of the greater
dilutions of effluent in those test chambers.
The bioassay system was equipped with an electrically operated warning
system which shut down all diluters, activated a light and bell alarm, and
turned on an air supply to each test tank in the event of extreme effluent
or dilution water temperature, unacceptably high concentrations of chlorine
in the chlorinated effluent, or the presence of chlorine in the dechlorinated
effluent stream. The various systems were also equipped with monitors so
that any failure in the supply of effluents or dilution water to the head
tanks or to the diluters and any malfunction of the diluters would activate
the system.
The bioassay laboratory lighting was from artificial sources only and was
requlated to approximate seasonal changes in day length. Light intensity
was gradually increased in the morning and decreased in the evening to
simulate dawn and dusk,1 respectively. A combination of General Electric
F40 daylight fluorescent bulbs, General Electric F40 plant light fluorescent
bulbs, and 40 watt incandescent bulbs was used for illumination.
Progeny of fathead minnows (£. promelas), obtained from stock cultures main-
tained at the U.S. E.P.A. National Water Quality Laboratory in Duluth, Min-
nesota, were used for the life cycle tests. These tests were started by
placing fifty 1-2 day old fry in each test aquarium and monitoring their
survival. Any test tank containing less than 15 living fry after 15 - 17
days was restocked with 15 - 17 day old fry which had been reared in 100
percent dilution water to bring the number of fish per aquarium back to 50.
This process was necessitated because of the high random mortality of the
original stock, which we concluded was caused by the scarcity of natural
food after the larvae had absorbed their yolk sac and become dependent upon
ingested food. The test animals were photographed at 30 and 60 days into
the test. The photographs were enlarged to determine the average lengths
of the survivors.
At 60 days into the test the fish population in each test tank was thinned
to a maximum of 15 apparently healthy fish, and five spawning substrates
(7.6 cm (3 in) lengths of 12.7 cm (5 in) asbestos drain tile cut in half)
were placed in each tank. These spawning tiles provided refuge for the
fathead minnows, sites for males to establish territories, and substrates
on which the females could deposit their eggs. When the fatheads were
mature enough 'for us to definitely determine their sex (170-204 days into
the test), each tank was thinned to no more than four males (to eliminate
territorial conflict), and the number of females was recorded.
During each day of the spawning period, all eggs produced in each tank were
removed, counted, and examined microscopically to determine their condition.
Those eggs that we felt, based upon past experience, would go on to hatch
if incubated under optimum conditions were considered viable eggs. The
number of viable eggs in each spawning was recorded within 24 hours of the
time those eggs were produced. Some of the eggs produced in each concen-
tration of each effluent type were incubated to determine the hatchability
63
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of eggs spawned in that particular environment. For our purposes, hatchabil-
ity was defined as the ability of eggs to hatch and produce living fry. If
fry only partially emerged from the egg, or hatched and died prior to the
end of the incubation period, they were not counted as living fry.
Fifty viable eggs from each spawning were incubated in mesh bottom egg cups
in the tank in which they were spawned. When the total number of spawnings
in any particular tank equaled the number of females in that tank, we incu-
bated only eggs from every third spawning, except that to minimize weekend
work, no eggs were incubated from those spawnings which occurred on Mondays
and Tuesdays.
Generally, five days of incubation were required for all viable eggs from a
spawning to hatch. The eggs were recounted each day of the incubation period
and those which had died were removed. After hatching began, the egg cup
was left untouched until all eggs had hatched. At that time the numbers of
dead eggs and living and dead fry were recorded. Data from one or more in-
cubation in each effluent type and concentration were utilized to calculate
the mean percentage hatchability.
In addition, when time and spawnings permitted, hatchability information was
obtained on eggs produced in high concentration effluent tanks and incubated
in dilution water tanks, and, conversely, on eggs produced in dilution water
tanks and incubated in high concentration effluent tanks.
If more than four of the fifty eggs in any one incubation were unaccounted
for, that incubation attempt was discarded. Loss of eggs and/or fry was
not uncommon due to their small size. The number of successful incubations
was also limited by spawnings that were deposited on the mesh bottom of the
egg cups, and intermittent periods when effluents were high in suspended
solids, which partially plugged the mesh bottoms and thereby precluded ade-
quate water transfer through the mesh screening.
Forty 1-2 day old fry from each reproducing adult tank were placed in fry
chambers receiving the same concentration of effluent as the adult tank in
which they were produced. Their lengths were also measured photographically
at 30 days, and they were directly weighed, measured, examined, and frozen
for future analyses at 60 days of age.
The fry were fed three times each day, once with live brine shrimp nauplii,
once with frozen trout starter mash, and once with a diatom culture. Juve-
nile and adult fish were fed twice each day, once with live brine shrimp
nauplii, and once with granular frozen trout food. These feedings were
occasionally supplemented with feedings of live Daphnia magna. Excess food
and other debris were siphoned from the test chambers daily. Fry chambers
were not cleaned until the fish were thirty days old.
With few exceptions, the duration of acute toxicity tests was 96 hours.
Test animals for acute studies were either reared in the laboratory, purchased
from private sources, or obtained from State or Federal fish hatcheries. In
all cases they were held in the laboratory at test temperatures at least ten
64
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days prior to testing, or until they were determined to be disease free.
All data collected during the life cycle study were stored and analyzed on
a Xerox Sigma-6 computer. All water chemistry data, disinfectant residuals,
egg production and hatchability data, and fish, growth and mortality data
were keypunched weekly onto cards which were verified and processed. The
computer was programmed to provide printouts of means, standard deviations,
and ranges during the course of the study.
In addition, a two-way analysis of variance with unbalanced and nested
designs was performed on mortality, spawning, hatchability, and growth data
for all treatment types and concentrations using the Statistical Analysis
System (S.A.S.).17
RESULTS AND DISCUSSION
Water Chemistry
The results of residual chemical determinations in the various effluent streams
delivered to the adult test chambers are summarized in Table 14. in many cases
the standard deviation was high; this was particularly true for dechlorinated
samples. The occasional high sulfite residual levels (above 0.100 mg/1 only
17 times out of 208 samples) seemed to occur during those times of low total
suspended solids (3-11 mg/1), low volatile suspended solids (1-11 mg/1), and
low turbidity (6-14 Jackson turbidity units) values.
Ozone and bromine chloride residuals were more uniform than the sulfite re-
siduals, as evidenced by the standard deviations approaching more closely
the mean value for most concentrations, while chlorine residuals were the
most uniform with standard deviations approximately half the mean values.
The relatively narrow distribution of the chlorine residual determinations
can be explained by the fact that chlorine was the only disinfectant whose
concentration was regulated by residual control rather than by dosage control.
Chlorine residuals were adjusted on an hourly basis. This compensated for
changes in demand of the effluent, a feature which was lacking from the other
disinfection systems (see Section III).
Table 15 summarizes the residual levels measured in the fry tanks. In almost
every instance, residual levels were less, in some cases considerably less,
than those in the adult tanks (Table 14). This was attributed to differences
in the cleaning and feeding procedures between adult and fry chambers. The
adult tanks were fed granulated food and newly-hatched brine shrimp daily,
and excessive food and other debris were siphoned out. The fry tanks, on the
other hand, were not cleaned at all during the first thirty days of their use,
while a daily allotment of diatom culture, granulated food, and brine shrimp
were added as feed. As a result, an organic layer formed on the bottom of
the fry tanks on which the fry were frequently seen feeding. Although this
proved to be an effective feeding method, the presence of the excessive
material in the tank increased the demand for disinfectant, which in turn
resulted in lower residual levels.
65
-------
Table 14. THE MEAN RESIDUAL CHEMICAL LEVELS (mg/1), SAMPLE SIZES, AND STANDARD DEVIATIONS
MEASURED IN HEAD TANKS AND ADULT TEST CHAMBERS DURING THE LIFE-CYCLE TESTS
Effluent Stream
Chlorinated
Chlorine Residual
Sample Size
Standard Deviation
Sampling Site
Head Tank
1.357
182
0.435
20%
Effluent
0.101
200
0.064
14%
Effluent
0.067
156
0.038
9.8%
Effluent
0.035
103
0.020
6.9%
Effluent
0.024
105
0.015
4.8%
Effluent
0.022
105
0.015
3.4%
Effluent
0.011
102
0.008
' 2.4%
Effluent
0.008
101
0.002
Sampling Site
Dechlorinated
Sulfite Residual
Sample Size
Standard Deviation
Ozonated
Ozone Residual
Sample Size
Standard Deviation
Chlorobrominated
Bromine Chloride
Residual
Sample Size
Standard Deviation
Head Tank
1.572
175
1.13
0.027
182
0.025
0.478
185
0.476
100%
Effluent
0.027
248
0.119
0.012
250
0.016
0.119
173
0.128
50%
Effluent
0.012
100
0.060
0.005
100
0.003
0.032
64
0.034
25%
Effluent
0.005
97
0.021
0.003
101
0.003
0.017
103
0.016
12 . 5%
Effluent
0.004
96
0.011
0.002
97
0.002
0.007
103
0.006
6 . 25%
Effluent
0.005
101
0.021
0.001
97
0.002
0.005
103
0.005
3.12%
Effluent
0.001
99
0.004
0.001
99
0.002
0.004
104
0.004
1.56%
Effluent
0.000
99
0.000
0.001
98
0.002
0.003
103
0.003
-------
Table 15. THE MEAN RESIDUAL CHEMICAL LEVELS (mg/1), SAMPLE SIZES, AND STANDARD DEVIATIONS
MEASURED IN FRY TEST CHAMBERS DURING THE LIFE-CYCLE TESTS
Effluent Stream
Chlorinated
Chlorine Residual
Sample Size
Standard Deviation
Sampling Site
20%
Effluent
0.076
84
0.049
14%
Effluent
0.053
35
0.028
9QV
. O/o
Effluent
0.033
35
0.017
6.9%
Effluent
0.026
34
0.016
4.8%
Effluent
0.023
38
0.013
3.4%
Effluent
0.014
35
0.008
2.4%
Effluent
0.008
42
0.007
Sampling Site
Dechlorinated
Sulfite Residual
Sample Size
Standard Deviation
Ozonated
Ozone Residual
Sample Size
Standard Deviation
Chlorobrominated
Bromine Chloride
Residual
Sample Size
Standard Deviation
100%
Effluent
0.015
83
0.096
0.012
79
0.009
0.045
73
0.041
50%
Effluent
0.000
34
0.000
0.005
38
0.004
0.027
31
0.018
25%
Effluent
0.000
37
0.001
0.003
39
0.002
0.011
38
0.010
12.5%
Effluent
0.002
35
0.007
0.003
39
0.003
0.008
47
0.007
6.25%
Effluent
0.004
43
0.023
0.002
38
0.002
0.005
35
0.004
3.12%
Effluent
0.000
33
0.000
0.002
40
0.002
0.003
40
0.004
1.56%
Effluent
0.000
38
0.000
0.002
36
0.002
0.003
37
0.003
ON
-------
The average dissolved oxygen concentrations in the effluent head tanks
ranged from 2.54 mg/1 for the nondisinfected effluent to 8.15 mg/1 for the
ozonated effluent (Table 16). The dilution water averaged 3.80 mg/1 dis-
solved oxygen, and was the only liquid that was aerated to increase the
dissolved oxygen levels. The 100 percent through 12.5 percent effluent con-
centration tanks (adult and fry) on all life cycle test tables except the
chlorinated table were aerated to increase the dissolved oxygen levels. This
was not necessary for the chlorinated table because of the lower oxygen demand
due to higher effluent dilution factors. Thus, the lowest mean dissolved
oxygen level for a 100 percent effluent concentration was 3.93 mg/1 in the
chlorobrominated fry tanks, while the highest was 5.41 mg/1 in the 100 percent
chlorobrominated adult tanks. These values range between 47 percent and 65
percent of dissolved oxygen saturation at the test temperature of 25C. Since
fathead minnows are capable of surviving dissolved oxygen levels as low as
2 mg/1 for several days at 25C, the above values were probably within safe
limits for £. promelas.
The alkalinity, acidity, hardness, conductivity, and total ammonia nitrogen
values measured in the various streams are summarized in Tables 17 and 18.
According to McKee and Wolf-^ the results of these tests are within acceptable
tolerance limits for fish. The measured pH of the effluents in the head tanks
ranged from 6.9 to 8.0 and was usually between 7.4 and 8.0. The pH of the
contents of the fish tanks ranged from 7.0 to 8.2 and was usually between 7.4
and 8.0. In every instance, the mean pH values were lower and the mean acid-
ity values higher in the head tanks than in the highest effluent concentration
test chambers. These differences may in part be a result of the presence of
food, animals and their by-products. Also, aeration of the test tanks, which
received high effluent concentrations, to maintain satisfactory dissolved
oxygen levels may have contributed to the observed pH and acidity changes.
With the exception of the chlorinated and chlorobrominated test tanks for
which lower effluent concentrations were sampled, all water chemistry values
and water temperatures (Table 19) were similar for each of the effluent
streams. Thus any negative effects that were observed when each treatment
type was compared to the nondisinfected stream was attributed to the partic-
ular disinfection process applied to that effluent stream, since the values
of other chemical parameters measured were within safe limits.
MORTALITY
Mortalities of the first and second generation fish in the various effluent
streams are summarized in Tables 20-29. All survivor counts during the first
60 days of the test were made from photographs of the fish in each tank,
while the survivor counts after day 60 were determined through direct obser-
vations.
The first generation data included the additional variable of restocking
some of the tanks in each effluent stream on day 15, as shown in the respec-
tive tables. This restocking was necessitated by the nearly total mortality
observed in many tanks during the first two weeks of the study. Because this
early mortality occurred in a random pattern and because the growth of the
test fish was retarded during this interval, it was concluded that the major
68
-------
Table 16. THE MEAN DISSOLVED OXYGEN CONCENTRATIONS (mg/1)
MEASURED IN STORAGE TANKS AND TEST CHAMBERS
DURING THE LIFE CYCLE STUDIES
Dilution Water
M , i r r r t- - i Adult
Nondisiuiected „
n^ i i , i 11 Adult
Declilor iua ted _
Fry
rl ... t_ _,,._..., „-,-_:, Adult
Chlorobroininated •-• — — • •••
Fry
^ j Adult
Ozonated • •'• • •
Fry
riL-K-Li-ti-ut-prl Adult
Chlorinated. _' •• • •• — |
rry
Storage
Tank
3.80
2.54
2.54
4.10
4.10
3.23
3.23
8.15
8.15
Storage
Tank
L5.46
5.46
Nominal Percent Effluent Concentrations
100
4.87
4.14
5.37
4.75
5.41
3.93
4.70
4.91
20
5.46
4.00
50
4.64
5.14
5.02
5.69
4.82
4.41
4.29
5.04
14
4.76
4.43
25
4.95
4.57
4.90
4.55
4.50
4.50
4.49
4.56
9.8
4.62
5.00
12.50
4.79
4.23
4.61
5.45
4.16
4.26
4.46
4.65
6.86
4.56
5.06
6.25
4.66
4.68
5.08
4.44
4.62
4.15
4.77
4.73
4.80
3.12
4.69
4.79
5.21
5.31
5.06
4.72
5.49
5.28
3.36
4.79 5.37
4.85 i 5.23
1.56
5.45
5.32
5.87
5.16
5.51
5.14
5.40
5.40
2.35
5.46
5.22
0.00
5.45
4.50
5.49
L 5.04
5.38
4.70
5.89
5.90
0.00
5.49
4.81
OS
VO
-------
Table 17. THE MEAN WATER CHEMISTRY VALUES MEASURED IN HEAD TANKS
Head Tanks
Nondisinf ected
Mean
Sample Size
Standard Deviation
Chlorinated
Mean
Sample Size
Standard Deviation
Dechlorinated
Mean
Sample Size
Standard Deviation
Chlorobrominated
Mean
Sample Size
Standard Deviation
Ozonated
Mean
Sample Size
Standard Deviation
Dilution Water
Mean
Sample Size
Standard Deviation
Alkalinity
as mg/1
CaCCL
194
48
36.7
193
46
35.8
185
47
35.3
192
47
37.3
193
46
41.5
194
47
2.3
Acidity
as mg/1
CaC03
27
46
6.8
28
46
5.0
37
45
5.0
31
45
6.2
20
46
3.4
15
46
2.6
Hardness
as mg/1
CaCO
288
47
36.9
287
47
39.9
289
47
43.5
289
45
38.3
289
46
38.1
465
47
9.3
Conductivity
Micromhos/cm
911
46
73.9
908
45
85.3
918
44
64.5
911
45
73.9
911
45
71.1
859
47
20.4
Total Ammonia
Nitrogen
Crag/D
9.3
46
5.4
9.4
45
5.2
9.8
46
5.4
9.1
46
5.6
8.7
46
5.3
0.16
36
0.18
-------
Table 18. THE MEAN WATER CHEMISTRY VALUES MEASURED IN THE HIGHEST EFFLUENT
CONCENTRATION ADULT TEST TANKS CONTAINING LIVE FISH
Effluent Stream
Nondisinf acted
Mean
Sample Size
Standard Deviation
Chlorinated3
Mean
Sample Size
Standard Deviation
Dechlorinated
Mean
Sample Size
Standard Deviation
Chlorobrominated3
Mean
Sample Size
Standard Deviation
Ozonated
Mean
Sample Size
Standard Deviation
Dilution Water
Mean
Sample Size
Standard Deviation
Alkalinity
as mg/1
CaC03
189
48
36.6
191
37
5.2
180
47
35.4
184
29
20.0
187
47
36.4
193
48
2.5
Acidity
as mg/1
CaCO,
15
46
3.7
15
36
2.7
17
45
4.3
22
27
37.0
16
46
3.5
10
46
2.5
Hardness
as nig/1
CaCO_
386
47
36.4
437
37
7.2
293
47
52.4
369
28
10.0
285
47
35.8
466
46
8.1
Conductivity
Micromhos/cm
905
47
67.5
875
37
17.2
911
45
64.7
874
29
27.7
906
47
68.5
856
46
26.9
Total Ammonia
Nitrogen
(rag/1)
9.1
45
5.1
1.3
37
0.9
9.2
46
4.9
5.2
28
2.8
8.5
46
4.8
0.18
45
0.35
3The chlorinated test tank sampled was 14% effluent, the chlorobrominated test tank sampled was
50% effluent; and the remaining test tanks sampled were 100% effluent, with the exception of
dilution water. This accounts for the difference in hardness, conductivity, and ammonia nitrogen
levels.
-------
Table 19. MEAN WATER TEMPERATURES (°C)
MEASURED IN STORAGE TANKS AND ADULT TEST CHAMBERS
DURING THE LIFE CYCLE STUDIES
Effluent Type
Mean Temperature
In Storage Tank
Mean Temperature
In Aquariaa
NJ
Nondisinfected
Chlorinated
Dechlorinated
Chlorobrominated
Ozonated
26.5
27.3
27.3
26.4
26.2
24.9
25.1
24.7
25.1
24.9
All temperatures were measured In aquaria containing a 50 percent effluent concentration, except
in the chlorinated effluent stream where temperatures were recorded in an aquarium containing a
20 percent effluent concentration.
-------
cause of this mortality was an insufficient supply of food (microscopic
organisms) as the fish became dependent upon ingested food rather than
their yolk sacs. Those tanks that were restocked showed higher survival
rates at days 23 and 53 of the study than did comparable tanks that had
not been restocked. However, no difference in survival was detected
between restocked and nonrestocked tanks after the tanks were thinned to
15 fish each on day 53.
Nondisinfected Effluent
The only pattern of mortality observed in the first generation fish reared
in nondisinfected effluent was that observed during the first two weeks of
the study (Table 20). The greatest mortality during that period occurred
in the two highest effluent concentrations, 50 and 100 percent, and both
duplicates in these concentrations required restocking on Day 15.
The second generation of fish exposed to nondisinfected effluent showed a
variable rate of survival which, except for the undiluted effluent, was not
clearly dependent upon effluent concentration (Table 21). While only four
survivors were observed in the 100 percent effluent concentration, mortality
occurred early in life as in the first generation test animals which appar-
ently suffered from an inadequate food supply.
In considering these results, it is necessary to point out that the quality
of the effluent to which the two generations were exposed differed substan-
tially. For example, during the first two months of their lives the first
generation test animals were exposed to treated wastewater with mean monthly
suspended solids levels and total phosphate concentrations of approximately
20-40 mg/1 and 8 mg/1, respectively. This contrasts with the exposure of
second generation test animals of similar age to mean monthly suspended
solids and total phosphate concentrations of 10-15 mg/1 and Z. 2 mg/1,
respectively.
It appeared, then, that both first and second generation J?. promelas were
subject to significantly higher mortality during the first 15 or 30 days
of the test, respectively, when exposed to 100 percent nondisinfected ef-
fluent. This mortality probably resulted from a combination of factors,
including the supply of microscopic food organisms available to the young
fish as well as the generally unfavorable environmental conditions which
occurred in the 100 percent effluent concentrations.
73
-------
Table 20. NUMBER OF FIRST GENERATION P. PROMELAS SURVIVING IN NONDISINFECTED EFFLUENT
No. of fish alive
at day 23a
C.I.d for prob.
of survival
thru day 23
Survival/ 100
at day 53
C.I. for prob.
of survival
thru day 53
No. of fish alive
from day 53 until
day 330 or death6
Number of fish
alive at:
90 days
120 days
150 days
180 days
210 days
240 days
270 days
300 days
330 days
C.I. for prob.
of survival
day 53-330
Nominal Percent Nondisinfected Effluent
0.00
87
0.79-0.92
84
0.76-0.90
26
26
26
26
26
26
26
26
25
25
0.81-0.99
1.56
71b
0.61-0.79
71
0.61-0.79
27
27
27
26
26
26
26
26
25
25
0.76-0.99
3.12
,.b
64
0.54-0.73
63
0.53-0.72
23
23
23
23
23
23
23
23
22
22
0.79-0.99
6.25
76b
0.67-0.83
74
0.65-0.82
20
20
20
20
20
20
20
20
19
19
0.76-0.99
12.50
78b
0.69-0.85
78
0.69-0.85
23
23
23
23
23
23
23
23
23
23
0.86-1.00
25.00
79b
0.70-0.86
79
0.70-0.86
29
29
29
29
29
29
29
29
28
28
0.83-0.99
50.00
96C
0.90-0.98
89
0.81-0.94
25
25
25
25
25
25
25
25
25
25
0.87-1.00
100.00
92°
0.85-0.96
85
0.77-0.91
25
25
25
25
25
25
25
25
24
24
0.80-0.99
?From the original 100 fish that were stocked in each concentration
One of the duplicate tanks restocked on day 15
.Both of the duplicate tanks restocked on day 15
95 percent confidence interval for the true probability of survival
6 The number of fish in each effluent concentration was reduced to 30 on day 53 and excess males were
subsequently removed prior to the spawning season.
-------
Table 21. NUMBER OF SECOND GENERATION I>. PROMELAS SURVIVING
IN THE NONDISINFECTED EFFLUENT*
30 Days of Age
No. Surviving
Conf . Interval
60 Days of Age
No . Surviving
Conf . Interval
Nominal Percent of Nondisinfected Effluent
0.00
49
0.50-0.72
49
0.50-0.72
1.56
35
0.33-0.55
35
0.33-0.55
3.12
44
0.44-0.65
41
0.40-0.62
6.25
46
0.47-0.68
46
0.47-0.68
12.50
37
0.36-0.57
35
0.33-0.55
L_ 25.00
72
0.81-0.95
72
0.81-0.95
50.00
31
0.62-0.88
31
0.62-0.88
100.00
4
0.01-0.12
4
0.01-0.12
Ln
a40 fish were started in each of the two duplicate tanks of each effluent concentration, except for the
50 percent concentration for which only one tank with 40 fish was started.
395 percent confidence interval for the true probability of survival.
-------
Chlorinated Effluent
The first generation of fathead minnows reared in chlorinated effluent suf-
fered lethal effects in the two highest effluent concentrations between days
23 and 53 of the study (Table 22). This observation is important not only
because the highest concentration of chlorinated effluent to which fish were
exposed was only 20 percent, but also because both duplicate tanks of these
concentrations were restocked on day 15, an action that normally enhanced
survival at days 23 and 53.
Fish reared in both 20 and 14 percent chlorinated effluent concentrations
also exhibited a higher mortality after day 53, but the cause of that mor-
tality was apparently not excessively high levels of residual chlorine. On
days 72 and 73 of the study, 26 fish died in the 20 percent effluent concen-
tration tanks, and 12 died in the 14 percent effluent concentration tanks.
Although the highest total residual chlorine measured on those days was
0.188 mg/1 in the 20 percent effluent concentration tanks and 0.163 mg/1 in
the 14 percent effluent tanks, these temporary residuals were apparently not
extreme since no lethal effects were observed at other times when those re-
siduals were exceeded. However, from days 66 to 71 the fish had been exposed
to unchlorinated effluent because of mechanical problems. The ensuing
mortality brought on by renewed exposure to chlorine suggests that the test
animals had lest some of their tolerance to chlorine during that brief period
of no exposure. Our data and experience indicate that fathead minnows main-
tained in sublethal levels of chlorinated effluent develop a tolerance
which permits them to survive in concentrations of chlorinated effluent
which would normally be lethal to nonacclimated individuals of the same
species. Other data which tend to support this apparent acclimation phenom-
enon are documented and discussed in the section dealing with acute toxicity
studies (Section VI).
The second generation test fish exhibited lethal effects in the 20 percent
effluent concentration (Table 23). Fish reared in 14 percent effluent showed
a tendency toward reduced survival, although the 95 percent confidence inter-
vals for their probability of survival overlapped with those of fish reared
in less concentrated effluents.
The principal difference in the mortality patterns of the first and second
generation fish in 20 percent chlorinated effluent was that the first gener-
ation showed excessive mortality between 23 and 53 days of life, while the
second generation showed a lethal effect only during the first 30 days of
life. While a lethal effect may have been masked by the restocking of first
generation fish on day 15, it is known that the two generations of test
animals were exposed to different total residual chlorine levels. A minimum
mean total chlorine residual of 0.045 mg/1 was apparently necessary to exert
a lethal effect during the first 60 days of life. These findings agree well
with those of Arthur, et_ _al. ,1 who found that long-term exposure of f. prome-
las to a mean chlorine residual of 0.042 mg/1 was lethal, but a mean chlorine
residual of 0.014 mg/1 was not. However, in our study, older fathead minnows
survived mean total chlorine residuals as high as 0.074 mg/1 for 180 days
76
-------
Table 22. NUMBER Of FIRST GENERATION P. PROMELAS SURVIVING IN CHLORINATED EFFLUENT
No. of fish alive
at day 23*
C.I.e for prob.
of survival
thru day 23
No. of fish alive
at day 53a
C.I. for prob.
of survival
thru day 53
No. of fish alive
from day 53 until
day 330 or death^
Number of fish
alive at:
90 days
120 days
150 days
180 days
210 days
240 days
270 days
300 days
330 days
C.I. for prob.
of survival
day 53-330
Nominal Percent Chlorinated Effluent
0.00
66b
(0.000)°
0.56-0.75
64
(0.000)
0.54-0.73
26
26
(0.000)
26
(0.000)
26
(0.000)
26
(0.000)
26
(0.000)
25
(0.000)
25
(0.000)
25
(0.000)
25
(0.000)
0.75-0.99
2.35
7lb
(0.002)
0.61-0.79
69
(0.004)
0.59-0.77
23
23
(0.004)
23
(0.006)
22
(0.007)
22
(0.009J
22
(0.009)
22
(0.008)
22
(0.008)
22
(0.008)
22
(0.008)
0.79-0.99
3.36
52
(0.003)
0.42-0.62
52
(0.006)
0.42-0.62
24
24
(0.007)
24
(0.010)
24
(0.011)
24
(0.012)
23
(0.012)
23
(0.012)
23
(0.012)
22
(0.012)
22
(0.011)
0.74-0.98
4.80
67b
(0.007)
0.57-0.75
65
(0.016)
0.55-0.74
25
25
(0.020)
25
(0.023)
25
^0.024)
25
(0.026)
25
(0.025)
25
(0.025)
25
(0.023)
25
CO. 02 31
25
(0.022)
0.87-1.00
6.86
81°
(0.010)
0.72-0.87
80
(0.016)
0.71-0.87
21
21
(0.020)
21
(0.023)
20
(0.026)
20
(0.028)
20
(0.027)
20
(0.026)
20
(0.026)
20
(0.025)
20
(0.024)
0.77-0.99
9.80
930
(0.013)
0.86-0.97
85
(0.025)
0.77-0.91
24
23
(0.030)
23
(0.036)
23
(0.038)
23
(0.039)
22
(0.038)
22
(0.038)
22
(0.037)
22
CO. 036)
22
(0.035)
0.74-0.98
14.00
950
(0.020)
0.89-0.98
72
(0.045)
0.63-0.80
28
15
(0.057)
15
(0.063)
15
(0.067)
15
(0.074)
15
(0.074)
15
(0.073)
15
(0.073)
15
(0.071)
15
(0.067)
0.34-0.72
20.00
87C
(0.038)
0.79-0.92
43
(0.076)
0.34-0.53
30
1
(0.088)
1
(0.092)
1
(0.098)
1
(0.103)
1
(0.105)
1
(0.106)
1
(0.105)
1
(0.103)
1
(0.102)
0.01-0.17
fFrom the original 100 fish that were stocked in each concentration
One of the duplicate tanks restocked on day 15
.Both of the duplicate tanks restocked on day 15
Mean total residual chlorine (mg/1)
^95 percent confidence interval for the true probability of survival
The number of fish in each effluent concentration was reduced to 30 on day 53, and
subsequently removed prior to the spawning season.
excess males were
-------
Table 23. NUMBER OF SECOND GENERATION JP. PROMELAS SURVIVING
IN THE CHLORINATED EFFLUENT3
30 Days of Age
No. Surviving
Conf . Interval
X Residual, mg/1
60 Days of Age
No. Surviving
Conf . Interval
X Residual, mg/1
Nominal Percent Chlorinated Effluent
0.00
53
0.55-0.76
0.000
53
0.55-0.76
0.000
2.35
52
0.54-0.75
0.008
51
0.53-0.73
0.008
3.36
63
0.69-0.86
0.004
64
0.70-0.87
0.009
4.80
59
0.63-0.82
0.018
59
0.63-0.82
0.020
6.86
67
0.74-0.90
0.016
66
0.73-0.89
0.016
9.80
62
0.67-0.85
0.028
62
0.67-0.85
0.033
14.00
49
0.50-0.71
0.033
47
0.48-0.69
0.035
20.00
26
0.23-0.43
0.045
26
0.23-0.43
0.033
00
40 fish were started in each of the two duplicate tanks of each effluent concentration.
95 percent confidence interval for the true probability of survival.
tlean total residual chlorine.
-------
without any lethal effect, indicating that the chlorine tolerance of P_.
promelas increases with age and/or size.
Dechlorinated Effluent
Sixty-one percent of the first generation fish reared in the 100 percent
dechlorinated effluent died within the first 23 days of exposure and, by
day 53, mortality had increased to 64 percent (Table 24).
The second generation fish exhibited greater mortality in 100 percent de-
chlorinated effluent (Table 25). However, these test animals were exposed
to much lower mean sulfite residuals (0.005-0.010 mg/1) than the first
generation fish (0.042-0.080 mg/1).
The significance of the mortality observed in the 100 percent dechlorinated
effluent was obscured by the fact that similar mortality occurred in the 100
percent nondisinfected effluent and by the varied restocking histories of
different tanks.
Thus it is impossible to ascertain the true cause of the observed mortality
in the 100 percent dechlorinated effluent tanks. Since other investigators^
have found no lethal effect on fathead minnows with long-term exposure to
mean residual sulfite concentrations of 0.104 mg/1, it would appear that our
sulfite residuals did not account for the observed mortality.
While the lethality of 100 percent dechlorinated effluent is questionable,
it is probably not an important consideration since it has no application
to normal wastewater disposal practices. The important point to be gained
here is that dechlorination eliminated the lethal effect of the 20 percent
chlorinated effluent, and appeared to eliminate the toxicity of 50 percent
chlorinated effluent.
Chlorobrominated Effluent
First generation fish reared in 100 and possibly 50 percent chlorobrominated
effluent appeared to exhibit lethal effects at both 23 and 53 days of age
(Table 26), as evidenced by the reduced survival observed even after restock-
ing. Since the fish living in 100 percent chlorobrominated effluent were
the only test animals which clearly showed a lethal response to a 100 percent
effluent concentration between days 23 and 53, such mortality appeared to be
related to the bromine chloride concentration in the effluent. Almost all
of the mortality during this interval occurred over a two-day period when
high bromine chloride residuals, resulting from lower demand due to better
effluent quality, were measured in the test aquaria. The maximum bromine
chloride residual measured during this period was 0.651 mg/1 in the 100 per-
cent effluent fish tanks. That value was several times higher than the 96
hour TL50 values for fathead minnows discussed in the acute toxicity section
(Section VI) of this report.
On days 76 and 77 a mechanical failure of the bromine chloride feed system
resulted in a fish kill in the 100 percent effluent tanks, where bromine
79
-------
Table 24. NUMBER OF FIRST GENERATION P. FRCMELAS SURVIVING IN DECHLORINATED EFFLUENT
OO
O
No. of fish alive
at day 23*
C.I. for prob.
of survival
thru day 23
No. of fish alive
at day 53a
C.I. for prob.
of survival
thru day 53
No. of fish alive
from day 53 until
day 330 or deathf
Number of fish
alive at:
90 days
120 days
150 days
160 days
210 days
240 days
270 days
300 days
330 days
C.I. for prob.
of survival
day 53-330
Nominal Percent Dechlorinated Effluent
0.00
94C
(0.000)
0.88-0.97
81
(0.000)
0.72-0.87
24
24
(0.000)
24
(0.000)
24
(0.000)
24
(0.000)
24
(0.000)
24
(0.000)
24
(0.000)
24
(0.000)
24
(0.000)
0.86-1.00
1.56
60b
CO. 000)
0.50-0.69
49
(0.000)
0.39-0.59
24
22
(0.000)
22
(0.000)
22
(0.000)
22
(0.000)
22
(0.000)
22
(0.000)
22
(0.000)
22
(0.000)
. 22
(0.000)
0.74-0.98
3.12
56b
(0. 000)
0.46-0.65
56
(0.000)
0.46-0.65
26
26
(0.002)
26
(0.002)
26
(0.002)
26
CO. 001)
25
(0.001)
25
(0.001)
25
(0.001)
25
^0.001)
25
(0.001)
0.81-0.99
6.25
75C
(0.008)
0.66-0.82
61
(0.010)
0.51-0.70
23
23
(0.018)
23
(0.013)
23
(0.011)
23
(0.009)
23
(0.008)
23
(0.007)
23
(0.007)
23
(0.006)'
23
(0.005)
0.86-1.00
12.50
68b
(0.002)
0.58-0.76
64
(0.010)
0.54-0.73
24
24
(0.012)
24
(0.009)
24
(0.007)
24
(0.006)
24
(0.005)
24
(0.005)
24
(0.005)
24
(0.004)
24
(0.004)
0.86-1.00
25.00
83c
(0.000)
0.74-0.89
66
(0.000)
0.56-0.75
20
20
(0.011)
20
(0.009)
20
(0.008)
20
(0.007)
20
(0.007)
20
(0.006)
19
(0.006)
18
(0.005)
18
(0.005)
0.69-0.98
50.00
59
(0.000)
0.49-0.68
56
(0.000)
0.46-0.65
23
23
(0.027)
23
(0.021)
23
(0.016)
23
(0.020)
23
(0.018)
23
(0.016)
23
(0.014)
23
(0.014)
23
(0.012)
0.86-1.00
100.00
39c
(0.080)
0.30-0.49
36
(0.042)
0.27-0.46
21
21
(0.060)
21
(0.045)
21
(0.036)
21
(0.034)
21
(0.031)
21
(0.029)
21
(0.027)
21
(0.028)
21
(0.026)
0.84-1.00
the original 100 fish that were stocked in each concentration
One of the duplicate tanks restocked on day 15
TBoth of the duplicate tanks restocked on day 15
Mean residual sulfur dioxide as sulfite (mg/1)
,95 percent confidence interval for the true probability of survival
The number of fish in each effluent concentration was reduced to 30 on day 53, and excess males were
subsequently removed prior to the spawning season.
-------
Table 25- NUMBER OF SECOND GENERATION P. PROMELAS SURVIVING
IN THE DECHLORINATED EFFLUENT3
30 Days of Age
No. Surviving
Conf. Interval
X Residual, mg/1
60 Days of Age
No. Surviving
Conf. Interval
X Residual, mg/1
Nominal Percent Dechlorinated Effluent
0.00
45
0.45-0.66
0.000
43
0.43-0.64
0.000
1.56
37
0.36-0.57
0.000
37
0.36-0.57
0.000
3.12
56
0.59-0.79
0.000
58
0.62-0.81
0.000
6.25
45
0.45-0.67
0.000
44
0.44-0.65
0.000
12.50
51
0.53-0.73
0.004
47
0.48-0.69
0.002
25.00
67
0.74-0.90
0.000
66
0.73-0.89
0.000
50.00
39
0.87-1.00
0.000
39
0.87-1.00
0.000
100.00
3
0.01-0.10
0.010
3
0.01-0.10
0.005
00
a40 fish were started in each of the two duplicate tanks of each effluent concentration, except for the
50 percent concentration for which only one tank with 40 fish was started.
95 percent confidence interval for the true probability of survival
°Mean residual sulfite.
-------
Table 26. NUMBER OF FIRST GENERATION P. PROMELAS SURVIVING IN CHLOROBROMINATED EFFLUENT
00
NJ
No. of fish alive
at day 23a
C.I.e for prob.
of survival
thru day 23
No. of fish alive
at day 53a
C.I. for prob.
of survival
thru day 53
No. of fish alive
from day 53 until
day 330 or deathf
Number of fish
alive at:
90 days
120 days
150 days
180 days
210 days
240 days
270 days
300 days
330 days
C.I. for prob.
of survival
day 53-330
Nominal Percent Chlorobrominated Effluent
0.00
95b
(0.000)
0.89-0.98
88
(0.000)
0.80-0.93
24
24
(0.000)
24
(0.000)
24
(0.000)
24
(0.000)
24
(0.000)
24
(0.000)
24
(O.OOOJ
23
(0.000)
23
(0.000)
0.79-0.99
1.56
62c
(0.000)
0.52-0.71
60
(0.000)
0.50-0.69
22
22
(0.001)
22
(0.001)
22
(0.002)
22
(0.003)
22
(0.003)
21
(0.003)
20
(0.003)
20
(0.003)
20
(0.003)
0.71-0.98
3.12
67C
(0.001)
0.57-0.75
67
(0.002)
0.57-0.75
23
23
(0.002)
23
(0.002)
23
(0.003)
23
(0.004)
23
(0.004)
23
(0.004)
21
CO. 004)
20
(0.004)
20
(0.004)
0.67-0.97
6.25
74c
(0.000)
0.65-0.82
71
(0.002)
0.61-0.79
23
23
(0.003)
23
(0.004)
23
(0.004)
23
(0.005)
23
(0.005)
23
CO. 00 5)_
19
(0.005)
19
(0.005)
19
(0.005)
0.62-0.97
12.50
96b
(0.002)
0.90-0.98
92
(0.006)
0.85-0.95
23
23
(0.007)
23
(0.008)
23
(0.008)
23
(0.008)
23
(0.008)
23
(0.008)
23
(0.008)
23
(0.008)
23
(0.007)
0.86-1.00
25.00
53
(0.008)
0.43-0.62
51
(0.019)
0.41-0.61
20
20
(0.021)
20
(0.022)
20
(0.020)
20
(0.019)
20
(0.019)
20
(0.018)
20
(0.018)
20
CO. 017)
20
(0.017)
0.84-1.00
50.00
65b
(0.018)
0.55-0.74
62
CO. 043)
0.52-0.71
24
24
(0.048)
24
(0.052)
24
(0.043)
23
(0.039)
23
(0.038)
23
(0.036)
23
(0.035)
23
_{p.033)
23
(0.033)
0.79-0.99
100.00
52t>
(0.054)
0.42-0.62
31
(0.129)
0.23-0.41
30
26
(0.144)
26
(0.142)
0
(0.141)
0
(0.141)
0
(0.1411
0
(0.127)
0
(0.122)
0
(0.119)
0
(0.119)
0.00-0.13
^From the original 100 fish that were stocked in each concentration
Both duplicate tanks restocked on day 15
.One of the duplicate tanks restocked on day 15
Ttean residual bromine chloride (mg/1)
C95 percent confidence interval for the true probability of survival
fThe number of fish in each effluent concentration was reduced to 30 on day 53, and excess males were
subsequently removed prior to the spawning season.
-------
chloride residuals of 0.628 mg/1 were measured. The mortality in 100 percent
effluent on day 121 occurred after the fish were exposed to measured bromine
chloride residuals of 0.020 mg/1 following three and one-half days when,
because of mechanical failures in the bromine chloride dosing system, the
test fish were exposed to nondisinfected effluent. Since other instances of
exposure to much higher bromine chloride residuals were not lethal to fat-
head minnows, it appeared that, just as with chlorinated effluent, fish in
chlorobrominated effluent developed a tolerance to levels of residual bromine
chloride which might have killed nonacclimated fish. This acclimation was
apparently lost during the three and one-half day period when the fish were
not exposed to residual bromine chloride.
Mortality of the second generation fish (Table 27) was observed only in the
100 percent and the 3.12 percent effluent concentrations after 30 and 60 days
of exposure. Considering that the bromine residual in the 100 percent ef-
fluent concentration was almost ten-fold greater than that in the 3.12 percent
effluent concentration, the observed mortalities do not appear to be due to
residual bromine chloride. Unlike their first generation counterparts, second
generation fish in 50 percent effluent did not appear to suffer any lethal
effects. However, the latter had the advantage of being subjected to less
variation in residual bromine levels because the effluent quality was consis-
tently high during their exposure period. Thus, even though first and second
generation fish in 100 and 50 percent effluent were subjected to nearly the
same mean residual bromine concentrations during their first month of life,
the highest values to which first generation fish were exposed were several
times higher than those to which second generation fish were exposed. These
data suggest that long-term exposure of first and second generation fathead
minnows to chlorobrominated effluent will not be lethal except when fish are
temporarily exposed to unnecessarily high levels of residual bromine chloride
or are suddenly exposed to chlorobrominated effluent following a period of
nonexposure.
Ozonated Effluent
First generation fish exhibited no lethal effects attributable to ozonated
effluent (Table 28). Most of the mortality after 53 days occurred between
days 219 and 276 of the test. During that interval a total of 14 male and
4 female fathead minnows died for no apparent reason in the 1.56, 3.12, 6.25,
and 25.00 percent effluent concentrations. A similar pattern of mortality
occurred in the chlorobrominated effluent stream where a total of 8 males died
during the same interval in effluent concentrations of 1.56, 3.12, and 6.25
percent. This mortality did not appear to be the result of exhaustion from
spawning in either of these effluent streams, since the fish that died had
not been the most productive. The exact cause of this mortality was never
determined.
Second generation fish reared in the ozonated effluent and exposed to approxi-
mately the same residual ozone levels as the first generation test animals
also failed to exhibit a definite lethal response pattern (Table 29). These
data suggest that long-term exposure to ozonated effluent will not be toxic
or lethal to fathead minnows.
83
-------
Table 27. NUMBER OF SECOND GENERATION P_. PROMEIAS SURVIVING
IN THE CHLOROBROMINATED EFFLUENT3
30 Days of Age
No . Surviving
Conf. Interval
X Residual, mg/1
60 Days of Age
No . Surviving
Conf . Interval
X Residual, mg/1
Nominal Percent Chlorobrominated Effluent
0.00
71
0.80-0.94
0.000
69
0.77-0.92
0.000
1.56
61
0.66-0.84
0.003
60
0.65-0.83
0.002
3.12
19
0.16-0.34
0.005
19
0.16-0.34
0.004
6.25
64
0.70-0.87
0.006
62
0.67-0.85
0.006
12.50
76
0.88-0.98
0.006
73
0.83-0.96
0.006
25.00
44
0.44-0.65
0.012
42
0.42-0.63
0.014
50.00
36
0.77-0.96
0.020
36
0.77-0.96
0.024
100.00
34
0.32-0.53
0.045
33
0.31-0.52
0.034
oo
40 fish were started in each of the two duplicate tanks of each effluent concentration, except for the
50 percent concentration for which only one tank with 40 fish was started.
95 percent confidence interval for the true probability of survival.
°Mean residual bromine chloride.
-------
Table 28. NUMBER OF FIRST GENERATION P. PROMELAS SURVIVING IN OZONATED EFFLUENT
00
1
No. of fish alive
at day 23a
C.I.d for prob.
of survival
thru day 23
No. of fish alive
at day 53a
C.I. for prob.
of survival
thru day 53
No. of fish alive
from day 53 until
day 330 or death*
Number of fish
alive at:
90 days
120 days
150 days
180 days
210 days
240 days
270 days
300 days
330 days
C.I. for prob.
of survival
day 53-330
Nominal Percent Ozonated Effluent
0.00
97b
(0.000)c
0.92-0.99
95
(0.000)
0.89-0.98
22
22
(0.000)
22
(0.000)
21
10.000)
20
(0.000)
20
10.000)
20
(0.000)
20
(0.000)
1_8
(0.000)
18
(0.000)
0.85-1.00
1.56
79
(0.000)
0.70-0.86
76
CO. 000)
0.67-0.83
15
15
(0.000)
15
10.000)
15
^0.000)
15
(0.001)
15
(0.001)
14
(0.001)
12
(0.001)
12
(0.001)
12
(0.001)
0.52-0.95
3.12
56
(0.000)
0.46-0.65
56
(0.000)
0.46-0.65
26
26
(0.000)
26
(0.000)
26
(0.0011
26
(0.001)
26
(0.001)
19
(0.001)
19
(0.001)
19
(0.001)
19
(0.001)
0.53-0.88
6.25
58
(0.000)
0.48-0.67
55
(0.000)
0.45-0.64
20
20
(0.000)
20
(0.001)
20
^O.OOIL
20
(0.001)
20
(0.001)
18
(0.001)
16
(0.001)
15
(0.001)
15
(0.001)
0.51-0.91
12.50
64
(0.000)
0.54-0.73
63
(0.000)
0.53-0.72
23
23
(0.000)
23
(0.001)
23
(0.001)
23
(0.002)
23
(0.002)
23
(0.002)
23
(0.002)
23
(0.002)
23
(0.002)
0.86-1.00
25.00
71
(0.000)
0.61-0.79
69
(0.000)
0.59-0.77
29
29
(0.001)
29
(0.002)
28
(0.002)
28
(0.002)
28
(0.002)
27
(0.003)
26
(0.003)
26
(0.003)
26 -
(0.003)
0.73-0.97
50.00
43
(0.003)
0.34-0.53
43
(0.002)
0.35-0.59
25
25
(0.003)
. 25
(0.004)
25
(0.004)
24
(0.004)
24
(0.004)
24
(0.004)
24
(0.004)
24
CO. 004)
24
(0.005)
0.80-0.99
100.00
94b
(0.010)
0.88-0.97
93
(0.016)
0.86-0.97
21
21
(0.014)
20
(0.012)
20
(0.012)
20
(0.012)
20
(0.012)
20
(0.012)
20
(0.011)
20
(0.011)
20
(0.012)
0.77-0.99
fFrom the original 100 fish that were stocked in each concentration
Both of the duplicate tanks restocked on day 15
TMean residual ozone (mg/1)
95 percent confidence interval for the true probability of survival
eThe number of fish in each effluent concentration was reduced to 30 on day 53, and excess males were
subsequently removed prior to the spawning season.
-------
Table 29. NUMBER OF SECOND GENERATION JP. PROMELAS SURVIVING
IN THE OZONATED EFFLUENT3
30 Days of Age
No . Surviving
Conf . Interval
» f*
X Residual, mg/1
60 Days of Age
No . Surviving
Conf . Interval
X Residual, mg/1
Nominal Percent Ozonated Effluent
0.000
60
0.65-0.83
0.000
60
0.65-0.83
0.000
1.56
32
0.30-0.51
0.002
31
0.29-0.50
0.001
3.12
60
0.65-0.83
0.003
60
0.65-0.83
0.003
6.25
35
0.33-0.55
0.004
35
0.33-0.55
0.003
12.50
49
0.50-0.71
0.004
48
0.49-0.70
0.003
25.00
46
0.47-0.68
0.004
46
0.47-0.68
0.004
50.00
48
0.49-0.70
0.004
46
0.47-0.68
0.005
100.00
26
0.23-0.43
0.013
26
0.23-0.43
0.012
oo
40 fish were started in each of the two duplicate tanks of each effluent concentration.
' 95 percent confidence interval for the true probability of surviving.
Mean residual ozone.
-------
A Comparison of Treatments
The comparison of respective effluent concentrations in the various effluent
streams was complicated by several major factors. First, the history of all
tanks was not uniform since some had been restocked on day 15 of the test.
Second, the variability of the mortality data in identical dilution water
control tanks of the various treatment streams could not be ignored. While
every effort was made to provide similar environments in each dilution water
control tank, analysis of the survival data, and other data as well, showed
that statistical differences were occasionally observed in the data from
these controls. Another factor which caused difficulty in treatment compari-
sons was that, after 53 days of age, first generation test fish appeared to
be more affected by accidents or mechanical failures than by exposure to the
various treatment systems per se. Thus, first generation data for the period
53-330 days of age failed to demonstrate a consistent lethal effect by any
treatment, even though differences were noted among the various treatments.
Both duplicate 100 percent effluent tanks were restocked with first generation
fish in all effluent streams except the chlorinated stream, where the highest
effluent concentration was only 20 percent. The data in Tables 20, 24» 26»
and 28 showed that fish reared in 100 percent dechlorinated and 100 percent
chlorobrominated effluent exhibited lethal effects at 23 and 53 days of age,
but fish reared in 100 percent nondisinfected and 100 percent ozonated efflu-
ents did not. The data from the second generation test animals neither
supported nor refuted this observation due to the exceptionally high mortality
in the 100 percent nondisinfected effluent stream and the generally lower
chemical residuals to which the test animals were exposed.
Both duplicate tanks of the 50 percent nondisinfected and 50 percent chloro-
brominated effluent were restocked with first generation fish, and at 23 and
53 days of age fewer fish survived the chlorobrominated effluent. None of
the tanks containing 50 percent concentrations of dechlorinated or ozonated
effluent were restocked, and no difference in survival to age 53 days was
observed in these two streams. The second generation fish exposed to 50 per-
cent effluent concentrations displayed similar survival patterns in all
streams, although survival in ozonated effluent appeared to be lower than
in dechlorinated and chlorobrominated effluent.
The survival patterns of first generation fish In 25 percent nondisinfected,
dechlorinated, chlorobrominated, and ozonated effluent were similar over the
first 53 days of the test. Comparison of the latter survival patterns with
those of first generation fish exposed to the 20 percent chlorinated effluent
revealed two important points: (1) the chlorinated effluent was toxic, and
(2) the toxicity of the chlorinated effluent stream was completely eliminated
by dechlorination with SC^-
Survival of second generation fish in 25 percent nondisinfected effluent
exceeded survivals in 25 percent chlorobrominated and ozonated effluent con-
centrations. However, since this would not correlate with the expected
relationship between effluent concentration and mortality, i.e., higher mor-
tality in higher effluent concentrations, we concluded that random mortality
87
-------
accounted for the observed differences. The mortality of second generation
fish in 20 percent chlorinated effluent exceeded the mortalities observed
in any of the 25 percent effluent streams. However, this lethal effect vas
eliminated by dechlorination.
No significant trends in mortality were observed in any of the lower effluent
concentrations. Thus, only the 100 percent dechlorinated effluent, 100 and
50 percent chlorobrominated effluent, and 20 percent chlorinated effluent were
lethal to fathead minnows. The lethality of 20 percent chlorinated effluent
was eliminated by dechlorination, and the data in Table 24 suggested that the
lethal effects of chlorinated effluent concentrations of up to at least 50
percent were eliminated by S02« The excessive mortality in 100 percent de-
chlorinated effluent may have been the result of products formed by the addi-
tion of S02, by inadequate neutralization of residual chlorine, by compounds
formed in the chlorination process and not destroyed in the dechlorination
process, or by the inherent lethality that was observed in the 100 percent
concentration of the nondisinfected stream.
The findings on the chronic toxicity of chlorinated effluent agree well with
the work of Arthur, .^.al..1 who found that 5 and 10 percent effluent concen-
trations with mean total residual chlorine levels of 0.042 and 0.110 mg/1,
respectively, were lethal to fathead minnows, and that the application of
sulfur dioxide eliminated the toxicity of those effluents. Other investi-
gators have reported success in detoxifying chlorinated effluent with sodium
thiosulfate6 and bisulfite.3
GROWTH
When reviewing the growth data of all first generation test animals, some
consideration of the restocking history of the various tanks must be given.
A comparison of growth in restocked and nonrestocked duplicate tanks indi-
cated that restocked fish did not grow as well as their nonrestocked counter-
parts. Thus, an inherent bias for reduced length was assumed in any test
condition where restocking occurred on day 15. Further, one should note the
variability in growth of both generations of test animals in the dilution
water control tanks, (Tables 35-39) where environmental conditions were, for
all practical purposes, identical. One environmental variable which was not
measured was the quantity of microorganisms available as a food supply for
first generation fish. The importance of this factor was well illustrated
by the fact that when special attention was given to insure that an adequate
supply of microorganisms was available as food for the second generation fry,
they grew approximately twice as long in their first 30 days of life as the
first generation test animals grew in their first 23 days of life.
The first generation test fish reared in nondisinfected effluent showed no
significant differences in length at the termination Cday 330) of the study
(Table 30). However, the first generation fish reared in 50 and 100 percent
concentrations of nondisinfected effluent were significantly shorter in mean
length at 23 and 53 days of age than the fish reared in dilution water control
tanks. Similar retardation of growth was not exhibited by the second genera-
88
-------
Table 30. MEAN LENGTHS (IN mm) OF FIRST AND SECOND GENERATION £. promelas
REARED IN NONDISINFECTED EFFLUENT AND IN DILUTION WATER
00
vO
First Generation
1 Second Generation
Age of Fish
and Data
23 days
N
X
S.D.
S> *
S<
53 days
N
X
S.D.
S}
S<
330 days
N
X
S.D.
S>,
S <
30 days
N
5
S.D.
S>
S<
60 days
N
X
S.D.
S>
S<
Nominal Percent Noq
Dilution H_0
(A)
87
13.13
1.54
B-H
84
33.29
3.32
G.H
24
68.02
2.02
54
22.18
3.16
D
54
35.32
3.45
B.D
1.56
(B)
71b
11.86
2.22
G,H
A
71
32.70
5.00
G,H
25
69.50
9.47
35
22.07
2.35
D
35
38.23
2.78
A,G
3.12
(C)
64b
11.86
2.23
G,H
A
63
32.41
4.47
G,H
21
72.48
9.56
44
22.78
3.25
G
D
41
37.43
3.87
G
isinfected Effluent Concentration
6.25
(D)
76b
11.38
1.80
G,H
A
74
32.66
3.91
G,H
19
71.47
11.15
46
25.04
1.98
A-C.E-H
47
38.00
3.40
A,G
12.50
(E)
77b
11.55
2.26
G,H
A
78
31.58
3.92
G,H
23
68.07
10.85
37
21.34
2.94
D
35
35.73
2.84
25.0
(F)
79b
11.65
1.94
G,H
A
78
31.37
3.71
G,H
28
67.05
7.79
72
22.58
2.72
D
72
36.28
2.80
50.0
(G)
96C
9.86
1.37
A-F
89
28.92
3.04
A-F
25
68.02
9.35
31
20.15
1.77
l
C.D
31
33.65
1.91
B.D
100.0
(H)
92C
9.89
1.24
A-F
85
28.04
2.92
A-F
24
63.48
7.54
4
19.00
1.15
D
4
33.00
1.41
^Significantly different (P=0.05) by Scheffe's analysis of variance test.
One of the duplicate tanks restocked on day 15 of the test.
°Both of the duplicate tanks restocked on day 15 of the test.
-------
tion test animals reared in the two highest effluent concentrations, but
this observation should be viewed in light of the fact that only four fish
survived in the 100 percent effluent concentration. Thus, it appears that
the differences observed in the first generation fathead minnows were prob-
ably related to the different restocking histories of the dilution water
tanks, which were not restocked, and the 50 and 100 percent effluent tanks,
which were both restocked on day 15. Another possible factor, which might
have influenced the observed differences, was the poor quality of effluent
during the first few months of the project compared to the higher quality
effluent during the remainder of the project.
While the data were not always significantly different, fathead minnows
reared in the lower concentrations of nondisinfected effluent tended to
attain greater mean lengths than those reared in the higher concentrations.
A similar tendency toward greater growth at the lower effluent concentra-
tions was also seen in each of the disinfected effluent streams (Tables
31-34). The consistency of this pattern suggests that high concentrations
of either nondisinfected or disinfected effluent were detrimental to the
growth of £. promelas.
First generation fish exposed to various concentrations of chlorinated
effluent showed no significant differences in length at day 330 (termin-
ation) of the study (Table 31). However, those animals maintained in 20
and 14 'percent chlorinated effluent were significantly smaller than the
dilution water controls at 53 days and the fish in the lower effluent
concentrations at 23 and 53 days. Similarly, second generation test fish
were significantly shorter than their dilution water controls at 30 days
of age. Thus, mean total residual chlorine levels as low as 0.045 mg/1
were adequate to suppress the growth of fathead minnows. This threshold
value is less than that found by Arthur, et al.1 (0.079-0.096 mg/1) to
retard the growth of P_. promelas. The maximum total residual chlorine
level of 0.01 mg/1 recommended by Brungs? to protect the more resistant
fish species continuously exposed to chlorinated effluent appears to be
supported by this study.
The first generation of ]?. promelas reared in various concentrations of
dechlorinated effluent showed no significant differences in length at 330
days (termination) of the life-cycle test (Table 32). Although both the
first and second generation test animals exposed to 100 percent dechlori-
nated effluent were smaller than the other fish at 53 and 60 days of age,
respectively, the validity of the second generation data is questionable
since only three fish survived to the age of 30 days. This pattern is
almost identical to that observed in the nondisinfected effluent (Table 30)
where only four second generation fish survived in the 100 percent de-
chlorinated effluent. Thus, it appears that the factor(s) responsible
for growth inhibition and mortality in the dechlorinated effluent was
(were) also responsible for growth inhibition and mortality in the non-
disinfected effluent. This conclusion is further supported by the fact
that the maximum mean sulfite residual to which our fish were exposed
was 0.025 mg/1 (range of 0.000-0.610 mg/1) over the 330 day test period,
a concentration considerably below the mean sulfite residual of 0.104 mg/1
90
-------
Table 31. MEAN LENGTHS (IN mm) OF FIRST AND SECOND GENERATION P_. promelas
REARED IN CHLORINATED EFFLUENT AND IN DILUTION WATER
First Generation
| Second Generation
Age of Fish
and Data
23 days
N
*
S>Da
X Resid. d
53 days
N
X
S.D.
s>
Si
X Resid.
330 days
N
*
S.D.
s>
SC
X Resid.
30 days
N
X
S.D.
S>
!<
X Resid.
60 days
X
S.D.
S>
s<
X Resid.
Nominal Percent Chlorinated Effluent Concentration
Dilution H.O
(A) 2
66b
10.53
1.68
B.C.E
0.000
64
30.67
3.89
G,H
C
24
68.35
7.84
53
19.53
2.74
H
B,C,E,F
0.000
53
34.76
3.21
0.000
2.35
(B)
71b
12.39
1.95
D.F-H
0.002
69
31.92
4.13
F.G.H
C
0.004
20
69.63
10.64
0.008
52
22.78
2.00
A.G.H
0.008
51
36.18
2.82
0.008
3.36
(C)
52
12.68
1.39
D.F-H
0.003
52
34,54
3.29
A.B.D-H
0.006
22
69.34
10.37
0.011
63
23.43
1.96
A.D.E.G.H
0.004
64
35.95
2.97
0.009 J
4.80
(D)
67b
11.10
1.44
B,C,E
0.007
65
30.96
3.84
G,H
C
0.016
24
68.60
10.20
0.022
59
21.15
2.54
C
0.018
59
35.23
3.28
0.020
6.86
(E)
81b
12.82
2.22
A.D.F-H
0.010
80
31.83
3.90
F-H
0.016
18
70.78
10.05
0.024
67
21.42
2.87
A
C
0.016
66
34.92
3.80
0.016
9.80
(F)
93C
10.27
1.66
B.C.E
0.013
84
28.95
3.90
B,C,E
0.025
23
67.24
9.39
0.035
62
22.08
1.94
A.G.H
0.028
62
34.70
2.27
0.033
14.00
(G)
95°
10.37
1.71
B.C.E
0.020
71
27.80
3.67
A-E
0.045
13
70.08
9.79
0.067
49
17.95
3.27
B-F
0.033
47
34.13
3.48
0.035
20.00
(H)
87C
10.64
1.29
B.C.E
0.038
43
26.84
2.88
A-E
0.076
1
52.00
0.00
0.102
26
16.35
2.25
A-f
0.045
26
34.77
3.37
0.033
Significantly different (P=0.05) by Scheffe's analysis of variance test.
One of the duplicate tanks restocked on day 15 of the test.
°Both of the duplicate tanks restocked on day 15 of the test.
Residual chlorine in mg/1.
-------
Table 32. MEAN LENGTHS (IN mm) OF FIRST AND SECOND GENERATION P_. promelas
REARED IN DECHLORINATED EFFLUENT AND IN DILUTION WATER
vO
Age of Fish
and Data
! 23 days
1
N
X
' S.D.
s>a
B
_ ' a
N. r\
X Resid?
.3 i 53 days
en N
M
0
X
g i S.D.
& s>
•u
CO
Tl
b
•H
U
a
u
S
o
•o
o
u
IU
CO
s<
X Resid.
330 days
N
X
S.D.
S>
S<
X Resid.
30 days
N
X
S.D.
s>
I<
X Resid.
60 days
N
X
S.D.
s>
S <
X Resid.
Nominal, Percent Dechlorinated Effluent Concentration
Dilution H~0
(A) 2
94C
10.15
1.57
—
C,E,G
0.000
79
30.79
3.60
H
C
0.000
23
69.30
9.49
—
—
0.000
45
19.18
3.03
—
B-G
0.000
43
36.15
3.81
F,H
—
0.000
1.56
(B)
u
60b
11.18
1.85
F
G
0.000
49
32.91
3.09
F,H
—
0.000
21
69.50
11.10
—
—
0.000
36
23.63
3.10
A,H
0.000
37
36.60
3.46
F-H
—
3.12
(C)
h
56b
11.86
1.61
A.D.F.H
G
0.000
56
34.05
3.46
A.F.H
—
0.000
25
68.20
7.66
—
—
0.001
56
21.92
3.54
A,H
E
0.000
58
34.80
4.81
H
E
0.000 i 0.000
6.25
(D)
p
75°
10.37
1.43
—
C,G,
0.008
60
31.53
3.08
H
—
0.010
23
71.07
10.79
„
—
0.005
45
22.76
2.52
A,H
0.000
44
35.51
3.00
H
E
0.000
12.50
(E)
b
68
11.44
2.69
A,F
G
0.002
64
31.55
3.91
F,H
—
0.010
24
68.71
8.15
—
—
0.003
51
24.21
1.77
A.C.H
0.004
47
38.17
3.16
C.D.F-H
—
0.002
25.0
(F)
83°
10.04
1.37
—
B.C.E.G
0.000
65
29.09
3.06
—
B.C.E.G
0.000
18
69.58
8.52
—
—
0.005
67
22.58
2.20
A,H
0.000
66
33.70
2.58
—
A.B.E
0.000
50.0
(G)
59
13.72
1.42
A-F.H
—
0.000
56
32.51
1.89
F,H
—
0.000
100.0
(H)
39°
10.13
1.19
—
C,G
0.080
36
27.21
2.72
—
A-E.G
0.042
23 21
69.13 > 66.67
11.52 9.33
— ; — -
—
0.012
39
22.50
1.94
A,H
0.000
39
33.64
2.36
—
B,E
0.000 .
—
0.025
3
16.00
3.04
— .
B-G
0.010
3
27.33
2.25
•'" "
A-E
0.005
^Significantly different (P=0.05) by Scheffe's analysis of variance test.
One of the duplicate tanks restocked on day 15 of the test.
jBoth of the duplicate tanks restocked on day 15 of the test.
"Slg/l residual sulfite.
-------
Table 33. MEAN LENGTHS (IN mm) OF FIRST AND SECOND GENERATION £. promelas
REARED IN CHLOROBROMINATED EFFLUENT AND IN DILUTION WATER
vo
Age of Fish
and Data
First Generation
Second Generation
23 days
N
X
S.D.
S>j
S .
X Resid.
53 days
N
X
S.D.
S>
_s/
X Resid.
330 days
N
S
S.D.
S>
_s <
X Resid.
30 days
N
X
S.D.
S>
_s <
X Resid.
60 days
N
X
S.D.
s>
sC
X Resid.
Nominal Percent Chlorobrominated Effluent Concentration
Dilution U,0
(A)
95b
10.51
1.35
F
0.000
90
29.57
3.62
H
B,F
24
68.75
9.85
71
21.87
1.97
E
E
0.000
69
34.83
2.13
H
C
0.000
1.56
(B)
62C
11.35
1.54
G,H
F
0.000
60
31.97
4.05
G,H
0.000
20
67.98
9.84
0.003
61
23.53
2.25
F-H
0.003
60
35.13
2.48
H
C
0.002
3.12
-------
Table 34. MEAN LENGTHS {IN mm) OF FIRST AND SECOND GENERATION £. promelas
REARED IN OZONATED EFFLUENT AND IN DILUTION WATER
vO
Age of Fish
and Data
First Generation
I Second Generation
23 days
N
X
S.D.
*>*
S <
X Resid.c
53 days
N
1c
S.D.
s >
_s<
X Resid.
330 days
N
X
S.D.
S>
s C
X Resid.
30 days
N
X
S.D.
s y
sc
X Resid.
60jtays
N
X
S.D.
sC
-s>
X Resid.
Nominal Percent Ozonated Effluent Concentrations
Dilution Water
(A)
96b
11.18
1.76
B-6
0.000
95
30.03
3.63
B-F
0.000
17
69.88
10.64
0.000
60
21.04
3.21
G
B.D.F
0.000
61
36.59
4.04
0.000
1.5
(B)
7-9
13.67
2.52
A,H
0.000
76
33.26
4.10
A,H
0.000
12
75.33
12.25
0.001
32
24.13
3.85
G,H
0.002
31
36.823
4.52
0.001
3.1
(C)
55
13,71
2.38
A,H
0.000
56
33.92
4.58
A,H
0.000
19
62.79
5.74
0.001
60
22.47
2.28
G
0.003
60
34.44
2.73
D,F
0.003
6.2
(D)
58
14.25
2.24
A,H
0.000
55
34.24
6.16
AtH
0.000
14
68.07
9.47
0.001
35
24.16
2.59
G,K
0.004
35
37.09
3.15
C
0.003
12.5
(E)
64
14.49
2.64
A,H
0.000
62
32.99
3.21
A,H
0.000
23
68.74
8.81
0.002
49
21.98
2.35
G
0.004
48
36.78
2.78
0.003
25.0
(F)
71
14.67
2.11
A.H
0.000
69
33.23
3.58
A,H
0.000
27
65.22
7.53
0.003
46
23.85
3.02
G,H
0.004
46
36.99
3.15
C
0.004
50.0
(G)
43
14.01
1.78
A,H
0.003
43
31.86
4.20
H
0.002
24
68.46
8.92
0.005
48
18.37
3.52
•A— F
0.004
46
35.53
3.88
, 0.005
100.0
(H)
93b
11.26
1.29
B-G
0.010
93
29.23
3.24
B-G
0.016
20
68.60
8.22
0.012
26
20.67
2.03
B.D.F
0.013
26
36.87
2.81
0.012
^Significantly different (P=0.05) by Scheffe's analysis
Both of the duplicate tanks restocked on day 15 of the
cResidual ozone in mg/1.
of variance test.
test.
-------
(range of 0.020-0.700 mg/1) found by Arthur, «££!., to have no adverse
effects on fathead minnows. Thus, dechlorination with SC»2 appeared to
eliminate any inhibition of growth in fathead minnows that may have result-
ed from the chlorination process.
The fathead minnows that survived in chlorobrominated effluent and dilution
water exhibited no significant differences in length at the termination C330
days) of the life cycle test (Table 33)• As with the other effluent streams
that were tested, maximum growth occurred at some low to intermediate efflu-
ent concentration (3.12-25.0 percent). First generation fish reared in 100
percent chlorobrominated effluent were significantly smaller at 53 days than
those fish reared in the dilution water control or in any lower dilution of
chlorobrominated effluent. The same pattern was observed in second gener-
ation fish exposed to 100 percent effluent. Since the fish reared in 100 per-
cent concentrations of some of the other effluent streams, including the non-
disinfected stream, exhibited reduced growth, there was no reason to attribute
the reduction of the length of fish reared in 100 percent chlorobrominated
effluent to the disinfection treatment.
At the termination of the 330 day life cycle study no significant differences
were observed between the lengths of fathead minnows reared in dilution water
and those reared in ozonated effluent (Table 34). It is important to note
that no significant differences in mean length between fish exposed to dilu-
tion water and those exposed to 100 percent ozonated effluent were ever
recorded. As in all other effluent streams, the maximum growth of fathead
minnows always occurred in one of the lower effluent concentrations (1.50 to
25.00 percent) rather than in dilution water or 100 percent effluent.
Tables 35 to 39 summarize all of the foregoing bioassay data by displaying
effluent concentrations versus effluent type for each age group of test
animals. A comparison of both the statistically significant differences and
the trends in the growth of fish in the different effluent streams indicates
that both generations of P_. promelas reared in 100 percent ozonated effluent
grew larger than fish reared in all other 100 percent effluent concentrations.
This greater growth in undiluted ozonated effluent may have resulted from a
conditioning of the effluent as the result of the relatively high dissolved
oxygen concentration in the ozonated stream prior to delivery to the fish
tanks,
A second generalization that can be made from a study of Tables 35-39 is that
the growth of fathead minnows exposed to mean total chlorine residuals of
0.045 mg/1 or higher in 14 or 20 percent effluent concentrations during their
first two months of life was inferior to the growth of fish reared in compa-
rable concentrations of the other effluent streams. This growth retardation
effect was eliminated by dechlorination as evidenced by the fact that mean
lengths of fish in 50 and 100 percent dechlorinated effluent were comparable
to those in the respective concentrations of the other effluent streams.
Table 40 summarizes the weights of the first generation test animals measured
at the termination of the life cycle study (330 days). No significant differ-
ences were observed in either the lengths (Table 37) or weights of the first
95
-------
Table 35. MEAN LENGTHS (IN mm) OF FIRST GENERATION £. promelas
AT DAY 23 OF THE LIFE CYCLE TEST
Nominal Effluent
Concentration
and Data
Dilution Water N
X
S.D,
s> *
s
S<
X Resid.
3.12% N
X
S.D.
s>
S<
X Resid.
6.25% N
X
S.D.
s>
_ s<
X Resid.
12.50% N
X
S.D.
s>
_ s<
X Resid.
25.00% N
X
S.D.
S
s
X Resid.
50.00% N
X
S.D.
S >
_ s<
X Resid.
100.00% N
X
S.D.
Sl
s <
X Resid.
Nondis.
(A)
87
13.13
1.54
B.C.D.E
711
11.86
2.22
E
64f
11.86
2.23
E
76f
11.38
1.80
C
B,E
77r
11.55
2.26
B.D
E
79*
11.65
1.94
B,C
D,E
968
9.86
1.37
C,E
928
9.89
1.24
E
Eff
Chlor.
(B)
66f
10.53
1.68
A
a
52b
12.68
1.39
D
0.00?
8ic,f
12.82
2.22
A.C.D
E
0.010
95° '8
10.37
1.71
A.C.E
0.020
87e»B
10.64
1.29
A.D.E
0.038
a
a
uent Stream
Dechlor .
(C2
948
10.15
1.57
A.E
60r
11.18
1.85
E
0.000
56r
11.86
1.61
E
0.000
758
10.37
1.43
A,B,E
0.008
68f
11.44
2.69
B,D
E
0.002
838
10.04
1.37
A.D.E
0.000
59
13.72
1.42
A,D
0.000
398
10.13
1.19
E
O.OftO
Chlorobr .
ty
958
10.51
1.35
A
62£
11.35
1.54
E
0.000
67*
11.14
1.66
B.E
0.001
74*
10.93
1.98
B,E
0.000
968
10.40
1.39
A.C.E
0.002
53
12.89
1.59
A.B.C
E
0.008
65*
10.10
1.26
C,E
0.018
528
9.87
1.42
E
0.054
Ozon.
(E)
96*
11.18
1.76
C
A
79
13.67
2.52
A.C.D
0.000
55
13.71
2.38
A.C.D
O.QOQ
58
14.25
2.24
A.B.C.D
0.000
64
14.49
2.64
A,B,C,D
0.000
71
14.67
2.11
A.B.C.D
0.000
43
14.01
1.78
A.D
0.003
938
11.26
1.29
A.C.D
0.010
?No comparable chlorinated effluent concentration.
Nominal 3.36 percent chlorinated effluent concentration.
.Nominal 6.86 percent chlorinated effluent concentration.
Nominal 14.09 percent chlorinated effluent concentration.
,Nominal 20.00 percent chlorinated effluent concentration.
One of the duplicate tanks restocked on day 15.
ffioth of the duplicate tanks restocked on day 15.
Significantly different (P<0.05) by Scheffe's analysis of variance test.
96
-------
Table 36. MEAN LENGTHS (IN nun) OF FIRST GENERATION £. promelas
AT DAY 53 OF THE LIFE CYCLE TEST
Nominal Effluent
Concentration
and Data
Dilution Water N
X
S.D.
qx h
s
S <
X Resid.
3.12% N
X
S.D.
S>
S <
X. Resld.
6.25% N
X
S.D.
S>
S<
X Resid.
12.50% N
X
S.D.
S
S
X Resid.
25.00% N
X
S.D.
S2
S<
X Resid.
50.00% N
X
S.D.
S >
s<
X Resid.
100.00% N
X
S.D.
S>
S <
X Resid.
Effluent Stream
Nondis.
(A)
84
33.29
3.32
B.C.D.E
71'
32.70
5.00
63i
32. 41
4. 47
D
B
74*
32.66
3.91
D
78r
31.58
3.92
B
781
31.37
3.71
B,C
D,E
89g
28.92
3.04
C,E
858
28.04
2.92
D
Chlor.
(B)
64*
30.67
3.89
A •
a
52b
34.54
3.29
A,D
0.006
80e'*
21.83
3.90
E
0.016
71a,B,
27.80
3.67
A.D.E
0.045
43e>8
26.84
2.88
A.D.E
0.076
a
a
Dechlor .
(0
798
30.79
3.60
A
49f
32.91
3.09
0.000
56E
34.05
3.46
D
0.000
60g
31.53
3.08
E
0.010
64f
31.55
3.91
B
0.010
65*
29.09
3.06
B
A.D.E
0.000
56
32.51
1.89
A,D
0.000
368
27.21
2.72
0
0.042
Chlorobr .
(D)
908
29.57
3.62
A
601
31.97
4.05
0.000
67f
30.39
3.15
A.B.C.E
0.002
70f
30.71
3.92
A,E
0.002
928
30.01
3.15
B
E
0.006
50
34.11
3.20
A.B.C
0.019
62f
28.35
3.17
C,E
0.043
318
23.39
2.14
A.C.E
0.129
Ozon.
(E)
958
30.03
3.63
A
76
33.26
4.10
0.000
56
33.92
4.58
D
0.000
55
34.24
6.16
B,C,D
0.000
62
32.99
3.21
B,D
0.000
69
33.23
3.58
A,B,D
0.000
43
31.86
4.20
A,D,
0.002
93«
29.23
3.24
D
0.016
*No comparable chlorinated effluent concentration.
Nominal 3.36 percent chlorinated effluent concentration.
^Nominal 6.86 percent chlorinated effluent concentration.
Nominal 14.09 percent chlorinated effluent concentration.
^Nominal 20.00 percent chlorinated effluent concentration.
One of the duplicate tanks restocked on day 15.
?Both of the duplicate tanks restocked on day 15.
Significantly different (P(0.05) by Scheffe's analysis of variance test.
97
-------
Table 37, MEAN LENGTHS (IN mml OF FIRST GENERATION £. promelas
AT THE TERMINATION (DAY 330) OF THE LIFE CYCLE TEST
Nominal Effluent
Concentration
and Data
Dilution Water N
X
S-.D.
oV h
s>(h
1.56% N
X
S.D.
s>
s <
X Resid.
3.12% N
X
S.D.
s)
s<
X Resid.
6.25% N
X
S.D.
s>
S<
X Resid.
12.50% N
X
S.D.
S>
s<
X Resid.
25.00% N
X
S.D.
s>
s<
X Resid.
50.00% N
X
S.D.
S>
_ s<
X Resid.
100.00% N
X
S.D.
s>
s<
X Resid.
Effluent Stream
Nondis.
(A)
24
68.02
2.02
25f
69.50
9.47
21r
72.48
9.56
E
19*
71.47
11.15
23f
68.07
10.85
28t
67.05
7.79
25s
68.02
9.35
24*
63.48
7.54
Chlor .
(B)
24*
68.35
7.84
a
22"
69.34
10.37
0.011
18C>£'
70.78
10.05
0.024
13a'S
70.08
9.79
0.067
!«,&• '
52.00
0.00
0.102
a
a
Dechlor .
(9
23B
69.30
9.49
2lf
69.50
11.10
0.000
25'
68.20
7.66
0.001
23s
71.07
10.79
0.005
24'
68.71
8.15
0.003
188
69.58
8.52
0.005
23
69.13
11.52
0.012
21s
66.67
9.33
0.025
Chlorobr.
(D)
24B
68.75
9.85
20f
67.98
9.84
0.003
20£
67.45
8.88
0.004
17£
67.27
8.68
0.005
238
70.39
11.86
0.007
20
69.80
9.48
0.017
2 If
70.36
9.73
0.033
0s
0,119
OZOTl.
(E)
17S
69.88
10.64
12
75.33
12.25
0.001
19
62.79
5.74
A
0.001
14
68.07
9.47
0.001
23
68.74
8.81
0.002
27
65.22
7.53
0.003
24
68.46
8.92
0.005
20s
68.60
8.22
0.012
"No comparable chlorinated effluent concentration.
Nominal 3.36 percent chlorinated effluent concentration.
^Nominal 6.86 percent chlorinated effluent concentration.
Nominal 14.09 percent chlorinated effluent concentration.
^Nominal 20.00 percent chlorinated effluent concentration.
One of the duplicate tanks restocked on day 15.
8Both of the duplicate tanks restocked on day 15.
hSignlflcantly different (P <0.05) by Scheffe's analysis of variance test.
98
-------
Table 38. MEAN LENGTHS (IN mm) OF 30 DAY OLD SECOND GENERATION
P. promelas IN THE LIFE CYCLE TEST
Nominal Effluent
Concentration
and Data
Dilution Water N
X
S.D.
S>f
S
S <
X Resid.
3.12% N
X
S.D.
S >
s.<
X Resid.
6.25% N
X
S.D.
S>
S<
X Resid.
12.50% N
X
S.D.
S>
S <
X Resid.
25.00% N
X
S.D.
S N
1 <
X Resid.
50.00% N
X
S.D
s>
1 <
X Resid.
100.00% N
X
S.D.
s>
s<
X Resid.
Effluent Stream
Nondis.
(A)
54
22.18
3.16
B,C
35
22.07
2.35
E
44
22.78
3.25
46
25.04
1.98
B,C,D
37
21.34
2.94
B
C,D
72
22.58
2.72
B
31
20.15
1.77
C
4
19.00
1.15
D
Chlor .
(B)
53
19.53
2.74
A.D
a
Dechlor.
(0
45
19.18
3.03
A.D.E
36
23.63
3.10
0.000
56
- 23.43 21.92
1.96 3.54
0.004 0.000
67C 45
21.42 22.76
2.87 2.52
A,D,E A
0.016 • 0.000
49d 51
17.95 : 24.21
3.27 : 1.77
— ; A,B,E
A.C.D.E j —
0.033 I 0.004
26e
16.35
2.25
A,C,D,E
0.045
a
a
67
22.58
2.20
B
0.000
39
22.50
1.94
A,D,E
0.000
3
16.00
3.04
0.010
Chlorobr.
(D)
r 71
21.87
1.97
B,C
61
23.53
2.25
0.003
19
22.00
3.33
0.005
64
23.25
2.92
B
A
0.006
76
24.18
2.36
A.B.E
0.006
45
21.11
2.58
B
E
0.012
36
20.28
2.11
E
C
0.020
34
13.25
2.13
A,E
0.045
Ozon.
(E)
60
21.04
3.21
C
32
24.13
3.85
A
0.002
60
22.47
2.28
0.003
35
24.16
' 2.59
B
0.004
49
21.98
2.35
B
1 C,D
0.004
46
23.85
3.02
B,D
' 0.004
48
18.37
'[ 3.52
; C.D.
! 0.004
26
20.67
1 2.03
; D
i 0.013
?No comparable chlorinated effluent concentration.
Nominal 3.36 percent chlorinated effluent concentration.
CNominal 6.86 percent chlorinated effluent concentration.
Nominal 14.09 percent chlorinated effluent concentration.
6Nominal 20.00 percent chlorinated effluent concentration.
Significantly different (P<0.05) by Scheffe's analysis of variance.
99
-------
Table 39. MEAN LENGTHS (IN mm) OF 60 DAY OLD SECOND GENERATION
P. promelas IN THE LIFE CYCLE TEST
Nominal Effluent
Concentration
and Data
Dilution Water N
X
S.D.
ll'<
1.56% N
X
S.D.
S>
S<
X Resid.
3.12% N
X
S.D.
S>
S<
X Resid.
6.25% N
X
S.D.
S^
S<
X Resid.
12.50% N
X
S.D.
S^
s<
X Resid.
25.00% N
X
S.D.
s>
S<
X Resid.
50.00% N
X
S.D.
S>
_ s <
X Resid.
100.00% N
X
S.D.
S>
s<
X Resid.
Effluent Stream
Nondis.
(A)
54
35.32
3.45
35
38.23
2.78
D
41
37.43
3.87
C,E
Chlor .
(B)
53
34.76
3.21
a
64°
35.95
2.97
0.009
47 66C
38.00 34.92
3.40 3.80
B,C,D —
— A,E
0.0016
35
35.73
2.84
C
72
36.28
2.80
C
31
33.65
1.91
4
33.00
1.41
D
47d
34.13
3.48
C.D.E
0.035
26e
34.77
3.37
0.033
a
a
Dechlor .
(C)
43
36.15
3.81
37
36.60
3.46
0.000
58
34.80
4.81
A.D
0.000
44
35.51
3.00
A
0.000
47
38.17
3.16
A,B
0.002
66
33.70
2.58
A,E
0.000
39
33.64
2.36
0.000
3
27.33
2.25
E
0.005
Chlorobr.
(D)
69
34.83
2.13
60
35.13
2.48
A
0.002
19
38.45
2.92
C,E
0.004
62
35.85
3.13
A
0.006
73
36.62
3.19
B
0.006
43
35.62
3.33
0.014
36
32.64
2.41
E
0.024
33
22.99
5.26
A,E
0.034
Ozon.
(E)
61
36.59
4.04
31
36823
4.52
0.001
60
34.44
2.73
A,D
0.003
35
37.09
3.14
B
0.003
48
36.78
2.78
B
0.003
46
36.99
3.15
C
0.004
46
35.53
3.88
D
0.005
26
36.87
2.81
C,D
0.012
?No comparable chlorinated effluent concentration.
Nominal 3.36 percent chlorinated effluent concentration.
^Nominal 6.86 percent chlorinated effluent concentration.
Nominal 14.09 percent chlorinated effluent concentration.
^Nominal 20.00 percent chlorinated effluent concentration.
Significantly different (P\0.05) by Scheffe's analysis of variance.
100
-------
Table 40. MEAN WEIGHTS (IN GRAMS) OF FIRST GENERATION f_. promelaa
AT THE TERMINATION (330 DAYS) OF THE LIFE CYCLE TEST
Nominal Effluent
Concentration
and Data
Dilution Water
Na
X Weight
S.D.E
1.56%
N
X Weight
I.D.
X Residual
3.12%
N
X Weight
J5.D.
X Residual
6.25%
N
X Weight
S.D.
X Residual 1
12.50%
N
X Weight
S.D.
X Residual
25.0%
N
X Weight
S_.V.
X Residual
50.0%
N
X Weight
S^.D.
X Residual
100.0%
H
X Weight
£.D.
X Residual
Effluent Stream
Nondisinfected
24
3.25
1.69
25
3.56
1.98
21
4.10
2.10
19
3.86
2.14
23
3. A3
1.98
28
3.11
1.38
25
3.19
1.49
24
2.75
1.12
Chlorinated
24
3.51
1.71
d
22e
3.27
1.92
0.011
18f
3.71
1.85
0.024
138
3.67
2.00
0.067
lh
1.1
0.102
d
d
Dechlorinated
23
3.39
1.71
21
3.63
2.16
0.000
25
3.20
1.51
0.001
23
3.90
2.16
0.005
24
3.31
1.55
0.003
18
3.89
1.88
0.005
23
3.61
1.66
0.012
21
3.33
1.40
0.025
Chlorobrorainated
24
3.45
1.79
20
3.42
1.95
0.003
20
3.13
1.66
0.004
17
3.01
1.50
0.005
23
3.77
2.39
0.007
20
3.69
1.90
0.017
21
4.03
1.89
0.033
0
0.119
Ozonated
17
3.80
2.10
12
5.18
2.55
0.001
19
2.47
0.97
0.001
14
3.53
1.86
0.001
23
3.54
1.97
0.002
27
2.67
1.27
0.003
24
3.23
1.47
0.005
20
3.69
1.43
0.012
fw = Sample size.
S.D. =• Standard deviation.
*TX Residual in mg/1.
No comparable chlorinated effluent concentration.
^Nominal 3.36 percent chlorinated effluent concentration.
Nominal 6.86 percent chlorinated effluent concentration.
^Nominal 14.00 percent chlorinated effluent concentration.
Nominal 20.00 percent chlorinated effluent concentration.
101
-------
generation test fish. This suggests that time, and perhaps the improved
effluent quality, had a normalizing effect on the size of the test animals.
This observation lends further support to the conclusion that young JP. pro—
melas are more sensitive to adverse environmental conditions than older,
more mature members of the species.
REPRODUCTION
The effects of the various effluent streams on the reproductive functions
of the fathead minnow were analyzed from the standpoint of egg production
and egg hatchability. Egg production is defined as the numbers of viable
eggs produced per female or per spawning in each concentration of each
effluent stream. Hatchability refers to the percentage of eggs that hatched
in incubation attempts in the various concentrations of the different efflu-
ent streams. Significant differences (P=0.05) between concentrations within
the same effluent stream were determined by Tukey's two-tailed analysis of
variance test.
Table 41 summarizes the total number of eggs produced in all effluent con-
centrations and the mean residual disinfectant levels per concentration.
Egg production in nondisinfected effluent was maximum at an intermediate
effluent concentration (25 percent). The mean number of eggs produced per
female in the 25 percent nondisinfected effluent concentration was signifi-
cantly greater (P=0.05) than the mean number produced in the 100, 6.25 and
0.00 percent effluent concentrations. Thus, it appeared that the 100 per-
cent concentration of nondisinfected effluent exerted a negative effect on
the production of eggs by our test fish. The reductions in egg production
observed in the low effluent concentrations were probably not the result
of one or more factors present in the nondisinfected effluent, but may
have been related to lower levels of nutrients and/or planktonic food in
the low effluent concentrations and dilution water tanks.
A similar pattern of reduced egg production per female in the higher and
lower effluent concentrations was observed in the dechlorinated stream.
Since this reduction in egg production exhibited the same pattern as those
in the nondisinfected effluent stream, there was no reason to believe it
was related to the chlorination-dechlorination processes.
Comparison of egg production on the chlorinated effluent stream with egg
production in similar concentrations of the nondisinfected effluent stream
reveals that only the 20 percent chlorinated effluent adversely affected
viable egg production. Fish in those tanks were exposed to a mean chlorine
residual of 0.103 mg/1, which exceeded the TL50 values of 0.082 to 0.095
mg/1 found in the acute tests with, chlorinated effluent. This supports
the findings of Arthur, &t_ al_. ,1 who observed that spawning was completely
inhibited by a mean chlorine residual of 0.110 mg/1. The lower average
residual chlorine levels to which our fish were exposed (0.069-0.008 mg/1)
did not appear to have an adverse effect on egg production. Except for the
20 percent effluent concentration, there were no significant differences in
viable egg production observed among the lower concentrations of chlorinated
effluent.
102
-------
Table 41. MEAN NUMBER OF VIABLE EGGS PRODUCED PER FEMALE AND THE MEAN DISINFECTANT
RESIDUAL (mg/1) IN EACH CONCENTRATION OF EACH EFFLUENT STREAM
O
U)
Effluent
Stream
Nondisinfected
x eggs/female
Chlorinated3
x eggs /female
x residual
Dechlorinated
"x eggs/female
x residual
Chlorobrominated
x eggs /female
X residual
Ozonated
x eggs/female
x residual
Nominal percent effluent concentrations3
100.00
382
b
90
0,027
od
0.119
2054
0.011
50.00
1240
b
380
0.013
649
0.033
1182
0.004
25.00
2750
oc
0.103
2482
0.005
2788
0.017
1988
0.003
12.50
1584
981
0.069
2299
0.004
0
0.008
1063
0.002
9.80
b
1236
0.036
b
b
b
6.25
450
1245
0.025
1649
0.006
1281
0.005
1749
0.001
4.80
b
1940
0.023
b
b
b
3.12
1803
778
0.012
1385
0.001
2126
0.004
1007
0.001
1.56
1215
1606
0.088
1454
0.000
917
0.003
1098
0.001
0.00
715
1929
1359
955
806
Mean for All
Concentx ations
1315
1458
1538
1232
1420
The nominal effluent concentrations in the chlorinated stream were 20.00, 14.00, 9.80, 6.86, 4.80, 3.36, 2.35
and 0.00 percent.
No equivalent concentration
Only one fish survived to maturity
d
No fish survived to maturity
-------
Viable egg production In the dechlorinated stream was maximum in the 25
and 12.5 percent effluent concentrations. This pattern closely parallels
that seen in the nondisinfected stream. The considerable variability in
egg production observed in the dechlorinated stream (90-2482 viable eggs
per female) is similar to the variability in the nondisinfected stream
(382-2750 viable eggs per female) and in the dilution water control tanks
(715-1929 viable eggs per female). The similarity in the pattern of mean
egg production per female in the dechlorinated effluent to productivity in
the nondisinfected stream suggests that, at least in effluent concentrations
of less than 25 percent, the dechlorination process effectively eliminated
the adverse effects of chlorination reported by Arthur, et al.l
Fathead minnows reared in chlorobrominated effluent produced from zero to
2780 viable eggs per female. Egg production in the 25 percent effluent
level (0.017 mg/1 residual bromine chloride) was significantly higher than
the other effluent concentrations. No eggs were produced in 100 percent
effluent (0.119 mg/1 residual bromine chloride) because none of the test
fish survived to reproductive age. Fish exposed to 50 percent effluent
concentration (0.033 mg/1 residual bromine chloride) produced about half
as many viable eggs per female, as fish in the same concentration in the
nondisinfected stream. Although some fish survived exposure to 12.5 per-
cent effluent (0.008 mg/1 residual bromine chloride), no eggs were produced.
It appears unlikely that this lack of egg production was caused by excessive
levels of residual bromine chloride, since egg production was greater in two
higher effluent concentrations (25 and 50 percent).
Fish reared in the ozonated effluent stream exhibited the least variation
in egg production (1007-2065 viable eggs per female) with no statistically
significant differences occurring between any two concentrations. Even in
100 percent effluent egg production and viability were normal. In fact,
maximum egg production per female occurred in the latter effluent concen-
tration, in contrast to the pattern established in all other effluents.
The ozonation process apparently eliminated or significantly reduced the
inherent toxicity of the nondisinfected effluent.
As previously mentioned, the total number of eggs in each spawning was
determined within 24 hours after the eggs were deposited. The mean per-
centage of viable eggs was then calculated for all concentrations of each
effluent type. The mean percent viability in all effluent streams was
92.6 percent, while the range was 78.9 to 97.6 percent. Neither the con-
centration nor the type of effluent markedly affected percent viability,
as the variance among test streams was negligible.
The greatest mean number of eggs per spawning was produced by fish in non-
disinfected effluent and in ozonated effluent (respective means of 234 and
235 eggs per spawning) (Table 42). There were no significant differences
among the mean number of eggs per spawning in the various concentrations
of the nondisinfected effluent stream. In the chlorinated stream no
spawnings occurred in the 20 percent effluent concentration because only
one fish survived to reproductive age. Likewise no fish survived to
reproduce in the 100 percent chlorobrominated effluent concentration and,
104
-------
Table 42. MEAN NUMBER OF EGGS PER SPAWNING IN THE VARIOUS
CONCENTRATIONS OF EACH EFFLUENT STREAM
Effluent
Stream
Nondisinfected
x eggs /spawning
No. of spawnings
Chlorinated3
x eggs /spawning
No. of spawnings
Dechlorinated
x eggs/spawning
No. of spawnings
Chlorobrominated
U eggs/spawning
No . of spawnings
Ozonated
If eggs/spawning
No. of spawnings
100.00
181
31
b
146
6
Od
0
249
83
50.00
314
66
b
93
48
170
48
218
96
25.00
266
230
oc
0
198
159
259
136
333
146
Nominal
12.50
292
83
166
51
260
148
0
0
210
82
percent
9.80
^,
179
112
V,
h
•{)
effluent
6.25
160
31
176
78
171
136
184
100
218
99
concentr
4.80
Q
261
130
b
b
ations3
3.12
197
122
186
60
163
158
240
142
202
97
1.56
161
150
169
133
213
144
172
83
144
57
0.00
237
53
210
171
217
105
170
94
132
75
Mean for all
Concentrations
234
96
199
92
210
113
211
75
235
92
The nominal effluent concentrations in the chlorinated stream were 20.00, 14.00, 9.80, 6.86, 4.80, 3.36, 2.35,
and 0.00 percent.
No equivalent concentration
Only one fish survived to maturity
No fish survived to maturity
-------
while both sexes were present in the 12.5 percent chlorobrominated effluent
concentration, no spawnings occurred. The patterns of egg production per
spawning in the dechlorinated and ozonated effluent streams were generally
similar to the pattern observed in the nondisinfected effluent. These
results indicate that the mean number of eggs produced per spawning by fat-
head minnows Is not adversely affected by the disinfection processes studied.
Some of the viable eggs produced were incubated to determine their hatchabil-
ity (i.e., the percent of eggs per spawning that hatched and produced fry
that were living at the end of the five-day incubation period). Fry that
had hatched, or partially hatched, but that were not alive at the end of the
incubation period were not considered living fry.
The hatchability of eggs spawned and incubated in nondisinfected effluent
improved with decreasing effluent concentration, with optimum hatchability
(90 percent) occurring in 6.25 percent nondisinfected effluent (Table 43).
Hatchability in the 100 percent effluent concentration was significantly
less than in other concentrations of nondisinfected effluent. The hatch-
ability of eggs produced in dilution water and incubated in 100 percent
nondisinfected effluent was only 3 percent, while the hatchability of eggs
produced in 100 percent nondisinfected effluent and incubated in dilution
water was 39 percent (Table 44). The hatchability of eggs spawned in dilu-
tion water and incubated in 50 percent effluent, or eggs spawned in 50
percent effluent and incubated in well water, exceeded the hatchability of
eggs spawned and incubated in 50 percent nondisinfected effluent. Thus, the
100 percent concentration of nondisinfected effluent appeared to have an
adverse effect on the hatching of fathead minnow eggs.
There were no data available on the hatchability of eggs produced and incu-
bated in the highest concentration of chlorinated effluent (20 percent) due
to adult mortality in those tanks. Hatchability in the lower effluent con-
centrations (77-86 percent) was similar to hatchability in comparable non-
disinfected effluent concentrations. This supports the findings of Arthur,
_e_t _al.1 Eggs spawned in 14 and 9.8 percent chlorinated effluent and incubated
in well water had mean hatchability values of 70 and 90 percent respectively.
The mean hatchabilities of eggs spawned in well water and incubated in 20,
14, and 9.8 percent chlorinated effluent were 63, 66, and 59 percent respec-
tively, which were lower than values for eggs spawned and incubated in
similar concentrations of nondisinfected effluent (78-85 percent). The
values for eggs transferred from dilution water to 14 and 9.8 percent
chlorinated effluent were lower than eggs spawned and incubated in the
chlorinated effluent, which suggests that the eggs spawned and incubated in
the presence of chlorinated effluent may have developed a higher tolerance
to chlorine than eggs produced in well water and then exposed to chlorinated
effluent during incubation.
With the exception of the 100 percent concentration where only one incubation
was attempted, hatchability in all dechlorinated effluent concentrations
compared favorably with the hatchability recorded in respective concentrations
of nondisinfected effluent (Table 43). When eggs spawned In dilution water
were incubated in 100 percent dechlorinated effluent (Table 44) mean hatch-
106
-------
Table 43. PERCENT HATCHABILITY, MEAN DISINFECTANT RESIDUAL (mg/1) ,
AND INCUBATION ATTEMPTS IN THE VARIOUS EFFLUENT STREAMS
Effluent Type
Nondisinfected
% Hatchability
No. of incubations
Chlorinateda
% Hatchability
No. of incubations
Mean residual
Dechlorinated
% Hatchability
No. of incubations
Mean residual
Chlorobrominated
% Hatchability
No. of incubations
Mean residual
Ozonated
% Hatchability
No. of incubations
Mean residual
Nominal Percent Effluent Concentration3
100.00
29
4
0
1
0.012
b
0
0.069
64
10
0.010
50.00
61
11
50
8
0.014
73
10
0.028
59
13
0.006
25.00
78
35
b
0
0.114
82
29
0.001
80
25
0.014
80
28
0.004
12.50
85
13
86
9
0.072
77
32
0.001
b
0
0.006
85
16
0.003
9.80
82
22
0.034
6.25 ! 4.80
90
2
77
15
0.023
88
25
0.001
77
15
0.006
84
14
0.002
84
26
0.019
3.12
88
23
81
11
0.011
87
19
0.000
89
20
0.007
88
16
0.003
1.56
83
21
86
27
0.009
79
18
0.000
82
15
0.005
79
15
0.002
0.00
76
24
83
42
0.000
91
22
0.000
87
18
0.000
84
12
0.000
o
-J
The nominal chlorinated effluent concentrations were 20.0, 14.0, 9.8, 6.9, 4.8, 3.4, and 2.4 percent.
No spawnings occurred in this concentration.
-------
Table 44. PERCENT HATCHABILITY OF EGGS INCUBATED IN WATER
DIFFERENT FROM THAT IN WHICH THEY WERE SPAWNED
Effluent Type
Nondisinfected
Spawned in:
Incubated in:
% Hatchability:
No. of attempts:
Chlorinated
Spawned in:
Incubated in:
% Hatchability:
No. of attempts:
Dechlorinated
Spawned in:
Incubated in:
% Hatchability:
No. of attempts:
Chlorobrominated
Spawned in:
Incubated in:
% Hatchability:
No. of attempts:
Ozonated
Spawned in:
Incubated in:
% Hatchability:
No. of attempts:
Test
Group 1
dilution water
100% effluent
3
2
dilution water
20% effluent
63
18
dilution water
100% effluent
56
4
dilution water
100% effluent
67
13
12.5% effluent
dilution water
68
6
Test
Group 2
dilution water
50% effluent
87
3
dilution water
14% effluent
66
8
dilution water
50% effluent
82
5
25% effluent
dilution water
71
5
Test
Group 3
50% effluent
dilution water
87
7
dilution water
9.8% effluent
59
4
Test
Group 4
100% effluent
dilution water
39
4
14% effluent
dilution water
70
5
Test
Group 5
9.8% effluent
dilution water
90
6
o
oo
-------
ability (56 percent) was lower than the mean of eggs that were both spawned
and incubated in dilution water (84 percent) (Table 43), but higher than
eggs both produced and incubated in 100 percent dechlorinated effluent
(0 percent, based on one incubation attempt). Similarly, eggs spawned in
dilution water and incubated in 50 percent dechlorinated effluent showed
improved hatchability (82 percent) over those produced and incubated in
50 percent dechlorinated effluent (50 percent). Thus, egg hatchability in
dechlorinated effluent was similar to egg hatchability in nondisinfected
effluent.
Eggs produced and incubated in chlorobrominated effluent exhibited a hatch-
ability pattern similar to that of the nondisinfected stream, except for
the 100 and 12.5 percent effluent concentrations in which no spawnings
occurred (Table 43). Since no adults survived exposure to 100 percent
chlorobrominated effluent, no eggs were produced. There was no apparent
reason for the lack of egg production in the 12.5 percent effluent concen-
tration. The hatchability of eggs spawned in dilution water and incubated
in 100 percent chlorobrominated effluent was 67 percent. This result was
similar to that which occurred with eggs that were spawned in dilution water
and incubated in 100 percent dechlorinated effluent. The hatchability of
eggs spawned in 25 percent chlorobrominated effluent and incubated in dilu-
tion water was similar to the hatchability of eggs spawned and incubated in
the 25 percent chlorobrominated effluent. These findings indicate that
chlorobrominated effluent has no adverse effect on the hatchability of fat-
head minnow eggs.
Eggs spawned and incubated in various concentrations of ozonated effluent
had hatchability values similar to the respective concentrations of non-
disinfected effluent. While the mean hatchability in ozonated effluent was
higher than the mean hatchability in any other 100 percent effluent concen-
tration, limited sample sizes precluded an objective statistical analysis
of the data. However, ozonated effluent did not appear to have any adverse
effect on the hatchability of fathead minnow eggs.
The mean hatchability in each effluent treatment was calculated by dividing
the sum of the percent hatchability values in each concentration of a treat-
ment by the number of concentrations of the respective treatment for which
hatchability values were determined (Table 43). A statistical comparison
of the mean hatchability values for each effluent treatment showed that no
significant differences (p » 0.05) existed between the mean hatchability
values of any two treatment types.
In summary then, the reproduction studies show that egg production and egg
hatchability were reduced in the highest effluent concentrations of all but
the ozonated effluent stream. Intermediate effluent concentrations general-
ly tended to be optimum for egg productivity except in the ozonated effluent
where the greatest egg production occurred in the 100 percent concentration.
This difference may have been due to the fact that during part of the spawn-
ing period the ozonated effluent was filtered, while the other effluent
streams were not, or to the higher dissolved oxygen levels observed in the
ozonated stream prior to delivery to the fish tanks. Except for the 20
percent chlorinated effluent concentration where no fish survived to repro-
109
-------
duce, no adverse effects on reproduction occurred as the result of any
disinfection process.
110
-------
REFERENCES
1. Arthur, J. W., R. W. Andrews, V. R. Mattson, D. T. Olson, B. J. Halligan,
and C. T. Walbridge. Comparative Toxicity of Sewage Effluent Disinfec-
tion to Freshwater Aquatic Life. EPA Ecological Research Series
(EPA-600/3-75-012). 1975.
2. Arthur, J. W., and J. G. Eaton. Chloramine Toxicity to the Amphipod
Gammarus pseudolimneus, and the Fathead Minnow, Pimephales promelas.
Jour Fish Res. 28:1841-1845, 1971.
3. Esvelt, L. A., W. J. Kaufman, and R. E. Selleck. Toxicity Assessment of
Treated Municipal Wastewaters. Jour Water Poll Cont Fed. 45:1558-1572,
1973.
4. Tsai, C. F. Effects of Chlorinated Sewage Effluents on Fishes in Upper
Patuxent River, Maryland. Chesapeake Sci. 9:83-93, 1968.
5. Tsai, C. F. Changes in Fish Populations and Migrations in Relation to
Increased Sewage Pollution in Little Patuxent River, Maryland.
Chesapeake Sci. 11:34-41, 1970.
6. Zillich, J. A. Toxicity of Combined Chlorine Residuals to Freshwater
Fish. Jour Water Poll Cont Fed. 44:212-220, 1972.
7. Brungs, W. A. Literature Review of the Effects of Residual Chlorine
on Aquatic Life. Jour Water Poll Cont Fed. 45:2180-2193, 1973.
8. Mills, J. F. The Disinfection of Sewage By Chlorobromination.
Presented before the Division of Water, Air and Waste Chemistry,
American Chemical Society Meeting. Dallas, Texas. Arpil, 1973.
9. Mills, J. F. The Chemistry of Bromine Chloride in Wastewater Dis-
infection. Presented before the Division of Water, Air and Waste
Chemistry, American Chemical Society Meeting. Chicago, Illinois,
August, 1973.
10. Venosa, A. D. Ozone as a Water and Wastewater Disinfectant: A
Literature Review. In: Ozone in Water and Wastewater Treatment,
Evans, F. L. Ced.). Ann Arbor, Ann Arbor Science Publishers Inc.,
1972. p.83-100.
11. Rosenlund, Bruce. Disinfection of Hatchery Water Supply by Ozon-
ation and the Effects of Ozonated Water on Rainbow Trout. Paper
presented at the International Ozone Institute Workshop on Aquatic
Applications of Ozone. Boston, Mass. 1974.
Ill
-------
12. Standard Methods for the Examination of Water and Wastewater. 13th ed.
American Public Health Association, New York, N.Y., 1971. 874p.
13. Andrew, R. W., and G. E. Glass. Amperometric Titration Methods for
Total Residual Chlorine, Ozone and Sulfite. Draft Report, National
Water Quality Laboratory, Duluth, Minn., 1974.
14. Seligson, D., and H. Seligson. A Microdiffusion Method for the Deter-
mination of Nitrogen Liberated as Ammonia. Jour Lab and Clinical Med.
38:324-330, 1951.
15. Mount, D. E., and W. Brungs. A Simplified Dosing Apparatus for Fish
Toxicology Studies. Water Res. 1:21-29, 1967.
16. Drummond, R. A., and W. F. Dawson. An Inexpensive Method for Simu-
lating Diel Patterns of Lighting in the Laboratory. Trans Amer Fish
Soc. 99:434-435, 1970.
17. Barr, J. A., and J. H. Goodnight. A Users Guide to the Statistical
Analysis System. Raleigh, Sparks Press, 1972.
18. McKee, J. E., and H. W. Wolf. Water Quality Criteria. 2nd ed.
Sacramento, California State Water Resources Control Board, 1963.
112
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SECTION VI. ACUTE TOXICITY TESTS
INTRODUCTION
Acute toxicity tests are valuable for determining an organism's tolerance to
some lethal agent during a relatively short exposure time (usually one week or
less). Although acute toxiclty tests are not nearly as comprehensive as life
cycle tests, they do permit the rapid collection of toxicity data for many
species. Thus, concurrently with our life cycle toxicity tests, we conducted
acute toxicity tests on each effluent stream as time and the availability of
test animals permitted.
METHODS
Acute tests of 96 hours in duration were run using a variety of cold and warm
water fishes. Acute tests of 48 hours in duration were also run with the
freshwater macroinvertebrate Daphnia magna. In most cases, each species was
exposed to all five types of effluent available to us, i.e., chlorinated, de-
chlorinated, ozonated, chlorobrominated, and nondisinfected. In general,
procedures were followed as outlined by the Committee on Methods for toxicity
tests with Aquatic Organisms. Tests were conducted at 25C (±1C) for warm
water species, and at 14C (±1C) for cold water forms. Diluters similar to
those described for the life cycle tests were used to dilute the effluent and
deliver the proper concentrations to the test chambers. Dilution water was
identical to that used in the life cycle studies. During the tests, the
animals were exposed to 753-1346 lumen/sq m of light, with light intensity
increasing and decreasing gradually over 30-minute morning and evening periods,
respectively. Since the acute tests were conducted in the same area as the
life cycle studies, the photoperiod was varied from 10-14 hours of light per
day, depending upon the stage of the life cycle tests during which each acute
test was run.
Alkalinity, pH, acidity, hardness, conductivity, and ammonia analyses were
made once during each acute test on the dilution water, the effluent storage
tanks, the control (dilution water) test chambers, and the 100 percent efflu-
ent test chambers. Dissolved oxygen concentrations were measured in each
test tank at 0, 48, and 96 hours after the start of each, test period. The
results of these analyses are similar to the results obtained from the iden-
tical analyses conducted during the life cycle bioassays (Table 16).
Total residual chlorine, bromine chloride, ozone, and sulfite were measured
in the test chambers with a polarograph. using methods identical to those
described for the life cycle tests. Residuals from one duplicate test tank
were measured for each effluent concentration prior to the introduction of
the test animals. Immediately after the animals were introduced, the residual
113
-------
analyses were run on the other set of duplicate test chambers. Subsequentlys
residual disinfectant concentrations were measured at three and six hours
after starting time in the highest effluent concentration tanks in which
subjects remained alive. After 24 hours, residuals were determined in one
duplicate tank, of each effluent concentration containing living fish. For
the duration of the test, residuals were determined at least two times each
day in the highest effluent concentration tanks with living test subjects.
In the event of partial mortality of test animals in an effluent concentra-
tion, the residual in the next lower effluent concentration tank was also
measured.
The TL50 for each test species was calculated using the graphical interpola-
tion method described in Standard Methods2, All results of residual deter-
minations for each pair of duplicate test tanks were averaged together to
approximate a mean residual level for that concentration for the 96-hour
period.
The first two acute bioassays with chlorinated effluent and the first ten
with chlorobrominated effluent were conducted using diluters calibrated to
deliver to the duplicate test tanks 100 percent effluent, 100 percent
dilution water, and six intermediate concentrations, each having 50 percent
less effluent than the immediately preceding higher concentration. For the
remaining tests the diluters were recalibrated to deliver 100 percent efflu-
ent, 100 percent dilution water, and six intermediate test concentrations,
each having 40 percent less effluent than the immediately preceding higher
concentration. This change was made to minimize the difference in effluent
concentration between those tanks having 100 percent mortality and those
having 100 percent survival of test animals. Ideally under these conditions,
concentrations which would kill 50 percent of the test animals would be
maintained. However, under actual conditions, the Grandville effluent was
so variable that -maintaining a constant disinfectant residual concentration
was virtually impossible.
Test animals were either reared in the laboratory, purchased from private
sources, or obtained from State of Michigan or National fish hatcheries.
In all cases they were held in the laboratory at test temperatures and
lighting conditions for at least ten days prior to testing. During the
acclimation period, the test fish were observed for signs of disease or
parasites. Fish exhibiting symptoms of bacterial infections ware treated
with Neomycin, Tetracycline, Furox 50 or Furanace; fish having ectoparasites
were treated with formalin; and fish having fungal infections were treated
with Dexon.
Acute tests were started between 8 A.M. and 12 noon. Small beakers were
used to capture and transport the test animals to the test tanks where they
were randomly distributed. Ten animals were added to each test tank except
when numbers of animals were limited, in which case as few as five were used
per tank. Dead individuals were removed every half hour for the first 3 hours,
again at 6 hours, and daily thereafter.
Observations on the behavior and general condition of the test subjects were
also made at these times. With the exception of the first three tests with
114
-------
bromine chloride, standard lengths (±0.5 mm) and weights (±0.1 g) were re-
corded for each animal that died. Test subjects were not fed for 96 hours
prior to the beginning of the test, nor for the duration of the test.
Most acute tests were performed during the chronic testing period with the
same effluent used in the life cycle bioassays. However, some acute bio-
assays were conducted either before or after the chronic tests with effluent
receiving exceptionally high, doses of disinfectants or sulfur dioxide in an
attempt to produce a toxic response in the test animals. Thus, the reader
is cautioned against drawing any conclusions on the efficacy of the various
disinfection treatments from the residuals reported in the acute toxlcity
studies.
As previously mentioned, the quality of the effluent varied throughout the
study period and thus was not Identical for all acute tests. Also, the feed
systems for bromine chloride and ozone occasionally malfunctioned or failed
completely. We attempted to conduct our acute toxicity tests on chloro-
brominated and ozonated effluents during those periods of time when we had
the most confidence in the latter dosing systems. Any tests that we felt
contained an unreasonable amount of variation due to mechanical failure were
discontinued and not Included in this report. Although the residual disin-
fectant levels in our test tanks varied moderately, such variation probably
approximated a natural situation that exists at the discharge point of most
wastewater treatment plants.
RESULTS AND DISCUSSION
Acute Toxicity Tests with Nondisinfected Effluent
Nondisinfected effluent was examined for its acute toxic effects on all test
species studied in the project. Specific results are not listed, because
nondisinfected effluent produced no acute toxicity response in those species
of fish tested. However, the freshwater macroinvertebrate, Daphnia magna,
was unable to tolerate 100 percent nondisinfected effluent.
Acute Toxicity Tests with Chlorinated Effluent
The acute toxicity of chlorinated effluents on fishes is well documented.
Zillich3 reported 100 percent mortality of fathead minnows exposed to five
percent chlorinated effluent after 96 hours. In a literature review, Brungs
reported 96 hour TL50 values ranging from 0.014 mg/1 total residual chlorine
for rainbow trout to 0.19 mg/1 total residual chlorine for golden shiners.
McKee and Wolf5 reported various species of fish killed at total residual
chlorine levels ranging from 0.03 to 2.0 mg/1. They also discussed the
effects of pH, temperature, and dissolved oxygen on the toxicity of chlori-
nated effluents.
Chlorinated effluent was acutely toxic to all species of fish, tested (Table
45), with TL50 values for fish ranging from 0.045 mg/1 to 0.278 mg/1 total
residual chlorine, and greater than 50 percent mortality occurring in efflu-
ent concentrations of 3.12 percent to 60 percent. These results generally
115
-------
Table 45. RESULTS OF ACUTE TOXICITY TESTS WITH CHLORINATED EFFLUENT
Species
Fathead Minnow Test #1
Pimephales promelas
Fathead Minnow Test #2
Pimephales promelas
Pugnose Shiner
Notropis anogensus
Northern Common Shiner
Notropis cornutus
Western Golden Shiner
Notemigonus crysoleucas
Goldfish Test #1
Carassius auratus
Goldfish Test #2
Carassius auratus
Lepomis sp. Test #1
Lepomis sp. Test #2
Test
Temp
(C)
25
25
25
25
25
25
25
25
25
96 Hour
TL50a
(mg/1)
0.095
0.082
0.045
0.051
0.040
0.153
0.210
0.278
0.195
Comments
100% mortality at 0.145 mg/1* (12.5% effluent)
x length 32 mm, x weight 0.6 g
70% mortality at_0.099 mg/1 (3.12% effluent)
x length 26 mm, x weight 0.4 g
75% mortality at_0.057 mg/1 (21.6% effluent)
x length 43 mm, x weight 0.5 g
60% mortality at_0.057 mg/1 (21.6% effluent)
x length 49 mm, x weight 0.7 g
100% mortality at 0.047 mg/1 (21.6% effluent)
x length 98 mm, x weight 10.4 g
90% mortality at_0.264 mg/1 (21.6% effluent)
x length 40 mm, x weight 2.3 g
100% mortality at 0.270 mg/1 (60% effluent)
x length 55 mm, x weight 5.6 g
100% mortality at 0.370 mg/1 (36% effluent)
x length 50 mm, x weight 3.7 g
100% mortality at 0.276 mg/1 (21.6% effluent)
x length 50 mm, x weight 3.2 g
Total residual chlorine
(continued)
-------
Table 45. RESULTS OF ACUTE TOXICITY TESTS WITH CHLORINATED EFFLUENT
Page 2.
Species
Pomoxis sp_.
Lake Trout
Salvelinus namaycush
Rainbow Trout
Salmo gairdnerii
Coho Salmon
Oncorhynchus kisutch
Largemouth Bass
Micropterus salmoides
Yellow Walleye
Stizostedion vitreum
Daphnia magna Test #1
(3 days old)
Daphnia magna Test #2
(Less than 1 day old)
Test
Temp.
(C)
25
14
14
14
25
25
25
96 Hour
TL50a
(mg/1)
0.127
0.060
0.069
0.059
0.241
0.108
0.017
Comments
100% mortality at 0.183 mg/1 (7.8% effluent)
x length 67 mm, x weight 5.8 g
90% mortality at_0.078 mg/1 (7.8% effluent)
x length 38 mm, x weight 0.5 g
85% mortality at_0.087 mg/1 (7.8% effluent)
x length 55 mm, x weight 2.6 g
100% mortality at 0.087 mg/1 (7.8% effluent)
x length 44 mm, x weight 1.3 g
95% mortality at_0.320 mg/1 (60% effluent)
x length 71 mm, x weight 7.6 g
100% mortality at 0.181 mg/1 (60% effluent)
x length 70 mm, x weight 3.4 g
100% mortality in 5.5 hours at 0.220 mg/1
(13% effluent), and total mortality in 10.5 hours
at 0.070 mg/1 (4% effluent)
30% mortality at 0.011 mg/1 (23% effluent)
-------
agree with those of Arthur, et^ a]±., who reported TL50 values ranging from
0.08 to 0.154 mg/1 total residual chlorine.6»7 Salmonids and shiners gener-
ally exhibited the lowest tolerance of residual chlorine, while members of
the sunfish family exhibited the greatest tolerance. The typical signs of
stress in fish exposed to chlorinated effluent were gasping at the surface,
rapid gill movements, loss of equilibrium, hemorrhaging at the gills and
base of fins, loss, of slime coat, rapid erratic movements, and a passive
floating or lying on the bottom prior to death. These are similar to the
symptoms described by Zillich.-* in most cases, any mortality which occurred
during a 96-hour test was complete by the end of the first 48 hours. The
final two days of most acute tests were generally uneventful.
]). magna was more sensitive to residual chlorine toxicity than any species
tested. Total residual chlorine concentrations of 0.220 mg/1 and 0.070 mg/1
were lethal to 3-day old I), magna in 5.5 and 10.5 hours, respectively. In a
48-hour acute test with ID. magna less than 1 day old, a TL50 of 0.017 mg/1
total residual chlorine was observed. This was the lowest TL50 value of any
acute test. Thus, extremely low levels of chlorinated effluent may adversely
affect the survival of some invertebrates which are potential food supplies
for many species of fish.
Acute Toxicity Tests with Dechlorinated Effluent
The same species that were tested with chlorinated effluent were also tested
with chlorinated effluent that had been dechlorinated with sulfur dioxide
(Table 46). The results show that the dechlorination process effectively
detoxified the chlorinated effluent. Arthur, et ^1.,6 Coventry,8 Allen,'
and Zillich^ all reported that addition of sufficient quantities of sodium
thiosulfate or sulfur dioxide to chlorinated water or wastewater effectively
removed the toxic properties.
Acute tests conducted with dechlorinated effluent having sulfite residuals
in the range that we normally maintained (0.00-1.937 mg/1) caused little
mortality, and hence a TL50 value could not be calculated. The only TL50
values obtained for fish were for western golden shiners (4.82 mg/1) and
pugnose minnows (5.68 mg/1), which, in an attempt to determine lethal sulfite
concentrations, were exposed to elevated sulfite residual levels (9.52 to
10.44 mg/1). The mortality observed in these tests was at least partially
attributable to the depressed dissolved oxygen concentrations which occurred
in the highest effluent concentrations CO.95-1.6 mg/1 in 100 percent effluent)
In the remainder of the acute tests, fish survived in 100 percent dechlori-
nated effluent as well as they did in 100 percent nondisinfected effluent.
It is interesting to note that the salmonids and shiners, which were rela-
tively sensitive to chlorinated effluent, were able to survive in 100 percent
dechlorinated effluent with little mortality. I), magna was the only species
tested which exhibited mortality in 100 percent dechlorinated effluent at
normal sulfite residual levels. This is not surpising considering the sensi-
tivity of this species and the fact that two acute tests using this same
species in full-strength nondisinfected effluent resulted in 50 percent and
100 percent mortality, respectively.
118
-------
Table 46. RESULTS OF ACUTE TOXICITY TESTS WITH DECHLORINATED EFFLUENT
Species
Fathead Minnow Test #1
Pimephales promelas
Fathead Minnow Test #2
Pimephales promelas
Fathead Minnow (Fry)
Pimephales promelas
Pugnose Minnow
Opsopoeodus emiliae
Pugnose Shiner
Notropis anogenus
Northern Common Shiner
Notropis cornutus
Western Golden Shiner
Notemigonus crysoleucas
Goldfish Test #1
Carassius auratus
Goldfish Test #2
Carassius auratus
Lepomis sp.
Test
Temp.
(C)
25
25
25
25
25
25
25
25
24
25
96 Hour
TL50a
(mg/1)
5.68
4.820
Comments
No mortality at 0.041 mg/la (100% effluent)
No mortality at 8.417 mg/1 (100% effluent)
30% mortality at 2.10 mg/1 (100% effluent)
30% mortality at 4.36 mg/1 (60% effluent)
25% mortality at 0.364 mg/1 (100% effluent)
No mortality at 0.364 mg/1 (100% effluent)
40% mortality at_4.15 mg/1 (60% effluent)
x length 98 mm, x weight 16.5 g
No mortality at 0.033 mg/1 (100% effluent)
No mortality at 0.00 mg/1 (100% effluent)
No mortality at 0.671 mg/1 (100% effluent)
Residual sulfite
(continued)
-------
Table 46.
Page 2.
RESULTS OF ACUTE TOXICITY TESTS WITH DECHLORINATED EFFLUENT
N>
O
Species
Lake Trout Test #1
Salvelinus namaycush
Lake Trout Test #2
Salvelinus namaycush
Rainbow Trout
Salmo gairdnerii
Brown Trout
Salmo trutta
Coho Salmon
Oncorhynchus kisutch
Chinook Salmon
Oncorhynchus tshawytscha
Large Mouth Bass
Micropterus salmoides
Yellow Walleys
Stizostedion vitreum
Daphnia magna
Less than 24 hours old
Test
Temp.
(C)
14
14
15
14
15
25
24
25
96 Hour
TL50a
(mg/1)
0.018
Comments
No mortality at 1.973 mg/1 (100% effluent)
Nine juveniles (x length 43 mm, x wt. 0.80 g)
were exposed to dechlorinated effluent shortly
after introduction of the sulfur dioxide. In 81
hours one had died, and in 10% hours all were
dead. Residuals were not monitored during this
time period.
5% mortality at 0.287 mg/1 (100% effluent)
No mortality at 0.002 mg/1 (100% effluent)
No mortality at 0.287 mg/1 (100% effluent)
15% mortality at 0.002 mg/1 (100% effluent)
No mortality at 0.000 mg/1 (100% effluent)
60% mortality in 60% effluent, 30% mortality in
36% effluent. Residual levels were too incon-
sistent to calculate a TL50.
50% mortality at 0.018 mg/1 (100% effluent)
-------
With the exceptions of the two Instances noted above where sulfite residuals
were intentionally elevated to unusually high levels (9.52-10.44 mg/1), low
dissolved oxygen concentrations were not a cause for concern in our acute
tests. The mean dissolved oxygen levels in the 100 percent effluent concen-
trations for all the dechlorinated, nondisinfected, and chlorinated acute
tests were 4.8, 5.1, and 6.4 mg/1 respectively.
Acute Toxicity Tests with Chlorobrominated Effluent
Table 47 summarizes the results of acute toxicity tests performed with chloro-
brominated effluent. The chlorobrominated effluent was less toxic than chlo-
rinated effluent, as evidenced by the higher TL50 values. This supports the
findings of Mills, ^ who concluded that chlorobromination produced a less
toxic effluent because bromamines are less stable than chloramines and thus
do not persist as long. However, chlorobrominated effluent vas more toxic
than the other effluents tested, which contrasts with Zillich's-'- observation
that there is no appreciable difference in the toxicity of chlorobrominated
and nondisinfected wastewater. These apparently conflicting results are
probably due to differences in methodology. Zillich conducted his tests
under static conditions, while we utilized flow-through techniques.
Ten of the acute tests listed on Table 47 were conducted with effluent dosed
with 10.3 mg/1 of bromine chloride (the amount generally required to achieve
disinfection was 2.0-3.0 mg/1). This high feed rate was used to generate
sufficient toxicity data to compute TL50 values for bromine chloride, which
Is a relatively new and untested wastewater disinfectant. The signs of
stress associated with bromine chloride toxicity were identical to those
produced by chlorinated effluent.
Bromine chloride was found to be approximately half as toxic to fathead
minnows and shiners as chlorine (Table 45). Of the species tested in chloro-
brominated effluent at normal dosage levels (2-3 mg/1), fathead minnows, lake
trout, chinook salmon, and I), magna were sufficiently sensitive to permit the
calculation of TL50 values. The lowest chlorobrominated effluent concentra-
tion producing significant mortality was 36 percent (TL50 of 0.102 mg/1
total residual bromine chloride for lake trout), while effluent disinfected
with chlorine produced mortality in the same species at 3.12 percent effluent
(TL50 of 0.060 mg/1 total residual chlorine).
Acute Toxicity Tests with Ozonated Effluent
No mortality was observed in those acute toxicity tests conducted during
periods when ozone was effectively disinfecting the effluent. Thus, TL50
values could not be calculated for any test.
In experiments where fish were placed in aquaria receiving 100 percent
ozonated effluent within 10 minutes after the injection of ozone, goldfish
and fathead minnows survived residual ozone concentrations of 0.047-0.185
mg/1 for seven to fifteen days without any mortality (Table 48). However,
under similar conditions, lake trout fingerlings died within 5 hours when ,
the residual ozone concentration was 0.322 mg/1. Similarly, Arthur et. al
121
-------
Table 47. RESULTS OF ACUTE TOXICITY TESTS WITH CHLOROBROMINATED EFFLUENT
t-o
N3
Species
Fathead Minnow Test #1
Pimephales promelas
Fathead Minnow Test #2
Pimephales promelas
Fathead Minnow Test #3
Pimephales promelas
Fathead Minnow Test #4
Pimephales promelas
Fathead Minnow Test #5
Pimephales promelas
Fathead Minnow Test #6
Pimephales promelas
Northern Common Shiner
Test #1
Notropis cornutus
Northern Common Shiner
Test #2
Notropis cornutus
Test
Temp.
(C)
25
25
25
25
25
25
25
25
96 Hour
TL50a
(mg/1)
0.185b
0.173b
0.193b
0.148
0.133
0.120b
0.140b
Comments
100% mortality at 0.286 mg/la (25% effluent)
100% mortality at 0.246 mg/1 (50% effluent)
100% mortality at 0.328 mg/1 (25% effluent)
x length 31 mm, x weight 0.6 g
5% mortality at 0.082 mg/1 (100% effluent)
This test was 14 days in duration.
100% mortality at 0.321 mg/1 (50% effluent)
x length 23 mm, x weight 0.3 g
85% mortality at_0.175 mg/1 (50% effluent)
x length 28 mm, x weight 0.5 g
100% mortality at 0.161 mg/1 (60% effluent)
x length 45 mm, x weight 0.8 g
This test was only 24 hours in duration due to
brominator failure.
100% mortality at 0.211 mg/1 (100% effluent)
x length 50 mm, x weight 1.0 g
(continued)
Total residual bromine chloride
Tests in which high dosages of bromine chloride were intentionally applied to produce a toxic
response in the test animals.
-------
Table 47.
Page 2.
RESULTS OF ACUTE TOXICITY TESTS WITH CHLOROBROMINATED EFFLUENT
Species
Pugnose Shiner Test #1
No tr op is anogenus
Pugnose Shiner Test #2
Notropis anogenus
Western Golden Shiner
Notemigonus crysoleucas
Goldfish
Carassius auratus
Lake Trout
Salvelinus namaycush
Rainbow Trout
Salmo gairdnerii
Brown Trout
Salmo trutta
Coho Salmon
Oncorhynchus kisutch
Chinook Salmon
Oncorhynchus tschawytscha
Largemouth Bass
Micropterus salmoides
Test
Temp.
(C)
25
25
25
25
14
16
16
16
16
25
96 Hour '
TL50a
(ing /I)
0.109b
0.136b
0.090b
0.102
0.059
Comments
100% mortality at 0.161 mg/1 (60% effluent)
x length 44 mm, x weight 0.6 g
This test was only 24 hours in duration due to
brominator failure.
100% mortality at 0.211 mg/1 (100% effluent)
x length 47 mm, x weight 0.8 g
55% mortality at_0.095 mg/1 (50% effluent)
x length 96 mm, x weight 8.9 g
35% mortality at 0.127 mg/1 (100% effluent)
100% mortality at 0.154 mg/1 (36% effluent)
x length 39 mm, x weight 0.7 g
43% mortality at_0.153 mg/1 (100% effluent)
x length 73 mm, x weight 6.4 g
20% mortality at_0.066 mg/1 (100% effluent)
x length 53 mm, x weight 2.9 g
21% mortality at_0.153 mg/1 (100% effluent)
x length 65 mm, x weight 4.5 g
60% mortality at_0.066 mg/1 (100% effluent)
x length 63 mm, x weight 3.9 g
No mortality at 0.095 mg/1 (100% effluent)
NS
(continued)
-------
NJ
Table 47.
Page 3.
RESULTS OF ACUTE TOXICITY TESTS WITH CHLOROBROMINATED EFFLUENT
Species
Lepomis sp.
Northern Yellow Bullhead
Ictalurus natalis
Northern Black Bullhead
Ictalurus melas
Crayfish
Orconectes propinqus
Daphnia magna Test #1
Less than 24 hours old
Daphnia magna Test #2
Less than 24 hours old
Test
Temp.
(C)
25
25
25
25
25
25
96 Hour
TL50a
Cms/I)
0.177b
0.283b
0.047
0.055
Comments
No mortality at 0.063 mg/1 (100% effluent)
100% mortality at 0.285 mg/1 (25% effluent)
x length 95 mm, x weight 19.0 g
50% mortality at_0.283 mg/1 (25% effluent)
x length 99 mm, x weight 21.4 g
No mortality at 0.071 mg/1 (100% effluent)
90% mortality at 0.068 mg/1 (60% effluent)
70% mortality at 0.072 mg/1 (37% effluent)
-------
Table 48. RESULTS OF ACUTE TOXICITY TESTS WITH OZONATED EFFLUENT
Species
Fathead Minnow Test #1
Pimephales promelas
Fathead Minnow Test #2
Pimephales promelas
Pugnose Shiner
Notropis anogenus
Northern Common Shiner
Notropis cornutus
Goldfish Test #1
Carassius auratus
Goldfish Test #2
Carassius auratus
Goldfish Test #3
Carassius auratus
Lepomis sp.
Test
Temp.
(c)
18
18
25
25
25
25
16
25
96 Hour
TL50
(mg/D
b
Comments
Ten juvenile fatheads were exposed for 11 days to
100% ozonated effluent shortly after contact. The
mean residual during that period was 0.058 mg/l.a
There were no mortalities.
Ten males in spawning condition and five juveniles
were exposed for 15 days to 100% ozonated effluent
shortly after contact. The mean ozone residual
during that period was 0.047 mg/1, and there were
no mortalities .
No mortality at 0.016 mg/1 (100% effluent)
No mortality at 0.016 mg/1 (100% effluent)
No mortality at 0.007 mg/1 (100% effluent)
No mortality at 0.038 mg/1 (100% effluent)
Ten adult goldfish were exposed for 7 days to 100%
ozonated effluent shortly after contact. The mean
residual during that period was 0.185 mg/1. No
mortalities were attributed to ozone toxicity.
No mortality at 0.002 mg/1 (100% effluent)
Ui
(continued)
Tests conducted during periods when ozonation disinfected the effluent to project standards.
Residual ozone
b
-------
Table 48.
Page 2.
RESULTS OF ACUTE TOXICITY TESTS WITH OZONATED EFFLUENT
Species
Large Mouth Bass
Micropterus salmoides
Lake Trout Test #1
Salvelinus namaycush
Lake Trout Test #2
Salvelinus namaycush
Rainbow Trout
Salmo gairdnerii
Brown Trout
Salmo trutta
Coho Salmon
Oncorhynchus kisutch
Chinook Salmon
Oncorhynchus tschawytscha
Daphnia magna
(Less than 24 hours old)
Test
Temp.
(c)
25
14
14
15
17
15
17
25
96 Hour
TL50
(mg/1)
b
b
b
b
b
Comments
No mortality at 0.012 mg/1 (100% effluent)
This test was terminated after 72 hours when the
ozone generator failed.
No mortality at 0.016 mg/1 (100% effluent)
Nine fingerlings (x length 43 mm and x weight 0.83 g)
were exposed to ozonated effluent shortly after contact
The mean ozone residual was 0.322 mg/1. All test
animals died within 5 hours.
No mortality at 0.010 mg/1 (100% effluent)
This test was terminated after 48 hours when the
generator failed.
ozone
No mortality at 0.018 mg/1 (100% effluent)
No mortality at 0.010 mg/1 (100% effluent)
This test was terminated after 48 hours when the
generator failed.
ozone
No mortality at 0.018 mg/1 (100% effluent)
30% mortality at 0.030 mg/1 (100% effluent)
ro
-------
found that residual ozone concentrations of 0.2-0.3 mg/1 were lethal to
fathead minnows. While this indicates that high ozone concentrations in
effluent are lethal to fish, such high ozone residuals are unlikely to
occur in receiving waters. Our lake trout were exposed to undiluted ef-
fluent approximately 6 to 7 minutes after ozone injection. Under actual
operating conditions, the time between ozone injection and effluent dis-
charge will be greater and the effluent will be diluted by the receiving
waters.
Acclimation Tests
In several instances we observed that if residual chlorine or bromine chlo-
ride levels were relatively low at the start of an acute test, and then
gradually increased to a higher level, test animals were able to tolerate
higher residual levels for the duration of the test than they would have
been able to tolerate had they not had previous exposure. In other words,
they were achieving a certain degree of acclimation to chlorine or bromine
chloride in effluent. To test this hypothesis, we conducted two experiments,
one with fathead minnows exposed to chlorinated effluent, the other with
lake trout exposed to chlorobrominated effluent.
The first experiment consisted of eight individual tests utilizing five fat-
head minnows each. These tests were conducted over a 7-week period. Each
test group was exposed to one sub-lethal concentration of residual chlorine
for 1 week, then transferred to a slightly higher concentration for another
week. This procedure was continued until each group of fish was exposed to
chlorine concentrations which were higher than our TL50 values for fathead
minnows. At that time a control group of non-exposed fish was isolated in
the same test tank and the mortality of both groups was monitored. If that
group survived for 1 week, it was transferred to the next higher concentra-
tion.
The second acclimation experiment consisted of 4 tests, each utilizing 18
fingerling lake trout. The fish were exposed to sub-lethal concentrations
of chlorobrominated effluent for 4 to 9 days, then placed in effluent having
bromine chloride residual levels well above their TL50 values. Mortality
was monitored to ascertain the degree of acclimation achieved by the test
animals.
Table 49 indicates that fathead minnows previously exposed to sub-lethal
levels of residual chlorine were able to tolerate residual chlorine levels
greater than our observed TL50 values CO.082-0.095 mg/1). Fathead minnows
survived for 1 week at residual chlorine levels of 0.113, 0.116, 0.110,
0.134, and 0.138 mg/1, while all control fish, died in less than 68 hours.
Also, at higher residual levels CO.215 to 0.512 mg/1) previously exposed
fathead minnows survived 11 to 44 times longer C20-142 hours) than control
groups.
Our data also suggest that there is a direct relationship between increased
resistance of fathead minnows to high residual concentrations and length of
exposure time to sub-lethal chlorine levels. For example, in tests numbers
127
-------
Table 49. FATHEAD MINNOW ACCLIMATION TEST IN CHLORINATED EFFLUENT
Test No.
1
2
3
4
5
6
7
8
Week Number
1
A 0.056
B 6.3%
C NM
D
A 0.052
B 6.3%
C NM
D
A 0.021
B 3. 12
C NM
D
A 0.01B
B 3.12
C NM
D
A 0.007
B 1.6*
C NM
D
A 0.007
B 1.62
C NM
D
A
B
C
D
A
B
C
D
2
0.063
12.52
NM
0.064
12.52
NM
0.036
6.32
NH
0.047
6.32
NM
0.016
3.12
NM
0.029
3.12
NM
o.ooe
1.62
NM
0.012
1.62
NM
3
0.113
252
NM
20 hrs.
0.116
252
NM
68 hrs.
0.064
12.52
NM
0.069
12.52
NM
0.033
6.32
tK
0.043
6.32
NM
0.021
3.12
NM
0.022
3.12
NM
4
0.504
502
20 hrs.
1.5 hra,
0.512
502 '
20 hrs.
1 hr.
0.233
252
20 hrs.
0.215
252
28 hrs.
0.110
12.52
NM
0.113
12.52
NM
0.042
6.32
NM
0.070
6.32
NM
5
0.306
252
142 hrs.
4 hrs.
0.318
252
45 hra.
4 hrs.
0.134
12.52
NM
4 hrs.
0.138
12.52
NM
6
0.241
252
142 hrs.
0.224
252
(202)
9 hrs.
7
0.359
502
44 hrs.
1 hr.
A - Average total residual chlorine (mg/1) to which fish were exposed.
B • Percent effluent to which fish were exposed.
C « Time required for total mortality of all previously exposed test animals
(or percent of test animals dying during the one week exposure period).
NM indicates no mortality.
D • Time required for total mortality of test animals not previously exposed.
(Blanks indicate no fish tested.)
128
-------
three and four (Table 49), after 3 previous weeks of exposure to sub-lethal
chlorine residuals, fathead minnows survived for 20 hours and 28 hours when
subjected to 0.233 and 0.215 rag/1 residual chlorine, respectively. Tests
five and six indicate that fathead minnows exposed previously for 4 weeks
survived exposure to chlorine residual levels of 0.318 and 0.306 mg/1 for
45 and 142 hours, respectively. Tests numbers seven and eight show similar
trends.
Four separate acclimation tests were conducted with lake trout in chloro-
brominated effluent (Table 50). Test one involved 18 trout which were
initially exposed to an average of 0.068 mg/1 total residual bromine chlo-
ride for 4 days, and then were exposed to a residual bromine chloride
concentration of 1.066 mg/1. After 3 hours of exposure, 72 percent were
still alive. (Our TL50 value for lake trout in chlorobrominated effluent
was 0.102 mg/1.) In the second test lake trout were subjected to mean
bromine chloride residuals of 0.029 mg/1 for 9 days and then were placed
in a tank having 0.664 mg/1 residual bromine chloride. The first mortality
occurred at 9 hours, and all of the test animals were dead 4 hours later.
Unexposed lake trout (test three), which were subjected to effluent having
similar residual levels (0.647 mg/1), were all dead in 4-1/2 hours. The
fish in test four were exposed to 0.011 mg/1 bromine chloride for 9 days and
were then placed in effluent having 0.635 mg/1 residual bromine chloride.
They began dying in 6 hours and were all dead after 8 hours. This suggests
that group two was able to tolerate similar residual levels for a longer
period of time, because they were exposed to a slightly higher bromine chlo-
ride concentration (0.029 mg/1) than group three (0.011 mg/1).
The above data indicate that at least two species of fish, if previously
exposed to sublethal residual concentrations of chlorine or bromine chloride,
are capable of tolerating levels of chlorine and bromine chloride higher than
their 96-hour TL50 values for longer periods of time than fish which were not
previously exposed to either disinfectant.
SUMMARY
In summary, the results of our acute tests indicate that nondisinfected,
ozonated, and dechlorinated effluents were nontoxic to all fish species
tested, and toxic only in high effluent concentrations to D_. magna. Chloro-
brominated effluent, however, was toxic to all fish exposed to elevated doses,
but toxic to some fish species only in high effluent concentrations with
normal residuals. J). magna, an invertebrate which serves as food for fish,
was more sensitive than any fish species tested. Effluent disinfected with
chlorine was the most toxic of those we tested. Sufficient mortality to
calculate a TL50 value was recorded for each, species exposed to chlorine.
This was not true for any other effluent type. Also, residual levels and
effluent concentrations that produced mortality in fish and ]D. magna were
lower for chlorinated effluent than any other effluent tested.
129
-------
Table 50. LAKE TROUT FINGERLING ACCLIMATION TESTS IN
CHLOROBROMINATED EFFLUENT
OJ
o
Test
Number
1
2
3
4
A =
B =
C =
D =
Eighteen fish were jused in each test
(x length 39 mm, x weight 0.7 g)
A
B
C
D
A
B
C
D
A
B
C
D
A
B
C
D
0.068 mg/1 (12.5% effluent)
4 days
1.066 mg/1 (100% effluent)
72% survival after 3 hours, 22% survival after 5 hours.
Time to total mortality unknown.
0.029 mg/1 (6.3% effluent)
9 days
0.664 mg/1 (100% effluent)
Total mortality in 13 hours.
0.000 mg/1 (0% effluent)
0.647 mg/1 (100% effluent)
Total mortality in 4.5 hours.
0.011 mg/1 (1.6% effluent)
9 days
0.635 mg/1 (100% effluent)
Total mortality in 8 hours.
Total residual bromine chloride to which the lake trout were initially
Length of time the fish were exposed to the initial residual.
Total residual bromine chloride to which lake trout were subsequently
Elapsed time prior to the death of all test animals in the subsequent
exposed
exposed.
exposure
-------
REFERENCES
1. Methods for Acute Toxtcity Tests with Fish, Macroinvertebrates, and
Amphibians. Corvallis, National Environmental Research Center, U.S.
Environmental Protection Agency, 1974. 63p.
2. Standard Methods for the Examination of Water and Wastewater. 13th ed.
American Public Health Association, New York, N.Y., 1971, 874p.
3. Zillich, J. A. Toxicity of Combined Chlorine Residuals to Freshwater
Fish. Jour Water Poll Cont Fed. 44:212-220, 1972.
4. Brungs, W. A. Literature Review of the Effects of Residual Chlorine
on Aquatic Life. Jour Water Poll Cont Fed. 45:2180-2193, 1973.
5. McKee, J. E., and H. W. Wolf. Water Quality Criteria. 2nd ed.
Sacramento, California State Water Resources Control Board, 1963.
6. Arthur, J. W., R. W. Andrews, V. R. Mattson, D. T. Olson, B. J. Halligan
and C. T. Walbridge. Comparative Toxicity of Sewage-Effluent Disinfec-
tion to Freshwater Aquatic Life. EPA Ecological Research Series
(EPA 600/3-75-012). 1975.
7. Arthur, J. W., and J. G. Eaton. Chloramine Toxicity to the Amphipod
Gammarus pseudolimneus, and the Fathead Minnow, Pimephales Promelas.
Jour Fish Res. 28:1841-1845, 1971.
8. Coventry, F. L., V. E. Shelford, and L. F. Miller. The Conditioning
of a Chloramine Treated Water Supply for Biological Purposes.
Ecology. 16:60-66, 1935.
9. Allen, L. A., N. Blezard, and A. B. Wheatland. Formation of Cyanogen
Chloride During Chlorination of Certain Liquids and Toxicity of Such
Liquids to Fish. Jour Hyg. 46:184-193, 1948.
10. Mills, J. F. The Disinfection of Sewage by Chlorobromtnation.
Presented before the Division of Water, Air and Waste Chemistry,
American Chemical Society Meeting. Dallas, Texas, April, 1973.
11. Zillich, J. A. Preliminary Investigation of the Relative Toxicities
of Chlorine, Bromine, and Bromine Chloride. Midland, In House Report,
The Dow Chemical Company, 1971. 3p.
131
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1, REPORT NO.
EPA-600/2-76-156
3. RECIPIENT'S ACCESSION*NO.
4. TITLE AND SUBTITLE
DISINFECTION EFFICIENCY AND RESIDUAL TOXICITY OF
SEVERAL WASTEWATER DISINFECTANTS
Volume I. Grandville, Michigan
5. REPORT DATE
October 1976 (Issuing Date)
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
Ronald W. Ward,* Randall D. Giffin,** G. Michael
DeGraeve,* and Richard A. Stone**
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
**City of Wyoming, Michigan
1155 - 28th Street, S.W.
Wyoming, Michigan 49509
10. PROGRAM ELEMENT NO.
1BC611
11. KaKKKAQT/GRANT NO.
S802292
12. SPONSORING AGENCY NAME AND ADDRESS
Municipal Environmental Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, Ohio 45268
13. TYPE OF REPORT AND PERIOD COVERED
Final Report, Jan.-Nov. 1974
14. SPONSORING AGENCY CODE
EPA-ORD
15. SUPPLEMENTARY NOTES
Cecil W. Chambers and Albert D. Venosa, Project Officers, 26 W. St. Clair Street,
Cincinnati, Ohio 45268
*Grand Valley State Colleges. Allendale. Michigan 49401
16 ABSTRACT •
This study was conducted to determine the comparative effectiveness of chlorine,bromine
chloride, and ozone as wastewater disinfectants, and to determine any residual toxicity
associated with wastewater disinfection with these agents or with chlorinated waste-
water which had been dechlorinated with sulfur dioxide.
Streams of nondisinfected and chlorinated wastewater were pumped from the Grandville,
Michigan, Wastewater Treatment Plant to the project laboratory. Part of the chlorinated
wastewater stream was delivered directly to the toxicity laboratory for bioassay studie
while the remainder of the chlorinated stream was dechlorinated with sulfur dioxide
prior to its use in bioassay tests. A portion of the nondisinfected wastewater stream
was delivered to the toxicity laboratory for use in bioassays while the remaining por-
tion was split to receive bromine chloride and ozone prior to use in the bioassay
studies.
Total and fecal coliform densities, suspended solids, volatile solids, COD, ammonia
nitrogen, phosphate, turbidity, color, and pH were measured in the wastewater streams.
Each of the five wastewater streams was used in acute toxicity tests with several
species of fishes and the freshwater macroinvertebrate Daphnia magna, and in a life
cycle toxicity study with the fathead minnow, Pimephales promelas, as the test subject.
7.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.IDENTIFIERS/OPEN ENDED TERMS
c. COSATI Field/Group
Waste water, Disinfection, Disinfectants,
Bactericides, Effectiveness, Efficiency,
Chlorination, Chlorine, Dechlorination,
Sulfur dioxide, Bromine halides, Ozone,
Ozonization, Coliform bacteria, Toxicity,
Bioassay, Aquatic animals, Daphnia, Fishes
Wyoming (Michigan) parallel
streams, Acute bioassays,
Chronic bioassays,
Residual toxicity
13B
6F
8. DISTRIBUTION STATEMENT
Release unlimited
19. SECURITY CLASS (This Report)
UNCLASSIFIED
21. NO. OF PAGES
144
20. SECURITY CLASS (Thispage)
UNCLASSIFIED
22. PRICE
EPA Form 2220-1 (9-73]
132
1976 — 757-056/5420 Region 5-H
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