Environmental Protection Technology Series
          DISINFECTION  EFFICIENCY AND
                  RESIDUAL TOXICITY OF
SEVERAL  WASTEWATER DISINFECTANTS
         Volume II • Wyoming, 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 nine series. These nine broad cate-
gories were established to facilitate further development and application of en-
vironmental technology.  Elimination of traditional grouping was consciously
planned to foster technology transfer and a maximum interface in related fields.
The nine series are:

      1.   Environmental  Health Effects Research
      2.   Environmental  Protection Technology
      3.   Ecological Research
      4.   Environmental  Monitoring
      5.   Socioeconomic Environmental Studies
      6.   Scientific and Technical Assessment Reports (STAR)
      7.   Interagency Energy-Environment Research and Development
      8.   "Special" Reports
      9.   Miscellaneous Reports

This report has been assigned to the ENVIRONMENTAL PROTECTION TECH-
NOLOGY  series. This series describes research performed to develop and dem-
onstrate instrumentation, equipment, and methodology to repair or prevent en-
vironmental 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-600/2-77-203
                                           November 1977
DISINFECTION EFFICIENCY AND RESIDUAL TOXICITY
     OF SEVERAL WASTEWATER DISINFECTANTS

        Volume II - Wyoming, Michigan
                     by

               Ronald W. Ward
           Department of Biology
        Grand Valley State Colleges
         Allendale, Michigan  49401

             Randall D. Giffin
      City of Wyoming, Michigan  49509

            G. Michael DeGraeve
           Department of Biology
        Grand Valley State Colleges
         Allendale, Michigan  49401
             Grant No. S-802292
              Project Officer

              Albert D. Venosa
        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 constitute endorsement or recom-
mendation 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 preserva-
tion 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 communica-
tions 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 disinfection
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
chlorine, bromine chloride, and ozone as wastewater disinfectants, and to
determine any residual toxicity associated with wastewater disinfection with
these agents or with chlorinated wastewater which had been dechlorinated with
sulfur dioxide.

A stream of nondisinfected trickling filter effluent was pumped from the
Wyoming, Michigan Wastewater Treatment Plant to the project's water treat-
ment building.  The supply of effluent was split into four streams, three
of which were disinfected with either chlorine, bromine chloride, or ozone
and then delivered to the bioassay laboratory for residual toxicity tests.
The fourth stream was delivered directly to the bioassay laboratory for
testing.  In addition, a portion of the chlorinated effluent stream was
dechlorinated with sulfur dioxide and then pumped to the bioassay labor-
atory.

Total and fecal coliform densities, total 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 tested for
acute toxicity towards several species of fishes and invertebrates, and
chronic toxicity in a life cycle study with the fathead minnow, Pimephales
promelas, as the test subject.

Ozone was found to be nontoxic to fish at the concentrations normally
expected to exist in receiving waters from plants practicing ozone disinfec-
tion.  Sulfur dioxide eliminated the inherent toxicity of chlorine, and
bromine chloride was less toxic than chlorine.

The disinfection effectiveness of ozone was severely impaired by the lack of
a reliable method of pacing dosage with shifts in demand.  No problems were
experienced with the chlorination or sulfonation equipment.  Disinfection
by bromine chloride was impaired by occasional malfunctions in the dosing
equipment.

This report was submitted in fulfillment of Grant No. S-802292 by the City of
Wyoming, Michigan under the partial sponsorship of the U.S. Environmental Pro-
tection Agency.  Investigative research was performed jointly under subcontract
by the City of Wyoming, Michigan and Grand Valley State Colleges.  This report
covers the period from July 1975 to June 1976.
                                      IV

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                                CONTENTS


Foreword	  ill

Abstract	   iv

Figures	   vi

Tables	 viii

Acknowledgements	   xi


      I   Introduction	    1

     II   Conclusions	    4

    III   The Wastewater Treatment Systems and the
          Characteristics of the Wastewater Streams	    6


     IV   Disinfection Studies	   16

      V   Life Cycle Residual Toxicity Studies	   49

     VI   Acute Toxicity Tests	   88
                                     v

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                                    FIGURES

Number                                                                  Page

  1      Flow of effluent and well  (dilution) water  	  2

  2      Study period and monthly average turbidity  and total suspended
         solids levels in the nondisinfected trickling filter effluent..  8

  3      Study period and monthly mean COD values	  10

  4      Study period and monthly average ammonia nitrogen and total
         phosphate values	   11

  5      Correlation between chlorine residual concentration  (mg/1) and
         resulting total coliform density  (number/100 ml of chlorinated
         effluent)	  22

  6      Correlation between bromine chloride residual concentration
         (mg/1) and resulting total coliform density (number/100 ml
         of chlorobrominated effluent)	  23

  7      Correlation between ozone dosage  (mg/1) and resulting total
         coliform density (number/100 ml of ozonated effluent)	  24

  8      Correlation between turbidity (J.T.U.) and  chlorine dosage
         (mg/1) necessary to maintain a total residual chlorine level
         greater than 1.5 mg/1 after 30 minutes	  25

  9      Correlation between turbidity (J.T.U.) and  bromine chloride
         dosage (mg/1) necessary to maintain any titrable residual	  26

 10      Correlation between turbidity (J.T.U.) and  ozone dosage  (mg/1). 27

 11      Correlation between chemical oxygen demand  (mg/1) and chlorine
         dosage (mg/1) necessary to maintain a total residual chlorine
         level greater than 1.5 mg/1 after 30 minutes	 28

 12      Correlation between chemical oxygen demand  (mg/1) and bromine
         chloride dosage (mg/1) necessary to maintain any titrable
         residual	 29

 13      Correlation between chemical oxygen demand  (mg/1) and ozone
         dosage (mg/1)	 30
                                     vi

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                            FIGURES  (continued)


Number                                                                Page

 14      Correlation between suspended solids  (mg/1) and chlorine
         dosage  (mg/1) necessary to  maintain a total residual chlorine
         level greater than 1.5 mg/1 after 30  minutes	  31

 15      Correlation between suspended solids  (mg/1) and bromine
         chloride dosage  (mg/1) necessary to maintain any titrable
         residual	  32

 16      Correlation between suspended solids  (mg/1) and ozone dosage
         (mg/1)	  33

 17      Diagramatic representation  of significant difference  (least
         significant difference  (p _> 95%))  between total  and fecal
         coliform means	  35

 18      Monthly geometric means of  total coliform denisities  (MPN)
         for each stream	  38

 19      Monthly geometric means of  fecal coliform densities  (MPN)
         for each stream	  39

 20      Monthly frequencies that daily samples of disinfected
         effluent achieved the project total coliform standard	  40

 21      Monthly frequencies that daily samples of disinfected
         effluent achieved the project fecal coliform standard
                                    vii

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                                 TABLES

Number                                                                   Page

  1    Mean characteristics of the influent and the test streams
       during the study period June 16, 1975, through April 30,
       1976	   7

  2    Reduction of coliform numbers by chlorine, chlorine followed by
       dechlorination, bromine chloride, and ozone, June through
       December, 1975	  18

  3    Reduction in coliform numbers by chlorine, chlorine followed by
       dechlorination, bromine chloride, and ozone, January through
       April, 1976	  19

  4    Frequency that daily samples of disinfected  effluent achieved
       proj ect bacteriological standards	  37

  5    Characteristics of the dilution water	  50

  6    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	  52

  7    The mean residual chemical levels (mg/1), sample sizes, and
       standard deviations measured in fry test chambers during the
       life cycle tests	  53

  8    The mean dissolved oxygen concentrations (mg/1) measured in head
       tanks and test chambers during the life cycle studies	  55

  9    Mean water temperatures ( C) measured in head tanks and adult
       test chambers during the life cycle studies	  56

 10    Characteristics of the test streams measured in the head tanks...  57

 11    Characteristics of the test stream measured in the highest
       concentration adult test tanks	  58

 12    Mean metal and cyanide concentrations (mg/1) measured in the
       nondisinfected effluent during the life cycle studies	  60

 13    Number of first generation P^ promeals surviving in non-
       disinfected effluent	  61


                                     viii

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                             TABLES  (continued)

Number                                                                     Page

  14    Number of second generation  P_.  promelas  surviving in the
        nondisinfected  effluent	  62

  15    Number of first generation P_. promelas surviving in the
        chlorinated effluent	  63

  16    Number of second generation  P_.  promelas  surviving in the
        chlorinated effluent	  65

  17    Number of first generation P_. promelas surviving in the
        dechlorinated effluent	  66

  18    Number of second generation  P_.  promelas  surviving in the
        dechlorinated effluent	  67

  19    Number of first generation P_. promelas surviving in the
        chlorobrominated effluent	  68

  20    Number of second generation  P_.  promelas  surviving in the
        chlorobrominated effluent	  69

  21    Number of first generation P_. promelas surviving in the
        ozonated effluent	  71

  22    Number of second generation  P_.  promelas  surviving in the
        ozonated effluent	  72

  23    Mean lengths  (in mm)  of  first generation P_. promelas
        on day 30 of  the life cycle  test	  74

  24    Mean lengths  (in mm)  of  first generation P_. promelas
        on day 60 of  the life cycle  test	  75

  25    Mean lengths  (in mm)  of  first generation P_. promelas
        on day 120  of  the  life cycle test	  76

  26    Mean lengths  (in mm)  of  first generation P_. promelas
        at day 310  (termination) of  the life cycle test	   77

  27    Mean lengths  (in mm)  of  30  day  old second generation
        P_. promelas  in the life  cycle test	  78


  28    Mean lengths  (in mm)  of  60  day  old second generation
        P_. promelas  in the life cycle test	  79

  29    Mean number  of  viable eggs  produced per female and the mean
        chemical residual  (mg/1) in each concentration of each effluent
        stream	  81

                                       ix

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                             TABLES  (continued)

Number                                                                    'Page

  30    Mean number of eggs per spawning in the various concentrations
        of each effluent stream	  82

  31    Percent hatchability, mean chemical residual  (mg/1), and
        incubation attempts in the various effluent streams	  84

  32    Percent hatchability of eggs incubated in effluent but
        spawned in dilution water	  85

  33    Results of acute toxicity tests with nondisinfected effluent	  92

  34    Results of acute toxicity tests with chlorinated effluent	  94

  35    Results of acute toxicity tests with dechlorinated effluent	  97

  36    Results of acute toxicity tests with chlorobrominated effluent...  99

  37    Results of acute toxicity tests with ozonated effluent	 102

  38    Results of acute toxicity tests with ozonated activated sludge
        effluent	 104

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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. William
Stonebrook, Superintendent of the Wyoming Wastewater Treatment Plant, and
all of his staff, were greatly appreciated.  Dr. Gary Griffiths, 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, while 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
for the project.

Dr. William Brungs, Mr. Robert Andrew, and Mr. John Arthur, all of the
National Water Quality Laboratory in Duluth, Minnesota, and Drs. Quentin
Pickering and Timothy 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 Company, Drs. Allen Filbey and Michael McEuen of Ethyl
Corporation, and Dr. Harvey Rosen and other employees of Union Carbide.
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. Steve Boss, Mr. Terry
Cruzan, Mr. Dale DeKraker, Mr. Irwin Jousma, Mr. Richard Lincoln, Ms.
Patricia Matthews, Mr. Dennis Swanson, and Ms. Bonnie White, is gratefully
acknowledged.

The suggestions, assistance and critical evaluations provided by Messers.
Cecil W. Chambers and Albert D. Venosa, EPA Project Officers, were invalu-
able to the completion of this project.
                                     xi

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                                SECTION I

                               INTRODUCTION
AN OVERVIEW OF THE PROJECT

This report deals with the second half of a two-part project designed to
investigate the bactericidal efficacy of chlorine and alternative disin-
fectants in parallel on identical wastewater streams, and evaluate any
undesirable residual toxic effects associated with those alternative pro-
cesses.  The reader is referred to the INTRODUCTION of Volume I of this
report for an overview of the problem and related research.

This project was conducted at two study sites, the Grandville, Michigan,
Wastewater Treatment Plant and 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 facilities and treats an in-
fluent composed of 35-45 percent industrial wastes.  This study utilized
trickling filter effluent from the Wyoming plant.

The specific objectives of this study were to evaluate simultaneously the
efficacy of chlorine (C^) with and without sulfur dioxide (802) dechlori-
nation, ozone (03), and bromine chloride (BrCl) to disinfect identical
streams of a high industrial waste content wastewater that had been treated
by the trickling filter process, and to determine the extent to which the
treated effluents were toxic to aquatic life.

THE WYOMING STUDY SITE

The Wyoming Wastewater Treatment Plant includes both activated sludge and
trickling filter treatment facilities which may be operated in series or in
parallel.  The design capacity of the plant is 72,000 cu m/d  (19 mgd) and
the average inflow during the study period was 44,000 cu m/d  (11.6 mgd).

Wastewater flow to the trickling filter averaged approximately 5700 cu m/d
(1.5 mgd).  After final settling, nondisinfected effluent was pumped to the
water treatment building for disinfection with Cl^, BrCl, or  03 (Figure 1).
In addition, a portion of the chlorinated stream was dechlorinated.  Detail-
ed information on the flow rates, dose rates and resulting residual, and
other characteristics of the wastewater are presented in Section III of
this report, while bacteriological data are presented in Section IV.

The various effluent streams were pumped from the water treatment building

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SOURCE
    OF
 WATER
WATER    CONDITIONING    BUILDING
                        CHLORINATION

                       '30 MINUTE CONTACT
                       CHLOROBROMINATION

                        ;'|Q MINUTE CONTACT)
                           \30 MINUTE
                            CONTACT
                       OZONATION
                       (10 MINUTE  CONTACT)
                            1-2 MINUTE
                            CONTACT
                       IRON   REMOVAL

                           FILTER
                         1.0
        a NUMBERS ON LINE INDICATE THE APPROXIMATE FLOW RATE IN LITERS PER SECOND.
        b SOURCE OF EFFLUENT is DELIVERY LINE TO ONE OF THE MEAD TANKS IN THE BIOASSAY LABORATORY.
        c SAMPLING SITES FOR EFFLUENT ANALYZES.

        FIGURE  1=  FLOW   OF  EFFLUENT
                      AND  WELL   (DILUTION)       b-
                      WATER
                                                    OJ.
                                                                            O.I.
                                                    0.8.
    BIOASSAY    LABORATORY
                                                        HEAD   TANK
                                                                                 HEAD   TANK
                                                                            0.2
                                                         HEAD   TANK
                                                         HEAD   TANK
                                                                                 HEAD   TANK
                                                         HEAD   TANK
                                                    O.I
COLD  EFFLUENT

  HEAD  TANK
                                                                                                            LIFE CYCLE TABLE
                                                                                                             ACUTE TABLE®
                                                                                                            LIFE CYCLE TABLE
                                                                                                             ACUTE TABLE(S)
                                                                                                            LIFE CYCLE TABLE
                                                                                                             ACUTE TABLE(S) |
                                                                                                             IFE  CYCLE TABLE
                                                                                                             ACUTE TABLE(s)
                                                                                                            LIFE  CYCLE TABLE
                                                                                                             ACUTE TABLE(S)
                                                                                                            CHRONIC & ACUTE
                                                                                                                 TABLES
                                                                                                            ACCLIMATION TANKS
                                                                                                            COLD ACUTE TABLE
                                                                                    :OLD  ACCLIMATION
                                                                                         TANKS
                                                                                    COLD ACUTE TABLE

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to their respective head tanks in the bioassay laboratory which was located
in an adjacent building (Figure 1).  Well water was passed through an iron
removal filter in the water treatment building and delivered to a head tank
in the bioassay laboratory where it served as a dilution water supply.  The
various water supplies were adjusted to test temperature in their head tanks
and, after a detention time of 30-45 minutes, were then delivered by gravity
flow to proportional diluters on the acute and life cycle test tables.
Dilution water and seven concentrations of each effluent type were delivered
to aquaria in which test subjects were maintained.  Several species of
fishes and invertebrates served as subjects for acute toxicity studies, while
fathead minnows (Plmephales promelas) were the subjects in the life cycle
studies. Bioassay results are presented in Sections V and VI of this report.

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                                SECTION II

                                CONCLUSIONS

1.  With the exception of the residual toxicity to aquatic organisms im-
    parted to the effluent by some of the disinfection processes, no change
    in wastewater quality that might create an environmental problem under
    the typical conditions of effluent release to waterways was observed
    as the result of disinfection with chlorine, bromine chloride, or ozone,
    or dechlorination with sulfur dioxide.

2.  All of the disinfectants tested had the capability to reduce total and
    fecal coliform densities to project standards.

3.  While the efficiency of each of the disinfectants was directly related
    to effluent quality, ozone was most dependent and chlorine least depen-
    dent upon this factor.

4.  Malfunctions of the bromine chloride and ozone dosing systems demon-
    strated the need for technological improvement in these systems.

5.  Undiluted nondisinfected trickling filter effluent from the Wyoming
    Wastewater Treatment Plant was lethal to young fathead minnows (Pime-
    phales promelas).

6.  Any adverse affects on the growth or reproduction of fathead minnows
    were not attributed to any of the disinfectants tested.

7.  Chlorine was the only disinfectant tested which produced a lethal resid-
    ual toxicity effect in life cycle studies with fathead minnows.

8.  Life cycle studies showed that fathead minnows were more tolerant of
    residual chlorine in Wyoming effluent, which contained 35-45 percent
    industrial wastes, than in Grandville effluent, which was primarily
    of domestic origin.  For example, the probability of test animals
    surviving through 60 days of age was similar in mean residual chlorine
    concentrations of 0.076 mg/1 in a 20 percent concentration of Grandville
    effluent and 0.161 mg/1 in a 25 percent concentration of Wyoming ef-
    fluent.

9.  Dechlorination with sulfur dioxide eliminated the lethal effects of
    chlorinated effluent and produced no adverse effects on the survival,
    growth or reproduction of fathead minnows.

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10.   The only treatment-related mortality of fathead minnows in the life
     cycle study with chlorobrominated effluent resulted from an apparent
     synergistic effect between residual bromine chloride and some con-
     stituent of the wastewater.

11.   Fathead minnow survival was not adversely affected in the life cycle
     study with ozonated effluent.

12.   Chlorine was generally the most acutely toxic disinfectant tested as
     evidenced by its consistency in producing mortality in acute toxicity
     tests.  However, salmonid fishes and the pugnose shiner (Notropis
     anagenus) were more tolerant of higher concentrations of residual
     chlorine than of residual bromine chloride.

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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 disin-
fectant.  Conversely, the disinfectant may directly affect one or more of
the wastewater properties, such as dissolved oxygen or pH, or may render the
effluent toxic to aquatic organisms.

This section discusses the characteristics of the Wyoming wastewater and
the design of the test treatment systems and their effects on the charac-
teristics of the wastewater.

THE WYOMING WASTEWATER TREATMENT PLANT

The wastewater treatment plant at Wyoming, Michigan includes both trickling
filter and activated sludge systems, which may be operated singly, in series
or in parallel.  The influent wastewater is of mixed origin, with approxi-
mately 55-65 percent being from domestic sources and 35-45 percent from
light industry, metal plating plants, a dairy products processor, and com-
mercial establishments.  The plant was recently expanded to a capacity of
72,000 cu m/d (19 mgd) and the operational and equipment problems typically
associated with the new plant start-ups resulted in a highly variable ef-
fluent quality during this study.  The accumulation of sludge in the treat-
ment systems during the December-May period was especially troublesome and
resulted in reduced effluent quality from both the trickling filter and
activated sludge treatment systems.

The average plant inflow during the study period was 44,000 cu m/d (11.6 mgd)
with a mean biochemical oxygen demand of 321 mg/1.  About 5,700 cu m/d
(1.5 mgd) of this influent was passed through the trickling filter treatment
system and served as the source of the effluent for this study.  The char-
acteristics of the raw wastewater and test streams are summarized in Table 1.

Figure 2 illustrates the monthly and study period averages of total suspend-
ed solids in the nondisinfected trickling filter effluent.  Monthly mean
total suspended solids levels in the raw wastewater ranged from 204-422 mg/1
(x= 293 mg/1) and generally tended to increase during the study period.
The average removal of total suspended solids by the trickling filter was
74 percent.

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          Table 1.  MEAN CHARACTERISTICS OF THE INFLUENT AND THE TEST STREAMS
                  DURING THE STUDY PERIOD JUNE 16, 1975, THROUGH APRIL 30, 1976
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)
Total Phosphorus
(mg/1)
Dissolved Oxygen
(mg/1)
Plant
Influent
293
212
N.A.C
N.A.
N.A.
N.A.
14.8
7.5
N.A.
Test Stream
Nondis-
infected
75 ,
(57)b
55
(43)
97
(71)
275
(187)
67
(26)
146
(89)
9.3
(1.9)
6.2
(2.4)
1.1
(0.9)
Chlor-
inated
59
(50)
43
(35)
71
(45)
206
(117)
57
(18)
136
(80)
9.1
(1.9)
6.0
(2.2)
1.9
(1.2)
Dechlor-
inated
61
(49)
44
(34)
70
(46)
207
(122)
55
(17)
141
(79)
9.2
(1.9)
5.8
(1.5)
3.4
(1.1)
Chloro-
brominated
58
(48)
43
(37)
71
(47)
205
(123)
57
(20)
135
(79)
8.9
(1.9)
5.9
(2.2)
1.8
(0.9)
Filtered
19
(16)
15
(14)
48
(42)
146
(103)
86
(51)
97
(60)
9.3
(2.0)
5.0
(1.8)
0.5
(0.9)
Filtered &
Ozonated
21
(17)
16
(15)
29
(31)
85
(75)
36
(32)
94
(59)
9.3
(1.8)
5.1
(2.2)
9.0
(1.7)
aThe characteristics of the disinfected streams were measured at the sites shown in Figure 1.
b.
 Numbers in parentheses are standard deviations
f*
 Data not available

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oo
   300-r-
   290- -     Total Suspended  Solids fmg/l)
   280-}-     Turbidity  (JTU)
   270-
   260-
   250-
   240-
   230-
   220-
   210-
   200-
   190-
   180--
   170-
   160-
   150-
 3 140-
 £ 130-
 T 120-
 0 110-
^T 100-1- Study  Period Average: 97 JTU
 g   90--
    80--
                 70--
                 60--
                 50--
                 40--
                 30--
                 20--
                 10--
                  o--
                                                                              Study Period Average: 75 ms/1
             \
               ^
                  \
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I
                          June
                   July   August September October November December January February March
                           1975                                       1976
                                            April    May
             Figure  2.
            Study period and monthly average  turbidity and  total suspended solids  levels
            in the nondisinfected  trickling filter effluent.

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The chemical oxygen demand of the nondisinfected effluent averaged 146 mg/1
(Table 1 and Figure 3).  The mean effluent total phosphate and ammonia
nitrogen concentrations were 6.2 and 9.3 mg/1, respectively  (Figure 4).
The trickling filter averaged 17 percent removal of phosphate and 37 percent
removal of ammonia nitrogen.

Figures 2-4 illustrate the general decline in effluent quality during the
course of the study.  Other effluent parameters exhibited a  similar trend.
This change in quality caused a significant increase in disinfectant demand
in the effluent, thereby moderately affecting disinfection efficiency and
residual disinfectant  concentrations.

DESIGN OF TEST WASTEWATER TREATMENT SYSTEMS

A water well was drilled adjacent to the water treatment building to provide
chlorine-free dilution water for the bioassay laboratory.  After passage
through an iron removal filter in the water treatment building, well water
was pumped to a head tank in the bioassay laboratory (Figure 1).

After final settling,  nondisinfected effluent was pumped to  the water treat-
ment building and divided into four streams:  one was treated with Cl2» one
with BrCl, one with 03, and one was passed directly to the bioassay labor-
atory (Figure 1).  A portion of the chlorinated effluent from the end of
the chlorine contact chamber was dechlorinated with S02-  All five streams
were then pumped to respective head tanks in the bioassay laboratory.

Chlorine was injected  into the wastewater by an aspirator, using a Wallace
and Tiernan Model 20-055 chlorinator.  The chlorinated effluent flowed into
a steel contact  chamber having a 30-minute residence time at a flow rate of
2.2 I/sec  (35 gpm) .  The steel contact chamber was 3.7 m long by 1.2 m wide
by 0.9 m deep with steel baffles welded to the bottom and sides at intervals
of 1.2 and 2.4 m from  the end, and wooden baffles inserted from the top at
0.6, 1.8, and 3.0 m from the end.  This arrangement of baffles provided an
under-over-under flow  configuration.  A Fisher-Porter Anachlor continuous
titrator was installed in the line at the end of the Cl2 contact chamber to
continuously monitor the 30 minute residual Cl2 concentration.  The readouts
were used as the basis for the manual adjustment of Cl2 metering rates so
that a constant  Cl2 residual could be maintained.  The Cl2 residual was
normally checked at 1-2 hour intervals, and metering adjustments were made
as necessary.

The 30 minute residual Cl2 concentration was maintained at 2.0 mg/1 through-
out the study.   The average Cl2 dosage required to maintain  this residual
was 5.9 mg/1.

A portion of the chlorinated stream  (1.0 I/sec  (15 gpm)) was treated with
S02-  The S02 was injected into the  chlorinated effluent by  an aspirator
and regulated by a Wallace and Tiernan Model 20-055 chlorinator.  The  de-
chlorinated stream flowed into a fiberglass contact chamber  having a resi-
dence time of 10 minutes at a flow rate of 1.0  I/sec.  The contact chamber
was 1.8 m long by 0.6  m wide by 0.6 m deep and  contained three baffles to
produce an under-over-under flow configuration.

-------
300--
290--
280--
270--
260--
250--
240--
230--
220--
210--
200--
190--
180--
170--
160-
150-1-Study  Period Average:  146mg/l
140--
130--
 110--
 100--
 90--
 80--
 70--
 60--
 50--
 40--
 30-
 20-h
 10-
  o-h
                  I
I
I
  I
I
          June    July   August September October November December Jam
                         1975
                            ary
           February March
                1976
April   May
               Figure 3.   Study period and  monthly mean COD  values.

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13-

12-

11-
10-
 4-

 3

 2
Ammonia  Nitrogen-
   Total  Phosphate-
                    Study  Period
                    Averages 9.34mg/l

                                                        Averages6.22 mg/l
                     I
                 I
I
I
I
I
I
I
                   June     July    August  SeptemberOctober November December January  February  March   April
                              1975
                                                                            May
                                                        1976
  Figure  4.   Study period  and monthly average  ammonia nitrogen  and  total phosphate values.

-------
A Fisher-Porter Anachlor continuous titrator unit received flow of dechlori-
nated effluent from the end of the SC>2 contact chamber.  This analyzer was
modified to sound an alarm and shut off the dechlorinated effluent supply
to the bioassay test chambers in the event that residual Cl£ was present in
the dechlorinated effluent.

To further protect the bioassay test animals from accidental breakthrough of
Cl2 in the dechlorinated stream, the residual sulfite concentration was
maintained at approximately 5 mg/1.

Prior to February, liquid BrCl was vaporized by heat and fed to the effluent
by a Wallace and Tiernan Model 20-055 chlorinator.  From February until the
end of the study, BrCl was fed by a Capitol Controls prototype BrCl feeder
which vaporized the liquid BrCl in a constant temperature warm water bath.
The BrCl contact chamber was identical to the Cl2 contact chamber.  Chloro-
brominated effluent was delivered to the bioassay laboratory after a 10 min-
ute residence in the contactor.

A Fisher-Porter Anachlor continuous titrator was installed to measure resi-
dual BrCl after a five minute residence time.  Readings were made and dosages
were adjusted on the same schedule as the chlorination system.  The five
minute BrCl residual was maintained at 0.4-0.5 mg/1 by feeding an average
of 4.2 mg/1.

The ozonation system consisted of an Ingersoll-Rand Model ESV-NL air com-
pressor, a Pall-Trinity Model 35 HA1 dryer, a modified W. R. Grace Model
LG-16 ozonator, a W. R. Grace positive pressure injection contacting system,
and a contact chamber.  A Wellsbach Model CL-34-D19L ozone generator served
as a standby ozone source.  Air from the compressor was dried to a -50C
dew point prior to delivery to the 0^ generator.  Prior to 03 injection the
effluent was filtered through a Baker Model HRC Hi-Rate filter system to
remove suspended solids.  The effluent was then mixed with the ozonated air
at the top of a 3.7 m column through the 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, flowed upwards through a concentric 20 cm diameter circular
tank, down again through a concentric 30.5 cm circular tank, and emerged
from the bottom through a hose to a covered steel contact chamber (1.2 m
long x 1.2 m wide x 0.9 m deep).  Detention time was approximately 40 seconds
in the vertical column and 10 minutes in the steel contact chamber.  About
28 percent of the ozone that was introduced was lost in the off-gas from
the contactor.

Ozone dosages ranged from 4.5 to 12 mg/1, calculated on the basis of the
amount of ozone in the gas stream minus that lost in the off-gas in re-
lation to the liquid flow to the contactor.

The sampling sites for chemical analyses of the various effluent streams are
shown in Figure 1.  Identical samples were taken from each stream during the
period June 9, 1975, to April 30, 1976, five days per week, usually between
2 and 4 P.M.  Since residence times varied in the different contactor systems,
designated sample sites may have varied by as much as 30 minutes.  However,


                                      12

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such short term variation was probably of little significance compared to
the longer term variations in the effluent.

REACTIONS OF DISINFECTANTS

See pages 19-23 in Volume I of this report1.

MATERIALS AND METHODS

The materials and methods for chemical tests on the effluent streams were
identical to those described on pages 23 and 24 in Volume I of this report,
with the exception that residual C12 and BrCl were measured by the modified
amperometric titration method described on page 60 of Volume I.  Statistical
differences among respective mean test results were determined by subjecting
the data to a two-tailed t-test.

RESULTS AND DISCUSSION

Mean total and volatile suspended solids levels in all of the treated test
streams  (Table 1) were significantly lower (PX^O.Ol) than in the nondisin-
fected effluent stream.  The sedimentation which occurred in the contact
chambers and the filtration process accounted for most of the observed
decrease in suspended solids.  The small differences between the mean total
and volatile suspended solids levels in filtered effluent and ozonated
filtered effluent were not statistically significant.

The mean turbidity of each treated stream was significantly lower than that
of the nondisinfected effluent  (Table 1).  Again, the settling time in the
various contact chambers probably accounted for most of the observed de-
creases in turbidity.

Mean apparent and true color levels in all of the treated effluent streams
were significantly  (P/C 0.001) lower than those of the nondisinfected efflu-
ent.  Filtration had a significant  (P< 0.001) effect on mean true and
apparent color levels when contrasted with the respective means for the non-
disinfected stream.  Likewise,  the mean apparent and true color levels in
ozonated effluent were significantly different (P*l 0.001) from the respec-
tive means for filtered effluent.  Thus, in view of the varied contact and
settling times in the different treatment systems, ozonation appeared to
have the greatest potential of  the disinfectants tested to reduce color- r
levels, especially the true color of the effluent.  Other investigators
have similarly found ozone to be a good decolorizer.

None of the disinfection processes produced a significant change in mean
chemical oxygen demand  (COD) levels.  This contrasts with reports  »  ' »'
of COD reductions after ozonation.  Filtration produced a significant
   - 0.001) reduction in the mean COD of the nondisinfected effluent.
When  contrasted  with nondisinfected  effluent,  only the  filtered  test  stream
exhibited  a  significant (P< 0.001) reduction in mean total phosphate  con-
centration.
                                      13

-------
The mean dissolved oxygen (DO) levels in the chlorinated and chlorobrominated
effluent streams were not significantly different from the mean observed for
nondisinfected effluent.  However, the dechlorinated effluent had a signifi-
cantly higher mean DO (P£ 0.001) concentration than the nondisinfected ef-
fluent, primarily because of the aeration which occurred as chlorinated
effluent was pumped to the dechlorination treatment system.  While the DO
in the filtered effluent was significantly (P4[ 0.001) lower than the DO in
the nondisinfected effluent, ozonation significantly (P<. 0.001) increased
the mean DO of the filtered stream, as expected.

The pH of the effluent was not significantly altered by any of the disin-
fection systems.

The most notable difference between these data and those from the study
conducted at Grandville ((Volume I of this report) was the generally poorer
quality of the Wyoming effluent as evidenced by all of the wastewater char-
acteristics presented in Table 1.  This lower effluent quality had the
potential to affect the results of the bacteriological and bioassay tests.

CONCLUSIONS

The quality of the effluent tested in this study was substantially lower
than the quality of the effluent tested in the Grandville study.

The physical and chemical characteristics of the Wyoming effluent test
streams were affected by necessary differences in the dosing and contact
systems of the various disinfectants.  Nevertheless, none of the test treat-
ments produced adverse changes in the water quality parameters measured,
all treatments reduced effluent color, and ozonation improved effluent DO
concentrations.
                                     14

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                                REFERENCES

1.  Ward, R. W., R. D. Giffin, G. M. DeGraeve, and R. S. Stone.  Disinfec-
    tion Efficiency and Residual Toxicity of Several Wastewater Disinfec-
    tants.  Volume I Grandville, Michigan.  Environmental Protection
    Technology Series EPA-60012-76-156, 1976.

2.  Nebel, C. R., D. Gottschling, 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.  45:2493-2507, 1973.

3.  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.  39 pp.

4.  Snider, E. H., and J. J. Porter.  Ozone Treatment of Dye Waste.  Jour
    Water Poll Cont Fed.  45:886-894, 1974.

5.  Singer, P. C., and W. B. Zilli.  Ozonation of Ammonia in Wastewater.
    Water Res 9:127-134, 1975.

6.  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.  pp. 123-143.

7.  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.  pp. 61-82.
                                     15

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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 Wyoming, Michigan wastewater treatment plant,
which receives both industrial and domestic wastewater.  For a description
of the Wyoming study site and a detailed discussion of the effectiveness and
chemical reactivity of the disinfection processes, refer to Section III.

MATERIALS AND METHODS

The five tube-three dilution multiple tube fermentation method (MPN)  was
used to enumerate coliform bacteria in the five effluent streams.  For deter-
mining total coliform densities, Lauryl Tryptose broth (Difco) was used as
the presumptive medium^ and Brilliant Green Bile broth (Difco) as the con-
firmatory medium.  EC broth was used for fecal coliform determinations.
Grab samples of nondisinfected, chlorinated, chlorobrominated, and ozonated
effluent were collected daily in sterile bottles containing sodium thio-
sulfate.l

A number of bacteriological measurements were performed to determine if
significant diurnal fluctuations in coliform numbers occurred in the Wyoming
effluent.  When it was found that coliform levels were fairly constant
throughout the day, the sampling time was set for mid-afternoon to accom-
modate two working shifts.  Based on findings from the Grandville study,
samples were collected at the following locations:

     (1)  Nondisinfected wastewater - immediately prior to entering the
          treatment systems.

     (2)  Chlorinated effluent - at a site in the chlorine contact chamber
          corresponding to a detention time of 30 minutes (see figure  1).
                                      16

-------
     (3)   Dechlorinated effluent - at a site in the dosing unit immediately
          following the SCL injection point.

     (4)   Chlorobrominated effluent - at the end of the BrCl contact chamber,
          corresponding to a mean detention time of 30 minutes.

     (5)   Ozonated effluent - from a sample tap immediately following the
          contact unit, corresponding to a detention time of 1 to 2 minutes.

Data were tabulated and statistically analyzed to evaluate 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 coliforms per 100 ml.  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.

Chlorine and bromine chloride residuals and ozone dosages were measured as
described in Section III.  Halogen dosages were determined by cylinder weight-
loss and rotameter flow rates.

All data were subjected to statistical analyses.  Significant differences in
geometric mean coliform densities among the various disinfection systems were
determined by t-test, analysis of variance, and least significant difference
(a priori).

RESULTS

Observations of Accumulated Data

In order to determine and compare the relative bactericidal effects of each
disinfectant under similar conditions, coliform data were grouped into two
time segments during which wastewater quality was fairly consistent.  The
first segment, from June through December, 1975, was marked by relatively
low mean suspended solids, turbidity, and chemical oxygen demand (COD) values
(see figs. 2-4).  December was a transition month, as wastewater quality
began deteriorating.  However, for statistical grouping purposes, December
was included in the first segment because effluent quality more closely ap-
proximated the quality observed since June.  The second time segment, from
January through April, was marked by very poor wastewater quality.

Tables 2 and 3 summarize all disinfection data accumulated in each time
segment.  Geometric mean coliform densities computed over each entire time
segment are shown, along with the corresponding standard deviations.  The
dechlorinated effluent contained no detectable chlorine residual.  Ozone
residuals were not measured because of the lack of a reliable and quantita-
tive method of analysis.

Mean total and fecal coliform numbers in the nondisinfected effluent were
significantly higher (p20.99) in the first interval than in the second.
Further, when the mean coliform densities in the unfiltered, nondisinfected
                                      17

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            Table 2.  REDUCTION OF COLIFOEM NUMBERS BY CHLORINE, CHLORINE FOLLOWED BY DECHLORINATION, BROMINE CHLORIDE, AND OZONE
                                                             JUNE THROUGH DECEMBER, 1975
Treatment
Nondisinfected
Chlorinated
Dechlorinated
Chlorobrominated
Ozonated
Disinfectant Concentration
(mg/1)
Mean
Dose

4.4
b
4.2
7.4
Residual Concentration
Mean

2.0a

0.5C
d
S.D.

0.3

0.2

'Total Coliform Density
(MPN/100 ml)
No. of
Samples
119
124
121
100
116
Log Mean
MPN
6.48
2.15
2.26
2.45
2.30
S.D.
1.45
0.72
0.61
0.76
0.68
Geom. Mean
MPN
3.0 x 106
140
180
280
200
Fecal Coliform Density
(MPN/100 ml)
No. of
Samples
117
122
118
100
109
Log Mean
MPN
6.08
1.76
1.73
1.93
1.84
S.D.
0.54
0.64
0.59
0.82
0.62
Geom. Mean
MPN
1.2 x 106
58
54
86
69
oo
        Residual concentration after 30 minutes contact
        S0_ dosage was set to maintain an excess sulfite residual (refer to Section III)
       cResidual concentration after 5 minutes contact
        Residual not measured

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     Table 3.  REDUCTION IN COLIFORM NUMBERS BY CHLORINE, CHLORINE FOLLOWED BY DECHLORINATION, BROMINE CHLORIDE, AND OZONE
                                                      JANUARY THROUGH APRIL, 1976
Treatment
Nondisinfected
Chlorinated
De chlorinated
Chlorobrominated
Ozonated
Disinfectant Concentration
(mg/1)
Mean
Dose

8.6
b
8.1
8.8
Residual Concentration
Mean

2.1

0.6°
d
S.D.

0.7

0.7

Total Coliform Density
(MPN/100 ml)
No. of
Samples
72
71
62
47
69
Log Mean
MPN
6.26
2.32
2.76
2.76
3.95
S.D.
0.40
0.63
0.74
0.89
0.69
Geom. Mean
MPN
1.8 x 106
210
570
570
9000
Fecal Coliform Density
(MPN/100 ml)
No. of
Samples
69
69
63
44
64
Log Mean
MPN
5.91
2.00
1.92
1.81
3.69
S.D.
0.45
0.62
0.77
1.05
1.01
Geom. Mean
MPN
8.2 x 10"
100
84
64
4900
aResidual concentration after 30 minutes contact
 SO
„ dosage was set to maintain an excess sulfite residual (refer to Section III)
 Residual concentration after 5 minutes contact
 Residual not measured

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effluent were compared with the levels in the filtered, nondisinfected ef-
fluent (not shown in Tables 2 and 3), no significant difference between the
means was noted.  Thus, pressure filtration of the effluent did not result
in a significant removal in bacterial numbers.

The chlorine residual after 30 minutes contact time was maintained at great-
er than 1.5 mg/1 for the duration of the project.  The bromine chloride
residual after five minutes contact time was planned to be maintained at
0.4 to 0.5 mg/1 throughout the project.  To maintain these constant residual
levels, dosage was paced manually with demand from readings taken on Fisher-
Porter continuous chlorine residual analyzers (see Section III) and from
grab samples analyzed amperometrically.  However, it was found that the
fluctuations in the BrCl residual analyzer were too wide to enable contin-
uous maintenance of the desired residual level, presumably due to the
instability of BrCl in the presence of reducing agents.  Therefore, it was
assumed that maintenance of any residual BrCl after five minutes contact
time would result in the desired disinfection efficiency.

Halogen dosage was recorded by two different methods.  The data appearing
in Tables 2 and 3 were derived from cylinder weight loss readings.  This
method gives the mean halogen dosage for the duration of each time interval.
The other method involved taking gas and liquid flow rate readings from
rotameters located in the flow pacing equipment.  Prior to each sample
collection, the halogen dosage was adjusted based on the measured halogen
residuals and the dosage recorded.  When these data were collected and
analyzed, the resulting means were quite different from those depicted in
Tables 2 and 3.  Thus, the mean chlorine and bromine chloride dosages in
the first interval were 6.8 mg/1 and 4.5 mg/1, respectively, by the rota-
meter method (compare with 4.4 mg/1 for Cl£ and 4.2 mg/1 for BrCl in Tables
2 and 3).  In the second interval, mean Cl2 and BrCl dosages were 10.1 mg/1
and 13.4 mg/1, respectively, measured by the rotameter method (compare with
8.6 mg/1 for Cl2 and 8.1 mg/1 for BrCl in Tables 2 and 3).

The precision and accuracy of these measurements were dependent upon several
factors.  The rotameter floats tended to accumulate debris, causing a change
in float density and adherence to the rotameter walls.  Gas leaks occasion-
ally developed.  Disinfectant dosage control after normal working hours was
often less than adequate because of other operator responsibilities.  Daily
weight-loss readings were recorded from an inherently inaccurate cylinder
scale.  Thus, the dosage data reported above represent approximate means of
fairly imprecise readings and should only be construed as gross estimates
of the true dosage rates applied.

It is evident from Tables 2 and 3 that wastewater quality severely deteri-
orated in the second time interval.  Both primary and secondary clarifiers
were overloaded with solids, which could not be removed fast enough to
prevent a chronic solids carry over.  The monthly mean effluent suspended
solids and COD levels in this time interval were more than 100 mg/1 and
200 mg/1, respectively.  Eventually, the clarifiers went septic, causing
additional solids spill-over, as rising gas bubbles carried sludge to the
surface.
                                     20

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Figures 5, 6, and 7 illustrate the correlation between total coliform
numbers and chlorine residual, bromine chloride residual, and ozone dosage,
respectively, in the treated effluents.  The correlation coefficients  (r val-
ues) for C±2 anc* BrCl were significant at the p>0.99 level, due to the
large number of data points (n = 170, 154, and 81 for C±2> BrCl, and 03,
respectively).  This indicates that total coliform density was inversely
proportional to halogen residual.  However, the magnitudes of the corres-
ponding coefficients of determination (r^ values), which is a measure  of
the strength of a linear relationship, were quite low (0.055 for Cl2 and
0.098 for BrCl).  This means that only 5.5 and 9.8 percent of the variabil-
ity in coliform densities in the treated effluents were accounted for  by the
values of the chlorine and bromine chloride correlation coefficients,  res-
pectively.  Of course, some of the variation in effluent coliform levels was
undoubtedly due to variation in influent levels, as the latter were not
accounted for in the regression analyses.  Nevertheless, the trend exists in
the data suggesting that lower bacterial numbers will result from higher
halogen residuals in the effluent.

Examination of Figure 7 reveals no correlation between total coliform  num-
bers in the treated effluent and ozone dosage.  This is not unexpected,
since ozone dosage could not be controlled by residual.  In fact, much of
the data recorded in the second time segment (i.e., when wastewater quality
was poor) were obtained under conditions when the ozone generator, which
was operating at maximum capacity, could not produce sufficient ozone  to
meet the demand of the wastewater.

Figures 8 to 16 illustrate the effects of turbidity, chemical oxygen demand,
and suspended solids on disinfectant dosage necessary to maintain a given
residual  (except in the case of ozone, where no residual could be maintained).
It is clear that, in all cases involving the halogens, as the levels of key
water quality parameters increased, the halogen dosage had to be raised in
order to maintain the desired residual concentrations necessary to meet
project disinfection standards.  The correlation coefficients were all
statistically significant at the p>0.99 level.  However, no such conclusion
can be reached with ozone, because the correlation coefficients and the
corresponding coefficients of determination were too low and the Y-intercept
values were too high to provide a meaningful interpretation of the data.
This again is not unexpected because of the lack of adequate control of
ozone dosage as a function of demand.

A two-way analysis of variance was performed on all coliform data to test
two null hypotheses:  (1) there is no significant difference among treat-
ment means (i.e., chlorinated, dechlorinated, ozonated, and chlorobrominated
effluents) with respect to geometric mean coliform densities;  (2) there is
no significant difference in geometric mean coliform densities in the  treat-
ed effluents between the two time segments (i.e., June-December, and January-
April) .  Results from this analysis revealed that the mean coliform densities
among treatments (and, thus, disinfection efficiencies) were significantly
different (p>0.99).  Furthermore, mean coliform densities in all effluents
in the first time segment were significantly different  (p^0.99) from  the
mean coliform densities in the second time segment.  This indicates that
wastewater quality considerably affected the performance of all disinfectants.

                                     21

-------
100,000
                                        LOG(y)=2.846-0.285x

                                            r=0.2354 (p>0.99)

                                                 (n=!70)
 10,000 -
UJ
13


LL.
U.
UJ


O
LU
  1,000 -
                        •  *
O


U.
O



1


O

o
oc
LU
Q.

CO
S
CC

e
_j
o
o
    100-
     10-
                    12345

                            CHLORINE RESIDUAL (mg/l)


Figure 5.  Correlation between chlorine residual concentration (mg/l)

           and resulting total coliform density  (number/100 ml of

           chlorinated effluent).
                                   22

-------
100,000
                                            LOG(y)=3.057-0.509x
                                                 r=0.3!27 (p>0.99)
                                                     (n=!54)
 10,000 -
UJ
Iti
Q
UJ
   1,000 -
m
3
o
o
o
cr
UJ
0.
to
o
o
    100-
     10-
              .2     4     .6     .8     1.0     1.2    14
                       BROMINE  CHLORIDE RESIDUAL (mg/l)
                                              1.6
1.8    2.0
Figure  6.
Correlation between bromine chloride residual concentration
(mg/l) and resulting total coliform density (number/100 nil
of chlorobrominated effluent).
                                  23

-------
100,000
zl 0,000



U_
U.
iLl

o
llJ


I


§ 1,000

-------
             30
Ul
            ,20-
          Q
           OC
           O
          O
10-
 8-
 6-
 4-
 2-
 0
                                                                              r=0.4424lp>0.99)
                            50
                           100
           200
TURBIDITY (J.T.U.)
300
                Figure  8.   Correlation  between turbidity (J.T.U.)  and chlorine dosage (mo;/l) necessary
                            to maintain  a  total residual chlorine level > 1.5 mg/1 after 30 minutes.

-------
  30
1
UJ
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IT
O

O
                                                                   y=O.IOOx + l.07
                                                                   r=0.6829  (p>0.99)
O
£
8-

6-

4-

2-

0
                 50
                          100
           200
TURBIDITY (J.T.U.)
300
     Figure 9.
             Correlation between  turbidity (J.T.U.)  and bromine chloride dosage  (mg/1)
             necessary  to maintain  any  titrable residual after 5 minutes.

-------
  15-
01
£
O
Q


LU

O
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0  5
                                                             y=0.02!2x+727

                                                             r=02092 (p 20.95)
                  50
100
150
250
300
     Figure 10.
                                     200

                         TURBIDITY  (J.T.U.)

Correlation between turbidity  (J.T.U.)  and  ozone dosage (mg/1).

-------
N3
oo
          '20-
         8
QC
O
           10-

            8-

            6-

            4-

            2-

            0
                                                                          y=0.0432x+0.9487
                                                                          r=0.8036 (p>0.99)
                         100
                            200         300        400        500
                                CHEMICAL  OXYGEN  DEMAND (mg/l)
600
             Figure 11.
                 Correlation between chemical oxygen demand (mg/l)  and chlorine  dosage  (mg/l)
                 necessary to maintain a total residual chlorine level >1.5  ng/1  after 30
                 minutes.

-------
  30
O>
CO
§
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   6-
   4
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                                                                 y=0.062x4-1.417
                                                                 r=0.3952  (p>0.99)
—I	1	\	1—
 200         300         400         500
      CHEMICAL OXYGEN DEMAND  (mg/l)
                100
600
     Figure 12.  Correlation between chemical oxygen demand (mg/l) and bromine chloride
                 dosage (mg/l) necessary to maintain any titrable residual after 5 minutes.

-------
   15-
  10-
OT
O
O

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8  5
                                                            y=O.OII67x+6.821
                                                            r=0.3605 (p>.99)
                 100
600
     Figure  13.
            200        300        400        500

                CHEMICAL OXYGEN DEMAND (mg/l)

Correlation between chemical oxygen demand  (mg/l)  and ozone  dosage  (mg/l),

-------
           30
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                                                                         r=0.8193 (p>0.99)
                          50
                                                                             300
                        100          150         200         250
                            TOTAL  SUSPENDED SOLIDS (mg/l)
Figure 14.  Correlation between suspended solids  (mg/l)  and chlorine dosage (mg/l)
            necessary to maintain a total residual chlorine level >1.5 ng/1 after
            30 minutes.
350

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r-o
            30
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                           •
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Figure 15.
                           50
                             100         150        200         250

                                 TOTAL SUSPENDED SOLIDS (mg/l)
                                                   300
350
                          Correlation between suspended solids  (mg/l) and bromine chloride dosage

                          (mg/l) necessary to maintain any titrable residual after 5 minutes.

-------
UJ

LO
             15
           o>
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           UJ
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                                                                        y = 0.0396 x+7.14

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                             r

                             10
                              i            i             i            i

                             20          30         40          50

                                 TOTAL SUSPENDED SOLIDS (mg/l)
60
                Figure 16.  Correlation between  suspended  solids  (mg/l)  and  ozone dosage (mg/l)

-------
However, the analysis of variance also indicated a significant interaction
between treatments and time segments (p>0.99).  This means that the dif-
ferences observed among disinfectants were not consistent in both time
segments, suggesting wastewater quality affects performance of each disin-
fectant in different ways.  Indeed, this is not unexpected, since the re-
activities of each disinfectant with various demand materials are known to
be quite diverse.

Since the analysis of variance only indicated significant differences among
treatment means, it did not define which means were significantly different
or how many differences there were.  To test hypotheses about these differ-
ences, a Least Significant Difference test^ was performed on the same data.
Figure 17 illustrates diagrammatically the significant differences in total
and fecal coliform geometric means as determined by this method.  Any pair
of means within the range of any one line below it is not significantly
different (p>0.95).  In general, results from the fecal coliform data were
similar to those from the total coliform data.  Considering both total and
fecal coliform means, each mean in the first interval ("A") was lower than
its counterpart in the second interval ("B").  This difference was statis-
tically significant (p>0.95) for all total coliform treatments except chlo-
rination and all fecal coliform treatments except dechlorination.

In general, fewer differences among treatment means occurred in the first
time segment than in the second.  Thus, in the first interval, only two
pairs of total coliform means were significantly different (i.e., Cl2/BrCl
and S02/BrCl, the BrCl being greater than both), and only one pair of fecal
coliform means displayed a difference (i.e., S02/BrCl, the BrCl again being
the greater mean).  However, in the second interval, most coliform means
were significantly different.  Thus, only one pair of total coliform means
was not significantly different (i.e., S02/BrCl), and only two pairs of fecal
coliform means exhibited no significant difference (i.e., SC^/Clo and CL^/
BrCl).  Both the total and fecal coliform means from the ozone treatment in
the second interval were higher (p>0.99) than all other treatment means in
both intervals.  It appears that chlorine is least affected by changes in
wastewater quality, followed by bromine chloride and then ozone.  This is
undoubtedly due to the inherently greater ability to control chlorine dosage
as a function of chlorine demand and suggests the need to develop better
means of controlling bromine chloride and ozone in a similar fashion to
chlorine.

Regarding the untreated (i.e., nondisinfected) effluent, a t-test was per-
formed to determine if there was a significant difference in total and fecal
coliform levels between the two time segments.  Results indicated that both
total and fecal coliform means were significantly greater (p> 0.99) in the
first interval than in the second.  Thus, the gross deterioration in organic
quality of the Wyoming effluent from January to April, 1976, compared to
June to December, 1975, was not paralleled by a corresponding deterioration
in bacteriological quality.
                                      34

-------
       TOTAL COLIFORM  GEOMETRIC MEANS (xIN NUMBER PER 100 ml)0:
:i2A
X
140
S02A
X
180
O/A"
X
200
CI2B
X
210
BrCIA
X
280
                                                                  SOg'B*
                                                                    XXX
                                                                   570         570        9000
       FECAL COLIFORM  GEOMETRIC MEANS (x IN NUMBER PER 100 ml)0;
S02A*
x
54
CI2'A"
x
58
OsA
x
69
S02V
x
84
BrCIA"
x
86
CI2Brt
x
100
it \i
BrCIB
x
170
                                                                                             a o
                                                                                           O^B
                                                                                            x
                                                                                          4900
                       0 ANY PAIR OF  MEANS ENCLOSED BY THE RANGE OF ANY
                       ONE LINE IS NOT SIGNIFICANTLY DIFFERENT (p.> 0.95)
Figure 17.  Diagramatic representation of  significant differences (least significant difference,
            (p ^ 95%)) between total and fecal coliform means from chlorinated (Cl2), dechlorinated
            (802),  chlorobrominated (BrCl),  and ozonated (03) effluents taken from June through
            December, 1975 ("A")  and January through April, 1976 ("B").

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Frequency of Project Standards Achievement

Table 4 illustrates the frequency that each disinfectant reduced total and
fecal coliform densities to project standards within each time interval and
then combined for the entire project.  In the first interval, all treatments
were effective in enabling achievement of both bacteriological standards.
In the second interval, all process efficiencies diminished, the most dra-
matic decrease being exhibited by ozone.  The reason for this, as already
stated, was that the ozonation system in use did not have the capability to
respond to such an extreme increase in demand, as it was already operating
at maximum capacity.

It is clear from observation of Table 4 that the number of samples analyzed
from each treatment varied.  The reason for this was mechanical breakdowns
in the test equipment.  The BrCl dosing unit and accompanying residual
analyzer frequently malfunctioned.  Thus, when such an occurrence took
place, either a sample was not collected at all, or the analysis of a sample
obtained under such conditions was subsequently excluded from the overall
evaluation.  If the number of samples collected were not shown in Table 4,
one would receive the impression that BrCl was equally effective as Cl2 in
attainment of desired bacteriological goals, when in reality the only data
included in the evaluation were those obtained when the BrCl system was
functioning properly (approximately 80 percent of the time in the first
interval, and 66 percent of the time in the second interval).  The problem
with the BrCl dosing system was the accumulation of solid and semi-solid
contaminants in the evaporator, resulting in blockage of flow of the BrCl
gas.  This system malfunctioned substantially more often than any other
system, even after a newly-designed dosing apparatus had been introduced in
February, 1976.

The ozone generation system operated better than it had in the previous
study at Grandville.  Two minor electrical malfunctions occurred in October,
1975, and in February, 1976.  However, little experimental time was lost due
to the presence of a back—up ozone generation system.  Two serious mal-
functions in the air compressor occurred in September, 1975, and in March,
1976, resulting in a moderate amount of down time.

Observations of Data on a Monthly Basis

To inspect the temporal relationships of the performance of  the various
disinfection processes in a more typical fashion, mean coliform data are
presented in Figures 18 to 21 in terms of monthly intervals.  Additional
information is presented in Appendices 1-5, which show sample sizes, stan-
dard deviations, and arithmetic and geometric means.

Figures 18 and 19 show the monthly geometric means of total and fecal coli-
form densities, respectively, in all test streams.  All streams exhibit
moderately stable monthly total and fecal coliform means for the first
seven months of the study  (through December, 1975).

Starting in January, 1976, and continuing until the end of the experimental
period, a deterioration in bacteriological quality in all effluent streams

                                     36

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                  Table 4.  FREQUENCY3 THAT DAILY SAMPLES OF DISINFECTED EFFLUENTb
                                 ACHIEVED PROJECT BACTERIOLOGICAL STANDARDS

Time Interval
June to December, 1975
January to April, 1976

Combined

Total Coliforms
% of Samples Below 1000 per 100 ml
ci2
88
(124)c
83
(71)
86
(195)
so2
89
(121)
69
(62)
83
(183)
BrCl
85
(100)
72
(47)
81
(147)
°3
86
(116)
12
(69)
58
(185)
Fecal Coliforms
% of Samples Below 200 per 100 ml
ci2
81
(122)
68
(69)
77
(191)
so2
84
(118)
59
(63)
75
(181)
BrCl
77
(100)
68
(44)
74
(144)
°3
84
(109)
16
(64)
59
(173)
aFrequency calculated as number of samples which met coliform standards divided by total number of
 samples times 100%
 For disinfectant concentrations, refer to Tables 2 and 3

°Number of samples in parentheses

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      io6-
      10
        5_
      10*-
      I03-
   E
  O
  O
  cc
  UJ
  CL
  o:
  o
  o
  o
      I02-
      10' -
•	• NONDISINFECTED

•	• CHLORINATED

A	A DECHLORINATED

•	• CHLOROBROMINATED

O	O OZONATED
      10° •
            June July  Aug. Sept. Oct.  Nov.  Dec. Jan. Feb. Mar.  Apr.

Figure 18.  Monthly geometric means of  total coliform densities  (MPN)  for

            each  stream.

                                     38

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            I0e
            105-
            io4-
            10'-
        E
        O
        O
        tf)
        (E
        O
        2
        UJ
        u.
I02-
10'-
                        V
-• NONDISINFECTED
-• CHLORINATED
                                   A	A  DECHLORINATEO (S02)
                                   •	•  CHLOROBROMINATED
                                   O	O  OZONATED
Figure 19.
10°
      June  July  Aug. Sept. Oct.  Nov.  Dec.  Jan. Feb. Mar.  Apr.
 Monthly geometric means of fecal coliform densities (MEN) for
 each stream.
                         39

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100
                                      CHLORINATED
                                      DECHLORINATED  (S02)
                            •	• CHLOROBROMINATED
                            O	O OZONATEO
   JUN.
AUG.
SEP
OCT.
NOV.
DEC.
JAN.
FEB.
MAR.
APR.
  Figure 20.  Monthly frequencies  that daily samples of disinfected effluent  achieved  the
              project total coliforn  standard of  <1000 organisms per 100 ml samp.e.

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   100
 E
O
o

v.

O
o

CM
UJ

CD
0.


<
V)
UJ
o
tr
    80-
    60 -
    40-
    20-
     0
JUN.
                                •	• CHLORINATED


                                A	A DECHLORINATED  (S02)


                                •	• CHLOROBROMINATED


                                O	O OZONATED
               JLY
AUG.
SEP
OCT.
NOV.
DEC.
JAN.
FEB.
MAR.
                                                                                        APR.
     Figure  21.  Monthly frequencies that daily samples  of disinfected effluent achieved the

                 project fecal coliform standard of  <200 organisms per 100 tnl sanvole.

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except the nondisinfected is quite evident.  The increase in monthly coli-
form means is particularly marked in the ozonated effluent.  Coliform levels
in the chlorobrominated stream were generally higher than the chlorinated
and dechlorinated streams, but not in every instance.  Total coliform den-
sities in the dechlorinated stream were consistently higher than those in
the chlorinated stream, particularly from January, 1976, to April, 1976,
whereas the converse is true in the fecal coliform population.  This fact
is further supported by the previously discussed Least Significant Differ-
ence Test (Figure 17), in which it was determined that the mean total coli-
form density in the dechlorinated stream (S(>2 "B") was significantly higher
(p?0.95) than the chlorinated stream (C12 "B") , while the mean fecal coli-
form level in the dechlorinated stream (SC>2 "B") was significantly lower
than the chlorinated stream (C12 "B").

Figures 20 and 21 present the frequency, on a monthly geometric mean basis,
that samples from each treatment met project disinfection standards.  These
data closely parallel those discussed above.

Summary

Duration of the project was eleven months.  During the first seven months
of the project, wastewater quality was satisfactory.  As winter temperatures
approached, upsets occurred in the biological treatment system, giving rise
to increased organic demand in the effluent prior to disinfection.  Disin-
fection efficiencies of all experimental processes were affected to varying
degrees by the deteriorating conditions.  Bromine chloride and ozone dis-
played the greatest sensitivities to these shifts in demand.  Excellent
correlations were found between chlorine and bromine chloride dosages neces-
sary to maintain given residuals and turbidity, suspended solids, and chemi-
cal oxygen demand.  No correlation was found between ozone dosage and organic
demand due not only to the lack of an adequate means of controlling dosage
in accordance with such fluctuations, but also to the incapability of the
ozone generation system to meet the higher dosage requirements.

Malfunctions in the BrCl and 03 dosing systems occurred with greater fre-
quency than in the chlorination and dechlorination systems, suggesting the
need to improve tfhe engineering capability of these newer processes, as
well as to develop better means of controlling dosage as a function of
demand.  Thus, application of these newer technologies to wastewater disin-
fection is supportable in that they have demonstrated the capability to
reduce total and fecal coliform densities to acceptable levels.  Neverthe-
less , they have also demonstrated a tendency to require more maintenance
and repairs due to incomplete technology development.
                                     42

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                 Appendix 1
COLIFORM DENSITIES OF NONDISINFECTED EFFLUENT
       JUNE, 1975 THROUGH APRIL, 1976
                (number/100 ml)

June
July
August
September
October
November
December
January
February
March
April
Total Colif orm Density
No. of
Samples
18
14
17
17
18
16
19
14
19
23
16
Geometric
Mean
3.9 x 106
3.3 x 106
4.0 x 106
5.9 x 106
2.8 x 106
1.8 x 106
1.5 x 106
1.0 x 106
1.6 x 106
1.8 x 106
3.0 x 10
Fecal Colif orm Density
No. of
Samples
16
14
17
17
18
16
19
14
17
23
15
Geometric
Mean
1.7 x 106
9.6 x 105
2.9 x 10
2.9 x 106
5.3 x 105
6.2 x 105
8.2 x 105
4.8 x 105
8.2 x 105
8.4 x 105
1.3 x 10
                    43

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                Appendix 2
COLIFOEM DENSITIES OF CHLORINATED EFFLUENT
      JUNE, 1975 THROUGH APRIL, 1976
              (number/100 ml)

June
July
August
September
October
November
December
January
February
March
April
Total Coliform Density
No. of
Samples
18
16
17
18
18
16
20
14
18
23
16
Geometric
Mean
160
210
390
170
91
70
95
140
380
270
110
Fecal Coliform Density
No. of
Samples
17
16
17
18
18
16
20
14
17
23
15
Geometric
Mean
68
73
180
58
29
35
46
66
220
160
34
                   44

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                 Appendix 3
COLIFOKM DENSITIES OF DECHLORINATED EFFLUENT
       JUNE, 1975 THROUGH APRIL, 1976
               (number/100 ml)

June
July
August
September
October
November
December
January
February
March.
April
Total Coliform Density
No. of
Samples
18
16
17
18
18
15
19
12
16
20
14
Geometric
Mean
180
180
450
200
120
120
140
230
550
1000
590
Fecal Coliform Density
No. of
Samples
16
16
17
17
18
15
19
12
16
20
15
Geometric
Mean
59
44
180
76
26
31
47
55
220
88
39
                     45

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                   Appendix 4
COLIFORM DENSITIES OF CHLOROBROMINATED EFFLUENT
         JUNE, 1975 THROUGH APRIL, 1976
                 (number/100 ml)

June
July
August
September
October
November
December
January
February
March
April
Total Coliform Density
No. of
Samples
14
13
13
15
17
15
13
8
12
12
15
Geometric
Mean
320
310
350
370
250
220
190
420
500
450
920
Fecal Coliform Density
No. of
Samples
13
13
13
15
18
15
13
8
10
12
14
Geometric
Mean
120
110
220
140
42
41
70
210
180
140
160
                      46

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               Appendix 5
COLIFORM DENSITIES OF OZONATED EFFLUENT
     JUNE, 1975 THROUGH APRIL, 1976
             (number/100 ml)

June
July
August
September
October
November
December
January
February
March
April
Total Coliform Density
No. of
Samples
17
17
17
14
15
16
20
14
20
19
16
Geometric
Mean
210
430
98
320
210
110
200
1600
23000
7900
16000
Fecal Coliform Density
No. of
Samples
14
17
17
13
12
16
20
12
18
19
15
Geometric
Mean
67
130
60
130
47
27
79
300
21000
4300
9700
                   47

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                                REFERENCES

1.  Standard Methods for the Examination of Water and Wastewater. 13th ed.
    New York, American Public Health Association, 1971.  874 pp.

2.  Sokal, R. R., and F. J. Rohlf.  Biometry. San Francisco, W. H. Freeman
    and Co., 1969.  368 pp.
                                    48

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                                 SECTION V

                    LIFE CYCLE RESIDUAL TOXICITY STUDIES

INTRODUCTION

The residual toxicity of chlorinated wastewater effluent has been demonstrat-
ed in several recent studies. ~°  As a result of these findings, attention
has been given to either neutralizing chlorine residuals in treated effluents
or replacing chlorine with a disinfectant which has no residual toxicity.
The former approach has shown that residual chlorine toxicity can be elimi-
nated through the use of sulfur dioxide, ^»® bisulfite,3 or sodium thio-
sulfate.°  Among the alternative processes being considered for wastewater
disinfection are ozonation 8-10 an(j chlorobromination.8,11,12

The life cycle study reported herein was designed to determine whether various
chemical disinfectants, applied to a trickling filter effluent derived from
wastewater containing a large proportion of industrial wastes, imparted a
long-term residual toxic effect to fish.  Thus, the toxicity of parallel ef-
fluent streams treated with chlorine (C12), sulfur dioxide (802) dechlori-
nation, bromine chloride (BrCl), or ozone (0-) and a nondisinfected control
stream was examined.  The fathead minnow, Pimephales promelas, was the subject
of these tests.

METHODS AND MATERIALS

Most of the bioassay methods and materials were similar to those described in
the Grandville report.^  The following paragraphs include only highlights and
aspects unique to the Wyoming study site.

Chemical Analyses

Residual Cl2, BrCl, 03, and  sulfite (residual S02 was measured as sulfite)
were measured bi-weekly in the respective effluent storage tanks and daily
in one aquarium containing the highest effluent concentration (see Vol. I for
methods).  Heavy metal analyses were performed weekly on effluent samples
which were stored from the preceeding seven days.  A Perkin-Elmer model 403
Atomic Absorption Spectrophotometer was used for these determinations.  Also,
on Mondays through Fridays, three 8-hour composite samples from the preceed-
ing day were tested for cyanide concentrations using the colorimetric pyri-
dine-barbituric acid method. •"••*

Water Supplies

Detailed descriptions of the treatment site and the effluent flow schemes are
presented in Section III of this report.  Dilution water was obtained from a

                                      49

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well at the study site and had the characteristics shown in Table 5.  The pH
and conductivity of the well water were 7.4 and 1663 jimhos/cm, respectively.

               Table 5.  CHARACTERISTICS OF THE DILUTION WATER
Concentration
Analysis in mg/1
Hardness (as CaCOo)
Calcium
Magnesium
Sulfate
Chloride
Iron (prior to iron filtration)
NH3-N
Alkalinity (as CaCO,)
Acidity (as CaCOj
782
272
14
545
86
1.39
0.60
197
25
Bioassay Methods

The effluents and dilution  (well) water were delivered to headtanks where
they were heated to 25C for life cycle bioassays with P_. promelas.  The
residence time in the headboxes was approximately 45 minutes.

With the exception of the chlorinated effluent diluter system, all diluters
were calibrated to deliver 100 percent effluent, 100 percent dilution water,
and six intermediate nominal effluent concentrations of 50.00, 25.00, 12.50,
6.25, 3.12, and 1.56 percent.  The chlorinated effluent diluter was designed
to deliver nominal effluent dilutions of 50.00, 25.00, 18.75, 12.50, 6.25,
3.12, and 1.56 percent, and 100 percent dilution water.  Lower concentrations
of chlorinated effluent were used because of the previously demonstrated
toxicity of chlorinated effluent to aquatic life.l

The calibration of all life cycle diluters and flow-splitting cells was
checked volumetrically each week and adjusted, if necessary, to maintain
the proper effluent concentration and turnover time in each test chamber.
Due to the generally poor quality effluent, frequent adjustments of all di-
luters and flow-splitting cells were necessary to maintain proper calibration
throughout the entire study.  Periodically, it became necessary to completely
disassemble and clean each diluter system.

Those adult and fry tanks receiving the five highest effluent concentrations
were continuously aerated with oil-free air to prevent excessively low dis-
solved oxygen concentrations.

Progeny of fathead minnows  (P_. promelas) obtained from stock cultures main-
tained at the Environmental Research Laboratory-Duluth, were used for the
life cycle tests.  The tests were started by placing fifty 8-12 day old fry
in each aquarium and monitoring their survival.  The test animals were photo-
graphed at 30, 60, 120, and 310 days (adult termination) into the test.  The
photographs were enlarged to determine the lengths of the surviving test


                                     50

-------
animals.

If the total number of spawnings with at least 50 viable eggs exceeded 10
in a given tank, only eggs from every third spawning were incubated.  In
addition, when time and spawnings permitted, hatchability information was
obtained on eggs produced in dilution water tanks and incubated in high
concentration effluent tanks.

If more than 5 of the 50 eggs in any one incubation were unaccounted for,
that incubation attempt was discarded.  Loss of eggs and/or fry was not un-
common, due to their small size and the nature of the effluent.

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.  In an attempt to obtain more complete data on
second generation fish, some fry were transferred from the 100 percent
dilution water tanks in which they were spawned and incubated to high efflu-
ent concentration fry tanks for which no fry were produced in the respective
adult tanks.  The lengths of all fry were measured photographically at 30
and 60 days.  At 60 days the fry were discarded and the tank was then avail-
able for restocking.

All fish were fed twice daily, once with a mixture of live brine shrimp
nauplii and a commercial trout preparation, and once with live brine shrimp
nauplii only.  Excess food and other debris were siphoned from the adult test
chambers daily.  Fry chambers were not cleaned until the fish were 10 days
old.

Communication with local industry as well as close surveillance of disin-
fectant demand enabled the bioassay staff to forecast unusual effluent
conditions which might be temporarily lethal to the test fish.  In such
circumstances, the effluent supply to the test chambers was halted and the
fish were maintained with only a supply of dilution water.  As soon as the
effluent returned to its normal condition (generally less than 1 day) its
flow to test chambers was restored.

Dunnett's test   was performed on all growth data for all treatment types
and concentrations to determine significant (p = 0.05) differences between
fish reared in effluent and dilution water within a treatment type, and
between fish reared in similar concentrations of effluent and dilution water.

RESULTS AND DISCUSSION

The mean residual chemical levels in the head tanks, adult chambers, and
fry chambers of the various effluent streams are shown in Tables 6 and 7.
Frequent changes in the quality of the Wyoming effluent produced increased
variations in the residual chemical levels in all effluent streams.
                           ft
As in the Grandville study,  the C12 residual was the most consistent of
those measured.  This was partly due to the continuous residual Cl2 analyzer
placed on the chlorinated stream, which enabled operators to adjust dosage
frequently to achieve a predetermined residual.  Another factor contributing


                                      51

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         Table 6 .   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
Head Tank
1.432
85
0.619
Nominal Percent Effluent Concentration
50
0.299
6
0.124
25
0.085
213
0.071
18.75
0.059
114
0.050
12.5
0.040
114
0.030
6.25
0.018
81
0.015
3.12
0.012
83
0.010
1.56
0.010
81
0.008
Effluent Stream
De chlorinated
Sulfite Residual
Sample Size
Standard Deviation
Chlorobrominated
Bromine Chloride
Residual
Sample Size
Standard Deviation
Ozonated
Ozone Residual
Sample Size
Standard Deviation
Head Tank
1.851
85
1.868
0.089
77
0.093
0.008
81
0.015
Nominal Percent Effluent Concentration
100
0.010
156
0.050
0.008
6
0.005
0.003
173
0.003
50
0.048
140
0.140
0.008
207
0.015
0.002
119
0.002
25
0.005
115
0.026
0.005
114
0.005
0.002
108
0.002
12.5
0.002
80
0.006
0.005
104
0.006
0.002
77
0.002
6.25
0.000
84
0.001
0.010
54
0.019
0.002
75
0.002
3.12
0.000
66
0.000
0.006
64
0.006
0.002
75
0.002
1.56
0.000
80
0.000
0.005
79
0.010
0.001
74
0.002
Ui
NJ

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             Table   7.   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
Nominal Percent Effluent Concentration
50
0.055
117
0.096
25
0.041
59
0.085
18.75
0.027
56
0.025
12.5
0.021
54
0.012
6.25
0.012
47
0.012
3.12
0.010
49
0.006
1.56
0.006
53
0.006
Effluent Stream
De chlorinated
Sulfite Residual
Sample Size
Standard Deviation
Chlorobrominated
Bromine Chloride
Residual
Sample Size
Standard Deviation
Ozonated
Ozone Residual
Sample Size
Standard Deviation
Nominal Percent Effluent Concentration
100
0.215
116
0.478
0.003
113
0.009
0.001
115
0.002
50
0.117
58
0.266
0.005
54
0.009
0.001
56
0.002
25
0.037
50
0.121
0.003
48
0.005
0.001
49
0.002
12.5
0.000
51
0.000
0.003
52
0.008
0.001
49
0.001
6.25
0.000
50
0.002
0.002
47
0.004
0.001
48
0.002
3.12
0.074
51
0.470
0.002
46
0.004
0.001
46
0.001
1.56
0.001
52
0.004
0.003
47
0.004
0.001
46
0.002
Ui
LO

-------
to the relatively uniform Cl2 residuals was the reliability of the dosing
system which seldom malfunctioned.

The principal reason for the great variation in mean residual sulfite con-
centrations in the dechlorinated effluent was the lack of capability for the
continuous analysis of residual sulfite to assist in the adjustment of SO^
feed rates as demand varied.  A continuous analyzer for residual BrCl placed
in the contact chamber corresponding to a 5-minute detention time was used
to adjust the dose of BrCl applied to the chlorobrominated effluent stream,
but frequent failures of the analyzer or dosing system contributed to the
considerable variation of residual BrCl.

Ozone was generally applied at a constant rate, and thus residual Oo varied
considerably with demand.  Because of the limited capacity of the 63 gener-
ation-dosing system, it was often impossible to meet  the high Oo demand of
the wastewater.

With the exception of residual S02> residual chemical concentrations in the
fry chambers (Table 7) were lower than those observed in the respective
adult chambers.  This was partially the result of variations in effluent
demand.  In addition, some organic matter was allowed to accumulate in fry
tanks to promote the growth of microscopic organisms which constitute an
important fraction of the diet of young fish.  This accumulation of organic
matter in fry tanks tended to increase the chemical demand and to decrease
measured residuals.

The well water used to dilute the effluent entered its head box with a low
DO content and was constantly aerated to an acceptable DO concentration
(Table 8).  The nondisinfected effluent stream lacked the settling time of
a disinfectant contact chamber and carried a greater load of suspended solids
to its bioassay laboratory head tank.  The consequent accumulation of addi-
tional sludge in the head tank increased the oxygen demand and decreased the
DO concentration in that tank.  The dechlorinated effluent head tank exhibit-
ed the lowest mean DO concentration of any disinfected effluent head tank.
This lower DO was probably the result of the excess sulfite in this stream
reacting with oxygen to form sulfate, as well as the BOD produced in this
tank where biological oxidation of sludge was not inhibited by residual
disinfectant.

Aeration of the higher effluent concentration fish tanks provided an adequate
supply of DO for the test animals.  The lowest mean DO was 4.1 mg/1 in the
100 percent chlorobrominated fry chamber.  This is approximately 50 percent
saturation at the 25C test temperature  (Table 9 ) and was probably within
the safe limits of DO for P_. promelas.

Tables 10 and 11 summarize some of the characteristics of the various test
streams as measured in the respective head tanks and fish tanks.  According
to the tables presented in Thurston, et al.,+5 the mean total ammonia nitro-
gen concentrations recorded in the highest concentration fish tanks may have
been great enough to produce mortality in the test animals.  However, all
other characteristics were within the general tolerance limits of fish.16
                                     54

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                        Table  8.  THE MEAN DISSOLVED OXYGEN CONCENTRATIONS  (mg/1)
                                     MEASURED IN HEAD TANKS AND TEST CHAMBERS
                                           DURING THE LIFE CYCLE STUDIES
Stream
Dilution Water
Nondisinfected
Dechlorinated
Chlorobrominated
Ozonated
Head
Tank
4.7a
1.6
3.3
3.6
4.5
Test
Chambers
b
Adult
Fry
Adult
Fry
Adult
Fry
Adult
Fry
Nominal Percent Effluent Concentration
100
b
5.4
4.2
5.9
5.1
6.1
4.1
5.6
4.5
50
,
5.6
5.2
5.6
4.6
5.5
4.2
5.8
5.4
25

5.7
6.1
5.9
4.6
5.1
4.4
5.7
6.2
12.5

5.7
6.6
5.8
6.8
5.4
5.2
6.3
5.5
6.25

5.2
6.3
5.8
6.1
5.5
6.4
6.0
6.7
3.12

5.6
6.4
6.1
6.5
5.9
6.2
6.3
6.8
1.56

5.8
6.9
5.7
7.0
5.7
6.4
5.9
6.6
0.00

6.2
7.3
6.3
7.3
6.3
7.0
6.5
5.8
Ul
Ln
Stream
Chlorinated
Head
Tank
5.1
Test
Chambers
Adult
Fry

50
4.8
4.9
Nominal P<
25
5.5
6.1
18.75
5.7
6.2
srcent Effluent Concentration
12.5
6.1
6.3
6.25
6.3
7.3
3.12
6.2
6.8
1.56
5.8
6.6
0.00
6.2
6.8
        aThe dilution water head  tank and all test chambers with 6.25 percent or more effluent were
         continuously aerated.
         Mean dissolved  oxygen measurements  for 100 percent dilution water tanks are shown under the
         0.00 percent effluent column.

-------
      Table  9.   MEAN WATER TEMPERATURES ( C)
               MEASURED IN HEAD TANKS AND ADULT
           TEST CHAMBERS DURING THE LIFE CYCLE STUDIES
Effluent Type
Nondlsinfected
Chlorinated
Dechlorinated
Chlorobrominated
Ozonated
Dilution Water
Mean Temperature
In Head Tank
26.2
26.3
27.2
26.7
25.5
26.5
Mean Temperature
In Aquaria3
24.9
24.9
24.8
24.4
24.8
No data available
All temperatures were measured in aquaria containing a 50 per-
cent effluent concentration.
                              56

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Table 10.  CHARACTERISTICS OF THE TEST STREAMS MEASURED IN THE HEAD TANKS
Stream
Nondisinfected
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 CaCOo
(mg/ir
281
43
37.6
269
42
34.7
260
42
36.7
272
42
34.0
277
43
37.7
197
43
2.5
Acidity
as CaCO.,
(mg/l)J
28
38
6.0
28
38
5.8
41
36
9.1
29
38
6.7
58
38
154.9
25
38
4.9
Hardness
as CaCO-
(mg/ir
279
42
22.9
281
42
23.8
286
40
19.9
281
42
22.9
280
42
23.0
782
42
9.9
Conductivity
(Micromhos/cm)
1091
34
136.8
1101
34
133.1
1135
32
131.7
1090
34
130.1
1097
34
126.6
1663
33
251.0
Total Ammonia
Nitrogen
(mg/D
11.6
40
3.11
11.9
40
2.67
11.9
40
3.75
11.7
40
2.28
11.3
40
3.91
0.6
40
1.42

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                                Table 11.  CHARACTERISTICS OF THE TEST  STREAMS MEASURED
                                           IN THE HIGHEST CONCENTRATION ADULT TEST TANKS
Stream
Nondisinfected
Mean
Sample Size
Standard Deviation
Chlorinated
Mean
Sample Size
Standard Deviation
De chlor inated
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 CaCO-
(mg/1)
280
43
43.0
231
43
20.8
254
43
37.9
272
43
37.3
273
42
43.3
197
43
3.3
Acidity
as CaCO
(mg/ir
18
39
6.3
23
39
6.9
26
39
11.5
20
39
7.6
19
39
6.2
16
39
5.4
Hardness
as CaCO~
(mg/1)
280
42
22.8
534
42
25.7
284
40
21.8
281
42
21.7
283
42
22.9
784
42
12.3
Conductivity
(Micromhos/cm)
1087
36
112.1
1410
36
164.4
1105
34
128.0
1093
36
111.0
1088
35
105.2
1690
35
207.7
Total Ammonia
Nitrogen
(rng/1)
11.0
40
3.48
5.6
40
2.81
11.4
40
4.11
10.7
40
3.16
9.8
40
3.60
0.4
39
0.60
Ui
00

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The concentrations of selected metals  in  the nondisinfected  effluent were
generally typical for a wastewater  treatment plant receiving wastes from
plating industries (Table 12).   Some of the concentrations measured had the
potential to be lethal to aquatic life, especially in  the 100 percent ef-
fluent concentrations.  Through  cooperation with local industry and vigilance
in monitoring chlorine demand, laboratory personnel were generally able to
anticipate concentrations of metals which might be toxic to  the test animals.
When such conditions were expected, effluent was temporarily diverted around
the bioassay laboratory until tests indicated  that metal concentrations were
again safe for the test animals.  This procedure prevented mass mortality
from metal toxicity during  the life cycle study.

The chemical and physical characteristics of respective concentrations of
the different effluent streams were generally  uniform  and safe to the test
animals.  Thus, differences in results between test animals  in disinfected
effluent and those in nondisinfected effluent  were attributed to the disin-
fection process.

Mortality

First generation test fish  showed high mortality in 100 percent nondisinfect-
ed effluent during the first 30  days of the life cycle test  (Table 13) .  The
mortality of that period was directly  related  to relatively  high concen-
trations of heavy metals and cyanide in the effluent.   As a  result of this
high mortality, both 100 percent concentration tanks of nondisinfected
effluent were restocked on  day 31 with test animals of the same age as the
original test animals.

No other first generation mortality with  an obvious relationship to effluent
concentration occurred in nondisinfected  effluent.  The mortality observed
between days 120 and 240 in the  various concentrations of nondisinfected
effluent was the result of  a disease which was not related to effluent
quality.  This same disease-related mortality  was observed in all test
streams.

Second generation test animals exhibited  a high mortality, similar to that
observed in first generation fish,  when exposed to undiluted nondisinfected
effluent (Table 14).  Thus, it would appear that 100 percent nondisinfected
effluent was lethal to young fathead minnows even in the absence of any
chemical disinfectant.

First generation test fish  in 50 percent  chlorinated effluent died within
three days when exposed to  total residual chlorine concentrations of 0.2 to
0.4 mg/1 (Table 15).  These tanks were restocked on day 4, but once again
total mortality occurred within  24  hours.  First generation  fathead minnows
in 25 percent chlorinated effluent  (mean  total residual C12  concentration
of 0.165 mg/1) were visibly stressed during the first  30 days of the study
and also exhibited reduced  survival.   No  further mortality that could be
directly related to chlorinated  effluent  was observed  in any lower test
concentration for the remainder  of  the study.  These data  are presented  in
Table  15.
                                      59

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          Table 12.  MEAN METAL AND CYANIDE CONCENTRATIONS (mg/1)
   MEASURED IN THE NONDISINFECTED EFFLUENT DURING THE LIFE CYCLE STUDIES2

Number
of Samples
Mean
Concentration
Standard
Deviation
Minimum
Concentr at ion
Maximum
Concentr at ion
Chromium
287
0.084
0.085
0.010
0.570
Copper
287
0.045
0.036
0.000
0.480
Nickel
287
0.511
0.223
0.060
2.580
Zinc
287
0.149
0.082
0.010
0.540
Iron
287
1.031
0.865
0.070
5.640
Cyanide
136
0.159
0.416
0.000
9.900
Pleasured by atomic absorption spectrometry
                                     60

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        Table   13.  NUMBER OF FIRST GENERATION P.  PROMELAS SURVIVING IN NONDISINFECTED EFFLUENT

Survival/ 100
at day 30
C.I. for prob. of
survival thru
day 30
Survival/ 100
at day 60
C.I. for prob. of
survival thru
day 60
No. of fish kept
o> for further study
Number of fish
alive at : 90 days
120 days
150 days
180 days
210 days
240 days
270 days
310 days
C.I. for prob. of
survival
day 60-310
Nominal Percent Nondisinfected Effluent
0.00
97
0.92-0.99
97
0.92-0.99
30
29
21
20
16c
9
7
7
7
0.12-0.41
1.56
96
0.90-0.98
90
0.83-0.94
30
21
14
14
9c
0
0
0
0
0.00-0.11
3.12
99
0.95-0.99
99
0.95-0.99
30
16
15
15
7
7
7
7
7
0.12-0.41
6.25
94
0.88-0.97
93
0.86-0.97
30
30
30
30
250
14
lie
11
11
0.22-0.54
12.5
99
0.94-0.99
98
0.93-0.99
30
29
29
17
16
4
4
4
4
0.05-0.30
25.0
98
0.93-0.99
96
0.90-0.98
30
30
30
30
29
29
29
29
28
0.79-0.98
50.0
99
0.95-0.99
94
0.88-0.97
30
29
29
29
29
29
28
24
21
0.52-0.83
100.0
3
0.01-0.08
93a
0.86-0.97
30
30
30
22
20C
6
0
0
0
0.00-0.11
      100 percent concentration tanks were restocked  on  day  31
 The number of fish in each effluent concentration  was reduced  to  30  on day  60
^Excess  males removed from tanks

-------
                        Table 14.  NUMBER OF SECOND GENERATION P_. PROMELAS SURVIVING
                                           IN THE NONDISINFECTED EFFLUENT

30 Days of Age
Number
Surviving/Started
a
Confidence Interval
60 Days of Age
Number
Surviving/Started
Confidence Interval
Nominal Percent Nondisinfected Effluent
0.00
36/40
0.77-0.96
36/40
0.77-0.96
1.56
72/80
0.81-0.95
72/80
0.81-0.95
3.12
50/80
0.52-0.72
49/80
0.50-0.71
6.25
25/40
0.47-0.76
23/40
0.42-0.71
12.5
18/40
0.31-0.60
18/40
0.31-0.60
25.0
22/40
0.40-0.69
22/40
0.40-0.69
50.0
b

100.0
0/40
0.00-0.08

 95 Percent confidence interval for the true probability of survival

5No data available

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        Table  15.   NUMBER OF FIRST GENERATION P.  PROMELAS SURVIVING IN CHLORINATED EFFLUENT

Survival/100
at day 30
C.I.& for prob. of
survival thru
day 30
Survival/100
at day 60
C.I. for prob. of
survival thru
day 60
No. of fish kept
for further study
Number of fish
alive at: 90 days
120 days
150 days
180 days
210 days
240 days
270 days
310 days
C.I. for prob. of
survival
day 60-310
Nominal Percent Chlorinated Effluent
0.000
80
(0.002)a
0.71-0.87
79
(0.003)
0.70-0.86
30
30
(0.003)
29
(0.003)
29
(0.003)
27d
(0.003)
27
(0.003)
27
(0.003)
27
(0.002)
27
(0.002)
0.74-0.97
1.56
95
(0.017)
0.89-0.98
95
(0.016)
0.89-0.98
30
30
(0.015)
29
(0.014)
29
(0.014)
22d
(0.014)
22
(0.013)
18
(0.012)
17
(0.011)
16
(0.009)
0.36-0.70
3.12
38
(0.020)
0.29-0.48
38
(0.021)
0.29-0.48
30
30
(0.019)
29
(0.019)
29
(0.018)
221
10.018)
22
(0.017)
22
(0.016)
22
(0.014)
22
(0.012)
0.56-0.86
6.25
100
(0.026)
0.96-1.00
99
(0.032)
0.95-0.99
30
30
(0.033)
30
(0.031)
30
(0.029)
20^
(0.028)
20
(0.026)
20
(0.024)
20
(0.021)
20
(0.018)
0.49-0.81
12.5
76
(0.059)
0.67-0.83
75
(0.065)
0.66-0.82
30
30
(0.062)
30
(0.056)
30
(0.053)
25d
(0.051)
25
(0.049)
23
-------
First generation test fish in this study exhibited a greater tolerance to
chlorinated effluent than their counterparts in the Grandville study.  For
example, a 53 day mean total residual Cl^ concentration of 0.076 mg/1 in the
20 percent effluent concentration resulted in a 0.34-0.53 probability of
survival at Grandville (Table 22, Vol. I),8 while a 60 day mean total resid-
ual CIj concentration more than two times as great (0.161 mg/1) in the 25
percent effluent concentration resulted in about the same probability of
survival (0.37-0.56) at Wyoming.  While the cause of this difference in
tolerance is unknown, it may be related to natural variations in test repli-
cation, differences in the qualities of the raw wastewaters at the two plants,
differences between activated sludge and trickling filter effluents, or some
other factor(s).

Second generation test animals were exposed to low residual C^ levels and
showed no mortality related to residual Cl£ in the effluent (Table 16).
While total mortality of test animals did occur in the 50 percent effluent
concentration, the mean residual Cl2 present in that effluent concentration
was 0.008 mg/1, far less than the threshold value for lethality (0.045 ma/1)
that was observed in the Grandville study.8  Further, the total mortality of
second generation fish in the 50 percent concentrations of all disinfected
effluent streams supported the conclusion that mortality did not result from
the low levels of residual Cl£ to which second generation test animals were
exposed.

First generation test fish exposed to dechlorinated effluent survived much
better than those which were exposed to chlorinated or nondisinfected efflu-
ent (Table 17).  Dechlorination eliminated the toxicity of chlorinated ef-
fluent to young fathead minnows.  The improved survival over fish reared in
respective concentrations of nondisinfected effluent might be the result of
the greater aeration (and subsequent higher DO levels) of the dechlorinated
stream  (see Chapter III).  The mortality observed in the 100 percent concen-
tration of dechlorinated effluent after day 150 was the result of a disease
outbreak in the two undiluted effluent test chambers.  The limitation of the
spread of the disease to those two tanks was probably the result of stress
on those test animals in the undiluted effluent at a time when effluent
quality was very low (see Chapter III).

Second generation test fish did not survive in either 50 percent or 100 per-
cent dechlorinated effluent (Table 18).  However, since a similar pattern
of survival was also observed in second generation fish in nondisinfected
effluent (Table 14), the mortality in the 50 and 100 percent concentrations
of dechlorinated effluent was  probably the result of poor effluent quality
rather than the chlorination-dechlorination process.

In the chlorobrominated effluent stream, only the first generation fish
exposed to 100 percent effluent exhibited unusually low survival (Table 19).
This mortality appeared to result from low effluent quality rather than the
disinfection process, because similar mortality was observed in the nondisin-
fected effluent stream (Table 13) and because only very low (<0.017 mg/1)
BrCl residuals were present during the first 30 days of the test.  Likewise,
the survival pattern of second generation test fish (Table 20) was similar  to
that of second generation test animals reared in nondisinfected effluent,

                                      64

-------
                                  Table  16.  NUMBER OF SECOND GENERATION P_. PROMELAS SURVIVING
                                                       IN THE CHLORINATED EFFLUENT

30 Days of Age
Number
Surviving/Started
Confidence Interval
x Residual, mg/lc
60 Days of Age
Number
Surviving/Started
Confidence Interval
x Residual, mg/1
Nominal Percent Chlorinated Effluent
0.00
52/80
0.54-0.75
0.000
52/80
0.54-0.75
0.000
1.56
15/40
0.24-0.52
0.000
15/40
0.24-0.53
0.002
3.12
a

6.25
a

12.5
54/80
0.57-0.77
0.007
49/80
0.50-0.71
0.001
18.75
38/80
0.37-0.58
0.003
37/80
0.36-0.57
0.009
25.0
9/40
0.12-0.38
0.005
9/40
0.12-0.38
0.021
50.0
0/40
0.00-0.08
0.008

Ln
          ^o  data available
           95  Percent confidence interval for the true probability of  survival
          °Mean total residual chlorine (mg/1)

-------
         Table  17.  NUMBER OF FIRST GENERATION P. PROMELAS SURVIVING IN DECHLORINATED EFFLUENT


Survival/100
at day 30
C.I.b for prob. of
survival thru
day 30
Survival/100
at day 60
C.I. for prob. of
survival thru
day 60
No. of fish kept
for further study
Number of fish
alive at : 90 days
120 days
150 days
180 days
210 days
240 days
270 days
310 days
C.I. for prob. of
survival
day 60-310

0.00
75
(0.000)a
0.66-0.82
75
(0.000)
0.66-0.82
30
30
(0.000)
30
(0.000)
30
(0.000)
25<*
(0.000)
25
(0.000)
25
(0.000)
25
(0.000)
25
(0.000)
0.66-0.93

1.56
93
(0.000)
0.86-0.97
91
(0.000)
0.84-0.95
30
30
(0.000)
30
(0.000)
30
(0.000)
22d
(0.000)
22
(0.000)
22
(0.000)
22
(0.000)
22
(0.000)
0.56-0.86
Nominal F
3.12
96
(0.000)
0.90-0.98
96
(0.000)
0.90-0.98
30
30
(0.000)
30
(0.000)
30
(0.000)
24d
(0.000)
24
(0.000)
24
(0.000)
24
(0.000)
23
(0.000)
0.59-0.88
ercent Dech
6.25
99
(0.001)
0.95-0.99
98
(0.000)
0.93-0.99
30
30
(0.000)
29
(0.000)
29
(0.000)
26d
(0.000) j
26
(0.000)
26
(0.000)
26
(0.000)
26
(0.000)
0.70-0.95
lorinated Ei
12.5
74
(0.000)
0.65-0.82
63
(0.003)
0.53-0.72
30
30
(0.002)
30
(0.002)
30
(0.001)
28d
(0.001)
28
(0.002)
28
_£0.002)
28
(0.002)
26
(0.001)
0.70-0.95
'fluent
25.0
94
(0.000)
0.88-0.97
90
(0.007)
0.83-0.94
30
30
(0.006)
30
(0.005)
30
(0.004)
26d
(0.004)
26
(0.003)
26
(0.006)
26
(0.005)
25
(0.005)
0.66-0.93

50.0
96
(0.000)
0.90-0.98
94
(0.010)
0.88-0.97
30
30
(0.012)
30
(0.009)
30
L (0.008)
30
(0.007)
30
(0.013)
30
(0.039)
28
(0.051)
28
(0.048)
0.79-0.98

100
47
(0.019)
0.38-0.57
47
(0.016)
0.38-0.57
30
30
(0.012)
30
(0.009)
30
(0.009)
0
(0.010)
0
0
0
0
0.00-0.11
      residual sulfur dioxide as sulfite (mg/1)
 95 percent confidence interval for the true probability of survival
cThe number of fish in each effluent concentration was reduced to 30 on day 60
 Excess males removed from tanks

-------
                                 Table 18.  NUMBER OF SECOND GENERATION P_. PROMELAS SURVIVING
                                                     IN THE DECHLORINATED EFFLUENT

30 Days of Age
Number
Surviving /Star ted
Confidence Interval
x Residual, ing /I
60 Days of Age
Number
Surviving/Started
Confidence Interval
x Residual, mg/1
Nominal Percent Dechlorinated Effluent
0.00
27/40
0.52-0.80
0.000
27/40
0.52-0.82
0.000
1.56
31/40
0.62-0.88
0.000
31/40
0.62-0.88
0.009
3.12
63/80
0.69-0.86
0.000
51/80
0.53-0.73
0.000
6.25
31/80
0.29-0.50
0.000
31/80
0.29-0.50
0.000
12.5
a

25.0
21/40
0.37-0.67
0.135
21/40
0.37-0.67
0.000
50.0
0/40
0.00-0.08
0.388

100.0
0/40
0.00-0.08
0.616

ON
         ^o data available
          95 Percent confidence interval for the true probability of survival
         °Mean residual sulfite (mg/1)

-------
                       Table 19.  NUMBER OF FIRST GENERATION P.  PROMELAS  SURVIVING IN CHLOROBROMINATED EFFLUENT
00

Survival/100
at day 30
C.I.b for prob. of
survival thru
day 30
Survival/100
at day 60
C.I. for prob. of
survival thru
day 60
No. of fish kept
for further study
Number of fish
alive at: 90 days
120 days
150 days
180 days
210 days
2AO days
270 days
310 days
Nominal Percent Chlorobrominated Effluent
0.00
75
(0.008)a
0.66-0.82
75
(0.006)
0.66-0.82
30
30
(0.006)
16
(0.006)
16
(0.005)
lla
(0.005)
9
(0.004)
9
(0.004)
9
(0.003)
8
(0.003)
1.56
88
(0.005)
0.80-0.93
89
(0.011)
0.81-0.94
30
30
(0.010)
30
(0.010)
30
(0.009)
26d
(0.009)
26
(0.008)
26
(0.007)
26
(0.006)
11
(0.005)
C.I. for prob. of j :
survival '0.14-0.44! 0.22-0.54
day 60-310 •. 1
3.12
62
(0.006)
0.52-0.71
61
(0.007)
0.51-0.70
30
30
(0.006)
30
(0.007)
30
(0.006)
22d
(0.006)
10
(0.006)
10
(0.006)
10
(0.006)
10
(0.005)
0.19-0.51
6.25
90
(0.006)
0.83-0.94
90
(0.014)
0.83-0.94
30
20
(0.010)
15
(0.011)
15
(0.010)
12d
(0.010)
12
(0.010)
12
(0.010)
12
(0.009)
12
(0.007)
0.25-0.58
12.5
75
(0.007)
0.66-0.82
75
(0.009)
0.66-0.82
30
28
(0.008)
24
(0.007)
24
(0.007)
2ld
(0.007)
16
(0.007)
16
(0.007)
15
(0.006)
14
(0.006)
0.30-0.64
25.0
88
(0.009)
0.80-0.93
81
(0.009)
0.72-0.87
30
29
(0.009)
29
(0.008)
29
(0.008)
28d
(0.007)
28
(0.007)
26
(0.007)
26
(0.006)
26
(0.005)
0.70-0.95
50.0
96
(0.009)
0.90-0.98
87
(0.015)
0.79-0.92
30
22
(0.013)
22
(0.011)
22
(0.010)
21
(0.010)
21
(0.009)
15
(0.009)
14
(0.009)
11
(0.008)
0.22-0.54
100.0
0
(0.009)
0.00-0.03
0
0.00-0.03
0
0
o,
0
0
0
0
0
0
0.00-0.11
                   total residual bromine  chloride  (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 60
              Excess males removed from tanks

-------
                        Table 20.  NUMBER OF SECOND GENERATION £.  PROMELAS  SURVIVING
                                           IN THE  CHLOROBROMINATED EFFLUENT

30 Days of Age
Number
Surviving/Started
Confidence Interval
x Residual, mg/lc
60 Days of Age
Number
Surviving/Started
Confidence Interval
x Residual, mg/1
Nominal Percent Chlorobrominated Effluent
0.00
56/80
0.59-0.79
0.000
30/80
0.28-0.48
0.000
1.56
55/80
0.58-0.78
0.002
55/80
0.58-0.79
0.002
3.12
34/80
0.32-0.53
0.001
34/80
0.32-0.53
0.001
6.25
16/40
0.26-0.55
0.000
15/40
0.24-0.53
0.000
12.5
58/80
0.62-0.81
0.000
29/80
0.27-0.47
0.001
25.0
37/80
0.36-0.57
0.001
15/80
0.12-0.29
0.000
50.0
0/40
0.00-0.08
0.000

100.0
a

    data available
 95 Percent confidence interval for the true probability of  survival
°Mean total residual bromine chloride (mg/1)

-------
suggesting that the survival of fathead minnows was not affected by the
levels of residual BrCl to which they were exposed in this study.

First generation test animals in undiluted ozonated effluent exhibited mor-
tality only as the result of disease and aeration failures.  Thus, first
generation survival in ozonated effluent was superior to that in any other
effluent stream (Table 21).  The only other time that the survival of first
generation fathead minnows approached the survival level of subjects reared
in 100 percent ozonated effluent was in dechlorinated effluent (Table 17).
Since both of these effluent streams were subjected to greater mixing and
aeration during the treatment process, the improved survival that we observed
may have been related to the DO content of these effluent streams.  The sur-
vival of second generation test fish in ozonated effluent was poor (Table 22),
probably because of the poor quality of the effluent during the period when
those fish were cultured.

As in the Grandville study,8 undiluted nondisinfected effluent was lethal to
test subjects.  Further, no life cycle mortality was attributed to residual
03 and, in fact, ozonated effluent produced improved survival of young fat-
head minnows in 100 percent effluent concentrations.  Unlike the Grandville
findings, no evidence of death from residual BrCl was observed in the life
cycle study, presumably because of the very low BrCl residuals to which the
Wyoming test animals were subjected.

The fathead minnow test subjects were more tolerant of residual Cl£ in the
Wyoming study than in the Grandville study.  For example, test animals of
less than 60 days of age survived mean residual C^ concentrations as high
as 0.109 mg/1 in 18.75 percent effluent at Wyoming, while mean residual Cl£
concentrations as low as 0.045 mg/1 in 14 percent effluent were lethal in
comparable tests at Grandville.  The cause of this difference in tolerance
at the two test sites is unknown, and, because of the many variables poten-
tially involved, any attempt at an explanation would be pure speculation.
Factors which may have affected tolerance levels include:  (1) experience
and skill of laboratory personnel (greater at Wyoming than Grandville);  (2)
the characteristics of the raw wastewater stream (Grandville was primarily
domestic wastewater, while Wyoming included a large fraction of industrial
wastes);  (3) differences in wastewater treatment processes (activated
sludge at Grandville, trickling filter at Wyoming);  and (4) effluent quality
at the most sensitive life stage of the test animals (the quality of the
Grandville effluent was lowest at the beginning of the study and gradually
improved thereafter).  Finally, it is possible that the observed difference
is not a real difference, but merely the normal variation characteristic of
any replicated test.  The significance of the difference in sensitivity to
residual Cl? observed at the two test sites can only be determined through
additional study of the phenomenon.

One of the more definitive effects on mortality in the life cycle tests was
observed in the dechlorinated effluent stream.  Both the Grandville and
Wyoming data illustrate the effectiveness of S0£ in eliminating the lethal
effects of chlorinated effluent concentrations as great as 100 percent.
Further, during the course of this study, the dosing of S02 proved to be an
uncomplicated and reliable process.  This suggested that dechlorination with


                                      70

-------
        Table  21. NUMBER OF FIRST GENERATION P.  PROMELAS  SURVIVING IN OZONATED EFFLUENT

Survival/ 100
at day 30
C.I.D for prob. of
survival thru
day 30
Survival/100
at day 60
C.I. for prob. of
survival thru
day 60
No. of fish kept
for further study
Number of fish
alive at: 90 days
120 days
150 days
180 days
210 days
2AO days
270 days
310 days
C.I. for prob. of
survival
day 60-310
Nominal Percent Ozonated Effluent
0.00
96
(0.002)3
0.90-0.98
96
(0.002)
0.90-0.98
30
30
(0.002)
30
(0.002)
30
(0.002)
26d
(0.002)
26
(0.001)
26
(0.001)
25
(0.001)
25
(0.001)
0.66-0.93
1.56
97
(0.0021
0.92-0.99
94
(0.002)
0.88-0.97
30
30
(0.003)
30
(0.003)
30
(0.003)
22d
(0.003)
20
(0.003)
20
(0.002)
15
(0.002)
15
(0.002)
0.33-0.67
i
3.12
95
(0.003)
0.89-0.98
92
(0.003)
0.85-0.96
30
30
(0.003)
30
(0.003)
30
(0.003)
26d
(0.002)
26
(0.002)
26
(0.002)
26
(0.002)
26
(0.002)
0.70-0.95
6.25
100
(0.003)
0.96-1.00
95
(0.003)
0.89-0.98
30
28
(0.002)
28
(0.003)
28
(0.003)
18d
(0.003)
18
(0.002)
18
(0.003)
18
(0.002)
18
(0.002)
0.42-0.75
12.5
98
(0.003)
0.93-0.99
95
(0.003)
0.89-0.98
30
29
(0.003)
29
(0.003)
29
(0.003)
22d
(0.002)
22
(0.002)
22
(0.002)
21
(0.002)
20
(0.002)
0.49-0.81
25.0
95
(0.003)
0.89-0.98
93
(0.003)
0.86-0.97
30
30
(0.003)
30
(0.003)
30
(0.002)
24d
(0.002)
23
(0.002)
22
(0.002)
22
(0.002)
22
(0.002)
0.56-0.86
50.0 100.0
96 91
(0.004) ! (0.004)
0.90-0.98 0.84-0.95
!
91 i 84
(0.004) (0.004)
0.84-0.95
30
30
(0.003)
30
(0.003)
30
(0.003)
22d
(0.003)
22
(0.003)
22
(0.003)
18
(0.003)
15
(0.002)
0.33-0.67
0.76-0.90
30
30
(0.005)
28
(0.004)
27
(0.004)
25d
(0.004)
22
(0.003)
0
0
0
0.00-0.11
»
      residual ozone (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  60
 Excess males removed from tanks

-------
                        Table 22.   NUMBER OF SECOND GENERATION P. PROMELAS SURVIVING
                                               IN THE OZONATED EFFLUENT

30 Days of Age
Number
Surviving/Started
Confidence Interval
Q
x Residual, tng/1
60 Days of Age
Number
Surviving/Started
Confidence Interval
x Residual, mg/1
Nominal Percent Ozonated Effluent
0.00
49/80
0.50-0.71
0.000
49/80
0.50-0.71
0.000
1.56
43/80
0.43-0.64
0.001
43/80
0.43-0.64
0.001
3.12
49/80
0.50-0.71
0.001
49/80
0.50-0.71
0.002
6.25
31/80
0.29-0.50
0.000
31/80
0.29-0.50
0.000
12.5
0/40
0.00-0.08
0.000

25.0
a

50.0
0/40
0.00-0.08
0.000

100.0
0/40
0.00-0.08
0.001

 MJ data available
 95 Percent confidence interval for the true probability of survival
Tlean residual ozone (mg/1)

-------
S02 has the potential to be an effective and relatively simple method of
preventing any long-term effects which might be associated with the C19 re-
sidual in chlorinated effluent.

Growth

Tables 23-28 summarize the mean length data for first and second generation
test fish.  The data were statistically analyzed with Dunnett's test in which
the mean length of the fish in one concentration of a disinfected effluent
is compared with the mean length of  fish in the respective concentration of
nondisinfected effluent, and with the mean length of the fish in the dilution
water control tank on the same test  table.  Note that this analysis does not
compare data from respective effluent concentrations of the other disinfected
effluents.  For example, the mean length of fish in the 25 percent concen-
tration of ozonated effluent is statistically tested against the mean length
of fish in 25 percent nondisinfected effluent and the mean length of fish in
dilution water on the ozonated effluent test table, but is not statistically
tested against the mean lengths of fish reared in the 25 percent concentra-
tions of the other disinfected streams  (chlorinated, dechlorinated, and chlo-
robrominated).

Significant differences were observed in the mean lengths of first and second
generation test animals reared in dilution water (Tables 23, 27, and 28),  in
spite of the fact that the ecosystems within these tanks were so planned and
maintained as to be similar in all measured characteristics.

Uncontrolled variables which might have affected growth in the dilution
water control tanks included population size, the sex composition of a popu-
lation, and the development of natural populations of planktonic food organ-
isms within a test tank.

Inconsistent differences in mean length data were also observed between fish
reared in different concentrations of the same effluent stream and in their
dilution water controls.  These inconsistencies generally made it impossible
to identify trends in growth related to effluent concentration.

The inconsistencies mentioned above  greatly limit the conclusions which can
be drawn from the growth data.  Further, Dunnett's test applied to the mean
weights of fish in each concentration of each test stream at the termination
of the life cycle study failed to show relationships between effluent type
or concentration and body weight.  Thus, the statistical analyses of the
available data did not demonstrate that any of the effluent treatments tested
had any effect on the growth of fathead minnows.  However, the growth of life
cycle test fish in the higher concentrations of ozonated effluent, because
of its consistency and pattern, does deserve additional discussion.

As seen in Tables 23-26, the mean lengths of fish reared in 50 and 100 per-
cent concentrations of ozonated effluent tended to be greater than their
counterparts in any other effluent stream.  While these length differences
were not always statistically significant,  the consistency of the trend was
such as to suggest that fathead minnows achieved greater mean lengths  in
high concentrations of ozonated effluent than  in similar high concentrations


                                      73

-------
           Table 23.  MEAN LENGTHS (IN mm) OF FIRST GENERATION P_. PROMELAS
                               ON DAY 30 OF THE LIFE CYCLE TEST
Nominal Effluent
Concetration
and Data
Dilution Water N
X
S.D.
SDNDa
1.56% N
X
SDDWb
SDND
x Residual
3.12% N
X
S.D.
SDDW
SDND
x Residual
6.25% N
x
S.D.
SDDW
SDND
x Residual
12.50% N
x
S.D.
SDDW
SDND
x Residual
25.00% N
x
S.D.
SDDW
SDND
x Residual
50.00% N
x
S.D.
SDDW
SDND
x Residual
100.00% N
x
S.D.
SDDW
SDND
x Residual
Effluent Stream
Nondis.
(A)
97
22.7
2.82
96
21.2
3.45
Yes
98
23.4
3.18
No
94
22.1
2.80
No
99
22.4
2.40
No
98
22.0
3.27
No
99
21.2
2.73
Yes
60
14.5
1.67
Yes
Chlor.
(B)
80
20.9
3.24
Yes
95
22.1
2.69
Yes
No
0.017
38
25.5
2.93
Yes
Yes
0.020
100
21.5
2.35
No
No
0.026
76
21.1
2.35
No
Yes
0.059
46
17.2
2.42
Yes
Yes
0.165
N.D.C
N.D.
Dechlor .
(C)
75
20.2
2.75
Yes
93
20.7
3.29
No
No
0.000
96
20.9
3.06
No
Yes
0.000
99
20.4
2.24
.No
Yes
0.000
74
20.0
2.18
No
Yes
0.000
94
21.0
2.19
No
No
0.000
96
18.9
2.30
Yes
Yes
0.000
47
14.8
2.01
Yes
No
0.019
Chlorobr .
(D)
74
20.6
2.62
Yes
88
20.0
3.44
No
Yes
0.005
62
21.3
3.10
No
Yes
0.006
90
20.4
3.02
No
Yes
0.006
75
18.4
3.55
Yes
Yes
0.007
88
20.4
2.12
No
Yes
0.009
96
19.7
2.36
No
Yes
0.009
N.D.
Ozon.
(E)
96
19.6
2.69
Yes
97
20.3
3.09
No
No
0.002
95
20.4
3.09
No
Yes
0.003
100
20.6
2.95
No
Yes
0.003
98
20.6
2.92
No
Yes
0.003
95
20.9
2.99
Yes
Yes
0.003
96
21.5
3.22
Yes
No
0.004
91
19.1
2.05
No
Yes
0.004
Significantly different (P=0.05) by Dunnett's Test from the mean
in the same concentration in the nondisinfected effluent stream.
                                                                  length of fish reared

DSignificantly different (P=0.05) by Dunnett's Test from the mean length of fish reared
 in dilution water tanks of the respective effluent stream.
 No data available.
                                         74

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            Table 24.   MEAN LENGTHS (IN mm) OF FIRST GENERATION P_. PROMELAS
                                On DAY 60 OF THE LIFE CYCLE TEST
Nominal Effluent
Concentration
and Data
Dilution Water N
X
S.D.
SDND3
1.56% N
X
S.D.b
SDDW
SDND
x Residual
3.12% N
X
S.D.
SDDW
SDND
x Residual
6.25% N
x
S.D.
SDDW
SDND
x Residual
12.50% N
x
S.D.
SDDW
SDND
x Residual
25.00% N
x
S.D.
SDDW
SDND
x Residual
50.00% N
x
S.D.
SDDW
SDND
x Residual
100 . 00% N
x
S.D.
SDDW
SDND
x Residual
Effluent Stream
Nondis .
(A)
97
32.7
3.14
90
32.9
3.21
No
99
33.1
3.50
No
93
31.7
2.97
No
98
30.8
2.41
Yes
96
31.7
2.64
No
94
31.9
2.73
No
93
27.3
5.31
Yes
Chlor.
(B)
79
31.9
4.09
No
95
32.4
3.59
No
No
0.016
38
38.5
4.44
Yes
Yes
0.021
99
31.9
3.27
No
No
0.032
75
32.8
3.01
Yes
Yes
0.065
46
28.8
2.92
Yes
Yes
0.161
N.D.C
N.D.
Dechlor.
(C)
75
32.0
4.92
No
91
32.5
4.33
No
No
0.000
96
31.8
4.62
No
No
0.000
98
30.7
3.40
No
No
0.000
63
32.3
3.53
No
Yes
0.003
90
32.8
2.94
No
No
0.007
94
31.9
2.70
No
No
0.010
47
24.2
4.19
Yes
Yes
0.016
Chlorobr .
(D)
75
32.2
4.08
No
89
31.8
3.78
No
No
0.011
61
34.7
4.30
Yes
No
0.007
90
32.7
5.16
No
No
0.014
75
33.4
3.85
No
Yes
0.009
81
31.0
3.17
No
No
0.009
87
30.6
3.82
No
Yes
0.015
N.D.
Ozon.
(E)
96
31.1
3.78
No
94
31.5
4.17
No
No
0.002
92
32.2
4.40
No
No
0.003
95
32.0
4.35
No
No
0.003
95
31.4
4.05
No
No
0.003
93
33.3
4.10
Yes
Yes
0.003
91
34.9
4.13
Yes
Yes
0.004
84
33.6
3.27
Yes
Yes
0.004
Significantly different  (P=0.05) by Dunnett's Test from the mean
 in the same concentration in the nondisinfected effluent stream.
bSignificantly different  (P=0.05) by Dunnett's Test from the mean
 in dilution water tanks of the respective effluent stream.

 No data available.
length of fish reared

length of fish reared
                                         75

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            Table 25.   MEAN LENGTHS (IN mm) OF FIRST GENERATION £. PROMELAS
                               ON DAY 120 OF THE LIFE CYCLE TEST'
Nominal Effluent
Concentration
and Data
Dilution Water N
X
S.D.
SDND3
1.56% N
X
S.D.
SDDW
SDND
x Residual
3.12% N
X
S.D.
SDDW
SDND
x Residual
6.25% N
x
S.D.
SDDW
SDND
x Residual
12.50% N
x
S.D.
SDDW
SDND
x Residual
25.00% N
x
S.D.
SDDW
SDND
x Residual
50.00% N
x
S.D
SDDW
SDND
x Residual
100 . 00% N
x
S.D.
SDDW
SDND
x Residual
Effluent Stream
Nondis .
(A)
23
51.1
6.70
14
53.8
7.28
No
15
54.4
4.68
No
30
51.8
5.67
No
29
49.8
5.32
No
30
47.4
4.26
No
29
45.0
3.07
Yes
30
29.9
2.38
Yes
Chlor .
(B)
29
48.7
5.29
No
29
52.9
5.15
Yes
No
0.014
29
55.0
7.65
Yes
No
0.018
30
54.2
4.89
Yes
No
0.031
30
53.8
5.73
Yes
Yes
0.056
27
48.6
5.55
No
No
0.128
N.D.C
N.D.
Dechlor .
(C)
30
50.7
5.83
No
30
52.8
6.29
No
No
0.000
30
51.5
5.79
No
No
0.000
30
50.2
5.22
No
No
0.000
30
51.5
5.51
No
No
0.002
Chlorobr .
(D)
16
51.3
4.63
No
29
51.3
5.12
No
No
0.010
30
52.5
7.27
No
No
0.007
15
51.8
6.12
No
No
0.010
24
50.2
6.50
No
No
0.007
30 29
51.1 49.5
4.65 4.03
No No
Yes No
0.005 0.008
30
46.1
3.48
Yes
No
0.010
17
45.7
2.63
Yes
No
0.011
30 !
29.4
3.27 N.D.
Yes i
No
0.009
Ozon.
(E)
30
48.5
5.19
No
30
52.0
5.99
No
No
0.003
30
51.0
6.14
No
No
0.002
28
55.7
6.98
Yes
Yes
0.003
29
54.5
6.90
Yes
Yes
0.002
30
54.9
6.89
Yes
Yes
0.003
30
55.1
5.90
Yes
Yes
0.003
28
49.3
4.16
No
Yes
0.004
Significantly different (P=0.05) by Dunnett's Test from the mean length of fish reared
 in the same concentration in the nondisinfected effluent stream.
 Significantly different (P=0.05) by Dunnett's Test from the mean length of fish reared
 in dilution water tanks of the respective effluent stream.
°No data available.
                                          76

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            Table 26.   MEAN LENGTHS (IN mm)  OF FIRST GENERATION P.  PROMELAS
                          AT DAY 310 (TERMINATION)  OF THE LIFE CYCLE TEST
Nominal Effluent
Concentration
and Data
Dilution Water N
X
S.D
SDND
1.56% N
X
S.D
SDDWD
SDND
x Residual
3.12% N
X
S.D.
SDDW
SDND
x Residual
6.25% N
x
S.D.
SDDW
SDND
x Residual
12.50% N
x
S.D.
SDDW
SDND
x Residual
25.00% N
x
S.D.
SDDW
SDND
x Residual
50.00% N
x
S.D.
SDDW
SDND
x Residual
100.00% N
x
S.D.
SDDW
SONS
x Residual

Nondis .
(A)
7
66.7
8.44
N.D.C
7
74.0
3.61
Yes
11
66.9
9.73
No
4
67.8
10.75
No
28
61.3
4.58
No
21
53.7
2.74
Yes

N.D
Effluent Sti
Chlor.
(B)
27
64.9
9.39
No
16
67.4
8.07
No
0.009
22
68.1
10.55
No
No
0.012
20
68.9
9.62
No
No
0.018
23
66.7
9.84
No
No
0.040
24
63.1
5.92
No
No
0.085
N.D.

N.D
ream
Dechlor .
(C)
25
65.0
9.02
No
22
68.5
9.72
No
0.000
23
67.4
9.56
No
No
0.000
26
65.2
7.01
No
No
0.000
12
65.8
7.87
No
No
0.001
25
65.8
6.52
No
No
0.005 J
28
54.5
3.33
Yes
No
0.048

N.D

Chlorobr .
(D)
8
68.6
10.51
No
11
66.2
7.85
No
0.005
10
65.4
8.01
No
No
0.005
12
66.0
9.69
No
No
0.007
14
70.5
10.59
No
No
0.006
26
65.2
6.67
No
No
0.005
11
55.0
2.38
Yes
No
0.008

N.D

Ozon.
(E)
25
65.7
9.20
No
15
63.2
5.58
No
0.002
26
64.3
5.83
No
Yes
0.002
18
68.8
10.45
No
No
0.002
20
66.7
8.73
No
No
0.002
22
65.0
8.49
No
No
0.002
15
63.3
6.14
No
Yes
0.002

N.D.
aSignificantly different  (P=0.05) by Dunnett's Test from the mean length of
 in the same concentration in the nondisinfected effluent stream.

Significantly different  (P=0.05) by Dunnett's Test from the mean length of
 in dilution water tanks  of the respective effluent stream.

TJo data available.
fish reared


fish reared
                                           77

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       Table  27-  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.
SDNDS
1.56%

3.12%

6.25%

12.50%

25.00%
50.00%

100.00%

N
X
S.D.,
SDDW
SDND
x Residual
N
X
S.D.
SDDW
SDND
x Residual
N
x
S.D.
SDDW
SDND
x Residual
N
x
S.D.
SDDW
SDND
x Residual
N
x
S.D.
SDDW
SDND
x Residual
N
x
S.D.
SDDN
SDND
x Residual
N
x
S.D.
SDDW
SDND
x Residual
Effluent Stream
Nondis .
(A)
36
24.4
2.71
68
24.3
2.17
No
50
23.2
1.32
No
25
24.4
3.36
No
18
19.4
1.11
Yes
22
21.7
1.20
Yes
N.D.
N.D.
j Chlor. ' Dechlor.
! (B) (C)
Chlorobr. Ozon.
(D) , (E)
52 27 56
19.6 i 19.8 21.6
3.16 : 3.44 2.84
Yes Yes Yes
15 ,31 55
21.0 22.4 20.8
1.49 0.92 2.75
No Yes No
Yes Yes Yes
0.000 0.000 0.002
63
22.3
N.D.C 2.41
Yes
No
0.000
31
21.2
N.D. 3.23
No
Yes
0.000
54
22.5
1.61 N.D.
Yes
Yes
0.007
9
19.4
1.39
No
Yes
0.003 _,
N.D.
N.D.
21
15.5
1.32
Yes
Yes
0.135
N.D.
N.D.
34
22.9
1.68
No
No
0.001
49
20.9
2.62
Yes
43
19.2
1.80
Yes
Yes
0.001
49
23.4
4.18
Yes
No
0.001
16 I 31
20.7 i 23.4
0.98 ', 1.52
No ' Yes
Yes | No
0.000 l_ 0.000
58 i
22.0 i
2.61 ! N.D.
No
Yes
0.000
37
17.5
1.03
Yes
Yes
0.001
N.D.
N.D.
N.D.
N.D.
N.D.
 Significantly different  (P=0.05)  by Dunnett's  Test  from the mean length of  fish reared
 in the same concentration in the  nondisinfected effluent stream.

 Significantly different  (P=0.05)  by Dunnett's  Test  from the mean length of  fish reared
 in dilution water tanks  of the respective effluent  stream.

TJo data available.
                                          78

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        Table 28.  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.
SDNDa
1.56% N
X
S.D.
SDDW
SDND
x Residual
3.12% N
X
S.D.
SDDW
SDND
x Residual
6.25% N
X
S;D.
SDDW
SDND
x Residual
12.50% N
x
S.D.
SDDW
SDND
x Residual
25.00% N
x
S.D.
SDDW
SDND
x Residual
50.00% N
x
S.D.
SDDW
SDND
x Residual
100.00% N
x
S.D.
SDDW
SONS
X Residual
Effluent Stream
Nondis .
(A)
36
38.5
3.33
Chlor.
(B)
52
33.3
2.77
Yes
Dechlor .
(C)
27
34.2
3.09
Yes
Chlorobr .
(D)
30
34.2
3.44
Yes
72 15 ; 31 33
37.3 38.2 37.1 36.5
3.91 3.05 1.99 2.91
No Yes 1 Yes Yes
No i No No
0.002 i 0.009 0.002
49
39.4
3.62 N.D.C
No
23
38.2
2.82 N.D.
No
18
32.4
1.81
Yes
22
35.9
1.85
Yes
N.D.
N.D.
49
37.2
1.80
Yes
Yes
0.001
9
34.2
2.06
No
Yes
0.021
N.D.
N.D.
51 29
34.2 37.7
3.23 2.65
No Yes
Yes Ho
0.000 0.001
31
35.8
3.68
No
Yes
0.000
N.D.
21
29.2
1.58
Yes
Yes
0.000
N.D.
N.D.
15
36.1
1.85
No
No
0.000
29
33.4
1.86
No
No
0.001
15
28.8
0.98
Yes
Yes
0.000
N.D.
N.D.
Ozon.
(E)
49
33.9
3.53
Yes
43
34.8
3.57
No
Yes
0.001
49
37.1
4.88
Yes
Yes
0.002
31
39.2
2.54
Yes
No
0.000
N.D.
N.D.
N.D.
N.D.
Significantly different  (P=0.05) by Dunnett's Test from the mean length of fish reared
 in the same concentration in the nondisinfected effluent stream.

bSignificantly different  (P=0.05) by Dunnett's Test from the mean length of fish reared
 in dilution water tanks  of the respective effluent stream.

    data available.
                                          79

-------
of any of the other effluent streams tested.  This trend was also seen when
mean body weights at termination were compared.  No fish survived to day 310
in the 100 percent concentration of any effluent stream, but the mean weights
of fish in 50 percent ozonated effluent were at least 50 percent greater than
the mean weights of fish in any other 50 percent effluent concentration.  Due
to mortality, the second generation data (Tables 27 and 28) contribute no
information to this idea.  However, in the Grandville study,  mean lengths of
both first and second generation fish in 100 percent ozonated effluent, as
well as mean weights of fish in the 100 percent ozonated effluent, were
greater than the respective values for fish in comparable concentrations of
any other effluent stream.  Thus, the raw data from this project support the
idea that fathead minnows grew better in very high concentrations of ozonated
effluent than in comparable concentrations of the other effluent streams
tested.  Since growth was always greatest in intermediate effluent concentra-
tions, it appeared that the effect of ozonation was to reduce the growth
inhibiting characteristic(s) inherent in the effluent, rather than to
directly promote superior growth.  None of the measurements made in this
study indicate what growth inhibiting factor(s) may have been surpressed by
the ozonation process.

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 number 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 incu-
bation attempts in the various concentrations of the different effluent
streams.

Tables 29 and 30 summarize the spawning activity in all effluent concentra-
tions.  The inherently toxic nature of high effluent concentrations mani-
fested itself clearly with regard to egg production which was greatly reduced
at concentrations higher than 6.25 - 12.5 percent effluent  (Table 29).  Poor
survival of adult fish to reproductive age as a result of several outbreaks
of an unidentified disease contributed to reduced and variable egg production
in the low concentrations of nondisinfected effluent.  All of the fish died
prior to reproductive age in at least one of the duplicate tanks in all con-
centrations below 25 percent nondisinfected effluent.  The fish that did
produce eggs in all nondisinfected effluent concentrations except 6.25 per-
cent deposited considerable fewer viable eggs per female than were observed
for the same concentrations of nondisinfected effluent in the Grandville
study° and for similar concentrations in the work of Arthur, et_ aj_., 1 with
nondisinfected effluent.  The source of raw wastewaters in the latter two
studies was almost entirely domestic.

The fact that essentially no egg production was observed in the 25 and 50
percent effluent concentrations even though fish survived those exposures
indicates that the poor quality trickling filter effluent from wastewater
with 35-45 percent industrial input severely inhibited the reproductive
activities of the fathead minnow.  This inherent inhibitory effect of the
effluent, as well as the erratic impact of the unidentified disease upon a


                                      80

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               Table 29.   MEAN NUMBER OF VIABLE EGGS PRODUCED PER FEMALE AND THE MEAN CHEMICAL
                             RESIDUAL (mg/1)  IN EACH CONCENTRATION OF EACH EFFLUENT STREAM
oo
Effluent
Stream
Nondls infected
x Egg/female
Chlorinated
x Eggs/female
x Residual
De ch lor inat ed
x Eggs /female
x Residual
Chlorobrominated
x Eggs /female
x Residual
Ozonated
x Eggs/ female
x Residual
Nominal percent effluent concentrations
0.000
195a
386
0.002
1049
0.000
1211
0.003
333
0.001
1.56
Oc
1262
0.009
437
0.000
1176a
0.005
672
0.002
3.12
oa
46
0.012
1343
0.000
1059a
0.005
1383
0.002
6.25
569a
2
0.018
922
0.000
126
0.007
462
0.002
12.50
372a
0
0.040
oa
0.001
la
0.006
336
0.002
18.75
b
21
0.059
b
b
b
25.00
2
2
0.085
0
0.005
8
0.005
18
0.002
50.00
0
oc
0.299
0
0.048
0
0.008
31
0.002
100.00
0
b
oc
0.010
oc
0.009
oc
0.003
             One of the two duplicate tanks had zero survivors  at  termination of  the study
             No equivalent effluent concentration
            CA11 fish in this concentration died prior  to  reproductive  age

-------
                       Table 30.  MEAN NUMBER OF EGGS PER SPAWNING IN THE VARIOUS

                                      CONCENTRATIONS OF EACH EFFLUENT STREAM
oo
to
Effluent
Stream
Nondisinfected
x Eggs/spawning
No. of spawnings
Chlorinated
x Eggs/spawning
No. of spawnings
Dechlorinated
x Eggs /spawning
No. of spawnings
Chlorobrominated
x Eggs /spawning
No. of spawnings
Ozonated
x Eggs /spawning
No. of spawnings
Nominal percent effluent concentrations
0.00
15ia
23
130
72
163
110
279
24
116
60
1.56
13 Ob
17
190
81
183
41
190a
100
148
78
3.12
lla
2
94
7
231
105
190a
46
180
196
6.25
219a
49
16
3
187
139
124
23
170
29
12.50
1973
13
0
1113
1
41a
1
134
38
18.75
c
90
4
c
c
c
25.00
86
4
36
1
0
197
2
72
3
50.00
0
b
0
0
0
157
2
100.00
b
0
c
b
0
b
0
b
0
            aOne of the two duplicate tanks had zero survivors at termination of the study


             Both duplicate tanks had zero survivors at termination of the study


            °No equivalent effluent concentration

-------
number of test tanks in different  effluent  streams, made  it  impossible  to
assess the effect of any chemical  treatment upon  egg production.

Some of the viable eggs produced were  incubated to determine their hatch-
ability (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 were  not alive at the end of the
incubation period were not  considered  living  fry.

A summary of the results of tests  with eggs spawned and incubated in the
same effluent type and concentration are shown in Table 31.   This table is
incomplete because viable egg  production was  limited to only the lower
effluent concentrations.  However, Table 31 does  show  that,  with but one
exception (6.25 percent ozonated effluent), the highest percent egg hatch-
ability on each treatment table occurred in 100 percent dilution water.
Addition to the dilution water of  the  smallest amount  of  effluent, whether
it was disinfected or not,  inhibited egg hatchability.  Thus,  the reduced
hatchability recorded in this  table appeared  to result from  characteristics
of the raw effluent rather  than the chemical  treatment applied to any efflu-
ent stream.

The deleterious nature of the  raw  effluent  was further documented when eggs
spawned in dilution water were incubated in the higher concentrations of
the various effluent streams (Table 32). No  eggs hatched in the 12.5-100
percent nondisinfected effluent concentrations and no  significant hatchability
was observed in any disinfected effluent stream at concentrations greater than
12.5 percent.

In summary, the observations on the reproductive  activity of the test sub-
jects in this  study illustrated the harmful effects of the nondisinfected
effluent produced by the Wyoming plant, but did not identify any deleterious
effects attributable to the chemical treatments applied to the various test
streams.
                                      83

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                       Table 31.   PERCENT HATCHABILITY, MEAN CHEMICAL RESIDUAL (mg/1),
                                 AND INCUBATION ATTEMPTS IN THE VARIOUS EFFLUENT STREAMS
Effluent Type
Nondis infected
% Hatchability
No. of incubations
Chlorinated
% Hatchability
No. of incubations
Mean Residual
De chlorinated
% Hatchability
No . of incubations
Mean Residual
Chlorobrominated
% Hatchability
No. of incubations
Mean Residual
Ozonated
% Hatchability
No. of incubations
Mean Residual
Nominal Percent Effluent Concentration
0.00
68
4
82
19
0.000
86
38
0.000
89
12
0.001
86
19
0.000
1.56
45
4
76
28
0.002
73
9
0.000
75
25
0.002
81
13
0.001
3.12

0
46
2
0.002
78
25
0.000
69
11
0.004
81
38
0.001
6.25
57
11

0
0.004
61
24
0.000
76
2
0.002
93
5
0.001
12.50
0
5

0
0.005

0
0.002

0
0.003
65
8
0.001
18.75
a


0
0.005

a

a


a


25.00

0

0
0.009

0
0.006
10
1
0.002

0
0.001
50.00

0

0
0.000

0
0.087

0
0.003
48
1
0.001
100.00

0

a


0
0.009

0
0.000

0
0.001
oo
           equivalent  effluent concentration

-------
                            Table 32.   PERCENT HATCHABILITY OF EGGS INCUBATED IN EFFLUENT

                                                BUT SPAWNED IN DILUTION WATER
oo
Ul
Effluent Type
Nondisinfected
Incubated in:
% Hatchability:
No. of attempts:
Chlorinated
Incubated in:
% Hatchability:
No. of attempts:
Dechlorinated
Incubated in:
% Hatchability
No. of attempts:
Chlorobrominated
Incubated in:
% Hatchability:
No. of attempts:
Ozonated
Incubated in:
% Hatchability:
No. of attempts:
Test Group
1
100% effluent
0
6
50% effluent
0
4
100% effluent
0
20
100% effluent
0
16
100% effluent
0
6
i
Test Group
2
50% effluent
0
4
25% effluent
0
4
50% effluent
0
17
50% effluent
4.7
11
50% effluent
0
1
Test Group
3
25% effluent
0
2
18.75% effluent
8.4
5
25% effluent
0.7
3
25% effluent
2.6
9
Test Group
4
12.5% effluent
0
1

12.5% effluent
24
2
12.5% effluent
77.6
5


-------
                                 REFERENCES

 1.  Arthur, J. W., R. W. Andrews, V. R.  Mattson, D. T. Olson, B. J. Halli-
     gan, and C. T. Walbrldge.  Comparative Toxicity of Sewage Effluent
     Disinfection 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
     Gannnarus 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.  Ward, R. W., R. D. Giffin, G. M. DeGraeve, and R. S. Stone.  Disin-
     fection Efficiency and Residual Toxicity of Several Wastewater Disin-
     fectants.  Volume I Grandville, Michigan.  Environmental Protection
     Technology Series EPA-600/2-76-156, 1976.

 9.  Nebel, C. R., D. Gottschling, R. L. Hutchinson, T. J. McBride, D. M.
     Taylor, J. L. Pavoni, M. E. Tittlebaum, H. E. Spencer and M. Fleischman.
     Ozone Disinfection of Industrial-Municipal Secondary Effluents.  Jour
     Water Poll Cont Fed. 45:2493-2507, 1973.

10.  Greening, E.  Feasibility of Ozone Disinfection of Secondary Effluent.
     Chicago.  Document No. 74-3.  Illinois Institite for Environmental
     Quality, Project No. 20,028.  January, 1974.  39 pp.
                                      86

-------
11.  Mills, J. F.  The Disinfection of Sewage by Chlorobromination.  Present-
     ed before the Division of Water, Air and Waste Chemistry, American
     Chemical Society Meeting.  Dallas, Texas.  April, 1973.

12.  Mills, J. F.  The Chemistry of Bromine Chloride in Wastewater Disinfec-
     tion.  Presented before the Division of Water and Waste Chemistry,
     American Chemical Society Meeting.  Chicago, Illinois. August, 1973.

13.  Annual Book of ASTM Standards.  Designation D2036-74.  American Society
     for Testing and Materials, 1916 Race Street, Philadelphia, Pa. 1974
     pp. 500-514.

14.  Dunnett, C. W.  A Multiple Comparison Procedure for Comparing Several
     Treatments With A Control.  Journal of the American Statistical Ass.
     50:1096-1121. 1955.

15.  Thurston, R. V., R. C. Russo, and K. Emerson.  Aqueous Ammonia Equilib-
     rium  Calculations.  Tech. Rept. No. 74-1, Fisheries Bioassay Laboratory,
     Montana  State University, Bozeman, Montana. 1974.

16.  McKee, J. E., and H.  W. Wolf.  Water Quality Criteria. 2nd ed.  Sacra-
     mento, California State Water Resources Control Board, 1963.
                                      87

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                                SECTION VI

                           ACUTE TOXICITY TESTS

INTRODUCTION

Acute toxicity tests are used to determine the amount of toxicant necessary
to produce a given response (usually death) by a biological indicator pop-
ulation in a relatively short (normally 96 hours or less) period of time.
While some species of fish and invertebrates do not lend themselves well to
life cycle toxicity testing, almost any species that can be captured and
maintained in the laboratory can be tested in acute toxicity tests.  Results
are often expressed as the TL50 or LC50, which is that level of toxicant
required to kill half of the test animals during the exposure time under a
given set of experimental conditions.  The results of acute toxicity tests,
along with those from life cycle tests, are useful in predicting the poten-
tial impact of toxicants upon aquatic life.

MATERIALS AND METHODS

Acute toxicity tests of 48-96 hours in duration were conducted using a
variety of cold and warm water fishes.  In addition, a number of species
of aquatic insect larvae and several species of microinvertebrates were
used in 48-hour tests.  Materials and methods were identical to those used
in the Grandville study,  except for the differences noted below.

In test chambers where low dissolved oxygen levels were potentially detri-
mental, oil-free air was introduced for aeration.  The dissolved oxygen
concentrations in all test aquaria were usually measured at 0, 48, and 96
hours into each test period.  Due to industrial components in the effluent,
occasional flows of chemicals of an unknown nature entered the effluent
stream.  When possible, chemical analyses were performed to identify the
unknown substances.

The first three tests with fathead minnows using dechlorinated effluent,
as well as the fathead minnow test with chlorinated effluent, were con-
ducted using diluters calibrated to deliver to the duplicate test tanks
100 percent effluent, 100 percent dilution water, and six intermediate
concentrations, each diluted 50 percent more than the immediately pre-
ceding higher concentration.  For the remaining tests, the diluters were re-
calibrated to deliver 100 percent effluent, 100 percent dilution water, and
six intermediate test concentrations, each receiving a volume of effluent
equal to 60 percent of the immediately preceding higher concentration.
This calibration change was made to minimize the difference in effluent con-
centration between those levels producing 100 percent mortality and those
resulting in 100 percent survival of test animals.  Ideally, effluent con-

                                     88

-------
centrations would be maintained  at  levels  giving  nearly  50 percent mortality.
However, under actual experimental  conditions,  the Wyoming effluent quality
was so variable that maintenance of a  constant  disinfectant residual level
was virtually impossible.

Fish used in acute  tests were  either reared  in  the laboratory, purchased
from private sources, collected  from local streams, or obtained from State
of Michigan or national fish hatcheries.   Invertebrates were captured from
local lakes and streams, or obtained in pure culture from the Environmental
Research Laboratory - Duluth.  Test animals  were  held under experimental
temperature and light conditions for at least seven days prior to testing.
During the acclimation period, fish were observed for signs of disease or
parasites.  Fish exhibiting symptoms of bacterial infection were treated
with neomycin, tetracycline, terramycin, nitrofurazone, or furanace, while
those showing evidence of  ectoparasitic diseases  were treated with formalin
and/or malachite green.  Total lengths (±0.5 mm)  and weights (±0.1 g) were
recorded for each fish that died during the  test  period.

Acute toxicity tests with  invertebrates were started between 9 A.M. and
2 P.M.  The insect  larvae  and  other large  invertebrates were transferred
to the test tanks by hand.  Rocks were provided in the test chambers to
serve as shelter for those animals.  The smaller  invertebrates (copepods,
cladocera, ostracods, and  midge  larvae) were captured and transported to
the test chamber using small pipettes.  These subjects were exposed to ef-
fluent in test containers  which  were specifically designed for testing small
invertebrates.  A 160 mm length  of  16  mm O.D. glass tubing was immersed
approximately 80 mm into the desired effluent concentration.  The immersed
end of the tubing was fitted with a fine mesh (34 strands/cm) stainless
steel cap to prevent escape of the  invertebrates.  This glass cylinder was
suspended from an apparatus which caused it  to move vertically 18 mm through
the water column in an up  and  down  motion  eight times per minute.  Since
the tanks in which  the test containers were  immersed were being continuously
supplied by a flow-through diluter  system, the  invertebrates were exposed to
the desired test concentration under flow-through conditions.  This method
also facilitated recapture for mortality determinations.  Partial plugging
of the stainless steel screening was a frequent problem  in high effluent
concentrations.  The small size  of  the microinvertebrates made intermittent
mortality and behavior observations impractical.  Therefore, the TL50 was
calculated based upon mortality  counts made  when  the test was terminated.
Invertebrates were  observed under a dissecting microscope and were considered
dead when they gave no response  to  gentle  prodding.  Ten animals were used
per test chamber when possible,  but when numbers  of subjects were limited,
as few as five were used per  test container.

Most acute toxicity experiments  were run during the life cycle test period
with the same effluent used in the  life cycle tests.  However, after com-
pletion of the life cycle  study, additional  toxicity tests with 03 were
performed so that a TL50 could be calculated.  To accomplish this objective,
exceptionally high  03 residuals  were needed. Test aquaria were situated
immediately adjacent to the 03 contactor and were fed ozonated effluent
direct from the contactor.
                                      89

-------
ACUTE TOXICITY TESTS WITH NONDISINFECTED EFFLUENT
                2
As early as 1933  Hubbs expressed concern over man's failure to resolve the
problem of reducing the harmful effects of wastewater effluent on fish life.
He discussed the lack of legal limits of dissolved oxygen levels and bio-
chemical oxygen demand, the harmful effects on fishing, recreation, health,
and economics, and the potentially beneficial effects of proper sludge
handling as topics that required more attention.  He cited several instances
of fish kills that could be attributed to wastewater effluent (nondisin-
fected) and reported that the state of New York was conducting bioassay
studies to determine if there had been a violation of the law prohibiting
discharges injurious to fish life.
         3
Alabaster  studied a channel containing undiluted, nondisinfected waste-
water effluent.  The wastewater channel was connected to a relatively un-
polluted canal and fish could move freely between the canal and the channel.
Alabaster found coarse fish through the length of the wastewater channel,
but fishing success and concentrations of dissolved oxygen were highest
upstream from the discharge site and more than 2.5 miles downstream from
the discharge site.  Mortalities were often observed when caged fish were
placed in the channel within one mile downstream from the point of discharge,
although no comparable mortalities of free-living fish were ever observed.
Alabaster concluded that the potential for an aquatic environment to support
fish populations varied directly with dissolved oxygen levels, and that
free-living fish avoided areas where mean dissolved oxygen levels were less
than 1-2 mg/1 as the result of effluent discharge.
               4
Esvelt, et^ _al.,  found that primary effluent, derived either from domestic
or industrial wastewater sources, was toxic to golden shiners.  The TLSO's
ranged from 39 to 63 percent effluent.  They further found that the toxicity
was effectively removed by activated sludge treatment and, to a lesser
extent, by physical-chemical treatment, with acute test results ranging
from 100 percent survival in undiluted effluent to 50 percent survival in
76 percent effluent.

In a study evaluating the acute toxicity of nondisinfected municipal waste-
water effluent to several fishes and invertebrates, Arthur, e^t al_. ,-* ob-
served mortalities in excess of 50 percent of the test population in 2 of
12 tests, and greater than 20 percent in 6 of 12 tests.  They concluded that
the lethal responses were primarily the result of reduced dissolved oxygen
levels.

Gaufin  found that heavy organic enrichment from an effluent discharge
limited the distribution and species composition of the macroinvertebrate
community in an Ohio trout stream.  However, he also noted that approxi-
mately 11 miles downstream from the effluent discharge the stream had
recovered completely and displayed a varied and well-balanced aquatic com-
munity.

Acute toxicity tests with nondisinfected effluent from the Wyoming plant
resulted in mortality of eight of the nine fish species tested and one of
the seven invertebrate species tested, particularly in the higher effluent

                                     90

-------
concentrations  (Table 33).   The  toxic  nature of  the nondisinfected trickling
filter effluent was likely due to  the  extremely  poor  quality with respect
to soluble and  insoluble  organic constituents (see Section III) and the
unknown industrial components present  in  the source wastewater.  Even though
test tanks were cleaned daily, fish  tested  in 100 percent nondisinfected
effluent were usually not visible  due  to  the extremely turbid nature of the
effluent.  It was periodically necessary  to pass a net through the test tank
to determine if the test  specimens had died.

The toxic nature of the effluent prior to the addition of any disinfectant
is a factor worthy of consideration  when  reviewing the results of acute
tests with disinfected effluents.  In  the cases  of acute tests with de-
chlorinated and ozonated  effluents,  the mortality observed was usually at
a level comparable to that experienced by the same species in nondisinfected
effluent.  This information, coupled with our previous findings, suggests
that probably the effluent rather  than the  chemical treatment was the toxic
factor in those situations.

ACUTE TOXICITY  TESTS WITH CHLORINATED  EFFLUENT

The chlorinated effluent  was more  toxic to  fishes and invertebrates than
any other effluent tested.   The  signs  of  stress  typically evidenced by fish
exposed to chlorinated effluent  included  gasping at the water surface, rapid
gill movements, loss of equilibrium  and erratic  swimming movements, loss of
slime coat, hemorrhaging  at  the  gills  and base of fins, and passive floating
or resting on the bottom  immediately prior  to death.  Mortality of Daphnia
and copepods occurred in  effluent  concentrations as low as 4.7 percent.
However, a few  species of macroinvertebrates  survived well in effluent with
mean residual CL? levels  as  high as  1.491 mg/1.  Arthur, et_ al_. ,-* also
found macroinvertebrates  to  be less  sensitive to Cl£ than fish, although
sufficient mortalities occurred  in most of  his tests to enable calculation
of a seven-day  TL50.  The greater  toxicity  observed in our tests as com-
pared to Arthur's may be  the result  of the  greater hardness of our dilution
water.  Hardness might affect the  toxicity  of C19 as it does other materi-
                                                 7
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                   Table 33.  RESULTS OF ACUTE TOXICITY TESTS WITH NONDISINFECTED EFFLUENT
Species
Fathead Minnow #1
Pimephales promelas
Fathead Minnow #2
Pimephales promelas
Fathead Minnow #3
Pimephales promelas
Northern Common Shiner
No tr op is cornutus

Pugnose Shiner
Notropis anogenus
Rainbow Trout
Salmo gairdnerii

Brown Trout
Salmo trutta

Splake
Salvelinus sp.

Brook Trout
Salvelinus fontinalis

Lake Trout
Salvelinus namaycush
Test
Temp.
(C)
25
25
25
25
25
15
14
15
15
15
96 Hour
TL50
% Effluent



75
65
55
55
81
77
38
Inclusive
Dates
1-27 to
1-30-75
2-11 to
2-12-75
2-17 to
2-21-75
3-24 to
3-28-75
5-12 to
5-16-75
3-18 to
3-21-75
3-18 to
3-21-75
4-28 to
5-2-75
4-28 to
5-1-75
5-4 to
5-8-76
a
Comments
72 hour acute test; zero mortality
in 100% effluent
24 hour acute test; zero mortality
in 100% effluent
Zero mortality in 100% effluent
95% mortality in 100% effluent
100% mortality in 100% effluent
72 hour acute test; 60% mortality
in 60% effluent
72 hour acute test; 60% mortality
in 60% effluent
85% mortality in 100% effluent
100% mortality in 100% effluent
100% mortality in 60% effluent
NJ
       Percent mortality is given for the
        if a TL50 was obtained;  otherwise,
        tration.
   (continued)

lowest concentration which had greater than 50 percent mortality
 percent mortality is given for the 100 percent effluent concen-

-------
             Table 33 (continued).  RESULTS OF ACUTE TOXICITY TESTS WITH NONPISINFECTED EFFLUENT
Species
Chinook Salmon
Oncorhynchus tshawytscha
Mayfly larvae
Hexagenia Sp.
Mayfly larvae
Stenonema sp .

Caddisfly larvae
Hydropsychidae
Caddisfly larvae
Limnephilidae
Stonefly larvae
Plecoptera
Isopods
Ascellus sp.

Cyclopoid copepods
Test
Temp.
(C)
15
16
18
17
18
18
17
18
96 Hour
TL50
(% Effluent)
54
18






Inclusive
Dates
3-18 to
3-21-75
5-8 to
5-10-75
8-28 to
8-30-75
7-23 to
7-25-75
8-28 to
8-30-75
7-28 to
7-30-75
7-23 to
7-25-75
12-9 to
12-11-75
Comments
72 hour acute test; 65% mortality
in 60% effluent
60% mortality in 13% effluent
30% mortality in 100% effluent
15% mortality in 100% effluent
10% mortality in 100% effluent
24 hour acute test;
10% mortality in 100% effluent
15% mortality in 100% effluent
10% mortality in 100% effluent
vO
U>

-------
                     Table 34.  RESULTS OF ACUTE TOXICITY TESTS WITH CHLORINATED EFFLUENT
Species
Fathead Minnow
Pimep hales promelas
Pugnose Shiner
Notropis anogenus
Pugnose Minnow
Opsopoeodus emiliae

Goldfish
Carassius auratus

Goldfish
Carassius auratus

Brook Trout
Salvelinus fontinalis

Rainbow Trout
Salmo gairdnerii

Splake
Salvelinus sp .

Test
Temp.
(C)
25
26
25
13
25
16
16
15
96 Hour. TL50D
mg/1 C12
f% effluent")
0.120
(18%)
0.029
(75%)
0.085
(75%)
0.519
(55%)

0.068
(33%)
0.037
(19%)
0.071
(30%)
Inclusive
Dates
2-4 to
2-8-74
4-21 to
4-25-75
5-5 to
5-9-75
3-9 to
3-11-76
5-5 to
5-9-76
4-14 to
4-18-75
4-14 to
4-18-75
4-14 to
4-18-75
a
Comments
100% mortality at 0.300 mg/1
(25% effluent)
x length 17 mm, x weight 0.1 g^
100% mortality at 0.029 mg/1
(100% effluent)
x length 65 mm, x weight 3.1 g
100% mortality at 0.104 mg/1
(100% effluent)
x length 48 mm, x weight 1.0 g
60% mortality at 0.586 mg/1
(60% effluent)
Test terminated at 48 hours
x length 59 mm, x weight 2.6 g^
20% mortality at 0.071 mg/1
(100% effluent)
60% mortality at 0.075 mg/1
(36% effluent)
x length 42 mm, x weight 0.6 g
55% mortality at 0.041 mg/1
(22% effluent)
x length 53 mm, x weight 1.5 g
80% mortality at 0.095 mg/1
(36% effluent)
x length 53 mm, x weight 1.1 g
VD
-P-
                                                   (continued)
        Percent mortality is given for the lowest effluent concentration having greater than 50% mortality
         if a TL50 is obtained; otherwise, percent mortality is given for the 100% effluent concentration.
         Figures in parentheses are TL50 values calculated in terms of percent effluent

-------
Table 34 (continued).  RESULTS OF ACUTE TOXICITY TESTS WITH CHLORINATED EFFLUENT
Species
Chinook Salmon
Oncorhynchus tshawytscha

Mayfly larvae
Hexagenia sp.
Caddisfly larvae
Family Hydropsychidae
Caddisfly larvae
Family Limnephilidae
Stonefly larvae
Order Plecptera
Isopods
Ascellus sp.

Amphipod
Grammarus sp .

Snails
Goniobasis livescens

Daphnia magna
Calanoid copepod
Epischura lacustris

Assorted Calanoid and
Cyclopoid copepods
Test
Temp.
(C)
15
16
17
17
16
17
17
16
16
14
14
1 _- .-.- .„.!.
96 Hour TL50
mg/1 C12
(% effluent)
0.065
(28%)
0.357
(51%)


0.781
(60%)

1.102
(77%)

0.045
(4%)
0.063
(5%)
0.041
(4%)
Inclusive
Dates
4-14 to
4-18-75
5-5 to
5-7-75
7-21 to
7-23-75
9-8 to
9-10-75
8-6 to
8-8-75
7-16 to
7-18-75
7-21 to
7-23-75
8-6 to
8-8-75
10-14 to
10-16-75
9-17 to
9-19-75
10-8 to
10-10-75
Comments
100% mortality at 0.095 mg/1
(36% effluent)
x length 59 mm, x weight 1.8 g
70% mortality at 0.400 mg/1
(60% effluent)
30% mortality at 1.361 mg/1
(100% effluent)
Zero mortality at 1.491 mg/1
(100% effluent)
60% mortality at 1.411 mg/1
(100% effluent)
25% mortality at 1.624 mg/1
(100% effluent)
100% mortality at 1.361 mg/1
(100% effluent)
Zero mortality at 1.411 mg/1
(100% effluent) . Snails exposed to
high concentrations of chlorinated
effluent withdrew into their shells,
but emerged again when placed in well
water upon completion of the test.
100% mortality at 0.054 mg/1
(4.7% effluent)
100% mortality at 0.086 mg/1
(7.8% effluent)
67% mortality at 0.044 mg/1
(4.7% effluent)

-------
ACUTE TOXICITY TESTS WITH DECHLORINATED EFFLUENT

As in the Grandville study,  the mortality pattern of fish exposed to de-
chlorinated effluent was similar to the pattern in nondisinfected effluent
(Table 35).  It appears that the inherent toxicity of the effluent was the
likely cause of the mortality rather than residual sulfite.

All invertebrates exposed to dechlorinated effluent exhibited a response
similar to those tested in nondisinfected effluent, which again indicated
the high acute tolerance of many invertebrates to wastewater effluent.  In
fact, in most cases, there was slightly more mortality when invertebrates
were exposed to nondisinfected effluent as compared to those tested in
dechlorinated effluent.  Due to aeration of all concentrations, low dis-
solved oxygen levels did not appear to have been a factor affecting the
results of either the dechlorinated or nondisinfected acute tests (the mean
dissolved oxygen level of all 100 percent nondisinfected acute tanks was
6.0 mg/1, while the mean dissolved oxygen concentration of all 100 percent
dechlorinated acute test chambers was 6.5 mg/1).

ACUTE TOXICITY TESTS WITH CHLOROBROMINATED EFFLUENT

Most acute test fish in chlorobrominated effluent exhibited mortality
patterns and stress symptoms similar to those tested in chlorinated effluent
(Table 36).  An exception was the salmonid fishes which proved to be more
sensitive to chlorobrominated effluent than to chlorinated effluent.  Less
than half of the amount of residual BrCl was necessary to produce a TL50
when compared to similar tests run with chlorinated effluent, although TL50
values for chlorobrominated effluent expressed as percent effluent were
slightly higher than those for chlorinated effluent.  These results are in
contrast with our Grandville findings with chlorobrominated activated
sludge effluent,^ in which we found chlorobrominated effluent to be less
toxic than chlorinated effluent.  These results also disagree with Zillich's
conclusion that chlorobrominated effluent was no more toxic than nondisin-
fected effluent.  On the other hand, little mortality was recorded in
chlorobrominated acute tests with macroinvertebrates, while several species
were killed in sufficient numbers to calculate a TL50 when exposed to
chlorinated effluent.

The apparent difference between the toxic effects of chlorobrominated ef-
fluent at Grandville and at Wyoming might have been the result of an
additive or synergistic interaction involving BrCl and some other component
of the effluent.  On one occasion (and probably others, although compre-
hensive observations were available for only the one reported incident),
an apparently synergistic reaction involving BrCl and cyanide occurred.
Seven hours after the beginning of an acute toxicity experiment involving
exposure of fathead minnows to chlorobrominated effluent, no mortalities
or unusual responses had yet been observed.  However, the next morning,
after 22 hours of exposure, total mortality had occurred in the 100, 50 and
25 percent effluent tanks, and 90 percent mortality in the 12.5 percent
effluent tanks.  Ampereometric measurement of BrCl at that time indicated
only 0.127-0.015 mg/1 residual BrCl in the 100-12.5 percent effluent tanks,
although the titrations were erratic and unstable.  Immediately after the


                                     96

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            Table 35.  RESULTS OF ACUTE TOXICITY TESTS WITH DECHLORINATED EFFLUENT
Species
Fathead Minnow #1
Pimephales promelas
Fathead Minnow #2
Pimephales promelas
Fathead Minnow #3
Pimephales promelas
Fathead Minnow #4
Pimephales promelas
Northern Common Shiner
Notropis cornutus

Brook Trout
Salvelinus fontinalis

Brown Trout
Salmo trutta

Test
Temp.
(C)
25
25
25
25
15

14

14

96 Hour TL50b
mg/1 S02
(% effluent)


0.273
(74%)

0.117
(100%)

1.400
(77%)

0.117
(42%)

Inclusive
Dates
10-15 to
10-19-73
10-22 to
10-26-73
2-4 to
2-8-74
1-27 to
1-30-75
4-14 to
4-18-75

4-7 to
4-11-75

4-7 to
4-11-75

a
Comments
20% mortality at 0.001 mg/1
(100% effluent)
x length 21 mm, x weight 0.2 g
Zero mortality at 0.000 mg/1
(100% effluent)
100% mortality at 0.428 mg/1
(100% effluent)
x length 18 mm, x weight 0.2 g.
The dissolved oxygen level in the
100% effluent tanks was less than
1.0 mg/1 when the fish were found
dead. In this case, mortality was
probably the result of anoxia rather
than residual sulfur dioxide.
5% mortality in 100% effluent
x S02 residual 2.64 mg/1
50% mortality at 0.117 mg/1
(100% effluent)
x length 55 mm, x weight 1.5 jg^
100% mortality at 3.52 mg/1
(100% effluent)
x length 40 mm, x weight 0.6 g
95% mortality at 0.142 mg/1
(60% effluent)
2? length 45 mm, x weight 1.0 g
                                     (continued)

aPercent mortality is given for the lowest effluent concentration having greater than 50% mortality
 if a TL50 is obtained; otherwise, percent mortality is given for the 100% effluent concentration.
^Figures in parentheses are TL50 values calculated in terms of percent effluent for comparison
 purposes.

-------
CD
Table 35 (continued). RESULTS OF ACUTE TOXICITY TESTS WITH DECHLORINATED EFFLUENT
Species
Rainbow Trout
Salmo gairdnerii

Lake Trout
Salvelinus namaycush

Splake
Salvelinus sp.

Chinook Salmon
Oncorhynchus tshawytscha
Stonefly larvae
Order Plecoptera
Caddisf ly larvae
Family Hydropsy chidae
Amphipods
Gammarus sp .

Isopods
Ascellus sp.

Snails
Goniobasis lives cens

Cyclopoid copepods
Test
Temp.
(C)
16
16
14
14
18
17
18
17
17
17
96 Hour TL50
mg/1 S02
(% effluent)
1.185
(82%)
0.325
(77%)
1.670
(81%)
0.190
(63%)






•
Inclusive
Dates
5-12 to
5-16-75
5-10 to
5-11-76
4-7 to
4-11-75
4-7 tp
4-11-75
7-30 to
8-1-75
8-11 to
8-13-75
7-30 to
8-1-75
8-11 to
8-13-75
8-11 to
8-13-75
12-1 to
12-3-75
Comments
80% mortality at 1.894 mg/1
(100% effluent)
x length 64 mm, x weight 2.9 g
100% mortality at 0.488 mg/1
(100% effluent)
x length 56 mm, x weight 1.1 g
85% mortality at 3.52 mg/1
(100% effluent)
x length 47 mm, X weight 0.8 g
100% mortality at 3.11 mg/1
(100% effluent)
x length 57 mm, x weight 1.8 g
Zero mortality at 0.000 mg/1
(100% effluent)
Zero mortality at 0.000 mg/1
(100% effluent)
Zero mortality at 0.000 mg/1
(100% effluent)
Zero mortality at 0.000 mg/1
(100% effluent)
Zero mortality at 0.000 mg/1
(100% effluent)
10% mortality at 0.034 mg/1
(100% effluent)

-------
          Table 36.   RESULTS OF ACUTE TOXICITY TESTS WITH CHLOROBROMINATED EFFLUENT
Species
Fathead Minnow
Pimephales promelas
Fathead Minnow
Pimephales promelas
Pugnose Shiner
Notropis anogenus
Goldfish
Carassius auratus

Rainbow Trout
Salmo gairdnerii

Brook Trout
Salvelinus fontinalis

Splake
Salvelinus sp.

Lake Trout
Salvelinus namaycush

Chinook Salmon
Oncorhynchus tshawytscha
Test
Temp.
(C)
25
25
25
15
14
14
15
15
15
96 Hour TL50b
mg/1 BrCl
(% effluent)


0.010
(55%)

0.017
(28%)
0.020
(44%)
0.019
(35%)
46 Percent
Chlorobro-
minated
effluent
0.016
(28%)
Inclusive
Dates
1-28 to
1-29-75
2-18 to
2-21-75
4-28 to
5-2-75
4-26 to
4-28-76
4-21 to
4-25-75
4-21 to
4-25-75
4-21 to
4-25-75
4-26 to
4-28-76
4-21 to
4-25-75
a
Comments
See text
72 hour acute test
Zero mortality at 0.143 mg/1
(100% effluent)
60% mortality at 0.010 mg/1
(60% effluent)
x length 59 mm, x weight 2.0 g
Zero mortality at 0.000 mg/1
(100% effluent). Test terminated
after 54 hours.
100% mortality at 0.020 mg/1
06% effluent)
x length 56 mm, x weight 2.0 g
100% mortality at 0.019 mg/1
(60% effluent)
55% mortality at 0.019 mg/1
(36% effluent)
x length 53 mm, X weight 0.9 g
100% mortality at 0.002 mg/1
(60% effluent) . Insufficient amounts
.of bromine chloride were measured for
determination of a TL50 expressed as
mg/1 BrCl.
100% mortality at 0.019 mg/1
(36% effluent)
x length 64 mm, x weight 2.4 g
                                            (continued)

aPercent mortality is given for the lowest effluent concentration having greater than 50% mortality
 if a TL50 is obtained; otherwise, percent mortality is given for the 100% effluent concentration.

^Figures in parentheses are TL50 values calculated in terms of percent effluent.

-------
            Table 36 (continued).  RESULTS OF ACUTE TOXICITY TESTS WITH CHLOROBROMINATED EFFLUENT
Species
Mayfly larvae
Stenonema sp.

Caddisfly larvae
Family Limnephilidae
Stonefly larvae
Order Plecoptera
Snails
Goniobasis livescens

Midge
Tanytarsus dissimilis

Ostracods
Subclass ostracoda
Calanoid copepods
Cyclopoid copepods
Cladoceran
Simocephalus serrulatus
Test
Temp.
(C)
16
16
17
17
17
17
17
17
17
96 Hour TL50
mg/1 BrCl
f% effluent)






0.002
(4%)

0.023
(47%)
Inclusive
Dates
8-25 to
8-27-75
8-25 to
8-27-75
8-20 to
8-22-75
8-20 to
8-22-75
11-17 to
11-19-75
11-17 to
11-19-75
10-21 to
10-23-75
11-11 to
11-13-75
11-13 to
11-15-75
Comments
10% mortality at 0.032 mg/1
(100% effluent)
Zero mortality at 0.032 mg/1
(100% effluent)
Zero mortality at 0.035 mg/1
(100% effluent)
Zero mortality at 0.034 mg/1
(100% effluent). Snails exposed
to high concentrations of chloro-
brominated effluent withdrew into
their shells, but emerged again
when placed in well water.
Zero mortality at 0.037 mg/1
(100% effluent)
20% mortality at 0.037 mg/1
(100% effluent)
100% mortality at 0.006 mg/1
(7.8% effluent)
10% mortality at 0.030 mg/1
(36% effluent)
60% mortality at 0.026 mg/1
(60% effluent)
o
o

-------
dead fish were removed,  ten more  fathead minnows were placed in the 100 and
50 percent chlorobrominated effluent  tanks.  All of  the fish died within
27 minutes of exposure in  the  100 percent  effluent.  There was 90 percent
mortality in the 50 percent effluent  tank  after 6.5  hours of exposure.
Moreover, fathead minnows  exposed at  the same  time to the same concentra-
tions of nondisinfected  and dechlorinated  effluents  were not adversely
affected.  The BrCl residual level in the  100  percent tank at that time was
0.127 mg/1, and the mean residual level in the 50 percent tank was 0.101 mg/L
Both of these figures are  well below  TL50  values obtained in earlier work.
A cyanide analysis was performed  at the time of the  test mortalities.  It
was found that 1.10 mg/1 total cyanide was present in the undiluted effluent.
Perhaps the cyanide, or  cyanide in combination with  a metallic plating waste,
combined with the BrCl to  produce an  extremely toxic environment for the
fish.  We observed this  phenomenon on one  other occasion during preliminary
testing.

ACUTE TOXICITY TESTS WITH  OZONATED EFFLUENT

Acute toxicity tests with  ozonated effluent produced less mortality than
tests with any other type  of effluent  (Table 37).  However, the effluent
was filtered prior to ozonation,  and  this  may  have been a significant factor
contributing to the reduced toxicity  observed.  Acute toxicity of undiluted
ozonated effluent to cladocera was the only routine  test wherein sufficient
mortalities occurred to  enable calculations of a TL50.  It is unlikely that
the observed mortality was caused by  the residual ozone, the concentration
of which (0.001 mg/1) was  at the  threshold of  detectability.  These results
are in agreement with previous tests-'-  and  with the findings of Arthur, et
_al. ,-* who observed in six  different acute tests less mortality in 100 per-
cent ozonated effluent than in 100 percent nondisinfected effluent.  Evans'
suggested that ozone has the advantages of removing  taste, odor, and color,
addition of oxygen, and  reduction of  BOD and COD, all of which may have
contributed to the reduced toxicity observed in ozonated effluent.  However,
as early as 1933 Hubbs^  noted  that 0.10 mg/1 of 0^ in water was sufficient
to kill small fishes, and  that smaller quantities, perhaps less than 0.01
mg/1, "sufficed to throw the fish in  nervous movements, from which there
was no quick recovery".  Arthur,  et_ al.,5  also observed mortalities of
fathead minnows exposed  to 03  residual levels  of 0.20-0.30 mg/1.

Rosenlund   observed high  mortalities  when rainbow trout were exposed to
ozonated Lake Mohave water.  However,  the  03 residual at the head of his
experimental tanks ranged  from 0.01 to 0.06 mg/1, while the highest 03 resid-
ual measured in any of our routine test tanks  was 0.012 mg/1, with the
majority of the observed residual levels being considerably lower  (0.002 to
0.008 mg/1).

Some acute tests were conducted with  activated sludge effluent receiving
exceptionally high doses of 03.   Fish were exposed to the ozonated effluent
immediately after 03 addition.  The results of these tests  (Table  38) indi-
cate that 03, in concentrations ranging from 0.088 to 0.288 mg/1,  is  suf-
ficiently toxic to cause mortality.   Symptoms  of stress evidenced  by  test
fish included swimming at  the  surface, loss of equilibrium, and quiescent
periods at the surface or  bottom.   Lake trout  were least resistant, while


                                      101

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                Table 37.  RESULTS OF ACUTE TOXICITY TESTS WITH OZONATED EFFLUENT
Species
Fathead Minnow
Pimephales promelas
Fathead Minnow
Pimephales promelas
Northern Common Shiner
Notropis cornutus
frontinalis

Goldfish
Carassius auratus

Brown Trout
Salmo trutta

Splake
Salvelinus sp.

Lake Trout
Salvelinus namaycush

Test
Temp.
(C)
16
16
25
16
13
13
16
96 Hour TL50
mg/1 03
(% effluent)







Inclusive
Dates
5-20 to
5-21-76
5-26 to
5-27-76
4-7 to
4-11-75
5-20 to
5-24-76
3-24 to
3-26-75
3-24 to
3-26-75
6-18-76
a
Comments
C100% mortality at 0.225 mg/1
(100% effluent) . All dead within
20 hours.
x length 59 mm^ x weight 2.3 g
°Zero mortality at 0.059 mg/1
(100% effluent). Fish exposed for
a total of 27 hours.
40% mortality at 0.003 mg/1
(100% effluent)
C100% mortality at 0.288 mg/1
(100% effluent)
5? survival time = 62.7 hours
x length 77 mm, x weight 5.6 g
10% mortality at 0.008 mg/1
(100% effluent)
This test was 48 hours in duration
5% mortality at 0.007 mg/1
(100% effluent)
This test was 48 hours in duration
C100% mortality at 0.126 mg/1
(100% effluent)
x survival time =61 minutes
x length 58 mm, x weight 1.4 g
                                      (continued)
Percent mortality is given for the lowest effluent concentration having greater than 50% mortality
 if a TL50 is obtained; otherwise, percent mortality is given for the 100% effluent concentration.
 Figures in parentheses are TL50 values calculated in terms of percent effluent.
°Fish were exposed to ozonated effluent immediately after the ozone was introduced.

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                  Table 37.(continued).   RESULTS OF ACUTE TOXICITY TESTS WITH OZONATED EFFLUENT
Species
Lake Trout
Salvelinus namaycush

Lake Trout
Salvelinus namaycush

Chinook Salmon
Oncorhynchus tshawytscha
Stonef lies
Order Plecoptera
Caddisf lies
Family Hydropsychidae
Amphipod
Gammarus sp .

Isopods
Ascellus sp.

Snail
Goniobasis livescens
Cyclopoid
Copepod
Cladoceran
Simocephalus serrulatus
Test
Temp.
(C)
16
16
13
18
18
18
18
18
18
18
96 Hour TL50
mg/1 03
(% effluent)









0.001
(100%)
Inclusive
5-19-76
5-25 to
5-26-76
3-24 to
3-26-75
8-4 to
8-6-75
8-13 to
8-15-75
8-4 to
8-6-75
8-13 to
8-15-75
8-4 to
8-6-75
11-20 to
11-22-75
11-24 to
11-26-75
Comments
C100% mortality at 0.234 mg/1
(100% effluent)
x survival time = 42 minutes
100% mortality at 0.048 mg/1
(50% effluent)
x survival time = 4.4 hours
X length 61 mm, x weight 1.9 g
C100% mortality at 0.088 mg/1
(100% effluent). All dead within
21 hours.
x length 63 mm, x weight 2.0 &
5% mortality at 0.008 mg/1
(100% effluent). This test was
48 hours in duration.
Zero mortality at 0.005 mg/1
(100% effluent)
30% mortality at 0.009 mg/1
(100% effluent)
Zero mortality at 0.005 mg/1
(100% effluent)
10% mortality at 0.009 mg/1
(100% effluent)
Zero mortality at 0.005 mg/1
(100% effluent)
Zero mortality at 0.001 mg/1
(100% effluent)
50% mortality at 0.001 mg/1
(100% effluent)
o
u>

-------
                Table 38.  RESULTS OF ACUTE TOXICITY TESTS WITH OZONATED ACTIVATED SLUDGE EFFLUENT0
o
-C-
Species
Fathead Minnow
Pimephales promelas
Fathead Minnow
Pimephales promelas
Goldfish
Carassius auratus

Lake Trout
Salvelinus namaycush

Lake Trout
Salvelinus namaycush

Lake Trout
Salvelinus namaycush

Test
Temp.
(C)
16
16
16
16
16
16
Inclusive
Dates
5-20 to
5-21-76
5-26 to
5-27-76
5-20 to
5-24-76
5-19-76
5-25 to
5-26-76
6-18-76
Comments
100% mortality at 0.225 mg/1 (100% effluent)
All dead within 20 hours.
x length 59 mm, X weight 2.3 £
Zero mortality at 0.059 mg/1 (100% effluent)
Fish exposed for a total of 27 hours.
100% mortality at 0.288 mg/1 (100% effluent)
x survival time = 62.7 hours.
x length 77 mm, x weight 5.6 g
100% mortality at 0.234 mg/1 (100% effluent)
x survival time = 42 minutes.
100% mortality at 0.048 mg/1 (50% effluent).
x survival time = 4.4 hours.
x length 61 mm, X weight 1.9 g
100% mortality at 0.088 mg/1 (100% effluent)
All dead within 21 hours.
x length 63 mm, x weight 2.0 g
100% mortality at 0.126 mg/1 (100% effluent)
x survival time = 61 minutes.
x length 58 mm, x weight 1.4 g
            aThe characteristics  of  the  activated  sludge effluent were 49 mg/1 BOD,  67 mg/1  total  suspen-

            ded solids, 0.9 mg/1 total  phosphate,  7.9 mg/1 NH -N, and pH 7.5.

-------
goldfish appeared most  tolerant, which is  the  typical response pattern of
fish species to halogens  and  other  oxidants.   However, it is unlikely that
fish would encounter  such high 03 levels in a  receiving  stream.  Ozone re-
siduals dissipate rapidly,  as evidenced in this project  which reports that
after only five minutes of  contact  time the residual level was commonly
reduced by an order of  magnitude.

SUMMARY

The results of our acute  toxicity tests indicate  that the nondisinfected
effluent used in  these  studies was  inherently  toxic to most species of fish
tested, with TL50 values  ranging from 38-81 percent effluent.  Invertebrates
were generally more tolerant  of nondisinfected effluent  than were fishes.
Ozonation coupled with  filtration appeared to  eliminate  the toxicity asso-
ciated with the  effluent, although  exposure of fish to ozonated activated
sludge effluent  Immediately after 03 addition  caused mortality.  Salmonid
fishes were more  sensitive to chlorobrominated effluent  than to any other
effluent type, while  most invertebrates tested showed similar tolerance to
chlorobrominated  effluent as  to nondisinfected effluent. Chlorinated efflu-
ent was  the most  toxic  effluent type tested, with sufficient mortality
occurring in  all  but  four tests to  calculate  a TL50.  TL50 values calculated
in terms of percent  chlorinated effluent were  generally  lower than for any
other effluent  stream.   Dechlorination with  sulfur dioxide reduced the
toxicity of  the  chlorinated effluent to levels similar to nondisinfected
effluent for  both fish and invertebrates.
                                      105

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                                 REFERENCES

 1.   Ward,  R.  W.,  R.  D.  Giffin,  G.  M.  DeGraeve,  and R.  S.  Stone.   Disin-
     fection Efficiency  and Residual Toxicity of Several Wastewater Disin-
     fectants.  Volume I Grandville, Michigan.   Environmental Protection
     Technology Series EPA-600/2-76-156,  1976.

 2.   Hubbs, C.  L.   Sewage Treatment and Fish Life.   Sew Works Jour. 5:1033-
     1040,  1933.

 3.   Alabaster, J. S. The Effect of a Sewage Effluent  on  the Distribution
     of Dissolved Oxygen and Fish in a Stream.   Jour Animal Ecol.   28:283-
     291,  1959-

 4.   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.

 5.   Arthur, J. W., R. W. Andrews,  V. R.  Mattson, D. T. Olson, J.  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.

 6.   Gaufin, A. R.  The  Effects of Pollution on a Midwestern Stream.  Ohio
     Jour Sci.   197-208, 1958.

 7.   Mount, D.  I.   The Effect of Total Hardness and pH  on  Acute Toxicity
     of Zinc to Fish. Air and Water Poll Int Jour  10:39-56, 1966.

 8.   Zillich, J.  A.  Preliminary Investigation of the Relative Toxicities
     of Chlorine, Bromine and Bromine Chloride.   Midland,  In House Report,
     The Dow Chemical Company,  1971.  3 pp.

 9.   Evans, F. L.  (ed.).  Ozone in Water  and Wastewater Treatment.  Ann
     Arbor, Ann Arbor Science Publishers  Inc., 1972 pp. 83-100.

10.   Rosenlund, B.  Disinfection of Hatchery Influent by Ozonation and the
     Effects of Ozonated Water on Rainbow Trout.  In:  Aquatic Applications
     of Ozone, Blogoslawski, W. J., and R. G. Rice   (Eds.).  International
     Ozone Institute, Syracuse, 1975.
                                     106

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                                   TECHNICAL REPORT DATA
                            (f tease read Instructions on the reverse before completing)
   EPA-600/2-77-205
                                                           3. RECIPIENT'S ACCESSION-NO.
 4. TITLE AND SUBTITLE
   DISINFECTION EFFICIENCY AND RESIDUAL TOXICITY OF
   SEVERAL WASTEWATER DISINFECTANTS
   Volume II - Wyoming, Michigan
               5. REPORT DATE
               November 1977 (Issuing Date)
               6. PERFORMING ORGANIZATION CODE
 . AUTHOR(S)                            "   ~~~	~
   Ronald W. Ward*, Randall  D.  Giffin**, G. Michael
   DeGraeve*
               8. PERFORMING ORGANIZATION REPORT NO.
 9. PERFORMING ORGANIZATION NAME AND ADORES
   **City of Wyoming,  Michigan
   1155 28th Street,  S.W.
   Wyoming, Michigan  49509
               10. PROGRAM ELEMENT NO.

                   1BC611
               11-XXEROOBRKX/GRANT NO.

               S-802292
 12. SPONSORING AGENCY NA'ME AND ADDRESS
   Municipal Environmental Research Laboratory.-Cin., OH
   Office of Research  and Development
   U. S. Environmental Protection Agency
   Cincinnati, Ohio  45268
               13. TYPE OF REPORT AND PERIOD COVERED
                 Final,  July  1975-June 1976
               14. SPONSORING AGENCY CODE
                 EPA/600/14
 ,5.SUPPLEMENTARY NOTES  See also Volume I  - Grandville, Michigan, EPA-600/2-76-156, PB-
   262-245/AS.   Project Officer:  Albert D.  Venosa (513-684-7668).
   *Grand  Valley State Colleges, Allendale,  Michigan  49401	
  C/^OO^Q A /*T                         ^^^_    ^ ^^^™™	 , . 	       lm ,,   	f	 ^^™P«™ ...«.._   	
 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
 wastewater which had been dechlorinated with  sulfur dioxide.

 A stream of nondisinfected trickling filter effluent was  pumped from the Wyoming,
 Michigan Wastewater Treatment Plant to the project's water treatment building.  The
 supply of effluent was split into four streams, three  of  which were disinfected with
 either chlorine, bromine chloride, qr ozone and then delivered to the bioassay labor-
 atory for residual toxicity tests.  The fourth stream  was delivered directly to the
 bioassay laboratory for testing.  In addition, a portion  of the chlorinated effluent
 stream was dechlorinated with sulfur dioxide  and then  pumped to the bioassay labor-
 atory .

 Total and fecal coliform densities, total 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  tested  for acute toxicity towards
 several species of fishes and invertebrates,  and chronic  toxicity in a. life cycle
 study with the  fathead minnow, Pimephales promelas, as the test subject.	
 7.
                                KEY WORDS AND DOCUMENT ANALYSIS
                  DESCRIPTORS
                                              b. IDENTIFIERS/OPEN ENDED TERMS
                              COS AT I Field/Group
 Waste water,Disinfection, Disinfectants,
 Bactericides,  Effectiveness, Efficiency
 Chlorination, Chlorine, Dechlorination,
 Sulfur dioxide,  Bromine halides, Ozone
 OzonizationjColiform bacteria, Toxicity,
 Bioassay, Aquatic animals, Daphnia, Fishes.
   Wyoming  (Michigan)  par-
   allel streams,  Acute
   bioassays, Chronic  bio-
   assays, Residual Toxicity
                              13B
                               6F
 3. DISTRIBUTION STATEMENT

 Release to  Public
UNCLASSIFIED
             ASS (
              FIE
!1. NO. OF PAGES
  119
                                              20. SECURITY CLASS (Thispage)
                                                  UNCLASSIFIED
                                                                         22. PRICE
EPA Form 2220-1 (9-73)
107
                                                                    *U.S.6WBNIIHtT PRINTING OFFICE 1977-757-140/6585

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