EPA-600/3-75-012
November 1975
Ecological Research Series
COMPARATIVE TOXICITY OF
SEWAGE-EFFLUENT DISINFECTION TO
FRESHWATER AQUATIC LIFE
Environmental Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Duluth, Minnesota 55804
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development,
U.S. Environmental Protection Agency, have been grouped into
five series. These five broad categories were established to
facilitate further development and application of environmental
technology. Elimination of traditional grouping was consciously
planned to foster technology transfer and a maximum interface in
related fields. The five series are:
1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
4. Environmental Monitoring
5. Socioeconomic Environmental Studies
This report has been assigned to the ECOLOGICAL RESEARCH series.
This series describes research on the effects of pollution on
humans, plant and animal species, and materials. Problems are
assessed for their long- and short-term influences. Investiga-
tions include formation, transport, and pathway studies to
determine the fate of pollutants and their effects. This work
provides the technical basis for setting standards to minimize
undesirable changes in living organisms in the aquatic, terres-
trial and atmospheric environments.
This document is available to the public through the National
Technical Information Service, Springfield, Virginia 22161.
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EPA-600/3-75-012
November 1975
COMPARATIVE TOXICITY OF SEWAGE-EFFLUENT DISINFECTION TO
FRESHWATER AttUttift LIFE
by
John W. Arthur
Robert W. Andrew
Vincent R. Mattson
Donald T. Olson
Gary E. Glass
Barbara J. Halligan
Charles T. Walbridge
Environmental Research Laboratory
Duluth, Minnesota 55804
U.S. ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF RESEARCH AND DEVELOPMENT
ENVIRONMENTAL RESEARCH LABORATORY
DULUTH, MINNESOTA 55804
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DISCLAIMER
This report has been reviewed by the Environmental Research Laboratory,
U.S. Environmental Protection Agency, and approved for publication.
Mention of trade names or commercial products does not constitute
endorsement or recommendation for use.
ii
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ABSTRACT
Flow-through laboratory bioassays were conducted with a domestic
secondary sewage effluent that had been disinfected by chlorination,
by chlorination followed by dechlorination, and by ozonation. Effluent
without disinfection served as a control. Disinfection with chlorine and
ozone generally maintained the effluent at total coliform levels of less
than 1,000 per 100 ml. Lake Superior water served as the diluent source
for the experiments. Short-term (7-day) exposures were conducted with
13 species (seven fish and six invertebrates), and long-term (generation)
tests were performed with three species (one fish and two
invertebrates). In both series of tests the chlorinated effluent was
lethal at appreciably lower concentrations than any of the other three
effluent treatments. Fish were more sensitive than the invertebrates to
the chlorinated effluent in 7-day tests. The respective 7-day TL50
values of total residual chlorine to fish and invertebrates ranged from
0.08 to 0.26 and 0.21 to >0.81 mg/1. Residual ozone rapidly decreased in
the treated effluent and was not measurable in the test tanks. When
special short-term test procedures and shorter retention times for the
ozonated effluent were used, measured residual ozone was about as lethal
to fathead minnows as residual chlorine. The highest mean total residual
chlorine concentrations having no long-term adverse effect on fathead
minnows, amphipods, and Daphnia were 14, 12, and 2-4 yg/1., respectively.
No daphnids survived at approximately 10 yg/1. mean total residual
chlorine, a concentration that corresponds to a chlorinated sewage
concentration of about 2.5%. Residual sulfite at the concentrations
used to neutralize the chlorine residual dissipated readily and had no
measurable adverse effect on the species tested.
iii
-------
This report was submitted in fulfillment of Project Numbers 18050 WAP
and 16AAE by the Environmental Research Laboratory-Duluth (formerly
the National Water Quality Laboratory). Experimental work was completed
in November 1972.
iv
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CONTENTS
Page
Abstract iii
List of Figures vi
List of Tables vii
Acknowledgments ix
Sections
I Conclusions 1
II Recommendations 3
III Introduction 4
IV General Bioassay and Analytical Procedures 6
Disinfection 6
Analytical Methods 13
Results 15
V Acute Tests 22
Methods 22
Results 25
VI Chronic Tests 32
Methods 32
Results 35
Fathead Minnow Chronic Test 36
Amphipod Chronic Test 39
Daphnia Chronic Test 40
VII Discussion 44
VIII References 49
IX Appendices
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FIGURES
No. Page
1 Sewage Flow from Storage Tanks to Diluter Cells 7
2 Chronic Test Apparatus Used for Volumetrically 9
Dividing, Chlorinating, and Dechlorinating the
Sewage Effluent
3 Ozone Generator 11
4 Apparatus Used for Volumetrically Dividing and 12
Ozonating the Sewage Effluent in Chronic Tests
vi
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TABLES
No. Page
1 Chemical Characteristics of the Sewage Effluent 16
Collected at the Silver Bay, Minnesota, Municipal
Treatment Plant
2 Chemical Characteristics of the Sewage Effluent 17
in the Laboratory Storage Tanks
3 Total Residual Chlorine and Ozone Concentrations 18
Measured from Diluter Toxicant-Bearing Cells and
Ozone from Column During the Acute and Chronic
Tests
4 Concentrations of Total Coliforms in the Storage 20
Tanks and Diluter Cells During the Acute Tests
5 Concentrations of Total Coliforms in the Storage 21
Tanks and Diluter Cells During the Chronic Tests
6 Sources and Sizes of Animals Used for the Acute 23
Tests
7 Mortality of Seven Fish and Six Invertebrate 26
Species in Acute Tests of Various Concentrations
of Four Effluent Treatments after 7 Days'
Exposure
vii
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Page
8 Acute Toxicity of Chlorinated Effluent to 28
Seven Fish and Six Invertebrate Species
9 Hatchability of Fathead Minnow Eggs in 31
Dechlorinated and Ozonated Test Waters
10 Survival and Reproduction of Fathead Minnows 37
During 43 Weeks' Continuous Exposure to
Nondisinfected and Disinfected Effluents
11 Survival and Reproduction of Amphipods after 41
20 Weeks' Continuous Exposure to Nondisinfected
and Disinfected Effluents
12 Survival and Reproduction of Daphnia magna During 43
2 Weeks' Continuous Exposure to Nondisinfected
and Disinfected Effluents
viii
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ACKNOWLEDGMENTS
We would like to thank the staff of the Silver Bay, Minnesota, Sewage
Treatment plant for their generous support in providing equipment and
giving time for the effluent collections. Appreciation is expressed to
many members of the Environmental Research Laboratory-Duluth (formerly
the National Water Quality Laboratory) staff who provided assistance
during the course of this project. Mr. F. A. Puglisi devised and
constructed the special chemical metering apparatus and determined the
dose concentrations. Mr. 6. R. Endicott designed and constructed many
of the supportive components incorporated into the disinfection and
bioassay process. Bacterial analyses were performed by Ms. S. Bagley,
Mr. D. Herman, and Mr. R. M. Brosdal; Ms. V. Snarski and Mr. L. F. Mueller
performed the routine analyses and contributed bioassay assistance. Mr.
A. R. Carlson assisted with the acute tests. The effluent was transported
to the laboratory and maintained in the storage tanks by Mr. D. Beedy and
Mr. R. E. Lewis. Dr. T. W. Thorslund performed the statistical analyses
reported in section VI. In addition, we thank Ms. S. L. Forseth for
typing this manuscript.
ix
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SECTION I
CONCLUSIONS
1. Chlorinated effluent was lethal to the test animals at appreciably
lower concentrations than nondisinfected, dechlorinated, and
ozonated effluents in both short- and long-term tests.
2. Both ozone and chlorine generally maintained effluent disinfection
levels at <1,000 total coliforms per 100 ml.
3. Residual ozone rapidly disappeared from the effluent and was not
measurable in the test-tank waters.
4. During the short-term tests fish mortality was more rapid than
invertebrate mortality in the chlorinated effluent.
5. The highest mean total residual chlorine concentrations having no
long-term adverse effect on fathead minnows, amphipods, and Daphnia
were 14, 12, and 2-4 ug/1., respectively. This corresponds to a
concentration of 1.2 to 2.5% chlorinated effluent.
6. The lowest mean total residual chlorine concentrations having a
measurable long-term effect were 42 yg/1. for fathead minnows
(survival of initial animals), 19 yg/1. for amphipods (reproduction),
and approximately 10 pg/1. for Daphnia (survival of initial animals).
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7. Residual sulfite at the concentrations used to neutralize the
residual chlorine dissipated readily and had no adverse effect on
the species tested.
8. When special test procedures and shorter retention times for the
ozonated effluent were used, measured residual ozone was about
as lethal as residual chlorine to fathead minnows.
9. Amperometric procedures were developed and used for analyzing total
residual concentrations of chlorine, ozone, and sulfite. Total
residual chlorine concentrations as low as 10 yg/1. were readily
measurable with a lower limit of detection of 1 yg/1.
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SECTION II
RECOMMENDATIONS
Concentrations of total residual chlorine as low as 10 yg/1. had a
detrimental effect on survival and reproduction of aquatic life.
Ozonation and chlorination followed by dechlorination are suggested as
disinfection alternatives for protecting aquatic life. Neutralization
of chlorinated effluents by dechlorination is needed to alleviate
adverse effects on aquatic life in the discharge areas. Additional
investigations are needed to more fully define long-term no-effect levels
for sulfite and other dechlorinating agents in receiving waters and
the potential formation of harmful oxidation products by ozonation.
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SECTION III
INTRODUCTION
Federal and most state pollution control regulations require disinfection
of sewage effluents prior to discharge. By 1977 treatment of all
municipal effluents will be required to achieve a fecal coliform level of
less than 200 per 100 ml (0. S. Environmental Protection Agency, 1973).
Historically, chlorination has been a widely practiced means of disinfection
in this country. However, recent concern over the toxicity of chlorinated
effluents to aquatic life has stimulated a re-evaluation of effluent-
disinfection practices. The toxicity of chlorine to aquatic life in
laboratory and field situations has been summarized recently by Brungs
(1973). Besides the effects of chlorination, municipal wastewater
toxicity has also been attributed by Esvelt et al. (1973) and Tsai (1973)
to other components such as methylene blue active substances (MBAS),
ammonia, and turbidity.
Ozonation and chlorination followed by dechlorination are alternative
methods for disinfecting wastewaters. By means of short-term exposures
Zillich (1972) and Esvelt et al. (1973) compared the toxicity of
chlorinated and dechlorinated wastewaters to fish. In an earlier study
Kelly et al. (1960) showed that the feeding activity of oysters was
suppressed in both chlorinated and dechlorinated water but not in untreated
or ultraviolet-sterilized water. Ozonation is being widely studied and
rappears to offer many advantages over the use of chlorine (Sliter, 1974).
Although the effects of ozonating natural waters have been reported
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(Benoit and Matlin, 1966; Institute of Maritime Fisheries, 1972; MacLean
je_t al^., 1973), very little has been done to determine the toxicity of
ozonated effluents to higher aquatic life.
Collins and Deaner (1973) compared the toxicity of water immediately
above and at points 100-300 ft downstream from a chlorinated wastewater
plume. All king salmon fry died within 14 hr in the downstream waters
(total chlorine residuals of 0,2-0.3 mg/1.), and none died in the
upstream water. When chlorination was temporarily shut off at four
wastewater treatment plants by the Michigan Department of Natural
Resources (1971), the survival of brook trout and fathead minnows in
cages below these plants was usually much higher.
The need for additional information on the toxicity of effluent
disinfectants to aquatic life led to the present investigation. The
purposes of this study were to compare with short- and long-term flow-
through bioassays the toxicity of a domestic sewage effluent after (1)
chlorination, (2) chlorination followed by dechlorination (hereafter
called dechlorination), and (3) ozonation. At the same time tests were
conducted with the nondisinfected effluent. The purpose of the short-
term tests was to determine the lethality of these four effluent
treatments to several fish and invertebrate species. The intent of the
long-term tests was to establish concentrations of nondisinfected and
disinfected effluents having no effect on three aquatic species over a
life cycle.
The municipal effluent used in this study had the following necessary
characteristics:
1) it had been subjected to secondary treatment;
2) chemical characteristics were typical of secondary municipal
effluents throughout the country;
3) it did not contain toxic industrial wastes; and
4) its diluent water was similar in quality to the test water.
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SECTION IV
GENERAL BIOASSAY AND ANALYTICAL PROCEDURES
The secondary sewage treatment plant of Silver Bay, Minnesota, on the
north shore of Lake Superior (population 3,500) was chosen as the effluent
source. The municipality's water supply is taken directly from Lake
Superior, as is the water supply for the National Water Quality
Laboratory. Silver Bay's secondary sewage treatment plant employs a
trickling filter. The effluent disinfection treatments used in this
study were designed to approximate current field conditions.
DISINFECTION
Effluent was collected by pump from the treatment plant's final settling
basin before chlorination and was transported by truck to the National
Water Quality Laboratory, a distance of 80 km. The three cylindrical
steel tanks on the truck had a combined capacity for hauling 4,550 1.
Upon arrival at the laboratory the sewage effluent was pumped into three
1.8-m-diameter elevated tanks, each with a storage capacity of 1,900 1.
The stored sewage was kept at about 15° C by recirculating chilled lake
water through stainless steel coils in the bottom of the storage tanks.
The general effluent flow design for disinfection is given in Figure 1.
From the storage tanks sewage was pumped into two stainless steel head
boxes for distribution and for the disinfection steps before acute and
chronic bioassay. When acute and chronic tests were conducted simultaneously,
3,200-3,800 1. per day of effluent were needed. Between 1,100 and 1,700 1.
?
per day were used for the fathead minnow and amphipod chronic tests.
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Dechlor
Contoc
Chlorli
Conta
nation
Cell
Nondtein
Was
ration
:t Cell
f
Acute 1
Head Box
1
iHeater |
(to test temp)
1
ected
te
F
*^
Ozon
Coli
ition Ozon
mn Coli
. r
i
^v
Head Box
\
[Heater |
(to test temp)
•ir
ition Nondlsii
mn Wa
i r
B A
Chronic b
f ected
ste
Chlorinatlon
Contact Cell
Dechlorination
Contact Cell
S ri rS
B A B A B
oassavs I
A-Fathead minnow tests
B-Amphipod and Oaphnid tests
Figure 1. Sewage flow from storage tanks to diluter cells.
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A series of glass chambers (after Brungs and Mount, 1970) was designed
to separate the nondisinfected effluent from that being chlorinated and
dechlorinated. Details of this apparatus employed for the chronic tests
are shown in Figure 2. For chlorination, distilled water from a Mariotte
bottle entered near the bottom of a glass tube. The bottom of the glass
tube was equipped with a fritted glass disc for dispersing the entering
chlorine gas. This saturated solution of chlorine was added to the
nondisinfected effluent by a chemical-metering apparatus (Mount and
Brungs, 1967). The concentration of chlorine in this solution was
maintained at 3.5 + 0.6 mg/ml. The chlorine contact chamber had a
retention time of approximately 30 min. At the end of this chamber the
chlorinated effluent was again separated for dechlorination and for
delivery to the chlorinated effluent diluter. Sulfur dioxide gas was
used for dechlorination with a chemical-metering apparatus similar to
that used for chlorination. At the end of the dechlorination contact
cell (approximately 40-min retention time) the effluent flow was split
for delivery to the appropriate diluter.
The basic difference in the equipment used for acute tests was the
absence of modified diluters at the end of the dechlorinated contact
cell. A wooden enclosure surrounded each acute and chronic disinfection
apparatus for venting the surrounding air to the outside of the
laboratory. The chlorine dose was adjusted so that a total residual of
approximately 1 mg/1. (orthotolidine colorimetric determination) remained
after a 30-min retention time, which would result in a total coliform count
of less than 1,000 cells per 100 ml. To maintain this residual, 3.8 +
0.4 mg Cl/1. of sewage was used. The sulfur dioxide (dechlorination)
dose rate was adjusted to neutralize the chlorine residual and provide an
excess sulfite residual of approximately 4 ppm in case of an overdose
of chlorine.
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Nondblnf«ct«d Mwog* tffkitnt
f
V»nt
x— -^
-O"
1 r\.
\nl,
{ .
1
-z-r.
i:
l!
II
1^
\
^-Chlortnottd
fttt TS,SS?- '
opparatut
Cl*
To F.H.
dlhiftr
END VIEW
8am. - Amphlpod (8ommcru«)
F.H.- FathMd Mine*
Mehlerlntttd
II H
NondltlnftcMd
II
To Oom. To F.H. To Bom.
dlluNr • dlftittr dHurtr
NendMnf«ct«d
II
To F.H.
dllutar
Figure 2. Chronic test apparatus used for volumetrically dividing,
chlorinating, and dechlorinating the sewage effluent.
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For disinfecting the effluent by ozonation separate ozonators were
constructed for the acute and chronic tests. Ozone was produced by
passing dried air between concentric reactor tubes with an applied
voltage difference of 8,000 V. The voltage was obtained from a Jefferson
luminous tube outdoor transformer operated at 65 V (60 cps). The ozone
dose was adjusted by regulating the air-flow rate (98 + 18 cc/min) to
achieve a disinfection of usually less than 1,000 total coliforms per
100 ml. Details of the ozone generator are given in Figure 3. A
concentration of approximately 32 mg 03/1. in the dried air was introduced
into the bottom of a contact column (Figure 4). Sewage effluent entered
near the top of the contact column filled with glass marbles. The effluent
retention time in the acute and chronic ozone contact columns was 7—8
and 10-11 min, respectively. The vented air after sewage contact
contained 18 mg/1. residual ozone. To attain an effluent concentration
of 1 mg/1. residual ozone, an ozone dose rate of approximately 5.7+1
mg/1. of sewage was needed. The disinfected effluent was then split
for delivery to the appropriate diluter.
The diluters used for the acute and chronic bioassays were patterned
after Mount and Brungs (.1967) 1 the arrangement of diluter components was
similar to that given by Arthur and Eaton (1971). The percentage
sewage concentrations used for the tests are given in sections V and VI.
Vacuum manifolds from the diluters were connected in series, and the
acute and chronic diluters were separately connected to water aspirators.
Nondisinfected sewage overflowing into a plastic box (Figure 2) triggered
a microswitch, thereby opening a solenoid valve controlling the water
aspirator. This event cycled the diluters and simultaneously closed
solenoid valves delivering diluent waters. Both acute and chronic
diluter cycle rates were maintained at approximately 5 min. Flow-
splitting cells divided the test and control waters for transfer to the
.£
duplicate acute and chronic test chambers.
10
-------
1/2" QQ
stainless steel
tubing
Dry air flow
3/4" QQ
pyrex tubing
3/8" hole
In stainless
steel tubing
Aluminum foil
around pyrex
tubing
3/O note
In stainless steel
tubing
Plug
(stainless steel
solder)
l/2"-3/4" Swagelok
stainless steel
fining with
teflon ferrules.
Silicone rubber bead
around pyrex tubing
Air and 0-
Figure 3. Ozone generator.
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NondMnfoctod
— Mortilm
1 I
1
!
J
I
1
1
' j
i
1
(
)
AmpMpod (Oamroorua)
FoffiMOd Minnow
C—Clomp
a) SetanoU
To Gam.
tftattr
To Ra
dlkitar
Figure 4. Apparatus used for volumetrically dividing and ozonating
the sewage effluent in chronic tests.
12
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ANALYTICAL METHODS
Routine procedures (American Public Health Association, 1971) were
followed in characterizing this sewage effluent. Determinations of
dissolved oxygen (D.O.), pH, biological oxygen demand (B.O.D.)> total
Kjeldahl nitrogen, total phosphates, and total, settleable, suspended,
and volatile solids were made by treatment-plant personnel at the
Silver Bay sewage treatment plant. Routine determinations of storage
temperature, pH, D.O., total hardness, total alkalinity, total acidity,
and chemical oxygen demand (C.O.D.) were generally made on each batch
of sewage. Membrane filter techniques (American Public Health Association,
1971) were used throughout this study for determination of total and
fecal coliform counts in the diluter cells and storage tanks and for
determining the degree of disinfection achieved. These bacterial
determinations were generally made two to three times weekly during
the acute and chronic tests.
The variable chemical makeup of the effluent required daily adjustments
in the chlorinated metering apparatus and air flow to the ozonators.
Total chlorine residuals were measured both colorimetrically and
amperometrically (American Public Health Association, 1971).
Colorimetric determinations were made with a Hach water test kit. The
sensitivity for determining the amperometric endpoint was significantly
increased by using a polarograph and rotating platinum electrode. The
electrode system consisted of a synchronous motor electrode rotator
and a 2.5-cm-diameter platinum disc serving as the cathode. The anode
was composed of a 0.5-cm-diameter platinum circle placed in close
proximity to the cathode. The phenylarsine oxide titrant (5 x lO"1* N)
was added with a microburet to a 50-ml sample buffered to pH 4 with
sodium acetate-acetic acid and made 5 x 10~3 M with potassium iodide.
The sample was mixed with a magnetic stirrer rotated slowly to minimize
turbulence at the electrode surfaces. A polarograph sensitivity of 0.1
13
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ya/mm (10-25 ya full scale) provided sufficient discrimination of the
endpoint with additions of titrant as small as 0.01 ml. Ozone
concentrations were determined volumetrically by using the same
procedures. For both the residual chlorine and ozone measurements,
samples were titrated immediately after the iodide and buffers were
added. Complete descriptions of the analytical procedures are given by
Andrew and Glass (1974) .
Sulfur dioxide concentrations were measured as sulfite by using a
_4
solution of potassium iodate, 5 x 10 N, as the titrant and the
amperometric method already described to detect the endpoint. The
sample to be titrated was made approximately 0.2 M I^SOi,. and 5 x 10~ M KI.
As titrant is added the sulfite is oxidized to sulfate, and an excess
converts iodide to iodine for amperometric detection. Since iodide in
the presence of oxygen is oxidized gradually at low pH, the titrations
were performed within 3 min after the samples were taken.
Ammonia was determined colorimetrically with Nessler reagent and a
microdiffusion concentration technique (Seligson and Seligson, 1951) .
In this procedure a 2-ml sample was placed into a 50-ml sample bottle,
2 ml of saturated ^CO^ solution was carefully pipetted into the bottle
to form a layer under the sample, and the bottle was then quickly
stoppered with a receiving rod whose ground glass tip had been coated by
dipping into 0.5 M I^SO^. The ammonia diffused out of the sample and
onto the acid-coated rod tip as the sample bottle was rotated
horizontally at 50 rpm for 30 min. The receiving rod was transferred
into a colorimeter tube containing 10 ml. of Nessler reagent, mixed,
and after 10 min the optical absorbance at 420 nm was determined.
Standards over the range of 5-30 yg ammonia (as N2) were used.
14
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Sodium levels were measured to determine the concentration of effluent
actually delivered to the acute and chronic test tanks. A flame
photometric method was initially used. Later, sodium concentrations were
determined by direct aspiration of the aqueous samples into a Perkin-
Elmer 403 atomic absorption spectrometer (Perkin-Elmer Corporation, 1971).
RESULTS
The modified amperometric methods developed for this study were more
sensitive than procedures given by the American Public Health Association
(1971). Fifty-milliliter samples of measured concentrations of tap
water chloramines diluted serially five- to a hundredfold with distilled
water gave recoveries of 77-107%. This corresponded to a mean standard
error of approximately 15% in the range of 10-200 yg/1. total residual
chlorine. A thousandfold dilution, or approximately 1 yg/1., gave
recoveries of + 100% corresponding to the lower limit of detection.
Difficulties in measuring samples <10 yg/1. were partly due to inaccuracies
in delivering +0.01 ml of titrant. For the sulfite measurements,
titrations were reproducible and sensitive to 20 yg/1.
The effluent was not uniform and required daily monitoring. The
chemical characteristics of the effluent are shown in Tables 1 and 2.
In the treatment-plant analyses the greatest variations were found in
the measurements of solids and B.O.D. The largest variations in the
laboratory storage tanks were in total and fecal coliforms, C.O.D., acidity,
and dissolved oxygen. The ammonia nitrogen concentrations analyzed from
the diluters were similar, and all mean values were within 1 mg/1. of
the mean storage tank concentration.
The concentrations of residual chlorine, ozone, and sulfite maintained
in the effluent after disinfection are listed in Table 3. Total
residual chlorine and ozone analyses were done daily, and sulfite cell
15
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Table 1. CHEMICAL CHARACTERISTICS OF THE SEWAGE EFFLUENT COLLECTED AT THE SILVER BAY, MINNESOTA,
MUNICIPAL TREATMENT PLANT
Sewage flow (101* l./dey)
Influent
Solids (ng/1.)
Settleable
Total
Total suspended
Total suspended volatile
l.O.D (ng/1.)
Effluent
Solids (ng/1.)
Settleable
Total
Total suspended
Total suspended volatile
B.O.D (ng/1.)
Dissolved oxygen (ng/1.)
pH (unite)
KJeldahl nitrogen (ng/1.)
Total phosphates (as roM ng/1.)
Tear ant; Bantu
1971 -1
June
181
(126-254)
7.5
(3.5-15.5)
547
(402-984)
192
(93^342)
135
(78-194)
140
(87-213)
0.4
(0.1-1.6)
296
(256-380)
45
(26-67)
35
(22-48)
40
(24-61)
5.5
(4.5-6.1)
7.4
(7.2-7.6)
15.1
(14.8-15.4)
11.0
(8.5-13.3)
July
1ST
(126-262)
7,6
(4.0-12.0)
411
(259-619)
150
(84-250)
121
(77-174)
149
(92-180)
0.4
(0.1-1.0)
227
(85-300)
36
(25-60)
29
(22-45)
29
(13-41)
6.0
(5.5-6.5)
7.3
(7.1-7.5)
14.5
(12.5-15.4)
15.3
(11.3-19.9)
*"«•
fil
(117-157)
8.0
(4.0-11.5)
563
(449-869)
214
(148-354)
180
(119-293)
160
(129-184)
0.2
(0.1-0.4)
290
(240-568)
27
(24-33)
24
(20-26)
23
(19-28)
5.7
(5.2-6.0)
7.2
(7.0-7.4)
11.4
(10.4-12.3)
13.1
(10.8-17.3)
Sept.
131
(89-263)
9.3
(6.0-18.0)
473
(352-704)
165
(120-326)
137
(104-248)
168
(139-215)
0.2
(0.1-0.6)
245
(209-279)
30
(23-40)
26
(20-33)
30
(20-39)
5.0
(4.6-5.5)
7.1
(7.0-7.3)
13.7
(9.5-17.1)
14.5
(10.6-18.2)
Oct.
(136-460)
5.2
(0.6-12.0)
411
(309.-539)
108
(30-194)
90
(26-162)
130
(52-218)
0.2
(0.1-0,9)
276
(236-306)
26
(21-33)
20
(15-24)
32
(23-55)
5.2
(4,7-6.1)
7.2
(7.0-7.4)
13.6
(B.1-17-.6)
10.2
(5.5-1J.O)
Nov.
242
(159-528)
5,1
(0.3-11.0)
364
(262-460)
98
(36-203)
81
(32-169)
129
(60-275)
0.1
(0.1-1.0)
256
(210-282)
29
(21-43)
21
(3-34)
34
(25-60)
5.7
(5.2-6.3)
7.1
(7.0-7.3)
13.6
(12.3-15.1)
8.8
(5.8-11.3)
Dec.
IJf-,
(117-160)
7.5.-
(4.0-12.0)
429
(364-508)
143
(96-147)
117
(80-150)
168
(112-222)
0.4
(0.2-0.6)
242
(213-267)
34
(14-49)
29
(11-38)
32
(24-90)
5.5
(5.2-5.8)
7.1
(7.0-7.2)
16.1
(14.0-19.0)
7.3
(4.2-9.3)
Jan.
1ZU
(107-131)
8.8
(5.0-15.0)
439
(292-554)
145
(92-206)
124
(86-164)
163
(128-242)
0.3
(0.1-0.9)
219
(194-268)
31
(25-38)
27
(21-33)
33
(29-39) '
6.1
(5.7-6.9)
6.9
(6.8-7.1)
19.5
(15.8-22.1)
13.8
(8.3-18.1)
Feb.
(103-129)
8.6
(5.0-13.0)
442
(291-494)
141
(105-180)
121
(94-160)
155
(111-184)
0.2
(0.1-0.5)
225
(186-247)
35
(27-42)
31
(25-38)
50
(38-58)
6.2
(5.9-6.6)
6.9
(6.8-7.0)
18.4
(11.2-21.8)
13.2
(11.1-17.0)
Karch
(107-253)
7.8
(2.0-15.0)
412
(353-472)
117
(75-168)
96
(66-142)
140
(81-168)
0.3
(0.1-0.6)
265
(213-315)
46
(37-71)
39
(32-57)
60
(48-69)
6.9
(6.3-8.0)
6.9
(6.7-7.0)
20.6
(17.1-24.3)
13.8
(11.6-19.1)
April
(206-536)
3.7
(1.0-10.5)
303
(244-416)
61
(33-96)
48
(27-78)
91
(60-132)
0.5
(0.1-2.0)
249
(203-295)
36
(24-51)
30
(19-40)
48
(34-55)
8.7
(7.9-9.5)
7.3
(7.1-7.5)
14.4
(11.2-17.4)
7.8
(6.3-8.4)
July
(7§??46)
6.1
(1.5-12.0)
425
(320-566)
127
(22-278)
103
(18-210)
108
(46-183)
0.5
(0.1-1.8)
282
(217-357)
47
(23-97)
35
(20-68)
31
(40-62)
5.2
(4.6-6.1)
7.4
(7.2-7.5)
12.3
(9.5-16.5)
10.5
(8.8-11.6)
Oct.
(11$60)
8.4
(4.0-13.0)
417 '
(285-505)
136
(88-175)
117
(77-147)
143
(112-166)
0,2
(0.1-1.0)
248
(171-294)
26
(21-35)
22
(18-27)
25
(18-27)
5.6
(5.4-5.9)
7.6
(7.5-7.7)
14.0
(11.8-15.1)
10.5
(9.0-12.3)
*JUmge in
-------
Table 2. CHEMICAL CHARACTERISTICS OF THE SEWAGE
EFFLUENT IN THE LABORATORY STORAGE TANKS
Item
Temperature (°C)
pH
Dissolved oxygen (mg/1.)
Total hardness (mg/1.)
Total alkalinity (mg/1.)
Total acidity (mg/1.)
Ammonia nitrogen (mg/1.)
C.O.D. (mg/1.)
Sodium (mg/1.)
Total coliforms (per 100 ml)
Fecal coliforms (per 100 ml)
Mean
14.2
7.23a
4.7
124
131
16.7
6.9
71.1
17.8
676,000
57,800
N
87
95
91
95
96
95
49
126
31
144
69
Range
9.0-20.6
6.94-7.59
0.9-8.8
74-190
90-177
3.1-36.3
2.3-13.5
25.8-141.4
14.3-23.0
20,000-10,100,000
700-620,000
A median value.
17
-------
Table 3. TOTAL RESIDUAL CHLORINE AND OZONE CONCENTRATIONS MEASURED FROM DILUTEE TOXICANT-BEARING CELLS AND OZONE FROM COLUMN
DURING THE ACUTE AND CHRONIC TESTS
(mmierams/liter)
00
Test
organism
Flah
Brook trout
Coho salmon
Fathead ' minnow
Fathead minnow
White sucker
Walleye
Yellow perch
Largemouth basa
Invertebrates
Amphipod
Amphipod
Stonefly
Caddlsfly
Crayfish
Operculate snail
Pulmonate anall
Dlluter toxicant-bearinK cells (in mg/1.)
_ AmDerometric method
Residual sulflte
Mean
1.35
_
_
0.90
1.30
_
0,43
_ _
—
N
4
_
_
_
_
_
2
2
2
—
Range
0.6-2.21
_
_
_
_
_
0.8-0.9
1.1-1.4
__
0.36-0.48
„ _
„ _ .
—
Reaidual chlorine
Mean
1.63
1.74
1.24
1.99
1.52
1.98
2.10
1.97
1.77
2.22
1.73
1.74
1.98
2. OS
2.22
N
8
6
7
8
8
7
B
8
8
8
8
8
11
13
8
Range
Ac
1.39-1.94
1.48-2.03
0.39-2.73
1.69-2. 25
0.70-2.30
1.77-2.13
1.80-2.47
1.61-2.26
0.03-2.60
1.73-2.78
1.19-1.98
1.42-2.03
1.80-2.27
1.69-2.75
1.73-2.78
Residual ozone
Mean
ute tes
0.03
0.04
0.06
_
0.04
0.17
0.02
0.02
0.02
0.02
0.16
0.01
_
0.01
0.02
N
ts
5
7
6
_
5
7
8
1
6
6
7
3
^_
10
6
Range
0.01-0.04
0.01-0.08
0.01-0.07
_
0.02-0.08
0.05-0.30
<0. 001-0. 10
—
<0. 00 1-0. 04
<0. 001-0. 02
<0. 001-0. 62
0.004-0.02
_
<0. 00 1-0. 06
<0. 001-0. 18
Orthotolidine method
Residual chlorine
Mean
0.86
0.85
0.50
1.06
0.85
0.99
1.16
1.28
1.02
1.55
1.15
1.16
1.14
1.55
N
8
7
7
8
7
6
6
7
2
2
5
12
9
2
Range
0.71-0.99
0.58-0.98
0.17-1.50
0.65-1.25
0.70-1.05
0.89-1.14
1.00-1.40
1.06-1.48
0.75-1.30
1.55
1.10-1.27
0.80-1.35
0.65-1.55
1.55
Ozone column
Residual ozone (mg/1.)
Mean
a
_
_
_
_
1.95
2.07
2.32
1.73
2.20
2.32
N
_
_
_
_
_
4
3
6
5
__
9
6
Range
_
_
_
_
1.16-2.33
2.04-2.10
_
1.72-2.67
1.05-2.66
1.72-2.71
1.72-2.67
Fathead minnows
Amphipods
Daphnids
Teat 1
Test 2
4.30
2.70
_
25
6
_
0.1-14.3
0.2-5.2
_
_
2.30
2.16
2.26
2.84
332
146
14
11
<0. 001-3. 61
0.64-3.93
1.64-2.71
2.11-3.49
0.02
0.01
0.47
0.07
157
113
4
7
<0. 001-0. 17 )
<0. 001-0. 11 )
<0. 001-1.0
<0. 001-0. 32
1.02
102
—
0.05-3.15)
)
—
1.03
1.62
1.20
133
7
6
<0. 001-2. 89
0.50-2.37
0.45-2.87
*Not measured.
-------
measurements and ozone column measurements were generally made weekly.
Substantial amounts of residual ozone were lost in passage from the
ozane contact column to the diluter cells (Table 3). Approximately 50%
less total residual chlorine was measurable in the chlorinated diluter
cells by the orthotolidine method than by the amperometric method.
Excess total residual chlorine was not measurable in the dechlorinated
effluent during any of the acute tests despite variations in the residual
sulfite levels. On three occasions residual chlorine was measurable in
the dechlorinated effluent for the chronic tests (see section VI).
The mean concentrations of total coliforms in the effluent before and
after disinfection are given in Tables 4 and 5. After disinfection
the mean coliform concentrations were less than 10,000 per 100 ml in
13 of 15 acute tests. About 10 times more coliforms were present in
the dechlorinated than in the chlorinated effluent. Coliform concentrations
were generally higher in the ozonated effluent than in the chlorinated
effluent during the acute tests. During the fathead minnow and amphipod
chronic tests all mean total coliform concentrations after disinfection
were less than 10,000, and after the eighth week concentrations of coliforms
were generally less than 1,000 per 100 ml. A loss in measurable coliform
bacteria occurred during the flow of effluent from the storage tank
to the nondisinfected diluter cells (Tables 4 and 5); no single
component of the system was largely responsible. In the Daphnia
chronic tests the mean total coliform concentrations after disinfection
were <1,000 per 100 ml.
Disinfection of fecal coliforms in the acute and chronic test systems
was greater than 99»9%. Mean fecal coliform counts were generally
less than 10 per 100 ml in the treated effluents.
19
-------
Table 4. CONCENTRATIONS OF TOTAL COL I FORMS IN THE STORAGE TANKS AND MUTTER CELLS DURING THE ACUTE TESTS
(mean log counts per 100 ml)
Teat organism
Flah
Brook trout
Coho salmon
Fathead minnow
Fathead minnow
White sucker
Walleye
Yellow perch
Largemouth base
Invertebrates
Amphlpod
Anphlpod
Stonefly
Caddlsfly
Crayfish
Operculate snail
Fulmonate snail
Storage tank
Mean log
5.76
5.91
6.02
5.86
5.40
5.72
5.81
5.44
6.51
5.63
5.44
5.46
5.49
5.80
5.63
N
6
6
5
6
6
6
6
6
6
6
5
5
6
9
6
Range
5.45-6.07
5.43-6.49
5.30-6.40
5.62-6.30
5.15-5.76
5.40-6.27
5.34-7.00
4.85-5.94
5.63-7.20
4.95-6.13
4.95-5.88
4.85-5.91
5.15-5.76
4.95-6.36
4.95-6.13
Nondlsinfected
Mean log
5.49
5.71
5.59
5.83
5.24
5.63
5.59
5.50
6.18
5.44
5.31
5.57
5.21
5.73
5.44
N
6
6
6
6
6
6
6
5
6
6
5
5
6
9
6
Range
5.11-5.68
5.53-6.01
5.08-6.18
5.30-6.05
4.95-5.41
5.30-6.09
4.85-7.05
5.00-5.83
5.85-6.41
4.90-6.05
4.85-5.89
5.15-5.86
4.78-5.46
4.90-6.94
4.90-6.05
Dechlorlnated
Mean log
3.96
3.97
4.02
a
2.99
3.33
2.87
2.68
4.26
—
2.77
_
_
2.83
-
N
6
6
5
_
6
6
6
5
6
_
5
9
-
Range
3.60-4.41
3.36-5.31
3.60-4.43
_
2.62-3.59
2.91-4.19
2.66-3.11
2.26-3.04
3.28-5.52
^
2.40-3.85
—
2.08-3.67
-
Chlorinated
Mean log
3.22
3.07
4.24
2.01
1.95
2.15
1.66
1.40
3.34
1.99
1.58
2.29
1.43
1.99
1.99
N
6
6
5
6
6
6
6
6
6
6
5
5
6
9
6
Range
0.60-3.63
2.41-3.79
4.02-4.54
1.60-2.27
1.60-2.29
1.30-3.12
1.45-1.88
1.08-1.83
3.18-3.54
1.56-2.46
1.20-1.94
0.90-4.29
0.60-2.00
1.60-2.27
1.56-2.46
Ozonated
Mean log
2.41
3.14
3.98
—
1.54
2.69
2.36
2.03
3.82
1.99
_
2.28
-
N
6
6
6
_
6
6
6
6
6
_
5
9
-
Range
*
2.08-2.78
2.51-4.20
3.49-4.74
1.00-2.10
2.18-3.66
1.3074.09
1.48-3.63
3.34-4.24
1.00-2.36
_
1.00-3.94
-
NJ
O
Not measured.
-------
Table 5. CONCENTRATIONS OF TOTAL COLIFORMS IN THE STORAGE TANKS AND DILUTEE CELLS DURING THE CHRONIC TESTS
(mean 4-week log counts per 100 ml)
Test organism
and time (weeks)
Fathead .minnows
0-4
5-8
9-12
13-16
17-20
21-24
25-28
29-32
33-36
37-40
41-43
Amphipods
0-4
5-8
9-12
13-16
17-20
Storage tank
Mean log
5.46
5.82
5.57
5.62
5.63
5.29
5.53
5.51
5.56
5.66
5.64
5.72
5.70
5.35
5.65
5.33
N
4
9
11
9
6
10
12
9
10
9
7
9
9
8
8
8
Range
5.18-5.79
5.43-6.49
5.15-6.27
5.15-7.00
4.95-6.30
4.85-5.88
5.11-5.93
4.70-6.05
4.60-6.26
4.30-6.77
4.60-6.64
5.18-6.08
5.32-6.41
4.85-5.73
5.20-6.12
4.78-5.92
Nondisinfected
Mean log
5.51
5.46
5.43
5.36
5.16
4.99
5.31
5.25
5.07
5.36
5.59
5.33
5.39
4.97
5.44
5.01
N
4
9
11
9
6
10
12
9
10
9
7
9
9
8
8
8
Range
5.11-5.76
4.85-5.86
4.85-6.45
4.85-5.56
4.67-5.91
4.48-5.84
4.70-5.79
4.48-5.99
4.00-5.74
4.00-6.53
4.60-6.56
4.78-5.72
4.90-6.04
4.30-5.52
5.08-5.73
4.30-5.61
Dechlorinated
Mean log
3.95
3.58
2.76
1.89
1.70
1.91
1.75
1.42
1.67
1.66
1.76
4.00
3.28
1.34
1.92
1.79
N
4
9
11
9
6
10
12
9
10
9
7
Q
9
8
8
8
Range
3.48-4.45
2.15-4.85
0-4.26
3.04-1.48
1.00-2.62
1.00-3.81
0-4.31
0-2.36
0-2.59
0-3.73
0-3.04
2.63-4.84
2.85-3.90
0-2.00
1.48-2.72
1.00-2.20
Chlorinated
Mean log
2.20
2.02
1.51
1.60
1.87
1.25
1.51
1.72
0.93
1.45
1.45
2.24
1.29
0.26
1.51
1.13
N
4
9
11
9
6
10
12
9
10
9
7
9
9
8
8
8
Range
1.72-2.58
1.08-2.38
0-2.66
2.17-0.90
1.20-2.62
0.60-2.26
0.60-2.40
0.60-2.57
0-1.51
0-4.09
0-3.08
1.60-2.91
0-2.00
0-0.90
0.60-2.23
0.60-1.64
Ozonated
Mean log
3.68
2.79
2.55
2.05
2.12
1.75
1.30
1.57
1.56
2.23
2.56
2.83
2.56
1.58
1.96
1.43
N
4
9
11
9
6
10
12
9
10
9
7
9
9
8
8
8
Range
2.87-4.91
2.15-3.32
1.48-3.48
1.48-3.46
1.00-3.76
0-3.15
0-2.08
1.00-2.53
1.00-2.26
0-4.21
2.08-3.15
2.28-3.51
2.04-3.65
1.30-3.54
1.30-3.54
0-4.32
-------
SECTION V
ACUTE TESTS
METHODS
Modified proportional diluters served as the water-delivery system for
each of the four effluent treatments. The disinfection apparatus and
the diluters are described in section IV. The nominal percentage
sewage concentrations used for the nondisinfected, dechlorinated, and
ozonated treatment tests were 100, 75, 50, 25, and 12%. For the
chlorinated effluent tests, nominal sewage concentrations were set at
one-half of the other three treatments. The diluent source was raw
Lake Superior water piped directly to the laboratory. Duplicate test
chambers were used for the five effluent concentrations and the controls.
Diluter cycle rates were maintained at approximately 5 min throughout
the tests. The test tanks held 15 1. of°water and had a water-retention
time of approximately 5 hr. Durotest (Optima FS) and widespectrum
Gro-lux fluorescent tubes provided the light source, and a constant
16-hr photoperiod was used.
Acute exposure tests were conducted with seven fish and six invertebrate
species. Sources and sizes of the test animals are given in Table 6.
Before testing, all animals were acclimated in laboratory holding tanks
for 10 days or more within 2° C of test temperature. Ten organisms
randomly selected were introduced to each test tank so that 20 individuals
of each species were tested at each concentration. Any damagjad or
diseased individuals were rejected before testing. Complete
immobilization of the organisms was considered the endpoint and equated
22
-------
Table 6. SOURCES AND SIZES OF ANIMALS USED FOR THE ACUTE TESTS
to
u>
Test species
Fish
Brook trout
Coho salmon
Fathead minnow
White sucker
Walleye
Yellow perch
Largemouth bass
Invertebrates
Amphipods
(Gammarus pseudolimnaeus)
Stonefly
(Pteronarcys sp)
Caddisfly
(Hydropayche sp)
Crayfish
(Orconectea virilis)
Operculate snail
(Campeloma decisum)
Pulmonate snail
(Phym Integra)
n
Source
National Water Quality Laboratory, Duluth, Minnesota
Western Fish Toxicology Station, Corvallis, Oregon
National Water Quality Laboratory, Duluth, Minnesota
State Fish Hatchery, Grand Rapids, Minnesota
State Fish Hatchery, Aitken, Minnesota
Park Lake, Mahtowa, Minnesota
Federal Fish Hatchery, New London, Minnesota
Eau Claire River, Gordon, Wisconsin
Blackhoof River, Carlton County, Minnesota
Pequaywan Lake Outlet, St. Louis County, Minnesota
Eau Claire River, Gordon, Wisconsin
St. Crotx River, Gordon, Wisconsin
Sau Claire River, Gordon, Wisconsin
Mean
length (cm)
11.0
8.7
3.1
6.7
6.1
4.9
7.0
Adult stage
Nymph stage
Nymph stage
2.8
1.6
0.7
-------
with death. Animals were not fed during the experiments, except the
amphipods where leaves provided cover and food. Percentage mortality
of each test species was determined after 12-, 2k-, and additional
2U-hr intervals thereafter through 7 days. Tests with operculate
snails were extended for 1^ days to obtain a 50$ mortality level.
Standard graphical procedures were followed for determining the
concentrations resulting in 50# mortality levels (American Public
Health Association, 1971). Round stainless steel cages were used
for the stonefly and caddisfly tests. The cages were submerged
in the test tanks where the water entered and were covered with
stainless wire covers.
Routine chemical determinations were performed on the test-chamber waters.
Dissolved oxygen was generally determined every other day, and pH
analyses were made once a week on all test-chamber waters. The dissolved
oxygen measurements were made with a YSI oxygen meter. Water temperatures
were measured daily on two test chambers in each effluent treatment.
The flame photometric sodium method was used for monitoring the
nominal sewage concentrations delivered during these tests.
All residual chlorine and sulfite determinations were made on grab
samples collected at mid-tank depth with a volumetric pipette. Total
residual chlorine measurements were conducted daily during the 5-day
work week by alternating between the duplicate test chambers. Residual
ozone was not measurable in the highest concentration test-tank waters.
Residual ozone and sulfite measurements were taken in the test waters
only where additional tests were needed to establish lethal levels.
Three additional tests were conducted with the same general procedures
as above:
l) The sewage effluent was chlorinated to a residual level
of 16 mg/1. followed by neutralization with
24
-------
sulfur dioxide gas. Any associated toxicity from this
procedure was determined with fathead minnows at a test
temperature of 18° + 1° C.
2) Tests were also conducted on the concentrations of sulfite
lethal to fathead minnows and amphipods. The SO^ gas was
added to the nonchlorinated effluent. The test water
temperature was 19° + 1° C.
3) Fathead minnow eggs were obtained from control waters in the
chronic test and incubated in the two highest dechlorinated
and ozonated test concentrations (100 and 75%) and controls.
Procedures to determine hatchability differences followed the
recommendations of the National Water Quality Laboratory (1971). The
test water temperature was 24° + 1° C.
Two tests were also conducted to determine the concentrations of residual
ozone lethal to fathead minnows under flow-through conditions. Special
procedures were used for this test with the ozonated effluent. To
maintain a residual the ozonated sewage was conveyed directly from the
contact column to glass boxes each holding 650 ml. The boxes were
arranged in a five-step sequence, in which ozonated sewage flowed from
one box to the next. A diluter was not used for this test. Five
fathead minnows were placed in each of the glass boxes and tested at
a temperature of 23° + 1° C. Test concentrations were not duplicated.
RESULTS
After 7 days the chlorinated effluent was appreciably more toxic to the
test animals than the other effluent treatments tested (Table 7). More
than 50% of the fish tested died in chlerinated effluent concentrations
of 12-37%. Less than 50% of the operculate snails died in the
highest concentration of chlorinated effluent. Mean mortality in
the two highest dechlorinated and ozonated concentrations (100% and 75%)
was less than 50% and 20%, respectively. In the highest nondisinfected
25
-------
to
Table 7. MORTALITY OF SEVEN FISH AND SIX INVERTEBRATE SPECIES IN ACUTE TESTS OF VARIOUS CONCENTRATIONS OF
FOUR EFFLUENT TREATMENTS AFTER 7 DAYS' EXPOSURE
Cexpreased as percentage)
Test species
Fish
Brook trout
Coho salmon
Fathead minnow-Test 1
Fathead minnow-Test 2
White sucker
Walleye
Yellow perch
Largemouth bass
Invertebrates
Amphipod test 1
Amphlpod test 2
Stonefly
Caddisfly
Grayftshb
Opexculatc onall
Pulaonaf: snail
Hondls Infected
100%
90
0
0
-
40
25
5
0
25
-
45
80
-
0
-
75*
45
0
0
-
0
35
20
0
5
-
-
25
-
0
-
Dechlorlnated
100Z
0
0
0
-
0
20
0
0
45a
-
-
-
-
0
-
75%
0
0
0
-
0
20
5
0
15
-
-
-
-
0
-
Ozonated
100%
0
0
0
-
5
20
5
0
10
-
5
10
-
0
-
75*
0
0
0
-
0
0
0
0
5
-
5
5
-
0
-
Chlorinated
50%
100
100
-
100
100
100
100
100
-
100
100
95
50
10
40
37%
100
100
100
100
100
100
100
100
100
100
85
75
30
0
20
257,
100
100
100
100
100
100
60
50
100
95
70
85
5
10
10
12%
75
100
40
100
10
45
0
0
80
30
25
40
20
0
0
6%
0
35
0
15
10
0
0
0
35
5
5
5
5
0
0
"Survival of controls became, less than 90% after 72 hr.
Tests with crayfish and operculate snails extended through 12 days and 14 days, resnectlvely.
-------
effluent concentration more than 50% of the test animals died in 2 of
12 tests, and more than 20% died in 6 of 12 tests. This higher mortality
in the nondisinfected effluent may have been related to reduced dissolved
oxygen concentrations in these test waters.
Progressively higher concentrations of effluent delivered to the test
tanks reduced the dissolved oxygen concentration. The lowest dissolved
oxygen concentrations were found in dilutions of the nondisinfected
effluent. For any individual test the mean dissolved oxygen concentrations
were above 3.0 mg/1. (about 30% saturation). Occasional measurements
below this level were recorded in the nondisinfected tests with brook
trout, coho salmon, sucker, amphipod (test 1), and stonefly. Mean
dissolved oxygen measurements less than 50% saturation were typically
found in the three highest concentrations of nondisinfected effluent
and the two highest levels of dechlorinated effluent. Mean dissolved
oxygen concentrations were consistently above 50% saturation in the
ozonated and chlorinated test waters. The range in pH for all the test-
chamber waters was from 6.9 to 7.9 units.
The chlorinated effluent was more acutely ttgcic to fish than to
invertebrates. The 7-day TL50 concentrations of total residual chlorine
for fish and invertebrates ranged from 0.08 to 0.26 and 0.21 to >0.81 mg/1.,
respectively (Table 8). The mean total residual chlorine concentrations
throughout the entire test were used for calculating the TL50 values.
Brook trout and coho salmon were the most sensitive fish species, and
amphipods the most sensitive invertebrate. Flow-through acute tests were
not done with Daphnia magna. Fish died more quickly than invertebrates
in the chlorinated effluent. Most of the fish died in the first 24 hr of
the test, but 24-48 hr were needed to kill 50% of the amphipods, 72 hr
were needed for the stoneflies, and more than 6 days for the remaining
invertebrate species tested. Fifty percent of the operculate snails
(Campeloma decisium) died only after 14 days' exposure to the effluent.
27
-------
Table 8. ACUTE TOXICITY OF CHLORINATED EFFLUENT TO SEVEN FISH AND SIX INVERTEBRATE SPECIES
N)
00
Test species
Fish
Brook trout
Coho salmon
Fathead minnow-Test 1
Fathead minnow-Test 2
White sucker
Walleye
Yellow perch
Largemouth bass
Invertebrates
Amphipod-Test 1
Amphipod-Test 2
Stone fly
Caddis fly
Crayfish0
Operculate snailc
Pulmonate snail
Test
temperature
+ 1" C
14
14
17
18
16
12
17
17
17
18
18
18
17
18
18
Total residual chlorine TL50 concentrations (in uK/1.)
1-hr
a
_
_
>790
>560
,^__
>850
>574
-
>780
>740
___
—
12-hr
360
230
335
185
245
267
365
494
>810
>780
>740
>780
>810
>810
24 -hr
200
230
145
140
148
220
245
345
900
>810
>780
>740
>780
>810
>810
48-hr
175
105
145
88
140
173
205
310
630
660
>780
>740
>780
>810
>810
72-hr
175
102
140
86
138
150
205
300
470
255
480
>740
>780
>810
>810
96-hr
135
102
130
86
138
150
205
295
330
215
400
>740
>780
>810
>810
5-day ,
130
95
130
84
138
150
205
272
250
185
270
>740
>780
>810
>810
6-day
93
95
125
82
132
150
205
261
220
177
235
>740
>780
>810
>810
7-day
83
83
115
82
132
150
205
261
210
t
195
>550
780
b
>810
Mortality levels not recorded.
Survival of controls became less than 90%.
°Testa with crayfish and operculate snails extended through 12 and 14 days, respectively.
-------
Sodium analyses (used as a tracer for determining diluter accuracy)
were conducted during 7 of 13 acute tests. These analyses showed that
all of the chlorinated and dechlorinated test concentrations and the
highest three ozonated and two highest nondisinfected test concentrations
were delivered within 2% of the nominal concentration. The remaining
experimental nondisinfected and ozonated concentrations were within
5% and 10%, respectively.
Acutely lethal concentrations of S0£ were established by additions of
sulfur dioxide gas directly to the sewage effluent without prior
chlorination, but no toxicity was found after neutralization of sewage
chlorinated to high levels (16 mg/1.) followed by sulfur dioxide gas
neutralization. The amphipods were about five times more sensitive to
the measured residual sulfite than the fathead minnows. The 7-day TL50
values for fathead minnows and amphipods were 67 and 10 mg/1., respectively.
The dissolved oxygen concentrations bracketing these TL50 values for
fathead minnows and amphipods were 2.1-4.3 and 5.7-6.0 mg/1., respectively.
The bracketing pH levels were 6.3-6.8 and 6.5-7.7 mg/1. Thus suppressed
dissolved oxygen and decreased pH in the test waters may have contributed
to the toxicity.
Residual ozone concentrations between 0.2 and 0.3 mg/1. were lethal
to the fathead minnows. All deaths occurred in the first 1-3 hr of the
test. One test was continued for 96 hr, the Bother for 7 days. The
measured dissolved oxygen in the test cells where the deaths occurred
had a supersaturation level of 120-125%. However, the next lower
concentration had a similar dissolved oxygen supersaturation level
without loss of fish.
29
-------
Percentage hatch of fathead minnow eggs did not differ in the
dechlorinated, ozonated, and control waters (Table 9). Although egg
hatchability was the lowest in dechlorinated tank 100 A, the hatchability
in the duplicate chamber (100 B) was similar to that in all other test
chambers. Egg hatchability in the control waters was similar to that
in the dechlorinated and ozonated treatments.
30
-------
Table 9. HATCHABILITY OF FATHEAD MINNOW EGGS IN DECHLORINATED AND OZONATED TEST WATERS
(acute exposure system)
Nominal
percentage
sewage
concentration
A
100
B
A
75
B
A
0
(Control) B
Dechlorinated
Number
of groups
incubated
3
3
1
2
3
3
Mean
percentage
hatch
37.3
62.0
54.0
54.0
64.6
60.0
Range
8-60
44-76
-54-
34-74
46-88
36-82
Ozonated
Number
of groups
incubated
3
3
3
3
3
3
Mean
percentage
hatch
67.3
79.3
84.0
86.0
61.3
70.6
Range
58-86
70-80
74-100
66-98
0-98
64-84
-------
SECTION VI
CHRONIC TESTS
METHODS
Modified proportional diluters served as the water-delivery system, and
the diluent source was raw Lake Superior water. The nominal sewage
concentrations used for tests of the nondisinfected, dechlorinated, and
ozonated effluent were 20, 10, 5, 2.5, and 1.2%. For the chlorinated
effluent, the nominal sewage concentrations were set at one-half of the
other three treatments. Duplicate test chambers were used for each
experimental test concentration and for the controls. The diluters
cycled at approximately 5-min intervals throughout the tests. Flow-
splitting cells divided each test concentration at a 2-to-l ratio for
delivery to adult and progeny test chambers. Durotest (Optima FS) and
widespectrum Gro-lux fluorescent bulbs provided the light source for the
chronic tests.
The fathead minnow chronic test was started on June 3, 1971. Thirty-
five hatched fry, 1-20 days old, were randomly assigned to each adult (27-1.)
tank. Fry were obtained from National Water Quality Laboratory cultures.
.Mean test-chamber retention time was maintained at about 4 hr, and water
temperature was maintained at 24° + 2° C. Additional procedures for this test
followed the methods recommended by the National Water Quality Laboratory (1971),
The chambers were cleaned daily 1-2 hr after feeding, since excess food
temporarily lowered the residual chlorine test concentrations. The first
spawning was recorded on October 27. Hatched larvae were either discarded
or placed into corresponding progeny (14-1.) tanks. Spawning of the
minnows ceased on March 24, 1972, and the test terminated on April 1, 1972.
32
-------
Adult amphipods, Gammarus pseudolimnaeus Bousfield, were collected from
the Eau Claire River near Gordon, Wisconsin. Fully gravid females were
isolated in the laboratory and 35 newborn young, 1-6 days old, were
randomly placed on June 25, 1971, into each of 48 progeny tanks holding
7.4 1. of water. Test temperature was maintained at 17.5° + 1° C.
Mean retention time for the progeny chambers was approximately 4 hr.
After 60 days the amphipods were transferred to corresponding adult
tanks with a water capacity of 14.7 1. and impartially thinned to 15
individuals per tank. The photoperiod was the same as that used for the
fathead minnow chronic test. The amphipods matured to adults in early
September and started producing young that continued until test
termination on November 10-11, 1971. Measurements of head capsule length
after 30 days* exposure in the progeny chambers were used for determining
growth of the second generation young.
The diluters and test chambers housing the fathead minnows and amphipods
were separately encompassed by black plastic enclosures to minimize
external physical disturbances. Laboratory air temperatures generally
ranged from 17° to 24° C. The amphipod enclosures were air conditioned to
reduce air supersaturation in the test waters. However, during the 14th
week of the test air bubbles were observed clinging to the external
surfaces of the amphipods, making them more buoyant. Aeration of the test
water during this time eliminated the air-bubble problem.
Daphnia magna for the chronic tests were obtained from laboratory cultures.
Test temperature was maintained at 22° + 1° C. The adult amphipod chambers
and the same mean water-retention times were used for this test. Ten
1-day-old (16-24-hr) daphnids were placed into each chamber for tests
of 2 weeks' duration during July and October 1972. To maintain the
daphnids in the control tanks, food was continuously added with a
peristaltic (Durum Co.) pump to the diluent water head box. Food source
used for the July test was a 20:1 (31.5 g) mixture of Glencoe No. 2 trout
33
-------
Starter and powdered dry grass and cereal leaves (Cerophyl) prepared
according to procedures of Biesinger and Christensen (1972).
Fleischmann's active dry yeast (7-8 g) was used for the October test.
Each food mixture was suspended in 9 1. of lake water. Performance of
the pump in delivering the food into the diluent test water was monitored
daily, and calculated respective amounts of food present were 1.7 + 0.6
and 1.4 +0.7 mg/1. per day. Reproduction (parthenogenetic) occurred in
the test chambers after 1 week with a production of one or two generations
by termination. At test completion all test-chamber waters were
separately siphoned through a 60-wire-mesh stainless steel basket to
collect the daphnids for preservation in 70% ethanol. All daphnids
were counted in subsamples in petri dishes subdivided into 1-cm2 grids;
uniform microscope counting procedures were used.
Routine chemical analyses, in addition to those used for the acute tests,
were performed on the chronic test-chamber water (American Public
Health Association, 1971). For the fathead minnow and amphipod tests
dissolved oxygen analyses were usually conducted every other day; pH,
total hardness, total alkalinity, and acidity generally were measured twice
weekly; and water temperatures were recorded daily on two test chambers
in each effluent treatment. Sodium determinations were generally made
weekly with the flame photometric procedure until mid-November and
thereafter with the atomic absorption procedure. For the Daphnia tests
the only routine analyses conducted were for dissolved oxygen and pH;
they were done weekly on six test waters in each effluent treatment.
Samples for determining total residual chlorine, ozone, and sulfite
were collected from the test waters; protocol as described for the
acute tests was followed. Total residual chlorine analyses were conducted
daily during the workweek. The total residual sulfite procedure was not
developed until after the 14th week for the fathead minnow anclamphipod
tests, and then sulfite was measured weekly. As in the acute tests, residual
ozone was not measurable in any of the waters of the chronic tests.
34
-------
The amphipod and daphnid results were statistically analyzed with
computation procedures found in Biomedical Computer Programs (Dixon, 1971).
The production of Daphnia (young per adult) was compared with a 3-way
analysis of variance. The variables were treatment method, concentration,
and test trial. Fathead minnow survival and the amphipod reproduction
index were examined with a 2-way analysis of variance. Because the 20
and 0.6 nominal percentage effluent concentrations were not uniformly
used in all four of the effluent test treatments, these two concentrations
were not analyzed with these procedures. Comparisons of differences
among the disinfected and nondisinfected effluents and differences within
an effluent group were made according to Dunnett's (1955) method.
RESULTS
Results of the routine chemical analyses of fathead minnow and amphipod
test waters are given in appendix Tables 1-2. Increasing amounts of
sewage in the test tanks progressively lowered the dissolved oxygen
concentrations and pH and increased the levels of total hardness,
alkalinity, and acidity. Mean dissolved oxygen concentrations in the
test waters were all above 50% saturation. An individual measurement
less than 30% saturation occurred only once during the fathead minnow
test and was found in the highest concentration of nondisinfected
effluent (20%).
The concentrations of total residual chlorine and sulfite maintained in
the respective chlorinated and dechlorinated effluent concentrations are
presented in appendix Tables 3-4. The duplicate chambers in each of the
five concentrations of chlorinated effluent had slightly different mean
concentrations of residual chlorine. However, the calculated standard
deviations were generally similar for each corresponding duplicate tank pair.
Concentrations of total residual sulfite maintained in the dechlorinated effluent
test chambers were more variable than the residual chlorine concentrations.
35
-------
The dechlorination apparatus failed on two occasions during the fathead
minnow and amphipod chronic tests (on July 8-9 and September 5-6, 1971)
and on October 18 during the second Daphnia test. During these times
residual chlorine concentrations of 0.2-1.1 mg/1. were measured in the
dechlorinated effluent. During the September failure 0.05-0.15 mg/1.
residual chlorine was found in the highest dechlorinated test
concentration. No failures were recorded with the dechlorination
apparatus used for the acute tests.
Analysis of sodium in the adult test-tank waters showed that the mean
sewage concentrations actually delivered were generally within 5 and 2.5%
of the nominal levels set for the respective fathead minnow and amphipod
chronic studies. Individual determinations overlapped the next
corresponding nominal test level about 15% of the time in the two highest
ozonated effluent concentrations. No similar overlap occurred with the
other three effluent treatments.
Fathead Minnow Chronic Test
More fathead minnows died in dilutions of chlorinated effluent than in
the other effluent treatments (appendix Table 5). Most deaths occurred
during the first 10 weeks of the test. During this time minnow survival
was significantly lower (P<0.05) in the two highest chlorinated
concentrations (>_ 42 ug/1. mean total residual chlorine). Survival in
these two concentrations was also lower at test termination.
No spawning occurred in the highest chlorinated effluent concentration
(110 yg/1.)(Table 10). No differences were observed in the spawning
results at the other test concentrations. Six deformed females were
found at test termination. These deformed females were not limited to
any one effluent treatment or test concentration, but were included in
the spawning calculations. The low egg hatchability in chlorinated
36
-------
Table 10. SURVIVAL AND REPRODUCTION OF FATHEAD MINNOWS DURING 43 WEEKS'
CONTINUOUS EXPOSURE TO NONDISINFECTED AND DISINFECTED EFFLUENTS
Nominal
percentage
sewage
concentration
A
20
B
A
10
B
A
5
B
A
2.5
B
A
1.2
B
A
0
(Control) B
A
10
B
A
5
B
A
2.5
B
A
1.2
B
A
0.6
B
A
0
CControl) B
Final
number
surviving
(male/female)
4/7a
5/5
4/1
4/5
4/6
4/6
4/5
4/5
4/4
3/10
4/4
5/5
1/1
0/1
3/2
3/2
4/6
4/5
4/9
3/6
4/5
3/8
4/3
4/9
Number of
excess males
removed
(Nov-Jan)
3
3
5
7
5
1
4
2
2
0
4
1
0
0
0
0
2
4
2
0
4
3
3
1
Total eggs
and
spawnings
Nondlslnfected
6817 (50)
7377 (60)
244 (2)
8808 (64)
7822 (64)
10586 (90)
11546 (88)
9407 (62)
5426 (62)
16524 (132)
5950 (64)
9148 (74)
Chlorinated1"
0 CO)
0 (0)
7435 (55)
4833 (29)
7203 (72)
7193 (73)
7661 (74)
9626 (97)
7550 (69)
3726 (36)
8250 (68)
5038 (35)
Eggs
per
female
974
1475
244
1762
1304
1764
2309
1881
1357
1652
1488
1830
Q
0
3717
2416
1200
1439
851
1604
1510
466
2750
560
Spawnings
per
female
7
12
2
13
11
15
18
12
16
13
16
15
0
0
20
14
12
15
8
16
14
4
23
4
Egg Hatchabillty
Number
of
spawnings
22
12
1
14
17
15
17
11
16
22
14
12
Mean
percentage
hatch
66
53
80
64
82
78
59
71
72
61
84
69
—
14
9
24
22
18
19
18
13
15
11
—
75
20
86
87
83
75
81
85
83
71
37
-------
Table 10 (continued).
SURVIVAL AND REPRODUCTION OF FATHEAD MINNOWS DURING
43 WEEKS' CONTINUOUS EXPOSURE TO NONDISINFECTED AND
DISINFECTED EFFLUENTS
Nominal
percentage
sewage
concentration
A
20
B
A
10
B
A
5
B
A
2.5
B
A
1.2
B
A
0
(Control) B
A
20
B
A
10
B
A
5
B
A
2.5
B
A
1.2
B
A
0
(Control) B
Final
number
surviving
(male/female)
4/5
3/9
4/10
4/6
4/7
4/8
4/6
4/6
4/6
4/1
4/5
5/6
Number of
excess males
removed
(Nov-Jan)
4
0
0
3
2
3
1
1
4
4
0
2
Total eggs
and
spawnings
Dechlorinated
17579 (114)
12480 (109)
9238 (70)
10099 (77)
17110 (103)
7272 (75)
10291 (99)
7604 (62)
7018 (63)
1297 (12)
8418 (78)
3654 (26)
Eggs
per
female
3516
1387
924
1683
2444
909
1715
1267
1170
1297
1684
609
Spawnings
per
female
23
12
7
13
15
9
16
10
10
12
16
4
Em Hatchability
Number
of
jpawninRS
31
25
12
25
25
20
28
12
18
3
18
12
Mean
percentage
hatch
48
43
73
72
66
82
73
79
84
60
69
87
Ozonated
4/6
4/6
5/4
4/6
5/9
4/5
6/7
5/8
4/5
4/8
5/5
4/1
5
5
3
4
1
3
0
1
4
4
3
4
7033 (77)
8595 (81)
8039 (71)
8776 (67)
8175 (78)
10001 (101)
8822 (91)
5472 (56)
6342 (64)
9802 (89)
7061 (86)
4705 (25)
1172
1432
2010
1463
908
2000
1260
684
1268
1225
1412
4705
13
14
18
11
9
20
13
7
13
11
17
25
14
15
17
13
15
17
18
17
14
45
15
10
71
87
C3
60
73
73
75
54
53
71
69
82
alnitial number of fathead minnows per tank was 35, thinned to 15 individuals after 60 days.
bMean total residual chlorine levels for the six test concentrations (in ug/1.) were 110, 42, 14, 6, 3, and
nonmeasurable, respectively.
38
-------
chamber 5B may be due to factors other than residual chlorine. For
unknown reasons spawning by the minnows was not confined to the undersurface
of the concrete tiles. Thirty-six per cent of the spawning occurred on
the egg cup undersurfaces and on two or three occasions on standpipe
screens.
Overall egg hatchability was lower in the highest dechlorinated
concentration (Table 10). For comparison, seven groups of eggs spawned
on different days in one of the highest dechlorinated test chambers
were incubated in a control chamber. The average hatchability was 87%
(range 78-90%). By contrast, two groups of eggs from the control
chambers incubated in one of the highest dechlorinated chambers gave
hatchability values of 56% and 54%.
Survival data for the second generation of fathead minnow larvae after 30
days' exposure in progeny tanks are given in appendix Table 6. Two to five
trials were conducted at each effluent test concentration. Final
survival varied widely. The lowest survivals of minnow larvae occurred
in the two highest chlorinated concentrations containing mean total
residual chlorine levels of ^88 yg/1. Survival was reduced in mean chlorine
levels of 21 yg/1., but the variation in final survival was much greater
(2.5-47.5%).
Amphipod Chronic Test
Amphipod survival was noticeably lower after 10 weeks in chambers with the
highest chlorinated and dechlorinated effluent concentrations (appendix
Table 7). Poor survival in the highest dechlorinated concentration was
probably due to failures of the dechlorinator. Survival after 16 weeks
of the test was also lower in the second highest chlorinated concentration
•(54 yg/1. mean total residual chlorine).
39
-------
Reproduction was significantly reduced in the three highest chlorinated
effluent concentrations. Second generation young were not produced at
either the 123 or 54 ug/1. mean concentrations (Table 11). The
reproductive index was significantly lower in the 19 yg/1. chambers
as compared to the controls (P <0.05). The highest measured level of
residual chlorine having no effect on the amphipods was 12 yg/1.
Survival and growth of the second amphipod generation were similar after
30 days in all concentrations (appendix Table 8). The second generation
appeared to survive somewhat better overall (83.5%) than did the original
adults after 30 days (64.4%). Survival of both generations was
generally similar during the first 30 days of exposure.
Daphnia Chronic Test
Greater numbers of the Daphnia initially added to test chambers survived
to termination in the first of the two tests performed (Table 12).
Survival and reproduction of the daphnids were similar in the control
chambers with the two different food sources used. Elimination of the
daphnids during the first week of the second test at the highest
dechlorinated effluent concentration was probably related to failure
of the dechlorinator.
Daphnia did not survive the 2 weeks' exposure in the three highest
chlorinated effluent concentrations. In these three concentrations
death occurred during the first week of exposure, and no young were
recoverable at test termination. Increased daphnid numbers at 12 days in
a few of the disinfected and nondisinfected effluent concentrations were
due to counting errors. Calculation procedure used for determining young
per adult is given in Table 12, and the results were statistically analyzed.
The highest level of total residual chlorine having no significant
*
effect on Daphnia was 2-4 yg/1. (P <0.05).
40
-------
Table 11. SURVIVAL AND REPRODUCTION OF AMPHIPODS AFTER 20 WEEKS'
CONTINUOUS EXPOSURE TO NONDISINFECTED AND DISINFECTED EFFLUENTS
Nominal
percentage
sewage
concentration
A
20
B
A
10
B
A
5
B
A
2.5
B
A
1.2
B
A
0
(Control) B
Final
male/female
ratio
6/4b
6/9
4/6
7/8
5/7
7/8
7/5
5/5
7/7
4/7
5/7
7/4
Final
gravid
females
4
3
2
5
4
8
3
5
6
6
2
2
Total
number
young
produced
Nondlslnfe
431
682
731
585
783
649
883
399
393
117
56
19
Total
births
cted
15
14
17
13
20
15
23
13
14
6
3
1
Young
per
female
107.8
75.8
121.3
73.1
111.9
81.1
176.6
79.8
56.1
16.7
8.0
4.8
Births
per
female
3.8
1.6
2.8
1.6
2.9
1.9
4.6
2.6
2.0
0.9
0.4
0.25
Reproductive3
index*
4.8
1.9
3.2
2.3
3.4
2.9
5.2
3.6
2.9
1.7
0.7
0.8
Chlorinated0
A
10
B
A
5
B
A
2.5
B
A
1.2
B
A
0.6
B
A
0
(Control) B
0/0
0/0
3/2
4/1
6/5
4/6
2/7
5/11
4/7
6/8
6/6
8/5
0
0
0
1
;1
0
4
9
6
1
5
3
0
0
0
0
42
77
518
444
430
360
192
102
0
0
0
0
2
4
16
16
16
11
10
6
0
0
0
0
8.4
12.8
74.0
40.4
61.4
45.0
32.0
20.0
0
0
0
0
Q.4
0.7
2.3
1.5
2.3
1.4
1.7
1.2
0
0
0
1.0
0.6
0.7
2.9
2.7
3.2
1.5
2.5
1.8
41
-------
Table 11 (continued). SURVIVAL AND REPRODUCTION OF AMPHIPODS AFTER
20 WEEKS' CONTINUOUS EXPOSURE TO NONDISINFECTED
AND DISINFECTED EFFLUENTS
Nominal
percentage
sewage
concentration
Final
male /female
ratio
Final
gravid
females
Total
number
young
produced
Total
births
Young
per
female
Births
per
female
Reproductive3
index
Dechlorinated
A
20
B
A
10
B
A
5
B
A
2.5
B
A
1.2
B
A
0
(Control) B
2/5
0
8/4
7/4
8/3
3/6
4/11
8/9
6/6
8/6
8/6
9/4
4
0
4
3
3
5
7
8
5
6
5
4
151
48
341
422
283
487
740
907
482
501
290
183
4
2
8
11
8
11
22
23
13
16
12
7
30.2
—
85.2
105.5
94.3
81.2
67.3
100.8
80.3
83.5
48.3
45.8
0.8
—
2.0
2.8
-2.7
1.8
2.0
2.6
2.2
2.7
2.0
1.8
1.6
0
3.0
3.5
3.7
2.7
2.6
3.4
3.0
3.7
2.8
2.8
Ozonated
A
20
B
A
10
3
A
5
B
A
2.5
B
A
1.2
B
A
0
(Control) B
7/5
8/5
4/4
6/5
4/7
7/5
5/8
3/9
9/5
5/5
9/3
5/8
3
2
4
1
4
3
5
5
3
1
2
5
818
429
487
722
442
478
502
465
708
679
164
46
21
12
14
20
17
14
16
17
21
19
5
2
163.6
85.8
121.8
144.4
63.1
95.6
62.8
51.7
141.6
135.8
54.7
5.8
4.2
2.4
3.5
4.0
2,4
2.8
2.0
1.9
4.2
3.8
1.7 *
0.25
4.8
2.8
4.5
4.2
3.0
3.4
2.6
2.4
4.8
4.0
2.3
0.9
"Determined by adding total births (spawnings) plus final gravid females divided by final number of
surviving females.
''initial number of amphlpods per test was 35, thinned to 15 individuals after 60 days.
°Mean total residual chlorine levels for the six test concentrations (in ug/1.) were 123, 54, 19, 12, 8,
and nonmeasurable, respectively.
42
-------
Table 12. SURVIVAL AND REPRODUCTION OF DAPHNIA MAGNA DURING 2 WEEKS1
CONTINUOUS EXPOSURE TO NONDISINFECTED AND DISINFECTED EFFLUENTS
Hondia infected
F.£B(8tti 1
savage Survival after
concentration |7 "daV»T"l2 days
Total
umber
found
Young0
per
adult
Dechlo Inated
Survival after
7 days | 12 days
1 1 1
Total
nuaber
found
Youngb
per
adult
First t
Ozonated
Jurvlvi
7 days
eatc
1 Total
il aftea nuaber
12 dayij found
1
Young
per
adult
Chlorinated*
Nominal 1 1
Percentage 1 Total
sevaKe [survival after] number
concentration |7 daya|12 days! found
1 1 1
Joaaif
per
adult
A
20
B
A
10
B
A
5
B
A
2.5
'
A
1.2
B
A
0
(Control) B
A
20
B
A
10
" B
A
5.
B
A
2.5
B
A
1.2
B
A
0
(Control) B
10
10
10
10
10
10
9
7
a
9
10
a
9
9
7
J
o
7
7
9
7
10
10
4
8
8
8
9
7
10
9
6
8
9
a
8
9
a
6
2
5
5
8
9
a
10
10
4
1404
1101
977
1525
915
1850
1368
498
790
348
589
704
698
374
305
96
250
324
534
637
268
214
848
319
174
137
121
168
130
184
151
82
98
38
73
87
77
46
50
47
49
64
66
70
32
20
84
79
10
10
9
10
10
8
10
10
9
10
10
10
0
0
9
2
10
9
10
10
10
8
10
10
10
10
9
10
9
8
7
9
10
8
9
8
—
9
2
10
9
9
10
8
8
9
8
1380
928
1207
1496
1239
858
B34
812
893
832
595
597
0
0
351
106
713
721
391
855
577
455
371
402
137
92
133
149
137
106
US
89
88
103
65
74
S«conn
0
0
38
52
70
79
L2
1
' 84
71
56
40
49
10
10
10
8
10
10
10
7
10
9
10
10
4
9
6
6
7
9
10
9
8
9
8
10
9
9
9
9
8
6
a
6
a
4
9
7
4
9
5
6
6
9
10
6
B
a
6
10
1223
1100
841
757
1082
572
492
318
287
444
852
476
383
U
316
103
336
168
587
154
199
82
480
271
135
121
92
83
134
94
60
52
35
110
94
67
95
-1
62
16
55
18
57
25
24
9
79
26
A
10
B
A
5
B
A
2.5
B
A
1.2
B
A
0.6
B
A
0
(Control) B
A
10
B
A
5
8
A
2.5
B
A
1.2
B
A
0.6
B
A
0
(Control) B
0
0
0
0
0
0
5
9
9
9
7
10
0
0
0
0
0
0
0
5
10
10
10
7
—
—
—
3
8
5
6
7
10
—
-
—
5
9
10
10
7
0
0
0
0
0
0
171
596
398
211
565
983
0
0
0
0
0
0
0
382
326
743
938
676
0
0
0
0
0
0
56
74
79
34
80
97
0
0
0
0
0
0
0
75
35
73
93
96
JK*an total residual chlorine levels for the six test concentration* (in yg/1.) were: Teat 1-114, SO. 14. 4, 2. and nonaeasurable respectively:
Test 2- 136, 38, 7, 2, nonoeasurable, and nonaeasurable, respectively.
Young par adult • (total nuaber found ainua adult* alive at 12 days) divided by (adults alive at 12 days).
'Tor both tests the Initial nunber of daphnlds par test chamber vas 10.
43
-------
SECTION VII
DISCUSSION
Disinfection with chlorine and ozone effectively reduced coliform
bacterial levels by 99.9%. After retention, the levels of total and
fecal coliforms were usually below 1,000 and 100 per 100 ml, respectively.
The disinfected effluents delivered to the diluters for the bioassays
had respective mean residual chlorine, sulfite, and ozone levels of
1.0-2.1, 0.4-4.3, and 0.0-0.5 mg/1. Residual ozone rapidly disappeared
from the effluent since residual concentrations of 1.0-2.0 mg/1. were
measured from the bottom of the ozone contact column. Shuval et al.
(1973) found a marked regrowth of coliform bacteria in both chlorinated
and dechlorinated wastewaters. In our study coliform regrowth was not
clearly demonstrated, probably because of the short retention times employed,
Only the chlorinated effluent consistently showed damaging effects to all
test species. The 7-day TL50 concentrations of total residual chlorine
ranged from 0.08 to >0.81 mg/1. for the test animals. Fish were more
sensitive than the invertebrate species during short-term exposures to
chlorine. These results agree closely with Brungs1 (1973) conclusion that
salmonids are the most acutely sensitive to chlorine; next most sensitive
are warmwater fish and invertebrate species. The residual chlorine TL50
values for fathead minnows and amphipods agree closely with those given
by Arthur and Eaton (1971). Also, the acute toxicity of residual chlorine
for fathead minnows parallels that reported by the Michigan Department
of Natural Resources (1971) and Zillich (1972). The residual chlorine
96-hr TL50 for rainbow trout by the Michigan Department of Natural
44
-------
Resources (1971) at two plants was 0.023 mg/1., which is lower than that
reported for brook trout in the present study (0.083 mg/1.). For additional
comparisons the reader is referred to the review by Brungs (1973) and
recent compilations by Becker and Thatcher (1973).
This study also showed that the chlorinated effluent had long-term effects
on survival and reproduction of fathead minnows, amphipods (Gammarus
pseudolimnaeus), and Daphnia magna. Chlorinated effluent concentrations
of 10% and 5% greatly reduced survival of fathead minnows and eliminated
daphnid survival. Chlorinated effluent concentrations of 2.5%
significantly affected amphipod reproduction, and no daphnids survived
at this concentration. The highest mean concentrations of total
residual chlorine having no effect on fathead minnows, amphipods
(Gammarus), and Daphnia magna were 14, 12, and 2-4 yg/1., respectively.
Daphnia magna was the most sensitive species during life-cycle tests
in the chlorinated effluent. Daphnid populations that survived to
adulthood reproduced successfully. Arthur and Eaton (1971) exposed
Ga™n«rus pseudolimnaeus and fathead minnows to chlorinated (as chloramines)
lake water for 15 and 21 weeks, respectively. The effect concentration
for the amphipods was 3.4 yg/1. and 43 yg/1. for fathead minnows.
The.se authors also cited work by Biesinger that chloramine levels of
approximately 1 yg/1. were lethal to Daphnia magna in 3-5 days. The long-
term results from both of these studies are substantially in agreement.
From field surveys Tsai (1973) suggested that 50% and 25% reductions in
fish diversity could occur at total chlorine concentrations of 0.1 and
0.025 mg/1., respectively. Therefore, protection of aquatic life requires
the reduction of chlorine residuals to concentrations near the limit of
detection.
Dechlorination of the chlorinated effluent with sulfur dioxide provided
protection to the aquatic species tested. Short-term mortality in the
45
-------
highest dechlorinated concentration (100%) was always less than 50% for
the 13 species tested. Similarly, no long-term effects were noted on the
three species tested in the highest dechlorinated concentration (20%),
except those ascribed to failure of the dechlorinator. Residual sulfite
concentrations ranged from <20 to 700 yg/1. (as S02) in this chronic test
dilution. In addition, no fathead minnows died in the 7-day tests when
chlorination of the effluent to very high levels was followed by
neutralization with sulfur dioxide.
Other studies have also shown that chlorinated effluents are more toxic
than effluents that have been dechlorinated after chlorination. Onsite
tests by Zillich (1972) at two Michigan wastewater plants showed that
thiosulfate dechlorination effectively removed the toxicity from
chlorination. As we found in our study, he also found that dechlorinator
failure resulted in acute chlorine toxicity. Esvelt e_t al. (1973) have
shown that dechlorination of San Francisco wastewater with bisulfite also
removed the toxicity from chlorination. Collins and Deaner (1973) have
advocated sulfur dioxide as an inexpensive and effective means for
neutralizing chlorinated effluent toxicity.
Acute toxicity was observed when the effluent was purposely dosed with
elevated amounts of sulfur dioxide. The 7-day TL50 values for amphipods
and fathead minnows ranged from 10 to 60 mg/1. (as sulfite). At these
lethal levels the dissolved oxygen concentrations and pH were suppressed.
A principal disadvantage of sulfite dechlorination is the need to balance
dosages carefully to prevent toxic excesses of either chlorine or sulfite,
Municipal wastewater toxicity is not limited to chlorine. Esvelt et al.
(1973) have attributed the toxicity of municipal wastewaters to MBAS
and ammonia nitrogen (NHa-N) as well as to chlorination. Using an
effluent from an oxidation pond receiving primary effluent, they found
46
-------
that all fish survived 96-hr tests when the concentration of NHs-N averaged
6.0 mg/1. and the MBAS concentration was 2.0 mg/1. Fifty per cent of the
fish died when NH3-N and MBAS concentrations reached 19.4 and 4.8 mg/1.,
respectively.
Ammonia nitrogen concentrations in our effluent from the storage tanks
averaged 6.9 mg/1. Fish deaths in some of the acute nondisinfected tests
were ascribed to low dissolved oxygen levels, but concentrations of
MBAS were not measured.
Dunstan and Menzel (1971) reported that secondary-treated sewage provided
an excellent source for continuously culturing and maintaining marine
phytoplankton. Reproductive responses in the nondisinfected effluent by
both fish and invertebrates were generally similar to that of the controls.
This sewage source neither stimulated nor suppressed their survival and
fecundity.
No measurable toxicity to aquatic life was found from either short- or
long-term exposure to the ozonated effluent. Acute toxicity was only
demonstrable when special procedures were used to shorten the retention
time so that residual ozone could be maintained in the effluent
continuously. With these procedures residual ozone concentrations of
0.2-0.3 mg/1. were lethal to fathead minnows after 1-3 hours' exposure.
By comparison, 0.19-0.34 mg/1. of residual chlorine was lethal to fathead
minnows after 12 hours' exposure. Although the toxicity magnitude for
these two disinfectants appeared to be alike, residual ozone had the
advantage of rapid dissipation from the effluent.
Little has been published on the toxicity of ozone to aquatic life,
particularly in wastewaters. MacLean et al. (1973) reported effects
of ozonated seawater on oyster eggs. They found that fertilization
47
-------
occurred less readily, parthenogenesis was increased, and large numbers of
cleaving eggs had abnormal nuclei. In an early study Hubbs (1930)
concluded that the effects of ozone were cumulative and that concentrations
as low as 0.03 mg/1. were possibly lethal to minnows. In our study
lethal concentrations of ozonated effluent to fathead minnows were at
residuals approximately ten times higher than the concentrations
reported by Hubbs. Nightingale (1931) and Nebel ej: al. (1973) have
reported much higher fish survival in ozonated than in nondisinfected
effluents. Insufficient information is available at the present time for
suggesting criteria to protect aquatic life in natural water reaches
immediately adjacent to ozonation facilities.
Recommended criteria for chlorine have been proposed by Brungs (1973).
He concluded that for wastes treated continuously with chlorine the
more resistant organisms would be protected at residual chlorine
concentrations not exceeding 0.01 mg/1. and that concentrations not
exceeding 0.002 mg/1. would protect most aquatic life. Collins and
Deaner (1973) stated that dechlorination equipment should provide chlorine
concentrations in the final effluent of 0.1 mg/1. or less in accordance
with recent California requirements. Our results indicate that
continuous neutralization of the residual chlorine is necessary to
prevent harmful effects on sensitive organisms in the receiving water.
48
-------
SECTION VIII
REFERENCES
American Public Health Association. 1971. Standard methods for the
examination of water and wastewater. 13th ed. Washington, D.C. 874 p.
Andrew, R. W. and G. E. Glass. 1974. Amperometric titration methods for
total residual chlorine, ozone, and sulfite. Draft Report, National Water
Quality Laboratory, Duluth.
Arthur, J. W. and J. G. Eaton. 1971. Chloramine toxicity to the amphipod
Gammarus pseudolimnaeus and the fathead minnow (Pimephales promelas).
J. Fish. Res. Bd. Canada 28:1841-1845.
Becker, C. D. and T. 0. Thatcher. 1973. Toxicity of power plant chemicals
to aquatic life. For U.S. Atomic Energy Commission by Battelle, Pacific
Northwest Laboratories, Richland, WA, WASH-1249, UC-11.
Benoit, R. F. and N. A. Matlin. 1966. Control of Saprolegnia on eggs of
rainbow trout (Salmo gairdneri) with ozone. Trans. Amer. Fish. Soc.
95:430-432.
Biesinger, K. E. and G. M. Christensen. 1972. Effects of various metals
on survival, growth, reproduction, and metabolism of Daphnia magna. J.
Fish. Res. Bd. Canada 29:1691-1700.
Brungs, W. A. 1973. Effects of residual chlorine on aquatic life. J.
Water Pollut. Control Fed. 45:2180-2193.
Brungs, W. A. and D. I. Mount. 1970. A water delivery system for small
fish-holding tanks. Trans. Amer. Fish. Soc. 99:799-802.
Collins, H. F. and D. G. Deaner. 1973. Sewage chlorination versus
toxicity - A dilemma? J. Environ. Eng. Div., ASCE 99:761-772.
Dixon, W. J. (editor). 1971. Biomedical computer programs. Univ.
California Publications in Automatic Computation, Univ. California
Press, Berkeley. 600 p.
49
-------
Dunnett, C. W. 1955. A multiple comparison procedure for comparing
several treatments with a control. J. Amer. Stat. Assoc. 50:1096-1121.
Dunstan, W. M. and D. W. Menzel. 1971. Continuous cultures of natural
populations of phytoplankton in dilute, treated sewage effluent. Limnol.
Oceanogr. 16:623-632.
Esvelt, L. A., W. J. Kaufman, and R. E. Selleck. 1973. Toxicity
assessment of treated municipal wastewaters. J. Water Pollut. Control
Fed. 45:1558-1572.
Hubbs, C. L. 1930. The high toxicity of nascent oxygen. Physiol. Zool.
3:441-460.
Institute of Maritime Fisheries. 1972. Use of ozone in sea water for
cleansing shellfish. Effluent and Water Treat. J. 12:260-262.
Kelly, C. B., W. Arcisz, and M. W. Presnell. 1960. Bacterial
accumulation by the oyster Crassostrea virginica, on the gulf coast. U.S.
Dep. Health, Education, and Welfare, Public Health Service, Robert A. Taft
Sanitary Engineering Center Tech. Rep. F60-4. 26 p.
MacLean, S. A., A. C. Longwell, and W. J. Blogoslawski. 1973. Effects of
ozone-treated seawater on the spawned, fertilized, meiotic, and cleaving
eggs of the commercial American oyster. Mutation Res. 21:283-285.
Michigan Department of Natural Resources. 1971. Chlorinated municipal
waste toxicities to rainbow trout and fathead minnows. U. S. Environmental
Protection Agency, Washington, D.C. Water Pollut. Control Res. Ser.,
EPA-18050GZZ10/71, October 1971. 49 p.
Mount, D. I. and W. A. Brungs. 1967. A simplified dosing apparatus for
fish toxicology studies. Water Res. 1:21-29.
National Water Quality Laboratory. 1971. Recommended bioassay procedure
for fathead minnow Pimephales promelas Rafinesque chronic tests. In C. I.
Weber (editor), Biological field and laboratory methods for measuring the
quality of surface waters and effluents. U.S. Environmental Protection
Agency, Cincinnati. Env. Monitor. Ser., EPA-670/4-73-001, July, 1973.
Nebel, C., R. D. Gottschling, R. L. Hutchinson, R. L. McBride, D. M.
Taylor, J. L. Pavoni, M. E. Tittlebaum, H. E. Spencer, and M. Fleischman.
1973. Ozone disinfection of industrial - municipal secondary effluents.
J. Water Pollut. Control Fed. 12:2493-2507.
50
-------
Nightingale, H. W. 1931. Ozone treatment of waste sulphite liquor with
reference to the effects upon cutthroat trout, chinook salmon, and oysters.
Pacific Pulp Paper Ind. 5:23-28.
Perkin-Elmer Corporation. 1971. Analytical methods for atomic absorption
spectrophotometry. Norwalk, CT.
Seligson, D. and H. Seligson. 1951. A microdiffusion method for the
determination of nitrogren liberated as ammonia. J. Lab. Clin. Med.
38:324-330.
Shuval, A. I., J. Cohen, and R. Kalodney. 1973. Regrowth of coliforms
and fecal coliforms in chlorinated wastewater effluent. Water Res. 7:
537-546.
Sliter, J. T. 1974. Ozone: An alternative to chlorine? J. Water
Pollut. Control Fed. 46:4-6.
Tsai, C. 1973. Water quality and fish life below sewage outfalls.
Trans. Amer. Fish. Soc. 102:281-292.
U.S. Environmental Protection Agency. 1973. Secondarjr treatment
information, notice of proposed rulemaking. Federal Register
38:10642-10643.
Zillich, J. A. 1972. Toxicity of combined chlorine residuals to
freshwater fish. J. Water Pollut. Control Fed. 44:212-220.
51
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SECTION IX
APPENDICES
Page
1. Chemical Characteristics of Test-Chamber Waters 54
During the Fathead Minnow Chronic Test
2. Chemical Characteristics of Test-Chamber Waters 55
During the Amphipod Chronic Test
3. Total Residual Chlorine Concentrations in Test 56
Chambers During Fathead Minnow, Amphipod, and
Daphnid Chronic Exposures
4. Total Sulfite Concentrations in Test Chambers 57
During Fathead Minnow and Amphipod Chronic
Exposures
5. Number of Surviving Fathead Minnows after 10, 25, and 58
43 Weeks' Continuous Exposure to Nondisinfected and
Disinfected Sewage Effluent
6. Survival and Growth of Second Generation Fathead 59
Minnow Larvae after 30 Days' Continuous Exposure
to Nondisinfected and Disinfected Sewage Effluent
52
-------
7. Number of Surviving Amphipods after 4, 10, 16, and 60
20 Weeks' Continuous Exposure to Nondisinfected and
Disinfected Sewage Effluent
8. Survival and Growth of Second Generation Amphipods 61
after 30 Days' Continuous Exposure to Nondisinfected
and Disinfected Sewage Effluent
53
-------
Appendix Table 1. CHEMICAL CHARACTERISTICS OF TEST-CHAMBER
WATERS DURING THE FATHEAD MINNOW CHRONIC TEST
Nominal
percentage
aevage
concentration
20
10
5
2.5
1.2
0
(Control)
20
10
5
2.5
1.2
0
(Control)
20
10
5
2.5
1.2
0
(Control)
20
10
5
2.5
x.2
0
(Control)
20
10
5
2.5
1.2
0
(Control)
Nondlainf acted
Mean
7.32
7.45
7.56
7.61
7.64
7.66
5.1
6.2
6.4
6.8
7.0
6.8
60.8'
54.7
51.4
50.0
51.3
49.3
53.4
50.0
47.3
45.5
44.9
46.1
H
12
12
12
12
12
12
74
58
68
62
60
62
11
11
12
11
' 11
11
12
12
12
11
11
11
Range
7.15-7.46
7.20-7.63
7.31-7.70
7.30-7.86
7.35-7.88
7.46-7.92
2.5-7.4
3.9-7.6
4.7-7.8
4.8-7.7
5.8-8.0
4.6-8.0
54.0-70.0
47.0-64.0
48.0-56.0
49.0-51.0
47.0-60.0
47.0-50.0
50.0-63.0
46. 0-54. C
44.0-55.0
43.0-52.0
41.0-50.0
41.0-50.0
Dechlorlnated
Mean
7.18
7.34
7.47
7.57
7.66
7.69
N
12
12
12
13
12
12
Range
pH*
7.00-7.30
7.05-7.55
7.15-7.60
7.15-7.80
7.49-7.85
7.50-7.86
Chlorinated*
Mean
7.40
7.51
7.59
7.61
7.67
7.68
Dissolved oxygen (ng/1.)
5.3
6.0
6.6
7.1
7.1
7.2
69
65
59
55
59
64
3.0-7.4
2.9-7.9
4.3-7.9
6.4-7.9
6.0-8.2
5.9-8.2
6.6
6.9
7.0
7.2
7.2
7.2
N
12
12
12
13
12
12
68
64
66
61
64
62
Total hardness (as CaCOa* mg/1.)
59.8
55.7
51.8
49.8
50.6
48.3
11
11
12
12
11
11
53.0-66.0
52.0-70.0
47.0-58.0
45.0-54.0
47.0-55.0
46.0-51.0
59.9
53.4
52.1
50.3
51.2
49.0
11
11
12
12
11
11
Total alkalinity (as CaCO». ng/1.)
50.9
47.9
46.8
45.2
45.5
45.7
13
12
12
11
11
11
44.0-61.0
45.0-53.0
43.0-51.0
43.0-50.0
42.0-51.0
42.0-50.0
52.6
49.0
46.3
45.6
44.7
46.0
12
12
12
12
11
11
Range
7.16-7.60
7.29-7.74
7.35-7.78
7.19-7.83
7.50-7.85
7.46-7.84
4.9-8.1
4.8-8.0
6.1-8.0
6.6-8.2
6.4-8.1
5.3-8.0
53.0-66.0
48.0-60.0
48.0-56.0
48.5-52.0
47.0-55.0
45.0-53.0
47.0-62.0
46.0-59.0
42.0-53.0
43.0-50.0
40.0-50.0
42.0-50.0
Ozonated
Mean
7.16
7.26
7.38
7.41
7.43
7.46
5.5
6.5
6.9
7.0
7.0
6.9
60.1
53.5
52.8
50.0
51.6
49.8
52.8
48.6
46.4
45.4
45.4
46.5
N
12
12
12
13'
12
12
67
63
60
61
61
64
11
11
12
12
10 '
11
12
12
12
12
11
11
Range
6.83-7.50
6.95-7.50
7.06-7.62
7.04-7.68
7.05-7.64
7.15-7.75
2.9-7.5
4.1-8.1
4.9-8.6
5.3-8.6
5.7-8.5
5.7-8.5
53.0-72.0
49.0-57.0
48.0-57.0
47.0-5J.O
47.0-60.0
46.0-57.0
44.0-63.0
45.0-58.0
44.0-51.0
41.0-53.0
42.0-50.0
44.0-53.0
Acidity (nut/1.)
5.6
4.2
3.9
3.1
2.9
3.1
12
12
12
11
11
11
3.9-11.8
2.8-5.9
2.5-9.9
1.9-5.0
2.3-5.0
2.0-6.1
7.2
4.9
4.1
3.1
2.8
2.9
12
12
12
12
11
11
5.0-14.0
3.5-7.4
2.8-9.9
2.3-5.9
2.3-4.0
2.0-5.6
5.3
4.0
3.5
3.0
2.9
2.9
12
12
12
12
11
10
3.1-9.9
2.8-5.0
2.5-8.8
1.9-5.0
2.3-4.0
2.0-5.0
6.2
4.8
4.0
3.7
3.5
3.3
12
12
12
12
11
11
4.2-10.9
3J-6.9
2.5-9.9
2.3-6.4
2.5-6.4
2.0-5.9
"Nominal percentage swage concentrations were 10, 5,2.5, 1.2, 0.6, and 0, respectively.
bKsdlan values.
54
-------
Appendix Table 2. CHEMICAL CHARACTERISTICS OF TEST-CHAMBER
WATERS DURING THE AMPHIPOD CHRONIC TEST
Nominal
percentage
•wage
concentration
20
10
5
2.5
1.2
0
(Control)
20
10
5
2.5
1.2
0
(Control)
20
10
5
2.5
1.2
0
(Control)
20
10
5
2.5
1.2
0
20
10
5
2.5
1.2
0
(Control)
Nondis infected
Hean
7.30
7.47
7.53
7.63
7.70
7.69
6.8
8.3
8.7
8.7
8.9
9.0
62.3
59.0
52.2
50.5
51.8
50.7
57.8
49.7
46.4
45.1
44.8
44.9
IN
5
6
4
7
5
7
40
41
34
39
37
41
4
5
5
7
5
7
5
6
5
7
5
7
Range
7.11-7.45
7.35-7.72
7.44-7.80
7.45-7.78
7.44-7.82
7.53-7.82
4.8-9.1
7.3-9.8
7.8-9.9
7.4-9.9
8.0-10.2
8.3-10.2
59.0-67.0
54.0-68.0
50.0-54.0
45.0-54.0
50.0-55.0
45.0-60.0
1
53.0-70.0
45.0-55.0
45.0-50.0
43.0-48.0
42.0-47.0
42.0-49.0
Dechlorinated
Mean
7.19
7.39
7.67
7.62
7.69
7.70
N
4
6
4
7
5
7
Kange
7.09-7.38
7.34-7.63
7.62-7.79
7.52-7.83
7.53-7.85
7.50-7.83
Chlorinated
Hean
7.43
7.52
7.44
7.60
7.70
7.67
Dissolved oxvgen (mK/1.)
7.4
8.6
8.9
9.1
9.2
9.3
41
37
36
37
39
40
6.0-8.9
6.9-9.8
7.1-10.2
8.3-J.0.2
8.5-10.6
8.5-10.4
8.6
8.8
9.0
9.1
9.0
9.2
N
4
6
4
7
5
7
43
38
40
36
36
42
Total hardness (as CaCO^. mx/1.)
63.3
59.0
53.2
50.1
51.2
52.1
otal a]
50.8
47.2
47.0
43.4
45.6
44.1
4
5
5
7
5
7
Lfcallx
5
6
5
7
5
7
58.0-67.0
51.0-72.0
49.0-55.0
49.0-51.0
49.0-55.0
47.0-65.0
lity (as CaC
43.0-60.0
44.0-50.0
44.0-53.0
43.0-50.0
43.0-47.0
42.0-47.0
57.0
54.8
52.4
50.4
51.8
53.0
50.0
47.1
45.8
45.4
46.8
44.8
4
5
5
7
5
7
1 )
5
6
5
7
5
7
Range
7.32-7.58
7.45-7.63
7.63-7.94
7.29-7.74
7.54-7.85
7.52-7.83
7.7-9.7
8.3-9.8
8.5-9.9
8.4-10.0
8.4-10.0
8.5-10.0
55.0-60.0
50.0-58.0
48.0-56.0
48.0-55.0
49.0-54.0
46.0-68.0
48.0-55.0
45.0-50.0
44.0-48.0
42.0-52.0
45.0-49.0
41.0-47.0
Ozonated
. Jfean.
7.31
7.36
7.57
7.60
7.58
7.50
7.8
8.6
8.7
8.8
8.9
9.0
63.5
60.2
53.2
52.1
53.2
53.7
57.1
50.3
48.4
45.8
47.6
44.4
•I'
T
4
6
4
7
5
7
42
42
36
31
33
41
4
5
5
7
5
7
5
6
5
7
5
7
Ranee
7.24-7.46
7.22-7.54
7.50-7.68
7.40-7.82
7.48-7.67
6.98-7.86
6.2-9.6
7.6-9.7
7.9-9.8
8.2-9.4
8.2-9.8
8.1-9.9
57.0-70.0
52.0-80.0
53.0-55.0
49.0-55.0
so.o-SB.n
49.0-65.0
50.0-67.0
45.0-60.0
43.0-55.0
43.5-50.0
43.0-51.0
41.0-47.0
Acidity Com/1.)
6.1
3.8
2.8
2.7
2.5
2.9
5
6
5
7
5
7
5.2-7.3
3.0-4.0
2.5-3.3
2.0-3.3
2.3-3.0
2.0-5.1
6.7
4.5
2.9
2.8
2.8
2.6
5
6
5
7
5
7
5.0-8.5
4.0-5.4
2.3-4.2
2.0-4.2
2.3-3.9
2.1-4.0
4.2
3.2
2.8
i.4
2.6
2.7
5
6
5
7
5
7
3.3-5.0
2.8-4.0
2.5-2.8
1.5-2.8
2.5-3.5
2.3-3.9
5.6
4.4
3.J.
2.7
2.7
3.5
6
6
5
7
5
7
4.4-7.5
3'. 0-6.1
3.0-3.3
1.7-3.6
2.3-3.3
2.3-7.0
*Hoaln*l percentage sewage concentrations were 10, 5, 2.5, 1.2, 0.6, and 0, respectively.
*Wian values.
55
-------
Appendix Table 3. TOTAL RESIDUAL CHLORINE CONCENTRATIONS IN TEST CHAMBERS
DURING FATHEAD MINNOW, AMPHIPOD, AND DAPHNID CHRONIC EXPOSURES
(mlcrograms/liter)
Ul
Hoalnal
Mremtan
'•mie
conoiatrMlon
A
10
A
5
B
A
2.5
B
A
1.2
B
A
0.6
B
A
0
(Control) B
A
10
B
A
5
B
A
2.5
B
A
1.2
B
A
0.6
B
A
0
(Control) B
raehaad m
Hun
98 + 52*
123 + 58
45 + 24
40 + 21
17 + 12
11 + 9
8+10
5 + 6
4+8
2 + 5
OB
OB
117 + 38
129 + 41
SO + 18
58+15
20+11
18+11
12+11
12+9
8+9
7 + 9
OB
„
Adul
170
116
109
80
105
74
100
76
96
71
52
47
46
41
42
38
36
33
34
33
unn
t tank!
oab - 248
OB -.340
OB- 160
OB- 90
on- 57
OB- 42
OB- 35
OB- 42
OB- 39
OB- 35
40 - 262
10 - 203
14 - 106
27 - 89
OB- 53
OB- 40
OB - 50
OB- 40
OB- 35
OB - 40
Hun of
pail*
110
42
14
6
3
Lpnov tut
Hun
183+ 85
212 + 83
96 + 47
79 + 42
18+19
23 + 23
6±5
6+16
1 + 2
1+ 2
na
OB
ABphlpod tut
123
54
19
12
B
123 + 19
147 + 9
59 + 17
71 + 0
18+9
15 + 4
3+4
4 + 2
2 + 3
1 + 2
OB
na
N
Proi.
38
40
37
38
39
38
35
36
34
30
6
5
6
5
8
6
7
9
10
14
Ranu
nn - 420
32 - 420
na - 160
OB - 170
OB- 96
oa - 99
OB - 17
na- 99
na- 7
ua - 6
106 - 141
142 - 163
35 - 81
71
7-35
11 - 21
oa - 11
OB - 7
OB- 7
oa- 6
MUQ Of
pair*
198
88
21
6
1
135
65
17
4
2
0
Kuo
92 + 29
136 + 21
45 + 19
55 + 10
15+11
14 + 5
5 + 3
3 + 3
2+2
3+4
OB
OB
133 + 99
140 + 35
38+17
37 + 20
3 + 4
11 + 11
2+2
2 + 3
OB+ 1
BB+1
oa
N
7
6
5
5
5
5
6
6
5
5
5
6
5
6
a
11
8
6
5
6
hold tut
ROOM
Tut 1
60 - 127
106 - 159
29 - 71
42 - 69
na- 25
7-21
na- 8
OB- 8
OB - 4
Tut 2
57 - 303
81 - 177
19-64
11 - 64
- 12
na - 39
OB- 5
OB- 7
OB- 2
HMD of
oclr*
114
50
14
4
2
136
38
7
2
.,
"Hum + 1 BtnuUvd deviation.
*lot ..*Mur«bl«, !•*• than 1 ug/1.
-------
Appendix Table 4. TOTAL SDLFITE CONCENTRATIONS IN TEST CHAMBERS DURING FATHEAD MINNOW
AND AMPHIPOD CHRONIC EXPOSURES
(mlcrograms/liter)
Nominal
percentage
sewage
concentration
20
10
5
2.5
1.2
0
(Control)
Fathead minnow test
Mean
104 + 165a
95 + 146
35 + 61
tun
run
run
N
53
48
39
32
32
33
Range
nmb - 700
nm - 600
nm - 300
nm - 200
nm - 60
nm
Amphlpod test
Mean
172 + 163
123 + 51
40 + 36
nm
nm
nm
N
10
6
6
4
4
4
Range
nm - 400
60 - 200
nm - 80
nm - 20
nm
nm
aMean + 1 standard deviation .
Not measurable, less than 20 yg/1.
-------
Appendix Table 5. NUMBER OF SURVIVING FATHEAD MINNOWS
AFTER 10, 25, AND 43 WEEKS' CONTINUOUS EXPOSURE TO
NONDISINFECTED AND DISINFECTED SEWAGE EFFLUENT
Nominal
percentage
sewage
concentration
A
20
B
A
10
B
A
5
B
A
2.5
B
A
1.2
B
A
0
(Control) B
10a
weeks
Nondl
35
29
26
27
28
31
27
23
23
28
27
29
256
weeks
.sinfecte
15
12
11
15
15
11
14
11
11
14
11
11
43
weeks
.d
11
10
5
9
10
10
9
9
8
13
8
10
Nominal
percentage
sewage
concentration
A
20
B
A
10
B
A
5
B
A
2.5
B
A
1.2
B
A
0
(Control) B
10a
weeks
Dec
24
23
24
25
20
25
23
26
27
28
26
25
25b
weeks
-hlorinat
13
12
15
13
13
15
13
13
14
13
11
13
43
weeks
ed
9
12
14
10
11
12
10
10
10
5
9
11
Chlorinated0 Ozonated
A
10
B
A
5
B
A
2.5
B
A
1.2
B
A
0.6
B
A
0
CControl) B
8
1
5
4
20
19
15
12
13
20
18
24
2
1
5
4
13
15
14
10
13
14
11
15
2
1
5
5
10
9
13
9
S
11
7
13
A
20
B
A
10
B
A
5
B
A
2.5
B
A
1.2
B
A
Q
(Control) B
36
30
30
23
23
32
30
23
26
20
31
31
15
15
12
15
15
13
14
14
1*
15
13
12
10
10
9
10
14
9
13
13
9
12
10
5
"Each test chamber (N - 35) thinned to 15 fathead minnows after 60 days.
bEzcess males removed during the spawning period.
'Respective total residual chlorine levels ftn yg/1.) were 110, 42, 14, 6, 3,
and nonmeasurable.
58
-------
Appendix Table 6. SURVIVAL AND GROWTH OF SECOND GENERATION FATHEAD
MINNOW LARVAE AFTER 30 DAYS' CONTINUOUS EXPOSURE TO NONDISINFECTED
AND DISINFECTED SEWAGE EFFLUENT
percentage
aevage
concentration
A
20
B
A
10
B
A
5
B
A
2.5
'
A
1.2
B
A
0
(Control) B
Teat triala
i 1 2
Hondlaln
87.5
7.5
95.0
50.0
80.0
90.0
70.0
52.5
87.5
72.5
82.5
42.5
100.0
—
—
97.5
85.0
90.0
—
72.5
40.0
40.0
75.0
a
~
— •
—
—
45.0
—
—
12.5
—
Mean
percentage
Eect«a
65.0
72.5
88.1
64.4
68.1
50.5
Final
•can
velght (g)
0.05
0.07
0.03
0.04
0.05
0.05
Chlorinated11
A
10
R
A
5
B
A
25
B
A
1.2
B
A
0.6
B
A
0
(Control) B
—
5.0
0.0
47.5
15.0
92.5
42.5
90.0
75.0
85.0
42.5
—
0.0
—
27.5
2.5
~~
80.0
97.5
57.5
22.5
55.0
O."c
0.0
—
27.5
"~
92.5
—
55.0
—
0.0
1.2
24.0
76.9
80.0
52.0
0.02
0.05
0.05
0.05
0.04
Hoednal
percentage
aevage
concentration
A
20
B
A
10
B
A
5
B
A
2.5
B
A
1.2
B
A
0
(Control ) B
Final percentage surviving
IV (L triala
i
87.5
82. 5
45.0
55.0
25.0
60.0
45.0
47.5
90.0
84.0
52.5
45.0
Deehlorii
85.0
—
97.5
60.0
90.0
95.0
80.0
95.0
—
37.5
97.5
—
—
—
75.0
—
42.5
Mean
percentage
surviving
lated
85.0
64.4
58.3
68.5
88.3
55.0
Final
•ean
velght (g)
0.05
0.09
0.07
0.06
0.04
0.06
Ozonatcri
i
20
"'
A
10
B
' A
5
B
A
2.5
B
A
1.2
'
A
0
(Control) B
95.0
87.5
77.5
77.5
92.5
70.0
67.5
45.0
35.0
62.5
60.0
45.0
95.0
°"*.^
65.0
95.0
47.5
10.0
80.0
—
100.0
95.0
80.0
55.0
90.0
—
-
—
—
—
10.0
45.0
90.0
78.8
55.0
64.2
73.1
49.2
0.05
0.06
0.05
0.06
0.04
0.04
*Teac trial vu not conducted.
DHean total residual chlorina laval* for the six tot coacaatritioDi (In ng/1.) mr« 198. 88.
^nrvM war* taluo fron control tank*.
21, b. 1. and i
irable, respectively.
59
-------
Appendix Table 7. NUMBER OF SURVIVING AMPHIPODS AFTER 4, 10, 16, and 20 WEEKS'
CONTINUODS EXPOSURE TO NONDISINFECTED AND DISINFECTED SEWAGE EFFLUENT
Nominal
percentage
sewage
concentration
A
20
B
A
10
B
A
5
B
A
2.5
B
A
1.2
B
A
0
(Control) B
4
weeks
25
20
22
29
20
23
19
30
15
23
24
27
10a
weeks
londlsln
22
15
20
29
21
19
19"
28
19
18
21
32
16
weeks
Eected
12
15
14
15
13
14
14
10
13
10
11
11
20
weeks
10
15
10
15
12
15
12
10
14
11
12
11
Chlorinated
A
10
B
A
5
B
A.
2.5
B
A
1.2
B
A
0.6
B
A
0
(Control) B
21
2-i
29
25
21
27
22
25
28
19
24
25
9
10
20
19
23
25
19
25
20
13
22
21
0
1
6
6
11
10
10
14
12
15
10
15
0
0
5
5
11
10
9
16
11
14
12
13
Nominal
percentage
sewage
concentration
A
20
B
A
10
B
A
5
B
A
2.5
B
A
1.2
B
A
0
(Control) B
4
weeks
10
14
17
19
28
23
27
16
27
24
22
28
10a
weeks
Dechlor
11
14
21
21
21 "
20
29
14
26
23
20
25
16
weeks
tnated
7
1
12
12
12
11
14
15.
13
14
13
13
20
weeks
7
0
12
11
11
9
15
17
12
14
14
13
Ozonated
I.
7.0
B
A
10
B
A
5
B
A
2.5
B
A
1.2
B
A
0
(Control) B
18
16
30
21
23
16
31
25
21
17
16
22
18
18
28
19
17
16
21
24
20
13
22
21
13
14
10
9
13
10
14
12
15
£
13
14
12
13
8
11
11
12
13
12
14
10
12
13
"Each test chamber (N - 35) thinned to 15 amphlpods after 60 days.
""Respective total residual chlorine levels (In ug/1.) were 123, 54, 19, 12, 8, and nonmeasorable.
60
-------
Appendix Table 8. SURVIVAL AND GROWTH OF SECOND GENERATION AHPHIPODS AFTER 30 DAYS' CONTINUOUS EXPOSURE TO
NONDISINFECTED AND DISINFECTED SEWAGE EFFLUENT
Nominal
percentage
sewage
concentration
f
A
20
B
A
10
B
A
5
B
A
2.5
B
A
1.2
B
A
0
(Control) B
Nondis Infected
Percentage
survival
90
100
88
98
58
100
53
94
93
71
70
c
Average
final head
length
(mm)
0.6
0.6
0.6
0.6
0.5
0.6
0.5
0.6
0.7
0.6
0.6
—
Dechlorlnated
Percentage
survival
88
66
89
41
97
98
91
90
90
91
89
100
Average
final head
length
(mm)
0.7
0.6
0.7
0.6
0.7
0.7
0.7
0.7
0.7
0.7
0.6
0.5
Ozonated
Percentage
survival
65
85
95
98
86
75
86
75
67
93
37
88
Average
final head
length
(mm)
0.8
0.8
0.8
0.8
0.8
0.5
0.7
0.7
0.6
0.7
0.6
0.6
Nominal
percentage
sewage
concentration
A
10
B
A
5
B
A
2.5
B
A
1.2
B
A
0.6
B
A
0
(Control) B
Chlorinated*
Percentage
survival
88b
K
82b
96b
b
95°
50
85
90
92
86
92
95
79
Average
final head
length
(mm)
0.7
0.6
0.8
0.7
0.7
0.7
0.6
0.7
0.6
0.6
0.6
0.6
total residual chlorine levels for the six concentrations (In yg/1.) were 135, 65, 17, 4, 2, and nonmeaeurable, respectively.
Teat animals taken from control tanks.
Teat not conducted.
-------
TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
. REPORT NO.
EPA-600/3-75-012
3. RECIPIENT'S ACCESSION>NO.
4. TITLE AND SUBTITLE
COMPARATIVE TOXICITY OF SEWAGE-EFFLUENT
DISINFECTION TO FRESHWATER AQUATIC LIFE
5. REPORT DATE
November 1975 (Issuing Date)
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
John W. Arthur, Robert W. Andrew, Vincent R. Mattson,
Donald T. Olson, Gary E. Glass, Barbara J. Halligan,
and Charles T. Walbridee
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Environmental Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Duluth, Minnesota 55804
10. PROGRAM ELEMENT NO.
1BA021; ROAP/Task 06AOJ/005
11. CONTRACT/GRANT NO.
None (in-house)
12. SPONSORING AGENCY NAME AND ADDRESS
Same as above
13. TYPE OF REPORT AND PERIOD COVERED
Final
14. SPONSORING AGENCY CODE
15. SUPPLEMENTARY NOTES
16. ABSTRACT Flow-through laboratory bioassays were conducted with a domestic secondary
sewage effluent that had been disinfected by chlorination, by chlorination followed
by dechlorination, and by ozonation. Effluent without disinfection served as a con-
trol. Disinfection with chlorine and ozone generally maintained the effluent at
total coliform levels of less than 1,000 per 100 ml. Lake Superior water served as
the diluent source for the experiments. Short-term (7-day) exposures were conducted
with 13 species (seven fish and six invertebrates), and long-term (generation) tests
were performed with three species (one fish and two invertebrates). In both series
of tests the chlorinated effluent was lethal at appreciably lower concentrations
than any of the other three effluent treatments. Fish were more sensitive than the
invertebrates to the chlorinated effluent in 7-day tests. The respective 7-day TL50
values of total residual chlorine to fish and invertebrates ranged from 0.08.to 0.26
and 0.21 to >0.81 mg/1. Residual ozone rapidly decreased in the treated effluent
and was not measurable in the test tanks. When special short-term test procedures
and shorter retention times for the ozonated effluent were used, measured residual
ozone was about as lethal to fathead minnows as residual chlorine. The highest mean
total residual chlorine concentrations having no long-term adverse effect on fathead
minnows, amphipods, and Daphnia were 14, 12, and 2-4 yg/1., respectively. No daphnids
survived at approximately 10 yg/1. mean total residual chlorine, a concentration that
corresponds to a chlorinated sewage concentration of about 2.5%.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.lDENTIFIERS/OPEN ENDED TERMS
c. COSATI Field/Group
Disinfection
Chlorination
Bioassay
Aquatic animals
Ozone
Minnows
Daphnia
Fresh water fishes
Dechlorination
Chlorine toxicity
Ozone toxicity
Aquatic life
Sewage effluent
Amphipoda
6A
6C
7B
7C
18. DISTRIBUTION STATEMENT
RELEASE UNLIMITED
19. SECURITY CLASS (ThisReport)
UNCLASSIFIED
21. NO. OF PAGES
71
20. SECURITY CLASS (Thispage)
UNCLASSIFIED
22. PRICE
EPA Form 2220-1 (9-73)
62
11.5. GOVERNMENT PRINTING OFFICE: 1975-657-695/5339 Region No. 5-11
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