600378023
March 1978
Ecological Research Series
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development, U.S. Environmental
Protection Agency, have been grouped into nine series. These nine broad cate-
gories were established to facilitate further development and application of en-
vironmental technology. Elimination of traditional grouping was consciously
planned to foster technology transfer and a maximum interface in related fields.
The nine series are:
1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
4. Environmental Monitoring
5. Socioeconomic Environmental Studies
6. Scientific and Technical Assessment Reports (STAR)
7. Interagency Energy-Environment Research and Development
8. "Special" Reports
9. Miscellaneous Reports
This report has been assigned to the ECOLOGICAL RESEARCH series. This series
describes research on the effects of pollution on humans, plant and animal spe-
cies, and materials. Problems are assessed for their long- and short-term influ-
ences. Investigations include formation, transport, and pathway studies to deter-
mine 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, terrestrial, and atmospheric environments.
This document is available to the public through the National Technical Informa-
tion Service, Springfield, Virginia 22161.
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EPA-600/3-78-023
March 1978
TOXICITY OF RESIDUAL CHLORINE COMPOUNDS TO AQUATIC ORGANISMS
by
Gary L. Larson, Charles E. Warren
Floyd E. Hutchins, Larry P. Lamperti
David A. Schlesinger, Wayne K. Seim
Department of Fisheries and Wildlife
Oak Creek Laboratory of Biology
Oregon State University
Corvallis, Oregon 97331
Grant Number
R802286
Project Officer
William A. Brungs
U.S. Environmental Protection Agency
Environmental Research Laboratory - Duluth
Duluth, Minnesota 55804
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DISCLAIMER
This report has been reviewed by the Environmental Research Laboratory-
Duluth, U. S. Environmental Protection Agency, and approved for publication.
Approval does not signify that the contents necessarily reflect the views
and policies of the U. S. Environmental Protection Agency, nor does mention
of trade names or commercial products constitute endorsement or recommendation
for use.
11
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FOREWORD
The advantages and disadvantages of chlorination for disinfection and
slime control have been the subject of intensive debate over the past several
years. Included in that debate are such considerations as the need for
disinfection in all cases, and the possibility of mitigating the adverse
effects of chlorination (while retaining the beneficial effects) by variations
in the dose regime.
This report presents results and interpretations of research on the
effects of chlorine and inorganic chloramines on several species of fish, a
crayfish, and laboratory stream communities. Among other things, it was
found that the relationship between the concentration-time integral of
exposure under a particular exposure regime and the toxic effects of that
exposure could be generalized to predict effects on fish under different
exposure regimes. These results should aid in establishing a basis for the
possible controlled use of chlorine to achieve its purpose with minimal damage
to aquatic life.
J. David Yount, Ph.D.
Acting Director
Environmental Research Laboratory
Duluth, Minnesota
iii
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ABSTRACT
Laboratory studies on the acute and chronic toxicity of chlorine and
inorganic chloramines to trout, salmon, minnows, bullhead, largemouth bass,
arid bluegill were conducted. Acute toxicity under continuous and inter-
mittent patterns of exposure as well as behavioral, reproduction,
development, and growth responses to low level exposures to residual
chlorine compounds were determined. But not all patterns of toxicant
exposure or all responses of all fish species were studied. Acute and
chronic toxicities of chloramines to crayfish were investigated.
Algae, invertebrates, including insects, and juvenile salmon were
exposed continuously to relatively low levels of residual chloramine
compounds in laboratory stream communities. The acute toxicities of
inorganic chloramines, as measured by 96-hour LC50 values, were less than
100 pg/1 for salmonids and were a function of life history stage, body
size, and some water quality conditions. Whereas adult trout may live
indefinitely at concentrations near 50 ug/1, the LC50 values for late
developmental stages—fry and very small juveniles—were not much above
this concentration. Effects on growth of alevins and juveniles had
threshold concentration values between about 10 and 22 ug/1, effects being
quite marked at 22 ug/1. In intermittent exposure to relatively high
concentrations of free residual chlorine, mortality was found to be a
rather consistent function of the area under the time-concentration curves
of exposure, for different forms, durations, and frequencies of such
patterns of exposure. Behavioral responses of fish, such as avoidance of
chlorinated water which could be advantageous in nature and lethargic
swimming, surfacing, and sinking to the bottom which would probably be
harmful were studied. Such behaviors occur not only at acutely toxic
concentrations but also at lower ones. It was necessary to introduce
concentrations ranging from 100 to 800 ug/1 of chloramine into laboratory
stream communities to maintain mean residual concentrations near 20 yg/1.
No marked effects on algal or insect abundances or on survival and production
of juvenile salmon were observed at this and lower concentrations in the
laboratory streams. It is doubtful that the amperometrically determined
residual concentrations of chloramines in the streams consisted predominantly
of inorganic chloramines, organic chloramines perhaps being an important
constituent under the stream conditions. Little is known of the toxicity
of organic chloramines. The amount of inorganic chloramines introduced to
maintain desired residual concentrations appears to have been a function
of the amount of organic material in the stream communities.
IV
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CONTENTS
Foreword ill
Abstract iv
Figures vi
Tables x
Introduction 1
Conclusions and Recommendations 4
Methods and Materials 7
Preparation and Introduction of Toxicants 7
Water Quality and Physical Conditions 8
Experimental Animals 8
Methods for Determining Acute Toxicity: Continuous Exposure .... 8
Methods for Determining Acute Toxicity: Intermittent Exposure ... 11
Methods for Determining Chronic Toxicity: Partial Chronic Tests . . 13
Methods for Determining Chronic Toxicity: Survival, Development
and Growth 16
Methods for Determining Effects on Behavior 21
Laboratory Stream Community Methods 25
Results and Interpretation . 27
Acute Toxicity of Chloramines When Exposure Continuous 27
Acute Toxicity of Free Chlorine When Exposure Intermittent 33
Chronic Toxicity of Chloramines: Partial Chronic Test Conditions . 41
Chronic Toxicity of Chloramines: Development, Survival, and Growth 54
Behavior Effects of Chlorine and Chloramines . 62
Continuous Exposure of Laboratory Stream Communities to Chloramines 70
Discussion 93
References 101
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FIGURES
Number Page
1 Schematic diagrams of the experimental apparatus used in the
avoidance experiment with largemouth bass. (A) top view of
the entire apparatus. (B) cross sectional view of the
middle of the test section 23
2 Acute toxicity of inorganic chloramines (TRC) to coho salmon
in 96-hour tests as affected by: (A) fish weight; (B)
temperature; (C) total alkalinity; and (D) pH. Toxicant
concentrations ( yg/1) on the abscissal axes in B, C, and D
are expressed as base 10 logarithms 29
3 Relationship between the mean dry weight (g) per fish and the
96-hour LC50 of inorganic chloramines (TRC) for juvenile
cutthroat trout 31
4 Examples of the time-concentration relationships of the square
pattern of exposure and of the high and low spike patterns
of exposure to.free residual (TRC) 35
5 Relationships between mortality of largemouth bass (in probits)
and the area under the time-concentration curve (expressed as
mg/1 x minutes) for fish subjected to square and high and low
spike exposures to free residual chlorine (TRC). Data points
with arrows pointing up indicate that all of the fish died;
those with arrows pointing down indicate that no fish died
during the test 37
6 Relationships between mortality of largemouth bass (in probits)
and the mean plateau concentration of free residual chlorine
(A and D), mean concentration for the entire exposure (B and
E), and area, expressed as mg/1 x minutes under the time-
concentration curve (C and F) for bass subjected to one or
two 90-minute square patterns of exposure, or to one 90-
minute or one 150-minute square patterns of exposure. Data
points with arrows pointing up indicate that all of the
fish died during the test 38
vi
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FIGURES
(continued)
Number Page
7 Relationships between mortality of largemouth bass (in
probits) and the mean plateau concentrations of free residual
chlorine (TRC) in 90-minute square patterns of exposure
for bass acclimated and exposed to the toxicant at either
13,1 .or 24.3 C and for bass acclimated to 13.1 C and
exposed to the toxicant at 24.3 C. Data points with
arrows pointing up indicate that all of the fish died;
data points with arrows pointing down indicate that no
fish died during the test .............. ., ..... ^9
8 Relationships between mortality of largemouth bass (in
probits) and the mean plateau concentrations of free
residual chlorine (TRC) in 90-minute square patterns of
exposure for bass weighing either 3,75 g or 5.93 g mean
dry weight per fish. Data points with arrows pointing
down indicate that no fish died; data points with arrows
pointing up indicate that all of the fish died during
the test .............. .............. 40
9 Relationships between mortality of fish (in probits) and the
mean plateau concentration of free residual chlorine
(TRC) during 90-minute square patterns of exposure of
bluegill (A), redside shiner (B) , and blackside dace
(C), Data points with arrows pointing up indicate that
all of the fish died; those with arrows pointing down
indicate that none of the fish died during the tests ...... 41
10 Relationship ^between the mean concentration of inorganic
chloramines (TRC) and the days of exposure 'until 50
percent mortality of adult crayfish kept under partial
chronic conditions during tests conducted in 1974-75 and
1975-76. The two data points with arrows pointing up
indicate that fewer than 50 percent of the crayfish had
died at that concentration by the end of the 365 day
exposure (1975-76) ....................... 53
11 Cumulative alevin mortality (sum of replicates) at 5-day
intervals at different concentrations of inorganic
chloramines (TRC) in the coho salmon embryo-alevin
experiment. Increased alevin mortality occurred at
indicated time of low dissolved oxygen ............. 56
12 Relationships between the mean dry weight of alevins and
the days from 95 percent alevin hatch at different
concentrations of inorganic chloramines (TRC) in the
coho salmon embryo-alevin experiment ............ 57
VI1
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FIGURES
(continued)
Number Page
13 Relationships between instantaneous growth rate and mean
dry weight of alevins at different concentrations of
inorganic chloramines (TRC) in the coho salmon embryo-
alevin experiment,. The days from 95 percent hatch are
indicated 58
14 Relationships between food consumption rate and relative
growth rate (A) and gross efficiency (B) for coho salmon
juveniles (from the embryo-alevin experiment) at dif-
ferent concentrations of inorganic chloramines (TRC).
Some groups of fish had been exposed to the toxicant
since the embryo life stage, others were exposed only
during the growth experiment. 61
15 Relationships between food consumption rate and relative
growth rate (A) and gross efficiency (B) for coho salmon
juveniles at different concentrations of inorganic
chloramines (TRC). 63
16 Relationships between the time to first occurrence of
bobbing behavior of largemouth bass and the mean con-
centration of free residual chlorine (TRC) in the aquaria.
-Mean concentration calculated for-the period until first
occurrence, during one and two 90-minute square patterns
of exposure. 55
-17 Relationships between the areas under the time-concentration
curves, expressed as mg/1 x minutes, and the occurrence
of five behavioral changes in two weight groups of bass
subjected to square and spike patterns of exposure
to free residual chlorine (TRC)* Open circles indicate
that the behaviors did not occur; dark circles indicate
that the behaviors occurred* Symbols: NS - near the
surface; T thrashing; L lethargic swimming; B bobbing;
and 0 -on aquarium bottom* 69
18 Relationships between the mean toxicant concentration in
the toxicant discharge area of the avoidance apparatus
and the avoidance index for largemouth bass subjected to
60-minute exposures to free residual chlorine or inorganic
chloramines, expressed as total residual chlorine (TRC). ... 71
19 The amount of exposure, expressed as mg/1 x minutes, to
which bass were subjected when exposed to either free
residual chlorine or inorganic chloramines in the avoidance
experiment. The dashed lines represent the relationship
for each toxicant. The solid line represents the
VI11
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FIGURES
(continued)
Number Page
(continued)
19 relationship for the pooled data since neither the slopes
nor the Y-intercepts of the individual toxicant lines
were different significantly (P > 0.05 ........... 72
20 Relationships between the mean daily concentrations of
chloramines (TRC) in laboratory stream renewal water and
the concentrations of the toxicant in one of the streams,
for selected periods in 1975. •,,.,..,, ..... 75
21 Relationships between the amount of illumination (in foot-
candles) at the surface of a laboratory stream and the
pH of the stream water (A) and the concentration of
residual chlorine compounds (TRC) in the water (B) from
approximately 0830 to 1830 hours on July 26, 1976 ...... 77
22 Relationships between the concentration of inorganic
chloramine (TRC) in renewal water that was necessary to
maintain a residual concentration of about 20 ug/1 and
the amounts of organic matter (A) and chlorophyll (B)
present in the stream, ................... 78
23 Densities of organic (g/m^) in the laboratory streams at
the end of each experimental period, from 1 through 6.
Each plotted value is a mean for three streams at a
given concentration (TRC) and time. .............. 80
_ 2
24 Densities of total chlorophyll (g/m ) in the laboratory streams
at the end of each experimental period, 1 through 9. Each
plotted value is a mean for three streams at a given
concentration (TRC) and time. ................. 81
25 Relationships between the production and biomass of coho
salmon in the laboratory streams^ The enclosed numbers
(1 to 9) refer to the experimental periods (see text).
Two possible levels of productivity are indicated by
curves A and B* ...................... 89
26 A graphical analysis, using the isocline method (Booty and
Warren, MS) of the relationship between the mean terminal
invertebrate biomass and fish biomass for each experimental
period. The identity of the prey isoclines (P) is organic
matter. The identity of the predator isoclines (M) is
mortality of fish. The enclosed numbers (1 to 9) refer
to the experimental periods. Each plotted value is a
mean for three streams at a given concentration and
time.
IX
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TABLES
Number Page
1 Sources of Species of Fis,h and the Crayfish used on Experiments. . 9
2 The Experiments in which each Species was used 9
3 Mean Dry Weight and Standard Deviation of Largemouth Bass Tested
in the Intermittent Exposure Experiments with Free Residual
Chlorine 12
4 Number and Weight of Fish and Water Quality Conditions in the
Intermittent Exposure Experiments with Free Residual Chlorine . 14
5 Species, Duration, and Size of Animals in Partial Chronic Tests . 15
6 Mean Concentrations (and Standard Deviations) of Inorganic
Chloramine, in Micrograms per Liter, to which Groups of Test
Organisms were Subjected during the Partial Chronic Experiments 17
7 Mean Concentrations (and Standard Deviation) of Residual Inorganic
Chloramines, in Micrograms per Liter, to which Groups of Coho
Salmon were Subjected during the Embryo-alevin Experiment ... 18
8 Mean Concentrations of Inorganic Chloramines, in Micrograms per
Liter, to which Groups of Coho Salmon were Exposed during the
Embryo-alevin and the Concentration (and Standard Deviation)
to which They were Subjected as Juveniles during the First
Growth Experiment 19
9 Mean Concentrations (and Standard Deviations) of Inorganic
Chloramines, in Micrograms per Liter, to which Groups of Coho
Salmon Juveniles were Exposed during the Second Growth
Experiment 20
10 Forms of Residual Chlorine Compounds Present in the Intermittent
Exposure Experiments and their Mean Percentage (and Standard
Deviation) 24
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TABLES
(continued)
Number Page
11 Life History Stage, Mean Dry Weight per Fish, Water Quality
Conditions, and the 96-hr LC50 of Inorganic Chloramines in each
Acute Toxicity Test with Coho Salmon. Dry Weights of Alevins
and Fry do not Include Yolk .................. 2^
12 Life History Stages, Mean Dry Weight per Fish, Water Quality
Conditions, and the 96-hr LC50 of Inorganic Chloramines in
each Acute Toxicity Test with Brook Trout and Cutthroat Trout.
Dry Weights of Alevins and Fry do not Include Yolk. ...... 30
13 Concentrations of Secondarily Treated Kraft Paper Mill Effluent
f.SKME) Between which the 96-hr LC50 Occurred in Chlorinated
SKME Acute Toxicity Tests with Coho Salmon ..... ..... 32
14 Mean Dry Weight (without Chelipeds) and Carpace Length per
Animal, with Quality Conditions, and Ranges of Concentrations
of Inorganic Chloramines Tested in Experiments in which
Insufficient Deaths Occurred to Estimate the 96-hr LC50 Value
for Crayfish .......................... ^
15 Mean Dry Weight per Fish and the Number of Deaths for Cutthroat
Trout Juveniles Subjected to Different Intermittent Square
Exposures of Free Residual Chlorine .............. 42
16 Mean Dry Weight per Fish, Temperature of Test Solutions, Toxicant
Exposures, and the Number of Deaths of Brown Bullhead Subjected
to 90-minute Square Exposures of Free Residual Chlorine .... 44
17 Concentrations of Inorganic Chloramines and the Number of Deaths
of Adult Brook Trout during the 1974-75 Partial Chronic
Experiment ...................... .... 45
18 Length and Wet Body Weight, and the Number of Eggs which were
Stripped from Female Brook Trout Exposed to Inorganic
Chloramines during the 1974-75 Partial Chronic Experiment ... 47
19 Influence of Continuous Exposure to Inorganic Chloramines on
the Percent Fertilization, Embryo Survival and Hatch, and
Alevin Survival and Growth of Brook Trout Progeny from
Adults Kept under Partial Chronic Test Conditions or from our
Brood Stock ............... . .......... 48
20 Number of Deaths of Adult Cutthroat Trout Exposed to Inorganic
Chloramines during the 1974 and 1974-75 Partial Chronic Tests . 49
xi
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TABLES
(continued)
Number Page
21 Influence of Inorganic Chloramines on the Food Consumption
(Expressed as Percent Wet Body Weight of each Group of
Fish) of Adult Cutthroat Trout for Three Days during the
1974 Partial Chronic Experiment 50
22 Spawning Performance and Deaths of Adult Crayfish Exposed to
Inorganic Chloramines during the 1974-75 and 1975-76 Partial
Chronic Experiments 51
23 Number of Molts and the Number of Deaths Associated with Molting
for Crayfish Exposed to Inorganic Chloramines during the
1975-76 Partial Chronic Test 52
24 Influences of Continuous Exposures to Inorganic Chooramines on
the Caloric Values (Calories/g) for Alevins with Yolk and
with the Yolk Removed during the Coho Salmon Embryo-alevin
Experiment 59
25 Comparison of Yolk Metabolism from January 28 to February 4 by
Control Alevins and Alevins Exposed to 47 yg/1 Inorganic
Chloramines in the Coho Salmon Embryo-alevin Experiment .... 60
26 Influence of Square and Spike Exposures of Free Residual Chlorine
on the Occurrence of the Bobbing and Resting Behavioral Respon-
ses and Death by Largemouth Bass in the Flowing Water
Intermittent Exposure Experiment 67
27 Concentrations of Free Residual Chlroine and Areas under the Time-
Concentration Curves using Square and Spike Exposures in
the Behavioral Experiment with Largemouth Bass Weighing either
7.37 or 14.65 g per Fish 68
28 Daily Mean High and Mean Low Temperatures (and Standard
Deviations) in the Laboratory Streams for each Experimental
Period in 1974 and 1976 73
29 Mean Concentrations (and Standard Deviations) of Residual
Chlorine Compounds in each Laboratory Stream during each
Experimental Period 74
30 Density of Organic Matter (g/nr) in the Laboratory Streams at
the End of Experimental Periods 1 through 6 79
2
31 Density of Invertebrates (g/m ) in the Laboratory Streams at the
End of each Experimental Period, Including Two Presamples. 82
xii
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TABLES
[continued)
Number Page
32 Mean Biomass, Growth Rate and Production of Coho Salmon
Exposed to Residual Chlorine Compounds in the Laboratory
Stream for Experimental Periods 1 through 9 84
xiii
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INTRODUCTION
Present and probable future use of chlorine for. disinfection and for
slime control presents a hazard to aquatic life that makes the establishment
and enforcement of adequate standards important. Effluent and receiving
water standards intended to provide protection for aquatic organisms ought
to ensure that concentrations of toxic materials permitted in receiving
waters are no higher than is consistent with the persistence of desired
aquatic communities and high levels of production of species man uses.
For lack of empirical and theoretical knowledge, few if any toxic substance
standards are known to provide such needed protection of aquatic resources.
Use of chlorine for disinfection and slime control and what is known of
its toxicity have led some to consider chlorine to be a general and
serious water pollution problem. And yet only recently have even modest
levels of research on this problem been forthcoming. The research here
reported is intended to extend the basis for setting standards to protect
some species of fish and invertebrates from direct and indirect effects
of chlorine and inorganic chloramines.
Chlorine in water may exist as free residual in the forms of
chlorine gas, hypochlorous acid, and hypochlorite ion, and as combined
residual—the chloro-derivatives. One important group of chloro-derivatives
includes the inorganic chloramines monochloramine (Nt^Cl), dichloramine
(NHC12), and trichloramine (NC^). One or more of these compounds are
formed when water containing ammonia is chlorinated. Monochloramine and
dichloramine are the predominant forms of inorganic chloramines found in
receiving waters. Total residual chlorine (TRC) is the sum of the concentra-
tions of free and combined residuals as measured by amperometric titration
analysis.
Residual chlorine compounds are known to be present in many receiving
waters in concentrations toxic to fish (Brungs, 1973). Through studies
conducted in natural streams receiving chlorinated effluents, the Michigan
Department of Natural Resources (Anonymous, 1971) found the 96-hour LC50
for rainbow trout (Salmo gairdneri) to be 22.8 pg/1 total residual chlorine;
concentrations high enough to cause mortality persisted for 0.8 mile down
stream from the point of discharge. Nonchlorinated effluent was not toxic
to the trout. In chlorinated sewage effluent, the 96-hour LC50 for fat-
head minnows (Pimephales promelas) was found to be between 80 and 190 yg/l
total residual chlorine (3.2 and 7 percent effluent); no mortality
occurred in 100 percent effluent that was dechlorinated (Zillich, 1972).
Arthur et al. (1971) found that growth and survival of fathead minnow
larvae were reduced at 108 pg/1 total residual chlorine in sewage effluent.
Tsai (1973) reported that a species diversity index for fish in rivers
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in Maryland, Virginia, and Pennsylvania dropped to zero below
chlorinated effluent outfalls when mean total residual chlorine concentrations
were about 250 yg/1. Sprague and Drury [1969) conducted laboratory experi-
ments in which concentrations as low as 1 ug/1 of TRC were avoided by
rainbow trout. Others have shown avoidance responses of various freshwater
and estuarine fishes at relatively low TRC concentrations under laboratory
and field conditions (Meldrim et al., 1974; Tsai and Fava, 1975; Bogardus,
et al., MS ; and Basch and Truchan, 1976). Not much is known about the
effects of intermittent exposure of fish to chlorine, but deaths and
abnormal behavior have been observed where chlorinated effluents were being
discharged intermittently (Basch and Truchan, 1976). Laboratory studies
have been conducted on the acute toxicity of chlorine to bluegill, yellow
perch, and several species of salmonids, as affected by temperature,
exposure duration, and exposure frequency (e.g. Stober and Hanson, 1974;
Brooks and Seegert, 1977, and Bass and Heath, in press). Tolerance of
fish to chlorine has been shown to decrease with increasing duration and
frequency of exposure.
There are sound reasons, we believe, for man not to alter aquatic and
other natural communities any more than absolutely necessary to ensure his
own well being. But activities of man that impinge upon aquatic systems
are likely to alter in some degree their communities. Even if we take the
perhaps too narrow a view that, at a minimum, man must protect fish and
other species of particular interest, it becomes necessary to maintain the
physical conditions and biological communities making possible the persistence,
the production, and acceptable yields of these species. For such species
of interest, receiving water standards for toxicants ought to insure not
only that reproduction, development, growth, and behavior of individuals
and the persistence and production of their populations are not directly
affected by toxicants, but also that biological communities providing
species of interest with food and other resources are maintained. Differ-
ent life history stages and biological responses of species of interest
are known to vary widely in sensitivity to toxicants, and such sensitivity
is also known to be a function of water quality conditions. All these
considerations should be borne in mind as the necessary context for
interpretation of any results obtained through research on particular
species.
The results and interpretations reported here, as well as any conclu-
sions and recommendation derived from them, are based on research on
the effects of chlorine and inorganic chloramines on several species of fish,
a crayfish, and laboratory stream communities. This research included
studies on the reproduction, development, growth, and survival of salmon
and trout exposed to chronically as well as acutely toxic concentrations
of chlorine and inorganic chloramines. The effects of water temperature,
pH, and total alkalinity on acute toxicity of inorganic chloramines were
also determined. Acute toxicity of chlorine to largemouth bass as well as
effects of chlorine and chloramines on behavior were determined, when the
fish were intermittently exposed. Both acute and chronic toxicity of
inorganic chloramines to crayfish were determined. Preliminary studies of
acute toxicity and behavioral effects of chlorine were conducted with
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other species of freshwater fish. Finally, we conducted laboratory stream
studies, in which communities of algae, insects, other invertebrates, and
juvenile salmon were exposed for relatively long periods of time to low
concentrations of residual chlorine compounds.
The above summary of the research conducted suggests that a rather
wide variety of methodological approaches was employed in determining
acute and chronic toxicity of chlorine and inorganic chloramines to aquatic
organisms. But the relatively limited support and time available for
this research prevented really adequate replication of experiments of
particular kinds, and thus many of the results should be viewed as being
of a preliminary nature. In planning and conducting this research, we and
the Environmental Protection Agency scientists advising us decided that,
in view of the breadth, complexity, and seriousness of chlorine toxicity
problems and the scarcity of pertinent information, it was most immediately
important to obtain information on a variety of responses of several
species to chlorine and inorganic chloramines under different sets of
conditions. Although we are not satisfied with the precision obtained in
most of the experiments, taken together the results of these experiments
rather clearly indicate exposures of aquatic organisms to these toxicants
that would be harmful. The conclusions and recommendations reflect this.
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CONCLUSIONS AND RECOMMENDATIONS
1. The acute toxicity of free chlorine and inorganic chloramines, as
determined by 96-hour continuous exposure to constant concentrations,
differs considerably among species, among individuals of different sizes,
and among different life history stages. Crayfish, for example, may have
96-hour LC50 values near 900 pg/1 inorganic chloramines, whereas such
values for salmon and trout are likely to be less than 100 pg/1. But
adult salmonids may live indefinitely at concentrations near 50 yg/1,
which are close to the LC50 values for fry and very small juveniles. And
adult crayfish may succumb to concentrations near 50 yg/1 when molting.
Chronic effects on growth and other responses of salmonids may occur at
concentrations near 20 Pg/1 or even lower. Thus the difference between
acutely toxic and chronically toxic concentrations of inorganic chloramines
is very great for some species and really quite small for others. When
considered along with differences in sensitivity of different life history
stages, this makes it apparent that information on acute toxicity—as
measured by 96-hour LC50 values—alone is quite inadequate for determining
levels of residual chlorine compounds that will not be deleterious to
aquatic life in receiving waters when exposure is more or less continuous.
2. Chlorination of some industrial effluents will present difficult and
perhaps unexpected problems in the toxicology of aquatic organisms. We
found that chlorination of a kraft pulp and paper effluent—which was
biologically stabilized and exhibited no toxicity in 96 hours at 100 percent
concentrations—rendered this effluent acutely toxic at concentrations
near 20 percent, even though only 1 mg/1 of inorganic chloramines was
added and no residuals were detectable in the test solutions. Certainly
chlorination of effluents should be undertaken only when there is a
demonstrable need and then only minimal effective treatments should be
employed, after possible toxic effects on aquatic organisms have been
evaluated.
3. Intermittent introduction of free chlorine, such as for one hour in
every 24 hours, has important power plant and other industrial applications
in slime organism control. There must be an infinite number of possible
concentration-time patterns and frequencies of introduction of chlorine for
such applications; some of these present serious toxic problems to fish
and other aquatic organisms in receiving waters. But so many different
patterns and frequencies of chlorine introduction are employed in slime
control, the difficulty of evaluation of toxic hazard is very great. We
have found that the area, expressed as mg/1 x minutes, under concentration-
time curves of exposure makes it possible to generalize to different
exposure regimes results obtained under particular regimes. Time,
concentration, and frequency of exposure boundary conditions of application
4
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of this method of expressing bioassay results should be further explored,
because it could be very useful in simplifying analysis, interpretation,
and application of studies on intermittent exposure to chlorine. We gain
the impression that unnecessarily high concentrations, durations, and
frequencies of chlorine treatment in slime control are often employed.
This is not only needlessly harmful to aquatic life but economically
wasteful. Properly controlled use of chlorine can, we believe, generally
achieve its purpose with little or no damage to fish populations.
4. Under some circumstances, relations between temperature and the
toxicity of free and other residual forms of chlorine are likely to be
important. Temperature is generally a factor in determining the toxicity
of substances to aquatic organisms. It is likely to be especially important
when organisms are exposed to toxicants at water temperatures higher than
the ones to which they have been acclimated. This may occur when chlorine
residuals are present in thermal discharges from power plants.
5. Especially under conditions of more or less continuous exposure of
aquatic populations to residual chlorine compounds, as occurs where
chlorinated sewage effluents are discharged, effects of quite low residual
concentrations on development, reproduction, growth, and production of
fish and other organisms are of concern. Our studies have shown that
threshold concentrations of inorganic chloramines having effects on the
growth of salmonid alevins and juveniles probably lie somewhere between 10
and 20 ug/1 of total residual chlorine as determined by amperometric
titration. Effects on growth are quite marked at concentrations near
20 v.g/1. Field studies by others indicate that salmonid populations may
be eliminated from areas where they are subjected to such levels of
exposure. Much more emphasis needs to be given to studies of reproduction,
development, and growth of fish and other organisms likely to be exposed
more or less continuously to such relatively low levels of residual
chlorine compounds. There is no way that 96-hour acute toxicity studies
can provide adequate information on which to base the protection of aquatic
organisms from such conditions.
6. Studies of the possible effects of various levels and durations of
exposure of fish to residual chlorine compounds are also important. Fish
have the capacity to avoid chlorinated waters under some conditions, and
this could enhance the possibility of their persistence. Laboratory studies
have demonstrated such capacity, but the extent to which fish are actually
able to avoid chlorinated discharges in nature cannot be determined by
extrapolation from laboratory studies. Careful field studies, perhaps
employing underwater observations, would be very worthwhile. We have
shown behavioral modification of fish by acutely toxic and lower concentra-
tions of chlorine and inorganic chloramines, under conditions of intermittent
exposure. Such behavioral modifications include lethargic swimming, sur-
facing, and sinking to the bottom. Similar observations have been made in
the field, and there is every reason to believe that these and other
behavioral modifications are deleterious to fish populations exposed
intermittently to relatively high concentrations of residual chlorine
compounds,
-------
7. We introduced continuously into laboratory stream communities—including
algae, invertebrates, and juvenile salmon—concentrations of inorganic
chloramines from about 100 to 800 yg/1. This was necessary to maintain
experimental mean residual concentrations up to about 20 yg/1, as determined
by amperometric titration. The introduced concentrations needed to maintain
desired experimental concentrations were seasonally very dependent on the
amount of organic material, especially algal materials, present in the
laboratory streams. Mean concentrations of 20 yg/1 TRC and less had little
if any apparent effect upon the algae, stream invertebrates, and juvenile
salmon, under the conditions in these streams. But we are uncertain as to
the forms of residual chlorine compounds that were present. It is unlikely
that inorganic chloramines were the main persistent form, probably more of
the TRC being present as organic chloramines, the toxicity of which we know
little. We do not wish to suggest that in nature reproduction and other
life processes of insects and other invertebrates would not be harmed by
concentrations near those we maintained. Insect reproduction, for example,
cannot be adequately evaluated by means of our laboratory streams as presently
designed and housed. Much more needs to be done to determine the occurrence
of different forms of residual chlorine under different conditions and the
effects of these on aquatic organisms, both in the laboratory and in the
field.
-------
METHODS AND MATERIALS
PREPARATION AND INTRODUCTION OF TOXICANTS
In most of our experiments with inorganic chloramines, test solutions
were formed by mixing solutions of sodium hypochlorite (NaOCl) and ammonium
chloride (NH^l) in a molar ratio of 0.7:1. The solution of each chemical
was prepared with well water in a 20- or 45-liter Mariotte bottle. By
connecting the atmospheres of the bottles containing the two chemicals, the
flows from the two bottles mixed at equal volumes on entering a mixing cham-
ber. Retention in the mixing chamber was about 3 hours, sufficient time for
no free residual chlorine to be detectable in any test solution of inorganic
chloramines entering the dilution systems. Test solutions of free residual
chlorine were prepared and introduced in the same way, except that hypochlor-
ite stock solutions were not mixed with other chemicals.
In the laboratory stream studies which required the introduction of high
concentration solutions, stock solutions of sodium hypochlorite were kept in
a collapsible bottle submersed in a cool water bath. This procedure reduced
exposure to air and minimized loss of chlorine gas. Stock solutions of
ammonium chloride were kept in a Mariotte bottle. The two solutions were
transferred with a tubing pump to a closed mixing bottle having a retention
time of several hours for formation of inorganic chloramines. The tubing
pump was controlled by a flowing-mercury switch located on the "dipping
bird" mechanism of a dilution system similar to that described by Mount
and Brungs (1967). To reduce gas formation in the closed mixing bottle,
NaOCl and NI^Cl concentrations were adjusted to achieve a pH of about 8.
This adjustment was necessary because a high NaOCl concentration has a pH
high enough for release of ammonia upon mixing with Nt^Cl. Conversely,
the presence of too much NffyCl produced acid conditions and the release of
chlorine gas.
Concentrations of free residual chlorine and inorganic chloramines
were determined using a Wallace and Tiernan amperometric titrator, the
titrant (0.0564N phenylarsene oxide) being added to the test solutions
with a micropipette. Concentrations were determined in micrograms per liter
(yg/1) or milligrams per liter (mg/1) of total residual chlorine (TRC).
Concentrations as low as 1 ug/1 of TRC could, with adequate precision, be
determined in test solutions.
Flowing water dilution systems were used for all experiments. In the
partial chronic and theembryo-alevin experiments, 2-liter proportional
diluters, similar in design to the one described by Mount and Brungs (1967),
were used. An 8-liter proportional diluter was used in combination with a
-------
modified Chadwick et al. (1972) type dilution system in the laboratory
stream studies. The dilution systems used in the acute toxicity, growth, and
behavior experiments were similar to the one described by Chadwick et al.
(1972).
WATER QUALITY AND PHYSICAL CONDITIONS
Well water was used for dilution in all experiments, except for the sand-
filtered Oak Creek water used in the laboratory stream studies and the
sand-filtered Willamette River water used in the acute toxicity studies
with pulp and paper mill effluent. The creek and river waters were pumped
through ultraviolet light to destroy pathogens. In order to keep the
temperature of the sand-filtered dilution waters well within the maximum
temperature range of creeks and rivers in western Oregon, water chillers
were used for cooling during summer. Temperature control of well water
was maintained with submersible heating elements located in the dilution
water reservoir boxes.
Temperatures of control water and test solutions were determined with
hand thermometers; pH was determined by means of an Orion Model 801 Analyzer.
Total alkalinity was determined by the bromcresol green-methyl red indicator
method. Dissolved oxygen concentrations were analyzed by the modified
Winkler method.
Except for the laboratory stream studies, all experiments were conducted
in laboratories having constant temperature control. Photoperiod was kept at
the regime for the Corvallis, Oregon, latitude (45°N) by adjustments made
every two weeks. The laboratory streams were kept under natural photoperiod,
but the quality and quantity of light were altered somewhat by the trans-
lucent plastic roof of the building housing the streams.
EXPERIMENTAL ANIMALS
The species of animals used in these studies included: coho salmon
(Oncorhynchus kisutch); brook trout (Salvelinus fontinalis); cutthroat
trout (Salmo clarki); crayfish (Pacifasticus trowbridgi); brown bullhead
(Ictalurus nebulosus); redside shiner (Richardsonius balteatus); blackside
dace (Rhinichthys osculus); bluegill (Lepomis macrochirus); and largemouth
bass (Micropterus salmoides). The locations from which these species were
obtained are listed in Table 1, and the experiments in which the species
were used are listed in Table 2.
METHODS FOR DETERMINING ACUTE TOXICITY: CONTINUOUS EXPOSURE
For 96-hour acute toxicity experiments, 10 fish, or 8 crayfish,
having nearly the same individual body weights were tested in each of twelve
45-liter glass aquaria. Small fish (alevins and fry) and the crayfish were
tested in 15 liters of control (well) water or test solutions to insure
adequate circulation of the water around the animals, since they remained on
or near the aquaria bottoms. Larger fish were tested in 30 liters of
solution. Two groups of animals were tested at each toxicant concentration
8
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TABLE 1. SOURCES OF SPECIES OF FISH AND THE CRAYFISH USED ON EXPERIMENTS
Species
Source and location of collection
Coho salmon
Brook trout
alevins and juveniles
Cutthroat trout
juveniles
adults
Crayfish
Redside shiner
Blackside dace
Bluegill, brown bullhead
and largemouth bass
Fall Creek Hatchery, Oregon Department of
Fish and Wildlife, Western Oregon
Raised from embryos obtained from Beities
Resort, Valley, WA.
Fall City Hatchery, Washington State Depart-
Department of Fisheries, Fall City, WA.
Black Creek, Western Oregon
Alsea River and Fall Creek, Western Oregon
Esmond Creek, Western Oregon
Alsea River, Western Oregon
Farm ponds near Corvallis, OR.
TABLE 2. THE EXPERIMENTS IN WHICH EACH SPECIES WAS USED.
Experiment
Species
Acute Toxicity:
Continuous exposure -
Intermitcent exposure -
Chronic Toxicity:
Partial chronic -
Development, survival
and growth -
Behavior -
Coho salmon, brook trout, cutthroat trout,
and crayfish
Largemouth bass, bluegill, brown bullhead,
redside shiner, blackside dace, and
cutthroat trout
Cutthroat trout, brook trout, and crayfish
Coho salmon
Largemouth bass, bluegill, brown bullhead,
redside shiner, blackside dace, and
cutthroat trout
Laboratory Streams:
Coho salmon
-------
and for controls. Coho salmon alevins and brook trout alevins were tested
in darkness to simulate streambed conditions, and at 10 C, but other tests
were conducted in light and at 15 C unless stated otherwise. The flow
rate of control water or test solution into each aquarium was maintained
at 500-ml/rain.
The effects of temperature (10.8 and 15.0 C), total alkalinity (135
and 320 mg/1 as CaCO ), and pH (7.0, 7.5, and 8.1) on the acute toxicity of
chloramines to juvenile coho salmon were determined in separate tests. The
alkalinity of the well water was increased by the addition of a saturated
solution of sodium bicarbonate, changes of pH being made and controlled by the
addition of carbon dioxide or 0.2 N sodium hydroxide.
For tests with coho salmon, the 96-hour median lethal concentrations
(LC50) were determined by calculating the logit linear regression line (Ashton,
1972). Percentage of mortality was plotted on a logit scale and concentration
of toxicant was plotted as TRC on a base 10 log scale. A computer program
designed by D. A. Pierce (Department of Statistics, Oregon State University)
was used to test for significant differences (P <_0.05) between the
slopes and intercepts of logit regression lines. For tests with other
species, the 96-hour LCSO's were determined by calculating probit regression
lines (Finney, 1971). Probits of mortality were plotted against concentration
of toxicant on a base 10 log scale. Regression analyses were performed
with a Monroe Model 1766 calculator.
In 96-hour acute toxicity experiments with chlorinated and nonchlorin-
ated secondary-treated pulp and paper mill effluent (SKME), 10 juvenile
coho salmon of nearly equal body weight were tested in each of twenty-two
45-liter glass aquaria. Each aquarium contained 30 liters of control water
or test solution. Separate dilution systems were used for the chlorinated
and the nonchlorinated effluent in each test. The effluent was chlorinated
with a solution of inorganic chloramines (71 percent) and free residual
chlorine (29 percent) immediately before it entered the dilution system.
Concentrations of 18, 32, 56, and 100 percent chlorinated and nonchlorinated
SKME were used in the first three tests. The 18 percent concentration
was replaced by a 75 percent concentration in a fourth test. Two replicates
were tested for each set of control and treatment conditions. The flow
rate to each aquarium was maintained at 330 ml/min. The concentrations of
chlorinated SKME between which the 96-hour LC50 values occurred were esti-
mated by plotting percent mortality against percent chlorinated SKME on a
base 10 log scale.
The effluent was from a kraft liner board mill and had 24 hours of
primary treatment, which was followed by 7 days of secondary treatment in
biological stabilization ponds. An additional 10 days of biological treat-
ment in a small stabilization pond—operated by the National Council for
Air and Stream Improvement (NCASI)—was provided to produce an effluent of
high and and relatively consistent quality, one that might be characteristic
of stabilized kraft process effluents produced by this mill in the future.
The BOD of the SKME was determined by personnel of the NCASI.
10
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The animals used in the acute toxicity tests with chlorinated well
water and SKME were acclimated to the laboratory conditions for at least
3 days prior to being tested. During the acclimation period, the fish
were fed either Oregon moist pellet or Tubifex. The crayfish were fed a
mixture of alfalfa and Oregon Test Diet (Lee et al., 1967], The fish and
crayfish were not fed for 24 hours prior to testing and during the tests.
METHODS FOR DETERMINING ACUTE TOXICITY: INTERMITTENT EXPOSURE
Fish were subjected to short-term exposure to residual chlorine,
of which 97.04 percent was free residual chlorine. Two types of time-
toxicant concentration exposures were tested. These will be referred to
as square and spike exposure patterns. They represent the extremes of
time-concentration relationships found in the field at the points of discharge
of intermittently chlorinated waters (G. Nelson, EPA, personal communication).
The square exposure patterns were produced by adding the toxicant to the
aquaria at constant rates for predetermined periods of time. Chlorine
concentrations reached a plateau level 20 minutes after the toxicant flow
as initiated. With but one exception, the toxicant flow was terminated at
60 minutes, and the toxicant was completely flushed from the aquaria after
an additional 30 minutes. Thus, total exposure time was 90 minutes. For
bioassays in which spike exposure patterns were employed, both toxicant and
dilution water flows were manipulated to achieve the desired time-
conncentration curves. Each spike exposure pattern was characterized by a
rapid rise to a peak toxicant concentration, followed immediately by a
rapid decline in concentration. Spike patterns having different peak
concentrations and different durations were used in the tests. One pat-
tern peaked in concentration 5 minutes after initiation of toxicant flow,
and others peaked at 29 and 22 minutes. Total exposure times for these
three spike exposures were 51, 60, and 63 minutes, respectively. The
concentrations of total residual chlorine for each exposure pattern were
determined at 2 to 10 minute intervals. The fish, which had been fed a
daily ration of Oregon moist pellet, were starved for 24 hours prior to
being tested and during the tests.
Most of the intermittent exposure tests were conducted with largemouth
bass in aquaria. But channels in which the fish were forced to swim against
a current were used in some tests. And other species were also studied in
aquarium experiments. In each of the aquarium tests, six bass of nearly
the same weight were acclimated to the test conditions (except for toxicant)
for 30 minutes in a 45-liter glass aquarium containing 30 liters of water.
These fish were then exposed to the toxicant for a predetermined period o£
time, after which they were maintained in water without toxicant for observa-
tion for the duration of the 96-hour test. The quality of the well water
used in these studies was as follows: dissolved oxygen, 7.4 mg/1; hardness,
128 mg/1; total alkalinity, 148 mg/1; pH 7.94; and temperature, 24.3 C,
with the exception of one experiment in which temperature was varied.
11
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Previous studies at our laboratory had shown that the tolerance of
echo salmon for residual chlorine in acute toxicity tests is a function of
body size (Larson et al., in press). Preliminary tests were conducted
to determine if body weight affected the tolerance of the bass for
short-term exposures of chlorine. The tested fish were divided into
two weight classes, one being 3.87 ± 0.15, the other 5.93 ± 0.28 g/fish,
dry weight. Groups of each weight class were subjected to one 90-minute
square pattern of exposure. The LC50, based on observation of mortality
but not exposure for 96 hours, for the two classes differed by approximately
1.2 mg/1 TRC (mean plateau concentration from 20 to 60 minutes), the smaller
fish being the more sensitive. On the basis of these preliminary results,
fish weight was standardized in each experiment, although the weights
varied from test to test (Table 3}.
TABLE 3. MEAN DRY WEIGHT AND STANDARD DEVIATION OF LARGEMOUTH BASS TESTED IN
THE INTERMITTENT EXPOSURE EXPERIMENTS WITH FREE RESIDUAL CHLORINE.
''ExperimentMean dry weightStandard
per fish (g) deviation
Weight effects (90-minute square exposure)
Class 1 3.87 0.15
Class 2 5.93 0.28
Exposure regime
90-minute square exposure 8.60 0.20
53-minute low spike exposure 8.50 0.20
63-minute high spike exposure 8.51 0.24
Exposure duration
90-minute square exposure 8.92 0.36
150-minute square exposure 8.92 0.53
Exposure frequency
one 90-minute square exposure 6.18 0.19
two 90-minute square exposures 6.15 0.32
Temperature effects (90-minute square exposure)
13.1 C (acclimation and test) 4.53 0.36
24.3 C 4.64 0.28
13.1 24.3 C 4.62 0.13
In one experiment, groups of bass were exposed to 51- or 63-minute spike
patterns of exposure, or to the 90-minute square pattern of exposure. The
mean plateau concentrations of TRC for square exposure patterns ranged
from 2.35 to 3.32 mg/1. The peak TRC concentrations were from 8.21 to
11.93 mg/1 and from 5.73 to 9.06 mg/1 for the 51- and 63-minute spike
patterns of exposure, respectively. The effects of the three types of
exposures were compared on the basis of areas under the time-concentration
12
-------
curves. These areas were measured using a compensating planimeter and
expressed as mg/1 x minutes of total residual chlorine. The curves for
all tests were graphed using the following scale: a 10 minute exposure
to 1 mg/1 TRC equalled 5.9 cm.
The effect of exposure frequency on survival was examined by subjecting
some groups of bass to single 90-minute square patterns of exposure,
while other groups were subjected to two such exposures, a two-hour
recovery period separating the exposures. The effect of exposure duration
on survival was investigated by subjecting groups of bass to either
a 90-minute or a 150-minute square pattern of exposure. For the latter,
toxicant flow into the aquaria was terminated at 120 minutes.
In other experiments, cutthroat trout, brown bullhead, bluegill,
blackside dace and redside shiner were tested separately. Groups of
each species of fish were subjected to one 90-minute square pattern of
exposure to free residual chlorine and then kept in freshwater for the
remainder of the 96-hour tests. The number of tests, range of fish
weights, number of fish used in each aquarium, and the water quality in
each test are shown in Table 4.
METHODS FOR DETERMINING CHRONIC TOXICITY: PARTIAL CHRONIC TESTS
The main objective of partial chronic experiments is to determine,
for the species of interest, the life history stages most sensitive to a
toxicant when constant exposure has occurred over all life stages. The
method requires a period of exposure of separate groups of sexually
maturing adult organisms to different concentrations of a toxicant and
subsequent exposure of embryo, alevin, and juvenile life stages to the
same toxicant concentrations. The experimental animals used in our
partial chronic experiments with chloramines were brook trout, cutthroat
trout, and crayfish (Table 5), Because it was not possible to collect
brook trout and crayfish from the field until midsummer, the gonadal
products of these species were near maturity at the beginning of each
experiment.
Ten stainless steel aquaria, two at each toxicant concentration and
two well water controls, were used in each partial chronic experiment
with fish. Each aquarium was 90 cm long, 30 cm wide, and 38 cm deep.
In experiments with trout, each aquarium contained 81 liters of water 30
cm deep. Other aquaria used in the crayfish tests contained 27 liters
of water 10 cm deep.
In tests with trout, the aquaria were covered with screens for
shading. Spawning trays, either pans of gravel or pans covered with
plastic screening upon which rocks had been glued, were placed in the
aquaria. Fertilized eggs from each group of fish were placed in a cup
positioned at the water surface in the same aquarium. The cups were so
designed that water flow was upward and around developing embryos and
alevins (Larson et al., in press).
13
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TABLE 4. NUMBER AND WEIGHT OF FISH AND WATER QUALITY CONDITIONS IN THE INTERMITTENT EXPOSURE EXPERIMENTS
WITH FREE RESIDUAL CHLORINE
-
Species
Cutthroat trout
Brown bullhead
Redside shiner
Blackside dace
Bluegill
No.
of
tests
6
2
4
10
8
5
8
Mean
dry weight
per fish
(g)
2.21
0.43
0.36
0.99
0.02
0.33
0.85
Standard
deviation
0.25
0.04
0.04
0.05
0.003
0.05
0.08
No. of fish
tested in each
aquarium
CC)
5
4
4
6
10
6
6
Acclimation
and test
temperature
12.5
12.5 + 22.0
24.0 •* 22.0
20.6
19.6
19.6
25.2
PH
8.1
8.1
8.0
8.0
8.0
8.0
8.1
Total
alkalinity
(rog/1)
150
150
150
158
155
155
150
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TABLE 5. SPECIES, DURATION, AND SIZE OF ANIMALS IN PARTIAL CHRONIC TESTS.
Species Test
no.
Brook trout 1
*->
2
Cutthroat trout 1
2
Crayfish 1
2
Duration of experiment
December 4
August 9,
January 14
August 23,
August 20,
Aueust 14,
, 1973 to January 11, 1974
1974 to March 12, 1975
to August 5, 1974
1974 to May 26, 1975
1974 to May 1, 1975
1975 to August 14, 1976
Mean wet weight
or mean carapace
length per animal
65.4 g
57.7 g
79.4 g
79.9 g
41.1 mm
40.5 mm
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In tests with crayfish, each animal was isolated by partitioning
each aquarium into 10 compartments with plastic screens. Only during
the mating period were male and female crayfish placed together, each
male being placed with a particular female. After mating, the animals
were again isolated. The-number of females extruding clusters of embryos
was recorded. The survival of adults and embryos was checked daily.
In each partial chronic experiment, four concentrations of total
residual chlorine plus a well water control were tested, and each was
replicated (Table 6). Monochloramine was the predominant species of
chlorine in each experiment, this averaging about 90.3, 87.8, and 90.1
percent of the total residual chlorine in experiments with brook trout,
cutthroat trout, and crayfish, respectively. Concentrations of the toxicant
in the test aquaria were determined 3 to 7 times each week during each
experiment.
In tests with trout, water temperature was approximately 12 C in July
and August, and near 10 C during other months. In crayfish tests, the water
was heated in summer to approximately 16 C, gradually decreased to ambient
(10 C) level by January, and then gradually increased to 16 C by the follow-
ing summer. The pH ranged from 7.0 to 8.1 in tests with trout and ranged
from 7.5 to 8.1 in crayfish tests. Total alkalinity ranged from 130 to 150
mg/1 (as CaCOg) in all tests. Dissolved oxygen concentrations were always
slightly less than 100 percent of the air saturation level. Illumination at
the water surface of the test aquaria was about 175 lux.
Adult trout were fed Oregon Test Diet and adult crayfish were fed a
mixture of Oregon Test Diet and alfalfa. The diet of the crayfish was supple-
mented with pieces of frozen herring.
METHODS FOR DETERMINING CHRONIC TOXICITY: SURVIVAL, DEVELOPMENT, AND GROWTH
It is not always possible, or even appropriate, to subject a species to
a toxicant under chronic or partial chronic test conditions. It may be more
practical or appropriate to expose particular life stages of the species to a
toxicant at concentrations and for periods of time similar to exposures these
stages would have in such tests. For anadromous salmonid species, for exam-
ple, it would seem appropriate to conduct tests on the freshwater life stages,
because it is during this period that the species are most likely to be
exposed to particular toxicants. We determined for coho salmon—whose
progeny spend about one year in fresh water before migrating to sea—the
effects of exposures to inorganic chloramines on the survival, development,
and growth of embryos and alevins, and on the growth of juveniles, which were
not at the migratory stage of their development.
Coho salmon embryos and alevins were exposed to concentrations of total
residual chlorine of 47 yg/1 and lower (Table 7). Survival and development
of the embryos, size of alevins at hatching, and time of hatching, as well as
subsequent survival and growth to yolk sac absorption were determined.
Immediately after eggs were fertilized in well water on November 22, 1973,
about 300 embryos (estimated by volume) were placed in each of 12 chambers
designed so that water flowed continuously upward from the chamber bottoms to
16
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TABLE 6. MEAN CONCENTRATIONS (AND STANDARD DEVIATIONS] OF .INORGANIC CHLORAMINES, IN MICROGRAMS PER
LITER, TO WHICH GROUPS OF TEST ORGANISMS WERE SUBJECTED DURING THE PARTIAL CHRONIC EXPERIMENTS
Group
A
B
C
D
E
Brook
1973-74
0
(control)
5
11
21
44
.3 (1.0)
.0 (1.6)
.7 (2.9)
.7 (5.3)
trout
0
5
10
21
50
1974-75
.4 ( 5.0)
.7 ( 3.7)
.8 ( 7.2)
.1 (15.7)
Cutthroat
1974
0
4.3 C 1-
9.2 ( 3.
23.1 ( 7.
54.9 (19.
Trout
1974-75
3)
0)
0)
9)
0
5.9
10.5
18.8
48.4
( 2.4)
( 3.3)
( 6.4)
(16.2)
Crayfish
1974-75 1975-76
0
38.7 (15.
80.1 (33.
170.3 (73.
373.4(124.
0
9) 25.2
7) 48.6
5) 99.4
0) 194.0
C 5.4)
( 9.5)
(20.4)
(46.2)
-------
pass around the developing embryos (Larson et al., in press). Each of four
test concentrations and for the two controls were replicated (Table 7). The
chambers were kept in light proof boxes to approximate natural streambed
light conditions. Temperature was maintained between 9.8 and 10.5 C; total
alkalinity and pH were about 135 mg/1 and 7.5.
TABLE 7. MEAN CONCENTRATIONS (AND STANDARD DEVIATION) OF RESIDUAL INORGANIC
CHLORAMINES, IN MLCROGRAMS PER LITER, TO WHICH GROUPS OF COHO
SALMON WERE SUBJECTED DURING THE EMBRYO-ALEVIN EXPERIMENT.
Group
•A
B
C
D
E
F
TRC
(Mg/D
0 (well
0
5.2 (1.0)
11.3 (1.9)
22.7 (3.3)
47.0 (6.1)
water control)
The instantaneous growth rates of the alevins in each chamber were
determined by randomly removing 25 alevins each week, from January through
February 11, and then dividing them into five groups of five fish each. The
yolk sacs were removed from the alevins and then each group of animals was
dried at 70 C for 7 days before dry weight was determined. Instantaneous
rates (k) were calculated for each group using the following formula:
log W0 - log W,
se 2 se 1
k =
T - T
12 ll
•
where W2 was the final dry weight of each group of five fish at time T~ and
W, was the initial dry weight at time T-.
The caloric content of alevins (without yolk) was determined on January
28 and February 4 and 11, and that of the yolk was determined on the two
February sampling dates, by means of a Parr Oxygen Calorimeter and standard
methods. To determine the content in the yolk, one group of five alevins
was removed from each chamber on each sampling date, dried, weighed, and
then oxidized in the calorimeter. The caloric content in the yolk was
determined by the difference in the content of alevins without yolk and
those with yolk.
At the conclusion of above experiment, each of the remaining groups of
fish (now fry) was transferred to a 10-liter plastic aquarium and exposed
to the concentration of toxicant to which it had been previously exposed.
These fish were fed a daily ration of Oregon Test Diet and were later used
in the first growth experiment with juveniles, which began on May 1, 1974.
18
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One purpose of the first growth experiment was to compare effects of
the toxicant on the growth of juvenile fish exposed continuously to the
toxicant since fertilization to effects on fish exposed only during the
growth test. A second purpose of this experiment was to determine if there
were any residual effects of the toxicant on the growth of fish in water
containing no toxicant after these fish had been exposed to the toxicant
since fertilization. To do this, control fish from the embryo-alevin experi-
ment were divided into eight lots of 15 fish each [Deferred to as Group A)
and exposed to one of three concentrations of toxicant or well water
or control conditions (Table 8). Furthermore those fish which had been
exposed to 5 or 11 yg/1 total residual chlorine in the embryo-alevin experi-
ment were divided into two lots of 15 fish each (referred to as Group B) and
exposed to nearly the same concentrations of the toxicant in the growth test.
In both groups (A and B), one lot of fish at each toxicant concentration and
control was fed Oregon Test Diet at a rate of 4 percent of their initial dry
weight each day, while the other lot was fed at a rate of 8 percent. The
initial weights were estimated from the weight of a lot of 15 fish that had
been dried at 70 C for seven days at the beginning of the experiment.
Finally, a third group (C) of fish was used to estimate the residual effects
of the toxicant on the growth of fish that had been exposed to 5, 11, or
23 yg/1 total residual chlorine in the embryo-alevin experiment. This was
done by placing one lot of 15 fish from each concentration and control into
water containing no toxicant and feeding them at the 8 percent ration level
(Table 8). The temperature of the well water in these tests was maintained at
10 to 11 C, and total alkalinity and pH were about 135 mg/1 and 7.5.
TABLE 8. MEAN CONCENTRATIONS OF INORGANIC CHLORAMINES, IN MICROGRAMS PER
LITER, TO WHICH GROUPS OF COHO SALMON WERE EXPOSED DURING THE EMBRYO-
ALEVIN AND THE CONCENTRATION (AND STANDARD DEVIATION) TO WHICH THEY
WERE SUBJECTED AS JUVENILES DURING THE FIRST GROWTH EXPERIMENT.
Group Embryo-alevin test First growth test
A 0 (control]
B 5.2
11.3
C 0
5.0
11.3
22.7
0 (control
4.9 (2.8)
10.9 (4.2)
22.3 (8.9)
4.9 (2.8)
10.9 (4.2)
0
0
0
0
19
-------
The mean relative growth rate of each lot of fish in the 21-day
experiment was determined with the following formula:
GR
0.5 cwj + w2) (T2 -
where W2 was the final dry weight at time ^2 an^ ^i was tne estimated
initial dry weight at time Tj. At the end of the experiment, the fish
were not fed for 24 hours before being sacrificed, dried at 70 C for seven
days, and then weighed. Gross efficiency of food conversion for each lot
of fish was calculated by dividing the dry weight of food eaten into the
change in dry weight of the fish during the test. Food consumption was
determined by subtracting the dry weight of uneaten food from the dry weight
of the food offered to each lot of fish. Each day the uneaten food was
removed from each aquarium before that dayfs ration was fed to the fish.
The purpose of the second growth test, which began on January 10,
1975 and lasted for 14 days, was to determine the effects of residual
chlorine on the growth of juvenile coho salmon not previously exposed to
the toxicant. These fish were older and larger than those used in the
previous growth test. The fish were divided into 12 lots of eight fish
each and exposed to either well water (control) or to one of three concentra-
tions of inorganic chloramines (Table 9). At each treatment, one lot was
fed a ration (Oregon Test Diet) of 3 percent of their estimated initial dry
body weight, another was fed a 6 percent ration, and a third lot was fed to
repletion each day. The methods for feeding, removing the uneaten food,
and for calculating the growth of each lot of fish were the same as those
described above, except the fish were starved for 48 hours before each lot
was sacrificed, dried, and weighed. Quality of the well water used in this
study was as follows: temperature, 15 C; total alkalinity, 135 mg/liter;
and pH, 7.5.
TABLE 9. MEAN CONCENTRATIONS (AND STANDARD DEVIATIONS) OF INORGANIC CHLORA-
MINES, IN MICROGRAMS PER LITER, TO WHICH GROUPS OF COHO SALMON
JUVENILES WERE EXPOSED DURING THE SECOND GROWTH EXPERIMENT.
Group TRC
(yg/D
A 0 (control)
B 5.2 (1.2)
C 9.9 (3.6)
D 23.2 (8.0)
20
-------
METHODS FOR DETERMINING. EFFECTS ON BEHAVIOR
Observations on changes in behavior of bass were made during each
acute toxicity test under conditions of intermittent exposure to free
residual chlorine. Most of the observations were made during the exposure
period, other observations being made at 24-hour intervals while the
fish were held in toxicant-free water for the remainder of the 96-hour tests.
The time of first occurrence of particular behavioral responses was usually
recorded. These responses are described in the appropriate section under
Results and Interpretation. In tests with cutthroat trout, brown bullhead,
bluegill, blackside dace, and redside shiner, qualitative observations of
behavior Were made according to the procedures outlined for bass.
During the intermittent exposure experiments, the behavior of largemouth
bass was altered even when exposures to chlorine were not acutely toxic.
Thus we attempted to determine the threshold concentrations at which the bass
exhibited particular behaviors, when subjected to intermittent exposure to
chlorine (97 percent free residual) in glass aquaria. The test conditions
were those described in the section entitled Methods for Determining Acute
Toxicity : Intermittent Exposure. The behavior of individual bass was con-
tinuously observed during each exposure, either a 90-minute square pattern
or a 63-minute spike pattern, and then for one to two minutes at 24-hour
intervals for the remainder of the 96-hour tests. In most cases, two fish
were tested together, one having a dry weight of about 14 grams and the
other about 7 grams. Paired fish were kept together in a test aquarium for
five days prior to being tested. The fish were fed a daily ration of
Oregon moist pellets supplemented with pieces of frozen herring, but they
were not fed for 24 hours before being tested and during the tests.
Effort expended by fish to maintain their position in flowing water
could affect their tolerance to toxicants. We subjected bass, having
nearly the same body weight as those used in the intermittent exposure
regime experiment to similar expo'sures to chlorine under conditions in
which the fish were forced to swim against a current. In each test,
individual bass were acclimated to the test conditions (except for toxicant)
for 60 minutes, and then the fish were subjected to either a 90-minute
square pattern of exposure or a 60-minute spike pattern of exposure. For
the latter, peak concentrations of total residual chlorine occurred at 29
minutes after the tests were started. After being subjected to either type
of exposure, the fish were kept in the experimental apparatus for 24 hours
before being transferred to 45-liter glass aquaria in which the toxicant was
not present for the remainder of the 96-hour tests. The behavior of the
bass was continuously observed during the exposures and for one to two
minutes at 24-hour intervals thereafter.
The experimental apparatus used in these studies is shown in Figure 1.
It consisted of a half-round aluminum trough, painted light blue, with two
straight sections connected at the ends with semi-circular sections. The
fish were tested in a straight section of the trough. Two stainless steel
screens, one at each end of the test section, restricted the movements of the
fish to within the section. A piece of styrofoam was placed over a portion
21
-------
of the test section to provide cover for the fish. Water current in the
trough was maintained at 13.4 centimeters per second with a submersible
pump. Toxicant was added upstream from the test section for these experiments
on the effects of swimming activity on tolerance of the fish for chlorine.
The animals were continuously subjected to the toxicant during each exposure
and were unable to avoid exposure.
Although aquatic organisms are often unable to avoid exposure to toxicants
in nature, there is evidence that fish may be able to avoid or move out of
areas receiving intermittent discharges of chlorine (Basch and Truchan,
1976). In view of this, the following experiment was conducted with largemouth
bass given the opportunity of either remaining in intermittent discharges
of chlorine or moving into water containing no toxicant. Our purposes
were to determine if bass would avoid such exposure conditions and, if so,
whether or not such avoidance was correlated with the concentrations of the
toxicant. Exposures to either inorganic chloramines or free residual
chlorine were tested in order to determine if fish responded differently to
these forms at similar concentrations of total residual chlorine.
In this experiment, bass having a mean dry weight per fish of 9.18 grams
were tested individually on six successive days. The experimental apparatus
was designed so that the fish had freedom of movement between a section
into which the toxicant was introduced and a section without toxicant (Fig. 1).
During each of the first three days each bass was acclimated to the apparatus
for 30 minutes and then observed for 60 minutes in order to establish the
average expected time the fish spent in the two sections of the apparatus.
During the next three days, the same procedure was followed each day, except
that toxicant was introduced during the 60-minute observation (exposure)
period in each test. Then, after toxicant was no longer present, the
section of the apparatus in which each fish was located 15 minutes after
each 60-minute exposure was recorded. All observations were made through a
small opening in a black plastic sheet which surrounded the apparatus.
Each fish was kept in a separate 45-liter glass aquarium for one week prior
to being tested and during periods between tests. The fish were fed a
daily ration of Oregon moist pellets, supplemented with pieces of frozen
herring, about 24 hours before being tested. At the conclusion of each
series of tests on a fish, it was starved for an additional 24 hours and
then sacrificed, dried, and weighed.
Changes in the amount of time each fish spent in the two section of
the apparatus, between tests without toxicant and those with toxicant, were
determined using the following formula (after Tsai and Fava, 1975):
T
Percent change = 100(1 ")
T
c
22
-------
\ WATER MANIFOLD i
SC
1 TOXICANT MANIFOLD
^ INFLOW PIPE
=>=»
REEN-*.,
i^-
FRESH WATER AREA
FLOW — *•
^5-TOXICANT DELIVERY TUBES
•£T TOXICANT
DISCHARGE
1ST AREA
c*
TEST SECTION
• . - IS? m .
0"^*
*f SCREEN
^
_ SECONDARY OUTFLOW
O*-S- RIPE
PRIMARY OUTFLOW
PIPE
TROUGH
FLOW
B
TOXICANT DELIVERY TUBES
Figure 1. Schematic diagrams of the experimental apparatus used in the
avoidance experiment with largemouth bass; (A) top view of the entire
apparatus;; (B) cross sectional view of the middle of the test section,
23
-------
where Tt was the total time spent in the discharge section divided by
the observation (exposure) time (60 minutes) in each test with toxicant,
and Tr was the mean time spent in the toxicant section divided by the obser-
vation time in each test without toxicant. In addition to determining
changes in the time fish spent in the two sections, a comparison was also
made between the actual amount of exposure each fish received and the total
possible exposure in each test with toxicant. This was represented by
means of the following formula:
T x T
s e
Exposure =
where T was the time spent in the toxicant section, Te was the total pos-
sible exposure (expressed as" mg/1 x minutes) under the time-concentration
curve, and Tc was the total possible time of exposure (60 minutes).
In each toxicant test, the maximum concentration of inorganic chlora-
mines or free residual chlorine in the toxicant section was reached within
a minute after the test began, and the concentration dropped to zero within
a minute after toxicant introduction was terminated. The water current in
the control and toxicant sections was about 4 cm per second, a velocity
found on the basis of dye studies to be sufficient to keep toxicant out
of the control section. The concentrations of toxicant in the toxicant
section of the apparatus were determined by analysis of samples taken from
the primary outflow pipe (Fig. 1) at 1 to 10-minute intervals during each
60-minute test with toxicant. Samples of test solutions used in free
residual chlorine experiments contained a small percentage of inorganic
chloramine, but solutions of inorganic chloramines did not contain detectable
amounts of free residual chlorine (Table 10).
TABLE 10. FORMS OF RESIDUAL CHLORINE COMPOUNDS PRESENT IN THE INTERMITTENT
EXPOSURE EXPERIMENTS AND THEIR MEAN PERCENTAGE (AND STANDARD
DEVIATION).*
Form of
residual chlorine
Percent of total residual chlorine
Tests using free
residual chlorine
Tests using
inorganic chloramines
Free chlorine
Monochloramine
Di chloramine
94.2 (1.1)
5.1 (1.1)
0.7 (0.7)
0
97.2 (1.2)
.2.8 (1.1)
Average water quality of the well water in each test was as follows:
Temperature, 25C; pH, 7.9; dissolved oxygen, 7.5 mg/1; total alkalinity
(as CaCO ), 145 mg/1, and hardness, 126 mg/1.
24
-------
LABORATORY STREAM COMMUNITY METHODS
Communities of aquatic plants and animals in laboratory streams have
provided a useful way to study possible influences of toxicants on
communities in receiving waters. In this portion of our program, 12 separate
stream communities were established to determined the influence of inorganic
chloramines on the biomasses of algae and invertebrate animals and on the
production of juvenile coho salmon at TRC concentrations of about 20yg/l
or less.
The laboratory streams used in these studies were similar in design
to those described in detail by Mclntire et al. (1964). Each stream was
contained in a plywood trough 3.3 m long, 66 cm wide, and 25 cm deep, divided
into two channels by a median plywood partition open at each end to permit
the water to circulate. The floors of the troughs were covered with gravel
and stones in such a way as to form a pool at each end alternating with
riffles along the straight channels. Electrically driven paddle wheels
provided current velocities up to about 24 cm/sec over the riffles. The
water in each stream was exchanged at about 2 liters per minute with sand-
filtered creek water.
Benthic organisms were initially established in the streams by repeated
stocking with organisms collected from nearby creeks, by egg deposition of
adult insects, and possibly by some immigration of algal cells and inver-
tebrate animals through the water supply. Colonization began in September
1974, and by April of the following spring the stream communities were well
established. The salmon were not introduced into the streams until
after the colonization period,
Benthic organisms, including macro-invertebrates and periphyton, were
sampled at approximately three-week intervals. A 0.093 m (1 ft2) section
of stream bottom was sealed off with two partitions and the enclosed sub-
strate was scrubbed to remove attached organisms. After cleaning, the
gravel was removed and the water containing organisms was siphoned into a
100-micron mesh plankton net. The sample was then divided into two parts
with a soil-type sample splitter. Half of the sample was returned to the
stream, the other half being retained and frozen until the macro-invertebrates
could be removed.with the aid of a binocular microscope. The remaining
sample materials were then analyzed for chlorophyll A, B, and C and for the
amount of organic matter, determined by loss-on-ignition, by means of methods
described by Strickland and Parsons (1968).
Juvenile coho salmon were each marked by a "cold brand" (Everest and
Edmundson, 1967) to identify individual fish. From a group of fish accli-
mated to the dilution water for at least two weeks, fish were selected for
uniformity in body size. These were starved for 48 hours to eliminate
differences in the weights owing to stomach contents, anesthetized in MS-222,
blotted dry, and weighed in a tared water bath on a top-loading balance
25
-------
accurate to 0.05 grams. Weighed fish were then distributed to each of the
12 streams at the beginning of the experiments. At the end of each experi-
ment, and at about 21 day intervals, the fish in each stream were removed
and reweighed. Mean relative growth rates and production rates (growth
rate x mean biomass) were calculated.
26
-------
RESULTS AND INTERPRETATION
ACUTE TOXICITY OF CHLORAMINES WHEN EXPOSURE CONTINUOUS
Over the past two decades, the 96-hour acute toxicity bioassay has
become the most generally employed means of evaluating and setting standards
for toxic materials introduced into aquatic systems. It is generally recog-
nized that concentrations of toxic substances near the 96-hour median
lethal concentration (LC50) cannot be permitted over appreciable areas or
for more than a few hours, if aquatic life is to be protected. In conse-
quence, there has developed a tendency to adopt some fractional proportion
of the 96-hour LC50 of particular toxic substances in standards for the
protection of particular aquatic species. There is no sound empirical or
theoretical basis for this practice, because the relationships between
concentrations leading to death in a short period of time and those affect-
ing important biological responses such as reproduction and growth have not
been and cannot be expected to be generally representable by simple coeffi-
cients or "application factors" (Warren, 1971), It is only in the absence
of adequate empirical and theoretical knowledge that use of application
factors can be justified as a necessary expedient in the first stages of
dealing with pressing problems of water pollution control. Nevertheless,
acute toxicity bioassays have had and will continue to have an important
role in the solution of these problems. They do provide one possible means
for biologically assaying the relative toxicities of substances and effluents
and for determining the relative sensitivity of different species and life
history stages to acutely toxic conditions. And they also permit some
evaluation of the effects of other water quality conditions on the toxicity
of substances and effluents. The acute toxicity of chloramines to different
life stages of coho salmon, brook trout, and cutthroat trout under conditions
of continuous exposure are considered in this section. Influences of other
water quality conditions on acute toxicity of chloramines are also considered,
In the next section, the acute toxicity of free residual chlorine to large-
mouth bass and other species of fish when exposure was intermittent will be
discussed.
The acute toxicity of chloramines to coho salmon, brook trout, and
cutthroat trout, as measured by 96-hour LCSO's, is a function of the develop-
mental stage and body size of the fish. Alevins (yolk sac extending outside
the body cavity} are more tolerant than are late fry (yolk sac inside the
body cavity) and early juvenile stages (Fig. 2A; Table 11, 12). Median
lethal concentrations of chloramines for very small juvenile coho salmon
ranged from 57 to 66 yg/1 (Table 11). With increasing body size, the
96-hour LCSO's for juveniles of this species increased to about 80 yg/1,
near the tolerance level of the alevin stage (Fig. 2A; Table 11). In
developing from alevin through fry and then into juvenile stages, brook
27
-------
TABLE 11. LIFE HISTORY STAGE, MEAN DRY WEIGHT PER FISH, WATER QUALITY CON-
DITIONS, AND THE 96-HR LC5Q OF INORGANIC CHLORAMINES IN EACH
ACUTE TOXICITY TEST WITH COHO SALMON. DRY WEIGHTS OF ALEVINS AND
FRY DO NOT INCLUDE YOLK.
Life
stage
Alevin
Alevin
Fry
Juvenile
Juvenile
Juvenile
Juvenile
Juvenile
Juvenile
Juvenile
Juvenile
*
Juvenile
Mean
dry weight
per fish
Cg)
0.0238
0.0549
0.0727
0.123
0.114
0.227
0.210
0.234
0.240
1.150
1.550
1.530
Temp.
CC)
9.9
10.2
10.3
10.5
15.0
10.8
15.0
15.0
15.0
15.0
15.0
15.0
15.0
15.0
Alkalinity
Cmg/1)
135
135
135
135
135
135
135
135
135
135
316
135
322
135
pH
7.4
7.6
7.4
7.6
7.6
7.5
7.0
7.0
7.5
8.1
7.0
7.5
8.2
7.5
8.1
7.5
7.5
7.4
96-hr LC50
TRC
Cvg/iD
83
80
79
66
62
57
57
64
72
72
72
53
72
72
60
71
65
82
82
82
82
81
* 1973 year-class; all other groups from 1974 year-class
28
-------
0.01 0.10 1.00
Mean Dry Weight Per Coho Salmon (g)
0>
o>
2
c
Q_
90r-
80
70
60
50
40
30
20
10
B
O O'
090
O I5.0°C
• K).8°C
1.71 1.73 1.75 1.77 1.79
Log,0 TRC
1.81
90r
>* 80
o 70
o 60
^ 50
*0 40
S- 30
I 10
_ c
(ED O
O O
O 135 mg/1 as CaC03
• 320mg/lasCaC03
1.84
1.86
1.88
1.90 1.92
Log|Q TRC
1.94 (.96
1.98
90 r
>. 80
2 70
o 60
J 50
*5 40
3. 30
20
a>
CL-
D
1.74 1.76
• PH 8.1
O pH 7.5
A pH 7.0
1.78
1.80
1.82 1,84
Log |o TRC
1.86 1.88 1.90 1.92
Figure 2. Acute toxicity of inorganic chloramines (TRC) to coho salmon in 96-hour tests as affected
by: (A) fish weight; (B) temperature; (C) total alkalinity; and (D) pH. Toxicant concentrations (yg/1)
on the abscissal axes in B, C, and D are expressed as base 10 logarithms.
-------
trout exhibited the same pattern of initial decrease and then increase
in tolerance to chloramines (Table 12). Figure 3 clearly illustrates
the increase in tolerance of juvenile cutthroat trout with increase in
body weight.
TABLE 12. LIFE HISTORY STAGES, MEAN DRY WEIGHT PER FISH, WATER QUALITY CON-
DITIONS, AND THE 96-HR LC50 OF INORGANIC CHLORAMINES IN EACH ACUTE
TOXICITY TEST WITH BROOK TROUT AND CUTTHROAT TROUT. DRY WEIGHTS
OF ALEVINS AND FRY DO NOT INCLUDE YOLK.
Life
Species Stage
Brook trout Alevin
Alevin
Alevin
Fry
Juvenile
Juvenile
Cutthroat trout Juvenile
Juvenile
Juvenile
Juvenile
Juvenile
Mean dry
wt per fish
(g)
.006
.006
.009
.012
.041
.983
.546
.761
.803
1.255
1.297
Temp Total
(CJ alkalinity pH
10.1
10.6
10.8
10.8
11.3
11.1
15.1
12.3
12.7
12.3
10.1
148
142
180
180
150
130
145
155
143
155
150
7.7
7.8
7.8
7.8
7.8
7.7
7.7
7.7
7.7
7.7
7.7
96-hr LC50
TRC (yg/1)
insufficient
deaths
105.5
90.6
81.8
90.6
88.4
74.5
81.7
83.1
94.7
94.0
Temperature was not found to affect the acute toxicity of chloramines to
juvenile coho salmon significantly (P > 0.05), in tests conducted at 10.8 C
and 15.0 C (Fig. 2B). Total alkalinity, in tests conducted at 135 mg/1 and
320 mg/1, did not affect significantly the toxicity of chloramines to the
juvenile salmon (Fig. 2C). Although there was no significant difference in
the acute toxicity of chloramines tested at pH 7 and pH 7.5, toxicity was
significantly higher at pH 8.1 (Fig. 2D).
Extensive monitoring of stabilized kraft pulp and paper mill effluents
(SKME) at our laboratory has demonstrated that, when biologically stabilized
to levels of BOD near 15 mg/1, these effluents extremely rarely result in
any mortality in 96 hours, even when tested at 100 percent concentration
(Robinson-Wilson and Seim, MS). But the addition of 1 mg/1 of chloramines
to SKME in one test resulted in acute toxicity of the effluent at a dilution
of 18 percent by volume (Table 13)0 In this and the other three acute toxicity
tests conducted, the results were inadequate to estimate 96-hour LCSO's, but
the effluent was rendered toxic. In three of the tests the concentration
range within which the LCSO's occurred could be estimated (Table 13). After
mixing with the effluent, no residual chloramines could be detected by
araperometric titration, when chloramines were added at concentrations of 1.0,
1.7, and 2.3 mg/1.
30
-------
~ iuu
3
o
OC 90
1-
o
lO
o
-I 80
i
(D
* 70
/
t
•
t J
•
^^r
. L 1 1 1 1 1 1 1 I , 1
0.2 1.0 3.0
MEAN DRY WT / FISH (g)
Figure 3. Relationship between the mean dry weight(g) per fish and the
96-hour LC50 of inorganic chloramines (TRC) for juvenile cutthroat trout,
31
-------
u*
to
TABLE 13. CONCENTRATIONS OF SECONDARILY TREATED KRAFT PAPER MILL EFFLUENT (SKME) BETWEEN WHICH THE
96-HR LC50 OCCURRED IN CHLORINATED SKME ACUTE TOXICITY TESTS WITH COHO SALMON.*
Starting
date of
test
7-17-75
7-20-75
7-30-75
8-6-74
Mean dry wt
per fish
(g)
0.14
0.72
0.60
1,10
Milligrams
of chlorine
added per
liter of
100% SKME
1.0
1.7
1.7
2.3
Concentrations of
chlorinated SKME
Mean temperature (C)
River Non-chlorinated' chlorinated
water 100% SKME 100% SKME
19.1 22.1 22.0
19.1 23.6 22.0
16.9 20.6 19.8
17.7 17.9 17.8
between which
the
96-hr LCSO occurred
(%)
18 - 32
56 -100
insufficient deaths
56 -100
t
pH ranged from 7.8 to 8.3, dissolved oxygen from 6.1 to 8.9 mg/1, and BOD from 9 to 17.6 mg/1
over all tests.
-------
Adult crayfish are much more tolerant of high concentrations of chlora-
mines than are fish in 96-hour tests. In two tests, we were unable to
estimate the 96-hour LCSO's even though the highest concentration tested
was 749 ug/1 (Table 14}.
ACUTE TOXICITY OF FREE CHLORINE WHEN EXPOSURE INTERMITTENT
When chlorine is employed for slime control in power generation facili-
ties or industrial processes, its introduction is generally intermittent,
with some regular intervening period. This is. unlike the continuous intro-
duction of this toxicant generally practiced in sewage disinfecting appli-
cations. The concentration of chlorine introduced, the duration of introduc-
tion, and the frequency of introduction are parameters that can be varied
so as to achieve effective slime control. Because concentration, duration,
and frequency of chlorine introduction will determine the time pattern of
concentrations of chlorine in natural waters and thus exposure of aquatic
organisms to this toxicant, it is important to determine what, if any,
pattern of utilization of chlorine for slime control could minimize or
eliminate harmful effects on fish and yet achieve its objectives. In slime
control applications, the total amounts of chlorine employed, when introduc-
tion is intermittent, may not be great and, when mixed in most receiving
waters, may not result in concentrations high enough to be either acutely
or chronically toxic to fish and organisms in their food chains. But in
the immediate area of effluent introduction, there may occur concentrations
of chlorine and derivative compounds high enough to be acutely toxic on
short exposures, to have deleterious effects on the behavior of fish, or to
have chronic effects . For these reasons, we have evaluated the effects on
largemouth bass of free chlorine at different concentrations and exposure
durations and frequencies. Acute toxicity—as measured by survival—and
some behavioral effects were investigated with largemouth bass. Prelimi-
nary acute toxicity and behavioral studies with bluegill, blackside
dace, redside shiner, brown bullhead, and cutthroat trout were also conducted,
In this section, the acute toxicity of chlorine under conditions of
intermittent exposure will be considered; results and interpretation of
behavioral studies will be presented in a later section.
One of the major problems of general application of the results of
bioassays of the acute toxicity of chlorine to fish under conditions of
intermittent exposure is that the concentration, duration, and frequency
of exposure of fish to chlorine are highly variable at different locations,
because very different sets of values of these parameters are employed for
slime control at different power plant installations. Conceivably, bioassays
would need to be employed to determine the effects of slime control practices
at each power facility. This would require a rather extensive bioassay
undertaking, and the results might not prove to be very useful for setting
general standards for the protection of fish. Figure 4 illustrates three
general time-concentration curves representing patterns of chlorine concentra-
tion that may occur in receiving waters, with a single application of chlorine
treatment for slime control. The curves in Figure 4 also illustrate the
main time-concentration patterns of chlorine to which we exposed test fish,
with single or multiple exposures. We have named these patterns high-
spike pattern of exposure (peak about 10 mg/1 TRC, mainly free chlorine),
33
-------
TABLE 14. MEAN DRY WEIGHT (WITHOUT CHELIPEDS) AND CARPACE LENGTH PER ANIMAL, WITH QUALITY CONDITIONS,
AND RANGES OF CONCENTRATIONS OF INORGANIC CHLORAMINES TESTED IN EXPERIMENTS IN WHICH
INSUFFICIENT DEATHS OCCURRED TO ESTIMATE THE 96-HR LC50 VALUE FOR CRAYFISH.
Test
Mean dry
Date weight per
animal
(g)
Mean carapace
length
per animal
(mm)
Temp. pH
(C)
Alkalinity
(mg/1)
Dissolved
oxygen
(yg/i)
TRC
(yg/i)
9-19-74
0.928
23.4
15.1
7.7
140
9.5 306 - 530
9-26-74
2.540
33.6
15.3
7.
143
,.0 449 - 749
-------
HIGH SPIKE
EXPOSURE
LOW SPIKE
EXPOSURE
SQUARE
EXPOSURE
40 60
TIME (min)
100
Figure 4. Examples of the time-concentration relationships of the square
pattern of exposure and of the high and low spike patterns of exposure to
free residual (TRC),
35
-------
low-spike pattern of exposure (peak about 6 mg/1 TRCJ, and square pattern* of
exposure (plateau about 3 mg/1 TRCJ. Mortality of fish can be related to
peak concentrations, plateau concentrations, mean concentrations, or the
time-concentration area of exposure (mg/1 x minutes). The first three alter-
natives are quite acceptable for reporting the results of particular experi-
ments or exposure patterns, but results so reported cannot be generally
applied to other patterns. We will present evidence that the last procedure—
relating mortalities to the time-concentration area of exposure—yields very
similar results for different exposure patterns, at least for those we tested.
This result could be very important for generalisation of the results of
tests conducted on particular patterns and setting general standards based on
such tests. More extensive work should be conducted to confirm this and to
clearly establish its parametric boundaries, if reliable application of this
procedure is to be made.
Figure 5 presents the results of an experiment in which different groups
of largemouth bass were exposed to what we have called high-spike pattern of
exposure, low-spike pattern of exposure, and square pattern of exposure. The
peaks of the high-spike patterns ranged from 8,21 to 11.93 rag/1 of chlorine;
those for the low-spike patterns from 5.73 to 9,06 mg/1; and the mean
plateau concentrations for the square patterns ranged from 2.35 to 3.32 mg/1.
Even so, the relationships between mortality of the largemouth bass and
the area under the time-concentration exposure curve were not significantly
different for the three quite different exposure patterns (Fig. 5).
Similarly, mortality of largemouth bass was closely related to total
area beneath time-concentration exposure curves, when the fish were exposed to
two 90-minute square patterns or to one 90-minute and one ISO-minute square
pattern, with two-hour intervals between exposures (Fig. 6 C, F). This was so
even though clearly distinct mortality-concentration relationships were found
when the mortalities were graphed in relation to mean plateau concentration
(Fig. 6 A, D) and to mean concentration during the entire exposure (Fig, 6 B, E)
Again, area under the time-concentration exposure curve appears to be a way
of generalizing the results of different patterns of exposure to
chlorine. It should, however, again be noted that the interval between these
exposures was only two hours, which was clearly not enough time for recovery
from chlorine intoxication of the exposed fish. Were much longer recovery
periods to have occurred, it is quite likely that relationships between
mortalities and total areas under successive exposure curves would not be
so well defined.
Both test temperature and body size were shown to influence the toler-
ance of largemouth bass to chlorine when exposed to a single square pattern.
The fish were much more tolerant at 13.1 C than at 24,3 C, and were least
tolerant when acclimated at 13.1 C and exposed to chlorine at 24.3 C (Fig. 7).
And bass having a mean weight of 5,93 grams were more tolerant to chlorine
than bass weighing 3.75 grams (Fig. 8].
Mortality of cutthroat trout occurred, under conditions of the square
pattern of exposure, when the fish were exposed just once for 90 minutes to
a pattern having a mean plateau concentration of about 985 ng/1 (Table 15).
36
-------
5 5
.0
o
oL
>- 4
I-
4
D
on
D SQUARE
O LOW SPIKE
• HIGH SPIKE
100
T
L I i I
i i
I
. i . .
150 200
mg/ I x minutes
250
Figure 5. Relationships between mortality of largemouth bass (in probits)
and the area under the time-concentration curve (expressed as mg/1 x minutes)
for fish subjected to square and high and low spike exposures to free residual
chlorine (TRC), Data points with arrows pointing up indicate that all of the
fish died; those with arrows pointing down indicate that no fish died during
the test.
37
-------
er (A) .
1j> 2 90/rtin /
2 Exposures o
2 /
£5- y
>• o
3. /
< 4- 0
C
o:
O
s, i
A 4 , « r- (B) A A A*
/
^
// 9^/77>/7
/ Exposure
0
/
1 III
7 .7
/ /
-o ,•
1 /
1 1 1
A
A
I I
I III]
r- (D)
00 W
J3
O
S. 5
/ 150m in
Exposure
4-
oc
O
I I I
2 34
TRC (mg/l)
5 6
r- (F)
• o
o
2 3
TRC (mg/l)
100 150 200 300 500
mg/l x minutes
700
Figure 6. Relationships between mortality of largemouth bass (in probits) and the mean plateau
concentration of free residual chlorine (A and D), mean concentration for the entire exposure (B and E),
and area, expressed as mg/l x minutes under the time-concentration curve (C and F) for bass subjected to
one or two 90-minute square patterns of exposure, or to one 90-minute or one 150-mnute square pattern
of exposure. Data points with arrows pointing up indicate that all of the fish died during the test.
-------
o
11
I3.I-*24.3°C
A
O
o o
O 24.3°C
o
T 5 T
2? I 5
TRC ( mg/I)
I I I
10
20
Figure 7. Relationships between mortality of largemouth bass (in probits}
and the mean plateau concentrations of free residual chlorine (TRC) in
90-minute square patterns of exposure for bass acclimated and exposed to the
toxicant at either 13.1 or 24.3 C and for bass acclimated to 13.1 C and
exposed to the toxicant at 24.3 C. Data points with arrows pointing up
indicate that all of the fish died; data points with arrows pointing down
indicate that no fish died during the test.
39
-------
rt A* O U3 P >T1
a4 o ^ o a H.
"Elffl MORTALITY (probits)
i— • a w c n> "^
H* O. ft Ot>
H-oq fQ S • . -
000
H> PJ ^ W 01 '
rt^ja a ya l\j
rt n> We a '
(D rt i-J H" (U
a- s a PJ rt
Hi PJ (D rt H.
P- rt p >T3 (D O
(A 3 PI » 3
3* 3 rt C in
O F^j pf £3"*
^ w 4i» o« cn -si
1 T^ T 1
1
a. H a o H-
h" HI *"% *"( O ^O -- ^*^
(D H- saw ^^"3^
&, w S tfl o ^ (S
a4 (D o v ^\J
a. H- o a a
C a.0q H) rt rt
H* (C rt 0) p CD
3 tX_ >< rt (D
)q *« « "73 'o H* 3 a
0 O 0 _ ^J
?&H£3 1 5 w
0> rt HI >-i 4 ^U
PJ H- (t» O rt -».
rt M HJ ft) rj
0) H3 B4 Hj M * *
WO- O H) H.
rt h" H HJ rt jmwi*
* 3 OX
rt o cr* (D ^
(fl p fli O -1
rt tn 4 H> -f
^ W w O *fN
h>. tn »-i *••
rt *ti ? H- (a ^
a* o n> D-- 4 ^^*
H. iu. e erg N.
(U 3 CTQ &] (D «MB 4*>
4 rt »* (-• S j-
4 U) M. 0 ^—"
o a 0 c
rt CD O
"X* 3* N- H cr"
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H. pi Er 3
^F
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rt
CD
-------
For 53 minute exposures once each day for four days, mortality occurred at
mean plateau concentration of about 745 yg/1, (Table 15}. Effects of chlorine
intoxication can apparently be cumulative under conditions of intermittent
exposure, even when the interval between exposures is as long as one day.
The median lethal concentrations, based on deaths occurring within 96
hours after a single square pattern exposure of 90 minutes, were determined
for bluegill, redside shiner, and blackside dace. Determined in this way
from the mean plateau concentrations, the LC50 for the bluegill at a
temperature of 25.2 C was 2.32 mg/1 chlorine, and for the redside shiner
at 20.6 C it was 1.6 mg/1 (.Tig. 9). Blackside dace having a mean weight of
0.33 gram had a LC50 of approximately 700 yg/1 at 19.6 C, and those having
a mean weight of 0.02 gram had a LC50 of about 400 jag/1, the large dace
being more tolerant to chlorine under these conditions. The estimated mean
concentrations of TRC for the entire exposure at the LC50!s for bluegill,
shiners, 0.33 gram, dace and 0.02 gram dace were 1.62, 1.13, 0.55 and 0.27
mg/1, respectively. Similarly, the estimated areas (expressed as mg/1 x
minutes) under the time-concentration curves at the LCSO's for these fishes,
in the same order, were 145.5, 101.5, 41.9, and 25.1. The median lethal
concentration for brown bullheads acclimated at 24 C and tested at 22.5 C
was about 4100 yg/1, no deaths occurred when the fish were acclimated at
12.5 C and tested at 22 C and a chlorine concentration of about 4800 yg/1
(Table 16).
CHRONIC TOXICITY OF CHLORAMINES : PARTIAL CHRONIC TEST CONDITIONS
In the vicinity of sewage treatment plants, fish and other aquatic
organisms may be exposed continuously to varying concentrations of chlora-
mines. This, of course, raises the question of whether or not there are
chronic effects of these chlorine compounds on the survival, reproduction,
development, and growth of fish exposed to relatively low concentrations
over rather long periods of time. Experiments in which adult aquatic organ-
isms are maintained in constant concentrations of toxicants during gonadal
maturation and reproduction and then the embryos and early juvenile stages
develop and grow under these conditions have been called "partial chronic
toxicity tests." In a "full chronic test," the resulting young would be held
under the sa^ie conditions until they matured and reproduced, which would
require experiments several years in duration for trout and crayfish, with
which we performed only partial chronic tests. Continuous exposure to
nearly constant concentrations of toxicants throughout the life history of
fish probably rarely if ever occurs in nature. But, for some species of
organisms, such tests do provide a means for determining some of the pos-
sible chronic effects of low levels of toxicants.
Two partial chronic tests were conducted with brook trout, two with
cutthroat trout, and two with crayfish. The periods of these six experiments
have already been presented in Table 5. Means and standard deviations
of chloramine concentrations present during these experiments were given in
Table 6. Neither species of trout spawned under the chloramine test or
control conditions. But eggs and sperm were collected from the exposed brook
41
-------
TABLE 15. MEAN DRY WEIGHT PER FISH AND THE NUMBER OF DEATHS FOR CUTTHROAT TROUT JUVENILES SUBJECTED
TO DIFFERENT INTERMITTENT SQUARE EXPOSURES OF FREE RESIDUAL CHLORINE. MEAN PLATEAU CON-
CENTRATIONS WERE CALCULATED FROM 10 TO 30 MINUTES AND FROM 20 TO 60 MINUTES FOR THE 53- AND
90-MINUTE EXPOSURES, RESPECTIVELY. MEAN CONCENTRATIONS REPRESENT THOSE FOR THE ENTIRE
EXPOSURES. AREAS UNDER THE TIME-TOXICANT CONCENTRATION CURVES ARE EXPRESSED AS yg?1 TRC
X MINUTES.
N>
Test
1
2
3
Mean plateau
Replicate Exposure concentration
aquarium regime of TRC
(yg/D
*
A one 53-minute exposure 744
per day for 4 days
B one 53-minute exposure 743
per day for 4 days
A one 53-minute exposure 1067
B one 53-minute exposure 1077
A one 90-minute exposure 987
B one 90-minute exposure 985
Mean Area Mean dry Number
concentration (mg/1 x weight of fish Number
of TRC minutes) per fish in each of
(yg/1) (g) aquarium deaths
389# 82.72* 2.05 5 2
422 89.50 1.82 5 3
631 33.39 2.46 5 0
615 32.54 2.34 5 2
606 54.58 2.16 5 1
609 54.75 2.41 5 1
For test 1, concentration given is the mean for all exposures.
For test 1, concentration given is the mean for all exposures.
For test 1, area given is the sum of all exposures.
-------
.o
h. 5
o.
a:
o
Blueqill
Redside
Shiner
B
o.st
Blackside
Dace
0.02.g/fish
Group 2
0.33 g/fish
0.2
To
.5
1.0 1.5
TRC (mg/l)
Figure 9. Relationships between mortality of fish (in probits) and the mean plateau concentration of
free residual chlorine (TRC) during 90-minute square patterns of exposure of bluegill (A), redside
shiner (B), and blackside dace (C). Data points with arrows pointing up indicate that all of the fish
died; those with arrows pointing down indicate that none of the fish died during the tests.
-------
TABLE 16. MEAN DRY WEIGHT PER FISH, TEMPERATURE OF TEST SOLUTIONS, TOXICANT EXPOSURES, AND THE NUMBER
OF DEATHS OF BROWN BULLHEAD SUBJECTED TO 90-MINUTE SQUARE EXPOSURES OF FREE RESIDUAL
CHLORINE.
Test
1
2
3
4
5
6
Acclimation
temperature
(C)
12,5
12.5
24.0
24.0
24.0
24.0
Test
temperature
(C)
22.6
22.1
22.6
22.4
22.5
22.4
Mean plateau
concentration
of TRC
Cvg/D
410.8
4815
3274
4123
4823
4580
Mean
concentration
of TRC
(yg/i)
2481
2885
2038
2766
2774
2503
Area
(mg/1 x
minutes)
223.23
259.67
183.40
249.00
249.67
225.27
Mean dry
weight
per fish
(g)
0.45
0.40
0.41
0.31
0.37
0.35
No. of fish
in each
aquarium
4
4
4
4
4
4
Number
of
deaths
0
0
0
2
3
3
-------
trout, and fertilized eggs were held through hatching and then alevin growth
under the same test conditions. Some of the crayfish spawned, but hatching
was not successful, because fungus developed on egg masses under both control
and test conditions. In spite of our inability to complete partial chronic
tests according to specifications, information on survival of adult trout
and crayfish and development of reproductive products during such long
exposures to relatively constant concentrations of chloramine is of con-
siderable value. Of the total residual chlorine present in all tests,
about 90 percent was in the monochloramine form.
The first test with adult brook trout began on December 4, 1973 (Table
5), at which time the fish were already in spawning condition, this test
thus not meeting specifications for a "partial chronic test." The fish
did not spawn on the provided substrate, and their gametes were found to^
be overripe in mid-January, when the test was terminated. Mortality during
this 38 day experiment was quite low, even at the highest test concentrations
of 21.7 and 44,7 yg/1 total residual chlorine (TRC) and was probably not caused
by the toxicant.
The second test with brook trout began on August 9, 1974, and extended
to March 12, 1975. Mortality was higher during this test, but the pattern
of mortality indicated that even at 50 yg/1 survival was little affected by
the toxicant (Table 17). Fish in all test groups except those held at
50 yg/1 TRC were in spawning condition by early December. Two females^from
the 50 yg/1 test group were ripe by early February, but sucessful ferti-
lization was not accomplished with eggs collected from this test group of
fish.
TABLE 17. CONCENTRATIONS OF INORGANIC CHLORAMINES AND THE NUMBER OF DEATHS
OF ADULT BROOK TROUT DURING THE 1974-75 PARTIAL CHRONIC EXPERIMENT.
Concentration
TRC
(Pg/1)
0 (control)
5.4
10.7
21.8
50.1
Replicate
aquaria
A
B
A
B
A
B
A
B
A
B
No. of fish
in each
aquarium
7
7
7
7
7
7
7
7
7
7
Number
of
deaths
5
0
2
5
0
2
1
5
2
1
45
-------
Although some digging in the spawning gravel was observed in early
December, no egg deposition took place. When it became apparent that suc-
cessful spawning behavior was not likely to occur, eggs were stripped from
two or three females from each test concentration (except 50 yg/1) and
control group, and these were fertilized with milt from males from the same
test conditions. Fish that were held under replicate conditions, and were
ripe but were not artificially spawned, did not spawn in the test chambers
even though they were held there until March 12, 1975. Variation in the
number of eggs recovered from females under different test conditions appeared
to be more related to the size of the females than to toxicant concentration
(Table 18).
Fertilization was 100 percent for eggs from control fish and from those
at 5.4, 10.7, and 21.8 yg/1 TRC, but embryo mortality was relatively high
in all groups (Table 19). Mortality of alevins—after hatching—was low
in controls (4.5 percent), at 5.4 yg/1 (0.4 percent), and at 10.7 yg/1
(0 percent), but was 17.4 percent at 21.8 yg/1 TRC (Table 19). And yolk
utilization and growth of alevins at 21.8 yg/1 was lower than for the controls
and those at 5.4 Pg/1 (Table 19). Results very similar to these were
obtained when fertilized eggs of brook trout from our brood stock were
introduced into the test chambers for incubation and alevin growth (Table 19).
Embryo and alevin mortality in the latter tests was higher at 21.8 and
50.1 yg/1 than for controls and those at 5.4 and 10.7 yg/1 TRC. And alevin
yolk utilization and growth were lower at 10.7 yg/1 and 21.8 yg/1 than in
the control group (Table 19). All alevins died at 50.1 yg/1.
The two extended tests, with cutthroat trout, much as those with
brook trout, failed to achieve the specifications and objectives of "partial
chronic tests." Only a few of the cutthroat trout came into breeding
condition in late April, in both tests, and these failed to spawn.
Cutthroat mortality was quite high but apparently was unrelated to toxicant
concentration up to about 50 yg/1 TRC (Table 20). Much as with the brook
trout, we obviously failed to provide suitable conditions for spawning or
even for survival over very extended periods. This is, perhaps, not surpis-
ing, in view of the close confinement of about seven rather large fish,
in each aquarium, even though others have reported some success with brook
trout under specified partial chronic conditions (McKim and Benoit, 1971).
In the brook trout tests, fish held at the highest concentration of
chloramine were observed to consume less food. This was also observed in
the extended experiments with cutthroat trout. Food consumption of the
cutthroat trout was measured on three different days, and this confirmed
the casual observations that consumption was much lower at concentrations of
chloramines near 50 yg/1 (Table 21).
46
-------
TABLE 18. LENGTH AND WET BODY WEIGHT AND THE NUMBER OF EGGS KHICH WERE
STRIPPED FROM FEMALE BROOK TROUT EXPOSED TO INORGANIC CHLORAMINES
DURING THE 1974-75 PARTIAL CHRONIC EXPERIMENT. THE EGGS WERE
STRIPPED ON DECEMBER 16, 1974, EXCEPT FOR THOSE FISH EXPOSED TO
50.1 pg/1 WHOSE OVA WERE NOT RIPE UNTIL FEBRUARY 7.
ConcentrationLengthWetNo. of
TRC (cm) weight eggs *
Cng/1]
0 Ccontrol)
5.4
10.7
21.8
21.0
20.5
23.0
16.2
16.3
18.9
17.3
16.0
18.0
103
94
123
38
40
61
43
45
53
260
187
214
46
60
122
24
66
98
50.1 19.2 51 220
* At least two males were used to fertilize the eggs in each case except
the 50.1 Pg/1 fish which were necessarily fertilized with milt from only
1 control male since there were no ripe males in the 50.1 Pg/1 aquaria on
February 7.
47
-------
00
TABLE 19. INFLUENCE OF CONTINUOUS EXPOSURE TO INORGANIC CHLORAMINES IN THE PERCENT FERTILIZATION, EMBRYO SURVIVAL AND
HATCH, AND ALEVIN SURVIVAL AND GROWTH OF BROOK TROUT PROGENY FROM ADULTS KEPT UNDER PARTIAL CHRONIC TEST
CONDITIONS OR FROM OUR BROOD STOCK. NINETY-FIVE PERCENT OF EACH GROUP OF EMBRYOS FROM ADULTS IN THE PARTIAL
CHRONIC TEST HATCHED BY FEBRUARY 7, BUT RANGED FROM FEBRUARY 7 TO 9 FOR GROUPS OF EMBRYOS FROM THE BROOD STOCK.
Concentration
TRC
(Mg/D
No.
of Percent
Embryo
mortality
eggs fertilized No. %
Embryo
hatch
No.
Off -spring
0
(control)
5.4
10.7
21.8
50.1
447
320
146
164
220
100
100
100
100
0
226
56
104*
72
-
50.6
17.5
71.2
43.9
-
221
264
42
92
-
Off -spring
0
(control)
5.4
10.7
21.8
50.1
52
36
27
64
79
100
100
100
100
100
12
7
11
32
32
23.1
19.4
40.7
50.0
40.5
40
29
16
32
47
*
Alevin mortality
Percent of hatch Percent
swim-up state of hatch
No. (March 7-10) (March 12)
Alevins (March 12)
mg dry wt Percent
per alevin yolk
(w/o yolk) (wet wt)
from partial chronic test
49.4
82.5
28.8
56.1
-
from brood
76.9
80.6
59.3
50.0
10 4.5 4.5
1 .4 .4
0* 0
16 17.4* 17.4
-
stock
2 5.0 5.0
0* 0
00 0
9 28.1* 28.1
10.1 2.0
8.0 1.7
-
7.0 15.8
-
14.0 16.7
12.0 20.3
7.0 42.4
59.5 47** 100.0 100. 0
Embryos were infested with fungus.
# Until swim-up stage (March 7-10), alevins then escaped from test chambers,
Alevins were lethargic and usually remained either near or on the bottoms of the test chambers,
Before dying these alevins were lethargic and remained on the bottoms of the test chambers
-------
TABLE 20. NUMBER OF DEATHS OF ADULT CUTTHROAT TROUT EXPOSED TO INORGANIC
CHLORAMINES DURING THE 1974 AND 1974-75 PARTIAL CHRONIC TESTS,
Concentration Repl icate
TRC aquaria
Oig/U
1974
0 (control) A
B
4.3 A
B
9.2 A
B
23.1 A
B
54.9 A
B
1974-75
0 (control) A
B
5.9 A
B
10.5 A
B
18.8 A
B
48,4 A
B
No. of fish
in each
aquarium
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
Number
of
deaths
2
4
2
4
1
2
2
1
5
5
2
4
4
5
2
5
6
4
5
3
49
-------
TABLE 21. INFLUENCE OF INORGANIC CHLORAMINES ON THE FOOD CONSUMPTION
(EXPRESSED AS PERCENT WET BODY WEIGHT OF EACH GROUP OF FISH)
OF ADULT CUTTHROAT TROUT FOR THREE DAYS DURING THE 1974 PARTIAL
CHRONIC EXPERIMENT. A SEVEN PERCENT FOOD RATION WAS FED DAILY
TO EACH GROUP OF FISH (A § B) AT EACH TOXICANT CONCENTRATION AND
CONTROL. FOOD WAS AVAILABLE FOR 24 HOURS.
TRC
(ug/D
0
4.3
9.2
23.1
54.9
March
A
3.4
1.5
2.2
1.4
0.1
27
B
0.7
2.9
1.3
0.3
0.2
April 3
A B
3.4 1.1
2.5 2.5
2.8 2.8
1.4 2.1
0.2 0.2
April 5
A B
3.1 2.1
1.3 1.1
2.2 1.8
1.5 1.4
0 0
Fungal infection of developing embryos prevented successful completion
of the two "partial chronic tests" we performed with crayfish. But, in some
respects, the crayfish experiments were more successful than those Conducted
with trout. Egg deposition, spermatophore placement, and fertilization of
eggs occurred at all concentrations of chloramines tested except the highest,
373 yg/1 TRC (Table 22). And mortality data provided good evidence that
levels of chloramines lethal to crayfish subjected to long-term exposure are
certainly no higher and probably are lower than those for trout (Table 22),
even though crayfish are very much more tolerant to chloramines than are
trout when exposures is for 96 hours (Table 14). Mortalities of crayfish at
relatively low concentrations of chloramines were often associated with
molting, especially at the chloramine concentration of 48.6 yg/1 (Table 23).
During the first experiment conducted from August 20, 1974 to May 1,
1975 (254-day exposure) all crayfish died that were exposed to 170 and 373
yg/1 TRC (Table 22). Those exposed to the latter concentration died within
a few weeks. As these animals (group 1) died, they were replaced (group 2),
and the replacements also died after a few weeks of exposure (Table 22).
Extensive mortalities occurred among crayfish exposed to 80.1 and 38.7 yg/1
TRC, 75 and 60 percent respectively. Three control crayfish died, one death
resulting from an unsuccessful molt; males killed the other two females
during mating.
50
-------
TABLE 22. SPAWNING PERFORMANCE AND DEATHS OF ADULT CRAYFISH EXPOSED TO INORGANIC CHLORAMINES DURING THE 1974-75 AND 1975-76
PARTIAL CHRONIC EXPERIMENTS. A AND B REFER TO REPLICATE AQUARIA AT EACH TOXICANT CONCENTRATION AND CONTROL.
Inorganic chloramine concentrations (TRC, yg/1)
during Test 1 (1974-75)
373,0
0 38.7 80.1 170.3 Group 1 Group 2
AB ABAB AB ABA. B
No. crayfish
per tank 10 10 10 10 10 10 10 10 10 10 10 10
Spermatophore
depositions 55 55 55 55 43 03
Egg «
depositions 53 55 54 55 11 10
Adult mortality
254 days of exposure
Males 00 33 32 55 55 55
Females 1**2 22 24 45 55 55
365 days of exposure
Males - - -- -- -- -- --
Females - - -- -- -- -- - -
Inorganic chloramine concentrations
during Test 2 (1975-76)
0 25,2 48
ABABA
88888
4 4 4 3* 4
44434
00000
00010
10014
02123
,6 99.4
B A
8 8
*
3 4
3 4
0 2
1 2
4 4
(TRC, Mg/1)
194.0
BAB
888
*
244
2 3* 4
444
444
444
1 female died after an unsuccessful molt before the spawning period.
2 females killed by males during mating.
1 female killed by male during mating.
Unsuccessful molt.
-------
TABLE 23. NUMBER OF MOLTS AND THE NUMBER OF DEATHS ASSOCIATED WITH MOLTING
FOR CRAYFISH EXPOSED TO INORGANIC CHLORAMINES DURING THE 1975-76
PARTIAL CHRONIC TEST. DEATHS OCCURRED EITHER DURING MOLTING OR
PRIOR TO HARDENING OF THE NEW CARAPACES.
Concentration
TRC
(lig/1)
0 (control)
25.2
48.6
99.4*
194.0*
Replicate
aquaria
A
B
A
B
A
B
A
B
A
B
No. of crayfish
in each
aquarium
8
8
8
8
8
8
8
8
8
8
No.
molting
8
5
8
6
8
7
1
0
1
1
Deaths
associated
with molting
0
0
1
2
7
7
1
0
0
0
* Most of these crayfish had died prior to the major molting period
(spring, 1976).
During the first 254 days of the second test—conducted from August 14,
1975 to August 14, 1976 (365-day exposure)—all the animals exposed to
194 yg/1 died, 75 percent died at 99.4 yg/l> and 6 percent died at 48.6 and
25.2 yg/1 (Table 22). No control crayfish died. For the entire 365 days of
exposure, however, all the animals exposed to 99.4 yg/1 died, 94 percent
died at 48.6 yg/1 and about 19 percent died at 25.2yg/l and in the controls
(Table 22). The days of exposure to 50 percent mortality of the crayfish
decreased with increasing toxicant concentrations in both tests, as is
clearly shown in Figure 10.
During the first experiment, one control crayfish and two crayfish exposed
to 38.7 yg/1 TRC molted in September. The former died but the latter molted
successfully. During the second experiment, nearly all molting occurred
in the spring of 1976, after 254 days of exposure. Most crayfish exposed
to 48.6 yg/1 TRC died during the molting process, while crayfish exposed
to 25.2 yg/1 had fewer mortalities that could be associated with molting
(Table 23). Deaths among control animals were not attributable to molting.
In both experiments, nearly all the males successfully deposited
spermatophores (Table 22). Only those exposed to 373 yg/1 TRC during the
first experiment had a reduction in performance—particularly the second
group—as compared to the controls. Two males of the second group deposited
spermatophores on the heads of females. Egg depositions occurred about 10
52
-------
H
<
cr
o
^
H
UJ
o
a:
UJ
a.
o
m
o
o
365
300
200
100
80
60
40
20
u
8
—
O
• to
^^
.
to
o
• 1974-75 »
0 1975-76 I
„ 1 1 1 1 1 1 1 I 1 . . I
10
20
40 60 80 100
TRC
200
400
Figure 10. Relationship between the mean concentration of inorganic
chloramines (TRC) and the days of exposure until 50 percent mortality of
adult crayfish kept under partial chronic conditions during tests conducted
in 1974-75 and 1975-76. The two data points with arrows pointing up indicate
that fewer than 50 percent of the crayfish had died at that concentration by
the end of the 365 day exposure [1975-76).
53
-------
days after spermatophore depositions in each experiment and were apparently
not affected by the toxicant at concentrations between 25.2 and 194.0 yg/1
(Table 22). Only those females exposed to 373 yg/1 TRC performed poorly,
most dying prior to egg deposition. Embryo survival was poor for all test
and control groups of crayfish. In nearly all cases, the embryos became
infested with fungus and died, even those of control crayfish.
CHRONIC TOXICITY OF CHLORAMINES: DEVELOPMENT, SURVIVAL, AND GROWTH
Successful "partial chronic tests" permit some evaluation of the effects
of toxicants on gonadal maturation, reproductive behavior, embryonic develop-
ment, growth, and subsequent maturing and reproduction of progeny.
But successful reproductive behavior of adult fish cannot always be expected
under conditions specified for partial chronic tests, this often vitiating
the approach. And, of course, partial chronic tests are hardly appropriate
for anadromous species such as Pacific salmon. For these species, studies
should be conducted on the effects of toxicants on life history stages likely
to be exposed to toxicants.
We conducted experiments with embryos, alevins, and juveniles of coho
salmon, an anadromous species. Effects of relatively low, constant concentra-
tions of chloramines on embryo and alevin survival and on alevin and juvenile
growth were determined. Fish remaining from the experiment on embryo and
alevin survival and alevin growth were retained for studies on the effects
of different concentrations of chloramines on growth of juveniles at differ-
ent ration levels. Control fish from the embryo-alevin experiment were
separated into groups to be tested at different chloramine concentrations,
so that growth of juveniles having had no prior exposures to chloramines
could be studied. In addition, the growth of juveniles developing from
embryos and alevins exposed to various concentrations of chloramines was
studied at these levels of toxicant exposure. Some juveniles from each
prior test condition were held in water containing no toxicant in order to
determine whether or not there were residual effects on growth. A second
experiment on the effects of different concentrations of chloramines on the
growth of juvenile coho salmon fed different ration levels was conducted
with juveniles raised at our hatchery and not previously involved in any
experiment.
The experiment on the effects of continuous exposure to constant
concentrations of chloramines on embryonic survival and hatching and on the
survival and growth of alevins of coho salmon was begun on November 22,
1973. The hatching process was about 95 percent complete under all test
conditions by January 7, 1974. At the test concentrations of 0 [control A),
0 (control B), 5, 11, 23, and 47 jag/1 TRC, survival of embryos was appar-
ently unaffected by chloramines, total mortality among 600 embryos in each
control and toxicant concentration being 75, 26, 32, 40, 54, and 26,
respectively. Hatching did not appear to be affected by the presence of
chloramines at these concentrations.
54
-------
The activity and behavior of alevins was affected by exposure to
chloramine concentrations of 23 and 47 ug/1 but not by exposure to concentra-
tions of 5 and 11 ug/1. Within one week after hatching, alevins exposed to
47 ug/1 became lethargic, and many were lying on their sides on the chamber
bottoms. By January 22, most of these alevins were lying on their sides
and exhibited an abnormal, spiral, swimming behavior when disturbed. This
syndrome began to appear in alevins exposed to 23 pg/1 TRC by January 22
and was general in this group by January 28.
Mortality among alevins at all test conditions was very low during the
first two weeks after hatching, as is shown in Figure 11. But by January
28, mortality of alevins at 47 ug/1 TRC had increased markedly, an increase
that continued and reached 70 percent by February 7. During this entire
time, mortality of controls and those alevins held at chloramine concentra-
tions up to and including 23 ug/1 remained very low (Fig. 11). Substantial
mortality occurred in all groups including controls on February 9, as a
result of water flow interruption and low concentrations of dissolved oxygen
(Fig. 11). Little or no further mortality occurred in any of the test groups
by February 11, except among alevins at 47 ug/1 where mortality reached 97
percent.
The growth of alevins was severely affected by chloramines at concen-
trations of 23 and 47 ug/1 but not at 5 and 11 ug/1, as shown in Figure 12.
On January 7, at which time hatching was approximately 95 percent complete
in all test groups, the sizes of alevins in all groups were very nearly the
same, about 0.01 gram dry weight per individual, exclusive of yolk. This,
of course, is strong evidence that embryonic growth had not been affected by
chloramines at the concentrations tested. But, as already noted, subsequent
alevin growth was severely affected at 23 and 47 pg/1, weights of these fish
being only about 80 and 45 percent those of controls by February 11 (Fig. 12)
The relative growth rates (instantaneous coefficients of growth) of
fish and other animals are generally a negative function of body weight.
Thus, as body weight* increases with growth through time, relative growth
rates decline. The relationships between relative growth rate and body
weight (exclusive of yolk materials) for alevins tested at chloramine con-
centrations of 5 and 11 ug/1 were not significantly different from those
of control groups (Fig 13). Relative growth rates at given body weights
(reached at different times) of alevins tested at 23 and 47 ug/1 TRC were
much lower than those of the other groups.
Determination and comparison of the caloric or heat value of alevin
tissue and yolk material permitted evaluation in equivalent energy terms
of yolk utilization for growth and for metabolism. Caloric values per gram
of alevin tissue (exclusive of yolk materials) were essentially the same for
all test conditions (Table 24). But total caloric value in grams of alevin
tissue and yolk together was higher at 23 and 47 ug/1 TRC than at the lower
test concentrations and for the controls. Total yolk utilization, yolk
utilization for growth, and yolk utilization for metabolism were clearly
less for alevins held at 47 ug/1 TRC than for those held under control
conditions (Table 25).
55
-------
in
ON
JU O H- T1
rt 0 3 H-
3- rtqg
p. O CD p
3 H H>
P* tfl <^ CD
p. pj (u
r) i— • i— • i— <
W 3 t/l K-"
r+ 0
CD 3 P
® n *V. -
H-&&I §;• ^
O CD P VN v ft
0 I 4 rt S»«Sr
Hi P CD H- "^T v^.
•- 3 < X
I-- CD rt O ^
Q < fn
£ P- O P ***
3 O i-
P- 3 O
M- CD O -> i-t 4 3 tM
H - ^00
era o *•< ^
CD HH Hi
3 3 ' — i fk »
• n H- w iv
H 3 C /.i
CD o a VX
P w era o
i^a: r\>
£ P o 3 oo
p. i
x ^ d i O)
LJ* ^ *-" %PVP
COOP
^ 0 ^-^
1 IB- i\J
p r*
CD 2 g.
3 «
CUMULATIVE ALEVIN MORTALITY
( at 5 day intervals )
^ l\) 4* O> 00 O
3 o o o o o
1 1 1 1 1 1 1 1 1 1 1
k
^H *^ ^^ ^^ ^* ^ ^^1
^F ^^^
^ M% ^^ ^H ^^ ^^ ^^»
V N 01 o o O
l& 33
i^H ^^ ^S ^^m
o 2. S
^^
^ OD > ^
^f "*~*
*
fll
^
HP ^
"flp ^
•
k*S ^
cT 1^ >• Tl ^ LOW U.U.
o • >•* a >
-------
0.081-
0.06
o>
0.04
X
e>
UJ
on
0.02
0.01
0
TRC(|jg/l)
o Control A
• Control B
D5
• II
A 23
A 47
14 21
JAN
28
i
FEB
II
14
21
28
35
DAYS FROM 95% HATCH
Figure 12. Relationships between the mean dry weight of alevins and the
days from 95 percent alevin hatch at different concentrations of inorganic
chloramines (TRC) in the coho salmon embryo-alevin experiment.
57
-------
O.IOr-
From 95%
Alevin Hatch
O Control A
• Control B
0 5
• II
A 23
A 47
0.01 0.02
0.03 0.04 0.05 0.06
DRY WEIGHT (g)
0.07 0.08
Figure 13. Relationships between instantaneous growth rate and mean dry
weight of alevins at different concentrations of inorganic chloramines (TRC)
in the coho salmon embryo-alevin experiment. The days from 95 percent hatch
are indicated.
58
-------
TABLE 24. INFLUENCES OF CONTINUOUS EXPOSURES TO INORGANIC CHLORAMINES ON THE
CALORIC VALUE (CALORIES/G) FOR ALEVINS WITH YOLK AND WITH THE
YOLK REMOVED DURING THE COHO SALMON EMBRYO-ALEVIN EXPERIMENT.
SAMPLES WITH YOLK CONSISTED OF 5 ALEVINS WHEREAS SAMPLES WITHOUT
YOLK CONSISTED OF 25 ALEVINS.
Concentration
TRC
(Vg/D
0 A
0
0 B
0
5
5
11
11
23
23
47
47
January 7
without
yolk
5447
5242
5320
5279
5253
5308
5297
5320
5237
5227
5332
5368
January 28
with
yolk
5770
5648
-
-
-
-
-
-
-
-
5949
5935
without
yolk
5302
5254
-
-
-
-
_
-
-
-
5406
5589
February 4
with
yolk
5432
5404
5501
5553
5436
5456
5536
5325
5698
5720
5944
5883
without
yolk
5390
5440
5462
53S9
5504
5423
5445
5540
5521
5618
5553
5509
In the first experiment on the growth of juvenile coho salmon at
different concentrations of chloramines and at different ration levels, fish
previously involved in the embryo and alevin experiment were used. In one
part of this growth experiment, the growth of fish originally developing
under control conditions of no exposure to the toxicant was then determined
under different concentrations of the toxicant as well as under continued
control conditions. In this series of groups, some effect of 11 pg/1 TRC
was apparent on the growth of juveniles fed at the 8 percent ration level, of
which these fish consumed only about 83 percent (Fig* 14). Juveniles in
this group having no previous exposure to chloramines and then exposed
to 22 vg/1 consumed much less food at both ration levels than did those
in any of the other treatments. These fish used for growth with much less
efficiency the food they did consume and exhibited much less growth than
did those in the other groups (Fig. 14). The growth of juveniles having
originally developed at 5 and 11 pg/1 TRC was not affected when these fish
were further tested at these concentrations, some selection or acclimation
apparently having occurred (Fig. 14). Growth was high in those groups of
fish that had been exposed to 0, 5, 11, or 23 ug/1 inorganic chloramines
(TRC) during development and then were transferred to water containing no
toxicant during the first growth experiment (data not shown).
-------
TABLE 25. COMPARISON OF YOLK METABOLISM FROM JANUARY 28 to FEBRUARY 4 BY CONTROL ALEVINS AND ALEVINS EXPOSED
TO 47 wg/1 INORGANIC CHLORAMINES IN THE COHO SALMON EMBRYO-ALEVIN EXPERIMENT, YOLK UTILIZATION
RATE, GROWTH RATE AND TOTAL METABOLIC RATE ARE EXPRESSED AS CALORIES PER KILOCALORIE OF ALEVIN PER
DAY.
Total
residual
chlorine
(iig/i)
0 A
0 B
47
47
Total
yolk
utilized
(cal)
143
141
69
63
Increase
in calories
per a levin
(cal)
58
75
39
29
Mean
calories
per alevin
(kcal)
.347
.335
.191
.190
Efficiency
of yolk
utilization
for growth
(*)
40.6
53.2
56.5
46.0
Yolk
utilization
rate
(cal/kcal/d)
58.8
60.1
51.6
47.4
Growth
rate
(cal/kcal/d)
23.9
32.0
29.1
21.8
Total
metabolic
rate
(cal/kcal/d)
35.0
28.2
22.5
22.3
-------
O
20
10
O o
TRC
pq/i
Control
5
II
22
Exposed
Only As
Juveniles
Exposed
From
Embryos
20
30
40
50
60
70
UJ
en
0)
40r-
^ 30
o
z
UJ
y 20
10
o
20
(B)
30
40
50
60
70
FOOD CONSUMPTION (mg/g/day)
Figure 14. Relationships between food consumption rate and relative growth
rate W and gross efficiency (B) for coho salmon juveniles (from the
embryo-alevin experiment) at different concentrations of inorganic
chloramines (TRC). Some groups of fish had been exposed to the toxicant
since the embryo life stage, others were exposed only during the growth
experiment.
61
-------
In a second experiment on the effects of chloramines on the growth
of juvenile salmonids at different ration levels, the fish used had not
been previously involved in any experiment and thus had no history of expo-
sure to chloramines. In this experiment, marked effects of 23 yg/1 TRC on
the food consumption, growth, and efficiency of food utilization for growth
were observed (Fig. 15). This experiment tended to confirm the effects on
food consumption, growth, and efficiency of food utilization for growth
observed at 22 ug/1 TRC in the previous experiment. No effects of concen-
trations of 5 and 10 yg/1 TRC on food utilization and growth were demonstrated
in this experiment.
BEHAVIORAL EFFECTS OF CHLORINE AND CHLORAMINES
The behavior of bass and other species subjected to intermittent expo-
sure to both lethal and nonlethal concentrations of the chlorine was observed
to be markedly modified. Other laboratory and field investigations have shown
behavioral changes to occur in fish subjected to intermittent exposure to
chlorine, and it is of importance to describe such behavioral changes and
relate them to effective chlorine concentrations.
In aquarium tests, largemouth bass swam slowly and deliberately, had slow
opercular movement, and seldom "coughed," during the 30-minute acclimation
period before chlorine was introduced in each test. The behavior of the
fish changed when chlorine was introduced at concentrations near median
lethal ones. Behavioral changes usually occurred in the following sequence:
[a) increased rates of swimming, opercular movement, and coughing; (b)
reduced swimming activity with the animals "nearing the surface;11 (c) rapid
swimming with "thrashing" at the water surface and some jumping; (d) "lethar-
gic swimming" and frequent collisions with aquaria walls and other fish; (e)
dorsal portion of heads "bobbing" into the atmosphere; (f) resting "on the
bottoms" of the aquaria, accompanied by heavy and pulsating opercular move-
ment; and (g) turned over or belly up. Most of the behavioral changes occur-
red in all the tests, even when exposures to test solutions were not lethal.
But despite the consistency of the order in this behavioral sequence, par-
ticular behavioral changes often failed to occur. For example, behaviors
(f) and (g) did not occur when the fish were subjected to test solutions
that were not acutely toxic. And even at the highest concentrations, some
of the behaviors (e.g., bobbing) did not always occur. In such cases, the
fish appeared to pass through the behavioral sequence so rapidly that some
behavioral changes were skipped. Fish not dying from exposure to chlorine
usually exhibited normal behavior within 24 hours after exposurfe.
Judging the time to first occurrence of each kind of behavior was rather
subjective, but this time appeared to decrease with increasing toxicant
concentration. Only the first occurrence of a behavior in each group of fish
was recorded. It was not possible to relate times to occurrence to areas
under the time-concentration curves, because the bass exhibited a particular
response at different times when the cures were of different height, even
when areas under the cures were the same. Thus our preliminary results
were expressed as mean exposure concentration to the time of first
occurrence of a behavior. When largemouth hass were exposed to two 90-
minute square patterns of exposure to chlorine, with a two-hour
62
-------
O
cr
o
3 30
o
uj 20
o
u.
Lu
U 10
0
20
/
(B)
i
30 40 50 60 70 80
FOOD CONSUMPTION (mg/g/day)
90
Figure 15. Relationships between food consumption rate and relative growth
rate (A) and gross efficiency (B) for coho salmon juveniles at different
concentrations of inorganic chloramines (TRC).
63
-------
intervening period, the time to "bobbing" decreased as the toxicant concen-
tration increased in the first exposure (Fig. 16). In the second exposure,
bobbing occurred earlier than in the first, at nearly equal toxicant con-
centrations. Two groups did not exhibit bobbing in the second exposure.
These had been exposed to acutely toxic concentrations in the first expo-
sure, and some of the fish were on the bottoms of the aquaria at the start
of the second exposure. The other fish in the aquaria were active, but
their behavioral changes progressed quickly through the sequence, and bobbing
behavior appeared to be skipped during the second exposure.
As in the tests with bass, the sequence of behavioral changes for each
of the other species subjected to intermittent exposures to chlorine was
not always complete, particularly at concentrations at which no fish died.
The following description of the behavior of each species during the tests
represents behavior occurring at or near the lethal concentration, or in
the case of the bullhead, the highest toxicant concentrations used.
The behavioral changes of the trout were similar to those of the bass,
"thrashing" and "bobbing" frequently occurring. The bluegill also responded
in much the same manner as did the bass, except the bluegill did not thrash
or jump. The shiner and dace differed from the above in their behavioral
changes. During exposure, the swimming activity of these normally active
swimmers continuously declined. Neither species exhibited "nearing surface,"
thrashing, or jumping, and only the shiner exhibited the bobbing behavior.
For the shiner, resting on the bottom of "the aquarium did not occur until they
were very near "turnover;" in the dace this behavior occurred well in
advance of turnover. Some of the behavioral changes of the bullhead were
markedly different from those of the other species. Prior to toxicant intro-
duction, bullhead swam actively throughout the aquaria. In each test with
toxicant, behavioral changes of bullhead acclimated to 24 C and tested at
22 C usually occurred in the following sequence: (1} active swimming near
the bottom of the aquaria; (2} active swimming near the surface of the water;
(3) lethargic swimming followed by maintaining a vertical [head-up) position
at the water surface; (4) resting on the bottom; (5) sporadic swimming, and
(6) again resting on the bottom, accompanied by either stopped or greatly
reduced opercular movement. The toxicant concentrations tested were not
sufficiently high to induce "turnover" during the exposures. Those bullhead
acclimated to 12.5 C and tested at 22 C exhibited the same sequence of behavior,
except the vertical position did not occur. These fish appeared to progress
through the sequence faster than the groups acclimated to 24 C and tested at
2.2 C.
Some behavioral observations were made on largemouth bass held in a cur-
rent of flowing water and exposed to chlorine. During acclimation periods
and control tests, the bass spent nearly all of their time under the cover
provided. Within a few minutes after the addition of the toxicant, however,
the fish typically began swimming throughout the test chamber but would return
to the cover for short periods. If the concentration of chlorine was suffi-
ciently high, the fish first "bobbed" and then "rested" with their caudal
peduncle pressed against the downstream screen. Control fish were not observed
to do this. Occurrence of the behavioral patterns outlined for the aquarium
64
-------
50
£ 40
3
U
u
o
0>
E
20
10
• One Exposure
o First of Two Exposures
• Second Exposure
o
o
o
o
o
0
I 2 3
TRC (mg /1)
Figure 16. Relationships between the time to first occurrence of bobbing
behavior of largemouth bass and the mean concentration of free residual
chlorine (TRC) in the aquaria. Mean concentration calculated for the period
until first occurrence, during one and two 90-minute square patterns of
exposure.
65
-------
studies was difficult to judge, bobbing and resting being the easiest to
detect. Bobbing appeared to occur at about the same exposure (mg/1 x min) in
the square and spike patterns of exposure, but resting appeared to occur at
a lower exposure in spike than in square patterns (Table 26}.
Most of the behavioral changes that occurred at acutely toxic
concentrations of chlorine also occurred at some sublethal concentrations.
These results suggest that chlorinated discharges that are not acutely
toxic could adversely affect the behavior and thus affect survival of bass.
Unfortunately, very little is known about the extent to which fish are
exposed to residual chlorine in the chlorinated discharge plumes of power
generation plants. Certainly the location of the fish in such plumes could
influence their ability to escape before serious modification of their
behavior occurred. In view of these potential impacts of chlorine on the
behavior of fish distributed at different locations in plumes of chlori-
nated discharges, we conducted two additional behavioral experiments with
bass subjected to intermittent exposure to residual chlorine.
In one experiment, we attempted to determine the threshold concentrations
of chlorine at which particular behavioral changes occurred in bass unable to
avoid exposure. In a second experiment, we determined if bass would move out
of or avoid water containing chlorine or chloramines. We also determined
the extent to which such avoidance was correlated with the concentrations
and species (inorganic chloramines or free residual chlorine) of total
residual chlorine.
In each test in the first experiment, two bass of different mean dry
weights (7.37 and 14.63 g/fish) were subjected together to either the square
or the spike pattern of exposure to sublethal concentrations of chlorine.
At the highest exposures, expressed as mg/1 x minutes (Table 27), the sequence
of behavioral changes was consistent with that occurring in the acute toxicity
tests described earlier, except that none of the fish turned over (belly up)
or died (Fig. 17). The threshold concentrations at which each behavior
occurred were difficult to determine, but "nearing the surface" occurred at a
lower exposure than did the other behaviors, in each type of exposure for
each size group of fish. Nonetheless, an important aspect of these results
is that the five behaviors occurred when the fish were subjected to the
square and spike patterns of exposure at sublethal levels. For fish of the
sizes used here, the median lethal level would have been at an exposure of
more than 148 mg/1 x minutes. In these tests, however, most of the changes
of behavior occurred from exposures of less than about 102 mg/1 x minutes.
Based upon these results, it seems possible that bass, of the sizes tested
here, could undergo considerable changes in behavior, if they were not soon
able to escape from chlorinated discharge plumes. This could be so even
for discharges that are not acutely toxic to the fish.
Before the results of the second experiment are presented, it is impor-
tant that we make clear what we will mean by the expressions "move out"
and "avoid." In each test, the bass showed a definite preference to position
themselves in the toxicant discharge area (Fig. 1), so long as toxicant was
not being introduced. During tests without toxicant, all of the fish (which
66
-------
TABLE 26. INFLUENCE OF SQUARE AND SPIKE EXPOSURES OF FREE RESIDUAL CHLORINE ON THE OCCURRENCE OF THE BOBBING AND RESTING BEHAVIORAL
RESPONSES AND DEATH BY LARGEMOUTH BASS IN THE FLOWING WATER INTERMITTENT EXPOSURE EXPERIMENT.
Mean
plateau
concentration
TRC (yg/1)
0
0
267
368
564
759
986
1095
1224
1297
1492
1493
1510
1560
1689
1850
1968
Square
Mean
concentration
TRC (yg/1)
0
0
160
254
395
565
735
800
895
970
1092
1074
1121
1177
1252
1375
1469
Exposure
Area
(rag/1 x Occurrence (+ or -J
minutes) Bobbing Resting Death
0 -
0 -
14.41
22.88 - - -
35.60 + - -
50.85 + - -
66.11 +
72.04 + +
80.51 + + -
87.29 + +
98.31 + +
98.48 + -*• -
100.85 + + -
105.94 + +
112.72 + +
123.74 + + +
132.21 + + +
Spike Exposure
Mean
plateau
concentration
TRC (yg/1)
0
0
940
1330
1660
1800
2070
2100
2790
3280
3440
3880
4800
5250
Mean
concentration
TRC (yg/1)
0
0
352
484
619
673
807
834
1090
1291
1332
1534
1910
2072
Area
(rag/1 x
minutes)
0
0
22.21
30.51
38.99
42.38
50.85
52.55
68.65
81.36
83.90
96.62
120.35
130.52
Occurrence (+ or -)
Bobbing Resting Death
_
_
*
+
_
+
+
+ +
+ +
+ +
+ +
+ t
+ +
+ +
+ +
* Fish was extremely active during the early portion of the test and appeared to he exhausted during the rest of the test,
-------
TABLE 27. CONCENTRATIONS OF FREE RESIDUAL CHLORINE AND AREAS UNDER THE TIME-CONCENTRATION CURVES
WITH SQUARE AND SPIKE EXPOSURES IN THE BEHAVIORAL EXPERIMENT WITH LARGEMOUTH BASS
WEIGHING EITHER 7.37 OR 14.65 G PER FISH.
00
Square Exposure
Mean
plateau
concentration
TRC Og/1)
140
280
351
431
542
705
1081
1230
1437
2138
2130
Mean
concentration
TRC (pg/1)
94
198
254
311
396
509
791
942
1073
1554
1563
Area
(mg/1 x
minutes)
8.48
17.80
22.88
27.97
35.60
45.77
71.19
84.75
96.62
139.84
140.69
Spike
Mean
plateau
concentration
TRC (yg/1)
400
855
1260
1650
2030
3010
5980
6980
Exposure
Mean
concentration
TRC (yg/1)
121
283
431
565
686
1090
2072
2246
Area
(mg/1 x
minutes)
7.63
17.80
27.12
35.60
43.22
68.65
130.52
141.53
-------
a\
GROUP I (7.37g/fish)
GROUP 2 (I4.63g/fish)
0
40
SQUARE EXPOSURE (mg/l x minutes)
80 120 160 0 40 80
120
160
NS-OOM* •
T -O OOO0 •
L -O OOOO •
B -O OOOO O
o LO 0000 o
0 40
NS-0 • •••
T -0 O O • •
L -O O OO0
B -0 O 0 • •
0 LQ O O O •
• • • •• NS
O • • M T
• • • M L
0 • • M B
• • • W 0
-OOO** • • • • W
-O OOO* • • • • «•
-O OOOO • • • • W
-O OOOO O O O • W
-ooooo o • • • ••
SPIKE EXPOSURE {mg/l x minutes)
80 120 160 0 40 80 120 160
• • • NS
• • • T
• • • L
• • • B
• • • 0
1 1 1 1 1 1 1 1
-0 •••• • • *
-O OOOO • • •
-oooo* • • m
-o 0000 o o o
-o 00*0 • • •
Figure 17. Relationships between the areas under the time-concentration curves, expressed as mg/l x
minutes, and the occurrence of five behavioral changes in two weight groups of bass subjected to square
and spike patterns of exposure to free residual chlorine (TRC). Open circles indicate that the
behaviors did not occur; dark circles indicate that the behaviors occurred. Symbols: NS -near the
surface; T - thrashing; L -lethargic swimming; B - bobbing; and 0 -on aquarium bottom.
-------
were tested individually) spent nearly all of the time in the discharge
area of the testing apparatus. During most toxicant tests, the fish moved
out of this area, only to return at irregular intervals for short periods
of time before moving out again. In nearly all tests, the fish returned
to the toxicant discharge area within 15 minutes after toxicant introduction
was terminated. Thus, in the experiment reported here, we determined the
effects the toxicant had on keeping the bass out of the discharge area. We
did not determine avoidance in the same way as did Sprague and Drury (1969).
In this experiment we found that bass spent less time in the discharge
area when exposed to free residual chlorine than when exposed to inorganic
chloramines, at similar concentrations expressed as total residual chlorine
CFig. 18). The amount of time s,pent out of the discharge area increased
significantly with increasing concentration of either toxicant (regression
analysis, P < 0.05). The slopes of the two regression lines were not sig-
nificantly different (analysis of covariance, P > 0.05), but the Y-intercepts
were significantly different (P < 0.05). Although the exposed bass moved out
of the discharge area at all concentrations of free residual chlorine tested,
the threshold for such movement when exposed to inorganic chloramines was
about 0.09 mg/liter total residual chlorine (Fig. 18). Furthermore, none
of the fish in these tests died or showed any of the behavioral changes
described for the above experiment, even though some of the concentrations
would have been acutely toxic to the fish, if they had not moved out of the
discharge area.
In each toxicant test, the bass did not move out of the discharge area
immediately after the toxicant was introduced. These delays and the repeated
returns to the discharge area mentioned above resulted in greater exposures
(expressed as mg/1 x minute) to the toxicants as the concentrations increased
(Fig. 19). The slopes of the regression lines for inorganic chloramines
and free residual chlorine were significant (P < 0.05). But neither the
slopes nor the Y-intercepts of the two lines were significantly different
(P > 0.05). Thus the data were pooled and a single regression line was
calculated (Fig. 19).
CONTINUOUS EXPOSURE OF LABORATORY STREAM COMMUNITIES TO CHLORAMINES
Twelve laboratory stream communities were maintained from May 1, 1975
through June 24, 1976. For purposes of conduct of experiments and analysis
and representation of results, this entire period was separated into nine
shorter experimental periods, which will be referred to by numbers 1 through
9. These periods are shown in Table 28, which presents data on laboratory
stream temperatures for the various experimental periods. The twelve
laboratory stream communities were divided into four groups of three each,
our intention being that one group would provide replicate controls and
each of the other three groups would provide replicates at chloramine
concentrations near 25, 12, and 6 vtg/1. It is our belief that the experi-
ments were less than satisfactory, for a number of reasons. First, we
experienced difficulty in developing stream communities that were as
productive for coho salmon juveniles as we had wished. And variance
in productivity among replicates at particular treatment levels was high.
Further, it was extremely difficult to maintain total chlorine residuals
70
-------
100
80
60
u 40
Q
O
> 20
0
-20
INORGANIC CHLORAMINES
A FREE RESIDUAL CHLORINE
0.04
0.10
0.50
TRC (mg/l)
1.0
4.0
Figure 18. Relationships between the mean toxicant concentration in the toxicant discharge area of the
avoidance apparatus and the avoidance index for largemouth bass subjected to 60-minute exposures to
free residual chlorine or inorganic chloramines, expressed as total residual chlorine (TRC).
-------
60
40
20
I0
o»
m AX* A
A'/
A FREE RESIDUAL CHLORINE
£ • INORGANIC CHLORAMINES
L.I l
J I I
O.I 1.0
TRC (mg/l)
Figure 19. The amount of exposure, expressed as mg/l x minutes, to which
bass were subjected when exposed to either free residual chlorine or
inorganic chloramines.in the avoidance experiment. The dashed lines
represent the relationship for each toxicant. The solid line represents the
relationship for the pooled data since neither the slopes nor the Y-intercepts
of the individual toxicant lines were different significantly (P >0.05).
72
-------
near the values we sought for particular treatments, as can be seen from
Table 29. Finally, it is not at all clear that the total residual chlorine,
as measured by amperometric titration, represented inorganic chloramines.
Around 50 percent of the total residual chlorine appeared as dichloramine,
even though we had expected nearly all the residual chlorine to appear as
monochloramine under the test conditions. As we will later note, this sup-
posed dichloramine fraction, in these streams having high amounts of organic
materials, may have been organic chloramines, of which little is known
regarding toxicity.
TABLE 28. DAILY MEAN HIGH AND MEAN LOW TEMPERATURES (AND STANDARD DEVIATIONS)
IN THE LABORATORY STREAMS FOR EACH EXPERIMENTAL PERIOD IN 1975 AND
1976.
Experimental
period
Inclusive
dates
High
temperature
C
Low
temperature
C
1975
1 5-1 to 5-28 14.03 (1.67) 9.47 (1-17)
2 6-6 to 6-26 16.29 (2.56) 12.01 (1.11)
3 6-27 to 8-5 18.34 (2.35) 13.54 (1.71)
4 8-17 to 9-8 15.59 (1.12) 12.64 (1.12)
5 9-10 to 9-30 16.06 (0.76) 12.89 (1.06)
6 10-8 to 10-31 11.79 (1.39) 10.10 (1.24)
1976
7 4-12 to 5-10
8 5-11 to 6-2
9 6-4 to 6-24
13.19 (2.83)
14.44 (1.67)
16.72 (1.44)
9.58 (2.10),
9.67 (1.19H
13.06 (0.54)
But in spite of these difficulties, a summary of the results is probably
worthwhile. Relatively large amounts of chloramines were introduced into
these laboratory stream communities over a long period of time, and yet there
appeared to be little if any effect on the stream communities. The variability,
even within treatments, that so plagued us experimentally was surely less than
occurs in stream communities exposed to chlorine compounds in nature. And
it is with nature, or something very like nature, that we must be most
concerned in setting stream standards for the protection of aquatic life.
The concentrations of chloramines we found necessary to introduce with
the exchange water to maintain residual concentrations near those we desired.
in the stream varied greatly among the different seasons of the year. In
August and September, introduced concentrations ranged from about 400 to
800 yg/1 TRC (Fig. 20). But in May, June, October, and December, these
introduced concentrations ranged from about 100 to 400 yg/1. As we will
show, these periods roughly coincided with periods during which large amounts
73
-------
TABLE 29. MEAN CONCENTRATIONS (AND STANDARD DEVIATIONS) OF RESIDUAL CHLORINE COMPOUNDS IN EACH LABORATORY STREAM DURING EACH
EXPERIMENTAL PERIOD. SAMPLES WERE TAKEN DAILY IN MIDMORNING.
Experimental
period
1
2
3
4
5
6
7
8
9
TRC
S.D.
TRC
S.D.
TRC
S.D.
TRC
S.D.
TRC
S.D.
TRC
S.D.
TRC
S.D.
TRC
S.D.
TRC
S.D.
Low concentration
S3 S5 N5
Grand
mean
1.2
(1.7)
3.0
(2.2)
1.5
(2.1)
1.2
(2.1)
4.0
(5.2)
3.1
(1.4)
2.2
(1.7)
4.8
(2-0)
5.5
(3.6)
1,0
(2.1)
3.0
(2.1)
1.1
(1.6)
1.0
(1.5)
1.9
(2.5)
4.1
(2.8)
4.0
(2-7)
4.0
(1.8)
4.6
(2.5)
4.8
C4.4)
3.0
(2.9)
1.5
(1.4)
1.4
(2.0)
2.1
(2.6)
2.8
(1.7)
3.3
(2.4)
3.6
(.1-8)
5.0
(2.7)
2.3
3.0
1.4
1.2
2.7
2.9
3.2
4.1
5.0
Medium concentration
S2 N2 NJ
Grand
mean
4.9
(4.0)
9.7
(4.5)
5.4
(4.1)
3.4
(3.2)
6.6
(6.5)
12.4
(3.7)
8.9
(4.7)
11.0
(4.1)
11.4
(4.0)
5.3
(3.2)
9.4
(6.0)
6.3
(4.6)
5.7
(6.6)
7.4
(6.6)
13.1
(6.5)
7.8
(4.2)
8.5
(2.9)
9.7
(3.7)
3.6
(4.0)
8.1
CS.l)
6.3
(4.4)
2.5
(2.6)
6.6
(6.5)
13.0
(5.6)
7,6
(4-4)
7.7
(2.8)
8.1
(4.0)
4.6
9.1
6.0
3,9
6.9
12.8
8.1
9.1
9.7
High concentration
SI S4 N4
12,9
( 9.8)
24.8
(ll.D
17,7
C12.0)
17.9
(15.2)
26.2
(16,1)
26.7
( 8.0)
20.8
( 9.2)
24.7
C 7.6)
22.4
( 8.2)
10.9
C 8,6)
20.8
16,9
(10,9)
10,7
(11.0)
23.2
(15,1)
26.3
(12.8)
20,6
C 8,8)
22.2
C 7,8)
25,9
(9,3)
7.2
(7.8)
21.5
(11.2)
15.1
( 9.4)
11.9
(11.1)
23,4
(14,1)
41.7
(50,0)
18,1
( 8.1)
21.8
( 9-3)
23,1
Grand
mean
10.3
22.4
16,6
13.5
24.3
31,5
19,8
22.9
24,5
Control
Nl N6 S6
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
-------
60
50
*^~
o>
a.
~ 40
UJ
o:
fc 30
UJ
X
H
z 20
o
cr
10
— O MAY
A JUN
• AUG
_ A SEP
n OCT
• DEC ns
/ s
0 /m
P / n'' A '
/ m/§' *'
- / / s' U A S
~ £* U /
1 DX^A * {
Q Q * / A A^
~ D *• Ax -7 y
r\ •* ^A x^* A /
9 !AxxA A *
fffi
•
^ /
/ A
' ^
1 1
700 800 9C
TRC ADDED (pg/l)
Figure 20. Relationships between the mean daily concentrations of chloramines (TRC') in laboratory
stream renewal water and the concentrations of the toxicant in one of the streams, for selected
periods in 1972.
-------
and lesser amounts, respectively, of organic matter were present in the
streams.
Even over a 24-hour period in any one stream, during which the
concentration of chloramines introduced was maintained quite constant, the
concentration of total residual chlorine changed dramatically in a cyclical
manner, as can be seen in the example given in Figure 21. For different
months, the amount of chloramines necessarily introduced to maintain a
desired concentration of total residual chlorine in a particular stream
was roughly correlated with the amounts of organic matter and chlorophyll
present (Fig. 22). Mean concentrations of total residual chlorine main-
tained at the three treatment levels, for the entire experimental period,
were 20.6, 7,8, and 2.9 \igfl (Table 29}, and these are the treatment
levels to which we will refer in the ensuing discussion.
Total organic matter, exclusive of macroinvertebrates and fish,
varied considerably between replicates within each treatment (Table 30).
But the major variation in organic matter was between seasons, the
highest mean values being maintained during experimental periods 3 and 4
(late June to early September), as can be seen from Figure 23. The
stream communities exposed to the highest concentrations of total residual
chlorine (20.6 ug/1) tended to have the lowest amounts of organic matter
(Table 30; Fig. 23), whatever may have been the causal relationships.
The major differences and changes in total chlorophyll concentration
(chlorophylls a, b, and c) were also seasonal and did not appear to be
consistently associated with differences in chloramine treatments (Fig.
24). None of this is to say that chlorine treatment did not have effects
on the algal communities, such as effects on species composition.
But we did not determine algal species composition.
A reasonably diverse assemblage of species of macroinvertebrates colo-
nized all streams, the major tendency being an increase in their total bio-
mass throughout the entire period of experiments (Table 31). All major
groups eventually appeared in all streams, and differences in their abundances
were not clearly associated with differences in concentration of total resid-
ual chlorine. Very high biomasses of an isopod, Asellus, developed in one
stream at the high concentration of total residual chlorine—the first stream
to be colonized by this species. But eventually all streams were to be
colonized by this species, wiii.ch became moderately abundant in most streams.
It is unlikely that abundance of this organism was closely related to the
presence or absence of chlorine compounds.
The same major groups of insects appeared in all streams: Chironomidae,
Coleoptera, Ephemeroptera, Odonata, Plecoptera, and Trichoptera. With the
possible exception of the Ephemeroptera, their occurrence and abundance was
not clearly related to the concentration of total residual chlorine.
Ephemeroptera appeared less often in samples from treatment streams than
in those from control streams. Within each treatment level and control,
33 benthic samples were taken, 11 from each stream. Of these 33 samples
from each treatment and control level, Ephemeroptera were absent from 9, 9,
6, and 0 samples from high, medium, and low concentration treatment and
control streams, respectively.
76
-------
I
a
9.5
9.0
8.5
8.0
1430
1130
0930
1030
0830
I
I
0 500 1000 1500 2000 2500 3000
ILLUMINATION (foot candles)
o
a:
40
35
30
25
20
0
(B)
0930
1830
1230
1730
1330
1630
1430
i
i
500 1000 1500 2000 2500
ILLUMINATION (foot candles)
3000
Figure 21. Relationships between the amount of illumination (in foot-candles)
at the surface of a laboratory stream and the pH of the stream water (A) and
the concentration of residual chlorine compounds (TRC) in the water (B) froia
approximately 0830 to 1830 hours on July 26, 1976.
77
-------
40
04
V.
S 30
tr
UJ
o
20
< 10
O
0
1000
(A)
SEP
AUG
CJ
E 800
x.
o>
E
"i 600
'l.
0.
O 400
o:
o
x
° 2001-
(B)
OCT
MAY
• CHLOROPHYLL a
o CHLOROPHYLL
a + b+c
0 200 400 600 800
INPUT CONCENTRATION OF TRC (pg/l)
Figure 22. Relationships between the concentration of inorganic chloramine
(TRC) in renewal water that was necessary to maintain a residual concentration
of about 20 vg/1 and the amounts of organic matter (A) and chlorophyll (B)
present in the stream.
78
-------
TABLE 30, DENSITY OF ORGANIC MATTER C
-------
50
00
o
OJ
o:
UJ
40
30
<
5
y 20
o
o:
0 10
1
0 123456
EXPERIMENTAL PERIOD
_ o
Figure 23. Densities of organic (g/m ) in the laboratory streams at the end of each experimental
period, from 1 through 6,
(TRC) and time.
Each plotted value is a mean for three streams at a given concentration
-------
2.4
00
CM
e 2.0
.6
-J
5!
X
a.
o
X
o
<
H
O
Z
UJ
0.8
0.4
0
O CONTROL
A 2.9 pg/I
n 7.8 pg/1
V 20.6pg/1
1
3456
EXPERIMENTAL PERIOD
8
Figure 24. - Densities of total chlorophyll (g/m ) in the laboratory streams at the end of each
experimental period, 1 through 9.
concentration (TRC) and time.
Each plotted value is a mean for three streams at a given
-------
-TABLE 31. DENSITY OF INVERTEBRATES (G/M2) IN THE LABORATORY STREAM AT THE END OF EACH EXPERIMENTAL PERIOD, INCLUDING TWO PRESAMPLES,
00
Concentration of residual chlorine conpounds (TRC, ug/1)
Taxa
Insects
Gastropoda (Physa)
Isopoda (Asellus)
Ostracoda
Tubificidae (Tubifex)
Miscellaneous
Total
0 2.9
April 24, 1975
9.50 7.52
7.8 20.6
(PreBample)
4.94 6.71
0
May
5.04
0.02
2.9
28, 1975
7.27
0.25
7.8
(Period
10.48
<0.01
0.02
<0.01
20.6
I)
4.83
0.20
0.03
0 2,9
June 25, 197S
2.73 3.12
3.07 0.03
7.8 20.6
(Period 2)
3.51 3.46
0.17 -0.02
0.09
<0.01
9.50 7.52
4.94 6.71
September 3t 1976 (Period 4)
Insects
Gastropoda (Physa)
Isopoda (Asellus)
Ostracoda
Tubificidae (Tubifex)
Miscellaneous
Total
Insects
Gastropoda (Physa]
Isopoda (Asellus]
Ostracoda
Tubificidae (Tubifex)
5.17 4.53
0.45 2.88
1.10 2.35
0.13 0.02
6.85 9.78
May 12, 1976
1.71 6.48
6.69 6.08
0.15 1.03
2.50 0.83
0.02
4.87 4.66
0.65 2.64
5.62
3.22 2.30
0.04 0.32
0.28
8.78 15.82
(Period ?)
7.99 2.21
5 . 62 1 . 80
0.81 3.20
1.48 1.22
0.10 2.40
5.06
7.52
October St 1375
3.35
1.34
9.34
0.10
<0.01
14.13
June
1.28
6.94
0.76
1.07
5.06
5.79
10.02
20.87
8, 1976
2.32
7.18
1.76
1.17
10.50
5.06
(Period S)
4.51
1.60
0.09
2.84
0.18
9.22
(Period
1.40
7.25
8)
0.76
5.18
1.61 56.75
4.26
0.31
2.12
0.52
5.80 3.15
3.68 3.57
November 3, 1975 (Period 8)
3.56 4.27
1.16 1,93
0.01
3.21 1.18
0.01 0.64
7.94 8.03
June 29t 1376
2.39 1.10
1.50 0,74
1.31 3.02
11.92 12.96
2.81 0.20
2.20 9.31
3.04
0.30 0.23
5.31 12.78
(Period 9)
1.84 2.62
8.06 3.12
2.59 9.13
0.41 0.23
0.28
0 2.9
7.8
20.6
August £, 1975 (Period 3)
3.15 3.18
0.73 0.85
0.06 0.30
0.12
0.23
3.94 4.68
April 24 ,
5.80 3.70
0.26 0.43
1.27
3.87 1.08
0.04
9.93 6.52
2.78
0.15
0.12
0.14
3.19
3.08
2.27
1.47
0.23
0.02
7.07
1976 (Preeample)
2.24
4.16
0.07
2.36
8.83
3.03
0.55
10.65
0.21
0.05
14.49
Miscellaneous
Total
0.02
11.05 14.46 16.00 10.83
10.05 12.43 14.83 65.33
0.22
17.12 17,82 13.12 15.38
-------
Juvenile coho salmon were maintained in the laboratory streams through-
out most of the entire experimental period. These fish were dependent for
their food upon organisms produced in the streams. And, of course, they were
continuously exposed to all other stream conditions, including the presence
of residual chlorine compounds in the streams. Coho salmon biomasses, produc-
tion rates, and relations with their food organisms were quite variable,
but probably no more so than in natural streams. And the interpretation
of such complex data requires the application of productivity theory, which
we will do in a very cursory way. But, however the data were to be inter-
preted, it is extremely doubtful that effects of chloramine compounds at
concentrations near those we sought to maintain could be shown to have
had much if any direct or indirect effect on the juvenile salmon.
The mean biomasses of salmon in the 12 laboratory streams were
similar during periods 1 through 5, although the high concentration
streams supported slightly less fish biomass during periods 1, 2, and
3 than did the other streams (Table 32). No data are shown for the high
chloramine concentration streams for period 5, because many of the fish
were killed by the short-term presence of free chlorine due to the failure
of the tubing pump supplying ammonium to the mixing chambers of the
diluter. Fish were restocked in all streams during the winter (period 6),
but because of the seasonally low invertebrate biomasses, and the large
size of available juvenile salmonids of the current year class, the
streams would not support a sufficient number of fish for a good experiment.
The period 7 experiment was delayed until higher invertebrate biomass
levels were present in May 1976. During periods 7, 8, and 9, the mean
fish biomass increased rapidly in the low and medium concentration
streams, but it declined or remained nearly constant in the control and
high concentration streams (Table 32). Mean salmon biomasses in control
streams were well below those in treatment streams during these 1976
periods.
The production rate of fish has been shown to increase to some maximum
as fish biomass increases from zero to some intermediate level, but then
production rate declines with further increase in biomass (Warren, 1971).
Although such relationships are not always well defined, their examination
is usually instructive. Each production curve is descriptive, to some
extent, of a general level of productivity of the system—its capacity to
produce salmon, in the present instance. Two major production relations can
be roughly defined for the stream experiments on the effects of chloramines
(Fig. 25). Curve A, in Figure 25, describes the high spring-early summer
production relation occurring during the beginning of the study (periods
1 and 2). Only the streams having a high concentration of total residual
chlorine exhibited lower production during period 2. Fish in the low con-
centration streams during period 9 were at this high level of production.
As summer progressed into fall, production dropped in all streams to around
the level described by curve B. Control streams were even lower during
periods 7, 8, and 9, as were the high concentration streams during periods
7 and 8. Fish production in medium and low concentration streams
increased during the final period 9.
83
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TABLE 32. MEAN BIOMASS, GROWTH RATE AND PRODUCTION OF COHO SALMON EXPOSED TO
RESIDUAL CHLORINE COMPOUNDS IN THE LABORATORY STREAM FOR EXPERI-
MENTAL PERIODS 1 THROUGH 9.*
Mean
concentration Stream Mean biomass Growth rate
(Pg/1) (g/m2) (mg/g/day)
0 Nl
N6
S6
Mean
2.3 S3
S5
N5
Mean
4.6 S2
N2
N3
10.3 SI
S4
N4
Mean
0 Nl
N6
S6
Mean
3.0 S3
S5
N5
Mean
9.1 S2
N2
N3
Mean
22.4 SI
S4
N4
Mean
Experimental Period 1
5.82
4.86
5.30
5.33
5.26
6.39
4.87
5.51
5.60
5.59
4.68
5.29
6.33
5.16
3.77
5.09
Experimental Period 2
7.32
7.14
6.66
7.04
5.99
7.21
7.88
7.03
6.54
7.59
7.41
7.18
6.22
6.61
5.95
6.26
26.4
22.8
23.2
(24.4)
21.1
29.2
15.8
(22.5)
22.5
23.0
17.7
(21.3)
27.4
20.3
22.0
(23.7)
20.7
18.9
14.5
(19.1)
3.8
19.2
26.4
(18.5)
12.2
24.7
22.3
(21.1)
7.7
12.5
5.7
( 9.3)
Production
(mg/m2/day)
154
111
123
129
111
184
77
124
126
129
83
113
174
105
83
121
159
142
102
134
25
146
219
130
84
197
174
152
50
89
36
58
84
-------
TABLE 32. CONTINUED
Maan
concentration
(ug/D
Stream
Mean biomass Growth rate
(g/m ) (mg/g/day)
Production
(mg/m2/day)
Experimental Period S
0
Mean
1.4
Mean
6.0
Mean
16.6
Mean
0
Mean
1.2
Mean
3.9
Mean
13.5
Mean
Nl
N6
S6
S3
S5
N5
S2*
N2
N3
SI
S4
S5
Nl
Nfe
S6
S3
S5
N5
S2
N2
N3
SI
S4
N4
9.68
8.66
8.91
9.08
6.59
9.48
9.29
8.45
7.38
11.05
8.90
9.98
7.23
8.89
7.61
7.91
Experimental Period 4
7.67
7.16
6.99
7.27
6.57
6.95
7.16
6,89
7.09
7.71
7.31
7.37
7.49
8.06
7.02
7.52
4.8
0.7
7.1
(4.3)
3.2
4.9
5.9
(4.9)
7.7
- 1.7
(3.5)
2.7
8.9
10.4
(4.7)
9.5
3.4
-3.0
C4.1)
-0.9
3.1
1.9
C1.6]
-0.7
10.8
10.5
C 7.2)
7.6
16.1
2.9
(9.7)
46
7
63
39
20
47
57
41
85 -
- 15
35
20
80
13
38
73
25
- 8
30
- 6
23
17
11
- 2
82
78
53
60
133
25
73
85
-------
TABLE 32. CONTINUED
Mean
concentration
Cug/1)
Stream
Mean biomass
(g/m2)
Growth rate
Production
Cmg/m2/day)
2.7
6.9
24.3
2.9
12.8
31.5
Mean
Mean
Mean
Mean
Mean
Mean
Nl
N6
S6
S3
S5
N5
S2
N2
N3
Experimental Period 5
8.26 -2.9
7.45 0.3
6.79 -2.5
7.50 (-1-0)
6.18 -8.3
8.27 13.0
7.21 -4.9
7.22 ( 2.4)
8.74 2.2
8.15 -3.0
8.45 (2.8)
N4
N1<
N6
S6
N5
S2#
Experimental Period 6
10.53
11.16
10.93
10.87
11.48
10.42
10.95
N4
-16
2
-_8
- 7
-33
108
-23
17
50
-_2
24
Mean
86
-------
TABLE 32. CONTINUED
Mean
concentration
Cug/D
0
Mean
3.2
Mean
8.1
Mean
19.8
Stream
Nl
N6
S6
S3
S5
N5
S2
N2
N3
SI
S4
N4
Mean biomass
(g/m2)
Experimental Period
5.37
4.58
4.71
4.89
5.75
6.05
4.97
5.59
5.85
5.05
5.72
5.54
5.26
4.72
4.85
Growth rate
(mg/g/day)
7
5.6
-3.8
-0.4
CQ<7)
12,7
13, Q
1,8
C 9,6]
10.. 7
1.9
10.2
C 7.9)
5.2
1.4
-2.7
Production
(mg/m2/day)
30
-17
- 1
4
73
79
_9
54
63
10
!§.
44
27
7
-13
Mean
Mean
4.1
Mean
9.1
Mean
22.4
4.94
C 1-4)
Nl
N6
S6
S3
S5
N5
S2
N2
N3
SI
S4
N4
Experimental Period 8
5.41 -6.8
3.70 -1.5
4.48 -3.8
Mean
4.53
7.86
7.46
4.68
6.67
7.52
5.09
6.90
6.50
5.24
4.74
4.54
4.84
(-4.3)
-1.5
2.7
-7.9
(-1.4)
8.4
-1.1
3.8
(4.5)
-7,2
5.1
-2.1
(-1.5)
-37
- 5
-17
-20
-12
20
-10
63
- 1
29
-38
26
-12
- 7
87
-------
TABLE 32. CONTINUED
Mean
concentration Stream
Og/D
0 Nl +
N6+
S6
Mean
5.0 S3
S5
N5
Mean
9.7 S2
N2
N3
Mean
24.5 SI
S4
N4
Mean
Mean biomass
(g/m2)
Experimental Period
3.53
2.35
4.71
3.53
8.59
10.01
4.50
7.70
8.95
5.88
8.37
7.73
5.45
5.22
4.88
5.18
Growth rate
(mg/g/day)
9
-29.3
-29.6
8.4
(-15.9)
9.5
22.2
18.6
( 16.8)
7.6
12.6
13.3
(11.0)
20.1
7.4
8.7
(12.5)
Production
(mg/m2/day)
-139
- 69
40
- 56
82
222
84
129
68
74
112
85
109
38
43
63
Mean growth rate for fish in the three streams at each toxicant concentra-
tion and control for each experimental period = mean production/
mean biomass.
Insufficient number of fish recovered from stream.
2 or more fish not recovered from stream.
88
-------
CO
<£>
eg
0.165
0.15
0.12
0.09
2 0.06
o
§ 0.03
cr
o.
en
-0.03
-0.06
O CONTROL
A 2.9pg/l
D 7.8 pg/1
V 20.6
\
0
1.0
3.0 5.0 7.0
MEAN FISH BIOMASS (gVm2)
9.0
Figure 25. Relationships between the production and biomass of coho salmon in the laboratory streams
The enclosed numbers (1 to 9) refer to the experimental periods (see text). Two possible levels of
productivity are indicated by curves A and B.
-------
Except for period 2, salmon production in the three high concentra-
tion streams was as high or higher than that in control streams. Production
in low and medium concentration streams was higher than in other streams
during periods 7, 8, and 9. The presence of some ammonium derived from
the introduction of the toxicant able to enrich these systems could be
speculated.
Relationships between a predator species and its prey—coho salmon and
invertebrates in the present case—are extremely complex. Generally,
for fixed values of some external variable or variables (organic matter
in this instance) prey density is related to predator density by a "prey
isocline" defined by a series of possible equilibrium points. Other
levels of organic matter can theoretically be shown to parameterize
other relationships of this sort. Another set of isoclines, "predator
isoclines," defining relations between predator and prey can be shown to
be parameterized by predator loss terras. Theory for this has been
explicated by Booty and Warren (MS), but this is too involved to go
through here. Even so, some representation of fish and invertebrate
data in terms of such relations may be worthwhile, for it shows that
even with more sophisticated interpretations, no consistent effects of
residual chlorine on fish and invertebrate could be shown (Fig. 26).
The descending "prey isoclines," P^ through P$t are identified with
values for organic matter, as estimates of the energy and material
availability to the predator-prey systems. Mean values for organic
matter for stream data points along the lines P^, Pj, ?2, and
P! are respectively, 25.7, 19.3, 12.8 and 8.6 g/m , isocline P^
partially defining high level of productivity for the salmonids. The
"predator isoclines," MI through M4, can be parameterized by different
rates of mortality of the predator, salmon, MJ being at a low mortality
rate, M4 being at a high rate. Examining mortalities during the experi-
ment for data points along the predator lines, mean values of fish
deaths per period per stream for Mj, M2, MS, and M4 were 0.2, 0.4, 0.7
and 3.6 respectively. M4 included some points for the late fall when
mortality was too high to allow a meaningful calculation of fish pro-
duction, as noted earlier.
Such a theoretical interpretation allows us to relate densities of
salmonids to densities of invertebrates by two series of relationships, the
first parameterized by organic matter, the second by fish mortalities.
Although streams having low concentrations of total residual chlorine tended
to have coordinate values for invertebrates and salmonids located about
?3 or above, and about M3 or above, the distribution of coordinate values
does not clearly show residual chlorine to have had a definite impact on
the stream community.
In the growth studies of juvenile coho salmon in aquaria, which we
reported earlier, growth was clearly depressed at concentrations of
chloramines near 20 yg/1 TRC. We might then have expected growth and
production of juvenile salmonids in the laboratory streams to have been
depressed at mean concentrations near 20 yg/1 TRC, but this does not appear
90
-------
(SI
w 9.0
a
GQ
CO
u.
6.0
<
| 3.0
cc
UJ
<
UJ
0
M4
O CONTROL
A 2.9pg/l
D 7.8 pg/I
V 20.6ug/l
5.0 10.0 15.0 20.0 25.0
MEAN TERMINAL INVERTEBRATE BIOMASS (q /m2)
Figure 26. A graphical analysis, using the isocline method [Booty and Warren,
MS) of the relationship between the mean terminal invertebrate biomass and
fish biomass for each experimental period. The identity of the prey isoclines
(P) is organic matter. The identity of the predator isoclines (M) is
mortality of fish. The enclosed numbers (1 to 9) refer to the experimental
periods. Each plotted value is a mean for three streams at a given
concentration and time.
91
-------
to have occurred. Variability in stream conditions and data may have been
sufficient to obscure such a depression of growth and production, were it
to have occurred. But we are more inclined to believe that no appreciable
differences in production owing to the presence of residual chlorine com-
pounds occurred. Differences in stream conditions make any simple extrapo-
lation of the aquaria data hazardous. But beyond this, uncertainty of the
meaning of the values for total residual chloramines—which were determined
by araperometric titration—in the streams may be the biggest consideration.
The concentrations of inorganic chloramines may have been considerably lower
than the analyses suggest. Even so, it is of considerable interest that
exchange flows of water containing up to about 800 yg/1 TRC had little if
any effect on the invertebrates and salmonids in the streams, whatever may
have been the residual concentrations maintained in the streams.
92
-------
DISCUSSION
Chlorine is exceedingly important for the disinfection of water
supplies and sewage effluents and has important applications in controlling
undesirable biological growth in power generation cooling facilities and
other industrial applications. But the properties of chlorine and some
of its compounds that make it valuable in such applications also make it a
potentially serious toxic hazard to aquatic organisms in receiving waters.
The setting of standards to protect aquatic life from toxic effects of
chlorine and chlorine derivates, standards that still permit necessary
uses of chlorine, presents an exceedingly complex problem, but perhaps
no more so than setting standards for most other toxic materials.
Ward (1974) has estimated that about 200,000 tons of chlorine are
used annually in wastewater treatment. It is our impression that unneces-
sarily large amounts of chlorine are used in sewage treatment and in slime
control in power generation facilities. More chlorine is often used in
sewage treatment than has been shown to be necessary for effective disinfec-
tion. Entirely too little effort has been expended to determine the minimum
levels of chlorine application that would effectively control slime. Use of
such knowledge could result in considerable economic savings as well as
minimize the danger to aquatic life. It is our present belief that effective
application of chlorine and the protection of aquatic life can usually be
achieved. But more yet needs to be known about chlorine application
technology, chemistry, and effects on aquatic life. Still, even now,
enough is known to permit much better use and control of chlorine.
What is toxic about chlorinated effluents? This is not an easy question,
and it has no single answer. The chemistry of chlorine in effluents and in
receiving waters is highly complex, and it is not very well understood. Some
investigators report that free residual chlorine is more toxic than
inorganic chloramines (Merkens, 1958), but others believe the reverse to
be true [Holland et al., 1960). Differences in water quality, such as
pH, temperature, organic load, ammonia content, affect the chemistry of
the chlorinated effluents. In addition to this, chlorinated effluents
may contain small amounts of numerous chlorine containing organic compounds
(Jolly, 1975). Very little is known about these compounds or their
toxicity. The point we are making is that the conditions under which
fish and other aquatic organisms are exposed in nature are complex,
poorly understood, and probably make impossible any single adequate
universal standard for chlorine.
93
-------
Determination of levels of residual chlorine not likely to deleteriously
affect aquatic life is further complicated by different patterns of exposure
to the toxicants, different responses of various species of fish and
different life stages of particular species, and the many possible effects
on other organisms in aquatic communities. It is a problem faced in
setting standards for any toxic material, and it is not one for which we
are likely to develop entirely satisfactory solutions.
We have already mentioned that there is ample evidence that residual
chlorine can be acutely toxic to aquatic life, even at very low concentra-
tions, under conditions of continuous exposure (Brungs, 1976; Arthur et al.,
1975). Tsai (1973) has shown in the field that chlorination of sewage ef-
fluent has caused great reductions in the species diversity of fishes, his
index of diversity falling to zero where the mean concentration of residual
chlorine was about 250 Pg/liter. He also observed no fish in streams where
the mean residual was near or above 370 ug/liter. And he found brook trout
to disappear from streams receiving chlorinated sewage discharges, the
mean concentration of residual chlorine in these streams being about
20 yg/1.
The work presented in this report was an attempt to determine the
responses—survival, development, growth, behavior, and reproduction—of
fishes to residual chlorine as influenced by life stage, water quality,
and patterns of exposure. Possible effects of residual chlorine on
the structure of laboratory stream communities and the production of salmon
in the streams were also studied. As originally planned, a wide variety of
approaches was employed. This prevented as much replication of the research
as we would have liked. Nevertheless, taken as a whole, the work does
elucidate important problems of chlorine and chloramine toxicity to fish
and other aquatic organisms.
At acutely toxic concentrations of chloramines under conditions of
continuous exposure, we showed the tolerance of salmonids to be functions of
life stage and body weight. Others have also shown that body weight can
affect the median lethal levels for fish exposed to residual chlorine
(Rosenberger, 1971; Wolf, 1976). It is important that all life stages of
species likely to be exposed to residual chlorine be taken into account in
establishing standards for their protection, special emphasis being placed
on the most sensitive. It is not going to be generally adequate to base
standards only on the results of experiments on particular life history stages
of species of concern.
Based upon the complex chemistry of chlorine in water and effluents,
variations in water quality are apt to result in shifts in species of
chlorine present, shifts that could alter the toxicity of the solutions,
with or without change in concentration of total residual chlorine. Mer-
kens (1958) discussed this with respect to chlorinated effluents discharged
into rivers. In our experiments, we do not know why the toxicity of test
solutions to coho salmon juveniles increased with pH increase from
7.5 to 8.1. The expected increase in monochloramine did not occur with the
94
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increase in pH, the percentage of monochloramine being 83 at each pH. Cer-
tainly much more needs to be known about the chemistry of chlorine and about
entry and activity of residual chlorine compounds in fish. Some suggest
that the primary action of chlorine is gill damage (Bass, 1975), but
others believe this not to be the case (Fobes, 1971). The action and effects
of chlorine in fish are probably much more complex than now thought.
Buckley et al. (1976) found a decrease of hemoglobin and hematocrit to lev-
els indicative of anemia in coho salmon exposed for 12 weeks to 30 ug/1.
total residual chlorine in municipal sewage.
We found no difference in the acute toxicity of inorganic chloramines
to coho salmon juveniles at 10 and at 15 C, when the fish were acclimated
to these temperatures. Thatcher et al. (1976) obtained a similar
result with brook trout exposed to 10 and 15 C, and they concluded that
this was not a range of temperatures likely to cause thermal stress
influencing the toxicity of residual chlorine compounds. But they did
find that increasing the test temperature to 20 C, when acclimation
temperatures had been 7, 10, and 15 C, did increase the sensitivity of
the trout to residual chlorine. Sudden changes in temperature are
likely to greatly influence the toxicity of chlorine and derivative
compounds; and this can be an important problem when these are present
in thermal discharges.
Chlorination of complex industrial effluents presents extremely dif-
ficult problems of chemistry and toxicology. We found stabilized pulp and
paper mill effluents, which were not usually acutely toxic, to become acutely
toxic after chlorination with about 1 mg/liter of total residual chlorine.
Yet no residual chlorine was detectable in the test solutions in the aquaria.
Much work would be necessary to identify the toxic compounds. Reduction of
toxicity of such effluents may be possible through increased retention time,
as found by Watkins (1973), or by other means, even when the toxic compounds
are not known.
For a given species of fish, we would expect that the median lethal
levels of acutely toxic solutions under conditions of intermittent intro-
duction of residual chlorine to be higher than those for 96-hour continuous
exposure, because of the shorter duration of exposure. But analysis and
interpretation of results of intermittent exposure experiments present
problems. The usual method of representing toxicity in terms of toxicant
concentration does not permit results obtained from particular patterns
of concentration and time of exposure to be applied to any other patterns
to which organisms might be exposed. Results of experiments in which
fish are exposed to patterns of given form, duration, and frequency,
when reported in terms of peak or mean concentrations, are applicable
only to those conditions of exposure. Those investigating spike patterns
of exposure tend to report peak concentrations (i.e., Heath, in press),
and those investigating square patterns of exposure tend to report mean
plateau concentrations (i.e., Brooks and Seegert, 1977). But the precise
patterns and all patterns occurring in receiving waters can never be
duplicated and studied. We believe that further representation of data
resulting from intermittent exposure experiments on the basis of the
95
-------
areas (expressed as mg/liter x time) under concentration-time curves
could reduce problems of analysis, interpretation, generalization, and
application. We showed, for the range of chlorine concentrations
and shapes of curves that we studied, that the area under the curves
was a general representation of the toxicity of different patterns of
exposure. But the general usefulness of this sort of representation
should be investigated for a greater variety of patterns, concentra-
tions, and frequencies of exposure, so as to make boundary conditions of
its applicability clear. Intermittently discharged effluents generally
contain toxicants other than chlorine (Dickson et al., 1974), and this
may influence the general usefulness of this approach. Furthermore, we
used solutions containing mostly free residual chlorine, and work should
be done with inorganic chloramines, the latter being predominant in some
intermittent discharges (G. Nelson, EPA, personal communication).
Not only the toxic responses of fish to different patterns and durations
of exposure but also to different frequencies of exposure may be usefully
represented by total areas under time-concentration curves. In our one
and two 90-minute square patterns of exposure, we found that bass did not
recover much, if any, during the two-hour interval between the exposures.
Using this approach, the amount of recovery between exposures can be
directly determined when the duration between exposures increases. This
approach could add much generality to work on intermittent exposure to
residual chlorine compounds, and make it unnecessary to study all pos-
sible patterns, durations, and frequencies of exposures of fish and
other organisms.
Our work and that of others (Brooks and Seegert, 1977; Greg, 1974;
Stober and Hanson, 1974) indicate that temperature under conditions of
intermittent chlorination can have substantial effects on the toxicity
of residual chlorine compounds to aquatic organisms. This is especially
true if there is considerable difference between the temperature of
effluents and the temperature to which fish are acclimated (Stober and
Hansen, 1974), for the fish may move from cooler waters into heated
effluent plumes. Brooks and Seegert (1977) showed for alewife, after
acclimation for up to two weeks at 10, 20, 25, or 30 C, that the median
lethal level for one 30-minute exposure to residual chlorine decreased
with increasing acclimation temperature. Our own work and that of
Thatcher et al. (1976) indicate that a sudden increase in temperature
over the acclimation temperature decreases the tolerance of fish to
residual chlorine compounds. This must be taken into account in
research and application of results to problems associated with the
intermittent discharge of heated effluents containing residual chlorine
compounds.
The empirical rule that warm-water fishes are less sensitive to
residual chlorine than are cold-water species needs to be evaluated critically
(Brungs, 1976). There appears to be a continuum of sensitivity between these
two groups of fishes. But some species of minnows, not all of which are
actually "cold-water" species, may be at least as sensitive to intermittent
exposures to residual chlorine as are salmonids, as our results for the
blackside dace suggest.
96
-------
The significant feature of the partial chronic test is that the toxicity
of relatively low concentrations of toxicants can be determined for fish and
other organismss when exposure is continuous throughout development of life
history stages from gonadal maturation through juvenile growth. The success
of partial chronic tests, as they have been defined, is largely dependent
upon successful reproduction of the test species under test conditions. Arthur
and Eaton (1971) exposed fathead minnows (96-hour TL50 between 85 and
154 yg/1 total residual chlorine) for 21 weeks to residual chlorine. They
found no effects on adult survival at concentrations of 43 wg/1 and less,
but spawning was reduced somewhat at 43 yg/1 total residual chlorine. No
effects on reproduction were observed at 16 yg/l» In tests we conducted
with cutthroat trout, brook trout, and crayfish, reproduction was not
successful. But we did establish that the trout could tolerate very
long exposures to concentrations as high as 50 yg/1 total residual
chlorine (96-hour LC50 for juveniles was less than 100 yg/1). We did
notice, however, that at the highest test concentration, about 50 yg/1,
the trout did not eat as well as those fish exposed to lower concentra-
tion of the toxicant and to control conditions. In the tests with
crayfish (96-hour LC50 greater than 749 yg/1), those exposed to about
50 yg/1 lived for many months, but then most died during molting. The
sensitivity of trout and crayfish to residual chlorine may not be very
different under long-term exposure, even though median lethal levels
determined only for 96 hours are very different for most life history
stages.
When it is not possible or appropriate to conduct partial chronic
tests on a species, it may be useful to determine growth and other
responses of particular life history stages to relatively low concen-
trations of toxicants. In some of our tests with coho salmon, we
examined the effects of concentrations of about 50 yg/1 and less of
total residual chlorine (mainly monochloramine) on the survival,
development, and growth of embryos and alevins. The highest concen-
tration had no measurable effect on embryo survival or the size of the
alevins at hatching. The high concentration was, however, lethal to the
alevins. The survival of alevins was not affected at about 23 yg/1
total residual chlorine, but their growth rates were substantially
reduced as compared to alevins exposed to concentrations of about llyg/1
and less and to control conditions. Such reductions of growth could
delay the emergence of fry from gravel beds and could reduce their
ability to compete for food and space in streams. Similarly, the growth
of juveniles was reduced at 22-23 Vg/1, the threshold being between
11 and 22-23 yg/1. Such reductions in growth rate could have a sub-
stantial impact on the size of juveniles at smolting. Reduced size of
seaward migrants has been shown to be correlated with increased mor-
tality rates of salmon (Ricker, 1972). Such studies provide useful data
upon which to establish effects on fish of sublethal concentrations of
residual chlorine known to occur in natural streams (Tsai, 1973).
A number of investigators have reported behavioral changes in fish sub-
jected to either continuous or intermittent exposure to residual chlorine
compounds (Dandy, 1972; Basch and Truchan, 1976; Brooks and Seegert, 1977).
Although it is difficult to relate these behavioral changes to the
97
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persistence and status of fish populations in nature, such changes at
least indicate the fish are stressed. We found the behavior of coho
salmon alevins to be altered substantially when they were exposed to
about 23 yg/1 of total residual chlorine. The changes were probably
sufficiently great to interfere with intragravel bed movement and
later emergence of fry. Lethargic swimming and bobbing of largemouth
bass, as a result of their exposure to free residual chlorine, could be
detrimental to their survival by affecting their ability to avoid
obstacles and predators and escape from chlorinated discharge plumes.
Basch and Truchan (1976) noted that in some cases salmonids exhibited
considerable distress at the water surface when exposed to intermittent
chlorinated discharges from power generation plants. Others have
observed birds feeding on fish floundering at the water surface below
outfalls of chlorinated discharges from power plants (Brungs, 1977}.
Such behavioral responses as lethargic swimming, thrashing, and bobbing
observed in our work with bass may be indicative of those noted in field
studies. It is important to point out that in our studies bass exhibited
these behavioral changes as a result of sublethal as well as lethal
intermittent exposures to residual chlorine. Changes of behavior could
serve as a sensitive index of sublethal exposure of fish to residual
chlorine compounds, and the responses themselves may lead to reduction
in the survival of fish.
Avoidance responses could have much to do with the actual exposure
of fish to chlorine compounds in nature. Unfortunately, laboratory
studies of avoidance, for dimensional and other reasons, may not be at
all appropriate and are certainly difficult to interpret. And adequate
field studies of avoidance behavior would be difficult to conduct.
Sprague and Drury (1969) found that rainbow trout avoided as little as
1 yg/1 total residual chlorine. Yet, the trout did not avoid the much
higher concentration of 100 yg/1, and this could be given the rather unlikely
interpretation "preference." Fava and Tsai (1976) noted a similar
response of blacknose dace to free residual chlorine but not to inorganic
chloramines. Meldrin et al., (1974) did not observe such an apparent
"preference" response, nor did we. This illustrates a problem of all
laboratory studies of avoidance behavior. Design o£ apparatus, conduct
of experiments, and recording of behavior, as well as the ways in which
observed behavior could be interpreted offer so many possibilities as to
make extrapolation to nature extremely doubtful. Even so, such studies
do indicate that fish do have the ability to avoid chlorinated waters
under some conditions.
One aspect of this work needs attention. Tsai and Fava (1975) and we
have shown that tested fish delay in moving out of chlorinated solutions.
We showed that this resulted in bass receiving increased exposures
as the toxicant concentration increased. Yet, even at the highest acutely
toxic concentration of residual chlorine tested, the fish moved out of the
solution soon enough not to receive lethal exposures to the toxicant. Delayed
response behavior by fish in the field could be deleterious to their survival,
even if conditions were such as to make avoidance possible. If the fish
were exposed in an area over which chlorine compounds were widely distributed,
98
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or if exposures were such as to adversely affect their behavior, delay in
avoidance behavior could lead to increased mortality. In our avoidance
study with largemouth bass we found a greater response to free residual
chlorine than to inorganic chloramines at the same concentration of total
residual chlorine. Tsai and Fava (1975) found just the reverse situation
when working with the blacknose dace.
In our laboratory stream studies, we found it necessary to introduce
100 to 800 yg/1 total residual chlorine, in an exchange flow of two liters
per minute, so as to maintain desired concentration of about 20 pg/1 or
less in the streams. Seasonal variation in the needed rate of intro-
duction of chloramines was correlated with the amount of organic matter
present in the streams. Our amperometric titration results indicated
that from 50 to 80 percent of the residual chlorine compounds detected
in the streams was dichloramine. But J. D. Johnson (personal communica-
tion) believes that dichloramines should not have been present at the
stream pH levels we recorded and that what we detected as dichloramines
could have been organic chloramines.
That streams containing as much organic matter as did our laboratory
streams should have such a high chlorine demand is not particularly sur-
prising. But attempts to account for this demand, without extensive empirical
and theoretical investigation, are not apt to be very revealing. Loss of
chloramines to break-point effects does not appear likely on the basis of
present understanding of this difficult chemistry, because the chlorine/
ammonia ratio of solutions added to the streams was slightly less than
1:1 (Sawyer, 1967). The demand of the laboratory streams for chloramines
may have resulted from the formation of inorganic salts and chlorine
containing organic compounds. Some losses of residual chlorine could
have occurred through the volatilization of trichloramine, but mono-
chloramines and dichloramines are not very volatile (J. D. Johnson,
personal communication.) We can only conclude that, even though rela-
tively high concentrations of residual chlorine were introduced into
the streams to obtain desired residuals of 20 ug/1 or less, the forms
in which detected residuals were present and thus their possible
toxicity to organisms in the streams was quite uncertain.
Our studies of organic matter, chlorophyll, macroinvertebrates, and
juvenile salmon growth and production in the stream communities did not
demonstrate any marked effects of introduction of chloramines. We would
have expected, on the basis of our aquarium studies, some effects of residual
chlorine concentrations near 20 ug/1 on the growth and production of the
salmon. But in the aquarium studies, the fish were exposed to residual
chlorine mainly in the form of monochloramine, and we know practically
nothing about what residual compounds actually constituted the determined
stream concentrations. The 24-hour cycle in residual chlorine we observed
in the streams may also have made conditions there less deleterious to the
fish. The difficulty we found in applying simple aquarium experiments even
to controlled laboratory stream experiments raises some question as to how
adequately we can generally apply laboratory results to natural stream
conditions, which must usually be still more complex. Along with more
99
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laboratory investigation of problems of chlorine toxicity, much more
extensive and detailed investigation of the forms of residual chlorine
present in stream waters under different conditions and of the apparent
effects of these on populations of fish and other stream organisms would
certainly be well advised. Only with more information of this sort can
we expect to set standards adequate for the protection of aquatic life and
still permit legitimate use of chlorination.
100
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REFERENCES
1. Anonymous. Chlorinated Municipal Waste Toxicities to Rainbow Trout
and Fathead Minnows. Michigan Dept. Natural Resources. EPA Water
Control Research Series, 18050GZZ10/71. 1971. 49 pp.
2, Arthur, J. W. and J. G. Eaton. Chloramine Toxicity to the Amphipod
Gammarus pseudolimnaeus and the Fathead Minnow (Pimephales promelas).
J. Fish. Res. Bd. Canada 28:1841-1845. 1971.
3. Arthur, J. W., R. W. Andrew, V. R. Mattson, D. T. Olson, G. E. Glass,
B. J. Halligan, and C. T. Walbridge. Comparative Toxicity of Sewage-
effluent Disinfection to Freshwater Aquatic Life. EPA-600/3-75-012,
U.S. Environmental Protection Agency, Duluth, Minn., 1975. 62 pp.
*
4. Ashton, W. D. The Logit Transformation: With Special Reference to
Its Uses in Bioassay. Hafner Publishing Company, New York. 1972.
5. Basch, R. E., and J. G. Truchan. Toxicity of Chlorinated Power Plant
Condenser Cooling Waters to Fish. EPA-600/3-76-009, U.S. Environmental
Protection Agency, Duluth, Minn. 1976. 105 pp.
6. Bass, M. L. A Study of Lethality and Toxic Mechanisms of Intermittent
Chlorination to Freshwater Fish. Ph.D. Thesis, Virginia Polytechnic
Institute and State University, Blacksburg, Virginia. 1975. 70 pp.
7. Bass, M. L. and A. G. Heath. Cardiovascular and Respiration Changes
in Rainbow Trout, Salmo gairdneri, Exposed Intermittently to Chlorine.
Water Research. In press.
8. Bogardus, R. B., D. B. Boies, and D. A. Etnier. The Avoidance Responses
of Selected Wabash River Fishes to Mono-chlorainine. Wapora Inc.,
Charleston, Illinois. Manuscript. 13 pp.
9. Booty, W. M., and C. E. Warren. A General Theory of Productivity and
Resource Utilization. Department of Fisheries and Wildlife, Oregon
State University, Corvallis, Oregon. Manuscript.
10. Brooks, A. S.,and G. 'L. Seegert. The Effects of Intermittent Chlori-
nation on the Biota of Lake Michigan. Special Rept. No. 31. Center
for Great Lakes Studies, The University of Wisconsin--MiIwaukee,
Milwaukee, Wisconsin. 1977. 167 pp.
101
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36. Sawyer, C. N. Chemistry for Sanitary Engineers. Pages 246-256.
McGraw-Hill. 1967. 518 pp.
37. Sprague, J. B., and D. E. Drury. Avoidance Reactions of Salmonid Fish
to Representative Pollutants. In: Advances in Water Pollution
Research, Vol. I. Proceedings Fourth International Conference. Perga-
mon Press, Oxford. 1969. pp. 169-179.
38. Stober, Q. J., and C. H. Hanson. Toxicity of Chlorine and Heat to
Pink Salmon (Oncornynchus gorbuscha) and Chinook Salmon (CK tshawytscha)
Transactions American Fish. Soc. 103:569-576. 1974.
39. Strickland, J. D. H., and T. R. Parsons. A Practical Handbook of
Seawater Analysis. Pages 185-196. In: Fisheries Res. Bd. Canada
Bulletin 167, Ottawa. 1968. 311 pp.
40. Thatcher, T. 0., M. J. Schneider, and E. G. Wolf. Bioassays on the
Combined Effects of Chlorine, Heavy Metals, and Temperature on Fishes
and Fish Food Organisms. Part I. Effects of Chlorine and Temperature
on Juvenile Brook Trout (Salvelinus fontinalis). Bull. Environmental
Contamination and Toxicology 15 (1):40-48. 1976.
41. Tsai, Chu-Fa. Water Quality and Fish Life Below Sewage Outfalls.
Transactions American Fish. Soc. 102(2):281-292. 1973.
42. , and J. A. Fava. Chlorinated Sewage Effluents and
Avoidance Reaction of Stream Fish. University of Maryland, Technical
Report No. 35, College Park, Maryland. 1975. 59 pp.
43. Ward, P. S. Chlorine for Effluents in Short Supply. J. Water Poll.
Control Fed. 46(1):2-4. 1974.
44. Warren, C. E. Biology and Water Pollution Control. W. B. Saunders Co.,
Philadelphia. 1971. 434 pp.
45. Watkins, S. H. Coliform Bacteria Growth and Control in Aerated
Stabilisation Basins. EPA-660/2-73-028, U.S. Environmental Protection
Agency, Corvallis, Oregon. 1973. 281 pp.
46. Wolf, E. G. Combined Effects of Waste Heat and Chlorine on Juvenile
Rainbow and Eastern Brook Trout. Battelle Pacific Northwest Labora-
tory Annual Report for 1975, Part 2 Ecological Sciences. 1976. pp.
67-68.
47.• Zillich, J. A. Toxicity of Combined Chlorine Residuals to Freshwater
Fish. J. Water Pollution Control Fed. 44(2):212-220. 1972.
104
*U.S, GOVERNMENT PRINTING OFFICE : 1978 0-72 0-3 3 5/6 06S
-------
TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPORT NO.
EPA-600/3-78-023
3. RECIPIENT'S ACCESSION NO.
4. TITLE AND SUBTITLE
Toxicity of Residual Chlorine Compounds to Aquatic
Organisms
5. REPORT DATE
March 1978 issuing date
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
Gary L. Larson, Charles E. Warren, Floyd E. Hutchins,
Larry P. Lamperti, David A. Schlesinger, Wayne K. Seim
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Department of Fisheries and Wildlife
Oak Creek Laboratory and Biology
Oregon State University
Corvallis, Oregon 97331
10. PROGRAM ELEMENT NO.
1BA608
11. CONTRACT/GRANT NO.
Grant #R802286
12. SPONSORING AGENCY NAME AND ADDRESS
13. TYPE OF REPORT AND PERIOD COVERED
Environmental Research Laboratory-Dul, MN
Office of Research and Development
U.S. Environmental Protection Agency
Duluth, Minnesota 55804
14. SPONSORING AGENCY CODE
EPA-600/03
15. SUPPLEMENTARY NOTES
16. ABSTRACT
Laboratory studies on Che acute and chronic toxicity of chlorine and inorganic chloramines to
trout, salmon, minnows, bullhead, largemouth bass, and bluegill were conducted. Acute toxicity under
continuous and intermittent patterns of exposure as well as behavioral, reproduction, development, and
growth responses to low level exposures Co residual chlorine compounds were determined. But not all
patterns of toxicant exposure or all responses of all fish species were studied. Acute and chronic
toxicities of chloramines to crayfish were investigated. Algae, invertebrates, including insects, and
juvenile salmon were exposed continuously to relatively low levels of residual chlorainine compounds
in laboratory stream communities. The acute toxicities of inorganic chloramines, as measured by
96-hour LC50 values, were less than 100 ug/1 for salmonids and were a funcCion of lifis history stage,
body size, and some waCer qualiCy conditions. Whereas adult trout may live indefinitely at concen-
trations near 50 pg/1, the LC50 values for late developmental stages—fry and very small juveniles—
were not much above this concentration. Effects on growth of alevins and juveniles had threshold
concentration values between about 10 and 22 ug/1, effects being quite marked at 22 pg/1. In inter-
mittent exposure to relatively high concentrations of free residual chlorine, mortality was found
to be a rather consistent function of the area under the time-concentration curves of exposure, for
different forms, durations, and frequencies of such patterns of exposure. Behavioral responses of
fish, such as avoidance of chlorinated water which could be advantageous in nature and lethargic
swimming, surfacing, and sinking to the bottom which would probably be harmful were studied. Such
behaviors occur not only at acutely toxic concentrations but also at lower ones. It was necessary
to introduce concentrations ranging from 100 to 800 Pg/1 of chloramine into laboratory stream
communities to maintain mean residual concentrations near 20 pg/1. No marked effects on algal or
insect abundances or on survival and production of juvenile salmon were observed at this and lower
concentrations in the laboratory streams. It is doubtful that the amperometrically determined
residual concentrations of chloramines in the streams consisted predominantly of inorganic chloramines,
organic chloramines perhaps being an important constituent under the stream conditions. Little is
known of the toxicity of organic chloramines. The amount of inorganic chloramines introduced to
maintain desired residual concentrations appears to have been a function of the amount of organic
material in the stream communities.
17.
KEY WORDS AND DOCUMENT ANALYSIS
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Chlorination
Chlorine
Toxicity
Bioassay
Aquatic animals
Fishes
Disinfection
Condenser tubes
Cooling water
Cooling towers
Fouling prevention
Sewage
Sewage treatment
Aquatic bioassays
Chronic bioassays
Toxicity tests
Brocides
57F
57H
57Y
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117
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