EPA-600/3-76-116
December 1976
Ecological Research Series
VALIDITY OF LABORATORY TESTS FOR
PREDICTING COPPER TOXICITY IN
STREAMS
•I
Environmental Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Duluth, Minnesota 55804
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development, U.S. Environmental
Protection Agency, have been grouped into five series. These five broad
categories were established to facilitate further development and application of
environmental technology. Elimination of traditional grouping was consciously
planned to foster technology transfer and a maximum interface in related fields.
The five series are:
1,. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
4. Environmental Monitoring
5. Socioeconomic Environmental Studies
Th is report has been assigned to the ECOLOGICAL RESEARCH series. This series
describes research on the effects of pollution on humans, plant and animal
species, and materials. Problems are assessed for their long- and short-term
influences. Investigations include formation, transport, and pathway studies to
determine the fate of pollutants and their effects. This work provides the technical
basis for setting standards to minimize undesirable changes in living organisms
in the aquatic, 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-600/3-76-116
December 1976
VALIDITY OF LABORATORY TESTS FOR PREDICTING
COPPER TOXICITY IN STREAMS
By
Jack R. Geckler (Deceased)
William B. Horning
Timothy M. Neiheisel
Quentin H. Pickering
Ernest L. Robinson
Newtown Fish Toxicology Station
Environmental Research Laboratory-Duluth
Cincinnati, Ohio 45244
Charles E. Stephan
Environmental Research Laboratory-Duluth
Duluth, Minnesota 55804
ENVIRONMENTAL RESEARCH LABORATORY-DULUTH
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
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. Mention
of trade names or commercial products does not constitute endorsement or
recommendation for use.
11
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FOREWORD
The research described in this report was completed to determine if
laboratory-based predictions of the effects of a pollutant in a natural
waterway are valid. The effort was one of the largest of its kind that we
know of and required many years and resources to complete.
While many new questions were raised, the work has served two purposes.
First, we found that even though this stream was a complex system, our
laboratory-based estimate of effects from the exposure concentration was not
far off but somewhat low. Second, the observations made during the study
provided a basis for establishing priorities for further research in many
areas thereby improving the effectiveness of other programs.
We feel the results of this study provide to EPA confidence that the
bioassay data base being generated in our laboratories can be used with
confidence as a basis for regulatory action in natural waterways.
Donald I. Mount, Ph.D.
Director
Environmental Research Laboratory-Duluth
Duluth, Minnesota
iii
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ABSTRACT
A field study was conducted on Shayler Run, in Clermont County, Ohio, to
determine the effects of copper on the stream biota. Copper was added to the
stream for 33 months to maintain a concentration of 120 yg/Z., a concentration
that was expected to adversely affect some species of fish and not others. This
natural stream received sewage effluent containing a variety of compounds known
to affect acute copper toxicity. All but one abundant species of fish in the
stream and four of the five most abundant macroinvertebrates were adversely
affected by exposure to copper. Direct effects on fish were death, avoidance,
and restricted spawning.
To determine the usefulness of laboratory toxicity tests when establishing
water quality criteria for an aquatic ecosystem, acute and chronic tests with
copper were conducted at the Newtown Fish Toxicology Station and on-site at
Shayler Run with stream species and the fathead minnow. The acute toxicity of
copper varied widely because of water quality variations in the stream. The
chronic tests underestimated the in-stream toxicity by about two times because
only the effects of copper on survival, growth, and reproduction were measured
but avoidance was not and it was a significant effect in the stream. Agreement
between the predictions from laboratory toxicity tests and the observed effect
is surprisingly close considering the measurement errors involved.
iv
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CONTENTS
Foreword 0 « 0 ill
Abstract „ „ iv
Figures . . . „ 0 0 , . . . ,vii
Tables 0 . . x
Acknowledgments xiv
Dedication xv
I Executive Summary „ . . . „ 1
II Conclusions 2
III Recommendations 4
Part A: Introduction to the Study
IV Introduction 5
V Description of the Study Area 7
VI Streamside Laboratory Facilities .... 16
VII Toxicant-Metering System 18
VIII Water Quality „ . 21
IX Stream-Copper Analysis 23
Part B: Field Studies
X Effects of Copper on Stream Fish 28
Introduction 28
Methods „ 28
Observations and Results 31
XI Effects of Copper on Stream Benthic Communities . . . . „ 60
Introduction . . . „ o .... 60
Methods „ . . 60
Results and Discussion . . . . „ 63
XII Fish-Stomach Analysis „ . „ . „ . . . . 85
Introduction 0 .... 85
Methods 85
Results and Discussion 86
Part C: Laboratory Studies
XIII Acute Studies 102
Introduction 102
Methods 103
Results 105
Discussion 128
v
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XIV Chronic Studies 131
Introduction 131
Methods 131
Results 137
Discussion ..... 162
Part D: General Discussion
XV General Discussion 167
References 172
Appendices .175
vn.
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FIGURES
Number Page
1 Aerial view of Shayler Run study area, looking downstream 8
2 Location map of Shayler Run 9
3 Sampling stations and gradient for Shayler Run 10
4 V-notch gaging weir 0 „ -Q
5 Control weir screens and V-notch gaging weir 12
6 Upstream log barrier 14
7 Upstream log barrier covered with debris and logs 15
8 Interior of pole building wet laboratory showing chronic
test setup 17
9 Schematic of toxicant-delivery system 0 . . . . 19
10 Schematic of fish-fry trap 30
11 Numbers of mature bluntnose'minnows from (A) biannual fish
collections and (B) weir-screens collections 37
12 Numbers of mature striped shiners from (A) biannual fish
collections and (B) weir-screen collections . ...».» 38
13 Numbers of mature stonerollers from (A) biannual fish
collections and (B) weir-screen collections 39
14 Numbers of mature rainbow darters from (A) biannual fish
collections and (B) weir-screen collections » 40
15 Numbers of mature creek chubs from (A) biannual fish
collections and (B) weir-screen collections ......<> 41
16 Numbers of mature fantail darters from (A) biannual fish
collections and (B) weir-screen collections 42
17 Numbers of mature orangethroat darters from (A) biannual fish
collections and (B) weir-screen collections 43
18 Numbers of mature green sunfish from (A) biannual fish
collections and (B) weir-screen collections 44
vli
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19 Daily collections of various fish species from the weir screens during
1970, and stream temperatures and dosing regimes at the time of
collection ............................. 48
20 Rock-filled basket sampler for collecting macroinvertebrates
and the wire mesh screen used to cover sampler when removing
it from the stream ......................... 61
21 Isopoda (sowbugs): basket-sampler collections ........... • 69
22 Isopoda (sowbugs): natural substrate collections ........... 70
23 Isopoda (sowbugs): weir-screen collections .............. 72
24 Ephemeroptera (mayflies): basket-sampler collections ......... 73
25 Ephemeroptera (mayflies) : weir-screen collections . . ........ 74
26 Ephemeroptera (mayflies): natural substrate collections . . . . „ . . 76
27 Amphipoda (scuds): weir-screen collections .............. 78
28 Chironomidae (Chironomids) : basket-sampler collections ........ 79
29 Chironomidae (Chironomids): natural substrate collections ...... 80
30 Psephenidae (riffle beetles): basket-sampler collections ....... 82
31 Psephenidae (riffle beetles): natural substrate collections ..... 83
32 Trichoptera (caddisf lies) : natural substrate collections ....... 84
33 Total numbers of macroinvertebrates found in orangethroat
darter stomachs ....................... „ . . . 89
34 Isopoda (sowbugs) found in orangethroat darter stomachs ........ 90
35 Ephemeroptera (mayflies) found in orangethroat darter
stomachs .......................... _. . . . 91
36 Copepoda found in orangethroat darter stomachs ............ 92
37 Chironomidae (Chironomids) found in orangethroat darter
stomachs .............................. 94
38 Total numbers of organisms found in green sunfish stomachs ...... 95
39 Isopoda (sowbugs) found in green sunfish stomachs ........... 96
40 Ephemeroptera (mayflies) found in green sunfish stomachs ....... 97
41 Chironomidae (Chironomids) found in green sunfish stomachs ...... 99
viii
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42 Fish found in green sunfish stomachs „ .<,....<>.. o 100
43 Terrestrial organisms found in green sunfish stomachs . 0 ...... 101
IX
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TABLES
Number gage
1 Percentage of Time That Copper was Added to Shayler Run During
the Exposure Period 20
2 Background Copper Concentrations in Shayler Run Water, 1968-73 25
3 Monthly Average Copper Concentrations in Exposure Section of
Shayler Run . . „ 27
4 Total Number of Individuals Collected from Shayler Run in the
Biannual Fish Collections, 1968-71 33
5 Age-group 0 and Mature (In Parentheses) Fish Collected from
Shayler Run in Biannual Fish Collections, 1968-71 . . „ 36
6 Data Relevant to the Fish Collections on the Weir Screens, Shayler
Run, 1970-72 . . „ „ 46
7 Number of Age-group 0 and Adult Fish Collected from Shayler Run
on Weir Screens „ 47
8 Number of Young-of-the-Year Orangethroat Darters Collected on
Weir Screens, Shayler Run, 1970-72 50
9 Number of Fish Spawnings Observed in Control and Exposure
Areas of Shayler Run During Three Seasons of Copper
Introduction „ 0 52
10 Number of Fry Collected on Weir Screens in Shayler Run, 1970 55
11 Species and Numbers of Fry Collected on Weir Screens in Shayler
Run, 1971 oo „ . 56
12 Species and Numbers of Fry Collected in Fry Traps in Shayler
Run, 1971 o . . . . o o 58
13 Species of Macroinvertebrates Collected by All Methods in
Shayler Run During 1969-71 „ . . „ 64
14 Numbers of Macroinvertebrates Collected in June and July from
Paired (A and B) Rock-filled Basket Samplers in Shayler
Run, 1969-71 „ „ . . 66
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15 Total Numbers of Macroinvertebrates In All Collections from Paired
Rock-Filled Basket Samplers in Shayler Run, 1969-71 67
16 Total Macroinvertebrates Collected from Weir Screens in Shayler
Run During 1970 and 1971 0 „ 71
17 Total Macroinvertebrates Collected from Natural Substrates in
Shayler Run During 1969-71 75
18 Number of Organisms in the Stomachs of Orangethroat Darters,
Shayler Run, 1968-71 87
19 Number of Organisms in the Stomachs of Green Sunfish, Shayler
Run, 1969-71 88
20 Summary of LC50 Values Based on Total Copper for the Bluntnose
Minnow 106
21 Summary of LC50 Values of Copper for the Bluntnose Minnow in
Shayler Run Water 108
22 Summary of Chemical Analysis of Test Water in Tests Reported in
Table 21 109
23 Summary of Static Bioassay with Fathead Minnows in Shayler Run
Water 110
24 Chemical Analysis of Test Water in Tests Reported in Table 23 Ill
25 Total Copper LC50 Values for the Bluntnose Minnow in Shayler
Run Water Upstream and Downstream from the Sewage Treatment
Plant 113
26 Total Copper LC50 Values for the Bluntnose Minnow in Various
Dilutions of Shayler Run Water 114
27 Effect of Hardening Shayler Run Water on the Toxicity of Total
Copper to the Bluntnose Minnow 115
28 Effect of Hardening Standard Water on the Acute Toxicity of
Total Copper to the Bluntnose Minnow 117
29 Effect of Added Phosphate on the LC50 of Copper to the Bluntnose
Minnow 118
30 Acute Toxicity to the Bluntnose Minnow of Copper in Standard
Water 119
31 Relative Sensitivity of Different Species of Fish to Copper in
Standard Water 121
32 Relative Sensitivity of Six Species of Fish to Copper in Shayler
Run Water, November 12, 1969 „ 122
xi
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33 Relative Sensitivity of Six Species of Fish to Copper in Shayler
Run Water, November 19, 1969 123
34 Relative Sensitivity of Six Species of Fish to Copper in Shayler
Run Water, December 15, 1970 124
35 Copper Concentration (in milligrams per liter) in Exposure Chamber
for Tests Reported in Table 34 125
36 Relative Sensitivity of Eight Species of Fish to Copper in Shayler
Run Water, May 6, 1971 126
37 Copper Concentrations (in milligrams per liter) in Exposure Chamber
for Tests Reported in Table 36 ° 127
38 Relative Sensitivity of Five Species of Fish to Copper in Shayler
Run Water, May 8, 1972 129
39 Sources of Fish for Streamside Chronic Tests 136
40 Weekly Chemical Analyses of the Water in the Exposure Chambers for
the NFTS Prespawning Exposure Chronic Tests With Copper 138
41 Total Copper Concentrations in Weekly Composite Samples from the
NFTS Prespawning Exposure Toxicity Tests 139
42 Hatchability of Eggs from the NFTS Prespawning Exposure Chronic
Tests 140
43 Number of Spawns and Eggs from Fathead Minnows with a 6-Month
Prespawning Exposure to Copper 141
44 Number of Spawns and Eggs from Fathead Minnows with a 3-Month
Prespawning Exposure to Copper 142
45 Number of Spawns and Eggs from Fathead Minnows with No Prespawning
Exposure to Copper 143
46 Combined Egg Production by Fathead Minnows in the Six Chambers for
each Concentration of Copper „ 144
47 Measured Total Copper Concentrations in Duplicate Test Chambers of
Test System I - Fathead Minnow Chronic Test 146
48 Measured Total Copper Concentrations in Duplicate Test Chambers of
Test System II - Fathead and Bluntnose Minnow Chronic Test 147
49 Measured Total Coppper Concentrations in Duplicate Test Chambers of
Test System III - Bluntnose Minnow Chronic Test j_48
50 Measured Total Copper Concentrations in Duplicate Test Chambers of
Test System IV - Fathead and Bluntnose Minnow Chronic Test 149
xii
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51 Measured Total Copper Concentrations in Duplicate Test Chambers of
Test System V - Green Sunfish Chronic Test 150
52 Summary of Exposure Conditions for Streamside Chronic Tests 151
53 Spawning and Egg Production by Fathead Minnows in Chronic Test
System I „ . „ „ 153
54 Spawning and Egg Production by Fathead and Bluntnose Minnows in Chronic
Test System II 154
55 Spawning and Egg Production by Fathead Minnows in Chronic Test
System IV .„...„ ..„<>.. o . o ........ o 156
56 Spawning and Egg Production by Bluntnose Minnows in Chronic Test
System III 158
57 Spawning and Egg Production by Bluntnose Minnows in Chronic Test
System IV 160
58 Spawning and Egg Production by Green Sunfish in Test System V 161
59 Summary of Streamside Chronic Test Data 164
Xlll
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ACKNOWLEDGMENTS
The authors sincerely acknowledge the help of Dr. William A. Brungs and
Dr. Donald I. Mount in the planning and early conduct of this research project.
In addition to that participation by those former directors of the Newtown
Fish Toxicology Station, we sincerely acknowledge the support provided by the
staff of that facility. Special mention is made of the efforts of the late
Dr. William H. Irwin, whose advice on ecological aspects and analyses of
pre-exposure fish collections were much appreciated, and to Rosemary Swantack,
who helped in typing and assembling the drafts of the report. The untiring
efforts of Marion Cast during the field and on-site portions of the study are
sincerely appreciated. The efforts of Jim Dryer and Greg Marsh performing
most of the chemical analyses are gratefully acknowledged.
We thank Dr. T. W. Thorslund of the Environmental Research Laboratory-
Duluth, Duluth, Minnesota, for his statistical advice and analysis of
egg-production data. The assistance of John G. Eaton, also of the Environmental
Research Laboratory-Duluth, Duluth, Minnesota, in preliminary planning and
initial field work is appreciated.
The support and assistance provided by other staff members from the
Environmental Research Laboratory-Duluth, Duluth, Minnesota and the Environmental
Monitoring and Support Laboratory, Cincinnati, Ohio, are gratefully
acknowledged.
We also gratefully acknowledge the excellent cooperation of another Federal
agency. The construction and operation of the stream gaging facility by the U.S.
Geological Survey was critical to the success of this study. Without their
participation and guidance, this field investigation could not have succeeded.
xiv
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DEDICATION
We dedicate this publication to Jack Geckler whose untimely death at
the end of the study was a loss to science as well as to his personal friends.
Without Jack's keen ability to
remember what his eyes saw, the study
would have lost much of its value. We
are thankful Jack was able to complete
data collection and the draft report,
and we truly appreciate his hard work
and diligence in seeing the study
through.
We also want to recognize the
support and patience of Jack's family
that helped him so much, especially
his loving wife, Norma. We have tried
to make this report a tribute to Jack
in the hope that it will help Norma
endure her loss and provide Chuck,
Linda and Brenda a glimpse of their
father's interests and abilities
that they can never fully know.
xv
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SECTION I
EXECUTIVE SUMMARY
This report describes the results of a study completed by the Newtown Fish
Toxicology Station to determine if laboratory estimates of safe concentrations of
pollutants are valid in real streams. Copper was used because the laboratory data
base was adequate and drinking water supplies would not by jeopardized by the
concentration that was expected to adversely affect some fish species and not
others.
For 26 months before copper was introduced into the study area, fish,
macroinvertebrate, and periphyton populations were sampled to determine baseline
data. Spawning and behavioral activities of fish were observed throughout the
entire study area. During the 33 months of copper addition, field observations,
biannual fish collections, fry collections, and spawning observations were made to
evaluate the direct effects of copper on the stream fish populations.
Macroinvertebrates were collected to determine the effects of copper on their
populations.
Death, avoidance of copper, and restriction of spawning areas were the direct
effects observed on the fish resulting in a general decline in fish populations
and reduction of food-organism populations. Indirect effects on fish as a result
of the effects of copper on the aquatic food chain were not demonstrated.
Copper toxicity varied widely, depending on stream flow stage, season, and
water quality. A copper concentration that was not lethal under one stream stage and
water-quality condition was rapidly lethal under other circumstances. The tests
underestimated the total effect, however, because they did not include avoidance
of copper by the fish. Thus, the bioassay data appear to be unconservative when
used for estimating safe levels of a toxicant.
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SECTION II
CONCLUSIONS
GENERAL
Laboratory-derived data can be used to predict toxic effects in a natural
stream situation. In general, the toxicity of copper was underestimated by the
laboratory data because avoidance by fish to this metal was not measured in the
laboratory tests.
Chronic laboratory toxicity tests with fish can be conducted with natural
waters of varying quality, but they are more difficult than tests with water of
a more consistent quality.
Indirect effects on fish, as a result of the effects of copper on the
aquatic food chain, could not be demonstrated in this study.
Other laboratory tests, such as those based on behavioral responses, may be
required in addition to those based on survival, growth, and reproduction, to
better predict the effects of a toxicant on a natural ecosystem.
The quality of the field portion of the study was excellent; the data showed
the same overall effects of copper on the biota from year to year. Various facets
of the study complement each other.
SPECIFIC
On the basis of acute relative sensitivity tests and a fathead minnow chronic
test, it was predicted that sunfish would be unaffected and the other fish species
would be affected by copper. The predictions were accurate for all fish species
except the orangethroat darter.
The order of sensitivity for the various stream species was not consistent
from One acute toxicity test to another, but the differences were not great. The
data indicate that the order to sensitivity differs with different water quality.
On the basis of chronic toxicity tests, copper was two to three times less
toxic to fathead minnows in Shayler Run water, which averaged 270 mg/Z. hardness
(84-356 mg/Z..), than in standard (laboratory) water (200 rag/Z. hardness).
The safe copper concentration for bluntnose minnows in Shayler Run was
underestimated by at least two times with the laboratory chronic toxicity tests
conducted in Shayler Run water.
The striped shiner, rainbow darter, creek chub, fantail darter, bluntnose
minnow, and stoneroller populations in Shayler Run all showed a reduction
attributable to copper.
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Because of varying water quality, a copper concentration that is near the
maximum acceptable toxicant concentration (MATC) may be near the 96-hr LC50 at
certain times during the year.
An estimated value for a long-term safe concentration of a toxicant using an
application factor approach cannot be based on a single acute toxicity test when
dealing with variable water quality.
Detoxitfying agents had a major effect on acute copper toxicity, but only
minor effects on chronic toxicity, based on values for total copper.
The effect of alkalinity and hardness on acute copper toxicity to fish was
not as important as effects of other detoxifying agents.
There is evidence that copper affected the spawning location of green and
longear sunfish in Shayler Run. Limited laboratory evidence indicates that, when
confined, these fish will spawn at higher copper concentrations than were introduced
into the stream.
Copper had a concentration-related effect on the number of eggs produced per
female in the laboratory tests.
The maximum acceptable toxicant concentration (MATC) is limited by egg
production. Short exposures to copper concentrations above the MATC reduce egg
production.
Avoidance of copper by the fish was an important effect in Shayler Run
during the first year of the study, but not during succeeding years, probably
because the more sensitive individuals had been removed from the populations.
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SECTION III
RECOMMENDATIONS
Bioassays should be used to establish water quality standards, when possible,
because they provide accurate data to predict effects on aquatic life.
Water quality standards should include the effects of various water quality
conditions on toxicity at various stream stages and seasons of the year.
Even short excursions of pollutant concentration above the MATC should
not be permitted unless specific data prove they will not be detrimental.
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PART A — INTRODUCTION TO THE STUDY
SECTION IV
INTRODUCTION
Water quality criteria for aquatic life are often developed on the basis of
toxicity tests performed in the laboratory, and water quality standards for
protection of aquatic life are largely based on these criteria. Some investigators
question the validity of the application of laboratory results to a natural
situation. Since validity depends on many factors, as discussed by Stephan and
Mount (1973), a simple answer to the question is not possible. Extrapolation of
results from laboratory tests to different situations is not unique to aquatic
toxicology, only the specific circumstances are different.
The usefulness of laboratory aquatic toxicity tests can be studied with
artificial streams, such as planned by Merna and Eisele (1973). Although artificial
streams and model ecosystems are elaborate laboratory toxicity tests, they do not
necessarily accurately simulate natural situations. Theoretically, the best, but
practically the most difficult, way is to compare the results of laboratory
exposures with the results of field exposures in a natural situation, as done by
Sprague and Drury (1969). In this way the effects of natural stresses, such as
changes in temperature, flow, water quality, species competition, affects on the
food web, and behavioral reactions, can be taken into account. Studies in natural
situations are more likely to produce qualitative, circumstantial evidence rather
than unequivocable, quantitative results. Slightly modified natural situations,
such as those used in this study, are designed to produce quantitative
cause-and-effect data under real-life conditions. Many aspects of the field
portion of this study were based on experiences of Larimore et_ a\_, (1959).
The major purpose of this study was to evaluate the usefulness of laboratory
toxicity tests in predicting water quality criteria for protection of aquatic
life. The study was designed so that the results from standard laboratory and
streamside laboratory chronic tests could be compared with results of long-term
stream exposure to a toxicant. The streamside chronic tests varied from the
standard laboratory chronic test in that water quality, temperature, and
photoperiod were not controlled. The streamside tests were conducted on site, in
a temporary building, using control and exposure waters and mixtures of the two.
Several resident and one non-resident fish species were tested during the study.
Although the study was not designed to obtain information concerning the
validity of the application factor hypothesis (Mount and Stephan, 1967), information
relative to it was gained from this study. Effects other than those studied in
the chronic tests could occur in the field exposure, so emphasis was placed on
stream observations. This single study cannot answer all questions, but it is a
major contribution towards the validation of the use of laboratory results in
establishing water quality criteria for protection of aquatic life.
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Stream selection criteria were: (1) adequate stream flow, (2) little chance
of uncontrolled pollution, (3) limited public access, (4) no direct public use of
water in the stream, (5) presence of riffles and pools, (6) proximity to the
Newtown Fish Toxicology Station in Cincinnati, Ohio, and (7) diverse aquatic flora
and fauna. Approval by The State of Ohio Department of Health, the Ohio Department
of Natural Resources, and landowners was also needed. Shayler Run, near
Cincinnati, Ohio, was chosen and met most of the requirements, except that at times
heavy runoff was known to cause excessive flooding and a small domestic sewage
treatment plant (extended aeration) was located 4.4 km upstream from the test site.
Copper was chosen as the toxicant for this study for the following reasons:
(1) both chronic and acute laboratory tests had been performed successfully with
fish; (2) exposure concentration could be rapidly measured; (3) the stream exposure
concentration would be less than 1.0 ppm, the maximum recommended for drinking
water by the U. S. Environmental Protection Agency, thereby posing no hazard to
nearby wells; (4) a sufficient range in sensitivity was exhibited by the stream
species; (5) it is a common persistent pollutant; and (6) it does not markedly
bioconcentrate.
Chronic tests with fathead minnows and copper had been conducted with a hard
water of 200 ppm hardness of CaCOs (Mount, 1968) and a soft water of 30 ppm
hardness (Mount and Stephan, 1969). Because of the higher hardness and alkalinity
and the presence of detoxifying agents from the sewage treatment plant, the
maximum acceptable toxicant concentration (MATC) of copper for fathead minnows in
Shayler Run water was predicted to be about 75 yg/£. The MATC is defined as the
highest toxicant concentration that has no adverse effect on survival, growth, and
reproduction of the test organism. Preliminary acute mortality tests indicated
that most of the important fish species in Shayler Run were about as sensitive
to copper as were fathead minnows except sunfish, which were much less sensitive.
We therefore concluded that a concentration of 120 Mg copperII. probably would
adversely affect the more sensitive species, but not the sunfish. Higher
concentrations would be lethal to some stream species when hardness, alkalinity,
and the concentration of detoxifying materials were lower because of rain. Thus,
120 Mg/2-. seemed to be a concentration of copper that would be adverse to some
species, but not to others, and would allow us to evaluate its effect on sensitive
and resistant species of fish.
The field portion of the study lasted for 4 years and 9 months. This time
span was divided into two periods: the pre-exposure period lasted from December
1967 to February 16, 1970; the exposure period lasted from February 16, 1970, to
October 31, 1972. The chronic toxicity studies continued until January 18, 1973.
Limited stream sampling for chemical parameters continued through May, 1973.
Part A of this report deals with the rationale of the project, description
of the field study site, physical alterations made on this site, toxicant metering
system, general water quality, and stream copper concentrations over the period of
exposure. Part B reports findings relative to the effects of copper on stream
fish and benthic communities and the analyses of stream fish stomach contents.
Part C presents the results of laboratory tests done in conjunction with the
field study before and during the stream exposure. Part D is a discussion that
relates the field studies to the laboratory studies.
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SECTION V
DESCRIPTION OF THE STUDY AREA
Shayler Run, located in Clermont County, Ohio, is a tributary of the East
Fork of the Little Miami River. The use of the watershed is predominently urban,
but areas adjacent to the study portion of the stream are wooded or used for grazing
(Figure 1). The laboratory site on the stream is latitude 39° 06' 46", longitude
84° 13' 24", and is 3.5 km from the mouth (Figure 2). The drainage area is 30.58
km , and the stream gradient in the study stretch is 26.8 m/km. The stream
consists of a series of limestone riffles and shallow pools, with a minimal amount
of sedimentation. At normal stream flow the pools are 3-9 m wide and 1-3.5 m deep.
Riffles comprise more than 50% of the stream length at normal stream flow and
are 3-12 m wide.
The only continuous flow (1890 kilpliters./dayX originates from a small domestic
sewage treatment plant that, during low flow periods, may contribute as much as
90% of the stream discharge. Data from macroinvertebrate collections obtained
between April 1969 and February 1970 indicate that the effluent from the sewage
treatment plant had minimal, if any, effect on the aquatic biota in the section of
stream considered in this report. Flow from Arrowhead Lake is only intermittent.
The U. S. Geological Survey designed, constructed, installed, and maintained
a stream gaging system approximately 4.4 km downstream from the waste treatment
plant outfall (Figure 3). This system consisted of a gaging pool and V-notch weir
(Figure 4), bubble gage (water-stage servo-manometer with gas purge system), and
a Stevens water-level recorder. The gaging system was completely installed by
July 1968 and functioned accurately throughout the study period.
The study area was divided at the V-notch weir into an upstream control area
(487 m long) and a downstream exposure area (902 m long). Copper was added to
the stream at the weir, which is designated as the zero point for both elevation
and distance. The remaining 2.7 km of the stream below the exposure area to the
confluence with the East Fork of the Little Miami River was called the recovery
area. This stretch of stream was similar to the experimental area, but of lower
gradient. In this area the copper concentration was less than that in the exposure
area and decreased with distance downstream. Limited sampling in this area
provided some information on repopulation of the stream by macroinvertebrates that
were adversely affected by copper in the exposure area.
Fish weirs were constructed to prevent upstream migration and to capture fish
moving downstream from both the control and exposure areas (Figure 3 and 5). They
were constucted of concrete, steel, and stainless steel wire screen and were
placed at the upper and lower ends of the 0.8-km exposure section. These weirs
were a modification of a Wolf-type fish-counting fence described by Clay (1961),
but were more permanent structures.
-------
Figure 1. Aerial view of Shayler Run study area, looking downstream.
-------
LATITUDE 39° 06' 46"
LONGITUDE 84° 13' 24'
SCALE
0 1km
3.94 cm 1 km
LABORATORY BUILDING
Figure 2. Location map of Shayler Run.
-------
z
o
UJ
>
RECOVERY]
AREA
Biannual Fish
Collection Areas
Fry Trap •
Chemical Station A
Macro in vertebrate
Station o
300
METERS
LOG BARRIER
H
2.7 km cc
LU
DC
i
oc
O
<
UJ
Figure 3. Sampling stations and gradient for Shayler Run.
-------
:
Figure 4. V-notch gaging weir.
-------
Figure 5. Control weir screens and V-notch gaging weir.
-------
When stream flow exceeded 1.7m /sec, the screen could not be kept clean, and
water flowed over the top allowing unmeasured fish passage. From March through
November a fine stainless steel screen with openings of 0.5 mm was used to
capture both fish fry and macroinvertebrates. From November through February a
coarser screen with 2.2-mm openings was installed in place of the finer screen to
reduce maintenance time. This coarser screen captured young-of-the-year fish.
Log barriers were constructed approximately 120 m upstream from both control
and exposure fish weirs to protect the stainless steel screens from damage from
large floating debris during heavy runoff. These barriers (Figures 6 and 7) were
constructed of 15.2-cm steel casings driven into the stream bed and filled with
reinforcing rods and concrete. Three lengths of 2.5-cm steel cable were then
attached to the castings and anchored to a concrete deadman on each side of the
stream. Vertical pieces of cable were placed at equal distances on three
horizontal cables. The barriers were placed at a 45-degree angle to the stream
flow so that debris would be shunted to one side. The log barriers efficiently
protected the fish weirs.
The locations of the sampling stations for chemical analysis and for fish,
macroinvertebrate, and periphyton collections, are shown in Figure 3. Station 1
was located just upstream from the V-notch weir, and station 2 was. 15 m
downstream from the V-notch weir. The Baldwin Road bridge was 850 m downstream
from the V-notch weir, near the downstream log barrier. The other chemical
sampling stations, 3, 4, 5, and 6, were located 130, 410, 620, and 795 m downstream
from the V-notch weir, respectively. The control riffle and control pool stations
were 350 and 275 m, respectively, upstream from the V-notch weir; the exposure
pool and exposure riffle were 200 and 525 m, respectively, downstream from the
V-notch weir. Fry traps were placed 50 (#3), 125 (#2), and 225 m (#1) above and
250 (#4), 475 (#5), and 775 m (#6) below the V-notch weir. Basket samplers for
collection of macroinvertebrates were located as follows: (1) control area—
midstream at fry trap 3; (2) upper exposure area—30 m downstream from chemical
station 3; (3) lower exposure area—20 m downstream from chemical station 6;
and (4) recovery area—2 km downstream from V-notch weir. The areas for sampling
natural substrates for macroinvertebrates were the exposure riffles, 70 m
downstream from chemical station 3 and 100 m downstream from chemical station 5,
and in a recovery riffle, 2.2 km downstream from the V-notch weir (not shown on
Figure 3).
Periphyton samples were collected from natural and artificial substrates
located in the control riffle and control pool, and from station 4 (pool) and the
exposure riffle in the exposure area.
Stream-flow recording began on August 1, 1968, after completion of the gaging
pool and weir and the calibration of the system. Charts and data were analyzed
by the U. S. Geological Survey and recorded in Water Resources Data for Ohio, Part 1,
for 1970, 1971, and 1972 (Appendix Table 1).
Daily mean flow was 50% lower during the pre-exposure period than during the
exposure period. After periods of heavy runoff the stream flow decreased to 0.25
m3/sec within 24 hr after the crest had passed the V-notch weir.
13
-------
- it '• '•
1 0 \ '• ' ! <
~- • ^ -
••-.> - ---,-•• ,, .
• •»>*-
• • '.« • -^ -^J
^r^-'' « * - ,i • ^
Figure 6. Upstream log barrier.
-------
Figure 7. Upstream log barrier covered with debris and logs.
-------
SECTION VI
STREAMSIDE LABORATORY FACILITIES
A 12.2- by 2.4-m mobile laboratory trailer and a 6.1- by 9.1-m sheet metal
pole building with an attached 12.2- by 3.0-m outside screened porch constituted
the laboratory facilities. These were located adjacent to the stream, beside
the V-notch weir. The mobile laboratory trailer was used for routine
chemical analyses, for processing some of the biological samples, and as an office.
The fish holding tanks and toxicity testing systems were located in the pole
building and its attached porch (Figure 8). This building also housed the copper
stock-supply reservoir. The pole building was the only wet laboratory area during
the first year of the test.
Two 314 Z-./min self-priming centrifugal cast iron pumps continuously supplied
water for the test systems. One pump supplied control water from just above the
V-notch weir. The other pump supplied exposure water, nominally dosed at 120 yg/Z.
copper, from station 3, 130 m below the V-notch weir where thorough mixing had
taken place. .The water from each pump was pumped through polyethylene pipes to
separate manifolds in the wet laboratory and was distributed to the flow-through
test systems, water baths, and holding tanks. The pumps had 6.3-mm intake
strainers and flow switches to turn them off automatically when flow was
interrupted, thus preventing damage to the pump heads.
During the first year and a half of the study a constant pressure regulator
and strainer assembly was used to maintain a constant flow for the test systems.
For the last year and a half, water was pumped to small-volume headboxes, located
in the rafters of the wet laboratory, to maintain a constant pressure.
16
-------
Figure 8 Interior of pole building wet laboratory showing chronic test setup,
-------
SECTION VII
TOXICANT-METERING SYSTEM
Initial addition of copper to the stream, beginning February 16, 1970, was
done with a gear-type pump having a manually controlled pumping rate. Staff
personnel were required to adjust the pumping rate, as stream flow varied, to
maintain the desired copper concentration in the stream. Use of an automatic
toxicant-metering system began on May 18, 1970, and was continued for the
remainder of the study. This system was designed so that, at stream discharges of
0.25 m3/sec and below, copper sulfate solution would be metered to the stream to
maintain a nominal concentration of 120 yg/Z-. at station 3, the point of thorough
mixing (Figure 9)- The stream-gaging apparatus was used to control the pumping
rate. The stream was not dosed at flows above 0.25 m3/sec because of the large
quantity of copper sulfate required.
Technical grade CuSO^ • 5H20 was dissolved in deionized water in a 2,000-Z.
fiber glass tank. Aeration was used to mix the solution that contained 3.06 g
Cu/l. The stock solution was then automatically siphoned to a 2,000-1. feed tank
for the pump.
During the exposure period, February 16, 1970, to October 31, 1972, copper was
added 75% of the time. The dosing pattern varied from month to month (Table 1).
For any given month over the 33-month period, the amount of time that dosing
occurred varied considerably since no toxicant was added at flows greater than
0.25 m /sec.
18
-------
TOXICANT LINE TO STREAM
V-NOTCH
WEIR
Figure 9. Schematic of toxicant-delivery system.
-------
TABLE 1. PERCENTAGE OF TIME THAT COPPER WAS ADDED TO SHAYLER RUN DURING THE EXPOSURE PERIOD
Year
1970
1971
1972
• —
Jan.
-
83
62
Feb.
95
21
36
March
57
46
17
. i
April
57
97
30
1
May
90
88
58
June
96
98
93
July
98
87
96
Aug.
84
95
64
Sept.
100
74
85
Oct.
98
100
90
Nov.
91
97
-
Dec.
73
59
-
Average
85
79
63
t
-------
SECTION VIII
WATER QUALITY
TEMPERATURE
Temperatures for Shayler Run during the 3 months of copper addition were
recorded continuously from the gaging pool. The minimum and maximum daily
temperatures normally occurred at 6 a.m. and 6 p.m., respectively. Data were
taken from daily records at four different times, 6 a.m., 12 N, 6 p.m., and 12 M,
for calculating the daily means. Monthly means were calculated from the daily
means (Appendix Table 2). Temperature regimes for the 3 years were similar.
CHEMICAL
Five different sets of measurements were performed periodically to determine
the chemical characteristics of Shayler Run water collected at station 1, which
is located upstream from the point of copper addition. Analyses for pH, alkalinity,
hardness, and dissolved oxygen constituted set 1 and were performed on-site
(Appendix Tables 3, 4, 5, and 6).
Set 2 measurements were performed at the Newtown Fish Toxicology Station on
samples generally less than 4 hr old. Analyses were pH, alkalinity, hardness,
specific conductivity (SC)., total solids (TS), dissolved solids (DS), calcium (Ca),
magnesium (Mg), orthophosphate-phosphorus (OP-P), and total phosphorus (TP)
(Appendix Table 7). Recommended methods were used (U. S. Federal Water Pollution
Control Administration, 1969), except that dissolved solids were defined as those
that passed through a 0.45-y membrane filter.
Set 3 measurements were performed on a portion of each sample used in set 2.
Analyses were potassium (K), sodium (Na), chloride (CHD), nitrate-nitrogen
(N03-N), nitrite-nitrogen (N02-N), ammonia-nitrogen (NH3-N), organic-nitrogen
(ORG-N), total Kjeldahl-nitrogen (TKN), total phosphorus (TP), total organic carbon
(TOG), calcium (Ca), and magnesium (Mg) (Appendix Table 8).
These analyses were performed under the supervision of Robert T. Williams
by the Waste Identification and Analysis Section of the Waste Water Research
Division of the Municipal Environmental Research Laboratory, U. S. Environmental
Protection Agency, Cincinnati, Ohio, (formerly known as the Advanced Waste
Treatment Research Laboratory, U. S. Environmental Protection Agency, Cincinnati,
Ohio), on water samples from Shayler Run that were generally less than 8 hr old.
Recommended methods were used (U. S. Federal Water Pollution Control Administration,
1969), except that chloride (CHD) was measured with an automatic titrator and
silver nitrate.
21
-------
Thirteen measurements, constituting set 4, were made for pesticides. These
were DDT, DDE, ODD, chlordane, dieldrin, endrin, heptachlor, heptachlor epoxide,
aldrin, BHD, lindane, endosulfan, toxaphene, and methoxychlor. Values for these
pesticides were generally less than detection limits, which were between 50 and
1000 ng/Z. for toxaphene and between 3 and 36 ng/Z. for the others. These
measurements were performed under the supervision of James J. Lichtenberg by the
Pesticides Identification Group of the Environmental Monitoring and Support
Laboratory, U,, S. Environmental Protection Agency, Cincinnati, Ohio, (formerly
known as the Analytical Quality Control Laboratory, U. S. Environmental Protection
Agency, Cincinnati, Ohio), using recommended methods (U. S. Environmental
Protection Agency, 1971). Four samples were also analyzed for malathion,
parathion, methyl parathion, trithion, methyl trithion, fenthion, ethion, DEF,
and dimethoate. The concentrations were always less than the detection limits,
which were between 10 and 50 ng/Z. Fifteen samples were analyzed for trace metals
(Appendix Table 9). These constituted set 5 measurements and were performed
under the supervision of John F. Kopp of the Metals Analyses Group of the
Environmental Monitoring and Support Laboratory, U. S. Environmental Protection
Agency, Cincinnati, Ohio, (formerly known as the Analytical Quality Control
Laboratory, U. S. Environmental Protection Agency, Cincinnati, Ohio), using an
emission spectro-chemical method (American Society for Testing and Materials,
1971).
22
-------
SECTION IX
STREAM-COPPER ANALYSIS
The backround concentration of copper (Cu) in Shayler Run water was measured
with a solvent extraction and atomic absorption spectrophotometric procedure like
that used by Brungs et_ a^L. (1973). Samples were acidified with 3 drops of
concentrated nitric acid per 100 ml. One to three hundred milliliters were poured
into a tared separatory funnel and weighed, and 2 ml concentrated nitric acid were
added per 100 ml. The sample was then extracted three times with 25 ml of a
solution made by dissolving 4 g diethylammonium diethyldithiocarbamate (Stary,
1964) in 1 gal distilled-in-glass chloroform, shaking the extractions for 2 min,
30 sec, and 30 sec, respectively. The extracts and any interfacial cuff were
collected in a beaker and (a) evaporated to dryness on a steam bath, or (b) 1 ml
of bromine or peracetic acid solution was added to destroy the volatile copper
complex before the solution was evaporated to dryness on a hot plate. The
peracetic acid solution was prepared fresh daily by mixing 2 ml of 30% hydrogen
peroxide with 10 ml of glacial acetic acid (Patchett and Batchelder, 1960). The
sides of the beaker were washed down with concentrated nitric acid, and the solution
was evaporated to dryness. The residue was dissolved in 0.15% concentrated nitric
acid and analyzed on a Perkin-Elmer model 303 atomic absorption spectrophotometer
with an air-acetylene flame and a Boling burner. Standards prepared in the
laboratory by dissolving reagent-grade copper sulfate in 0.15% concentrated nitric
acid in distilled water were checked against purchased copper standard solutions.
Between January 21, 1969, and November 2, 1972, 100 recoveries, using 300 ml
Shayler Run water spiked with 10-20 yg Cu/l., averaged 95.2% with a standard
deviation of 3.5% after three values of 132, 60, and 58% were discarded as outliers.
Sixty-two recoveries, using 100 ml Shayler Run water spiked with 30-60 yg Cu/l.,
averaged 98.1% with a standard deviation of 4.4% after one value of 75% was
discarded as an outlier. The outliers were all more than four standard deviations
from the respective means0
Between March 10, 1970, and December 3, 1971, 52 samples of undosed Shayler
Run water containing between 1 and 17 yg Cu/l. (average 6) were analyzed in
duplicate and seven in triplicate. The ratio of the higher result to the lower
in each set averaged 1.08 and ranged from 1.00 to 1.40 after two values of 2.07
and 2.54 were discarded as outliers. The coefficient of variation (relative
standard deviation) of the method calculated from the replicates averaged 7% and
ranged from 0% to 23% after two values of 49% and 61% were discarded as outliers,
when Cochran's test for homogeneity of variances was used (Guenther, 1964).
Between October 28, 1970, and August 19, 1971, 50 samples of dosed Shayler
Run water containing between 15 and 70 yg Cu/l. (average 40) were analyzed in
duplicate. The ratio of the higher result to the lower in each set averaged 1.02
23
-------
and ranged from 1.00 to 1.09. The coefficient of variation of the method
calculated from the replicates averaged 1.6% and ranged from 0% to 6%.
The results of all measurements of the backround level of copper in undosed
Shayler Run water are given in Table 2. These values were not corrected for the
recovery percentages. Set 1 of the measurements was performed on grab samples
collected almost weekly at station 6 until July 30, 1968, and at station 1
afterwards. Set 2 of the measurements was performed in triplicate on grab samples
in conjunction with some of the recoveries.
The other sets of measurements were performed on composite samples formed
by combining equal volumes of water taken on 7 consecutive days from the control
test containers from chronic tests conducted at streamside. Sets 4A and 4B were
taken from two different chronic tests conducted simultaneously. For the 20 weeks
for which samples were collected for both sets, the ratio of 4A to 4B averaged
0.92, with a standard deviation of 0.23, and ranged from 0.65 to 1.52.
Similarly, sets 5A and 5B were taken from two different simultaneous chronic
tests. For the 16 weeks for which samples were collected for both sets, the ratio
of 5A to 5B averaged 1.06, with a standard deviation of 0.31, and ranged from
0.49 to 1.78.
Samples for sets 6A and 6B were taken from one chronic test, whereas samples
for set 6C were taken from a simultaneous test. Although set 6A was analyzed by
the extraction procedure, sets 6B and 6C were analyzed by direct aspiration. For
the duplicate analyses performed on 22 samples, the ratio of 6A to 6B averaged
1.46, with a standard deviation of 0.49, and ranged from 0.76 to 2.83. For the
20 weeks for which samples were analyzed for both sets, the ratio of 6B to 6C
averaged 1.04, with a standard deviation of 0.36, and ranged from 0.50 to 1.80.
To determine the concentration of copper in the exposure area of Shayler Run
when the stream was being dosed as desired, water samples were collected in
125-ml polyethylene water-tight bottles that had been rinsed with 10% concentrated
nitric acid. Normally, samples were collected in Monday, Wednesday, and Friday
if the stream was being dosed. Whenever dosing was interrupted for any reason,
samples were not collected for 24 hr after dosing was begun again. Three sampling
schedules were used during the study. From February 16, 1970, to September 15,
1970, about 20% of the sample collections consisted of duplicate samples at stations
3, 4, 5, and 6, and the rest of the collections consisted of single samples at
each of these stations. From September 15, 1970, to June 1, 1971, collections
consisted of duplicate samples at stations 3 and 6 and single samples at stations
4 and 5. From June 1, 1971, to October 31, 1972, collections consisted of
duplicate samples at stations 2, 3, and 6 and single samples at stations 4 and 5.
Samples were taken near the middle of the stream, below the surface and upstream
of the collector. Samples were transported to the laboratory, acidified with 4
drops of concentrated nitric acid, and shaken. They were then analyzed for copper
by direct aspiration atomic absorption spectrophotometry and were compared against
standards containing similar levels of copper prepared in 0.15% concentrated nitric
acid in distilled water.
Between May 10, 1970, and January 11, 1973, 57 measurements of the accuracy
of the direct aspiration method were made at each of two levels by adding equal
amounts of copper to both acidified Shayler Run water and acidified distilled
water. For 100 yg Cu/l. the increase in instrumental response for the Shayler
24
-------
TABLE 2. BACKGROUND COPPER CONCENTRATIONS IN SHAYLER RUN WATER, 1968-73
(yg/l.)
Set
1
2
3
4A
4B
5A
5B
6A
6B
6C
Beginning
dates
5-29-68
3-30-71
1-21-70
9-30-70
10-14-70
4-28-71
4-28-71
1-12-72
1-12-72
1-26-72
Ending
dates
10-13-69
12-3-71
9-9-70
4-21-71
3-3-71
8-25-71
8-25-71
10-4-72
1-18-73
8-23-72
Number of
samples
59
7
32
26
20
16
16
22
34
20
Mean
5.0
4.4
6.9
6.3
5.5
6.4
6.4
6.7
9.2
8.3
Standard
deviation
3.2
2.9
2.3
1.9
1.5
3.1
3.1
3.6
3.8
2.5
Range mg/Z.
1-18
3-6
4-14
3-10
3-9
4-15
4-17
4-21
5-24
5-14
-------
Run water, compared to that for the distilled water, averaged 96.4% with a standard
deviation of 3.1%, after one value of 115% was discarded as an outlier. For 400
yg Cu/l. the increase for the Shayler Run water, compared to that for the
distilled water, averaged 96.6% with a standard deviation of 2.0%.
For 680 sets of duplicate samples of dosed Shayler Run water containing
between 30 and 263 yg Cu/l., obtained from stations 2, 3, 4, 5, and 6 between
February 16, 1970, and October 31, 1972, the ratio of the higher result to the
lower averaged 1.04 and ranged from 1.00 to 1.56 after one value of 2.27 was
discarded as an outlier. The coefficient of variation calculated from these
duplicates averaged 4.8% and ranged from 0% to 30% after one value of 55% was
discarded as an outlier.
The monthly averages of the measured copper concentrations in dosed Shayler
Run water are given in Table 3. These values were not corrected for the results
of the method of known additions. The results of 30 of the more than 2,100
analyses were discarded because they were not consistent with the rest of the
values obtained for the same day. Only four values were above 195 yg/Z. (258 and
263 at station 2 on July 28, 1971, and 234 and 239 at station 2 on June 21, 1972),
and these were discarded. No values were below 30 yg/Z. The monthly standard
deviations averaged 11.1 and ranged from 1 to 28. They tended to be higher in
the summer, lower at the downstream stations, and higher near the beginning of
the project.
2.6
-------
TABLE 3. MONTHLY AVERAGE COPPER CONCENTRATIONS IN EXPOSURE
SECTION OF SHAYLER RUN
(yg/Z.)
Month
1970
Feb.
March
April
May
June
July
Aug.
Sept.
Oct.
Nov.
Dec.
1971
Jan.
Feb.
March
April
May
June
July
Aug.
Sept.
Oct.
Nov.
Dec.
1972
Jan.
Feb.
March
April
May
June
July
Aug.
Sept.
Oct.
2 3
111.0
103.8
109.4
122.5
106.8
104.1
113.5
131.5
132.5
114.7
116.8
117.4
134.7
108.8
109.0
105.9
135.9 123.6
138.3 130.5
140.6 122.3
117.4 108.6
135.8 122.6
120.1 112.6
98.7 96.4
102.6 99.3
114.2 111.4
96.2 97.3
108.6 102.2
113.5 108.2
120.6 115.2
151.3 137.6
154.7 130.7
168.6 141.5
142.5 131.0
Station
4
106.9
97.0
102.6
101.8
84.8
81.4
83.9
99.2
119.5
111.0
108.8
115.4
128.2
103.1
98.6
93.3
99.6
100.2
96.7
102.7
108.0
103.3
91.0
94.1
106.6
88.6
94.3
101.5
99.9
104.0
97.8
110.9
117.6
5
101.2
90.2
88.9
79.3
53.3
54.2
57.3
67.2
96.6
98.8
102.4
111.3
119.1
95.2
86.1
79.4
70.8
73.3
65.1
86.5
90.9
90.2
83.6
89.0
105.1
87,8
86.6
88.1
81.7
75.8
63.3
72.5
98.2
6
99.
87.
85.
75.
5
4
7
0
48.8
51.
52.
58.
83.
94.
96.
111.
111.
92.
81.
76.
69.
64.
60.
73.
86.
86.
82.
88.
102.
83.
81.
81.
75.
64.
56.
62.
94.
2
1
1
7
4
3
2
4
5
3
5
1
9
5
5
6
2
8
0
1
8
4
4
1
5
1
2
6
6/33
89.3
86.3
79.3
61,8
47.6
48.8
44.5
44.4
64.0
82.4
83.4
96.3
85.0
74.9
73.3
53.3
51.0
48.5
73.9
70.8
76.0
86.0
85.1
86.3
73.1
64.6
49.1
42.9
44.1
72.2
Number
of days
sampled
15
10
11
15
12
12
10
13
12
13
10
10
2
6
11
10
13
10
12
7
12
13
8
7
4
8
3
9
12
13
7
7
8
aPercent copper in the stream at station 6 compared to station 3, the point of thorough
toxicant mixing.
-------
PART B — FIELD STUDIES
SECTION X
EFFECTS OF COPPER ON STREAM FISH
INTRODUCTION
Many collections and observations were made to evaluate the effects of the
addition of copper on population density, reproduction, growth, survival, and
avoidance or other behavioral reactions of fish. The purposes of the biannual
fish collections were to ascertain base population levels in control and exposure
areas before and after introduction of copper; to determine in the spring whether
mature fish were present in these areas before their spawning period; and to
determine in the fall the degree of spawning success and survival of young.
Since fish weirs were installed to isolate the- exposure area, changes in
fish activity resulting from the addition of copper would be evident. If fish
avoided copper in the stream or their activity increased or both, greater numbers
would be expected on the exposure weir. A species comparison of fish captured
could then be made with those captured on the upstream control weir. The purpose
of spawning observations was to obtain data relating to the effects of copper, if
any, on fish spawning success and spawning behavior.
Sections in this part of the report will deal with the collections and
observations used to evaluate the effects of copper on the fish populations. In
Part D of this report these field results will be related to laboratory results
obtained before and during the study.
METHODS
Biannual Fish Collections
Eight biannual (spring and fall) collections were made, starting in the spring
of 1968 and ending in the fall of 1971. Four collections were made before the
introduction of copper, and four collections were made during the exposure period.
The collections were made in the same pool and riffle areas each time (Figure 3).
Collections were made with an electrofishing apparatus consisting of an
electric seine, 7.6 m long, similar to the one described by Funk (1957). A
variable voltage pulsator (Coffelt Electronic Co., Model No. III-C) was used, and
the power supply was a Model 9A 115-1A Homelite generator with a continuous output
of 3,000 watts. The variable voltage pulsator supplied both alternating current
(AC) and direct current (DC), but AC was more successful in this stream and was
used for all collections. The AC voltage could be varied between 0 and 280 with
this unit. Since all fish captured were removed for further analysis, little
concern was given to killing or injuring the specimens with an electric charge.
The maximum electric output of the system was always used.
28
-------
Block seines made from 4.76-mm stretch mesh were placed at the upstream and
downstream limits of each section of stream being fished to prevent escapement.
Collections were made with a crew of six men only during periods of low flow and
when the water was clear. Two passes were made over each area, and an attempt was
made to capture all fish seen. During the initial collection period a third pass
of an area produced less than 2% of the total catch from the two previous passes
and was subsequently abandoned.
All collected specimens were preserved with acetic acid formalin and alcohol
(AFA), and specimens 60 mm and longer were injected with AFA so that stomach
contents would be well preserved for future analysis. Fish were identified to
species, enumerated, and measured. For the eight most abundant species length-
frequency graphs were prepared as an aid in determining age-group 0. Age-group 0
was defined as those individuals that were less than 1 year old in a fall
collection and between 7 and 13 months old in a spring collection. All fish that
were older at the designated time were considered adults.
Weir-Screen Collections
Daily collections were made from the weir screens, beginning 2 days before
the introduction of copper sulfate to the stream. During the period of exposure
February 16, 1970, through October 31, 1972, few fish other than fry were taken
on the screens between June and February. During May many predators and scavengers
consumed fish captured on the screens. Data presented are from the dates within
the period February 15 to May 28. The initial sampling dates were dependent upon
flooding and icing conditions and the ending dates upon activities of predators
and scavengers.
Fry Collections (Traps and Weirs)
Fry samples were collected during the exposure period from fish weirs and fry
traps to obtain information relating to reproduction and young-of-the-year growth
rates in control and exposure areas.
The fry were captured from both areas for identification. This sampling was
only qualitative. Fry captured on the fish weirs could not be compared because
the collections of fish from the weirs did not include all species of fry observed
in the study stream. Many of the specimens were partially decomposed, and
scavengers or predators removed fry from the weirs.
In 1971 fish-fry traps were designed and built to obtain both quantitative
and qualitative samples and specimens in better condition, but too few fry were
captured in the exposure area for quantitative estimates. These traps
were constructed of fine mesh brass window screen and aluminum framing (Figure 10).
The trap was placed at the stream's edge, where the waters were shallow and of
low velocity and the bottom substrate was composed of fine sand and some silt.
A 2-m lead touched the shore above the water's edge, and a short lead (1 m) was
placed at right angles to the shore. An opening of approximately 15 cm remained
between the short lead and the shore. At the termination of a 4-hr sampling
period, a piece of sheet metal was placed across this opening and anchored in the
stream bottom. The fry were then removed from the trap with a fine-mesh dip net
and preserved. In the laboratory they were identified to species, measured for
length, and counted,,
29
-------
STEEL ROD
FISH FRY TRAP
Figure 10. Schematic of fish-fry trap.
30
-------
Three similar locations in both the control and exposure area were selected
for trap placement (Figure 3). All traps were fished for 4 hr over the same
general period. Twenty-two collections were taken starting on May 19, 1971, and
ending on August 30, 19710
Fish-Spawning Observations
Fish spawning was observed for the years 1968 through 1972. Considerable
time was spent during the pre-exposure period observing spawning activity of
various species, but the number of spawns was not recorded. For every spawning
activity observed and recorded, a confirmation of the spawn was made by examining
the eggs for fertility and age. For species that spawn in groups, such as
stoneroller, creek chub, striped shiner, and white sucker, each group was recorded
as one spawning. The range in number of individuals per group for the above four
species was 20-30, 5-10, 20-25, and 10-15, respectively. For bluntnose minnows,
which spawn on the underside of rocks, one group of eggs was recorded as one
spawning.
Spawning observations were usually made every other day, but varied with
stream conditions. During the exposure period observations were normally made
for equal lengths of time (1 to 1-1/2 hr) in both control and exposure areas when
a species was known to be spawning. More time and effort were spent on species
that have secretive spawning habits, such as the bluntnose minnow, and emphasis
was placed on finding first spawning of each species. On numerous occasions
spawning activity was prevented by an abrupt decrease in temperature.
OBSERVATIONS AND RESULTS
Avoidance
On February 22, 1970, approximately 400 fish were seen concentrated at a point
in the exposure area where a small spring-fed stream enters. Most were striped
shiners, stonerollers. and bluntnose minnows, but a few sunfish and darters were
also present. The copper concentration in the area where the fish were seen was
77 yg/Z.., whereas the copper concentration at midstream at this location was 121
Pg/Z-. The temperature of the spring water was 2° C, and the stream was 1° C at the
mouth of a tributary in the control stretch. No fish of any species were observed
and the temperature of the tributary was 3° C, indicating that the congregation
of fish was not caused by a temperature preference. Few fish were seen in the
control area other than darters, which raced away when stream rocks were disturbed,
and schools of striped shiners at the heads of deeper pools. On the other hand,
numerous fish were observed in the exposure area located at the very edge of the
stream or in small shallow backwater pools. The copper concentration in one of
these pools measured 3 Vg/l., but in midstream at this location it was 125 vg/l.
A decision was made to introduce control water 130 m downstream from the point
of copper introduction to verify the apparent avoidance. No fish had been observed
at this site on February 22. On February 23, 3 mature creek chubs, 10 mature
stonerollers, 8 rainbow darters, 20 bluntnose minnows, and 3 mature striped shiners
were observed at the location of this discharge. At the spring outfall in the
exposure stretch for that date, estimates were 300-400 mature striped shiners,
40-50 stonerollers, 200-300 bluntnose minnows, and a few sunfish. By the first
of May and thereafter, no concentrations of fish were observed at the spring
outfall or along the stream edge or in backwaters, and those fish that were
31
-------
observed in the exposure area were in what would be considered their normal habitat,
suggesting that either the animals adapted to the copper in the stream or they
were under a greater physiological stress at the low water temperatures and short
photoperiodo
For the first week of dosing, no fish were observed dead or in distress;
however, on February 23, 7 days after the start of exposure, and for approximately
1 week thereafter, fish were observed dead or in distress in the exposure area, but
not in the control area. The following fish were observed for that period:
Dead Distressed
Stoneroller 20 13
Hog sucker 9 2
Johnny darter 4 1
Bluntnose minnow 2 7
White sucker 1 1
Carp - 1
Green sunfish 1 -
Since no fish were observed dead or in distress in the control area, copper
apparently caused the above effects, especially to the more sensitive stonerollers,
hog suckers, johnny darters, and bluntnose minnows.
In other tests stonerollers were shown to be the most sensitive and died in
the greatest numbers. The actual number observed was small when compared to the
stream population, Since numerous crayfish in the exposure area consumed dead
fish most readily, the number of fish killed by copper was probably greater than
observed.
Biannual Fish Collections
Thirty-four species of fish were collected during the study period. The
totals for the control and exposure areas for these eight collections are
presented in Table 4. The first eight species listed lend themselves to more
detailed analysis since adequate populations of these were present in both control
and exposure areas during the pre-exposure period. The ninth species listed,
longear sunfish, was not common in the control area, but was common in the exposure
area in adequate numbers for evaluation. The last 25 species were considered rare
or not common to the stream. Some of them may have escaped from upstream ponds
that drained to Shayler Run.
Table 5 presents the number of age-group 0 and mature fish by species for
control and exposure areas from the biannual collections. The mature portion of
the fish population was more stable than the age-group 0 portion during the
pre-exposure period. This would be expected in a natural population because of
the various stresses on spawning success and the vulnerability of eggs and young
to flooding and predation. Figures 11A through ISA present graphically the
numbers of adults of the eight most numerous species collected from control and
exposure areas.
Except in the 1970 fall collection, bluntnose minnow adults (Figure 11A)
show a general decline in the exposure area during the exposure period when
compared with the pre-exposure period. Further, the population of adult bluntnose
32
-------
TABLE 4. TOTAL NUMBER OF INDIVIDUALS COLLECTED FROM SHAYLER RUN IN THE
BIANNUAL FISH COLLECTIONS, 1968-71
OJ
Species
1. Bluntnose minnow
Pimephales notatus (Rafinesque)
2. Striped shiner
Notropis chrysocephalus (Rafinesque)
3. Creek chub
Semotilus atromaculatus (Mitchill)
4 Stoneroller
Campos tema anomalum (Rafinesque)
5. Rainbow darter
Etheostoma caeruleum Storer
6. Fantail darter
Etheostoma flabellare Rafinesque
7. Orangethroat darter
Etheostoma spectabile (Agassiz)
8. Green sunfish
Lepomis cyanellus Rafinesque
9. Longear sunfish
Lepomis megalotis (Rafinesque)
10. Johnny Darter
Etheostoma nigrum Rafinesque
11. Blacknose dace
Rhinichthys atratulus (Hermann)
Location
April
'68
Control 335
Exposure! 298
|
C i 356
_c
E
C
E
C
E
C
E
346
136
118
101
139
96
162
C 166
E ' 193
Sept.
'68
2,057
465
2,565
596
287
434
779
551
57
187
66
131
1
C
E
C
E
C
E
C
E
C
E
98
83
78
133
47
83
19
20
2
1
183
72
78
156
8
248
23
3
63
11
'
March
'69
922
765
1,325
1,121
213
104
1,026
184
53
153
61
205
129
231
24
74
0
58
23
8
154
20
Sept.
'69
1,643
429
436
493
262
133
531
496
14
60
19
75
137
50
31
39
0
125
18
18
49
18
Feb.
16-1970
copper
first
introduced
to
exposure
area
May
'70
648
152
190
217
289
42
112
51
8
11
30
19
13
45
36
70
1
78
36
31
22
1
1
Oct.
'70
818
838
360
268
423
157
1,322
182
46
28
136
281
148
128
128
46
0
79
76
62
108
12
April
'71
315
51
72
113
178
133
312
76
9
22
45
121
58
138
17
52
0
36
46
29
74
14
Oct.
'71
352
257
i
236 !
73
570
116
402
34 !
5 \
3
52
65 i
159
247
42
70
5
32
60
20
90
10
-------
TABLE 4 (continued). TOTAL NUMBER OF INDIVIDUALS COLLECTED FROM SHAYLER
IN THE BIANNUAL FISH COLLECTIONS, 1968-71
RUN
Species3
12. White sucker
Catostomus commersoni (Lacepede)
13. Rosefin shiner
Notropis ardens (Cope)
14. Northern hog sucker
Hypentelium nigricans (Lesueur)
15. Bluegill
Lepomis macrochirus Rafinesque
16. Golden redhorse
Moxostoma erythrurum (Rafinesque)
17. Black bullhead
Ictalurus melas (Rafinesque)
18. Gizzard shad
Porosoma cepedianum (Lesueur)
19. Spotted sucker
Minytrema melanops (Rafinesque)
20. Carp
Cyprinus carpio Linnaeus
21. Golden shiner
Notemigonus crysoleucus (Mitchill)
22. Spotfin shiner
Notropis spilopterus (Cope)
23. Silver jaw minnow
Ericymba buccata Cope
Location
C
E
C
E
C
E
C
E
C
E
C
E
C
E
C
E
C
E
C
E
C
E
C
E
i
April
'68
68
84
6
17
11
17
17
13
26
19
22
20
0
0
0
0
15
0
1
1
1
0
3
2
Sept.
'68
64
53
4
177
5
9
38
226
0
5
0
18
0
3
0
2
0
0
0
0
0
0
0
6
March
'69
10
35
0
11
0
6
1
0
0
0
0
1
0
0
0
0
0
1
0
0
0
0
0
14
Sept.
'69
0
3
0
199
0
23
19
112
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Feb.
16-1970
copper
first
introduced
to
exposure
area
May
'70
30
14
0
4
2
0
45
6
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
Oct.
'70
17
8
0
26
4
5
287
15
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
April
'71
12
16
0
1
6
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
o
I
Oct.
•71 :
90
129
0
12 :
4 ;
0
o :
o :
o !
0
0
0
0
0
0
0
0
o
0
0
0
0
0
0
-------
TABLE 4 (continued). TOTAL NUMBER OF INDIVIDUALS COLLECTED FROM SHAYLER RUN
IN THE BIANNUAL FISH COLLECTIONS, 1968-71
Ul
^Bailey,
Control.
Exposure.
al_. (1970).
Species3
24. Fathead minnow
Pimep hales promelas Raf inesque
25. Yellow bullhead
Ictalurus natalis (Lesueur)
26. Brown bullhead
Ictalurus nebulosus (Lesueur)
27. White crappie
Pomoxis annularis Rafinesque
28. Rock bass
Ambloplites rupestris (Rafinesque)
29. Smallmouth bass
Location
C
E
C
E
C
E
C
E
C
E
C
Micropterus dolomieui Lacepede ] E
30. Spotted bass C
Micropterus punctulatus (Rafinesque) E
31. Largemouth bass | C
Micropterus salmoides (Lacepede) ; E
32. Greenside darter
Etheostoma blennioides Rafinesque
33. Emerald Shiner
Notropis antherinoides Rafinesque
34. Northern redbelly dace
Chrosomus eos Cope
C
E
C
E
C
E
April
'68
0
1
0
6
2
1
0
0
0
3
1
4
1
3
0
2
3
2
0
0
0
0
Sept.
'68
0
0
1
25
1
11
3
0
1
6
0
61
14
18
0
4
0
0
0
0
0
0
March
'69
0
0
0
8
0
1
0
0
0
0
0
24
0
0
0
0
0
0
0
0
0
0
Sept.
'69
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Feb.
16-1970
copper
first
introduced
to
exposure
area
May
'70
11
0
1
1
0
0
19
0
0
0
0
0
0
0
3
2
0
0
0
1
0
0
Oct.
'70
1
0
0
1
0
0
8
3
0
0
0
0
0
0
0
1
1
7
0
0
April
'71
Oct.
'71
0
0
0
3
0
0
0
0
0
0
0
0
0
0
0
0
0
2
0
0
i
o! o
°! !
o
0
0
0
0
0 '
0
0
0
0
0
0
0 :
o !
0 :
o ;
0
3 i
1
0
0
0
0
-------
TABLE 5. AGE-GROUP 0 AND MATURE (IN PARENTHESIS) FISH COLLECTED FROM SHAYLER RUN IN
BIANNUAL FISH COLLECTIONS, 1968-71
Species Location
Bluntnose minnow Control
Exposure
Striped shiner Ca
Eb
Creek chub C
E
Stoneroller C
E
Rainbow darter C
E
Fantail darter C
E
Orangethroat darter C
E
Green sunfish C
E
Pre-exposure period
April
'68
153(182)
173(125)
44(312)
42(304)
65 ( 71)
46( 72)
35( 66)
47 ( 92)
62 ( 34)
104 ( 58)
57(109)
72(121)
69( 32)
72( 20)
26 ( 52)
43( 73)
Sept.
'68
1,902(155)
320(145)
2,489( 76)
411(185)
170(117)
268(166)
688 ( 91)
202(349)
33 ( 24)
30(157)
17 ( 49)
41( 90)
170( 13)
41( 31)
20( 58)
17(139)
March
'69
845( 77)
570(195)
1,298( 27)
774(347)
161 ( 52)
29( 75)
940( 86)
137( 47)
33 ( 20)
78( 75)
24 ( 37)
83(122)
107 ( 22)
149( 82)
19( 5)
Sept.
'69
1,444(199)
125(304)
152(284)
5(488)
193( 69)
33 ( 96)
375(156)
138(358)
7( 7)
5( 55)
3( 16)
9( 66)
118( 19)
24 ( 26)
2( 29)
34( 40) 14( 25)
Feb.
16-1970
copper
first
introduced
to
exposure
area
Exposure period
May
'70
386(262)
112 ( 40)
49(141)
40(177)
128(160)
20( 22)
50( 62)
23 ( 28)
2( 6)
7( 4)
16 ( 14)
11 ( 8)
10( 3)
36( 9)
24 ( 12)
45( 25)
Oct.
'70
544(274)
575(263)
21(339)
104(164)
301(122)
104 ( 53)
981(341)
81(101)
12( 34)
6( 22)
73( 63)
233 ( 48)
100 ( 48)
103 ( 25)
5(123)
5( 41)
April
'71
221 ( 94)
42 ( 9)
30( 42)
5(108)
127 ( 51)
92 ( 41)
207(105)
61 ( 15)
K 8)
12 ( 10)
14( 31)
83 ( 38)
40( 18)
91 ( 47)
0( 17)
9( 43)
Oct.
'71
226(126)
241 ( 16)
69(167)
52 ( 21)
492 ( 78)
72( 44)
232(170)
17( 16)
4( 1)
0( 3)
11 ( 41)
24 ( 41)
142 ( 17)
235(.12)
32 ( 10)
46( 24)
,Control.
b
Exposure.
-------
600
500
400
300
200
100
Control Area —
Exposure Area-
S = Spring
F = Fall
I
I
V- T
\
\
B\
I
\
NOTE: I
Points adjusted
for equal sampling
periods each spring
\
S6B F68 S69 F69 S70 F70
BIANNUAL FISH COLLECTION
S71 F71 S70 S71 S72
FISH WEIR COLLECTION
Figure 11. Numbers of mature bluntnose minnows from (A) biannual fish
collections and (B) weir-screen collections.
-------
700
600
LO
O>
Control Area-
Exposure Area
S = Spring
F = Fall
S68
F68
Figure 12.
F69 S70 F70
BIANNUAL FISH COLLECTION
S71
F71
> NOTE:
\ Points adjusted
\ for equal sampling
* periods each spring
\
\
\
S70 S71 S72
FISH-WEIR COLLECTION
Numbers of mature striped shiners from (A) biannual fish
collections and (B) weir-screen collections.
-------
600
500
400
300
200
100
Control Area-
Exposure Area
S = Spring
F= Fall
1 NOTE: '
Points adjusted
for equal sampling
periods each spring
S68 F68 S69 F69 S70 F70
BIANNUAL FISH COLLECTION
S71 F71 S70 S71 S72
FISH-WEIR COLLECTION
Figure 13. Numbers of mature stonerollers from (A) biannual fish
collections and (B) weir-screen collections.
-------
350
300
250
200
150
100
50
1 1 1 1 1 1 1 1
1
_
5
_ ^
\
exi
— t—
ae.
\—
c/>
Lkl
AN 1
/ \ ><
/ \ LlJ
/ S
/ \
/ \
~ / N
/ \
/ V
/ ^..
t *^
^^
~ \
\
L S
^""""""""^^^^^w^l^ \
" ***^ '
1 1 ^
Control Area
Exposure Area
S = Spring
f - Fall
A
j^L
^^^ ^^^^^
^S~~ "*~ ""^*^^«
-^•^" 1 ^"^**=«i=d
1 1 485 1
\
1 NOTE:
I Points adjusted
' for equal
\ sampling periods
Jeach spring.
\
\
B \
\
\
1
\
\
I
\ __
\
\
\
\
\
\
\
\
\
\
\
\
\
\
I _
i
?
I
* 1 ^
S68
F68
S69
F69
S70
F70
S71
F71
S70
S71
S72
Figure 14<
BIANNUAL FISH COLLECTION FISH-WEIR COLLECTION
Numbers of mature rainbow darters from (A) biannual fish
collections and (B) weir-screen collections.
-------
200
150
100
50
CO
C3
Q_
X
I
I
Control Area -
Exposure Area.
S -- Spring
F -- Fall
I
NOTE:
Points adjusted
for equal sampling
periods each spring.
I
I
S68 F68 S69 F69 S70 F70
BIANNUAL FISH COLLECTION
S71 F71 S70 S71 S72
FISH-WEIR COLLECTION
Figure 15. Numbers of mature creek chubs from (A) biannual fish
collections and (B) weir-screen collections.
-------
300
250
200
Control Area-
Exposure Area-
S-Spring
F --Fall
100
50
I
NOTE:
Points adjusted
for equal sampling
each spring.
S68
F68
S69 F69 S70 F70 S71 F71 S70 S71 S72
BIANNUAL FISH COLLECTION FISH-WEIR COLLECTION
Figure 16„ Numbers of mature fantail darters from (A) biannual fish
collections and (B) weir-screen collections.
-------
OJ
Control Area
Exposure Area
S68
S69
F69 S70 F70
BIANNUAL FISH COLLECTION
S71
F71
NOTE:
Points adjusted
for equal sampling
periods each spring.
S70 S71 S72
FISH-WEIR COLLECTION
Figure 17. Numbers of mature orangethroat darters from (A) biannual fish
collections and (B) weir-screen collections.
-------
Control Area
Exposure Area-
_ S = Spring
n F = Fall
~~1I
NOTE:
Points adjusted
for equal sampling
periods each spring.
S68
S69
F69 S70 F70
BIANNUAL FISH COLLECTION
S71
F71
S70 S71 S72
FISH-WEIR COLLECTION
Figure 18. Numbers of mature green sunfish from (A) biannual fish
collections and (B) weir-screen collections.
-------
minnows in the spring and fall collections of 1971 was considerably lower than
that of the lowest collection during the pre-exposure period.
The numbers of adult striped shiners (Figure 12A), stonerollers (Figure 13A),
and rainbow darters (Figure 14A) represented in the exposure collections for the
dosing period show a definite decline without any appreciable recovery when
compared with numbers for the predosing period.
Creek chub and fantail darter adults (Figures 15A and 16A) in the exposure
area display a general decline in numbers in the collections during the exposure
period when compared with those for the pre-exposure period. Adult orangethroat
darter and green sunfish populations (Figures 17A and 18A) did not appear to be
directly affected by copper during the exposure period.
Weir-Screen Collections
Information is presented in Table 6 that pertains to the addition of copper
to the stream, the flooding of the screens, and the number of fish collections
from the weir screens during the exposure period between February 15 and May 28,
1970-72. Data for adults of the eight most abundant fish species are graphically
expressed in Figures 11B through 18B.
During the first spring of copper introduction greater numbers of adult
bluntnose minnows, striped shiners, stonerollers, rainbow darters, and fantail
darters were captured on the exposure screens than on the control screens (Table
7). In the following two springs, however, when the copper was still being
added, the numbers of adults captured from control and exposure areas were
similar. This may have been due to acclimation to copper or to fewer fish in the
exposure area. Young-of-the-year bluntnose minnows and rainbow darters also
contributed large numbers of individuals to collections from the exposure screens
during the first spring of dosing.
Numbers of adult green sunfish, longear sunfish, and orangethroat darters
on both screens were low. However, the numbers of young-of-the-year orangethroat
darters on the exposure screens substantially increased during the springs of
1971 and 1972 compared to 1970. In fact, the number of young-of-the-year captured
by the exposure screen during the spring of 1972 on 21 collection days was
approximately seven times greater than the number captured in 1970 on 53 collection
days. The downstream movement of young-of-the-year orangethroat darters from
both the control and exposure areas increased during the three springs of exposure
(Table 8). The number of individuals captured on the exposure screens
progressively increased each year during the period of exposure, even on those
days when copper was not added to the stream. Thus this migration of
young-of-the-year orangethroat darters was not directly related to copper
exposure. For example, in the spring of 1972, 293 specimens were captured on the
exposure weir screen in 21 days of copper exposure. In contrast, 221 specimens
were captured from the same weir in 15 days when no copper was being added. The
cause of this movement is not known; it may be a normal downstream migration.
Results of daily weir-screen collection for 1970 (Figure 19) present a more
detailed account of fish movement and how the various species responded to copper.
The bluntnose minnow and, to a limited degree, the stoneroller gave an initial
response to copper within the first 48 hr of exposure by moving out of the
exposure area onto the screens. These two species, plus the striped shiner,
A R
-------
TABLE 6. DATA RELEVANT TO THE FISH COLLECTIONS
ON THE WEIR SCREENS, SHAYLER RUN, 1970-72
Year 1970 1971 1972
Dates of collections 2/15-5/12 2/25-5/28 3/11-5/3
No. of days copper
was added 58 69 23
No. of times weirs flooded 567
Total hours of flooding 140 114 163
No. of fish collections
when adding copper 53 56 21
No. of fish collections
when not adding copper 8 16 15
46
-------
TABLE 7. NUMBER OF AGE-GROUP 0 AND ADULT FISH COLLECTED FROM SHAYLER RUN ON WEIR SCREENS0
Species Location
Bluntnose minnow Control
Exposure
Striped Shiner Cc
Ed
Creek chub C
Stoneroller C
jr
Rainbow darter C
E
Fantail darter C
E
Orangethroat darter C
E
Green sunfish C
E
Long ear sunfish C
E
Dosing
Spring 1970(53)b
Age-group
0 Adult
56 74
460 579
5 69
34 674
17 178
6 60
32 152
67 561
Spring 1971(56)°
Age-group
0 Adult
34 9
20 15
2 19
0 138
1 30
1 37
89 59
91 35
8 3 ; 4 3
260 415 j 3 9
19 7 15
86 315 90 84
11 4
38 13
10 2
89 42
12 12 I 0 26
7 10 i 0 10
4 3 ' 1 0
26 4 25
Spring 1972(21)D
Age-group
0 Adult
10 14
32 8
0 12
5 17
0 35
0 3
11 25
14 3
1 5
8 0
3 3
11 6
31 7
293 21
1 2
0 9
0 0
1 0
Not dosing
Spring 1970(8)b
Age-group
0 Adult
2 7
3 7
0 6
0 5
2 12
0 1
0 29
0 6
1 1
0 3
0 2
0 3
0 1
4 3
1 0
0 0
0 0
1 0
Spring 1971(16)°
Age-group
0 Adult
8 5
2 0
0 9
0 14
1 10
1 0
77 28
25 11
0 0
8 5
12 10
46 26
11 3
56 37
0 17
0 4
0 0
1 1
Spring 1972(15)"
Age-group
0 Adult
3 2
1 1
0 15
0 2
1 25
0 1
27 22
1 4
3 5
2 2
14 12
29 28
21 8
221 3
3 16
1 5
0 0
1 0
^The number of days of collection are different for each year. Therefore the columns cannot be directly compared.
Indicates number of days of collecting.
^Control.
Exposure.
-------
FftllAKl Vinci
II II II !l 1 11 fi
-i 1 1 1 1 r
RAINBOW DARTER FISH WEIR DAILY COLLECTION TOTALS 1970
FAKTAIL DARTER FISH WEII DHL) CDUECTION TOTALS 1971
CREEK CHUB FISH WEIR DAILY COLLECTION TOTALS 1970
CONTROL AREAS
EIPOSURE
JI.Hi Ifln.L
Figure 19. Daily collections -of various fish species from the weir screens during
1970, and stream temperatures and dosing regimes at the time of collection.
48
-------
«»•»•«' Nm
IS II 23 28 S
- r
.
.
.O-Q
10 IS
APRIL
!0 25 30 1 i 14 19
1 • ' • IT
n 1
BLIINTNOSE MINNOW EISN WEIR DAILf COLLECTION TOTALS 1970
L
JlLl,! -
T — 1 1 1 1
niFi.LLn
— 1 T
STUPED SHINER MSH XEIB Dili! COLLECTS TOHIS 11
-
ID , J
n
1
1
1!
li
I'll '144
w
n fill
y o III
1
i n
1
T
r
MAY
It 1) < 9 12
1 1 1
-
_J
fl
iJ
-
„
III
•
u
1 ' 1
|
ntt\
ll
Li,
m
-
1
I 1 I I I T
STQNEROUER fISH WEIR DAILY COLLECTION TOTALS 1970
I in Tfll I
n r
T1IIPMATOIES
. HtX
TEHPEHATUIE HO BOSH6 ICCIME
Figure 19. Daily collections of various fish species from the weir screens during
1970 and stream temperatures and dosing regimes at the time of collection.
49
-------
TABLE 8. NUMBER OF YOUNG-OF-THE-YEAR ORANGETHROAT DARTERS
COLLECTED ON WEIR SCREENS, SHAYLER RUN, 1970-72
Spring Spring Spring
1970 1971 1972
Control weir
Dosing 11 (53)S 10 (56)3 31 (21)a
Not dosing 0(8) 11 (16) 21 (15)
Exposure weir
Dosing 38 (53) 89 (56) 293 (21)
Not dosing 4(8) 56 (16) 221 (15)
o
Number of collectings.
50
-------
rainbow darter, and fantail darter, began a mass exodus from the exposure area on
April 7 or 8, 1970. The increase in numbers of specimens captured on the exposure
weir screen was correlated with an increase in water temperature. The average
daily stream temperature increased from 5° C on April 6 to 11° C on April 9.
The rainbow darter, fantail darter, stoneroller, and striped shiner were just
starting their spawning activities at this time. It is believed that with increased
spawning activity, plus the increased activity caused by copper exposure, more
stress was placed on the population, and a behavioral change or avoidance reaction
occurred.
To a limited degree creek chubs responded similarly. However, this was the
only species for which more specimens were captured from the control weir than
from the exposure weir. During the first week of exposure creek chubs gave no
indication of downstream movement. From April 7 through 22 downstream movement
of this species from the exposure area increased slightly (Figure 19)„ At the
same time the bluntnose minnow, striped shiner, stoneroller, rainbow darter, and
fantail darter were moving downstream, but the numbers of creek chubs were much
Iower0 On April 14 a definite movement of creek chub downstream from the control
area began. This movement was not exhibited by any of the other stream fish
species. The creek chub is a high gradient stream species and is normally
associated with the uppermost headwaters (Breder and Rosen, 1966). It has a
tendency to migrate downstream for spawning since favorable spawning sites for
this species are more abundant in lower stream gradient. This would partially
explain the capture on the control weir-screen of numerous individuals in advanced
spawning conditions„ These fish could easily be stripped of eggs or milt» During
May a number of the specimens captured on the control weir-screen were "spent"
after spawning and were-damaged or had fungus infections, apparently from their
aggressive spawning behavior. Copper probably did not cause major downstream
movement by this species.
Spawning Observations
Thirteen species of fish were observed spawning during the pre-exposure period
in both control and exposure areas of the stream. Six of these, bluntnose minnow,
striped shiner, creek chub, stoneroller, green sunfish, and white sucker, were
common in both control and exposure areas, and their spawning activities in the
two areas were similar before copper exposure began. Longear sunfish were scarce
in the control area and fairly abundant in the exposure area before the
introduction of copper. The control area was probably a marginal habitat for this
species.
Only 11 species were observed spawning in Shayler Run during the exposure
period (Table 9). Fantail darters and rainbow darters were observed spawning in
both control and exposure areas during pre-exposure, but not during the exposure
period. Bluegill and largemouth bass are considered pond or lake species, and
blacknose dace was not common in the exposure area. These three species,
therefore, will not be considered in detail.
Stoneroller, striped shiner, and creek chub spawned abundantly throughout the
study area before copper exposure,, However, no spawnings were observed for these
species in the exposure area during the exposure period (Table 9). The total
number of spawnings observed in the control area during the exposure period was
66, 30, and 34 for the stoneroller, striped shiner, and creek chub, respectively.
51
-------
TABLE 9. NUMBER OF FISH SPAWNINGS OBSERVED IN CONTROL AND EXPOSURE AREAS OF
SHAYLER RUN DURING THREE SEASONS OF COPPER INTRODUCTION
Ln
to
Species
Bluntnose minnow
Striped shiner
Creek chub
Stoneroller
Rainbow darter
Fantail darter
Orangethroat darter
Green sunfish
Longear sunfish
Blacknose dace
White sucker
Bluegill
Largemouth bass
Control
area
26
16
4
6
2
19
4
0
0
27
4
1970
Exposure
area
1
0
0
0
2
35
78
0
0
5
0
Control
area
22
8
15
47
7
30
2
10
16
0
0
1971
Exposure
area
1
0
0
0
2
13
33
0
3
0
0
1972
Control Exposure
area area
0 1
6 0
15 0
13 0
3 2
28 20
5 32
1 0
0 0
2 0
0 0
Total
Control Exposure
area area
48
30
34
66
12
77
11
11
16
29
4
3
0
0
0
6
68
143
0
3
5
0
All of the listed species were observed to spawn throughout the control and exposure areas for the 2 years before copper was
.introduced, but the number of spawns were not recorded.
Species for which spawning was not observed during the exposure period.
-------
During the 1970 spawning season prespawning activity was observed for each
of the above three species in the exposure area. A group of stonerollers was
observed cleaning a nesting site and displaying spawning activity at a point
approximately 695 m below the V-notch weir, but no eggs were deposited. This
activity was observed only once. Four groups of striped shiners displayed
spawning activity in the exposure area at three locations. Two of these were
observed approximately 25 m below the point of copper introduction; one group was
observed at the downstream end of the exposure biannual fish-collecting pool, 200
m below the V-notch weir; and the other group was located 620 m below the V-notch
weir (Figure 3). No eggs were found at any of the above locations, and the
activity did not persist more than 2 days. Three large male creek chubs prepared
and defended nests approximately 25 m downstream from the V-notch weir, but no
females were ever observed with them and the nest had no eggs.
During the 1972 spawning period a limited number of spawning observations
were made in the recovery area between the downstream fish weir and the East Fork,
Little Miami River. Striped shiners were observed spawning in this area on four
occasions. The most upstream point of their spawning was approximately 1,340 m
downstream from the V-notch weir, where concentrations of copper for the period
ranged from 62 to 69 ug/Z. Stonerollers were observed spawning on two occasions
at a point 2,550 m downstream from the V-notch weir in the recovery area, where
the copper concentration ranged from 40 to 53 yg/Z-. Creek chubs, however, were
not observed spawning in the recovery area in 1972, even though observations were
made during their most active spawning period.
Bluntnose minnow spawned well in the control area, but spawning was very
limited in the exposure area during the exposure period. There were 48 spawnings
in the control area but only three in the exposure area (Table 9). No spawnings
were observed in the control during the 1972 season because of heavy rains and
turbid water. Numerous fry of this species were observed in the control
area in 1972, however, indicating that spawning had occurred. The three spawnings
that were observed in the exposure stretch during the three seasons were located
at the extreme downstream portion, where the copper concentration during the
spawning period averaged 60 yg/Z. and ranged from 35 to 77 yg/Z. The numbers of
eggs in these three spawns ranged between 200 and 250 and were probably the result
of a limited number of females. In contrast, the numbers of eggs observed in the
average control area spawnings were from 1,500 to 3,000. During the 1970 spawning
season (on three occasions) bluntnose minnow males were observed guarding nesting
sites in the exposure pool; no eggs were found at these locations, and the males
left after a few days.
Heavy rains and turbid waters during April 1970 and 1972 prevented spawning
observations of white suckers, but during 1971 they were observed spawning in the
control area 16 times (Table 9). In the exposure area only three spawnings were
observed for this species. These three spawnings were confined to the extreme
downstream portion, where the average copper concentration during this period was
99 yg/Z. These spawning sites were poor for white suckers. The nests were against
the exposure weir stop logs, in water depths of 0.45-0.6 m, and the bottom was
composed of silt, sand, and leaf detritus. In contrast, white sucker spawning
sites in the control area, which are considered good spawning habitats, consisted
of a fine gravel and fragmented limestone bottom substrate and flowing water of
0.3 m or less in depth. Habitats of this type were available in the exposure area,
but were not used. The spawn from these exposure area nests developed to an
advanced embryonic stage, but did not hatch. Failure of hatching is not attributed
53
-------
to copper, but probably to inadaquate water circulation around the eggs and thus
insufficient oxygen.
During the two pre-exposure spawning seasons, green sunfish spawned in all
the pools in both the control and exposure areas. Likewise, longear sunfish
spawned in all the exposure area pools, but spawning of this species in the
control area was limited and the population was usually low (Table 4). For the
three spawning seasons during the exposure period, green sunfish spawned well in
both the control and exposure areas, 77 and 68 spawnings, respectively (Table 9).
The longear sunfish during this same period had limited spawnings in the control
area, as was expected. There were 11 compared to 143 in the exposure area. Adult
individuals of both species were present throughout the exposure area during their
spawning seasons. Spawning of these sunfish species was restricted, however, to
the downstream half of the exposure area, where the average copper concentration
during the spawning season was 90 yg/£. or less. During the first year of
exposure, prespawning activity was observed for both green sunfish and longear
sunfish in the exposure pool (Figure 3), which is in the upstream half of the
exposure area where the average copper concentration is greater than 90 yg/Z.
This activity consisted of male individuals cleaning and guarding nesting sites.
No females were observed in spawning activity on these sites, however, nor were
eggs found. This activity lasted only a few days, and the nests were abandoned.
Restriction of spawning of green and longear sunfish to the lower half of the
exposure area reduced the production potential for young based on area alone, and
more than likely reduced their total production in the exposure area.
Orangethroat darters were observed spawning throughout the control and
exposure areas during the three spawning seasons of exposure. Their spawning was
not restricted by copper concentration in the exposure area. Much more spawning
activity was observed than was confirmed by actual observation of eggs. The eggs
of this species were exceedingly hard to find, since they were buried in fine
gravel and sand.
Fry Collections
Many of the fry data obtained from the 1970 fish-weir collections were
estimates, especially for the control weir (Table 10). Because the specimens were
in poor condition, only white sucker fry were identifiable. Therefore, the
collections only show the effect of copper on total number of fry and white sucker
fry captured on the exposure weir.
Eight times as many white suckers were captured on the control weir as on the
exposure weir, 5,768 and 764, respectively,, The weirs were flooded Dn April 23,
1970, for a 72-hr period and again on May 2, 1970, for 23 hr. White suckers had
spawned in the control area before these dates, and white sucker fry were observed
only in the control area on April 29, 1970. Thus, it is likely that the white
suckers captured in the exposure weir were produced in the control area and were
washed downstream during the flooding. The white sucker data as well as the
reduced total numbers of fry captured on the exposure weir compared to the control
weir, 858 versus 11,088, respectively, indicated that spawning in the exposure
area was adversely affected by copper. The situation was repeated in 1971 (Table
11).
The fry data presented for 1971 are actual values (Table 11). The specimens
were separated into the following categories: white sucker, sunfish, bluntnose
54
-------
TABLE 10. NUMBER OF FRY COLLECTED ON WEIR SCREENS IN SHAYLER RUN, 1970
Date
White '
1970 sucker
4/22
4/29 60
4/30 600
5/1 3,860
5/4 300
5/5 22
5/6 180
5/7 51
5/8 628
5/11 5
5/18
5/19 20
5/21 30
5/22
5/29
6/10
6/12
6/25 12
7/15
9/9
Totals 5,768
Control screei
Unidentified
fry
4
495
1,000
980
1,170
1,300
302
27
' 29
13
5,320
White
Total sucker
60
600
3,860 200
300 125
26 39
180 21
51
628 68
500 300
1,000
1,000
1,200
1,300
302
27
29
25 11
11,088 764
cposure screen
Unidentified
fry
25
'
5
4
8
10
423
94
Total Remarks
Weir flooded: 4/23
200
125 Weir flooded: 5/2
64
21
68
300
5
4
8
10
53 Weir flooded: 7/8
Weir flooded: 8/7
Weir flooded: 8/20
858
Al1 bullhead fry.
-------
TABLE 11. SPECIES AND NUMBERS OF FRY COLLECTED ON.WEIR SCREENS IN SHAYLER RUN, 1971
1971
5/5a
5/11
5/17
5/24
5/28b
6/2
6/4
6/7
6/11
6/13
6/18
6/21
White
sucker : Sunfish
2
9
92
3
15
265
11
5
3
i
5 i 192
3,994
1,820
6/25
3,260
1
6/28
7/15
7/19
8/6
155
1,650
318
13
i
8/30
Species
total 402 11,410
Control screen
Bluntnose
minnow
Darter
40
! 2
Bullhead
i i
60
i
270 20
1 !
i
1,760
128 |
!
Unidentified
fry
4
6
1
5
10
Total
0
2
0
18
98
4
15
305
21
207
3,994
Exposure screen
White
sucker ' Sunfish
2
t
2
t
5
Bluntnose
minnow Darter
1
10
1,880 !
3,550
155
3,410
446
1 14
64
440 30
120 60
i
1,751 ' 60
'
764 ,
I
1,160 : 20
569
120 .
2
Bullhead
Unidentified
fry
2
Total!
ol
0
o!
2
4
0
0
5
1
64
480
180
1,811
i 766
'
: 1,180
569
120
0 ! 10
10
|
|
2,218 42 20 27 14,119 ; 19 4,999
i i
170 2 2 0 5,192
i ]
^5/6 and 5/7 - Weir flooded.
5/25 - Weir flooded.
-------
minnow, darter, bullhead, and unidentified fry, the latter probably composed of
creek chub, striped shiner, stoneroller, and blacknose dace. The fry collections
from control and exposure fish weirs, in 1971, 11, 410 and 4,999, respectively,
were dominated by sunfish. The control sunfish were predominently green sunfish
rather than longear sunfish based on the difference in the number of spawns
observed for the two species (Table 9). It is apparent that in 1971, when fry
specimens were in good condition and could be identified, unidentified fry were
not very abundant in the weir collections—only 27 specimens in the control and
none in the exposure. Evidently the species represented by these fry did not move
downstream during normal stream flow (2.55 m3/sec or less) as did the white sucker,
sunfish, and bluntnose minnow fry.
In 1971 white sucker fry were first observed in the control area on May 4;
they were abundant on May 5. No fry of this species had been observed in the
exposure area before or on these dates. The fish weirs were flooded on May 6 for
7 hr and again on May 7 and 8 for another 42 hr. White sucker fry were first
observed in the exposure area on May 10.
Bluntnose minnow fry were first observed in the control area in 1971 on May
23, but none were observed in the exposure area before or on this date. On May 25
the weirs were flooded. On May 29 the first bluntnose minnow fry were observed
in the exposure area. Thus, bluntnose minnows were probably produced in the
control area and were washed downstream during flooding of the weir.
Sunfish fry were not exposed to high water and flooding of the weirs during
the 1971 spawning period. Reproductive success of this species in the control and
exposure areas is accurately indicated by the weir-screen data. Spawning success
of sunfish in the exposure area was not drastically affected by the addition of
copper.
In 1971 fry traps captured fry of 12 species (Table 12). Traps 1, 2, and 3
are control-area traps, traps 4, 5, and 6 are exposure-area traps (Figure 3). The
fantail darter occurred in large numbers in both the control and exposure
stretches, but only three fry were captured by these traps.
In the control area bluntnose minnows, striped shiners, creek chubs,
stonerollers, orangethroat darters, blacknose dace, and white suckers spawned
successfully, and their young were well represented in the fry-trap collections.
Although rainbow darters and johnny darters were not observed spawning, sufficient
numbers of these species were taken in the traps to indicate that spawning had
occurred. Longear sunfish fry were not collected, but the adult population was
low and only limited spawning of this species was observed in the control area in
1971. Green sunfish, on the other hand, spawned abundantly in the control area,
but their young were not well represented in the trap collections.
In the exposure area only three species of fish were known to have spawned
successfully: the green sunfish, longear sunfish, and orangethroat darter. They
were well represented in the fry-trap collections. Spawning of bluntnose minnows
was observed in the exposure area only once in 1971. Few fry of this species were
taken in the exposure area traps when compared to the number captured in the
control area. The majority, 22 of 32, were captured in trap 4, which is
approximately 550 m upstream from the only observed spawning location (station 6
near the downstream fish weir) for this species. Probably most of the bluntnose
minnows and all of the creek chubs, rainbow darters, blacknose dace, johnny
57
-------
TABLE 12. SPECIES AND NUMBERS OF FRY COLLECTED IN FRY TRAPS IN SHAYLER RUN, 197T
Ul
CO
Species
Bluntnose minnow
Striped shiner
Creek chub
Stoneroller
Rainbow darter
Fantail darter
Orangethroat darter
Green sunfish
Longear sunfish
Johnny darter
Blactcnose dace
White sucker
Total for all species
Control traps
#1
456
41
267
20
13
39
31
31
192
1,090
#2
619
177
12
12
12
2
10
49
27
920
#3
851
100
105
13
6
24
5
19
57
133
1,313
Species
total
1,926
141
549
45
31
75
7
60
137
352
3,323
#4
22
2
4
2
16
6
21
1
1
75
Exposure traps
Species
#5 #6 total
2 8 32
4 5 11
1 5
1 3
5 16 37
4 50 60
31 66 118
156
1
52 81 134
100 232 407
aTwenty-two sampling periods, 5/19-8/30, 1971.
-------
darters, and white suckers captured in the exposure area were spawned in the
control area and were transported to the exposure area during flooding of the fish
weirs on May 6, 7, 8, and 25, 1971. All of these species were present in the
control area before flooding, but they were not observed in the exposure area
until after flooding. No striped shiners or stonerollers were taken in traps
in the exposure area during this period. Seven times more fry were captured in
the control area than in the exposure area, indicating that the addition of
copper caused a reduction on reproduction in the exposure area.
59
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SECTION XI
EFFECTS OF COPPER ON STREAM BENTHIC COMMUNITIES
INTRODUCTION
The purposes of this portion of the study were to investigate the effects of
copper on the macroinvertebrates of the stream and to relate, if possible, these
effects to the feeding habits of the stream fish through analysis of stomach
contents. In addition, sampling was done from July 17, 1968, through June 14,
1971, to evaluate the effects of copper on the periphyton community.
During the early pre-exposure period, between May 1968 and February 1969,
various apparatus and methods for collecting qualitative and quantitative
macroinvertebrate samples were tried to determine baseline population levels..
Since the stream riffles are composed of large limestone rubble and the bottom
of the pools is bedrock, conventional samplers, such as the Ekman or Petersen dredge,
Surber sampler, or multiplate sampler, could not be used. The basket sampler
was the most efficient sampler tested and was used to collect macroinvertebrates
during the pre-exposure period from March 7, 1969, to February 16, 1970.
During the exposure period macroinvertebrates were collected from February
16, 1970, through July 15, 1971. Samples were taken from (1) artificial substrates,
created by suspending rock-filled baskets; (2) the natural substrate; and (3)
weir screens in both the control and exposure areas.
METHODS
Basket Samplers
Basket samplers used for collecting macroinvertebrates were the same as
those described by Mason et_ jal_. (1967, 1970). They were filled with 36 limestone
rocks, 6.5-9.5 cm in diameter. The baskets were permanently attached to
specially designed "T's" made of black iron pipe, which were inserted over iron
stakes driven into the stream bottom (Figure 20). When in position approximately
9 cm above the bottom, the baskets were always directed downstream and could
move freely so that silt and sand would not accumulate in or around them, and they
were always covered with water. Paired samplers were positioned approximately
75 cm apart in pools immediately downstream from riffles at stations located
in midstream, 50 cm above copper introduction; in the upper exposure area, 160
m downstream from copper introduction; in the lower exposure area, 815 m
downstream from copper introduction; and in the recovery area, 2 km downstream
from copper introduction (Figure 3).
Basket samplers were left in place for 6-week periods and were removed as
close to the end of these periods as stream and weather conditions permitted.
60
-------
Figure 20. Rock-filled basket sampler for collecting macroinvertebrates and the
wire-mesh screen used to cover sampler when removing it from the stream.
-------
They were removed from the stream with the aid of a cylinder made of U. S.
standard number 30 wire mesh (50cm long, 25 cm ID), which fitted over the
baskets to prevent loss of organisms (Figure 20). The rocks were dumped into a
tub partially filled with stream water and scrubbed with a stiff brush to remove
organisms. The samples were concentrated in a U. S. standard number 30 sieve
(0.59-mm mesh opening), preserved with 70% ethanol in plastic containers, and
stored in the laboratory for later sorting and identification. The cleaned
samplers were then reset at the same locations.
Natural Substrate Sampling
Natural substrate sampling was done in a control riffle immediately
downstream from the control pool, 275 m upstream from copper introduction; in the
two exposure riffles, 200 m downstream from copper introduction and 720 m
downstream from copper introduction; and in a recovery riffle, 2.2 km downstream
from copper introduction (Figure 3). Natural substrate samplings were usually
collected on the same day that basket samplers were removed.
Rocks were selected randomly for 10 min, were placed in a tub partially
filled with water, and were scrubbed clean with a stiff brush to remove the
organisms. These samples were processed in the manner previously described for
artificial substrate samples.
Weir-Screen Collections
Samples were collected from the control and exposure weir screens on the days
when flow was 0.25 m3/sec or less and copper was being added. The material
containing macroinvertebrates was gently hosed from the screens with water into
the weir troughs and collected in the wire-mesh baskets (0.5-mm openings) at the
ends of the troughs. This material was then put in 1-gal jars and immediately
preserved in 7% formalin. The samples were thoroughly washed in tap water
in the laboratory and stained with a solution of 200 mg/Z-. rose bengal in 70%
ethanol. These samples contained large volumes of leaves, stems, bud scales, and
seed pods, and staining improved the thoroughness and rate of sorting the
organisms (Mason and Yevich, 1967).
An additional technique was used to separate macroinvertebrates more
effectively from debris. Small portions of the sample were washed with tap
water in a U. S. standard number 30 sieve to remove excess stain and were placed
on a strip of fine mesh wire screen that was then placed in a shallow white
porcelain pan containing a small amount of water. This created a contrasting
backround against which the stained organisms could easily be seen and picked
from the debris. When samples contained excessive debris, aliquots were examined.
All specimens from the various sampling techniques were identified to family.
Organisms found in large numbers were identified to genus and species in most
cases. Macroinvertebrates collected by all the techniques are recorded as
numbers per sample.
Macroinvertebrate samples were collected by hand and dip nets on May 28 and
June 13, 1969. by William R. Mason, Jr., and Phillip A,, Lewis of the Environmental
Monitoring and Support Laboratory, U. S. Environmental Protection Agency,
Cincinnati, Ohio, (formerly known as the Analytical Quality Control Laboratory,
U. S. Environmental Protection Agency, Cincinnati, Ohio). The organisms were
62
-------
identified and sent to this laboratory to used as a reference collection (Table
13). Specimens of amphipods were identified by Dr. John R. Holsinger of Old
Dominion University, Norfolk, Virginia. His identifications were corroborated
by Dr. E. L. Bousfield of the National Museum of Natural Sciences, Ottawa, Ontario.
In early summer 1970 a copper-concentration gradient became evident in the
exposure and recovery areas when the water temperature had increased. Additional
information could be gained in this gradient, so additional natural substrate
stations were established in the lower exposure and recovery areas. Basket
samplers in the recovery area in June 1970 were destroyed by vandalism and
replaced, and the first collection from this area was made in late August.
Periphyton samples were collected for approximately 3 years from natural and
artificial substrates in both riffle and pool in the control and exposure areas.
The control riffle and pool were 350 and 275 m, respectively, upstream from the
point of copper introduction; the exposure pool and riffle were 410 and 200 m,
respectively, downstream from the point of copper introduction. The parameters
examined included cell density, dry weight, ash-free weight, chlorophyll content,
biomass-to-chlorophyll a_ ratio, pheophytin-to-chlorophyll a_ ratio, species
composition, and species diversity.
RESULTS AND DISCUSSION
Pre-exposure macroinvertebrate samples from the control and exposure areas
contained organisms representing 15 and 14 orders, respectively. These were
composed of sufficient numbers of taxa to indicate that the test area had
recovered from any deleterious effects of the Shayler Run Waste Treatment Plant
on the stream. The species list presents all of the organisms collected and
identified from all of the sampling procedures used during the study (Table 13).
The number of macroinvertebrates collected in each basket of the paired
samplers at three locations in June and July is shown in Table 14. The data from
the paired samplers were combined for evaluation of the macroinvertebrate
populations at each station and are recorded as numbers per station (Table 15).
Voids in these data are the result of vandalism, flood damage, or poor
preservation.
Natural substrate sampling yielded organisms that were not normally collected
by basket samplers and provided additional information for evaluation of the
macroinvertebrate population. Macroinvertebrate organisms collected from weir
screens were those that were transported by stream currents. This was an
excellent sampling of classical drift organisms, since the screens collected
organisms from the entire stream flow.
The predominant groups of macroinvertebratess collected from Shayler Run,
Isopoda, Ephemeroptera, Amphipoda, Chironomidae, Psephenidae, and Tricoptera, are
dealt with in detail below. The other groups shown in the tables represent too
small a percentage of organisms to warrant further discussion.
Isopoda (sowbugs)
Only one species, Lirceus fontinalis, was present in the stream.
Basket-sampler collections showed that the population of sowbugs in the exposure
area during the pre-exposure period was as great as, if not greater than, it was
63
-------
TABLE 13. SPECIES OF MACROINVERTEBRATES COLLECTED BY ALL
METHODS IN SHAYLER RUN DURING 1969-71
Organisms
Diptera (True Flies)
Chironomidae (Midges)
Ablabesmyria sp. 1
Calopsectra glabreseens
C. (poss.) macrosandalum (Kieff)
Chironomous attenuatus Walker
C. plump sa Linnaeus
~C_. riparius Qr.
Conchapelopis sp.
Coryneura (Thienemaniella) Xena
Cricotopus biqinetus gr.
C. ceris
C. slossonae
C. trifaciatus
Cryptochironomus argus
Diamesa longimanus
Glyptotendipes lobeferus (Say)
Metriocnemus sp ,
Orchocladius nivoriundus
0. sordidella
Paratendipes sp .
Pentaneura flavifrons (Joh.)
P. monilis
Polypedilum convictus
P. trituro
Procladius riparius
Pseudochironomus sp .
Tanytarsus (Paratany tarsus) sp.
T. (Cladotanytarsus) sp . 3 Roback
Tribelos sp.
Ceratopogonidae (Biting Midges)
Atrichopogon sp.
Bezzia varicolor
Probezzia g_labra (Coq.)
Chaoboridae (Phantom Midges)
Chaoborus sp.
Culicidae (Mosquitoes)
Simuliidae (Blackflj.es)
blrauilum sp.
Tabanidae (Horseflies)
Stratiomyiidae (Soldierf lies)
Euparyphus ^reylockensis
Odonotomyia cincta
Stratiomys sp.
Tipulidae (Craneflies)
Antocha saxicola
Megistocera longipennis
Tipula sp.
Psychodidae (Mothflles)
Psychoda alternata (Say)
Ephemeroptera (Mayflies)
Baetidae
Baetis sp.
Neocleon alamance
Ephemeridae
Hexa^enia limbata
Caenidae
Caenis sp.
Heptageniidae
Stenonema (Femoratum) scitulum
S. interpunctatum
S. tripunctatum
Coleoptera (Beetles)
Dytiscidae (Predaceous Water Beetles)
Deronectes sp .
Laccophilus terminalis
Elmidae (Riffle Beetles)
Narpus sp.
Neoelmis sp.
Optioservus sp.
Promoresia elegans
Stenelmis lateralis
S. markeli
Zaitzevia sp .
Gyrinidae (Whirligig Beetles)
Haliplidae (Crawling Water Beetles)
Peltodytes simplex
Hvdrophilidae (Water Scavenger Beetles)
Enochrus sp.
Hydrochus sp .
Psephenidae (Water Penny)
Psephenus herricki
Control
"69 '70 '71
X X
XXX
X
X
X
X
X X
X X
X
X X
X X
X
X X
XXX
XXX
X X
X
XXX
X
XXX
X
X X
XXX
XXX
X
X
X
X
X X
X
X X
X
XXX
X
X X
X X
X X
X X
X
xxx
xxx
X X
xxx
X X X
xxx
x x :;
X
X
X
x x
X
xxx
xxx
X
X X
X X
X
X X X
Collectto
Upper
exposure
'69 '70 '71 '
X X X :
X X
X '
X X
X
x
X
A X
X
X X
xxx
X
x x
X
X
X
x i
X X
xxx
X
X
X
X
X
X X
X
X
X X
xxx
X X
X X
X X
X
X
xxx
xxx
X
X
X
X X
xxx
Lower
exposure
'69 '70 '71
X
X
X
XXX
X
X
X
X X
xxx
X X
X X
X X
X
xxx
xxx
Recovery
'69 '70 '71
X X
X X
X X
X X
X
: X X
X X
1
-------
TABLE 13 (continued). SPECIES OF MACROINVERTEBRATES COLLECTED BY
ALL METHODS IN SHAYLER RUN DURING 1969-71
Collection areas
Upper Lower
Control exposure exposure
Organisms '69 '70 '71 '69 '70 '71 '69 '70 '71
Tricnoptera (Caddisflies)
Glossosomatidae X X
Hydropsy chidae
Cheumatopsyche sp. XXX XX
Hydropsyche bifida Gr. XXXXXXXKX
Hydroptilidae
Hydroptila agrosa X ' X X
H. waubesiana X ' X
Stactobiella sp. X X
Philopo tarn idae
Chimarra sp. X X | X
Rhyacophilidae ',
Agapetus sp. X
Leptocerus sp. X
Limnephilidae
Linephilus sp. XX.
Odonata (Dragonf lies and Damself lies)
Aeshnidae
Aeschna sp. X
Dromogotnphus spoilatus X X :
Leucodrinia cf. intacta X
Plathemis lydia > ,
Coenagrionidae
Argia moes ta X
Nehallenia sp. X
Macromiidae
Macromia magnifica X •
Hemiptera (Aquatic and Semi-aquatic Bugs) \
Belostomatidae (Giant Water-bugs) .
Lethocerus americanus X
Gerridae (Water Striders)
G err is remigis X X
Metrobates sp. X XX
Rheumatobates r ileyi X XXX'
Tenagogonus gillettie X \
Naucor idae (Creeping Water -bugs) X !
Corixidae (Water Boatmen)
Sigara alternata X X
Gelastocoridae (Toad -shaped Bu£,s) j
Megaloptera (Dobsonf lies and Fish flies)
Corydalidae •. j
Corydalus cornutus . X
Recovery
'69 '70 '71
X X
X
X X
X
X
Plecoptera (Stoneflies)
Perlodidae
Isoperla sp.
Collembola (Springtails)
Isotomidae
Isotomurus palustris
Amphipoda (Scuds)
Crajigonyx anomalus (Hubricht)
C_. setodactylus (Bousfield)
S_ynurel_la_ dentata (Hubricht)
Decapoda (Crayfish)
Qrconectes rusticus (Girard)
Isopoda (Sowbugs)
Lerceus frontinalis
Ostracoda (Shrimps)
Mysidacea
Oligochaeta (Worms)
ha id idae
_S_t_ylarig_ lacuscris
P r i s t i na sp ,
Lumbriculidae
Rhynchelmis sp.
Tubificidae
Branchiobdellidae
Cambarincola sp.
Planariidae (Fla tworms)
Dugesia tigrina
Gastropoda (Snails)
Lymnaidae
Lymnaea sp.
Pseudosucc inea columella
Physidae
P hy sa g y r ina
Planorbidae
Gjrra u 1 us c ir c um s t r ia ^u_s
Pleuroceridae
Pleurocera^ sp.
Pelecypoda (Clams)
Sphaeriidae
65
-------
TABLE 14. NUMBERS OF MACRO INVERTEBRATES COLLECTED IN JUNE AND JULY FROM
PAIRED (A and B) ROCK-FILLED BASKET SAMPLERS IN SHAYLER RUN, 1969-71
Date Area
1969
6/4 Control A
Control B
Upper Exposure A
Upper Exposure 8
Lower Exposure A
Lower Exposure B
7/17 Control A
Control B
Upper Exposure A
Upper Exposure B
Lower Exposure A
Lower Exposure B
1970
6/1 Control A
Control B
Upper F.xposure A
Upper Exposure B
Lower Exposure A
Lower Exposure B
7/14 Control A
Control B
Upper Exposure A
Upper Exposure B
Lower Exposure A
Lower Exposure B
1971
6/3 Control A
Control B
Upper Exposure A
Upper Exposure B
Lower Exposure A
Lower Exposure B
7/15 Control A
Control B
Upper Exposure A
Upper Exposure B
Lower Exposure A
Lower Exposure B
10 00
0- 3
0 0
M ^
3,589
1,155
6,001
4,398
3,729
3,723
13
4
22
62
13
49
407
504
14
0
3
3
2
2
0
0
1
0
125
140
7
8
0
0
1
0
0
0
0
0
o to
-H 3
,c u
Q. tft
20
10
12
4
6
15
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
0
0
0
n) .-.
M to
o o;
^H -D
o ^
37
42
17
20
16
49
10
5
16
39
3
25
4
7
1
1
1
0
2
5
0
5
1
0
14
33
0
3
0
0
1
0
1
2
2
1
OJ ^
o -^
e >•
"D.-^
bj
15
31
200
138
34
48
340
291
463
315
95
386
COPPER
37
23
3
5
9
0
148
153
1
1
2
7
26
21
0
0
1
0
57
41
3
0
30
26
S|
_£ to
'M "°
H ^
2
7
118
70
13
5
0
0
4
1
22
19
to
•H 3
Q ^J
~
267
0
405
269
3
26
17
20
4
0
33
30
u ^H
SIM
C
-o oo
M
"O
0
0
0
2
2
0
2
1
1
0
18
10
o
to
a.
2
0
6
3
5
3
1
1
1
5
1
1
fO .-^
•H to
TO n
.0 oj
^ ^H
H -^
36
47
21
21
122
127
8
2
4
2
0
0
tu w
x e
o o
DO 3
•H - —
0
25
0
40
121
20
22
0
0
0
0
230
1
e j
§ H H
w o
3,993
1,292
6,820 m
5,046 UJ
3,950
4,018
391 (1)
324
515 ...
424 l ;
52]5 (16>
z
O J
M <;
H H
1
5,285
11,873
7,968
716
940
952
EXPOSURE STARTED 2/16/70
0
0
0
0
0
1
0
0
1
2
0
1
0
0
0
0
0
0
0
0
0
0
0
0
14
71
17
18
100
10
18
12
9
13
5
0
80
35
93
117
62
68
7
6
33
12
23
9
0
0
0
0
0
0
0
0
0
0
0
0
1
2
0
0
1
1
0
0
10
8
8
0
1
3
1
0
0
1
0
0
0
0
1
3
4
7
0
0
3
2
1
0
1
1
1
5
0
3
7
6
1
4
3
4
0
0
0
0
7
3
0
0
0
0
0
0
0
0
0
0
1
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
464
611
43
31
115
18
173
176
11
21
10
11
257
241
100
128
67
71
67
47
23 (2>
64
41
1,075
74
133
349
32
21
498
228
138
114
73
105
66
-------
TABLE 15. TOTAL NUMBERS OF MACROINVERTEBRATES IN ALL COLLECTIONS FROM
PAIRED ROCK-FILLED BASKET SAMPLERS IN SHAYLER RUN, 1969-71
"w
[1
to ^ «| aj In
2 "S 1 3 'e
•0 00 Q.-J |j E 0
Q. -Q ) l-i U-i ICO
03 ; « >. t| o ^
Collection — '
Da t e Ar ea
1969
4/17 Control 733
Upper Exposure 491
Lower Exposure 1,432
6/4 Control 4,744
Upper Exposure 10,399
Lower Exposure 7,452
7/17 Control 17
Upper Exposure 84
Lower Exposure 62
8/28 Control 5
Upper Exposure 13
Lower Exposure 105
10/9 Control 0
Lower Exposure 11
11/18 Control 11
Upper Exposure 372
Lower Exposure 409
1970
2/16 Control 236
Upper Exposure 44
Lower Exposure 32
J: -
D.
U
69
469
288
46
338
82
631
TIB
481
883
674
226
562
398
53
196
201
36
4
56
5 ^
11
335
225
267
672
22
37
0
61
18
7
16
8
26
0
3
12
10
0
TO
I- kJ
1 s.
O 'H
Q
7
0
1
0
2
7
0
4
2
2
1
2
0
3
0
0
1
4
4
9
4 0
-a ^
0 w
sl
<
1
7
12
30
16
21
0
0
0
0
0
0
0
0
0
0
0
7
16
12
^
•3 aJ
-H -f -a
TO 'H
u —1
C C
Q 0
0 TO°
i
0
31
44
9
188
18
0
5
41
0
0
2
0
0
0
2
1
8
0
0 0
1
1
2
0
2
2
3
1
28
7
25
67
0
34
2
0
2
0
0
0
£ 3i
tt) 3
£• a
E 3
0
0
1
0
1
0
0
1
1
0
0
0
0
0
0
0
0
0
0
tui
"
u ^
0
0
0
2
9
8
2
6
2
2
1
0
0
0
0
0
0
0
0 ' 0
0 0
-a ^
o- -H
TO f .,
S ~
TO U)
^ e
ii S ' o o o 3
^ | O H ^-
0 13
0 0
3 8
0 ! 25
6 i 161
0 | 42
1 ' 0
0 ' 0
13 231
0 3
0 0
5 66
0 0
0 ' 12
0 i J
0 0
2 11
0 0
0 . 0
0 0
1
0
3
83
42
249
10
6
C
4
0
58
0
0
71
2
0
1
0
0
0
=5 ~
0 'H
i yi ^ TO
« « « »
^ ] u &
! ~
0
0
0
0
0
0
0
0
2
0
0
0
0
0
0
0
0
0
0
0
0
0
-J
H
o
849
0 | 1,355
0 | 2,037
0
0
U
0
0
0
o
5,285
11,873
7,968
716
940
952
949
0 t 829
1
0
0
0
0
1
652
949
'558
76
576
651
0 :' 320
0 t 77
0
Exposure started 2/16/70
4/9 Control 180
Lower Exposure 68
6/1 Control 911
Upper Exposure 14
Lower Exposure 6
7/14 Control 4
Upper Exposure 0
Lower Exposure 1
8/26 Control 34
Upper Exposure 0
Lower Exposure 0
Recovery 0
10/12 Control 0
Upper Exposure 0
Lower Exposure 0
Recovery 0
11/24 Control ' 0
Upper Exposure 1
Lower Exposure 0
Recovery 0
1971
3/2 Control 94
Upper Exposure 30
Lower Exposure Vandalism
Recovery 55
4/21 Control 30
Upper Exposure 9
Lower Exposure 0
Recovery 1
6/3 Control 265
Upper Exposure 15
Lower Exposure 0
Recovery 1
7/15 Control 1
Upper Exposure 0
Lower Exposure 0
Recovery 0
34
56
134
60
8
9
301
2
9
886
6
17
284
671
0
24
516
16
8
0
24
132
27
70
77
9
8
23
47
0
1
5
98
3
56
102
0 5 0
134 | 3
0
85
35
106
30
20
5
36
16
13
f,
6
38
6
0
51
25
11
0
43
23
76
31
42
49
67
115
209
130
9
12
45
32
13
10
0
0
/t
0
2
0
0
0
1
0
0
2
0
0
1
2
0
1
0
0
0
0
1
0
0
0
1
0
0
1
0
0
0
22 . 12
4 4?
10
0
0
0
0
0
0
0
0
0
1
0
1
0
0
0
0
0
0
7
4
96
10
2
1
7
3
1
21
2
6
31
0 '| 0
6 0
0 2
1 0
0
0
0 j 1
9 0
2 3
0 1
j
0 0
1 2
i ; 6
o ; o
0
0
4
0
0
0
0
0 0
0 ! 0
0
0
22
4
0 1
0
1
0 0
0 i 0 0
0 i 0 4
19
3
34
7
9 i 0 i 0 | 4
0 0
0 1 0
14
1
0
0
1
57
12
4
0
0
0
0
0
0
0
0
0
0
0
0
33
21
8
0
8
45
2
0
8
1
1
0
2
0
0
0 1
0 1
0 7
1 2
0 17
i
2
13
14
1
0
2
1
2
1
0
1
0
2
3
0
51
33
74
2
]
0
4
0
0
0
0
0
0
0
3
3
19
0
0
0
0
0
2
0
4
4
1
0 ; 0
o : 2
0 0 ! 2
0
0
3
0
6
0
10
3
0
1
3
3
0
2
3
0
18
8
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
2
0
0
0
0
2
3
9
0
0
0
0
0 8
0 1
0 1
0
0
0 0
0 • 0
0
0
0
3
13
5
7
0
o i o i o
0
0
0 0
0
0
0
0
0
1
0
0
0
1
10
1 ' 6
r -
22
6
1
0
2
11
0
5
2
1
2
6
4
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
o
4
3
5
0
2
0
0
0
0
0
0
0
0
0
0
6
1
2
3
7
0
3
5
1
0
0
1
0
0
0
0
0
0
0
10
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
1
0
0
3
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
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
102
248
315
354
1,075
74
133
349
32
21
1,004
31
84
336
693
44
54
539
72
43
20
49
415
152
366
171
74
60
109
498
228
138
30
114
73
105
124
67
-------
in the control area (Table 15, Figure 21). During the exposure period sowbugs
were practically eliminated from both the exposure and recovery areas (Figures 21
and 22), whereas the population was maintained in the control area.
The weir screens were sampled often during the first year of exposure (Table
16, Figure 23); all the sowbugs captured were medium to large. The drift rate of
sowbugs from the exposure area was nearly three times greater than it was from
the control area during the period of February 23 to April 8, 1970. This was
expected since a greater population was present. However, the exposure drift rate
declined to a level lower than that of the control by April 22, 1970, as the
population in the exposure area became depleted (Table 15).
No young and only a few adult sowbugs were collected in the exposure and
recovery areas after June 1970, indicating that those animals that were transported
downstream during high water did not survive in either exposure or recovery area.
Further support for this conclusion is indicated by a test conducted during April
and May 1971, in which 40 mature sowbugs were placed in each of two chambers at
the streamside laboratory. A continuous-flow system was used whereby one chamber
received control water and the other received water with a nominal concentration
of 120 Ug/£. of copper. During this test newly hatched sowbugs died within 2 days
in the exposure chamber, whereas newly hatched young in the control chamber were
never observed dead. At the termination of the study no young sowbugs were found
in the exposure chamber, but in the control chamber 1,298 young were present.
Thus, reduction in the population of sowbugs in the exposure and recovery areas
during the period of exposure is attributed to copper through increased drift rate
and death of the young.
Ephemeroptera (mayflies)
Seven species of mayflies were present in the various collections (Table 13).
Data from basket samplers (Table 15, Figure 24) indicate that during the
pre-exposure period the mayfly population was as great in the exposure area as
in the control area. For the first month of copper exposure, the drift rates
from the control and exposure areas were low and similar, as evidenced by the
number of mayflies captured on the weir screens (Table 16, Figure 25). No
mayflies were taken on either screen in the collection on March 17, 1970. On
April 8, 1970, the number of mayflies captured on the exposure screens increased
considerably, 334 and 5 on the exposure and control screens, respectively. Rising
water temperature may have increased the sensitivity of the organisms to copper,
which could account for the increased drift rate. The mean daily stream
temperature from March 17 through April 6 ranged between 3° and 5° C. By April 8
the mean daily stream temperature had increased to 9° C, and it increased
progressively over the next few weeks to 14° C by April 22. On this date 33
mayflies were captured on the control screen and 21 on the exposure screen. For
the remainder of the exposure period greater numbers were captured on the control
screens in most cases. Evidently the number of mayflies present in the exposure
area had been reduced because there were fewer in the exposure basket samplers
and natural substrate samples as well (Tables 15 and 17, Figures 24 and 26).
Reduction may have been a result of increased drift rate or number of deaths,
although deaths were not noticeable.
Peak population levels of mayflies occur normally in the summer and early
fall. The low copper concentrations at the sampling stations in the recovery
area probably permitted reproduction and survival of mayflies, whereas the copper
68
-------
Upper lower
Exposure Exposure
AREAS
4-17-69 6-469
7-17-69
Figure 21. Isopoda (sowbugs): basket-sampler collections.
-------
100
LLJ
O
UJ
>
50
o
LU
Q.
500
400
D
Q
5300
O
CC
200
100
D I
Control Upper Lower Recovery
Exposure Exposure
AREAS
2-16-VOl 6-10-70
8-26-70 10-19-70
6-3-70 7-15-71
Figure 22. Isopoda (sowbugs): natural substrate collections.
-------
TABLE 16c TOTAL MACROINVERTEBRATES COLLECTED FROM WEIR SCREENS IN
SHAYLER RUN DURING 1970 AND 1971
Exposure
Expoiur.
I , IMS
3.058
i? !
2,96!
b, %5
71
-------
n
n
Q 300
z
LL
O
£5 200
CD
100
I
CONTROL EXPOSURE
AREAS
n. Q.
0 0 0 0 Q 0
o_ n
Q
o£
<
-------
—1
OJ
Control Upper Lower
Exposure Exposure
4-17-69 6-4-69
7-17-69 8-28-69 10-9-69 11-18-69 2-16-70 4-9-70
6-1-70
7-14-70 8-26-70 10-12-70 11-24-70 3-2-71
4-21-71
6-3-71
7-15-71
Figure 24. Ephemeroptera (mayflies): basket-sampler collections.
-------
o
z
O
in
100
UJ
>
UJ
cc
O
LU
O-
50
_. n n. . _ • fi
500
< 400
D
Q
>
O 300
cc 200
UJ
CO
100
Q
UJ
Qo
Or-
I
CONTROL EXPOSURE
AREAS
2-23-TO 2-25-70 2-28-70 3-9-70 3-11-70 3-12-70 3-17-70 4-8-70 4-22-70 5-1-70 5-8-70 5-29-70 6-25-70 7-15-70 9-9-70 4-20-71 6-4-71 7-15-71
o
o
Figure 25. Ephemeroptera (mayflies): weir-screen collections.
-------
TABLE 17. TOTAL MACROINVERTEBRATES COLLECTED FROM NATURAL SUBSTRATES
IN SHAYLER RUN DURING 1969-71
c. £
~ ?
Col U-cClon
ArutiR
Control 464
Control 100
I'ppvr fixposun- 18
Control 3
UIIPLT F-xnopuru' 0
l.ow..-r Exposure 0
RL-i-nut-ry 0
Control 4
UpplT DtpUS-Uri: 0
U>WL-r Exp.ihiiri.' 1
Kfuwi-rv 0
Ojiurol 189
1'1'pLT L.xposuru 0
U-wt-r t.-.posiiri- 0
Rc.-.,vL.rv 2
C-.tiitn>l 63
L'ppu-r Kxposun- 0
Lowt-r Kxposuri' 0
Rui ovi-rv 0
rnntr.il 4
I'ppor l-.\pnsuri- 1
LUWLT Exposure i
Rc-i iivt-rv Pour
Control 21
Upper Exposure 2
Luwi-r r.xposurL- 1
Ri-. nvt-rv 0
Control 21 1
llpiu-r i:-
0
9
1
0
6
0
3
10
2
h
1 1
60
0
0
0
K-
0
1
1
LI
1
260
2,486
10
L7
0
0
11
£"-
^f)
urc st
1
0
0
0
0
0
0
0
0
0
0
0
0
l(
0
0
0
o
0
0
-
0
0
0
[)
0
0
0
0
0
0
0
7
C .0
g- ~
'i-
216
irced
,,
0
1
1
24
33
133
0
i
1 5
205
4
9
91
59
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1 12
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4
9
4
8
68
L3
9
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32
9
5
T
C. -—
L. ^
1
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/I6/70
0
0
0
3
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1 1
0
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0
0
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4
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0
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1 3
0
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23
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0
66
52
73
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31
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0
243 ,
0
0
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Ifi5
12
31
13
0
2
12
28
86
94
13
14
151
1
5
3
1
0
2
24
0
0
35
98
4
0
1 10
-. --
-o --c
5
1
0
0
1
0
0
1
3
12
9
4
9
24
7
5
7
3
0
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0
-
0
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0
4
0
0
0
1
4
1
2J!
E =
~
0
0
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0
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0
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0
2
1
0
0
0
0
0
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0
0
0
-
0
0
0
0
0
0
0
0
1
4
i 1
- '-
* ~
2
0
0
0
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10
0
5
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0
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1
0
0
1
0
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0
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0
-
7
2
0
7
0
5
2
5
1
8
1 I
11
" S
0
0
0
0
0
0
3
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0
0
13
0
0
0
6
1
0
0
I
0
0
3
-
1
0
n
0
0
0
0
0
0
0
0
1?
% z
0
0
0
0
0
0
0
0
0
0
0
0
0
0
2
0
0
0
0
0
0
0
0
1
0
0 i
0
0
0
0
0
0
0
1 t-
: %
a n
_ _
0
0
3
0
0
0
0
2
1
2
0
0
0
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0
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0
0
0
0
0
0
0
0
0
0
0
4
"I
0
ul
y
OJ 0
•"•
0
4
0 '
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
1 '
o'
0
0
0
0
0
0
0
0
0
0
0
ll
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0
0
0
0
0
0
0
0
0
0
0
0
0
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0
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0
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•^
(2
864
3,342
194
249
186
83
269
354
183
70
14h
697
230
258
403
305
62
189
JS3
18
56
24
-
158
112
235
287
335
2,920
6,285
270
559
52
31 1
75
-------
>•
O
o
tu
cc
ULJ
CC
O
cc
LU
Q_
100
50
500
400
> 300
Q
£ 200
co
100
0
—
n
|n
o {•:•:•
I
Control Upper Lower Recovery
Exposure Exposure
AREAS
0 0 Xv
O 0 0 0
2-16-701 MO-70 7-16-70 8-26-70 10-19-70 11-25-70 3-2-71 4-21-70 6-3-70 7-15-71
Figure 26. Ephemeroptera (mayflies): natural substrate collections.
-------
concentrations in the exposure area were too high. The average copper
concentrations at the upper exposure, lower exposure, and recovery sampling
locations for the period June through September 1970 were 114, 58, and 17 ug/Z.,
respectively. Mayflies are both terrestrial and aerial as adults and thus have
the potential to deposit eggs in all portions of Shayler Run. The relatively few
mayflies captured in the exposure area from July 1970 until the end of the study
are believed to have been produced in the control area, and the young were probably
transported downstream during high water. The reduction in the mayfly population
in the exposure area is attributed to copper.
Amphipoda (scudjQ
This group of organisms was represented by three species in the stream,
Crangonyx anomalus, jC. setodactylus, and Synurella dentata. Their normal
habitat is shallow, slow-moving water where aquatic vegetation and organic detritus
are present. Collections from basket samplers and natural substrates (Tables 15
and 17) indicate that scuds were more abundant during the spring and early
summer, and the population levels in the control and exposure areas were
similar. Also, there was little reproduction or survival during 1970 and 1971,
but the cause is not known.
Results from the weir-screen collections (Table 16, Figure 27) show a
considerable difference in the numbers of scuds moving downstream from the exposure
area and the numbers from the control area during the first few months of exposure.
For the period of February 23 to May 8, 1970, there was a 25-fold difference
(3,937-157) between the number of scuds collected on the exposure screens and the
number on the control screens. The high drift rate from the exposure area is
attributed to the addition of copper.
Chironomidae
The population of Chironomids in the stream was composed of 30 species
(Table 13). Peak population levels normally occurred in May, June, and July
(Tables 15, 16, and 17, Figures 28 and 29). Like mayflies, the adults are both
terrestrial and aerial and may deposit eggs in surface water.
Basket-sampler collections made before exposure to copper showed that the
Chironomid population in the exposure area was somewhat greater than that of the
control area. The frequency of occurrence (Figure 28) of Chironomids per
individual samples averaged less than 10% for the control station and the two
exposure-area stations. During the exposure period, however, there was a
substantial increase in the Chironomid population of the exposure area compared
to that of the control. The average frequency of occurrence per sample increased
to 15% for the control, but there was a major increase at the exposure stations
to 60% and 43% for upstream and downstream stations, respectively. The percentage
increase shows the dominance of Chironomids in the exposure area during the
exposure period over some of the more common forms that had been adversely affected
by copper, such as isopods and mayflies. It is reasonable to believe that, with
the reduction in numbers of other common forms of macroinvertebrates and the
reduction in the exposed fish population, Chironomids that could withstand the
copper concentration had less competition for food and space along with less
predation and therefore, flourished during this period.
77
-------
OC
u- 100
50 -
Z
in
<-> 0
cc
500
00
- 300
Q
Z
cc
111
CO
2
3100
Z
JJ
_d
_d
1]
I
CONTROL EXPOSURE
AREAS
oo
2-23-70 2-25-70 2-28-70 3-9-70 3-11-70 3-12-70 3-17-70 4-8-70 4-22-70 5-1-70 5-8-70 5-29-70 6-25-70 7-15-70 9-9-70 4-20-71 6-4-71 7-1511
O
UJ
Q o
O r-
< "^
CD
•a.
-------
o
z
LLJ
O
LLJ
100
LLJ
>
50
Z
LLJ
O 0
DC
LLJ
Q_
200
150
^ 100
0
en
LLJ
CO
JJl
i_
Z
J=l
1
_O
I
Control Upper Lower Recovery
Exposure Exposure
AREAS
J^
I feo
4-17-69 6-4-69 7-17-69 8-28-69 10-9-69 11-18-69 2-16-70 I 4T70
7-14-70 8-26-70 10-12-70 11-24-70 3-2-71 4-21-71 6-3-71 7-15-71
Figure 28. Chironomidae (Chironomids): basket-sampler collections.
-------
00
o
Upper Lower Recovery
Exposure Exposure
AREAS
° 2-16-70 6-10-70 7-16-70 8-26-70 10-19-70 11-25-70 3-2-71 4-21-70 6-3-70 7-15-71
Figure 29. Chironomidae (Chironomids): natural substrate collections.
-------
Psephenidae (riffle beetles)
Only one species of riffle beetle, Psephenus herricki, was present in the
collections, and it was common during the pre-exposure period in the control area
and in even greater numbers in the exposure area (Tables 16 and 17, Figures 30 and
31). Riffle beetle larvae adhere very tightly to rocks in shallow, swift-moving
waters, and thus they contributed little to the number of drift organisms
collected on the weir screens (Table 16). The adults are semi-aquatic and can fly.
Eggs, therefore, could be deposited in any area of the stream regardless of
copper concentration.
During the exposure period, from June 1970 onward, the population of riffle
beetles was reduced in the exposure area as compared to the population curing the
pre-exposure period (Table 15, Figure 30). The population in the control area
during the exposure period was comparable to the pre-exposure level. The recovery
area station had a population during the exposure period greater than that at the
exposure stations and somewhat similar to that at the control station. This
response was similar to that of mayflies, in that the population of riffle beetles
was greater in the recovery area where the copper concentration was lower. It
appears that copper adversely affected the riffle beetle population in the
exposure area.
Trichoptera (caddisflies)
Few caddisflies were collected in basket samplers as compared with natural
substrate sampling. The results from rock scrubbing were similar to those of
Anderson and Mason (1968) when they made comparisons of sampling success by
Ekman dredge and basket sampler. The data indicate that caddisflies were not
greatly affected by copper (Figure 32, Tables 15, 16, and 17).
Periphyton
Of the various parameters examined, species composition was found to be the
most sensitive and informative measure of the effects of copper on the periphyton
community. Two of the dominant species of algae were eliminated from the
periphyton: the diatom Cocconeis placentula var. euglypta, and the filamentous
green alga, Cladophora glomerata. Cocconeis placentula, which commonly contributed
85-98% of the summer diatoms in the exposure area before the addition of copper,
was replaced by three species of diatoms—Nitzschia palea, Navicula minima, and
and ^N. seminulum var. hustedtii. Other species of algae that were more abundant
in the treated area than in the control area of the stream were the filamentous
blue-green alga Schizothrix calcicola and the desmids Cosmarium granatum and C^.
subprotumidum.
A manuscript presenting more detailed information concerning effects of copper on
the Shayler Run periphyton community Is being prepared by C. I. Weber and B. J.
McFarland of the Environmental Monitoring and Support Laboratory, U. S.
Environmental Protection Agency, Cincinnati, Ohio.
81
-------
u
z
a
LU
< 50
_i
ui
en
I »
oc
200
m
OO
ro
D I
Control Upper Lower Recovery
Exposure Exposure
AREAS
4-17-69 64-69 7-17-69 8-28-69 10-9-69 11-18-69 2-16-701 4-9-70 6-1-70 7-14-70 8-26-70 10-12-70 11-24-70 3-2-71 4-21-71 6-3-71 7-15-71
Figure 30. Psephenidae (riffle beetles): basket-sampler collections.
-------
00
o
LLJ
O
UJ
DC
LL.
UJ
>
O
cc
O
z
O
oc
UJ
m
S
100
50
500
400
300
200
100
1
0 I
Control Upper Lower Recovery
Exposure Exposure
AREAS
2-16-70 6-10-70 7-16-70 8-26-70 10-19-70 11-25-70 3-2-71 4-21-70 6-3-70 7-15-71
Figure 31. Psephenidae (riffle beetles): natural substrate collections,
-------
>-
o
00
-P-
12100
a
UJ
oc
u.
Ill
> 50
I-
01
oc
5 0
o
oc
500
5300
o
cc
LLJ •
m'
100
2-16-70
_n
Control Upper Lower Recovery
Exposure Exposure
AREAS
6-10-70 7-16-70 8-26-70 10-19-70 11-25-70 3-2-71 4-21-70 6-3-70 7-1571
Figure 32. Trichoptera (caddisflies): natural substrate collections.
-------
SECTION XII
FISH-STOMACH ANALYSIS
INTRODUCTION
Continuous observations of feeding habits reveal that selectivity of organisms
by fishes, even within the limits of a single ration and absolutely stable
conditions, is not a constant value, and any quantitative measurement is a
function of the food consumed (Ivelev, 1961). It was assumed that if
macroinvertebrates were reduced or eliminated by copper, the number of fish
dependent upon this food source in the exposure area would be reduced by migration,
starvation, and predation. If fish were to survive, a shift in diet to other
food materials would have to occur. The phenomena of electivity of feeding based
upon a preference shown by the stream fishes and upon the degree of accessibility
of food organisms could not be assessed under natural conditions. Therefore the
predominant organisms found in fish stomachs were compared with data obtained
from the artificial and natural substrates used during the study. This is in
keeping with the proven results that show that electivity values in any biological
system are the ratio between the concentrations of the ingredients making up the
food complex.
It was thought originally that five or six fish species would lend themselves
to stomach-content analysis, but this was not the case. To be useful the species
would have to be common in both the control and exposure areas throughout the
study period, and their stomach contents would have to be identifiable. The two
species that met these requirements were the green sunfish and the orangethroat
darter, both of which spawned rather well in the exposure area.
METHODS
Fish specimens analyzed for stomach content were from the eight biannual fish
collections discussed earlier. Ten specimens each of green sunfish and
orangethroat darters were chosen from both control and exposure area collections.
Fish were selected so that the lengths of individual specimens within each group
from control and exposure collections were duplicated as close as possible. The
orangethroat darters were analyzed for all eight biannual fish collections, four
pre-exposure collections and four exposure-period collections. Green sunfish
stomach contents, however, were only analyzed for the two 1969 pre-exposure
collections and the following four exposure-period collections. More than 85%
of the stomach contents from both species could be identified for all specimens.
Since all fish of 60-mm total length or greater were injected with AFA
after collecting, preservation of the stomach contents was excellent.
Total lengths of each fish were recorded, and their stomachs were removed
intact. Stomach contents were removed with fine forceps and placed in a petri
dish containing distilled water. The materials were examined with the aid of a
85
-------
stereomicroscope or a compound microscope when necessary at magnifications from
10X to 1000X. Organisms found in the stomach materials were identified and
enumerated.
RESULTS AND DISCUSSION
The organisms found in the stomachs of orangethroat darters and green sunfish
are listed in Tables 18 and 19, respectively. The numbers shown for each group
of macroinvertebrates are the totals from 10 stomachs. The group classified as
other aquatic macroinvertebrates consists of the following: cranefly, stonefly,
leech, and nematode. Based on their occurrence, fish and terrestrial invertebrates
were not of major importance in the diet of orangethroat darters during either the
pre-exposure or the exposure period.
Orangethroat Darter
At least 9 of the 10 stomachs per sample contained food, including the samples
from the exposure area during the exposure period. In general, more organisms
were present in orangethroat darter stomachs in the exposure-period collections
than in the pre-exposure collections for both the control and exposure samples
(Table 8, Figure 33). Based on pre-exposure data, the dominant food items 'for
this species of fish were sowbugs, mayflies, copepods, and Chironomids. This
would be expected since sowbugs, mayflies, and Chironomids were the most common
benthic organisms in the stream during the pre-exposure period.
During the exposure period sowbugs and mayflies were practically eliminated in
the exposure area, and this, in turn, was reflected in the stomach contents of
the orangethroat darters collected in the fall of 1970 and in both spring and fall
of 1971 (Table 18, Figure 34 and 35). The sowbugs present in the stomachs from
the fish in the exposure area for this period were found only in the spring
collections, and these specimens were small early instars that could have passed
through the weir screens. The mayflies, which were present in the orangethroat
darter stomachs from the exposure area for the May 15, 1970, collection, were
probably produced in the exposure area.
The mayfly population in the exposure area was still fair into June 1970. In
the next three collections only two mayfly specimens were found in orangethroat
darter stomachs from the April 7, 1971, collection, and these may have been washed
from the control area during high water.
During the spring months copepods contributed large numbers to the diet of
orangethroat darters (Table 18, Figure 36), except for the 1969 spring collection
which was made earlier than the other spring collections when the normal spring
population increase of copepods had not yet occurred. The water temperature was
still low (3° C), and the fish probably were not feeding heavily. During the
exposure periods copepods made up a higher percentage of the spring diet of the
orangethroat darter in both the control and exposure area.
The effects of the copper exposure on copepods in the stream are not known.
Copepods were not normally captured or studied during the project, and therefore
population comparisons were not possible. Because of their small size, many of
the specimens found in the stomachs may have originated in the control area and
passed through the weir screens.
86
-------
TABLE 18. NUMBER OF ORGANISMS IN THE STOMACHS OF ORANGETHROAT DARTERS, SHAYLER RUN, 1968-71
Collection area
Aquatic macroinvertebrates
Diptera (true flies)
Chironomidae (midges)
Simuliidae (blackflies)
Coleoptera (beetles)
Psephenidae (water penny)
Elmidae (riffle beetles)
Ephemeroptera (mayflies)
Trichoptera (caddisf lies)
Amphipoda (scuds)
Copepoda (Copepods)
Decapoda (crayfish)
Isopoda (sowbug)
Ostracoda (shrimps)
Oligochaeta (worms)
Other aquatic macroinvertebrates
Pisces (fish)
Terrestrial invertebrates
Total
Number of empty stomachs
Length of fish-range mm
Pre-exposure period
Spring
4/11/68
iH
O
M
4-1
c
O
u
78
1
2
2
5
70
37
7
3
1
206
1
45-56
a
en
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X
w
44
1
2
1
5
13
12
21
9
108
0
45-57
Fall
9/26/68
t— i
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u
c
0
cj>
41
1
1
32
14
22
25
136
0
45-56
0)
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3
w
0
ex
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w
Spring
3/18/69
rH
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c
0
o
54 | 14
1
1
1
18
3
6
5
51
139
0
45-58
1
1
2
4
2
3
3
31
1
44-52
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p
3
en
o
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w
9
1
2
1
3
1
3
1
21
1
46-55
Fall
9/26/69
M
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46
28
1
31
1
1
108
0
48-58
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7
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Exposure
Spring
5/15/70
i-H
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3
89
57
11
1
274
0
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207
3
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8
6
136
64
2
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1
433
0
45-53
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10/6/70
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10
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2
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1
48-58
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70
1
5
76
1
48-58
period
Spring
4/7/71
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40
15
2
8
223
12
11
1
312
0
41-57
cu
P
0
W
o
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37
2
3
9
108
1
1
2
1
164
1
44-56
Fall
10/5/71
T-H
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4J
C
O
u
48
1
32
1
1
20
1
3
107
0
42-52
QJ
£
3
CO
o
ex
iB
122
1
2
1
1
127
1
40-54
O3
-------
TABLE 19. NUMBER OF ORGANISMS IN THE STOMACHS OF GREEN SUNFISH, SHAYLER RUN, 1969-71
00
00
Pre-exposure period Exposure
Spring
3/18/69
o
J-J
o
Collection Area °
Aquatic macroinver tebrates
Diptera (true flies)
Chironomidae (midges) 6
Tipulidae (craneflies) 4
Coleoptera (beetles)
Psephenidae (riffle beetles) 1
Ephemeroptera (mayflies) 9
Trichoptera (caddisflies)
Amphipoda (scuds) 15
Copepoda (Copepods)
Decapoda (crayfish)
Isopoda (sowbugs) 8
Oligochaeta (worms) 4
Odonata
Zygoptera (damsel flies)
Anisoptera (dragonf lies)
_ , . , T
Pisces (fish) 1
Terrestrial invertebrates
Total 51
Number of empty stomachs 0
Length of fish-range mm 103-152
Fall Spring
9/21/69 5/15/70
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4-11-68 9-26-68 3-18-69 9-26-69
SPRING
5-15-70
FALL
10-6-70
SPRING
4-7-71
FALL
10-5-71
Figure 33. Total numbers of macroinvertebrates found in orangethroat darter stomachs.
-------
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4-11-68 9-26-68 3-18-69 9-26-69 5-15-70 10-6-70 4-7-71 10-5-71
Figure 34. Isopoda (sowbugs) found in orangethroat darter stomachs.
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Figure 35. Ephemeroptera (mayflies) found in orangethroat darter stomachs.
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4-11-68 9-26-68 3-18-69 9-26-69 5-15-70 10-6-70 4-7-71 10-5-71
Figure 36. Copepoda found in orangethroat darter stomachs.
-------
The chironomids as a group provided the greatest numbers of individuals in
the diet of the orangethroat darter in both the control and exposure areas for
the pre-exposure and exposure periods (Table 18, Figure 37). In general,
chironomids were not adversely affected by the addition of copper to the stream,
and the stomach analyses indicate that they composed a larger portion of the diet
of the orangethroat darter in the exposure area during the exposure periods.
Green Sunfish
The results of the green sunfish stomach analysis are presented in Table 19.
The data were treated as they were for the orangethroat darter. The group
classified as other aquatic macroinvertebrates consists of the following:
Stratiomyidae, Elmidae, Plecoptera, Megaloptera, Hirudinea, Ostracoda, Physa, and
salamander larvae. All of these were found in stomachs of green sunfish from the
control area only, except the salamander larvae of which only one specimen was
observed from the stomach of a green sunfish from the exposure area.
A few empty stomachs were found during the pre-exposure-period collections.
A greater number of empty stomachs occurred during the exposure period, but the
numbers were similar for both the control and exposure specimens. No relationship
between empty stomachs and exposure to copper was found.
The numbers of organisms present in green sunfish stomachs in the
pre-exposure collections were similar for control and exposure-area specimens
(Table 19, Figure 38). However, the numbers of organisms present in green
sunfish stomachs were from two to five times greater in control specimens than in
exposure specimens in collections during the exposure period.
During the pre-exposure period five groups of food organisms were present in the
control fish stomachs at levels greater than 10% of the total numbers. These were
Chironomids, sowbugs, mayflies, scuds, and riffle beetles. During the same
period, however, only mayflies, scuds, and sowbugs were above the 10% level in
the stomachs of exposure-area fish.
The reduced population of sowbugs and mayflies in the exposure area is
reflected by the stomach contents of exposure-area green sunfish for the same
period. In the first exposure-period collection, 19 sowbugs were found in fish
stomachs from the exposure area. For the remaining three collections, only one
or two were found per collection (Figure 39). During the pre-exposure period
mayflies made up approximately 20% of the food organisms in the diet in both
control and exposure areas. During the exposure period this level was maintained
for the control area (Figure 40), but only two specimens of mayflies were
present in green sunfish stomachs from the exposure-period collections. This
supports the observations during the macroinvertebrate study.
Scuds normally occurred in stomachs of fish collected in the spring, but not
in those collected during the fall. Scuds found in the fish stomachs during the
pre-exposure spring collection were all adults, since reproduction of this form
had not yet occurred. Specimens of scuds from green sunfish stomachs during the
exposure period were of mixed sizes in control-area fish stomachs, but were all
small (early instars) in the stomachs from exposure-area fish. Adult scuds were
either not present or at least were very scarce'in the exposure area. It is also
possible that the newly hatched young present in the exposure-area fish stomachs
were not produced in the exposure area, but were from the control area.
93
-------
00
PERCENT OF
MACKOIN VERTEBRA
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200
150
Q 100
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0 CONTROL
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SPRING FALL SPRING FALL I SPRING FALL SPRING FALL
4-11-68 9-26-68 3-18-69 9-26-69 5-15-70 10-6-70. 4-7-71 10-5-71
Figure 37„ Chironomidae (Chironomids) found in orangethroat darter stomachs.
-------
150
100
50
Q CONTROL AREAS
• EXPOSURE
i
3-18-69 9-26-69 | 5-15-70 10-6-70 4-7-71 10-5-71
Figure 38. Total numbers of organisms found in green sunfish stomachsc
-------
100
C3
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100
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[i
D CONTROL
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3-18-69 9-26-69
5-15-70 10-6-70 4-7-71 10-5-71
Figure 39. Isopoda (sowbugs) found in green sunfish stomachs.
-------
CO
100
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3-18-69 9-26-69
n n ru II
aCONTROL AREAS
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n n. n
5-15-70 10-6-70 4-7-71 10-5-71
Figure 40. Ephemeroptera (mayflies) found in green sunfish stomachs.
-------
Chironomids, even though present in both control and exposure areas
throughout the study period, were not as important in the green sunfish diet as
they were in the diet of the orangethroat darter (Tables 18 and 19, Figure 41).
In one collection during the exposure period, however, Chironomids composed as
much as 54% of the total organisms present in stomach contents of green sunfish
in the exposure area.
Fish composed a portion of the diet of green sunfish during the study. Even
though few were actually present in stomachs, a number of these specimens were
large when compared to other food forms and thus did constitute a considerable
volume of the diet (Table 19, Figure 42). The number of fish present in the
stomachs was similar in control-area and exposure-area sunfish for both the
pre-exposure and exposure periods. If common food organisms such as sowbugs,
mayflies, and scuds were reduced, a shift in diet to fish might occur. Such a
shift would be mitigated because there was also a major reduction in the
population of fishes in the exposure area during the same period.
Terrestrial organisms were not common in the stomachs of green sunfish
during the pre-exposure period (Table 19, Figure 43). During the exposure period,
however, a greater number were present in the stomach contents, especially for the
exposure-area specimens. This is not unexpected since common aquatic food
organisms of green sunfish had been reduced. Even though only a limited shift in
food type did occur in the exposed green sunfish population, if copper were
added to the stream for a longer period, food could become the limiting factor
for the population of green sunfish in the exposure area.
98
-------
100
50
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J
100
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ea
n
HI CONTROL
• EXPOSURE
AREAS
CO
CD
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3-18-69 9-26-69
5-15-70 10-6-70 4-7-71 10-5-71
Figure 41. Chironomidae (Chironomids) found in green sunfish stomachs,
-------
o
o
C/9
OO
100
50
_TL
100
.CO
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X
I 50
3-18-69 9-26-69
1
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D CONTROL
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AREAS
Ih TL
5-15-70 10-6-70 4-7-71 10-5-71
Figure 42. Fish found in green sunfish stomachs.
-------
CO
CO
CM
100
50
D CONTROL
• EXPOSURE
AREAS
3-18-69 9-26-69 5-15-70 10-6-70_ 4-7-71 10-5-71
Figure 43. Terrestrial organisms found in green sunfish stomachs.
-------
PART C — LABORATORY STUDIES
SECTION XIII
ACUTE STUDIES
INTRODUCTION
In preparation for long-term exposure of the stream to a constant
concentration of copper, laboratory toxicity tests were conducted to evaluate
acute copper toxicity to fish in Shayler Run water. The data from these toxicity
tests were to be used not only for the evaluation of acute toxicity, but also for
predicting "safe" and "unsafe" concentrations in Shayler Run. To evaluate acute
copper toxicity in Shayler Run water, two major objectives were pursued: (1) to
determine the effect of changes in water quality of Shayler Run on acute toxicity,
and (2) to determine the relative sensitivity of several common stream species.
*
Because many water quality factors influence copper toxicity, it was
necessary to know how acute toxicity of copper in Shayler Run water varied with
the varying water quality of the stream. Most of these toxicity tests were
conducted with unmodified Shayler Run water. A few were made, however, with water
from the Newtown Fish Toxicology Station (NFTS) laboratory (standard water) and
with modified Shayler Run water. The bluntnose minnow, a common Shayler Run fish
species, and the fathead minnow were used in these toxicity tests. Static tests
were used so that more bioassays could be conducted and so that large volumes of
water would not be needed.
To determine the copper concentration to be used in the stream exposure, it
was desirable to know what concentrations of copper in Shayler Run water might
be acutely lethal to the more sensitive species. Also, the acute toxicity data
were to be used for predicting direct chronic toxicity of copper in the Shayler
Run exposure to species with which streamside chronic tests could not be
conducted. This was to be done by assuming that the order of sensitivity of the
stream species to copper would be the same in both short-term and long-term
exposures.
The model for predicting long-term direct toxicity to the species in the
stream exposure was the laboratory chronic toxicity studies of Mount (1968) and
Mount and Stephan (1969). They proposed that the application factor for copper
be calculated by dividing the maximum acceptable toxicant concentration (MATC)
of a chronic test by the LC50 concentration of an acute test, using the same
species of fish in the same dilution water. They suggested that the application
factor might be applicable to other species of fish. The application factor
assumes that the relative sensitivity of various species in terms of the
MATC's should be the same as the relative sensitivities of the species in terms
of the LC50 in the same water. The application factor does not assume that the
relative sensitivities of different species must be the same in all waters.
102
-------
The relative sensitivity studies were made with flow-through testing to
more closely duplicate stream conditions. Sensitivity of fish species was
determined and evaluated in terms of the 96-hr LC50 values of copper. These
species were tested at different times in the standard water, and a series of
three tests was conducted with different species at the same time in Shayler
Run water.
METHODS
Water Quality Studies
These static acute mortality tests were conducted according to routine
bioassay methods recommended by the American Public Health Association (1965).
The physical and chemical characteristics of Shayler Run water and copper
concentrations were measured as reported in Part A (Water Quality-Chemical).
Copper was added to the test chamber from a stock solution of reagent-grade
CuSCv 5H20 dissolved in distilled water. At least five concentrations with a
0.5 dilution factor were tested; 10 fish were used for each concentration. The
experimental design of these bioassays gave estimated LC50 values by using
graphical interpolation. In addition, some of the LC50 values were calculated
by probit analysis using a computer program based on Chapter 18 "Assays Based on
Quantal Response" in "Statistical Methods in Biological Assay" (Finney, 1971).
The bluntnose minnow was used as the test species for the toxicity tests
conducted at the NFTS, because it was the most abundant species in Shayler Run
and is adaptable to laboratory conditions. All test fish were young-of-the-year
individuals collected from Shayler Run. The test fish were held in Shayler Run
water at the natural stream temperature, and the fish along with the stream
water were brought to the laboratory on the day of testing. Initially the fish
averaged 25 mm in length; at the end of the testing period they averaged 50 mm.
For a given bioassay the size of the largest fish was not more than 1.5 times
the length of the smallest. Initially, 10 fish were tested in 2 1. of solution. As
the season progressed and the fish grew, the volume of the test solution was
increased so that the weight of fish in the test containers did not exceed 1 g/Z.
and dissolved oxygen concentrations were maintained above 4 mg/Z.. The volume
for 10 fish was increased to 3, 6, and 10 1. The test fish were added to the
3.8-1. or 19-1. wide-mouth glass jars immediately after the preparation of the
test solutions. Tests lasted 48 hr, even though most of the deaths occurred in
24 hr at the test temperature of 24±2° C.
The usual source of Shayler Run water was from just above the V-notch weir,
the point of copper introduction A few bioassays were conducted with Shayler
Run water upstream from the entrance of the effluent from the sewage treatment
plant and with standard laboratory water. The majority of the bioassays were
conducted in unmodified stream water; however, to investigate sources of
variation of copper toxicity, the stream water was modified for some tests.
For the tests conducted on site, the fathead minnow was used. The fish
were obtained from the Newtown Fish Farm, Ohio Department of Natural Resources,
Division of Wildlife, and were acclimated for at least 2 weeks in stream water.
These fish ranged from 20 to 71 mm total length, but for an individual test the
difference in length between the largest and the smallest fish was less than two
times. The test chambers were 19-Z. wide-mouth jars and contained 10 1. of
103
-------
unaerated stream water. The chambers were placed in a flow-through water bath
supplied with stream water, and the bioassay was run at ambient stream water
temperature. Five fish were randomly assigned to duplicate chambers to give a
total of 10 fish per concentration. Numbers of dead fish were recorded every
24 hr for 7 days. Dissolved oxygen, pH, alkalinity, and hardness were measured
at the beginning of each test before fish were added and at the end. One hour
after copper was added, samples for dissolved copper analysis were taken from
one of each set of duplicate test chambers. At the end of the static tests the
test solutions were acidified, and samples were taken for total copper analysis.
Relative Sensitivity Studies
Standard Water—
Two flow-through exposure systems were used to test the acute toxicity of
copper in the standard water. A proportional diluter (Mount and Brungs, 1967)
was used to deliver a control and five toxicant concentrations with a dilution
factor of 0.6. The glass test chambers were 30 by 30 by 30 cm high and
were calibrated by means of a standpipe to contain 15 1. of test solution. The
water from the diluter was divided to deliver 500 ml to each duplicate tank.
A chemical-metering device was used to deliver reagent-grade copper sulfate
(CuSOti-St^O) solution from a Mariotte bottle. The standard dilution water was
a mixture of spring water and carbon-filtered demineralized Cincinnati tap water
maintained at a hardness of 200 mg/Z.. (as CaCOg). During the tests the
characteristics of this water were as follows: hardness, 196-205 mg/Z-. (as
CaCOs); alkalinity, 148-161 mg/Z.. (as CaC03); pH, 7.9-8.1; and temperature
23-25° C.
The fathead minnows, bluegills, and brown bullheads were obtained from
ponds at the Newtown Fish Farm. All other species were collected from Shayler
Run and nearby streams. Test fish were held in 50-gal glass aquaria receiving
flowing water similar to that used during the testing. All fish were acclimated
in the laboratory to the exposure temperature of 24°± 1° C for at least 30 days.
Bluegills and brown bullheads were larger, and only five fish per duplicate
test chamber were used. All other species were tested with 10 individuals per
duplicate chamber. The LC50 values were calculated by graphical analysis
(American Public Health Association, 1965) for each duplicate series of 10 fish
per concentration; for the bluegills and brown bullheads, the duplicates were
combined for the calculation. The LC50 values and 95% confidence limits were
also calculated from the data on the combined duplicates. Total copper
concentrations were measured from daily grab samples. In some cases these grab
samples were composited for the duration of the test, and the composite
concentration was measured.
Shayler Run Water—
A series of flow-through tests was done at the NFTS for appraisal of acute
copper toxicity in Shayler Run water. The dilution water was hauled over a
2- to 3-day period and stored in an underground 19-kiloliter polyester-lined
tank and a 11.4-kiloliter cement cistern. The water was piped to an indoor
stainless steel headbox for use during the test. Test water was aerated
continuously in these tanks and in the indoor stainless steel headbox. From
this headbox a pipe manifold delivered water to three proportional diluters.
104
-------
For the two series of tests conducted during November 1969, the diluters
delivered a control and five test concentrations to each exposure system. A
dilution factor of 0.6 was used in this series. In the other tests the diluters
delivered a control and six concentrations with a dilution factor of 0.5.
All diluters delivered 500 ml per cycle to the duplicate exposure chambers,
which received 120-180 Z „ per day. These glass exposure chambers were 30 cm by
60 cm by 30 cm high and were calibrated to contain 30 1. Test concentrations
were randomly assigned to the chambers arranged in two rows. The source of test
animals was the same as those used in the standard dilution water. For the test
that started in November 1969, the fish were held in flowing standard laboratory
water for 20 days and then acclimated for 10 days in Shayler Run water. For the
tests started on November 15, 1970, and May 6, 1971, the fish were held at the
stream in continuous-flow tanks with aeration. They were held for at least 30
days and were fed Oregon Moist fish food and frozen brine shrimp. Three days
before the test they were brought into the NFTS laboratory and held in stream
water to be used during the test. For the last test the fish were held in standard
water for 4 months, after which the fish were held in Shayler Run water for 2 days
before testing.
Because it had been shown that the acute toxicity of copper in Shayler Run
water varied greatly with water quality, the species were tested simultaneously
in the three exposure systems with 10 fish per test concentration. Routine
chemical analyses were made as in the tests with standard water. In addition,
some dissolved copper measurements were made.
In conjunction with the continuous-flow testing, a static reference bioassay
was conducted using the bluntnose minnow or the fathead minnow. Duplicate
series of five fish per 10 Z. were used in dilution water and at a temperature
similar to those of the continuous-flow tests.
RESULTS
Water Quality Studies
Three series of acute toxicity tests were conducted to develop information
on the variation of copper toxicity due to the varying water quality of Shayler
Run. For the first series, weekly bioassays were conducted at the NFTS
laboratory with stream water at laboratory temperatures The results of these
static toxicity tests and characteristics of the dilution water are given in
Table 20. The nominal copper LC50 values for the bluntnose minnow varied from
a high of 21 mg/Z. to a low of 0.57 mg/Z.; very few fish died after 24 hr of
exposure. Hardness varied from 322 mg/Z. (as CaC03) to 134 mg/Z. and alkalinity
varied from 220 mg/Z. (as CaC03) to 98 mg/Z. Both hardness and alkalinity were
lower under high flow conditions.
Agreement was good between the LC50 values calculated by graphical
interpolation and by probit analysis, but because only 10 fish were used per
concentration with a dilution factor of 0.5, the 95% confidence limits were
rather large. The test conducted on July 21 was set up with four replicates of
10 fish per concentration to check replication of results. The agreement of
LC50 values of these four bioassays was excellent, but because of the lack of
two partial kills, probit analysis could not be made0
105
-------
TABLE 20. SUMMARY OF LC50 VALUES BASED ON TOTAL COPPER FOR THE BLUNTNOSE MINNOW
Date
7/15/69
7/21/69
7/23/69
7/25/69
7/29/69
8/4/69
8/6/69
8/11/69
8/18/69
8/20/69
8/21/69
Graphical [
interpolation Probit analysis
LC50 (mg/Z.) LC50 (mg/Z.)
24-hr
14
21
19
19
19
5.8
6.0
8.0
48-hr 24-hr
14 14.5
21
19
19
19
5.8 5.8
6.0 , 5.5
8.0
19 19
13 13
7.0
6.4
7.0
6.4
0.57 0.57
3.0
8/25/69 9.0
9/2/69
9/8/69
9/9/69
9/16/69
9/24/69
9.0
2.0
5.6
3.2
4.0
3.0
9.0
9.0
2.0
5,, 6
3.2
4.0
5.7
-
12.1
8.0
6.5
0.66
3.7
8.6
7.3
2.3
4.5
3.3
3.2
95% 1
Confidence i
limits
11.3-23.7
48-hr
14.5
i
1
I
4.8-7.1
4.4-6.9
4.4-7.4 i
-
8.8-16.5
5.8-11.3
4.7-9.2
0.47-0.95
5.8
95%
Confidence
limits
11.3-23.7
4.8-7.1
5.5 4.4-6.9
5.5 4.3-7.1
12.1 , 8.8-16.5
7.5 i 5.5-10.3
6.5 4.7-9.2
0.66
2.7-4.9 ; 3.7
6.2-12.3 8.6
5.3-10.2 j 7.3
1,6-3.3 j 2.3
3.1-6.9 4.5
1.7-4.8 ! 3.3
2.1-3.2 !; 3.2
0.47-0.95
2.7-4.9
6.2-12.3
5.3-10.2
1.6-3.3
3.1-6.9
1.7-4.8
2.1-3.2
(
i
Shayler Run dilution water
Mean daily
stream flow
(m^ /sec)
0.0156
0.0204
0.1048
0.0312
0.0173
0.1104
0.0108
0.0453
0.0878
0.2237
0.0595
0.1756
0.0280
0.0850
0.0470
0.0116
Hardness
(mg/Z. as
CaCOq)
286
305
134
274
302
312
322
236
258
170
218
276
300
222
246
316
Alkalinity
(mg/Z. as
CaCO,)
202
219
98
208
220
206
222
176
186
144
168
216
214
170
204
218
0.0523 324 212
; i
: ' i
PH
8.1
7.9
7.7
8.0
7.9
7.9
8.0
7.8
8.0
7.9
8.1
8.0
8.1
7.9
7.8
7.8
7.9
Calcium
(mg/Z.)
74
_
-
75
82
83
-
62
70
47
61
77
81
54
62
76
77
Magnesium]
(mg/Z.)
16.6
_
-
15.6
19.2
18.6
-
14.5
15.9
10.6
14.3
18.3
19.0
14.8
16.7
21.0
21.0
o
-------
Under low-flow stream conditions LC50 values were much higher than expected.
Toxicity appeared to be related to stream flow; copper was less toxic (LC50 values
higher) during low-flow conditions. A correlation analysis was made comparing LC50
values to stream flow, and the relationship was significant (P=0.05).
Because of the great variation in LC50 values, additional chemical
characteristics were measured to find other variables that might be related to
toxicity. The results of the second series of bioassays with the bluntnose
minnow are given in Table 21. A summary of additional chemical analysis of the
test water for these bioassays is given in Table 22. The stream flow during
October 1969 was the lowest mean monthly flow of any month during the study.
The mean daily flow never exceeded 0.017 m3/sec. Despite the stability of stream
flow, hardness, and alkalinity, the 24-hr LC50 values for total copper averaged
8.7 mg/Z. and varied from 0.75 mg/Z. to 22 mg/Z.
Stepwise regression analysis of the 24-hr LC50 values (graphical
interpolation) and the chemical and physical measurements were made. There was
a significant (P=0.01) correlation of LC50 values with total phosphate and
potassium. During this period of low stream flow the correlation coefficient for
LC50 values and stream flow was not significant (P=0.05).
The third series of bioassays was conducted with the fathead minnow at the
streamside laboratory, and the 96-hr and 7-day LC50 values are given in Table
23. A summary of additional chemical analysis of test water for these bioassays
is given in Table 24. The 96-hr and 7-day LC50 values for nominal total copper
varied widely, approximately 25 and 45 times, respectively, 23.6-0.92 mg/l.
and 23.6-0.56 mg/l. The LC50 values based on dissolved copper, however, varied
only threefold, 0.52-1.40 mg/Z.s for 96 hr and fourfold 0.36-1.40 mg/Z., for 7
days. At temperatures above 10° C the lethal threshold for copper was apparently
reached in 96 hr, since no additional deaths occurred after that time. However,
at temperatures below 10° C test fish were still dying at the end of the test
period of 7 days
Higher LC50 values for both total and dissolved copper, indicating low
toxicity, generally occurred during the summer and low-flow periods, whereas
lower LC50 values, indicating high toxicity, occurred during the winter and
spring and higher stream-flow periods. The higher LC50 values were also
generally associated with higher values of measured chemical characteristics
attibutable to effluent from the sewage treatment plant. During low-flow periods
the effluent from the treatment plant made up 80-90% of the stream flow at the
gaging station.
Stepwise regression analysis of total copper 96-hr LC50 values and water
quality characteristics indicated a significant (P=0.01) correlation of LC50
values with sodium (Na), total phosphate (TP), potassium (K), nitrite (N03),
temperature, chlorine (Cl), total solids (TS), and conductivity. Stream flow
was not significant (P=0.05). For dissolved copper LC50 values there was no
significant (P=0.01) correlation with any of the water quality characteristics
measured; however, TP and TS were significantly (P=0.05) correlated with
dissolved copper LC50 values.
Two tests were made to compare copper toxicity in water from different
locations in Shayler Run: upstream from the sewage treatment plant and
downstream at the regular sampling station. During normal flow the downstream
water had a higher hardness and alkalinity than Shayler Run water above the
107
-------
TABLE 21. SUMMARY OF LC50 VALUES OF COPPER FOR THE BLUNTNOSE MINNOW IN SHAYLER RUN WATER
o
00
Date
10/7/69
10/8/69
10/9/69
10/10/69
10/13/69
10/14/69
10/15/69
10/16/69
10/17/69
10/20/69
10/21/69
10/21/69
10/22/69
10/23/69
10/27/69
10/29/69
11/1/69
11/3/69
11/5/69
11/7/69
11/17/69
LC50 Graphical interpolation
Nominal total
copper
24-hr
6.3
9.0
4.7
11
5.7
10
8.0
12
9.7
8.0
14
21
22
8.9
11
9.2
2.0
0.75
2.8
1.6
4.0
48-hr
6.3
9.0
4.7
11
5.7
10
8.0
11
9.7
7.0
12
21
19
8.0
11
6.3
1.5
0.75
2U5
1.6
4.0
Dissolved
copper
24-hr
0.43
0.42
0.30
0.32
0.33
0.42
0.33
0.37
0.34
0.40
0.39
LC50 (mg/Z.) Probit analysis
Nominal total copper
24-hr
6.5
-
4.9
7.9
5.7
8.7
9.2
12.1
8.3
8.0
14.2
-
-
-
11.4
8.6
2.4
0.72
2.9
1.8
4.7
95%
Confidence
limits
4.9-9.9
3.5-6.8
5.4-11.8
4.2-10.7
6.0-14.8 '
6.3-13.7
9.3-15.9
6.2-11.2
48-hr
6.5
-
4.9
7.9
5.7
8.7
9.2
11.0
8.3
5.8-11.4 , 7.5
10.6-28.7 12.1
;
8.5-16.0 -
6.2-12.3
1.6-3.6
1.1-3.0 ,
2.1-4.7
1.2-2.4
2.9-7.9
-
14.7
-
11.4
7.1
2.0
0.66
2.7
1.8
4.7
957.
Confidence
limits
4.9-9.9
3.5-6.8
5.4-11.8
4.2-10.7
6.0-14.8
6.3-13.7
7.4-14.3
6.2-11.2
5.5-10.6
9.2-16.3
10.2-22.8
8.5-16.0
5.2-9.8
1.3-3.0
1.0-2.4
2.0-3.7
1.2-2.4
2.9-7.9
Physical and chemical analysis
Instantaneous
stream flow
(m /sec )
0.0178
0.0204
0.0178
0.0178
0.0261
0.0241
0.0187
0.0178
0.0161
0.0153
0.0510
0.0269
0.0300
0.0241
0.0187
0.1674
0.0374
0.0490
0.0413
0.0347
0.0312
Hardness
(mg/Z.
as CaCOq) j
Alkalinity
(mg/Z.
as CaCO,)
320 j 226
324 226
324 226
320 228
318 213
318
314
318
324
339
310
310
302
296
332
340
296
306
308
314
315
210
210
214
218
218
197
214
212
198
220
212
198'
189
207
209
220
PH
8.2
8.2
8.1
8.0
8.0
8.0
8.0
7.9
8.1
8.1
8.1
8.1
8.1
8,0
8.1
8.2
8.2
8.1
7.8
8.2
8.3
-------
TABLE 22. SUMMARY OF CHEMICAL ANALYSIS OF TEST WATER IN
TESTS REPORTED IN TABLE 21
Date
10/7/69
10/8/69
10/9/69
10/10/69
10/13/69
10/14/69
10/15/69
10/16/69
10/17/69
10/20/69
10/21/69
10/21/69
10/22/69
10/23/69
10/27/69
10/29/69
11/1/69
11/3/69
11/5/69
11/7/69
11/17/69
Ca
(mg/Z.)
93
96
96
96
93
93
93
93
93
94
89
89
88
81
90
92
83
78
83
85
87
Mg
(mg/Z.)
20.3
20.7
20.3
20.2
20.0
20.0
20.0
20.3
20.3
21.4
19.3
19.4
19.2
18.7
20.9
20.9
19.0
18.0
19.9
19.8
20.4
TP
(mg/Z.)
7.4
7,6
7.4
8.0
7.3
8.0
7.8
7.6
7,8
7.6
9.0
9.3
8.9
8.4
7.7
7.9
7.1
4.8
5.8
5.5
5.3
K
(mg/Z.)
10.0
10.6
10,5
10.4
11.9
11.7
11.1
9.5
9.6
9.8
13.2
13.4
12.9
11.3
10.9
10.8
11.0
8.0
8.3
8.7
7.8
Na
(mg/Z.)
71
71
71
73
72
68
66
70
68
72
67
66
62
57
62
60
63
38
47
53
50
Cl
(mg/Z.)
80
76
80
89
77
77
74
80
82
87
81
73
68
61
68
71
71
41
54
59
53
Total
organic
carbon
(mg/Z.)
5.4
5.4
6.2
6.4
6.4
6.2
7.0
5.8
6.0
5.8
8.6
8.4
7.0
8.6
5.2
6.8
7.6
7.4
6.2
6.0
4.8
N03 N
(mg/Z.)
10.5
10.0
10.4
10.2
9.9
10.1
9.7
9.4
9.6
11.2
11.7
10.6
10.0
10.7
12.5
12.6
10.5
5.3
7.0
7.3
8.8
109
-------
TABLE 23. SUMMARY OF STATIC BIOASSAY WITH FATHEAD MINNOWS IN
SHAYLER RUN WATER
Date
2/25/70
3/11/70
3/25/70
4/1/70
4/8/70
4/15/70
4/22/70
4/30/70
5/13/70
5/20/70
•7/1/70
7/8/70
7/16/70
7/23/70
7/29/70
8/19/70
9/9/70
9/30/70
10/13/70
10/28/70
11/27/70
12/2/70
1/6/71
1/20/71
2/3/71
2/17/71
3/4/71
3/25/71
4/2/71
5/13/71
5/19/71
6/2/71
6/16/71
6/30/71
7/13/71
7/28/71
Possibly
bpH for v.
Instantaneous
stream flow
mVsec
0.143
0.165
0.170
0.199
0.113
0.159
0.320
0.937
0.073
0.021
0.014
0.039
0.029
0.027
0.025
0.026
0.026
0.170
0.040
0.110
0.059
0.237
0.099
0.055
1.98
0.159
0.159
0.059
0.208
0.096
0.041
0.088
0.023
0.033
0.025
diseased greater
Hardness
(mg/Z. as
CaCO, )
280
280
244
212
260
302
224
228
150
310
308
336
280
280
266
310
324
290
260
240
242
308
206
262
322
210
260
252
312
272
276
284
252
298
282
284
Alkalinity
(mg/Z. as
(CaCO,)
190
184
160
132
170
198
160
158
96
210
230
180
180
170
206
220
172
184
212
170
210
140
190
242
130
174
180
212
200
208
206
176
210
188
194
Temperature
range
°C
4-7
2-4
5-8
7-12
10-15
12-17
15-21
12-23
19-24
16-21
22-30
20-27
20-23
24-27
22-27
20-25
18-24
13-17
0-14
11-19
5-li
1-13
1-5
1-4
1-15
1-5
1-8
2-11
10-19
11-26
14-25
20-28
20-26
22-28
21-28
18-24
than expected dead in low concentrat
intration nearest to LC50.
Control
pH
8.0
8.0
8.2
8.5
8.1
8.0
8.0
8.1
8.1
8.2
8.3
8.3
8.4
8.2
8.4
8.4
8.3
8.2
7.6
8.2
8.0
8.0
8.2
8.1
8.0
8.1
8.1
7.9
8.1
8 1
8.4
8.1
8.1
8.2
8.1
8.3
ions - VE
Nominal
96-hr LC50
Ong/Z.)
4.9
3.3
1.6
2.0
4.5
16
8.3
5.0
2.8
9.0
8.7a
21
12
10
20
19a
18a
3.15a
22.2
14. 5a
<0.65
4.67
0.92
1.19
2.83
1.45
1.58
1.00
5.33
1.02
4.16
>8
10.55
22.2
21.8
23.6
ilue used in data
copper
168-hr LC50
(mE/Z.)
2.6
1.7
1.2
1.6
2.4
13
0.9
0.80
2.8
9.0
3.9a
12
11
9.5
16.5
17. 4a
16.3a
2.21a
22.2
14. 5a
<0.65
3 19
0.56
0.40
1.41
0.89
0.82
0.75
5.00
<1.0
3.63
7.22
10.55
22.2
21.8
22.2
analysis.
PHb
8.0
8.0
8.0
8.5
8.0
8.0
7.9
7.9
7.3
7.2
7.3
7.5
7.5
7.4
7.9
7.5
7.9
7.4
7.5
8.1
7.8
8.2
8.1
8.0
8.0
7.9
7.8
7.6
8.1
8.0
7.7
7.6
7.4
7 5
7.6
Dissolved
96-hr LC50
(ms/Z.)
0.75
0.75
0.66
0.95
>0.80
1.06
0.82
0.94
0.81
0.97a
>0.80
0.78
0.64
>0.61
>0.81a
>0.78a
u.49a
1.09
0.92a
<0.64
0.75
0.60
0.68
0.92
0.69
••0.82
0.58
0.76
<0.56
0.65
0.83
0.83
1.40
0.96
0.82
copper
168-hr LC50
(ing/ 1.)
0.58
0.63
0.59
0.67
0.80
0.90
0.80
0.94
0.76
>0.84a
>0.80
0.75
0.64
>0.61
>0.81a
>0.78a
0.43a
1.09
0.92a
<0.65
0.65
0.44
0.36
0.56
0.46
0.56
<0.58
0.74
<0.56
0.65
>0.83
0.83
1.40
0.96
0.79
110
-------
TABLE 24. CHEMICAL ANALYSIS OF TEST WATER IN TESTS REPORTED
IN TABLE 23
Date
2/25/70
3/11/70
3/25/70
4/1/70
4/8/70
4/15/70
4/22/70
5/13/70
6/10/70
7/1/70
7/8/70
7/16/70
7/25/70
7/29/70
8/19/70
9/9/70
9/30/70
10/13/70
10/28/70
1/6/71
1/20/71
2/3/71
2/17/71
3/4/71
3/25/71
4/21/71
5/13/71
5/19/71
6/2/71
6/16/71
6/30/71
7/14/71
7/28/71
T-P
(mg/Z.)
£.1
1.4
0.7
0.4
1.1
1.4
1.3
0.8
3.6
3.4
3.4
6.6
4 .1
5.4
5.0
7.5
5.5
0.6
1.1
2.8
0.3
0.7
0.9
2.9
1.5
2.1
3.8
4.3
4.9
4. 6
solids
(ms/z.)
439
389
348
297
792
404
358
508
478
619
447
533
648
506
478
534
326
403
490
400
405
391
450
426
432
460
569
586
i,142
solids
440
384
340
290
318
372
340
492
461
609
434
521
607
492
481
526
312
415
491
371
393
379
451
412
415
426
540
502
534
conductivity
(mhos)
651
659
604
429
522
689
496
806
824
911
742
825
916
680
570
672
504
633
806
596
656
661
756
621
678
671
866
765
820
(mg/Z.)
i. J
1.6
l.U
0.6
1.2
'0.1
0.6
0.5
3.2
0.9
2.9
5.0
3.4
7.6
5.6
i.9
6.8
1.1
1.,
0.8
1.2
0.6
0.7
1.3
1.4
1.7
4.4
4.4
4.0
7.0
7.0
NO^-N
(nw/Z.)
0.2
4.9 58
5.8 44
3.8 38
2.6 32
<*.<« 35
b.j 38
J.D 31
3.2 20
/.4 57
10.3 71
13. / 80
9.7 50
10.8 62
8.8 55
ti.it 56
B.6 82
/.:> 58
9.8 59
b2
3.4 31
b.l 35
3.* 55
i.y 63
4.4 35
J.fo
l.i 51
4.0 34
j.3 38
5.8 66
b.U 39
7.3 68
7.0 61
9.3
Ca
78
75
68
60
80
84
68
61
82
90
96
78
82
76
90
96
85
73
89
68
82
94
67
78
75
89
80
88
72
89
83
102
Mg
17
15
13
12
14
15
13
12
15
19
22
17
17
16
19
21
19
17
20
14. J
17.7
21.6
U.J
16.6
Ib. 1
21.0
18.3
19.4
16. 0
20.0
18.1
23.0
111
-------
sewage treatment plant (Table 25). Bioassays were conducted in unmodified
upstream and downstream water. In addition, the harder downstream water was
diluted with demineralized water to prepare a modified water similar in
hardness to the upstream water. The first bioassay gave total LC50 values of
less than 1.0 mg/Z. of copper in the upstream water and the diluted and softened
downstream water (Table 25); the lowest concentration tested was 1.0 mg/Z. A
similar test was conducted with water collected July 29, 1969. The bioassay in
unmodified downstream water gave an LC50 value of 8.0 mg/Z. (Table 25). The
LC50 values in the upstream water and in the diluted downstream water were 0.35
and 0.50 mg/Z., respectively.
Additional tests were made to evaluate the effect of dilution of Shayler
Run water on copper toxicity. For the first test, demineralized water was
used to prepare three dilutions of stream water. The 24-hr LC50 values for
copper ranged from 19 mg/Z. for unmodified stream water to 3.6 mg/Z. for the
greatest dilution (Table 26). Bioassays in the intermediate dilutions had
similar LC50 values of 10 and 9.1 mg Cu/Z. This dilution not only lowered total
hardness and alkalinity, but also diluted other detoxifying agents in the
stream water. The LC50 value of 3.6 mg/£; was much higher than would have been
predicted since the standard water (200 mg/Z. hardness) gave an LC50 value of
0.45 indicating that other detoxifying agents were present. At the low flow
of this sampling period, most of the flow was due to the flow from the sewage
treatment plant.
The second test, September 18, 1969, was conducted in stream water collected
at a flow of 0.085 m3/sec and falling from a high flow of 00170 m3/s'ec. Thus,
there was much dilution of the sewage treatment plant effluent. For this test
the stream water was diluted with a 1-to-l dilution of demineralized water with
a reconstituted water similar in hardness and alkalinity to the stream water.
The LC50 value in the unmodified stream water was 17 mg/Z. of copper. In the
stream water diluted with reconstituted water, the LC50 value was 0.78 mg/Z.,
and copper was even more toxic in the stream water diluted with demineralized
water. Apparently the rain had greatly reduced the concentration of detoxifying
agents contributed by the sewage treatment plant, and the copper was much more
toxic in this dilution water than in the first test.
A dilution water containing calcium chloride, magnesium sulfate, and sodium
bicarbonate in demineralized water was prepared with similar hardness, alkalinity,
and pH as the stream water. The stream water was diluted with this water to 75%,
50%, and 25% stream water. Toxicity increased with decreasing amounts of
Shayler Run water. The LC50 values varied from 11 mg/Z. in the unmodified stream
water to 2.9 mg/Z. in the test water containing the smallest percentage of
Shayler Run water. These tests indicated that some chelating agent or agents
from the sewage treatment plant were being diluted, and thus copper toxicity
was increased.
Three tests were conducted to determine the effect on copper toxicity of
added hardness at a constant alkalinity. These results are summarized in Table
27. Calcium chloride and magnesium sulfate were used to maintain the
calcium-magnesium ratios in Shayler Run water. The first test was conducted in
rain-diluted Shayler Run water. Of all the unmodified stream water bioassays,
copper was the most toxic in this dilution water, with an LC50 value of 0.57
mg/Z. The LC50 value of 0.76 mg/Z. indicated, at most, only minor effect of
112
-------
TABLE 25. TOTAL COPPER LC50 VALUES FOR THE BLUNTNOSE MINNOW IN SHAYLER RUN
WATER UPSTREAM AND DOWNSTREAM FROM THE SEWAGE TREATMENT PLANT
Date
7/25/69
Source of
dilution water
Upstream
Downstream, unmodified
Hardness
(mg/Z.) as
CaCOq)
173
268
Calcium
(mg/Z.)
48
75
Magnesium
(mg/Z.)
10.6
15.6
Alkalinity
(mg/Z.. as
CaC03)
24-hr LC50
(mg/Z.)
<1.0
6.0
Downstream, diluted
7/29/69 Upstream
Downstream, unmodified
Downstream, diluted
175
196
300
196
53
82.5
131
19.2
156
220
148
0.35
8.0
0.50
-------
TABLE 26. TOTAL COPPER LC50 VALUES FOR THE BLUNTNOSE MINNOW IN VARIOUS DILUTIONS OF SHAYLER RUN WATER
Date
8/4/69
9/18/69
9/3/69
Test water
Graphical
interpolation
(LC50 ag/l.)
24 -hr
Unmodified Shayler Run water 19
Shayler Run water diluted
with demineralized water
Nominal hardness (mg/Z. as
48-hr
19
Probit analysis
LC50 (mg/Z.)
95%
confidence
48-hr limits
17.3 14.5-20.6
i
CaC03) j
250 i 10
200
150
Unmodified Shayler Run water
50% Shayler Run water
50% Reconstituted water
50% Shayler Run water
50% Reconstituted water
9.6 7.7 6.2-9.7
9.1 ] 9.1
3.6
17
3.3
17
0.78
0.25
0.78
0.25
1
Unmodified Shayler Run water : 11
i
Shayler Run water diluted
with reconstituted water
75% Stream water ; 6.7
50% Stream water 3.6
11
6.7
3.6
25% Stream water 2.9 '. 2.9
1
6.8 5.2-9.0
3.2 2.4-5.6
Hardness
(mg/Z. as
CaCO,)
316
255
205
158
222
220
112
292
300
302
296
Alkalinity
Ca Mg (mg/Z. as
(mg/Z.) (mg/Z.) CaCO,)
83 18.6 206
69 15.2 174
53 11.5 138
43 10.0 110
54 14.8 170
176
90
81 18.3 204
204
81.7 19.5 200
81 19.5 202
PH
7.8
7.8
7.8
7.7
7.9
7.9
8.0
7.9
-------
TABLE 27. EFFECT OF HARDENING SHAYLER RUN WATER ON THE TOXICITY OF
TOTAL COPPER TO THE BLUNTNOSE MINNOW
Date Dilution water
8/20/69 Unmodified stream water
Modified stream water
230 hardness
295 hardness
8/21/69 Unmodified stream water
Modified stream water
8/25/69 Unmodified stream water
Modified stream water
320 hardness
370 hardness
Graphical
interpolation
LC50 (mg/Z.)
24-hr 48-hr
0.57 0.57
0.85 0.85
1.7 1.6
3.0 3.0
4.0 3.5
9.2 9.2
Probit analysis
LC50 (mg/7, .)
95%
conf idence
24-hr limits
0.66 0.47-0.95
0.94 0.66-1.3
1.6 1.1-2.3
3.0 2.2-4.1
3.7 2.7-4.9
9.2 9.2 j
8.0 8.0
95%
confidence
48-hr limits
0.66 0.47-0.95
0.94 0.66-L.3
1.6 1.1-2.3
3.0 2.2-4.1
3.4 2.5-4.6
Hardness
(mg/l. as
CaCO,)
170
234
302
218
284
276
330
370
Alkalinity
(mg/i. as
CaCO?)
150
146
144
180
178
216
208
210
PH
7.9
7.9
7.9
8.1
8.0
8.0
7.9
8.0
Hardness expressed in mg/i. as CaC03-
-------
added calcium and magnesium. For the bioassay of August 21, 1969, the stream
flow had fallen to 0.059 m3/sec, with a corresponding hardness of 218 mg/Z. (as
CaCOs). The bioassay in the unmodified stream water gave an LC50 value of 3.0
mg/Z-. With added calcium and magnesium the LC50 value was 4.0 mg/Z. For the
third test the stream flow had fallen to 0.02 m3/sec. The LC50 value in the
unmodified water was 9.2 mg/Z. In the intermediate hardened water of 330 mg/Z..,
a similar LC50 value of 9.2 mg/Z. was obtained. In the hardest water the LC50
value was lower—8.0 mg/Z. This lower value resulted from the death of one
more test fish in the test concentration of 8.0 mg/Z. These three toxicity
tests indicate that added hardness (calcium and magnesium) had only a small
effect on the toxicity of copper in Shayler Run water. Apparently the reduction
of copper toxicity at lower stream flows was due to the greater contribution
of agents in the effluent from the sewage treatment plant during this time and
not to variation in alkalinity or hardness.
A supplemental test was conducted with standard water to evaluate the
effect of added calcium and magnesium. This standard water was diluted with
demineralized water, and calcium and magnesium salts were added to obtain test
waters of different hardness but similar alkalinity. In addition, the regular
standard water was used. The LC50 values in the dilution water with a constant
alkalinity ranged from 0.15 mg/Z. in the lowest hardness water to 0.26 mg/Z. in
the higher hardness dilution waters (Table 28). In the standard water with an
alkalinity if 154 mg/Z. the LC50 value was 0.29 mg/Z. These tests in standard
laboratory dilution water indicated that the great variation in acute toxicity
values obtained from Shayler Run water could not be explained in terms of
hardness.
Toxicity tests were carried out to determine the effect of added phosphate
on the toxicity of copper to the bluntnose minnow (Table 29). In the first
test 7.6 mg P/Z. were added as dibasic sodium phosphate to stream water. The
total copper LC50 value in unmodified stream water was 5.6 mg/Z., and in the
bioassay conducted with the added phosphate the copper was less toxic with
an LC50 value of 20 mg/Z. (Table 29). In the second test bioassays were
conducted with unmodified stream water containing 7.4 mg/Z» total phosphate
to which 0, 125, and 500 mg/Z. of pyrophosphate were added. Copper was less
toxic in the water with the added phosphate and was still less toxic in the
higher concentration of phosphate. The third test was conducted in standard
water, which is low in total phosphate (0.2 mg/Z. or less). Again, copper was
less toxic in the water with added pyrophosphate. The difference in the LC50
value for total copper was greater than tenfold between the standard water
and the water with the higher concentration of added pyrophosphate. Dissolved
copper measurements were made, and the range in LC50 values of dissolved
copper was about as great as the range for total-copper LC50 values.
Apparently much of the pyrophosphate-copper complex goes through the
0.45-millipore filter. However, the toxicity of this complex appears to be
greatly reduced.
Bluntnose minnows were brought into the laboratory on August 13, 1969.
They were held and tested in standard water. Five toxicity tests over a 2-month
period showed that the sensitivity of these fish to copper did not change with
being held in the laboratory. The LC50 values were similar in all tests (Table
30).
116
-------
TABLE 28. EFFECT OF HARDENING STANDARD WATER ON THE ACUTE TOXICITY OF TOTAL COPPER TO THE BLUNTNOSE MINNOW
Date
Dilution water
8/29/69 Unmodified standard water - 200 hardness^
LC50 values
(mg/Z.)
24-hr
0.29
Modified standard water
125
175
225
275
325
hardness
hardness
hardness
hardness
hardness
0.15
0.20
0.18
0.26
0.26
48-hr
Hardness
(mg/Z. as
0.29
208
1
0.15 | 132
0.20
0.18
0.26
0.26
182
233
282
337
Alkalinity
(mg/Z. as
CaCOO
154
100
99
99
99
98
Ca Mg i
(mg/Z.) (mg/Z.)! pH
' ff
25 15.5
I
44 27.3
7.7
7.7
7.6
1 7.6
. t
64 36.1 ! 7.7
Hardness expressed in mg/Z. as CaC03-
°200 hardness diluted with demineralized water to 125 hardness.
"Calcium chloride and magnesium sulfate added to 125 hardness water in the same ratio of Ca/Mg.
-------
TABLE 29. EFFECT OF ADDED PHOSPHATE ON THE LC50 OF COPPER TO THE BLUNTNOSE MINNOW
00
Graphical interpolation Probit analysis
LC50 (mg/Z.) LC50 (rog/Z.) Dilution water
Total copper
Total ; Dissolved | 95%
copper { copper j confidence
Date Test conditions 24-hr
9/9/69 Unmodified Shayler Run water 5.6
(Na2H P
-------
TABLE 30. ACUTE TOXICITY TO THE BLUNTNOSE MINNOW
OF COPPER IN STANDARD WATER
LC50 values (mg/Z.)
Date 24-hr 48-hr 96-hr
8/29/69
9/16/69
10/2/69
10/9/69
10/27/69
0.29
0.26
0.29
0.26
0.28
0.29
0.26
0.26
0.28
0.29
0.26
0.26
0.28
119
-------
Relative Sensitivity Studies
The results of copper-toxicity tests with standard water on 10 fish species
are summarized in Table 31. The 24-, 48-, and 96-hr LC50 values of measured
total copper are given for graphical interpolation, and, in addition, the 96-hr
LC50 value and 95% confidence limits were obtained by using probit analysis on
the data of the combined duplicates. There were no partial kills in either
bluegill test, so it was not possible to use our program of probit analysis.
Of the 10 species tested the bluegill was the most resistant. The striped shiner
and the orangethroat darter were more sensitive than the bluegill, but more
resistant than the other seven species. Agreement was excellent between the two
methods of calculating LC50 values, based on 20 test fish per concentration and
a dilution factor of 0.6.
The LC50 values obtained in the first series of tests with Shayler Run water
are shown in Table 32. The 96-hr LC50 values (graphical interpolation) varied
from 4.8 mg/Z-o for the rainbow darter to 11 mg/Z. for the fathead minnow. The
measured LC50 values were about 10 times the values for those species tested
in the standard water (hardness, 200 mg/Z. as CaC03). However, the dissolved
copper values were similar to the total copper values found in the standard water.
At concentrations of less than 1 mg Cu/Z. in the standard water, we found that
about 85% of the total copper was "dissolved." The dissolved copper LC50 of
0.38 found in the static reference bioassay was similar to the value in this
flow-through test.
A week later the preceding test was repeated with fish from the same stock.
Hardness, alkalinity, pH, and temperature of the dilution water were similar
in this and the preceding series of bioassays. In all cases the LC50 values for
each species were higher for this test than in the preceding test (Table 33).
Also, the LC50 value of the bluntnose minnow in the static test was higher. In
both tests the rainbow darter was the most sensitive species, and the striped
shiner and fathead minnow were the most resistant. For this test the LC50
values of the orangethroat darter, brown bullhead, and bluntnose minnow were all
within the confidence limits of these three species.
The results of the toxicity test started on December 15, 1970, are given
in Table 34; some of the results of copper analyses for this test are given in
Table 35. The bluegill was the most resistant species, and the stoneroller was
the most sensitive. The 96-hr LC50 values for both total and dissolved copper
were over 10 times higher for the bluegill than for the stoneroller. On the
basis of 96-hr LC50 values for total copper, the rainbow darter was more
sensitive than the orangethroat darter, and their confidence limits did not
overlap. However, their 96-hr LC50 values for dissolved copper were not
different.
The acute toxicity of copper in Shayler Run water for the test started on
May 6, 1971, is summarized in Table 36. The results of the copper analysis are
given in Table 37. The 96-hr LC50 (total copper) value ranged from 16 mg/Z. for
the bluegill to about 5 mg/Z.. for the striped shiner. The LC50 value (dissolved
copper) was lower for the rainbow darter than for the orangethroat darter. The
phosphate concentration was higher in this test water than in the water of the
preceding or following test. The static reference test gave a high 96-hr LC50
value of 19 mg/Z.
120
-------
TABLE 31. RELATIVE SENSITIVITY OF DIFFERENT SPECIES OF FISH TO COPPER IN STANDARD WATER
LC50 - (mg//..) measured total copper
Graphical interpolation Probit analysis - 96-hr
Species
Stoneroller
Creek chub
Rainbow darter
Blacknose dace
Bluntnose minnow
Fathead minnow
Fathead minnow
Brown bullhead
Striped shiner
Orangethroat darter
Striped shiner
Bluegill
Bluegill
Date
5/20/69
5/26/69
5/20/69
5/26/69
5/12/69
6/16/69
9/15/69
10/8/69
4/1/69
6/2/69
5/12/69
9/21/69
9/29/69
Duplicate
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
24-hr
>0.40
>0.40
>0.48
>0.48
0.40
0.46
0.70
0.52
0.26
0.40
0.58
0.76
0.60
0.52
>0.65
0.89
0.83
1.1
0.93
1.6
2.0
13
14
48-hr
0.36
0.37
0.37
0.38
0.34
0.40
0.46
0.38
0.26
0.31
0.58
0.75
0.60
0.47
>0.65
0.83
0.79
1.0
0.93
1.6
2.0
13
12
95% confidence
96-hr LC50 limits
0.31 0.29 0.25-0.29
0.31
0.33 0.31 0.27-0.35
0.29
0.34 0.32 0.26-0.38
0.36
0.33 0.32 0.28-0.36
0.33
0.26 0.34 0.29-0.40
0.31
0.42 0.44 0.38-0.52
0.47
0.55 0.49 0.41-0.59
0.42
0.52 0.54 0.43-0.77
0.83 0.79 0.67-0.92
0.76
1.0 0.85 0.75-0.98
0.71
1.6 1.9 1.6-2.2
2.0
8.3
10
Average Average
length weight
of test of test
fish (ran) fish (g)
60
64 4.0
41
47 1.1
84 6.7
56 1.6
47
39
55
44 0.9
55 1.7
103 18.6
101 19.2
-------
TABLE 32. RELATIVE SENSITIVITY OF SIX SPECIES OF FISH TO COPPER IN SHAYLER RUN WATER, NOVEMBER 12, 1969
LC50
Measured total
(mg/Z.)
Species
Rainbow darter
Brown bullhead
Bluntnose minnow
Orangethroat darter
Striped shiner
Fathead minnow
24-hr
14
>11.1
10
9.9
9.4
13
48-hr
5.9
5.8
10
9.9
8.4
11
Graphical interpolation
copper
96-hr
4.8
5.2
6.8
7.1
8.4
11
LC50
Measured dissolved copper
(mg/Z.)
96-hr
<0.36
<0.38
0.39
<0.72
0.72
0.54
Probit analysis
96-hr LC50
Measured total copper
(rag/Z.) Average
95% length
confidence G(Heterogenity of test
LC50 limits factor) fish (mm)
5.2 4.2-6.4 0.31 46
5.1 3.9-6.1 0.34 52
7.3 5.9-8.8 0.21 39
44
47
9.6 7.8-12 0.20 44
Average
weight
of test
fish (g)
1.2
1.4
0.43
^Dilution water - hardness = 314 mg/Z. as CaC03; alkalinity = 206 mg/Z. as CaC03; pH
Static bioassa.y - 96-hr LC50 = 16 mg/Z. (nominal), 0.38 mg/Z. (dissolved).
.0; test temperature = 24 °C.
-------
TABLE 33. RELATIVE SENSITIVITY OF SIX SPECIES OF FISH TO COPPER IN SHAYLER RUN WATER,3 NOVEMBER 19, 1969
Graphical interpolation
LC50
Measured total
(mg/Z.)
h-1
K>
U)
Species
Rainbow darter
Orangethroat darter
Brown bullhead
Bluntnose minnox-j
Fathead minnow
Striped shiner
24-hr
18
20
16
15
16
16
48-hr
12
17
15
15
16
16
copper
96-hr
5.3
9.4
12
13
15
16
LC50
Measured dissolved copper
(mg/Z.)
Probit analysis
96-hr LC50
Measured total copper
(mg/Z.)
95%
confidence G(Heterogenity
96-hr
<0.59
^0.59
0.57
0.62
1.0
1.1
LC50
5.5
9.8
11
11
13
limits
3.1-6.9
7.8-12
8.1-13
8.6-14
11-17
factor)
0.
0.
0.
0.
0.
46
25
32
26
29
Average
length
of test
fish (mm)
46
44
53
40
42
50
Average
weight
of test
fish (g)
1.0
0.8
1.5
0.6
0.6
0.9
Dilution water - hardness = 303 mg/Z. as CaC03; alkalinity = 206 mg/Z. as CaC03; pH
Static bioassay - 11/21—96-hr LC50 = 20 mg/Z. (nominal), 0.45 mg/Z. (dissolved).
11/23—96-hr LC50 = 18 mg/Z. (nominal), 0.47 mg/Z. (dissolved).
.0; test temperature = 24 °C.
-------
TABLE 34. RELATIVE SENSITIVITY OF SIX SPECIES OF FISH TO COPPER IN SHAYLER RUN WATER, DECEMBER 15, 1970
LC50
(ing/;.)
Spec iss Duplicate 24 -hr 48-hr 96-hr
StoneroUor A 11 5.0 1.5
B 11 5.2 0.87
Bl.unl.nuse minnow A 3.3 3.0 2.7
B 3.0 2.7 2.7
Striped shiner A 13 9.0 4.2
B 13 8.4 2.7
K.jinbow darter 6.0 5.5 4.5
B 11 9.5 7.1
Fathead minnow1' A 11 9.5 3.2
B .11 8.5 7.1
BluegilL A 18 17 17
B 17 17 17
1
24 -hr
1.5
0.91
0.53
0.51
2.8
2.8
0.84
1 .3
>0.91
>0.91
4.8
4.4
LC50 LC50
(mfi/£.) (mB/Z.)
952
48-hr 96-hr 36-hr limits
0.55 0.36 i.« i. 1-1.9
0.54 0.31
0.51 0.50 2.6 2.1-3.3
0.49 0.49
] .4 0.63 6.4 4.8-8.3
1.2 0.59
0.78 0.67 b.7 4.7-9.5
0.79 0.61
0.80 0.69 8.3 6.7-1 1
0.72 0.61
4.4 4.4
4.4 4.1
Probit analysis
LC50
(niR/t.)
95%
96-hr limits factor)
1 ,t L. 1-1 .9 0. 10
2.4 2.0-3.0 0. 10
3.4 2.6-4. i 0.11
4.3 3.9-6.0 0.15
6.9 5.7-8.3 0.16
LC50
(IDR/I.)
95%
96-hr limits factor)
0.34 0.31-0. 38 0.11
0.48 0.45-0.51 0.11
0.63 0.56-0.71 0. 17
0.63 0.56-0.74 0.24
0,67 0.63-0.74 0.12
-------
TABLE 35. COPPER CONCENTRATIONS (IN MILLIGRAMS PER LITER) IN EXPOSURE
CHAMBER FOR TESTS REPORTED IN TABLE 34a
Nominal
concentration
Control
0.125
0.250
0.500
1.00
2.00
4.00
Control
2.00
4.00
8.00
16.00
32,0
64.0
Control
0.50
1.00
2.00
4.00
8.00
16.00
24 -hr
Total Cu
0.
0.
0,
0.
0.
1.
0.
0.
1.
2.
4.
6.
11.
23.
0.
0.
0.
2.
3.
7.
9.
A
017
088
236
436
893
76
450
C
014
11
70
31
47
0
6
E
Oil
364
884
02
68
15
94
B
-
0.092
0.216
0.444
0.880
1.76
3.63
D
-
1.12
2.78
3.98
6.17
10.9
37.3
F
-
0.346
0.876
1.97
3.70
7.32
9.73
Sample
Dissolvec
A
0.011
0.078
0.198
0.269
0.351
0.438
0.555
C
0.009
0.386
0.573
0.649
0.931
1.99
11.2
E
0.008
0.228
0.299
0.373
0.503
0.649
0.943
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
2.
12.
0.
0.
0.
0.
0.
0.
Cub
B
-
081
191
268
348
422
587
D
-
408
573
663
909
12
0
F
72-hr
Total Cu
0
0
0
0
0
1
3
0
0
2
4
6
9
23
i °
233
300
369
503
618
931
0
0
2
3
6
11
A
.010
.089
.240
.458
.937
.84
.70
C
.010
.841
.60
.37
.33
.92
.1
E
.007
.341
.863
.00
.77
.96
.9
B
-
0.092
0.233
0.450
0.981
1.87
3.70
D
-
0.841
2.69
4.02
6.50
11.9
120.0
F
-
0.332
0.854
1.89
3.74
7.13
12.0
Sample ,
Dissolved Cu
A
0.023
0.091
0.205
0.278
0.361
0.457
0.522
C
0.020
0.887
0.530
0.596
0.849
1.88
10.1
E
0.012
0.229
0.308
0.390
0.470
0.583
0.888
B
0.095
0.199
0.272
0.372
0.462
0.530
D
-
0.394
0.535
0.609
0.863
2.02
10.6
N
F
0.208
0.322
0.385
0.470
0.578
0.849
aGrab samples were collected 24 and 72 hr after test began. Three diluters were used in duplicate:
,A and B, C and D, E and F.
The part that passes through a 0.45-micron filter.
125
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TABLE 36. RELATIVE SENSITIVITY OF EIGHT SPECIES OF FISH TO COPPER IN SHAYLER RUN WATER,& MAY 6, 1971
0\
Species
Strioed shiner
31untr.ose minnow
Orangethroat darter
Johnny darter
Rainbow darter
Fathead minnow
Cteek chub
Bluegill
Duolica te
A
B
A
E
A
B
A
B
A
B
A
B
24-hr
5.1
5.7
8.3
8.0
8.3
8.3
9.3
10
12
10
12
11
16
16
LC50
(ng/Z.)
48-hr
5.1
5.6
8.0
8.0
6.1
6.1
8.0
8.0
10
10
12
H
16
16
Graphic;! 1
copper
96-hr
4.5
3.3
6.8
4.4
5.4
5.6
6.8
8.0
10
9.6
12
11
16
16
Inter pola t ion
1
I
1
: 24-hr
! 0.63
0.79
0.73
] 0.66
: 1.4
! I-4
• 0.83
' 0.98
1
: 1.1
0.95
1.1
, 1.0
i 4.3
4.3
LC5Q
(mg/Z.)
48-hr
0.63
0.7;
0.66
0.66
0.94
0.94
0.66
0.66
0.95
0.95
1.1
1.0
4.3
4.3
?robit analysis
96-hr 96-hr
(mg/Z.) (ag/Z.)
95X
confidence G (Heterogenicy
96-hr LC50 limits factor)
0.61 4.0 0.05-8.8 0.87
0.68
0.60 5.0 4.0-6.2 0.14
0.51
0.72 5.4 5.0-5.8 0.23
0.79
0.61
0.66 5.9 4.5-7.7 0.22
0.85
0.88
1.1
1.0
4.3
95%
confidence G(Heterogenity
LC50 limits factor)
0.68 0.59-0.79 0 15
0.57 0.51-0.64 0.17
0.76 0.70-0.85 0.14
0.61 0.54-0.74 0..26
4.3
aDilution water - hardness ' 316 mg/'Z. as CaC03; alkalinity = 214 mg/Z. as CaC03; pH = 8.2; test temperature = 19 "C.
^Static bioassay - 96-hr LC50 = 19 mg/Z. (nominal), 1.3 mg/Z. (dissolved).
-------
TABLE 37. COPPER CONCENTRATIONS (IN MILLIGRAMS PER LITER) IN EXPOSURE
CHAMBER FOR TESTS REPORTED IN TABLE 36a
Nominal
concentration
Control
0.125
0.250
0.50
1.0
2.0
4.0
Control
2.0
4.0
8.0
16.0
32.0
64.0
Control
0.5
1.0
2.0
4.0
8.0
16.0
24-hr
Total Cu
A B
0.011
0.072
0.189
0.356
0.777
1.533 1.533
3.000
C D
0.010
1.118 1.229
4.889
5.286
6.023
10.200
23.240
E F
0.011
0.367
0.962
2.370 2.261
4.611
7.P80
11.703
Sample
Dissolved Cu
A B
-
0.087
0.200
0.316
0.438
0.533
0.608
C D
-
0.411
0.600
0.672
0.980
2.128
-
E F
96 -hr
Total Cu
A B
0.010
0.090
0.216
0.416
0.829
1.680 1.680
3.400
C D
0.013
0.753 0.847
2.625
4.806
6.240
11.540
22.690
E F
; 0.010 -
0.243 - 0.451
0.333 - 1.051
0.427 - 2.500 2.375
0.530 - 4.167
0.684 8.050
1.161 11.340
Sample ,
Dissolved Cu
A B
-
0.090
0.208
0.297
0.400
0.494
0.530
C D
0.370
0.541
0.595
0.892
1.811
9.135
E F
-
0.262
0.328
0.411
0.481
0.628
1.061
aGrab samples were collected 24 and 96 hr after test began. Three diluters were used in duplicate:
A and B, C and D, E and F.
The part that passes through a 0.45-micron filter.
127
-------
Table 38 lists the LC50 values obtained from the toxicity test started on
May 8, 1972. One diluter failed to perform satisfactorily in these tests. The
creek chub was the most sensitive species, and the striped shiner was the most
resistant.
DISCUSSION
Water Quality Studies
The results of the water quality studies are discussed more fully in the
general discussion (p. 168).
Copper was much less acutely toxic in Shayler Run water than would be
predicted on the basis of hardness and alkalinity. Much of this reduced
toxicity was related to copper detoxifying materials contributed by the effluent
from the sewage treatment plant. Bioassays indicated that added phosphate
detoxifies copper. The added phosphate from the sewage treatment plant could
account for part of the reduced toxicity of copper in Shayler Run.
Even with this reduced toxicity, the acute toxicity of copper varied
greatly with the changing water quality of Shayler Run. Acute bioassays have
been and will continue to be an important tool for the establishment of water
quality and effluent criteria. Our study indicates that varying toxicity due
to varying water quality can be an important consideration in the establishment
of water quality criteria for the protection of aquatic life.
Relative Sensitivity Studies
The wide range in sensitivity or resistance of different stream species to
copper was an important consideration in the decision to use this metal as the
toxicant in the Shayler Run study. The most striking result of the relative
sensitivity studies was the high resistance of the bluegill to copper. The
96-hr LC50 (total measured copper) value for the bluegill was more than 10 times
that of any other species tested in standard water. However, this variation of
LC50 values was less than the variation of LC50 values of copper for both the
bluntnose minnow and the fathead minnow due to water quality effects on copper
toxicity. In the two tests in Shayler Run water in which the bluegill was used,
it was the most resistant species.
In the bioassays conducted with standard water, the stoneroller, creek chub,
rainbow darter, and bluntnose minnow were the most sensitive species, and their
LC50 values were similar. The 96-hr LC50 values of these species were within
the 95% confidence limits of each other. In the one toxicity test with Shayler
Run water in which the stoneroller was used it was the most sensitive species.
The relative sensitivity of the creek chub varied greatly in the two Shayler Run
water tests. Except for the bluegill, the creek chub was the most resistant
species in the bioassays conducted in May 1971; it was the most sensitive species
in the May 1972 test.
In the standard water the bluntnose minnow was more sensitive than the
fathead minnow. The 96-hr LC50 value for the bluntnose minnow was 0.34 mg/l.
(0.29-0.40), and for the two fathead minnow bioassays it was 0.44 rag/I.
(0.38-0.52) and 0.49 mg/l. (0.41-0.59). These LC50 values for the fathead minnow
are similar to the value reported by Mount (1968) for the fathead minnow tested
128
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TABLE 38. RELATIVE SENSITIVITY OF FIVE SPECIES OF FISH TO COPPER IN SHAYLER RUN WATER, MAY 8, 1972
Graphical interpolation
Species Duplicate
Creek chub A
B
Rainbow darter
Fathead minnow A
B
Orangethroat darter
Striped shiner A
B
aDilution water - hardness = 274 mg/Z.
Rf-at-lr bioassav - 96-hr LC50 = 11 me
Measured
24-hr
1.2
1.1
>4.9
6.0
6.0
9.9
6.3
6.3
as CaC03;
Cu/Z. {nomi
LC50
total
(mg/Z.
48-hr
1.2
1.1
>4.9
5.2
6.0
8.3
6.3
6.3
copper
96-hr
1.2
1.1
2.6
4.5
5.3
5.8
6.0
6.0
LC50
Measured dissolved
(mg/Z.)
24-hr 48-br
0.36 0.36
0.34 0.34
= 0.59 >0.59
0.69 0.65
0.69 0.69
1.9 0.98
0.98 0.98
0.98 0.98
alkalinity = 202 mg/Z. as CaC03; pH = 8
nal).
Probit analysis
96-hr ! 96 -hr
copper Measured total copper j Measured dissolved copper
(mg/Z.) | (mg/Z.)
957. 1 95%
confidence G (Heterogenity confidence G(Heterogenity
96-hr LC50 limits factor) LC50 limits factor)
0.36 1.1 1.0-1.3 0.27
0.34
0.48 2.8 1.7-7.0 0.44
0.62 4.1 1.3-9.3 0.73
0.67
0.70
0.81 5.0 1.3
0.81
0.34 2.8
0.50 0.44-0.62 0.37
0.61 0.55-0.68 0.12
0.69 0.59-1.2 0.39
3; test temperature = 17 °C.
-------
in Newtown Laboratory water (hardness, 200 mg/Z. as CaC03). He carried out
three flow-through tests and obtained a 96-hr value of 0.47 mg/Z. The LC50
values of copper for the bluntnose minnow in a test conducted in Shayler Run
water were always lower than those for the fathead minnow. Most of the important
species of fish in Shayler Run appeared to be at least as sensitive as the
fathead minnow.
The 96-hr LC50 of the rainbow darter was about half that of the orangethroat
darter in standard water. In four of the five tests in Shayler Run water, the
rainbow darter was the more sensitive of the two species. In the bioassays with
Shayler Run water, so much copper was precipitated on the bottom and in
suspension above the bottom that it was impossible to see the darters that stayed
on the bottom of the tank. The bottom of the tank had to be searched with a
net to see if the fish were alive or dead.
Except for the bluegill, which was always the least sensitive species, the
order of sensitivity of the species was not constant in the different dilution
waters. Part of this change in order of sensitivity probably was due to the
difficulty in the care and handling of some stream species.
The relationship of the LC50 values of the fathead minnow and the bluegill
is a good example of the difficulty in comparing acute toxicity values of
species tested in different dilution waters. Both of the species were easy to
maintain under our laboratory conditions. In all cases the LC50 value of the
bluegill was higher than that of the fathead minnow. However, because of water
quality, the ratio between the values for these two species varied greatly. In
the tests with standard water the LC50 (total measured copper) for the bluegill
was about 20 times greater than that for the fathead minnow. In Shayler Run
water (Tables 34 and 36) the LC50 value for the bluegill was only two times
greater.
Another problem encountered in this comparative sensitivity study was the
inablility to distinguish and measure the toxic form or forms of copper. As a
first approach to measure "toxic" copper, dissolved concentrations were measured
In standard water about 85% or more of the total copper was dissolved. As shown
in Tables 35 and 37, at high concentrations only about 10% of the total measured
concentration was dissolved. As the total copper concentration decreased, the
proportion of dissolved copper increased until at the lowest concentrations the
copper is about all "dissolved." As shown in both the flow-through and static
bioassays, there is less variation in LC50 values for dissolved c«pper than in
values for total copper. This suggests that insoluble copper is relatively
nontoxic.
130
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SECTION XIV
CHRONIC STUDIES
INTRODUCTION
To obtain laboratory test results for comparison with results from the
field studies at Shayler Run, chronic toxicity tests with fish were conducted at
both the Newtown Fish Toxicology Station (NFTS) and the streamside laboratory
facilities adjacent to Shayler Run. At the NFTS laboratory before the field
studies, three chronic tests with fathead minnows were conducted to determine the
effect on reproduction of exposure to copper for different lengths of time before
spawning. The three tests were performed at the same time and arranged to give
6 months, 3 months, and 0 months of exposure to copper before spawning. The
tests were carried out because in the long-term stream exposure the different
species of fish would be exposed to copper for different periods of time before
spawning. The effect on reproduction was studied because Mount (1968) and
Mount and Stephan (1969) had found that the MATC for the fathead minnow was
established on the basis of egg production. Two flow-through acute toxicity
tests were also conducted with the chronic tests so that an application factor
could be calculated.
At the streamside laboratory, chronic tests with the fathead minnow and four
resident stream species were conducted concurrently with the field studies. The
tests were performed to obtain chronic toxicity data when the fish in the
laboratory were under conditions as similar as possible to those in the stream
so that a direct comparison of the results of the laboratory and the field
exposure could be made. The tests used stream water from the control area and
from the copper-treated area and were conducted under ambient conditions. A
fry-growth and survival study was also conducted during the streamside studies
with white sucker and creek chub fry.
METHODS
Newtown Fish Toxicology Station (NFTS) Chronic Tests
The methodology and design of the tests were similar to those described by
Mount (1968). The three tests were conducted at the same time, November 1968
through October 1969. Copper was introduced immediately in the first test, 3
months later in the second, and immediately after the first spawning in the third,
giving 6 months', 3 months', and 0 months' exposure to copper before spawning.
The three exposure systems each consisted of a proportional diluter (Mount and
Brungs, 1967) delivering 1 1. of control water and 1 1. each of six copper
concentrations per cycle to duplicate exposure chambers. The exposure chambers
were all glass and measured 30 by 60 by 30 cm high and contained 30 Z. of water.
The flow rate was about 7 tank-volumes of water a day. A dilution factor of
131
-------
0.6 was used to give nominal concentrations of 100, 60, 36, 22, 13 pg/£. copper
and control water. The concentrations were arranged randomly in each row of
duplicate exposure chambers in one exposure system, and the same random
arrangement was used in the other two systems.
The dilution water and the supply systems were the same as described by
Mount (1968). Test temperature was controlled by a heater in the water-supply
headbox and was modified by room temperature. Temperature was recorded by
means of three 7-day indicating and recording thermographs, each with a probe
in one chamber of an exposure system. For the first 4 months the average
temperature was 23° C with a maximum of 26° C. During February the heater was
off for 4 days, and the temperature averaged 15° C with a minimum of 11° C.
There was an unanticipated early spawning on April 6. At that time the
temperature was lowered to 19° C to approximate more closely the planned
prespawning exposure periods to copper. The mean temperature slowly increased
to 24° C by May and averaged 24.5° C during June, July, and August, when most
of the spawning occurred. The mean temperature slowly dropped during September
and reached 20° C at the end of the test.
Six pieces of half-tile were placed in each exposure chamber for spawning
substrates. Light was provided by cool-white flourescent ceiling fixtures
controlled by two time switches. Early in December the day length was reduced
from 16 to 10 hr. The day length was increased 1 hr every month until 16 hr
were obtained in June, maintained for 2 months, and then decreased 1 hr every
month.
All test fish were reared from eggs spawned in the laboratory by the fathead
minnows obtained from the Newtown Fish Farm. Eggs were hatched, and fry were
reared for about 4 weeks before they were randomly introduced into the exposure
chambers. The fish were fed a commercial dry trout food, and live organisms in
the water supply supplemented the diet. Excess food and waste products were
siphoned from the exposure chambers as necessary. When a disease broke out, all
42 exposure chambers were given the same treatment. Potassium permanganate was
used to control protozoan infections, and tetracycline and neomycin were used to
control bacterial infections.
During the spawning season all tiles were examined in the early afternoon
for eggs. Egg handling and hatching procedures were the same as described by
Mount (1968). Hatchability was calculated as the percentage of larvae hatching
from 50 eggs after 7 days of incubation.
Routine analyses of the test water from the exposure chambers were made in
each set of duplicates every other week. Methods described by the American
Public Health Association (1965, 1971) were used to measure oxygen in all seven
chambers; hardness (EDTA) in the control chamber only; and pH, alkalinity, and
acidity in two chambers. In addition, hardness was measured in the dilution
water every weekday. The stock solution of copper for the toxicant-metering
system was made as previously described by Mount (1968). During copper dosing,
water samples for copper analysis were removed daily from each chamber and
composited for 7 days. Each set of duplicate chambers was sampled on alternate
weeks. After acidification, these samples were analyzed for total copper by
means of the atomic absorption spectrophotometric procedure described in Section
IX (p. 24 ).
132
-------
Reproduction data from the three long-term exposures were subjected to a
two-way analysis of variance using transformed values of Vx + Vx+I where x_ is
the mean number of eggs per female. The program used was "Analyses of Variance
for Factorial Design" (Dixon, 1974).
Newtown Fish Toxicology Station (NFTS) Acute Tests
The acute toxicity of copper to fathead minnows from the same source as
those used in the long-term test was determined with flow-through tests in NFTS
water. The diluter was calibrated for a 0.75 dilution factor and delivered test
concentrations to duplicate exposure chambers containing 10 fish each. Two tests
were conducted: one with 6-week-old fish that averaged 22 mm in length, the
other with 6-month-old fish that averaged 55 mm in length and 1.5 g in weight.
Streamside Chronic Tests
Procedures similar to those described for the NFTS tests were used for
chronic tests with minnows and darters, and procedures similar to those described
by Eaton (1970) were used for the chronic test with sunfish. Control water and
water from the exposure area of the stream or control water dosed with copper
in the laboratory were used for the tests. Five test systems were used for the
streamside chronic tests over the 3-month study period; as many as three species
were exposed in a test system. Roman numerals were used to designate the test
systems. The numbers were assigned consecutively over the study period, I through
V. Fathead minnows were exposed in test systems I, II, and IV; bluntnose minnows
in test systems II, III, and IV; green sunfish in test system V; and johnny and
fantail darters in test system III. Fish in test systems II through V were
exposed to copper when copper was being metered to the stream. Fathead minnows
in test system I were continuously exposed to a constant concentration of copper
in stream water so a comparison could be made with fish intermittently exposed
to copper in the other tests.
A proportional diluter (Mount and Brungs, 1967) was used in test system I
to dose stream water from the control area with a stock solution of copper to
obtain concentrations of 600, 350, 200, 120, 60, and 30 yg/Z. copper and control
stream water. In test systems II and III, modified proportional diluters were
used to dilute the nominally dosed 120 yg/Z. copper water from the exposure area
with water from the control area to give nominal concentrations of 120, 60, and
30 yg/Z. copper and control stream water. In test system IV a modified
proportional diluter was used to give concentrations of 120 and 60 yg/Z. copper
and control water, and a toxicant-metering device, patterned after McAllister
jet al. (1972), was used to give a nominal 240 yg/Z. copper concentration. For
test system V a modified proportional diluter and metering device, as described
for test system IV, was used when the test was started inside the laboratory
during the winter. A proportional mixing of continuous flows of control water
and exposure water and the continuous addition of copper solution from a Mariotte
bottle and capillary-tube metering system to control water was used when the test
was moved outside to give concentrations of 240, 120, and 60 yg/Z. copper and
control water. All test concentrations were set up in duplicate. Although
toxicant concentrations were constant in test system I, they were not continuous
or constant in test systems II through V since copper was not delivered to the
test systems when copper was not being metered to the stream.
Glass tanks, 60 by 30 by 30 cm, were used for tests inside the laboratory
with bluntnose and fathead minnows, and for the start of tests with bluntnose
133
-------
minnows and darters in test system III. Plywood tanks were used for tests
conducted outside and for the sunfish tests which required larger tanks. Plywood
tanks, 120 by 60 by 30 cm, divided into 60- by 40-cm sections by plastic screens,
were used to test the bluntnose minnow, the johnny darter, and the fantail darter
in test system III. In test system V green sunfish were tested in 240- by 60- by
45-cm plywood tanks. In an attempt to lessen territorial fighting and loss of
test fish, two partial dividers, 40- by 20- by 25-cm concrete blocks set on the
long edge and extending across the tank from one side, were placed in each green
sunfish tank. Smith (1975) suggests that there is considerable aggressive
behavior and territorial fighting in green sunfish brought from the field into
the laboratory. The sunfish tanks were also covered with nets, attached to one
side of the tanks and weighted on the other side, to keep fish from jumping out
or over to another tank. Water depth in the glass tanks was approximately 15 cm.
Water depth in the 120- by 60- by 30-cm plywood tanks for test system III was
15 cm, and the depth in the sunfish tanks for test system V was 30 cm.
The flow of water for all of the tests was between 4 and 6 tank-volumes per
day. When water flow to the tanks stopped because of clogged lines or pump
failure, the tanks were aerated. During the first year and a half the air was
turned on manually. After headboxes were installed, floats in the headboxes
attached to microswitches turned on the air when the flow of water stopped. The
test temperature followed ambient stream temperature ±1° C except in the
bluntnose chronic test system III, in which electric immersion heaters in the
headboxes and aquarium heaters in the tanks were used to maintain water
temperature at about 24° C.
Spawning substrates for the minnows and darters and egg handling and hatching
for the minnows were described by Mount (1968). Spawning substrates and egg
hatching and handling for the sunfish were similar to those described by Eaton
(1970). Fry growth chambers for sunfish were 30- by 15- by 30-cm glass tanks with
screened ends placed in the sunfish tanks. Six spawning substrates were placed
in each minnow and darter chamber, and three spawning substrates were placed in
each sunfish tank.
The photoperiod for all tests, except for those in test systems I and IV, was
the natural photoperiod for the season. Translucent green fiber-glass panels in
the roof of the laboratory allowed passage of light from the outside. Cool-white
flourescent lights, turned on after sunrise and off before sunset by a timer,
also provided light to test systems in the laboratory. Test system I was enclosed
in opaque black plastic and had incandescent lights turned on by a timer to
approximate the natural photoperiod pattern of southern Ohio. The bluntnose test
in system IV, after 16 hr of daylight was reached naturally, was held at a 16-hr
photoperiod by setting the timer for the laboratory lights to extend the
spawning period for the bluntnose minnow.
Fish used for the chronic tests were seined or trapped from Shayler Run
except for fathead minnows, which were obtained from the Newtown Fish Farm.
Bluntnose minnows exposed in test system IV in one set of the nominal 120 yg/£.
copper duplicates were from the exposure area of the stream. Green sunfish
exposed in test system V in the two high nominal concentrations of 120 and 240
yg/Z. copper were also from the exposure area. These fish were collected from
the exposure area to determine the effect of prior and long-term exposure on
their response in the tests. The green sunfish had been potentially exposed to
copper in the exposure section of the stream for 2 years. The bluntnose
134
-------
minnows had potentially been exposed to copper for 6 months before the test,
and their parents had potentially been exposed to copper. The species, source,
and age of fish used in the streamside chronic tests are summarized in Table 39.
The fish were held after collection from the field until sufficient numbers
were available for testing. During holding and testing the fish were fed once
a day ad libitum with dry trout food and frozen brine shrimp, or live Daphnia
when available. The tanks were cleaned twice a week or more often if necessary.
When external parasites and bacterial or fungal infections were observed, fish
were treated. All tanks in a test system W£re treated similarly. Fungal
infections were treated with 20 mg/l. Dexon . A mixture of formalin (20 mg/l.)
and DexoiP* (20 mg/l.) was used for parasites, and a mixture of tetracycline
(20 mg/l.), neomycin (12 mg/l.), and Dexori® (20 mg/l.) was used to treat bacterial
infection. Dexort^ was included for the treatment of parasitic and bacterial
infection because secondary fungal infections usually were observed with disease
or injury, or both. Problems with fungus were probably related to the high
amount of organic material present in the water from the sewage treatment plant.
Dissolved oxygen, pH, alkalinity, hardness, and aciditiy were determined on
grab samples of dilution water 5 days a week and on one of the duplicate control
concentrations each week. Additional chemical analyses were made on other
dilution water characteristics at less frequent intervals.
Water samples for copper analysis from the chronic tests were 7-day composited
samples and were collected from one chamber of a set of duplicates on alternate
weeks. Some grab samples were also taken from chronic tests in systems IV and V.
Total copper and dissolved copper were determined weekly on chambers containing
a nominal copper concentration of 120 yg/Z.
Streamside Fry Growth and Survival
White sucker and creek chub fry were collected from Shayler Run and exposed
in the streamside laboratory in flow-through systems to either control water or
exposure water to determine the effects of copper on growth and survival. Two
water-delivery systems (Brungs and Mount, 1970) delivered control or exposure
water to duplicate sets of twelve 60- by 30- by 30-cm glass tanks. The water
depth in the tanks was 15 cm, approximately 27 I. of water. The flow of water
to the tanks was 6 tank-volumes per day. The drains of the tanks were covered
with a fine-mesh stainless steel screen to prevent loss of fry.
The fry were captured in a soft nylon mesh dip net in the control area of the
stream. They were sorted and identified, and fry of both species were
randomized into five groups of 20-30 fry, depending on the number captured. Four
of the groups were assigned to duplicate sets of control and exposure tanks. The
remaining group was anaesthetized, and the lengths were measured to the nearest
millimeter with a ruler. The average length calculated for that group was used
as the initial length of the fry for the study. At 2 weeks, 4 weeks, and 8
weeks the fry were removed from the tanks, anaesthetized with MS 222, measured,
and returned to the tanks. From the measurements an average length for each
combined duplicate was obtained, and a t-test was used to determine if there was
a significant difference between control and exposure groups of fry.
Temperature of the test water varied with the stream water temperature, and
the photoperiod was the natural photoperiod for that time of year. The fish
135
-------
TABLE 39. SOURCES OF FISH FOR STREAMSIDE CHRONIC TESTS
Test Species
Fry growth and survival Creek chub
White sucker
Chronic tests
System I Fathead minnow
System II Fathead minnow
Bluntnose minnow
System III Bluntnose minnow
1 i
CP\ Johnny darter
Fantail darter
System IV Fathead minnow
Bluntnose minnow
Control
60
120
240
120 E
System V Green sunfish
Control
60
120
240
Source Age-life stage
Control area of Shayler Run Fry
Newtown Fish Farm 5 months
Newtown, Ohio 10 months
Control area of Shayler Run Early young-of-the-year
Control area of Shayler Run Early young-of-the-year
Young-of-the-year
Young-of-the-year
Newtown Fish Farm Young-of-the— year
Control area of Shayler Run Late young-of-the— year
Exposure area of Shayler Run Early young-of-the-year
Control area of Shayler Run Adults
Exposure area of Shayler Run Adults
Size (mm)
14-21
12-13
•^
32-38
40-50
40-50
40-50
30-40
30-50
30-40
30-40
45-50
100-120
100-120
-------
were not fed during the study so that fry in the test would receive as nearly as
possible the same food as fry in the stream. Loose material was siphoned from
the tanks every week or after high stream flows when large amounts of suspended
materials settled out.
RESULTS
Newtown Fish Toxicology Station (NFTS) Tests
The results of the routine chemical analyses of water from the exposure
chambers are given in Table 40; the copper concentrations in the 7-day composited
samples are given in Table 41. Survival of fathead minnows was not affected by
continuous exposure to increasing concentrations of copper up to 100 pg/Z. In the
acute mortality tests there was 5% mortality at 160 pg/Z. and 17.5% mortality
at 280 pg/Z. Survival of the fish in the long-term tests was related to disease,
as fish died in several of the exposure chambers, but in no case was death related
to copper concentration. The deaths that occurred after the number of animals
was reduced by thinning were probably related to handling the fish.
Survival of the eggs that were spawned and incubated at the experimental
concentrations was not adversely affected by copper (Table 42). In all three
systems hatchability varied from 87% to 97%, all within the normal range of
variation found at the NFTS. Thus, the length of time the adult fish were exposed
to copper had no influence on hatchability of eggs.
The effect of copper on the fathead minnow reproduction is summarized in
Tables 43, 44, and 45. During May at a photoperiod of 14 hr of light, spawning
started in all systems. The first spawning in system III was on May 4, and
copper was introduced on May 16 before the second spawning occurred. The last
spawning was on September 22 at a photoperiod of 14 hr and mean temperature of
22° C. The test was terminated on October 23, 1969.
The reproduction data were examined by statistical analysis of variance for
factorial design. The two controlled variables were length of time of copper
exposure and copper concentration. The assumption was made that the measured
concentrations at the same nominal concentrations for the three tests were not
different. The statistical analysis indicated that the length of time of
exposure to copper had no significant effect on the number of eggs per female.
However, the effect of copper concentration was highly significant. To test
which concentrations were different from the control, the data were analyzed with
a one-sided Dunnett test. The analysis indicated that all nominal concentrations
of 36 pg/Z. (37 pg/Z., measured) and higher caused a significant reduction in
egg production (P=0.05). Nominal concentrations of 22 pg/Z. (24 pg/Z. measured)
and lower had no significant effect. In summary, the statistical analysis
indicated that the length of time the fish were exposed to copper had no effect
on reproduction; that copper concentrations of 22 pg/Z. (nominal) and lower had
no effect on reproduction; and that copper concentrations of 36 pg/Z. (nominal)
and higher had a significant effect on reproduction.
Since the effect of pre-exposure to copper was not significant, the number
of eggs per female for all six replicate chambers is summarized in Table 46. Egg
production in the control and low copper concentration was less than that in
16 pg/Z. However, there is a straight-line relationship between the mean number
of eggs per female and the log of copper concentrations between 16 and 98 pg/Z.
137
-------
TABLE 40. WEEKLY CHEMICAL ANALYSES OF THE WATER IN THE EXPOSURE CHAMBERS FOR
THE NFTS PRESPAWNING EXPOSURE CHRONIC TESTS WITH COPPER
OJ
CO
Characteristic
Total hardness
(mg/Z. as CaC03)
Alkalinity
(mg/Z. as CaC03)
Dissolved oxygen
(mg/Z.)
Acidity
(mg/Z.)
PH
(6-month
Test 1
prespawning exposure)
Number of
analyses Mean ± S.D.
18
99
340
91
98
204 ± 5.8
158 ± 8.1
7.8 ± 0.79
8.0 ± 3.4
7.9C
(3-month
Test 2
prespawning exposure)
Number of
analyses Mean ± S.D.
15
96
337
94
95
203 ±5.1
158 ± 7.3
7.8 i 0.71
8.1 ± 3.0
7.9C
(0-month
Test 3
prespawning
Number of
analyses Mean
14
97
336
93
98
204
157
7.7
8.7
7.8
exposure)
± S.D.b
± 6.1
± 7.0
± 0.72
± 2.6
c
.Additional analyses of water leading to the test systems indicated: total hardness = 202 ± 6.1 (N=236); conductivity = 470 i 50 (N=43).
S.D. - standard deviation.
CMode.
-------
TABLE 41. TOTAL COPPER CONCENTRATIONS IN WEEKLY COMPOSITE SAMPLES FROM
THE NFTS PRESPAWNING EXPOSURE TOXICITY TESTS
U)
Nominal
copper
concentration
(ug/M
Control
8
13
22
36
60
100
Test 1
(6-month prespawning exposure)
Duplicate
A
B
A
B
A
B
A
B
A
B
A
B
A
B
Number of
samples
24
24
24
23
24
23
24
23
24
24
24
24
23
23
Mean ± S.D."
{VR/I.)
4.9
4.2
12
12
16
17
25
26
38
38
57
62
97
101
± 2.9
± 1.3
i 1.6
± 1.5
± 2.0
± 1.8
± 2.3
± 3.1
± 4.8
± 2.7
± 7.4
± 4.5
± 9.3
i 6.1
Test 2
(3-month prespawning exposure)
Number of
samples
17
16
17
17
17
17
17
17
17
17
17
17
17
17
Mean ± S.D."1
(UK/Z.)
4.2
4.2
11
11
15
16
22
24
36
36
57
58
96
96
± 1.1
± 1.5
± 1.2
± 0.9
± 1.4
± 1.4
± 1.6
+ 3.3
r 2.8
± 1.7
± 4.1
i 3.3
± 7.8
± 10
Test 3
(0-month prespawning exposure)
Number of Mean ± S.D.a
samples (yg/Z..)
10
10
11
10
11
10
10
10
10
10
11
10
11
10
3.5
4.3
10
11
16
16
22
23
38
38
61
64
98
101
± 0.4
± 1.8
± 1.3
± 1.2
± 1.9
± 2.5
± 2.6
± 2.3
± 4.7
± 3.2
± 6.4
± 5.8
± 11
± 10
S.D. - standard deviation,
-------
TABLE 42. HATCHABILITY OF EGGS FROM THE NFTS PRESPAWNING EXPOSURE CHRONIC TESTS
Nominal
copper
concentration
(l^g/Z-.)
Control
8
13
22
36
60
100
Duplicate
A
B
A
B
A
B
A
B
A
B
A
B
A
B
(6-month
Number of
eggs
incubated
500
700
500
550
500
399
550
450
500
561
495
250
0
150
Test 1
prespawning
Number
hatched
456
630
460
520
468
360
531
422
474
544
453
224
0
143
exposure)
Percentage
hatched
91
90
92
95
94
90
97
94
95
97
92
90
_
95
(3-month
Number of
eggs
incubated
500
500
500
500
500
550
500
350
400
550
250
450
202
49
Test 2
prespawning
Number
hatched
450
435
468
470
465
517
482
334
371
505
234
407
196
39
exposure)
Percentage
hatched
90
87
94
94
93
94
96
95
93
92
94
90
97
90
(0-month
Number of
eggs
incubated
460
500
500
500
400
500
308
500
497
550
257
485
200
0
Test 3
prespawning
Number
hatched
419
479
450
459
375
479
285
434
437
511
241
439
177
0
exposure)
Percentage
hatched
91
96
90
92
94
96
93
87
88
93
94
91
89
-
-------
TABLE 43. NUMBER OF SPAWNS AND EGGS FROM FATHEAD MINNOWS WITH A
6-MONTH PRESPAWNING EXPOSURE TO COPPER
Nominal
copper
concentration
(vs/l.)
Control
8
13
22
36
60
100
Duplicate
A
B
A
B
A
B
A
B
A
B
A
B
A
B
Number
of
females
10
15
7
8
11
9
14
17
13
12
12
11
15
16
Number
of
males
6
5
5
4
5
6
6
4
5
7
5
8
4
5
Number
of
spawnings
49
106
65
60
100
92
52
102
45
73
34
8
1
5
Number
of
spawnings/
female
4.9
7.1
9.3
7.5
9.1
10.2
3.7
6.0
3.5
6.1
2.8
0.7
0.1
0.3
Total
eggs
produced
5,700
23,557
12,361
10,052
15,881
12,609
5,937
13,453
5,189
7,196
2,558
708
11
463
Number
of eggs/
spawning
116
222
190
168
159
137
114
132
115
99
75
88
11
93
Number
of eggs/
female
570
1,570
1,766
1,257
1,444
1,401
424
791
399
600
213
64
1
29
-------
TABLE 44. NUMBER OF SPAWNS AND EGGS FROM FATHEAD MINNOWS WITH A
3-MONTH PRESPAWNING EXPOSURE TO COPPER
Nominal
copper
concentration
(ug/i.)
Control
8
13
22
36
60
100
Oup lica te
A
B
A
B
A
B
A
B
A
B
A
B
A
B
Number
of
females
13
13
8
17
14
9
4
7
11
13
14
12
15
11
Number
of
males
7
7
6
3
5
10
6
2
10
7
6
6
4
9
Number
of
spawnings
106
33
30
75
102
59
35
31
26
56
10
25
11
2
Number
of
spawnings/
female
8
2
3
4
7
6
8
4
2
4
0
2
0
0
.2
.5
.8
.4
.3
.6
.8
.4
.4
.3
.7
.1
.7
.2
Total
eggs
produced
18
5
3
11
16
8
6
6
2
6
2
,664
,558
,318
,199
,558
,281
,255
,268
,416
,676
715
,854
544
70
Number
of eggs/
spawning
176
168
111
149
162
140
179
202
93
119
72
114
49
35
Number
of eggs/
female
1,436
428
415
659
1,183
920
1,564
895
220
513
51
238
36
6
-------
TABLE 45. NUMBER OF SPAWNS AND EGGS FROM FATHEAD MINNOWS WITH
NO PRESPAWNING EXPOSURE TO COPPER
Nominal
ropiK't
cone unLra t Ion
(MK//-.)
Control
8
13
22
36
60
100
!)up 1 icate
A
B
A
B
A
B
A
B
A
B
A
B
A
B
Number
or
1 ema 1 es
13
13
1 1
12
15
J3
1 1
13
14
12
12
16
15
12
Number
or
males
6
6
9
6
5
6
8
6
6
7
8
5
4
8
Number
oT
spawnings
99
63
54
96
84
87
29
83
52
86
1 1
37
7
0
Number
of
spawnings/
T ema 1 e
7
4
4
8
5
6
2
6
3
7
0
2
0
.6
.8
.9
.0
.6
.7
.6
.4
.7
.2
.9
.3
.5
0
Total
eggs
produced
14
8
6
12
10
13
3
12
4
8
2
,099
,930
,705
,329
,796
, 142
,855
,884
,992
,798
841
,698
866
0
Number
or eggs/
spawning
142
142
124
128
129
151
133
155
96
102
76
73
124
0
Number
or eggs/
rema 1 e
1 , 084
687
610
1,027
720
1,011
350
991
356
733
70
169
58
0
-------
TABLE 46. COMBINED EGG PRODUCTION BY FATHEAD MINNOWS IN THE SIX
CHAMBERS FOR EACH CONCENTRATION OF COPPER
Copper concentration
(tag/I.)
Nominal
Control
8
13
22
36
60
100
Mean
measured
4.2
11
16
24
37
60
98
Total number
Eggs
76,508
55,964
77,267
48,652
35,787
10,674
1,954
Males
37
33
37
35
42
38
34
Females
77
63
71
66
68
77
84
Eggs per female
Mean
number
894
888
1,088
836
526
139
23
Percentage
of control
100
99
122
94
59
16
3
144
-------
The 96-hr LC50 values and 95% confidence limits were 0.49 (0.41-0.63) rag
Cu/Z. for 6-week-old fry and 0.46 (0.39-0.54) mg Cu/Z. for the 6-month-old
subadult fish, which indicates little effect of age on toxicity.
Streamside Tests
Analytical—
No measured dissolved oxygen concentrations were below 5 mg/Z. in the test
chambers in any chronic test. Hardness of the dilution water varied from 88 to
352, 110 to 356, and 84 to 330 mg/Z., respectively, for the 3 months of the study.
Alkalinity varied from 56 to 248, 66 to 256, and 50 to 236 mg/Z0, respectively,
and pH from 7.5 to 8.5, 7.7 to 8.5, and 7.6 to 8.4, respectively. The chemical
characteristics of the control test chambers are given with the results for each
test. Some less frequently measured characteristics of the dilution water are
reported in Appendix Tables 2 to 9. The total copper concentration for each of
the chronic tests is reported in Tables 47 through 51. These values are the
average measured total copper values of the composite samples of the test
concentrations for the whole test period when the water was being dosed with
copper. The average concentrations during the spawning periods of the combined
composite samples for both duplicates are also listed. In winter and spring the
copper concentration of the tests was lower since the stream was not dosed
continuously because of high water. No attempt was made to adjust the values
for average measured copper for samples lost, improperly taken or contaminated,
or not measured. During the last 2 years of the study some of the weekly
composite samples were discarded if the test was not dosed for more than 2 days
for the week. The measured values for these samples probably would have been
low, near the backround value. Because most of the samples not measured
probably would have been low, the average copper value given in the tables
would be high. The fish in the test therefore would have been exposed to a
slightly lower average value of copper than the measured average value indicated.
The total numbers of samples not measured are reported in the tables. The
average measured total values of copper will be used in the discussion of the
results.
Fathead Minnow Chronic Test (Continuous Exposure)—Test System I--
The first fathead minnow chronic test was in test system I. Only fathead
minnows were used. They were continuously exposed to nearly constant
concentrations of copper for 9 months from January 13, 1970, to September 16,
1970 (Table 52). The temperature during the test ranged from 0° to 30° C. The
hardness ranged from 148 to 340 mg/Z., and the alkalinity ranged from 76 to 244
mg/Z., with means of 274 and 183 mg/Z., respectively. The pH ranged from 7.6
to 8.6. The average measured copper concentrations for the weekly composite samples
were 561, 316, 180, 118, 66, 33, and 6.8 yg/Z. (control) (Table 47). For 11 samples
the mean dissolved copper in streamwater dosed with 118 mg/ Z. copper was 87% of
the total (range 66 to 104). This test was similar to chronic tests with copper
and fathead minnows conducted at the NFTS, in which the fish were exposed continuously
to a constant concentration of copper except for the varying quality and temperature
of the stream water.
Some fish died during the test because of disease„ Deaths attibutable to
copper were observed in the three highest copper concentrations. These deaths
were attributed to the effect of copper because there was no evidence of
145
-------
TABLE 47. MEASURED TOTAL COPPER CONCENTRATION IN DUPLICATE TEST CHAMBERS OF
TEST SYSTEM I - FATHEAD MINNOW CHRONIC TEST3
Nominal
copper
concentration
(U8/M
Control
30
60
120
200
350
600
Duplicate
A
B
A
B
A
B
A
B
A
B
A
B
A
B
Number
of weekly
composites
analyzed
14
14
15
15
16
15
16
15
16
15
16
15
16
15
Mean measured
copper
concentration
(Mg/Z.)
7.2
6.5
31
35
64
67
120
116
180
181
313
319
557
565
Standard
deviation
2.7
1.7
2
5
6
7
8
10
14
16
20
18
33
25
Range
(Mg/Z.)
5-14
4-11
28-35
29-50
55-76
58-88
107-141
101-143
156-208
135-201
287-346
290-351
487-610
525-617
Data from Brungs et al. (1974).
-------
TABLE 48. MEASURED TOTAL COPPER CONCENTRATIONS IN DUPLICATE TEST CHAMBERS OF
TEST SYSTEM II - FATHEAD AND BLUNTNOSE MINNOW CHRONIC TEST
Species
Fathead
minnow
Bluntnose
minnow
Nominal
copper
concentration
(us/Z..)
Control
30
60
120
Control
30
60
120
Duplicate
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
Number of
weekly
composites
analyzed
2
2
3
4
3
4
3
4
2
3
3
4
3
4
3
4
Mean measured
copper
concentration
9.5
4.3
29.0
27.5
49.3
49.7
86.3
97.0
5.4
7.1
28.3
30.2
51.3
52.3
85.7
95.2
Standard
deviation
8.7
3.9
12.7
7.1
23.7
18.2
4.1
6.7
3.9
14.4
7.9
27.2
16.0
Range
(yg/Z..)
7-12
4-5
19-35
26-34
35-59
44-60
60-106
83-122
5-6
5-12
21-34
26-34
35-62
47-64
55-107
84-119
Number of
samples
discarded
1
2
0
0
0
0
0
0
1
2
0
0
0
0
0
0
-------
TABLE 49. MEASURED TOTAL COPPER CONCENTRATION IN DUPLICATE TEST CHAMBERS OF
TEST SYSTEM III - BLUNTNOSE MINNOW CHRONIC
J>
00
Nominal
copper
concentration
(ng/Z.)
Control
30
60
120
Duplicate
A
B
A
B
A
B
A
B
Number of
weekly
composites
analyzed
19
19
20
20
20
20
21
21
Mean measured
copper
concentration
(Pg/l.)
6.3
6.5
30.5
30.0
57.3
53.4
102.7
98.1
Test period
Standard
deviation
2.3
2.5
10.4
10.1
19.9
19.4
37.5
35.2
Spawning period
Range
(Ug/Z.)
4-13
4-15
6-46
7-46
13-87
10-85
11-166
14-166
Number of
samples
discarded
2
2
1
1
1
1
0
0
Number of
weekly
composites
analyzed
8
8
8
8
Mean0 measured
copper
concentration
Cvs/Z.)
7.5
32.8
58.0
107
Standard
deviation
4.1
3.7
5.7
12.9
Range
(Mg/Z.)
5-15
29-38
51-65
94-135
Average for,.combined duplicates.
-------
TABLE 50. MEASURED TOTAL COPPER CONCENTRATION IN DUPLICATE TEST CHAMBERS OF
TEST SYSTEM IV - FATHEAD AND BLUNTNOSE MINNOW CHRONIC TEST
Nomina 1
copper
concent rat ion
Species (vf.ll.)
Fathead Control
minnow
60
120
Bluntnose Control
60
120
120
240
aPeriod of 12/22/71 to 11/1/72
Perioa ct 6/18/72 to 9/6/72 fc
LAvera£e for combined duplicate
Test period"
Number of Mean measured
week] y copper
composites concentration
Duplicate
A
B
A
B
A
B
A
B
A
B
A
B
AE
BE
A
B
for both fathead
, .
analyzed
6
5
6
5
6
5
10
9
11
9
9
8
9
6
16
14
and bluntnose
(ut.ll.)
9.1
6.7
53
41
105
78
10.3
9.3
43.1
45.1
92.8
95.6
96.2
87.0
190
195
minnows .
Standard
deviation
2.7
0.8
12.8
17.6
17.1
27.7
5.5
3.1
11.3
9.3
12.3
32.4
21.7
31.1
53.7
46.2
Range
(ug/Z.)
8-13
6-8
43-78
21-60
84-131
42-108
5-24
5-14
17-57
30-57
67-104
41-135
60-142
42-11 1
90-279
97-245
Spawning period
Number of Mean1" measured
Number of weekly copper
samples composites concentration Standard Range
discarded analyzed (ug/Z.) deviation (ug/Z.)
4 5 8.8 2.7 6-13
5
4 5 59.1 11.5 46-78
5
4 5 112.5 12.8 98-131
5
6
6 See Table 57
6
7
7
9
5
6
-------
TABLE 51. MEASURED TOTAL COPPER CONCENTRATION IN DUPLICATE TEST CHAMBERS OF
TEST SYSTEM V - GREEN SUNFISH CHRONIC TEST
Test period
N'omina 1
copper
concert ra tion
(ug/Z.)
Control
60
120
240
Duplicate
A
B
A
B
A
B
A
B
N'umber of
weekly
composites
analyzed
9
7
10
7
10
7
9
7
Mean measured
copper
concentration
(pg/Z.)
7.1
9.0
50.4
45.6
97.3
84.4
166
162
Standard
deviation
2.5
2.4
15.4
13.2
28.5
30.6
55.5
58.6
Range
(ug/Z.)
5.0-7.9
6-14
18-69
29-62
37-126
47-124
47-224
70-236
Number of
samples
discarded
5
5
4
5
4
5
5
5
Number of
weekly-
composites
analyzed
4
4
4
4
Spawning period
Mean" measured
copper
concentration
(ug/Z.)
7.6
65.0
118.9
205.2
Standard
deviation
2.0
9.0
11.9
19.7
Range
(MS/Z.)
6-11
52-72
104-130
190-234
Average for conbined duplicates.
-------
TABLE 52. SUMMARY OF EXPOSURE CONDITIONS FOR STREAMSIDE CHRONIC TESTS
Chronic
test
System I
System II
System III
System IV
System V
Species
Fathead minnow
Fathead minnow
Bluntnose minnow
Bluntnose minnow
Darters
Fathead minnow
Bluntnose minnow
Green sunfish
Number
of fish
per chamber
20
20
20
25
25
20
20
11-15
Date
test
began
1-13-70
4-29-70
4-29-70
9-24-70
9-24-70
12-22-71
12-22-71
1-25-72
Date
spawning
started
5-24-70
5-24-70
5-11-70
6-1-71
no
6-18-72
7-4-72
7-14-72
Date
spawning
ended
8-24-70
7-15-70
7-15-70
8-6-71
spawning
9-6-72
1-18-73
8-12-72
Date test
terminated
9-16-70
7-15-70
7-15-70
9-3-71
9-3-71
10-1-72
1-18-73
8-30-72
Range of
temperature
during test
(° C)
0-30
11-30
11-30
0.5-30
0.5-30
0-29
0-29
0-29
Range of
temperature
during spawning
(° C)
16-30
11-30
11-30
17-30
17-30
14-29
14-29
15-29
Percentage
of exposure
time
during test
-
95
95
80
80
60
53a
56
Percentage
of exposure
time
during spawning
-
95
95
93
84K
86b
84
From 12-22-71 to 11-1-72, after 11-1-72 only the high concentration was dosed.
From 7-4-72 to 11-1-72.
-------
disease on other fish in the chamber from which the dead fish were removed and
in other chambers of the test at that time. Within the first month of exposure,
38 of 40 fish died in the 561 pg/Z. concentration, and 20 of 40 fish died in the
316 pg/Z. concentration. By the end of the test 100% of the original fish
placed in the 561 pg/Z. copper concentration had died, and all but one of a
second batch of fish placed in that concentration had also died. By the end
of the test 75% of the fish at 316 pg/Z. copper had died, and some deaths were
attributable to copper in the 180 pg/Z. copper concentration. No effect of
copper on length or weight was found at the end of the test, possibly because
of the small numbers of fish.
The first spawning in the test occurred on May 24, 1970, and the last
spawning occurred on August 24, 1970. Exposure to copper resulted in complete
blockage of spawning at concentrations of 561, 316, and 180 pg/Z. (Table 53).
Analysis of variance indicated that there was a significant difference at the
0.05 level in eggs per female among the different copper concentrations. A
sequential variant of the Q-test (Hartley, 1955) indicated that at the 0.05 level
the number of eggs per female was significantly different from the control at
118 Pg/Z. copper, but not at 66 pg/Z.
Egg hatchability was not affected at any test concentration of copper even
at 561 pg/Z., where all adult fish died. In 6-10 batches of 50 eggs, better
than 90% hatch was obtained in all concentrations of copper (6.8 (control)-llS
pg/Z,,) in which eggs were produced. Duplicate batches of control eggs
transferred to all other test concentrations had hatches of better than 90%.
In this test the most sensitive adverse effect was a decrease in number of
eggs per female, and thus the MATC was between 66 and 118 pg copper/Z.
Fathead Minnow Chronic Test (for Development of Methods)—Test System II—
The second exposure of fathead minnows in the streamside laboratory was in
test system II. Fathead minnows were exposed to copper for 2 1/2 months, April
29, 1970, to July 15, 1970. Copper was present in the test system 95% of the
time during the test (Table 52). This test was primarily a methods-development
test, because it was the first streamside test in which the copper-treated
water from the exposure section of the stream was used for obtaining the test
concentrations of copper. The nominal and measured copper concentrations are
shown in Table 48. The fish used for this test were adults 10-11 months old,
because younger fish would not have been mature enough to spawn. Temperature
during this test ranged from 11° to 30° C (Table 52) and pH from 8.1 to 8.6.
Hardness ranged from 230 to 346 mg/Z. and alkalinity from 160 to 232 mg/Z., with
means of 307 and 289 mg/Z., respectively.
There were no copper-related deaths in this test, although a few fish died
as a result of disease in all concentrations. No effect on growth was determined
because of the loss of the fish at the termination of the test before they could
be weighed, measured, or sexed.
The first fathead minnow spawning occurred on May 5, 1970, after only 26
days of exposure to copper. The fish spawned in all test concentrations until
the test was terminated. No effect of copper was apparent on total number of
eggs, number of spawns, or eggs per spawn. The average measured high
concentration for the test was 92 pg/Z. (Table 54). Egg hatchability, based on
152
-------
TABLE 53. SPAWNING AND EGG PRODUCTION BY FATHEAD MINNOWS IN CHRONIC TEST SYSTEM
Ui
LO
Mean measured
copper
concentration
(VK/1.)
7.2 (control)
6.5 (control)
31
35
64
67
120
116
180
181
313
319
557
565
Duplicate
A
B
A
B
A
B
A
B
A
B
A
B
A
B
Number of
females
during
spawning
9
9
3.5
10
9
3.2
6
10
9
3
3
1
1
0
Number
of
males
2
2
4
2
1
2
7
5
3
1
0
3
0
0
Number
of
spawnings
46
74
38
69
60
52
1
13
0
0
0
0
0
0
Spawnings
per Total Eggs per Eggs per
female eggs spawning female
5.1 7,576 162 842
8.2 13,755 186 1,528
JO. 9 5,218 137 1,491
6.9 9,533 138 953
6.7 7,632 127 848
6.3 5,125 99 621
0.2 88 1
1.3 1,578 121 158
_ _ _ _
- - -
_ _
- - -
_ _
Data from Brungs et_ al_. (1974).
These numbers have been adjusted to account for those females that died during the spawning season.
-------
TABLE 54. SPAWNING AND EGG PRODUCTION BY FATHEAD AND BLUNTNOSE
MINNOWS IN CHRONIC TEST SYSTEM II
Ln
Species
Fathead minnow
Bluntnose minnow
Nominal
copper
concentration
(Mg/Z.)
Control A
Control B
30 A
30 B
60 A
60 B
120 A
120 B
Control A
Control B
30 A
30 B
60 A
60 B
120 A
120 B
Mean measured
copper
concentration
(yg/l.)
9.5
4.3
29.0
27.5
49.3
49.7
86.3
97.0
5.4
7.1
28.3
30.2
51.3
52.3
85.7
95.2
Number of
spawnings
34
30
50
12
38
51
4
38
21
5
8
36
37
17
15
10
Total
eggs
6,912
5,072
7,806
2,538
6,018
9,051
349
4,113
2,212
493
536
3,018
4,289
1,731
1,231
611
Eggs per
spawning
203
169
156
211
158
177
87
108
105
98
67
85
115
101
82
61
-------
4^10 batches of 50 eggs per concentration, was high for all concentrations.
Ninety-one per cent of all eggs hatched, and the lowest average hatch for any
copper concentration was not less than 87%.
Fathead Minnow Chronic Test (Intermittent Exposure)—Test System IV--
The third exposure with fathead minnows was in test system IV. The test
was started December 2, 1971, and ended October 1, 1972. The temperature during
the test ranged from 0° to 29° C and the PH from 8.0 to 8.2. Hardness ranged
from 156 to 300 mg/Z-., and alkalinity ranged from 120 to 230 mg/Z., with means
of 258 and 179 mg/Z.., respectively. The mean measured copper concentrations for
the entire test were 91, 47, and 8.0 yg/Z. (Table 50). The test fish were exposed
to copper 60% of the time during the entire test and 84% of the time during
spawning (Table 52).
No deaths attributable to copper occurred during the test, although some
fish died early in the test because of disease. Copper had no effect on growth
as measured by the size and weight of the fish at the end of the test.
The first spawning of the fathead minnow occurred on June 18, 1972 and the
last on September 6, 1974. The fish spawned in all test concentrations. There
was, however, a significant reduction at the 0.05 level in eggs per female at
112.5 yg/Z-. copper, the average concentration during spawning (Table 55). Based
on spawning results, the safe concentration for the fathead minnow in this test
would have been between 112.5 and 59.1 yg/Z-., the average measured copper
concentration during spawning (Table 55).
Bluntnose Minnow Chronic Test (for Development of Methods)—Test System II
Bluntnose minnows were exposed for the first time in test system II. This
was a methods-development test, as discussed earlier for the fathead minnow in
test system II. The exposure conditions and water chemistries for this test
were the same as described for the fathead minnow test in system II.
There were no copper-related deaths in this test, although some fish died
as a result of disease in all concentrations. No effect on growth could be
measured because of the loss of the fish at the termination of the test before
they were weighed, measured, or sexed.
The first spawning occurred on May 11, 1970, after only 13 days of exposure
to copper. Fish spawned in all concentrations until the test was terminated.
No effect was apparent on total eggs, number of spawns, or eggs per spawn. The
average measured high concentration of copper for the test was 90 yg/Z-. (Table
54).
Egg hatchability based on 5-11 batches of eggs per concentration averaged
86% for all eggs hatched, and the lowest average hatch for any concentration
was 82%. Success of hatch was not related to copper concentration.
Bluntnose Minnow Chronic Test (Intermittent Exposure)—Test System III
The second exposure of bluntnose minnows to copper was in test system III,
beginning September 24, 1970, and ending September 3, 1971. The test was
conducted outside during the spawning period although the fish were initially
155
-------
TABLE 55. SPAWNING AND EGG PRODUCTION BY FATHEAD MINNOWS IN CHRONIC TEST SYSTEM IV
Nominal
copper
concentration
(ug/Z.)
Control A
Control B
60 A
60 B
120 A
120 B
Mean measured
copper
concentration
(ug/Z.)
9.1
6.7
53
41
105
78
Mean measured
copper
concentration
during spawning
(ug/Z.)
8.8
59.1
112.5
Number
of
males
1
1
9
6
2
4
Number of
females
during
spawning
8
7
6
11
6
10
Number
of
spawnings
47
54
35
53
21
24
Spawnings
per
f ema le
5.8
7.7
5.8
8.0
3.5
2.4
Total
eggs
13,714
13,927
5,971
10,271
2,774
4,352
Eggs per
spawning
291
257
170
193
132
181
Eggs per
female
1,714
1,989
995
1,711
462
435
Average for combined duplicates.
-------
exposed inside the streamside laboratory during the winter. The fish were
young-of-the-year from the early (May-June 1970) spawn of the year. The fish
were exposed to copper 80% of the time during this test (Table 52). The
temperature range during the test was 0.5° to 30° C, and the pH range was 8.0, to
8.3. Hardness ranged from 220 to 324 mg/Z., and alkalinity ranged from 156 to
240 mg/Z., with means of 272 and 189 mg/Z., respectively. The measured copper
concentrations were 100, 55, 30, and 6.4 yg/Z. (control) (Table 49).
Copper had no effect on growth or death of the test fish. Very few deaths
occurred among the bluntnose minnows in this test compared to the number of fish
deaths in the other chronic tests.
The fish spawned in all test concentrations of copper. The first spawn was
June 1, 1971, and the final spawn was August 8, 1971. During spawning the fish
were exposed to copper 93% of the time (Table 52), with an average high copper
concentration of 107 yg/Z. (Table 56). Based on spawning, the MATC was above
107 yg/Z.
Bluntnose Minnow Chronic Test (Extended Spawning)— Test System IV--
Bluntnose minnows were exposed for the third time in test system IV. The
test started December 22, 1971, and ended January 18, 1973. During the test the
temperature ranged from 0° to 29° C and the pH from 7.8 to 8.3. Hardness ranged
from 156 to 328 mg/Z. and alkalinity ranged from 102 to 230 mg/Z., with means of
255 and 173 mg/Z., respectively. To November 1, 1972, the average measured copper
concentrations were 193, 91, 94, 44, and 10 yg/Z. (Table 50), and the fish were
exposed to copper 53% of the time during that period (Table 52). After November
the copper concentrations of 94 yg/Z. and below received only control water, while
the 193 yg/Z. concentration was dosed intermittently to determine if spawning
could be blocked after it had started.
Deaths attributable to copper occurred among the bluntnose minnows in this
test. All 20 fish in each duplicate, at the 193 yg/Z. concentraton, died within
70 days of the beginning of the test. Of the 15 additional fish added to each
duplicate tank, two died in one tank at 193 yg/Z. copper and three died in the
other before the end of the test. These deaths also were apparently due to copper.
The range of the measured copper values during the entire test was 78 to 279 yg/Z.
copper for weekly composite samples. A single grab sample on June 12, 1972, gave
a value of 320 yg/Z. copper.
Because the bluntnose minnows in most concentrations did not start spawning
well during the normal spawning period, the test was extended after September by
maintaining spawning temperatures and photoperiod (Table 52). On November 1,
1972, treatment of the stream with copper was stopped. Since the test system
used copper-treated water from the exposure area of the stream to obtain
concentrations of 94 yg/Z. and below, exposure of fish at those concentrations
was also stopped. Exposure of fish to 193 yg/Z. was also stopped, although the
toxicant-delivery system used for that concentration depended only on the flow
of control water. Eleven days after copper exposure stopped, the bluntnose
minnows in one tank of the 193 yg/Z. concentration (218 yg/Z. average copper
concentration during the July 14 to November 1 spawning period) started to spawn,
and 30 days later the fish in the other tank began to spawn. The spawning
continued in both tanks until December 21 when copper was again introduced.
Spawning stopped in the 193 yg/Z. chambers the day after copper introduction and
157
-------
00
TABLE 56. SPAWNING AND EGG PRODUCTION BY BLUNTNOSE MINNOWS IN CHRONIC TEST SYSTEM III
Species
Bluntnoso.
minnow
Nominal
copper
c one e.n t r a t io n
(Mg/Z.)
Control A
Control. B
30 A
30 B
60 A
60 B
120 A
120 B
Moan measured
copper
concent ration
(ug/Z.)
6.3
6.5
30.5
30.0
57.3
53.4
102.7
98.1
Mean measured
copper
concentration
during spawning
(ug/Z.)
7.5
32.8
58 -
107
Number
of
males
9
7
4
5
6
11
12
6
Number of
females
during
spawning
9
16
15
19
17
10
12
17
Number
of
spawnings
6
22
29
26
12
28
3
26
LSpawnings
per
female
1.5
1.3
1.9
1.4
0.7
2.8
0.2
1.3
Total
eggs
405
1,283
1,988
2,111
1,100
1,801
62
1,834
Eggs per
soawning
67
58
68
81
91
64
20
70
Eggs per
female
45
80
133
111
64
180
5
107
Average for combined duplicates.
-------
did not begin again before January 18, 1973, when the test was terminated (Table
57). The average measured copper concentration during the period, December 1 to
January 18, was 130 yg/Z,., and the range was from 78 to 195 yg/Z. The fish in
other concentrations, exposed only to untreated control water, continued to
spawn. On the basis of spawning and survival, the safe concentration of copper
in this test for bluntnose minnows was between 113 and 193 yg/Z. since the fish
spawned at an average copper concentration of 113 yg/Z., and spawning could
apparently be blocked at 193 yg/Z. at which concentration death had also occurred
early in the exposure.
In one tank at 91 yg/Z. copper concentration (113 yg/Z. average copper
concentration during July 14-November 1 spawning period), the fish from the exposure
area had been spawning since July 14, 1972 (Tables 50 and 57). Analysis of variance
for the eggs-per-female data showed no significant differences among concentrations in
any spawning period. Thus, the performance of the bluntnose minnows from the
exposure area in the one set of duplicates at 91 yg/ I. copper was not statistically
different from the fish from the control area used for the set of duplicates at
94 yg/Z. copper and other test concentrations. There was, however, poor
duplication at all concentrations. The fish from the exposure area appeared to
have spawned better. If they did spawn better, however, it was probably because
they were larger when the test started, had matured earlier, and had started
spawning earlier than the bluntnose minnows from the control area rather than
because if any increase in resistance owing to previous exposure to copper or the
exposure of their parents.
Green Sunfish Chronic Test—Test System V
Green sunfish were exposed to copper at the streamside laboratory only in
test system V and in a preliminary spawning study. In the system V test they
were exposed from January 25, 1972, to August 30, 1972 (Table 52). The temperature
range during the test was 0-29° C, and the pH ranged from 8.0 to 8.3. Hardness
ranged from 154 to 325 mg/Z. and alkalinity from 98 to 236 yg/Z- with means of
264 and 180 mg/Z., respectively. The measured average copper concentrations were
164, 91, 48, and 8.0 yg/Z. (Table 51). Copper was added only 56% of the time
during the entire test period and 84% of the time during spawning.
No deaths were attributable to copper during the test, but may fish died as
a result of secondary bacterial and fungus infection of wounds suffered during
territorial fighting. All green sunfish in one control had died before spawning
had started.
No conclusion on the effect of copper on growth of green sunfish was reached
because of the limited number of fish left at the end of the test.
Spawning was poor in the 91 yg/Z. copper concentration, (119 yg/Z. average
spawning concentration, Table 58, spawning probably was not related to copper
concentration. Spawning in the 164 yg/Z. copper concentration (205 yg/Z. average
spawning concentration) apparently was not different from the control or the 47
yg/Z. copper concentration. Since fish from the control area were used in the low
concentrations of this test and fish from the exposure area in the high concentrations,
the test was different from the other chronic tests. Apparently spawning was not
affected in green sunfish that were exposed to copper in the exposure area of the
stream for 2 years and were transferred to the same or a higher concentration.
159
-------
TABLE 57. SPAWNING AND EGG PRODUCTION BY BLUNTNOSE MINNOWS IN
CHRONIC TEST SYSTEM IV
Nominal
copper
concentration
(us/Z.)
Control A
Control B
60 A
60 B
120 A
120 B
120 AE
120 BE
240 A
240 B
Percentage of Mean measured
time of copper Number
during test (pg/Z.) males
Spawning period
86 8.9 6
2
86 59 8
5
86 113 7
8
86 113 1
4
86 218 4
7
Number
of
females
7-14-1972
4
8
8
13
9
6
6
4
9
5
Spawning period 11-1-1972 to
Control A
Control B
60 A
60 B
120 A
120 B
120 AE
120 BE
240 A
240 B
0 NMb 6
2
0 8
5
0 7
8
0 1
4
0 4
7
4
8
8
13
9
6
6
4
9
5
Spawning period 12-21-1972 to
Control A
Control B
60 A
60 B
120 A
120 B
120 AE
120 BE
240 A
240 3
Control A
Control B
60 A
60 B
120 A
120 3
120 AE
120 BE
240 A
240 B
Average value
0 9.5C 6
2
0 8
5
0 7
8
0 1
4
80 130 it
7
Entire spawning
53 --10 6
2
53 XiO 8
5
53 ^60 7
8
53 --65 1
4
53 -VL26 4
7
for combined duplicates.
4
8
8
13
9
6
6
4
9
5
Number
of
spawnings
Spawnings
per
females
Total
eggs
Eggs
per
spawning
Eggs
per
female
to 11-1-1972 (copper)
3
2
1
0
7
1
8
2
0
0
0.8
0.3
0.1
0
0.8
0.2
1.3
0.5
0
0
11
167
63
0
357
7
510
226
0
0
4
4
63
0
51
7
64
113
0
0
3
21
8
0
40
1
85
57
0
0
12-21-1972 (no copper)
0
6
0
6
4
2
10
1
11
7
1-18-1973 (copper
3
3
3
7
1
6
12
5
0
0
0
0.8
0
0.5
0 4
0.3
1.7
0.3
1.2
1.4
240 vt.ll.)
0.8
0.4
0.4
0.5
0.1
1.0
2.0
1.3
0
0
0
541
0
499
61
52
1,356
31
1,041
300
195
334
32
708
22
392
1,915|
452
0
0
0
90
0
83
15
26
136
31
95
43
65
111
11
101
22
65
160
90
0
0
0
68
0
38
7
9
226
8
116
60
49
12
4
55
2
65
319
113
0
0
period, 7-14-1972 to 1-18-1973
^
8
8
13
9
6
6
4
9
5
6
11
4
13
12
9
30
8
11
7
1.5
1.4
0.5
1
1.3
1.5
5.0
2.0
1.2
1.4
206
1,042
95
1,207
440
451
3,778
709
1,041
300
34
95
24
93
36
50
126
89
95
43
51
131
12
92
49
75
629
178
116
60
Not measured - assumed to be backround.
Value for combined duplicate controls A and B.
160
-------
TABLE 58. SPAWNING AND EGG PRODUCTION BY GREEN SUNFISH IN CHRONIC TEST SYSTEM V
Nominal
copper
concentration
(Mg/Z.)
Control A
Control B
60 A
60 B
120 A
120 B
240 A
240 B
Mean measured
copper
concentration
(pg/Z.)
7.1
50.4
45.6
97.3
84.4
166
162
Mean measured
copper
concentration
during
spawning
(yg/Z.)
7
All fish
65
118
205
.6
died
.0
.9
.2
Number
of
males
2
7
7
5
4
5
6
Number of
females
during
spawning
2
2
3
5
5
5
7
Number
of
spawnings
2
2
3
1
0
9
2
Spawnings
per
female
1
1
1
0.2
0
1.8
0.3
Total
eggs
11,548
3,923
7,215
996
0
33,972
6,431
Egg
;s per
spawning
5,
1,
2,
3,
3,
774
961
405
996
0
774
215
Eggs per
female
5,774
1,961
2,405
199
0
6,794
918
Average for combined duplicates.
-------
In a preliminary spawning test with sunfish the previous year, longear sunfish
and green sunfish were taken from the exposure area and exposed to copper at a
nominal concentration of 120 yg/Z. in test tanks in the laboratory for 2 weeks
before spawning. The longear sunfish spawned an average of 1,377 and 1,315 eggs
per.female in duplicate chambers containing seven females in each chamber. In
that test the green sunfish, treated in the same way as the longear sunfish,
failed to spawn. The green sunfish were in 240- by 30- by 30-cm tanks in this
preliminary test. Territorial disputes and overcrowding in the smaller tanks
were assumed to be the reasons for the lack of green sunfish spawning in the
preliminary study.
Egg hatchability, fry growth, and survival studies were attempted with the
green sunfish eggs and fry from the chronic test in system V. However, low
hatchabiblity, because of fungus infection, and poor fry survival in all test
concentrations precluded any conclusion relating to the effect of copper on these
life stages.
Johnny and Fantail Darter Chronic Tests—Test System III—
The test conditions, copper concentrations, and water quality characteristics
for johnny darters and fantail darters were the same as those described for the
bluntnose minnow in test system III (Tables 49 and 52). The deaths that occurred
in both species throughout the test were the result of disease and were unrelated
to copper. Neither species spawned in any copper concentration, possibly because
of overcrowding or other unacceptable test conditions. Analyses of fish lengths
and weights at the end of the test showed no effect on growth related to copper
concentration. Thus, copper had no effect on the growth and survival of the
darters at a concentration of 107 yg/Z.
Fry Growth and Survival in Streamside Tests —
The white sucker test was run for 2 months. After 4 weeks the same number of
fry, 33, were alive in control and exposure tanks. The test was started with a
total of 40 fry for each treatment. The average length of fry was 20.2 mm for
the combined control duplicates and 16.4 mm for the combined exposure duplicates.
The 3.8-mm difference was significant at the 0.05 level. After 8 weeks, survival
was so low in both sets of tanks that the fish were not measured.
The creek chub test was run for only 4 weeks and terminated because of low
survival in both control and exposure concentrations. At 2 weeks, however, all
40 fry were alive in both control and exposure concentrations, and there was a
significant difference (P=0.05) in length. The length of fry was 20.7 mm for the
combined control duplicates and 18.6 mm for the exposure duplicates.
DISCUSSION
Newtown Fish Toxicology Station (NFTS) Tests
The length of time that fathead minnows were exposed to copper before spawning
had no influence on the effect of copper on egg production. Apparently the
reduction in egg production due to copper is the result of exposure of the sexually
mature fish during the spawning season, i.e., only the spawning season is the
critical time of copper exposure.
162
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The results of this chronic study were in excellent agreement with those
reported by Mount (1968). In both studies the fathead minnow was exposed to copper
in a standard (200 mg/Zc, hardness) water at the NFTS laboratory. Mount found that
a copper concentration of 33 yg/Z. completely blocked spawning and that a copper
concentration of 14.5 yg/Z. had no effect on spawning. In the present study a
measured copper concentration of 37 yg/Z. significantly (P=0.05) reduced egg
production, and the effect of a copper concentration of 12 yg/Z. was not
significantly (P=0.05) different from the control. The intermediate concentration
of 24 yg/Z. in this test also was not significantly (P=0.05) different from the
controls.
The 96-hr LC50 values for the acute tests in this study were also in
excellent agreement with the value reported by Mount (1968). The 96-hr LC50
value was 460 yg/Z. copper for subadult fish and 490 yg/Z. copper for 6-week-old
fry. The mean 96-hr LC50 reported by Mount for three tests with adult minnows
was 470 yg/Z. copper. Thus the application factor that lies between 0.05 and
0.08, based upon a MATC value between 37 and 24 yg/Z. and a continuous-flow 96-hr
LC50 value of 460 yg/Z. found in this study, was in excellent agreement with the
application factor reported by Mount (1968) that lies between 0.03 and 0.08,
based upon a MATC value between 33.0 and 14.5 yg/Z. and a continuous-flow 96-hr
LC50 value of 470 yg/Z.
Although the "unsafe" copper concentration was similar in the two studies,
Mount (1968) found no spawning in 33 yg/Z. whereas in the present study only
41% reduction in eggs per female in 37 yg/Z. was found. The difference may be
due in part to the use of many more females in the present study. In chronic
studies of this design, production of the individual female (except for zero eggs
per female) cannot be quantified because only the mean of eggs per female per
exposure chamber is determined. Mount (1968) exposed a total of eight females in
the duplicate chambers, whereas 68 females were exposed in six exposure chambers
in this study. Perhaps the large sample size was responsible for the graded
effect, and the small samples size gave an all-or-none response.
Although the MATC value for copper was in agreement with the value reported
by Mount, analyses of the results of the study were similar to Mount's
because the data for all three exposures could be combined—six exposure chambers
per concentration. If the analyses of variance had shown the results for the
three exposures to copper to have been different, the data from each would have
been analyzed separately. In that case the MATC values for the individual
exposures would have been greater than the MATC found by Mount. Thus, with only
a duplicate the ability of the test to detect statistically significant
differences was lessened. The MATC values for the individual chronic tests were
between 38 and 59 yg/Z. copper, 36 and 58 yg/Z. copper, and 38 and 62 yg/Z. copper
for the 6-month, 3-month, and 0-month pre-spawning exposures, respectively.
These values are about two times higher than the MATC value when the data for the
three tests are combined.
Streamside Tests
The effects of copper on survival, growth, and reproduction of fathead and
bluntnose minnows, green sunfish, and two species of darters as determined in
the streamside chronic tests are summarized in Table 59. In general, copper had
no effect on growth or egg hatchability. Effects on reproduction were the most
sensitive indicator of toxic effects of copper studied in the chronic tests.
163
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TABLE 59. SUMMARY OF STREAMSIDE CHRONIC TEST DATA
Chronic
test
system Species
Measured
copper
concentration
Mortality (pg/I.)
Measured
copper
concentration
Growth (pg/£.)
Measured
copper
concentration
Spawning (ug/7,.)
Measured
copper
Eggs per concentration
female (iig/l.)
Measured
copper
Egg concentration
hatchability (jjg/Z.)
I Fathead minnow
II Fathead minnow
Bluntnose minnow
III Bluntnose minnow
Darters
IV Fathead minnow
Bluntnose minnow
100%
25%
Slight
No effect
No effect
No effect
No effect
No effect
100%
561
316
181
92
91
100
100
91
193
Insufficient data
No data
(specimens lost)
No
No effect
No effect
No effect
No effect
data
100
100
100
193
Blocked
No effect
No effect
No effect
Did not
No effect
Blocked
561
316
181
92
91
107
spawn
113
218
Reduced 118
No data
(specimens lost)
No data
No effect 107
Reduced 113
No effect 113
No effect 561
No effect
No effect
92
91
Not done
Not done
Not done
Green sunfish
No effect 164
Insufficient data No effect 205
No effect
205
Insufficient data
-------
Higher concentrations killed fish. A possible effect on fry growth at
exposure-water concentrations of copper was indicated in the tests on fry growth
and survival.
The statistical analysis of eggs-per-female data showed that in the fathead
minnow chronic test in system I at 65 yg/Z. copper, and in the fathead minnow
chronic test in system IV at 59 yg/Z. copper (the average copper concentration
during spawning), egg production of the fathead minnows was not different from
the control. At 118 yg/Z. copper in the test in system I and at 112 yg/Z. in the
test in system IV, egg production was significantly different (P=0.05). The
close agreement of these values indicates that there is little difference
between the effect of continuous exposure to copper, as in test system I at an
average concentration of 118 yg/Z. copper, and the effect of intermittent
exposure to copper at an average concentration of 112 yg/Z. copper during
spawning, as in test system IV. These results are also in general agreement with
the laboratory fathead minnow study on the effects of different lengths of
exposure to copper before spawning, which showed that the effect of copper is an
acute effect on egg production during spawning. The fathead minnows in test
system IV were exposed to copper less than 60% of the time before spawning.
Although results of streamside fathead minnow chronic tests in test systems
I and IV indicate that the number of eggs per female was reduced between 66 and
118 yg/Z. copper and 59 and 112 yg/Z., respectively, the results of bluntnose
minnow tests in systems III and IV indicate that reproduction of the bluntnose
minnow was not reduced between copper concentrations of 58 and 107 yg/Z. and
59 and 113 yg/Z., respectively. These results contrast with the results of acute
mortality tests with both stream and laboratory water, in which bluntnose minnows
were more sensitive to copper than fathead minnows. It also contrasts with
results of the chronic tests in which the extended mortality was greater for the
bluntnose minnows. In the bluntnose minnow chronic test in system IV; at 193
yg/Z. average copper concentration, all of the bluntnose minnows died within 70
days. In the fathead minnow test in system I, only a few fish died at 181 yg/Z.
copper. The results also disagree with those of a just-completed bluntnose
minnow chronic toxicity test with copper in the NFTS laboratory that appears to
show a graded response to increasing copper concentrations and an effect on eggs
per female in the same range of copper concentrations as that found for the
fathead minnow in the laboratory. The apparent reversal of the order of sensitivity
in the streamside chronic tests in relation to the effect of copper on
reproduction possibly results from a difference in the ability to detect a change
in reproduction in the two species with the design of the test. A bluntnose
minnow spawn averages about half the number of eggs in a fathead minnow spawn,
and the number of eggs per spawn appears to be more variable. Thus a difference
in number of eggs would be more difficult to detect in bluntnose minnows than
in fathead minnows. Also, the spawning conditions in the streamside tests may
not have been optimum for bluntnose minnow spawning.
In the streamside chronic tests green sunfish were more resistant than
bluntnose and fathead minnows. Green sunfish spawned at an exposure concentration
of 164 yg/Z. copper and an average 205 yg/Z. copper concentration during
spawning. This order of sensitivity agrees with that found in acute mortality
tests with sunfish and minnows in NFTS standard (200 mg/Z. hardness) water and
stream water.
In the streamside chronic tests apparently there was a seasonal difference
in mortality related to water quality. Most of the deaths attributable to
165
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copper in the fathead minnow chronic test in system I and the bluntnose minnow
chronic test in system IV occurred near a copper concentration of 190 yg/Z. or
higher and during winter and early spring when acute static tests generally
indicated increased copper toxicity. This may have been a result of the initial
loss of the most sensitive individuals in the test population. However, in the
bluntnose minnow chronic test in system IV, only 5 of the 30 bluntnose minnows
in the second batch died when placed in the high concentration of copper, 193
yg/Z., as compared to the death of all 40 in the initial batch. The presence
of smaller amounts of detoxifying materials, which decrease the toxicity of copper,
at this time would explain the difference in survival of the two batches of fish.
No acute tests, however, were run concurrently with the bluntnose minnow chronic
test in system IV.
The MATC values for copper in the streamside fathead minnow chronic tests
systems I and IV were between 65 and 117 yg/Z. copper and between 59 and 112 yg/Z.
copper, respectively. These MATC's were different from the MATC of between 24
and 37 yg/Z. found in this study and the MATC of between 14.5 and 33 yg/Z. copper
found by Mount (1968) in constant-quality NFTS standard (200 mg/Z. hardness) test
water and the MATC value of between 11 and 18 yg/Z. copper in constant-quality
(30 rag/I. hardness) water determined by Mount and Stephan (1969). In fathead
minnow streamside chronic tests in test systems I and IV, the average hardness was
274 and 272 mg/l., respectively. The change in hardness from 200 to 270 mg/Z.
probably did not raise the MATC by a factor of four for the streamside tests. The
higher values for the streamside chronic tests probably represented detoxification
of copper by a complexing or preciptiating agent in the stream water. As
suggested by the affects of water quality on the acute tests, some detoxifying
agent from the sewage treatment plant may have been responsible for the
detoxification of the copper.
The average of the eight lowest total copper LC50 values (arbitrarily one-third
of the total number of values), from the acute toxicity tests w^ith -fathead minnows in
stream water was 1.2 mg/Z. copper. The application factor ranges, calculated from
the 96-hr static LC50 value of 1.2 mg/l. copper and the MATC values for fathead
minnows in streamside chronic tests in test systems I and IV, were 0.05 to 0.10
and 0.05 to 0.09, respectively. These are in good agreement with the values
found by Mount (1968) and those found in this study for constant-quality NFTS
standard (200 mg/Z. hardness) water.
The application factor ranges of 0.05 to 0.10 and 0.05 to 0.09 for the
continuous exposure of fathead minnows to copper in varying-quality water in test
system I and for the intermittent exposure of fathead minnows to copper in
varying-quality water in test system IV are also within the overall range of
application factors of 0.02 to 0.24 found with fathead minnows, brook trout, and
bluegills in continuous laboratory exposures in different constant-quality soft
waters (Mount and Stephan, 1969; McKim and Benoit, 1971; Benoit, 1975).
166
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PART D — GENERAL DISCUSSION
SECTION XV
GENERAL DISCUSSION
The field study indicated that the fish in Shayler Run, except for the
orangethroat darter, were adversely affected in some manner by the addition of
120yg/Z-. of copper to the stream. Death, avoidance, and restricted spawning
were the direct effects found. Fish deaths were observed only during a 1-week
period beginning 7 days after initial introduction of copper, when a total of
62 fish (7 species) were observed dead. The number of fish killed was probably
greater than the number recorded, however, because crayfish were observed eating
dead fish. These early deaths were probably the result of sudden stress on the
populations, which killed the most sensitive individuals.
Within the first 48 hr after initial addition of copper, bluntnose minnows
and, to a lesser degree, stonerollers responded by moving downstream out of the
exposure area. During the week after the initial copper introduction, streamside
observations also indicated that an avoidance reaction to copper occurred with
some species of fish. Large numbers, mainly striped shiners, stonerollers,
bluntnose minnows, and a few sunfish and darters, were found congregating in
areas where the copper concentration was less than 100 yg/Z-. as a result of
the influx of spring or tributary water. As the stream warmed in the spring,
bluntnose minnows, stonerollers, striped shiners, rainbow darters, and fantail
darters began a mass exodus from the exposure area onto the downstream screens.
This coincided with the onset of sexual maturation and spawning activity
for the latter four species.
Copper adversely affected almost every common species of fish that spawned
in the exposure or recovery areas by restricting the areas in which they could
spawn. The single exception was the orangethroat darter, which spawned
throughout both the control and exposure areas during the three seasons that
copper was added to Shayler Run. During the exposure period bluntnose minnows
spawned only in the lower exposure area where the total copper concentration
averaged 60 yg/Z- and ranged from 35 to 77 pg/Z-., even though they had spawned
throughout the exposure area before the introduction of copper. Green sunfish
and longear sunfish spawned throughout the exposure area during the spawning
period before the introduction of copper, but in the succeeding 3 years these
fish were limited to spawning areas where the copper concentration was 90 yg/Z-.
or less. An additional indication of apparent reproductive impairment by copper
was a sevenfold reduction in the number of fry in the exposure area.
The macroinvertebrate populations in the exposure area were generally
reduced by copper. Four of the five most abundant forms, scuds, sowbugs,
mayflies, and riffle beetles, were essentially eliminated. Chironomids were the one
group that was not adversely affected; they flourished in the exposure area.
Even though a dietary shift was evidenced by the green sunfish and the
orangethroat darter, indirect effects on the fish populations through effects
on the aquatic food chain could not be demonstrated.
167
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Results of the field studies were consistent from year to year, considering
the variations in stream flow, the varying amounts of time that copper was
actually being added to the stream, and other unmeasured factors. Each year
the fish populations generally declined, the macroinvertebrate populations were
reduced or essentially eliminated, and the fish spawning was generally eliminated
or reduced in the exposure area throughout the exposure period. Each of the
various types of fish and macroinvertebrate collections complemented one another,
showing essentially the same overall effect of copper on the biota of Shayler Run.
A wide range in sensitivity to copper was apparent in the different fish
species. However, the relative order of sensitivity may have changed with
changes in water quality or season. The bluegill was always the most resistant
species. In general, the 96-hr LC50 values of most of the common stream species
were lower than, but similar to that of the fathead minnow.
The acute bioassay results at both the Newtown Fish Toxicology Station
(NFTS) and the streamside laboratories varied because of water quality variations
of Shayler Run water. The 24-hr LC50 values varied from a high of 22 mg/Z. to
a low of 0.57 mg/Z. for the bluntnose minnow. At the test temperature of 24° C,
very few additional fish died after the first 24 hr. Even with the steady
low-stream flow during October 1969 and the resulting steady water quality of
alkalinity and hardness, the LC50 values varied from 22 to 0.75 mg/Z.. The 96-hr
LC50 values of total copper for the fathead minnow varied from 23.6 to' 0.92
mg/Z..; the 7-day LC50 values varied from 23.6 to 0.56 mg/Z-. Thus the acute
toxicity based on total copper in Shayler Run water varied about fortyfold for
both species. In the initial toxicity tests with Shayler Run water, it was
apparent that not only was there great variation in copper toxicity, but also
that toxicity was less (LC50 values higher) than would be predicted on the
basis of hardness, alkalinity, and pH of the stream water. This low toxicity
was verified by more tests with the bluntnose and the fathead minnows. The
mean 96-hr LC50 value of total copper to the fathead minnow was 8.9 mg/Z-. in
Shayler Run water, with a mean hardness of 271 mg/Z-. and mean alkalinity of
183 mg/Z-. This mean value was much higher than the 96-hr LC50 value of 1.8
mg/Z-. reported by Pickering and Henderson (1965) in a dilution water that was
higher in both hardness and alkalinity.
On the other hand, the variation of LC50 values of dissolved copper tested
in Shayler Run water was much less than the variation of total copper values.
For the bluntnose minnow, the dissolved copper 24-hr LC50 values varied only
from 0.42 to 0.30 mg/Z-., whereas the 96-hr LC50 values for the fathead minnow,
tested at ambient stream temperatures, varied from 1.4 to 0.52 mg/Z. The
constancy and smallness of these dissolved copper LC50 values, as compared to
total copper values, indicate that insoluble copper, as measured, is relatively
nontoxic. However, this does not mean that all copper that passes through a
0.45-micron filter is equally toxic since the copper associated with
pyrophosphate that passes through the filter is not as toxic as other forms of
copper that are present in the dissolved copper fraction.
It was suspected that the Shayler Run sewage treatment plant was discharging
materials that were detoxifying copper in Shayler Run water. Bioassays, using
diluent water from above and below the entrance of the effluent, indicated that
copper was much less toxic in Shayler Run water below the plant. Additional
toxicity tests, in which Shayler Run water was diluted with a reconstituted water
similar in hardness and alkalinity, indicated that the reduction in toxicity was
168
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not due to changes in hardness or alkalinity, but to some other detoxifying agent
or agents being diluted.
Effluents from sewage treatment plants normally contain substantial
concentrations of phosphates. Most of the total phosphorus measured in Shayler
Run was orthophosphate. Orthophosphates react with copper to form an insoluble
complex. Stepwise regression analyses indicated that there is a significant
(P=0.01) correlation of total copper LC50 values and total phosphorous
concentrations for both the fathead minnow and bluntnose minnow. Because this
does not necessarily indicate a cause-effect relationship of high LC50 values
and high phosphate concentrations, tests were conducted to determine the effect
of added phosphates on the toxicity of copper. Results indicated that the added
phosphates reduced the toxicity of total copper in both standard water and
Shayler Run water. It is probable that the reduced toxicity of total copper in
Shayler Run was due in part to the added phosphates from the sewage treatment
plant.
Estimation of an unsafe level of copper (120 yg/Z.) on spawning in the
stream, by extrapolating from results of laboratory chronic tests with fathead
minnows and acute tests with fathead minnows and stream species in
constant-quality NFTS standard (200 mg/Z. hardness) water, was in agreement with
results found for the minnow species in the stream. None of the minnow species
spawned very successfully in the exposure area during the exposure period.
Striped shiners and creek chubs were observed in prespawning activities in the
exposure area, but no spawning was observed. A few bluntnose minnow eggs were
observed in the extreme lower end of the exposure area. The average copper
concentration during spawning seasons for the 3 years of exposure was
approximately 106 yg/Z. in the upper exposure area at station 3 and 60 yg/Z. in
the lower exposure area at station 6.
The results of the streamside bluntnose minnow laboratory chronic tests
were not in close agreement with the predicted unsafe concentration or with what
occurred in the stream. The bluntnose minnow in the streamside tests spawned
in water from the exposure area as well as in the control water. Spawning of the
bluntnose minnow was blocked in 218 yg/Z. copper in the extended chronic test
in test system IV. This concentration was approximately two to three times the
predicted unsafe level of copper and two to three times the concentration at
which some bluntnose minnow spawns occurred in the exposure area. The reason for
this apparent lack of agreement is not known. Considering however, that a
0.5 dilution factor was used for the test concentrations and that spawning was
completely blocked at 218 yg/Z., there may have been a partial effect at some
intermediate concentration which was not tested. The actual unsafe concentration
in the chronic test may have been lower than 218 yg/Z. and nearer the 120 yg/Z.,
the predicted unsafe concentration. Additionally, other biological and
physical factors in the stream that were not present in the streamside chronic
tests may have had adverse effects on bluntnose minnow spawning.
169
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The spawning observations on the green sunfish and longear sunfish in the
stream suggest an effect of copper on spawning other than an effect on egg
production. The green sunfish spawned at 205 yg/Z. copper during the spawning
period in the chronic test in test system V, and the longear sunfish spawned at
107 yg/Z. in a preliminary test. In the stream they were not observed to spawn
at copper concentrations above approximately 90 yg/Z. The streamside laboratory
tests and the predictions from the laboratory acute tests indicate that green
sunfish could spawn at the highest concentration of copper in the exposure area.
Since both sunfish species had spawned throughout the exposure area before the
introduction of copper, the apparent restriction of the spawning area was possibly
a behavioral response, which would not have been found or predicted from the
chronic tests. Because there were effects in the stream not measured in the
laboratory tests, these tests tended to underestimate the toxicity of copper in
the stream. Based on extrapolation between species from a continuous to an
intermittent exposure and from constant-quality to varying-quality water, the
choice of 120 yg/Z. was a relatively good estimate of the actual toxic copper
concentration observed in the streamside laboratory chronic tests and in the
stream.
The application-factor hypotheses as proposed by Mount and Stephan (1967) is
a conceptual tool for estimating long-term safe conditions for fish. The
application factor is defined as the maximum acceptable toxicant concentration
(MATC) determined from a chronic toxicity test divided by the acute 96-hr LC50
value. They suggested that the application factor that is experimentally
determined for a specific toxicant and species of fish might be applicable to
the same toxicant and to other species of fish in other kinds of water.
This study on Shayler Run gave us an excellent opportunity to develop
information concerning the usefulness of the application-factor concept as applied
to a natural stream. The variable acute toxicity results found in Shayler Run
water illustrate a practical problem in estimating safe concentrations of
pollutants whose acute toxicity varies with water quality. The LC50 value to be
used for estimating safe concentrations is important. Brungs et a1. (1976)
discuss the problems of calculating an application factor for copper from toxicity
tests with the fathead minnow in a water of variable quality. They calculated an
application factor of 0.04-0.07 based on low total copper LC50 values and an
application factor of 0.07-0.13 based on dissolved copper measurements. They
conclude that if dissolved metal concentrations were used to calculate mortality
results and application factors, the predictive ability of the factors would
improve.
The use of the application factor of copper for predicting safe
concentrations for the bluntnose minnow in Shayler Run is also complicated by the
variable water quality which causes varying acute toxicity values. For a series
of toxicity tests, (Table 20) the LC50 values varied from 21 to 0.57 mg Cu/l.,
with a mean LC50 value of 7.9 mg Cu/l. Applying the application factor of
0.05-0.08 (found in our standard water test) to the mean total copper LC50 value
would give an estimated safe concentration of 400-630 yg/Z, The high,
concentration was found to be lethal during water quality conditions of low
detoxifying conditions. Using the minimum total copper LC50 value of 0.57 mg/Z-.
the estimated safe concentration in Shayler Run would be 29-47 yg Cu/l.
170
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On the basis of the relative sensitivity tests, the estimated safe
concentrations for many of the common stream species is similar to that calculated
for the bluntnose minnow. The estimated safe concentration for the sunfish,
however, would be much higher. Using the application factor of 0.07-0.13 based
on dissolved copper measurements, the estimated safe concentration for the
sunfish is 300-560 yg Cu/l. (dissolved) based on the mean dissolved copper
96-hr LC50 value of 4.3 mg Cull, for the bluegill.
Because of the effluent from the sewage treatment plant, Shayler Run probably
has higher concentrations of materials that detoxify copper than would be found
in most bodies of water. Thus, the variation in acute toxicity of copper
probably is greater than would be found in many streams. The water quality
studies emphasize, however, the importance of varying water quality in predicting
safe concentrations for aquatic life.
171
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172
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inney, D. J. 1971. Statistical method in biological assay. Cambridge University
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unk, J. L. 1957. Tbe Missouri Conservation Commission's electric seine. Iowa
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Guenther, W. C. 1964. Analysis of variance. Prentice-Hall, Inc., Englewood Cliffs,
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re-establishment of stream fish and invertebrates affected by drought. Trans.
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to facilitate sorting benthic samples. Trans. Am. Micro. Soc. 86:221-223.
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on the survival, growth, and reproduction of brook trout (Salvelinus fontinalis).
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Mount, D. I. 1968. Chronic toxicity of copper to fathead minnows (Pimephales
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174
-------
APPENDICES
Table Page
1 Shayler Run Discharge at Gaging Weir, 1968-72 176
2 Monthly Temperatures for Shayler Run, 1970-72 177
3 Monthly pH in Shayler Run, 1970-73 . . . „ „ . . 178
4 Alkalinity of Shayler Run, 1970-73 179
5 Hardness of Shayler Run, 1970-73 180
6 Dissolved Oxygen Content of Shayler Run, 1970-73 181
7 Chemical Characteristics of Shayler Run Water Based on
Weekly Grab Samples and Expressed as Monthly Means, 1968-72 182
8 Chemical Characteristics for Shayler Run Based on Weekly Grab
Samples and Expressed as Monthly Means, 1970-73 187
9 Metal Concentrations in Shayler Run, 1968-72 190
175
-------
APPENDIX TABLE 1. SHAYLER RUN DISCHARGE AT GAGING WEIR, 1968-72a
(IN CUBIC METERS PER SECOND)
Month
Jan.
Feb.
March
April
May
June
July
Aug.
Sept.
Oct.
Nov.
Dec.
Yearly
1968
Mean Maximum Minimum Mean
0
0
0
0
0
0
0
0.061 0.283 0.012 0
0.014 0.031 0.010 0
0.016 0.027 0.010 0
0.056 0.368 0.018 0
0.297 2.322 0.034 0
0
.408
.210
.153
.552
.317
.037
.033
.038
.026
.017
.144
.191
.189
1969
Maximum Minimum
4.
1.
1.
2.
1.
0.
0.
0.
0.
0.
2.
2.
4.
248
076
218
152
982
105
340
241
105
042
294
180
248
0.014
0.079
0.048
0.093
0.042
0.014
0.011
0.011
0.010
0.013
0.022
0.045
0.010
Mean
0
0
0
1
0
0
0
0
0
0
0
0
0
.294
.498
.799
.985
.239
.051
.057
.108
.032
.077
.131
.498
.394
1970
Maxinum
3.143
2.549
3.568
18.887
1.444
0.187
0.453
0.821
0.139
0.340
0.708
3.936
18.887
Minimum
0.048
0.085
0.082
0.085
0.028
0.017
0.018
0.014
0.018
0.021
0.042
0.048
0.014
Mean
0.428
1.354
0.657
0.118
0.280
0.052
0.045
0.079
0.524
0.068
0.101
0.799
0.377
1971
Maximum
4.021
6.768
2.917
0.425
1.897
0.190
0.178
0.425
2.152
0.113
0.708
4.870
6.768
Minimum
0.057
0.051
0.142
0.051
0.051
0.023
0.020
0.020
0.034
0.048
0.045
0.102
0.020
1972
Mean Maximum Minimum
0.416 1.614 0.079
0.600 2.265 0.068
0.847 3.908 0.198
1.065 5.154 0.165
0.685 3.341 0.057
0.180 2.039 0.028
0.041 0.113 0.022
0.030 0.190 0.018
0.124 1.189 0.015
Peak instantaneous discharge 67.961 m^/sec, April 2, 1970.
-------
APPENDIX TABLE 2. MONTHLY TEMPERATURES FOR SHAYLER RUN, 1970-72
(IN DEGREES CENTIGRADE)
Month
Jan.
Feb.
March
April
May
June
July
Aug.
Sept.
Oct.
Nov.
Dec.
Mean
1
2
5
12
20
23
24
23
21
13
8
5
1970
Maximum
2
7
9
23
28
30
30
28
26
19
13
13
Minimum
0
0
1
3
11
16
18
19
16
8
1
0.5
Mean
1
2
6
13
17
24
24
22
20
15
9
7
1971
Maximum
5
8
14
21
26
30
29
27
29
24
19
14
Minimum
0.5
0
0
5
10
18
18
17
14
10
4
3
Mean
6
4
6
14
18
19
23
23
20
14
9
7
1972
Maximum
13
8
12
21
24
26
29
28
24
20
15
11
Minimum
1
0
2
10
12
14
17
18
15
7
5
4
-------
APPENDIX TABLE 3. MONTHLY pH - IN SHAYLER RUN, 1970-73C
00
Month
Jan.
Feb.
March
April
May
June
July
Aug.
Sept.
Oct.
Nov.
Dec.
1970
Minimum Maximum
7.7
7.7
7.5
7.6
8.0
8.0
7.9
8.1
8.0
7.7
8.0
7.9
8.0
8.0
8.0
8.3
8.4
8.4
8.4
8.4
8.5
8.3
8.2
8.3
1971
Minimum Maximum
7.9
7.9
7.7
7.9
7.8
8.0
8.0
8.1
8.0
8.1
8.1
8.0
80
. Z
8.2
8.1
8.1
8.3
8.4
8.5
8.5
8.3
8.3
8.3
8.3
1972
Minimum Maximum
8.1
7.9
7.9
7.9
8.0
8.0
8.0
7.9
7.9
7.9
7.9
7.6
8.3
8.2
8.3
8.3
8.3
8.4
8.4
8.3
8.2
8.0
8.0
8.2
1973
Minimum Maximum
7.9 8.2
7.8 8.1
8.0 8.4
8.0 8.3
8.0 8.8
Sampling occurred between 8:00 and 10:00 a.m.
-------
APPENDIX TABLE 4. ALKALINITY OF SHAYLER RUN, 1970-73"
(IN mg/Z.. AS CaC03)
Month
Jan.
Feb.
March
April
May
June
July
Aug.
Sept.
Oct.
Nov.
Dec.
1970
Mean Minimum
204
151
142
155
188
212
191
184
206
202
202
200
168
78
92
56
96
160
88
80
164
164
150
134
Maximum
248
204
192
198
220
228
232
220
220
226
230
230
Mean
178
157
150
206
193
192
179
200
174
230
225
177
1971
Minimum
102
90
66
172
146
164
142
160
118
216
150
94
Maximum
230
248
196
224
222
216
198
222
226
250
256
226
Mean
177
170
154
150
165
207
205
200
177
210
137
126
1972
Minimum
136
,100
98
68
110
172
180
142
82
204
66
50
Maximum
236
230
188
210
226
228
224
220
206
214
186
170
1973
Mean Minimum Maximum
159 72 230
149 71 194
150 93 199
152 141 171
199 175 240
Sampling occurred between 8:00 and 10;00 a.m.
-------
Co
O
APPENDIX TABLE 5. HARDNESS OF SHAYLER RUN, 1970-73"
(IN mg/l. AS CaC03)
Month
Jan.
Feb.
March
April
May
June
July
Aug.
Sept.
Oct.
Nov.
Dec.
Mean
309
244
226
226
275
306
278
253
310
292
284
286
1970
Minimum
264
168
160
88
148
198
124
112
236
230
210
212
Maximum
350
316
288
302
320
352
336
310
350
342
316
320
Mean
253
234
221
298
266
277
271
287
236
310
316
240
1971
Minimum
168
150
110
254
210
236
204
214
186
300
224
148
Maximum
316
344
276
326
322
314
300
316
310
328
356
300
1972
Mean Minimum
255
250
231
205
242
289
301
307
272
305
178
180
200
160
148
102
170
236
270
222
144
298
106
84
Maximum
326
330
276
300
320
312
312
324
330
310
240
236
1973
Mean Minimum Maximum
219 118 282
203 114 268
206 134 276
207 190 244
260 220 284
'Sampling occurred between 8:00 and 10:00 a.m.
-------
00
APPENDIX TABLE 6. DISSOLVED OXYGEN CONTENT OF SHAYLER RUN, 1970-73C
(IN rog/l.)
Month
Jan.
Feb.
March
April
May
June
July
Aug.
Sept.
Oct.
Nov.
Dec.
Mean
12.
12.
11.
10.
8.
7.
6.
6.
5.
7.
9.
10.
1970
Minimum
6
4
3
2
8
0
9
7
9
6
2
4
12.0
10.5
8.0
8.6
7.0
6.0
5.0
5.8
5.3
6.2
7.3
8.2
Maximum
12.9
13.0
12.9
12.1
10.2
7.4
8.0
7.8
7.7
8.8
12.0
11.8
1971
Mean Minimum
11.7
11.7
10.9
8.7
7.8
6.4
6.7
7.3
7.3
9.0
10.6
11.2
10.3
10.8
9.4
7.3
6.6
5.9
6.0
6.5
4.8
7.8
8.2
10.0
Maximum
12.4
12.2
13.0
10.3
9.3
8.0
8.2
8.4
8.2
9.8
12.2
12.8
Mean
12.0
12.6
11.2
10.4
9.2
7.6
7.2
6.9
7.4
8.4
10.1
12.0
1972
Minimum
10.6
11.9
9.5
9.6
8.1
6.7
6.1
6.1
7.0
7.8
8.6
9.7
Maximum
13.2
13.4
13.1
11.1
10.6
9.4
8.6
8.0
8.1
9.3
11.2
13.4
1973
Mean Minimum Maximum
12.8 12.0 13.8
12.0 10.6 14.4
11.2 9.1 13.3
12.2 10.1 14.2
8.2 6.0 9.7
aSampling occurred between 8:00 and 10:00 a.m.
-------
APPENDIX TABLE 7. CHEMICAL CHARACTERISTICS OF SHAYLER RUN WATER BASED ON WEEKLY GRAB
SAMPLES AND EXPRESSED AS MONTHLY MEANS, 1968-72
Year /Month
1968
Jan.
Feb.
March
April
May
June
July
Aug.
Sept.
Oct.
Nov.
Dec.
1969
Jan.
Feb.
March
PH
7.8
8.0
7.8
7.9
7.8
8.1
7.8
7.8
7.8
7.6
7.8
7.6
7.7
7.8
7.7
Alkalinity
mg/Z. as
CaCOi
194
232
127
158
175
210
193
220
220
196
186
158
238
162
208
Hardness
mg/Z., as
CaC03
306
326
200
225
250
311
278
289
328
301
272
268
328
237
308
SC TS DS Ca Mg OP-P TP
umho/cm mg/Z. mg/Z. mg/Z. mg/Z. mg/Z. mg/Z-
595
679
469
518
550
787
840
842
1,060
956
743
532 378 366
580 457 446
468 361 346 95
567 475 471 89 23
-------
APPENDIX TABLE 7 (continued). CHEMICAL CHARACTERISTICS OF SHAYLER RUN WATER BASED ON WEEKLY
GRAB SAMPLES AND EXPRESSED AS MONTHLY MEANS, 1968-72
OO
Year /Month
1969
April
May
June
July
Aug.
Sept.
Oct.
Nov.
Dec.
1970
Jan.
Feb.
March
April
May
pH
7.7
7.7
7.8
7.6
7.8
8.1
8.1
8.0
8.1
7.8
8.0
8.3
8.1
Alkalinity
mg/Z. as
CaCO,
174
174
218
218
180
219
210
176
194
148
139
163
186
Hardness
mg/2., as
250
347
302
296
193
327
307
269
284
243
212
234
264
SC
umho/cm
502
518
781
883
722
772
930
841
853
862
678
530
526
613
TS
mg/Z.
364
370
505
552
538
617
634
501
467
468
384
340
431
411
DS
mg/Z.
356
348
505
535
504
587
620
492
447
464
383
277
322
401
Ca
mg/Z.
81
93
93
84
73
81
92
82
74
80
67
56
70
77
Mg OP-P TP
mg/Z. mg/Z. mg/Z.
19
21
21
18
17
18
21
19
17
18 1.9
14 1.0
12 0.6
13 1.1 1.5
14 1.9 1.9
-------
APPENDIX TABLE 7 (continued). CHEMICAL CHARACTERISTICS OF SHAYLER RUN WATER BASED ON WEEKLY
GRAB SAMPLES AND EXPRESSED AS MONTHLY MEANS, 1968-72
Year /Month
1970
June
July
Aug.
Sept.
Oct.
Nov.
Dec.
1971
Jan.
Feb.
March
April
May
June
July
PH
8.1
8.1
8.1
8.0
8.0
8.1
8.0
8.1
8.0
8.2
8.1
8.1
8.0
8.0
Alkalinity
mg/£. as
CaCO}
206
197
198
205
193
191
174
196
174
160
236
200
191
172
Hardness
mg/Z., as
CaC03
304
280
.268
308
276
257
247
272
251
230
312
278
275
269
SC
umho/cm
872
795
750
858
644
602
585
632
602
563
740
690
767
788
TS
mg/Z.
570
496
501
593
500
406
414
402
401
352
485
452
499
603
DS
mg/Z.
560
481
487
577
496
397
354
396
377
335
470
428
477
515
Ca
mg/Z-
89
84
84
93
83
78
77
81
74
66
89
81
84
83
Mg
18
18
18
20
19
18
17
18
16
15
21
19
19
18
OP-P
mg/Z.
3.8
4.3
3.9
5.4
5.3
2.3
2.0
1.2
1.3
0.7
2.6
2.6
3.8
5.2
TP
mg/Z.
4.0
4.4
4.0
5.4
5.1
2.5
2.3
1.6
1.6
1.0
2.6
2.7
3.8
5.4
-------
APPENDIX TABLE 7 (continued). CHEMICAL CHARACTERISTICS OF SHAYLER RUN WATER BASED ON WEEKLY.
GRAB SAMPLES AND EXPRESSED AS MONTHLY MEANS, 1968-72
Year /Month
1971
Aug.
Sept.
Oct.
Nov.
Dec.
1972
Jan.
Feb.
March
April
May
June
July
Aug.
Sept.
Oct.
PH,
8.2
8.1
8.1
8.2
8.2
8.2
8.2
8.4
8.1
8.1
8.1
8.1
8.1
8.0
8.1
Alkalinity
mg/Z. as
CaC03
191
180
227
222
177
177
162
166
142
194
215
198
192
181
207
Hardness
mg/Z. , as
CaCO,
279
248
306
294
240
246
243
241
198
262
275
298
310
280
300
SC
umho/cm
725
617
797
780
553
614
623
612
477
616
594
813
1,009
866
813
TS
mg/Z-
545
345
480
473
354
374
396
356
442
397
479
556
651
587
537
DS
mg/Z.
496
397
468
467
340
356
366
339
280
367
461
517
620
558
527
Ca
mg/Z-
82
75
89
80
66
69
75
69
53
70
92
87
84
74
87
Mg
18
15
20
19
15
16
16
16
12
16
19
19
20
19
19
OP-P
mg/Z.
4.2
2.2
3.3
4.7
0.6
0.9
0.9
0.5
0.6
0.9
2.4
3.4
5.1
8.9
6.2
TP
mg/Z.
4.2
2.2
3.3
4.8
0.7
0.9
1.0
0.6
0.6
1.0
2.4
3.4
5.3
9.0
6.2
-------
00
APPENDIX TABLE 7 (continued). CHEMICAL CHARACTERISTICS OF SHAYLER RUN WATER BASED ON WEEKLY
GRAB SAMPLES AND EXPRESSED AS MONTHLY MEANS, 1968-72
Year /Month
1972
Nov.
Dec.
Alkalinity
mg/Z. as
pH CaCO^
8.1 126
8.1 137
Hardness
mg/Z., as
CaCCh
185
188
SC
umho/cm
446
350
TS
mg/Z.
292
338
DS
mg/Z.
272
263
Ca
mg/Z.
56
57
Mg
tng/Z.
11
12
OP-P
mg/Z.
0.4
0.6
TP
mg/Z.
0.6
0.8
-------
APPENDIX TABLE 8. CHEMICAL CHARACTERISTICS FOR SHAYLER RUN BASED ON WEEKLY
GRAB SAMPLES AND EXPRESSED AS MONTHLY MEANS, 1970-73
00
Year /Month
1970
Jan.
Feb.
March
April
May
June
July
Aug.
Sept.
Oct.
Nov.
Dec.
1971
Jan.
Feb.
March
April
Na
mg/Z.
46
34
21
21
26
54
51
48
65
54
24
20
24
23
24
36
K
mg/Z.
5.0
4.4
4.2
3.8
4.0
8.2
10.7
7.8
9.0
8.4
4.6
4.3
5.1
3.8
3.5
5.1
CHD
mg/Z.
81
61
38
31
34
66
64
54
76
60
43
39
37
41
34
50
N03-N N02-N
mg/Z. mg/Z.
1.4 0.3
1.5 0.1
1.2 <0.1
0.5 <0.1
1.6 <0.1
4.1 <0.1
2.9 <0.1
3.4 <0.1
7.5 <0.1
5,5 <0.1
2.0 0.2
2.5 0.1
1.4 0.1
1.1 <0.1
0.6 0.1
3.0 <0.1
NH3-N
mg/Z.
2.2
0.4
0.4
0.9
0.5
<0.1
<0.1
0.2
0.1
1.4
1.8
1.6
2.6
1.9
0.9
0.4
ORG-N
mg/Z.
0.8
0.7
0.8
0.4
0.4
0.6
0.4
0.3
0.9
0.4
<0.1
0.3
0.5
0.6
0.5
0.4
TKN TP
mg/Z. mg/Z.
1.9
1.0
0.6
1.0
1.8
3.4
3.9
3.8
0.5 5.0
1.4 4.6
1.9 2.4
1.7 1.9
2.6 1.6
2.5 1.1
1.3 1.1
0.9 3.0
TOC Ca Mg
mg/Z. mg/Z. mg/Z.
6.6
5.7
8.0
5.5
6.0
5.6
6.2
4.3
6.0
7.3
7.0
7.3
5.4
7.4
5.1
5.7
-------
APPENDIX TABLE 8 (continued). CHEMICAL CHARACTERISTICS FOR SHAYLER RUN BASED ON WEEKLY
GRAB SAMPLES AND EXPRESSED AS MONTHLY MEANS, 1970-73
Year/Month
1971
May
June
July
Aug.
Sept.
Oct.
Nov.
1 — i
00
oo Dec.
1972
Jan.
Feb.
March
April
May
June
July
Aug.
Sept.
Na
34
44
51
50
32
44
39
19
23
28
20
16
24
34
46
60
49
K
mg/Z.
5.2
6.2
7.9
7.9
5.2
6.2
6.3
3.9
3.8
3.6
2.2
2.6
5.0
5.0
7.6
10.9
10.8
CHD
mg/Z.
49
56
62
62
39
46
46
27
33
46
37
24
28
47
60
82
67
N03-N N02-N
mg/Z. mg/Z.
1.9 0.1
4.1 0.1
7.2 <0.1
4.2 <0.1
1.6 <0.1
2.3 <0.1
2.8 <0.1
1.4 <0.1
0.8 <0.1
1.8 <0.1
1.2 <0.1
0.8 0.2
1.5 <0.1
3.4 0.1
6.0 0.1
12.0 <0.1
6.8 O.I
NH3-N
mg/Z.
0.4
0,2
0.2
0.5
<0.1
0.1
0.4
0.3
0.4
0.5
0.1
0.1
0.2
0.3
0.1
0.4
0.1
ORG-N
mg/Z.
0.3
0.8
1.8
0.9
0.7
0.3
0.4
0.4
0.1
0.8
0.2
2.8
0.4
0.6
0.7
0.8
1.0
TKN
mg/ Z.
0.9
1.0
1.9
1.4
0.7
0.4
0.8
0.7
0.5
1.3
0.5
2.2
0.6
0.9
0.8
1.1
1.1
TP
me/Z.
2.9
3.6
4.9
4.4
2.2
3.1
3.8
0.9
1.0
1.1
0.6
0.7
1.2
3.0
4.2
6.8
6.5
TOC
mg/Z.
5.9
5.7
11.3
7.6
6.1
7.0
7.0
7.3
5.7
9.1
6.5
13.4
7.5
8.6
8.3
9.1
8.4
Ca Mg
mg / Z . mg / Z .
82
75
90
89
71
73
75
69
53
70
92
87
84
74
18
16
20
20
16
16
16
16
12
16
19
19
20
19
-------
APPENDIX TABLE 8 (continued). CHEMICAL CHARACTERISTICS FOR SHAYLER RUN BASED ON WEEKLY
GRAB SAMPLES AND EXPRESSED AS MONTHLY MEANS, 1970-73
00
Year /Month
1972
Oct.
Nov.
Dec.
1973
Jan.
Feb.
March
Na K CHD
mg/Z. mg/Z. mg/Z.
47 8.4 56
15 2.8 22
15 2.9 22
20 2.2 26
20 3.2 30
19 2.0 30
N03-N N02-N NH3-N ORG-N
mg/Z. mg/Z. mg/Z. mg/Z.
5.4 0.1 0.6 1.0
1.3 <0.1 0.1 0.8
2.9 <0.1 0.5 0.6
1.2 <0.1 0.9 0.5
1.0 0.1 1.1 <0.1
1.2 <0.1 0.4 0.3
TKN TP TOG
mg/Z. mg/Z. mg/Z.
1.6 5.3 7.5
0.9 0.7 7.1
1.1 0.7 6.9
1.0 1.0 5.8
0.9 0.9 5.2
0.7 0.5 5.1
Ca Mg
mg/Z. mg/Z.
87
56
57
56
65
58
19
11
12
15
15
13
-------
APPENDIX TABLE 9. METAL CONCENTRATIONS IN SHAYLER RUN, 1968-72
(AS yg/Z.)
Metal
Zn
Cd
Ar
B
P
Fe
Mo
Mn
VO
0 Al
Be
Cu
Ag
Ni
Co
Pb
Cr
V
Ba
Sr
5-17-68 10-30-68
<21 46
<21 <19
^107 <50
205 830
610 3,700
<]_~L 9
<43 61
<11 <9
<43 <37
<0.21 <0.19
<11 27
<2.1 <1.9
<21 <19
<21 <19
<43 <37
<11 <9
<43 <37
33 30
190 380
2-18-69
<20
<20
<100
260
940
130
110
48
190
1.0
13
<2.0
<20
30
60
19
<40
33
184
7-2-69
<23
<23
<50
490
1,200
240
55
70
245
<0.23
<11
<2.3
<23
<23
<45
<11
<45
38
320
10-21-69
125
<36
<50
1,360
7,150
820
<71
<18
750
<0.36
18
<3.6
<36
<36
<71
<18
<71
25
170
5-14-70
63
<17
<50
205
<85
1,165
<33
37
1,230
<0.17
25
<1.7
7
3
<50
<8
<33
36
155
11-5-70
<23
<23
-------
APPENDIX TABLE 9 (continued). METAL CONCENTRATIONS IN SHAYLER RUN, 1968-72
(AS yg/Z.)
Metal
Zn
Cd
Ar
B
P
Fe
Mo
Mn
Al
Be
Cu
Ag
Ni
Co
Pb
Cr
V
Ba
Sr
3-12-71
<13
<13
<63
25
<63
590
<25
15
620
<0.13
<6
<1.3
<13
<13
<25
<6
<25
16
81
5-19-71
<22
<22
<50
300
585
17
<43
<11
<43
<0.22
1,000
<0.1
<20
<2
<20
<20
<40
<20
<40
24
5-12-72
<20
<20
<80
100
&0
20
<20
>1,000
<0.1
<20
<2
<20
<20
<40
<20
<40
20
6-1-72
<20
<20
<80
280
120
<20
<20
>1,000
<0.1
<20
<2
<20
<20
<40
<20
<40
20
-------
TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPORT NO.
EPA-600/3-76-116
3. RECIPIENT'S ACCESSION-NO.
4. TITLE AND SUBTITLE
VALIDITY OF LABORATORY TESTS FOR PREDICTING COPPER
TOXICITY IN STREAMS
5. REPORT DATE
December 1976 issuing date
6. PERFORMING ORGANIZATION CODE
7.AUTHORO) jack R. Geckler, William B. Horning, Timothy
M. Neiheisel, Quentin H. Pickering, Ernest L. Robinson,
Charles E. Stepban
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Newtown Fish Toxicology Station
Environmental Research Laboratory-Duluth
3411 Church Street
Cincinnati, Ohio 45244
10. PROGRAM ELEMENT NO.
1BA608
11. CONTRACT/GRANT NO.
NA
12. SPONSORING AGENCY NAME AND ADDRESS
Environmental Research Laboratory-Dul-uth, MN
Office of Research and Development
U.S. Environmental Protection Agency
Duluth, Minnesota 55804
13. TYPE OF REPORT AND PERIOD COVERED
Final Report 12/1967-5/1973
14. SPONSORING AGENCY CODE
EPA/600/03
15. SUPPLEMENTARY NOTES
16. ABSTRACT
A field study was conducted on Shayler Run, in Clermont County, Ohio, to determine
the'effects of copper on the stream biota. Copper was added to the stream for 33
months to maintain a concentration of 120 yg/Z-., a concentration that was expected to
adversely affect some species of fish and not others. This natural stream received
sewage effluent containing a variety of compounds known to affect acute copper toxici-
ty. All but one abundant species of fish in the stream and four of the five most
abundant macroinvertebrates were adversely affected by exposure to copper. Direct
effects on fish were death, avoidance, and restricted spawning.
To determine the usefulness of laboratory toxicity tests when establishing water
quality criteria for an aquatic ecosystem, acute and chronic tests with copper were
conducted at the Newtown Fish Toxicology Station and on-site at Shayler Run with
stream species and the fathead minnow. The acute toxicity of copper varied widely
because of water quality variations in the stream. The chronic tests underestimated
the in-stream toxicity by about two times because only the effects of copper on
survival, growth, and reproduction were measured. Agreement between the predictions
from laboratory toxicity tests and the observed effect is surprisingly close
considering the measurement errors involved.
7.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.IDENTIFIERS/OPEN ENDED TERMS C. COSATI Field/Group
*Copper
*Toxicity
*Fresh water fishes
^Invertebrates
*Aquatic Biology
Water pollution
*Field test
*Acute Laboratory tests
*Chronic
Median lethal concentra-
tion
Macroinvertebrates
Shayler Run
06S
06F
06T
RELEASE TO PUBLIC
. SECURITY CLASS (This Report)
UNCLASSIFIED
21. NO. OF PAGES
208
0 SECURITY CLASS (This page)
UNCLASSIFIED
22. PRICE
EPA
(9-73)
192
ft U.S. GOVERNMENT PRINTING OFFICE: 1977-757-056/5470
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