EPA- 660/3-73-004
September 1973
Ecological Research Series
WATER QUALITY REQUIREMENTS
OF AQUATIC INSECTS
Office of Research and Development
U.S. Environmental Protection Agency
Washington, D.C. 20460
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and
Monitoring, 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
H. Environmental Monitoring
5. Socioeconomic Environmental Studies
This report has been assigned to the ECOLOGICAL
RESEARCH series. This series describes research
on the effects of pollution on humans, plant and
animal species, and materials. Problems are
assessed for their long- and short-term
influences. 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.
EPA REVIEW NOTICE
This report has been reviewed by the Office of Research and
Development, EPA, and approved for publication. Approval
does not signify that the contents necessarily reflect the
views and policies of the Environmental Protection Agency,
nor does mention of trade names or commercial products
constitute endorsement or recommendation for use.
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EPA-660/3-73-004
September 1973
WATER QUALITY REQUIREMENTS
OF AQUATIC INSECTS
By
Ardetx R. Gaufin
University of Utah
Salt Lake City, Utah 84112
Project 18050 FLS
Project Officer
Dr. Alan V. Nebeker
Environmental Protection Agency
National Environmental Research Center
Corvallis, Oregon 97230
Prepared for
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, D.C. 20460
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ABSTRACT
The larvae of twenty species of aquatic insects (Diptera, Epheme-
roptera, Plecoptera, and Trichoptera) and the scud (Amphipoda)
were exposed to high water temperatures, low dissolved oxygen con-
centrations, and low pH to determine their tolerance of these three
environmental factors. The temperature at which 50% of the speci-
mens died after 96 hours exposure ranged from 11.7° C for the may-
fly, Cinygmula par Eaton, to 32.6° C for the snipe fly, Atherlx
var iexjata Wa1ker. The mayfly, Ephemerella doddsi Needham, was
most sensitive to low dissolved oxygen levels with a 96-hour TLm
of 5.2 mg/1. Acroneur i a pac i flca Banks, a stonefly, was the most
resistant with a TLm^> of 1.6 mg/1. Median tolerance levels for
pH ranged from pH 2.7 for the caddis fly, Limnephilus ornatus Banks,
to 7.2 for the scud, Gamma r us 1 i rhnaeu s Smith. Longer term bioassays
clearly indicated increased sensitivity and mortality of the test
specimens with increased length of exposure to each of these
factors.
This report was submitted in fulfillment of Contract Number
14-12-438 under the sponsorship of the Water Quality Office,
Environmental Protection Agency.
ii
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CONTENTS
Section
I . Conclusions '
I I. Recommendations 3
III. Introduction ^
IV. Studies on the Tolerance of Aquatic Insects to 6
Low Oxygen Concentrations
Introduction «
Materials and Methods 7
Short-Term (Acute) Bioassays Conducted at 9
University of Montana Biological Station
Results 9
Discussion ' 1
Long-Term Bioassays Conducted at the University 2k
of Montana Biological Station and the University
of Utah
Results 2/t
Discussion 29
V. Studies on the Tolerance of Aquatic Insects to 32
Heated Waters
Introduction 32
Materials and Methods 33
Results 3*»
Discussion 3^
Long-Term Thermal Bioassays Conducted at 38
Biological Station
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Section Pa9e
Studies on the Tolerance of Great Basin Aquatic 39
Insects to Heated Waters
Materials and Methods 39
Results *»0
Emergence 40
VI. Studies on the Tolerance of Aquatic Insects to 4A
Low pH
Introduction 44
Materials and Methods 45
Results 46
Discussion 47
Tolerance Limits of Great Basin Aquatic Insects 51
to Sulfuric and Hydrochloric Acid
Materials and Methods 51
Results 51
Long-Term Continuous Flow Bioassays 54
Discussion 55
VII. Acknowledgments 58
VIM. References (Literature Cited) 59
IX. Appendices - Supplementary Tables 64
VI
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FIGURES
1. 96-Hour TLm Results - Oxygen ^
2. Representative TLm96 Graphs - Temperature 37
3. PH Values - % Survival After 96 Hours 5°
vii
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TABLES
Number Title Page
1. Test Organisms, TLm, Saturation (Oxygen) and 10
Water Flow
2. Gill Beats Per Minute, Ephemere11 a grand?s 12
3. Long-Term Dissolved Oxygen Bioassays - Montana 2k
4. Long-Term Dissolved Oxygen Bioassays - Utah 25
5. Minimal D.O. Survival Levels 26
6. Average Minimum Dissolved Oxygen Requirements 27
7. Temperature Values - TLm9° (Montana) 36
8. Long-Term Thermal Bioassays (Montana) 38
9. Thermal Values - TLm96 (Utah) 42
10. Long-Term Thermal Bioassays (Utah) 43
11. PH Values - TLm96 49
t'
12. pH Values - Long-Term Exposure 50
13. Sulfuric Acid Bioassays - TLm96 Values 53
14. Hydrochloric Acid Bioassays - TLm96 Values 53
15. Long-Term Bioassays Results - pH 54
VI I 1
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CONCLUSIONS
' Acroneuria padf ica, a stonefly, was the most resistant form
tested to low oxygen concentrations with a TLm° of 1.6 mg/1; ,
Ephemerella doddsi, a mayfly, was the most sensitive with a TLm
of 5.2 mg/1.
2. The group most tolerant of low dissolved oxygen levels was the
Trichoptera (2.86 mg/1).
3. Water flow is very important in determining dissolved oxygen
limits. The mean TLm^° for 10 species of aquatic insects tested
at 500 cc/min flow was 3.6A mg/1; the mean for 10 species at 1000
cc/min was 2.55 mg/1.
4. Increased sensitivity and mortality of test specimens occurred
with increased length of exposure to low oxygen levels. Whereas,
50% of the specimens of Acroneu ri a pacif i ca survived an oxygen con-
centration of 1.6 mg/1 f b>~ Tf "days , the mi n i ma 1 oxygen level for 50%
survival at 111 days was 5.8 mg/1.
5' Atherix variegata, a Dipteran, was the most tolerant of high water
temperatures with a TLm^6 of 32.6° C; Cinygmula par, was the most
sensitive with a TLm96 of 11.7° C.
6. Acclimation to colder temperatures in nature results in increased
sensitivity to exposure to elevated temperatures. For example, speci-
mens of the stonefly, I s QCJ e n u s a e s t i v a 1 i s , f. r om Utah were much more
tolerant than Montana specimens with a TLm-' of 24.2° C in comparison
to a TLm9° of 16.1° C for Montana specimens.
7. Increased mortality of test specimens occurred with increased
length of exposure to high temperatures. The TLm9° for specimens
of the stonefly, Pteronarcella badia, from Montana was 24.4° C.
In comparison, 50% of the specimens succumbed to a temperature of
18.1° C in 2k days.
8. Exposure to sublethal temperatures increases growth rate and
emergence. P terona rce 11 a badji a\_, a stonefly, which normally emerges
in mid June, emerged early in February after exposure to a tempera-
ture of 17° C for 29 days.
9. Limnephi 1 us orna^tus, a caddis fly, was the most tolerant of low
pH 1 eve1s with a TLm^ of 2.7; Gammarus 1imnaeus, the scud, was
the most sensitive with a TLm-'" of 7.2.
1
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10. The results of both short and long-term bioassays Indicated
that mayflies are most sensitive to low pH levels with stoneflies
being moderately sensitive and caddis flies least sensitive.
11. Exposure for short periods to pH levels well below those normally
found in nature may not be harmful. Longer exposure, however, may
have decidedly detrimental effects on molting, growth, and emergence.
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RECOMMENDATIONS
1. To maintain a well-rounded diversified population of cold water
aquatic insects, maximum temperatures, minimum dissolved oxygen
levels, and the pH range should not exceed the requirements of
cold water fishes, such as trout and salmon. While some aquatic
insects can tolerate dissolved oxygen levels as low as 1.6 mg/1
for short periods, concentrations of 6.0 mg/1 are required for
long-term survival. Temperatures during the winter months must
be maintained at normal seasonal levels to prevent premature
emergence. Temperatures above 65° F during the summer months
are considered the maximum for maintaining many species of stone-
flies, mayflies, and caddis flies. A pH range of 6.0 - 8.5
should protect most cold water lotic insects.
2. Since aquatic insects are much more sensitive during molting
and emergence, further research should be undertaken to determine
the effects of these and other environmental factors on the most
sensitive stage of the most common species of aquatic inverte-
brates .
3. Inasmuch as there is considerable variation in the environ-
mental requirements of different species of aquatic insects, further
research is needed on a country-wide basis to set criteria for
the protection of both cold water and warm water species in various
types of habitat.
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INTRODUCTION
Industrial and population expansion in many areas has resulted in
badly polluting our streams. Among the results of pollution is
the reduction or even the depletion of dissolved oxygen in the
water. The amount of oxygen dissolved in water was cited by Reish
and Richards (1966) as perhaps the single most important environ-
mental factor for the survival, growth, and reproduction of aquatic
animals. The oxygen content of the water during nymphal growth was
considered by Per Brinck (19^9) as one of the most important factors
in the distribution of stoneflies. In his studies in south Swedish
waters he showed that sections of streams with a low oxygen content
(below 40$ saturation) had an insignificant or no stonefly fauna.
Water temperatures have a profound and diverse effect on aquatic
life. Uncontrolled high water temperatures may have a directly
lethal effect and serve as a barrier to movements of river-migrant
fishes. Continuously high water temperatures may prevent production
of desirable game fishes and other aquatic species and result in their
eventual elimination. High water temperatures may cause extensive
ecological changes in rivers and lakes and drastically alter the biota.
Limited quantities of warm water, however, may produce desirable
changes in selected localized situations.
In coal mining regions of the United States water pollution by acid
mine drainage constitutes a problem of major importance. Pollution
by acids may be sufficient to not only make the water of a stream
unfavorable for the growth and development of fish and aquatic inverte-
brates but there may also be a directly lethal effect.
Those discharging wastes into our waters need to know the requirements
of aquatic life in order to ascertain the amount of waste which can
be introduced into our streams without jeopardizing the conditions
necessary to maintain aquatic life. Water quality criteria for the
protection of aquatic life must be established, but there is a lack
of agreement among workers as to just what these criteria should be
or how they shoud be applied. Any criteria that are established must
be based on a knowledge of habitat requirements for those forms
inhabiting the particular body of water under consideration. Such
criteria must encompass all environmental factors necessary for the
survival, growth, reproduction, and well being of the aquatic organisms.
Each species should be evaluated at the various stages in its life
history if such criteria are to serve their purpose.
Biological examinations have been used for many years to assess the
degree of pollution of our lakes and streams. Immature aquatic
insects have been used extensively as biological indicators because
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of their sensitivity to changes in their environment, the length
of their life cycles, and their lack of mobility in comparison to
fish. Over the years fairly extensive lists of aquatic insects
indicative of various degrees of water pollution have been pub-
lished by such workers as Kolkwitz and Marsson (1909), Fair and
Whipple (19^8), Liebmann (1951), Gaufin and Tarzwell (1953, 1956),
Hynes (i960), and others. Disagreement over the exact status of
many of these organisms exists because of differences in chemical
and physical conditions at the time of sampling and insufficient
knowledge concerning the environmental requirements of the organisms
col lected.
Our knowledge of the requirements of individual species of aquatic
insects is extremely limited. For many of our North American species
life cycles are unknown, immature stages are undescribed, and the
total span of emergence periods unrecorded. Only a few species have
been the subject of detailed study.
Laboratory experiments have been conducted at the University of Utah
for the last ten years in order to better isolate, understand, and
interpret some of the environmental factors which have an important
effect on the behavior and physiological reactions of stoneflies. The
specific objectives of this work have been to determine the effects
of low dissolved oxygen concentrations at various temperatures and
water flow on the gross activity of stoneflies, to determine the
minimum dissolved oxygen concentrations at which exposure for a pro-
longed period of time can be endured without lethal effects, to deter-
mine metabolic levels of various species of stoneflies, and to deter-
mine the food habits of as many species as possible.
Ecological studies of the environmental requirements of various species
of aquatic insects in the Intermountain Region have been conducted at
the University of Utah since 19*»6. Considerable data has been collected
as to the effects of pollution on the biota present in a number of
streams. Considerable data as to the biota present, productivity, and
chemical-physical characteristics of a number of streams, such as the
Prove, Weber, and Jordan Rivers in Utah; Colorado River in Colorado,
and Bitterroot River in Montana, have been accumulated.
This report summarizes three years of research which focused upon the
effects of low dissolved oxygen levels, high temperatures, and an acid
environment on aquatic insects. The objectives of the research were
to determine lethal and sublethal levels, and acute and long-term
effects of these factors on the survival, growth, reproduction, and
behavior of 20 species of aquatic insects and the scud, Gammarus
1imnaeus. Gammarus 1imnaeus in this report is considered as a sub-
species of Gammarus lacustris.
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STUDIES ON THE TOLERANCE OF AQUATIC INSECTS
TO LOW OXYGEN CONCENTRATIONS
Introduction
Oxygen is a basic need of aquatic insects, yet information concern-
ing their exact oxygen requirements is known for but a very few
species. Gaufin and Tarzwell (1956) pointed out that if the oxygen
requirements of different species of aquatic insects were better
known, it should be possible to estimate in retrospect, with con-
siderable accuracy, what oxygen levels have existed in a given aquatic
environment during the life history of the organisms. Thus aquatic
insects could be used as an excellent index of water quality.
The literature is extensive on oxygen consumption by various animals,
yet such values are meaningful only for the particular conditions of
measurement. The conditions under which such measurements were made
are important because the rate of oxygen consumption is influenced
by several internal and external variables. The rate of oxygen con-
sumption is influenced by activity, temperature, nutrition, body
size, stage in life cycle, season, and time of day, as well as by
previous oxygen experience and genetic background (Prosser and
Brown, 1961). The highest respiratory rates usually occur in the
small, very active forms; whereas, the lowest occur in the large
relatively sedentary forms.
Wigglesworth (1950) and Edwards (19^*6) summarized much of the work
that has been done on respiration rates of insects. The majority
of the publications on immature aquatic insects has been on European
species. Extensive work on individual, immature, aquatic insects
was done by Balke (1957) on European species of the orders Neuroptera,
Odonata, Plecoptera, and Trichoptera. The difficulty in selecting
a suitable and adequate method for the measurement of the respira-
tory rate in a particular species of aquatic insect was evaluated
by Kamler in 19&9. An analysis of the various factors which influ-
ence the oxygen requirements and respiratory rates of benthic inver-
tebrates is presented in "The Ecology of Running Waters" by Hynes
(1970). The oxygen consumption of ten of the most common species
of stoneflies of the western United States and the factors which
modify their metabolic rate are discussed by Knight and Gaufin
(1966). The oxygen requirements of immature aquatic insects in
relationship to their classification as index organisms are thor-
oughly evaluated by Olson and Rueger (1968). Their statistical
analyses of oxygen consumption rates by twelve representative
-------
species of aquatic insects of the upper Great Lakes Region consti-
tute very valuable data for establishing water quality criteria for
the protection of aquatic life.
The principal objectives of the studies presented in this report were
to determine the oxygen requirements of representative species of
aquatic insects of the Intermountain Region and to determine their
relative sensitivity to low oxygen concentrations. Oxygen levels
necessary for survival and the long-term effects of low oxygen con-
centrations on molting, growth rates, time of emergence and behavior
patterns were investigated.
This report summarizes the results of acute, short-term 96-hour tests
(TLm96) used in screening 20 species of aquatic insects to determine
their relative sensitivity to low oxygen concentrations. In addi-
tion, the longer term effects of low oxygen levels on the survival,
molting, growth, time of emergence, and behavior patterns of 21
species are considered. The 96-hour TLm (Standard Methods, 1965)
was used as a measure of survival' in the tests. This report encom-
passes work conducted at the University of Montana Biological
Station during 1968-70 and at the University of Utah in 1966, 1970-
71.
Materials and Methods
The organisms used in the tests were all insects except for one
species of Amphipoda. All organisms were collected from streams and
ponds in northwestern Montana and in northern Utah. Care was taken
to ensure that the organisms for a test were all collected from the
same area at the same time. The specimens were kept in well oxy-
genated holding tanks for three days prior to testing. Only speci-
mens of the same age group were utilized. These were generally of
the oldest year class present. Test procedures were those outlined
in Standard Methods (1965).
De-oxygenated water was obtained from degassing equipment as des-
cribed by Mount (1965). Modifications included a cooling system
and an oxygen "ladder." The ladder is constructed of single pane
glass and cemented with silicone sealant. The ladder is 5~l/2 feet
long, 7 inches wide and 7 inches deep. It is divided into 15 com-
partments each separated by a glass partition 2 inches high. The
remainder of the divider is composed of fiberglass screen with a
1 mm mesh opening.
The de-oxygenated water comes from the degasser through plastic tub-
ing, passes through the cooler and then enters one end of the ladder
which is elevated above the outlet end. As the water flows over the
2-inch compartment dividers its oxygen content increases. Rates of
increase are dependent upon rate of inflow and the angle of inclina-
tion of the ladder. At an inclination of 40° from the horizontal
-------
and a flow rate of 1000 cc/min the oxygen increase per chamber is
about 0.5 mg/1 at 10° C.
Ten organisms were placed in each of seven test chambers and
observed twice daily. Point of death was determined by lack of
response when stimulated. Small rocks were placed in the test
chambers to which the organisms could cling.
The flow rate was checked weekly and varied plus or minus 25 cc/min.
The temperature was taken daily with a pocket thermometer. A varia-
tion of plus or minus 0.5° C occurred. Oxygen concentration was
taken daily using the modified Winkler method, utilizing a 50 ml
sample. Variations of plus or minus 0.2 mg/1 occurred.
Water used in the tests at the Biological Station was unchlorinated
well water with the following chemical composition: pH 7.8; total
hardness, 135 mg/1; temperature, 6.4° C; turbidity, 0-5 J.T.U.;
carbon dioxide, 1-2 mg/1.
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SHORT-TERM (ACUTE) BIOASSAYS CONDUCTED AT UNIVERSITY
OF MONTANA BIOLOGICAL STATION
Results
Nineteen species of aquatic insects and one species of Amphipoda
were studied to determine their 96-hour median tolerance limit
(TLm). Eight species of Plecoptera were tested. The mean TLm for
this group was 3.04 mg/1 of oxygen. Acroneuria pacifica Banks had
the lowest Tim, 1.6 mg/1 at a flow rate of 1000 cc/min (Table 1).
The highest TLm was obtained with Pterpnarcys^ californica Newport
(3.9 mg/1) at a rate of 500 cc/min~The TLm for this species
decreased to 3.2 mg/1 at a flow of 1000 cc/min. All of the speci-
mens of Arcynopteryx para 11lei a Prison survived at oxygen concentra-
tions of 2-5.00 mg/1 at a flow of 1000 cc/min. All of the test
species were stream forms.
Four species of mayflies (Ephemeroptera) were examined. Two
species were lotic forms, Hexagenia limbata Guerin and Callibaetis
montanus (Eaton). Their TLm's were 1.8 mg/1 and 4.4 mg/1 respec-
tively. The lentic forms tested were Ephemerella doddsi Needham
and Ephemerella grandis Eaton, with D.O. values of 5.2 mg/1 and
3.0 mgTT respec11ve1y. The mean for the group was 3.6 mg/1.
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Table 1
Test Organisms, TLm in mg/1, Per Cent Saturation and Water Flow
in cc/min
Organi sms
PLECOPTERA
Acroneuria pacifica Banks
Arcynopteryx aurea Smith
Arcynopteryx parallela Prison
Diura know!toni (Prison)
Nemoura cinctipes Banks
Pteronarcys californica Newport
ii ii it
Pteronarcella bad?a (Hagen)
TLm Saturation
Flow
EPHEMEROPTERA
Callibaetis montanus
Eaton
Ephemerella doddsi Needham
Ephemerella grand is Eaton
Hexagon ia 1imbata Guerin
TRICHOPTERA
Brachycentrus occidental is Banks
Drusinus sp.
Hydropsyche sp.
Lepidos toma sp.
Limnephi1 us ornatus Banks
Neophylax sp.
Neothremma alicia Banks
DIPTERA
Simulium vittatum Zetterstadt
AMPHIPODA
Gammarus
1.6
3.3
100%
Survival
3.6
3.3
3.9
3.2
2.4
4.4
5.2
3.0
1.8
30%
Survival
1.8
3.6
80%
Survival
3.4
3.8
1.7
14
29
2-5 mg/1
32
29
34
28
21
38
46
27
15
2-4 mg/1
15
32
3-4 mg/1
30
33
14
1000
1000
1000
500
1000
500
1000
1000
500
500
1000
1000
500
1000
500
1000
500
500
500
limnaeus Smith
3.2
80%
Survival
28
3 mg/1
500
500
10
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Seven species of Trichoptera were tested and all were from lentic
environments. Several of these organisms could not be identified
to the species level. Ninety percent of the specimens of Brachy-
centrus occidental is Banks survived at oxygen concentrations of 2-4
mg/1 and a flow rate of 500 cc/min. Neothremma a 1icia Banks, a small
species (5 mm), had the lowest TLm of 1.7 mg/1. Neophylax sp. had
the highest TLm of 3.8 mg/1. The mean for the entire group was 2.86
mg/1.
One Dipteran was tested (Sjmulium vittatum letterstadt) and had a
TLm of 3.2 mg/1. One Amphipoda was examined (Gammarus limnaeus
Smith) with a survival of 80% at 3 mg/1 of oxygen and a flow rate
of 500 mg/1.
The mean TLm for all organisms tested was 3-1 mg/1. The mean for all
organisms tested at a flow of 1000 cc/min was 2.55 mg/1 and 3.64 mg/1
at a flow of 500 cc/min. The lowest TLm recorded was 1.6 mg/1 for
Ac roneur i a pa c i f i ca, or 14% oxygen saturation. The highest TLm was
5.2 rag/1 for Ephemerella doddsi. or 46% oxygen saturation.
Discussi on
Of the organisms tested the group most tolerant to low dissolved
oxygen (D.O.) values was the Trichoptera (2.86 mg/1). All of the
Trichoptera tested, except Hydropsyche, were cased forms and all came
from lentic environments. All the organisms except Drusinus sp. were
tested at a flow rate of 500 cc/min. Higher flow rates would probably
reduce the Tim of many of the forms.
Acroneuria pacifica, a predacious stonefly, was the most resistant
formtested with a TLm of 1.6 mg/1 (14% saturation). The largest
organism tested, P te ron a r cy s ca1i f o r n 1 ca , showed a decrease in TLm
as the flow rate increased (3.9 mg/1 to 3.2 mg/l).
The mayfly, Ephemerella doddsi, had the highest TLm of 5.2 mg/1 (46%
saturation) at 500 cc/min"! fiTis species is found in fast streams
attached to rocks.
It has been shown by Knight and Gaufin (1963, 1964) that rate of water
flow is very important in determining tolerance limits. This was
again demonstrated by Pteronarcys californica as did the ranges and
means for the flow rates"! The TLm range for 11 species tested at
500 cc/min was 1.7 mg/1 to 5.2 mg/1, with a mean of 3.64 mg/1. At
1000 cc/min the range for 10 species was 1.6 mg/1 to 3-3 mg/1 with a
mean of 2.55 mg/1, a substantially lower value.
Behavior of the organisms during testing was of interest. All of
the Plecoptera initiated "push-up" movements upon introduction to the
test chambers. Most species ceased this motion after several hours
but Pteronarcys californica continued these movements periodically
11
-------
Pteronarcys californica also assumed a posi-
Nemoura
>narcys
in the
throughout the test.
tfon half out of the water in the low oxygen chambers.
cinctipes assumed a stilted position upon death.
Number of gill beats per unit time was indicative of oxygen concen-
tration. Gill beats in Ephemerella grand is were counted after 12
hours in the test chambers and results are given in Table 2. Each
value is the mean number of beats for the ten organisms in each
chamber.
Table 2
Gill beats/minute for Ephemerella grand is Eaton
Oxygen cone.
2.k mg/1
3.0 "
3.6 "
k.6 "
5.0 "
6.0 "
Beats
176
132
192
184
160
100
Rhythm
steady
ii
erratic
Except at the lowest D.O. concentration, the gill beat decreased as
the oxygen increased. The rhythm of gill beats also became erratic
as the oxygen increased.
The high TLm of the pond mayfly, Callibaetis montanus, was surprising.
It had the second highest TLm of all species tested (k.k mg/1). Another
lotic species Hexagenia limbata had a low TLm of 1.8 mg/1.
probably be explained by its acclimation to lower oxygen
tions in its normal environment.
This could
concentra-
in response to the low oxygen values the Trichoptera undulated their
abdomens in their cases. Simulium vittatum congregated on the chamber
walls where the flow was the greatest. Gammarus limnaeus showed no
behavioral response to the low oxygen values
12
-------
96-Hour TLm Results
Oxygen
100
80
3
CO
20-
1000 cc/min
Acroneuria pacifica
D.O. 123^567
mg/1
* Replicate tests
100
80.
5 60J
20
D.O. 1
mg/1
1000 cc/min
Arcynopteryx aur_ea_
Figure 1
13
-------
ID
CO
96-Hour TLm Results
Oxygen
1000 cc/min
Arcynopteryx parallela
100..
8a
60-
40-
20-
D.O.
mg/1
\ i
'5 '6 >
100-
SO-
ls 60.
3
co
20.
D.O.
mg/1
1000 cc/min
Pteronarcella bad I a
12
-------
96-Hour TLm Results
Oxygen
100 -I
80
> 60
>
i_
3 i _
20
D.O.
mg/1
1000 cc/min
Pteronarcys californica
/
23^567
* Repl Icate tests
1000 cc/min
Nemoura cinctipes
100 _
so ;
^ 60
>
>
3 ko
Ij) *TW
20-
D.O
mg/
/*
/
/
i ii i i i (
j 123^567
15
-------
96-Hour TLm Results
Oxygen
ro
>
°>
>_
n
CO
*e
ioa
80
60
40
20
D.O.
mg/1
1000 cc/mln
Hexageni a limbata
**
3
Vt
a*
loo., ^
80~
60_
4d
20-
D.d
mg/
.-^ ^ _/ *
/ / *
/ / ^^
/ /
.^-"'
j '1 2 3 i» 5 '6 7
* Replicate tests
1000 cc/min
Ephemeral la grand is
* Replicate tests
16
-------
96-Hour TLm Results
Oxygen
to
3
CO
100-
80!
60.
40.
20-
1000 cc/min
Lepidostoma sp.
D.O.
mg/1
100.
80-
60-
40.
20.
1000 cc/m?n
Drusinus sp.
T
3
T r
6 7
D.O.
mg/1
T"
2
17
-------
ID
3
to
96-Hour TLm Results
Oxygen
100-
80.
60.
40.
20-
D.d
mg/1
500 cc/min
Pteronarcys californica
'123456
* Replicate tests
100-
80
| 6^
20
D.O.
mg/1
500 cc/min
Diura know1 toni
18
-------
96-Hour TLm Results
Oxygen
500 cc/min
Ephemerella doddsi
_
"m
*>
3
CO
a*
100
80.
.
60.
40.
20-
D.(
/ *
/I
.,* 0
.^s'
f /
//
/ I
r/
1 *T T-* r r r
3. 1 2 3 H 5 6 7
mg/1
* Replicate tests
0!
100-
80.
60.
20-
D.O.
mg/1
500 cc/min
Callibaetis montanus
: 3 A 5 6
* Replicate tests
19
-------
10
3
CO
100
80
60
40.
20.
D.O.
mg/1
96-Hour TLm Results
Oxygen
500 cc/min
Limnephilus sp.
i '3 4 5 I F
* Replicate tests
to
100^
80
60
40
20
500 cc/min
Hydropsyche sp.
1 r
D.O. I 2
mg/1
* Replicate tests
20
-------
(0
3
CO
96-Hour TLm Results
Oxygen
500 cc/min
Neothremma sp.
100-,
80-
6a
40
20
CO
3
CO
CO-
10Q
80
6G
21
D 0.
mg/1
D.O. 1
mg/1
500 cc/min
Neophylax sp.
21
-------
96-Hour TLm Results
Oxygen
-------
96-Hour TLm Results
Oxygen
500 cc/min
Gammarus limnaeus
100
8o4
(0
> 60J
20
D.O. 1
mg/1
r
2
23
-------
LONG-TERM BIOASSAYS CONDUCTED AT THE
UNIVERSITY OF MONTANA BIOLOGICAL STATION
AND THE
UNIVERSITY OF UTAH
Results
Eight species of aquatic insects from northwestern Montana were
studied to determine their tolerance levels and behavior patterns
when exposed to low oxygen levels over longer periods of time than
96 hours. Five of these species and an additional 13 species from
northern Utah were also tested for periods of time ranging from 4
to 104 days to determine their long-term reactions (Tables 3,4,5).
Table 3
Long-Term Dissolved-Oxygen Bioassays Conducted at University
of Montana Biological Station
Species
PLECOPTERA
Pteronarcella bad!a (Hagen)
Pteronarcys ca1i forni ca Newport
Arcynopteryx aurea Smi th
Acroneuria pacifica Banks
EPHEMEROPTERA
Ephemerella grand is Eaton
TRICHOPTERA
Brachycentrus occidental is Banks
Hydropsyche sp.
DIPTERA
Atherix variegata Walker
AMPHIPODA
Gammarus limnaeus Smith
Minimum
D.O. level
(mg/1)
4.4
4.8
k.B
5.8
4.6
3-2
4.8
2.4
2.8
Survival
50%
40%
30%
50%
30%
50%
30%
90%
50%
Survival
time
(days)
69
97
12
111
30
120
50
40
20
Flow rate of 1000 cc/min
-------
Table 4
Long-term Dissolved-Oxygen Bioassays Conducted at the
University of Utah (50% + Survival)
Minimum Survival
D.O. level % time
Species _ (mg/1) Su rv i va 1 (days)
PLECOPTERA
Acroneuria pacifica Banks 3.0 50% 24
Brachyptera nj jj[nj?_enn i s (Banks) 2.3 60% 4
Isoperla fTTlva Claassen 2.3 50% 13
EPHEMEROPTERA
Ephemerella grand is Eaton 3.3 50% 18
Rhithrogena rpbusta Dodds 3.3 50% 7
TRICHOPTERA
Brachycentrus occidental is Banks 2.6 80% 91
Rhyacophila sp. I.1* 50% 45
ArctopsycTie" grand i s (Banks) 3.1* 50% 26
Parapsyche eisis Milne 5.2 60% 30
D I PTERA
Atherix variegata Walker 2.4 90% 97
Holorusia sp. 2.0 60% 86
0 DO NAT A
Argia vivida Hagen 3.0 50% 56
EnaTlagma anna Williamson 1.4 50% 21
Flow rate of 1000 cc/min
25
-------
Table 5
Long-Term Dissolved-Oxygen Bioassays Conducted
at the University of Utah (Minimum D.O. with Survival)
Minimum Survival
D.O. level % time
(mg/1) Survival (days)
Species - * - - '
PLECOPTERA
Acroneuria pacifica Banks 3.0 20% k\
Arcynopteryx parallela Prison 3-4 10% 8
Brachyptera nigrlpennis (Banks) 3'. 7 20% 9
Isoperla fulva Claassen 2.1 10% 27
Pteronarcella bad! a (Hagen) 2.0 30% 30
EPHEMEROPTERA
Baetis bicaudatus Dodds 3.8 10% 3
EphemerelTa grand"! s Eaton 3-5 50% 21
TRICHOPTERA
Parapsyche el sis Milne 4.8 40% 16
DIPTERA
Atherix variegata Walker 1.7 70% 90
Bibiocephala sp. 3-4 *»0% 21
ODONATA
Argia vivida Hagen 1.7 10% 100 days
EnaTTagmTanna Williamson 1.1 20% 35 days
Flow rate of 1000 cc/min
26
-------
Table 6
Average Minimum Dissolved Oxygen Requirements
of Different Groups of Aquatic Invertebrates*
Average Average
Montana survival Utah survival
species (days) species (days)
Plecoptera 4.9 mg/1 62 2.8 mg/1 1^
Ephemeroptera 4.6 mg/1 30 3.3 mg/1 10
Trichoptera 4.0 mg/1 85 3.1 mg/1 48
Diptera 2.4 mg/1 40 2.2 mg/1 92
Odonata 2.2 mg/1 39
Amphipoda 2.8 mg/1 20
* Averages based on 50% + survival for time indicated.
The results of the longer term bioassays clearly indicate increased
sensitivity and mortality of test specimens with increased length
of exposure to low oxygen levels. For example, while 50% of the
specimens of Ac roneur i a pacifica in Montana survived an oxygen con-
centration ofT.fc mg/1 for 4 days, the minimal dissolved oxygen
level for $0% survival at 111 days was 5.8 mg/1. Similarly, 50%
of the specimens of Arcynopteryx aurea survived in an oxygen concen-
tration of 3.3 mg/1 for 4 days but only 30% survived at a dissolved
oxygen level of 4.8 mg/1 for 12 days. This increased sensitivity
can be explained partly on the basis of physiological reactions such
as debilitation due to lack of food and fungus infection. For example,
60% of the larvae of the crane fly, Holorusia sp., survived for 86
days at a dissolved oxygen level of only 2.0 mg/1. Shrinkage of the
bodies of the larvae due to starvation and infection with fungus
caused a rapid die-off after 86 days.
Of the eight species of aquatic insects tested at the Biological
Station the carnivorous stonefly, Acroneuria pacifica, had the
highest TLm with a 50% death rate at an oxygen level of 5.8 mg/1
for 111 days. The most tolerant species was the Dipteran, Atherix
variegata, with 90% of the specimens surviving for 40 days at an
oxygen concentration of 2.4 mg/1. This species was also the most
tolerant of the Utah forms tested with 90% of the specimens surviving
at the same oxygen level for 97 days. The higher oxygen requirement
of Acroneuria pacifica under long-term conditions may be partially
27
-------
due to its food requirements. Inasmuch as this species is carni-
vorous, lack of a varied animal diet may have reduced its ability
to tolerate low oxygen levels for extended periods of time.
A comparison of the long-term median tolerance limits of the same
species of aquatic insects from Montana and Utah shows considerable
variation. Fifty percent of the specimens of the stonefly, Acroneuria
pacifica, from Montana died at a dissolved oxygen level of 4.4 mg/1
in 69 days. The same percentage of Utah specimens survived at a much
lower dissolved oxygen concentration, 3.0 mg/1, but for only 24 days.
A mayfly, Ephemere11 a grandis, was tested from both Montana and Utah
with similar results. Thirty percent of the Montana specimens sur-
vived at a dissolved oxygen level of 4.6 mg/1 for 30 days while fifty
percent of the Utah specimens survived at a dissolved oxygen concen-
tration of 3-3 mg/1 but for only 18 days. The differences in toler-
ance limits between the same species may have been much less if the
tests had been conducted under exactly the same conditions in the
two locations. Time did not permit this being done, so it was decided
to run the Utah tests at lower oxygen levels in order to determine
maximum survival rates at these much lower oxygen limits.
An evaluation of the average minimum dissolved oxygen requirements of
the different groups of aquatic invertebrates tested shows the may-
flies to be most sensitive, stoneflies next, and the caddis flies,
fresh water shrimp, true flies, and damselfly, following in that
order. While two species of mayflies could tolerate as low a dis-
solved oxygen concentration as 3.3 mg/1 for 10 days, a levet of 4.6
mg/1 was required for 50% survival at 30 days. Three species of stone-
flies from Utah survived at a dissolved oxygen concentration of 2.8
mg/1 for 14 days with 50% surviving, but an average oxygen concentra-
tion of 4.9 mg/1 was required for 30-50% survival for 62 days. The
caddis flies tested also indicated higher oxygen levels were necessary
with longer exposure with a minimum of 4.0 mg/1 being required for 50%
survival for 84 days.
The true flies, fresh water shrimp, and damselflies displayed a much
greater tolerance than the previous three groups to low oxygen levels.
Fifty percent of the specimens of these three groups were able to sur-
vive at dissolved oxygen levels ranging from 2.2 to 2.8 mg/1 for
periods ranging from 20 to 92 days.
While the principal objective of this project was to determine the
minimal dissolved oxygen levels required for both short and long-term
exposure, mere survival without growth and metamorphosis occurring
would eliminate a species of aquatic insect eventually. While not
all of the species tested molted or emerged during the study, many
species did. All of the species on which bioassays were run for
over 30 days molted one or more times at the oxygen levels required
for 50% survival. Species such as the stoneflies, Brachyptera nigri-
penms»
Ephemerella grandis, and the damselfly, Enallagma anna, emerged
Pteronarcys cal ifornica^, and Pteronarcel la badja; the may
ams
28
mgn-
tayfly,
-------
during the tests at oxygen concentrations of 4.8 mg/1 or below. None
of the caddis flies or Dipterans emerged inasmuch as only larvae and
not pupae were used for testing purposes.
Discussion
Dissolved oxygen is an aquatic constituent which is rarely avail-
able in excess at all times. Many aquatic animals possess varied
adaptations which facilitate the acquisition of oxygen when it
becomes scarce. Diffusion, along with special ventilation mechanisms,
provide extensive absorbing surfaces, in the case of stoneflies, for
the absorption of oxygen from the environment. An adaption utilized
by the nymphs of Pte rona rcys ca1if o r n ica, when environmental oxygen
becomes reduced, i s Txxfy undu1 at i ons wh i ch attempt to destroy the
oxygen gradient that develops around the body and gills. Of particu-
lar interest is the variation in the rate of these undulatory move-
ments with year class. The undulations of the smaller nymphs of
this species (year I, 17-18 mm long), in studies conducted at the
University of Utah in 19&3-65, were more rapid than that of the
larger (year II, 30 mm long).
The respiratory mechanism possessed by different species of aquatic
insects greatly influences their ability to withstand low oxygen
concentrations. In work conducted by Knight and Gaufin (1966) at the
University of Utah the value of gills in enabling some species to
better withstand low dissolved oxygen levels was clearly demonstrated.
The nymphs of Pteronareel la bad i a, Isoperla f u 1va, and Acroneuria
pacifica were al1 exposed to an environment of reduced dissolved
oxygen of 1.0 cc/1 and water flow of 0.004 feet/second, at 10° C.
The forms possessing gills exhibited quite similar mortalities during
the exposure period. Pteronarcella badia nymphs exhibited a 13 per-
cent mortality after 2k hours and 48 hours of exposure, and 29 per-
cent at the end of 72 hours, with no further mortality for the remain-
der of the exposure period. Ac roneur i a pac i f i ca showed the same mor-
tality as Pteronareel la badia after 72 hours of exposure. After 96
hours exposure Acroneuria pacifica displayed a 25 percent mortality.
No further mortality was noted for the remainder of the experimental
period. Eighty percent of the Isoperla fulva nymphs, a species with-
out gills, died within 2k hours. After 144 hours of exposure all
had succumbed. The increased mortality shown by the Isoperla fulva
nymphs may have been due to their smaller size and the fact that they
were year class I, as opposed to year class II in the gilled forms.
Isoperla fulva has only a one-year life cycle so it was impossible
to compare, nymphs of similar size.
In view of the above a second evaluation was carried out comparing
nymphs of Acroneuria pacifica (gills) to those of Arcynopteryx
parallela (no thoracic gill~s~). The nymphs were tested at a tempera-
ture of 15.6° C with a water flow of 0.25 feet/second and a dissolved
29
-------
oxygen concentration of 1.0 cc/1. The nymphs of both species were
between 25 and 30 mm in length. In general, the results of this
test, as in the case of the previous one, indicated that forms which
lack gills are more sensitive to reduced dissolved oxygen than forms
possessing gills. No mortality of Acrpneurla pacifica nymphs occurred
during the experimental period while nymphs of Arcynopteryx parallela
showed an 82 percent mortality after 10 hours of exposure and 88.5 per-
cent mortality at the end of 2k hours. After 3*> hours of exposure all
the nymphs were dead.
The metabolism of poikilotherms rises with temperature about two and
one-half times per 10° C change in temperature (Prosser and Brown,
ibid.). With this metabolic increase in response to increased environ-
mental temperature, increased oxygen consumption results. The increase
in oxygen consumption with increased water temperature would cause an
aquatic insect subjected to the higher temperature (15.6° C) to incur
an oxygen debt at a higher dissolved oxygen concentration than one
subjected to a similar situation except exposed to a reduced tempera-
ture (10° C). Stoneflies, mayflies, and caddis flies do not have an
apparent ability to get along without oxygen for an extended period.
They do survive for a short period in greatly reduced oxygen by
greatly reducing their activity, and they use energy apparently pro-
duced by the anaerobic phase of glycolysis. If the oxygen supply is
not restored within a certain time, the specimens die from asphyxia-
tion.
In the work conducted to date by the author and his colleagues there
has been a great difference in the dissolved oxygen concentration at
which initial mortality of test organisms was recorded. This differ-
ence was greatly influenced by the temperature difference in the
experimental environment. In a natural situation resulting in the
gradual reduction of dissolved oxygen over a short period of time due
to intermittent discharges of organic oxygen-demand ing wastes, the
onset of stonefly mortality would be influenced by the existing water
temperature. Providing the water flow and other variables remained
constant, one could expect the aquatic insects subjected to an environ-
mental temperature of 10° C to withstand reduced oxygen concentrations
about 2.k times lower than similar specimens exposed to a water tem-
perature of 15.6° C. In a hypothetical situation, based on the work
of Knight and Gaufin (1966), a stream possessing a temperature of
15.6° C and a dissolved oxygen concentration of 0.6 cc/1 would have
a stonefly mortality of 18 percent while a stream similar in all
respects except possessing a water temperature of 10° C would
exhibit 100 percent survival. Thus the water temperature of a
stream is a very important factor in the survival of aquatic insects
when they are subjected to a reduction in dissolved oxygen over a
short period of time.
The rate of water flow in a stream also is a very important factor
to be considered in the survival of aquatic insects when they are
30
-------
exposed to low oxygen concentrations. Knight and Gaufin (1966)
showed that a gradual reduction of dissolved oxygen with water
flow of 0.06 ft/sec produced an approximate 50 percent stonefly
mortality while a similar situation provided with a water flow of
0.25 ft/sec resulted in 100 percent survival.
In the present study the mean oxygen concentration required for
50% survival by 11 species of aquatic insects at a flow rate of
500 cc per minute was 3.64 mg/1. The mean for 10 species at a
flow rate of 1000 cc per minute was considerably lower or 2.55 mg/1
31
-------
STUDIES ON THE
TOLERANCE OF AQUATIC INSECTS TO HEATED WATERS
Introduction
By 1980, it is estimated that around 200 billion gallons of cooling
water will be needed daily, about one-sixth of the nationwide annual
runoff, to meet projected steam electric power station needs based on
once-through cooling (Pitcon, I960). Water used for cooling purposes
in industrial processes may be so hot and in such quantity that it
may substantially raise the temperature of a receiving stream. Limited
quantities of warm water, however, may produce desirable changes in
selected localized situations. The. requirements of the organisms in
a stream must be known before realistic water quality standards can
finally be adopted for their protection.
Literature concerning the effects of heated waters on aquatic insects
is limited in extent and comparability. The effects of heated efflu-
ents on aquatic life have been reviewed in two recent comprehensive
bibliographies, Mihursky and Kennedy (196?) and Raney and Menzel
(1967). The effects of heated discharges on water quality and assimi-
lation, aquatic organisms, and water uses have been thoroughly reviewed
by Parker and Krenkel (1969). The temperature requirements of fish
and other aquatic life were reviewed by Tarzwell (1968). Nebeker and
Lemke (1968) tested the relative sensitivity of twelve species of
aquatic insects to heated water in the laboratory. The lethal tempera-
ture at which 50% of the test specimens died after 96 hours exposure
(TLm96) ranged from 21 C for winter stoneflies to 33 C for dragon-
flies. An excellent review of temperature effects on aquatic insects
was presented by Tremblev (1965). Studies conducted by the Philadelphia
Academy of Science (1968) on the effects of heated water on the insect
fauna of the Potomac River have shown significant reductions in the
diversity and numbers of organisms below a steam electric power plant.
Coutant (1962) found substantial reductions in the volume and numbers
of macroinvertebrates in the Delaware River in sections receiving
heated water.
This section of the report summarizes the results of acute, short-term
96-hour tests (TLrn^) used in screening 15 species of aquatic insects
to determine their relative sensitivity to heated water. The 96-hour
TLm (Standard Methods, I960) was used as a measure of effect in these
tests. Long-term studies dealing with the effects of temperature on
the reproduction, molting, emergence patterns, feeding rates, and long-
term survival of aquatic insects were also conducted and will be con-
sidered in subsequent pages.
32
-------
Materials and Methods
Test chambers consisted of oblong stainless steel tanks 90 cm long,
18 cm wide, and 17.5 deep. Similar tanks were utilized by Nebeker
and Lemke (1968) in their studies on the tolerance of aquatic insects
to heated waters at the National Water Quality Laboratory at Duluth,
Minnesota. Fiberglass screening was employed to subdivide the tanks
into three test cages 15 cm long, 17.5 cm wide, and 11 cm deep. Rocks
were placed at the bottom of each cage to form a natural substrate for
the aquatic organisms. The fresh water source was introduced at the
forward end of the tank, which gradually slopes 7.5 cm to the over-
flow drain.
Five chambers were employed for temperature testing and one for a
control, with the control maintained at the initial acclimation
temperature. The oblong tanks were used as artificial streams
where various water flows could be maintained with a stream of water
and with paddle wheels.
The water used for all testing and for the holding tanks was obtained
from the University of Montana Biological Station water system. This
water originates in a spring, is chlorine-free, and has a constant
temperature of 6.4 C* 0.1 C. The pH is 7.8- 0.1. Total hardness is
near 135 ppm and the CO2 varies from 1 to 2 ppm (C02 and total hard-
ness expressed as ppm of CaCOj). The dissolved oxygen level is con-
sistently 100% of saturation or higher.
The test organisms, except for species of Simu1iurn, Hexagenia, Atherix,
and Gammarus, were collected from Rock Creek, a trout stream located
southeast of Missoula, Montana. Simulium and Hexagenia were collected
from Mud Creek, a slow flowing meadow creek, Atherix from the Clark's
Fork of the Columbia River, and Gammarus from a spring-fed pond near
Bigfork, Montana. All test organisms were mature larvae. The test
organisms were placed in large, vigorously aerated, fiberglass hold-
ing tanks for a minimum of three days prior to testing. Fresh water
was added at a rate of 3 to 5 liters per minute to insure a constant
temperature and a fresh water supply.
Desired temperatures in the test chambers were obtained by manual
regulation of mixing faucets. Temperatures were allowed to stabilize
over a period of 2k hours to insure uniformity. If the system remained
stable during this 24-hour period, the test was initiated.
Experimentation began with an initial series of temperatures usually
ranging from 10 to 25 C. The specimens were placed in an aerated
water bath, and the temperature gradually raised (2 to 4 C per hour)
to the appropriate test temperature before they were transferred to
the appropriate test chambers. This procedure was followed to insure
against nebulous results induced either by thermal "shock" from immedi-
ate transfer from one temperature to another or by the complete accli-
mation that can accompany a very gradual increase in temperature.
33
-------
In the test chambers the paddle wheels created a turbulence and
helped maintain a dissolved oxygen level of 100% saturation or
higher. A liberal fresh water supply was provided (at least 2
liters per minute) for the removal of toxic waste. Temperature
values were taken at least four times daily and if any value varied
by more than 0.5 C, the test was discarded. If any of the control
organisms died, the test was terminated.
The temperature at which 50% of the organisms died was obtained by
a modification of the straight line graph interpolation method as
outlined in Standard Methods (I960).
Results
Late instar larvae of 15 species of aquatic insects and one species
of amphipod were tested to determine their tolerance of high water
temperatures. A marked difference in sensitivity was apparent
(Table?) in the different species. A mayfly, Cinygmula par Eaton,
died at 11.7 C and was the most sensitive of all the species tested.
This species is found in very cold clear mountain streams in Montana.
The fresh water shrimp, Gammarus limnaeus Smith, proved to be sur-
prisingly sensitive to temperature increases, exhibiting a 96-hour
TLm of only 14.5 C. Ephemeral la doddsi Needham, a small, widely
distributed mayfly characteristic of cold turbulent streams in the
Intermountain Region, was also very sensitive with a TLm value of
15.4 C. A lotic species of mayfly, Hexagenia limbata Guerin, was
much more tolerant than other mayflies tested with a TLm of 26.6 C.
Considerable difference in susceptibility to temperature increases
existed between the three species of stoneflies tested. Isogenus
aestivalis (Needham and Claassen) was quite sensitive, 50% dying at
16 C, while Pteronarcella badia (Hagen) and Pteronarcys callfornica
Newport, two closely related species, survived increases to 24.6 and
26.6 C respectively. Six species of caddis flies were tested and
clearly reflected thermal differences in their habitat requirements.
Parapsyche el sis Milne, which is largely restricted to cold, fast
flowing mountain streams, had a TLm of 21.8 C while Hydropsyche sp.
taken from a slow flowing stream draining a marshy lake was very
tolerant with a TLm of 30.1 C. Atherix variegata Walker, the snipe
fly, was the most tolerant of all species tested with a TLm of
32.6 C. No dragonfly or damselfly nymphs were tested because a
thick ice and snow cover coating their habitats early in the winter
prevented collecting large enough numbers for testing purposes.
Discussion
The rate of development and the time of emergence of aquatic insects
is directly influenced by the temperature. An increase in water
temperatures in the winter above 5 C might completely eliminate
34
-------
winter stoneflles belonging to the family Capniidae.
Many species of stoneflies, mayflies, and caddis flies emerge in
late spring before stream temperatures reach high summer levels.
An artificial increase in stream temperatures during the winter
would very likely cause these species to develop more rapidly,
emerge earlier, and be killed by cold air temperatures, and may
substantially reduce the population or eliminate the species.
The stonefly Isogenus aest i va1i s and mayfly Cinygmula par are
largely restricted to clear, cold water streams in the Intermoun-
tain Region and even a slight increase in water temperature may
have an adverse effect on their survival. By comparison the
snipe fly, Atherix variegata, is often found in open sections of
streams which warm up during the summer months and this species
is decidedly temperature tolerant.
Two of the species of stoneflies tested, Pteronarcella badia and
Pteronarcys californica, are common in medium to large streams
in the western United States and are comparatively temperature
tolerant. These species require two and three years respectively
to complete their life cycle and have become adapted to the warmer
waters of late summer which many aquatic insects avoid by emerging
in the spring.
35
-------
Table 7. Temperatures (°C) at which 50% of the test species died
after 96 hours exposure (TLrrp"), Bigfork, Montana, 1968-69
Species tested
OIPTERA
Atherix variegata
Walker
Simul ium sp.
TRICHOPTERA
Parapsyche els is
Milne
Limnephilus ornatus
Banks
Neothrema alicia
Banks
Drusinus sp.
Brachycentrus occ i denta 1 i s
Banks
Hydropsyche sp.
PLECOPTERA
Isogenus aestivalis
(Needham and Claassen)
Pteronarcel la badia
(Hagen)
Pteronarcys californica
Newport
EPHEMEROPTERA
Cinygmula par
Eaton
Ephemeral la doddsi
Needham
Ephemerella grand is
Eaton
Hexagenia limbata
Guerin
AMPHIPODA
Gamma r us limnaeus
Test 1
32.6
25.0
21.8
24.5
25.8
27.2
29.7
30.0
16.0
24.4
28.0
11.7
15.4
21.5
26.1
14.5
Test 2
32.2
25.2
21.6
25.0
26.0
27.4
* *
30.1
16.3
24.6
26.4
*
15.5
* *
27.1
14.6
Mean Average
Test 3 TL^ group TLm
32.4
25.1
.... 21.7
24.75
25.9
.... 27.3
.... 29.7
30.05
16.15
24.2 24.4
26.6 27.0
11.7
15.45
21.5
.... 26.6
14.55
28.7
26.5
22.55
18.82
14.55
Smith
36
-------
100
03
1 50
3
to
0
~\ \
\ \
\ ^ i
\
\ l
\ I
5 10 1*5 20 25 30 35
0 C
96
Ephemerella doddsi , dashed line, TLm = 15-5 C
Ephemerella grandis, solid line, TLm5 = 21.5 C
100J
50-
>
-1_
3
\
V
'
51 13]%ib125130
0 c
35
Pteronarcella badia, dashed line, TLm9 = 2A.6 C
Pteronarcys californica, solid line, TLm9 = 26.6 C
Fig. 2 Straight-line interpolation graphs of representative TLm
96's
37
-------
LONG-TERM THERMAL BIOASSAYS
CONDUCTED AT BIOLOGICAL STATION
Long-term tests were conducted at the Biological Station through
March, 1970, at which time a breakdown in the heating system necessi-
tated transferring the work to the University of Utah. In the work
conducted in Montana a species of stonefly, Pteronarce11 a bad i a, was
most sensitive with 50% of the test spec i mens succumb i ng to a tempera-
ture of 18.1° C in 2k days. Brachycen t rus occ i denta1i s, a case making
caddis fly,was least sensitive withstanding a temperature of 26° C for
45 days. The sensitivity of the former species to longer term exposure
was a decided contrast to its tolerance of temperatures as high as
24.6° C for short-term exposures. Since the specimens involved in the
longer term tests were collected during the winter months, it is pos-
sible that acclimation to low winter temperatures increased the sen-
sitivity of the specimens tested. The tolerance of the four species
tested is summarized in the following Table 8.
TABLE 8
Long-Term Thermal Bioassays
Biological Station (Thru March 23, 1970)
50% Survival
Species 24 days 30 days 25 days 45 days 12 days
Pteronarce11 a bad i a 18.1° C
(Hagen)
Pteronarcella badia 20.5° C
(Hagen)
Pter onarcys ca1i forn ?ca 20° C
Newport
Brachycentrus occidental is 26° C
Banks
E ph erne re11 a grandi s 21.5 C
Eaton
38
-------
Specimens of Pteronarcys californica clearly showed the effects of
exposure to higher temperatures on their developmental rate. This
species normally emerges in Montana streams in mid June. Three
specimens emerged on January 5, 1970, after being exposed to a
temperature of 18.4° C for 25 days.
STUDIES ON THE TOLERANCE OF GREAT
BASIN AQUATIC INSECTS TO HEATED WATERS
Acute, short-term 96-hour tests were also conducted at the University
of Utah during 1970 with 8 species of aquatic insects to determine
their relative sensitivity to heated water. Longer term studies
were also conducted to determine the long-term survival of 16 species
of aquatic insects and the effects of elevated temperatures on their
molting and emergence patterns.
Materials and Methods
Test chambers consisted of stainless steel tanks 36 inches long,
7 inches wide, and 7 inches deep. These were immersed in two large
refrigerated water baths. Eight of these tanks were used for screen-
ing temperatures ranging from 14.5° C to 29° C. A ninth tank was
used as a control with a temperature of 10° C. A stainless steel
700 watt National Appliance Company heater was placed in each tank
for raising the water temperature to the desired level. The tempera-
ture in each tank was controlled by a National Appliance Company
thermostatic unit. A paddle wheel was used for circulating the
water in each test chamber.
The water used for all testing and for the holding tank was obtained
from artesian wells which supply the University of Utah with culinary
water. The water is non-chlorinated and varies little chemically
throughout the year. The dissolved oxygen content varies between
7.0 to 9.0 ppm; COo between 0 - 1 ppm; pH 7.8 - 8.2; carbonates 0.0;
and bicarbonates 165.0 to 225.0 ppm.
The test organisms were collected from streams in the Wasatch and
Uintah Mountains within a radius of 50 miles from the University of
Utah. All organisms tested were mature larvae. The specimens were
maintained in a large, vigorously aerated, fiberglass holding tank
for a minimum of three days prior to testing. In conducting the
tests 20 specimens of each species were held in small fish breeder
nets suspended in each test chamber. A fresh water supply of approxi-
mately 2 liters per minute was provided for the removal of toxic
wastes. Temperature readings were taken several times daily with
any variation being maintained at * 1.0° C. A YS1 Model 47 Scanning
Tele-Thermometer was used for recording temperatures.
39
-------
Results
A marked difference in the sensitivity of the various species tested
was apparent in both the acute and long-term studies. The mayfly,
Ephemerella doddsi. died at 16.0 C in 96 hours and was the most
sensitive of the species tested. This value was close to the 96-
hour TLm of 15.4 C obtained for the same species in Montana. The
snipe fly, Atherix variegata, was the most tolerant species tested
with all specimens surviving for 96-hours at a temperature of 29.0
C. This corresponded to the 96-hour TLm of 32.6 C obtained with
Montana specimens. However, specimens of the stonefly, Isogenus
aestivalis. from Utah were much more tolerant than Montana specimens
with a 96-hour TLm of 2k.2 C in comparison with a 96-hour TLm of
16.1 C for the latter specimens. Acclimation to the colder tempera-
tures encountered in Montana streams may account for the difference.
The results of the 96-hour tests conducted at the University of Utah
are given in Table 9.
Long-term thermal bioassays were conducted with 16 species of aquatic
insects with all species showing increased sensitivity with time of
exposure. (Table 10). Bibiocephala grandis, a Dipteran, found only
in cold torrential streams of the Intermountain Region, was the most
sensitive species with only 60% survival after 3 days at a temperature
of 15 C. Atherix variegata, the snipe fly, and Brachycentrus occiden-
talis, a caddis fly, were the most tolerant with 50? of the specimens
surviving at 28 C for 46 days and H days respectively.
Emergence
The effect of elevated temperatures on growth rate and time of emer-
gence was clearly shown by the research conducted at the University
of Utah. Six species emerged in the laboratory prior to the natural
period of emergence found in the region. Five Plecoptera and one
Odonate emerged early in response to increased temperature. The
organisms were primarily affected by the length of the exposure
period and the temperature level. Each organism reacted in a pat-
tern dissimilar to the emergence of the other species.
Acroneuria pacifica began to emerge approximately three months prior
to its normal period. The first four specimens emerged on April 12,
1971, after 4 weeks at 18° C. A total of nine specimens emerged.
The first Arcynopteryx parallela emerged at 15° C on February 16,
1971, after being exposed for six weeks. Emergence commenced approx-
imately two months early. The activity increased in intensity with
the longevity of exposure. Twenty-three adults emerged over a nine-
week period.
40
-------
Arcynopteryx signata started to emerge five days after being sub-
jected to 18° C. The first adult appeared on April 2k, 1971,
approximately one month prior to normal emergence activity. A
total of nine specimens emerged over a period of 2,5 weeks.
After 2.5 weeks exposure to 20° C, Isoperla fulva began emergence
on April 6, 1971, Six specimens emerged over a five-week period.
Initial emergence of PteronarceHa bad?a occurred on February 2,
1971, at 15° C four months prior to the normal emergence period.
This organism was subjected to heated water for a period of 4
weeks prior to adults appearing. Emergence continued over a
fifteen-week period with a total of forty-seven specimens emerging
A raj a vivida began emerging on April 12, 1971, after 3-5 weeks at
24 C. Normal emergence in this region occurs in early June.
Twenty-nine adults emerged over a four-week period.
-------
Table 9. Temperatures (°C) at which over 50% of the test species
survived after 96 hours exposure (TLm^"), University of
Utah, Salt Lake City, Utah, 1970.
Species tested
PLECOPTERA
Acroneuria pacifica Banks
Isogenus aestivalis
(Needham and Claassen)
Arcynopteryx parallela
Prison
EPHEMEROPTERA
Ephemeral la dodds i
Needham
Survival Temperature
70*
50%
70%
50%
60%
27.0
24.2
23.0
18.0
22.0
16.0
(Winter
test)
TRICHOPTERA
Brachycentrus occidental is
Banks
Arctopsyche grand is (Banks)
60%
70%
40%
29.0
28.0
20.0
DIPTERA
Atherix variegata Wa1ker
Ho1orusia grand is
100%
80%
0%
29.0
26.0
28.0
-------
Table 10
Long-Term Thermal Bioassays
University of Utah
1970-71
Species tested
PLECOPTERA
Acroneuria pacifica Banks
Arcynopteryx si gnata (Hagen)
Arcynopteryx pyaTfela Prison
Brachyptera nigripennis (Banks)
Isoperla fulva Claassen
PteronarcelTa badia (Hagen)
Exposure
time
Temperature (days)
15° C
15° C
15° C
14.5° C
18° C
17.5° C
31
14
41
5
11
38
Survival
50%
60%
55%
50%
40%
45%
EPHEMEROPTERA
Ephemerella grand is Eaton
Rhi throgena robust a' Dodds
17.5° C
15° C
18
4
50%
50%
TRICHOPTERA
Arctopsyche 9randi s (Banks)
Brachycentrus occidental is
Banks
Paj-apsyche elsjj^ Mi Ine
Rhyacophila fuscula
18.0° C
28.0° C
15.0° C
15.0° C
23
14
14
39
50%
45%
40%
40%
DIPTERA
Bibiocephala grand is
Atherix variegata Walker
Ho1 orusia grandis
15.0° C
28° C
24° C
3
46
31
60%
50%
45%
ODONATA
Argia vivida
18° C
29
50%
43
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STUDIES ON THE TOLERANCE OF AQUATIC INSECTS TO LOW pH
Introduction
In coal mining regions of the United States water pollution by acid
mine drainage constitutes a problem of major importance. Drainage
from many bituminous coal mines contains large quantities of sulfuric
acid as the result of the chemical and biological oxidation of sulfur
compounds associated with the coal seams. Streams receiving such
drainage may have a pH as low as 3.0 to 4.0. Pollution by acids
may be sufficient to not only make the water of a receiving stream
unfavorable for the growth and development of fish and aquatic inver-
tebrates but there may also be a directly lethal effect.
Numerous field studies have demonstrated the deleterious effects of
acid mine drainage on receiving waters. Lackey (1939) reported that
the number of species of microscopic forms in any given habitat at or
below a pH of 3.9 was very small. Parsons (1956) found a relatively
small number of benthic invertebrates in a central Missouri stream
below an acid strip mine. Harrison (1958) found a very restricted
flora and fauna in a stream near Johannesburg, South Africa, in which
the pH was between 3.7 to 4.3. A dramatic indication of the effects
of acid mine pollution in Pennsylvania occurred in July, 1964, in the
form of a massive fish kill in Slippery Rock Creek. Flushing out of
pockets of mine acid from strip cuts and abandoned deep mines follow-
ing a heavy rainfall killed thousands of fish and invertebrates in a
receiving stream.
A review of the literature revealed that few laboratory studies have
dealt with the effects of low pH on the biota of streams, particularly
with the bottom fauna. Stickney (1922) conducted a series of labora-
tory experiments on the relation of a species of dragonfly to acid
and temperature.
Research by Jewell (1922) indicated that fish can live in water having
a minimum pH of 4.4 with a pH of 4.3 being lethal. Bell and Nebeker
(1969) tested 10 species of aquatic insects and obtained TLnK" values
ranging from pH 4.65 for mayflies to pH 1.5 for caddis flies.
This report summarizes the results of acute short-term 96-hour tests
(TLm^6) and long-term continuous flow tests used in screening 19 species
of aquatic invertebrates to determine their relative tolerance to low
pH. The 96-hour TLm (Standard Methods, I960) was used as the measure
of effect in these tests. Further long-term studies dealing with the
effects of low pH on factors such as molting, adult emergence, repro-
duction, and long-term survival are being conducted. The test species
included the stoneflies, Acroneuria pacifica Banks, Arcynopteryx
44
-------
parallel a Prison, Ispgenus aes t i va1i s (Needham and Claassen),
Pteronarcys californica Newport, Pteronarcella badia (Hagen);
mayfTie's ,Cinyqmu1 a par Eaton, Ephemeral la grandis Eaton, Heptagenia
sp., Ephemerella doddsT Needham, HexagehTa" limbata Guerin, Ritnrogena
robusta Dodds, Leptophlebia sp.; caddis flies, Brachycentrus occi-
dental is Banks, Cheufflatopsyche sp., Hydropsyche sp.; true flies,
AtherTx variegata Walker, Simulturn vittatum Zetterstadt. and fresh
water shrimp, Gammarus limnaeus Smith.
Materials and Methods
All tests were conducted in fiberglass tanks measuring 252 cm long,
21 cm wide, and 25 cm deep. The tanks were partitioned with glass
plates into six test chambers each, measuring 36 cm long, 21 cm wide,
and 16 cm deep. These chambers were further subdivided into three
test cages measuring 13 cm long, 21 cm wide, and 16 cm deep. Each
chamber was furnished with a glass overflow tube capped with a fiber-
glass plug to prevent the loss of any test organisms. Rocks were
placed in the bottom of the cages to form a natural substrate for
the organisms.
Concentrated sulphuric acid was used exclusively in all tests. A
proportional diluter (Mount and Brungs, 196?) was used to deliver the
solutions of various pH to the test chambers. Mixing tanks were
added to the diluter to insure thorough mixing of the acid and water
prior to delivery to the test chambers. A mariotte bottle was used
to deliver the 10:1 solution of acid and water to the diluter. Prior
to each test the diluter was cycled for 24 hours to insure stabiliza-
tion. If, at the end of the 24-hour cycling period, no malfunctions
occurred, the test was initiated.
The test organisms, with the exception of Gammarus, Simulium, and
Leptophlebia, were collected from Rock Creek, a trout stream located
southeast of Missoula, Montana. Simulium and Leptophlebia were col-
lected from Mud Creek, a small slow-flowing stream located north of
the Biological Station in Flathead County, Montana. Specimens of
Gammarus were collected from a spring fed, heavily vegetated pond
located near BJgfork, Montana. All test organisms used were mature
larvae or nymphs. The organisms were held in large, vigorously
aerated fiberglass holding tanks for a minimum of three days prior
to testing. Fresh water was added to the holding tanks at a rate
of three to five liters per minute to insure constant temperature
and fresh water supply.
The water used for all tests and for the holding tanks was obtained
from the Biological Station water system. This water originates in
a spring; is chlorine free; is constant at 6.4 degrees C and pH of
7.8i .1; and is chemically stable. The total hardness remains con-
sistently near 135 ppm; the CC^ from 1 to 2 ppm; (Cn2 and hardness
expressed as CaCO^). Dissolved oxygen levels remain constantly at
100% saturation or greater.
-------
In all tests, pH values in the 2, 3, 4, 5, and 6 ranges were used.
The acclimation pH of 7.81 .1 was used as the control pH value.
The water in each test chamber was aerated to insure dissolved
oxygen saturation and to create turbulence. The diluter was cali-
brated to cycle every three minutes to insure a liberal fresh water
supply and a constant temperature of 9.5° C. The test organisms were
transferred immediately from the holding tanks into the test cages.
No attempt was made to decrease pH values down to the test value to
prevent shock.
During the test period, pH values were recorded four times daily
with a Corning Model 12 pH meter. If any of the pH values varied
by more than .25 pH units the test was discarded. If any of the
organisms at the control pH died, the test was also discarded. All
tests, except that with Rhithrogena, were duplicated. Tests with
Gamma r us were quadruplicated.
The pH values at which 50% of the test organisms died were obtained by
using a modification of the straight line graph interpolation method
as outlined in Standard Methods (I960). The mean of each duplicate
test was plotted as the final TLm value for each test organism.
Results
Late instar larvae and nymphs of 19 species of aquatic invertebrates
were tested to determine their relative tolerance to low pH. Tables
Hand 12 show that considerable difference in tolerance occurs between
the different species. In comparison with the results obtained by
Bell and,Nebeker (1968) on 11 species of aquatic insects from Minnesota
the TLnr are decidedly higher. This may be due to acclimation to
differences in pH in the streams from which the species were obtained.
The minimum pH encountered in the Montana streams from which the test
specimens were collected was 6.8 while in Minnesota much more acid
streams exist. The caddis fly, Limnephilus ornatus, was the most
tolerant species tested with a 96-hour TLm of 2.83 while the amphipod,
Gammar us 1 i mnaeus , was very sensitive with a 96-hour TLm of 7.2?.
S i mu 1 i urn sp. proved to be moderately tolerant with a TLm9^ of 3.64.
Al 1 four species of stoneflies tested, P te rona r ce 1 1 a bad j a , Pteronarcys
cal ifornica, Arcynopteryx paral lela , and Isogenus aestiv'a'lis are mod-
erately tolerant with TLm^& values of 4.37, 4.6, 5.33, and 5-24 respec-
tively. It is interesting to note that the sensitivity of Pteronarcys
cal ifornica compares closely with that of Pteronarcys dorsata tested
by Bell and Nebeker. The latter species had a TLm^& of 4.25. The
mayflies tested proved to be more sensitive than the stoneflies with
Ephemerel la doddsi being most tolerant with a TLm° of 5.13 and
Rhithrogena" robusta being least tolerant with a TLm^6 of 6.35.
The results of long-term continuous flow bioassays confirmed the
relative sensitivity of the orders of aquatic insects tested with the
mayflies being most sensitive, thfr stonef 1 ies moderately sensitive,
-------
and the caddis flies least sensitive. The number of deaths of each
species, however, increased with time of exposure with 50% of the
specimens of such species as Acroneuria pacifica succumbing within
90 days and a like percentage of Ephernere 11 a grand!s dying within 68
days.
D i scuss i on
In general, the test organisms died at pH values below those normally
found in the field. While numerous papers have been published dealing
with the hydrogen ion concentration of natural waters, the role of pH
in fresh water ecology is still something of a mystery. Some ecolo-
gists have maintained that pH of natural waters is a supreme control-
ling factor determining the presence and distribution of aquatic
organisms but this viewpoint is not generally recognized. However,
it is fairly clear that acids can affect aquatic insects by bringing
about changes in the conditions of existence and rate of growth, by
being directly lethal if present at high enough concentrations, and
by being harmful because they have anions of high toxicity or by
having marked toxic properties as undissociated molecules.
The mayflies which were tested in this study were less tolerant than
the genera reported by Bick (1953). He listed the genera Stenonema,
Bae t i s, Blasturus, Callibaetis , and Para1eptoph\eb i a , as being present
in streams having pH values of 4.0 to 5.0. While the first 4 genera
are found in Montana, the species of mayflies tested in the present
study are more characteristic of fast flowing, cold mountain streams
fed by snow melt or springs. Hydrogen ion concentrations as low as
Bick reported are very unlikely to occur under such conditions.
The stoneflies tested were moderately tolerant to low pH values but
a pH of 4.5 would undoubtedly eliminate them completely on long-term
exposure. Leuctra nymphs have been collected in Glacier National
Park in a smalV stream feeding McGee's meadow at pH values of 6.7 to
6.8. A decline in pH to 6.0, however, was at least partially respon-
sible for eliminating this species lower in the stream.
Of the caddis flies tested, Limnephilus ornatus was most tolerant with
a 96-hour TLm of 2.83. While this species was taken from a slow moving
stream with a pH of 7.2 or above, closely related members of the
family Limnephi1idae often occur in acid bogs. The TLm" value of
3.35 for Hydropsyche sp. corresponds closely with the pH values of
4.0 to 5.7 reported by Bell and Nebeker (1959) for Hydropsyche
betteni. The case making caddis fly, B rachycent rus occ i den ta1i s ,
which had a 90-day TLm of 4.5, while moderately tolerant, was much
more sensitive than B rachycent rus ame r i canu s tested by Bell and
Nebeker (TLirr pH 1.5). Ace 1 imat i on is probably responsible for this
difference with B. occidenta 1is occu r r i ng in streams in Montana with
a pH of 7-8 or aFove whi1e the Hatter species occurs in distinctly
acid streams in Minnesota.
-------
The results of the bioassays indicate that the species tested can
live for short periods of time at pH values below those normally
found in the field. Longer exposure, however, may have decidedly
detrimental effects on molting, growth, and reproduction as well
as survival for longer periods of time.
-------
Table 11. pH values at which 50£ of the test species died after
96 hours exposure (Tlnr* ), Flathead Lake, Montana, 1968-69
Species tested pH Values Mean
EPHEMEROPTERA
Epheme_re_na_ cjoddsj. Need ham 4.95 5.13
5.35
Leptophlebia sp. 5.30 5.21
5-11
Hexagenia 11mbata Cuerin 6.40 5.90
5.40
Cinygmula par Eaton 6.25 6.04
6.00
Rhlthrogena robusta Dodds 6.35 6.35
Heptagenia sp. 6.25 6.18
6.11
PLECOPTERA
Arcynopteryx parallela Prison 5.50 5.33
5.16
Pteronarcys caUfornica Newport 5.12 4.60
4.19
Pteronarcella badia (Hagen) 4.90 4.37
4.19
Isogenus aestiva1is 5.40 5.15
(Needham and Claassen) 4.90
TRICHOPTERA
Limnephilus ornatus Banks 2.72 2.83
2.94
Hydropsyche sp. 3.60 3.34
3.10
DIPTERA
Simulium vittatum Zetterstadt 3.68 3.64
3.59
AMPHI PODA
Gammarus limnaeus Smith 7.31 7.29
7.28
7.20 7.27
7.34
49
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Table 12. pH values at which 50$ of the test species died after
long-term continuous exposure
Species tested
EPHEMEROPTERA
Ephemeral la grand is
Eaton
Hexagenia limbata
Guerin
90 day
TLm
Exposure time
68 day
TLm
k& day
TLm
33 day
TLm
5.8
5.5
70 of 90
survived
PLECOPTERA
Acroneuria paci f i ca
Banks
P te rona rcys ca1i forn i ca
Newport
Pteronarcella bad!a
(Hagen)
5.8
4.95
4.52
TRICHOPTERA
Brachycentrus occidental is
₯
iks
Banks
Cheumatopsyche sp.
4.3
4.52
DIPTERA
Atherix variegata
Walker
4.2
50
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TOLERANCE LIMITS OF GREAT BASIN AQUATIC INSECTS
TO SULFUR 1C AND HYDROCHLORIC ACID
All three phases of this project dealing with the water quality require-
ments of aquatic insects were transferred from the Montana Biological
Station to the University of Utah in June, 1970. This proved to be
advantageous in extending the scope of the work to include other
species of insects and water with different chemical characteristics.
The research conducted at the University of Utah showed significant
differences in sensitivity between some of the species which were
tested in both locations.
Materials and Methods
Bioassays were conducted in a constant temperature room where the test
species were first retained in untreated water while acclimatizing
to laboratory conditions before being transferred to the bioassay
aquaria. All temperatures were controlled thermostatically at 8° C.
The bioassay equipment consisted of 12 glass aquaria with approxi-
mately two gallon capacities each. Acid concentrations ranging from
pH 2.0 to 6.0 were used in the experiments.
Each acid was tested separately using non-exposed specimens for each
bioassay and all tests were conducted in duplicate for 96-hour periods,
Test specimens were observed at 24-hour intervals and recorded as the
number unaffected, the number affected, and the number dead.
Results
A wide range in pH existed for both acids with TLm° values ranging
from pH 2.8 to 5.7 in sulfuric and pH 2.7 to 5.6 in hydrochloric acid.
The average of the combined response from the duplicated tests illus-
trated a similar lethal effect of the acids at most pH values among
the majority of the test species. A difference, however, was noted
in the sub-lethal response where many specimens exhibited a more notice-
able change in behavior and response to sulfuric acid than was observed
in the hydrochloric acid tests.
The calculated TLnr values showed Holorusia spp. most tolerant to
sulfuric acid with TLm9° at pH 2.8 and Arctopsyche gj-andis most
tolerant to hydrochloric acid with TLm9^ at 2.7.The least tolerant
species was Gammarus lacustris to both acid solutions with TLm" at
pH 5.7 in sulfuric and TLm^o at pH 5.6 in hydrochloric acid.
51
-------
The species used in the study adapted to laboratory conditions within
a short period of time and molting was observed among many of the
specimens. Near the end of the study many of the mature nymphs of
Ephemeral la grandis grand is displayed signs of emergence and before
the bioassays were completed some emerged as adults.
The bioassays are indicative of short-term exposure under ideal
laboratory conditions. The response produced is due to the influ-
ence of the acids without the consideration of other stress factors
that could result from conditions in the natural environment.
-------
Table 13
Values for Su If uric Acid Bioassays
H
Acid moles/
liter
.
Holorusia spp .................. 2.8 l.6xlO'3M
Arctopsyche grandis ............... 3. A 3.9 x 10"4M
Eohemerel 1 a grand i s grand i s ........... 3.6 2.5 x 10" M
Pteronarcella bad! a ............... 3.7 2.0 x 10" M
Acroneuria pacifica ............... 3-8 1.6 x 10 M
* i^aa a^ini »- ii N __ |,
Ephemerella doddsi ............... 3.8 1.6 x 10 M
A_rcy_nopteryx parallel a ............. *' 8-° * 10"5M
Rhithrogena robusta ............... ^.3 5-0 x 10 M
Isoperla fulva ................. *.5 3-3 x lO^M
Gamma rus lacustris ............... 5.7 2.0 x 10 M
Table 1*
TLm^^ Values for Hydrochloric Acid Bioassays
Acid moles/
£H_ liter
Arctopsyche grandis. .............. 2.7 2.0 x 10~3M
Holorusia spp .................. 3-2 6.3 x lO^M
Acroneuria pacifica. ............... 3.6 2.5 x IQ-jjM
Ephemerella grandis grandis ........... 3.7 2.0 x 10" M
Ephemerella doddsi ............... 3.8 1.6 x 1(T M
Rhithrogena robusta ............... ^ 8-° * 10"5M
Pteronarcella badi a. ... ........... ^ 3.9 x lO^M
Arcynopteryx parallela ............. *-6 2.5 x lO^M
isoperla fulva . ................ *-* 2.5 x lO^M
Gammarus lacustris ............... 5.6 2.5 x 10" M
53
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Long-Term Continuous Bioassays
Long-term continuous flow bioassays were initiated at the University
of Utah in January, 1971, utilizing the same methods and the equipment
used at the Montana Biological Station. The test species included
the stoneflies. Acroneuria pacifica Banks. ArcYnopteryx parallela
Prison, and PteronarceTTa~badia {Hagen); mayflies. Ephemerella dodds?
Needham, Ephemerella grandis Eaton, and Rhithrogena robustaL Dodds;
caddis flies. Arctopsyche grandis (Banks). Brachycentrus occidentalis
Banks; and Rhyacophi1 asp".; and the damsel fly. Argia sp.The mayfly,
Rhithrogena robusta, a species found only in cold, clear, well aerated
streams in the Intermountain Region, was most sensitive with all speci-
mens dying at a pH of 5.7 and only two surviving at pH 6.1, after 12
days. The stonefly, Acroneuria pacifica, was moderately tolerant with
4 of 10 specimens surviving at a pH 6.1 for 50 days.
Table 15
Long-Term Bioassay Results at Low pH
Species tested
EPHEMEROPTERA
Ephemerella dodds i Needham
Ephemerella grandis Eaton
Rhithrogena robusta Dodds
pH range
No. surviving
4.5 5.0 5.6 6.0
0013
4.6 5.1 5.7 6.1
0 0 3 4
4.6 5.1 5.7 6.1
0002
Exposure
Control time
10
10
10
16 days
26 days
12 days
ODONATA
Argia vivida
3.0 3.4 4.1 6.1
2 6 8 10
PLECOPTERA
Acroneuria pacifica Banks 4.5 5.0 5.6 6.1
0114
Arcynopteryx parallela Prison 4.5 5.1 5.6 6.1
0010
10 16 days,
Bio. not
completed
10 50 days
Pteronarcella badia (Hagen)
TRICHOPTERA
Arctopsyche grandis (Banks)
Brachycentrus occidentalis
Banks
Rhyacophila fuscula
0 43 days
1 emr.7 emer.10 emer.
3.3 3.8 4.3 6.1
3.1 3.4 4.2 6.1
0048
3.1 3.4 4.2 6.1
00 2 4
3.1 3.4 4.2 6.1
0013
10
10
10
10
33 days
43 days
39 days
30 days
54
-------
Discussion
In comparing the sensitivity of the same species of aquatic inverte-
brates from Montana and Utah to low pH levels, the 96-hour TLm values
of the latter species are considerably lower (Tables '3, 14, 15). For
example, 50% of the specimens of Ephemerella doddsi from Montana
died at pH 4.95 within 36 hours; whereas, a like number of the same
species from Utah withstood a pH of 3.8. Similar differences can be
seen with several other species. This difference is probably due to
two factors. First, the 96-hour TLm values obtained at Montana were
with a continuous flow diluter, while the tests at Utah were conducted
under static conditions. Secondly, the water at the Biological Station
is softer and less buffered with a calcium carbonate content of 135
ppm in comparison to a carbonate alkalinity of +200 for Utah well
water.
While considerable variability in tolerance levels existed between
the various species of aquatic invertebrates tested, pH levels
below 6.0 appear to be injurious to mayflies of the Intermountain
Region, and pH 5.5 would eliminate the more common stoneflies. A
number of caddis flies can tolerate pH levels below 4.0 for short
periods of time but a pH of 4.5 would be harmful under long-term
conditions. The scud, Gammarus j_a_custris, was most sensitive with
a 96-hour TLm of 5.7 for specimens from Utah compared to 7.27 for
Montana specimens. This great difference is difficult to explain
but a pH of at least 6.0 or above appears necessary to protect this
species.
55
-------
Arcynopteryx
100 parallela
90
80
70
60
50
W
30
20
10
0
.3 TLm
Pteronarcella
"EaHTa
4.5 TLm
Pteronarcys I soge_nus_
californica aestivalis
4.6 TLm
.2 TLm
7 6 5 * 3 2 765*32 765^32 765^32
CInygmu1 a
Ephemerella
doddsi
5.2 TLm
Heptagenia sp. Hexagen i a
6.16 TLm
7 b 5 4 3 2 765^32 7 6 5 * 3 2 765^32
pH Values
Survival After 96 Hours
Figure 3
56
-------
Leptophlebia sp. Rhi throgena^ Hydropsyche sp.
robusta
100
90:
80
70
60
50
40
30
20
10
0
.2 TLm
6.1 TLm
3.3 TLm
7 6 5 4 3 2 765^32 7 6 5 * 3 2
Limnephilus
ornatus
Simulium
vittatum
Gammarus
1imnaeus (lacustri s)
pH Values
Survival After 96 Hours
57
-------
ACKNOWLEDGMENTS
This project was conducted at both the University of Montana Bio-
logical Station and the University of Utah. Consequently personnel
from both institutions contributed considerably to completion of
the work. Thanks are extended to Robert Clubb, Roy Houberg, Garth
Morgan, Robert Newell, and Phillip Tourangeau for their involvement
and help in various aspects of the work. Special recognition is
extended to Stephen Hern, Wilbur Schraer, and Robert Yearsley for
their assistance during most of the period of study and for fulfil-
ling major roles in the research. The latter two utilized the
research for graduate dissertations dealing with the effects of
low pH and high temperatures on aquatic insects.
Mrs. Norma Fernley provided invaluable assistance in editing and
preparing financial reports and the final research report.
During the three years of the project Dr. Alan V. Nebeker served
as Project Officer and offered many helpful suggestions during the
course of the study. The research itself was made possible by not
only financial support through research contract #14-12-^38 from
the Environmental Protection Agency but also from assistance from
graduate trainees on a Training Grant in Water Pollution Ecology
from the same agency.
-------
LITERATURE CITED
DISSOLVED OXYGEN STUDIES
American Public Health Association. 1965. Standard methods for the
examination of water and wastewater. Twelfth Edition New York,
N.Y. pp 769.
Balke, E. 1957. Der 02 Konsum und die Tracheen-Innenflache bei durch
Tracheen Kiemen atmenden Insekten larven in Abhangig Keit von der
Korpergrosse. Z. Vergl. Physiol. 40:415-439.
Brinck, Per. 1949. Studies on Swedish Stoneflies (Plecoptera) . Lund
Berlingeka Boktryckeiret, Sweden.
Dodds, G. S. and F. L. Hisaw. 1924. Ecological studies of aquatic
insects. II. Size of respiratory organs in relation to environ-
mental conditions. Ecology. 5=262-271.
Edwards, G. A. 1946. The influence of temperature upon the oxygen
consumption of several arthropods. Jour. Cell. Comp. Physiol.
27:53-60.
Fair, G. M. and M. C. Whipple. 1948. Revision of the Microscopy of
Drinking Water. 4th ed. John Wiley, New York.
Fox, H. M. 1936. Oxygen consumption of mayfly nymphs in relation to
available oxygen. Nature (London) Dec. 1015.
Gaufin, A. R. and C. M. Tarzwell. 1952. Aquatic invertebrates as
indicators of stream pollution. Pub. Health Reprt., 67:57-64.
Gaufin, A. R. and C. M. Tarzwell. 1956. Aquatic macroinvertebrate
communities as indicators of organic pollution in Lytle Creek.
Sewage and Wastes. 28:906-924.
Gaufin, R. F. and A. R. Gaufin. 1961. The effects of low oxygen con-
centrations on stoneflies. Proc. Utah Acad. Sci., Arts and Let.
38:57-64.
Knight, A. W. and A. R. Gaufin. 1963. The effect of water flow,
temperature, and oxygen concentration on the Plecoptera nymph
Acroneuria pacifica Banks. Proc. Utah Acad. Sci., Arts and Let.
40 (2):175-lS4T
1964. Relative importance of varying
oxygen concentration, temperature, and water flow on the mechanical
activity and survival of the Plecoptera nymph Pteronarcys califor-
nica Newport. Ibid 41 (l):l4-28.
1965. Function of stonefly gills
under reduced dissolved oxygen concentration. Ibid 42 (2):186-190.
59
-------
Knight, A. W. and A. R. Gaufin. 1966. Oxygen consumption of several
species of stoneflies (Plecoptera) . J. Insect Physiol. 12:347-355-
Hynes, H. B. N. I960. The Biology of Polluted Waters. Liverpool
University Press.
Hynes, H. B. N. 1970. The Ecology of Running Waters. Univ. of
Toronto Press. 555 pages.
Kamler, E. 1969. A comparison of the closed bottle and flowing
water methods for measurement of respiration in aquatic inverte-
brates. Pol. Arch. Hydrobiol. 16(29) :31-49.
Kolkwitz, R. and M. Mars son. 1909. Ockologle der tierschen Sap rob i en.
Int. Revue ges. Hydrobiol. Hydrogr. 2:126-152.
Liebmann, H. 1951. Handbuch der Frischwasser und Abwasserbiologie.
Vol. 1. R. Oldenbaurg. Hunchen.
Madsen, B. L. 1968. The distribution of nymphs of Brachyptera rj
Mort. and Nemoura flexuosa Aub. (Plecoptera) in relation to
oxygen. Oikos 19:304-310.
Mount, D. I. 1964. Additional information on a system for controlling
the dissolved oxygen content of water. Trans. Am. Fish. Soc.,
93(0:100-103.
Olson, T. E. and M. E. Rueger. 1968. Relationship of oxygen require-
ments to index organism classification of immature aquatic insects.
Jr. Water Poll. Control Fed. 40(5):Rl88-R202.
Petty, W. C. 1967. Studies on the oxygen requirements of two species
of stoneflies (Plecoptera). Unpubl. Master's thesis, Dept. of
Zoology and Entomology, Univ. of Utah, Salt Lake City, pp 73.
Philipson, G. N. 1954. The effect of water flow and oxygen concentra-
tion on six species of caddis fly (Trichoptera) larvae. Proc. Zool ,
Soc. London (Brit.) 124:547-564.
Prosser, C. L. and Brown, F. A., Jr. 1961. Comparative Animal Physi-
ology (2nd Ed.). W. B. Saunders, Philadelphia.
Reish, D. J. and T. L. Richards. 1966. A technique for studying the
effect of varying concentrations of dissolved oxygen on aquatic
organisms. Int. J. Air. Wat. Pollut. 10:69-71.
Roeder, K. D. 1953. Insect Physiology. John Wiley and Sons, Inc.,
New York, 1100 pp.
Wigglesworth, V. B. 1950. The Principles of Insect Physiology. E.P.
Dutton and Co. New York, N.Y.
60
-------
LITERATURE CITED
THERMAL STUDIES
Coutant, C. C. 1962. The effect of a heated water effluent upon the
macroinvertebrate fauna of the Delaware River. Proc. Pennsylvania
Acad. Sci. 36:58-71.
Kennedy, V. S.f and J. A. Mihursky. 1967. Bibliography on effects
of temperature in the aquatic environment. Univ. of Md., Nat.
Res. Inst.; Contribution No. 326.
Nebeker, A. V., and A. E. Lemke. 1968. Preliminary studies on the
tolerance of aquatic insects to heated waters. J. Kansas
Entomol. Soc. 41(3):413-418.
Parker, F. L., and P. A. Krenkel. 1969. Thermal Pollution: Status
of the Art. Vanderbilt Univ. Dept. of Env. and Water Resources
Engr. Report No. 3. Chapt. 3. pp 1-65.
Patrick, R. 1968. Potomac River Surveys. 1968 River Survey Report
for the Potomac Electric Power Company. The Academy of Natural
Sciences of Philadelphia. 85 pp.
Pitcon, W. L. I960. Water use in the United States 1900-1980. Water
and Sewerage Division, U. S. Dept. of Commerce.
Raney, E. C., and B. W. Menzel. 1967. A bibliography: Heat efflu-
ents and effects on aquatic life with emphasis on fishes. Cornell
Univ. Water Resources and Marine Sciences Center. Philadelphia
Electric Co. and Ichthyological Assoc. Bulletin No. 2. 469 pp.
Tarzwell, C. L. (Chntn.), et_ a_K 1968. Water quality criteria for
fish, other aquatic life, and wildlife. Report, National
Technical Advisory Committee to the Secretary of Interior and
the Federal Water Pollution Control Administration. U. S. Dept.
of Interior, Washington, D. C. pp 26-110.
Trembley, F. J. 1965. Effects of cooling water from steam-electric
power plants on stream biota, jn, Proceedings of Third Seminar
on Biological Problems in Water Pollution. USPHS Publication
No. 999-WP-25; pages 334-345. R. A. Taft San. Eng. Ctr.,
Cincinnati, Ohio.
61
-------
LITERATURE CITED
ACID STUDIES
American Public Health Association, Inc. I960. Standard Methods
for the Examination of Water and Waste Water, llth Edition.
Amer. Publ. Health Assoc., Inc., New York. 686 pp.
Bell, H. L. and A. V. Nebeker. 1969. Preliminary studies on the
tolerance of aquatic insects to low pH. J. Kansas Entomol. Soc.
42(2):230-236.
Bick, G. H., L. E. Hornuff and E. N. Larobremont. 1953. An ecological
reconnaissance of a naturally acid stream in southern Louisiana.
Jour. Tenn. Acad. Sci., 28(3) :221-23K
Harrison, A. D. and J. D. Agnew. 1962. The distribution of inver-
tebrates endemic to acid streams in the western and southern
Cape Province. Ann. Cape Prov. Mus. Reprint No. 121:273-291.
Jewell, M. E. 1922. The fauna of an acid stream. Ecology, 3:22-28.
Lackey, J. B. 1938. The flora and fauna of surface waters polluted
by acid mine drainage. Public Health Rept. (U.S.), 54:740-747.
Mount, D. I., and Brungs, W. A. (1967). A simplified dosing
apparatus for fish toxicology studies. Water Research 1:21-29.
Parsons, J. D. 1956. The effects of acid strip mine pollution on
the ecology of a central Missouri stream. Ph.D. Dissertation,
Univ. of Missouri.
Pennsylvania Dept. of Health, Bureau of Env. Health, Div. of Sanitary
Engineering. 1967. Report to the Sanitary Water Board on
pollution of Slippery Rock Creek. Publ. 17. 109 pp.
Stickney, F. 1922. The relation of the nymphs of a dragonfly
(Libellula pulchella Drury) to acid and temperature. Ecology,
3(3):250-254.
62
-------
LIST OF PUBLICATIONS
RESULTING FROM PROJECT
Gaufin, A. R. and S. Hern. 1971. Laboratory studies on tolerance
of aquatic insects to heated waters. J. Kansas Entomol. Soc.
M» (2)-.240-245.
Gaufin, A. R., S. Hern and G. Yearsley. 1972. Studies on the
tolerance of aquatic invertebrates to low pH. Submitted to
J. Kansas Entomol. Soc.
Gaufin, A. R. and R. Clubb. Studies on the oxygen requirements
of aquatic insects. In manuscript form.
63
-------
APPENDICES
Arcynopteryx parallela
LONG TERM TEMPERATURE TOLERANCE
3 ? n c Aquarium
semi- Days of ^
weekly exposure Control 2 3 4
Temp Number Temp Number Temp Number Temp Number
0 C alive ° C alive ° C alive ° C alive
19-5 20
20.0 2
18.5 2
19.0 2
18.5 2
19.0 2
19.0 2
18.0 2
18.5 1
20.0 0
-09-71
-12-71
-15-71
-19-71
-22-71
-26-71
-29-71
2-02-71
2-05-71
2-09-71
2-12-71
2-16-71
2-19-71
2-23-71
2-26-71
J-02-71
J-05-71
5-09-71
M2-71
0
3
6
10
13
17
20
2k
27
31
34
38
41
45
48
52
55
59
62
13.0
13.5
12.5
13.5
13.0
13.0
13.0
13.0
13.0
13.0
13.0
13.0
13.0
13.0
13.0
13.0
13.0
13.0
13.0
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
14.5
14.5
14.5
15-0
15.0
15.0
15.0
15-0
15.5
14.5
14.5
15.0
15.0
15.0
15.0
15.0
15.0
15.0
15.0
20
20
20
20
20
20
18
15
15
15
14
12
11
8
5
5
5
3
0
18.0
18.0
17.5
18.0
18.0
18.0
18.0
17.5
17.5
17.5
17.5
17.5
17.5
18.0
18.0
18.0
18.0
18.0
20
9
6
6
6
5
5
2
2
2
2
2
2
2
1
1
1
0
64
-------
Arcynopteryx signata
LONG TERM TEMPERATURE TOLERANCE
Date
semi-
weekly
4-20-71
4-23-71
4-27-71
4-30-71
5-04-71
5-07-71
5-11-71
5-14-71
5-18-71
5-21-71
Days of
exposure Control
0
3
7
10
14
17
21
24
28
31
Temp
0 C
12.5
12.5
12.0
12.0
11.5
12.0
12.0
13.0
12.0
13.0
Number
al i ve
10
10
10
10
10
10
10
10
10
10
A q u a r 1 u m
2 3
Temp
0 C
14.5
15.0
15.0
15.0
14.0
15.0
15.0
15.0
14.0
14.0
Number
al ive
10
8
6
6
6
1
1
1
I
0
Temp
0 C
18.0
18.0
18.0
18.0
18.0
18.0
18.0
18.0
Number
al ive
10
6
4
4
4
4
1
0
4
Temp
0 C
19.0
18.0
18.0
19.0
20.0
Number
al ive
10
7
3
3
0
65
-------
PteronarceIIa badia
LONG TERM TEMPERATURE TOLERANCE
Date
semi- Days of
weekly exposure
Control
Temp Number
-09-71
-12-71
-15-71
-19-71
-22-71
-26-71
-29-71
2-02-71
2-05-71
2-09-71
2-12-71
2-16-71
2-19-71
2-23-71
2-26-71
3-02-71
3-05-71
3-09-71
3-12-71
3-16-71
3-19-71
3-23-71
3-26-71
3-30-71
4-02-71
4-06-71
0
3
6
10
13
17
20
24
27
31
34
38
41
45
48
52
55
59
62
66
69
73
76
80
83
87
0 C
13.0
12.5
12.5
13.5
13.0
13.0
13.0
13.0
13.0
13.0
12.0
13-0
13.0
13.0
13.0
13.0
13.0
13.0
13.0
13.0
12.0
13.0
13.0
13.0
13.0
13.0
a 1 i ve
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
2
A q u
Temp Number
8 C
14.5
14.5
14.5
15.0
15.0
15.0
15.0
15.0
15.5
14.5
14.5
15.0
15.0
15.0
15.0
15.0
15.0
15.0
14.5
15.0
15.0
15.0
15.0
15.0
15.0
15.0
alive
20
20
20
20
18
18
18
18
16
16
16
16
16
12
10
9
7
7
5
5
5
4
4
4
1
0
a r i u m
3
Temp Number
0 C
18.0
18,0
17.5
18.0
18.0
18.0
18.0
17.5
17.5
17.5
17.5
17.5
17.5
18.0
18.0
18.0
18.0
18.0
18.0
18.0
18.0
18.0
18.0
alive
20
19
19
17
17
17
17
17
15
12
11
9
7
7
4
4
1
1
1
1
1
1
0
4
Temp Number
0 C
19.5
20.0
18.5
19.0
18.5
19.0
18.5
18.0
18.5
18.5
19.0
18.4
18.5
19.0
20.0
18.5
19.0
al ive
20
20
20
20
16
15
12
9
5
3
2
2
2
1
0
66
-------
Isoperla fulva
LONG TERM TEMPERATURE TOLERANCE
Date
semi-
weekly
-15-71
-19-71
-22-71
-26-71
-29-71
2-02-71
2-05-71
2-09-71
2-12-71
2-16-71
2-19-71
2-23-71
2-26-71
3-02-71
Date
semi-
weekly
1-15-71
1-19-71
1-22-71
Days of
exposure
0
4
7
11
14
18
21
25
28
32
35
39
42
46
Days of
exposure
0
4
7
Control
Temp
0 C
12.5
13.5
13.0
13-0
13.0
13.0
13.0
13.0
12.0
13.0
13.0
13.0
13.0
13.0
Aquar
5
Temp
0 C
24.0
24.5
24.0
Number
alive
20
20
20
20
20
20
20
20
20
20
20
20
20
20
ium
Number
al ive
20
2
0
2
Temp
0 C
14.
15.
15.
15.
15.
15.
15.
14.
14.
15.
15.
15.
15.
15.
5
0
0
0
0
0
5
5
5
0
0
0
0
0
A q
Number
al ive
20
9
8
8
2
2
2
1
1
1
1
1
1
0
u a r
i
3
Temp
0 C
17.
18.
18.
18.
18.
17.
17.
17.
17.
17.
17.
18.
5
0
0
0
0
5
5
5
5
5
5
0
u m
Number
al ive
20
9
8
8
5
5
5
5
2
1
1
0
4
Temp Number
0 C
18.
19.
18.
19.
al ive
5 20
0 3
5 3
0 2
19.0 2
17.
18.
18.
19.
0 2
5 1
5 1
0 0
67
-------
Ephemere11a g rand is
LONG TERM TEMPERATURE TOLERANCE
Date
semi- Days of
weekly exposure
Control
Aquari
3
um
Temp No. Temp No. Temp No. Temp No. Temp No.
0 C alive ° C alive ° C alive ° C alive * C alive
1-29-71
2-02-71
2-05-71
2-09-71
2-12-71
2-16-71
2-19-71
2-23-71
2-26-71
3-02-71
3-05-71
3-09-71
0
4
7
11
14
18
21
25
28
32
35
39
13.0
13.0
13.0
13.0
13.0
13.0
13.0
13-0
13.0
13.0
13.0
13.0
10
10
10
10
10
10
10
10
10
10
10
10
15.0
15.0
15.5
14.5
14.5
15.0
15.0
15.0
15.0
15.0
15.0
15.0
10
10
10
10
10
10
10
7
5
2
1
0
18.0
17.5
17.5
17.5
17.5
17.5
17.5
18.0
18.0
18.0
18.0
18.0
10
7
6
6
6
5
3
3
1
1
1
0
19. 0
17.0
18.5
18.5
19.0
18.5
10
7
6
6
6
0
24.0
24.0
24.0
24.0
24.0
10
7
6
2
0
Date
semi- Days of
weekly exposure
6
7
A q u a r i
8
i u m
9
1-29-71
2-02-71
2-05-71
0
4
7
Temp No. Temp No. Temp No, Temp No.
0 C alive ° C alive ° C alive ° C alive
26.
26.
26.
0
0
0
10
2
0
28
27
.0
.5
to
0
29
29
.0
.0
10
0
30
30
.0
.0
10
0
68
-------
Brachycentrus occidental is
LONG TERM TEMPERATURE TOLERANCE
Date
semi- Days of
weekly exposure Control
Temp
0 C
1-05-71
1-08-71
-12-71
-15-71
-19-71
-22-71
-26-71
-29-71
2-02-71
2-05-71
2-09-71
2-12-71
2-16-71
2-19-71
2-23-71
2-26-71
3-02-71
3-05-71
3-09-71
3-12-71
3-16-71
3-19-71
3-23-71
3-26-71
3-30-71
4-02-71
0
3
7
10
14
17
21
24
28
31
35
38
42
45
49
52
56
59
63
66
70
73
77
80
84
87
13
13
13
13
13
13
13
13
13
13
13
13
13
13
13
13
13
13
13
13
13
13
13
13
13
13
.0
.0
.5
.0
.5
.0
.0
.0
.0
.0
.0
.0
.0
.0
.0
.0
.0
.0
.0
.0
.0
.0
.0
.0
.0
.0
No.
al ive
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
Temp
0 C
18.0
18.0
18.0
17.5
18.0
18.0
18.0
18.0
17.5
17.5
17.5
17.5
17.5
17.5
18.0
18.0
18.0
18.0
18.0
18.0
2
No.
al ive
20
20
20
19
14
14
14
14
14
11
9
7
5
5
5
4
3
3
3
3
A q u a r
3
Temp
0 C
26.0
26.0
26.0
25.5
26.0
26.0
26.0
26.0
26.0
26.0
24.0
26.0
26.0
26.0
25.0
25.0
25.0
26.0
26.0
26.0
25.0
26.0
26.5
25.5
25.5
25.5
No.
al ive
20
20
19
16
15
14
14
8
8
6
6
6
5
4
4
2
2
2
2
2
2
2
1
1
1
0
u m
Temp
0 C
27.0
28.0
28.0
28.0
28.0
28.0
28.0
28.0
27.5
27.0
27.0
27.5
27.5
27.5
27.0
27.0
28.0
27.0
27.5
26.5
27.0
27.5
28.0
4
No.
al ive
20
18
17
11
9
4
3
2
2
2
2
2
2
2
2
2
2
1
1
1
1
1
0
5
Temp No .
0 C alive
28.5 20
29.0 2
29.0 2
28.5 1
28.5 1
29.0 1
29.0 0
29.0 0
69
-------
Rhyacophlla fuscula
LONG TERM TEMPERATURE TOLERANCE
Date
semi-
weekly
2-19-71
2-23-71
2-26-71
3-02-71
3-05-71
3-09-71
3-12-71
3-16-71
3-19-71
3-23-71
3-26-71
3-30-71
4-02-71
4-06-71
4-09-71
4-13-71
4-16-71
4-20-71
4-23-71
Days of
exposure Control
0
4
7
11
14
18
21
25
28
32
35
39
42
46
49
53
56
60
63
Temp
0 C
13.0
13.0
13.0
13.0
13.0
13.0
13.5
12.0
12.0
13.0
13.0
13.0
13.0
13.0
13.0
13.5
12.5
12.5
13.0
Number
alive
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
2
Temp
c
15.0
15.0
15.0
15.0
15.0
15.0
14.5
15.0
15.0
15.0
14,5
15.0
15.0
15.0
15.0
15.0
15.0
14.5
15.0
A q
Number
alive
10
10
10
9
9
9
7
7
7
7
4
4
2
0
u a r
3
Temp
0 C
18.0
18.0
18.0
18.0
18.0
18.0
18.0
18.0
18.0
18.0
18.0
18.0
18.0
18.0
18.0
18.0
i u m
Number
al ive
10
10
10
9
9
9
9
9
8
7
7
7
7
3
3
0
4
Temp
0 C
19.0
19.0
20.0
18.5
20.0
19.0
19.0
18.5
18.0
19.0
19.0
19.5
19.5
20.0
20.0
19.0
19.0
Number
alive
10
10
9
9
9
9
9
8
8
5
4
3
2
1
1
1
0
Date
semi-
weekly
2-19-71
2-23-/1
2-26-71
3-02-71
3-05-71
3-09-71
Days of
exposure
0
4
7
11
14
18
23.0
23-0
23.5
24.0
24.0
24.0
5
10
7
6
3
1
0
Aquarium
6
25.0 10
25.0 0
7
27.0 10
27.0 0
70
-------
Arctopsyche grandis
LONG TERM TEMPERATURE TOLERANCE
Date
semi- Days of
weekly exposure Control
Aquarium
3 4
-o4-7i
-08-71
-12-71
-15-71
-19-71
-22-71
-26-71
-29-71
2-02-71
2-05-71
2-09-71
2-12-71
2-16-71
2-19-71
2-23-71
2-26-7 \
3-02-71
3-05-71
3-09-71
3-12-71
3-16-71
3-19-71
3-23-71
3-26-71
3-30-71
0
4
8
11
15
18
23
26
30
33
37
40
44
47
51
54
59
62
66
69
73
76
80
83
87
Temp
0 C
13.0
13.0
13.5
12.5
13.5
13.0
13.0
13.0
13.0
13.0
13.0
12.0
13.0
13-0
13.0
13.0
13.0
13.0
13.0
13.0
12.0
12.0
13.0
13.0
13.0
No.
al ive
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
Temp
0 C
14.5
15.0
14.5
14.5
15.0
15.0
15.0
15.0
15.0
15.5
14.5
14.5
15.0
15.0
15.0
15.0
15.0
15.0
15.0
14.5
15.0
15.0
15.0
15.0
15.0
No.
al ive
20
18
18
18
18
18
18
17
13
11
10
9
8
8
7
7
7
5
3
3
3
3
3
3
0
Temp
0 C
18.0
18.0
18.0
17.5
18.0
18.0
18.0
18.0
17.5
17.5
17.5
17.5
17.5
17.5
18.0
18.0
18.0
18.0
18.0
18.0
No.
al ive
20
20
20
20
18
15
10
9
7
4
2
2
0
Temp
0 C
19.3
19.0
20.0
18.5
19.0
18.5
19.0
18.5
18.0
18.5
18.5
20.0
No.
al i ve
20
17
8
6
4
3
2
2
2
1
1
0
Temp
0 C
25.0
24.0
24.0
24.0
24.5
24.0
23.5
24.0
24.0
No.
al ive
20
2
2
2
1
1
1
1
0
71
-------
Arpia vivjda
LONG TERM TEMPERATURE TOLERANCE
Date
semi- Days of
weekly exposure Control
3-25-71
3-26-71
3-30-71
4-02-71
4-06-71
4-09-71
4-13-71
4-16-71
4-20-71
4-23-71
4-27-71
4-30-71
5-04-71
5-07-71
5-11-71
5-14-71
0
1
5
8
12
15
19
22
26
29
33
36
40
43
47
50
Temp
0 C
13.0
12.5
13.0
13.0
13.0
13.0
13.5
12.5
12.5
12.5
12.0
12.0
11.5
12.0
12.0
13.0
Number
alive
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
A q u a r
2 :
Temp
0 C
15.0
14.5
15.0
15.0
15.0
15.0
15.0
15.0
14.5
15.0
15.0
15.0
15.0
?5.0
15.0
Number
alive
10
9
8
5
5
4
4
2
2
0
Temp
0 C
18.0
18.0
18.0
18.0
18.0
18.0
18.0
18.0
18.0
18.0
18.0
18.0
18.0
18.0
18.0
18.0
u m
)
Number
alive
10
10
10
10
10
10
10
10
10
5
5
3
2
?
1
0
4
Temp
0 C
20.0
19.0
19.5
20.0
20.0
20.0
19.0
18.0
18.0
18.0
18.0
19.5
Number
alive
10
10
8
8
6
6
6
6
5
2
J
0
Date
semi- Days of
weekly exposure
Aquarium
3-25-71
3-26-71
3-30-71
4-02-71
4-06-71
4-09-71
4-13-71
4-16-71
4-20-71
4-23-71
4-27-71
4-30-71
0
1
5
8
12
15
19
22
26
29
33
36
Temp Number Temp Number
0 C alive ° C alive
24.0
24.0
24.0
24.0
24.0
24.0
24.0
24.0
24.0
24.0
23.5
24.0
10
10
10
10
8
8
5
4
4
2
1
0
26.0
25.
25.
25.
25.
25.
25.
25.0
25.0
25.0
25.0
26.0
.5
.5
.5
.5
.5
.5
10
8
8
8
8
7
3
2
2
1
1
0
72
-------
Bibiocephala grandis
LONG TERM TEMPERATURE TOLERANCE
Date
semi- Days of
weekly exposure Control
1-19-71
1-22-71
1-26-71
1-29-71
2-02-71
0
3
7
10
14
Temp
0 C
13.5
13.0
13.0
13.0
13.0
Number
al ive
10
10
10
10
10
A q
2
Temp
0 C
15.0
15.0
15.0
15.0
15.0
Number
al ive
10
6
4
2
0
u a r
«
Temp
0 C
18.0
18.0
i u m
I
Number
al ive
10
0
I
Temp
0 C
19.0
18.5
19.0
19.0
4
Number
al i ve
10
4
1
0
Date
semi- Days of
weekly exposure
Aquarium
7
1-19-71
1-22-71
0
3
Temp
0 C
24.0
24.0
Number
alive
10
0
Temp
0 C
26.0
26.0
Number
al ive
10
0
Temp
0 C
28.0
28.0
Number
al i ve
10
0
Temp
0 C
28.5
29.0
Number
al ive
10
0
73
-------
Holorusia grandis
LONG TERM TEMPERATURE TOLERANCE
Date
semi-
weekly
Days of
exposure Control
Temp Number
-09-71
-12-71
-15-71
-19-71
-22-71
-26-71
-29-71
2-02-71
2-05-71
2-09-71
2-12-71
2-16-71
2-19-71
2-23-71
2-26-71
3-02-71
3-05-71
3-09-71
3-12-71
3-16-71
3-19-71
3-23-71
0
3
6
10
13
17
20
2k
27
31
34
38
41
45
48
52
55
59
62
66
69
73
0 C
13.0
13.5
12.5
13.5
13.0
13.0
13.0
13.0
13.0
13.0
13.0
13.0
13.0
13.0
13.0
13.0
13.0
13.0
12.5
12.0
12.0
13.0
alive
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
2
Aquarium
3 4
Temp Number
0 C
24.0
24.0
24.0
24.5
24.0
23.5
24.0
24.0
24.0
24.0
24.0
24.0
24.0
23.0
23.5
25.0
24.0
24.0
24.0
24.0
24.5
25.0
alive
20
20
20
17
15
15
13
12
9
9
8
8
6
6
5
k
3
2
2
1
1
0
Temp Number Temp Number
0 C
26.0
26.0
26.0
26.0
26.0
26.0
26.0
26.0
26.0
25.0
26.0
26.0
26.0
alive ° C alive
20 28.0 20
18 28.0 0
18
14
13
11
9
6
3
2
1
1
0
74
-------
Appendices
Chemical Characteristics
Montana Biological Station
Spring Water
September 15, 19&9
Aluminum
Barium
Ca Bicarbonate
Ca Carbonate
Carbon Dioxide
Chlorides
Chromium
Copper
Hardness (Total)
Hydrogen Sulfide
Fe (Ferric)
Fe (Total)
Fe (Ferrous)
Manganese
Nit. (Ammonia)
Nit. (Nitrate)
Nit. (Nitrite)
Oxygen (Dissolved)
pH Value
Phenol
Phosphate
Silica
Sulphate
Temp. Water
Turbidity
PPM
Trace
3.0
135.0
0.0
1-2
1.5
.037
.02
135
0.0
.09
.12
.03
.05
.21
Trace
0.0
8.0
7.8
None
.11
6.0
6.0
75
-------
REACTION OF PTERONARCYS CALI FORMICA
TO LOW OXYGEN CONCENTRATIONS
Date Initiated - Dec. 1,1970 Flow - 1000 cc/min Temp. - 10° C
Compartment
1
3
5
7
9
D.O.
Concentration
Days of Survival
4 days 11 26_ 49_ 5_8_ 97
111
2
2
3
4
4
6
.0
.8
.6
.0
.8
.4
60%
100
100
80
100
100
40
100
100
80
100
100
40
80
80
80
100
100
0
60
60
80
100
100
0
20
40
80
100
100
0
0
20
20
40
80
0
0
20
20
40
0
REACTION OF EPHEMERELLA GRAND IS
TO LOW OXYGEN CONCENTRATIONS
Date Initiated - Dec. 2,1970 Flow - 1000 cc/min Temp. - 10° C
Compartment
2
4
6
8
10
13
D.O.
Concentration
Days of Survival
4 days 12 25 30
2.4
3.0
3-6
4.6
5.0
6.0
103
50
70
100
80
90
0
20
60
70
60
90
0
20
20
60
50
80
0
0
0
0
0
20
REACTION OF BRACHYCENTRUS OCCIDENTAL IS
TO LOW OXYGEN CONCENTRATIONS
Date Initiated - Oct. 16,1970 Flow - 500 cc/min Temp. - 10° C
Compartment
2
4
6
8
10
12
D.O.
Concentration
Days of Survival
4 days 9_ 3£ Q 44_ 52_ 56 70 92 102 120
90%
90
90
100
100
100
90
90
90
100
100
100
90
90
70
TOO
100
100
90
90
70
100
100
100
90
90
60
100
100
100
90
90
60
90
100
100
90
90
60
90
100
100
90
90
60
90
90
100
90
80
60
90
90
90
70
80
60
70
90
90
70
50
50
70
80
80
76
-------
REACTION OF ACRONEURIA PACIFICA
TO LOW OXYGEN CONCENTRATIONS
Date Initiated - Dec. 1,1969 Flow - 1000 cc/min Temp. - 10° C
D.O. Days of Survival
Compartment Concentration 4 days J2_ 26_ 49_ 58_ 77 99_ HI
1 1.6 100% 90 70 20 10 10 10 10
3 3.2 100 100 90 90 90 60 40 30
5 4.4 100 100 100 70 70 60 30 30
7 5.8 100 100 100 80 80 70 60 50
9 6.4 100 100 100 50 50 50 30 30
lit 8.4 100 100 100 90 90 90 70 50
REACTION OF ATHERIX VARIEGATA
TO LOW OXYGEN CONCENTRATIONS
Date Initiated - Dec. 2.19&9 Flow - 1000 cc/min Temp. - 10° C
D.O. Days of Survival
Compartment Concentration 4 days 15 30 40
2 2.4 30% 90 90 90
4 4.0 100 100 100 100
6 5.6 100 100 100 100
8 6.0 100 100 100 100
10 6.8 100 100 100 100
13 8.0 100 100 100 100
REACTION OF RHYACOPHILA FUSCULA
TO LOW OXYGEN CONCENTRATIONS
Date Initiated - Jan. 12,1970 Flow - 1000 cc/min Temp. - 10° C
D.O. Days of Survival
Compartment Concentration 4 days £
2 2.4 20% 20
4 4.0 20 20
6 5.6 60 0
8 6.0 80 20
10 6.8 100 40
13 8.0 100 50
77
-------
Q%
0
0
90
80
0
0
0
70
70
0
0
0
40
60
0
0
0
20
30
0
0
0
20
30
REACTION OF HYDROPSYCHE SP.
TO LOW OXYGEN CONCENTRATIONS
Date Initiated - Dec. 1,1969 Flow - 1000 cc/min Temp. - 10° C
0.0. °ays of Survival
Compartment Cbhcentratlon 4 days 12 26 49 50
1 1.6
3 2.4
5 3.2
7 4.0
9 4.8
14 8.0 90 90 90 90 50
REACTION OF ARCYNOPTERYX AUREA
TO LOW OXYGEN CONCENTRATIONS
Date Initiated - Dec. 8,1970 Flow - 1000 cc/min Temp. - 10°
D.O. Days of Survival
Compartment Concentration 4 days J£ 26^
2 2.0
4 2.8
6 3.6
8 4.4
10 4.8
13 5.6
REACTION OF PTERONARCELLA BAD IA
TO LOW OXYGEN CONCENTRATIONS
Date Initiated - Dec. 12,1970 Flow - 1000 cc/min Temp. - 10° C
D.O. Days of Survival
Compartment Concentration 4 days J6^ 35 57_ 6_9_
10%
20
70
40
70
100
0
10
40
20
30
90
0
0
0
10
0
0
2
4
6
8
10
13
2.0
2.8
3.6
4.4
4.8
5.6
60%
60
60
90
50
100
20
50
60
90
20
100
0
30
50
90
20
90
0
10
30
90
20
80
0
10
30
50
20
40
78
-------
REACTION OF NEMOURA CINCTIPES
TO LOW OXYGEN CONCENTRATIONS
Date Initiated - Dec. 19,1970 Flow - 1000 cc/min Temp. - 10° C
D.O.
Compartment Concentration
1
3
5
7
9
14
1.6
2.4
3.2
4.0
4.8
8.0
Days of Survival
4 days 8
0%
20
40
80
100
100
0
0
0
10
20
40
REACTION OF ARCYNOPTERYX PARALLELA
TO LOW OXYGEN CONCENTRATIONS
Date Initiated - Feb. 17,1970 Flow - 1000 cc/min Temp. - 10° C
Compartment
1
3
5
7
9
14
D.O.
Concentration
1.6
2.4
3.2
4.0
4.8
8.0
Days of Survival
4 days 22 34
60%
100
100
100
100
80
0
50
60
80
80
80
0
0
0
10
10
10
79
-------
0
o
100
0
0
100
0
0
100
25
15
60
55
5
kO
REACTION OF GAMMARUS LACUSTRIS SARS
TO SULFUR1C ACID
RESPONSE pH 2.0 pH 3-0 pH *t.O PH 5.0 pH 6.0
2k Hours _A Qn
% Unaffected 0 0 0 50 80
% Affected 0 0 25 25 0
% Dead 100 100 75 25 10
48 Hours
% Unaffected
% Affected
% Dead
72 Hours
% Unaffected
* Affected
% Dead
96 Hours
3 Unaffected 0 0 0 15 »
% Affected 0 0 0 5 5
% Dead 100 100 100 80 *K>
REACTION OF GAMMARUS LACUSTRIS SARS
TO HYDROCHLORIC ACID
0
o
100
0
0
100
0
0
100
15
5
80
55
5
kQ
RESPONSE
pH 2.0 pH 3.0 pH k.Q pH 5-0 pH 6.0
2k Hours Qrt
% Unaffected 0 0 0 75 80
% Affected 0 0 45 10 20
* Dead 100 100 55 15 0
k% Hours
% Unaffected 0 0 0 55 90
* Affected 0 0 25 5 0
% Dead 100 100 75 *0 10
72 Hours
% Unaffected 0 0 0 30 75
% Affected 00500
% Dead 100 100 95 70 25
96 Hours
% Unaffected
% Affected
% Dead
80
0
0
100
0
0
100
0
0
100
20
0
80
75
0
25
-------
REACTION OF HOLORUSIA SPP.
TO SULFUR 1C ACID
RESPONSE
2k Hours
% Unaffected
% Affected
% Dead
48 Hours
% Unaffected
% Affected
% Dead
72 Hours
% Unaffected
% Affected
% Dead
96 Hours
% Unaffected
% Affected
% Dead
pH 2.0
45
20
35
0
15
85
0
0
100
0
0
100
pH 3.0
70
30
0
70
20
10
70
15
15
60
0
40
PH 4.0 PH 5.0
95
5
0
95
5
0
90
5
5
80
5
15
100
0
0
100
0
0
85
5
10
85
5
10
pH 6.0
100
0
0
100
0
0
100
0
0
100
0
0
REACTION OF HOLORUSIA SPP.
TO HYDROCHLORIC ACID
RESPONSE
24 Hours
% Unaffected
% Affected
% Dead
48 Hours
% Unaffected
% Affected
% Dead
72 Hours
% Unaffected
% Affected
% Dead
96 Hours
% Unaffected
% Affected
% Dead
75
15
10
100
0
0
100
0
0
100
0
0
pH 2.0 pH 3.0 pH 4.0 pH 5.0 pH 6.0
25
15
60
5
5
90
0
0
100
0
0
100
60
15
25
55
5
40
30
5
65
100
0
0
100
0
0
85
0
15
95
0
5
95
0
5
95
0
5
100
0
0
100
0
0
100
0
0
81
-------
REACTION OF RHITHROGENA ROBUSTA DODDS
TO SULFUR 1C ACID
RESPONSE
2k Hours
% Unaffected
% Affected
% Dead
48 Hours
% Unaffected
% Affected
% Dead
72 Hours
% Unaffected
% Affected
% Dead
96 Hours
% Unaffected
% Affected
% Dead
pH 2.0
0
0
100
0
0
100
PH 3.0
0
35
65
pH 4.0 pH 5.0 pH 6.0
0
0
100
75
20
5
35
0
65
80
10
10
80
5
15
100
0
0
0
0
100
0
0
100
70
10
20
80
5
15
100
0
0
0
0
100
0
0
100
70
10
20
80
5
15
100
0
0
100
0
0
REACTION OF RHITHROGENA ROBUSTA DODDS
TO HYDROCHLORIC ACID
RESPONSE
2k Hours
% Unaffected
% Affected
% Dead
48 Hours
% Unaffected
% Affected
% Dead
72 Hours
% Unaffected
% Affected
% Dead
96 Hours
% Unaffected
% Affected
% Dead
pH 2.0
0
0
100
0
0
100
0
0
100
0
0
100
pH 3.0 pH 4.0 pH 5.0 pH 6.0
0
5
95
60
15
25
85
10
5
100
0
0
0
0
100
0
0
100
0
0
100
60
5
35
45
0
55
80
0
20
80
0
20
95
0
5
45
0
55
80
0
20
95
0
5
95
0
5
82
-------
REACTION OF EPHEMERELLA DODDSI
TO SULFUR 1C ACID
NEEDHAM
RESPONSE
2k Hours
% Unaffected
% Affected
% Dead
48 Hours
% Unaffected
% Affected
% Dead
72 Hours
% Unaffected
% Affected
% Dead
96 Hours
% Unaffected
% Affected
% Dead
pH 2.0
0
0
100
0
0
100
0
0
100
0
0
100
pH 3-0
0
25
75
0
15
85
0
15
85
0
0
100
pH 4.0 pH 5.0
pH 6.0
85
15
0
70
15
15
60
5
35
60
5
35
100
0
0
95
5
0
95
5
0
90
5
5
100
0
0
100
0
0
95
0
5
95
0
5
REACTION OF EPHEMERELLA DODDSI NEEDHAM
TO HYDROCHLORIC ACID
RESPONSE
24 Hours
% Unaffected
% Affected
% Dead
48 Hours
% Unaffected
% Affected
% Dead
72 Hours
% Unaffected
% Affected
% Dead
96 Hours
% Unaffected
% Affected
% Dead
pH 2.0
0
0
100
0
0
100
0
0
100
0
0
100
pH 3.0
0
0
100
0
0
100
0
0
100
0
0
100
pH 4.0 pH 5.0
85
10
5
60
0
40
60
0
40
60
0
40
100
0
0
100
0
0
90
5
5
85
0
15
pH 6.0
100
0
0
100
0
0
100
0
0
100
0
0
83
-------
REACTION OF ARCTOPSYCHE GRAND IS (BANKS)
TO SULFUR 1C ACID
RESPONSE pH 2.0 pH 3.0 pH 4.0 pH 5.0 pH 6.0
24 Hours
% Unaffected 0 40 75 100 100
% Affected 25 50 25 0 0
% Dead 75 10 0 0 0
48 Hours
% Unaffected 0 40 75 100 100
% Affected 0 40 25 0 0
% Dead 100 20 0 0 0
72 Hours
% Unaffected 0 20 75 95 95
% Affected 0 30 15 0 0
% Dead 100 50 10 5 5
96 Hours
% Unaffected 0 15 55 90 95
% Affected 0 25 10 0 0
% Dead 100 60 35 10 5
REACTION OF ARCTOPSYCHE GRAND IS (BANKS)
TO HYDROCHLORIC ACID
RESPONSE pH 2.0 pH 3.0 pH 4.0 pH 5.0 pH 6.0
24 Hours
% Unaffected 15 85 100 100 100
% Affected 15 10 0 0 0
% Dead 70 5 0 0 0
48 Hours
% Unaffected 0 65 85 100 100
% Affected 05000
% Dead 100 30 15 0 0
72 Hours
% Unaffected 0 65 85 100 100
% Affected 05000
% Dead 100 30 15 0 0
96 Hours
% Unaffected 0 65 85 95 100
% Affected 0 5 00 0
% Dead 100 30 15 50
84
-------
REACTION OF ARCYNOPTERYX PARALLELA PRISON
TO SULFUR 1C ACID
RESPONSE
2k Hours
% Unaffected
% Affected
% Dead
1*8 Hours
% Unaffected
% Affected
% Dead
72 Hours
% Unaffected
% Affected
% Dead
96 Hours
% Unaffected
% Affected
% Dead
pH 2.0
0
0
100
0
0
100
0
0
100
0
0
100
pH 3.0 pH k.O pH 5.0
0
5
95
0
0
100
0
0
100
0
0
100
55
25
20
50
20
30
25
20
55
90
10
0
100
0
0
85
0
15
PH 6.0
100
0
0
100
0
0
45
25
30
100
0
0
100
0
0
100
0
0
REACTION OF ARCYNOPTERYX PARALLELA FRISON
TO HYDROCHLORIC ACID
RESPONSE
2k Hours
% Unaffected
% Affected
% Dead
48 Hours
% Unaffected
% Affected
% Dead
72 Hours
% Unaffected
% Affected
% Dead
96 Hours
% Unaffected
% Affected
% Dead
PH 2.0
0
0
100
0
0
100
0
0
100
0
0
100
pH 3.0
0
0
100
0
0
100
0
0
100
0
0
100
pH k.Q
55
10
35
35
0
65
35
0
65
5
0
95
pH 5.0 pH 6.0
100
0
0
95
0
5
95
0
5
75
0
25
100
0
0
100
0
0
100
0
0
100
0
0
85
-------
REACTION OF PTERONARCELLA BAD IA (HAGEN)
TO SULFUR 1C ACID
RESPONSE
2k Hours
% Unaffected
% Affected
% Dead
48 Hours
% Unaffected
% Affected
% Dead
72 Hours
% Unaffected
% Affected
% Dead
96 Hours
% Unaffected
% Affected
% Dead
pH 2.0 pH 3.0 pH 4.0 pH 5.0 pH 6.0
0
0
100
0
0
100
0
0
100
0
0
100
0
40
60
0
0
100
0
0
too
0
0
100
65
35
0
65
25
10
55
15
30
95
5
0
95
5
0
90
5
5
100
0
0
100
0
0
55
15
30
95
5
0
100
0
0
100
0
0
REACTION OF PTERONARCELLA BADIA (HAGEN)
TO HYDROCHLORIC ACID
RESPONSE
2k Hours
% Unaffected
% Affected
% Dead
48 Hours
% Unaffected
% Affected
% Dead
72 Hours
% Unaffected
% Affected
% Dead
96 Hours
% Unaffected
% Affected
% Dead
pH 2.0
0
0
100
0
0
100
0
0
100
0
0
100
pH 3.0 pH 4.0 pH 5.0 pH 6.0
10
10
80
0
0
100
0
0
100
0
0
100
85
15
0
35
0
65
100
0
0
100
0
0
100
0
0
70
5
25
100
0
0
100
0
0
100
0
0
15
0
85
90
0
10
100
0
0
86
-------
REACTION OF ISOPERLA FULVA CLAASSEN
TO SULFUR 1C ACID
RESPONSE
2k Hours
% Unaffected
3 Affected
% Dead
48 Hours
% Unaffected
% Affected
% Dead
72 Hours
% Unaffected
% Affected
I Dead
96 Hours
% Unaffected
% Affected
% Dead
pH 2.0
0
0
100
0
0
100
0
0
100
0
0
100
pH 3.0 pH 4.0 PH 5.0 pH 6.0
0
0
100
0
0
100
0
0
100
0
0
100
15
35
50
10
10
80
10
10
80
10
10
80
95
5
0
95
0
5
80
5
15
80
5
15
100
0
0
100
0
0
100
0
0
100
0
0
REACTION OF ISOPERLA FULVA CLAASSEN
TO HYDROCHLORIC ACID
RESPONSE
2k Hours
% Unaffected
% Affected
% Dead
48 Hours
% Unaffected
% Affected
% Dead
72 Hours
% Unaffected
% Affected
% Dead
96 Hours
% Unaffected
% Affected
% Dead
pH 2.0
0
0
100
0
0
100
0
0
100
0
0
100
pH 3-0
0
0
100
0
0
100
0
0
100
0
0
100
pH 4.0 pH 5.0
15
10
75
15
5
80
0
0
100
0
0
JOO
95
0
5
75
0
25
75
0
25
75
0
25
pH 6.0
100
0
0
100
0
0
100
0
0
100
0
0
87
-------
REACTION OF ACRONEURIA PACIFICA BANKS
TO SULFUR 1C ACID
RESPONSE
2k Hours
% Unaffected
% Affected
% Dead
48 Hours
% Unaffected
% Affected
% Dead
72 Hours
% Unaffected
% Affected
% Dead
96 Hours
% Unaffected
% Affected
% Dead
pH 2.0 pH 3-0 pH 4.0 pH 5-0 pH 6.0
0
0
100
0
0
100
0
0
100
0
0
100
0
0
100
0
0
100
65
35
0
0
0
100
0
0
100
65
35
0
65
10
25
55
5
40
85
15
0
85
15
0
85
10
5
85
10
5
100
o
o
100
0
0
100
0
0
100
0
0
REACTION OF ACRONEURIA PACIFICA BANKS
TO HYDROCHLORIC ACID
RESPONSE
2k Hours
% Unaffected
% Affected
% Dead
48 Hours
% Unaffected
% Affected
% Dead
72 Hours
% Unaffected
% Affected
% Dead
96 Hours
% Unaffected
% Affected
% Dead
PH 2.0 pH 3.0 pH 4.0 pH 5.0 pH 6.0
0
0
100
0
0
100
0
0
100
0
0
100
20
25
55
0
0
100
0
0
100
0
0
100
90
10
0
85
0
15
85
0
15
100
0
0
100
0
0
100
0
0
100
0
0
90
10
0
100
0
0
100
0
0
100
0
0
100
0
0
-------
REACTION OF EPHEMERELLA GRAND IS GRAND IS EATON
TO SULFUR 1C ACID
RESPONSE
2k Hours
% Unaffected
% Affected
% Dead
48 Hours
% Unaffected
% Affected
% Dead
72 Hours
% Unaffected
% Affected
% Dead
96 Hours
% Unaffected
% Affected
% Dead
pH 2.0
0
0
100
0
0
100
0
0
100
0
0
100
pH 3.0 pH 4.0
40
15
45
0
0
100
0
0
100
0
0
100
65
25
10
60
15
26
60
15
25
60
15
25
PH 5.0
95
5
0
90
0
10
90
0
10
90
0
10
pH 6.0
100
0
0
100
0
0
100
0
0
100
0
0
REACTION OF EPHEMERELLA GRAND IS GRAND IS EATON
TO HYDROCHLORIC ACID
RESPONSE
24 Hours
% Unaffected
% Affected
% Dead
48 Hours
% Unaffected
% Affected
% Dead
72 Hours
% Unaffected
% Affected
% Dead
96 Hours
% Unaffected
% Affected
% Dead
pH 2.0 pH 3.0 pH 4.0 pH 5.0 pH 6.0
0
0
100
0
0
100
0
0
100
0
0
100
35
10
55
0
0
100
0
0
100
0
0
100
95
5
0
95
5
0
65
0
35
65
0
35
100
0
0
85
0
15
85
0
15
85
0
15
100
0
0
100
0
0
100
0
0
95
0
5
89
-------
SELECTED WATER
RESOURCES ABSTRACTS
INPUT TRANSACTION FORM
ort No.
J. Accession No.
w
Title
Water Quality Requirements of Aquatic Insects
7. Author(s)
Arden R. Gaufin
9, 0 r
Department of Biology, University of Utah, Salt Lake City,
Utah 84112 and University of Montana Biological Station,
Bigfork, Montana 59911
12, Sponsoring Organization
5. Report Due
6,
J. Performing Orgtaizxtioa
Report No.
10. Project No.
18050 FLS
It. ContracttGrantNo.
13. Type < ' Repor and
Period Covered
15. Supplementary Motes
Environmental Protection Agency report number,
EPA-660/3-73-004, September 1973.
Abstract jne iarvae Of twenty species of aquatic insects (Diptera, Ephemeroptera,
Plecoptera, and Trichoptera) and the scud (Amphipoda) were exposed to high water temper-
atures, low dissolved oxygen concentrations, and low pH to determine their tolerance of
these three environmental factors. The temperature at which 50% of the specimens died
after 96 hours exposure ranged from 11.7° C for the mayfly, Cinygmula par Eaton, to
32.6° C for the snipe fly, Atherix variegata Walker. The mayfly, Ephemeral!a doddsi
Needham, was most sensitive to low dissolved oxygen levels with a 96-hour TLm of 5.2 mg/1
Acroneuria pacifica Banks, a stonefly, was the most resistant with a TLm 96 of 1.6 mg/1.
Median tolerance levels for pH ranged from pH 2.7 for the caddis fly, Limnephi 1 us omatus
Banks, to 7.2 for the scud, Gammarus limnaeus Smith. Longer term bioassays clearly
incidated increased sensitivity and mortality of the test specimens with increased length
of exposure to each of these factors.
To maintain a well-rounded diversified population of cold water aquatic^ trisects,
maximum temperatures, minimum dissolved oxygen levels, and the pK range should not exceed
the requirements of cold water fishes, such as trout and salmon. While some aquatic
insects can tolerate dissolved oxygen levels as low as 1.6 mg/1 for short periods,
concentrations of 6.0 mg/1 are required for long-term survival. Temperatures during the
winter months must be maintained at normal seasonal levels to prevent premature emergence
Temperatures above 65° F during the summer months are considered the maximum for main-
taininq many species of stoneflies. mayflies, and caddis flies. A pH range of 6.0 - 8.5
efHH$efit most cold water lotic insects.
Water pollution, Water Quality, Aquatic Insects, Thermal Pollution, Dissolved Oxygen, pH.
17b. fdentifiers
Pollution Evaluation, Water Quality Criteria, Receiving Waters, Water Quality Require-
ments.
17c. COWRR Field A Group
IS. Availability
19, SfurityC iss.
(Keport)
20. Security Class.
21. ?,--..»/
Pages
22. Price
Send To:
WATER RESOURCES SCIENTIFIC INFORMATION CENTER
U.S. DEPARTMENT OF THE INTERIOR
WASHINGTON, O. C. 2O24O
Abstractor
Institution
WREtC IO2 (REV JUNE 1971!
------- |