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

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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

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 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

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                      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

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 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

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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

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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

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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

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                 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

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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

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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

-------
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

-------
       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

-------
                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-
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     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

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                     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

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                          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

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             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!

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