Tennessee
Valley
Authority
Division of Environmental
Planning
Chattanooga TN 37401
TVA/EP-78/09
United States
Environmental Protection
Agency
Research and Development
Office of Energy, Minerals, and
Industry
Washington DC 20460
EPA-600/7-78-128
July 1978
Effects of Thermal
Discharges
on Aquatic Insects
in the Tennessee
Valley

Interagency
Energy/Environment
R&D  Program
Report

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                 RESEARCH REPORTING SERIES

 Research reports of the Office of Research and Development, U.S. Environmental
 Protection Agency, have been grouped into nine series. These nine broad cate-
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 planned to foster technology transfer and a maximum interface in related fields.
 The nine series are:

      1.  Environmental Health Effects Research
      2.  Environmental Protection Technology
      3.  Ecological Research
      4.  Environmental Monitoring
      5.  Socioeconomic Environmental Studies
      6.  Scientific and Technical Assessment Reports (STAR)
      7.  Interagency Energy-Environment Research and Development
      8.  "Special" Reports
      9.  Miscellaneous Reports

 This report has been assigned to the INTERAGENCY ENERGY-ENVIRONMENT
 RESEARCH AND DEVELOPMENT series. Reports in this series result from the
 effort funded under the 17-agency Federal Energy/Environment Research and
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 essary environmental data and control technology. Investigations include analy-
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This document is available to the public through the National Technical Informa-
tion Service, Springfield, Virginia 22161.

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                                             EPA-600/7-78-128
                                             TVA/EP-78/09
                                             July 1978
EFFECTS OF THERMAL DISCHARGE ON AQUATIC INSECTS
            IN THE TENNESSEE VALLEY
                      by
   Kenneth J. Tennessen and Johnny L. Miller
    ,  Division of Environmental Planning
          Tennessee Valley Authority
         Muscle Shoals, Alabama  35660
     Interagency Agreement No. D8-E721-DR
            Project No. E-AP 80-BDR
         Program Element No. INE-625A
                Project Officer

                Clinton W. Hall
   Office of Energy, Minerals, and Industry
     U.S. Environmental Protection Agency
             Washington, DC  20460
                 Prepared for
    OFFICE OF ENERGY, MINERALS AND INDUSTRY
      OFFICE OF RESEARCH AND DEVELOPMENT
     U.S. ENVIRONMENTAL PROTECTION AGENCY
             WASHINGTON, DC  20460

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                              DISCLAIMER
     This report was prepared by the Tennessee Valley Authority and has
been reviewed by the Office of Energy, Minerals, and Industry, U.S.
Environmental Protection Agency, and approved for publication.  Approval
does not signify that the contents necessarily reflect the views and
policies of the Tennessee Valley Authority or the U.S. Environmental
Protection Agency, nor does mention of trade names or commercial products
constitute endorsement or recommendation for use.
                                 ii

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                               ABSTRACT
     The primary objectives of this research project are (1)  to determine
the thermal tolerances of selected aquatic insects and (2)  to investigate
growth and emergence of selected aquatic insects in the vicinity of Ten-
nessee Valley Authority electric generating plants.   The information may
help establish thermal effluent limits to protect the aquatic ecosystem.

     The burrowing mayfly Hexagenia bilineata (Say)  and the midge
Coelotanypus sp. were chosen for study because they are abundant and
they occur in areas affected by thermal discharges from Tennessee Valley
Authority electric generating plants.

     The thermal{plume in the vicinity of Johnsonville Steam  Plant
(TRM 98 to 101)
only during the
were subjected
 'Humphreys County, Tennessee, reached the river bottom
 fall and winter  (1976-1977).  Therefore, benthic insects
.o above-ambient  temperatures during the coldest part of
the year.  In early spring, H. bilineata nymphs collected from the
area influenced by the thermal plume were larger on the average than
those collected from the ambient station.  However, growth at the ambient
station accelerated during late spring, and adult emergence occurred
almost simultaneously at both stations.

     Tolerance of the immature stages of both study species to thermal
shock was great; abrupt changes in temperature of 20°C (at low accli-
mation temperature of 10 and 15°C) resulted in low percentages of
mortality although sample numbers were low.

     Another stage in the life cycle that is subjected to ATs resulting
from thermal plumes is the egg.  The optimum range of constant tempera-
tures for development of H. bilineata eggs is from 31 to 34°C; the
upper limit for egg development is near 37°C.  Eggs subjected to a brief
(5 to 15 rain) shock of 10°C at the time of oviposition yielded a mean per-
centage of hatching comparable to that of the control treatment.  However,
a shock of 15°C resulted in a greatly reduced mean percentage of hatching.

     No difference in fecundity of adult females was found between the
ambient and thermal plume stations at Johnsonville Steam Plant.  Adult
males emerging from the heated discharge channel were significantly
larger on the average than males from the ambient station.

     A drift study conducted at TVA's John Sevier Steam Plant using damsel-
fly nymphs (Enallagma spp.) and mayfly nymphs (Stenonema spp.) in a thermal
plume resulted in little or no mortality at ATs that normally result from
the heated water.

     This report was submitted by the Tennessee Valley Authority, Division
of Environmental Planning, in partial fulfillment of Energy Accomplishment
Plan 80-BDR under terms of Interagency Agreement D8-E721-DR with the
Environmental Protection Agency.  Work was completed in October 1977.
                                iii

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                                CONTENTS
                                                                  Page
Abstract	    iii
List of Figures	    vii
List of Tables	    x
List of Abbreviations and Symbols	    xi
Acknowledgments  	    xii

     1.  Introduction  	     1
     2.  Conclusions 	     2
     3.  Recommendations 	     3
     4.  Experimental Procedures 	     4
           Field studies	     4
           Laboratory studies  	•	     9
     5.  Results and Discussion	    12
           Temperature profiles at Johnsonville Steam
             Plant and implications for aquatic insects  ....    12
           Growth and emergence time of
             H. bilineata	    12
           Size and fecundity comparisons  of
             H. bilineata	    28
           Thermal tolerance of immature aquatic insects ....    28
           Effects of entrainment within thermal plume
             on aquatic insects	    33
           Egg development and thermal tolerance 	    36

References	    48
Glossary	    49

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                            LIST OF FIGURES
Number
          Outline map of Kentucky Lake in vicinity of
          Johnsonville Steam Plant showing locations of
          the five sampling stations 	
          Emergence trap used to collect emerging
          mayflies 	
          Flotation device used to hold aquatic insects
          for drift study  	
   4      Plexiglas flow-through container for culturing
          H. bilineata eggs	10

   5      Vertical temperature profiles at five stations
          in vicinity of Johnsonville Steam Plant,
          Kentucky Lake, from October 1976 to March 1977 ....   13

   6      Vertical temperature profiles at five stations
          in vicinity of Johnsonville Steam Plant,
          Kentucky Lake, from April to August 1977	14

   7      Vertical temperature profiles at five stations
          in vicinity of Johnsonville Steam Plant,
          Kentucky Lake, from August to October 1977 	   15

   8      Substrate temperatures at two stations near
          Johnsonville Steam Plant, Kentucky Lake,
          from October 19, 1976, to September 21, 1977 	   16

   9      Average head width and fore wing pad length
          of H. bilineata nymphs collected at stations
          1 and 3 near Johnsonville Steam Plant at
          3-week intervals from March 31 to August 3, 1977 ...   18

  10      Comparison of frequency (%) of H. bilineata
          nymphs in various size classes (based on head
          width) at stations 1 and 3 near Johnsonville
          Steam Plant on March 31, 1977	19

  11      Comparison of frequency (%) of H. bilineata
          nymphs in various size classes (based on head
          width) at stations 1 and 3 near Johnsonville
          Steam Plant on April 19, 1977	20
                                 vii

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Number                                                           Page


  12      Comparison of frequency (%) of H.  bilineata
          nymphs in various size classes (based on head
          width) at stations 1 and 3 near Johnsonville
          Steam Plant on May 12, 1977	    21

  13      Comparison of frequency (%) of H.  bilineata
          nymphs in various size classes (based on head
          width) at stations 1 and 3 near Johnsonville
          Steam Plant on June 2, 1977	    22

  14      Comparison of frequency (%) of H.  bilineata
          nymphs in various size classes (based on head
          width) at stations 1 and 3 near Johnsonville
          Steam Plant on June 22, 1977	    23

  15      Comparison of frequency (%) of H.  bilineata
          nymphs in various size classes (based on head
          width) at stations 1 and 3 near Johnsonville
          Steam Plant on July 13, 1977	    24

  16      Comparison of frequency (%) of H.  bilineata
          nymphs in various size classes (based on head
          width) at stations 1 and 3 near Johnsonville
          Steam Plant on August 3, 1977	    25

  17      Comparison of frequency (%) of H.  bilineata
          nymphs in six size classes of  head width
          (interval =0.5 mm)  from March to  August 1977
          at  Stations 1 and 3  near Johnsonville Steam Plant  .  .    26

  18      Percentage of H.  bilineata nymphs  having black
          wing  pads at stations 1 and 3  on three sampling
          dates,  1977	    27

  19      Scatter diagram of relationship between
          abdomen length and number of eggs  in
          H.  bilineata females   	    30

  20      Predicted and observed development times (in
          days)  for H.  bilineata eggs cultured  at  seven
          nearly constant (±1°C)  temperatures 	    38

  21      Average percentage of hatching of  H.  bilineata
          eggs  due  to  main  effects  of shock  temperature (A),
          shock duration (B), and day after  oviposition (C)  .  .    44
                                 viii

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Number                                                           Page
  22      Surface response curves for shock temperature and
          shock duration (interaction) effects on mean per-
          centage of hatching of H. bilineata eggs over a
          4-day hatching period	    45

  23      Effect of shock temperature on mean cumulative
          percentage of hatching of H. bilineata eggs
          exposed to thermal shock immediately after
          oviposition	    46

  24      Interaction of shock duration and hatching day
          on the mean percentage of hatching of H. bilineata
          eggs exposed to thermal shock immediately after
          oviposition	    47
                                  ix

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                            LIST OF TABLES
Number
   1      Mean Values (x),  Standard Deviations  (s),  and
          Number of Observations (n) for Male and Female
          H. bilineata Wing Length, Abdomen Length,  and
          Egg Count at Stations 1 and 3 near Johnsonville
          Steam Plant	    29

   2      Results of t-Tests Comparing Mean Size and
          Fecundity of H. bilineata Subimagoes  at Stations
          1 and 3 near Johnsonville Steam Plant	    31

   3      Number of H. bilineata Nymphs Surviving
          Four Experimental Thermal Shocks  	   32

   4      Number of Coelotanypus sp. Larvae Surviving
          Four Experimental Thermal Shocks  	   34

   5      Number of Damselfly and Mayfly Nymphs Surviving
          after 8 Hours of  Exposure in the Thermal Plume
          and at Ambient Station near John Sevier Steam
          Plant, July 20, 1976	   35

   6      Mean Cumulative Percentage of Hatching
          Through 4 Days of H.  bilineata Eggs Cultured
          at 8 Constant Temperatures	   37

   7      Summary of Hatching Results at Constant
          Temperature and Tests for Significance of
          Difference Between Means as Determined by
          Duncan's Multiple Range Test  	   39

   8      Percentages of Hatching of H.  bilineata Eggs
          Exposed to Thermal Shocks (ATs of 5,  10, and
          15°C)  for Three Durations (5,  10,  and 15 min)
          Immediately after Oviposition 	   40

   9      Analysis of Variance  of 3-Factor Thermal
          Shock Experiment  on H.  bilineata Eggs	   42

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                   LIST OF ABBREVIATIONS AND SYMBOLS
AT     — change in temperature above ambient
°C     — degrees Celsius
df     — degrees of freedom
DO     — dissolved oxygen
h      — hour
HRM    — Holston River mile
km     — kilometer
m      — meter
min    — minute
ml     — milliliter
mm     — millimeter
n      — number of individuals in a sample
P      — probability
r      — correlation coefficient
s      — standard deviation
TRM    — Tennessee River mile
TVA    — Tennessee Valley Authority
x      — mean value
                                 xi

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                            ACKNOWLEDGMENTS
     The cooperation of several people in TVA's Water Quality and
Ecology Branch, Division of Environmental Planning, is gratefully
acknowledged:  Mr. Barry 0. Bell, who assisted in collecting the field
and laboratory data; Ms. Sylvia A. Murray, who helped in the experimental
design of temperature effects on mayfly eggs, helped with the analyses,
and critically reviewed the manuscript; and Mr. Thomas W. Toole, who
calculated statistical parameters for the fecundity data and generated
the regression equation for egg development.  We especially thank
Dr. Richard D. Urban for his initial planning of the thermal effects
research on aquatic insects and for advice and criticism throughout
the study.  Mr. James R. Marsh, Laboratory Branch, is also acknowledged
for his fine work in the design and fabrication of the emergence traps
and flow-through egg containers.
                               xii

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                               SECTION 1
                             INTRODUCTION
     The continued increase in demand for electric power increases the
amount of heated water discharged from fossil-fueled electric generating
plants, which in turn further alters the temperature regimes of aquatic
environments.  How much can temperatures be altered before changes occur
in the biology of aquatic plants and animals?  What are the limits on
thermal discharge that will not jeopardize a balanced indigenous flora
and fauna?

     The theory that diversity in an ecosystem ensures stability is
generally supported by results of ecological research.  Insects are a
major component of diversity in aquatic ecosystems, and their importance
in the food we)b and energy flow is well documented.  Yet relatively
little is known about the thermal tolerances of aquatic insects,
especially species in the southeastern United States.

     In this study, the thermal tolerances of selected species of
aquatic insects in the Tennessee River Valley were investigated.
Emphasis was placed on those stages of the life cycle that are subject
to thermal discharge from Tennessee Valley Authority (TVA) electric-
generating plants.  These stages include the eggs, the immatures
(stationary or drifting), and the stage of emergence to the adult
insect.

     Several questions were asked about what actually happens in a field
situation:

     1.  What stages of the selected species' life cycle are influenced
         by thermal discharge?

     2.  What magnitudes of change in temperature above ambient (AT)
         do these stages experience?

     3.  What are the thermal tolerances of these stages?

     4.  Does entrainment within a thermal plume affect survival?

     5.  How do elevated temperatures affect growth rate, emergence
         time, and fecundity?

     The species chosen for study were the burrowing mayfly, Hexagenia
bilineata (Say) (Ephemeroptera:  Ephemeridae) and the midge, Coelotanypus
sp. (Diptera:  Chironomidae), both of which are important food  items for
fish.  Both species are sufficiently abundant to facilitate experimenta-
tion, and both occur in areas subject to thermal discharges.  Also,
mortality of these species due to laboratory conditioning is low.  Dam-
selflies of the genera Enallagma and Ischnura and mayflies of the  genus
Stenonema were also studied.

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                                 -2-
                               SECTION 2
                              CONCLUSIONS
     Results from this study support other reports that insects inhabiting
large rivers and reservoirs are quite tolerant of thermal shock.  Eggs of
the burrowing mayfly Hexagenia bilineata developed and hatched (mean
hatching percentage was 63%) after a 15-min exposure to 40°C (ambient =
30°C, AT = 10°C) immediately after oviposition.  However, a 5- to 15-min
exposure to 45°C (AT = 15°C) resulted in a low mean percentage of hatch-
ing (13%).  A constant temperature of 37°C limited egg development.
Nymphs of H. bilineata and larvae of the midge Coelotanypus sp. withstood
thermal shocks as high as 20°C in the laboratory at low acclimation
temperatures (5 to 15°C).

     Although acute effects from moderate thermal shocks in the labora-
tory were minimal, effects on several aspects of the life cycle of H.
bilineata were found.  Nymphal growth was greater in areas where the
thermal plume extends to the bottom during the winter and early spring
than in ambient areas.  However, development in ambient areas accel-
erated in late spring so that adults emerged at about the same date as
those in thermal areas.  Size and fecundity of female H. bilineata
emerging from thermal plume areas and from ambient areas did not differ
significantly, although males from thermal plume areas may be significantly
larger.   These differences in the mayfly's biology found to date were not
expected to adversely affect the species in the study area.  Damselfly
and mayfly nymphs (Enallagma and Stenonema.) were held in the thermal plume
at John Sevier Steam Plant, resulting in no significant mortality,
compared with controls.

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                                -3-
                               SECTION 3
                            RECOMMENDATIONS
     The effect of various acclimation temperatures on the thermal toler-
ance of aquatic insects should be investigated further; results may he
useful for predicting seasonal effects.  Tolerance to cold shock should
be determined.  Organisms that survive experimental heat and cold shocks
should be observed afterward for latent mortality and chronic effects.
The experiment on the effect of thermal shock on H. bilineata eggs
should be repeated with better control.  The study of the effects of
thermal discharges at Johnsonville Steam Plant on the time of emergence,
size, and fecundity of H. bilineata should be continued for another
year.         j

     Establishing temperature limits to protect aquatic insects will
require study of species from other orders because tolerances no doubt
differ.  From the information gathered in this study, it appears that
thermal plumes extending to the river bottom that are below 37°C in
summer would fully protect the most sensitive stages of H. bilineata.

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                                 -4-


                                SECTION 4



                         EXPERIMENTAL PROCEDURES
      Workers  use two approaches  to  study the  effects of temperature
 increase.   In the field,  qualitative  and quantitative  changes in the
 fauna are  monitored, and  results are  usually  reported  as changes in
 population structure and  diversity  indexes.   In  the laboratory, various
 stages in  the life cycle  are  subjected  to different temperature regimes
 to  determine  thermal tolerances.  Rarely have these approaches been
 combined.   This  study used  both  approaches.   The field studies were
 aimed at discovering (1)  which species  and which stages of the life
 cycles of  those  species are influenced  by a thermal plume and (2)
 whether the plume affects growth, time  of emergence, and fecundity.
 Laboratory experiments were devised to  investigate implications from
 field data under controlled conditions.
FIELD STUDIES
     Field studies were conducted at TVA's Johnsonville Steam Plant and
vicinity  (TRM 98  to  101), on Kentucky Lake in Humphreys County,
Tennessee, and at TVA's John Sevier Steam Plant on the Holston River
(HRM 106.5 to 106) in Hawkins County, Tennessee.
Stages of Life Cycle Subjected to Thermal Plume

     To determine whether the benthic, immature stages of H. bilineata
and Coelotanypus sp. are subjected to the thermal plume from Johnson-
ville Steam Plant, monthly vertical temperature profiles were determined
with a calibrated thermistor.  Temperatures were measured at 1-m
intervals at five stations (Figure 1) between October 1976 and October
1977.  Bottom temperatures indicate whether the thermal plume reached
the benthic habitat and reveal the magnitude of ATs experienced by the
benthos.

     Mating swarms of H. bilineata were observed in the vicinity of
Johnsonville Steam Plant on June 5, 1977, around dusk.  Mated females
were observed to determine whether oviposition takes place in the
discharge channel, which would expose eggs to above-ambient temperatures.

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                              -5-
                     TRM 99+
                                                 -N-
                TRM
                                     DISCHARGE CHANNEL

                                       JOHNSONVILLE
                                         STEAM PLANT

                                      INTAKE CHANNEL
                                           '.70
Figure 1.  Outline map  of Kentucky Lake in vicinity of Johnsonville
          Steam Plant  showing locations of the five sampling stations.

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                                 -6-
 Determination of Growth,  Time of Emergence, and Fecundity

      To compare growth rates  of H. bilineata nymphs in ambient tempera-
 tures with those in thermal plume temperatures, five samples were taken
 with a Ponar dredge at stations 1 and  3  (TRM 100.6 and TRM 99.5 respec-
 tively) at 3-week intervals from March 31, 1977, to August 24, 1977.
 The sediments were field-washed on a 48-mesh sieve, and the mayfly
 nymphs were preserved in  an 80% ethanol  solution.

      Nymphal head capsule widths and wing pad lengths were measured
 with an ocular micrometer on  a binocular microscope.  Because individual
 instars cannot be exactly determined for H. bilineata,1 size categories
 of  0.2 mm were established  for head capsule width.  Data were trans-
 formed to percentages of  each size category, and histograms were con-
 structed.   Mean head width  and mean wing pad length were calculated for
 each station and sampling date.

      Time of emergence of H.  bilineata at stations 1 and 3 was deter-
 mined by trapping emerging  subimagoes  with 1-m2 floating traps (Figure
 2).   Three traps were anchored at each station from June 1 to June 6,
 1977;  trapped adults  were collected and  preserved in the mornings.

      Fore  wing and abdomen  lengths were  measured with a millimeter rule.
 Egg  count  for each female was estimated  by stirring the eggs in 10 ml of
 distilled  water and pipetting a 1-ml subsample for counting.  A linear
 correlation of size and fecundity was  drawn for each station, and the
 two  stations  were compared.   Student's t-test was used to compare the
 mean lengths  of wing  and  abdomen and mean egg counts between the two
 stations.
Entrainment

     A drift study was conducted at John Sevier Steam Plant on July 20,
1976, to determine the effect of the thermal plume on survival.  This
site was chosen because of the distance the thermal plume extends down
the Holston River (over 8 km) and the usually high ATs (6 to 10°C) that
occur.  These two factors create conditions for a maximum-exposure field
test.

     The experimental insects, damselfly nymphs of the genus Enallagma
and mayfly nymphs of the genus Stenonema, were collected from the
cooling-water intake channel.  Ten insects were placed in each Plexi-
glas box attached to a flotation apparatus (Figure 3).  One flotation
apparatus was anchored in the thermal plume, and another was anchored in
the ambient temperature area near the right bank.  After 6 h of exposure,
the insects were removed, and the dead organisms were counted.

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                             -7-
Figure 2.  Emergence trap used to collect emerging mayflies

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ALUMINUM
   ANGLE BAR
                                     EYE BOLT
                                        AND SNAP
POLYURETHANE
   FLOAT
                                                               ALUMINUM
                                                                 PLATE
                                              h-6-in.3  HOLDING CAGE
                                                                           00
       Figure 3. Flotation device used to hold aquatic insects for drift study.

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                                -Q-
LABORATORY STUDIES
Testing Immature Stages for Thermal Tolerance

     Larvae of Coelotanypus sp. were collected from Shoal  Creek (near
TRM 264.5) in Lauderdale County, Alabama, on November 18,  1976.  The
larvae were acclimated to 15°C for three days in the laboratory.
After the acclimation period, larvae were shocked at three different
temperatures—25, 35, and 40°C--which exposed them to ATs  of 10,  20,
and 25°C, respectively, above the control temperature of 15°C.   Ten
larvae were used for each treatment.  Mortality was checked at  15-
min intervals for 2.5 h.  Death was assumed when larvae did not respond
when squeezed gently with a pair of forceps.  At the end of the test the
larvae were held at 25°C for 8 h to check for latent mortality.

     Nymphs jof H. bilineata were collected from impounded  areas of
Second Creek (near TRM 275) near Highway 72 West, Lauderdale County,
Alabama, on November 22, 1976; substrate temperature was 10°C at time of
collection.  The nymphs were acclimated in aerated aquariums in sub-
strate from their natural habitat at 10°C for 8 days. The  nymphs were
then subjected to ATs of 10, 20, and 30°C; a control group was  held at a
constant temperature of 10°C.  The treatment consisted of  ten nymphs per
replicate and three replicates per treatment.  The number  of dead were
counted at 15-min intervals for 3.5 h.  Dissolved oxygen was determined
with an oxygen meter (YSI model 54).
Thermal Tolerance of H. bilineata Eggs

     Two experiments were conducted:  (1) Eggs were cultured at dif-
ferent constant temperatures to determine the upper limit and the optimum
range for development; and (2) eggs were exposed during oviposition to
several ATs for various durations to simulate deposition of eggs in a
thermal plume before they sink to an ambient temperature.


Experiment (1).  Constant Temperature and Development—

     About 300 mated females were collected under lights between 9 and
10 p.m. along Brush Creek (near TRM 264.5) in Lauderdale County, Alabama,
on July 8, 1977.  The females were placed in 27°C water, where they
released their eggs.  The eggs were transported to the laboratory,
where 1-ml aliquots (700 to 2000 eggs) were placed in Plexiglas flow-
through chambers (Figure 4).  Twenty chambers were placed in each of
eight aquariums that were half-filled with 27°C water.  Each aquarium
was placed in a refrigerator-size incubator set at one of eight tempera-
tures— 19, 22, 25, 28, 31, 34, 37, and 40°C.  The aquariums were gently
aerated, and the temperatures were allowed to equilibrate overnight.  By
the next day the eggs had adhered to the bottom of the chambers.  The
aquariums were then filled, and the water was circulated with Dynaflow-II
motor filters.

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                                 -10-
Figure 4.  Plexiglas flow-through container for culturing H. bilineata
           eggs.

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                                -11-
     Wheu hatching began, five chambers were removed each day for four
days, and the number of eggs hatched and the number not hatched were
recorded.  The data were transformed to percentage hatched and analyzed
by a 2-factor analysis of variance.
Experiment (2).  Egg Tolerance to Brief Thermal Shock—

     About 200 females were collected around 9 p.m. at Shoal Creek
(near TRM 264.5) in Lauderdale County, Alabama.  About 50 individuals
were simultaneously placed in each of four large glass dishes containing
water of four different temperatures—30, 35, 40, and 45°C.  These
temperatures subjected the females to ATs of 0 (control), 5, 10, and
15°C.  After 3 min these females were removed; 5 min after oviposition
some of thejjeggs from each dish were transferred to separate, labelled
containers of 30°C water.  The same was done after 10 and 15 min of
exposure, yielding 12 treatments of varying temperature and duration.
The eggs that had been transferred were then transported to the labora-
tory and placed in flow-through containers as described in experiment (1),
except that all incubators were set to maintain a temperature of 30°C.

     When hatching began, three containers (replicates) per treatment
were removed each day for four days (total 144 samples).  Data were
collected, transformed to percentage hatched, and analyzed by a 3-factor
analysis of variance.

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                                 -12-


                                SECTION 5



                         RESULTS AND DISCUSSION
 TEMPERATURE  PROFILES  AT JOHNSONVILLE  STEAM PLANT AND IMPLICATIONS FOR
 AQUATIC INSECTS
      Surface temperature  data  from October  1976 to October 1977 indicate
 that the  thermal plume  extended  from  1 to 2 km downstream from the plant.
 Surface temperatures at station  5 ranged from 0 to 3.0°C above ambient
 throughout  the year.  These  results agree with a mathematical model of
 the  plume.2

      The  vertical  temperature  profiles revealed that the depth to which
 the  thermal plume  extended was variable (Figures 5 through 7), depending
 on time of  year.   Throughout late fall and  winter at station 3 and
 throughout  winter  at station 4,  the thermal plume extended to the river
 bottom (Figure 5).  During this  5-month period, the substrate, which is
 inhabited by benthic insects,  was about 4 to 6°C above ambient (Figure
 8).   The  greatest  AT measured  was 6.5°C, which occurred in February
 1977.  Beginning in April 1977,  the depth of the thermal plume diminished,
 and  bottom  temperatures in the discharge channel were at or very near
 ambient throughout the  spring, summer, and  early fall (Figures 6 and 7).

      These  temperature  data  indicate  that the organisms inhabiting the
 substrate of the discharge channel were living at temperatures 4 to
 6.5°C above ambient throughout the coldest  part of the seasons.  The
 average bottom temperature at  station 3 from November 17 to March 31 was
 15.2°C, compared with 10.6°C at  station 1 (average based on five samples).
 Growth of H.  bilineata  nymphs  becomes negligible at 10°C, according to a
 field study,3 and  at 14°C, according  to a laboratory study.4  Tempera-
 ture  of the substrate at station 3 was never below 10°C on any of the
 sampling  dates, whereas at station 1  it was well below 10°C on December
 22,  1976, and February  16, 1977.
GROWTH AND EMERGENCE TIME OF H. BILINEATA
     If growth of H. bilineata nymphs becomes negligible as temperatures
approach a low of 10°C, nymphs inhabiting heated areas, where tempera-
tures stay above 10°C (Figure 8), should be larger than those in ambient
areas by the end of winter.  Therefore, nymphs in heated discharge areas
should mature faster, and it is plausible that they would emerge sooner
as adults.

-------
0 l »*»>''
1 .
2.
H*s!
6_
7.
8


5


A

i /
/
0 10 20

J 1,2
_ i

-
10/19/76
_
.




\
\

30 0 10
TEMPERATURE
0 1,25 4,3
1 .
2_
a 5.
6.
7.
8















.
-
2/16/77
-
_







, 21

[
X
.
;
1 11/17/76
/
1



i



20 30 0
(°C)
125 43









.
-
3/16/77
-
-





\

5 43


/






12/22/76



10

1,









20 30

t$ 43


i





3/31/77



                  20
30        0        10

        TEMPERATURE (°c)
20
30
10
20      30
Figure 5.  Vertical temperature profiles at five stations in vicinity of Johnsonville Steam Plant,
           Kentucky Lake, from October 1976 to March 1977.

-------
UJ
a
0
1.
2.
3-
IK
5.
6.
7.
8
i >
i

VI 9/77
*-?
i '•
•
1
j
?"T



k ft

\ 3
/
    0
    1.
    2
I"  3J
 «  5J
    6.
    7.
    8
              10
            6/22/77
              10
20
20
30
                              1,2 5 A3
30
                                                 5/12/77
           10
20
                                            TEMPERATURE (°c)
                            7/13/77
 0        10       20
TEMPERATURE (°c)
30
         30
                                                               6/2/77
10
                                                       8/3/77
                    10
20
         20
30
        Figure  6.   Vertical temperature profiles at five stations in vicinity of Johnsonville Steam Plant,
                    Kentucky Lake, from April to August 1977.

-------
0  T
1

2

3  -



5  .

6

7  J

8
       8/31/77
              20
 9/21/77
10
20
                                                           215
                                                                     30
                                         TEMPERATURE  (°L  )
                         10/19/77
                                               213,4,5
20
30
Figure 7.  Vertical temperature profiles at five stations in vicinity of Johnsonville Steam Plant,
           Kentucky Lake, from August to October 1977.

-------
        ___   STATION 3
  OCT
    9
NOV
 17
DEC
 22
FEE
 16
MAR
 16
MAR
 31
APR
 19
MAY
 12
JUNE
  2
JUNE
 22
JULY
 13
AUG
 3
AUG
 31
                                     SAMPLING DATE (1976-77)
Figure 8.  Substrate temperatures at two stations near Johnsonville Steam Plant,
           Kentucky Lake, from October 19, 1976, to September 21, 1977.   No data
           were obtained in January 1977.
SEPT
 21
OCT
 19

-------
                                -17-
     Measurements of head capsules and wing pads of H.  bilineata nymphs
near Johnsonville Steam Plant show that mean size was greater at station
3 (in the heated discharge channel) than at station 1 from March 31  to
June 2 (Figure 9), with the exception of mean head width on June 2.
Frequency histograms of nymphs in size classes of 0.2-mm head width
showed higher percentages of large nymphs at station 3 from March 31 to
June 2 (Figures 10 through 13).  After June 2, mean head width was slightly
greater at station 1 (Figure 9).  The percentage of nymphs with head widths
greater than 2.8 mm on June 22 was higher at station 1 (Figure 14);  on
July 13 and August 3, these percentages were again higher at station 3
(Figures 15 and 16).  Mean wing pad length was greater at station 3  on
all sampling dates except June 22 (Figure 9).  The total number of
nymphs collected (n) on successive sampling dates decreased, probably
because of drift and increased predation by fish.

     The differences in percentages of nymphs in different size classes
(based on OJ5-mm intervals of head width) and the changes in these
percentages through time are shown in Figure 17.  A greater percentage
of nymphs were in the larger size classes (2.0 to 3.5 mm) at station 3
from March 31 to at least June 2.  Nymphs at station 1 first reached
the 3.0- to 3.5-mm size class sometime between May 12 and June 2. How-
ever, on June 2, nearly equal percentages of nymphs were in the 3.0- to
3.5-mm class at the two stations.

     These data on nymphal head size through time indicate that, although
the nymphs in the heated discharge channel were larger on the average
than those at the unheated station throughout most of the spring, nymphal
growth at station 1 accelerated, yielding a mean size comparable to
that at station 3 by June 2.

     Emergence to the subimago  (adult) stage occurs shortly after the
wing pads become thickened and black.  No emergence occurred in the
vicinity of Johnsonville Steam Plant before the first week of June 1977.
On June 2, 1977, 14 of 57 nymphs (19.7%) collected at station 3, as
compared with 1 of 27 (3.7%) at station 1, had thick, black wing pads
(Figure 18).  A chi-square test showed the difference to be significant
(P < 0.05) although the number of observations was low.  The differences
in the percentages for June 22 and July 13 are not significant.

     Emergence to the adult occurred about two or three days earlier at
station 3.  On June 2, one adult was seen at station 3 in the discharge
channel; no adults were seen at station 1.  On the night of June 4,  a
large number of emerging mayflies were collected in emergence traps  at
station 3.  No mayflies were trapped at station 1, although subimagoes
were present along the nearby bank on the morning of June 5.  Subimagoes
were also present 2 to 6 km downstream from the steam plant.  Another
large emergence at station 3 occurred on the night of June 5; an emer-
gence also occurred at station 1, but again no adults were found in the
emergence traps.  Therefore, subimagoes were later netted at station 1
for size and fecundity comparisons with those trapped at station 3.

-------
  5.0
  4'0 -
  2.0

  0.0
                                                                               STATION 1
                                                                    ......  STATION 3
  4.0


  3.0
g
x 1.0


   0.0
                              1
            1
             1
              _L
                  MAR
                   31
                 APR
                  19
MAY
 12
JUNE
  2
JUNE
 22
JULY
 13
             JL
AUG
 3
 Figure 9.
Average head width and fore wing pad length of H.  bilineata nymphs collected  at
stations 1 and 3 near Johnsonville Steam Plant at 3-week intervals from March 31

to August 3, 1977.
                                                                                                           oo
                                                                                                            i

-------
    50
    40
                                                       STATION 1 (n = 144)
                                                                    STATION 3 (n = 144)
    30
uj   20
a
m
    10
Figure  10.
                      SIZE CATEGORIES OF HEAD WIDTH  (mm)



Comparison of frequency (%)  of H. bilineata nymphs in various size classes (based

on head width) at stations 1 and 3 near Johnsonville Steam Plant on March 31,  1977

-------
   50
   40
   30
   20
S
8E
    10
                                                             I
STATION 1 (n = 102)
                                                                 STATION 3 (n = 144)
                                                                                                            O
                                                                                                            I
                                     SIZE CATEGORIES OF HEAD WIDTH  (mm)
 Figure  11.  Comparison of  frequency  (%) of H. bilineata nymphs in various size classes (based
             on head width) at stations 1 and 3 near Johnsonville Steam Plant on April 19, 1977.

-------
   50
   40
   30
                                                                     STATION 1 (n = 69)
                                                                     STATION 3 (n = 110)
   20
   10
    0
                                                                                                           i
                                                                                                           to
Figure 12.
                         SIZE CATEGORIES OF HEAD WIDTH (mm)



Comparison of frequency (%) of H. bilineata nymphs in various size classes (based

on head width) at stations 1 and 3 near Johnsonville Steam Plant on May 12,  1977.

-------
   50
   40
   30
>-


S  20
   10
                               STATION 1.  (n = 28)
                               STATION 3  (n =  71)
                                                                                                            to
                                                                                                            N>
                                                                                                            I
                                     SIZE CATEGORIES OF HEAD WIDTH (mm)
 Figure 13.  Comparison of frequency  (%) of H. bilineata nymphs in various size classes  (based
             on head width) at stations 1 and 3 near Johnsonville Steam Plant on June 2, 1977.

-------
    50
    40
    30
5
H
     20
     10
           I
                               STATION 1 (n * 51)
                               STATION 3 (n = 48)
                  CO
                   •

                  o


                   I


                  IO
                   •

                  o
                                 C3O
          I


         oo
          •

         o
 I

cvj
                                                                                                               NJ
                                                                                                               co
                                                                                                               i
0
CVJ
1
CO
,J
CVJ
CVJ
1
0
cvj
•*
CVJ
1
CVJ
cvj*
«3
CVJ
1
*!•
CVJ
CO
CVJ
1
10
cvj
O
CO
1
00
cvj
CVJ
CO
1
o
ro
  Figure  14.
                     SIZE  CATEGORIES OF HEAD WIDTH (mm)



Comparison of frequency  (%) of H. bilineata  nymphs in various  size classes (based

on head width) at stations 1 and 3 near Johnsonville  Steam Plant on June 22,  1977.

-------
     50
      40
      30
      20
      10
                                                       I  STATION 1 (n = 24)




                                                           STATION 3 (n = 37)
                                     SIZE CATEGORIES OF HEAD WIDTH (mm)
                                                                                                           to
                                                                                                           .p"
Figure 15.  Comparison of frequency  (%) of H. bilineata nymphs  in various  size  classes  (based

            on head width) at stations 1 and 3 near Johnsonville Steam Plant on July  13,  1977.

-------
       60
       40
       30
   a
       20
       10
                                                        I
                                                                          STATION 1  (n = 16)
                                                                          STATION 3- (n = 40)
                                                            j
                                                                           CM



                                                                            I



                                                                           •si-

                                                                            •

                                                                           CM
1


00
 •

CM


 I


UD
 •

CM
                                                                                                             N5
                                                                                                             Ul
                                                                           O
                                                                            •

                                                                           CO


                                                                           I


                                                                           co
                                                                            •

                                                                           CM
Figure 16.
                          SIZE CATEGORIES OF HEAD WIDTH  (mm)




Comparison of frequency  (%)  of H.  bilineata nymphs  in various size classes (based

on head width) at stations  1 and 3 near Johnsonville Steam Plant on August 3, 1977

-------
                                 -26-
  to
  3
  o
  CO
  o;
  ff

  b
  o
                                           	STATION 3
               WR   APR    MAY  JUNE  JUNE  JULY   AUG
               31    19    12    2    22    13     3


Figure 17.  Comparison of frequency (%) of H.  bilineata nymphs in six
            size classes of head width (interval = 0.5 mm) from March
            to August 1977 at stations 1 and 3 near Johnsonville
            Steam Plant.

-------
                                                                    STATION 1

                                                                    STATION 3
                                  JUNE 22      JULY  13
Figure 18.  Percentage of H. bilineata nymphs having black wing pads  at stations  1  and 3 on
            three sampling dates, 1977.

-------
                                 -28-
      That elevated water temperatures can induce  early emergence of
 aquatic insects,  by as much as six months,  has  been established.5'6
 In contrast to these reports,  a dragonfly has been shown to  develop  in
 the laboratory at a rate similar to that in the field,  despite higher
 temperatures during the winter.7  A field study showed that  Ephemerop-
 tera, Trichoptera, and Megaloptera below a  power  station in  England  do
 not emerge earlier than those  in ambient areas.8   Adult H. bilineata
 live only two or  three days, in which time  they must mate and lay eggs.
 Therefore, synchronization of  emergence  to  the  adult stage is critical
 for propagation of the next generation.   H.  bilineata  near Johnsonville
 Steam Plant completed development sooner in the spring of 1977 in areas
 receiving thermal effluent, but evidently did not emerge appreciably
 earlier in these  areas than in ambient areas.   Some combination of
 extrinsic factors, rather than just temperature,  must  control the timing
 and hence synchronization of emergence.   Although daylength  may be an
 important cue,  the exact mechanism is unknown.9
 SIZE AND FECUNDITY COMPARISONS OF H. BILINEATA
     The mean values  for H. bilineata wing length, abdomen length, and
egg  count  are given in  Table  1.  The number of eggs per female was posi-
tively  correlated with  abdomen length.  The correlation coefficient r for
females at station 1  was 0.58, slightly higher than that found at station
3  (0.52);  both values are  significant (P = 0.01).  However, the variation
in egg  count  for a particular abdomen length was very large (Figure 19).

     Results  of t-tests on mean size and fecundity between stations 1
and  3 are  given in Table 2.   Males at station 1 were significantly
smaller on the average  than those at station 3.  Females differed in
average abdomen length, but not in average wing length or number of
eggs.   Unfortunately, the  specimens from station 1 were collected on a
later date (June 25,  1977) than those from station 3 (June 4 and 5,
1977).  Possibly, a seasonal  decrease in size, a phenomenon known to
occur in Hexagenia spp.,9  is  responsible for the differences in male
size.   This study will be  repeated to ensure collection of individuals
from the two  stations on the  same date.
THERMAL TOLERANCE OF IMMATURE AQUATIC INSECTS
H. bilineata Nymphs

     Results of the thermal shock experiment are given in Table 3.  All
nymphs survived the control and the 10°C AT, and 29 of 30 survived the
20°C AT after 4 h of exposure.  Mortality was high at the 30°C AT; 23
nymphs died within the first 15 min of exposure.  Dissolved oxygen (DO)
was probably not a factor in mortality, because nymphs can survive at
DO levels less than 6.5 ppm at lower temperatures.

-------
                                -29-
TABLE 1.  MEAN VALUES (x), STANDARD DEVIATIONS (s), AND NUMBER OF OBSERVA-
TIONS (n) FOR MALE AND FEMALE H. BILINEATA WING LENGTH, ABDOMEN LENGTH,
AND EGG COUNT AT STATIONS 1 AND 3 NEAR JOHNSONVILLE STEAM PLANT
                 Wing length  (mm)    Abdomen length  (mm)
            	Male	Female    Male	Female	Egg count

                         Station 1  (6/25/77)


            x    14.07      17.63      9.93         12.71        3779.79

            s      1.143       0.992     1.201          1.369       1058.24

            '•n    29         24       29            24           24
                      Station 3  (6/4/77-6/5/77)

            x     14.95       17.73    11.41         13.82      3548.61

            s      1.36        1.73     1.28          1.76      1623.17

            n     44         108       44           108          108

-------
           10
       o
       o
       o
      UJ
      fe
                                  -30-
                    DISCHARGE, STATION 3

                      JUNE 4 and  5 (s = 1618,6)


                    CONTROL, STATION 1

                      JUNE 25 fe  = 1058.2)
                       L
•    *




t
             10  11    12    13    14    15   16    17    18   19
                              FEMALE ABDOMEN LENGTH (mm)
Figure  19.   Scatter diagram of  relationship between abdomen length and

             number  of eggs in H.  bilineata females.

-------
                                -31-
TABLE 2.  RESULTS OF t-TESTS COMPARING MEAN SIZE AND FECUNDITY OF H.
BILINEATA SUBIMAGOES AT STATIONS 1 AND 3 NEAR JOHNSONVILLE STEAM PLANT

Character
Male wing length
Male abdomen length
Female wing ^length
Female abdomen length
Egg count
df
71
71
130
130
130
Value of t
2.88**
4.95**
1.62
2.89**
0.07
P
<0.01
<0.01
0.11
<0.01
>0.95

-------
            TABLE 3.  NUMBER OF H. BILINEATA NYMPHS SURVIVING FOUR EXPERIMENTAL THERMAL SHOCKS


Thermal shock Dissolved Replicate
temperature3 (°C) oxygen (ppm) number
10 (AT =0) 9.2 1
2
3
20 (AT = 10) 8.6 1
2
3
30 (AT = 20) 7.4 1


2
3
40 (AT = 30) 6.5 1
2
3
Number of survivors
15 rain
10
10
10
10
10
10
10


9
10
1
2
4
30 min
10
10
10
10
10
10
10


9
10
0
0
3b
45 min
10
10
10
10
10
10
10


9
10


1
60 min
10
10
10
10
10
10
10


9
10


1
of exposure duration of
90 min
10
10
10
10
10
10
10


9
10



120 min
10
10
10
10
10
10
10


9
10



180 min
10
10
10
10
10
10
10


9
10



240 min
10
10
10
10
10
10
10 ,
OJ
to
9 '
10



a
 Acclimation temperature was 10°C.



 Removed and slowly brought to 25°C.

-------
                               -33-
     The nymphs exposed to 20 and 30°C ATs immediately ceased moving;  3
min later the nymphs exposed to 20°C AT showed gill and leg movements,
but no movement was seen in the nymphs exposed to 30°C AT.   At all three
higher ATs, air bubbles appeared on the gills of many nymphs.   At the
15-min check, most of the nymphs exposed to 30°C AT were floating; those
alive showed very little gill movement.  The three nymphs still living
after 30 min were slowly brought to 25°C; two died within 15 min, but
one survived.

     Although the number of nymphs available for the tests was low,
results indicate that H. bilineata can tolerate relatively high thermal
shocks (at least 20°C) for short periods when acclimated to a low tempera-
ture.  Determination of the effects of various acclimation temperatures
on thermal tolerance would be helpful for establishing seasonal tolerances.

Coelotanypus! sp. Larvae
             \
     Results of the thermal shock experiment on Coelotanypus sp. larvae
are given in Table 4.  Survival after 2.5 h was high (90 to 100%) at all
four temperatures.  After the larvae were slowly brought to 25°C, some
mortality was observed in larvae exposed to shock; the highest mortality
(40%) occurred in the 25°C AT treatment.  The effects of acclimation
temperature on survival of these larvae should also be determined.
EFFECTS OF ENTRAINMENT WITHIN THERMAL PLUME ON AQUATIC INSECTS
     Results of the field study at John Sevier Steam Plant on July 20,
1976, are presented in Table 5.  The difference in temperature between
the thermal plume station and the ambient station was 6 to 7°C through-
out the 8-h test.  Of the two damselfly nymphs that died, one had been
partially eaten and was therefore probably killed by another damselfly.
The other had drowned as it tried to emerge to the adult stage without a
suitable support.  The single dead mayfly may have been killed by a
damselfly nymph present in the test basket.

     The thermal plume from John Sevier Steam Plant sometimes extends
several kilometers down the Holston River.10  An insect drifting the
length of the plume could be exposed to the heated water for many
hours.  Although the test was limited, the data indicate that damsel-
fly nymphs of the genus Enallagma and mayfly nymphs of the genus
Stenonema, common inhabitants of the Holston River, would probably
experience little or no mortality due to the heated water.  A simulated
laboratory study11 showed that mayflies of the genus Isonychia and
caddisflies of the genus Hydropsyche were not adversely affected until
shock temperatures neared the upper lethal limits.

-------
       TABLE 4.   NUMBER OF COELOTANYPUS SP.  LARVAE SURVIVING FOUR EXPERIMENTAL  THERMAL  SHOCKS


Thermal shock
temperature3 (°C)
15 (AT
25 (AT
35 (AT
40 (AT
= 0)
= 10)
= 20)
= 25)
15 min
10
10
10
10
30 min
10
10
10
10
Number of survivors of exposure duration of
45 min
10
10
10
9
60 min
10
10
10
9
75 min
10
9
10
9
90 min
10
9
10
9
120 min
10
9
10
9
150 minb
10
9
10
9
210 min
10
9
10
8
10 h
10
9
9
7
48 h
10
8
8
6

Acclimation temperature was 15°C.



Temperature in each treatment was brought slowly to 25°C.
•o
I

-------
                               -35-
TABLE 5.  NUMBER OF DAMSELFLY AND MAYFLY NYMPHS SURVIVING AFTER
8 HOURS OF EXPOSURE IN THE THERMAL PLUME AND AT AMBIENT STATION
NEAR JOHN SEVIER STEAM PLANT, JULY 20, 1976
                                                               »*
                              Number of survivors per replicate
                                    Thermal plume       Ambient
  Insect                      	station          station
	1234	1     2

Damselflies                    8    10    10    10      10    10


Mayflies                      10    10                   9
o
 10 nymphs per replicate.

-------
                                 -36-


 EGG DEVELOPMENT AND THERMAL TOLERANCE
      The results of egg hatching at seven constant temperatures are
 summarized in Table 6.   No eggs hatched at 40°C,  and although  eggs
 hatched at 37°C, the percentage was low (21%)  and the development
 time was comparatively  long (minimum of 12 days).   The shortest develop-
 ment time was 8 days, both at 31 and 34°C.   These data agree with
 Fremling's report4 of 8 days for development at 32°C and  indicate a
 range of optimal temperatures for development.  The relationship between
 temperature and incubation time is shown in Figure 20.  The regression
 equation is Y = 139.44  - 8.437t + 0.1349t2,  where Y is development time
 in days and t is temperature.  The observed values lie close to and twice
 coincide (at 25 and 37°C) with the predicted values.

      The average cumulative percentages of hatching (at the end of the
 4-day sampling period)  at 31 and 34°C are significantly different at the
 1% level,  as determined by Duncan's multiple range test (Table 7).  The
 abrupt drop in average  cumulative percentage of hatching  from  34 to 37°C
 indicates  that 34°C is  the peak temperature  for speed of  development.  (See
 Davidson12 for discussion on speed of development.)

      Embryos never developed in eggs cultured  at  40°C.  After 5 days at
 this constant temperature,  the cytoplasm was concentrated near the
 center of  the oval eggs and the ends were translucent.  After 2 weeks
 at 40°C, most of the eggs were almost entirely transparent, containing
 only small scattered pieces of opaque material.  At  37°C, most of the
 eggs that  did not hatch after 2 weeks were  opaque,  but very few embryos
 could be seen.   Some eggs had the cytoplasm  centered,  with the ends
 translucent.   Dissolved oxygen was maintained  at  the  saturation level
 for each temperature and therefore was not a limiting factor.

      Because 34°C is close  to the upper temperature  limit for develop-
 ment of H.  bilineata eggs,  30°C was chosen as  the  culture temperature to
 follow the thermal shocks.   Results of the thermal  shocks simulating
 females laying eggs  in  a thermal plume are given  in Table 8.  Dissolved
 oxygen values  were above 8.0 ppm.   The hatching percentages were highly
 variable,  partly because of the variable  number of eggs placed in the
 containers.   Some containers  had fewer than  100 eggs, whereas others
 contained  over 2000.  These discrepancies resulted  from the total number
 of  eggs the  females  laid in each treatment.  Especially notable was the
 comparatively  low number of eggs  laid  in  the 45°C water.

     The average  percentages  of hatching  in  the control treatments (AT =
 0°C) were  low  compared  with the  percentages  reported  for  the constant
 temperatures 28  and  31°C.   Reasons  for this  discrepancy are not clear;
 it  is doubtful that  the physical  transfer of the eggs caused the lower
percentage of hatching.    Possibly the  later  date of collection (August 10
 compare^ with July 8) was an  important factor.

     The 3-factor  analysis  of variance  is summarized  in Table 9.   The
three main effects—shock temperature,  shock duration, and hatching day
 (day after oviposition)—were partitioned by one-degree-of-freedom tests
for significant responses.  All  interactions were found to be significant.

-------
                                -37-
TABLE 6.  MEAN CUMULATIVE PERCENTAGE OF HATCHING THROUGH 4 DAYS
OF H. BILINEATA EGGS CULTURED AT 8 CONSTANT TEMPERATURES3


Day after
oviposition
1-7
8 |
9 ;
10
11
12
13
14
15
16
17
18
19
20
21-28
29
30
31
32
Mean cumulative hatch (%) at constant temperature of
40°C 37°C 34°C 31°C 28°C 25°C 22°C 19°C
74.8 62.5
93.1 85.9
94.7 87.8 26.1
95.4 89.1 76.1
9.5 87.6
14.4 88.1 34.8
19.8 81.6
21.0 85.1
88.0
12.7
40.7
66.7
81.6
2.5
12.0
24*. 1
35.8
3Each value is the mean of  five  replications.

-------
 32


 30


 28


 26


 24


 22


 20


18


16
        p   14
            12
                                  -38-
        UJ   g



            6
            2  _
            0
                    19
                                         PREDICTED VALUES

                                         OBSERVED VALUES
                                 Y = 139.44 - 8.437t + 0.1349t
              22    25
28    31
34    37
                            CULTURE TEMPERATURE (<>c)
Figure 20.
Predicted and  observed  development  times  (in  days)  for
H. bilineata eggs  cultured  at  seven nearly constant
(±1°C)  temperatures.

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                               -39-
TABLE 7.  SUMMARY OF HATCHING RESULTS AT CONSTANT TEMPERATURE
AND TESTS FOR SIGNIFICANCE OF DIFFERENCE BETWEEN MEANS AS
DETERMINED BY DUNCAN'S MULTIPLE RANGE TEST
   Constant
temperature (°C)	Mean cumulative hatch  (%)

    19                                             35.8 a

    22                                             81.6 b

    25                                             88.0 c

    28                                             88.1 c

    31                                             89.1 c

    34                                             95.4 d

    37                                             21.0 e
3Means followed by different letters  are significantly different
 at the 1%  level.

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                                      -40-
TABLE 8.  PERCENTAGES OF HATCHING OF H. BILINEATA EGGS EXPOSED TO THERMAL SHOCKS
(ATs OF 5, 10, AND 15°C) FOR THREE DURATIONS (5, 10, AND 15 MIN) IMMEDIATELY
AFTER OVIPOSITION

Pulse Pulse Date
temperature duration hatched
(°C) (min) (1977)
30
(AT =0) 5 8/18
8/19
8/20
8/21
10 8/18
8/19
8/20
8/21
15 8/18
8/19
8/20
8/21
35
(AT =5) 5 8/18
8/19
8/20
8/21
10 8/18
8/19
8/20
8/21
15 8/18
8/19
8/20
8/21
40
(AT = 10) 5 8/18
8/19
8/20
8/21
10 8/18
8/19
8/20
8/21
Hatching percentage (%)
by replicate number
1

35.79
60.44
37.09
43.75
44.32
13.04
58.00
45.05
14.20
45.29
34.29
47.22

16.98
53.62
71.43
83.23
29.69
62.37
51.49
88.17
15.38
78.32
89.91
82.79

7.81
43.52
29.23
62.92
10.96
52.26
69.92
59.84
2

27.91
90.91
44.95
49.49
85.71
22.62
51.43
21.26
8.13
27.03
75.14
34.62

28.07
56.18
90.11
79.79
7.84
62.50
30.69
79.02
5.71
79.31
87.57
90.25

11.70
41.09
35. C8
63.32
8.46
52.36
36.87
71.54
3

13.16
42.80
39.47
44.09
27.50
53.42
43.33
64.00
16.44
18.93
75.82
41.77

20.83
59.26
91.49
92.04
28.27
78.72
38.89
87.93
2.80
79.80
89.87
88.65

13.59
42.63
46.13
58.73
6.88
35.39
64.35
54.13
Total
(%)

76.86
194.15
121.51
137.33
157.53
89.07
152.76
130.32
38.77
91.25
185.25
123.61

65.88
169.06
253.03
255.06
65.80
203.59
121.07
255.12
23.89
237.43
267.35
261.69

33.10
127.24
111.04
184.97
26.30
140.01
170.53
185.51
Average
(%)

25.62
64.72
40.50
45.78
52.51
29.69
50.92
43.44
12.92
30.42
61.75
41.20

21.96
56.35
84.34
85.02
21.93
67.86
40.36
85.04
7.96
79.14
89.19
87.23

11.03
42.41
37.01
61.66
8.77
46.67
56.84
61.84

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                                      -41-






TABLE 8 (continued)

Pulse Pulse Date
temperature duration hatched
(°C) (min) (1977)
15 8/18
8/19
8/20
8/21
45
(AT = 15) 5 8/18
1 8/19
8/20
| 8/21
10 8/18
8/19
8/20
8/21
15 8/18
8/19
8/20
8/21
Hatching percentage (%)
by replicate number
1
12.32
42.03
60.53
73.52

0.00
22.84
0.00
63.48
2.91
0.00
1.01
3.25
3.54
3.39
12.39
5.35
2
9.76
35.96
64.01
69.72

0.00
11.54
6.90
14.05
1.36
0.00
7.69
9.35
3.13
2.53
9.18
3.67
3
11.53
43.00
67.05
54.72

0.86
12.16
27.27
11.54
0.41
0.00
1.79
2.97
33.33
3.43
14.39
3.40
Total
(%)
33.61
120.99
191.59
197.96

0.86
46.54
34.17
89.07
4.68
0.00
10.49
15.57
40.00
9.35
35.96
12.42
Average
(%)
11.20
40.33
63.86
65.99

0.29
15.51
11.39
29.69
1.56
0.00
3.50
5.19
13.33
3.12
11.99
4.14

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                               -42-
TABLE 9.  ANALYSIS OF VARIANCE OF 3-FACTOR THERMAL SHOCK EXPERIMENT ON
H. BILINEATA EGGS


Source of variation
Replications
Shock temp., T (t = 4)
Linear response
Quadratic response
Cubic response
Shock duration, D (d = 3)
Linear response
Quadratic response
Hatching day, H (h = 4)
Linear response
Quadratic response
Cubic response
TD (t-l)(d-l)
TH (t-l)(h-l)
DH (d-l)(h-l)
TDK (t-l)(d-l)(h-l)
Error
df
2
(3)
1
1
1
(2)
1
1
(3)
1
1
1
6
9
6
18
96
Mean square
11.51
(17,046.26)
25,134.29
25,183.05
821.46
(175.79)
8.61
342.97
(8,869.24)
23,008.16
3,094.33
505.25
363.32
1,538.56
549.84
479.18
75.373
F

333.47**
334.11**
10.90**
0.114
4.55**
305.26**
41.05**
6.70**
4.82**
20.41**
7.30**
6.36**


-------
                              -43-


     The mean percentages of hatching due to the three main effects  are
shown in Figure 21.  The average percentage of hatching after a shock of
15°C was much lower than that after lesser shocks (Figure 21A).  Shock
duration appeared to have no effect on hatching response when averaged
over all treatments (Figure 21B).  Cumulative percentage of hatching
increased through time as expected (Figure 21C).

     Partitioning the main effect of shock temperature showed that there
was a highly significant linear response of hatching to shock temperature,
but also that the response was curvilinear (Table 9).  These results indi-
cate that the increase in hatching at the 5°C AT, as shown in Figure 21A,
is significant.  The main effect of shock duration showed a significant
quadratic response when partitioned.  The reason for the lower hatching
response to the 10-min treatments is unknown.  The main effect of hatch-
ing day showed significant linear, quadratic, and cubic responses.  Hatch-
ing increased sharply from day 1 to day 2, but then tended to reach a
plateau (Figure 21C).

     The interaction between shock temperature and shock duration is
illustrated in Figure 22.  Hatching success responded in different pat-
terns at each shock duration, which showed the complex relationship
between these two variables.  Increased shock duration for the 15°C AT
treatment decreased the hatching success, although the response is
curvilinear.  The interaction between shock temperature and hatching day
revealed a lower hatching success at 15°C AT for each hatching day
(Figure 23).  At the 0, 5, and 10°C ATs, the percentage of hatching
increased appreciably through the 4-day observation period, but at the
15°C AT the percentage increase was much less.  The response of hatching
to the interaction between shock duration and hatching day was highly
variable (Figure 24).  Shock duration apparently affects the percentage
of hatching differently on each day that eggs hatch.

     The significant 3-way interaction (Table 9) indicates that the rela-
tionship between shock temperature, shock duration, and day after oviposi-
tion, as they affect egg development, is complex.  The percentages obtained
in the experiment were quite variable, and trends are difficult to define.
Shock durations of 5 to 15 min did not appear to affect hatching success
significantly unless the temperature change was at least 15°C  (ambient was
30°C); in this case, longer shock duration lowered the hatching responses,
although there was a slight cumulative increase in the response through
the 4-day observation period.

-------
    100






     90





     80




     70





     60



•  Ll- on
<  o 30





     20





     10
                 A
                                         B
          0
          10     15
              PULSE  T (°C)
                                                         10
15
                                                   PULSE

                                                 DURATION (min)
                                                                   HATCHING DAY
Figure 21.
Average percentage of hatching of H. bilineata  eggs  due  to  main effects of shock

temperature (A), shock duration  (B), and  day  after oviposition (C).

-------
                               -45-
                                                   15
                     SHOCK DURATION  (min)
Figure 22.  Surface response curves for shock temperature and shock
            duration (interaction) effects on mean percentage of
            hatching of H. bilineata eggs over a 4-day hatching period.

-------
    100

     90


     80


     70


o    60
z
1—4

§    50
u_
o
LU    40

I
B    30
a
|    20


     10

      0
                                        -— 8lH DAY AFTER OVIPOSITION

                                        — 9TH DAY AFTER OVIPOSITION

                                         — 10TH DAY AFTER OVIPOSITION

                                        '— llTH DAY AFTER OVIPOSITION
                                                                                0V
                     0
              Figure 23.
       5          10

    THERMAL SHOCK (°c)
15
Effect of shock temperature on mean cumulative percentage
of hatching of H. bilineata eggs exposed to thermal shock
immediately after oviposition.

-------
                                 -47-
  100


   90


   80
I70
u. 60
o
LU
   "

   30
   20
                                          SHOCK DURATION
     5 min
    10 min

    15 min

                                         10
11
                  DAY AFTER OVIPOSITION

  Figure  24.   Interaction of  shock  duration and hatching day on the mean
               percentage  of hatching of  H.  bilineata  eggs exposed to
               thermal  shock immediately  after  oviposition.

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                                -48-


                               REFERENCES
  1.   Horst,  T.  J.   1976.   Population dynamics of the burrowing mayfly
      Hexagenia  limbata.   Ecology 57:199-204.

  2.   Orlob,  G.  T.,  I. P.  King,  and  W. R. Norton.  1975.  Mathematical
      simulation of  thermal discharges from Johnsonville Steam Plant.
      An unnumbered  report prepared  for TVA by Resources Management
      Association, 3706 Diablo Blvd.,  Lafayette, Calif.

  3.   Hunt, B. P.  1953.   The life history and economic importance of a
      burrowing  mayfly, Hexagenia limbata, in southern Michigan lakes.
      Mich. Conserv. Dept.,  Bull.  Inst. Fish. Res. 4:1-151.

  4.   Fremling,  C. R.  1967.  Methods  for mass-rearing Hexagenia mayflies
      (Ephemeroptera:  Ephemeridae).  Trans. Amer. Fish. Soc. 96:407-410.

  5.   Nebeker, A. B.  1971.  Effect  of high winter water temperatures
      on adult emergence of aquatic  insects.  Water Res. 5:777-783.

  6.   Coutant, C. C.  1967.  Effect  of temperature on the development
      rate of bottom organisms.  In Biological effects of thermal discharges,
      pp. 11-12.  Ann. Rep., Pacific N.W. Lab., U.S. AEC, Div. Biol.
      Medicine.

  7.   Miyakaua, K.  1969.  A study of the life history of Pseudothemis
      zonata  (Burm.) (Odon., Libellulidae).  II.  Immature Stage.
      Kontyu 37:409-422.

  8.   Langford, T. E.  1975.  The  emergence of insects from a British
      river, warmed by power station cooling-water.  Part II.  The
      emergence patterns of some  species of Ephemeroptera, Trichoptera
      and Megaloptera in relation  to water temperature and river flow,
      upstream and downstream of  the cooling-water outfalls.  Hydrobiologia
      47:91-133.

  9.  Fremling, C. R.  1973.  Environmental synchronization of mass
     Hexagenia bilineata  (Ephemeroptera) emergence from the Mississippi
     River.  Verh. Internat. Verein. Limmol. 18:1521-1526.

10.  Ungate,  C.  D.  1977.  John Sevier Steam Plant hydrothermal investi-
     gations.  Part I;  Thermal discharge effects near the plant.  TVA
     Report No.  32-14-1, April 1977.

11.  Sherberger, F.  F., E. F. Benfield,  K. L. Dickson, and J. Cairns, Jr.
     1977.   Effects of thermal shocks on drifting aquatic insects:  A
     laboratory simulation.  J.  Fish. Res. Bd.  Can.   34:529-536.

12.  Davidson,  J.  1942.   On the speed of development of insect eggs
     at constant temperatures.   Aust. J.  Exp. Biol.  Med.  Sci. 20:233-239.

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                               -49-


                               GLOSSARY


ambient:  Surrounding environmental condition.

entrainment:  Transport by the flow of a liquid.

fecundity:  Ability to produce offspring; reproductive potential.

instar:  A stage in the life cycle of an insect between molts.

subimago:   Immature adult stage of mayflies, duller and darker than
     adult to which it molts.

thermal plume:  Warm water discharged from once-through cooling by
     electric generating plants; boundary is 2°C above
     ambient isotherm.

-------
                                            -50-
                                      TECHNICAL REPORT DATA
                               (Please read Instructions on the reverse before completing)
  1. REPORT NO.

    EPA-600/7-78-128
                                                                3. RECIPIENT'S ACCESSION>NO.
  4. TITLE ANDSUBTITLE
    EFFECTS OF THERMAL DISCHARGE ON AQUATIC INSECTS  IN THE
    TENNESSEE VALLEY
                                                               5. REPORT DATE
                                                                   July 1978
                                                         6. PERFORMING ORGANIZATION CODE
  7. AUTHOR(S)

    K. J.  Tennessen and J. L. Miller
                                                               8. PERFORMING ORGANIZATION REPORT NO
                                                             TVA/EP-78/09
 9. PERFORMING ORGANIZATION NAME AND ADDRESS

    Division of Environmental Planning
    Tennessee  Valley Authority
    Chattanooga, TN  37401
                                                         10. PROGRAM ELEMENT NO.

                                                             INE-625A
                                                         11. CONTRACT/GRANT NO.

                                                             80 BDR
  12. SPONSORIN
      U.S.
        AGENCY NAME AND ADDRESS
        nvironmental Protection Agency
Office of Research & Development
Office of Energy,  Minerals & Industry
Washington,  D.C.   20460	
                            13. TYPE OF REPORT AND PERIOD COVERED
                                                                14. SPONSORING AGENCY CODE

                                                                      EPA-ORD
 15. SUPPLEMENTARY NOTES
      This project is part of the EPA-planned and coordinated Federal Interagency
      Energy/Environment  R&D  Program.
 16. ABSTRACT

         The.Tennessee Valley Authority (TVA)  conducted  studies to  (1) determine the
    thermal tolerances of selected  aquatic  insects and  (2)  investigate growth  and
    emergence  of those insects in the vicinity of TVA electric generating plants.
    Results of the study  will be used to help  establish  thermal effluent limits to
    protect the aquatic ecosystem.

         Tolerance of the immature  stages of Hexagenia bilineata  (Say) and Coelotanypus
    sp.  to thermal shocks (ATs) of  up to 20°C  was found  to  be great.   However,  eggs
    subjected  to a shock  of 15°C resulted in a greatly reduced mean percentage  of
    hatching.   No difference in fecundity of adult females  was found  between ambient
    and  thermal plume stations.  Adult males from the heated discharge channel  were
    significantly larger  on the average than adult males from the ambient station.

         A drift study of Enallagma spp. and Stenonema spp. in a thermal plume  showed
    little or  no mortality at ATs that normally result from the heated water.
 17.
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tartar Crfl*
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