EPA-R3-73-041
^co'°9'ca' Research Series
MAY 1973
Thermal Effects on
Eggs, Larvae and Juveniles
of Bluegill Sunfish
Office of Research and Monitoring
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
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was consciously planned to foster technology
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2. Environmental Protection Technology
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U. Environmental Monitoring
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This report has been assigned to the ECOLOGICAL
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EPA-R3-73-041
May 1973
THERMAL EFFECTS ON EGGS, LARVAE AND
JUVENILES OF-BLUEGILL SUNFISH
A. Banner
and
J. A. Van Arman
Contract No. 14-12-913
Project 18050 GAB
Project Officer
Dr. Kenneth E. F. Hokanson
National Water Quality Laboratory
6201 Congdon Boulevard
Duluth, Minnesota 55804
Prepared for
OFFICE OF RESEARCH AND MONITORING
U. S. ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, D. C. 20460
For sale by the Superintendent of Documents, U.S. Government Printing Office, Washington, D.C. 20402
Price $2.10 domestic postpaid or $1.75 OPO Bookstore
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EPA REVIEW NOTICE
This report has been reviewed by the Environmental Protection
Agency and approved for publication. Approval does not 'sig-i
nify that the contents necessarily reflect the views and
policies of the Environmental Protection Agency, nor does men-
tion of trade names or commercial products constitute endorse-
ment or recommendation for use.
11
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ABSTRACT
Bioassay experiments were conducted to determine thermal tolerance
of early life history stages of bluegill sunfish. Bluegill eggs
hatched at temperatures from 18 to 36C during two incubation tests.
Maximal hatch occurred at 22.2 and 23.9C. Lower TL5Q temperature
for hatch of normal fry was 21.9C and upper TLrn temperature was
33.8C. U
Juvenile bluegills acclimated to 12.1C had a lower 96-hour TL50 of
3.2C and an upper 96-hour TL5Q of 27.5C. Juveniles acclimated to
32.9C had a lower 96-hour TL5Q of 15.3C and an upper 96-hour TL50
of 37.3C. TLcjQ increased with increasing temperature of acclimation.
For juveniles acclimated to a given temperature, upper TL5Q decreased
with longer exposure.
A preliminary test determined ranges of thermal tolerance for sac-fry
and swim-up fry. In another preliminary test, juvenile bluegills
were acclimated to 12.1, 19.0, 26.0 or 32.9C, and reared at a series
of test temperatures for three to six weeks to define optimal tempera-
ture ranges for growth and survival.
Additional research determined conditions for the culture of Lepomis
macrochirus, including spawning induction, hatching, and growth of
larvae and juveniles.
This report was submitted in fulfillment of Project 18050-GAB, Con-
tract 14-12-913, under sponsorship of the Office of Research and
Monitoring, Environmental Protection Agency.
111
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CONTENTS
Section Page
I CONCLUSIONS 1
II RECOMMENDATIONS 3
III INTRODUCTION 5
IV MATERIALS AND METHODS
Field Collections 9
Temperature Records 9
Water Quality 9
Incubation Tests 10
Thermal History of Adults 10
Injection Procedures 10
Fertilization 10
Bioassay Procedure li
Analysis of Results 12
Juvenile Bioassay 12
V RESULTS
Water Quality 15
Incubation Tests 17
Juvenile Bioassay 17
VI DISCUSSION
Incubation Tests 29
Juvenile Bioassay 30
VII ACKNOWLEDGMENTS 33
VIII REFERENCES 35
IX GLOSSARY 39
X APPENDICES 43
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FIGURES
No.
1. Relative percent normal hatch (see text) of bluegill
eggs at various temperatures during two incubation tests.
Each temperature included two replicate culture chambers
with 50 eggs per chamber. Dashed lines indicate TLrr,
limits. 20
2. The 1-hr ( ) and 96-hr ( ) TL5Q temperatures of
juvenile bluegills acclimated to stock temperatures of
12.1, 19.0, 26.0, or 32.9C. 26
VI
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TABLES
No.
1. Thermal history of adult female bluegills used in the
Three incubation tests. All animals were exposed to
a 16L-8D photoperiod. 11
2. Number of parents, number of eggs, and the temperature
at which parents and eggs were held prior to and dur-
ing fertilization for the two incubation tests. 11
3. Trace-chemical composition of water from the thermal
testing facility based on a single determination.
This analysis was performed by Precision Analytical
Laboratories, Inc., North Miami, Florida. 15
4. Results of routine water quality monitoring during
the incubation and bioassay experiments. Mean (ppm
unless otherwise specified) and range are given for
N = 3 replicates of each analysis. 16
5. Total percent hatch, percent hatch of normal larvae,
and relative percent hatch (see text) for bluegill
eggs. Percentages represent the combined results from
two replicates at each test temperature. 18
6. Percent survival of juvenile bluegills at various test
temperatures. Survival at 0.83 and 1.10 hrs. were
averaged to determine the 1-hr TLrn. 21
7- The upper and lower TL^g values for bluegills (Repli
cates combined). Confidence limits (95% CL) and slope
function of the mortality curve (S) were calculated
for each TL5Q. Animals were acclimated to 12.1, 19.0,
26.0 or 32.9C before the experiment. 25
VII
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SECTION I
CONCLUSIONS
1. Based on two experiments, bluegill eggs that are spawned at 26C
have a lower mean TL^Q of 21.9C and an upper mean TL^Q of 33.8C.
2. Juvenile bluegills have a greater thermal tolerance range than
eggs. The upper 96 hr. TL50's were 2'7i5C and 37.3C for juveniles
acclimated at 12;1C and 32.9C respectively, and the lower 96 hr.
TL^Q'S were 3.2C and 15.3C for juveniles at the same acclimation
temperatures. TL5Q temperatures for juvenile bluegills increase
with increasing temperature of acclimation. Upper TLcjg decreases
and lower TL^Q increases with increasing exposure duration.
3. Adult bluegills can be induced to spawn in laboratory aquaria by
manipulation of temperature and photoperiod. Injection of female
bluegills with carp pituitary induces ovulation and enables ex-
periments to be conducted with eggs of known history from several
sets of donor parents simultaneously.
4. Preliminary experiments indicate that fry survive for 96 hrs. at
temperatures of 11 to 34C. Fry therefore have greater thermal
tolerance than eggs and less thermal tolerance than juveniles, but
fry survive poorly in the life support conditions of our testing
facility.
5. The range of temperatures for optimal growth and survival of juveniles
was less than the range defined by upper and lower TLgQ. An
optimal range was not clearly defined because of inadequate life
support conditions.
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SECTION II
RECOMMENDATIONS
Preliminary tests conducted with sac-fry and swim-up fry indicate that
sac-fry were more resistant to thermal stress than eggs. Swim-up fry
were more sensitive to high temperature than any other stage, perhaps
because they have a shorter time to feed successfully before starvation.
Results of the sac-fry tests should be verified by experiments with
larger numbers of test animals and more intermediate temperatures.
The test with swim-up fry should be repeated, with more test animals,
and with animals that are reared to swim-up stage at higher stock
temperatures. Larvae should be fed more frequently (4 to 5 times
daily) beginning as soon as they reach swim-up stage.
Tests should be conducted to establish the effects of parental thermal
history on the thermal tolerance of offspring. Adults maintained at
three stock temperatures, e.g., 22, 26, and 30C should be induced to
spawn and thermal bioassays conducted on their progeny.
Hormone injections affect egg viability and hence may affect their
observed temperature tolerance. Experiments might be conducted to
compare temperature tolerance of eggs from normal spawns with the
tolerance of eggs from hormone-induced spawns.
Optimal limits for growth and survival of juvenile bluegills were
not well defined for most stocks. This experiment should be repeated,
eliminating some of the extreme temperatures that showed large negative
changes in biomass, and including more of the intermediate temperatures.
In particular, to define the optimal range for summer acclimated animals,
the 32.2C stock should be tested between 18 and 35C.
Swim-up fry may be most sensitive to high temperature because the
demands for food must be met adequately during a short critical period
(Toetz, 1966). Swim-up fry may also be more sensitive than other stages
to water quality conditions and the turbulent environment of the culture
baskets. Tests with swim-up fry should be repeated with an improved
life support system using other kinds of food (besides Artemia nauplii
and limnoplankton) and with different feeding rates.
The water quality and life support characteristics of the thermal test
facility can be improved by developing better methods to control dissolved
oxygen and pH, and to eliminate toxic metals and metabolites. Subsequent
experiments at ASI have yielded better survival of bluegill fry in tap-
water treated by charcoal filtration and in water treated by deionizing
resins and reconstituted with reagent grade inorganic salts. Chemicals
must be carefully selected to avoid addition of toxic heavy metals present
as contaminants.
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Confinement of fish in culture baskets is necessary t6 prevent escape
of fish, facilitate counting and permit replicates within a given tem-
perature chamber. Experiments are needed to define optimal container
size and fish density within containers in the temperature chambers
to prevent crowding.
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SECTION III
INTRODUCTION
The disposal of waste heat, arising from industrial processes and "the
production of electric power, constitutes a major pollution problem
with pronounced effects on aquatic organisms (Kennedy and Mihursky,
1967; Raney and Menzel, 1969). The need for electric power is increas-
ing rapidly. Parker and Krenkel (1969) predict that by the year 2000,
the equivalent of the total flow of surface waters in the United States
will be required for cooling purposes. If in meeting electrical produc-
tion requirements it is necessary to discharge large quantities of
heated water, the effects of this water on aquatic ecology must be as-
sessed so that practical guidelines and restrictions can be developed.
Fish constitute the highest trophic level in the ecology of most fresh
waters and are a source of food and recreation for man. A population
of fish in a lake or stream, however, is a complex phenomenon. Predic-
tion of the effects of environmental changes on this population requires
both laboratory and field experiments. Carefully controlled laboratory
experiments are necessary to define precise limits of tolerance, whereas
field observations are necessary to determine whether the guidelines
developed in the laboratory are relevant to natural populations.
To determine effects of temperature on a fish population, animals must
be tested at all stages of their life cycle. The temperature history
of the animals prior to testing, as well as the duration of exposure to
temperature extremes, influence the thermal tolerance (Brett, 1958).
Results of temperature bioassays are thus most meaningful when the
acclimation temperature and duration of exposure have been controlled
in the laboratory.
The responses of eggs to temperature stress and the limits for survival
or the "optimal range" can be determined in several ways (Hokanson,
McCormick, and Jones, 1973). The most generally acceptable criterion
for determining survival limits is the range over which 50% of the
animals survive (American Public Health Association, 1971). To determine
egg survival, total percent hatch, percent hatch of normal eggs, percent
fertilization (Lindroth, 1946), percent abnormal hatch (Price, 1940), or
percent of fertilized eggs surviving to hatch (Hubbs, 1962) may be
measured. However, percent normal hatch is the most meaningful of these
criteria with respect to natural populations and is the end result of
the processes of fertilization and egg survival.
Eggs may be exposed directly to test temperatures or they may be temper-
ed at various rates to the test temperatures. If eggs are tempered to
test temperatures, the rate of tempering becomes critical. The embryo
then has an opportunity to develop to a stage with a different tolerance,
and the rate at which this development occurs adds an experimental vari-
able. Direct exposure is desirable because the eggs are exposed to
temperature extremes sooner. Direct exposure to temperature stress
(thermal shock) may reduce survival of eggs (Kelley, 1968) but if this
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stress is applied early in embryonic development, preferably prior to
the first cell division, then egg survival is not reduced (Gorodilov,
1969; Tatarko, 1968).
Eggs can be obtained from ripe adult fish collected in the field. How-
ever, the temperature history of adults is unknown, collection of eggs
or adults produces a stress that may affect egg viability, and ,collec-
tion or holding of large numbers of individuals may be necessary to
obtain a few ripe parents. If animals are obtained ripe and "stripped"
in the field, it is difficult to precisely control temperature during
fertilization and transport of eggs to the laboratory. For these rea-
sons, it,is desirable to induce fish to reproduce in the laboratory.
Fish can usually be induced to spawn in laboratory,aquaria by manipula-
tion of light, temperature, and photoperiod. 'However, such spawns are
unpredictable and require many large aquaria to obtain sufficient eggs
for testing. The use of pituitary hormone injections facilitates the
production of viable eggs, but the viability of these eggs may be less
than viability of eggs that are spawned naturally (Pickford £ Atz, 1957).
Testing of larvae and juveniles should be performed with fish that have
been acclimated in the laboratory to stock temperatures that represent
the range found in the natural environment. Gradual change in tempera-
ture during acclimation permits metabolic changes, similar to changes
that may occur in response to seasonal temperature changes in the envi-
ronment. Fish that have been acclimated to appropriate temperatures can
then be challenged with a direct temperature change to estimate the
effects of addition or removal of heated water, which might occur during
operation of a power facility.
Proper evaluation of the effects of temperature requires that other
variables which may affect survival must be controlled, especially
oxygen, food, crowding and water quality (Fry, 1947).
Short-term survival at test temperatures indicates the ability of fish
to resist thermal stress but does not indicate whether the population
will continue to survive with prolonged temperature alteration. Long-
term studies are necessary to measure the effects of temperature on
growth rate, mortality, and overall population biomass.
Fish have an optimal physiological range of thermal tolerance (Fry, 1951)
within which maximum growth and activity occur. The optimal physiologi-
cal lange may bracket an optimal temperature for various metabolic activi-
ties, the temperature for optimal growth is dependent upon the amount
of food available (Brett, Shelbourne, and Shoop, 1969). The temperature
for optimal growth shifts to lower values as the amount of food is re-
duced. Measurement oftiie optimal physiological temperature therefore
requires that food be present in excess. The optimal physiological
temperature may regulate diurnal and seasonal behavior patterns of some
fishes to increase food conversion when the food supply is limited (Brett,
1971).
The bluegill sunfish, Lepomis macrochirus, is an important forage, food,
and game fish in North American fresh waters. Bluegills are generally
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considered a warm water species but they are widely distributed in the
United States and southern Canada. In the United States, L^. macrochirus
ranges from Minnesota to southern Florida and has been introduced into
Hawaii (Eddy, 1969) and Puerto Rico (Erdman, 1967) . L_. macrochirus
thus survives in a wide range of environments and may be ideally suited
for survival in, or introduction into, heated water effluents in tem-
perate zones.
The thermal tolerance of bluegills has been studied previously to some
extent. The relationship between zoogeography and thermal tolerance
(Hart, 1952) and the upper limits of thermal tolerance (Cairns, 1956)
have b'een- determined for'juvenile and adult bluegills. The limit of
tolerance for juvenile bluegills exposed briefly to elevated tempera-
tures* has also been tested (lezzi, Filson and Myers, 1952).
The objectives of this study were to determine the optimal temperature
ranges and tolerance limits for incubation of L. macrochirus eggs and
for survival of juveniles. Techniques were developed to induce spawning
of bluegills in the laboratory and to determine favorable conditions for
rearing of larvae in our test facilities. Preliminary tests were con-
ducted to determine the approximate temperature tolerance of bluegill fry
and the approximate temperature range for optimal growth of juvenile
bluegills. A bibliography of the literature relevant to thermal toler-
ance, spawning induction^ ecology, and'larval rearing of L. macrochirus
is included as an appendix.
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SECTION IV
MATERIALS AND METHODS
Field Collections
Adult bluegills were collected from canals near State Road 27, nine
miles southwest of Florida City, Florida; from Drainage Canal No. 35B
between U. S. Highways 27 and 441 in Water Conservation Area 2A of the
Central and South Florida Flood Control District; from Welaka Fish
Hatchery, Putnam County. Florida; and from Lake Apopka, Orange County,
Florida. Fish were captured by hook and line, by electro-shocking, or
by seining, and transported to the laboratory. Newly collected fish
were tempered from ambient field temperature to 28C (IC/hr) and trans-
ferred to 3.4 kiloliter tanks for quarantine. After one week, females
were placed in 2.9 kiloliter aquaria and males were placed in 209-
liter aquaria at 28C. Several males and females were sacrificed from
each monthly field collection to estimate the gonadosomatic index (GSI =
gonad wt/total fish wt x 100) of the wild population.
Temperature Records
During the incubation and bioassay experiments, the temperature of each
culture chamber in the thermal testing facility was recorded automati-
cally at two minute intervals. Temperature of each culture chamber was
also determined daily, to +0.1C, with a mercury thermometer. The daily
range, daily mean, grand mean, and 95% confidence limits of all means
were obtained from the recorded temperature re'cords after correction by
reference to the manual readings.
During the bioassay experiments, test containers generally remained
within a +Q.3C range of their nominal temperatures. Temperatures re-
ported in the results section represent the grand mean and 95% confidence
limits.
Water Quality
Water from the Boca Raton municipal water treatment plant was used during
the bioassay tests. This water was dechlorinated by aeration for two
weeks prior to the test, and was adequate to support the growth of nauplii
and adults of Daphnia magna for at least one .week.
A detailed analysis of water in the testing facility was performed during
the bioassay experiments. Levels of ionic components were measured by
techniques described in Standard Methods for the Examination of_ Water
and Wastewater, Twelfth Edition, 1965.
In addition to the detailed analysis, water quality was monitored daily
during the bioassay experiments using reagents and techniques prepared
by the Hach Chemical Company, Ames, Iowa. Water samples were collected
daily from test chambers at three temperatures (high, low and inter-
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mediate). Each sample was analyzed for changes in pH, ammonia or
dissolved oxygen that would result from organic decomposition in the
biological filter, autotrophic tanks or test chambers. Levels of
nitrites and nitrates were monitored at irregular intervals to check
for gradual enrichment of the water.
Incubation Tests
Two egg incubation tests were conducted to determine the upper and
lower limits of temperature for hatch of normal bluegill fry (after
Hokanson et^ al_. , 1973) . Upper and lower TL5Q were defined by incubating
eggs at test ""Temperatures ranging from 16 to 38C at 2C intervals.
Thermal History of Adults. For the preliminary incubation test (Appen-
dix D), conducted in June, 1971, adult bluegills collected from the
field were held for 8 days at 26C. These adults were pooled with ani-
mals that had been maintained in the laboratory for spawning-induction
experiments (Appendix A) under a variety of temperature conditions. All
animals were held under a 16L-8D photoperiod. Incubation test 1 was
conducted in July, and incubation test 2 was conducted in August,
1971. Females used for tests 1 and 2 were held in the laboratory at
23.8 to 26.4C for 20 to 122 days prior to the start of the incubation
experiments.
Injection Procedures. The development of the spawning inducation tech-
nology is given in detail in Appendix A. Carp pituitary glands (Stoller
Fish Industries, Ames, Iowa) were suspended (10 mg/ml) in fish Ringer's
solution (Humason, 1967). Carp pituitary suspensions were injected into
female bluegills with 1 ml plastic syringes and 24 gauge needles. Egg
development was monitored prior to each injection. A 2 mm 0.,D. glass
catheter attached to a syringe (Pennell,1964) was inserted into the
genital pore, eggs were withdrawn, suspended in water, and examined under
a microscope.
During the holding period, females were injected intraperitoneally with
2 mg carp pituitary every other day, to induce formation of premature
eggs. Injections to induce egg production began 30 to 44 hours before
eggs were needed for incubation tests. Twenty females that had premature
eggs were injected intramuscularly (below the base of the dorsal fin)
with 1 mg carp pituitary every four hours and these injections were con-
tinued until eggs matured. The thermal history of females used in the
first and second incubation tests is summarized in Table 1.
Fertilization. Fertilization was attempted when eggs reached maturity.
When 50% of the eggs from three or more females could be fertilized,
eggs from these females were stripped into a shallow dissecting tray
(30 x 21 x 5 cm) containing 350 ml of water from the holding tank. The
bottom of the tray was covered with 2.5 cm. x 2.5 cm pieces of Plexiglas®,
which formed a substrate to which the eggs adhered. Sperm from four or
five males were uniformly distributed over the eggs. The tray was
10
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floated in a water bath maintained at 25.0 or 26.OC. Eggs and sperm
were allowed to mix for two minutes, then were rinsed in water from
the holding tank. This served to remove extra sperm and non-adhesive
eggs. Conditions under which the eggs were fertilized are given for
each test in Table 2.
Table 1. Thermal history for adult female bluegills used
in the two incubation tests. All animals were
exposed to a 16L-8D photoperiod.
Test 1 Test 2
Collection Temp (C) 23-28 23-28
Holding
Duration (days) 20-108 38-122
Mean Temp (C) 24.5 24.7
Egg Production
Duration (hrs) 24 28
Mean Temp (C) , 26.0 ,25.0
Table 2. Number of parents and the temperature at which
parents and eggs were held prior to and during
fertilization for the two incubation tests.
Test 1 Test 2
No. males 4 5
No. females 6 3
Mean Temp (C) 26.0 25.0
Bioassay Procedure. The Plexiglas® squares, with adherent eggs, were
immediately transferred to 257-ml glass jars filled with water from
the holding tank. Each jar was placed in a randomly assigned culture
chamber of the thermal testing facility within ten minutes after
fertilization. The eggs in the jars were tempered rapidly (12C/hr) to
the temperature of the chamber. The Plexiglas® square was then trans-
ferred from the jar to a culture basket in the test chamber (two
baskets per chamber). Excess eggs were removed from the plastic squares
so that each square held 25 eggs. Duplicate test chambers were main-
tained at each test temperature giving N = 100 eggs for each temperature,
Odd-numbered chambers were assigned to one experimental replicate (A)
and even-numbered chambers were assigned to the second replicate (B).
Dead eggs and dead, normal, and deformed fry were counted and
removed daily. Normal eggs were transparent and yellowish-white.
Dead eggs were translucent to opaque. Fry that had noticeably curved
spines or swollen bodies (edema) were considered to be deformed.
11
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Analysis of Results. Total percent hatch and percent hatch of normal
fry were determined at each test temperature. A given test was con-
sidered valid, when more than 50% of the eggs hatched in a replicate at
any test temperature. Relative percent hatch was then calculated for
each test. The hatch at optimal temperature•(temperature which gave
the maximum percent hatch of normal fry) was assigned the value of 100%
and numbers of hatched fry at other temperatures in the same test were
designated as proportions of the maximum percent hatch of normal fry
(after Hokanson et_ _al_. , 1973).
Upper and lower TI^Q values were determined for each experiment. Rela-
tive percent hatch of normal fry was first graphed as a function of
temperature. A linear equation was fitted to two points (above and be-
low the temperature for 50% hatch) by the method of least squares. The
upper and lower TL^Q values were determined from the linear equations.
Juvenile Bioassay
A bioassay test was conducted to determined the range of thermal toler-
ance for juvenile bluegills. Juvenile bluegills (mean wt = 0.3 gm,
range = 0.1 to 2.1 gm) were obtained from the Federal Hatchery at
Richloam, Georgia. Fish were randomly assigned to four groups; accli-
mated at 0.5C/day to stock temperatures of 12.1, 19.0, 26.0 and 32.9C;
and maintained at these temperatures for 8 days. Ten fish were randomly
assigned from these stocks and transferred directly to each culture bas-
ket. Culture baskets were floated in 75.8-liter fiberglass culture
chambers (see Appendix C). Chambers were maintained at various test
temperatures in the thermal test facility. The temperature range to
which each stock was exposed is given in the results.
For the stocks that were acclimated to 12.1, 19.0, or 26.OC, two repli-
cate 'culture chambers were held at each temperature from 7 to 33C, and
one culture chamber per test temperature was used at 3 to 6C. For the
stock acclimated to 32.9C, one culture chamber was used at 39C, while
two culture chambers were used at each of the remaining test temperatures.
When two chambers were available at a given temperature, the odd-numbered
chamber was designated as one replicate (A) and the even-numbered chamber
as the second replicate (B). Each replicate contained two culture bas-
kets (10 animals per basket) with fish from a given stock.
For temperatures at which only a single chamber was available, this
chamber contained four culture baskets with fish from a given stock.
Two adjacent protectors were assigned as one replicate (A) and the remain-
ing two protectors were assigned as the second replicate (B).
Fish were fed to satiation at 0800, 1200, and 1700 hrs daily with adult
Artemia, Daphnia, and Tetramin®. Dead fish were counted and removed
after 0.83, 1.10, 24, 48, 72, and 96 hours. Samples taken at 0.83 and
1.10 hrs were averaged to determine mortality at one hour. Results of
12
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bioassays were analyzed to determine TL^Q temperatures, slope function
(S) of the TLrn curve, and 95% confidence limits for each stock at 1,
24, 48, 72, and 96 hours (Litchfield and Wilcoxon, 1949).
13
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SECTION V
RESULTS
Water Quality
Results of detailed analyses of water in the thermal test facility are
presented in Table 3. Results of routine water quality analyses are
presented in Table 4. Routine analyses indicated that pH varied between
7.4 and 8.3. Dissolved oxygen (expressed as % air saturation) ranged
between 71 and 145%, and ammonia (un-ionized NH ) reached a maximum of
0.095 ppm. Concentrations of nitrite reached a maximum of .008 ppm
while nitrate had a maximum concentration of 2 ppm.
Table 3. Trace-chemical composition of water from the
thermal testing facility based on a single
determination. This analysis was performed
by Precision Analytical Laboratories, Inc.,
North Miami, Florida.
Sensitivity of Concentration
Component Test (mg/1) (mg/1)
Calcium 0.01 38.50
Magnesium 0.01 2.84
Potassium 0.01 0.90
Sulfate 0.1 4.5
Sulfide 0.05 <0.05
Nitrate 0.05 <0.05
Nitrite 0.05 <0.05
Ammonia 0.01 <0.01
Phenol 0.001 < 0.001
Chloride 0.1 38.0
Fluoride 0.01 <0.01
Cyanide 0.005 < 0.005
Iron 0.001 0.045
Copper 0.01 0.10*
Zinc 0.001 0.001
Cadmium 0.01 0.03*
Chromium 0.01 <0.01
Lead 0.001 < 0.001
Alkalinity 1-0 51
*These levels may be harmful to fish (see discussion)
15
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Table 4. Results of routine water-quality monitoring
during bicascay experiments. Mean (ppm unless
otherwise specified) and range are given for
N = 3 replicates of each analysis.
Incubation Test No. 1
Diss. Oxygen
Day pH (% Sat.) Ammonia (NH3) Nitrite Nitrate
No. Mean Range Mean Range Mear} Range Mean Range Mean Range
0
1
2
3
4
7.7 7
7.9 7
7.7 7
7.8 7
7.8 7
.5-7.9
.8-8.0
.6-7.7
.7-7.8
.7-7.9
105
110
98
109
109
93-117 .009
93-132 .013
82-117 .007
93-118 .019
93-118 .023
.006-
.005-
.004-
.008-
.002-
Incubation Test No.
1
2
3
7.5 7
7.5 7
.5-7.6
.4-7.6
*
105
105
105
93-117 .011
93-117 .008
93-117 *
.004-
.006-
*
Juvenile Bioassay (26, 33C
1
2
3
4
7.9 7
7.7 7
7.9 7
8.2 8
.8-7.9
.6-7.8
.8-8.0
.1-8.3
101
104
106
115
80-119 .027
80-134 .016
80-134 .010
93-132 .047
Juvenile Bioassay
0
1
2
3
4
8.0 7
8.0 7
8.2 8
7.9 7
7.7 7
.9-8.1
.9-8.2
.2-8.2
.9-8.0
.7-7.8
91
88
92
90
87
74-108 .012
74-104 .016
77-102 .018
74-104 .012
71-102 .007
Juvenile Bioassay
0
1
2
3
4
8.0 8
7.7 7
8.1 8
7.9 7
7.7 7
.0-8.0
.6-7.7
.1-8.2
.9-7.9
.7-7.8
102
99
107
117
99
80-122 .013
82-116 .009
94-125 .017
82-145 .013
82-116 .039
.018-
.005-
.005-
.015-
.013
.021
.010
.029
.044
2
.012
.012
.000
.001
.001
.001
.001
.001
.000
0-0
0-.001
0-.001
0-.001
0-.001
0-.001
0-0
*
0
1
0
0
0
0
0
0-0
0-2
0-0
0-0
0-0
0-0
0-0
*
Stocks)
.042
.031
.018
.095
.004
.003
.005
.001
.003-.
.001-.
.003-.
.000-.
005
005
008
003
0
0
0-0
*
*
0-0
(12C Stock)
.006-
.008-
.011-
.006-
.004-
.028
.028
.028
.022
.012
.004
.000
*
.003-.
*
*
.000-.
006
001
0
1
*
0-0
*
*
0-2
(19C Stock)
.007-
.003-
.009-
.008-
.005-
.021
.018
.029
.019
.024
.003
.004
.005
.004
.001-.
.003-.
.001-.
.003-.
*
005
005
005
005
0
0
0
0
0-0
0-0
0-0
0-0
*
''Samples not analyzed
16
-------
Incubation Tests_
The total percent hatch, percent normal hatch, and relative percent
normal hatch are presented in Table 5. The relative percent normal,
hatch was calculated by setting the maximum percent normal' hatch to
100 and adjusting all other values accordingly. The relative percent
normal hatch values are plotted against actual test temperatures in
Figure 1. These values were used to calculate the upper and lower
temperature TLrQ values.
During a preliminary incubation test (Appendix D), egg viability
was low and hatching rates differed greatly between replicates. The
first incubation test showed close agreement between replicates with
regard to temperature for maximal hatch. Replicate A had a maximal
hatch (greater than 50%) at 27.8C, while replicate B had a maximal
hatch (greater than 50%) at 26.OC. When results of both replicates
were combined, maximal percent hatch was at 22.2C, but did not reach
50%. There was a 1 to 5% abnormal hatch at all test temperatures.
The upper TL50 was 34.5C and the lower TL5Q was 21.2C.
During the second test, maximal percent hatch occurred at 23.9C for
replicate A, and at 28.OC for replicate B. Both replicates had no
hatch at 18.1C and replicate A had no hatch at 35.9C. When results
of both replicates were combined, maximal hatch was at 23.9C, and
exceeded 50%. The percent abnormal hatch ranged from 0 to 4% and
was observed primarily at extreme temperatures. The upper TLrn was
33.2C and the lower TL5Q was 22.6C.
Juvenile Bioassay
Survival of juvenile bluegills at the various test temperatures is
shown in Table 6. Prior to the test, juveniles were acclimated to
stock temperatures of 12.1, 19.0, 26.0 or 32.9C. Animals from each
stock were then distributed to a series of test temperatures (Table 6)
designed to bracket the upper and lower limits of thermal tolerance.
The TLrn temperatures for.juvenile bluegills, as defined by statistical
analyses of the survival data, are presented in Table 7. For the stock
acclimated to 12.1C, a lower TL50 was not defined at 1, 24, 48, or 72
hours (TLqn was less than the minimum test temperature of 3.0C), but
TL50 was 3.2C (with 95% condifdence limits of +0.56C) after 96-hour expo-
sure. The upper one-hour TL5Q for the 12.1C stock was 28.9 +0.43C. The
TL5Q temperature decreased at 48 and 72 hours and reached a minimum of
27.5 +_ 0.58C after 96 hours.
The stock acclimated to 19.OC had a lower one-hour TL50 of 4.0 +_ 0.36C.
This value increased, with longer exposure times, to a maximum of 6.3 +
0.41C after 96-hour exposure. The upper TL was 34.7 +_ 0.31C after one
hour and decreased to 33.0 + 0.26C after 96 hours.
17
-------
Table 5. Total percent hatch, percent hatch of normal larvae,
and relative percent normal hatch (see text) for
bluegill eggs. Percentages represent the combined
results from two replicates at each test temperature,
Incubation Test No. 1
Mean
Temperature
(+ 95% CL)
17.9 ± 0.20
17.9 ± 0.20
20.1 ± 0.27
20.1 ± 0.29
22.2 ± 0.16
22.2 ± 0.16
24.0 ± 0.77
24.0 ± 0.91
26.0 ± 0.36
26.1 ± 0.35
27.9 ± 0.26
27.6 ± 1.04
29.9 ± 0.28
30.3 ± 0.37
32.0 ± 0.23
32.1 ± 0.46
33.9 ± 0.37
34.1 ± 0.41
35.9 ± 0.37
35.9 ± 0.36
38.2 ± 0.48
38.2 ± 0.45
Number
Temperature
Observations
96
96
96
96
96
96
96
96
96
96
96
96
96
96
96
96
96
96
96
96
96
96
Replicate
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
Total
Hatch*
n
\J
9
t*
46
27
38
42
34
35
34
6
0
Normal
Hatch
n
\J
44
24
36
37
33
32
29
4
0
Relative
Normal
Hatch
n
\j
100
55
82
84
76
73
66
9
0
(based on relative percent
normal hatch)
Upper 34.SC
Lower 21.2C
18
-------
Table 5 (cont'd)
Incubation Test No. 2
Mean
Temperature
C± 95% CL)
18.1 ± 0.38
18.1 ± 0.62
20.2 ± 0.36
20.1 ± 0.48
22.1 ± 0.34
22.3 ± 0.40
23.8 ± 0.65
24.0 ± 0.15
26.1 ± 0.12
26.2 ± 0.22
28.0 ± 0.20
28.0 ± 0.18
30.0 ± 0.20
29.9 ± 0.32
31.7 ± 0.82
31.7 ± 1.12
34.0 ± 0.19
33.9 ± 0.41
35.8 ± 1.08
35.9 ± 1.03
38.0 ± 0.50
38.0 ± 0.32
Number
Temperature
Observations
96
96
96
96
96
96
96
96
96
96
73
73
96
96
96
96
96
96
96
96
96
96
Replicate
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
Total
Hatch
0
5
24
59
44
53
41
49
24
1
0
Normal
Hatch
0
5
21
58
43
53
41
49
20
1
0
Relative
Normal
Hatch
0
9
36
100
74
91
71
85
34
2
0
TLrn (based on relative percent
normal hatch)
Upper 33.2C
Lower 22.6C
19
-------
Test No. 1
20
22
24
26 28 30 32
TEMPERATURE °C
34
36
38
100-
18
20
Test No. 2
22
24 26
28
TEMPERATURE °C
Figure 1. Relative percent normal hatch (see text) of bluegill eggs at
various temperatures during two incubation tests. Each tem-
perature included two replicate culture chambers with 50 eggs
per chamber. Dashed lines indicate TL5Q limits.
20
-------
Table 6. Percent survival of juvenile bluegills at various
test temperatures. Survival at 0.83 and 1.10 hrs
were averaged to determined the 1-hr TL5Q.
Stock Acclimated to 12.1C
Temperature No. Temperature Time (hrs)
Mean ± 95% CL Observations Replicate 0.83 1.10 Z4 48_ 12_ 96_
3.0 + 0.32 96
96
4.0 + 0.42 96
96
5.2+0.80 96
96
6.1+0.40 96
96
6.9 ± 1.31 96
96
7.9 + 0.26 96
7.9 + 0.36 96
26.1 + 0.22 96
25.9 ± 0.40 96
27.0 + 0.28 96
26.9 + 0.40 96
28.0 + 1.40 96
27.8 + 1.32 96
29.1 + 0.20 96
28.9 + 0.24 96
29.9 + 0.26 96
30.0 + 0.27 96
31.0 + 0.13 30
31.1+0.12 30
32.1+1.80 30
32.2+0.12 30
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
95
95
100
95
100
100
100
100
95
100
100
100
65
80
50
70
70
60
65
45
15
40
5
15
5
0
95
95
100
95
100
95
100
100
95
100
100
100
65
80
50
55
70
50
65
40
10
35
5
5
5
85
80
70
90
85
85
75
80
95
90
95
85
60
75
50
55
60
45
60
35
5
10
0
0
0
75
65
65
80
85
85
70
70
95
90
80
85
60
70
50
55
60
45
60
35
5
10
-
-
-
65
60
55
75
80
75
70
70
95
85
80
85
60
65
50
55
55
40
55
35
5
10
-
50
40
50
75
80
70
60
60
75
75
65
70
60
60
50
55
55
40
55
30
5
10
21
-------
Table 6. (cont'd)
Stock Acclimated to 19.OC
Temperature
Mean ± 95% CL
3.2 ± 0.50
4.1 t 0.78
4.8 ± 0.73
6.0 ± 0.19
6.8
7.0
7.8
8.0
9.1
8.9
9.8
9.8
30.1
30.1
30.9
30.8
31.8
31.9
32.9
32.9
33.9
33.8
34.9
34.7
0.84
0.28
0.34
0.56
0.30
0.36
0.28
0.28
0.32
0.50
0.20
1.11
0.47
0.31
0.48
0.54
0.21
0.23
0.51
0.09
No. Temperature
Observations
30
96
96
96
96
96
96
96
96
96
96
96
96
96
96
96
96
96
96
96
96
96
96
96
Replicate 0.83
Time (hrs)
1.10 24 48 72 96
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
0
0
75
45
95
85
90
100
100
95
100
90
100
100
100
100
100
100
100
100
100
100
95
100
60
85
50
30
.
-
60
45
95
80
90
100
100
95
100
90
100
100
100
100
100
100
100
100
100
100
95
100
55
65
10
10
25
10
80
25
80
90
70
95
90
80
95
95
100
95
100
100
85
90
95
70
75
75
15
10
10
0
25
5
55
15
65
45
60
60
80
75
90
95
95
90
KlO
100
80
90
95
65
70
70
15
10
10
-
15
5
45
10
65
30
55
5-5,
80
75
90
90
90
85
100
100
80
85
95
65
70
70
10
10
10
10
5
20
10
55
20
55
55
80
70
90
90
80
85
100
100
80
80
95
65
60
65
10
10
10
22
-------
Table 6. (cont'd)
Stock Acclimated to 26.OC
Temperature No. Temperature 'Time (hrs)
Mean ± 95% CL Observations Replicate 0.85 1.10 24 48 72 96
5.6 ± 0.81
6.0 ± 0.19
6.8 ± 0.84
7.0 ± 0.28
7.8 ± 0.34
8.0 ± 0.56
9.1 ± 0.30
8.9 ± 0.36
9.8 ± 0.28
9.8 ± 0.27
10.9 ± 0.38
11.0 ± 0.16
12.0 ± 0.44
11.8 ± 0.64
13.0 ± 0.28
13.0 ± 0.26
26.0 ± 0.22
26.0 + 0.20
33.9 + 0.76
33.9 ± 0.40
34.9 ± 0.68
34.8 ± 0.82
36.0 ± 1.02
35.8 ± 1.20
37.0 ± 0.53
37.2 ± 0.49
37.7 ± 0.04
37.8 ± 1.43
30
30
24
24
72
72
96
96
96
96
96
96
96
96
96
96
96
96
96
96
96
96
96
96
96
96
96
96
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
10
0
65
65
85
80
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
90
10
50
5
65
50
85
80
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
13
90
5
15
0
0
5
60
35
70
80
95
90
95
100
95
95
100
100
100
100
100
95
85
100
90
70
5
25
0
0
-
-
0
5
0
20
45
80
85
95
95
95
95
100
100
100
90
90
90
75,
100
85
60
5
20
-
5
-
10,
20
80
80
95
90
95
90
100
95
100
90
90
90
55
55
85
50
5
15
. 0
10
15
80
75
90
85
95
90
100
90
100
90
90
85
50
90
85
45
5
15
23
-------
Table 6 (cont'd)
Stock Acclimated to 32.9C
Temperature
Mean ± 95% CL
No. Temperature
Observations
10.0 ±
10.0 ±
11.2 ±
11.1 ±
11.9 ±
11.9 ±
13.0 ±
12.8 ±
13.9 ±
13.9 ±
14.8 ±
14.9 ±
15.9 ±
15.9 ±
17.0 ±
17.0 ±
19.9 ±
20.2 ±
32.9 ±
32.9 ±
36.0 +
35.7 ±
37.0 ±
37.0 ±
37.8 ±
38.0 ±
38.9 ±
0.18
0.19
0.16
0.16
0.40
0.28
0.24
0.46
0.13
0.14
0.49
0.30
0.23
0.20
0.18
0.70
0.20
0.32
0.28
0.20
0.86
1.17
0.59
0.61
0.50
0.36
0.15
30
30
24
24
24
24
72
72
48
48
96
96
96
96
96
96
96
96
96
96
96
96
96
96
96
96
30
Replicate
Time (hrs)
0.83 1.10 24 48 72 96
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
25
75
100
95
100
100
100
100
100
100
100
100
100
100
95
100
100
100
100
100
100
100
100
100
100
100
65
80
0
5
75
85
100
100
65
70
90
75
100
100
100
100
95
100
100
100
100
100
100
100
100
100
95
95
15
55
0
0
0
0
35
50
90
0
90
80
90
100
90
95
100
95
80
95
100
70
85
85
25
0
0
0
-
5
0
0
-
40
25
85
95
85
95
100
90
80
95
100
45
85
80
15
-
-
_
0
-
-
25
25
85
95
85
95
90
90
75
95
100
35
75
80
10
-
-
25
25
85
95
85
95
90
90
75
95
100
15
70
80
10
24
-------
Table 7- The upper and lower TL$Q values for bluegills
(Replicates combined). Confidence limits
(95% CL) and slope function of the mortality
curve (S) were calculated for each TL^o- Ani-
mals were acclimated to 12.1, 19.0, 26.0 or
32.9C before the experiment.
Exposure
Time
Upper TLsQ
95% CL
S
Lower TL5Q
95% CL
S
Upper TL50
95% CL
S
Lower TLso
95% CL
S
Upper TL50
95% CL
S
Lower TLso
95% CL
S
Upper TLso
95% CL
S
Lower TLso
95% CL
S
* Plot of
** Plot of
1 hr
Stock
28.9
±0.43
1.06
(not defined,
TL50 < 3C)
Stock
34.7
±0.31
1.03
4.0
±0.36
1.22
Stock
37.4
±0.30*
1.02
7.0
±0.20
1.12
Stock
39.0
±0.23*
1.01
10.3
±0.20*
1.05
Two points, no
24 hr
Acclimated to
28.5
±0.37
1.05
(not defined,
TL50 < 3C)
Acclimated to
33.0
±1.67
1.04
4.7
±0.41
1.19
Acclimated to
36.4
±1.27
1.03
8.2
±0.25
1.12
Acclimated to
37.5
**
1.01
13.7
±1.60
1.09
X2 test for "
48 hr
12. 1C
28.5
±0.37
1.05
(not defined
TL50 < 3C)
19. OC
33.0
±1.04
1.04
5.8
±0.48
1.39
26. OC
36.1
±0.34
1.02
9.5
±0.27
1.09
32. 9C***
37.4
±0.18*
1.01
15.0
±1.55
1.05
goodness of
two uoints , no values between 16 and 84%
72 hr
27.7
±0,57
1.11
, (not defined
TL50 < 3C)
32.9
±0.88
1.04
6.1
±0.50
1.40
36.1
±1.00
1.03
9.6
±0.26
1.09
37.3
±0.18
1.01
15.3
±0.22*
1.03
fit"
, CL could not
96 hr
27.5
±0.58
1.11
, 3.2
±0.56
1.89
33.0
±0.26
1.03
6.3
±0.41
1.35
36.1
±1.10
1.04
9.8
±1.00
1.09
37.3
±0.19
1.01
15.3
±0.22*
1.03
be
calculated
*** One replicate at 36C excluded (see text)
25
-------
40 -
30 -
u
o
20 r
LU
Q_
10 -
10
20
30
40
ACCLIMATION TEMPERATURE (°C)
Figure 2. The 1 hour ( ) and 96 hour ( ) TLjjQ temperatures of
juvenile bluegills acclimated to stock temperatures of 12.1°
19.0°, 26.0° or 32.9°C.
26
-------
Juveniles acclimated to 32.9C prior to testing had a one hour lower
TLso of 10.3 +_ 0.20C and a 96-hour TL5Q of 15.3 +_ 0.22C. Fish in one
replicate at T6.0C had 70% survival by 24 hours and 15% survival after
96 hours, while fish in the second replicate had no mortality for 96
hours. There is no clear indication why the replicates differed so
markedly in survival. The replicate with poor survival was ignored
during calculation of upper TLsQ. The upper TLrg after one hour was
39.0 +_ 0.23C and decreased to 37.3 +_ 0.19C after 96 hours.
As the temperature of acclimation increased, juvenile bluegills
showed greater tolerance of high temperatures and reduced tolerance
of low temperatures. Figure 2 indicates the relationship between
one hour and 96-hour TL^Q temperatures and temperature of acclimation.
The TL.5Q temperatures for 24, 48 and 72 hour exposures were between
these values. Within a given stock, the high-temperature TL^g tem-
perature decreased and the lower TL^Q increased with greater exposure
duration.
27
-------
SECTION VI
DISCUSSION
Incubation Tests
The percent hatch of bluegill eggs at each temperature, and the tem-
perature for optimal embryonic development varied between experiments,
but differences between replicates were greater than differences be-
tween tests. Genetic differences among parents, if they existed, were
masked by the larger variation within and between experiments and the
procedure of pooling gametes from several parents for each test. Dif-
ferences in hatching rate between replicates decreased in the first
test relative to the preliminary test and in the second experiment
relative to the first, suggesting that the source of variability was
controlled in the latter tests.
The relationship between temperature and incidence of abnormal fry was
not clearly defined. Dead and abnormal fry were less abundant during
the second incubation experiment than during the first experiment (Fig.
1). In the first experiment, abnormal fry occurred at both intermediate
and extreme temperatures. During the second experiment, dead and abnor-
mal fry were absent at temperatures where total hatch was less than 10%
or at 28 to 30C. Differences in the incidence of abnormal fry and in
optimal temperature between experiments were probably due to variables
within the experimental design.
The dosage and timing of carp pituitary injections, and the time selected
for egg removal affect egg viability (Pickford and Atz, 1957) and hence
may affect the observed temperature tolerance. Variations in water
quality may also have contributed to the observed differences. The second
incubation test was conducted two weeks after the first test, when water
in the thermal testing facility was conditioned by previous test animals.
Concentrations of cadmium and copper were above safe limits (Table 3).
Cadmium is acutely toxic to bluegills at 1.94 ppm (Pickering and Henderson,
1965) and copper is acutely toxic at 1.25 ppm (Cairns and Scheier, 1968)
after 96-hour exposure. The chronic toxicity of cadmium and copper for
bluegills is not known. Chronic toxicity tests of cadmium with the
fathead minnow indicate a maximum acceptable toxicant concentration between
37 and 57jug/l (Pickering and Cast, 1972). Chronic toxicity tests of
copper with brook trout indicate a maximum acceptable toxicant concen-
tration between 9.5 and llAjig/l (McKim and Benoit, 1971). Assuming that
these values are similar to values for bluegills, the level of cadmium
in our water supply is marginally acceptable while the level of copper
could have had an effect on egg survival. Oxygen was super-saturated in
some parts of the system, but there was no evidence of gas embolism.
Ammonia was present at levels that cause gill damage in some fish (Burrows,
1964) and reduced growth in others (Kawamoto, 1961).
29
-------
Parent bluegills used in the two tests differed in thermal history.
Adult fish which supplied eggs for the second incubation test were held
longer under laboratory conditions and maintained at a more constant
temperature than were the fish for the first incubation test.
During the incubation tests, several females and males were stripped
simultaneously. Attempts were made to distribute eggs uniformly on
plastic squares, but lack of a completely random distribution was a
source of error- Distribution of eggs was more uniform when fewer
donors were used, as in experiment 2.
The 11,50 values observed in these tests correspond with the spawning.
temperatures observed in natural populations. Adult bluegills in
laboratory aquaria spawned at temperatures from 22C to 30C (Appendix A),
which corresponds to the temperature range for optimal hatch during.
the incubation tests. In the field, L_., macrochirus spawns at tempera-
tures from 20 to 30C (Breder, 1936; James, 1946; Morgan, 1951; Miller,
1963; and Clugston, 1966). The upper Tl^Q for bluegill eggs exceeds
the temperatures at which spawning normally occurs, while the lower
TL^Q corresponds with the lower temperature limit for spawning observed
in the laboratory.
Juvenile Bioassay
Juvenile bluegills are more mobile than larvae and may range over the
same environments as those preferred by adults.- However, juvenile blue-
gills frequently inhabit shallow inshore waters where food is abundant
and predators are less effective. Juveniles might be expected to have
thermal tolerance limits that are the same as or greater than adults.
lezzi, Filson, and Myers (1952) indicated that the rate of temperature
change and the duration of exposure to high temperature have significant
effects on observed TL^Q values for juvenile bluegills, especially
during short-term bioassays. In the experiments conducted at Aquatic
Sciences, Inc., the one hour TL^Q temperatures for animals acclimated
to 32.9C and transferreddirectly to test temperatures were within 0.7C
of the maximum sublethal temperature following a l-4ir exposure, .as
determined by lezzi, e_t _al. (1952), for animals acclimated to 32.2C and
tempered at 3.3C/min. Cairns (1956) has shown that if bluegills are
tempered to test temperatures at 2C/day, they can tolerate temperatures
up to 39.2C (80% survival after one day).
During the experiments conducted at Aquatic Sciences, Inc. juvenile
bluegills that were obtained during the summer from Richloam, Georgia
and acclimated to 19.OC had an upper 24-hr TL50 of 33.OC. These
fish were less sensitive to high temperature than juvenile bluegills
from Welaka, Florida that were acclimated to 20C (Hart, 1952). Lower
TL50 for the fish tested at Aquatic Sciences were similar to those
determined by Hart (1952) at all acclimation temperatures.
30
-------
Differences in acclimation, photoperiod (Roberts, 1967) or size are
probably the most important factors that influence temperature tolerance.
Hart (1952) did not indicate the rate at which fish were tempered to
acclimation temperatures, but acclimation generally required 3 or 4 days.
In the experiments conducted at Aquatic Sciences, fish were adjusted to
acclimation temperature at 0.5C/day and maintained at that temperature
for 8 days prior to testing, so that acclimation lasted from two to six
weeks. Increased acclimation time may explain the apparent increase in
temperature tolerance of bluegills collected from Richloam, Georgia.
Seasonal changes in temperature tolerance of fishes result in increased
tolerance of high temperature during summer months and decreased toler-
ance of high temperature during winter (Fry, 1947). The experiments
by Hart (1952) were conducted with fish collected and tested during the
winter months at Welaka, Florida. The fish tested at Aquatic Sciences
were collected in the late summer and tested during early fall.
Hart (1952) observed an apparent increase in thermal tolerance with
increasing size. The fish used in our experiments were considerably
smaller, and yet showed a greater tolerance of high temperature than
the fish tested by Hart (1952). Subsequent tests (Appendix D) suggested
that small bluegills were less resistant than large bluegills to low
temperature stress.
Water quality and life support conditions during our experiments were
less than optimal. The concentrations of cadmium and copper in our water
supply may have had adverse effects on juveniles. Ammonia at the levels
measured in our system has been claimed to have adverse effects on the
growth and survival of other fish (Burrows, 1964; Kawamoto, 1961).
Fish densities of 10 animals per 0.96 liter culture basket were shown
to have adverse effects on survival of juveniles at high temperatures
when tested for 16 days (Appendix B). This crowding level probably had
little effect on survival of bluegill during the bioassay test, but may
have significantly reduced growth and survival during the growth test
(Appendix D). Larger culture baskets should be used for the determina-
tion of growth and long-term survival. A density of 10 animals per
basket was necessary to obtain sufficient experimental animals to allow
for natural mortality and sampling during the growth study.
31
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SECTION VII
ACKNOWLEDGMENTS
The staff of Aquatic Sciences appreciates the assistance of Paul
Barrett (Fare-General Corporation, Florida City, Florida), Dr. W.
R. Courtenay (Florida Atlantic University, Boca Raton, Florida),
and the Federal Fish Hatcheries at Welaka, Florida and Richloam,
Georgia for help in obtaining test animals. Dr. W. P. Davis (cur-
rently of the Mediterranean Marine Sorting Center, Tunis), and Dr.
R. G. Domey (the University of Texas Medical School, San Antonio,
Texas) aided in the design of experiments.
Mr. M. Beach (currently with the Department of Natural Resources,
Tallahassee, Florida), Mr. J. Peterson (AEO Systems, Inc., Lantana,
Florida), and Miss G. Klein (Oceanography Mariculture Incorporated,
Riviera Beach, Florida) aided in the performance of these experi-
ments .
Detailed analyses of water quality were performed by Precision
Analytical Laboratories, Inc., North Miami, Florida. Mr. Gregg
Stanton (Harbor Branch Foundation, Vero Beach, Florida) helped with
statistical analysis of the temperature data.
The financial assistance of the Environmental Protection Agency has
made this investigation possible. Dr. K. E. F. Hokanson (National
Water Quality Laboratory, Duluth, Minnesota) aided in the design of
experiments and in the preparation of this manuscript.
33
-------
SECTION VIII
REFERENCES
American Public Health Association. 1971. Standard Methods 'for the
Examination of Water and Wastewater, 12th Ed., APHA, New York., 874 pp.
Breder, C.M. 1936. The reproductive habits of North American sun-
fishes (family Centrarchidae). Zoologica, 21:1-48.
Brett, J. R. 1958. Some principles in the thermal requirements of
fishes. Quart. Rev. Biol., 31:75-87.
Brett, J. R. 1971. Energetic responses of salmon to temperature. A
study of some thermal relations in the physiology and freshwater
ecology of sockeye salmon (Oncorhynchus nerkaj. Amer. Zool., 11:99-113.
Brett, J. R., J. E. Shelbourne, and C. T. Shoop. 1969. Growth rate
and body composition of fingerling sockeye salmon, Oncorhynchus nerka
in relation to temperature and ration size. J_. Fish. Res. Bd. Canada,
26:236-3-2394. ~~
Burrows, R. E. 1964. Effects of accumulated excretory products on
the utilization of oxygen by some freshwater fish. U/S_. Dept. of the
Interior, Bur, of Sport Fish, and Wildlife. Res'. Rept. No. 66,~2 pp.
Cairns, J. 1956. Effects of heat on fish. Indust. Wastes, 1:180-183.
Cairns, J. and A. Scheier. 1968. A comparison of the toxicity of
some common industrial waste compounds tested individually and combined.
Progr. Fish-Cult., 30:3-8.
Clugston, J. P. 1966. Centrarchid spawning in the Florida Everglades.
Quart. J_. Fla. Acad. Sci. , 29:137-143.
Eddy, S. 1969. The Freshwater Fishes, 2nd Ed., Wm. C. Brown Co.
Dubuque, Iowa, 186 pp.
Erdman, D. S. 1967. Inland Game Fishes of Puerto Rico. Commonwealth
of Puerto Rico, Dept. of Agriculture, San Juan, Puerto Rico, 86 pp.
Fry, F. E. J. 1947. Effects of the environment on animal activity.
Publ. Ontario Fish. Res. Lab., No. 68, 62 pp.
Fry, F. E. J. 1951. Some environment relations of the speckled trout
(Salvelinus fontinalis) . Proc. N_.E_. Atlantic Fish. Conf. , May 1951,
29 pp.
35
-------
Gorodilov, Y. N. 1969. Study of the sensitivity of fish to high
temperature during embryogenesis. I. Changes in sensitivity to
high temperature effects of developing eggs of autumn spawning fish
species." Tsitilogiya (USSR), 11:169-180.
Hart, J. S. 1952. Geographic variations of some physiological and
morphological characters in certain fresh water fish. Publ. Ont. Fish.
Res. Lab., No. 72, 77 pp.
Hokanson, K. E. F., J. H. McCormick and B. R. Jones. 1973. Temperature
requirements for embryos and larvae of the northern pike, Esox lucius
(Linnaeus). Trans. Amer. Fish. Soc. , 102:89-100.
Hubbs, C. 1962. Developmental temperature tolerance of Texas and
Arkansas-Missouri Etheosfoma spectabile (Percidae Osteichthyes).
Ecology, 43:742-744^
Humason, G. L. 1967. Animal Tissue Techniques. W. H. Freeman and Co.,
San Francisco, Calif., 569 pp.
lezzi, T., J. A. Filson and C. S. Myers. 1952. Effects of temperature
changes and temperature levels on Lepomis macrochirus macrochirus (blue-
gill sunfish) and on Salmo fario (brown trout). Pennsylvania Dept.
Public Health, Indust. Wastes Div. (Mimeographed report), 17 pp.
James. M. F. 1946. Histology of gonadal changes in the bluegill,
Lepomis macrochirus (Rafinesque), and the largemouth bass, Huro salmoides
(LacepedeJ!J. Morph., 79:63-92.
Kawamoto, N. Y. 1961. The influence of excretory substances of fish on
their own growth. Progr. Fish-Cult., 23:70-75.
Kelley, J. W. 1968. Effects of incubation temperature on survival of
largemouth bass eggs. Progr. Fish-Cult., 30:159-163.
Kennedy, V. S. and J. A. Mihursky. 1967. Bibliography of the effects
of temperature in the aquatic environment. Univ. of Maryland, Natural
Resources Institute, Contribution No. 326. (Cited in Parker and Krenkel,
1969).
Lindroth, A. 1946. The biology of fertilization and development in the
pike. Mitt. Anstalt fur Binnenfischerei, Drottningholm, No. 24, 173,pp.
Litchfield, J. T. and F. Wilcoxon. 1949. A simplified method of
evaluating dose-effect experiments. J_. Pharm. Exp. Therap. , 96:99-113.
McKim, J. M. and D. A. Benoit. 1971. Effects of long-term exposures
to copper on survival, growth, and reproduction of brook trout (Salvelinus
fontinalis). J_. Fish. Res. Bd_. Canada, 28:655-662.
Miller, II. C. 1963. The behavior of the pumpkinseed sunfish, Lepomis
gibbosus (L.) with notes on the behavior of other species of Lepomis
and the pigmy sunfish, Elassoma evergladei. Behaviour, 22:88-151.
36
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Morgan, G. D. 1951. The life history of the bluegill sunfish, Lepomis
macrochirus, of Buckeye Lake (Ohio). Denison Univ. Bull. Sci. Lab.,
49:21^59.
Parker, F. L. and P. A. Krenkel (eds.) . 1969. Engineering aspects of
Thermal Pollution. Proc. Nat. Symposium on_ Thermal Pollution. Vander-
bilt University Press, Nashville, Tennessee", 351 pp.
Pennell, D. A. 1964. Development of eggs of the bluegill (Lepomis
macrochirus) with emphasis on the fertility of the eggs after ovulation.
National Science Foundation Undergraduate Research Program (unpublished
manuscript}, 18 pp.
Pickering, Q. H. and M. H. Cast. 1972. Acute and chronic toxicity of
cadmium to the fathead minnow (Pimephales promelas j . 3_. Fish. Res . Bd.
Canada, 29:1099-1106. (i, ~
Pickering, Q. H. and C. Henderson. 1965. The acute toxicity of some
heavy metals to different species of warm water fishes. In: Proc.
19th Indust. Waste Conf. Purdue Univ. 49:578-591.
Pickford, G. E. and J. W. Atz.-"l957. The Physiology of the Pituitary
Gland of Fishes. New York Zoological Society; N. Y., 613 pp.
Price, J. W. 1940. Time-temperature relations in the incubation of
the whitefish, Coregonus clupeaformis ,(Mitchill) . J_. Gen_. Physiol. ,
23:449-468. ~
Raney, E. C. and B. W. Menzel. 1969. Heated effluents and effects on
aquatic life with emphasis on fishes. A bibliography. Cornell Univ.
Water Resources and Marine Sci. Center, Philadelphia Electric Company-
and Ichthyological Associates, Bulletin No. 2, 470 pp.
Ricker, W. E. 1958. Handbook of Computations for Biological Statis-
tics of Fish Populations^Bulletin No. 119, Fish. Res. Bd. Canada,
Ottawa, 300 pp.
Roberts, J. L. 1967. Metabolic compensations for temperature in
sunfish. • In:: Molecular Mechanisms of Temperature Adaptation,
C. L. Prosser (ed.). American Association for the Advancement of Science,
Washington, D. C., Publ. No. 84, 390 pp.
Tatarkp, K. I. 1969. Sensitivity of pond carp to elevated temperature
at different periods of embryonic development. Gidrobiol. Zh_. Ac ad.
Nauk. Ukr. (USSR), 4:34.
Toetz, D. W. 1966. The .change from endogenous to exogenous sources of
energy in bluegill sunfish larvae. Invest. Indiana Lakes and Streams,
7:115-146.
37
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SECTION IX
GLOSSARY
acclimation - compensatory changes occurring in an organism in response
to variation of a. single environmental factor (usually in the lab-
oratory) .
acclimatization - compensatory changes that occur in an organism under-
going multiple natural changes in its climatic, physical and biotic
environment.
adaptation - any alteration or response of an organism which favors
survival in a changed environment.
autotrophic - needing only inorganic compounds for nutrition.
biomass - the weight of living matter.
catheter - tubular device for insertion into canals, vessels or body
cavities to permit injection or withdrawal of materials.
95% confidence limits - a range of values surrounding a sample mean
within which 95% of future measurements will fall.
culture basket (protector) - 0.96 liter cylindrical polystyrene vessel.
Openings on the side of the basket are covered with 253 micron mesh
screen to permit passage of water.
culture chamber - a 75.8 liter fiberglass container, filled with water
and fitted with brackets to support eight culture baskets.
electro-shocking - a technique used in collecting fish by which fish are
stunned with an electric current and netted from the water.
endogenous food source nutrient source obtained by metabolizing reserve
energy sources within the body.
exogenous food source - food obtained from outside the body.
food conversion - the ratio of increase in dry body weight of an organism
to the dry weight of food consumed.
garnetogenesis - the nrocess by which egg and sperm cells are produced
within the body.
gonadosomatic index (GSI) - the ratio of gonadal weight to whole body
weight, an index of gonadal maturity.
39
-------
goodness-of-fit - a statistical test to determine the degree to which
a given set of data approximates some hypothetical distribution, such
as a straight line.
incubation - the period of egg development between fertilization and
egg hatching.
induced spawning - manipulation of external and/or internal environ-
mental factors to cause fish to spawn.
instantaneous biomass change - the difference between rates of instan-
taneous growth and instantaneous mortality.
instantanteous growth (g) - defined as the natural logarithm of the
ratio of final weight over initial weight divided by the time period.
g = [LnOV./W)]/t, where Ln = natural logarithm. W = weight after
time t and W = initial weight.
instantaneous mortality [i] - defined as the natural logarithm of the
ratio of the number of surviving animals over initial number of
animals, divided by the time period. i = [Ln(Nt/N )] /t, where
Nt = number of surviving animals after time, t and NQ = initial
number of animals.
intramuscular within the muscle tissue.
intraperitoneal - within the peritoneum or internal body cavity.
juvenile a stage in the life history of an animal when morphological
characters of an adult are present but it is not sexually mature.
mature eggs - refers to eggs that are capable of fertilization to produce
viable offspring.
metabolic rate the rate of energy expenditure of an animal, usually
expressed per unit weight.
method of least squares - a method of fitting a curve to a set of data
so that the sum of the squares of the distance of the points from
the curve is minimized.
nauplius - the first free-living stage in the development of some
crustaceans, e.g., anostracans, cladocerans, barnacles.
pituitary - endocrine gland beneath the floor of the brain of verte-
brates. Anterior lobe controls hormone production by thyroid,
gonads, and adrenal glands.
premature eggs - are eggs that have increased in size to a point where
they are almost mature but are still not capable of fertilization.
40
-------
random assignment - placement of animals 'into an experiment in such a
manner as to simulate a chance distribution and hence to produce
unbiased statistical data.
relative percent hatch the number of animals that hatch at the
optimal temperature is assigned the value 100%. Hatch at the
remaining temperatures are assigned as relative proportions of
the hatch at the optimal temperature.
respiration - the sum of enzymatic reactions, both oxidative and non-
oxidative, by which energy is made available for biological work.
sac-fry - the just-hatched larvae of fishes in which the external
yolk-sac is still present.
slope function - expresses the ratio between dosages and mortality.
The dosages necessary to produce 84%, 50% and 16% mortality are
defined as ED84, ED50 and ED16 respectively. Slope function (S) =
[(ED84/ED50) + (ED50/ED16)]/2.
standard length - the length of fishes measured from the mouth to the
end of the caudal peduncle (exclusive of caudal fin).
stock - a group of fish that has been maintained in the laboratory under
carefully-defined environmental conditions.
stripping - a method of removing gametes from fish by_gently squeezing
the abdomen, forcing eggs or sperm out of the gential pore.
swim-up fry - stage in fish development when larvae are first capable
of actively swimming. The yolk sac has generally been absorbed by
this point.
thermocline - a sharp temperature discontinuity in a fresh water lake,
where warm surface waters grade rapidly into cold waters in the deeper
areas.
TL50 - median tolerance limit. The amount'or level of toxicant causing
50% mortality in a population at some specified time, e.g., 96 hours.
ventilation - circulation or movement of fluid medium (air or water)
of the external environment across respiratory exchange surfaces.
yolk-sac - sac containing a store of food material that hangs from
the ventral surface of vertebrate embryos (elasmobranchs, teleosts,
reptiles, birds).
zoogeography - branch of biogeography concerned with the geographical
dTstribution of animals and especially the determination of areas
characterized by specific groups of animals.
41
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SECTION X
APPENDICES
_ O
A. Development of Spawning Induction Techniques
Table A-l: Effects of 16L-8D photoperiod at 25C on gonadal
maturation of bluegill sunfish, relative to con-
trols that received 8L-16D photoperiod at 22C. 48
Figure A-l: Mean diameter of eggs sampled each day from two
female bluegills (A, B) maintained for forty
days in an aquarium with three males at 16L-8D
photoperiod and cycled temperature (C) . *0il
globules 51
Figure A-2: Mean egg diameter of samples from female blue-
gills injected (4) daily with carp pituitary
(CP) or human chorionic gonadotropin (HCG).
Fish were maintained in a 1.9 kiloliter aquarium,
with a 16L-8D photoperiod, at the indicated
temperature cycle for 30 days 55
Table A-2: Effects of bi-daily hormone injections on egg
production by female L_. macrochirus. Two fish
were given each injection, a sham injection (0.8
ml Ringer's), or were uninjected (control) and
observed daily for 20 days. The percentage of
observations was recorded in which eggs with oil
globules (A) or eggs that were 0.85 mm or greater
in diameter (B) were obtained. 59
Figure A-3: Percent fertilization £ ) of eggs obtained
from four female Lepomis macrochirus^ injected
(4 ) with carp pituitary (1 mg/4 hrs). Female
A also received 1,000 IU of Penicillin G with
each injection. 61
Figure A-4: Percentages of fertilization ( ) and hatch
( ) of eggs from three female Lepomis macro-
chirus injected (4") with carp pituitary (1.0 mg/
8 hrs). Female B received 1,000 IU of Penicillin
G with each injection. One female of this group
(not shown) failed to produce eggs. 62
Figure A-5: Percentages of fertilization ( ) and hatch
( j of eggs from three female Lepomis macro-
chirus injected (40 with carp pituitary (2 mg/8
hrs). Female B received 1,000 IU of Penicillin G
with each injection. One female (not shown) failed
to produce eggs. 63
43
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Page
B. Rearing and Life Support
Table B-l:
Figure B-l:
Table B-2:
Figure B-2:
Figure B-3:
Percent survival o£ L^. macrochirus sac-fry
after 15 days. Fry were reared in 20.3 cm
bowls with aeration (A); no aeration (NA); 10%
(10WC) or 60% (60WC) water changes daily at 20,
23, or 25C. Animals were fed at time of swim-
up stage (FO) or two days later (F2).
Prototype of the bluegill thermal testing
facility.
66
Survival of bluegill sac-fry at four temperatures.
Diets consisted of mixed Artemia nauplii,
limnoplankton, and rotifers (combination) or
Artemia nauplii. 67
Percent survival of juvenile L_. macrochirus
at various temperatures with 5, 10, 15, or 20
animals per test container (0.96 liter protectors).
Fish were acclimated to 25C, transferred directly
to test temperatures, and observed for mortality
for the next 16 days. 68
70
Survival of Lepomis macrochirus sac-fry at 26C
in the prototype of the'thermal testing facility.
Protectors received 250 ml/min of water from the
top ( ) or the bottom { ). 71
C. Thermal Test Facility
Figure C-l: Floor Plan of the Thermal Testing Facility
Figure C-2:. Culture Chamber and Temperature Control
Apparatus
75
77
Figure C-3: 0.96 liter culture basket (protector) for confine-
ment of eggs and larvae during the bioassay tests. 79
D. Preliminary Bioassay Experiments
Table D-l: Thermal history for adult bluegills used in the
preliminary incubation test. All animals were
exposed to 16L-8D photoperiod. 81
Table D-2: Routine water chemistry data: juvenile growth
test. 83
44
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Page
Table D-3:
Table D-4:
Table D-5:
Table D-6:
Figure D-l:
Figure D-2:
Figure D-3:
Figure D-4:
Figure D-5:
Percent hatch of bluegill eggs at various
temperatures.
Percent survival of bluegill sac-fry exposed to
various temperatures for 96 hours. Eggs were
incubated at 22, 26, or 34C until they hatched,
and the newly hatched larvae were transferred
directly to test temperatures.
Percent survival of fry, reared to the swim-up
stage at 22C and transferred directly to
various test temperatures. (N = 20 fry per test
temperature)
87
88
Instantaneous rates of growth (weight), mortality,
and change in population biomass of juvenile
bluegills, previously acclimated to four stock
temperatures. Instantaneous rates (Ricker, 1958)
were multiplied by 100 to obtain percent change. 89
Instantaneous rates of mortality, growth and
change in population biomass for juvenile blue-
gills. 93
Growth in length of juvenile bluegills. Fish
were acclimated to 12.1°C and then exposed to
various test temperatures. 98
Growth in length of juvenile bluegills. Fish
were acclimated to 19.0°C and then exposed to
various test temperatures. 99
Growth in length of juvenile bluegills. Fish
were acclimated to 26.0°C and then exposed to
various test temperatures. 100
Growth in length of juvenile bluegills. Fish
were acclimated to 32.9°C and then exposed to
various test temperatures. 101
E. References
105
45
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APPENDIX A
Development of Spawning Induction Techniques
Phase 1. Effects of External Environment on Gonad Maturation
Procedures
Adult bluegills were collected from canals near State Road 27, nine
miles south of Florida City, Florida, on 17 November, 1970, and quaran-
tined in the laboratory for two weeks. Mean GSI values were determined
for 5 female fish (0.56%) and four males'(0.40%). All fish were sam-
pled, but failed to release gametes. Three conditions were tested for
their effects on gonadal maturation:
1) the presence of males on development of ovaries
2) a 16L-8D photoperiod at 25C relative to a 8L-16D
photoperiod
3) population density, i.e., one male with one female,
versus one male with two females.
Fish were held in 95, 190, or 333 liter aquaria. All fish were fed
three times daily with trout chow (Ralston Purina Co.), earthworms
(Lumbricus sp.) and freshwater shrimp (Palaemonetes paludosus). Behav-
ioral observations were made daily. After two months, all females were
sacrificed to determine the GSI. Males were stripped to determine
whether sperm could be obtained.
Results
Results of this experiment are summarized in Table A-l. Exposure to
16L-8D photoperiod at 25C led to gonadal development and nest digging
and increased aggressive behavior among males.
Gonadal development of females was induced in the presence or absence
of males. When one male was present with one female, gonadal develop-
ment of the female was less than with more than one female. When one
male was present with two females at 16L-8D photoperiod and 25C, gonads
of one female developed (dominant female) while those of the second
fish did not. Males maintained at 16L-8D photoperiod and 25C released
sperm when stripped, while males held at 8L-16D photoperiod and 22C did
not release sperm. Females maintained at 8L-16D photoperiod and 22C
showed no increase in GSI.
Discussion
Males reached sexual maturity when maintained in the presence of females
at 25C and 16L-8D photoperiod. Optimal maturation of females, however,
47
-------
Table A-l. Effects of 16L-8D photoperiod at 25C on gonadal
maturation of bluegill sunfish, relative to con-
trols that received 8L-16D photoperiod at 22C.
Test Animals
No . No .
Males Females
Test Conditions
Tank Temp
Size (L) Photoperiod (C)
Results
Females*** Males****
(GSI) (Sperm Release)
0
95
16L-8D
25
3.3
2.0
95
16L-8D
25
4.1
0.5
95*
16L-8D
25
1.0
333
16L-8D
25
1.
333
16L-8D
25
0.8
Controls
95
8L-16D
22
0.5
0.4
190
8L-16D
22
0.7
0.7
Male separated from the females by an opaque partition.
The second female released eggs when stripped, and was presumed
to have a GSI of about 4.0. This female was saved for further
experimentation.
GSI = gonadosomatic index - (ovary wt/body wt) x 100.
+ = released sperm, (-) = failed to release sperm.
48
-------
might require that females be kept together, away from males, to reduce
the effects of social hierarchies and to permit uniform development.
Hierarchy formation among females was a factor that needed further study.
In addition, there was no indication yet whether spawning could be in-
duced out -of season, or how spawning and the formation of hierarchies
might be affected by hormonal injections. The following experiments were
designed to gather data on these points and extend spawning technology.
Phase 2. Induction of Spawning in the Laboratory
Procedures
Adult female and male bluegills were collected from the field in Octo-
ber, 1971. Thirteen fish sampled from this group included seven females
(Mean GSI = 0.27) and six males (GSI = 0.1). Fifty animals were main-
tained for three weeks in the laboratory with simulated summer environ-
ment (16L-8D photoperiod at 27-29C). Twenty-three fish were selected
from this group after three weeks and placed in a separate 2.9 kiloliter
aquarium with 16L-8D photoperiod at 28C. Six weeks after the initial
collection, four fish from the tank were sampled. Two males released
sperm when squeezed and were not" sacrificed. Two females had GSI values
of 0.15 and 0.50.
Males had thus achieved some degree of maturity, but females were unde-
veloped. For some spring and summer-spawning fishes, spawning may be
triggered by a sharp increase in temperature (Prosser and Brown, 1961) .
Since a sharp increase to perhaps 35 or 36C would probably harm blue-
gills, the fish were first acclimated to a lower temperature (ambient =
24C) for three weeks and then the temperature was rapidly increased
(IC/hr) to 30-32C.
After the temperature was increased (now nine weeks since the fish were
collected) the population density in the test aquarium was reduced to 10
fish, 5 males and 5 females. The aquarium substrate was covered with a
thin layer of black gravel to make nest-digging activity more conspicu-
ous. In a further attempt to stimulate spawning, six of the ten fish
were injected with carp pituitary (1.5 mg) combined with 0.25 cc of
.Combistrep® antibiotic solution (Pfizer Pharmaceutical Co.). A second
injection of carp pituitary and Combistrep® was administered two days
later, and a third injection of 0.3 mg.of carp pituitary (without
Combistrep®) was given two days after the second injection.
Results
On the day following the first injection, one female control fish was
observed digging a nest. Three male fish died following the second in-
jection (Mean GSI = 0.22). Two females died following the third injec-
tion (GSI = 4.7 and 7.2). A large nest (0.6 m diameter) was dug by one
male fish in the aquarium one week after the third injection. Spawning
occurred on 28 December 1970, approximately 10.5 weeks after the fish
49
-------
were first collected. Several thousand viable eggs were present in
this spawn.
The five fish in the tank now included one dominant male, which main-
tained the nest and one dominant female (the same fish that dug a nest
on the day after the first injection). Nest-digging by centrarchids
under natural conditions is generally confined to males; this activity
was frequently engaged in by female bluegills in the laboratory, both
in heterosexual and homosexual aggregations. Another female fish stayed
in a corner of the aquarium near the water surface. Two other fish dug
small nests in the corners but did not engage in spawning. Once again,
hierarchy was established.
These five remaining fish were maintained-on a 16L-8D photoperiod at a
mean temperature of 30.1C for four weeks. After four weeks, tempera-
ture in the tank was reduced to ambient (•£ 24C) for one week and again
elevated to 29.5C for 10 days. Spawning''occurred 6.5 weeks after the
first spawn, within 10 days of the temperature increase, and without
the aid of hormones.
Discussion
Spawning of bluegills was induced in the laboratory by manipulation of
temperature and photoperiod. These spawns may be triggered by cycled
temperature. The conditions under which the animals were held were
not conducive to egg maturation in.more than one female at a time. Eggs
were difficult to collect from spawns that occurred in 'large aquaria,
and removal of the adhesive eggs from gravel required more than the ten
minutes following fertilization.
The dosages of carp pituitary were lethal to five of the six fish.
Three injected males died after the second injection and did not show
increased gonadal development. Two females which died following the
third injection had nearly mature gonads with GSI values of 4.5 and 7,2.
At this point, it was known that viable eggs were not continually avail-
able from a given female. Further experiments were necessary to deter-
mine whether simultaneous ripening of sever.al females could be triggered
by temperature manipulation. In addition we needed to define the period-
icity of the ovarian cycle and to systematically define the course of
final egg development on the basis of observable changes in egg appear-
ance. These observations would then enable us to predict when fish were
capable of producing ripe eggs so that several fish could be stripped
simultaneously.
50
-------
1.2
1.1
1.0
0.9
0.8
0.
0.6
0.5
0.4
0.3
2
=3 0.7 -
o u-v
O 0.8
LU
0.7
< 0.6
g 0/5
0.4
0.3
u
°^
uu
&.
Qi
LU
Q_
30
28
26
24
22
spawn
spawn
10
15
20
25
30
35
40
n
5
10
15
20
DAYS
25
35
40
Figure A-l. Mean diameter of eggs sampled each day from two female
bluegills (A, B) maintained for forty days in an aquarium
with three males at 16L-8D photoperiod and cycled temperature
(C). * Oil globules.
51
-------
Phase 5. Periodicity of the Ovarian Cycle and the
Course of Egg Development in Female Bluegills
Procedures
The 2.9 kiloliter aquarium containing five adult bluegills was main-
tained with 16L-8D photoperiod. Temperature was cycled between 23 and
28.5C for six weeks. The two apparent females were checked every 1-4
days for the release of viable eggs, or sampled for eggs at various
stages of development. Females were carefully netted from the tank and
handled without the use of anesthetics. Eggs were removed from the geni-
tal opening with a catheter and observed under a microscope. Three to
four eggs were measured with an ocular micrometer to ± .01 mm and the
mean of these values was determined. Eggs were observed for the presence
and appearance of an oil globule, coloration, texture, and appearance of
the cytoplasm.
Results
Fish spawned three times during the six week experiment [Fig- A-l). Two
spawns (on days 14 and 33) occurred within 5 to 7 days of the return to
elevated temperature. One spawn, on day 19, occurred during a tempera-
ture minimum. Mean egg diameters ranged between 0.34 and 1.22 mm. Lack
of a consistent pattern of egg diameter in relation to spawning may re-
flect inadequate frequency of sampling during periods of rapid develop-
ment, or small numbers of eggs collected with the catheter and used in
the calculation of each mean.
The ovarian cycle, as monitored by egg diameter, appears to be character-
ized by a series of peaks at intervals of 2 to 11 days. The mean period-
icities and standard deviations were 4.11 ± 1.54 days for one fish and
6.35 ± 2.42 days for the second fish. Egg diameter by itself was a poor
predictor of spawning activity. Eggs were at least 0.70 mm diameter on
the day prior to spawning. In all cases, however, distinct oil globules
appeared in the eggs at least two days prior to spawning. The complete
cycle, from one spawn to the next, was completed in 14 days.
Discussion
The results of Phase 3 substantiate previous observations (Phases 1 and
2) that, when two females are placed in a tank with one dominant male,
one or the other female will spawn, but not both females simultaneously.
From the eggs sampled during these and subsequent experiments, and from
animals collected from the field, the cycle of egg development was de-
termined from observed characteristics of the eggs. Immature (pre-
spawning) eggs were less than 0.85 mm in diameter, dark brown, and gran-
ular. Premature eggs, which appeared two to three days before spawning,
were 0.85-0.92 mm in diameter with central "submerged" or multiple oil
globules and had a dense granular appearance. As eggs matured, the oil
globules coalesced and migrated to the egg surface, while the egg dia-
meter generally increased to 0.92-1.10 mm. Mature eggs (capable of
fertilization) were slightly granular, perfectly spherical and strongly
52
-------
adherent to surfaces. Mature eggs ranged from 0.85-1.36 mm diameter.
Eggs became over-ripe within 24 hours after becoming mature. Over-ripe
eggs were completely clear and non-spherical.
Cycled temperature may be conducive to spawning, but a sharp rise in
temperature does not in itself trigger a spawn. Controlled temperature
alone could not be used to produce several ripe females simultaneously.
Phase 4. Use of Hormone Injections to Induce Egg
Maturation in Female Bluegill Sunfish
Human chorionic gonadotropin (HCG) has been used to induce ovulation in
bluegills during the spawning season (Pennell, 1964) and carp pituitary
glands (CP) have been used to :induce ovulation and spawning in a variety
of fish (Pickford and Atz, 1957). Daily injections of HCG and carp
pituitary at various dosage levels, were tested for-their effects on egg
development. In most fish, levels of HCG and CP were increased with
time, following the recommendation of Garland Pardue, Auburn University
(personal communication). Results from injected fish were compared with
a control fish (iminjected) and"a fish that received 100 IU of HCG
throughout the experiment.
Procedures
Hormones and pituitary gland suspensions were injected into bluegills
with 1 ml plastic syringes and 24 gauge needles. Injections were given
intraperitoneally. Fish in each experimental group were marked by in-
jection of approximately 0.1 ml of India ink beneath the dermis.
Human chorionic gonadotropin (HCG) was obtained from Sigma Chemical Co.,
St. Louis, Mo. HCG was suspended in fish Ringer's solution (1000 lU/ml)
Lyophilized carp pituitary glands (CP) were purchased from Stoller Fish
Industries, Ames, Iowa, and were suspended (10 mg/ml) in fish Ringer's
solution. Injections were administered each day for 27 to 31 days. Egg
development of each fish was checked daily with a catheter. Adult blue-
gills '(150-350 gm) were maintained in 2.9 kiloliter aquaria with a
16L-8D photoperiod and temperature was cycled between 23 and 29C. The
following series of injections were administered intraperitoneally:
Carp pituitary'
1) 1 mg/day for nine days, 2 mg/day for eight days,
4 mg/day for 14 days
2) 1 mg/day for seven days, 2 mg/day for 23 days
HCG
1) 100 lU/day for 27 days
2) 100 lU/day for 3 days
3) 100 lU/day for 5 days, 300 lU/day for 4 days,
53
-------
500 lU/day for 1 day, 800 lU/day for 5_days,
900 lU/day for 1 day, 1000 lU/day for 7 days
Carp pituitary followed by HCG
1) 1 mg CP for 7 days, 2 mg CP for 16 days,
400 IU HCG for 8 days
Control fish (not injected)
Results
The mean diameter of eggs sampled from all fish ranged from1 0.4 to 1.10
mm during the experiment (Fig. A-2). Injection of 1 mg CP/day for 7
days followed by 2 mg CP/day for 23 days induced production of prema-
ture eggs in one fish. This fish produced eggs above 0.70 mm in mean
diameter for 70.4% of the test period and oil globules were present for
55.6% of the test period. The fish that received increasing dosages of
HCG released eggs that were larger than 0.70 mm during 75% of the test
period (100 IU HCG followed by .200 IU) and 71.4% of the test period
(100 IU to 1000 IU in increasing dosage's), but none of thes.e eggs dev-
eloped oil globules. The fish that received 100 IU of HCG/day through-
out the experiment had eggs above 0.70 mm for 67.9% of'the test period
and these eggs had oil globules present on two days (7.1%). The fish
that received 1, 2 and 4 mg of CP failed to release eggs through much
of the experiment and the eggs did not develop oil globules. The fish
that was injected with CP followed by HCG maintained eggs above 0.70 mm
for 75.9% of the time. Oil globules were present in the eggs 34.4%
of the time, but oil globules were not seen after the fish received
injections of HCG.
Discussion
Previous research (Phases 2, 3) established that eggs were 0.7 mm or
larger in mean diameter on the day prior to spawning, while the smallest
fertilized eggs that were collected from nests were 0.85 mm in diameter.
Oil globules were present in the eggs for two to four days before spawn-
ing occurred. These criteria could thus be used to evaluate the effects
of hormone injections on egg maturation.
Since eggs within the ovary do not develop uniformly, a sample with a
mean diameter of 0.70 generally contains a mixture of eggs ranging from
0.85 mm down to 0.60 in diameter. Fish were considered premature when
the diameter of more than half of the eggs in a sample exceeded 0.85 mm.
Hormone injections of 1 mg CP followed by 2 mg CP were best suited to
the induction of egg maturation, but this alone did not induce complete
maturation. Injections of HCG led to an increase in egg size but were
not conducive to the formation of oil globules. In fact, when HCG in-
jections were administered after CP, oil globules diminished.
54
-------
o
o
<
LU
oU
LU
Q_
Eggs above 0.70 mm 59.1%
No oil globules present
25' ' ' '3b
Eggs above 0.70 mm 75.9%
Oil globules present 34.5%
0
30
Figure A-2. Mean egg diameter of samples from female bluegills injected
( I ) daily with carp pituitary (C.B)or human chorionic gona-
dotropin (HCG). Fish were maintained in a 1.9 kiloliter
aquarium, with a 16L-8D photoperiod, at the indicated tem-
perature cycle for 30 days.
55
-------
1.1-1
i.o-
0.9-
0.8-
0.7-
0.6
0.5-
0.4.
0.3'
0.2'
1.1
1 mg
^C^.
day
• i
2mg
C.P..
day
CD
O
Eggs above 0.70 mm 70 A%
Oil globules present 55.6%
,\
0
1.0-
0.9-
0.8-
0.7-
0.6-
0.5-
0.4-
0.3-
0.2
0
u
o
30-
28-
26-
S 24-
22-
10 15 20 25 30
Eggs above 070mm 67.9%
i I g lobu les present 7.1%
10
Eggs above 070mm
No Oil globules
HCGIHCG
500J800~*'
1U/ IU
I I I
20
900 T 1000
IU/ IU
25 30
0 5 10
Figure A-2. (Continued)
i i i i i i i i
15 20
DAYS
25
30
56
-------
1.11
i.o-
0.9-
0.8-
0.7-
0.6-
0.5-
0.4-
0.3-
0.2
1.1
22
Eggs above 0.70 mm 40%
No oil globules
CONTROL FISH (NOT INJECTED)
10
15
20
25
30
Eggs above 0.70 mm 75%
No oil globules
Figure A-2. (Continued)
57
-------
Further experiments were still needed to refine the use of hormone and
pituitary injections for inducing gonadal ripening and to develop reli-
able methods for inducing the final stages of egg maturation. Also,
fish used in previous experiments spawned at temperatures from 23 to
30C, but the optimum temperature conditions were still not defined.
Phase 5. Effects of Carp Pituitary, HCG, and Manipulation
of Temperature on the Development of Premature Eggs
Procedures
Twelve female bluegills were placed in each of five .2.9 kilo liter aqua-
ria with 16L-8D photoperiod. Fish were given a cycled temperature
(20-28C) or were held at relatively constant temperature within the
following ranges: 27-31C, 27-29C, 28-30C, and 23-26C. Injections of
carp pituitary (1, 2 or 4 mg), HCG (400 IU, or fish Ringer's solution
(0.8 cc, sham) were given once every two days. Uninjected controls
were also held in each tank. Eggs were sampled daily and fertilization
was attempted with all mature eggs.
Results
Results of this experiment are summarized in Table A-2. No mature eggs
were obtained from any of the fish during this experiment. Fish in all
experimental and control groups developed premature eggs with oil glob-
ules, and fish that received 2 mg carp pituitary per day maintained pre-
mature eggs longer than did the remaining fish. HCG, once again, in-
duced eggs to increase in size but was not conducive to oil globule for-
mation. Fish held at fairly constant temperatures (26-29C) maintained
premature eggs and eggs with oil globules for longer than did fish at
lower or higher temperatures or fish that were exposed to a temperature
cycle.
Discussion
A temperature cycle was not necessary to induce egg maturation, at least
to the premature stage. The optimal temperature for egg maturation was
26-29C. Injections of 2 mg CP/day produced better egg development than
injections of 4 mg/day or injections of 400 ID/day of HCG. Injections
of 2 mg every other day was less damaging to the fish than were the
daily injections used in Phase 3.
Tests were still needed to determine reliable techniques for inducing
the final phases of egg maturation.
58
-------
Table A-2. Effects of bi-daily hormone injections on
egg production by female L. macrochirus.
Two fish were given each Injection (see
text for code), a sham injection (0.8 ml
Ringer's), or were uninjected (control) and
observed daily for 20 days. The percentage
of observations was recorded in which eggs
with oil globules (A) or eggs that were 0.85
mm or greater in diameter (B) were obtained.
Temperature
Range (C)
27-31
26-29
26-29
20-28
28-30
23-26
Mean
Percent
Egg
Condition HCG
A
B
A
B
A
B
A
B
A
B
A
B
35
32
12
72
30
77
12
52
27
75
23
62
.0
.5
.5
.5
.0
.5
.5
.5
.5
.5
.5
.0
CP
2 mg
32.5
57.5
5V. 5
92.5
55.0
67.5
45.0
60.0
15.0
47.5
41.0
65.0
CP
4 mg
5.0
30.0
32.5
52.5
40.0
80.0
40.0
57.5
15.0
47.5
26.5
53.5
Sham
2
5
2
32
10
47
12
40
10
65
7
38
.5
.0
.5
.5
.0
.5
.5
.0
.0
.0
.5
.0
Control
17
37
0
97
10
62
12
37
10
31
10
53
.5
.5
.5
.0
.5
.5
.5
.0
.0
.0
.2
•Mean
Percent
18
32
31
69
29
67
24
49
15
53
33
54
.5
.5
.0
.5
.0
.0
.5
.5
.5
.2
.7
.3
59
-------
Phase 6. Induction of Ovulation by
Injections of Carp Pituitary
Procedures
One female, held in the laboratory at 23C, had large (0.87 mm), prema-
ture eggs. This fish was given three intramuscular injections with
1.0 mg carp pituitary at 0800, 1200 and 1700 for one day in an attempt
to induce development of mature eggs. At each injection, eggs were
sampled and mixed with sperm.
Additional females were held at 26C and given bi-daily intraperitoneal
injections of 2 mg carp pituitary to induce development of premature
eggs. Six females with premature eggs were given the following intra-
muscular injections of carp pituitary (two fish per type of injection):
a) 1 mg every four hours; b) 1 mg every eight hours; and c) 2 mg every
eight hours. One of the females in each regime was given 1000 units
of Penicillin G with each carp pituitary injection to reduce bacterial
infection. Eggs were sampled prior to each injection. Percent ferti-
lization, when fertilization was attempted, was recorded after one hour.
Series a) thru c) were replicated. After females produced fertile eggs,
they continued to be sampled at two-hour intervals to determine the
length of time that eggs remained viable.
Results
The single female, injected at 0800, 1200 and 1700, ovulated, producing
mature eggs within 30 hours of the first injection. The hatch from
these eggs was 92%. All females injected with 1 mg/4 hours (Fig. A-3)
produced mature eggs within 26-44 hours (from 7 to 11 injections). Vi-
able eggs were obtained for 8 to 16 hours after ovulation.
Carp pituitary injections of 1 mg/8 hr (Fig. A-4) produced mature eggs
in 3 of 4 females. Eggs matured within 32 to 48 hours following the
first injection and remained viable for up to 20 hours. Greater than
50% hatch was obtained from eggs released during the first six hours.
Injection of 2 mg carp pituitary/8 hr (Fig. A-5) induced the production
of mature eggs in 3 of 4 females within 24 to 36 hours after the first
injection. The eggs remained fertile for 12 to 18 hours. Percent hatch
was greater than 50% for eggs released during the first six hours.
Discussion
The final stages of egg maturation could thus be induced by frequent in-
tramuscular injections of carp pituitary. Eggs ripened in 24 to 44
hours after the first injection. Adults did not survive more than two
days after they were injected and stripped. Fish secreted excessive
mucous. The animals developed ulcerated lesions at the injection sites,
perhaps due to secondary bacterial infection or perhaps as an allergic
reaction to carp pituitary. In any case, concomitant injection of anti-
biotics did not improve survival.
60
-------
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Figure A-3. Percent fertilization ( ) of eggs obtained from four female Lepomis macrochirus
injected (1) with carp pituitary (1 mg/4 hrs.,). Female A also received 1000 !U
of Penicillin-G with each injection.
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Figure A-4. Percentages of fertilization ( )
and hatch ( ) of eggs from three
female Lepomis macrochirus injected
( i ) with carp pituitary (1 .0 mg/
8 hrs.). Female B received 1000
IU of Penicillin-G with each injec-
tion. One female in this group (not
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Figure A-5. Percentages of fertilization ( .)
and hatch ( ) of eggs from three
female Lepomis macrochirus injected
( | ) with carp pituitary (2 mg/8 hrs.).
Female B received 1000 IU of Peni-
cillin-G with each injection. One
female (not shown) failed to produce
eggs.
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63
-------
The time needed to obtain fertile eggs ranged from 24 to 40 hours after
injection. This indicated that considerably more than 4 animals should
be selected in order to have several females with fertilizable eggs
available simultaneously. Depending upon the number of fish available
with mature eggs, 20-33 females were injected to ripen 3 to 7 females
simultaneously.
A reliable technique was now available for obtaining mature eggs from
several female bluegills simultaneously. The final procedure consists
of six steps.
1) Fish collected from the field are returned to the laboratory, ac-
climated to captivity, and sexed as well as possible on the basis
of morphology, coloration, and behavior.
2) Males are kept at 16L-8D photoperiod at 26C, with as many as 20
fish per 2.9 kiloliter aquarium. Ripening requires up to two
months for fish collected outside the spawning season.
3) Females are kept isolated from males and given a 16L-8D photo-
period at a temperature of 26-28C. The production of premature
eggs may require up to three months.
4) Females are injected intraperitoneally with 2 mg carp pituitary
every other day, and egg development is checked daily.
5) When a sufficient number of females have developed premature eggs,
20-30 such females are selected and injected intramuscularly With
1 mg carp pituitary every four hours. Eggs are sampled at each
injection interval and examined for size, appearance, adhesive-
ness, and fertilization-ability until several females have mature
eggs simultaneously.
6) Females producing eggs with greater than 50% fertilization at the
previous sampling interval are stripped and eggs are fertilized
with milt from four or more males.
64
-------
APPENDIX B
Rearing and Life Support
Growth and survival of young bluegills were determined under a variety
of culture conditions. Studies were conducted with eggs spawned in
the laboratory. Juveniles were collected in the field near Florida
City, Florida or in Conservation Area 2A of the Florida Flood Control
District or were obtained from Federal Fish Hatcheries at Welaka,
Florida and Richloam, Georgia.
Phase 1. Air Supply, Water Exchange, Feeding and Temperature
Procedures
Sac-fry were obtained from eggs spawned in the laboratory at 23C. Rear-
ing tests were conducted for 15 days in 20.3 cm diameter culture bowls;
each treatment had two replicates. Combinations of the following treat-
ments were tested: aeration (through 2.5 cm-long airstones) versus no
aeration; 10% water change daily versus 60% water change daily; food
(newly-hatched Artemia salina nauplii) offered at time of swim-up
stage versus food offered two days later; temperature of 20C, 23C
(ambient), or 25C.
Results
The best conditions for survival of L_. macrochirus fry (Table B-l) were:
1) absence of a supplementary air supply, 2) 10% water change daily, 3)
ambient temperature (23C), and 4) introduction of food two days after
swim-up stage.
Phase 2. Diet and Temperature
Procedures
Eggs were collected from a natural spawn in the laboratory at 26C and
tempered at 8C/hour to temperatures of 15, 20, 25, and 30C. Sac-fry
were maintained at these temperatures and fed at 0800, 1200, and 1600
daily when they reached swim-up stage. Survival at the various tempera-
ture levels was compared using two diets: 1) nauplii of Artemia salina
(360 microns by 135 microns); and 2) a mixed diet of Artemia nauplii,
cultured rotifers, and limno-plankton (65-270 micron fraction) collected
from the field.
Results .
Larvae that were fed a mixed diet of rotifers, Artemia, and limno-
plankton died within two days at 25C and within three days at 20C (Fig.
B-l). In contrast, more than 90% of the larvae that were fed only
Artemia at these temperatures survived after eight days. At 15 and 30C,
65
-------
e B-l. Percent survival of
L. macrochirus sac-fry after
15 days. Fry were reared in 20.
tion (A); no aeration (NA) ; 10%
water changes daily at
fed
at time of swim-up
3 cm bowls with aera-
(10WC) or 60% (60WC)
20, 23, or 25C. Animals were
stage
(FO) or two days
later (F2) .
Primary
Variable
NA
A
FO
F2
10WC
60WC
20C
23C
25C
Other
Conditions
10WC, FO, 20C
10WC, FO, 20C
NA, 10WC, 23C
NA, 10WC, 23C
NA, FO, 23C
NA, FO, 23C
NA, FO, 10WC
NA, FO, 10WC
NA, FO, 10WC
No . Fry
Tested
600
600
150
150
300
300
100
100
100
Percentage
Survival
16.9
3.6
19.5
24.0
21.5
12.0
21.0
42.0
3.0
Combined
Variables
VI V2
FO 20
23
25
F2 20
23
25
10WC FO
F2
60WC FO
F2
Other
Conditions
NA, 10WC
NA, 10WC
NA, 10WC
NA, 10WC
NA, 10WC
NA, 10WC
NA, 23C
NA, 23C
NA, 23C
NA, 23C
No . Fry
Tested
100
100
100
50
50
50
150
50
150
50
Percentage
Survival
21.0
^2.0
3.0
22.0
46.0
19.5
46.0
12.0
32.0
66
-------
ioo -i
60 —
C£
D
(SI
40 -
20 -
0
Artemia nauplii
combination
Artemia nauplii
combination
Artemia nauplii
Dcombination
Artemta nauplii
combination
8
Figure B-l . Survival of bluegill sac-fry at four temperatures. Diets consisted of mixed Artemia nauplii, limno-
plankton, and rotifers (combination) or Artemia nauplii.
67
-------
survival of larvae that were fed Artend a alone was not significantly
greater than survival of larvae that were fed the mixed diet.
Phase 3. Population Density
Procedures
Population densities of 5, 10, 15, and 20 juvenile bluegills per 0.96
liter culture basket were evaluated to determine, acceptable densities
for maintenance of animals during the bioassay tests. Juvenile blue-
gills ranging from 2.5 to 5 cm in total length were obtained in
February, 1971, from the Federal Hatchery at Richloam, Georgia. Fish
were placed in culture baskets and transferred directly from 27C to
aquaria maintained at temperatures ranging from 10 to 40C at 5C incre-
ments. Fish were fed frozen adult Artemia three times daily. The ex-
periment was ended after 16 days.
Results
Survival of juvenile bluegills (Table B-2) at high population densities
(15 and 20 per culture basket) was significantly lower at 30 and 35C
than survival at low densities (1Q and 5 per culture basket. Mortality
at 5 and 10 animals per protector and high temperature was greater than
expected, and may indicate an effect of crowding. The sampling fre-
quency and sample size required by the growth experiment made it neces-
sary to select a density of,10 animals per protector for use during the
bioassays despite the possible adverse effect of density.
Table B-2. Percent survival of juvenile L. macrochirus
at various temperatures with 5, 10, 15 or
20 animals per test container (0.96 liter
protectors). Fish were acclimated to 25C,
transferred directly to test temperatures,
and observed for mortality for the next
16 days.
Temp. No. of Animals per Protector
5 10 15 20
Percentage Survival
10
15
20
25
30
35
40
0
100
100
100
80
80
0
0
100
100
100
90
50
0
0
93
80
80
47
0
0
0
90
80
45
65
0
0
68
-------
Phase 4. Prototype of the Thermal Testing Facility
Procedure
A prototype of the thermal testing facility was constructed to test
the system design (Fig. B-2). Water was recirculated at the rate of
1.5-liters per minute through the 75.8-liter system. Water was
treated by passing it through a biological filter and an autotrophic
compartment containing Anacharis. From these conditioning tanks,
water flowed into two 75.8-liter culture chambers containing eight
1-liter culture baskets. Each culture basket contained 25 newly-
hatched sac-fry and received water at a rate of 250 ml/min. Water
was delivered through the bottom of protectors in one chamber and
through the top in the other. Larvae were fed Artend a nauplii three
times daily, beginning two days after they reached swim-up stage.
Results
Survival of sac-fry in the prototype facility after 15 days was
greater when water was delivered into the top of the culture basket
(Fig. B-3) than when water was delivered through the bottom of the
protector. The top-delivery system was selected for use in the
culture chambers of the thermal test facility.
69
-------
A. Autotrophic Chamber
B0 Biological Filter Chamber
C. Mixing Apparatus
D. Header Chamber
E. Overflow Tubes
F. Metering Valve
G. Pressure Manifold
H. Delivery Tube
I. Manifold Outlet
J. Culture Chamber
K. Overflow
L. Collecting Tank
M. Culture Basket
N. Pump
O. Aspirating Nozzle
Figure B-2. Prototype of Bluegill thermal testing facility.
70
-------
120 —
ioo H
<
I 80-1
60 -
40 -
20 -
'0'
I I I
5
10
I I I
15
DAYS
Figure B-3. Survival of Lepomis macrochirus sac-fry at 26 C in the prototype of the
thermal testing facility. Protectors received 250 ml/min of water from
the top ( ) or the bottom ( ).
71
-------
APPENDIX C
Thermal Test Facility
Design Specifications. A special testing facility was designed and con-
structed to perform the bluegill bioassays. Design specifications for
the facility were:
1. Capability to establish the temperature in each culture vessel at
any specified temperature over the range from 3 to 40C;
2. Capability to maintain the temperature in a given chamber within
+0.3C of the specified temperature for extended periods of time (up
to 6 weeks);
3. Continuous recording of the temperature in each culture vessel;
4. Capacity to provide suitable life support conditions for eggs, laf-
vae, and juveniles of the test species allowing adequate survival;
growth, and monitoring;
5. Capacity to maintain consistent water quality over extended periods
of time (up to 6 weeks);
6. Controlled flow rate of water into each culture basket;
i
7. Use of simulated natural daylight (Duro-Test Corp. Vita-Lite® flutfres^
cent tubes) with photoperiod corresponding to the light cycles for
the area from which the adult specimens were collected.
Theoretical Basis. Several alternative designs were considered to meet
the design specifications, but the need to precisely control the temp-
erature of a relatively large volume of moving water led to the dev-
elopment of a temperature-adjustment mechanism based on mixing water of
different temperatures. Water from reservoirs maintained at 4C, ambient
(25C), and 40C could be combined in a known ratio within a header tank
above each culture basket by means of precision mixing valves, to produce
water temperatures that are about 1C below the desired test temperatures',
Temperatures below 6C require additional cooling supplied by a refrigera-
tion coil set in the individual header tanks.
Approximate temperatures in the header tanks can be achieved by mainte-
nance of constant temperatures in the reservoirs and by maintenance of
a constant room temperature. Precise control of temperature in each cul-
ture chamber can be regulated by a heater located in the header tank and
controlled by a thermoregulator in the culture vessel.
To prevent mixing of test animals within the system and to facilitate
observation and sampling of test animals, eggs, larvae and juveniles can
73
-------
be confined in small containers. This confinement, however; requires an
adequate exchange of water to maintain oxygen levels and to remove waste
products. Mesh-covered culture baskets immersed in a large water bath
would provide confinement and have the thermal-stability of a large test
container. Several baskets (protectors) in a given culture chamber can
be used to expose fish of different origin to identical temperature con-(
ditions.
Natural or municipal water supplies generally vary in chemical composi-
tion, even over short periods of time. A closed, recirculating culture
system reduces the problem of outside contamination. However, water in
a closed system exhibits changes as a result of biological activity, in-
cluding increases in suspended solids, biological oxygen demand, and
nitrogenous compounds. Accumulation of these materials can be prevented
by various water-treatment methods such as mechanical filters, biologi-
cal filters, and tanks containing growing plants (autotrophie tanks).
Temperature of each thermal test unit can be continuously monitored by
thermistor probes connected to electronic recorders, while photoperiod
can be controlled by a single timer connected to lights above each test
unit.
Component Descriptions. The system occupies two floors of an air condi-
tioned warehouse. Experimental apparatus and monitoring equipment, are
located on the lower level. Water conditioning units are located on the
upper level. This arrangement provides for easy access to all components
and permits control of experimental apparatus without disrupt-ion of the
life-support elements of the system. An auxiliary diesel generator was
available in the event of electrical failure. The entire system exchanges
9.5 kiloliters of water per hour. Components of the system .are illus-
trated in Figure C-l.
A. Collecting Tank. Water from all culture chambers drains into a com-
mon fiberglass collecting tank. The temperature of the resulting mix-
ture approximates ambient temperature (24C). Water is pumped from
the collecting tank by an electric pump with a polyvinyl-chloride
(PVC) impeller to autotrophie tanks on the upper level.
B. Autotrophie Tanks. Four autotrophie tanks (in series) receive' wa-
ter from the collecting tank. The tanks are constructed of, marine
plywood coated with polyester resin. Each tank is 3.61 x 1.17 x
0.5 meters, contains 2.27 kiloliters of water and 5.7 kg (wet weight)
of Anacharis sp. Each tank is lighted by 24 full-spectrum Vita-
Lite® fluorescent bulbs. Water flows from the autotrophie tanks to
the biological filter. Plants in the autotrophie tank removed dis-
solved nutrients, especially nitrite, nitrate and phosphate, from
the water.
C. Biological Filter. The biological filter is 2.4 x 1.2 x 1.2 meters
and contains coarse (4-6 mm) calcareous (oolite) gravel as a sub-
strate for the growth of nitrifying bacteria. These bacteria metabo-
74
-------
A. Collecting Tank
B. Autotrophic Water Conditioning Tanks
C. Biological Filter
D . Pressure Filter
Chiller
Water Heater
G . Cold Header
H. Ambient Header
Hot Header
Power and Temperature Readout Console
K. Bench with 15 Culture Chambers
L. Bench with 21 Culture Chambers
E.
F .
I .
J .
Figure C-l . Floor plan of the thermal testing facility.
LOWER LEVEL
UPPER LEVEL
-------
lize ammonia and other toxic wastes to less toxic materials. When
filled with gravel substrate, the filter contains 1.4 kiloliters of
water.
D. Pressure Filter. Water is pumped from the biological filter to the
pressure filter by a direct-drive stainless steel pump. The pres-
sure filter is constructed of fiberglass, 0.74 meters in diameter,
0.96 meters in height, and contains diatomaceous earth capable of
removing particles 1.0 micron or larger. The pressure filter removed
suspended solids and large parasites from the water. Water from the
pressure filter flows by three routes: 1) through the chiller to the
cold header tank, 2) through the heater to the hot header tank, or
3) directly to the ambient header tank.
E. Chiller. The chiller cools water for the low-temperature reservoir.
Chilling 2.27 kiloliters of water per hour to 1C is accomplished
with a thirty-ton (90,720 kg-cal/hr) stainless steel chiller.
F. Hot Water Heater. The hot water heater raises the temperature of the
water for the high-temperature reservoir. Approximately 1.89 kilo-
liters of water per hour is heated to 40C in a 63,000 kg-cal/hr gas
water heater.
G. Cold, Ambient, Hot Water Headers. Three 1.143-kiloliter fiberglass
tanks provide a constant head of water pressure for hot, ambient, and
cold water supplied to the tank racks. Water in the headers receives
extensive aeration. Water is piped to the racks and metered to the
thermal control units above individual culture chambers. Air temper-
ature, water flow rate and hence rate of temperature change in the de-
livery pipes are constant.
H. Tank Racks. Seventy-two culture chambers and temperature control
units are arranged on two rows of tank racks. Each culture chamber
is lighted by full spectrum Vita-Lite® fluorescent lamps (Duro-Test
Corp.), regulated by an electric timer, to deliver a controlled photo-
period.
I. Temperature Control Units and Culture Chambers. Temperature control
units and culture chambers (Figure C-2) have the following components:
A. Valve for mixing waters of various temperatures and for regu-
lating water flow.
B. Header tank.
C. 500 watt heater
D. Heater controller
E. Flow-control outlets
F. 75.8-liter fiberglass culture chamber
76
-------
A.
B.
C.
D.
E.
F.
G.
H.
I.
J.
K.
L.
M.
NL
O.
Po
Valve
Header Chamber
500 Watt Heater
Heat Controller
Header Flow Control Outlets
Culture Chamber
Culture Basket
Thermal Monitor Sensor
Incoming Water
Header Overflow Standpipe
Bench Supports
Culture Chamber Standpipe
Unit Outflow
To Collecting Tank
Water Level
Delivery Tube
Figure C-2o Culture Chamber and Temperature Control Apparatus
77
-------
G. 0.96-liter, 253 micron mesh culture baskets (Figure C-3)
H. Temperature monitor sensor
Waters of different temperatures are mixed to provide water approxi
mately 1C below the required test level. High temperatures are
achieved by mixing hot with ambient water and low temperatures by
mixing cold with ambient water. The mixture is sprayed into the
header chamber to provide oxygen or to reduce supersaturation. The
procedure was not adequate to reduce super-saturation at the highest
temperatures, but was effective at lower temperatures. A 500 watt
heater in the thermal control header is linked by a feedback mechanism
to the heater control to provide the final adjustment to the desired
temperature level. Water flows from the header chamber through the
flow-control outlets at 250 ml/min to each of the eight 0.96-liter
culture baskets containing the test animals. Culture baskets (Figure
C-3) consisted of a polystyrene plastic cylinder (r=5.5 cm, h=10 cm)
with two 6 cm diameter holes drilled in opposite sides and covered
with 253 micron mesh Nytex® screen. Water overflows from the culture
chamber via a central standpipe and returns to the collecting tank.
Honeywell Electronik-16 Multipoint Recorders, located on the central
console (Figure C-l, J), provide chart records of temperatures
measured by the thermal sensor in each of the seventy-two culture
chambers.
78
-------
II cm,
10
cm
A. Polystyrene frame
B. Holes drilled in sides
C. Nytex® screen
Figure C-3. 0.96 liter culture basket (protector) for ccnfinement
of eggs and larvae during the bioassay tests.
79
-------
APPENDIX D
PRELIMINARY BIOASSAY EXPERIMENTS
INTRODUCTION
The thermal test facility (Appendix C) was used to conduct a series
of preliminary temperature bioassay experiments. A preliminary egg-
incubation experiment was performed as a test of the thermal facility,
to familiarize personnel with adult and egg handling procedures, and to
define the approximate range over which bluegill eggs would hatch.
Preliminary tests were conducted with bluegill sac-fry and swim-up fry
to determine whether the water quality, feeding schedule, diet, and
amount of food were adequate and to define approximate ranges for TL50
determinations and growth experiments.
A growth test was conducted with juvenile bluegills to define the temp-
erature limits at which growth and survival were sufficient to main-
tain a population. These limits could the.n be used in subsequent tests
to define the optimal temperature and the temperature range for optimal
growth and survival.
MATERIALS AND METHODS
The preliminary incubation test was conducted using the techniques des-
cribed in the main report. The adults used for the preliminary incu-
bation test were maintained under the temperature conditions shown in
Table D-l. Eggs from seven females were fertilized with the sperm from
four males at 25.1C. Results were analyzed as described in the main
report, except that replicates were treated separately.
Table D-l. Thermal history for adult bluegills used in the
preliminary incubation test. All animals were
exposed to 16L/8D photoperiod.
Collection Temperature (C) 23-26
Holding
Duration (Days) 8-82
Mean Temperature (C) 24.5
Egg Production
Duration (hrs) 24
Mean Temperature (C) 25.1
Bioassay Tests on_ Fry
Preliminary tests were conducted to determine maximum and minimum thermal
tolerances of bluegill sac-fry and swim-up fry. For these tests, only
a few animals were available for testing at any temperature. During
these experiments, temperatures were maintained to ± 1C (Means npt cal-
culated) and water chemistry measurements were not made. Eggs obtained
from females in the laboratory were incubated at 22, 26, and 34C. Newly
81
-------
hatched fry, and fry reared to the swim-up stage at 22C were transferred
directly to test temperatures in the thermal test facility. After lar-
vae had reached swim-up stage (See Appendix B), they were fed Artemia
nauplii (360-450 microns in length) three times daily- Dead fry were
counted and removed from test chambers after 24, 48, 72, and 96 hours.
Percent survival was calculated at each testtemperature.
Juvenile Growth Test
Juvenile bluegills (mean wt = 0.29 gm, range = 0.1 to 2.0 gm) from stocks
acclimated to 12.1, 19.0, 26.0 and 32.9C were transferred directly to a
series of test temperatures (Table D-6) in the thermal test facility-
Test temperatures were measured as during the juvenile bioassay. Mean
temperature ± 95% confidence limits are given for each replicate culture
chamber. Animals were maintained at various test temperatures for three
to six weeks. Juvenile bluegills were fed to satiation three times daily
(0800, 1200, 1600) with adult Artemia, Daphnia, and Tetramin®. These
feedings constituted a maximum ration, as some uneaten Artemia and Daphnia
remained in the protectors between feedings and overnight. Dead animals
were counted and removed daily. Prior to the bioassay test, an initial
sample of 28 to 115 animals was selected randomly, along with the test
animals at each stock temperature. Ten animals from each testtempera-
ture were sampled randomly at bi-weekly intervals for up to six weeks.
These fish were preserved in Bouin's solution (Humason, 1967), and were
later measured to determine growth rates. Standard length was determined
with calipers to the nearest 0.1 mm. Fish were weighed with an analytical
balance to the nearest 0.1 gm. Whenever five or fewer animals survived
at any test temperature, these animals were removed, preserved and
measured. Instantaneous mortality, instantaneous growth (weight), and
instantaneous change in population biomass (Ricker, 1958) were calculated
for each stock and test temperature.
RESULTS
Water Quality
The trace-chemical composition of the water used during these tests is
described in the main report. Routine water quality tests were not
performed during the preliminary egg incubation and fry bioassay tests.
Results of routine monitoring during the growth experiment are presented
in Table D-2. Dissolved oxygen levels ranged from 61% saturation to
supersaturation (145%). As mentioned before, improved control of oxygen
levels in the test system is necessary (see Appendix C). Ammonia levels
(un-ionized NH.j) were between .003 and .095 ppm. The sensitivity of the
ammonia test was 0.25 ppm of NKL, which represented as much as 0.039 ppm
of NH-J at high temperature and pH levels. The levels of NHj measured
were, in general, unacceptable to achieve optimal growth and development
(see Discussion in main report). Nitrite levels ranged from 0 to 0.010
ppm; pH ranged from 7.60 to 8.34 (except one reading of 6.94) and nitrate
ranged from 0 to 5 ppm.
Incubation Test
The percent hatch of bluegill eggs at various test temperatures is pre-
82
-------
Table D-2. Routine water chemistry data: juvenile growth test
Day
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
PH
Mean Range
Dissolved Oxygen
(% Saturation)
Mean Range
Ammonia
(Un-ionized
Mean Range
Nitrite
Mean Range
Nitrate
Mean Range
7.9
7.68
7.9
8.19
7.64
7.83
8.00
8.01
7.68
8.13
7.86
7.75
8.14
8.10
7.93
7.09
8.06
7.97
8.18
8.12
8.06
8.02
8.12
8.15
7.93
7.74
8.05
8.03
8.08
7.8-7.9
7.64-7.75
7.8-8.0
8.13-8.27
7.60-7.69
7.72-7.93
7.95-8.03
7.98-8.04
7.64-7.73
8.07-8.22
7.86-7.87
7.71-7.82
8.09-8.23
8.07-8.12
7.88-7.99
6.94-7.23
7.99-8.12
7.93-8.02
8.14-8.25
8.05-8.22
7.99-8.14
7.91-8.14
8.03-8.18
8.15-8.16
7.87-8.03
7.70-7.78
7.99-8.14
7.91-8.12
8.04-8.15
101
104
106
115
110
108
102
102
99
107
117
99
97
98
102
93
103
112
111
98
93
91
88
92
90
87
92
83
82
80-119
80-134
80-134
93-132
88-134
88-122
80-122
80-122
82-116
94-125
82-145
82-116
80-116
80-116
87-120
74-107
87-122
95-131
86-127
82-117
80-110
74-108
74-104
77-102
74-104
71-102
80-108
69-96
66-102
.027
.016
.010
.047
.006
.006
.013
.013
.009
.017
.013
.039
.027
.023
.012
.002
.015
.012
.030
.018
.017
.012
.016
.018
.012
.007
.018
.015
.018
.018-
.005-
.005-
.015-
.004-
.004-
.004-
.007-
.003-
.009-
.008-
.005-
.011-
.010-
.006-
.001-
.008-
.007-
.011-
.009-
.008-
.006-
.008-
.011-
.006-
.004-
.008-
.007-
.009-
.042
.031
.018
.095
.009
.018
.022
.021
.018
.029
.019
.024
.052
.035
.019
.004
.025
.020
.044
.031
.029
.028
.028
.028
.022
.012
.033
.025
.032
.004
.003
.005
.001
.002
.002
.002
.003
.004
.004
.004-
*
*
.009
*
*
*
.004
*
*
.003
*
.003
*
*
.000
*
.002
*
.003-
.001-
.003-
.0 -.
.001-
.001-
.001-
.001-
.003-
.001-
.003-
.008-
.004-
.003-
.003-
.005
.005
.008
003
.003
.003
.005
.005
.005
.005
.005
.010
.005
.003
.006
.0 -.001
.001-
.003
0
*
*
0
0
0
0
0
0
0
0
*
*
*
*
*
*
0
*
*
0
*
0
*
*
1
*
2
*
0-0
0-0
0-0
0-0
0-0
0-0
0-0
0-0
0-0
0-0
0-0
0-0
0-2
0-3
83
-------
Table D-2. (cont'd)
Da)
PH
Mean Range
Dissolved Oxygen
(% Saturation)
Mean Range
Ammonia
(Un-ionized
Mean Range
Nitrite Nitrate
Mean Range Mean Range
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
7.83
7.97
7.85
7.96
7.98
8.01
8.09
8.13
8.11
8.27
8.06
8.09
7.90
7.91
8.10
8.21
8.25
7.78-7.91
7.88-8.08
7.80-7.94
7.89-8.06
7.91-8.08
7.97-8.05
8.05-8.16
8.08-8.21
8.03-8.21
8.22-8.34
8.01-8.12
8.03-8.16
7.84-8.01
7.86-7.98
8.06-8.15
8.08-8.30
8.20-8.31
82
83
84
82
83
80
85
76
80
88
86
86
81
77
81
82
88
65-102
64-99
70-102
64-102
66-102
65-99
72-96
61-89
67-93
72-107
66-104
67-107
64-102
64-93
70-95
67-102
74-104
.010
.013
.010
.016
.013
.014
.017
.022
.014
.024
.016
.022
.012
.011
.018
.028
.023
.005-
.007-
.005-
.006-
.006-
.008-
.009-
.009-
.009-
.013-
.008-
.009-
.006-
.005-
.009-
.010-
.013-
.017
.023
.017
.033
.023
.022
.029
.041
.020
.039
.025
.041
.020
.020
.029
.054
.036
*
.002
*
.002
*
*
.001
*
.003
*
*
.002
*
*
.002
*
*
.001-
.001-
.0 -.
.003-
.001-
.001-
.005
.003
001
.003
.003
.003
2
0
1
0
2
0
*
0-3
*
0-0
*
*
0-2
*
0-0
*
*
0-5
*
*
0-0
*
*
Samples not analyzed
84
-------
sented in Table D-3. Eggs hatched at temperatures from 18.7 to 33.8C, but
failed to hatch at 16 or 35.8C. The temperatures for maximum hatch were
22.6C (replicate A) and 30.6C (replicate B), when both replicates were
combined, optimal temperature for hatch was 30.6C. The combined results
of the replicate A temperature chambers differed greatly from the com-
bined results obtained from replicate B chambers with respect to hatching
rate and optimal temperature for hatch. Both replicates had a low per-
cent hatch at 26.2C. Egg viability was low during this experiment, so
that a lower TL50 was not defined in replicate B. Temperatures were
recorded manually at 6-hr intervals. Temperature control was not adequate
since the confidence limits ranged from + 1.32 to + 4.46C during the ex-
periment .
Replicate A had an upper TL50 of 32.4C and a lower TL50 of 21.4C. Repli-
cate B had an upper TL50 of 31.6C, but lower TL50 was not defined. When
both replicates were combined, upper TL50 was 31.6C and lower TLSO was
22.6C. Upper TLSO values determined during the preliminary test were slight-
ly lower than the TLSO temperatures subsequently defined in incubation
tests 1 and 2 (see main report).
Bioassay Tests on Fry
Results of the preliminary tests were not intended to define TLSO temp-
eratures for sac-fry (Table D-4) from the three stocks, but rather to
define the temperature ranges necessary for subsequent testing. Max-
imum survival of fry that were hatched at 22C was 83% at 29C. Larvae
from this stock were exposed to temperatures from 9 to 37C, but only sur-
vived for 96 hours at temperatures from 16 to 32C.
Sac-fry that were hatched at 26C were exposed to temperatures from 8 to
38C survived for 96 hours at 12, 16 and 34C. These fry survived 96-hour
exposure to temperatures that were 4C lower and 2C higher than the temp-
eratures tolerated by the 22C stock. Sac-fry from the stock at 34C sur-
vived for 96 hours at temperatures from 18 to 24C. Sac-fry did not
survive at 14 and 16C and thus had poorer survival at low temperatures
than the stock at 26C. Larvae from the stock at 34C survived better at
38C than did larvae from the stock at 26C.
Preliminary tests with swim-up fry hatched at 22C (Table D-5) indicated
survival for 96 hours at temperatures from 11 to 26C. The low-temperature
TLSO was between 11 and 12C. The upper 96-hour TLSO was not defined be-
cause of poor survival of the fry at all test temperatures above 12C.
Juvenile Growth Tests
The juvenile growth test was conducted with fish that were acclimated to
12.1, 19.0, 26.0 or 32.9C. Instantaneous rates of mortality, growth (weight
of preserved fish), and change in population biomass (Ricker, 1958) for
juvenile bluegills are presented in Table D-6 and Figure D-l. The cal-
culated rates were multiplied by 100 to express the rates as percentages.
85
-------
Table D-3. " Percent hatch" of bluegill eggs at various temperatures.
Test Temp
Mean ±95%CL
18.
18.
20.
20.
22.
22.
23.
24.
26.
26.
27.
2S.
30.
30.
31.
32.
33.
33.
35.
9
5
4
6
6
6
6
0
1
3
5
1
4
•7
4
3
8
9
8
+ 1
± 1
± I
± 1
+. 1
+. 1
± 2
+_ 2
± 2
+_ 2
+. 3
+. 1
+. 3
^ 7
* 4
—
jt 2
_±_ •}
±. 2
.32
.43
.73
.47
.77
.76
.64
.66
.76
.53
.87
.90
.03
.72
.46
.87
.84
.54
.96
35.7 ± 2.17
Actual
Rep 11- No . Temp
cate Readings
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
16
16
16
16
14
14
14
14
14
14
10
10
10
10
10
10
10
10
10
10
Upper TL5Q
Replicate A
Replicate B
Combined
Total %
Hatch
2
0
2
10
46
14
36
22
12
10
32
32
36
80
36
10
4
18
0
0
32.3
31.6
31.7
Normal
% Hatch
2
0
2
10
46
12
26
20
2
4
18
32
34
72
36
10
2
12
0
0
Combined Actual
Total
Hatch
1
6
30
29
11
32
58
23
11
0
% Normal
% Hatch
1
6
29
23
3
25
53
23
7
0
Lower TL,-n
Replicate A
Replicate B
Combined
Relative %
Normal Hatch
4
0
4
14
100
17
57
28
4
6
39
44
74
100
78
14
4
17
0
0
21.5
(not defined)
22.3
Combined
Relative %
Normal Hatch
2
11
56
43
6
47
100
43
13
0
86
-------
Table D-4.Percent survival of bluegill sac-fry exposed to various
temperatures for 96 hours. Eggs were incubated at 22,
26, or 34C until they hatched, and the newly hatched
larvae were transferred directly to test temperatures.
22C stock N=12 larvae per test temperature
Test
Temp . (C)
9
11
12
13
14
15
16
17
19
22
25
27
28
29
30
31
32
33
35
37
0
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
Time
24
100
100
92
83
50
92
75
83
67
50
25
58
42
83
83
67
50
25
42
42
(hrs . )
48
0
25
83
17
25
50
58
67
25
50
8
58
42
83
50
67
50
17
0
0
72
0
0
0
8
33
58
50
8
25
0
42
42
83
50
50
25
8
-
96
_
_
0
0
50
25
8
17
-
17
25
83
42
50
17
0
-
-
26C stock N=5 larvae per test temperature
8
12
16
34
38
100
100
100
100
100
100
80
100
100
0
100
20
100
100
0
20
100
80
-
-
10
80
80
-
34C stock N=10 larvae per test temperature
14
16
18
20
22
24
38
40
42
100
100
100
100
100
100
100
100
100
0
40
90
90
90
80
80
0
0
10
70
90
90
80
10
-
-
10
70
90
80
80
0
-
0
70
70
80
80
-
-
_
87
-------
Table D-5.'Percent survival of fry, reared to the swim-up stage
at 22C and transferred directly to various test tem-
peratures. (N=20 fry per test temperature)
Test
Temperatures
Percent Survival at Time(hrs)
24 48 72
96
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
.31
32
33
34
35
36
38
39
40
42
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
0
0
0
0
20
60
100
55
55
70
50
50
45
55
50
45
50
50
45
30
40
0
40
30
15
60
0
25
5
0
0
0
0
0
0
0
60
100
50
50
70
50
50
40
55
50
45
50
50
45
30
40
_
30
30
15
40
_
5
5
0
_
_
_
-
-
-
50
100
45
50
40
50
45
40
55
50
35
50
50
40
25
20
_
25
25
15
0
_
0
5
0
_
_
_
-
_
15
100
45
50
30
50
45
40
55
50
35
35
20
35
5
20
0
0
0
_
_
0
88
-------
Table D-6. Instantaneous rates of growth (weight), mortality, and
change in population biomass of juvenile bluegills, pre-
viously acclimated to four stock temperatures. Instant-
aneous rates (Ricker, 1958) were multiplied by 100 to
obtain percent change.
Stock Acclimated to 12.1C
Mortality [Replicates Combined)
Test Temp
Mean±95%CL
3.0 ± 0.40
3.9 ± 0.56
4 9 ± 0.26
6.0 ± 1.21
6.9 ± 1.48
7.0 ± 1.07
7.9 ± 1.96
8.0 ± 1.98
12.0 ± 0.68
12.0 * 0.67
21.7 ± 0.80
21.6 ± 1.56
24.1 ± 0.46
24.0 ± 0.15
26.0 ± 0.23
26.0 ± 0.41
27.0 ± 0.34
27.0 ± 0.30
27.9 ± 0.25
27.9 ± 0.32
29.0 ± 0.22
28.8 ± 0.47
Repli-
cate
-
-
-
-
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
No . Temp
Readings
284
333
425
541
541
541
541
541
541
541
587
587
404
404
577
577
337
337
577
577
433
433
1
18.95
7.77
5.79
7.85
7.62
9.09
9.92
5.76
13.24
5.73
6.90
10.01
10.09
Week No.
2
26.76
9.86
7.56
2.07
1.15
0.46
0.00
0.00
1.48
4.08
1.05
0.00
3.03
3 4
*
*
4.41 *
0.00 *
0.00 *
0.00 *
o.oo *
0.00 *
*
18.80 *
*
0.00 0.00
15.32 *
Mean
-22.86
- 8.82
- 5.92
- 3.31
- 2.92
- 3.18
- 3.31
- 1.92
- 7.36
- 9-54
- 3.98
- 2.50
- 9.48
**
Growth
4.83
1.59
3.02
0.16
1.21
0.77
0.06
3.40
2.51
4.02
2.05
2.44
3.79
Biomass
Change
-18.03
- 7.23
- 2.90
- 3.15
- 1.71
- 2.41
- 3.25
+ 1.48
- 4.85
- 5-52
- 1.93
- 0.06
- 5.69
Based on an initial mean weight of 0.38 t 0.55 gm; N=105
All remaining fish were removed during the previous sampling period
89
-------
Table D-6. (cont'd)
Stock Acclimated to 19.OC
Mortality (Replicates Combined)
Test Temp
Mean+95%CL
5.8 +
6.0 +
7.0 +
7.0 +
8.0 +
8.0 +
9.0 +
9.0 +
.10.0 +
10.0 +
19.0 +
19.0 +
27.0 +
27.0 +
27.9 +
28.0 +
29.0 +
28.9 +
30.0 +
30.0 +
31.0 +
30.9 ±
31.9 +
32.0 +
32.9 +
32.8 +
1.16
0.22
C.45
0.23
0.85
0.82
0.80
0.82
1.00
0.33
0.41
0.39
0.30
0.28
0.28
0.28
0.26
0.40
0.30
0.39
0.40
0.40
0.39
0.40
0-35
0.59
Repli-
cate
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
No . Temp
Readings
263
263
643
643
878
878
878
878
901
901
911
911
669
669
669
669
669
669
669
669
669
669
577
577
601
601
1
21.
10.
0.
1.
4.
1.
2.
4.
0.
0.
3.
2.
7.
13
44
41
61
21
30
25
02
27
55
25
61
18
Week No.
2 3
5.
0.
0.
0.
0.
0.
2.
0.
2.
5.
10.
3.
2.
47
88
00
42
00
41
26
47
04
82
33
11
70
*
1.88
0.00
0.00
0.00
0.00
1.80
4.80
1.96
3.68
3.65
3.76
12.61
4
o.oo
0.00
0.73
0.00
0.00
0.00
2.61
3.58
10.38
0.00
2.36
16.22
5 Mean
13
* 3
* 0
0.00 0
0.00 0
0.00 0
1
* 2
* 1
* 5
* 4
* 2
* 9
.30
.30
.10
.55
.84
.34
.58
.98
.96
.11
.31
.96
.68
**
Growth
4.24
3.21
0.80
-0-54
0.87
1.86
3.94
4.14
3.74
4.17
5.53
3.92
6.37
Biomass
Change
- 9.06
- 0.09
+ 0.70
- 1-09
+ 0.03
+ 1.52
+ 2.36
+ 1.16
+ 1.78
- 0.94
+ 1.22
+ 0.96
- 3.31
** Based on an initial mean - weight of .25 + 0.31 gm; N=115
* All remaining fish were removed during the previous sampling period
90
-------
Table D-6. (cont'd)
Stock Acclimated to 26.OC
Mortality (Replicates Combined)
Test Temp
Mean+95%CL
10.0 +
10.0 +
11.0 +
11.0 +
12.0 +
12.0 +
13.0 +
13.0 +
26.1 +
26.0 +
33.9 +
34.0 +
35.0 +
35.0 +
36.0 +
35.9 +
0.36
0.48
0.41
0.38
0.38
0.43
0.32
0.37
0.28
0..44
0.47
0.38
0.94
0.84
1.78
2.11
Repli-
cates
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
No . Temp
Readings
680
680
973
973
973
973
973
973
985
985
649
649
649
649
335
335
1
5.
3.
2.
1.
0.
3.
6.
8.
17
01
63
52
92
14
94
56
2
5.88
0.64
0.00
0.00
0.89
0.67
1.10
1.13
Week No.
3
2.90
U.OO
0.00
0.00
1.10
2.98
0.00
16.48
2
n
0
0
0
1
4
4 5
.10 *
.00 d.OO
.46 0.00
.00 0.00
.00 3.65
.08 *
.86 *
*
6 Mean
4
0.00 0
0.00 0
0.00 0
3.52 1
1
3
8
.01
.61
.52
.25
.68
.97
.22
.72
**
Growth
1
0
0
0
3
3
3
0
.34
,55
.44
.17
.69
.81
.56
.55
Biomass
Change
- 2.67
- 0.06
- 0.08
- 0.08
+ 2.01
+ 1.84
+ 0.34
- 8.17
** Based on an initial mean weight of .23 + 0.29 gm; N=38
* All remaining fish were removed during the previous sampling period
91
-------
Table D-6. (cont'd)
Stock Acclimated to 32. 9C
Mortality (Replicates Combined)
Test Temp
Mean+95%CL
15.9
16.0
17.0
17.0
17.9
18.0
19.0
19.0
20.0
20.0
32.9
33.0
35.9
35.5
37.0
37.0
± o.
+ o.
± o.
± o.
+ o.
+ 0.
+ 0.
± 0-
+ 0.
+ o.
+ o.
+ 0.
+ 1.
+ 1.
+ 0.
± o.
31
26
27
31
35
25
38
18
29
33
36
48
27
62
51
62
Repli-
cates
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
No . Temp
Readings
955
955
1006
1006
1006
1006
1006
1006
1006
1006
985
985
658
658
335
335
2
2
1
1
1
2
12
8
1
.14
.54
. 32
.42
.31
.37
.49
.39
2
0.
1.
0.
0.
0.
1.
1.
8.
00
23
00
60
00
65
05
98
Week No.
3 4
0.00
0.52
0.84
0.00
0.00
0.76
0.00
*
0.
0.
0.
0.
0.
2.
3.
00
55
00
00
00
47
98
0
0
0
0
1
5
5
.00
.00
.00
.00
.15
.08
*
6
0.00
0.00
0.00
0.00
0.00
0.00
Mean
0.
0.
0.
0.
0.
2.
4.
8.
36
81
36
34
41
06
38
68
**
Growth
0
0
0
0
0
1
0
0
.38
.03
.59
.81
.56
.06
.66
.39
Biomass
Change
+ 0.02
- 0.78
+ 0.23
+ 0.47
+ 0.15
- 1.00
- 3.72
- 8.29
** Based on an initial mean weight of 0.29+0.39 gm; N=28
* All remaining fish were removed during the previous sampling period
92
-------
0
-3
6
9
12
15
18
21 -
24
0
o
8
7
6
5
3
2
+1
0'
O
+3
0
-3
1/1 D
O
Figure
T
"'IcT
15 20 25
TEMPERATURE °C
10 15 20 25
TEMPERATURE °C
' ' ' '35' ' ' '40
30
• i '
35
40
6.
9-
12-
15-
18.
21-
(
/
/
/
/
) 5 10 15 20 25 30 35 40
TEMPERATURE °C
STOCK ACCLIMATED TO 12.1°C
D1. Instantaneous rates of mortality, growth and change in population
biomass for juvenile bluegills
93
-------
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Figure Dl. continued
94
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Figure Dl. continued
95
-------
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(— T-T-T f-|- i i i i | i i i i | l l i i | 1 i 1 i | . l " « | I i i r t i « i '1
) 5 10 15 20 25 X 35 40
TEMPERATURE °C
^
) 5 10 15 2*0 25 & 35 40
TEMPERATURE °C
X
x\
\
\
) 5 10 15 20 25 X 35 -40
TEMPERATURE °C
STOCK ACCLIMATED TO 3?.9°C
Figure Dl. continued
96
-------
Juvenile stocks were introduced into the experiment on 15 September, 1971
(26.OC and 32.9C); 23 September, 1971 (19.OC); and 7 October, 1971 (12.1C),
All experiments were terminated on 26 October, 1971 due to a bacterial dis-
ease which may be a reflection of other sources of stress predisposing the
fish to infection. Fish that were acclimated to 12.1C prior to testing,
were maintained for three weeks in the growth experiment. The 19.OC stock
was exposed to test temperatures for five weeks, while the 26.0 and 32.9C
stocks were exposed to test temperatures for six weeks.
For the 12.1C stock, mean mortality rates were relatively high at all, tem-
peratures. Mortality was highest at the extreme low temperature (22.86%/
day at 3.0C) and at 26.OC (9.54%/day). Minimum mortality (1.92%) occurred
at 21.6C. Apparent growth rates were high at extreme temperatures. Ju-
venile bluegills showed an apparent increase in weight of 4.83%/day at
3.0C and 3.79%/day at 28.9C. Initial samples indicated that weight was
quite variable within the population. Ten animals did not constitute an
adequate sample. Growth rates were lowest (0.06%/day to 1.21%/day) between
6.0 and 12.0C and highest (2.05%/day to 4.02%/day) between 21.6 and 28.9C.
Net change in biomass was negative at all temperatures except 21.6C. No
population could survive with negative biomass gain and hence this result
must be considered atypical, reflecting stress from crowding and low water
quality.
The stock that was acclimated to 19.OC showed maximum mortality at extreme
low and extreme high temperatures (13.30%/day at 5.9C and 9.68%/day at
32.8C). Juveniles again showed an apparent increase in weight at extreme
temperatures (4.24%/day at S.9C and 6.37%/day at 32.8C). Growth rates were
least between 8.0C and 10.OC (-0.54 to 0.87%/day). Net change in biomass
was low (-9.06 to 0.70%/day) at 10.0 or less, 30.OC and 32.8C. Bluegills
maintained at temperatures of 8.0, 10.0 to 29.0, 30.0, 31.0 and 32.OC show-
ed a net increase in biomass. Maximum rate of biomass increase was 2.36%/
day at 27.OC.
The stock that was acclimated to 26.OC had maximum mortality at 36.OC
(8.72%/day) and at 10.OC (4.01%/day). Change in weight was 1.34%/day at
10.06. Minimal growth (0.17 to 0.55%/day) occurred at 11.0 to 13.OC and
at 36.OC. Maximum growth was 3.81%/day at 34.OC. Net change in biomass
was 0.06% to 2.67%/day at temperatures of 10.0 to 13.OC. Rate of biomass
change was negative (-8.17%/day) at 36.OC and was positive (0.34%/day) at
34.OC. The highest rate of biomass increase was 2.01%/day at 26.OC.
The stock that was acclimated to 32.9C showed highest mortality (2.06 to
8.68%/day) at 33.0 to 37.OC. Mortality was low (0.34 to 0.81%/day) at
temperatures from 16.0 to 20.OC. Maximum growth rate was 1.06%/day at
33.OC. Lowest growth rates were 0.03%/day at 17.0C, 0.38%/day at 16.OC,
and 0.39%/day at 37.OC. Net change in biomass was negative (-0.78%/day)
at 17.OC and positive (0.17%/day) at 18.OC. At_high temperatures (33.OC
and above) the rate of biomass change was negative.
Changes in body length' with time (Figures D-2 to D-5) did not show a con-
sistent pattern of response with increasing temperature. Some error due
to. sampling was present, since at several test temperatures, fish showed
a decrease in body length (Figure D-2b).
97
-------
40
-7 °°
Z. Q£
- £ 35 \
X LU
II
30-
29
0
401
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x
oo
35"
30 H
29
0
3.0'
24.0°
27.0°
14
DAYS
26.0
27.9
28
6.0°
14
DAYS
28
Figure D2C Growth \n length of juvenile bluegills. Fish were
acclimated to 12.1°C and then exposed to various test
temperatures.
98
-------
32.8C
31.0
29.0°
28.0°
30.0°
27.0°
19.0
0
DAYS
Figure D3. Growth in length of juvenile bluegills. Fish were acclimated to
19.0°C and then exposed to various test temperatures.
-------
40 -
t/>
35
II
30 •
25
Zoo
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LU
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il
30 i
25
34.0"
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14
28
DAYS
26.0
14
28
DAYS
42
42
Figure D40 Growth in length of juvenile bluegills. Fish were acclimated to
26.0° and then exposed to various test temperatures.
100
-------
25
0
33.0
DAYS
25
0
42
Figure D5. Growth in length of juvenile bluegills. Fish were acclimated
to 32.9 C and then exposed to various test temperatures.
101
-------
For fish acclimated to 12.1C (Figure D-2) and exposed to test temper-
atures at the upper limits of survival, change in length was maximal
at 21.6 during the first two weeks. Increase in length was greater
at 21.6 and 26.OC than at 27.9 or 28.9C. Growth was least at 24.0 and
27.OC. At the lower thermal limits, fish acclimated to 12C increased
in length for the first two weeks, but decreased in length at 7.0, 8.0,
and 12.OC during the second two weeks.
The stock that was acclimated to 19.OC (Figure D-3) showed an increase
in length for all temperatures at the upper limits of survival except
for the first sample taken at 32.OC. Growth rate increased with increas-
ing temperature except for animals tested at 30.0 and 32.OC. At the low-
er temperatures, changes in length were erratic. Two replicates at 19.0
reached the same size after 42 days, but the time-course of growth was
distinctly different.
The stock that was acclimated to 26.OC (Figure D-4) increased more rapid-
ly in length at 26.OC than at the higher temperatures tested. At low
temperatures, the stock that was acclimated to 26.OC showed no apprecia-
ble increase in length.
Fish acclimated to 32.9C (Figure D-5) prior to the growth test increased
more rapidly in length at 33C than at 35.7 or 37.OC. Animals exposed to
low temperatures increased consistently in length at 19.0, 18.0 and 20.OC,
while animals at 16.0, and 17.0C showed a decrease in length at one or
more sampling intervals.
DISCUSSION
Results of the preliminary bioassay tests indicated several problems with
the life support capabilities of the thermal test facility, and sampl-
ing for the juvenile growth study.
The preliminary incubation test was not considered adequate to define
upper and lower TL50. Egg viability was low. The percent normal hatch
was 5% at 26C and lower TL50 was not defined for replicate B because less
than 50% of the eggs hatched at all temperatures below 30C. Comparison
of the two replicates indicated large differences in TL50 and tempera-
ture for optimal hatch that could not be accounted for by the experimental
design. Finally, temperature control in the experimental chambers was not
adequate. In spite of the large differences in optimal temperature be-
tween replicates, and TL50 range for the two replicates was quite similar,
which agrees with observations by Hokanson, et al, 1973.
Poor egg viability and variation in hatching may have been due to poor
water quality, improper fertilization techniques, differences in handl-
ing by personnel or variable tempering rates to test temperatures (see
Discussion in main report). This variability, however, was reduced during
the subsequent incubation tests.
102
-------
Preliminary experiments with bluegill fry indicated that mortality rates,
especially at optimal temperatures, were too high to attribute to the
effects of temperature alone. Poor water quality (see main report), poor
feeding, or improper handling techniques contributed significantly to
fry mortality.
Bluegill sac-fry exhibited a slightly greater range of thermal tolerance
than eggs. Sac-fry leave the nest as swim-up fry approximately four
days after hatching at 23C and require an exogenous food supply within
six days after hatch. The period between initial swim-up and and yolk-sac
absorption is the "critical period" (Toetz, 1966). Thermal tolerance
of bluegill swim-up fry should be intermediate between tolerance of sac-
fry and tolerance of juveniles. However, an increase in temperature
increases metabolic rate, and may shorten the critical period for swim-
up fry, and intensify the need for these fry to have adequate food at
the proper time. For these reasons, fry should be fed frequently (at
least 4 to 5 times per day) during the critical period.
During the juvenile growth test, the span of temperatures tested was not
adequate to define an optimal range or optimal temperature, but was suf-
ficient to eliminate some of the extreme temperatures. However, the 12C
and 33C stocks showed high mortality during the course of the experiment.
Growth rates for most stocks were low.
Measurements of instantaneous growth showed large increases in weight
at low temperatures and negative growth at IOC for the 19C stock. These
results indicate some error due to sampling. The initial population was
quite variable in weight (0.1 to 2.1 gm) so that samples of 10 animals
were not representative. However, this type of sampling error does not
account for apparent weight increase at low temperatures for the 12, 19
and 26C stocks. The length data show that increase in weight at low
temperature is associated with an increase in body length. If animals
were not growing at extreme temperature, random error would produce as
many negative growth rates as positivie, and growth rates would not be
significantly different from zero. Large positive changes in weight
would occur at intermediate and high temperatures. The data suggests
instead that selective mortality of small juveniles was occurring at the
low temperatures. Animals that died of natural causes were not measured.
If the small animals died between sampling intervals, only the larger
animals remained to be sampled and measured at the next two-week interval.
The.32.9C stock should be retested at temperatures of 18 to 35C to define
optimal temperatures and optimal physiological range for the acestival
period when modification of thermal regimes by man's activity would be
most detrimental.
103
-------
APPENDIX E
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»U.S, GOVERNMENT PRINTING OFFICE: 1973 5U-156/333 1-3
-------
| I Access/on Number
w
n 1 Subject Field & Group
05C
SELECTED WATER RESOURCES ABSTRACTS
INPUT TRANSACTION FORM
Jj
Organization
Aquatic Sciences, Inc.
Boca Raton, Florida 33432
Title
THERMAL EFFECTS ON EGGS, LARVAE AND JUVENILES OF BLUEGILL SUNFISH,
10
Authorfe)
Banner, Arnold
Van Arman, Joel A.
16 I Pr°i°cl Doai&iatioa
EPA WQO Contract No. 14-12-913. Project No. 18050GAB
Note
22
Citation
Environmental Protection Agency report number,
EPA-R3-73-041, May 1973.
23
Desc'riptora (Starred First)
*Thermal stress, *sunfishes, *water temperature, fish reproduction, fish eggs, fish
larvae, fish growth stages, fish juveniles, temperature control, fish management,
fish kill, fish spawning, aquatic environment.
25
Identifiers (Starred First)
'Bluegill sunfish, fish spawning induction
27
Abstract Bioassay experiments were conducted to determine thermal tolerance of early life
history stages of bluegill sunfish. Bluegill eggs hatched at temperatures from 18 to 36C
during two incubation tests. Maximal hatch occurred at 22.2 and 23.9C. Lower TLcn tem-
perature for hatch of normal fry was 21.9C and upper TL$Q temperature was 33.8C.
Juvenile bluegills acclimated to 12.1C had a lower 96-hour TL50 of 3.2C and an upper 96-
hour TLcn of 27.5C. Juveniles acclimated to 32.9C had a lower 96-hour TLcn of 15.3C and
-50
an upper 96-hr
'50
of 37.3C. TL5Q increased with -increasing temperature of acclimation.
For juveniles acclimated to a given temperature, upper TL50 decreased with longer exposure
A preliminary test determined ranges of thermal tolerance for sac-fry and swim-up fry.
In another preliminary test, juvenile bluegills were acclimated to 12.1, 19.0, 26.0 or
32.9C, and reared at a series of test temperatures for three to six wks. to define opti-
mal temperature ranges for growth and survival.
Additional research determined conditions for the culture of Lepomis macrochirus, in-
cluding spawning induction, hatching, arid growth of larvae and juveniles.
This report was submitted in fulfillment of Project 18050-GAB, Contract 14-12-913, under
sponsorship of the Office of Research and Monitoring, Environmental Protection Agency.
Abmlnctor
J
oel Van Arman
Institution
Aquatic Sciences, Inc._
•m 102 IREV. juur 19691
SEND WITH COPY Or DOCUMENT, TO: <'• A T E R RESOURCES SCIENTIFIC INFORMATION CENTER
U.S. DEPARTMENT OF THE INTERIOR
. D. C. 20240
6PO: 1970 - 407 -att\
WASHING-TON.
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