WATER POLLUTION CONTROL RESEARCH SERIES 18050 EDP 12/71
The Use of Fish Movement Patterns
to Monitor Zinc
U.S. ENVIRONMENTAL PROTECTION AGENCY
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WATER POLLUTION CONTROL RESEARCH SERIES
The Water Pollution Control Research Series describes the
results and progress in the control and abatement of pollution
in our Nation's waters. They provide a central source of
information on the research, development and demonstration
activities in the Environmental Protection Agency, through
inhouse research and grants and contracts with Federal, State,
and local agencies, research institutions, and industrial
organizations.
Inquiries pertaining to Water Pollution Control Research
Reports should be directed to the Chief, Publications Branch
(Water), Research Information Division, R&M, Environmental
Protection Agency, Washington, B.C. 20^60.
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THE USE OF FISH MOVEMENT PATTERNS
TO MONITOR ZINC
John Cairns, Jr.
and
William T. Waller
Virgina Polytechnic Institute and State University
Biology Department and Center for Environmental Studies
Blacksburg, Virginia 24061
for the
ENVIRONMENTAL PROTECTION AGENCY
Project #18050 EDP
December 1971
For salo by the Superintendent of Documents, U.S. Government Printing Office, Washington, D.C. 20402 - Price 65 cents
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EPA Review Notice
This report has been reviewed by the Environ-
mental Protection Agency, and approved for
publication. Approval does not signify that
the contents necessarily reflect the view and
policies of the Environmental Protection
Agency, nor does mention of trade names or
commercial products constitute endorsement or
recommendation for use.
ii
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ABSTRACT
The feasibility of using fish movement patterns measured by light beam
interruption as a technique for continuous monitoring of the response
of fish to zinc was investigated. In conjunction with the monitoring
studies the growth and reproductive success of the B luegill sunfish
(Lepomis macrochirus) exposed to various fractions of the lowest
concentration of zinc detected by the monitoring apparatus were studied.
The monitoring apparatus does not in any way interfere with fish move-
ment within the test chamber and allows for the maintenance of fish for
long time periods. Under the conditions described the system detects
premortal aberrations in fish movement caused by zinc. The detection of
stress occurs in sufficient time to permit survival of the test fish if
stress conditions are reversed at the time of detection. The lowest
concentration of zinc detected by the system during a 96-hour exposure
was between 3.64 and 2,94 mg/1 Zn"*""1". The system's range of effective
measurement as related to turbidity is discussed. This method should
detect other toxicity equally well.
The growth and reproductive success of the bluegill was tested in
concentrations approximately equal to 1/10 and 1/100 the lowest
concentration of zinc detected by the monitoring system and 1/100 of
the 96 hour TL50 (median tolerance limit) determined under continuous
flow conditions. The growth and reproductive success in 1/100 the
lowest detected zinc concentration and 1/100 the 96 hour TL50 value
did not differ appreciably from the controls while a concentration of
approximately 1/10 the lowest detected zinc concentration in effect
eliminated reproduction in the bluegill.
iii
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CONTENTS
Section
I Conclusions
II Recommendations
III Introduction
IV Effects of Zinc on Fish Movement
V Growth and Reproductive Studies
VI Acknowledgments
VII Literature Cited
VIII Publications
Page
1
3
5
7
39
51
53
55
V
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FIGURE
Page
1 Block diagram of monitoring apparatus 9
VI
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TABLES
No. Page
1 Summary of general fish data: mean and standard deviation 11
of wet weight, standard length, and total length.
2 Chemical characteristics of dilution water. 13
3 Summary of statistical analyses of static experiments 1-4. 17
4 Summary of statistical analyses of static experiments 5-8. 19
5 Summary of statistical analyses of static experiments 9-10. 20
6 Summary of statistical analysis of continuous flow 22
experiment 11.
7 Summary of statistical analysis of continuous flow 23
experiment 12.
8 Summary of statistical analysis of continuous flow 24
experiment 13.
9 Summary of statistical analysis of continuous flow 25
experiment 14.
10 Summary of statistical analysis of continuous flow 26
experiment 15.
11 Summary of statistical analysis of continuous flow 27
experiment 16.
12 Summary of statistical analysis of continuous flow 28
experiment 17.
13 Summary of statistical analysis of continuous flow 29
experiment 18.
14-A Summary of statistical analysis of continuous flow 31
experiment 19; days 1-8.
14-B Summary of statistical analysis of continuous flow 32
experiment 19; days 8-10.
15-A Summary of statistical analysis of continuous flow 34
experiment 20; days 1-8.
VII
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No. Page
15-B Summary of statistical analysis of continuous flow 35
experiment 20; days 8-14.
15-C Summary of statistical analysis of continuous flow 36
experiment 20; days 14-20.
16 Zinc and dissolved oxygen concentrations, temperatures 43
in breeding tanks.
17 Survival of adult bluegills and weights, lengths and 44
condition of gonads of adults at end of breeding
experiment.
18 Spawning of adult bluegills and percentage hatch of 47
eggs at four zinc concentrations.
19 Survival and growth of bluegills in four zinc 48
concentrations.
VIII
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SECTION I
CONCLUSIONS
1. The monitoring system used in this study detects aberrations in
movement patterns of fish exposed to lethal and sublethal concentrations
of zinc.
2. The monitoring system does not detect aberrations in movement
patterns of bluegills exposed to concentrations of zinc which effectively
eliminate reproduction.
3. The system does not require direct contact with the fish being
monitored and therefore allows for the maintenance of the fish for
extended periods of time.
4. The monitoring system as described is limited in its applicability
to continuously monitor effluents unless turbidity is controlled.
5. The reproduction of bluegill sunfish under the conditions described
is effectively eliminated at a concentration of approximately .255 mg/1
Zn44".
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SECTION II
RECOMMENDATIONS
This laboratory study was designed to develop the equipment and
precedures necessary to test the hypothesis that fish movement patterns
could be used as a means of detecting premortal aberrations. This goal
was achieved, and it is recommended that further laboratory studies
using different toxicants and or combinations of stressors be carried
out to refine the techniques developed.
The relationship between the removal of suspended solids and toxicity
should be studied to determine if extending the effective range of
measurement of the instrument by removing the solids in turn alters
the predictability of the system.
In depth statistical evaluations should be undertaken to determine if
the sensitivity and speed of response through analysis can be facilita-
ted.
It is recommended that when growth and reproductive studies are
undertaken utilizing fish of the size range used in these experiments
that larger containers be employed to house the spawning fish. The
container should probably be at least twice the length of the tanks
employed in these studies.
In growth and reproductive studies several duplicates of each
concentration should be simultaneously analyzed to insure that the
apparent inherent biological differences in behavior and reproductive
potential are not interpreted as absolute differences due to the
toxicant being studied.
In these studies, contrary to that reported elsewhere, spawning was
not generally confined to a certain time interval within the photo-
period. Therefore checking the nests twice a day once in the morning,
and once in the evening is recommended as a means of eliminating the
loss of some spawnings due to hatching occurring prior to the time of
nest checking.
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SECTION III
INTRODUCTION
As the pollution problems associated with an expanding industrial base
and increasing population size become more acute, the need for an over-
all program of ecosystem management to minimize the adverse effects of
man's activities on the environment must be developed.
The report of the Council on Environmental Quality (1970) repeatedly
stresses the need for the development of predictive, simulative and
management capabilities to combat air and water pollution. These tech-
niques must be developed if the concept of multiple use set forth in the
Water Quality Act of 1967 (Public Law 89-234) is to succeed and if the
present practice of alteration without comprehension is to be eliminated.
A great deal of progress has been made in determining biologically safe
concentrations for fish (Mount and Stephan, 1967; Mount, 1968; Water
Quality Criteria, 1968; Sprague, 1969; Brungs, 1969; and Eaton, 1970).
There remains, however, at least one critical area of effluent quality
control which must be effectively monitored before the standards for
chronic exposure of fish to toxicants will prove effective. This
protection should include safeguards against the development of acutely
toxic conditions resulting from either industrial or municipal accidents
or from changes in the total environmental variation and industrial
processes.
If an industry conforms to the predetermined standards for chronic
exposure 363 days out of the year and over a period of two days due to
human error or a combination of human error and changes in the physical
and chemical characteristics of the receiving stream acutely toxic
conditions develop which are not detected in time to prevent deleterious
conditions from developingthen the standards for chronic exposure
alone can not protect the receiving stream. Also an industrial or
municipal effluent could foreseeably conform to the standards for chronic
exposure all the time and still not insure against the development of
acutely toxic conditions. For example, if an upstream industry changed
processes resulting in the release of a compound, at a predetermined
biologically safe concentration, which interacted synergistically with
an effluent from a downstream industry, also released at a predetermined
biologically safe concentration for fish, the result could be not only
chronic stresses but the development of acutely toxic conditions. The
standards for chronic exposure without sufficient protection against
the development of acutely toxic conditions will not maintain a vigor-
ously functioning aquatic ecosystem.
Continuous physical and chemical monitoring systems provide a partial
answer to this problem in that they may be used to detect almost
instantaneously a single environmental variable which has reached a
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lethal level. However, aquatic organisms respond to the collective
effect of the environmental factors and these effects can not be
predicted from the chemical-physical analyses alone without direct
feedback from the organisms.
Cairns (1970) and Cairns (in press) presents a plan for a systems
approach to aquatic ecosystem management on a regional basis. In order
to implement this plan both instream and inplant systems for continuous
biological monitoring must be developed (Shirer, et al^., 1968; Cairns,
e^al., 1970).
The objective of this study was to determine the feasibility of using
fish movement patterns monitored by light beam interruption as a
technique to detect premortal aberrations.
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SECTION IV
EFFECTS OF ZINC ON FISH MOVEMENTS
Three species of fish were tested under varied environmental conditions
in a total of twenty experiments to determine the feasibility of using
fish movement patterns as a means of detecting acute and chronic expo-
sure to pollutants.
Monitoring Apparatus; The design philosophy of the monitoring apparatus
was based on technical simplicity and reliability. Common five-gallon
tropical fish aquaria of about 41 cm length, 21.5 cm width, and 26 cm
height were used as the test tanks. Basically the monitoring units
consisted of light beams which were arranged to traverse the length of
the tanks near the bottom, middle, and just below the surface (Figure 1).
Photoresistors at the opposite end of the tanks were illuminated solely
by the lamp sources with the aid of collimating baffles. Interruption
of the light falling on the photoresistors operated relays, via two-
transistor amplifiers, which in turn advanced counters and deflected
pens of an event recorder. Six tanks, each with three light beam sen-
sors, were used in each experiment. For a detailed description of the
circuitry see Shirer, Carins, and Waller (1968).
Static Tests; Ten of the twenty experiments analyzed were carried out
under static test conditions. In these experiments the test tanks were
housed in a light-tight plywood chamber 2 x 1 x 2.5 meters. Ambient
illumination within the chamber was provided by a pair of 40 Watt
fluorescent tubes cycled daily for 12 hours darkness and 12 hours light
with a time switch.
Each tank, filled to a calibrated 17 liter mark, was equipped with an
air diffusion stone housed in a perforated plastic cylinder. The
plastic cylinder was located midway along the length of each tank and
contained the rising air bubbles. The cylinder served to eliminate any
erroneous signals which might have resulted from bubbles breaking the
light beam.
Synthetic dilution water was used for all static tests (Scheier and
Cairns, 1966). The chemical characteristics of the synthetic dilution
water are based on determinations made at the beginning and end of each
experiment. Dissolved oxygen was 7.5 mg/1 for all determinations.
The pH was always highest at the beginning of each test ranging from
7.0 - 7.8. For any given tank pH did not decrease by more than 0.5
during any experiment. Temperature always increased during an experi-
ment. Temperature ranged from 20 C - 25 C. Initial and final hardness
and methyl orange alkalinity values were constant for both factors and
were 51 mg/1 as CaC03 and 68 mg/1 as CaCO^ respectively.
Test Organisms; Two species of fish, the golden shiner Notemigonus
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Figure 1
Block diagram of
monitoring apparatus
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Fluorescent light
Lamps
Tank
Photoresistors
Time
switch
Counters Event recorder
ers
II II II
i
II 1 1 II
II 1 1 II
-
T
t
f
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crysoleucas (Mitchell) and the goldfish Carassius auratus (Linnaeus),
were tested under static conditions. Because of the difficulty in main-
taining stocks of golden shiners under static test conditions only a
single experiment using this species was completed. Test fish were
acclimated to experimental dilution water, temperature, and photoperiod
for a period of at least two weeks prior to their use in an experiment.
During this period they were fed a commercial food preparation (Tetra-
Min) ad libitum twice daily. The test fish were not fed during an
experiment.
Two days prior to the beginning of an experiment a single test fish was
placed in each of the six test chambers and allowed to readjust after
handling. After the two-day readjustment period recording was started
at 6:30 a.m. and continued for eight days. At 6:30 a.m. on the fifth
day the calculated concentration of toxicant was added in solution to
each of the experimental tanks; the control received the same volume of
dilution water minus the toxicant. The gradual addition of the toxicant
was accomplished by adding the solution via plastic tubing to each tank.
Mixing was quite rapid due to the air driven circulation of tank water.
At the beginning of the recording period the counts registered on the
electric counters for each of the three photocells in a single tank were
recorded in a notebook. Subsequent records were made every three hours
throughout the test period, except for a daily interval from 12:30 a.m.
to 6:30 a.m. during the dark cycle of the photoperiod. Cummulative move-
ment for the desired interval was obtained by subtracting two successive
readings. Upon completion of an experiment, total length and wet weight
was recorded for each fish (Table I). All tanks and equipment were washed
in EDTA between experiments.
Continuous Flow Experiments; Initial analyses of the movement patterns
recorded in the static tests revealed a high level of variance. Because
of the high level of variance and because continuous flow testing more
realistically approximated the conditions under which this apparatus
would be used, a second series of ten experiments using the bluegill
Lepomis macrochirus (Rafinesque) as the test species were performed. All
procedural changes incorporated in the continuous flow tests were done
in an attempt to increase reliability and reduce variance.
Monitoring Apparatus; The only change made in the monitoring apparatus
was the addition of red 650 mu filters to each of the light paths.
Although one can not say that visual detection of the beams would be
impossible at this level, the lighting conditions would provide reason-
able "darkness" i.e., at a level which would not exceed moonlight nights
(J. R. Brett, personal communication).
All movement experiments were carried out in an isolation room. The
isolation room was designed to reduce the effects of noise produced by
normal laboratory traffic. The experimental and stock tanks housed in
the isolation room were placed on a 35cm deep sand bed.
10
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TABLE 1. SUMMARY OF GENERAL FISH DATA
Expt. Weight
Number (grams)
Mean St. Dev.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
3.4
5.4
5.0
3.2
4.5
4.6
5.3
4.1
3.5
3.9
14.2
15.5
19.6
12.3
13.0
12.3
11.5
12.4
14.3
13.9
0.258
0.854
1.162
0.337
1.001
0.656
0.579
0.544
0.571
0.520
3.023
1.778
2.377
2,053
3.172
2.851
2.317
2.180
1.989
2.234
Standard Length
(cm)
Mean St. Dev.
~~
-
, ~
5.5
4.9
4.8
4.9
7.7
8.0
8.7
7.4
7.4
7.6
7.4
7.5
7.9
8.1
,
w
-,
..
0.213
0.233
0.242
0.147
0.432
0.273
0.297
0.322
0.561
0.450
0.404
0.460
0.281
0.368
Total Length
(cm)
Mean St. Dev.
7.9
7.9
8.2
7.2
7.7
7.9
8.2
8.2
7.3
7.6
10.2
10.6
11.3
9.7
9.5
9.9
9.7
9.6
10.2
10.3
0.568
0.531
0.564
0.334
0.606
0.206
0.459
0.729
0.278
0.524
0.567
0.350
0.312
0.436
0.739
0.588
0.470
0.563
0.337
0.444
11
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Two pieces of lucite, painted black, were cemented to the side and bottom
of each 17-liter stock and experimental tank to form the top and outside
wall of a 14 cm long by 6 cm open chamber. It was hoped that the chamber
would provide a refuge for the fish when stressed, thereby magnifying the
changes in normal movement patterns.
Both isolation rooms and the stock and growth and reproduction room were
maintained on the same simulated dawn-dusk photoperiod. A motor driven
dimming unit was used to increase light intensity at "sunrise" from zero
intensity to a maximum intensity of 100 foot candles, measured at the
water's surface of the experimental tanks. The increase in light inten-
sity from zero to full intensity occurred over a thirty-minute period.
The reverse sequence occurred during the simulated sunset, Forty-Watt
vita-lite fluorescent tubes provided the ambient illumination.
Dechlorinated municipal tap water flowed by gravity from a 500 gallon
reservoir to two smaller 189-liter reservoir. The smaller constant head
reservoir served as the immediate source of water for the movement exper-
iments, and final temperature adjustments were accomplished at this point.
Routine chemical analyses were performed daily on the water in the 189
liter reservoir (Table 2). Two modified Harvard 1210 variable speed
peristaltic pumps were used to provide flow to the experimental tanks.
Three tanks were serviced by each pump. A single hose for each tank was
anchored in the 189-liter reservoir five cm beneath the water surface.
The water was pumped up through a glass stopcock and flowmeter after
which the line split and passed through the head of the peristaltic pump.
After passing through the pump the line was rejoined and continued to a
small mixing vessel before continuing to the appropriate experimental
tank. The continuous flow line to each tank entered its respective tank
in one corner, the flow being released approximately one centimeter from
the bottom of the tank. A 0.64 cm effluent port was drilled through the
glass wall of each tank at the opposite end and side of influent water.
The effluent port was drilled at the 17-liter mark in each tank. The
effluent water for each tank was returned to the outside of the isolation
room for sample collection and if not used for that purpose was dis-
charged .
Flow rates were determined twice daily by collecting the effluent from
each tank five times for one-minute intervals. The mean flow rate for
this period was calculated and the flow to each tank adjusted accordingly.
An attempt was made to maintain all flow rates at 100 mls/min.
Toxicant introduction was accomplished by switching from the 189-liter
constant head reservoir to two 95-liter constant head containers in
which a calculated concentration of reagent grade ZnSO^^I^O had been
mixed. Care was taken to avoid temperature differences between the
regular dilution water and the batch mixed zinc solutions. The zinc
analyses x*ere determined using atomic absorption spectrophotometry made
at least twice daily. The initial measurement was made after nine hours
to insure that 95 - 99% particle replacement had occurred within the
test chambers. At the conclusion of each experiment the tanks and glass-
12
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TABLE 2. CHEMICAL CHARACTERISTICS OF DILUTION WATER.
Water Characteristics
Continuous Flow
Experiments
Temperature ( C)
PH
Total Hardness (mg/1 as CaCO )
M.O. Alkalinity (mg/1 as CaCO.,)
Number
of
Analyses
396
397
394
393
Mean
19.7
7.8
51
41.3
S.D.
1.79
0.26
10
8.8
Dissolved Oxygen 7.5 in all cases
Chlorine not detectable unless otherwise
noted
13
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ware were washed with EDTA and all tubing was replaced.
Water from the 189 liter reservoir also flowed into the stock and growth
and reproduction room where it was pumped to the elevated constant head
reservoir. Temperature adjustments were made at this point before the
water flowed by gravity to the five 189-liter stock tanks. Water also
flowed from the elevated constnat head box back into the isolation room
where it was distributed to the six 17-liter stock tanks.
Fish; The bluegill stock used in these experiments were obtained by
seining a pond located on the property of the Veterans Administration
Hospital in Salem, Virginia. In the laboratory the fish were maintained
under continuous flow conditions for a period of at least two weeks
before being moved to isolation room two. During acclimation fish were
fed Gordon's formula (Axelrod, 1952) once daily between the hours of
8:00 a.m. and 5:00 p.m. The exact hour of feeding was determined from
a random number table. This was done in an attempt to eliminate the
effect of anticipated feeding which may have developed had the fish been
fed at the same time each day (Davis and Bardach, 1964). Experiments 19
and 20 represent the only tests in which fish were fed. The methods for
bioassay fish (A.P.H.A., 1965) were adhered to as closely as possible.
Six test fish were moved from the community stock tanks and placed one
fish per tank in the 17-liter stock tanks located in isolation room two.
The fish were acclimated to conditions in these tanks for a period of at
least two weeks before being used in an experiment. Four days prior to
the beginning of an experiment the six fish located in the 17-liter
stock tanks were transferred to the experimental tanks. After a four-day
readjustment period recording was started and continued for a period of
eight days. At the beginning of the fifth day of recording the flow was
switched from the 189-liter constant head reservoir to the constant head
batch mixed zinc reservoirs.
Data recording was facilitated by the use of a 35 mm Ricoh Auto Shot
spring advance camera. The camera was tripped by a solenoid activated
by a time switch. Pictures of the electric counters (Figure 1) were
taken at hourly intervals throughout each 24-hour period except for the
hours divided by the simulated sunrise and sunset. During this period
pictures were taken on the half hour. At the end of an experiment the
fish were killed and wet weight, standard length, and total length
measurements were taken (Table 1).
Statistical Analyses; The statistical test used to analyze the fish
movement patterns was a two-sample test for homogeneity of variance. A
computer program written by Sokal and Rohlf (1969) was used to facilitate
handling the large volume of data. In all analyses each fish served as
its own control. For the experiments carried out under the static test
conditions day to day comparisons were made by using the six time inter-
vals for which records were made to obtain an estimate of the variance
in movement patterns for a given day. The variance for the data recorded
during day 1 was then compared to the variance recorded for day 2, the
14
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the largest variance value being divided by the smaller value. If the
test for homogeneity of variance was not significant the fish was not
considered to be showing abnormal movement patterns and the variance
estimate for day 2 was then tested for homogeneity against the variance
for day 3. If at any time the test for significance indicated hetero-
geneity the data for the last recorded day was dropped and the preceding
day was compared to the variance estimate for the next day. For example,
if the test for homogeneous variance between day 2 and day 3 indicated
the variances were heterogeneous, the variance estimate for day 3 was
dropped from the analysis and day 2 was compared to day 4. An analysis
showing heterogeneity was considered to indicate abnormal changes in the
movement patterns.
The data from the continuous flow tests were treated in the same manner
except the time interval for data collection was reduced from the day
to day comparisons in the static stest to comparisons made four times
during a 24-hour period. The level of significance used for all tests
was = .002.
Results and Discussion; xhe results presented here represent a progres-
sion from relatively crude static tests to more sophisticated continuous
flow tests. The results do not include fifteen tests which actually
formed the basis for the methods employed in the tests reported. The
preliminary studies included experiments in which more than one fish per
test chamber was used, more than three photocells per test chamber were
used, and various periods of acclimation were investigated as well as
different photoperiod regimes.
The criterion used to determine abnormal movement patterns was a positive
test for heterogeneity. The definition of "stress detection" is arbi-
trary and is based on the results obtained, rather than some pre-con-
ceived idea as to what might constitute "stress" in terms of fish move-
ment patterns as monitored by light beam interruption. For example, the
results presented in Tables 3-15 represent a series of experiments in
which the total movement of the fish (the sum of the light beam inter-
ruptions for the three photocells in a given tank) was used to obtain an
estimate of the variance used in the statistical test for heterogeneity.
Based on the results obtained from these experiments the definition of
"stress detection" is the occurrence of two or more positive tests for
heterogeneity (abnormal movement) during the same time interval.
Since light beam interruptions are recorded for three different photo-
cell levels within each tank there exists seven possible combinations of
photocell levels on which the variance estimates used to determine
abnormal movement can be based. The results presented here are based
entirely on the sum of the light beam interruptions for all three photo-
cells in a given tank. The advantages or disadvantages obtained from
analyzing the light beam interruptions from the three levels in all
possible combinations has not as yet been established but because there
is an observable tendency for bluegills under stress to increase activity
at the tops of the test chambers, the continuous flow data have been
15
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analyzed using light beam interruptions recorded for the top photocells
in each tank. The analyses in which the light beam interruptions re-
corded for the top photocells alone were used agree in general with
those obtained using the total light beam interruptions from all three
photocells with one major distinction. The definition for stress detec-
tion given for analyses based on the total light beam interruptions from
all three photocells does not give consistent or reliable results when
the light beam interruptions for the top photocells in each tank are
analyzed alone. It may be that analyses based on the- light beam interr-
uptions from the six possible combinations of photocell levels will each
have a different set of criteria on which stress detection is based. The
fact that the arbitrarily defined stress detection is dependent on which
combination of photocells are being analyzed is not important. What is
important is that once the criteria have been established for stress
detection using a certain combination of photocell levels, the results
based on this combination must give a consistent and reliable index of
premortal aberrations.
It should be noted that the system does not directly monitor the total
activity of the fish but rather reflects changes in the variances of
movement through the light beams as compared to the variance in light
beam interruptions recorded for a previous time interval. The magnitude
of change in the number of light beam interruptions does not necessarily
mean that a positive test for heterogeneity will be recorded. What is
necessary is an increase or decrease in the variance of one of the time
intervals compared.
Tables 3, 4, and 5 give the results obtained from static experiments 1
through 10. These results show that during the first four days of any
experiment the variance calculated from the number of light beam inter-
ruptions recorded for the test fish only periodically deviated from the
preceding day giving rise to a positive test for abnormal movement. A
positive test for abnormal movement is indicated by an asterisk in the
tables. The most important aspect of these analyses is that during the
first four days of these experiments, in only one special case was there
ever more than a single positive test for abnormal movement recorded dur-
ing a given time interval. For example, in Table 3, experiment 1, the
comparison of variances in light beam interruption for day 1 vs day 2,
fish number six, indicates a positive test for abnormal movement as shown
by the asterisk. During this same time interval (day 1 vs day 2) this was
the only analysis in which a positive test for abnormal movement was
detected.
The results from the second four days of experiments 1, 2, and 3 (Table
3) show that stress detection (the occurrence of two positive tests for
abnormal movement during the same time interval) occurred during the
first day of exposure to 7.5 mg/1 Zn*"*" for the golden shiner, and 15.5
mg/1 Zn*"1" and 7.5 mg/1 Zn4* for goldfish. For goldfish these were acute-
ly toxic levels as indicated by the deaths recorded on the following days.
The fact that stress detection occurred before the onset of death in both
cases indicated that the use of light beam interruption as a technique
16
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TABLE 3. STATISTICAL ANALYSIS OF LIGHT BEAM INTERRUPTIONS RECORDED
DURING STATIC EXPERIMENTS 1-4.
Day 1
vs
Day 2
Fish
1
2
3
4
5
6-C
1
2
3
4
5-C
6
1
2
3
4
5-C
6
0
Fish
0
0
0
*
0
0
0
0
0
0
0
0
0
0
0
0
Day 2 Day 3
vs vs
Day 3 Day 4
Experiment
0
No
0
died during day
0
0
0
0(lvs3)
Experiment
0
0
0
0
0
0
Experiment
0
0
0
0
0
0
0
0
0
0
No
0
0
0
0
0
0
No
0
0
0
0
0
0
Experiment No
1
2
3-C
4
5
6
0
0
0
0
0
Leak
0
0
0
0
0
in Tank
0
0
0
0
0
D.iy 4 Day 5 Day 6 Day 7
vs vs vs vs
Day 5 Day 6 Day 7 Day 8
. 1
i
i
i "S
TD
o
<
t.
N
1
. 2
i
T)
0)
TD
t)
4*
+
1
1
. 3
i
i
a
-------
for monitoring movement is sufficiently sensitive to detect premortal
signs of stress.
In experiments 4 through 10 (Tables 3, 4, and 5) no stress detection
occurred when goldfish were exposed to levels of zinc from 5.6 mg/1 to
1.8 mg/1 except in the special case recorded during experiment 6
(Table 4). Experiment 6 represents a weakness in the technique of
analysis, the correction of which involves programming a subjective
decision into the computer used for analysis. The statistical analysis
of the light beam interruptions for fish five revealed a positive test
for abnormal movement when day 1 was compared to day 2. This positive
test continued when day 1 was compared to day 3 and eventually lead to
stress detection by the system when day 1 was compared to day 4 for
fish five and abnormal movement was also recorded for fish 1 during
the same time interval. This was a false stress detection which must
occur only rarely if the system is to function properly. This type of
response for fish five was perpetuated after the comparison of day 1
and 2 by the rules governing the handling of data after a positive test
for abnormal movement had been recorded. This rule states that when two
intervals are compared and a positive test for abnormal movement is
recorded the data for the most recently recorded time interval is
dropped and the next time interval is compared to the last interval in
which normal movement was recorded. Obviously in this case the variance
calculated from the data recorded for day 1 was not in line with those
recorded for the successive time intervals. A programmable instruction
to the computer to eliminate this weakness would be based on the follow-
ing logic. If a positive test for heterogeneous variances is recorded
for a single fish for x number of comparisons in a row, during time
periods when no other analyses are indicating positive responses, then
re-evaluate the analysis in which the first heterogeneous variance was
recorded. The re-evaluation, in this case, would mean instead of drop-
ping the estimate of variance recorded for day 2 as was done during
the original analysis, drop the estimate for variance for day 1 and
proceed with the comparisons. This was done for these data and the
results showed homogeneous variances (normal movement patterns) for
all subsequent comparisons involving data from fish number five. As
noted in this discussion, x number of comparisons was used as opposed
to some specific number. Specifying a number of comparisons at this
point would be premature because of the limited number of instances in
which the perpetuation of heterogeneity has occurred.
Experiment 8 (Table 4) was carried out to determine the effects of
maintaining the test organisms for eight days under static test condi-
tions without feeding. In this experiment the fish were handled as in
the zinc addition experiments except that at the beginning of the fifth
day, instead of adding zinc in solution, the equivalent amount of dilution
water minus zinc was added to each test chamber. As the results show there
does not appear to be any tendency toward increased heterogeneity over
the eight-day test period.
The results from the continuous flow tests are presented in Tables 6
18
-------
TABLE 4. STATISTICAL ANALYSIS OF LIGHT BEAM INTERRUPTIONS RECORDED
DURING STATIC EXPERIMENTS 5-8.
Day 1
vs
Day 2
Day 2
vs
Day 3
Day 3
VK
Day 4
Experiment No. 5
Fish
1
2
3
4
5-C
6
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Experiment No. 6
1
2-C
3
4
5
6
0
0
0
0
0
0
0
0
0
*(lvs3)
0
0
0
0
0
*(lvs4)
0
Experiment No. 7
1
2
3
4-C
5
6
0
0
0
0
0
0
*
0
0
0
0
0
0(2vs4)
0
0
0
0
0
Experiment No. 8
1
2
3
4
5
6
0
0
0
0
0
0
0
0
0
0
*
0
0
0
0
0
*(2vs4)
0
Day 4
vs
Day 5
Goldfish 3.
i
i
a
0)
a
"O
4!
c
CsJ
1
1
0
0
0
*
0
0
Goldfish 3.
i
i
a)
*o
*fl
I
I
*
0
0
0
*(lvs5)
0
Goldfish 1.
i
a
0)
a
-a
_i_
c
N
1
1
0
0
0
0
0
0
Day 5
vs
Day 6
2 mg/1 Zn
0
0
0
0(4vs6)
0
0
2 mg/1 Zn
0(4vs6)
0
0
0
*(lvs6)
0
8 mg/1 Zn
0
*
0
0
0
0
Goldfish All Tanks
i
i
Q)
o
-------
TABLE 5. STATISTICAL ANALYSIS OF LIGHT BEAM
DURING STATIC KXI'liRIMiiNTS 9-30.
INTERRUPTIONS RECORDED
U;ty 1
vs
Day 2
Day 2
vs
Day 3
Day 3
vs
Day 4
Experiment No.
Fish
1
2-C
3
4
5
6
0
0
0
0
0
0
0
0
0
0
0
0
*
0
0
0
0
0
Experiment No.
1-C
2
3
4
5
6
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
9
i
i
o
13
o
t
c
1
10
-o
-------
through 15. These data are reported as first half day values, second
half day values, first half night values, and second half night values.
The segment referred to as first half day values was from 7:00 a.m. to
1:00 p.m., but because data were recorded at half hour intervals during
sunrise this interval includes seven record units on which the variance
estimate for this interval was based. Second half day values include the
time from 1: p.m. to 7:30 p.m. during which seven record units were made.
The first half night values include the time from 7:30 p.m. to 1:00 a.m.
and six record units, while the second half night values include the time
from 1:00 a.m. to 7:00 a.m. during which six record units were made.
The results presented in Table 6 (Experiment 11) indicate stress detec-
tion occurred during a time period when no zinc was being added to the
system, and would initially appear to be a false detection. However, the
reason for this detection was traced to a breakdown in the chlorine neu-
tralizing system. This breakdown probably occurred sometime during the
morning or early after noon of day 4 and by early evening the chlorine
content in the effluent from the test chambers reached 0.10 mg/1. The
chlorine neutralizing system was repaired and by 12:00 a.m. on day 4
the effluents from the test chambers was < 0.05 mg/1. Because the fish
had received this short term stress prior to the normal time for zinc
addition no additional stress was applied. The experiment was run to
normal termination to determine the recovery patterns of the fish. Once
the chlorine stress was removed the return to normal movement patterns
was quite rapid as can be seen from the lack of abnormal movement record-
ed for the second half night comparisons between day 3 and day 4.
The results presented in Tables 7 through 12 represent a series of exper-
iments designed to determine the lowest concentration of zinc detected by
the system under the conditions described. Each table includes a value
for the calculated concentration of introduced zinc and in parentheses
a mean measured concentration (atomic absorption).
The results from this series of experiments indicate that the lowest
detectable zinc concentration based on measured concentrations is bet-
ween 3.64 (Table 11) and 2.94 mg/1 Zn** for a 96-hour exposure (Table 12).
Some of the experiments in this series presented special problems and
interpretation and need further qualification. Tables 9 and 13 show the
results obtained from two experiments in which problems with the record-
ing equipment were encountered. These experiments are not consecutively
numbered but were carried out consecutively. The problem was due to the
loss of bias control in the main recorder, resulting in erroneous counts
in those cells for which no data are given. This equipment malfunction
was due to insufficient cooling in the main recorder, and the addition
of a fan to cool electrical components eliminated the problem.
The results from experiment 15 (Table 10) indicate that stress detection
occurred during the first half night values when day 4 was compared to
day 5. Comparing this response time to stress detection with that for
21
-------
TABLE 6. STATISTICAL ANALYSIS OF LIGHT BEAM INTERRUPTIONS RECORDED
DURING CONTINUOUS FLOW EXPERIMENT 11. iiLUEGILL ALL TANKS
CONTROLS.
Fish
1
2
3
4
5
6
1
2
3
4
5
6
Day 1
vs
Day 2
0
0
0
0
0
0
0
0
0
0
0
0
Day 2
vs
Day 3
0
0
0
0
0
0
0
0
0
0
0
0
Day 3
vs
Day 4
First
0
0
0
0
0
0
Second
0
0
0
0
0
0
Day 4
vs
Day 5
Day 5
vs
Day 6
Day 6
vs
Day 7
Day 7
vs
Day 8
Half Day Values
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Half Day Values
0
0
0
0
0
*
First Half Night
1
2
3
4
5
6
1
2
3
4
5
6
0
0
0
0
0
*
0
0
0
0
0
0
0
0
0
0
0
0(lvs3)
0
0
0
0
0
0
*
0
*
*
0
*
Second
0
0
0
0
0
0
0(3vs5)
0
0(3vs5)
*(3vs5)
0
0(3vs5)
Half Night
0
0
0
0
0
0
0
0
0
0
0
*(4vs6)
Values
0
0
0
0(3vs6)
0
0
Values
0
0
0
0
0
0
0
0
0
0
0
0(4vs7)
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
*
0
0
0
0
0
0
22
-------
TABLE 7. STATISTICAL ANALYSIS OF LIGHT BEAM INTERRUPTIONS RECORDED
DURING CONTINUOUS FLOW EXPERIMENT 12. BLUEGILL
7.5 mg/1 Zn** (5.93 mg/1 Zn4^).
Fish
1-C
2
3
4
5
6-C
1-C
2
3
4
5
6-C
1-C
2
3
4
5
6-C
1-C
2
3
4
5
6-C
Day 1
vs
Day 2
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
*
0
0
0
0
0
0
Day 2
vs
Day 3
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0(lvs3)
0
0
0
0
0
0
Day 3
vs
Day 4
First
0
0
0
0
0
0
Second
i
0 ^
0
-------
TABLE 8. STATISTICAL ANALYSIS OF LIGHT BEAM INTERRUPTIONS RECORDED
DURING CONTINUOUS FLOW EXPERIMENT 13. BLUEGILL
7.5 mg/1 Zn** (6.33 mg/1 Zn44") .
Fish
1-C
2
3
4
5
6
1-C
2
3
4
5
6
1-C
2
3
4
5
6
1-C
2
3
4
5
6
Day 1
vs
Day 2
0
0
0
0
0
*
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Day 2
VB
Day 3
0
0
8
0
0(lvs3)
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Day 3
VH
Day 4
First
0
0
8
0
o
Seconc
o !
0 "g
o 5
0 <
0 $
0 N
1
First
0
0
0
0
0
0
Second
0
0
0
0
0
0
Day
VB
Day
Half
0
*
8
0
0
I Hali
0
0
0
0
0
0
Half
0
0
0
0
0
0
Half
0
*
*
0
0
0
4 Day 5
vw
5 Day 6
Day Values
0
0(4vs6)
8
0
0
: Day Values
0
0
*
0
0
0
Night Values
0
0
0
0
*
0
Night Values
0
*(4vs6)
0(4vs6)
*
*
0
Day 6
vs
Day 7
0
0
B
0
*
0
0
*(5vs7)
it
*
0
0
0
0
Dead
Dead
0
0
*(4vs7)
0
Dead
Dead
0
Day 7
vw
Day 8
0
0
0
*(6vs8)
0
0
*(5vs8)
-
-
0
0
0
0
-
-
0
0
*(4vs8)
*
-
-
0
24
-------
TABLE 9. STATISTICAL ANALYSIS OF LIGHT BEAM INTERRUPTIONS RECORDED
DURING CONTINUOUS FLOW EXPERIMENT 14. BLUEGILL
5.6 mg/1 Zn++ (4.33 mg/1 Zn4"*").
Day 1
vs
Day 2
Day 2
vs
Day 3
Day 3
vs
Day 4
Day
vs
Day
4 Day 5
vs
5 Day 6
First Half Day Values
Fish
1
2
3
4
5
6-C
1
2
3
4
5
6-C
-
0
0
0
-
0
-
0
0
0
-
0
-
0
0
0
-
0
-
0
0
0
-
0
0
0
0
0
Second
i
- i
0 1
0 T3
0
+
0 ?
1
0
*
0
-
0
Half
-
0
0
0
-
0
First Half
1
2
3
4
5
6-C
1
2
3
4
5
6-C
_
0
0
0
-
0
*.
0
0
0
-
0
_
0
0
0
_
0
_
0
0
0
0
0
0
0
-
0
Second
_
0
0
0
-
0
_
0
0
0
-
0
Half
_
0
\j
0
-
0
0
0(4vs6)
0
0
Day Values
0
0
0
-
0
Night Values
_
0
0
0
-
0
Night Values
_
*
0
0
-
0
Day 6
vs
Day 7
*
*
0
_
0
-
0
0
0
0
0
0
0
-
0
Day 7
vs
Day 8
0(6vs8)
0(6vs8)
*
0
-
0
0
0
-
0
0
*
0
-
0
*(5vs7) 0(5vs8)
0
0
-
0
0
0
-
0
25
-------
TABLE 10. STATISTICAL ANALYSIS OF LIGHT BEAM INTERRUPTIONS RECORDED
DURING CONTINUOUS FLOW EXPERIMENT 15. BLUEGILL
5.6 mg/1 Zn++ (3.87 mg/1 En**).
Fish
1-C
2
3
4
5
6
1-C
2
3
4
5
6
1-C
2
3
4
5
6
1-C
2
3
4
5
6
Day 1
vs
Day 2
0
0
0
0
0
0
0
0
0
0
0
0
0
0
*
0
0
0
0
0
0
0
0
0
Day 2
vs
Day 3
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0(lvs3)
0
0
0
0
0
0
0
0
0
Day 3
vs
Day 4
First
0
0
0
0
0
0
Second
i
0 '
o |
o ,
0 c
0 ?
i
First 1
0
0
0
0
0
0
Second
0
0
0
0
0
0
Day
vs
Day
Half
0
0
0
0
0
0
Half
0
0
0
0
0
0
ialf
0
*
0
*
0
0
Half
0
0
0
0
0
0
4 Day 5
vs
5 Day 6
Day Values
0
0
0
0
0
0
Day Values
0
0
0
0
0
0
Night Values
0
0(4vs6)
0
0(4vs6)
0
0
Night Values
0
*
0
0
0
0
Day 6
vs
Day 7
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0(5vs7)
0
0
0
0
Day 7
vs
Day 8
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
26
-------
TABLE 11. STATISTICAL ANALYSTS OF LIGHT BEAM INTERRUPTIONS RECORDED
DURING CONTINUOUS FLOW EXPERIMENT 16. BLUEGILL
4.2 mg/1 Zn4^ (3.65 mg/1 Zn**).
Fish
1
2
3
4-C
5
6
1
2
3
4-C
5
6
Day 1
vs
Day 2
0
0
0
0
0
0
0
0
0
0
0
0
Day 2
vs
Day 3
0
0
0
0
0
0
0
0
0
0
0
0
Day 3
vs
Day 4
First
0
0
0
0
*
0
Second
0 -o
0 -B
0 <
0 +
0 c
o M
Day 4
vs
Day 5
Day 5
vs
Day 6
Half Day Values
0
0
0
0
0(3vs5)
0
Half Day
0
0
0
*
0
0
First Half Night
1
2
3
4-C
j
''
1
2
3
4-C
5
5
6
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
*
0
0
0
0
Second
0
0
0
0
0 i
0
0 '
0
0(3vs5)
0
0
0
0
0
0
*
0
0
0
Values
0
0
0
*(4vs6)
0
0
Values
0
*
0
0
0
I)
Day 6
vs
Day 7
0
0
0(5vs7)
0
0
0
0
0
0
*4vs7)
0
0
0
*(5vs7)
D
0
*
0
Day 7
vs
Day 8
0
0
*
0
0
0
0
0
0
*4vs8)
0
0
0
*(5vs8)
0
0
0((>v
0
!i«)
Half Night Values
0
*
0
0
0
0
0
0
0
0 (4vs6) 0
0
0
*
*
*
0
0
0
0
0
0
0(5vs7) 0
0(5vs7)
*(5vs7)
0
*(5vs8)
27
-------
TABLE 12. STATISTICAL ANALYSIS OF LIGHT BEAM INTERRUPTIONS RECORDED
DURING CONTINUOUS FLOW EXPERIMENT 17. BLUEGILL
3.5 ing/1 Zn"*4" (2.93 mg/1 Zn++) .
Fish
1
2
3-C
4
5
6
1
2
3-C
4
5
6
Day 1
vs
Day 2
0
0
0
0
0
0
0
0
0
0
0
0
Day 2
vs
Day 3
0
0
0
0
0
0
0
0
0
0
0
0
Day 3
vs
Day 4
First
0
0
0
0
0
*
Second
i
o -o
0 .g
0 <
o t
0 ^c
0 ?
i
Day 4
vs
Day 5
Day 5
vs
Day 6
Half Day Values
0
0
0
0
0
0(3vs5)
Half Day
0
0
0
0
0
0
First Half Night
1
2
3-C
4
5
6
1
2
3-C
4
5
6
0
0
*
0
0
0
0
0
0
0
0
0
0
0
0 (Ivs3)
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Second
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
*
0
0
Values
0
0
0
0
0
0
Values
0
0
0
0
0
0
Day 6
vs
Day 7
0
0
0
0(5vs7)
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Day 7
vs
Day 8
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Half Night Values
0
A
0
0
0
0
0
0(4vs6)
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
28
-------
TABLE 13. STATISTICAL ANALYSIS OF LIGHT BKAM INTERRUPTIONS RECORDED
DURING CONTINUOUS FLOW EXPERIMENT 18. BLUEGILL ALL TANKS
CONTROLS.
Fish
1
2
3
4
5
6
1
2
3
4
5
6
1
2
3
4
5
6
1
2
3
4
5
6
Day 1
vs
Day 2
-
0
0
0
0
0
0
0
0
0
0
_
0
0
0
0
0
_
0
0
0
0
0
Day 2
vs
Day 3
0
0
*
0
0
_
0
0
0
0
0
_
*
0
0
0
0
_
0
0
0
0
0
Day 3 Day 4 Day 5
vs vs vs
Day 4 Day 5 Day 6
First Half Day Values
_ _ _
000
000
*(2vs4) 0(2vs5) 0
000
000
Second Half Day Values
_ _ _
000
000
000
000
000
First Half Night Values
_ _ _
*(2vs4) 0(2vs5) 0
000
000
000
000
Second Half Night Values
_
000
000
000
000
000
Day 6
vs
Day 7
-
0
0
0
0
-
0
0
0
0
-
0
0
-
0
-
-
0
0
-
0
Day 7
vs
Day 8
0
0
0
_
-
0
0
-
0
-
-
-
-
^
-
-
-
-
0
-
29
-------
experiments 12 and 13 (Tables 7 and 8) in which the fish were exposed
to 7.5 mg/1 Zn~*~*~ shows that stress detection occurred in a shorter
period of time at the lower concentration. The quicker response time
at the lower concentration is probably the result of two factors. The
resistance of different individuals of the same species of fish are
known to be highly variable and this may have contributed to the more
rapid response. However, more than likely it was the result of an
attempt to meter toxicant into the continuous flow system during this
experiment. The results of zinc analysis showed that the variance in
concentrations reported for this experiment to be about 10 times greater
than the variances reported for those experiments in which batch mixed
zinc was used. The high variance in zinc concentrations was probably
responsible for the comparatively early stress detection reported in
this experiment.
Experiment 18 (13) shows the effects of maintaining fish under continuous
flow test conditions for eight days without feeding. Although some data
were lost near the end of this experiment the results indicate no ten-
dency toward increased abnormal movement through time.
Tables 14-A, 14-B, 15-A, 15-B, and 15-C represent the results from two
experiments in which the procedures for testing were modified to answer
specific questions concerning the practical use of the monitoring system.
In experiment 19 (Tables 14-A and 14-B) three basic questions were posed;
(1) could the time period between the transfer of fish to the test cham-
bers be reduced from the normal four-day period to two days, (2) what
effects would feeding have on the results, and (3) what effects would a
short term exposure to zinc over a six and one half hour period have on
the movement patterns.
In this experiment the time interval between fish transfer and initiation
of data recording was reduced to two days. The results show that the two-
day readjustment period is probably sufficient. During this experiment
the test fish were fed two pellets of Purina Trout Chow developer daily
at 11:00 a.m. Based on the results obtained feeding had no detectable
effect on the movement patterns recorded.
To answer the question concerning short term exposure to zinc, a batch
mixed zinc solution of 10 mg/1 Zn"^" was started through the system at
7:00 a*m. on day 5 of the experiment. At 1:00 p.m. the flow was returned
to the normal dilution water. The effluent samples collected at 1:00 p.m.
showed the following zinc concentrations in mg/1 Zn"^": Tank one, 7.88,
tank two, 7.91; tank three, not detected; tank four, 7.88; tank five,
7.96; and tank six, 7.79. By 12:00 a.m. on day 5 zinc analyses showed all
effluents contained <-0.5 mg/1 Zn*"*". The results from this short term
exposure showed that during exposure or after there were no abnormal
movement patterns recorded.
Experiment 20 (Tables 15-A, 15-B, and 15-C was run for a total of twenty
days during which the effects of only two days' adjustment after handling,
feeding during the experiment, and response to short term stress were
30
-------
TABLE 14-A. STATISTICAL ANALYSIS OF LIGHT BEAM INTERUPTIONS RECORDED
DURING DAYS 1-8 OF CONTINUOUS FLOW EXPERIMENT 19.
BLUEGILL INTERMITTENT Zn"*"'" STRESS.
Fish
1
2
3-C
4
5
6
1
2
3-C
4
5
6
1
2
3-C
4
5
6
1
2
3-C
4
5
6
Day 1
vs
Day 2
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
*
0
Day 2
vs
Day 3
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0(lvs3)
0
Day 3
vs
Day 4
First
0 -a"
0 -3
0 <
0 +
0 c
0 !
Second
0
0
0
0
0
0
First
0
0
0
0
0
0
Second
0
0
0
0
0
0
Day 4
vs
Day 5
Half Day
~~ 0
0
0
0
0
0
Half Day
0
0
0
0
0
0
Day 5
vs
Day 6
Values
0
0
0
0
0
0
Values
0
0
0
0
0
0
Day 6
vs
Day 7
0
0
0
0
0
0
0
0
0
0
0
0
Day 7
vs
Day 8
0
0
0
0
0
0
0
0
0
0
0
0
Half Night Values
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Half Night Values
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
31
-------
TABLE 14-B. STATISTICAL ANALYSIS OF LIGHT BEAM INTERRUPTIONS RECORDED
DURING DAYS 8-10 OF CONTINUOUS FLOW EXPERIMENT 19.
BLUEGILL INTERMITTENT Zn4"1" STRESS.
Fish
1
2
3-C
4
5
6
1
2
3-C
4
5
6
I
2
3-C
4
5
6
1
2
3-C
4
5
6
Day 8
vs
Day 9
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Day 9
vs
Day 10
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
First Half Day Values
Second Half Day Vnlues
First Half Night Values
Second Half Night Values
32
-------
re-examined. In addition, during one segment of the experiment the fish
were exposed to zinc until stress detection occurred. After stress
detection flow was returned to the normal dilution water and percent
survival and recovery patterns were examined.
The results given in Tables 15-A, 15-B, and 15-C agree with those
presented in Tables 14-A and 14-B concerning both feeding during the
experiment and the time between handling and data recording. In both
cases the effects of these changes in procedure were not detected in
the movement patterns. At 1:00 p.m. on day 7 of this experiment zinc
flow was started into the test chambers and continued until 7:30 p.m.
of the same day. The concentrations of zinc in mg/1 recorded in the
effluent at the time flow was returned to the normal dilution water
were: tank one, 13.32; tank two, <.08; tank three, 11.39; tank four,
12.72; tank five, 13.32; and tank six, 12.59. By 8:30 a.m. on day 8
of this experiment the effluent zinc concentrations were less than
0.300 in all cases. As was the case in experiment 19, this short term
stress did not cause stress detection during the period of zinc addition
or for the period following zinc addition.
To determine the percent survival and recovery patterns of the fish once
stress detection occurred, zinc flow was re-initiated at 1:00 p.m. on
day 13 of this experiment (experiment 20). Between 8:00 and 9:00 p.m.
on day 13 the zinc concentration reached a maximum of: 7.51 for tank
one; less than .05 for tank two; 7.49 for tank three; 7.52 for tank four;
7.49 for tank five; and 7.54 for tank six. The concentrations remained
at the above values until the statistical analyses showed stress detec-
tion during the first half night values on day 14. As soon as stress
detection occurred the flow was returned to normal dilution water. At
10:00 a.m. on day 15 zinc analyses showed the following effluent concen-
trations: 0.70 for tank one, 0.09 for tank two; 0.62 for tank three;
0.57 for tank four; 0.67 for tank five; and 0.44 for tank six. Stress
detection continued to be registered for two consecutive time intervals
following the initial detection, but after that no stress detection was
registered and the frequency of abnormal movement patterns returned to
pre-stress levels within 48 hours. To determine how the light beam
interruptions recorded for day 1 compared to those after the two stress
periods, a comparison was made between day 1 and day 20. The results
showed no abnormal movement patterns, indicating a complete recovery to
pre-stress levels. This does not, however, mean that the fish would
respond to a third stress period in the same way, nor does it indicate
an increase or decrease in sensitivity. In practical application after
stress detection the system should probably receive a new set of test
fish, added two a day until a complete exchange of fish had been made.
In this way the system need not be shut down, and the problems of
increased or decreased resistance to stress need not become a serious
consideration.
For the first ten days of experiment 20 from 1:00 p.m. to 7:00 p.m. the
number of light beam interruptions occurring during consecutive ten-
minute intervals was recorded manually for each photocell. The data
33
-------
TABLE 15-A.
STATISTICAL ANALYSIS OF LIGHT i'.EAM INTERRUPTIONS
RECORDED DURING DAYS 1-8 OF CONTINUOUS FLOW EXPERIMENT
20. BLUEGTLL (Zn"*4" ADDITION TO STRESS DETECTION).
Fish
1
2-C
3
4
5
6
1
2-C
3
4
5
6
Day 1
vs
Day 2
0
0
0
0
0
0
0
0
0
0
0
*
Day 2
vs
Day 3
0
0
0
0
0
0
0
0
0
0
0
0(lvs3)
Day 3
vs
Day 4
First
0
0
0
0
0
0
Second
0
0
0
0
0
0
Day
vs
Dav
Half
0
0
0
0
*
0
4 Day 5
vs
5 Day 6
Day Values
0
0
0
0
0(4vs6)
0
Day 6
vs
Day 7
0
0
0
0
*
0
Day 7
vs
Day 8
0
0
0
0
*(6vs8)
0
Half Day Values
0
0
0
0
0
0
First Half
1
2-C
3
4
5
6
1
2-C
3
4
5
6
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Second
0
0
0
0
0
0
0
0
0
0
0
0
Half
0
0
0
it
0
0
i
T3
0 <"
0 3
0 t
0 N
0 {
Night Values
0
0
0
0
0
0
Night Values
0
0
0
0(4vs6)
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
*
0
0
0
0
0
0
*
0
0
0
0
0
0
0
0
0
0
0(6vs8)
0
*
0
34
-------
TABLE 15-B.
STATISTICAL ANALYSIS OF LIGHT BEAM INTERRUPTIONS
RECORDED DURING DAYS 8-14 OF CONTINUOUS FLOW EXPERIMENT
20. BLUEGILL (Zn"*^" ADDITION TO STRESS DETECTION).
Fish
1
2-C
3
4
5
6
1
2-C
3
4
5
6
1
2-C
3
4
5
6
]
;M:
'i
/»
5
h
Day 8
vs
Day 9
0
0
0
0
0(6vs9)
0
0
0
0
0(7vs9)
0
0
0
0
0
0
0
0
0
0
0
0
0(7v:;9)
0
Day 9
vs
Day 10
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Day
vs
Day
First
0
0
0
0
0
0
Second
0
0
0
0
0
0
First
0
0
0
0
0
0
Second
0
0
0
0
0
0
10 Day 11
vs
11 Day 12
Half Day Values
0
0
0
0
0
*
Half Day Values
0
*
0
0
0
0
Half Night Values
o !
o -g
o 5
0 <
o t
0 Ǥ
1
1
Half Night Value.
0
0
0
0
0
0
Day 12
vs
Day 13
0
0
0
0
*
0(llvsl3)
*
0(llvsl3)
0
0
0
0
0
0
a
0
0
0
3
0
0
0
0
0
0
Day 13
vs
Day 14
0
0
0
0
0(12vsl4)
0
0(12 vs!4;
0
*
0
0
o
*
0
*
0
0
0
*
0
0
0
*
0
35
-------
TABLE 15-C. STATISTICAL ANALYSIS OF LIGHT BEAM INTERRUPTIONS
RECORDED DURING DAYS 14-20 OF CONTINUOUS FLOW EXPERIMENT
20. BLUEGILL (Zn"*4" ADDITION TO STRESS DETECTION).
Day 14
vs
Day 15
Day 15
vs
Day 16
Day 16 Day 17
vs
vs
Day 17 Day 18
First Half Day
Fish
1
2-C
3
4
5
6
*
0
*
0
*
0
0(l4vsl6)
0
0(14vsl6)
0
0(l4vsl6)
*
0
0
0
0
0
*(15vsl7)
Values
0
0
0
0
0
0(15vsl8)
Day 18
vs
Day 19
0
0
0
0
0
0
Day 19
vs
Day 20
0
0
0
0
0
0
Second Half Day Values
1
2-C
3
4
5
6
*
0
0(13vsl5)
0
0
0
*(14vsl6)
0
0
0
0
0
*!4vsl7)
0
0
0
0
0
0(14vsl8)
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
First Half Night Values
1
2-C
3
4
5
6
0(13vsl5)
0
0(13vsl5)
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Second Half Night Values
1
2-C
3
4
5
6
0(13vsl5)
0
0
0
0(13vsl5)
*
0
0
0
0
0
*(14vsl6)
0
0
0
0
0
*(!4vsl7)
0
0
0
0
*
0(14vsl8)
0
0
0
0
0(17vsl9)
0
0
0
0
0
0
0
36
-------
collected during this ten-day period were used to indicate the desir-
ability of reducing the time lag from the minimum five and one half
hour period used in the previous analyses to one hour. The results of
this initial limited analysis are sufficiently promising to warrant
further study. The monitoring system as it presently exists is not
sophisticated enough to monitor time intervals less than the hourly
intervals readily handled by the camera recording system. However,
plans are presently under way to completely automate the data acqui-
sition and analysis components of the system through direct interfacing
with a mini-computer. When complete automation is completed all time
intervals and photocell levels can be thoroughly investigated.
The monitoring equipment used in these experiments is limited by tur-
bidity. Turbidimetric determinations made under normal operating condi-
tions showed that arroneous counts were registered when the turbidity
of the water reached 15 APHA units with the 650 mu filters in place and
27 APHA units without the filters. This limited operating range presented
no problems in the laboratory studies but would require careful consid-
eration in field studies. The levels of turbidity over which the system
could operate can be increased significantly by increasing the lamp out-
put and providing better light beam-photocell alignment. Even with a
significant increase in operating range there would be conditions under
which effective measurements could not be made. The advantages gained
from continuous monitoring by this method would have to be weighed
against the potential loss of toxicity resulting from the reduction
or removal of suspended material.
37
-------
SECTION V
FISH REPRODUCTION AND GROWTH
Methods and materials; Bluegill sunfish were seined from a local pond
and held for several months in the laboratory in the same dilution
water, with the same photoperiod and water temperatures, as for the
experiments described in Section IV.
Starting April 13, approximately 200 fish (approximate total lengths:
8-15 cm; weights: 10-80 gms.) were brought into breeding condition by
exposing them to a photoperiod of 16 1/2 hours of light, water tempera-
tures of 31-32°C, and by feeding them twice daily with frozen Gordon
Formula (Axelrod, 1952) and once daily with live mealworms. The dim-
ming system described earlier simulated a 1/2 hour dawn starting at
6 a.m. and a 1/2 hour dusk starting at 10 p.m.
On May 4 most of the fish could be sexed by gently squeezing the sides
and observing whether eggs of milt was extruded, and three females and
one male were placed in each of twenty 20-gallon tanks (standard
aquaria, long type, Ramfab Aquarium Products cat. no. RA-20L).
One standard clay flowerpot (upper rim-to-rim diameter = 6 inches) was
placed on its side in each tank for the females to hide from the ag-
gressive attacks of the males. An artificial nest, described by Eaton
(1970) was also placed in each tank and five smooth pebbles, 2-3 cm in
diameter, were scattered on the bottom of each nest.
One toxicant delivery apparatus was used for each set of five tanks
receiving one concentration, and one water delivery apparatus was used
for five control tanks which received no added zinc. The toxicant
delivery apparatus combined a toxicant dipper and needle valve des-
cribed by Mount and Brungs (1967) with a water delivery system des-
cribed by Brungs and Mount (1970). The dilution water was the same as
that used in the experiments described in Section IV. The zinc con-
centrations for the reproduction study were based on the lowest con-
centration used in the fish breathing experiments; i.e. 2.5 mg/1. Tanks
6-10 received .250 mg/1 zinc (1/10 of 2.5 mg/1), tanks 16-20 received
.025 mg/1 zinc (1/100 of 2.5), tanks 11-15 received no added zinc and
served as controls. In addition, tanks 1-5 received 1/100 the 96-hour
TL50 (median tolerance limit) for adult bluegill sunfish exposed to
zinc in municipal tapwater: .075 mg/1. The initial zinc concentration
used in the stock jug of each toxicant delivery apparatus was calculated
to yield the desired concentration in the tanks. The concentrations in
the tanks were measured by atomic absorption spectropho tome try and were
lower than desired, so the concentrations of the stock solutions were
adjusted until the correct concentrations were obtained in the tanks.
The flow rate to each tank was approximately 100 ml/min. The water
entered at the top and front of the tank and was removed from the
39
-------
bottom carrying some detritus with it, by means of a sheathed standpipe
at the rear of the tank.
A plastic egg hatching box (20.5 cm long, 7.0 cm wide, and 15.5 cm
deep) hung on the front of each aquarium and was large enough to accept
three egg cups. The egg cups were made from Turtox plastic jars (5.5
cm o.d., 6.8 cm tall), with the bottoms removed. In use, each end of
the cup was covered with a piece of ladies' woven nylon hose held in
place with rubber bands. Each cup rested on an airstone cemented to
the bottom of the hatching box. A piece of plexiglas (20 cm long, 6.3
cm wide, and 0.6 cm thick) fit into grooves in the hatching box and
rested on top of the cups to keep them from floating.
Water siphoned from each tank into the hatching boxes and was returned
to the tank by an air lift. Another air lift delivered water from the
hatching box to a plastic rearing chamber (38.0 cm long, 30.5 cm wide,
and 17.5 cm deep) for rearing newly-hatched fry. Thus the eggs were
hatched and the fry reared in the same water as their parents. Water
drained from each rearing chamber through a rectangular opening (8.8 cm
wide, 1.8 cm high) into a trough. The bottom of the opening was 14.5
cm from the bottom of the pan, and the opening was covered by .8 mm
mesh nylon netting.
The fish in each tank were fed two grams of frozen Gordon Formula
(Axelrod, 1952) twice a day and eight live mealworms once a day. The
tanks were cleaned once a week by siphoning detritus from the bottom.
At 1 p.m. every day, the pebbles in each nest were removed and examined
closely for eggs. If eggs were present, a plastic chamber (same dimen-
sions as above) was filled with water from the tank and the nest was
removed from the tank and placed upside down in the chamber over an
airstone. A new nest was substituted immediately for the old one.
A subsample of 200 eggs was removed from the nest and placed in a
hatching cup, which in turn was placed in the hatching box. After 48
hours, the number of fry in both the egg cup and the chamber were
counted, by pipetting them into petri dishes and using a Dazor model
M209 fluorescent magnifier and a hand tally counter. The hatch in the
subsample of eggs in the cup was assumed to be proportional to the hatch
in the nest, and the numbers of fry and eggs in the cup were used to
back-calculate the number of eggs spawned in the nest:
Total No. Eggs _ No. Fry X (No. Eggs in Cup No. Fry in Cup) + No. Eggs
In Nest In Nest In Cup
When less than 200 eggs were spawned, the number of eggs in the nest and
the number of fry in the hatching chamber were counted directly, with-
out removing a subsample.
40
-------
Fish that were dead or that had lost their equilibrium were removed as
soon as they were noticed. In addition, six fish had an eye disease
that started as a white spot and gradually consumed the entire eye, and
these fish were also removed. Fish that were removed before the end of
the experiment were weighed, measured and sexedunless they were too
decomposed. Fish that were removed and could be sexed were replaced by
a fish of the same sex from a stock of ripe fish kept in dechlorinated
tapwater containing no added zinc, until July 20, when no further re-
placements were made. The breeding portion of the experiment termina-
ted August 19, when all the remaining adult fish were killed, weighed,
measured and sexed. The condition and weight of the genads was also
recorded.
Fifty fry from the first spawning in each tank were placed in the
rearing chamber for that tank. Newly-hatched brine shrimp were rinsed
in dechlorinated tapwater and placed in each rearing chamber twice a
day for the fish to feed upon.
A census of the rearing chambers of July 8 revealed that very few fish
were surviving, so changes were made in the apparatus and methods.
Some of the fry may have washed through the netting of the rearing
chambers, so the chambers were modified by drilling five 1.1 cm holes
on centers 11.5 cm above the bottom of the chambers and covering them
with a piece of woven nylon. All surviving bluegills were transferred
to the modified chambers on July 8 and 9, and all fry hatched after
July 9 were placed in chambers of the new design.
In addition, we learned that brine shrimp were too large for bluegill
fry and that the National Water Quality Laboratory, Duluth, Minnesota,
was successfully feeding plankton to baby bluegills (James M. McKim, III,
personal communication). Consequently, starting July 23, plankton was
obtained regularly from nearby ponds, and fed, after straining through
a 0.8 mm mesh net, twice a day to the fish. Samples of the plankton
were examined regularly under the microscope and never appeared to be
very rich, so the diet was supplemented with a pinch of TetraMin pow-
dered baby fish food twice a day. Newly-hatched brine shrimp were fed
to the fish starting approximately the third week of growth.
Since no spawnings ever occurred in some tanks, the rearing chambers
for these tanks received fry from other tanks. In addition to transfers
of fry made within chambers at the same zinc concentration, some fry
were taken from high zinc concentrations and put into chambers con-
taining low concentrations, and vice-versa.
The fry in each chamber were counted and total lengths determined 30,
60 and 90 days after introduction to the rearing chamber. Total lengths
were determined by placing each fish in a glass petri dish over a metric
ruler.
Dissolved oxygen concentrations in the breeding tanks were determined
by a YSI oxygen meter, and temperatures by a mercury thermometer. Since
41
-------
water from the breeding tanks was delivered directly to the rearing
chambers by air lifts, we assumed that the water characteristics in the
tanks and chambers were the same. This assumption was confirmed for
zinc by measuring zinc concentrations in the tanks and chambers at
random intervals, but D.O. and temperature were not measured in the
rearing chambers.
Results and discussion; Zinc concentrations, dissolved oxygen concen-
trations (D.O.), and temperatures in the breeding tanks are shown in
Table 16. On August 4, 1971, a new central air conditioning system be-
gan operating, and the room temperature was lowered 5.6°C in 12 hours.
As a result, the temperatures in the breeding tanks reached a new
steady state, approximately 4°C lower than the mean temperatures re-
corded earlier. Since a spawning occurred for the first time in tank 9
after the drop in temperature, and six additional spawnings occurred
in other tanks, the temperature drop did not seem to affect spawning.
However, eggs from a spawning on August 4 that were exposed to a drop
in water temperature of 12°C showed a very low percentage hatch (2%)
and were not included in the results.
The dechlorinated tapwater delivered to the control tanks (tanks 11-15)
contained a zinc concentration ranging from .002 - .062 mg/1. The mean
zinc concentrations and the standard diviations in all the tanks are
shown in Table 16. Nominal zinc concentrations will be used in the
rest of the text.
Data on the adult bluegills used as breeders are shown in Table 17 .
Although males and females were approximately the same size when they
were introduced to the breeding tanks on May 4, Table 17 shows that the
surviving males were generally heavier and longer than the surviving
females when the breeding portion of the experiment terminated August 19.
Also, mortality among females was proportionally greater than it was
among males. The disparity in growth and survival between males and
females was probably due to biting and butting attacks by the males.
Most of the dead females had tattered fins and scales missing from their
sides. Females that were in tanks with very aggressive males would feed
hesitantly, even when food was placed near their flowerpot shelters.
Most of the fish at all zinc concentrations were still ripe on August
19, and there were no trends in adult fish weights, lengths, survival,
or gonad weights that could be attributed to the effects of zinc.
The variation in female mortality from tank to tank probably indicates
some variation in aggressiveness from male to male, and variation in
aggressiveness among males may explain why there were some tanks at all
concentrations where no spawning occurred (Table 18). For example, the
male in tank 17 (a tank receiving .025 mg/1 zinc) killed, or contribu-
ted to the death of eight females in succession, and no spawning ever
occurred in this tank. Once a spawning did occur in a tank, it was high-
ly likely that several more would occur. In contrast to the multiple
spawnings obtained in the control tanks and tanks receiving zinc con-
42
-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
Table 16. Zinc and dissolved oxygen concentrations,
and temperatures in breeding tanks
Tank Zn*
.075
.075
.075
.075
.075
.250
.250
.250
.250
.250
.000
.000
.000
.000
.000
.025
.025
.025
.025
.025
Measured zinc con-
centrations (mq/1)
N
14
14
14
14
14
15
15
15
15
15
14
14
14
14
14
14
14
14
14
14
Mean
.071
.081
.079
.076
.074
.231
.232
.230
.234
.249
.028
.019
.019
.020
.017
.028
.040
.041
.035
.033
S.D.
.043
.047
.039
.032
.028
.055
.048
.045
.043
.044
.023
.007
.010
.014
.011
.012
.019
.018
.017
.012
P.O. (mg/1)
N
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
Mean
6.1
6.3
5.9
6.0
5.8
5.5
5.3
5.4
5.1
5.3
5.5
5.7
5.5
5.7
5.4
5.6
5.4
5.8
5.7
5.4
S.D.
0.5
0.2
0.7
0.7
0.5
0.6
0.7
0.7
0.9
0.8
0.6
0.5
0.6
0.5
0.8
0.7
0.7
0.5
0.5
0.7
Temperature ( C)
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
Mean
30.8
31.2
31.6
31.6
31.6
31.0
30,
30.
30,
30.1
30.
30.
30.
30.
30.4
30.5
30.4
30.4
30.4
29.9
S.D.
1.0
0
0
0
0.5
.5
.5
.5
0.4
0.8
0.8
0.9
0.8
0.9
0.6
0.7
0.9
0.9
0.6
0,
0,
0.8
0.5
* Nominal zinc concentrations (mg/1)
N-number of readings
43
-------
Table 17Survival of adult bluegills and weights, lengths,
and condition of gonads of adults at end of
breeding experiment
Tank
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
Zn
(mg/1 )
.071
.081
.079
.076
.074
.231
.232
.230
.234
.249
.028
.019
.019
.020
.017
.028
.040
.041
.035
.033
°No. males
removed be-
fore end of
experiment
0
0
*1
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
°No. females
removed be-
fore end of
experiment
*3
4
0
*1
1
5
0
2
3
0
4
1
3
*2
*2
0
*9
2
2
5
Wts. Standard
(gms) lengths (cm)
61.8
69.6
48.2
60.9
37.6
69.7
71.7
34.0
62.7
26.1
61.3
78.4
67.6
72.6
72.5
70.0
93.4
84.8
31.6
11.5
11.5
10.5
11.6
10.2
12.1
12.0
10.0
11.7
8.9
11.7
11.9
12.1
12.3
12.3
11.8
12.9
13.0
9.6
Males
No.
1
1
1
1
1
1
1
1
1
1
1
0
1
1
1
1
1
1
1
1
One fish removed because eye was diseased.
per tank contracted this disease.
No more than one fish
aFish with eye disease, dead fish, and fish that had lost their
equilibrium were removed immediately. A ratio of three females
and one male was maintained per tank by replacement of fish until
July 20, 1971.
values (with ranges in parentheses) are shown for tanks
containing more than one female.
44
-------
Fish removed at end of experiment
Females
Gonads
Wt(gms)
1.2
1.5
0.9
1.7
< .1
1.0
1.2
0.2
0.9
0.5
0.8
0.9
1.6
1.0
0.8
2.1
1.0
1.0
< .1
Wts. Standard
Condition*- (gms) lengths(cm) No.
R
N
R
R
R
R
R
R
R
N
13.1 7.2 1
0
24.2 16.6-33.6) 9.1(8.5-9.8) 3
31.2 23.0-39.4) 9.6(8.9-10.2) 2
29.4 23.7-35.0) 9.6(9.4-9.7) 2
0
29.1(22.0-35.4) 9.4(8.3-10.0) 3
44.6(38.3-51 .0)10.4(10.0-10.8)2
35.2(29.2-41.3 9.9(9.5-10.3) 2
36.2(25.2-46.4) 9.7(9.0-10.3) 3
0
31.7(24.6-35.2) 9.5(9.0-9.8) 4
38.4(34.2-42.7)10.1(9.6-10.6) 2
39.1 10.3 1
21.6 8.7 1
28.6(28.1-29.0) 9.5(9.3-9.7) 2
0
13.4 7.7 1
26.8 9.3 1
20.6(7.8-32.1) 8.5(6.6-9.7) 4
Gonads
Wt(gms) Condition*-
!./>
1.6(0.1-2.6)
2.3(1.3-3.3)
2.8(1.5-4.1)
1.3(0.5-2.2)
2.7(0.6-4.8)
2.4(2.3-2.6)
3.2(2.4-4.1)
2.6(2.0-3.3)
3.0(3.0-3.1)
1.7
0.6
1.6(1.3-1.9)
0.1
2.9
1.6(0.4-3.5)
1R
2R, IN
2R
2R
3R
1R, IN
2R
3R
j
4Rd
2R
1R
1R
2R
IN
1R
4Rd
CR = ripe, and indicates that milt was extruded when the sides of the
males were squeezed; or in the case of females, that the ovaries were
swollen and filled with pinhead-size eggs. N = not ripe, and indicates
that the testes weighed less than .1 gtn and no milt was extruded from
males; in the case of females, no eggs were visible. The number of
ripe and unripe females in each tank is shown.
An extra female was added to tanks 11 and 20.
45
-------
centrations of .075 and .025 mg/1, however, only a single spawning in
one tank occurred at a concentration of .250 mg/1 zinc (Table 18). Since
eggs or milt could be extruded from all the breeders at the beginning
of the experiment, and since most of the fish had ripe gonads when the
experiment terminated, the results in Table 18 indicate that a zinc
concentration of .250 inhibits spawning in ripe fish.
Table 18 also shows the percentage hatch in each tank. Where more than
one hatching was used, the mean percentage hatch is shown, with the
range in parentheses. Hatching data from eight spawnings were not used
because some of the eggs were hatching in the breeding tanks in less
than 24 hours at temperatures of 30-31 C. Attempts to remove eggs from
these nests by pipetting generally caused the egg membranes to rupture,
releasing the fry. In these cases, the percentage hatch in 48 hours of
the eggs that were transferred without repturing was confounded with
the 48-hour survival of the fry that were also unavoidably transferred
to the egg hatching cups. A hatch of 21% from one spawning in tank 16
that was heavily fungused was also excluded. The hatch obtained from
the one spawning at the highest zinc concentration was low (43%) , but
within the range of values in the other zinc concentrations.
The number of fry introduced to the old rearing chambers with 0.8 mm
mesh outlets and the number introduced to the modified chambers with
smaller mesh outlets are shown in Table 19. The introductions are
shown in chronological sequence from left to right across the rows.
For example, two introductions of fry were made to rearing chamber 4:
one introduction of 50 fry from tank 4, and a later introduction of
51 fry from tank 1, after inspection revealed that there were no sur-
vivors from the first introduction. Growth and survival data in the
right-hand portion of the table were always obtained on fish from the
last introduction reported in the left side of the table. Survival of
young bluegills at all zinc concentrations was poor. Mortality was
highest during the first weeks, and can probably be attributed to
starvation. The plankton collections that were fed to the young blue-
gills starting August 24 were never very rich, and the powdered baby
fish food may not have been utilized. Once the young were large enough
to feed on brine shrimp, survival improved as shown by the reduction
in mortality between 30, 60 and 90 days as compared to the mortality
between day 0 and day 30. At a zinc concentration of .250 mg/1, however,
no bluegills survived longer than 30 days. Fry obtained from eggs
spawned in a zinc concentration of .250 mg/1 and fry obtained from eggs
spawned in other zinc concentrations were placed in .250 mg/1 zinc.
Most of the fry from all these sources died within three days in 250 mg/1
zinc and were visible on the bottom of the rearing chambers. In addition,
fry taken from .250 mg/1 zinc and placed in control tank 11 showed poor
survival: only four fish survived for 30 days, one for 60 days, and
none for 90 days.
An experiment on the effects on young bluegill of momentary exposure
to a high zinc concentration was inadvertently conducted when a hose
46
-------
Table 18. Spawning of adult bluegills and percentage
hatch of eggs at four zinc concentrations
Tank
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
Mean
zinc
concen-
tration
.071
.081
.079
.076
.074
.231
.232
.230
.234
.249
.028
.019
.019
.020
.017
.028
.040
.041
.035
.033
Total
no. of
eggs
spawned3
8414
0
10647
4736
0
0
0
0
1009
0
7188
0
0
227
5849
4274
0
0
10202
0
Total
no. of
spawnings
3
0
8
5
0
0
0
0
1
0
4
0
0
2
7
3
0
0
5
0
Mean no.
of eggs
per
spawning0
2805
0
1331
1184
0
0
0
0
1009
0
:i797
0
0
114
985
1425
0
0
2040
0
Percen-
tage a h
hatch3' b
72 (71-72)
0
66 (49-78)
57 (44-68)
0
0
0
0
43
0
62 (35-76)
0
0
33
73 (47-96)
86
78 (65-90)
aNumber of eggs and percentage hatch were not determined for all
spawnings because of premature hatching, fungus infestation,
etc. (see text).
^Where more than one hatch was used, the mean percentage hatch
is shown, with the range in parentheses.
47
-------
Table 19. Survival and growth of bluegills in four
zinc concentrations
Rearing
chamber
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
Zn
mg/1
.071
.081
.079
.076
.074
.231
.232
.230
.234
.249
.028
.019
.019
.020
.017
.028
.040
.041
.035
.033
No. of fry
introduced
to old
rearing
chambers
50[l]a
50[3]
50[4]
50[15],51[15]
5o[n]
50[1 1]
50[15]
50[11]
No. of fry
introduced
to new
rearing
chambers
50[1]
50[4"
50f3"
51fT
,45[19]
50[3]
53[9]
58[9"
50[1"
51 [9!
50[11
,58[19]
],51[9]
50[15],40[14]
50[15]
50[16]
50[19]
56[16]
51 [19
50[19
]
]
Mean total lengths
30
21.8(5)b
13.2(4
(0)
21.0(4)
12.2(19)c
(0)
(0)
(0)
(0)
(0)
6.8(4)
9.5(2)
d
(0)
(0)
14.4(7)
Days
60
28.8(5)
20.3(3
(0)
24.5(4)
19.2(18)
(0)
(0)
(0)
(0)
(0)
10.0(1)
21.2(2)
20.0(2)
(0)
(0)
19.3(6)
(mm)
90
38.8(5)
34.3(3)
(0)
36.6(4)
26.9(14)
(0)
(0)
(0)
(0)
(0)
(0)
27.8(2)
33.0(1)
(0)
(o)
30.6(6)
a Numbers in brackets indicate the number of the tank where the fry
were obtained.
b Numbers in parentheses indicate the number of fish.
c At least 2 fish died as a result of a zinc soil! on day 22 (see
text).
d The fish in chamber 15 were not counted or weighed on day 30.
48
-------
separated from a connector and fell into rearing chamber 5 while a
stock bottle was being filled with concentrated zinc solution. The
hose was removed from the tank in a fraction of a second and the young
bluegill were transferred to the proper zinc concentration within two
minutes, but two of the 21 fish died within 15 minutes. During the
two minute period, the 22-day-old bluegills had been exposed to a zinc
concentration of 9.18 mg/1, although the concentration initially may
have been higher in portions of the chamber. There were 19 survivors
on day 30, 18 on day 60, and 14 on day 90. After the initial deaths,
survival in chamber 5 was comparable to survival in other tanks at
the same concentrations.
In summary, it appears that a zinc concentration of .250 mg/1 inhibits
spawning in adult bluegills brought into breeding condition in dechlor-
inated municipal water containing no added zinc, and causes complete
mortality of bluegill fry. Water containing no added zinc and zinc
concentrations of .075 and .025 mg/1 do not have these effects.
The lowest zinc concentration tested in the apparatus for monitoring
fish breathing was 2.55 mg-1. This concentration was detected by the
monitoring method, and the reproduction and growth experiment shows
that 1/100 or 1/34 of this concentration might be safe for chronic
exposure of bluegills, but that 1/10 of this concentration certainly
is not.
49
-------
SECTION VI
ACKNOWLEDGEMENTS
This research was carried out in the Aquatic Biology Laboratory of
the Biology Department and Center for Environmental Studies, Virginia
Polytechnic Institute and State University, Blacksburg, Virginia, 24061.
The guidance and help provided by Dr. Clyde Y. Kramer, of the Statistics
Department, Virginia Polytechnic Institute and State University is
gratefully acknowledged. Dr. Richard E. Sparks, Virginia Polytechnic
Institute and State University, worked on the reproduction and growth
studies. The cooperation and suggestions of the Environmental Protection
Agency Project Officer, Dr. James M. McKim, III, were greatly appreciated.
51
-------
SECTION VII
LITERATURE CITED
American Public Health Association. 1965. Standard Methods for the
Examination of Water and Wastewater. 12th ed., 769 pp. New York.
Axelrod, J. H. R. 1952. Tropical Fish as a Hobby. George Allen
and Unwin, Ltd. London. 264 pp.
Brett, J. R. 1968. Personal communication.
Brungs, William A. 1969. Chronic toxicity of zinc to the fathead
minnow, Pimephales promelas Rafinesque. Trans. Amer. Fish.
Soc. 98(2): 272-279.
Brungs, W. A., and D. I. Mount. 1970. A water delivery system for small
fish-holding tanks. Trans. Amer. Fish. Soc. 99(4): 799-802.
Cairns, J., Jr. 1970. New concepts for managing aquatic life
systems. Jour. Water Poll. Contr. Fed. 42(1): 77-82.
Cairns, J., Jr., K. L. Dickson, Richard E. Sparks, and William T.
Waller. 1970. A preliminary report on rapid biological
information systems for water pollution control. Jour. Water
Poll. Contr. Fed. 42(5): 685-703.
Cairns, Jr., Jr. (in press). Management problems in multiple use
of aquatic ecosystems. River Ecology and the Impact of Man.
Clarence A. Carlson (Ed.).
Davis, R. E. and J. E. Bardach. 1964. Time co-opdinated prefeeding
activity in fish. Animal Behaviour XIII (1): 154-162.
Eaton, S. G. 1970. Chronic malathion toxicity to the bluegill
(Lepomis macrochirus Rafinesque). Water Research. 4: 673-684.
Moore, J. G., Jr., Commissioner. 1968. Water Quality Criteria.
Report of the National Technical Advisory Committee to the
Secretary of the Interior. U.S. Govt. Printing Office. 234 pp.
Mount, Donald I. and Charles E. Stephen. 1967. A method for
establishing acceptable toxicant limits for fish- Malathion
and the butoxyethanol ester of 2,4-D. Trans. Amer. Fish.
Soc., 96(2): 185-193.
Mount, D. I., and W. A. Brungs. 1967. A simplified dosing apparatus
for fish toxicology studies. Water Research. 1: 21-29.
53
-------
Mount, Donald I. 1968. Chronic toxicity of copper to fathead
minnows (Pimephales promelas Rafinesque). Water Research,
2: 215-223.
Scheier, A. and J. Cairns, Jr. 1966. Persistence of gill damage in
Lepomis gibbosus following a brief exposure to alkyl benzene
sulfonate. Not. Nat. Acad. Nat. Sci. Philadelphia, No. 391:
1-7.
Shirer, H. W., J. Cairns, Jr., and William T. Waller. 1968. A
simple apparatus for measuring activity patterns of fishes.
Water Resources Bull. 4(3): 27-43.
Sokal, R. R. and F. James Rohlf. 1969. Biometry. W. H. Freeman
and Co. 776 pp.
Sprague, J. B. 1969. Measurement of pollutant toxicity to fish I.
Bioassay methods for acute toxicity. Water Research. 3: 793-
821.
Sparks, Richard E. (in prep.). Using the respiratory and cardiac
responses of bluegills (Lepomis macrochirus Rafinesque) to
monitor zinc concentrations in water.
Spoor, W. A., T. W. Neiheisel, and R. A. Drummond. 1971. An
electrode chamber for recording respiratory and other move-
ments of free-swimming animals. Trans. Amer. Fish. Soc.
100(1): 22-28.
Train, R. E. (Ed.). 1970. The First Annual Report of the Council
on Environmental Quality. U.S. Govt. Printing Office. 326 pp.
54
-------
SECTION VIII
PUBLICATIONS
Sparks, R. E., W. T. Waller, J. Cairns, Jr. and A. G. Heath. 1970.
Diurnal variation in the behavior and physiology of bluegills
(Lepomis macrochirus Rafinesque). The ASB Bulletin. 17(3):
90 (Abstract).
Cairns, J., Jr., R. E. Sparks and W. T. Waller. 1971. The relation-
ship between continuous biological monitoring and water quality
standards for chronic exposure. American Chemical Society,
Division of Water, Air and Waste Chemistry. Preprints of
Papers Presented at the 162nd National Meeting. 11(2): 55-62.
Cairns, J., Jr., R. E. Sparks and W. T. Waller. 1971. The relation-
ship between continuous biological monitoring and water quality
standards for chronic exposure. Abstracts of Papers. 162nd
National Meeting, September 12-17, 1971. Washington, D. C.
WATR division, abstract no. 19.
Cairns, J., Jr., R. E. Sparks and W. T. Waller. 1971. (manuscript
in press) The relationship between continuous biological
monitoring and water quality standards for chronic exposure.
Cairns, J., Jr., R. E. Sparks and W. T. Waller. 1971. (manuscript
in press) The use of fish as sensors in industrial waste lines
to prevent fish kills.
55
-------
SELECTED WATER
RESOURCES ABSTRACTS
INPUT TRANSACTION FORM
1. Report No.
2.
4. Title
THE USE OF FISH MOVEMENT PATTERNS TO MONITOR ZINC
7. Autbor(s)
Cairns, John, Jr.
Waller, William T.
9. Organization
Virginia Polytechnic Institute and State University
Biology Department and Center for Environmental Studies
12. Sponsoring Organization
15. Supplementary Notes
3. Accession No.
w
5. Report Date
6.
8. Performing Organization
Report No.
10. Project No.
11. Contract I Grant No.
18050 6bp
13. Type of Report and
Period Covered
16. Abstract -j^g feasibility of using fish movement patterns measured by light beam
interruption as a technique for continuous monitoring of the response of fish to zinc
was investigated. In conjunction with the monitoring studies the growth and reproductive
success of the Bluegill sunfish (Lepomis macrochirus) exposed to various fractions of
the lowest concentration of zinc detected by the monitoring apparatus were studied.
The monitoring apparatus does not in any way interfere with fish movement within the
test chamber and allows for the maintenance of fish for long time periods. Under the
conditions described the system detects premortal aberrations in fish movement caused
by zinc. The detection of stress occurs in sufficient time to permit survival of the
test fish if stress conditions are reversed at the time of detection. The lowest
concentration of zinc detected by the system during a 96-hour exposure was between
3.64 and 2.94 mg/1 Zri*"1". The system's range of effective measurement as related to
turbidity is discussed. This method should detect other toxicity equally well. The
growth and reproductive success of the bluegill was tested in concentrations
approximately equal to 1/10 and 1/100 the lowest concentration of zinc detected by
the monitoring system and 1/100 of the 96 hour TL50 (median tolerance limit) determined
under continuous flow conditions. The growth and reproductive success in 1/100 the
lowest detected zinc concentration and 1/100 the 96 hour TL50 value did not differ
appreciably from the controls while a concentration of approximately 1/10 the lowest
vine- f*rmrf*nl"rat-i nn "in pf-Fprt" pin tninafpfl rppTndiir t"i nn in the bluegill. _
17 a. Descriptors
*Water pollution control, industrial wastes , *Bioindicators , Fishkill, Fish
physiology
776. Identifiers
*Biological monitoring, Bluegill, Zinc, Lepomis macrochirus Rafinesque
17c. COWRR Field & Croup Q5G Q5C
18. Availability
19. Security Class.
(Report)
20. Security Class.
(Page)
Abstractor
21. No. of
Pages
22. Price
Institution
Send To:
WATER RESOURCES SCIENTIFIC INFORMATION CENTER
U.S. DEPARTMENT OF THE INTERIOR
WASHINGTON. D. C. 2O24O
WRSIC 102 (REV. JUNE 1971)
CPO 913.261
------- |