EPA-R3-73-045
Ecological Research Series
JUNE 1973
Effects of Copper
on the Locomotor Orientation
of Fish
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
been grouped into five series. These five broad
categories were established to facilitate further
development and application of environmental
technology. Elimination of traditional grouping
was consciously planned to foster technology
transfer and a maximum interface in related
fields. The five series are:
1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
4. Environmental Monitoring
5. Socioeconomic Environmental Studies
This report has been assigned to the ECOLOGICAL
RESEARCH series. This series describes research
on the effects of pollution on humans, plant and
animal species, and materials. Problems are
assessed for their long- and short-term
influences. Investigations include formation,
transport* and pathway studies to determine the
fate of pollutants and their effects. This wcrk
provides the technical basis for setting standards
to minimize undesirable changes in living
organisms in the aquatic, terrestrial and
atmospheric environments.
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EPA-R3-73-045
June 1973
EFFECTS OF COPPER ON THE
LOCOMOTOR ORIENTATION OF FISH
by
H. Kleerekoper
Texas A&M University
Department of Biology
College Station, Texas 77843
Project #R800995 (18050 DWQ)
Project Officer
Dr. James M. MeKim
National Water Quality Laboratory
Environmental Protection Agency
6201 Congdon Boulevard
Duluth, Minnesota 55804
For sale by the Superintendent of Documents, U.S. Government Printing Office, Washington, D.C. 20402
Price $1.25 domestic postpaid or $1 GPO Bookstore
Prepared for
OFFICE OF RESEARCH AND MONITORING
U.S. ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON..D.C,.20460
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EPA Review Notice
This report has been reviewed by the Environmental
Protection Agency and approved for publication.
Approval does not signify that the contents necessarily
reflect the views and policies of the Environmental
Protection. Agency, nor does mention of trade names or
commercial products constitute endorsement or recommenda-
tion for use.
ii
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ABSTRACT
The effects of copper ions at subacute concentrations on the loco-
motor orientation of goldfish (Carassius auratus), channel catfish
(Ictalurus punctatus), largemouth bass (Micropterus salmoides),
white sucker (Catostomus commersoni commersoni) and green sunfish
(Lejjomis cyannelus) were investigated in detail.
i I
In regions of water containing 11-17 yg/1 Cu (as CuCl2) in a
shallow gradient goldfish oriented toward the copper source
("attraction"). This response is reduced in a somewhat steeper
gradient. In steep gradients significant but no absolute avoidance
behavior occurred. Whether the response will be "avoidance" or
"attraction" seems to depend on the slope of the gradient to which
the fish is exposed. Even in steep gradients, the "avoidance"
behavior is reversed to "attraction" when the copper ions interact
with a temperature slightly higher (,4C) than that of the surrounding
copper free water. The interaction creates a new stimulus configura-
tion which is different from those formed by the two variables
separately,
The orientation of the largemouth bass is.not affected by copper ions
at the concentrations tested. Channel catfish are weakly attracted
by the copper-containing water and green sunfish significantly in-
crease time spent there. Suckers significantly but not absolutely
"avoid" such water through changes in turning behavior.
The report was submitted in fulfillment of Project Number R800995
(18050 DWQ) under the sponsorship of the Water Quality Office,
Environmental Protection Agency.
iii
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CONTENTS
Section
I
II
III
IV
V
VI
VII
VIII
IX
X
Conclusions
Recommendations
Introduction
Experimental Rationale
The Orientation of Goldfish (Carassius auratus)
in Response to a Shallow Gradient of a Sublethal
Concentration of Copper in an Open Field
Results
Discussion
The Locomotor Response of the Goldfish, Channel
Catfish and Largemouth Bass to a "Copper-
Polluted" Mass of Water in an Open Field
Conclusions
The Locomotor Response to a Steep Gradient of
1
3
5
7
27
37
39
45
63
XI
XII
XIII
XIV
XV
Copper Ions by the Goldfish 65
Interaction of Temperature and Copper Ions as
Orienting Stimuli in the Locomotor Behavior of
the Goldfish 73
The Locomotor Response to Copper Ions in the
White Sucker (Catostomus commersoni commersoni)
and Green Sunfish (Le ponds cyanellus 87
Acknowledgments 89
References 91
Publications 97
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FIGURES
PAGE
1 MONITOR TANK # 1 9
Z SCHEMATIC CROSS SECTION OF MONITOR TANK # 1 11
3 DIAGRAM OF INTERFACING BETWEEN THE MONITOR, COMPUTER,
AND PERIPHERAL DATA-PROCESSING COMPONENTS 15
4 SCHEMATIC CROSS SECTION OF MONITOR TANK # 2 21
5 SCHEMATIC VIEW OF THE WATER SUPPLY AND COPPER INFUSION
SYSTEM OF MONITOR TANK # 1 25
6 DISTRIBUTIONS OF SODIUM CHLORIDE IN THE TANK 1 AND
2 HRS. AFTER THE START OF THE INFUSION 29
7 BINOMIAL DISTRIBUTION OF THE SIGNIFICANT DEVIATIONS
OF HEADINGS ON EXPOSURE TO CuCl2 AS COMPARED WITH
NaCl OF THE SAME CHLORIDE CONCENTRATION 43
8 THREE-DIMENSIONAL REPRESENTATION OF MONITOR TANK
SHOWING THE ADVANCE OF THE CuCl2 BOUNDARY 47
9 MEAN INDEX OF ORIENTATION IN GOLDFISH FOR NINE TRACKS
AT 0, 50, AND 100 yg/1 GU++ 57
10 OVERALL MEAN INDEX OF ORIENTATION IN GOLDFISH FOR
0, 50, AND 100 yg/1 Cu+l- 59
11 ORIENTATION IN TRACKS (PREFERENCES FOR AN ANGLE
CATEGORY AND ITS 180° COUNTERPART) AT 50 AND 100
Ug/1 Cu++ IN GOLDFISH 61
12 THE RATIO OF THE TREATMENT MEAN TO THE CONTROL MEAN
FOR PERCENT ENTRIES INTO COMPARTMENTS WITH AND WITH-
OUT COPPER (MONITOR #2) 69
13 THE RATIO OF THE TREATMENT MEAN TO THE CONTROL MEAN
FOR PERCENT TIME SPENT IN COMPARTMENTS WITH AND
WITHOUT COPPER (MONITOR # 2) 71
14 ARRANGEMENT OF EXPERIMENTAL CONDITIONS OF TEMPERATURE
AND COPPER IN MONITOR #2 77
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PAGE
15 EFFECT OF HEAT ON THE MEAN PERCENT TIME SPENT IN
COMPARTMENTS WITH AND WITHOUT COPPER (MONITOR #2) 79
16 EFFECT OF HEAT ON THE MEAN PERCENT OF ENTRIES INTO
COMPARTMENTS WITH AND WITHOUT COPPER (MONITOR #2) 81
17 EFFECT OF COPPER ON THE MEAN PERCENT TIME SPENT IN
COMPARTMENTS WITH AND WITHOUT HEAT (MONITOR #2) 83
18 EFFECT OF COPPER ON THE MEAN PERCENT ENTRIES INTO
COMPARTMENTS WITH AND WITHOUT HEAT (MONITOR #2) 85
vii
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TABLES
Np_, Pagg
1 Chemical Characteristics of a Representative
Sample of the Laboratory Water 23
2 Number of Events on the X^ Y. Photocell Coordinates
for One Representative Experiment in Each of the
49 Areas of the Tank 32
3 Estimates of Polynomial Coefficients and Goodness
of Fit Tests for a Single Locomotor Characteristic
in One Representative Experiment 33
4 Frequency of Heading Class Versus Treatment for One
Representative Experiment 35
5 Test of the Wilcoxon Rank Sum Statistic of Changes
in Locomotor Characteristics Resulting From
Exposure to CuCl- 38
6 Significant Deviations in Headings Following the
Change 40
7 Wilcoxon Rank Sum Test for Responses by the Large-
mouth Bass, Channel Catfish, Goldfish to Copper 52
8 Summary of Results for Wilcoxon Rank Sum Test for
the Largemouth Bass, Channel Catfish, and Goldfish 54
9 Analysis of Variance on Headings of Goldfish in
0, 50, and 100 yg/1 Cu++ 55
10 Experimental Conditions of Temperature and Copper
in Monitor #2 74
viii
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SECTION I
CONCLUSIONS
In some species of fish the locomotor response to subacute
concentrations of copper ions results in changes in orientation.
The net result of these changes may be "attraction" or "avoidance",
orientation towards copper-containing zones of water. The evidence
indicates that the direction of the orientation is relatively independent
of the maximum concentration of the metal but is determined particularly
by the gradient of copper ions as the fish pass from laboratory water to
copper ion containing water. A shallow gradient leading to the zones of
maximum copper concentration elicits a clear "attraction", a medium steep
gradient decreases that response somewhat, and a steep gradient results
in significant "avoidance". However, in the latter condition, highly
significant "attraction" is again obtained when a temperature rise of ,4C
is associated with the copper ion. Copper and temperature interact to
elicit orientation response not produced by either copper alone or temperature
alone. Responses of this type can only be assessed through detailed
monitoring of locomotor characteristics and careful data analysis.
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SECTION II
RECOMMENDATIONS
It is recommended that the interaction of environmental variables
be emphasized in future studies. It is most unlikely that in natural
conditions variations in a single variable occur without simultaneous
variations of one or more other variables. Thus, new stimulus
configurations arise, the animal's response to which may be entirely
different from the response to variations of the individual components
of the configuration.
The "typical" experimental procedure: "all conditions constant except
the variable under study" ignores the possible interaction of variables
as they affect the animal's response.
The heat-copper interaction reported here provides clear evidence for
the need to analyze the effects of interaction of commonly occurring
environmental variables on the locomotor behavior of fish.
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SECTION III
INTRODUCTION
The very extensive literature on the effects of heavy metal ions on
fish deals almost exclusively with various aspects of the acute
effects of exposure to these substances. Only recently has attention
been directed to chronic effects of subacute concentrations of salts
of various heavy metals. Most of the available information in this
area refers to effects of exposure to heavy metals on the physiology
and viability of fish. Very scarce indeed is quantitative information
on the behavioral toxicology of these substances as it affects locomotor
behavior, particularly locomotor orientation. This gap in knowledge
is especially serious in the assessment of the potential harm to
fishes of heavy metals in the environment.
The potential danger for fish of any toxic substance in the environment
is greatly affected by the orientation behavior which the substance
elicits in the animals. A naive fish, that is a fish not previously
exposed to the substance under study, may spontaneously respond to
the perception of the toxic by orienting away from the source ("avoidance"
behavior), by approaching it ("attraction"), or-by "ignoring" it
insofar as orientation is concerned. Obviously, a polluting substance
responded to by effective "avoidance" orientation has a far lesser
harmful potential than one that elicits "attraction" or is responded
to indifferently.
The research here reported addresses itself to this and some other
problems.
The theoretical and experimental approaches were designed to acquire
quantitative information of the highest attainable reliability on the
locomotor orientation in various experimental conditions and to treat
this information with the best available statistical techniques so
that the results might not only inspire confidence but constitute
bases for generalization applicable to field conditions. In pursueing
this aim two major points had to be considered. 1. locomotor behavior
is highly variable over time, requiring the monitoring of the behavior
over.prolonged periods for all experimental conditions. 2. oriented
behavior only exceptionally displays an obvious directionality,
recognizable by more or less casual, descriptive techniques or by
direct observation over short periods. Some of the results reported
here make this clear. The orientation of the locomotor behavior of
fish is affected only subtly by changes in the environment. Changes
in frequency and sizes of turns, length of steps between turns, time
of sojourn in environmentally differents areas, velocity, and other
parameters constitute most often the only behavioral responses to
environmental change. The totality of these responses over time
-------
determine the final change in locomotor orientation, that is, whether
"avoidance" or "attraction" will occur. The experimental and analytical
complexities resulting from this behavior as it relates to the problems
with which we are concerned here are formidable. Consequently, much
preliminary experimentation, testing of statistical techniques on the
preliminary data acquired, designing and "debugging" of computer
programs constitute the massive background, not readily discernable
by the casual reader, of the research reported in the following sections
which represents 106 experiments with five species of fish.
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SECTION IV
EXPERIMENTAL RATIONALE
The experimental design of the research here reported aimed at
obtaining accurate quantitative descriptions of the locomotor
behavior of the experimental fish prior to and during the exposure
to subacute concentrations of copper ions in a free choice,
"open field" situation. The data treatment was designed to
assess the differences in the behavior as the fish moved from
laboratory water to copper-containing water. Locomotor behavior
can be quantitatively described when the following parameters
are known: 1. magnitude of turns; 2. length of straight
paths or steps between turns; 3. absolute orientation of the
fish at each displacement; 4. velocity.
Variability in time of these parameters requires long term
observations which can only be obtained through automatic
monitoring and data acquisition. Two monitoring tanks were used,
each yielding information of different but complementary nature
and requiring specific treatment of the data obtained from each
monitor.
The monitoring systems used in this research consist of: A. data
acquisition and processing equipment, and B. a number of
peripheral mechanisms to control and monitor the environmental
parameters to be investigated. Part A comprises: 1. two
experimental tanks which monitor the locomotor activity of a
fish, and 2. computation and data processing systems including a
number of computer programs.
Monitor Tank #1; It measures 5.00 x 5.00 x .5m and holds, embedded
in its floor, a square matrix of 1936 photoconductive cells on
10 cm centers, their light sensitive faces directed upwards (Fig. 1).
The cells are activated by collimated light which, from overhead,
illuminates, at 2500 K, the whole surface of the tank uniformly, and
at low intensity. Collimation is obtained through Fresnel lenses which
cover a flat black aluminum honeycomb, 5 cm deep, suspended horizontally
over the whole area of the tank (Fig. 2). The photoconductive cells
were selected for peak response in the far red so that the for the
fish eye visible part of the spectrum may be filtered out without
affecting the operation of the sensors. Interception of light by the
presence of the fish over a photocell increases the resistance of the
cell, an event which affects an electronic logic circuit, formed by
the x and y axes of the cell matrix. This circuit interfaces with
the data acquisition system outlined below.
-------
Figure 1
Monitor Tank # 1
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Figure 2
Schematic Cross Section of Monitor Tank #1
10
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u
u c
J (J
u
\
u o~ oo~
Square matrix of lamps — *
4
£
fe
f'1
-— -1
r
L_
5$
^
--u.
•L"
„ ^-"
/
, rresnei lenses y
1 T-pl 1- - "
1 '
) >V~- honeycomb collimator/
m
CO
ir
i
ceramic filter plates
_^D_
r^-.
i i i .1
logic interface to
computer
'^2S^S^^^^ I
I
square matrix of photocells on
ters
^L
-------
The tank is isolated from all control and processing equipment,
and is located in a room which, like the tank itself, is painted
a flat black throughout.
*
Data Acquisition and Processing; A small computer is interfaced
on-line with the cell matrix of the monitor and is programmed
to locate the position in the matrix of the photocell affected by the
presence of the fish. Simultaneously, a real time clock in the system
determines the time elapsed between the event in question and the
immediately preceding event. These three coordinates, x, y, and time,
form the basis for a series of computations by the computer which
determine direction and size of angles of turn, distance between
successive photocells shaded by the fish, velocity of locomotion,
absolute direction of the movements in the tank, length of step between
successive turns and "handedness" of the animals. Angle resolution is
better than 4° for 30 cm long goldfish. These basic computations could
be printed out on a teletypewriter but were more commonly recorded
on magnetic tape. In addition, the displacements of the fish could be
displayed on a cathode ray tube (Fig. 3).
The magnetic tape record was used, as needed,
1. To obtain from a plotter a graphic record of the locomotor
pattern during all or part of an experiment;
2. to feed back data to the computer for additional computations;
3. to feed back to an IBM 360 computer, available on campus, for
the execution of a variety of programs which were designed to analyze
the data collected from a variety of viewpoints, (see below).
Peripheral Equipment and jlechanisms to Control and Monitor Environ-
mental Conditions Related to the Problems of this Research
1. Water supply and flow control.
Water is admitted to the tank through any one of four peripheral
channels, located along the four walls of the tank, which are in
open communication with the tank through porous plates (Fig. 2).
It then enters the main body of the tank through the plates along
one side and leaves through the plates along the opposite side of
the tank. This arrangement brings about an almost perfect laminar
flow across the tank. The direction of flow and its rate can be
regulated through remote control from an adjacent room.
2. Temperature control.
The temperature of the supply water was controlled at 21 C + 1C.
12
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Treatment of Data from Monitor #1. The data obtained from the monitor,
recorded on magnetic tape, supplied basic information on the locomotor
behavior prior to and after the experimental change and provided a
plotter-produced tracing of the locomotor pattern. To this effect,
all or some of the following computer programs, were applied.
1. Event by event printout'including x, y, time, distance, velocity,
angle in tank, change in angle.
2. Hour by hour breakdown including turns per hour, percent right,
percent left, distance per hour, percent without turns, average length
of straight line, average velocity.
3. Frequency distribution of the changes in direction for a range
of 0 - 180° in 10° classes.
Frequency distribution of the velocities for a range of 0 - 95
cm/sec, in 5 cm/sec. classes.
4. Frequency distribution of the distances travelled between events
for a range of 0 - 120 cm in 10 cm classes.
5. F statistic for analysis of variance of group sizes from 2 to 50
for the first N events.
6. Revisitation of cells including a printout of all the cells in the
tank and the number of times each was fired plus a listing of the number
of cells fired one time, two times, three times, etc.
7. Frequency distribution of the changes in direction (range 0 -
180°) in 10° classes in one hour periods. These distributions could
be cumulated hour by hour.
Frequency distribution of velocities (range 0-95 cm/sec for 5 cm/sec
classes) in one hour periods. These distributions could be cumulated
hour by hour.
8. Contingency table on the relation of consecutive angles of turn.
9. For classes of 1000 events: the number of changes in direction,
the average change in direction without regard to sign, the average
length of a straight path and the average velocity,
10. Distribution of distances travelled with respect to orientation.
Orientation is expressed as 10° classes from 0 - 360° (actually
9.75° classes with 359.75° to 360° included with 0° class).
13
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Figure 3
Diagram of Interfacing Between the Monitor, Computer ,
and Peripheral Data-processing Components
14
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CRT display of
position of fish
matrix of 1936
photocells
directional control
of water flow
on line computer <*- real time clock
incremental
mag.recorder
plotter
input-output
teletypewriter
t
programming
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11. Distribution of distances travelled with respect to orientation.
Orientation is expressed as 10° classes from 0 - 360° (actually 9.75°
classes with 359.75° to 360° included with 0° class).
12. Correlation coefficient of incoming angle to outgoing angle in
10 cm distance classes for a range of 0 - 500 cm.
13. Orthogonal polynomial surface fit (see below).
''Block-time" listing
Any one of these programs, where applicable, could be restricted to
data from specific parts of the tank so that the spatial effects of
variable gradients, currents as well as that of artefacts (the walls
and corners of the tank) could be analyzed.
Special attention is called to program 13 which allowed us to test the
statistical significance of changes in any one of the locomotor
parameters mentioned with a very high degree of sensitivity and to
represent the change spatially as a surface fit representing the total
area of the tank.
In view of the important interpretative role this test played in the
research, an outline of the procedure follows.
The square tank was subdivided into a 7 x 7 area and for each sub-
division the following (X) characteristics are derived:
1) frequency
2) straight path length
3) other characteristics
The three-fold statistical objective was 1) to fit an empirical
polynomial model for each characteristic, 2) to test the equality of
such surfaces over different treatments, and 3) to interpret the
differences in surfaces provided they are statistically significant.
The above analysis was performed for each of the (X) characteristics
individually. Each phase is discussed separately below.
Polynomial Model; The data for a particular characteristic, call it
Z, consist of individual responses, Z-M , for each of the seven
equidistant X-^ coordinates of the tank and for each of the seven
equidistant Yj coordinates. This two-dimensional layout is statisti-
cally benign in permitting the use of two-dimensional orthogonal
polynomials. Such polynomials are not only computationally efficient
and accurate but also allow the order of the necessary fit equation
to be readily determined. Preliminary analyses of the various
characteristics suggested in most cases both the statistical necessity
16
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as well as sufficiency of a fifth order model. Hence the following
analysis assumes the fifth order equation; any other order would modify
subsequent discussion only slightly.
The resultant equation may be written as:
Zij = Boo + B10 Xi + B01 Yj + B20 Xj + Bll xi Xj
+ B Y2 + ... + B X^ +
4_ "R Y V^1 -4- "R V I A
+ B14 X± Y.J + BQ5 Y.. + ei;j
for i = 1, ..., 7 and j =1, ,.., 7
where
Z. . denotes the Z response of the i, j1-"- cell
X-£ denotes the i*-*1 position on the X axis
YJ denotes the j^ position on the Y axis and
eji denotes the error term of the i, jt" cell.
The variances of the observations are assumed equal and the covari-
ances slight, hence the ordinary least squares method is used to fit
the above 21 coefficients to the 49 data.
Surface Equality. The next phase of the statistical analysis tested
the null hypothesis of whether the three surfaces were equal for
different treatments. The standard procedure for linear regression
models (see e.g. Ostle (1963) p. 201) may be generalized to multiple
regression by 1) fitting the above equation to the three sets of 49
responses simultaneously and then 2) comparing that residual sum of
squares to the total of the individual sums of squares. An F test
was then performed which in this case has 42 and 84 degrees of freedom.
Region of Difference. The third phase was conducted upon the rejection
of the equality of surfaces hypothesis above the attempts to identify
the source of the significant differences. The treatment was applied
in such a manner that only certain of the 49 total cells were affected.
Based on this and prior knowledge, the tank was divided into several
regions of interest according to the concentration of treatment and
tank location. The third phase investigates whether the pattern of
residuals changed from region to region. An affirmative answer to
the above question suggested that such changes caused the difference
in polynomial fits.
Since fish activity varied a great deal from cell to cell, the cells
of a specific region were considered individually to overcome the
17
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variability by pairing. For a particular cell, the
response of the water treatment was compared with, the copper treatment.
Two paired non-parametric tests, the sign test and the signed rank
test, tested the hypothesis of a difference in response for that
given region. Also the homogeniety of the response over the different
regions was tested by the chi-square statistic.
Monitor // 2j^ This equipment is illustrated in figure 4. Cylindrical
tank (9) is made of acrylic and has double walls, 50 cm high, which
form a peripheral channel, 7.5 cm wide (17). The central remainder
of the tank has a diameter of 200 cm. Sixteen hollow dividers,
50 cm long; 5 cm wide, 35 cm high, extend radially from the inner
wall toward the center to create 16 peripheral compartments of
equal dimensions and an open central area. The dividers contain
light sources and photocell arrays with peak response at 700 nm
and form photoelectric gates which are activated by the passage
of a fish. The peripheral channel (17), receives fresh water of
which the ion content can be controlled and varied or synthetic
seawater. Either medium is treated through rapid and diatomaceous
earth filters. At the peripheral center of each compartment water
enters the equal rate from the channel (17) through siphons and leaves
centrally through pipe 18. Entries and exits of compartments by
the fish are monitored as to compartment number, solar time of entry
and time of exit, by the electronic interface and recorded through
a paper punch for computer processing (Kleerekoper, 1967, 1969,
Kleerekoper et_ ajU} in press).
The tank rests on a neoprene vibration-damping pad (11) on a platform
(12) . On the ceiling of the experimental room a bank of 36 fluores-
cent lamps (1) is mounted whose light passes through a diffuser (2)
of white translucent plexiglas and a cloth of white cotton (3).
Curtain 8 provides for a lightproof connection between tank and
"sky". No extraneous light enters the experimental room.
To produce temperature differentials between compartments heating
pads were used. Each pad is a nearly flat, black, silicone rubber
mat in which resistance wire is embedded the electrical power to which
was controlled.
Flow rate in each of 16 compartments was regulated individually.
Statistical Procedures, Monitor 2: Temperature and flow rates were
arranged so as to obtain a variation of conditions in the 16 compart-
ments; the percent distributions of the frequency of entry and time
spent in each compartment were analyzed in a fractional factorial
design.
Variability between fish was eliminated by blocking and bias due to
intangible physical factors was minimized through randomized assign-
ment of experiment conditions to the 16 compartments. Variability
18
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due to conditions was partitioned into contrasts and selected treat-
ment comparisons to isolate the effects of each.
Water Supply; Both montitors were supplied with continuously flow-
ing tap water filtered through activated carbon. A representative
chemical analysis of this water is shown in Table 1.
Introduction of Chemicals; Glass syringes, actuated by a multi-
channel infusion-withdrawal pump mechanism of continuously variable
speed under servo control, withdrew a. concentrated solution of either
sodium chloride or copper chloride from a supply vessel and delivered
the solution at the required rate through silastic tubing and stain-
less steel fittings into the monitors.
For monitor #1 the solutions were introduced through the perforated
water pipe supplying 1/3 of the length of one channel (Fig. 5).
Individual compartments of monitor #2 received the chemical solutions
through side arms of the glass water supply tubes.
Measurements of Ion Distribution in the Tank; Since a major aspect of
the research concerned the effects of copper ion distribution on the
orientation of locomotion, accurate information was required on the
distribution patterns of the ion in the tank. This was measured as
follows . Thermistors and conductivity cells of the flow-through type
were mounted horizontally in pairs (one thermistor and one cell), in
a rectangular matrix on 10 x 20 cm centers, on an open supporting rack.
The dimensions of the latter were those of the vertical cross section
of the water in the tank and allowed for a grid of 5 rows (10 cm interval)
and 23 columns (20 cm interval) of sensor pairs. These were connected
through an interface to the computer, which was programmed to record
electrolytic conductivity and temperature, together with the positions
of the sensors, on magnetic tape, and/or to print out these results on
the teletypewriter. The temperature data allowed for correction of the
conductivity readings of the individual cells when slight temperature
differences occurred. The data required for the construction of the
profiles of ion distributions were obtained by placing the grid of sensors
vertically in the tank and recording the conductivity and temperature
values registered by the 115 pairs of sensors, an operation which required
less than 5 minutes. Vertical conductivity profiles were recorded at
various distances from the water inlet. At the start of each recording,
sodium chloride solution was introduced into the water entering section B
(Fig. 5) of the water supply channel at a constant rate; Recordings
were repeated at 5 minute intervals.
The Experimental Fish; Prior to the experiments, the fish were kept
in the laboratory in large holding tanks in water at 18-20°C continuously
circulating through filters of activated carbon. The fish were fed
with "trout chow" while in the holding tanks but were not fed in the
monitor tanks, in which they remained only during the recording periods.
19
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Figure 4
Schematic Cross Section of Monitor Tank # 2
20
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\\\\\\>\\\\\\\\\\\\\\\\\\\\^^
\\\\\\\\\\
B r u ^ u
u
HhHh-II—I
I—I I—I t-H I—I I-H H
-------
The species used and the origin of the fish will be mentioned
separately for each series of experiments.
22
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TABLE 1. Chemical characteristics (mg/liter except as indicated)
of the laboratory water.
Carbonates (-CO )
Bicarbonates (-HCO-)
Chlorides
Sulphates (-SO )
Silica (SiO )
Ca^
iwr +*-
Mg
Na+
Hardness (CaCO )
Cu"
Zn"
Conductivity pmho
PH
10
152
47.5
10.7
18.5
1.8
0.2
99.4
5.4
0.006
0.002
162
8.4
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Figure 5
Schematic View of the Water Supply and Copper Infusio:
Svstem of Monitor # 1
24
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water supply
\
•• •
•
Infusion
pump
Stock solution
(NaCI or
Flow meter
perforated
baffles
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SECTION V
THE ORIENTATION OF GOLDFISH (CARASSIUS AURATUS) IN RESPONSE TO A
SHALLOW GRADIENT OF A SUBLETHAL CONCENTRATION OF COPPER IN AN OPEN
FIELD
Knowledge of the locomotor responses of fish to concentrations of heavy
metals was based mainly on observation of animals exposed to steep
gradients of these substances produced experimentally in narrow
troughs. The aim of this part of the study was to analyze the locomotor
response to shallow gradients of sublethal concentrations of copper
ions (as CuCl2> in an open field situation, in conditions of uniform,
slow flow (1.8 cm/s), by 30 cm long goldfish.
For this part of the study monitor #1 was used.
Glass syringes, actuated by a multi-channel infusion-withdrawal pump
mechanism of continuously variable speed under servo control withdrew
a concentrated solution of either sodium chloride or copper chloride
from a supply vessel and delivered the solution at the required rate
through silastic tubing and stainless steel fittings into the water
pipe (PVC) supplying 1/3 of the length of one channel (Figure 5).
The progression of sodium or copper chloride ions introduced into
section B of the water supply channel (Figure 5) is summarized in figure
6. Conductivity values were recorded in 5 minute intervals in the planes
indicated in the figure while the water traversed the tank at a rate
of 1.8 cm/s. Figure 6 represents the conductivity pattern at the end
of one and two hours following the introduction of the chloride with a
resolution of six micromhos. The lighter shade of grey corresponds
to the background conductivity of the supply water prior to the
introduction of the chloride, successively darker shades representing
increases in steps of six micromhos. The condition towards the end of
the third hour (not shown in the figure) indicated that only small pockets
of water remained unaffected by the chloride. During the first two
hours, however, the chloride-enriched water moved across the tank as a
mass whose boundaries are indicated in the profiles shown in figure 6
and which was flanked by "clean" water.
The information thus acquired allowed for the distinction and comparison
of the characteristics of locomotion within (Region B) and outside
(Region A) the water masses "polluted" by copper ions. Furthermore,
this information established that such comparison should be most
effective for data collected during a period not exceeding two hours
following the onset of the "pollution", that is, when polluted and non-
polluted regions in the tank were still well defined. The effects of
the movements of the fish on the patterns of the boundaries between
the two regions are unknown. However, the unidirectional, quasi laminar
flow should tend to continuously correct local disturbances produced
by fish.
27
-------
Figure 6
Distributions of Sodium Chloride in the Tank 1 and
2 Hrs. After the Start of the Infusion
28
-------
<
first hour
second hour
normal
water
NaCI-enriched
water
normal
water
NaCI
-------
Other Experimental Procedures: Seven experiments were done, each with
one naive goldfish, 30 cm long, purchased from a commercial grower in
Central Texas. Prior to the experiments, the fish had been kept in
the laboratory in large holding tanks in water at 18-20°C which continuously
recirculated through filters of activated carbon. The fish were fed
with "trout chow" while in the holding tanks, but not in the monitor
tank. Each of the seven experiments referred to in this paper consisted
of three consecutive eight^iour long recordings of the locomotion of
a single fish as follows: 1. with "normal" or "clean" water in the
entire tank to account for any effects of transfer to the monitor tank,
for behavior peculiar to an exploratory locomotor phase (Kleerekoper
et_ _al_., 1970), and of time of sojourn in the tank; 2. with NaCl solution
infused into the supply section B of the channel so as to make a chloride
concentration equivalent to that of a solution of copper chloride con-
taining 50 yg/1 Cu4"*"; and 3. with CuCl2 solution infused into section B
so as to make a concentration of 50 yg/1 Cu"*"*" in the supply water of that
section of the channel. Note that the maximum possible concentration
of copper as CuCl2 was 50 yg/1 found only at the boundaries formed
by the water exiting through the perforations in the baffle (Figure 5)
of section B and the water in the tank. Beyond these points the
concentration decreased, particularly at the interfaces with the
"clean" water which bounded the mass of advancing chloride-polluted
water. Calculations based on the actual electrolytic conductivity
profiles indicate that in large proportions of the mass of polluted
water the concentration did not exceed 50 J-tg/1 Cu^~ with very shallow
gradients leading to the clean water.
At the high electrolytic conductivity of the laboratory water, even
the maximum concentration of copper present in these experiments
had not caused mortality in controls after 30 days of exposure in a
separate test. The incipient lethal level (Fry, 1947) was not determined.
Treatment of Data: Changes in the locomotor behavior of the fish, how-
ever subtle, are reflected quantitatively in some or all of its loco-
motor characteristics (Kleerekoper et_ al_., 1969, 1970). This fact
was used: 1. to ascertain whether exposure to CuCl2, as compared to
NaCl of the same concentration, modified the locomotor behavior of the
fish; 2. to verify whether such modifications in locomotor
behavior were quantitatively related to the spatial distribution
pattern of Cu"*"*" ions in the tank; 3. to establish whether the
modifications resulted in a change in the orientation of the movements
and, if so, whether that change was correlated with the spatial
distribution of the copper ions; 4. to establish the effects of
transfer of the fish to the monitor tank, exploratory behavior, and
time in the absence of any "pollutants".
The locomotor characteristics considered in this study were number
of events (frequency of photocell responses triggered by presence
of fish); time accumulated in an area; distance accumulated; average
30
-------
velocity; number of turns; average size of turn; variance of size of
turn; and heading or orientation of movements.
Changes in any one of these characteristics were investigated by the
following statistical techniques: 1. fitting of empirical poly-
nomial models for each characteristic; 2. testing of equality of
such models over the different treatments, and 3. an interpretation
of the differences in the models, provided that the hypothesis of
model equality was rejected.
The data for a particular characteristic, say Z, consisted of
observations, Z^, for each point of a 7 x 7 grid of equidistant
X^ and YJ photocell coordinates representing locations in the tank.
Table 2 illustrates the data by listing the observations concerning
a single locomotor characteristic for a representative fish under
the NaCl and CuCl2 treatments. Preliminary analysis of all data
sets by two-dimensional orthogonal polynomials suggested that in
most cases a fifth order polynomial was statistically necessary and
sufficient to adequately describe the surfaces. The resultant equation
Zij = B00 + B10XiB01Yj + E20x2i + BllXiYj + B02Y2j + "-B05Y5j + £ij
where £.. denotes the error term. The ordinary least squares
procedur^ was used to provide the best estimates of the B-JJ coeffi-
cients. Table 3 contains both the B— estimates for the data of
Table 2 and a goodness-of-fit criterion.
The polynomial models, besides describing the data, were then used
to test the linear hypothesis that a NaCl model equalled the
corresponding CuCl2 model. The hypothesis was tested by an F
Statistic from the residual sums of squares (see e.g. Ostle, 1963,
p. 201). Table 3 supplied the necessary additional statistics for
the representative data above.
Upon rejection of the hypothesis of model equality, the third objective
was to identify the source of the inequality. The data were paired
to block out the effect of location of response; thus, a differential
response was determined for each location by subtracting the Z-j_j
observations between treatments. Since the treatment was concentrated
in only 25 of the 49 locations, the W^ differences of these 25
"treated" locations were compared with the 24 "control" locations by
the Wilcoxon rank sum statistic (Wilcoxon and Wilcox, 1964) . This non-
parametric statistic tested the hypothesis that the mean difference
in the treated region, say u^, equals the mean difference in the control
region, say u2. Rejection of HQIU^ - U2 established that the in-
equality of models was, at least partially, due to the treatment.
Abundant time series data were also available on the absolute
headings of each fish, indeed the total number of headings recorded
31
-------
Table 2. Numbers of events* on the X., Y. photocell coordinates for
experiment 172 in each of the 49 areas of the tank.
X.
1
2
Y, 3
j
4
5
6
7
1
2
YJ 3
j
4
5
6
7
58
20
26
21
13
9
29
34
22
25
20
17
9
42
35
8
17
20
20
14
44
29
19
30
26
20
12
14
Under
29
11
10
20
15
7
73
Under
21
8
10
21
18
8
27
NaCl
39
7
9
17
13
10
31
CuCl2
24
10
16
11
17
18
27
42
19
25
22
14
2
35
23
18
17
10
19
5
26
40
14
18
26
11
13
49
16
15
18
7
13
6
24
72
19
32
27
27
15
38
42
23
24
20
27
20
40
*Numbers of times any of the photocells of one area are triggered.
32
-------
Table 3. Estimates of polynomial coefficients (B ) and goodness of fit
tests for number of events in experiment 172. "^
Estimates for individual treatments Estimates for combined
Boo
B10
Boi
B20,
Bll
B02
B30
B21
B12
B03
B40
B31
B22
B13
B04
B50
B41
B32
B23
B14
B05
NaCl
24.18
0.93
-0.65
0.94
-0.28
2.90
1.12
-0.56
0.08
0.86
-0.00
1.35
0.00
-0.29
1.70
-0.05
-0.27
0.31
-0.56
0.06
-0.04
Total SS 11901.4
Residual SS 1312.2
F. statistic 11.30
CuCl2
19.76
-0.08
-0.26
1.09
0.13
1.14
1.74
-0.10
0.11
1.36
0.30
-0,17
0.11
-0.22
0.72
0.44
0.14
-0.28
0.24
-0.10
0.19
3689.1
857.1
4.63
treatments
21.97
0.42
-0.45
1.02
-0.08
2.02
1.43
-0.33
0.06
1.11
0.15
0.59
0.06
-0.25
1.21
0.19
-0.07
0.02
-0.16
-0.02
0.08
71434.8
60931.0
0.66
33
-------
varied from roughly 2,000 to 6,000. The frequencies of individual
headings were summarized for each fish with a 2 x 16 table according
to the treatment (NaCl vs. CuCl2) and consecutive orientation classes
of 22 1/2 degrees each. Table 4 illustrates for experiment 172 the
data as produced by the on-line computer.
Since exact angle resolution was not attained (in present experi-
mentation) , the angle classes are not equally probable; however,
subsequent tests are not biased by this fact. A y^ statistic was used
to test these multinomial distributions for homogeneity of the
populations of headings under NaCl and under CuCl9.
Upon rejection of the homogeneity hypothesis, a series of subsequent
X^ statistics identified which particular class or classes of headings
differed significantly. For each fish» each class was analyzed to
determine whether its proportion in the NaCl population of headings
equalled its proportion in the CuCl_ population.
Assuming now that the seven fish constitute a random sample from a
larger population of fish, the sign test indicates whether the popula-
tion as a whole prefers a specified orientation under one treatment
relative to that under the other. In the absence of such effect, the
observed relative frequency of u will exceed that of v with probability
0.5 Hence if the relative frequency of u exceeds that of v for all
seven fish there is evidence (at a = .01) that the population prefers
the specified orientation to that under u relative v.
34
-------
UJ
Table 4. Frequency of heading class v. treatment for fish in experiment 172.
Treatment
0-22
Classes of Headings in Degrees
23-45 46-67 68-90 91-112 113-135 136-157 158-180
NaCl
CuCl-
195
104
181-202
NaCl
CuCl2
181
99
35
42
203-225
36
27
84
62
226-248
51
60
36
37
249-270
39
25
120
74
271-292
97
138
29
25
293-315
41
35
81
89
316-337
79
75
53
43
338-360
28
33
-------
SECTION VI
RESULTS
The Effects of the Presence of Copper Chloride on the Locomotor
Characteristics of the Fish in the Tank as a Whole;
The results of the F tests of equality of the empirical polynomial
models demonstrated that, in six of the seven fish tested, all the
locomotor characteristics investigated were affected by substitution
of the sodium chloride by the copper chloride (?21.,5(> = 2>25 a = 0-D •
In one fish only (experiment #194), a single characteristic, time
accumulated in various areas of the tank, remained unaffected by the
change in condition.
The Changes in Locomotor Characteristic in Region B Resulting
from the Displacement of Sodium Chloride by Copper Chloride;
The results of the Wilcoxon rank sum test on these changes are re-
presented in Table 5 for seven naive fish. Increases and decreases
in the values resulting from the introduction of the copper ion into
that region are indicated by + and -; NS means that the changes are
not significant. As sodium chloride was being replaced by copper
chloride, the locomotor changes were statistically consistent for
three of the characteristics analyzed: Time accumulated, average size
of turn, and variance of the size of turn; all three increased highly
significantly in, respectively, four, five, and four of the seven fish
studied. In the remaining fish the test yielded nonsignificant
results. Throughout, three fish (experiment nos. 172, 190, 194)
behaved consistently with respect to all characteristics analyzed,
whereas, one fish (no. 173) showed inconsistency in only one characteristic f
Entirely inconsistent were the data from experiments nos. 197 and 198.
Changes in the Orientation of Locomotion in the Tank as a Whole in
Response to the Displacement of Sodium Chloride by Copper Chloride;
In all fish, the frequency distribution of all the headings during
copper chloride exposure was significantly different from the distribu-
tion during sodium chloride exposure (Table 6). The analysis of
individual headings reveals that, under copper chloride, in^five of
the seven fish the observed relative frequency of headings in the
226-248° arc significantly exceeded the corresponding frequency under
sodium chloride. Since the other two fish also exhibited the same
phenomenon (though not significantly), it is clear (a = .01) that
this characteristic is valid also for the population as a whole.
For no other angle category was this deviation consistent in the whole
sample of seven fish. That the phenomenon might extend to the whole
arc between 225-293° is suggested in the binomial distributions of
sixteen headings during exposure to sodium chloride (two hours) for
all seven fish.
37
-------
Table 5. Test of the Wilcoxon rank sum statistic of changes in
locomotor characteristics in region B of tank resulting from exposure
to CuCl2; +, increase; -, decrease; NS = not significant at a = .05
Experiment no.
Characteristic 170 172 173 190 194 197 198
No. of events
(frequency of + -\- + 4- -f - -
photocell
responses
Time accumulated
in region B + NS + + NS + NS
Distance
accumulated + -f + + +
Average
velocity NS NS - NS NS NS
No. of
turns + US + + NS NS
Average size
of turn + -f + NS NS +
Variance of
size of turn + + + + NS NS NS
38
-------
SECTION VII
DISCUSSION
The goldfish in these experiments perceived the copper ions present
in the "polluted" region of the tank at the prevailing concentration
of 5 Ug/1 and they responded with modification of their locomotor
characteristics in the tank as a whole as demonstrated by the analysis
of fitted surfaces. Within the polluted region, the orientation of
their movements deviated significantly in upstream direction as
compared to their orientation in NaCl of equivalent concentration.
This behavior resulted in an increase in time spent by the fish in
the copper—affected region. Since velocity was not affected in
five of the seven experiments and decreased in only one there is no
evidence for a stupefying effect of the copper ion on the fish (Jones,
1964). Furthermore, the total distance swum in the polluted region
actually increased for all the fish and the number of entries into
the polluted region increased for five of the seven fish, (Table 5).
Thus, the analysis must lead to the conclusion that copper chloride,
at the concentration which prevailed in the polluted region, was
either "attractive" to the goldfish or led to the entrapment of the
animal. The nature of the phenomenon becomes evident by an analysis
of the orientation behavior of the fish (Table 6, Figure 7). For the
sample of seven fish the positive deviations of the frequency distri-
bution of headings during the copper chloride exposure in comparison
with the same distribution in sodium chloride were in the 226-294°
sector, that is, in the general upstream direction in the tank, with
no consistent deviations in any of the remaining headings. A separate
analysis of only the downstream movements established that the deviations
of their headings were distributed at random. The interpretation
of these results is that the copper, in the conditions of the experiment,
released in the animals positive rheotaxis which was accompanied
neither by increased velocity nor increased turning frequency, changes
which are frequently associated with "alarm response". Distance,
on the other hand, increased in five of the seven experiments. Thus,
the significant and consistent preference for upstream swimming during
the copper pollution resulted in more extensive cruising and increased
time expenditure within the copper-affected region, as if the fish
had become entrapped there. The conclusions drawn seem to be at
variance with those of some other studies of the locomotor response
of fish to solutions of metal salts (Jones, 1947,1948; Sprague, 1963,
1964, 1968; Ishio, 1965; Burden, 1945; Hill, 1956; Syazuki, 1964;
Saunders and Sprague, 1967; Sprague et_ al_., 1965; Sprague and Ramsay
1965; Grande, 1967) which pointed to "avoidance" behavior, although
a few studies failed to show avoidance of heavy metal solutions
(Summerfelt and Lewis, 1967); in one instance "attraction" to a high
copper sulphate solution was reported (Jones, 1964). In the latter
case, the behavior was attributed to the stupefying effect of the
pollutant. It is to be noted,however, that the results reported here
and those in the literature are not readily comparable. The experimental
39
-------
Table 6. Significant deviations in headings following the change
180°
c
2;
10°
0°
?0°
No. of experiment
A
direction of flow
Headings in degrees
0-22 23-45 46-67 68-90 91-112 113-135 136-157 158-180
170
172
173
190
194
197
198
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
+
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
+
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
40
-------
of exposure from sodium chloride to copper chloride (a = 0.05)
181-202 203-225 226-248 249-270 271-292 293-315 316-337 338-360
+
-
NS
NS
NS
+
NS
NS
NS
NS
NS
NS
NS NS
+ NS
+ NS
+ NS
NS +:
+ NS
+ NS
4- NS
+
NS NS
NS +
NS NS
NS
NS
-
NS
+
NS
NS
NS
NS
-
-
NS
NS
41
-------
Figure 7
Binomial Distribution of the Significant Deviations
of Headings on Exposure to CuCl2 as Compared with
NaCl of the Same Chloride Concentration.
42
-------
o
o
5> O
•
O
ii
(/) O
C "5
2 >
o>
o
•
o
-5
0
•••••••I
en
o
i—i—i—i—i—r
i—i—r
180
Heading in degrees
360
-------
techniques used in the present study were not only very different
from those applied to the problem by other workers but the fish were
in an open field situation with a very shallow gradient as opposed
to the conditions prevailing in gradient tubes and troughs. Further-
more, the data permitted a much more detailed analysis of locomotor
behavior than had been possible hitherto. In addition, differences
in species (Ball, 1967), hardness of the water (Cairns and Scheier,
1957; Tarzwell and Henderson, 1960; Lloyd, 1960, 1961; Lloyd and
Herbert, 1962; Screenivasan and Soundar, 1963; Herbert and Wakeford,
1964), other aspects of water quality (Doudoroff and Katz, 1953;
Jones, 1939; Lloyd and Herbert, 1962; Mount, 1966; Malacca and Gruia,
1964), the interaction and additive effects of various ions (Southgate,
1932; Friedland and Rubleva, 1958; Bucksteeg et_ aU, 1955; Cherkinsky,
1957; Herbert, 1962; Herbert and Shurben, 1964; Brown e£ a^. 1969),
and acclimation (King, 1937; Paul, 1952; Afleck, 1952; Grande, 1967;
Lloyd, 1960; Pickering and Vigor, 1965; Schofield, 1965) are all
known to be important variables affecting the physiological and behav-
ioral responses to heavy metal pollution. Few of these variables
have been studied in any detail and the results of their interactions
are largely unknown. Should the locomotor behavior of the goldfish,
in response to sublethal concentrations of copper ions in an open
field, be representative of that displayed by other species, the
phenomenon described might be of considerable importance for the
assessment of the biological effects of copper pollution. In the
field, the behavior described might lead the fish towards an upstream
source of pollution, hence, into increasingly, higher concentrations
of the metal. At a critical concentration,dependent on species and
water quality, avoidance reactions (Sprague, 1963, 1964; Sprague
ej^ al^., 1965) might then replace the orientation behavior described
here. However, the response to a potentially harmful concentration,
reached through a shallow gradient, may be different from that elicited
by a steep gradient.
44
-------
SECTION VIII
THE LOCOMOTOR RESPONSE OF THE GOLDFISH, CHANNEL CATFISH AND LARGEMOUTH
BASS TO A "COPPER-POLLUTED" MASS OF WATER IN AN OPEN FIELD
The previous experiments established that in an open field, the
locomotor behavior of goldfish changed on entering a shallow gradient
of copper ions. The orientation of the fish in the copper-containing
water deviated consistently in the upstream direction and was due
to the copper rather than to the chloride ion. The concentration
in most of the mass was 11-17 yg/1 with isolated pockets containing
maximally 50 |ig/l Cu++ as CuCl2. As stated, these results are at
variance with those reported in the literature (Burden 1945; Grande
1967; Hill 1956; Ishio 1965; Jones 1947, 1948; Sprague 1963, 1964,
1968; Saunders & Sprague 1967; Syazuki 1964; Sprague & Ramsay 1965;
Sprague et_ al_., 1965) indicating distinct "avoidance" behavior,
when the fish are exposed to a steep gradient of copper ions
within a relatively restricted space.
The following experiments were designed to ascertain: 1. the behavior
of the fish when exposed to a steep gradient of copper ions in an
open field situation, and to determine whether, in such conditions,
"avoidance" behavior could be elicited; 2. the locomotor behavior
of the fish within a mass of water containing uniformly distributed
copper ions without possibility of "avoidance". The experiments
were done in monitor tank //I. The size of the perforations in the
baffles of the supply channel (B in Fig. 5) was increased resulting
in a different flow pattern and slope of the copper gradient. As
before, solutions of sodium or copper salts were delivered from a
supply vessel through silastic tubing and stainless steel fittings
by a chemical feeder into the water pipe (PVC) supplying the central
1/3 of the inlet channel (Fig, 5). When the chemicals were introduced
into section B only, the diluted solution traversed the main body of
"clean" water forming boundary diffusion gradients whose character-
istics were verified by determining the three-dimensional distribution
of the ions in the tank as described above (Section IV). The sodium
or copper salt-enriched water introduced into section B moved across
the tank as a mass whose boundaries are indicated in figure 8, During
the first two hours the ions affected about 1/3 of the mass of the
tank water. Because of the very distinct definition of that volume
of water affected by the ions (region B) and that unaffected (region A)
after two hours of recording, locomotor characteristics in these two
regions could be accurately analyzed and compared during that period.
After the initial two hours the copper ion gradually contaminated
the whole water mass of the tank.
Thirty-four experiments were done, each with a naive fish. Twenty
of the test fish were goldfish (Carassius auratus), 28-30 cm long and
obtained commercially in Texas, seven were largetnouth bass (Micropterus
salmoides), 28-30 cm long, aad seven were channel catfish (Ict_alurus
45
-------
Figure 8
Three-dimensional Representation of Monitor Tank
Showing the Advance of the CuCl2 Boundary
46
-------
water supply
\
i
'
Infusion
pump
Stock solution
(NaCI or CuCL)
Flow meter
perforated
baffles
-------
punctatus) , 32-35 cm long. The latter two species were obtained from
the State Fish Hatchery at Huntsville, Texas. The goldfish were held,
prior to experimentation, in large community tanks and the bass and
catfish were kept individually in smaller tanks. The water was
18-20 degrees C and was recirculated through carbon filters . The
goldfish and catfish were fed "trout chow" and the bass live minnows.
Feeding was daily except when the fish was in the monitor tank.
Each experiment consisted of three consecutive parts: 1. a twenty-
four hour recording period with laboratory water; 2. an eight
hour period with Nad solution infused into the supply section B
so as to make a chloride concentration equivalent to that of a solution
of copper chloride containing 50 yg/1 Cu"1"4", and 3. an eight hour
period with CuC^ solution infused into section B so as to make a
concentration of 50 yg/1 Cu**. The concentration of copper as CuCl2
was 50 yg/1 only at the boundaries formed by water exiting from
section B and the surrounding water. Beyond these points the
concentration decreased. The concentrations of ions exiting section B
was empirically verified by atomic absorption analyses. Ten goldfish,
seven bass and seven catfish were tested in this manner. In addition,
ten goldfish were tested under a regimen of 100 yg/1. The chemical
characteristics of the water supply to the monitor tank are indicated
in Table 1.
Treatment of _da_ta_; The analyses of locomotor behavior were those
as described (Section IV) with some additions. The locomotor
characteristics considered were: activity as indicated by the number
of events (frequency of photocell responses); time accumulated in
an area; distance accumulated; average velocity; number of turns;
average size of turn; variance of turn size, and heading or orientation
of movements.
Changes in any of these characteristics were expressed by the statisti-
cal techniques described and summarized as follows:
1. An F test of the linear hypothesis that a polynomial model for
NaCl data equalled the corresponding model for CuCl^ data.
2. If this hypothesis "as rejected, the source of the inequality
was tested using the Wilcoxon rank sum test. The NaCl response
was subtracted from the CuCl2 response for each photocell, thereby
minimizing effects of location, and the differences in region A
compared with those in region B. This test of mean differences
between the two regions, if rejected, establishes that the inequality
of the models is, at least partially, due to the treatment.
3. A x^ statistic was used to test the multinomial distributions of
headings, in 20 degree categories, for homogeneity of the populations
of headings under NaCl and under CuC^. Upon rejection, a series
of subsequent y^ statistics identified the particular classes of
headings differing significantly. A sign test ascertains whether the
48
-------
population as a whole prefers a specified orientation under one
treatment relative to the other.
In addition to these procedures the following analyses were made:
1. An analysis of exploratory behavior as defined by Kleerekoper
et_ al_. (1972) which essentially determines, on a temporal scale, the
changes in location of a center of gravity of locomotion by computing,
for each sequential time period, the mean and variance of the mean x
and y coordinates of the animal's locomotion in the monitor tank.
2. An analysis of the orientation response to copper ion by means of
an analysis of variance: each data set was grouped into a frequency
distribution with 18 angle categories of 20° interval width. Two such
distributions were obtained for each fish; the first summarized
orientation over a two-hour control period whereas the second applied
to a subsequent two-hour period of copper treatment. Let Xj_; i = 1,...,
18; denote the observed frequencies in the control period and Y-^;
i = 1, ..., 18; the frequencies during the copper treatment. Inasmuch
as the X-L and Y'i categories are unequally probable in a bounded
square grid, the data were transformed according to
Z. = (Y. - X.) / (Y. + X.).
Conceptually the Z-j_ variates, which can range from -1 to +1, represent
an index for the preference of angle category i under the treatment
and have expectation 0 for each angle category under the null
hypothesis of the absence of a treatment effect.
This analysis was performed on 28 goldfish with two groups of ten
randomly chosen for the 50 pig and 100 yg copper treatment and eight
remaining under control conditions (0 yg) to detect possible time trends.
A two way factorial analysis of variance analyzed the data to detect
differences in the mean response of the fish, the angles, and/or their
interaction. The fish variation was partitioned into four independent
sources; among treatment concentrations and within each of the three
concentrations (0,50, and 100 Ug) . Preliminary scanning revealed an
apparent correlation between angle categories 180° apart. Therefore,
defining a "track" as an angle category and its 180° counterpart, the
variation among angles was partitioned into a variation among the nine
tracks and within each of the nine tracks. Finally, the above partition-
ings suggested dividing the interaction sum of squares into the 40
components obtained by crossing the four sources of fish variability
by the ten sources of angle variability.
3. A comparison of locomotor behavior during a two hour control
period and the final two hours of CuCl2 introduction, when the chloride
had become distribututed throughout the monitor tank, to analyze changes
brought about by the fish finding itself in contaminated water from
49
-------
which it could not escape. The locomotor parameters utilized in
this aspect of the study were those itemized earlier (Kleerekoper et
aJU 1970).
Results: The results refer to two experimental conditions: I. the
fish could move freely between the zone affected by copper chloride
(region B) and that containing sodium chloride of the same concentra-
tion (region A), and II. all the water in the monitor tank contained
copper chloride and, hence, there was no free choice for the fish.
I. Exposure to either copper chloride or sodium chloride by free
choice.
1. Polynomial Model
The results of the F test of equality of the empirical polynomial
models demonstrated that in all of the 34 fish tested, all the
locomotor characteristics investigated were affected by substitution
of the sodium chloride by the copper chloride (a = 0.1).
Two of the seven bass, three of the seven catfish and seven of the
ten goldfish showed a consistently non-significant difference in all
locomotor parameters in region B following the displacement of the
sodium chloride by 50 yg/1 of copper chloride (see methods, Wilcoxon
rank sum test, p. < .05). The remaining individuals tended to
increase (+) the number of events, the time spent, the distance swum
and the number of turns made in region B relative to region A
(Tables 7 and 8).
The same technique applied to ten goldfish tested at 100 yg/1 of
copper ion indicated that four individuals were unaffected. The
other six goldfish were inconsistent, but two of them tended to
increase time spent, distance swum and number of turns made in the
mass of water containing the copper ion (Table 7).
2. Exploratory Behavior
The presence of the copper ion altered the center of gravity of
locomotion in the tank as a whole as specified by the analysis of
exploratory behavior (see methods). Six of the seven bass, all of
the catfish, and five of the ten goldfish displayed a significant
change in either the mean of variance of the x or y coordinate in
50 yg/1 of copper ion. Five of the ten goldfish tested at 100 y/1
were significantly affected.
3. Headings (Orientation in the tank as a whole)
A comparison of the headings between the exposure to sodium and copper
ions (see methods) for the three species tested at 50 yg/1 indicated
that for the bass there was no change in the distribution, for six
50
-------
of the seven catfish there was a significant increase in the 100° -
170° arc, and for eight of the ten goldfish a significant increase in
the 110° direction. Eight of the ten goldfish tested at 100 yg/1
increased the number of headings in the copper in the 10° direction.
The analysis of variance performed on the 18 headings (nine tracks)
tabulated for goldfish in control periods (0 yg/1, eight fish), in
50 yg/1 (ten fish) and in 100 yg/1 (ten fish) revealed that all sources
of variation were significant (p < .01, Table 9) . The mean index for
each track in each of these groups is shown in Figure 9. The overall
mean index did not change between controls (1305 + .0510) and 50 Ug/1
(.1294 + .0375), but was significantly less (-.0076 + 0.0704) in 100
yg/1 (Wilcoxon rank sum test, p < .05, Figure 10) . The partitioning of
the main sources of variation into their components indicated that in
50 yg/1 three tracks (Figure 11, broken lines) were significantly
increased compared to those in the sodium ion, forming an arc from
140° - 220° and 320 - 40° (resultant shown as heavy black line) . In
100 yg/1 the only track which changed significantly was composed of the
50° and 230° directions. In all cases the size of the index in either
component of the track was not significantly different. The dispersion
among tracks was greater in the 100 pg/1 group than in either of the
other two (Figure 10) .
II. Exposure to copper chloride without choice
The effects of this condition were analyzed by comparing the data from
the seventh and eighth hours of the initial control periods and the final
two hours of copper ion introduction which were the corresponding seventh
and eighth hours of recording after the commencement of the copper
chloride introduction into the monitor tank. By this time the ion had
been carried by the water flow completely across the tank to the exit
side. Only turning behavior was altered significantly. The mean angle
size of turn decreased in two and increased in three of the five goldfish
tested at 50 yg/1 but it increased in all five goldfish tested in
100 yg/1. Accordingly, these latter individuals consistently increased
the number of smaller-sized angles of turns.
51
-------
TABLE 7
Wilcoxon Rank Sum Test (significant at p < .05)
Each column represents an individual which showed a significant change
PARAMETER
Bass
(50 ug)
Catfish
(50 yg)
No. of events + + - + NS + NS NS NS
time accumulated NS + NS + NS + + NS NS
cilst. accumulated + + - 4- NS + NS NS NS
average velocity + NS - NS + NS - NS +
no. of turns + + - + NS + NS NS +
average turn size NS NS NS NS + NS NS - NS
variance of turn NS NS NS NS + NS - NS
size
SUMMARY
00500 Q120
+ 44042 4102
NS 33235 2555
52
-------
Goldfish
(50 yg)
+ + +
NS NS NS
+ NS +
NS NS NS
+ + NS
NS NS NS
NS NS NS
000
322
A 5 5
Goldfish
(100 yg)
NS NS
+ NS
+ NS
NS +
+ NS
NS NS
NS NS
0 0
3 1
4 6
+ NS
+ NS
+ NS
NS NS
+ -
NS NS
NS NS
0 1
4 0
3 6
NS
NS
NS
NS
NS
NS
NS
4 3
0 0
3 4
53
-------
TABLE 8
Summary of results for Wilcoxon rank sum test (50 yg/1)
Number of fish in brackets
PARAMETER
variance of turn
size
BASS(7)
0
CATFISH(7)
GOLDFISH(10)
POOLED
no . of events
time accumulated
dist. accumulated
average velocity
no. of turns
average turn size
+
3
2
3
2
3
1
-
1
0
1
1
1
0
+
1
2
1
1
2
0
-
0
0
0
1
0
1
+
3
0
2
0
2
0
-
0
0
0
0
0
0
-
7
4
6
3
7
1
-
1
0
1
2
1
1
0
-------
TABLE 9
ANALYSIS OF VARIANCE (p < .01)
Source of variation d.f. MS
FISH
Among concentrations
Within control (0 yg/1)
Within 50 yg/1
Within 100 yg/1
HEADINGS
Among tracks
Within tracks
FISH X HEADINGS
Among concentrations X
Headings
Within control (0 yg/1) X
Headings
Within 50 yg/1 X
Headings
Within 100 yg/1 X
Headings
27
2
7
9
9
17
8
9
459
34
119
153
153
2.1018
1.0300
1.6600
0.6255
4.1588
0.0205
0.0413
0.0021
0.0270
0.0185
0.0183
0.0441
0 .0186
55
-------
Figure 9
Mean Index of Orientation in Goldfish for Nine Tracks
at 0, 50, and 100 Ug/1 Cu4*
56
-------
.3
x.2
.1
-.1
50ug/l
Oug/l
100 ug/l
I
1
•
2
3
4
5
6
7
8
9
Track
-------
Figure 10
Overall Mean Index of Orientation in Goldfish for
0, 50, and 100 yg/1 Cu4*
58
-------
.15
.1
.05
.05
0 50 100
UQ/I Cu* *
59
-------
Figure 11
Orientation in Tracks (Preferences for an Angle
Category and its 180° Counterpart) at 50 and 100
yg/1 CU++ in Goldfish
60
-------
90
180°-
-0
50ug/l
270
i
direction
of
flow
180°-
100 ug/l
270
i
-0'
61
-------
SECTION IX
CONCLUSIONS
The orientation of none of the largemouth bass was affected by the
presence of copper ions at the experimental concentration. However,
all but one of the seven catfish and eight of the ten goldfish changed
their mean orientation as well as the "tracks" of locomotion highly
significantly in the copper-affected region of the tank. The above
changes, rather than indicate even a weak degree of "avoidance" of the
copper source, point to movement in the direction of the source but to
a lesser degree than that in shallower gradients as documented earlier
(Kleerekoper et_ al_., 1972). In the goldfish, when surrounded by copper-
containing water without access to "clean" water, only turning behavior
was affected and no change in orientation could be observed.
It is concluded that the characteristics of the copper gradient between
"clean" and "polluted" water rather than the eventual highest concen-
tration of the ion affect the orientation and locomotor behavior generally
of the catfish and goldfish.
63
-------
SECTION X
THE LOCOMOTOR RESPONSE TO A STEEP
GRADIENT OF COPPER IONS BY THE GOLDFISH
The studies reported in the previous sections on the locoraotor response
by fish to gradients of sublethal concentrations of copper ions in an
open field brought evidence for significant changes in orientation in
the direction of the copper source.
Furthermore, at the same maximum concentration, the slope of the copper
gradient to which the fish were exposed seemed to affect the response.
Strong "attraction" toward the copper source in a very shallow gradient,
lesser attraction in a somewhat steeper gradient. The aim of the
following experiments was to verify whether avoidance behavior might
be elicited by confronting the fish with a steep gradient, in a res-
tricted field, in conditions otherwise similar to those which resulted
in "attraction".
Methods
The experiments were carried out in monitor tank #2. In the experi-
ments here reported, carbon-filtered water at 21°C was led through
glass tubes to the periphery of each of the 16 compartments at a rate
of 200 ml/min.
By means of an infusion withdrawal pump, CuC^ stock solution was
infused into the water supply leading to any one compartment at a
rate of 0.92 ml/min to produce there the desired concentration of
copper. Experiments were done at .005 (7) .01 (9) 0.25 (8),
and 10.0 (6) ppm Cu++ in addition to controls (5). In all experi-
ments but those at the 0.25 ppm concentrations, a single fish was
introduced into the tank once the desired copper concentration in the
compartment had been reached. At the 0.25 ppm concentration, the fish
entered the tank at the beginning of the introduction of the copper
chloride whose full concentration in the compartment was reached in about
two hours. Copper levels were monitored by atomic absorption analysis.
In each of the 35 experiments, done between July and September 1971,
the movements of a single fish were monitored during eight hours
following its release in the tank.
The 22 goldfish (28-30 cm) used in these experiments had been obtained
from a commercial grower in central Texas and kept during several
months in large holding tanks supplied with recirculating carbon-filtered
water. The source and the temperature of this water were the same as
those of the water used for the experiments. The fish were fed daily,
in the morning, with a dry "trout food" but received no food in the
monitor tank.
65
-------
The frequency of exposure to copper solutions is shown below.
Number of fish Number of times fish were exposed
to copper ions
12 1
1 2
2 3
6 4
1 5
The discrepancy between the number of exposures and the number of
experiments reported is due to the occasional occurrence of periods
of inactivity of the fish in the course of an experiment.
Treatment of data
The percent distributions of the frequency of entry and time spent in
each compartment were obtained for each experiment. All these dis-
tributions were rotated so as to line up to the copper-releasing
compartments which were then considered to be the same in the subsequent
analyses.
A separate two-way analysis of variance for each variable was used to
test the significance of differences between compartments and of the
treatment by compartment interaction. The two-way table had slightly
varying cell sizes due to a recording failure of 2.5%; hence, the
approximate, unweighted means analysis was used.
In order to isolate the copper effect alone, the treatment by compart-
ment interactions was further studied by constructing contrasts. Copper
solution was infused into one compartment only but a certain amount
of spreading of the copper ion must be expected at the exit of the
compartment, prior to the outflow of water through the standing pipe.
A copper zone, defined as the copper-releasing compartment and one
compartment on either side of it, took into account this lateral spread
and hence a table of means was prepared which compared for each treatment
level the mean percent entries in the copper zone to the mean of the
thirteen remaining compartments. A contrast then tested the interaction
of these means over increasing concentrations of copper.
Interactions of the percent time spent variable were studied in like
manner. However, since there is little back-flow into neighboring
compartments, the percent time spent in the copper-releasing compartment
was compared to the mean percent in the fifteen remaining compartments.
Results
In Figure 12 the ratios of the treatment mean to the control mean for
the percent compartment entries are illustrated for the copper zone
66
-------
and the remaining compartments for concentrations of 0.05, .01,
.025, and 10.0 ppm. Figure 13 illustrates the ratios for the time
spent in the copper compartment and the remaining compartments. With
increasing copper concentration the fish entered the copper zone less
frequently and spent less time in the compartment into which the copper
was infused.
Discussion
The results demonstrate a significant degree of "avoidance" of the
copper zone dependent on the copper ion concentration. However, even
at the highest concentration tested (10.0 ppm), there was no evidence
for an absolute avoidance of the copper-containing compartments. Since
both the relative number of entries into the copper zone and the time
spent there per entry were affected, it appears that the perception
of the copper may either elicit a change in orientation resulting in
avoidance of the copper zone or an escape response once the zone has
been entered.
In previous experiments carried out at similar copper concentrations
and temperature the fish had been kept in the large monitor tank (#1)
(5.0 x 5.0m) which allowed for unrestricted locomotion within the con-
finements of the walls. Unlike the conditions of the experiments here
reported, the boundaries of the copper-affected zone consisted of
shallow gradients. The fish could swim into and through these gradients
in all directions, entering and leaving the copper-affected zone. Under
these conditions significant orientation against the current of the
copper "polluted" water was observed (Kleerekoper £t_£l_-» 1972).
In the subsequent experiments in a steeper gradient, with goldfish,
largemouth bass, and channel catfish the fish still oriented toward
the copper source but to a lesser degree (Timms et_ al_., 1972). It
is apparent from those results and from the ones here reported that
at similar copper concentrations and within a similar temperature range
the orientation response is, at least in part, dependent on the steep-
ness of the gradient encountered by the fish. The available information
leads to the conclusion that significant "attraction" by copper-
"polluted" water can take place when the exposure occurs through a
shallow gradient: "avoidance" or "escape" response may result when a
similar concentration of copper is encountered through a steep gradient.
A gradient of an intermediate slope still "attracts" the fish but to a
lesser degree than a steep gradient.
67
-------
Figure 12
The Ratio of the Treatment Mean to the Control Mean
for Percent Entries into Compartments With and Without
Copper (Monitor # 2)
68
-------
1.20
1.10
Ratio of Treatment Mean to Control Mean for
Percent Entries
i.oo
.7 -
Remaining Compartment
Copper Zone
69
-------
Figure 13
The Ratio of the Treatment Mean to the Control Mean
for Percent Time Spent in Compartments With and Without
Copper (Monitor #2)
70
-------
Ratio of Treatment Mean to Control Mean for
Percent Time Spent
i.t
Remaining Compartment
.7 -
.6 -
.5 -
Copper Compartment
71
-------
SECTION XI
INTERACTION OF TEMPERATURE AND COPPER IONS AS
ORIENTING STIMULI IN THE LOCOMOTOR BEHAVIOR
OF THE GOLDFISH
The previous work on the orientation response of goldfish to copper
ions distributed within discrete zones in a mass of copper free water
demonstrated that the same copper concentration may elicit either
"attraction" or "avoidance" behavior. The response exhibited seems to
depend on the steepness of the gradient leading from the mass of copper
free water to that containing the maximum copper concentration.
Clearly, the interaction between the copper ion proper and the gradient
creates stimulus conditions which cannot be accounted for by the
concentration of copper ions alone. The aim of the following experiments
was to ascertain whether a similar interaction occurs between temperature
and copper in their combined effect on the orientation of locomotion
in the goldfish.
By means of an infusion withdrawal pump, CuCl2 stock solution was
infused into the water supply leading to each of two of the 16
compartments of monitor #2 so as to produce there a copper concentration
of 0.01 ppm CufH', with negligible variation due to the above-mentioned
fluctuation in flow rate of the water supply. All copper concentrations
were checked by the atomic absorption technique.
To produce a temperature differential between compartments of 0.4C,
the maximum which could be obtained without raising the temperature
of neighboring compartments, heating pads were used. Each pad shaped
to the contour of the compartment, was a nearly flat, black, silicone
rubber mat in which resistance wire was embedded.
Copper infusions and heating pads were arranged so as to obtain the
conditions illustrated in Table 10 and figure 14.
In all experiments the relative orientation of the five conditions
was the same, but before each experiment their absolute position in
the tank was changed randomly.
A single fish was introduced into the tank after the desired copper
concentration and temperature increase (0.4C) had been attained in
the selected compartments. In each of the 16 experiments, done during
July and August 1972, the movements of a single fish were monitored
during 24 hours following its release in the tank.
The 16 goldfish (25-33 cm) were obtained from a commercial grower
in central Texas and kept during several months in large holding
73
-------
Table 10. Designation of the five experimental conditions in the
monitor tank.
Condition
A
B
C
D
E
Number of Compartments
Affected
one
one
one
one
twelve
I i
Heating pad Cu
functional + present +
non functional- not present -
not present 0
0
74
-------
tanks supplied with recirculating, carbon-filtered water which was
1-1.4 C cooler than the water used for the experiments.
A plastic bag containing a fish in water from its holding tank
was suspended for 25-30 minutes in the water of the monitor tank
prior to the release of the animal to allow gradual adjustment to
temperature differences.
The percent distributions of the frequency of entry and time spent
in each compartment were obtained for each experiment. All these
distributions were rotated so as to line up the four compartments
representing conditions A to D, which were then considered to be the
same in subsequent analyses. A two-way analysis of variance for
frequency and time spent was used to test whether there were significant
differences between compartments, and a condition x fish interaction.
Variability between fish was eliminated by blocking and bias due to
intangible physical factors was minimized through a randomized assignment
of experimental conditions to the compartments. The variability due
to conditions was partitioned into contrasts and selected treatment
comparisons to isolate the effects of each condition. In addition,
the means for time spent and entries were plotted to obtain the
effect of copper and of heat. (Figures 15 to 18).
Results and Discussion; The effects of the copper ion and of the
temperature differential, illustrated separately in figures 15 to 18
are based on an analysis of variance.
Whereas the presence of the copper ion in the absence of heat
(condition A) brings about a significant decrease in both percentage
of entries and time spent per entry, temperature rise in the presence
of copper (condition C) produced a significant increase in both the
percentage of entries into compartments and the time spent per entry.
Although temperature rise in the absence of the copper ion leads to
a significant increase in both percent entries and time spent per
entry, the same temperature rise in the presence of copper further
increases the percentage of both variables significantly. Figures
15 to 18 clearly demonstrate that the described effects are distinct
from those produced by the presence of the heating per se.
It is concluded from the above evidence that in the experimental
conditions described, the fish display a trend to "avoid" the copper-
containing compartments by a relative decrease in both the number of
entries and in the time spent in a compartment per entry. However,
when the presence of copper is associated with a 0,4 C rise in temperature,
"avoidance" changes to "attraction", with significant increase in the
percentage number of entries as well as in the time spent per entry
in the copper and heat compartment.
75
-------
Figure 14
Arrangement of Experimental Conditions of Temperature
and Copper in Monitor #2
76
-------
Figure 14. Arrangement of experimental conditions in the monitor
tank.
E
FUNCTIONAL
PAD
B
FUNCTIONAL
PAD,
NO COPPER
D
NON FUNCTIONAL
PAD,
NO COPPER
NON
FUNCTIONAL
PAD
77
-------
Figure 15
Effect of Heat on the Mean Percent Time Spent in
Compartments With and Without Copper (Monitor #2)
78
-------
10.0
9.0
8.0
LU
Q-
7.0
Lul
O
or
LU
CL
6.0
5.0
4.0
EFFECT OF HEAT ON MEAN
PERCENT TIME SPENT
WITH COPPER (•)
B
WITHOUT
COPPER («)
OFF
ON
HEAT CONDITION
79
-------
Figure 16
Effect of Heat on the Mean Percent of Entrlns into
Compartments With and Without Copper (Monitor #2)
80
-------
9.0
8.0
en
UJ
UJ
LU
O
or
UJ
Q-
7.0
6.0
5.0
4.0
EFFECT OF HEAT ON MEAN
PERCENT ENTRIES
WITH COPPER (•)
B
WITHOUT
COPPER (*)
OFF
ON
HEAT CONDITION
81
-------
Figure 17
Effect of Copper on the Mean Percent Time Spent in
Compartments With and Without Heat (Monitor #2)
82
-------
9.0
EFFECT OF COPPER ON MEAN
PERCENT TIME SPENT
8.0
V
FUNCTIONAL PAD (*)
UJ
Q.
CO
UJ
7.0
UJ
o
a:
UJ
a_
6.0
5.0
NON FUNCTIONAL PAD
4.0
OFF
ON
COPPER CONDITION
83
-------
Figure 18
Effect of Copper on the. Mean Percent Entries into
Compartments With and Without Heat (Monitor #2)
84
-------
9.0
EFFECT OF COPPER ON MEAN
PERCENT ENTRIES
8.0
CO
UJ
or
h-
2
UJ
7.0
B
FUNCTIONAL PAD
C)
UJ
UJ
Q.
6.0
5.0
NON FUNCTIONAL
PAD (•)
4.0
OFF
ON
COPPER CONDITION
85
-------
Although there is an extensive literature on the effects on locomotor
orientation in fish of either temperature or heavy metal ions
individually, no reference could be found to interaction between
temperature and these ions, such as that described here.
86
-------
SECTION XII
THE LOCOMOTOR RESPONSE TO COPPER IONS IN THE WHITE SUCKER
CATOSTOMUS COMMERSONI COMMERSONI) AND GREEN SUNFISH (LEPOMIS CYANELLUS)
White_ Sucker
The experiments done on the goldfish, channel catfish and largemouth
bass were repeated on the white sucker (14 fish) in monitor #1.
The basic locomotor behavior of this species is sufficiently different
from the others to require a different statistical treatment of the
data. The fish display a high degree of thigmotaxis which results in
the locomotor pathway being almost exclusively along the walls of the
monitor tank. In the virtual absence of locomotion in the remainder
of the tank the usual statistical treatment by which the locomotion
in equal areas of the monitor is compared had to be modified. Analysis
of variance and treatment contrasts for the control, NaCl, and CuCl2
conditions were applied. Each condition lasted eight hours.
Resuitsi With exception of length and frequency of straight pathways
(or steps), the presence of the copper ion did not significantly
affect the locomotor characteristics of the white sucker. The length
of the straight pathways became significantly shorter in the copper
region while their frequency increased highly significantly.
The data indicate that the time spent by the fish in the copper region
increased. However, the peculiar locomotor behavior of this species
makes the interpretation of the data analysis more difficult than with
the other species studied.
Green Sunfish
Twelve fish of this species were tested in monitor #1 using the
experimental method and data treatment described in Section X at a
concentration of .050 ppm as CuCl2. The fish spent more time in the
copper-containing region than in the "non-polluted" region of the tank.
Three locomotor characteristics were affected by the presence of the
copper: the frequency of turning and of straight paths increased.
Introduction of the copper ion resulted in a significant increase in
the time spent by the fish in the copper-affected region while the
frequency of turning also increased. The remainder of the locomotor
characteristics analyzed was not affected with exception of the total
activity which consistently increased during the eight hour intervals
of copper chloride infusion.
87
-------
SECTION XIII
ACKNOWLEDGEMENTS
The research was done in the Fish Behavior Laboratory of the Depart-
ment of Biology, Texas A&M University, College Station, Texas 77843.
The statistical techniques were elaborated by Dr. J. H. Matis, of the
Institute of Statistics of the same university; all computer programs
were designed by Dr. P. J. Gensler, of the Department of Computing
Science, West Texas State University, Canyon, Texas 79105. The
various phases of the experimentation were carried out by Mr. G. F.
Westlake*, Dr. A. M. Titnms**, and Mr. J. B. Waxman. The important
contributions made by all these collaborators in this research and
the cooperation of Dr. W. A. Spoor and Dr. J. M. McKim, Environmental
Protection Agency Project Officers is gratefully acknowledged.
Present addresses:
* Center for Environmental Studies
Virginia Polytechnic Institute and
State University
Blacksburg, Virginia 24061
** Department of Zoology
University of Western Ontario
London 72, Ontario, Canada
89
-------
SECTION XIV
REFERENCES
1. Afleck, R.J., "Zinc poisoning in a trout hatchery," Austral.
J. Marine Freshwater Res. 3, p 162 (1952).
2. Ball, I.R., "The relative susceptibilities of some species
of freshwater fish to poisons, II. Zinc," Water Res. 1, pp 777-
783 (1967) .
3. Brown, V.M., Jordan, D.H.M..and Tiller, B.A., "The acute toxicity
to rainbow trout of fluctuating concentrations and mixtures of
ammonia, phenol, and zinc," J. Fish Biol., 1, pp 1-9 (1969).
4. Bucksteeg, W., Thiele, H., and Stoltzel, K., "Die Beeinflussung
von Fischen durch Giftstoffe aus Abwassern," Vom Wasser,_22,
pp 194-211 (1955).
5. Burden, W.D., "Development of shark deterrent," Air Surgeon's
Bui., 2, p 344 (1945).
6. Cairns, J. Jr., and Scheier, A., "The effects of temperature and
hardness of water upon the toxicity of zinc to the common blue-
gill (Lepomis macrochirus)," Not. Natl. Acad. Nat. Sci. Phila.
299, pp 1-12 (1957) .
7. Cherkinsky, S.N., "The theoretical basis of hygienic standariza-
tion of simultaneous pollution of water courses with several
harmful substances," Gig. Sanit., 22, pp 3-9 (1957).
8. Doudoroff, R., and Katz, M., "Critical review of literature on
toxicity of industrial wastes and their components to fish,
II. Metals as salts," Sewage Industr. Wastes, 25, pp 802-839 (1953)
9. Friedland, S.A., and Rubleva, M.N., "The problems of hygienic
standards for water simultaneously polluted with several harmful
substances," Gig. Sanit., 23, pp 12-16 (1958).
10. Fry, F.E.J., "Effects of the environment on animal activity,"
Publ. Ontario Fish. Res. Lab., 68, pp 62 (1947) .
11. Grande, M., "Effect of copper and zinc on salmonid fishes,"
in: Advances in Water Pollution Research, Proc. 3rd Inst. Conf.
Munich, Germany, Vol. 1, pp 75-96 (1967).
12. Herbert, D.W.M., "The toxicity to rainbow trout of spent still
liquors from the distillation of coal," Ann. Appl_._ Biol. 50,
pp 755-777 (1962) .
91
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13. Herbert, D.W.M., and Shurben, D.S., "The toxicity to fish of
mixtures of poisons, I. Salts of ammonia and zinc," Ann. Appl.
Biol., 53. pp 33-41 (1964) .
14. Herbert, D.W.M., and Wakeford, A.C., "The susceptibility of salmo-
nid fish to poisons under estuarine conditions. I. Zinc sulphate,"
Int. J. Air Water Pollut., 8, pp 251-256.
15. Hill, R.N., "Victory over the shark," Nat. Hist., 65, p 352
(1956) .
16. Hoglund, L.B., "The reactions of fish in concentration gradients5"
Fish. Bd. Sweden Kept., 43, pp 4-147 (1961).
17. Ishio, S., "Behaviour of fish exposed to toxic substances,"
Advances in Water Pollution Research, Proc. 2nd Int. Conf. Water
Pollut. Res. Tokyo, Vol. 1, pp 19-33, Pergamon Press, London,
(1965).
18. Jones, B.F., Warren, C.R., Bond, C.E., and Doudoroff, P., "Avoidance
reactions of salmonid fishes to pulpmill effluents," Sewage
Industr. Wastes, 28, pp 1403-1413 (1939) .
19. Jones, J.R.E., "Antagonism between salts of the heavy metals in
their toxic action on the tadpole of the toad (Buffo bufo bufo L.),"
J. Exp. Biol., 16, pp 313-333 (1939).
20. Jones, J.R.E., "The reactions of Pygosteus pungitius L. to toxic
solutions," J. Exp. Biol., 24, pp 110-222 (1947).
21. Jones, J.R.E., "A further study of the reactions of fish to
toxic solutions, J. Exp. Biol., 25, pp 22-34 (1964).
22. Jones, J.R.E., "Fish and River Pollution, Butterworths, London,
p 203 (1964).
23. King, W., "Mortality of hatchery trout, Great Smoky Mountain
National Park," Prog. Fish. Cult. 29, p 14 (1937) .
24. Kleerekoper, H., "Some effects of olfactory stimulation on
locomotor patterns in fish," Olfaction and Taste II_; Proc. 2nd
Int. Symposium, Tokyo, 1965, Pergamon Press, Oxford and New
York, pp 625-645 (1967) .
25. Kleerekoper, H., "Olfaction in Fishes", Indiana U. Press,
Bloomington, London, pp 222 (1969) .
26. Kleerekoper, H., Timms, A.M., Westlake, G.F., Davy, F.B.,
Malar, T., and Anderson, V.M., "Inertial guidance system in
the orientation of the goldfish (Carassius auratus)," Nature 223,
pp 501-502 (1969).
92
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27. Kleerekoper, H., Timms, A.M., Westlake, G.F., Davy, F.B., Malar, T.,
and Anderson, V.M., "An analysis of locomotor behaviour of
goldfish (Carassius auratus)," Anim. Behav., 18, pp 317-330
(1970).
28. Kleerekoper, H., Westlake, G.F., Matis, J .H., and Gensler, P.J.,
"Orientation of goldfish (Carassius auratus) in response to a
shallow gradient of a sublethal concentration of copper in an
open field," J. Fish. Res. Bd. Canada, 29, pp 45-54 (1972).
29. Lloyd, R., "The toxicity of zinc sulphate to rainbow trout,
Ann. Appl. Biol. 48, pp 84-94 (1960).
30. Lloyd, R., "The toxicity of mixtures of zinc and copper sulphates
to rainbow trout (Salmo gairdnerii Richardson)," Ann. Appl. Biol.
49_, pp 535-538 (1961).
31. Lloyd, R., and Herbert, D.W.M., "The effect of the environment
on the toxicity of poisons to fish," J. Inst. Public Health Eng.,
6^, pp 132-145 (1962).
32. Malacca, L., and Gruia, E., "Contributions to the study of the
toxic effect of heavy metals on some aquatic organisms," Inst.
Hydrotech Res. Sci. Sess. Sect., 4» pp 47 (1965) .
33. Mount, D.I., "The effect of total hardness and pH on acute
toxicity of zinc to fish," Air Water Pollut., 10, pp 49-56 (1966).
34. Ostle, B., "Statistics in Research," Iowa State U. Press, Ames,
Iowa (1963).
35. Paul, R.M., "Water pollution: A factor modifying fish populations
in Pacific coast streams", Sci. Monthly 74, p 14 (1952).
36. Pickering, Q.H., and Vigor, W.N., "The acute toxicity of zinc
to eggs and fry of the fathead minnow", Progr. Fish. Cult. 27,
pp 153-157 (1965).
37. Saunders, R.L., and Sprague, J.B., "Effects of copper-zinc
mining pollution on a spawning migration of Atlantic salmon
(Salmo salar)," Water Res. 1, pp 419^32 (1967).
38. Schofield, C.L., Jr., "Water quality in relation to survival
of brook trout (Salvelinus fontinalis Mitchill)," Trans. Amer.
Fish. Soc. 94, pp 227 (1965) .
39. Screenivasan, A., and Soundar, R., "Toxicity of zinc to fish,"
Current Sci. 32, p 363 (1963).
93
-------
40. Southgate, B.A., "The toxicity of mixtures of poisons", Quart.
J. Pharm., 5, pp 639-648 (1932).
41. Sprague, J.B., "Avoidance of sublethal mining pollution by
Atlantic salmon," Proc. 10th Ontario Industr. Waste Conf.
Ontario Water Resources Commission Toronto, pp 221-236 (1963).
42. Sprague, J.B., "Avoidance of copper zinc solutions by young
salmon in the laboratory," J. Water Pollut. Contr. Fed., 36,
pp 990-1104 (1964).
43. Sprague, J.E., "Avoidance reactions of rainbow trout in zinc
sulphate solutions," Water Res. 2, pp 367-372 (1968).
44. Sprague, J.B., and Ramsay, B.A., "Lethal levels of mixed copper
zinc solutions for juvenile salmon," J. Fish. Res. Bd. Can. 22,
pp 425-432 (1965) .
45. Sprague, J.B., Elson, P.F., and Saunders, P.L., "Sublethal
copper-zinc pollution in a salmon river: A field and laboratory
study," Int. J. Air Water Pollut. 9, pp 531-543 (1965).
46. Summerfelt, R., and Lewis, W.M., "Repulsion of green sunfish by
certain chemicals," J. Water Pollut. Contr. Fed., 39, pp 2030-
2038 (1967) .
47. Syazuki, K., "Studies on the toxic effects of industrial waste
on fish and shellfish," J. Shimonoseki Coll. Fish., 13, pp 157-211
(1964) .
48. Tarzwell, C.M., and Henderson, C., "Toxicity of less common
metals to fishes," Industr. Wastes 5, p 12 (1960).
49. Timms, A.M., and Kleerekoper, H., "Locomotor responses of
blinded goldfish (Carassius auratus) to remote perception of
barriers," J. Fish. Res. Bd. Can., 27. pp 1103-1107 (1970).
50. Timms, A.M., and Kleerekoper, H., "The locomotor responses of
male Ict_alurug_ pun c tat us, the channel catfish, to a pheromone
released by the ripe female of the species," Trans . of the^
American Fish Soc., 101, No. 2 pp 302-310 (1972).
51. Timms, A.M., Kleerekoper, H., and Matis, J., "Locomotor response
of goldfish, channel catfish, and largemouth bass to a "copper-
polluted" mass of water in an open field," J. Water Resources
Research, 8, No. 6, pp 1574-1580 (1972).
94
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52. Westlake, G.F., and Kleerekoper, H., "Evidence for a memory
process in turning behavior of free-swimming goldfish," Can.
J. Zool., 48, pp 813-815 (1970).
53. Westlake, G.F., Kleerekoper, H., and Matis, J., "The locomotor
response to a steep gradient of copper ions by the goldfish,"
J. Water Resources Research (In Press).
54. Wilcoxon, F., and Wilcox, R.A., "Some rapid approximate
statistical procedures," Lederle Labs., Pearl River, N.Y.,
(1964) .
95
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SECTION XV
PUBLICATIONS
Kleerekoper, H., Westlake, G, F., Matis, J. H. and Gensler, P. J.,
"Orientation of goldfish (Carassius auratus) in response to a shallow
gradient of a sublethal concentration of copper in an open field,"
J. Fish. Res. Bd. Canada, 29, pp 45-54 (1972).
Kleerekoper, H., Waxman, J, B,, and Matis, J., "Interaction of tempera-
ture and copper ions as orienting stimuli in the locomotor behavior of
the goldfish (Carassius auratus), " J. Fish. Res. Bd. of Canada
(In Press).
Timms, A. M., Kleerekoper, H., and Matis, J., "Locomotor response of
goldfish, channel catfish, and largemouth bass to a "copper-polluted"
mass of water in an open field," J. Water Resources Research, 8,
No. 6, pp 1574-1580 (1972).
Westlake, G, F., Kleerekoper, H., and Matis, J., "The locomotor response
to a steep gradient of copper ions by the goldfish," J. Water Resources
Research (In Press) .
97 OU.S. GOVERNMENT PRINTING OFFICE: 1973 514-156/344 1-3
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SELECTED WATER
RESOURCES ABSTRACTS
INPUT TRANSACTION FORM
2. Report No,
2.
W
-1. . Title
Effects of copper on the locomotor orientation of fish
5,
8.
- Anthor(s)
Kleerekoper, H.
torn, .
tW.
10.
9. Organization
Texas ASM University
Department of Biology
32. \ Si'tnsorir Organ -.lion ,t
25. Supplementary Noses
Environmental Protection Agency Report No. EPA-R3-73-045, June 1973
R800995 (18050DWQ)
1 Typ ' Rep and
Porioa Cc jred
16. Abstract xhe effects of copper ions at subacute concentrations on the locomotor
orientation of goldfish (Garassius auratus), channel catfish (Ictalurus punctatus).
largemouth bass (Micropterus ealmoides). white sucker (Catostomus commersoni commersoni)
and green surifish (Lepomis cyannelus) were investigated in detail. In regions of
water containing 11-17 yg/1 CU-H- (as CuClz) in a shallow gradient goldfish oriented
toward* the copper source ("attraction"). This response is reduced in a somewhat
steeper gradient. In steep gradients significant but no absolute avoidance behavior
occurred. Whether the response will be "avoidance" or "attraction" seems to depend
on the slope of the gradient to which the fish is exposed. Even in steep gradients,
the "avoidance" behavior is reversed to "attraction" when the copper ions interact
with a temperature slightly higher (.4C) than that of the surrounding copper free water.
The interaction creates a new stimulus configuration which is different from those
formed by the two variables separately. The orientation of the largemouth bass is
not affected by copper ions at the concentrations tested. Channel catfish are weakly
attracted by the copper-containing water and green sunfish significantly increase
time spent there. Suckers significantly but not absolutely "avoid" such water through
changes in turning behavior. The report was submitted in fulfillment of Project
Number R800995 (18050DWQ) under the sponsorship of the Water Quality Office,
Environmental Protection Agency.
17a. Descriptors
*Copper
*Locomotion in fish
*0rientation in fish
*Avoidance behavior in fish
*Interaction of temperature and copper
ions
*Effect of gradient slope on orientation
*Temperature and orientation
17b. Identifiers
goldfish
White sucker
channel catfish
green sunfish
largemouth bass
Lepomis cyanellus
Carassius auratus^
Catostomus commersoni commersoni
Micropterus salmoides
Ictalurus punctatus
17c. COWRR Field £ Group ~~
15. Security Classt '.•
•(Report) , •,
• ~Q, • Security Cists.
'
21. No. of,
•:. : Pages
:32. : Price
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WASHINGTON. D. C. 2O24O
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