EPA-600/3-76-069
September 1976
Ecological Research Series
ASSAYS OF TOXIC POLLUTANTS BY FISH BLOOD
Environmental Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Narragansett, Rhode Island 02882
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development, U.S. Environmental
Protection Agency, have been grouped into 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 work provides the technical
basis for setting standards to minimize undesirable changes in living organisms
in the aquatic, terrestrial, and atmospheric environments.
This document is available to the public through the National Technical Informa-
tion Service, Springfield, Virginia 22161.
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EPA-600/3-76-069
September 1976
ASSAYS OF TOXIC POLLUTANTS BY FISH BLOOD
W. A. Curby, R. D. Winick, and E. C. Moy
Sias Research Laboratories
Brookline, Massachusetts 02146
Grant No. 800355
Project Officer
William S. Hodgkiss
Environmental Research Laboratory
Narragansett, Rhode Island 02882
ENVIRONMENTAL RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
NARRAGANSETT, RHODE ISLAND 02882
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DISCLAIMER
This report has been reviewed by the Environmental Research
Laboratory, U.S. Environmental Protection Agency, and approved
for publication. Approval does not signify that the contents
necessarily reflect the views and policies of the U.S. Environ-
mental Protection Agency, nor does mention of trade names or
commercial products constitute endorsement or recommendation
for use.
ii
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ABSTRACT
We hav,e developed a biological multichannel analyzer which,
using a sensor that operates on the Coulter principle, measures
and distributes mixed cell populations by cell size. It provides
an analog distribution and digital printed readout for future an-
alysis. Although primarily a pulse height analyzer (applied suc-
cessfully to studying bacteria, mammalian blood and inert particles)
it operates as a pulse shape analyzer if the instant at which each
pulse height is read is varied. This technique, applied to the
peripheral whole blood from freshly sacrificed Fundulus heteroclitus
shows the alterations with time and the variations caused by trace
amounts of cadmium and copper in the aquatic environment. The size
frequency distribution patterns of each trace element environment
differ from each other, and each, markedly from the norm.
We have investigated and recorded the response of F_._ hetero-
clitus whole blood cells from fishes living in several aquatic
environments of fixed pK and dissolved oxygen and temperature. We
compared these data with those obtained from fish subjected to
dissolved traces of chemical pollutants. In final fullfillment of
mix grant, we have delivered an advanced model of the multichannel
analyzer to the U.S. Water Quality Laboratories in Narragansett,
Rhode Island. The Fish Blood Analyzer is produced by Grumman
Health Systems.
111
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Section
I
II
III
IV
V
VI
VII
VIII
IX
CONTENTS
Page
Introduction 1
Materials and Methods: General Test Procedures 9
Results: Controls
Results: Freshwater Adaptation Experiments
Results: Cadmium Experiments
Results: Copper Experiments
Freshwater vs. Saltwater
Results: Copper Experiments
5ppm and Recovery Tests
Discussion
Bibliography
12
15
23
28
40
57
65
v
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FIGURES
Page
1 Biological Mulitchannel Analyzer 5
2 Analyzer Storage 6
Output from Storage
3 Sensor and Secondary Pulses 7
Representative Pulse Shape
4 Triggering Delays 8
Electronic Pulse and Cell Shape
5 Block Diagram of Multichannel Analysis 62
System for Fish Blood
6 Block Diagram of GHS Fish Blood Analyzer 63
7 GHS Fish Blood Analyzer 64
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TABLES
Page
I Fish in CdCl2 and Reversed 24
II Fish in CdCl2 and Reversed:
By Channel (Peak) Number
vii
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ILLUSTRATIONS AND PHOTOGRAPHS
Page
Photomicrograph : Control 13
Electronic Printout: Control. 14
Photomicrograph: Freshwater 2, 4, 6 Hours and Phase-over 18
Electronic Printout: Freshwater 4 Hours and Phase-over 19
Photomicrograph: Freshwater Adapted 20
Electronic Printout: Freshwater Adapted 20
Photomicrograph: Distilled Water 1, 3, 4 Hours 21
Electronic Printout: Distilled Water 1, 3, 4 Hours 22
Photomicrograph: CdCl2 3.5 and 5.75 Hours 26
Electronic Printout: CdCl2 3.5 and 5.75 Hours 27
Photomicrograph: CuCl2 in Freshwater and Saltwater 31
24 Hours, l.Sppm
Electronic Printout: CuCl2 in Freshwater and Saltwater 32
24 Hours, l.Sppm
Photomicrograph: CuCl2 in Freshwater and Saltwater 33
96 Hours, l.Sppm
Electronic Printout: CuCl2 in Freshwater and Saltwater 34
96 Hours, l.Sppm
Photomicrograph: CuCl2 in Freshwater and Saltwater 35
1 Hour, 2ppm
Photomicrograph: CuCl2 in Freshwater and Saltwater 36
3 and 5 Hours, 2ppm
Electronic Printout: CuCl2 in Freshwater and Saltwater 37
1, 3 and 5 Hours, 2ppm
Photomicrograph: CuC^ in Freshwater and Saltwater 38
24 Hours, 2ppra
Electronic Printout! CuCl2 in Freshwater and Saltwater 39
24 Hours, 2ppm
Photomicrograph: CuCl2 in Saltwater 45 Minutes, 5ppm 46
viii
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Page
Photomicrograph: CuCl2 in Saltwater IHour 3ppm 47
4.5 Hours, 5ppm
Electronic Printout: CuCl2 in Saltwater 4.5 and 5 Hrs 48
5ppm
Photomicrograph: CuCl2 in Saltwater 24 Hours, 5ppm 49
Photomicrograph: CuCl2 in Saltwater 44 Hours, Sppra 50
Electronic Printout: CuCl2 in Saltwater 24 and 44 Hours 51
Photomicrograph: CuCl2 in Saltwater 24 Hours, Spptn 52
Returned to Unpolluted Saltwater 10 Hours
Photomicrograph: CuCl2 in Saltwater 24 Hours, Sppra 53
Returned to Unpolluted Saltwater 1 Week
Electronic Printout: Return 10 Hours 54
Return 1 Week
Hypothetical Model: Effects of Cu"1"* Exposure on Fish 61
Erythrdcytes
IX
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INTRODUCTION
Fundulus heteroclitus exhibits rapid physiological responses to
changes in environmental conditions. We have been particularily inter-
ested in the ability of the peripheral blood cells to cope with various
environmental insults, i.e. changes in salinity and exposure to heavy
metals. In studying the responses of the blood cells, we have examined
the cell populations by classical methods such as microscopic study of
both fresh and Wright stained preparations. More importantly, we have
examined the blood cell populations using a Biological Multichannel
Particle Size Analyzer (BMA). The BMA electronically monitors the cell
population, giving a size and frequency distribution. Our tests have
confirmed our ability to use this electronic assay system to pick up
the morphological changes that are seen in the photomicrographs of both
the phase contrast studies and the Wright stained cells. The importance
of using a multichannel analyzer, such as the BMA, to detect changes in
the blood cell population lies in its rapidness and sensitivity. The
BMA can sample within seconds a statistically more appropriate number
of cells than more classical methods of differential counts from slides.
Further, the BMA can indicate changes in blood cell morphology, as well
as shifts in the composition of the components of the blood cell popula-
tion. These changes are indicative of the sublethal effects of an en-
vironmental pollutant. The sublethal effects of a pollutant may be
just as damaging to the continuing exsistence of a population as an out-
right fish kill. The impairment of breeding potential, the greater vul-
nerability to other environmental stresses, and the reduced viability
of the offspring, all these factors conspire in diminishing the popula-
tion's ability to florish or even to survive in its natural habitat.
The Biological Multichannel Analyzer (BMA)
In the search for more rapid and accurate methods for studying the
dynamics of cellular growth and changes and rates of changes in sizes,
shapes and numbers of cells in living populations of organisms, we devel-
oped a system built around a transducer operating on the differential
conductivity principle of Coulter. Through proper interfacing, we have
matched the particle sensor to a 200 multichannel analyzer. The proto-
type BMA system is presently in use at the Naval Biomedical Research
Laboratory, Oakland, California. An advanced model of the system is
at the Naval Weapons Laboratory, Dahlgren, Virginia.
The BMA system is computer compatible and capable of rapidly de-
fining the size and frequency distribution of a population of organisms
and particulate material contained in fluid media. The system can dis-
play population distributions over size ranges of interest to us on a
minute by minute basis if desired.
Figure 1 shows the analysis unit for the studies to be reported
here. The sensor is a manometer element of the type used in the
Coulter particle counter, and operates on the Coulter principle viz.,
-------
particles are carried in a conductive fluid through an aperture through
which an electric current is flowing. Each time a particle passes through
the aperture, the current flux density is altered in the aperture. This
alteration in flux density causes a change in potential across the aperture.
The change in potential is amplified and measured in subsequent circuitry.
The resulting output from the sensor during the passage of several spheri-
cal particles moving at a constant flow velocity (controlled in the system)
produce a series of voltage pulses which are approximately 20 microseconds
in duration and have an amplitude which is proportional to the size of the
particles.
Under constant flow rate conditions, it is possible to measure and
record pulses generated by particles in fixed intervals of time. The
equipment can store the information from one sample of fluid at a time
within its core memory and must be emptied after a digital or analog
form representation of the data contained in the memory has been made.
It is possible, however, in more complex equipment to produce a dupli-
cate of the data going into the core storage in the memory bank of a
time-shared computer or to record the data on magnetic tape. The system
couples the Coulter sensor through the proper interfacing into a Nuclear-
Chicago 200 Channel Multichannel Pulse Height Analyzer. The pulses
produced by the sensor are amplified and fed into a secondary pulse
generator which produces fixed duration uniformly shaped pulses propor-
tional in height to the input pulses. These pulses are measured and
assigned a position on the X axis of the analyzer. This axis is div-
ided into 200 parts or channels. Each time a pulse of the same height
is measured, one more number is added to that particular channel. Thus,
the storage will show the number of pulses (particles) which have been
assigned to a given channel at any instant in the sampling period. At
the end of the sampling period, the storage will contain the total count
(Px) for pulses having any given pulse height within a pulse height
range selected by the operator to be contained within the 200 channels
of the storage. The data included in this report have been recorded in
analog form using a Mosley X-Y plotter.
In Figure 2, we have attempted to clarify the concept by showing a
representation of what information would be contained in the first 100
channels of storage for a hypothetical sample. The stored information
would be the digital record of the number represented as equal blocks
in each channel, as shown in the upper graph. The analog output from
storage would be made by reading out a voltage proportional to the num-
ber of blocks in each channel starting at channel one and ending with
channel 100. The completed read-out as made on the X-Y Plotter is shown
in the lower graph. It should be noted that in the lower graph the
ordinate and abscissa definitions have changed. The Y axis reads the
particles counted since there is a one to one relationship to the pulses
generated by the sensor provided certain conditions are met. The X axis
absolute scale will change depending on the values of the control para-
meters set into the system. For any operating conditions set into the
equipment, a calibration curve is made using latex spheres of known sizes
as standards.
-------
For ease of comparison, we calibrate on the basis of particle diameter
rather than volume. A calibration on the basis of volume is possible when-
ever it is desired.
Total counts can be obtained from the X-Y plots using the following
relationship;
xmin
Where P = the total particle count
Px« the particle count per x increment
and
S = scale factor (as set within the equipment)
The x increment is usually taken as the width of one channel, in which
case for dilute solutions the integration constant may be taken as unity.
xm±n and ^ are determined by the region of interest, ^^ £ 0 and xmax
£ maximum number of channels in a memory block.
Because of the properties of the fish blood, pulse shape analysis
must be done. The system of pulse height analysis using the Coulter
transducer requires for proper operation, that the flow rate through the
counting aperture be absolutely precise. This means that the durations
of pulses generated by spheres of equal size will be equal. The 30 micron
aperture wafer thickness is usually 40 - 50 microns. The flow rate which
is normally available using the Coulter manometer with a 30 micron aperture
is such that pulses of 20 microseconds duration (base to base) are gen-
erated when 1 micron spheres traverse the aperture. The geometry and flow
rate characteristics are such that pulses of the same order duration are
produced by 2.7 micron diameter spheres passing through a 100 micron aper-
ture.
The pulses produced by spheres have bilateral symmetry peaking at 10
microseconds. The BMA equipment is normally set to trigger the production
of a 2 microsecond square wave pulse having an amplitude which is linearly
proportional to the voltage of the pulse produced by the sensor at the
exact instant of the triggering (Figure 3).
As the diameter of the sphere increases, the pulse duration increases,
but with a much smaller magnitude than the pulse amplitude at the gain
settings normally used for routine analysis.
Figure 3B shows the type of pulse normally seen when samples of
human erythrocytes are diluted on 0.9% physiological saline (particle-free)
and passed through a 100 micron aperture. The resulting pulse train is
made up of a population of pulses which differ from one another only
-------
slightly. With the BMA equipment set to trigger at 8 microseconds into
the pulse (DM = 8 raocroseconds), secondary voltage pulses of amplitudes
proportional to the sensor voltage at point "a" will be produced. If
the delay trigger is set to produce a secondary pulse 25 microseconds
after the initiation of the sensor pulse, pulses proportional to the sensor
voltage at point "b" will be produced. It will be noted that due to
the fact that uniform pulses are being produced by the sensor, the
resultant change in the delay of secondary pulse -triggering results only
in a shift of the apparent diameter to the right (a-»a? in Figure 4A).
This change in the apparent diameter is artificial since the size
index of a particle (be it diameter, volume, cross-sectional area or
any other dimension upon which a calibration curve was produced from
a known particle population calibration standard) will be different for
each delay trigger setting. In the data herein presented, all size
calibration data are based on an 8 microsecond delay and all X axis
scales are given relative to the DM 8 microsecond calibration value. In
practice, reference of pulse distributions to a single trigger delay is
useful since the changes in size indices of particles giving characteristic
complex pulse shapes will be reflected in shifts in the BMA plotted size
distributions from those produced by the population of the same component
particle mixes prior to changes in the size indices. Since the assay
data must be based upon differences from normals, and since the normals
are catalogued emperically, the system requires only absolute precision
to be effective. The system does have the requisite precision for its
utilization as an extremely sensitive differential pulse shape analyzer.
Interpretation of shape by two point (or three point) variation of
trigger delay can be made only if an analysis of the shapes of the
pulses in the array being studied can be made. We have made subjective
observations of the sensor pulse arrays and have observed at least three
generally occurring pulse shapes. Figure 4B illustrates these and in-
dicates the probable form of the particles producing each.
The power of this equipment lies in its high sensitivity (changes
as small as 10 cells/sample volume of 0.05ml can be easily seen), pre-
cision and rapid analysis interval (less than 15 seconds/sample actual
sample analysis and storage time).
-------
Figure 1
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FIGURE 2
GRAPH A
CL
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MI
CD "
z> -
Ar
E;;;!;::;3
10
sJALYZER STORAGE
j(» INFORMATION IN CORE
:::i STORAGE IN BCD FORM'
:;:|i) 1. CHANNEL NUMBER
i 2. SUM OF COUNTS
I;;;;; IN THAT CHANNEL
:= FOR EVERY CHANNEL.
50 100
GRAPH B
h-
IDH
LU
O
fe
CHANNEL NUMBER
(PULSE HEIGHT)
OUTPUT FROM STORAGE
( ANALOG FORM )
AS READ OUT ON AN
X - Y PLOTTER
PARTICLE SIZE
(FROM CALIBRATION CURVE)
-------
FIGURE 3
UJ
e>
§
§
UJ
I
UJ
cr
8 Microsecond Trigger
a
5 ' 10 15 20
TIME (microseconds)
PULSE Q = SENSOR PULSE
PULSE b = SECONDARY PULSE
UJ
e
UJ
1
UJ
cc
100 jj APERTURE
b
10 2025
TIME (microseconds )
60
REPRESENTATIVE PULSE SHAPE *
HUMAN ADULT MALE ERYTKROCYTE
( In 0.9% w/v Saline)
Q, B Microsecond Delay
D. 25 Microsecond Delay Trigger
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FIGURE 4
o
o
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fe
£
I y
PARTICLE DIAMETER
Q. 8 usec. Trigger
Q. 25 ^sec Trigger on a
Symmetrical! Highly Uniform
Pulso Population
Q 25 ysoc Trigger on a Semi-
random Mixed PuSso Popukrticn
B
*
c:
0 tO 20 SO 40 SO CO
TIME
a, o
c,
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MATERIALS AND METHODS: GENERAL TEST PROCEDURES
Daily Maintenance
The populations of Fundulus heteroclitus used in these experiments
were obtained from the Environmental Protection Agency headquarters in
West Kingston, Rhode Island. They were caught offshore of Naragansett
Bay just prior to their transportation to our laboratories in Brookline,
Massachusetts. They were transported in large plastic containers fitted
with air bubblers. The two hour trip never affected the fish adversely.
The Fundulus population was maintained in 50-gallon glass tanks
filled with artificial seawater (Triton)*. The water was filtered con-
tinuously through polyester fiber in Dynaflo water filters**. The
water was aerated by plastic bottom filters attached to an air pump
with a non-oil compressor. Plastic cooling coils kept the temperature
of the tanks at 17°C (±0.5°C). The water running through the coils was
cooled by a large Thermovac refrigeration unit***.
The pH of the water, buffered wuth NaHC03, was kept at 6.8 (iO.2).
The temperature of the tanks was monitored continuously by Weather-Hawk
recording thermometers****. Other parameters measured included specific
gravity, room temperature, relative humidity, and osmolarity. The fish
were fed once daily with frozen brine shrimp.
The fish kept in the tanks at our laboratory seemed quite healthy.
They ate well, swam through the tank energetically, and on only one
occassion were they affected by external parasites or fungus. The male
fish went into their breeding colors - the black spot on the dorsal fin,
the silver striping on the sides, and the yellowish belly. The popula-
of Fundulus carried over the winter and did reproduce in the spring.
However, the offspring did not survive; this is not surprising consid-
ering the number of fish present in the large holding tanks.
Freshwater Experiments
Since |\_ heteroclitus is a euryhaline fish and there had been prob-
lems with metals precipitating out in saltwater, it was decided to adapt
some of the saltwater fish to a freshwater environment. It was found
at the beginning that putting fish directly into freshwater from the salt-
water tank caused an 100% mortality within a few days. Subsequently, a
phase-over brackish tank (14£> was used to lessen the physiological shock
of the transfer.
Specifically, a 20-gallon tank was filled with artificial seawater
* Aquarium Systems Inc., Eastlake, Ohio
** Metaframe Corp., Maywood, New Jersey
*** Thermovac Industries Corp., Copiaque, New York
**** Taylor Instrument Companies, Asheville, North Carolina
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in half the concentration of the saltwater control tank. This tank was
buffered with NaIIC03 to a pH of 6.8 (tO.2). There were no cooling coils
in this tank, but the slightly higher temperature did not seem to bother
the fish, as there were no mortalities. After a minimum of one week in
the brackish tank, the fish were transferred to a 50-gallon freshwater
tank. This tank was equipped with cooling coils. With the initial phase-
over into brackish water, the fish, though obviously stressed in the fresh-
water, did not show the high mortality seen previously.
Freshwater experiments were designed to indicate the degree of ad-
aptation to the new environment. Fish from the phase-over 14%salinity
tank were sacrificed after a week in that environment. Similarily, after
a minimum of a week in the freshwater tank, fish were sacrificed to ex-
amine the condition of the circulating blood. The success of this phase-
over method of adaptation can be seen by comparison with the experiments
done placing fish directly into freshwater
Copper and Cadmium Experiments
Tests with Cu"*""1" and Cd"1"1" consisted of both short-term, long-term,
and recovery experiments. Metal ion tests were conducted in both fresh-
water and saltwater. The concentrations were kept sublethal (5 ), no
more than 5ppm for either Cu*"1" or Cd was used.
The primary test involving Cd"1"*" was a chronic exposure to Ippm for
ten months. The fish were maintained at the EPA headquarters in West
Kingston, R.I., and were brought up to these laboratories for testing.
Some of these fish were placed back in unpolluted saltwater for periods
of time from 21 - 42 hours, and then sacrificed.
II
The Cu tests were conducted in both freshwater and saltwater.
Test fish were placed in 1-gallon glass jars equipped with air bubblers.
Observations of fish behavior and appearance were made throughout the
exposure period. A stock solution of CuC^ was made up so that an ac-
curate measure of the concentration in the test jar could be made. The
experimental jars were monitored with a Lament Copper Test Kit*. In
the recovery experiments, the fish were put into 20-gallon saltwater tanks
after their exposure to Cu4"1".
The 1-gallon glass jars in these experiments were filled and then
immersed in a 15-gallon tank. Since the tank was equipped with cooling
coils, by immersing the jars in the tank, the water in the experimental
jars could be maintained at the temperature of the control tank. After
the experiments, these jars were cleaned thoroughly.
* LaMotte Chemical, Chestertown, Maryland
10
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Biological Multichannel Analyzer (BMA) and Photomicroscopy
At the time of sacrifice, fish were selected at random from the
saltwater control tank or the experimental jar/tank. As rapidly as
possible, the fish was sacrificed by making a transverse cut two-thirds
of the body length back from the head. Blood was collected from the
caudal artery in heparinized capillary tubes. One sample was put into
50ml 0.9% particle-free physiological saline to be monitored on the
BMA. Other samples went onto microscope slides for phase contrast obser-
vations and for Wright staining.
The sample diluted in 50ml physiological salin was monitored on the
BMA as soon as possible after the sacrifice of the fish, usually within
a minute. The technical explanation of the mode of action of tl.e BMA
is found in the Introduction. Using a sensor with a 30/x aperture, 0.05ml
from each sample was read at 4, 8 and 25/*second triggering delay intervals,
A printout was made of each distribution for the three triggering delay
intervals. A peak channel was determined, and the total cells sampled
were recorded using the 8/u.second delay setting. The BMA was calibrated
using a variety of polystyrene spheres and plant pollens on the 8 second
delay setting. In this way, the printouts could be interpreter by cor-
relating channel number with cell width in microns. The lower limit of
the printout was estimated at 3jn. , the upper limit at 10/A . Human blood
cells, which are 5 - 6^ in diameter, when monitored on the calibrated
electronic settings for fish blood came up midway in the 0-200 channel
printout range. This is another indication of the correctness of our es-
timated limits.
The slides of the blood cell samples made up for photomicroscopy
were allowed to air-dry. In the latter part of this research, phase
contrast microscopy was done in conjunction with the Wright staining.
Slides were Wright-stained using a commercial kit made by Gugol Clini-
Tex, Inc.* The stained cells were examined under oil immersion at
930X. Photomicrographs were made of representative fields; the negatives
were made into 5x7 prints. Measurements of cell length and width, and
nucleus length and width were made from the prints. The measurements
were analyzed statistically to obtain averages, variance, standard dev-
iation, and standard error. Calibrated polystyrene latex spheres were
used as size references, and a stage micrometer was used to obtain size
information on the cells studied under the microscope. Calibrated photo-
micrographs were also raade of the cells under phase contrast microscopy.
* Gugol Science Corp., North Elmsford, New York
11
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RESULTS
Controls
Specimens taken from the saltwater holding tank at random were sac-
rificed during each experiment to check the stability of the fish population^
Photomicrographs :
The circulating blood of ₯_._ heteroclitus is composed predominantly
of mature nucleated erythrocytes. These erythrocytes are elliptical in
shape with a centrally located nucleus. The fixed erythrocytes measure
between lOju. and 11^. in length, and 5/*> to 7^. in width.
Phase contrast optics indicate a smooth erythrocyte membrane (page 13)
Round dense granules appeared in many cells on the inner edge of the cyto-
plasmic membrane. The nuclei of the erythrocytes under phase contrast
optics were of an oval shape. The nuclei were dense and concentrated in
the center with a less dense nuclear membrane.
The photomicrographs of the Wright stained cells reveal the uniform
size and regular shape of the erythrocytes (page B ) . The cytoplasm of
the erythrocytes stained pale pink, the nuclei stained a dark magenta.
Granules were present in some cells on the inner edge of the cytoplasmic
membrane. The nuclei of these cells consist of densely packed and darker
stained chromatin clumps.
Electronic Printouts:
The BMA printouts of the saltwater control cells have shown a con-
sistent picture of normal population distributions (page ]& ) . The three
triggering delay intervals each have a distinct curve as well as a speci-
fic relationship with one another. Generally, the 4>usec triggering delay
interval was a single steeply risng and falling curve. The distribution
of the §A*sec triggering delay interval had a primary peak which fell
slightly and then rose again to a smaller secondary peak; this interval
had a broader distribution than the ^xsec interval. The 25»*sec trigger-
ing delay interval had the broadest of the three distributions. Like the
8/usec interval, the 2§Ksec interval had primary and secondary peaks in
approximately the same positions as the §txsec interval. In the control
population, the peak of the 4^.sec interval and the primary peaks of the
^usec and 25psec intervals all overlapped each other.
Just as there are fairly specif ic parameters for the normal popula-
tion as described above, there are also certain common deviations seen
in an abnormal experimental situation. In an abnormal population, instead
of overlapping one another, the peaks of the three triggering delay inter-
vals will separate from each other. As the peaks begin to shift apart,
there is an associated reduction and finally disappearance of the secondary
peaks of the §«Asec and 25/Asec triggering delay intervals. We have been
able to associate this peak shifting and peak reduction with changes in the
integrity of the cell membrane as well as changes in the proportions of cell
types in the circulating blood of chemically insulted fish.
12
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13
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CONTROLS
75-i
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RESULTS
Freshwater Adaptation Experiments
The freshwater experiments provided information on the ability of
Fundulus heteroclitus to cope with an environmental stress for which it
has the physiological capacity to adapt to. The osmotic stresses of a
freshwater insult were easier to interpret than the more complicated
actions of metal ions on the circulating blood.
A variety of adaptation tests v/ere conducted - short-term tests in
which the fish were in freshwater for less than 24 hours, longer tests
lasting up to one week, and extended tests in which the fish were fresh-
water adapted for over one week. Our objective in these tests was to
determine how long it took a population of F. heteroclitus to overcome
the initial stresses of the freshwater environment. Adaptation was eval-
uated by 1) an appraisal of the cells seen in the photomicrographs in-
cluding the statistical information from the photomicrographs, 2) a de-
termination of the extent to which the electronic printouts resembled
the printouts of the control fish, and 3) the judgement of an experienced
observer of a return to more normal behavior and appearance of the adapted
fish.
The first freshwater experiments were done by placing the fish directly
from the saltwater tank into buffered distilled water. This change was
too abrupt and would have resulted in 100% mortality within a few days if
the fish had not been sacrificed. The fish displayed common stress reac-
tions: remaining motionless at the bottom or very top of the tank, swim-
ming on the side or belly-up, and finally hemorrhaging around the mouth
and gills followed within hours by death.
The extreme reaction caused by the abrupt environmental change was
compared to a gradual phase- over of the fish from saltwater to freshwater.
As described in the Methods section, a 14%0salinity tank was set up to
reduce the stress of the fish. The fish remained in this tank for a min-
imum of a week before being placed in the freshwater tank; there were no
mortalities in the 14%osalinity tank.
None of the fish put into the freshwater tank from the 14% salinity
tank showed any signs of internal hemorrhaging, though a certain number
died during the first week, which was the most critical period. A number
of common stress reactions and behavior patterns were observed to be ex-
hibited by the fish in the freshwater. The first reaction of the fish
was to sink to the bottom of the tank and remain nearly motionless; a
smaller number would float near the surface, also remaining motionless.
Any swimming was feeble and only for short periods of time, there was none
of the constant swimming about throughout the tank that is characteristic
of normally active fish. When being fed, the fish had no sense of the
food in the water; they did not swim up to the surface when fed. Very
often, there were color changes in the fish, many becoming splotchy with
15
-------
white spots on the darker body.
After about a week the fish appeared more normal in behavior and
appearance. They swan freely throughout the levels of the tank. Before
feeding, the fish swan to the front of the tank seemingly in anticipation
of being fed. This reaction has been observed often and was one indica-
tion of a healthy fish. After 2-3 weeks in the freshwater tank, the elec-
tronic tests conducted indicated that the circulating blood in these out-
wardly healthy fish was similar to that seen in the controls.
After 3-4 weeks in the freshwater tank, some fish were tested by
placing them in jars of fresh buffered distilled water. We wished to
determine whether the fish were really adapted to the freshwater or
whether the fish had changed some chemical or physical properties of the
freshwater they were in that eased their adaptation and resulted in nearly
0% mortality. If the fish were again put into fresh distilled water,
would they exhibit the same stress reactions and greater mortality?
Photomicrographs:
Phase contrast microscopy was not done at the time of these ex-
periments.
The Wright stained photomicrographs of the fish placed directly in
freshwater without a gradual phase-over reveal striking abnormalities from
the normal blood picture. The cells became swollen and in many instances
the cytoplasmic membrane broke down leaving enlarged free nuclei (page 18).
Another distinct feature was the large per cent of thrombocytes in the
circulating blood. Thrombocytes are involved in the clotting process in
fish. Since the fish showed signs of hemorrhaging around the mouth and
gills, it was not surprising to see the great number of thrombocytes.
These cells may join to form a thrombocytic network which is formed by the
cohesive properties of the cytoplasm of the thrombocytes.
The photomicrographs also indicated an altered permeability of the
cell membrane to the Wright stain. The cells of the fish exposed to
freshwater for short periods of time (up to 2 hours) did not take up the
stain normally (page 18). The nuclei were indistinct, rather than appearing
dense as they usually do. The longer the fish remained in the freshwater,
the more distinct the nuclei appeared in the photomicrographs. To ex-
plain this change in permeability, it was theorized that during the first
few hours in freshwater, the cells are undergoing either partial or total
hemolyzation due to the changes in the osmotic pressure. It
was this process which affected the cell membranes.
During the gradual phase-over to freshwater, fish were sacrificed
after a minimum of one week in the 14%0 salinity tank. The cells in the
photomicrographs were not as uniform in size and shape as the control cells,
but otherwise they appeared normal, i.e. there was no swelling and there
were a normal number of thrombocytes frage 18 )
16
-------
After 8-10 days in the freshwater tank, the fish appeared much less
stressed, and some of the fish were sacrificed to try to determine the
degree of adaptation. The cells measured in the photomicrographs differed
from the control cells in that there was an average increase of ILL in the
length of the freshwater adapted cells. There were few free nuclei and
an average number of throrabocytes (page 20) .
Finally, when fish were put into the fresh distilled water from the
freshwater tank, the cells and nuclei became swollen in the first hour
(page 21). As the exposure was continued, the cells and nuclei became less
swollen in all dimensions and the variance decreased (page 21) .
Electronic Printouts:
There are several features that distinguish the printouts of the cells
of fish placed directly into freshwater with no phase-over period. There
was often a large pre-peak build up in the 4 sec triggering delay interval
(page 19). The build up occurred from the lower limit of the printout and
merged with the primary peak. The size range of this large population
was 3-4 microns in width; this probably represented the thrombocyte popula-
tion seen in the photomicrographs. Another feature of these printouts
was the reduction or disappearance of the secondary peaks of the 8>*sec and
25^/sec triggering delay intervals .
Fish sacrificed from the 14%0 salinity phase-over tank had printouts
resembling the controls (page 19). The distribution of the 4/isec triggering
delay interval was more spread out than the control; this indicated that
there was quite a variance in the cells seen at this interval. The fyusec
and 2^usec triggering delay intervals were fairly normal, both intervals
having some distinguishable secondary peak.
The fish adapted to freshwater were much more normal than those fish
placed directly in freshwater with no phase-over, in terms of what the
electronic printouts revealed (page 20). In the 4/«sec triggering delay
interval there was a slight pre-peak build up and the distribution was quite
broad; but the deviation from the control situation was not so obvious. The
and 25>isec triggering delay intervals were both normal.
It appeared from these freshwater experiments that the changes which
occurred due to exposure to freshwater were reflected primarily in changes
in the distribution made at the 4^sec triggering delay interval.
17
-------
?
m
v
*~ * ''''
* **
'
%
- 2 HOURS
FRESWATER - " IDURS
FRF.S»WVTER - 6 itxiRS
TATI
18
-------
FRESHWATER - L\ HOURS
25^8
FRESHWATER PHASE-OVER TANK
-------
CONTROL VS. KKi.:,iiUATKR ADAPTED
CONTROL
A F.W. Adapted
FRESUWATtR ADAPTED
-------
DISTILLED WATER vs. Cud2/DISTILLED WATER
y,
if*
00 *
%
«
I
CONTROL
DISTILLED WATER - 1 HOUR
DISTILLED WATER - 3 HOURS
%
DISILLED WATER- 4 HOURS
21
-------
DISTILLED WATER
10/29/71-4 FfW. Adapted
NJ
ho
25 sec
8 sec
4 sec
10/29/71-3 Distilled H?Q 1 hr 10/29/71-9 f?^Dljtllled HgO^? hrs 10/29/71-10 ^Distilled H20 4 hrs
-------
RESULTS
Cadmium Experiments
The primary experiment involving cadmium was a test done in collabora-
tion with the EPA headquarters in West Kingston, Rhode Island. At the EPA
facilities, Fundulus heteroclitus was exposed to Ippm Cd"*"*" for 10 months.
After the 10 months, the fish were transported to our laboratory to be
evaluated. Some of the fish were evaluated directly from the water they
were transported in and some of the fish were placed in unpolluted salt-
water for various intervals from 20.75 hours to 42 hours. The blood of
these fish was examined on the BMA. This results of this chronic exposure
test and the subsequent return of a small number of the fish to unpolluted
saltwater are summarized in Tables I'and II.
A short-term exposure to CdCl2 was conducted at our laboratory. There
was a problem in this experiment and the validity of the results are ques-
tionable. However, it served to point out some of the problems involved
in determining the exact amount of a metal -dissolved in either freshwater
or saltwater. When the CdCl2 was added to the freshwater experimental tank
a certain amount precipitated out as CdOE^. Thus, it was difficult to de-
termine with any accuracy the amount of free Cd"*"*". The fish used in this
cadmium experiment were fish taken from the freshwater adaptation tank. During
the time of the test, the fish.remained normal in both behavior and appear-
ance. The saltwater controls and freshwater adapted fish were also normal in
both behavior and appearance. Fish were sacrificed after 3.5 and 5.75 hours
in the experimental tank.
Photomicrographs:
The dimensions of the cells and nuclei of the experimental fish in the
short-term exposure were similar to the fish from their "parent" environ-
ment, the freshwater adapted tank (page 2Q.
It appeared from the statistical data as well as the appearance of the
cells in the photomicrographs that there was little if any effect on the cells
and the nuclei from the cadmium. Since most of the cadmium had precipitated
out as CdOH2, this non-effect would not be surprising.
Electronic Prinouts:
I i
The printouts for the fish exposed to Cd for 3.5 and 5.75 hours in
the three triggering delay intervals were fairly normal (page 27). In the
4i*sec triggering delay interval, the single peak was slightly more spread than
usual indicating more variance in cell widths than normally found. The
8j*sec and 25/tsec triggering delay intervals were normal in all cases; that is,
a major primary peak followed by a smaller secondary peak with the 25/tsec
interval distribution broader than the 3>usec interval.
As noted in the photomicrographs, there did not seem to be any disruption
in the normal cell state caused by the cadmium. The problem, as stated be-
fore, was determining how much Cd was available in solution.
23
-------
TABLE I
FISH - IN CADMIUM AND REVERSED
Number
Control #23
Cd-1
Cd-2
Control #26
Cd-3
Control #28
Cd-«f
Cd-5
Cd-6
Cd-7
Cd-8
Cd-9
Cd-10
Control #30 PM
Control #29 AM
Cd-11
Cd-12
Cd-13
CA-lk
Cd-15
Control #31
Cd-R-1
Cd-R-2
Control #32
Cd-R-3
Cd-R-'i
Cd-R-5
Metal ppm
Cd Ippm
Cd Ippm
Cd 1 ppm
Cd Ippm
Cd Ippm
Cd Ippm
Cd Ippm
Cd Ippm
Cd Ippm
Cd 1 ppm
Cd Ippm
Cd Ippm
Cd Ippm
Cd Ippm
Cd Ippm
Salt H_0
Salt H|O
Salt H-0
Salt HTO
Salt H_0
Time Exposed
10 months
10 months
10 months
10 months
10 months
10 months
10 months
10 months
10 months
10 months
10 months
10 months
10 months
10 months
10 months
20 3/4 hrs
21 hrs
41 3/4 hrs
41.9 hrs
42 hrs
Jt lisec
Normal
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
Cd
X
X
X
X
X
X
X
X
X
X
8 Vsec
Normal
X
X
X
X
X
X
X
X
X
X
X
X
Cd
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
25 Vsec
Normal
X
X
X
X
X
X
X
X
X
X
X
X
%
Cd Observation
X 25vsec No i
X
X
X
X
X
X
X
X
X
X
X
X
X
X
-------
TABLE II
FISH - IN CADMIW1 AND REVERSED
By Channel (Peak) Number
Ul
Number
Control #2?
Cd-1
Cd-2
Control #26
Cd-3
Control #28
Cd-4
Cd-5
Cd-6
Cd-7
Cd-8
Cd-9
Cd-10
Control #30 PM
Control #29 AM
Cd-11
Cd-12
Cd-13
Cd-l4
Cd-15
Control #31
Cd-R-1
Cd-R-2
Control #32
Cd-R-3
Cd-R-4
Cd-R-5
Metal ppm
Cd 1 ppm
Cd Ippm
Cd Ippm
Cd 1 ppm
Cd Ippm
Cd Ippm
Cd Ippm
Cd Ippm
Cd Ippm
Cd Ippm
Cd Ippm
Cd Ippm
Cd Ippm
Cd Ippm
Cd Ippm
Salt H-0
Salt H|0
Salt H-0
Salt HTO
Salt HTO
Time Exposed
10 months
10 non<. is
10 months
10 months
10 months
10 months
10 months
10 months
10 months
10 months
10 months
10 months
10 months
10 months
10 months
20 3/4 hrs
21 hrs
41 3/4 hrs
41.9 hrs
42 hrs
4 Vsec
Normal
51
52
58
58
56
50
54
53
52
54
58
41
53
50
40
4l
44
43
Cd
65
63
65
60
71
65
75
6l
65
b lisec
Normal
74
88
82
83
80
78
85
74
70
77
70
70
Cd
88
84
96
90
104
94
103
103
115
91
91
92
103
109
120
25 Vsec
Normal
74,112
81,
79,
79,
77,
79,
81,
70,
66,
75,
68,
66,
Cd Observation
93 25 Vsec No 2
80,
89,
88 25 Vsec No 2
98 " "
92,
97,
93,
93,
83,
91,
90,
108,
110 25VsecNo?
121 " " »
-------
CdCl2 in FRKSHWATER
*.
ifc ^'
^^^ ^^^^r
CONTROL
CdCl2 - 3.5 HOURS
\ %
FRESHWATER-ADAPTED
CJC12 - 5.75 HOURS
26
-------
CdCl2/ DISTILLED WATER
8/25/71-1 Control
25 sec
8 sec
8/25/71-5 Control -
8/25/71-4 F.W.
8/25/71-3 CdCl, 3.5 hrs
8/25/71-6
CdCl2 5.75 hrs
-------
RESULTS
Copper Experiments - Freshwater vs. Saltwater
Tests were conducted to compare the ability of freshwater adapted
and normal saltwater fish to cope with a Cu*4" insult. Freshwater adapted
fish were placed in jars containing l.Sppm Cu"4"1" diluted in freshwater. Salt-
water fish were taken from the control tanks and put into jars of saltwater
containing l.Sppm Cu"1"*- Some fish were sacrificed after 24 hours in the
test environment; others were left fro a 96 hour period. During this 96
hour period, one fish in Cu++/freshwater died, another was nearly dead.
The fish in Cu**/freshwater did not eat during the test period; not
did they seem to sense the presense of food in the water. The eyes of
these fish were much darker than normal. However, these fish did swim about
the jar and did not seem sluggish. The fish in Cu^/saltwater appeared
and behaved normally throughout the test period.
Another series of freshwater vs. saltwater tests were conducted using
a slightly higher concentration of Cu (2ppm). Fish were sacrificed at
intervals of 1, 3, 5, and 24 hours after exposure. The appearance of the
test fish during the first few hours of the experiment remained normal. The
fish behaved fairly normally, though perhaps more sluggish than usual. After
24 hours,-however, the behavior of the fish in the test jars was abnormal.
They either remained motionless on the bottom of the jars or swam rapidly
and erratically with jerky motions. When fed, the fish in Cu++/freshwater
did not eat or seem to sense the food in the water; the fish in Cu /salt-
water did eat.
At the time of sacrifice, the blood of some of the fish in Cu++/fresh-
water was abnormally dark and viscous. Only one of the fish in Cu /salt-
water showed this same phenomenon.
Photomicrographs:
11 i
After a 24 hour exposure to l.Sppm Cu , the cell size values in both
freshwater and saltwater experimental fish corresponded well to those
of the fish from the "parent" tanks. Generally, the cells of the test
fish were more irregular in size and shape than the controls (page 31).
The greatest difference between the Cu^/freshwater and Cu"H"/saltwater fish
was in the nuclear staining. The nuclei of the fish in Cu**/saltwater
showed what may be described as light-spot staining; that is less dense spots
or streaks in the nucleus. This phenomenon was consistent in all fish from
the Cu /saltwater environment.
At 96 hours in l.Sppm GU++, the cell dimensions of both sets of ex-
perimental fish were still within the size range of the fish from the
"parent" tanks. However, there was a definite change in the nuclei of the
experimental fish. The lengths of the nuclei in both sets of fish (fresh-
water and saltwater) were longer than the controls ( page 33. The nuclei
of the fish in Cu-H-/saltwater still exhibited light-spot staining. The cell
28
-------
shapes of these fish as well as the Cu /freshwater fish were also ir-
regular. One of these fish, which was nearly dead at the time of sac-
rifice, had cells and nuclei which were greatly disrupted; this disruption
consisted of swelling and bursting cells, a phenomenon seen often in dying
fish.
The data from the photomicrographs seem to indicate that whatever
happens on a cell membrane or cytoplasmic level, at this copper con-
centration, happens within a few hours after the exposure to copper. After
24 hours, it appears that the nucleus is the focus of change.
In the previous experiments, the effects of exposure to copper for a
few hours was not tested. Since some important membrane phenomena may
have been missed, tests were conducted to determine the more immediate effects
of 2ppm Cu"*~*" of the fish cells. Comparing the experimental fish with the
fish from the '{parent" tanks, a number of striking differences were found.
While the cell widths of both sets of experimental fish remained the same
as the controls, the cell length changed strikingly (35, 36 ). The situa-
tion of cells swelling, which has often been seen, did not occur here; the
cells changed in length only. Thus, another process seemed to be taking
place. The cells of the Cu /freshwater fish were shorter and rounder than
normal, and the cells of the Cu '"/saltwater fish were more elongated. Further,
the changes did not appear to increase with time elapsed. The greatest
change seemed to occur between the first and second hours.
After 24 hours in a 2ppm Cu"*""1" environment, the size values were within a
range of values shown by the fish from the "parent" tanks. The difference
was that the variance had increased. The cell length was again the most
affected dimension; the cell lengths of the fish in Cu /freshwater were still
relatively small; and those of the fish in Cu++/saltwater had decreased
considerably and come into line with the controls (page 38).
It was noted in the introduction to this section that several fish were
observed to have dark, viscous blood at the time of sacrifice. These
fish had blood cells that were extremely short and rounded. These observa-
tions appear to indicate that there is possible interference with the oxy-
genation of the blood. This phenomenon seemed to affect the fish in the
Cu"H'/freshwater environment to a greater extent than the fish in Cu++/salt-
water.
Electronic Printouts:
In discussing the mensurational data from the photomicrographs, it
was noted that the cell sizes did not vary greatly from the values of the
controls. In analyzing the printouts, it will be remembered that it is the
cell width which is the critical dimension.
In the case of the exposure to l.Sppm Cu"*""1" for 24 and 96 hours, it
was the cell nuclei which were the focus of change. Although the cells were
less regular in size and shape than the controls, the cell size values
were within the limits of the controls. The electronic printouts reflected
this situation (32, 34). While the peak channel does not change greatly
29
-------
the distributions do differ from the normal, indicating changes in shape
not size.
In an exposure of 1 hour to 2ppm Cu^"*" , the cell length was the
affected dimension. But since the electronic printouts are a reflection
of cell width, and this dimension did not change, the printouts did not
show an abnormal size distribution. Fish subjected to 2ppm Cu"*""1" for 3 and
5 hours did show abnormal electronic assays (page 37).
All of these electronic distributions must be studied on terms of the
relationships of the triggering delay intervals to one another. In a
nomal cell state, the major peaks of the three triggering delay intervals
fall almost in the same channel, this indicates similarity of size within
a cell population. But in a changed cell state, as the secondary peak
of the S^sec triggering delay interval begins to disappear, the peak of
the 4,iAsec triggering delay interval shifts away from the main peak of the
8^/tsec triggering delay interval. The 25^*sec triggering delay interval
follows the same pattern as the §tusec interval.
In the freshwater vs. saltwater experiments, the longer the fish were
exposed to Cu the greater the number of fish which had separated peaks
in the electronic printouts. The same held true the greater the concentra-
tion of Cu4"1" which was used. In addition, when comparing the Cu++/freshwater
and Cu+^/saltwater environments, the fish in the Cu /freshwater were the
first to show secondary peak reduction and peak separation. It appeared
that the freshwater adapted fish when subjected to further environmental
stress were more vulnerable to the added insult.
30
-------
CuCl2/SALTWATER vs. CuCl2/FRESHWATER
1
m * +
W--
ft
CONTROL
CuCl2/SALTWATER - 24 HOURS
Ir
0
*9 +
FRESHWATER-ADAPTED
CuCl2/FRESHWATER - 24 HOURS
31
-------
to
25 sec
8 sec
CuClj/FRESIIWATER VS. CuClj/SAUWATER
1.Sppra
3 Cud, in F.W. 24 hrs 11/19/71-5 CUC12 in F.W. 24 hrs 11/19/71-10 CuCl2 in F.W. 24hrs
CuCl2 in S.W. 24 hrs 11/19/71- / |6 CuCl, jn s.W. 24hrs 11/19/71-11 CuCl; in S.W. 24 hrs
-------
CuCl2/SALTWATER vs. CuCl2/FRESHWATER
CONTROL
CuCl2/FRESHWATER - 96 HOURS - NEARLY DEAD
CuCl2/FRESHWATER - 96 HOURS
CuCl2/SALTWATER - 96 HOURS
33
-------
CuCl2/FRKS!WATER VS. CuCK/SALTHATER - l.Sppra
in F.H. 96hrs nearly dead
25 sec -
8 sec
4 sec
in S.W. 96 hrs 11/22/71-5 )CuCl2 in 5.W. 96 hrs
in F.W. 96 hrs H/22/71-4CuCl2
-------
CuCl2/SALTWATER vs. CuCl2/FRESHWATER
e*
...
>
9
«
ft
-
>
i
CONTROL
FRESHWATER-ADAPTED
CuCl2/FRESHWATER - 1 HOUR
CuCli/SALTWATER - 1 HOUR
35
-------
CuC IT/SALTWATER vs. CuCl i/FRESHUAi l.K
^ V
IT 9
CuCl,/FRKSHWATKR - 3 HOURS
CuCl2/SALTWATER - 3 HOURS
CuCl ) /FRESHWATER - 5 HOURS
~
I
/SALTWATER - 5 HOURS
* *
36
-------
U)
25 sec
8 sec
sec
CuCl2/FRESHWATER VS. CuC^/SAWHATF.R - 2ppm
12 in F.W. 1 hr ACuCl? in F.W. 3 hrs
C^uCl2 In F.W. 5 hrs
-------
A
CONTROL
*
t-f
CONTROL
CuCl ,/FRtSHWAIER ~ ^ HOURS
CuCl ?/SALTV)ATER - ?4 HOURS
38
-------
U)
CuCl2/FRESHWATER VS. CuCl2/SALTWATER
2ppm
12/3/71-3 / \CuCl2 in F.W. 24 hrs
25 sec
8 sec
4 sec
12/3/71-1 | \ Control
CuCl, in S.W. 24 hrs 12/3/71-s Cud, in F.W. 24 hrs 12/3/71-6
- - ----- ' "
Jn S W 24 hrs
* *
-------
RESULTS
Copper Experiments - 5ppm and Recovery Tests
For this series of experiments, a concentration of 5ppm copper was
used to provoke a quick and dramatic response to the pollutant. The ex-
perimental protocol was similar to that described before. One gallon glass
jars were filled with saltwater, the experimental fish x^ere placed in the
jars, and finally, the CuCl2 was added to the jars. The recovery exper-
iments used the saine test set-up. After exposure to Cu"1"*, the fish were
placed in an unpolluted 15-gallon saltwater tank for a specified recovery
period.
Exposure times varied from 45 minutes to 44 hours. The 45 minute ex-
posure was designed to characterize the early effects of the metal on the
cells. Short-term exposures were continued for 4.5 and 5 hours.
The progression of exposure times was continued with a 24 hour ex-
posure. The results up to this point did not indicate a clear series of
of reactions. The 45 minute exposure to Cu"1"*" resulted in a greater dis-
ruption of the normal cell state than the 4.5 hour exposure to the metal.
This may indicate a dramatic initial reaction which looses its effect after
a time; that is a reaction only involving the blood cell membrane.
We wished to determine whether this immediate effect was the only
effect. Therefore, the time exposure to Cu was increased to determine
whether the reaction to Cu"*"*" was only transient.
After 24 hours, the cells were greatly disrupted. To estimate whether
this damage would progress further, the exposure was extended to 44 hours.
Short-term reversal experiments were carried out to get information
on the ability of the fish to recover from an insult by a metal. A 24 hour
exposure to the metal was chosen, as we had observed this time was long
enough to cause definite damage to the cells, but not long enough to kill
the fish. It was found that exposure for 24 hours to CuCl2 caused internal
not just superficial injury to the cells. We wished to learn whether this
kind of injury permanently or only temporarily altered the affected blood
cells. Fish in a natural environment exposed to chronic, not lethal, levels
of a pollutant may seem normal in behavior and appearance, but in fact may
be damaged in a way that interfers with reproductive or social behavior or
their ability to withstand any further environmental stress. It was hoped
that the reversal experiments would provide information on 1) the fish's
ability to recover from a heavy metal insult, 2) the level of metal in
the water with which the fish are unable to cope, and 3) the exposure time
in the polluted water after which the fish cannot recover.
Two recovery intervals were tested. After a 24 hour exposure to Cu"*"1",
fish were placed in the saltwater recovery tank for 10 hours and sacrificed;
others were left for one week and then sacrificed.
40
-------
Photomicrographs:
After a 45 minute exposure, both phase contrast studies from fresh
blood and bright-field studies of the Wright stained cells showed many
irregularities in cell shapes.
In phase contrast microscopy, some cells appear in a "u" or "v" for-
mation, others showed a saucer effect or were in other abnormal shapes
(page 46). Many still had the smooth edge on the plasma membrane, others
showed a jagged sword effect. The nuclei appeared normal, but as they had
not been satined no details could be seen. An uneven cytoplasmic trans-
parency was observed which was not present in the control erythrocytes,
and at the same time some cells had an opaque appearance.
In the photomicrographs, many of the Wright stained appeared abnormal
in shape (page 46). The cytoplasmic membrane had lost its rigor and a num-
ber of odd-shaped cells were evident. Although about half the cells have
retained the typical oval shape, many of the cells were swollen and rounded,
some had a sickle shape. The cytoplasm was much more transparent than in
the controls, and some showed an uneven clearing as in the phase contrast
studies. The nuclei exhibited the same granular mottled appearance seen
in the control cells. The nuclei of these cells were slightly more rounded
than the controls. Some of the nuclei were off-center. A few of the nuclei
were lobed.
In this 45 minute exposure to Cu , the greatest disturbance seemed to
be to the cytoplasmic membrane. The effects of the metal ranged from a
distortion of the cell shape to a total breakdown of the cytoplasmic membrane.
The nuclei seemed little affected by the metal.
Most of the cells exposed to Cu"*"*" for 4.5 to 5 hours maintained their
cytoplasmic membrane intact. Most of the cells have retained an oval shape,
though some were quite swollen (page 47). Some of the cells exhibited pro-
trusions of the cytoplasm similar to pseudopodia. In a few of the cells,
the cytoplasm appeared to be breaking down. Most of the nuclei seemed gen-
erally unaffected. A few of the nuclei were more round than oval, and some<
were not centrally located. The stain was taken up uniformly, except in
the swollen cells in which the nuclei were lighter in color.
After 24 hours, the phase contrast studies of the erythrocytes from
fresh blood showed definite atypical characteristics. Some had long points
or spear shapes, others had clear white holes resembling vacuoles in the
cytoplasm in a characteristic pattern (page 49). In many cells, the plasma
membrane did not stand out as in the phase contrast control cells. Some of
the cells had sickle shapes clearly outlined with the phase optics. The
nuclei were of the normal oval shape. With phase contrast studies, white
clearings were visible in some cell nuclei disclosing mottling of the chroma-
tin, while others exhibited full clearing of the nucleus.
It can be seen in the photomicrographs of the Wright stained cells
that after 24 hours in Cu"H~,the cytoplasmic membrane had lost its rigor.
41
-------
The edges were jagged and irregular (page 49). In many cases, the cyto-
plasm barely surrounded the nucleus. The cytoplasm of many cells seemed to
have a cohesive property causing some of the cells to become aggregated.
Although the cytoplasraic membrane appeared to have lost its rigidity and
the cytoplasm appeared to be breaking down, there were few free nuclei.
The nuclei varied greatly in size and shape. In some of the photomicro-
graphs (not included) the cells and nucleihad swollen enormously. The
nuclei had stained unevenly; the erythrocytes showed large variations in
the transparency of the cytoplasm. It would seem that the effects at this
24 hour exposure went beyond the transient effects to the membrane, since
the nuclei were also affected by the copper.
After a 44 hour exposure to CuCl2, the same clearly defined pattern
of deleterious effects is observed from the phase contrast photomicrographs.
A sickle cell phenomenon was visible with the cells having a smooth plasma
membrane (page 50). White clearings were consistently observed in the
cytoplasm usually in two's with one clear spot or vacuole at each end of
the cell. Some irregularily shaped cells differing from the above were
present, a few with jagged plasma membranes. Mixed in with the other cells
were normal appearing cells. In some cases, the nuclei were more trans-
parent than those observed in the controls. The mottled chromatin was
visible.
The Wright stained cells in the photomicrographs showed a greater
disturbance of the normal state of the blood cells than the 24 hour ex-
posure to CuCl2 (page .50). The cytoplasraic membrane again was seen to
have lost its rigor. The cytoplasm protruded, similar to pseudopodia, in
certain instances'. In other cells, the cytoplasm appeared to be breaking
down and freeing the nucleus. The cytoplasm of some of the cells seemed
to have meshed together. Several cells resembling thrombocytes were seen
in the photomicrographs ( page 50- The nuclei were stained unevenly, some
nuclei were swollen and rounded. The most swollen nuclei were those whose
cytoplasm had begun to break down.
In the first reversal experiment, fish were exposed to CuCl2 for 24
hours and then returned to unpolluted saltwater for 10 hours. The phase
contrast photomicrographs showed theerythrocytes to have a similar morph-
ological structure to those observed after 24 hours in CuCl2, i.e. irregular
shapes, plasma membrane indentations, atypical edges, and a sickling pheno-
menon. The return for 10 hours to the normal slatwater environment did not
restore the red blood cell population to that seen in a normal control-( page
52 ). A typical thrombocytic network was seen in fresh blood preparations
in phase contrast photomicrographs (page 52). Pseudopods of thrombocytes
linked with the pseudopods of other thrombocytes. Erythrocytes that came
into contact with the thrombocytic network became enmeshed.
The photomicrographs of the Wright stained cells showed very swollen,
rounded cells. There were very few of the normal oval shaped cells. The
cytoplasmic membrane, though not normal, seemed to have regained some rigor
(page 52). Numerous cells still exhibited the cytoplasmic protrusions and
jagged irregular edges, suggesting an injured plasma membrane unable to
42
-------
return to its normal shape. The cytoplasm of many of the cells was quite
transparent. The nuclei were also much more rounded than oval. Some were
quite swollen and these did not take up the stain well; they were much
lighter in color. All the nuclei had the granular appearance seen in the
normal nuclei. The chief difference between these cells and those seen after
an exposure of 44 hours was that there were fewer free nuclei and the cell
cytoplasm, though extremely swollen, at least had remained intact. The fact
that there was some rigor to the membrane may indicate a step towards re-
covery, though the cells seen in the photomicrographs were far from normal.
The cytoplasm and the nuclei were still affected by the copper insult. The
numerous cells with a frayed and irregular cytoplasmic membrane also in-
dicated the inability to return to a normal condition in this 10 hour re-
covery period.
The return to unpolluted saltwater after a 24 hour exposure to CuCl2
was continued for one week. Although some normal survivor erythrocytes
were present, as in all studies, nevertheless, phase contrast photomicro-
graphs revealed cells which continued to show injury. The beginning of a
recovery was evident in the disappearance of the sickled shapes, irregular
edges and plasma membrane injuries observed in the cells studied after 24
hours in a CuCl2 environment without restoration to normal saltwater ( page
53). However, most of the cells lacked the true normal control oval shape.
Signs of persistent internal injury were evident in most of the cells. Ir-
regular white clearings or vacuolations appeared in the cytoplasm of most
of the cells. Irregular sword-shaped plasma membranes similar to those seen
in the membranes of erythrocytes after one hour in a copper environment were
still present in some cells. This might suggest an attempt at plasma mem-
brane restoration. The nuclei of many cells instead of revealing the dark
opaque effect of the normal control cells were transparent. This may in-
dicate a clearing of the chromatin clumps and suggest a permanent copper
involvement with the nuclear material. In the phase contrast photomicro-
graphs, erythrocytes containing clear cytoplasm and some cytoplasmic in-
clusions were present. The thrombocytic network was seen again.
The high-dry photomicrograph (page 53) was of particular interest in
that it provided an overall idea of the RBC population. This photomicro-
graph depicted a very mixed population of both immature, mature and non-
viable erythrocytes. This was an unusual situation in that normally there
were few immature cells in the circulating blood (Ref. 11).
Some of the Wright stained cells of these fish seemed to show signs
of recovery. The cytoplasmic membrane shapes of these cells, though still
atypical, appeared to have approached the uniform shape of the controls
(page 53). The membranes appeared to be faint and jagged at the edges. In
general, the cells resembled those exposed to CuCl2 for 45 minutes. The nuclei
were more oval than previously seen. Most of the nuclei were centrally loc-
ated, another indication of a return of the red blood cell population towards
normalcy. There were few free nuclei. The Wright stain seemed to have been
taken up uniformly by the cell nuclei. After a 24 hour exposure to 5ppm CuCl2
followed by a return to unpolluted saltwater for one week, signs of recovery
and restoration of the erythrocyte population of the fish to normal shapes
were evident; but the cells still showed signs of internal injury.
43
-------
Electronic Printouts:
The DMA was not available to assay the fish exposed to CuCl9 for 45
-oc *
minutes.
Just as the photomicrographs gave a fairly normal picture of the cell
condition at an exposure of 4.5 hours, so the BMA printout of this interval
appeared quite normal (page 48). The peaks of the three triggering delay
intervals all overlapped, and the 8j*sec and 25/*sec intervals both had well-
defined secondary peaks. The printout of the 5 hour exposure was a markedly
abnormal printout (page 48). These findings indicate that the effect varied
with time among the individual fish.
The BMA printout of the 24 hour exposure to CuCl2 was abnormal. It
correlated with the condition of the cells seen in the photomicrographs
(page 51). The 4>sec triggering delay interval had two distinct peaks, the
second being slightly larger than the first. The first peak appeared at
channel 11, corresponding to an apparent diameter of about 3.4/x., the average
width of the nuclei. As can be seen in the photomicrographs (page 49), there
were few free nuclei; instead, the cytoplasm had broken down and was hugging
the nuclei. Thus, since there was no swelling of the nuclei, the pulses gen-
erated corresponded to the width of the average nucleus. This peak was only
present in the 4^sec interval because the cells were only as long as the
nuclei and would not be seen in the S^sec interval. The second peak of the
4/Asec triggering delay intervalhad shifted upward on the scale to a point
slightly larger than was usual. This peak probably represented the cell
population whose cytoplasm had remained intact though swollen. The 8/isec
interval had a peak similar to the second peak of the 4/*sec interval; this
indicated that the majority of the cells generating these pulses were fairly
round or slightly oval. Since a similar peak was not seen in the 25/csec
interval, these cells were probably not very long, but quite wide. The
distribution seen in the 25^sec interval pointed to a segment of the popula-
tion of erythrocytes of large and long cells; cells such as these were seen
on the slides of the Wright stained cells.
The electronic assay of the exposure to CuCl- for 44 hours was abnormal,
but quite different from the printout of the 24 hour exposure test ( page 5$
The 4^isec triggering delay interval had lost the first peak that came up
in the low channels. This may indicate that any free nuclei in the popula-
tion were quite swollen; this was usually the way the nuclei appeared. The
curve of the 4>»sec interval was extremely broad indicating a great variation
in the population sizes seen in this triggering delay interval. Usually the
pulses observed at this interval were uniform, that was not the case here.
In addition, there was a distinct population of very large cells as evidenced
in the small secondary curve of this triggering delay interval. The peak
of this interval was distinct from the S^sec and 25^sec delay triggered peaks,
and was one indicator of an abnormal cell situation. The peaks of the S^sec
and 25^ysec triggering delay printouts had shifted quite far up scale in-
dicating that a large segment of the population was swollen and rounded, a
fact borne out by the photomicrographs. This was an abnormal printout with
44
-------
characteristics seen often before - the separation of the peak of the 4 sec
triggering delay interval from the primary peaks of the 8 sec and 25 sec
triggering delay intervals; and the corresponding disappearance of the
secondary peaks of the 8j/.sec and 25/^sec intervals. The electronic printout
showed a general swelling of the cell population.
A representative printout of the 10 hour return to saltwater was
similar to the printout of the 44 hour exposure to CuCl-, with no return
(page 54). The broad 4/-sec interval distribution with its small secondary
peak was found here as it was in the 44 hour exposure test. The 4txsec delay
interval was separate from the Sj^sec and 25/Asec triggering delay intervals,
and the secondary peaks of the S^isec and 25/Msec intervals were gone. This
was theuusual pattern indicative of the abnormal copper-induced cell state.
The electronic printout described a population of large, swollen cells.
Though the printouts of the return to unpolluted saltwater for one week
were not normal, they indicated cells that had returned to a more normal
size-shape distribution. Printout "a" showed the peaks of the three trig-
gering delay intervals overlapping, one of the signs of a more normal con-
dition. There was a slight suggestion of a secondary peak in the S^sec
interval, and the 25/jusec interval had a much more pronounced secondary peak.
Printout "b" showed the typical abnormal pattern in the peak separation and
the disappearance of the secondary peaks in the S^ysec and 25/*sec triggering
delay intervals. But the hopeful sign here was that the peaks had shifted
down scale indicating the absence of large swollen cells seen before. The
printouts revealed the same preliminary signs of recovery seen in the photo-
micrographs (page 54 ).
45
-------
»
^ ^ y
* ^
-, >-
* "^fc^
"
IM1ASF.
CuCl2-5ppn 45 mins: 5/22/73
STAINED
i ft,
i ft * *
_
:.
+ i'
* *
=f *
-------
CuCl2-5ppn 1 hr: 5/15/73
PHASr
CuCl2-5ppm ^..5 hr: 5/21/7 !
^
^
47
-------
CUCL2- 5PPM
00
73.
4JS HOURS
5 HOURS
-------
f
,*'*f,-
v If
PHASE
CuCl2-5ppm 2A hrs:5/22/73
STAINED
,7'V*V K »
? *^^ * , ^ *
» *». * .*»»»S'
,!«** »'**,!
» * :;* V» *
*
«»_ .1
t
49
* * ' ***%
* ^ %**?* ^ ^ %%^>
-------
* .
*
PHASli
STAINED
v
'«*
CuCl2-5ppm 44 hrs: 5/29/73
'*
?-
50
-------
- 5PPM
24 HOURS
44 HOURS
-------
PHASI-:
STAINED
CuCl2-5ppm 24 hrs: 5/29/73
RETURNED SALTWATER: 10 lirs
.
4
^
52
-------
-
*
^c--\. ^T- (.
«0*W*/a
;%^S^!
^'Isr
, ;.. " -
' . -% ifV
»* v *
3
'.
^*\
<%
i#
HFCII DRY
PHASE
CuCl2-5ppm 24 hrs: 6/4/73
RETURNED SALTWATER: 1 Week
V**
**
'
*
*
PHASE
STAINED
53
-------
CuCU 5PPM 24HRS: RETURN SALTWATER
RETURN: io MRS
RETURN: i WEEK
RETURN:! WEEK
-------
DISCUSSION
Electronic Assay of Circulating Blood
In detecting changes in the normal cell state, the electronic print-
outs were examined in terms of changes in the distributions for the three
triggering delay intervals, and how these three distributions related to
one another. One aspect studied was how changes in one distribution re-
lated to changes in the other two. Further, these changes in the printouts
were related to Changes in cell membrane rigidity and changes in the gen-
eral composition of the circulating blood.
Fish showing a normal cell state had electronic distributions in which
the 4^*.sec triggering delay interval was one sharp peak; the S^sec triggering
delay interval was broader and had a smaller secondary peak following the
first principal peak; and the 25/*sec triggering delay interval was the broad-
est and, like the §wsec
intervals shifted. The distributions for the 25/Asec triggering delay in-
terval seemed to follow that for the S^sec interval. The peaks of the
and 25jssec intervals did not shift apart. The secondary peak of the
interval rarely disappeared.
In general, the peak channel for the normal distributions of the
and 8/Asec triggering delay intervals will differ by only five channels
As the distributions began to change, the average peak channel
difference becomes 10 (0.35,1*). When two distinct peaks could be observed,
the channel difference was at least 12 (0.42>.), and shifts of up to 70 (2.45,40
channels have been observed. The peak channel differences seem to fall into
one of two categories - those that have a channel difference of less than
15, and those that differ by 30 or more. There seem to be few middle values.
In terms of cell types, what does this represent? The classification
of circulating blood components of fish is unclear. In this report, we have
adopted Gardner's classification ( 7 ) of the formed elements of the peri-
pheral blood of F. heteroclitus. This is a recent work specifically ex-
amining F. heteroclitus. The blood components described by Gardner include
mature nucleated erythrocytes, thrombocy tes , small and medium lymphocytes,
and eosinophils. The predominant cell type is the erythrocyte. Thrombocy tes ,
the second most common cell type, are found in three forms, 1) the prothrombo-
cyte, 2) intermediate forms, and 3) the mature thrombocy te (the latter being
the most common of the three). In fresh preparations, the erythrocytes often
exhibited a slight tail, appearing tear-shaped or triangular; this is in con-
trast to the familiar oval shape found in the Wright stained preparations.
55
-------
Thrombocytes have the ability to extend pseudopods and form cohesive thrombo-
cytic networks. In fresh preparations, mature thrombocytes, with no ex-
tended pseudopods are large (10/L. diameter) rounded or somewhat oval in shape;
the intermediate forms are more elongate or spindle-shaped. The lymphocytes
and eosinophils, making up a small percent of the circulating blood popula-
tion, will not be discussed further.
The electronic analysis of these components carried out on the BMA
translated the cells into a population of electronic pulses. The shape of
the pulse indicated the size and shape of an individual cell; and the com-
posite of these pulses are accumulated by the BMA.
Any change in the membrane rigidity or composition of the cell pop-
ulation would be reflected in the BMA printout. In the freshwater ex-
periments, for example, several fish showed internal hemorrhaging. When
blood from such fish was examined on the BMA, there was a large shift to
the lower, channel range in the 4ju.sec triggering delay interval distribution.
This build up was found to represent a great number of thrombocytes; these
included many intermediate forms. Free nuclei, which are in the same size
range as thrombocytes, were also identified.from the Wright stained photo-
micrographs. The peak shifting and secondary peak reduction seen in the
printouts of the metal-stressed fish reflect the alteration in membrane con-
figuration and rigor, as evidenced by both the fresh and fixed preparations
examined microscopically.
The fact that fish adapted to freshwater showed a higher proportion
of peak separations may indicate that this adaptation to freshwater may
not be complete. The peak shifting may also indicate that slight disruptions
of the blood cell state result in peak separation. If this is the case,
then this may prove helpful in determining sublethal but deleterious levels
of metal pollutants. The separation of the peaks with the corresponding
secondary peak reduction may be the first indication of an abnormal cell
state.
56
-------
DISCUSSION
Furululus heteroclitus is a euryhaline fish capable of adjusting to
a wide range of osmotic conditions. During the time we conducted fresh-
water adaptation studies with F.^ heteroclitus, we observed a pattern of
behavior in response to the stress of the environment. The more abrupt
the change in ambient salinity, the more intense the reactions were. On
introduction to the new environment, the fish would remain motionless either
on the bottom of the tank or at the surface of the water; some fish would
swim on their side. A certain number of the fish would float belly-up
indicating that death would soon follow. The more gradual the introduction
to a 0%.salinity environment, i.e. periods of time spent in tanks of 14 %o
salinity, the less intense were their reactions. Further, the mortality
dropped from 95%, which occurred when the fish were moved from 20% salinity
to 0%*salinity directly, to about 5% mortality, which occurred when the
fish spent a .minimum of one week in the 14%0salinity tank before being put
in 0%<- salinity.
Freshwater adapted fish used in metal pollutant tests were less able
to adjust to the new experimental situation than test fish taken from the
slatwater tank. The freshwater adapted fish appeared the worst affected
by the metal insult. They were the most sluggish, did not eat when fed or
seem to sense the food in the water; they were the first to die in the
adverse conditions. What this may mean in a larger sense, is that even fish
in the wild that may appear to be adapted to slightly deleterious conditions
are less able to withstand any subsequent change in the environment. Such
fish would be more susceptible to a lower< level of pollutant in the water
than would fish in a "healthy" environment.
Other researchers investigating the ability of Fundulus species to
acclimate to changes in salinity found that these fish had a greater capacity
for regulation against a dehydrating hyperosmotic medium than a hyposmotic
one ( 9). Further, Garside ( 8) found that the upper lethal temperature
that could be tolerated by Fundulus was lowest at 0%0salinity, and highest
at isosmotic salinity of 14%0. The acquisition and retention of osmotically
active substances and the excretion of excessive 1^0 seemed to create greater
stress than the elimination of excess salts? This seems to agree with our
findings of an increased vulnerability of the freshwater adapted fish when
insulted by a metal pollutant.
The sublethal effects and mechanisms of actions of heavy metals on
fish tissues are quite various and complex. Some of the often cited re-
sponses include coagulation or precipitation of mucous on the gills and/or
cytological damage to the gills. This creates a breakdown in gas exchange
at the gills followed by hypoxia at the tissue level ( 2, 10 ). Another
aspect of heavy metal poisoning is a neurotoxic phenomenon which affects
the olfactory and lateral-line systems ( 6 ). Heavy metal ions are potent
enzyme inhibitors; the toxicity of the metal cations is a function of their
electron-attracting properties which combines them with the functional groups
particularly the sulfhydral groups on enzymes ( 1, 3 ).
57
-------
Our tests deal only with the circulating blood cells and their im-
pairment. A model is presented which attempts to explain the response of
the blood cell population to an insult by a metal, in this case, copper.
Throughout the experiments using copper as the heavy metal pollutant,
a definite progression of responses had been observed. The first effect of
the metal appeared to be on the membrane and involved a change from cells
having a smooth surface to one in which the surface of the cell became
jagged and irregular. The normal integrity of the membrane appeared to be
lost and the cell shapes became quite irregular. These responses were
noticed even after a 45 minute exposure to copper.
Rothstein (15 ) has reported that heavy metals at low levels change
the permeability properties of membranes which alters the chloride equil-
ibrium between erythrocytes and plasma. The loss of membrane rigor as
observed in the sickling phenomenon, the irregularity of shapes and the
serrated edges observed in our studies may be the result of an osmotic im-
balance caused by such membrane property changes.
Other investigators have found erythrocyte membrane proteins which
are contractile proteins, similar to actin and myosin ( 13). Changes in
size and membrane deformability have been reported to be dependent on the
conformational state of a membrane fibrous protein (14 ). This protein
is controlled by intracellular ATP and Ca** levels. When Ca"*""*" levels are
increased, both a conformational change of the membrane (contraction) and
a decrease in membrane permeability are observed. Sr"*"*" and fir*"*" have the
same effect as Ca"1"1" in causing conf ormational changes of the membrane. On
the basis of these results it would seem highly reasonable that Cu"*""*" would
have the same effects on the red blood cell membrane as those produced by
the other divalent cations (12). Thus, Cu"*"*" would cause an alteration in
the membrane's configuration and a loss of "rigidity"; allowing the red
blood cell to assume numerous abnormal shapes.
After 24 hours exposure to CuCl2 (5ppm), vacuoles or circular white
clearings became evident in the cytoplasm of the erythrocytes. These
clearings persisted in both the longer exposures to CuCl2 and in the short-
terra reversal tests. Generally, one or two clearings were found in a cell.
The cause or function of these clearings has not been determined. They may
be indicative of an erythrocyte that is no longer viable ( 11). Similar
phenomena were reported in the renal epithelium of F. heteroclitus, and the
same effect was observed in the gills (5 ).
The nuclei showed the effects of the exposure to 5ppm CuC^ after the
membrane became visibly affected. The initial reactions seen in the nuclei
were the loss of the central location in the cell. The nuclei became lobed.
The Wright stain was taken up unevenly with some of the nuclei apppearing
quite opaque. As the cytoplasm began to break down, the nuclei started to
swell, in some cases becoming nearly twice their normal size. In the phase
contrast studies, at the longer exposures to 5ppm CuCl2, the nuclei began
to show the clearings or vacuolations seen in the cytoplasm.
58
-------
Restoration to unpolluted saltwater for 10 hours after a 24 hour ex-
posure to 5ppm CuCl2 did not restore the normal erythrocyte shapes; pro-
longation of the return to the normal slatwater environment for one week
still did not produce normal-shaped cells. Our observations of the cells
which had had a one week restoration in normal saltwater after 24 hours in
5ppm CuCl2 showed indications of a return to a more normal state. Whether
this reversal was due to new cells being put into circulation rather than
a reversal of existing cells, however, was not clear. In the phase contrast
studies, there was a disappearance of the sickle shapes and irregular mar-
gins. The plasma membrane injuries seemed less severe. There were still
internal vacuolations present, and the nuclei still showed some transparency.
In the Wright stained cells, the cytoplasmic membrane appeared to have re-
gained some of its original rigor; though the membrane still showed jagged,
irregular edges. The nuclei were centrally located and took up the stain
evenly.
An increase in the thrombocytic series was observed in the blood of
F. heteroclitus after the specimen had been 1) placed in freshwater from a
saltwater tank, 2) placed in unpolluted saltwater f6r 10 hours after 24
hours in 5ppm CuCl2, and 3) placed in unpolluted saltwater for one week after
24 hours in 5ppm CuCl2- Dawson (4 ) reported a similar thrombocytic re-
sponse in the catfish after several weeks exposure to lead. In the phase
contrast studies of fresh blood of F. heteroclitus, thrombocytic networks
were observed after the specimens from the two recovery experiments were sac-
rificed. Thrombocytes were observed with extruded cytoplasmic pseudopods
bound together by the cohesive property of their cytoplasm with erythrocytes
caught in the mesh. Gardner and Yevich (7 ) in their studies of blood morph-
ology of Cyprinodontiform fishes observed in some freshwater preparations
an intricate thrombocytic network which they indicated served to function in
the blood clotting process. Their studies concerned the normal blood and
the phenomenon was observed in the fresh preparations when air was trapped
beneath the coverslip. In our studies, this phenomenon was only observed
under the environmental conditions stated above, and care was taken to ob-
viate the possibility of air under the coverslip of all slides. The pheno-
menon is suggestive of an environmental disturbance in the blood due possibly
to the copper.
Observations were made on the viscosity and color of the blood at the
time of sacrifice and when the blood was transferred to the slides in hep-
arinized capillary tubes. The blood of specimens tested in freshwater, tested
in a copper environment for 24 hours or longer, and tested in the reversal
experiments was much darker than normal; this blood was a dark purple-red
instead of the bright red of normal blood. This phenomenon of dark blood
suggests that less oxygen is getting into the blood, or that there is a low-
ered oxygen acceptance capability of the blood. Further, as the blood was
transferred to the slides, there was a decided resistance to flow which in-
creased as the exposure to copper increased. Hughes (10 ) has suggested that
interference with the gas exchange at the gill surfaces may result in tissue
hypoxia. He suggests that a reduction in erythrocyte flexibility, effectively
increasing the blood viscosity, and changes in cell volume are associated
with increases in blood C02 levels.
59
-------
Granulated erythrocytes were found in the circulating blood of fish
exposed to 5ppm CuCl2 for 24 hours and then returned to unpolluted salt-
water for 10 hours. An identical phenomenon has been recorded by Gardner
and Yevich ( 7 ) in teleosts undergoing increased erythropoietic activity
in early summer. The phenomenon may be similar to that found in human
blood production when under conditions causing increased erythropoiesis.
Immature red blood cells are released directly into the circulation. This
phenomenon happens either because of an increased call by the organism to
make up for a loss of functional erythrocytes in the circulation or because
of an alteration in the normal red blood cell production mechanism.
The results of our investigations in conjunction with the findings of
other researchers indicate a complex series of reactions are taking place
within fish sublected to copper ions in their environment. It appears that
a rapid effect (Less than 2 hours) takes place in the circulating blood cells
of the insulted fish. This is followed by a period of adaptation (4 to 6
hours) in which reserve cells are probably put into circulation to replace
the cells disabled by the CuCl2- Although it is not clear from the present
data, it appears that once a blood cell is altered it recovery (return to
normal appearance and function) is never complete. We have constructed a
model which appears on page 61.
Our previous studies showed that in the case of F. heteroclitus, fresh-
water adaptation took approximately 16 days and that even then the fish were
more susceptible to toxic insult. Our reversal studies indicate that the
blood cell populations of fish receiving transient (24 hour) exposure to
CuCl2» although more normal than at the time of insult, are still partially
aberrant. Since we are dealing with total populations of blood cells, it
is not possible to know whether the changes we see are due to a reversal
in the condition of the individual cells, or whether we are seeing a steady
replacement of new unaltered red blood cells. The indications are that the
latter is the case.
The final phase of the grant involved the design, calibration and
delivery of an advanced fish blood analyzer. This was produced by the
Grumman Health Systems (Woodbury, New York). Figure 5 shows a simple
block diagram of the system, and Figure 6 shows a block diagram of the
Grumman Health Systems Fish Blood Analyzer. One GHS Fish Blood Analyzer
is presently in use at our laboratories and another is in use at the
U.S. Water Quality Laboratories (EPA) in Narragansett, Rhode Island.
Figure 7 is a photograph of the GHS Fish Blood Analyzer. A detailed
instruction manual ("Fish Blood Analyzer Instruction Manual", Sias
Medical Research Laboratories, 1973, pages 1-82) was delivered with
the machine in November, 1973.
60
-------
Cu"1"*" Insult
Mature, Stored
, Erythrocytes
Intermediate
Erythrocyte Forms
I
I Erythrocyte Destruction
1 or Deformation
5J \
\
Multipotential^> Committed
Stem Cell
Stem Cell
*S
Circulating
""^Erythrocyte Population
Hb Concentration: Lower
02-Hb Capacity: Lower
: Normal Pathway
: Pathway of Cu"*"4"
Exposed Erythrocytes
HYPOTHETICAL MODEL:
Effects of Cu*"*" Exposure on Fish Erythrocytes
Immediate: (1)
5-10 Hrs (2)
Days or
Weeks
(3)
Erythrocyte destruction or deformation resulting in an
eventual decrease in either the effective erythrocyte
volume or Hb-concentration.
As a result of (1) , a. signal is received by the sensor
organ which stimulates the release of stored erythrocytes
and many intermediate erythrocyte forms into the general
circulation in an attempt to maintain homeostatic balance.
As a result of (1), increased amounts of erythropoietin
will be activated in an attempt to increase the numbers
of stem cells that will differentiate toward erythrocytes.
However, before any appreciable effects resulting from
this pathway can be seen, a considerable period of time
will pass.
61
-------
FIGURE 5
BLOCK DIAGRAM OF MULTICHANNEL
ANALYSIS SYSTEM FOR FISH BLOOD
SENSOR
AMPLIFIER
DISCRIMINATOR
PULSE
DELAY
TRIGGER
PHA
MONITOR
SCOPE
X-Y
PLOTTER
DIGITAL
STORAGE
DIGITAL
PRINTER
TRIGGER INSTANT
PULSE HEIGHT
ANALYZER
ANALOG TO
DIGITAL
CONVERSION UNIT
-------
FIGTJEE 6
CT\
-------
FIGURE 7
FISH BLOOD ANALYZER
n noo
n n
n c
-------
BIBLIOGRAPHY
1. Biesinger, K.E., "Effects of Various Metals on Survival, Growth,
Reproduction and Metabolism of Daphnia ma^na", JOURNAL OF THE
FISHERIES RESEARCH BOARD OF CANADA, 29:1691 (1972).
2. Burton, D.T., Jones, A.K. and Cairns Jr., J., "Acute Zinc Toxicity
to Rainbow Trout (Salmo gairdneri): Confirmation that Death is Re-
lated to Tissue Hypoxia", JOURNAL OF THE FISHERIES RESEARCH BOARD
OF CANADA, 29:1463 (1972).
3. Christensen, G.M., "Effects of Metal Cations and Other Chemicals
upon the in vitro Activity of Two Enzymes in the Blood Plasma of
the White Sucker, Catostoraus commersoni (laCepede)", CHEMICO-
BIOLOGICAL INTERACTIONS, 4:351 (1971/1972).
4. Dawson, A.B., "The Hemopoietic Response in Catfish, Ameiurus
nebalosus, to Chronic Lead Poisoning", THE BIOLOGICAL BULLETIN,
LXVII:335 (1935).
5. Eisler, R. and Gardner, G., "Acute Toxicology to an Estuarine Teleost
of Mixtures of Cadmium, Copper and Zinc Salts", JOURNAL OF FISH
BIOLOGY, 5:131 (1973).
6. Gardner, G. and LaRoche, G., "Copper Induced Lesions in Estuarine
Teleosts", JOURNAL OF THE FISHERIES RESEARCH BOARD OF CANADA, 30:363
(1973).
7. Gardner, G. and Yevich, P., "Studies on the Blood Morphology of Three
Estuarine Cyprinodontiform Fishes", JOURNAL OF THE FISHERIES RESEARCH
BOARD OF CANADA, 26:433 (1969).
8. Garside, E.T. and Chin-Yuen-Kee, Z.K., "Influence of Osmotic Stress on
Upper Lethal Temperatures in the Cyprinodontid Fish, Fundulus heteroclitus",
CANADIAN JOURNAL OF ZOOLOGY, 50:787 (1972).
9. Garside, E.T. and Jordan, C.M., "Upper Lethal Temperatures at Various
Levels of Salinity in the Euryhaline Cyprinodontids Fundulus hetero-
clitus and F. diaphanus after Isosmotic Acclimation", JOURNAL OF THE
FISHERIES RESEARCH BOARD OF CANADA, 25:2717 (1968).
10. Hughes, G.M., "Respiratory Responses to Hypoxia in Fish", AMERICAN
ZOOLOGIST, 13:475 (1973).
11. Dr. D. Kenney, personal communication, Center for Blood Research,
Boston, Massachusetts.
12. Dr. F. Lionetti, personal communication, Center for Blood Research,
Boston, Massachusetts.
13. Ohnishi, T., "Extraction of Actin and Myosin-like Proteins from Erythro-
cyte Membrane", JOURNAL OF BIOCHEMISTRY, 52:307 (1962).
65
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14. Palek, J., Curby, W.A., and Lionetti, F., "Relation of Ca"1"1"-Activated
ATPase to Ca"H"-Linked Shrinkage of Human Red Cell Ghosts", AMERICAN
JOURNAL OF PHYSIOLOGY, 220:1028 (1971).
15. Rothstein, A., "Cell Membrane as Site of Action of Heavy Metals",
FEDERATION PROCEEDINGS, FED. OF THE AM. SOCIETY FOR EXPERIMENTAL
BIOLOGY, 18:1026 (1959).
66
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TECHNICAL REPORT DATA
rcoil liiit/uctians on llie reverse be/ore completing)
I. rucoRi NO.
JP£l600/ ? -7 6-3069
4. TlTLE ANDSUD1ITLE
ASSAYS OF TOXIC POLLUTANTS BY FISH BLOOD
3. RECIPIENT'S ACCESSION NO.
5. REPORT DATE
September 1976 (Issuing date)
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
W. A. Curby, R. D. Winick, and E. C. Moy
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORG "VNIZATION NAME AND ADDRESS
Sias Research Laboratories
Brookline, Massachusetts 02146
10. PROGRAM ELEMENT NO.
1BA608
11. CONTRACT/GRANT NO.
800355
12. SPONSORING AGENCY NAME AND ADDRESS
Environmental Research Laboratory
Office of Research and Development
U. S. Environmental Protection Agency
Narragansett, Rhode Island 02882
13. TYPE OF REPORT AND PERIOD COVERED
Final
14. SPONSORING AGENCY CODE
EPA-ORD
1B. SUPPLEMENTARY NOTES
16. ABSTRACT
We have developed a biological multichannel analyzer which, using a sensor that
operates on the Coulter Principle, measures and distributes mixed cell populations
by cell size. It provides an analog distribution and digital printed readout for
future analysis. Although primarily a pulse height analyzer (applied successfully
to studying bacteria, mammalian blood and inert particles) it operates as a pulse
shape analyzer if the instant at which each pulse height is read is varied. This
technique, applied to the peripheral whole blood from freshly sacrificed Fundulus
heteroclitus shows the alterations with time and the variations caused by trace
amounts of cadmium and copper in the aquatic environment. The size frequency
distribution patterns of each trace element environment differ from each other,
and each, markedly from the norm.
We have investigated and recorded the response of F_._ heteroclitus whole blood
cells from fishes living in several aquatic environments of fixed pH and dissolved
oxygen and temperature. We compared these data with those obtained from fish
subjected to dissolved traces of chemical pollutants. In final fulfillment of
our grant, we have delivered an advanced model of the multichannel analyzer to
the U. S. Water Quality Laboratories in Marragansett, Rhode Island. The Fish
Blood Analyzer is produced by Grumman Health Systems.
17.
a.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
Particle Shape
Particle Size
Cytology
Bioassay
Marine Animals
Metals
b.lDENTIFIERS/OPEN ENDED TERMS
COSATl I-'ield/Group
06F
1M. UlSmiUUTION STATEMENT
RELEASE' TO PUBLIC
19. SECURITY CLASS (This Report)
UNCLASSIFIED
21. NO. OF PAGES
77
20.!
22. PRICE
EPA Form 2220-1 (9-73)
67
CUSGPO: 1976 757-056/5419 Region 5-11
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