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.,

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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.

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     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

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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).

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Figure 1

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FIGURE 2
     GRAPH A
CL
U_ "
O
cr
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)

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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
               UJ
               _i
               o

               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

-------
                  %
I
     *>    .       9  #
                                            r,ymm


                                                    ^v  £>

                                                                         «
                                                                        •
                                                                w
         tfk
                                                                    in
   CONTROL. 5/15/73   P1IASC                       COIITROI.:  5/15/73  PHASE
   I'DNTROL: 3/21/71   STAINED                     CONTROL: 5/21/73  STAINF.D
                                                                       #

10.
                                      13

-------
CONTROLS
   75-i

-------
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

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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

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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

-------
               »

       •
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      *             ^
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     "—
IM1ASF.
         CuCl2-5ppn 45 mins: 5/22/73
STAINED
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                    _ •
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-------
CuCl2-5ppn 1 hr: 5/15/73
PHASr
                           CuCl2-5ppm ^..5 hr: 5/21/7 !
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      •

                    ^
                                      47

-------
           CUCL2- 5PPM
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              73—.
           4JS HOURS
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                                50

-------

     - 5PPM
24 HOURS
44 HOURS

-------
PHASI-:
STAINED
                            CuCl2-5ppm 24 hrs: 5/29/73

                            RETURNED SALTWATER: 10 lirs


            .

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                                       52

-------
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               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

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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

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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

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     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

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     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

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                                                               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

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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

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                             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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