WATER POLLUTION CONTROL RESEARCH SERIES • 18050EBK02/71
      Response of Teleost Fish
       to Environmental Stress
ENVIRONMENTAL PROTECTION AGENCY • WATER QUALITY OFFICE

-------
         WATER POLLUTION CONTROL RESEARCH SERIES
 The Water Pollution Control Research Series describes
the results and progress in the control and abatement
of pollution in our nation's waters.  They provide a
central source of information on the research, develop-
ment, and demonstration activities in the Water Quality
Office, Environmental Protection Agency, through inhouse
research and grants and contracts with Federal, State,
and local agencies, research institutions, and Industrial
organizations.

Inquiries pertaining to Water Pollution Control Research
Reports should be directed to the Head, Project Reports
Office, Environmental Protection Agency, Room 1108,
Washington, E.G.  202U2.

-------
      RESPONSES OF TELEOST  FISH TO ENVIRONMENTAL STRESS
                                 by
          L.  S.  Smith, J. B.  Saddler, R. C. Cardwell,
           A.  J.  Mearns, H. M.  Miles, T. W. Hewconib,
                        and K. C.  Hatters
                  Fisheries Research Institute
                    University of Washington
                   Seattle, Washington  98105
                             for the


             FEDERAL WATER QUALITY ADMINISTRATION

                ENVIRONMENTAL PROTECTION AGENCY
                       Grant No. 18050 EBK

                         February,  1971
For sale by the Superintendent of Documents, U.S. Government Printing Office, Washington, D.C. 20402 - Price $1.25

-------
                 EPA Review Notice

This report has "been reviewed "by the Water
Quality Office, EPA, and approved for publication.
Approval does not signify that the contents
necessarily reflect the views and policies of
the Environmental Protection Agency, nor does
mention of trade names or commercial products
constitute endorsement or recommendation for
use.

-------
                              ABSTRACT

A floating laboratory was built for conducting multiparameter
physiological studies on salmon in marine, estuarine, and fresh
waters.  New methods were developed using a swimming chamber-
respirometer for adult salmon.  Normal values were measured for a
variety of physiological functions, then repeated on salmon migrat-
ing through an urban estuary characterized by sewage pollution and
low DO.  Effects seen included decreased swimming stamina and
respiratory efficiency, decreased oxygen consumption and increased
lactate, decreased urine flow and ammonia excretion, especially in
the presence of environmental ammonia.  Longer term disruptions in
hematology and lipid metabolism were seen.  Most of the effects
occurred at DO concentrations just below 5 mg/liter, except for
synergistic effects between ammonia and low DO at somewhat higher
concentrations.

This report was submitted in fulfillment of Grant No. 18050 EBK
under the sponsorship of the Federal Water Quality Administration.
Key Words:  Fish physiology, fish migration, environmental effects,
            Pacific salmon, oxygen sag.
                                 3.3.3.

-------
                             CONTENTS
Section
                                                                  xlii
   I    CONCLUSIONS
  II    RECOMMENDATIONS
 III    INTRODUCTION:  STUDYING THE ENVIRONMENTAL PHYSIOLOGY         1
                       OF SALMON
            The General Concept of a Floating Laboratory             1
            The Duwamish Estuary                                     2
            Training Aspects                                         5
  IV    MATERIALS AND METHODS                                        7
            Designing, Building and Using a Floating  Laboratory      7
            A Modified Version of the  Blazka Respirosseter and       11
               Exercise Chamber of Large Fish
                 Design                                               ^
                                                                     14
                 Operation
             Anesthesia, Multipararoeter Sampling, and Surgery        16
                 Problems of Sampling Live Swimming Fish             16
                 Problems of Preparing Fish for Physiological
                   Monitoring
    V    STRESS DURING OUTMIGRATION OF SMOLTS                        20
              Ionic Regulation  in  Migrating Juvenile Coho Salmon      20
                                                                     20
                 Introduction
                 Materials and Methods                               20
                 Results                                             21
                                                                     22
                 Discussion
                     Preraigratory Blood Characteristics             22
                     Adaptation  to  Sea Water                         25
                     Relationship of the Sea Water Experiment to the
                       Ecological Situation                           36
                                   v

-------
Section                                                            Page
             Lipid Composition and Hematology of Juvenile
               Chinook Salmon (Oncorhynchus tshawytscha) Before
               and After Migrating Through Duwamish Estuary          36

                 Introduction                                        36

                 Materials and Methods                               37

                     Sampling of the Fish                            37

                     Lipid Analysis                                  37

                     Identification of the Fatty Acids               37

                     Hematology                                      37

                 Results                                             39

                     Hematology                                      HO

                 Discussion                                          50

    VI   NATURAL STRESS DURING SPAWNING MIGRATION OF ADULTS          55

             Normal Hematological Variations During the Spawning
               Migration of Chinook Salmon                           55

             Renal Function in Migrating Adult Coho Salmon           61

                 Introduction                                        61

                 Methods and Materials                               61

                     Location and Collection of Animals              61

                     Preparation of an Experimental Animal           61

                     Inulin and PAH                                  62

                     Collection and Processing of Blood Samples      62

                     Plasma Ultrafiltration                          63

                     Collection and Processing of Urine Samples      63

                     Analysis of Urinary Precipitate                 63

                     Calculations                                    63

                 Results                                             63

                     Urine Flow Rate                                 63
                                   vr

-------
Section                                                            Page
                    Glomerular Filtration Rate                       66

                    PAH Clearance                                    66

                    Magnesium Clearance                              66

                    Urine Ion Concentrations                         66

                    Urine Ion Excretion Rates                        66

                    Protein Binding of Plasma Ions                   70

                    Filtered Ion Load                                70

                    Ion Load and Clearance Ratios Based on
                      PAH Clearance                                  73

                    Ion Load and Clearance Ratios Based on
                      Magnesium Clearance                            73

                    Filtration Ratios                                73

                    Filtration Fraction                              73

                    Blood Parameters                                 75

                    Urinary Precipitate                              75

                 Discussion                                           75

                    Maintenance of Internal  Homeostasis              75

   VII   EFFECTS OF MAN-MADE  STRESSES  ON ADULT SALMON                77

             Stress During the Migration of Adult Salmon              77

             Effects of Low Levels  of  Dissolved Oxygen on Chinook
               and Coho Salmon in the  Duwamish Estuary                78

                 General Considerations                                78

                 Materials and Methods                                78

                 Results                                              79

                    Swimming Stamina                                  79

                    Respiratory Changes                               80
                                VII

-------
Section                                                            Page
                    Changes in Levels of Lactate in the
                      Blood                                          80

                    Excretion of Lactate                             87

                    Ammonia Excretion                                87

                    Rate of Urine Production                         89

 VIII  ACKNOWLEDGMENTS                                               90

   IX  REFERENCES                                                    91

    X  APPENDIX A - LIST OF PUBLICATIONS FROM THE PROJECT            96

       APPENDIX B - SUPPLEMENTARY TABLES                             98

-------
                        FIGURES
                                                           Page

     R. V. Kumtuks and purse seine vessel anchored in
     the Duwamish Waterway (September, 1969) 	      3
2    Site of domestic sewage outfall approximately
     1,000 ft upstream from R. V. Kumtuks in the Duwamish
     Waterway	      4

3    Drawings of port and starboard sections of the
     R. V. Kumtuks	      8

t    Drawings of bow and stern sections of R. V. Kumtuks      9

5    Upper deck facilities of R. V. Kumtuks	     10

6    Lower deck facilities of R. V. Kumtuks	     11

7    Diagram of modified Blazka respirometer used for in-
     vestigating physiological responses of salmon
     swimming in water of low dissolved oxygen  	     15

8    Adult male coho salmon swimming in respirometer
     after surgical implantation of various cannulae and
     catheters	     17

9    Installation of catheter behind the gill in adult
     male coho  salmon.  Buccal  catheter is visible
     arising  from the snout of  the fish	     19

10  Seasonal changes in hematocrit and stream  temperature  23

11  Observations of hematocrit and total dissolved
      solids  in migrating -juvenile coho salmon   .  .  .
26
 12   Observations  of  sodium  and  chloride concentrations
      in the blood  plasma of  migrating juvenile coho
      salmon     	    27

 13   Observations  of  calcium and magnesium concentrations
      in the blood  plasma of  migrating juvenile coho
      salmon     	    28

 11   Observations  of  potassium concentration in  the blood
      plasma of migrating juvenile  coho  salmon  .....    29

 15   Changes  in the hematocrit and percentage of total
      dissolved solids in the blood plasma of juvenile
      coho salmon with adaptation to  sea water  	    30
                           IX

-------
                                                                 Page
16   Theoretical flow  rates of plasma water in juvenile
     coho salmon during the adjustive phase .........     32

17   Changes in concentrations of sodium and chloride in the
     blood plasma of juvenile coho salmon with adaptation
     to sea water  .......... • ..........     33

18   Changes in concentration of potassium in the blood
     plasma of juvenile coho salmon with adaptation to sea
     water   ........................      34
19   Changes in concentrations of calcium and magnesium in
     the blood plasma of juvenile coho salmon during
     adaptation to sea water ................      35

20   The Duwamish Estuary and Green -Duwamish River.  ....      38

21   Differences in the electrophoretic composition of the
     plasma proteins in chinook salmon at various stages of
     their life cycle  ...................      59

22   Oxygen consumption* extraction coefficient, and venti-
     lation volume with change in environmental oxygen con-
     centration in swimming coho salmon  ..........      81

23   Changes in blood lactate levels in four coho salmon
     (Onoorhynchus kisutch) swimming various periods (bar)
     in "a" elevated (65% saturated) and "b" low (50%
     saturated) dissolved oxygen concentration  .......      82

24   Temporal variations in blood lactate concentrations in
     three coho salmon swimming at 56 cm/sec in aerated
     (100% saturation = "a") and hypoxic (50% saturation
     = b) water .......................      84

25   Relation between blood lactate , dissolved oxygen and
     duration of swimming in four coho salmon from the
     Duwamish Waterway, 3 to 4 kg adult fish    .......      85

26   Relation between blood lactate, dissolved oxygen,
     oxygen consumption and duration of swimming in 3 to 4
     kg adult coho salmon and steelhead trout   .......      86

27   Upper graph indicates average changes in blood
     lactate levels of adult coho salmon ( Oncorhynchus
     kisutch) contacting a low dissolved oxygen (50%
     saturation) while swimming at 56 cm/sec.  Lower
     graph indicates variations in total ammonia excretion
     in swimming adult coho salmon subjected to various
     levels of dissolved oxygen  ......... .....      88

-------
                              TABLES

No.

  1    Correlation matrix of physical and blood parameters
       of freshwater residents (April 12 through May 10,
       (1967)                                                       24

  2    Percentage composition of basic ingredients in Oregon
       moist pellet and Abernathy dry pellet diets.  Oil content
       was derived for both diets from fish, soybean, and
       cottonseed oils                                              39

  3    Fatty acid contents of Oregon moist and Abernathy dry
       pellet diets expressed in per cent and mg weight             41

  4    Proportions of the 28 fatty acids found in Soos Creek
       Hatchery juvenile chinook salmon fed Oregon Moist Pellets    42

  5    Percentage fatty acid composition of juvenile, hatchery-
       reared chinook salmon retained in freshwater and fed
       Abernathy pellets                                            43

  6    Concentrations (in mg) of 28 fatty acids found in juvenile
       chinook salmon cultured at Soos Creek Hatchery on Oregon
       Hfcist pellets                                                44

  7    Concentrations (in mg) of 28 fatty acids found in juvenile
       chinook salmon retained in freshwater and fed Abernathy
       dry pellet diet                                              15

   8    Percentage fatty acid composition of 28 fatty acids found
       in juvenile  chinook  salmon that had been released from
       Soos  Creek Hatchery  and captured in the West Waterway of
       the Green-Duwamish river estuary                             16

   9    Fatty acid concentrations of juvenile chinook salmon that
       were  released from Soos Creek  Hatchery and  captured in the
       West  Waterway of the Green-Duwamish river estuary            17

  10    Percentage composition of 28 fatty acids of juvenile chinook
       salmon that  had been released  from Soos Creek Hatchery,
       captured  in  the estuary, and retained and cultured aboard
       the R. V. Kumtuks on Abernathy pellets                       18

  11    Concentrations of 28 fatty acids of juvenile chinook salmon
       that  had  been released from Soos Creek Hatchery, captured
       in the estuary, and  retained and cultured aboard the R. V.
       Kumtuks on Abernathy pellets                                 49

  12    Hematological characteristics  of juvenile chinook salmon
       cultured  on  different diets and residing in different
       environments                                                51
                                 XI

-------
No_._

 13    Percentage composition of the six major fatty acids in
       juvenile chinook salmon cultured on different diets
       and residing in different environments                       52

 m    Concentrations (in mg) of the six major fatty acids in
       juvenile chinook salmon cultured on different diets
       and residing in different environments                       53

 15    Hematological characteristics of sub-adult chinook and
       coho salmon, and adult sockeye salmon captured at the
       entrance to the Strait of Juan de Fuca.  Values are given
       as means ± standard errors.                                  57

 16    Hematological variations in adult Green-Duwamish River
       chinook salmon at various stages of maturation.  Values
       are given as means +^ standard errors                         58

 17    Symbols used in the equations for the calculation of the
       renal parameters (Koch, 1965)                                6H

 18    Means, standard deviations  (S.D.), and sample sizes (n),
       pooled for renal parameters in all salt water fish and
       in all freshwater fish                                       67

 19    Correlation coefficient matrix with sample sizes of sodium,
       potassium, calcium, magnesium, and chloride excretion rates
       for the pooled freshwater samples                            71

 20    Correlation coefficient matrix  (n=19)  of sodium, potassium,
       calcium, magnesium, and chloride excretion rates for the
       pooled salt water samples                                    72

 21    Percentages of protein bound sodium,  potassium, calcium,
       magnesium, and chloride in  blood pasma from  fish M          70

 22    Correlation coefficients  (r) and sample size (n) for
       correlations between plasma total solids and the
       filtered load of sodium, potassium, calcium, magne-
       sium,  and chloride                                           73

 23    Total  ion load and  clearance ratios based on the magne-
       sium clearance of fish  H  in salt water and  calculated
       from the mean plasma ion  concentrations and  excretion
       rates  of the  pooled samples in salt water and freshwater     7H

  24   Relation between dissolved oxygen concentrations
       and fatigue in swimming adult coho salmon                   79
                                 xii

-------
                            SECTION I


                           CONCLUSIONS


1.  A floating laboratory was built as a 98.5'  x 35'  self-contained
(but not self-propelled) barge for studying the environmental physiology
of salmon migrating through the protected waters of Oregon, Washington,
British Columbia, and Alaska.  The idea of taking the laboratory to the
fish proved advantageous in many respects, but particularly served to
minimize problems of handling stress (to which salmon are highly suscepti-
ble) and also problems of simulating in a fixed laboratory the specific
quality characteristics of a particular stream and estuary.

2.  A number of new techniques were devised and used to make repeated
physiological measurements from active salmon.  Since salmon in their
natural environment rarely stop swimming, a realistic assessment of the
problems they face in polluted estuaries had to be made on the basis of
data from swimming salmon.

3.  We described for the first time several normal physiological
functions of salmon in clean water for comparison with salmon in polluted
water in the areas of metabolism, excretion, osmoregulation, and blood
circulation.  Surprising as it may seem, since salmonids are among the best
studied of the commercially important fish, little is known of the basic
physiology of most of their organ systems, and therefore norms had to be
measured first before any interpretation of data from polluted waters
could be made.

4.  Normal osmoregulatory changes as young salmon migrate from
freshwater into sea water and the adults return to the stream take place
with a minimum of stress to the fish.  Outmigrants reach osmotic equilib-
rium with sea water  in  30 to  36 hours and appear not to need any gradual
adaptation to the change in salinity.  Inmigrant adults are relatively
impermeable  to   freshwater and their kidneys readily readapt to freshwater.
While tissues of spawning salmon become increasingly watery with continued
residence in the stream, this is not normally the result of kidney failure
or  incomplete readaptation to freshwater, as has sometimes been suggested.

5.  Urine output is  decreased to about half by decreases in environ-
mental oxygen or increased stressful activity.  In sea water, one of the
major urine  constituents is magnesium ion, and therefore, continued
depression of urine  production could lead to problems of magnesium toxicity
(which would produce muscular paralysis).  We identified the potential
problem, but did not determine its magnitude.  There is also possible
accumulation of  other toxic products when kidney function is depressed,
especially if the environment contains toxic products and is low in DO at
the same time.

6.  Ammonia  is the primary excretory product resulting from the normal
breakdown of proteins in fish and is excreted by the gills.  Ammonia
                               xiii

-------
excretion can be either depressed or elevated by decreased environmental
DO, but the mechanisms governing which direction the effect will be are
presently not understood.  However, it would be beneficial to the well-
being of the fish if the water quality standards for DO were^slightly
higher for waters containing ammonia than for those lacking it.

7.   We do not know exactly how a salmon chooses to try to surmount
the respiratory problems it faces when trying to enter a river blocked by
an area of low DO.  It appears that salmon stay in the bottom (salt water)
layers as long as possible and swim upstream as far as the river and tidal
currents and the available oxygen supply will allow.  We now do know,
however, most of the respiratory choices available to a salmon facing low
DO and have considerable experience with salmon choosing from among the
alternatives while in our swimming chamber.  We are now ready to make
physiological interpretations of the estuarine behavior of these fish as
soon as we can get the depth-sensitive sonic tag now being developed by
the Bureau of Commercial Fisheries.

8.  In the past, the appearance of lactate in blood and muscle has
been empirically associated with anaerobic metabolism and large amounts of
lactate have been associated with delayed mortality.  Our experiments
demonstrated that some lactate always occurred in the blood of even rested
salmon and that lactate increased proportionally to increased activity or
decreased DO.  The effects of increased activity and decreased DO together
were additive.  Thus blood lactate was an excellent indicator of immediate
environmental stress, but responded to environmental changes so rapidly
that great precautions were needed to prevent lactate changes due to the
stress of capture and handling.

9.  Production of lactate is a highly inefficient means of obtaining
energy which is used when the quantities of oxygen available to the tissues
are insufficient to meet the demand.  Lactate accumulation also produces
toxicity and pH problems, for which one solution is to excrete the lactate
and throw away the chemical energy it contains.  Thus production of urine
containing lactate could amount  to a continuous  "energy leak"  in salmon
experiencing continuous low DO.   We observed this phenomenon very carefully
because adult salmon on their spawning migration do not feed and could
exhaust their energy reserves before spawning.  However,  the loss of lactate
rarely exceeded 100 pgm/kg/hr or about 75 mg/kg/month.  Since an adult coho
or Chinook  salmon may start its  spawning migration with 20-30% of  its  body
weight as  lipids stored for energy reserve, this urine  loss  of lactate
cannot be  considered serious.   It is probable that  lactate  is  lost from
the  gills,  but  we have not yet  completed the  experiments  to  test this
hypothesis.

10.   A major problem  in  assessing the effects of environmental  stress
in migrating adult  salmon  is that fatal degrees of deterioration  in^
physiological condition take place naturally.   All Pacific  salmon  die  after
spawning,  usually  from diseases to which they become increasingly  less
resistant  as the migration stress progresses.   The effects  of estuarine
pollution  on adult  salmon  is  to increase the rate  rather than the  kind of
                                xiv

-------
deterioration.  We have described many of the hematological changes which
occur during both the downstream and upstream migrations in both polluted
and clean waters.  The comparisons between all variables, species, and
conditions are beginning to merge into a concept of a generalized response
for most kinds of stress.  The general concept of stress, in turn, is
producing ideas of how to treat fish to alleviate the effects of stress,
so that eventually wild fish can be treated for stress, diseases, etc.,
like any other of our better-managed game animals.

11. Our lipid research has had many facets.  Most basic has been the
description of typical fatty acid composition of structural and reserve
lipids of muscle, liver, and blood in all species and ages of salmon.  With
five species of salmon, 28 fatty acids, and many changes in their propor-
tional composition and distribution, this alone was a large job.  Once
completed, however, it became possible to demonstrate that outmigrant
juvenile salmon may have numerous behavioral and physiological problems
before they learn to recognize and adapt their metabolism to the new kinds
of food in their new marine environment.  This seemed to be a more serious
problem than adapting to the salinity.  Wild food organisms contain a much
smaller amount of saturated fatty acids than most hatchery foods, and the
conversion to eating wild food causes major reapportionment of the structural
fatty acids, especially 22:6 (22 carbon atoms, 6 unsaturated bonds).  Overall,
the study of lipids has assisted our assessment of chronic stress in terms
of the disruption of growth in juvenile fish and loss of reserve energy in
the adults -  i.e., the physiological cost of the stress.
                             xv

-------
                           SECTION II

                         RECOMMENDATIONS

     We tested adult coho salmon under circumstances closely resembling
the estuary and found that the existing standard of 5 mg 02/liter is
approximately correct.  Most of the fish which had physiological
difficulties did so at DO's below H.5 mg/liter.  In addition, we
identified synergisms between environmental DO and ammonia, environ-
mental DO and blood lactate, and between environmental DO and activity
levels.  These synergisms may later be elaborated to the point where it
can be demonstrated that the required DO level for salmonid streams should
be increased to 5.5 or 6.0 mg/liter when more than a few ppm of ammonia
are present (as occurs in the effluent of many sewage treatment plants).
                                 xvii

-------
                           SECTION III


 INTRODUCTION;   STUDYING THE ENVIRONMENTAL PHYSIOLOGY OF SALMON


     The time once was when the criteria for water quality consisted
partly of whether fish or any other organism could live in a specified
water sample for 96 hours or not.  While such tests are occasionally
still useful, today most people concerned with water quality recognize
that there are many degrees of well-being between living and dying.  Sub-
lethal stresses can eventually be just as devastating to a species as
a sudden death and much more subtle because they can slowly accumulate
unnoticed until a point-of-no-return is eventually passed.

     The projects to be described in this report represent a beginning^
in understanding the many interactions between salmon and their estuarine
environment in the urban setting of Seattle's industrialized waterfront.
Rather than choose a single indicator function of stress, we measured a
combination of functions encompassing several of a salmon's major functional
systems which relate to water quality - respiration, blood circulation,
osmoregulation, and lipid metabolism.  Thus we could observe the inter-
actions between organ  systems as well as between each organ system and
the environment.  The  results reported here demonstrate that we pioneered
in at least two areas: Descriptions of normal physiological functions in
adult salmon which relate directly to water quality  (kidney functions,
blood lactate  dynamics)  which had never before been described, and
developing techniques  for making a variety of measurements in salmon
swimming in  their normal estuarine environment.

     This report  describes  the  main points investigated during the four
years of the project.   Most of the material  here  has been or is  in press
 in scientific journals in  expanded  form and more  will  continue to appear
 for the next 1-2  years as  additional  graduate  students finish theses
 begun  under FWQA  sponsorship.

                The General Concept  of a Floating  Laboratory

     Pacific salmon are a  valuable  resource whose decline has  coincided
with the degradation of our aquatic environments.  The life  functions^of
 salmon  are known  mostly in terms of their freshwater stages.   Little  is
 known of their estuarine and marine environmental requirements for survival
 and reproduction.  Criteria for improving these environments for salmon are
 not well defined.  Thus the idea was  proposed to the Federal Water
 Pollution Control Administration (F.W.P.C.A.), U.S.A.  in 1966  to begin a
 study  of several  basic physiological functions of salmon which^are vital
 to their transition between fresh and salt water in the estuaries^of
 Puget  Sound.  These estuaries were considered as a crucial point in the
 salmon's lifelong migration where natural stressers  are greatest and
 mankind's additional stressers would most likely be overwhelming.

     we will use stresser to mean the agent or agents provoking stress re-
 sponses in animals.  Stress represents the sum of morphological, physiol-
 ogical, and biochemical changes resulting from the actions of  the stresser.

-------
     One problem was how to study the environmental physiology of a fish
whose annual migrations might exceed several thousand miles and whose
estuarine problems would probably be different in every estuary.  Further,
a proper physiological study required instrumentation characteristic of
a laboratory while the environmental aspects of the program demanded a
field orientation.  The eventual result was the design and construction
of a self-contained laboratory aboard a 98.5-foot (30.0-meter) steel barge
named the R.V. Kumtuks which could be anchored in most of the protected
waterways of Puget Sound, British Columbia, and southeastern Alaska along
the migration routes of the salmon.  We decided to take the laboratory to
the fish and study them in their chosen waters rather than bring the fish
to a laboratory and attempt to simulate natural conditions  (Fig. 1).
                      The Duwamish Estuary

     An important site for studying estuarine problems faced by salmon
was the Duwamish Estuary of the Green River which passes through the
primary industrial section of Seattle, Washington, and empties into
Elliott Bay.  The river is of moderate size, the flow varying from about
200 c.f.s. in the late summer to over 4,500 c.f.s. during the winter
rains.   The river flow is augmented by the effluent from the sewage
lagoon of the suburban city of Auburn, the sewage treatment plant which
serves the suburban city of Renton and southeastern Seattle, and, up
until November, 1969, by the effluent of Seattle's Diagonal Street
Sewage Treatment Plant and some temporarily-diverted raw sewage  (Fig. 2).
After that date, 95 per cent of the raw sewage and other wastes in Seattle
downstream from the Renton Treatment Plant were diverted to Seattle's
new METRO  treatment plant whose effluent goes directly into Puget Sound.

     The most significant biological resources of the Duwamish are the
runs of coho and chinook salmon.  Juvenile fish migrate downstream in
May, return from 2 to 3 years later as adults to Elliott Bay in August,
and ascend the river to spawn in September during the first autumn rains.
Most of the fish ascend the river to Soos Creek (Green River) Hatchery,
operated by the Washington State Department of Fisheries upstream from
the city of Auburn.  The salmon are avidly pursued by numerous sports-
fishermen who angle in Elliott Bay and the lower estuary by day and night;
the city's street lights make night fishing possible.  There are also
other fish present in the estuary including sole, rockfish, and hake •
    2Data from U.S. Geological Survey, Water Resources Division, Seattle,
Washington.
    3Municipality of Metropolitan Seattle.

-------
Fig. 1.   R.v. Kumtuks and purse seine vessel anchored in the Duwamish Waterway (September, 1969).

-------
	
  Fig. 2.
Site of domestic sewage outfall approximately 1,000 ft upstream from R.V  	
in the Duwamish Waterway.  White arrow indicates sewage outfall; black arrows
point to plume created by effluent.
                                                                                     Kumtuks

-------
      The major problems of the estuary are also biological in nature.
The once large amounts of organic material discharged into the river
produced a heavy biochemical oxygen demand (BOD), particularly in the
lower river and upper estuary.   The levels of dissolved oxygen (DO)
were drastically reduced in the surface waters, often nearly to zero,
especially at night when there  was no oxygen production by algae.  In
addition, the river channel is  dredged for several miles upstream,
creating a long tongue of rather static salt water beneath the freshwater
which appears to collect organic matter and to have chronically low DO
levels (Salo, 1969).  From the  results of our experiments on swimming
stamina in salmon (described below), we believe that a major cause of
the observed low DO was biological - the combination of algal blooms and
raw sewage discharges.  Experiments there did not detect any toxic products
affecting the fish.  Now that the major BOD source has been removed from
the river by diversion of sewage to a new treatment plant, there still
remains, we believe, a problem  of the salt wedge collecting dead and
dying river algae in the dredged part of the lower river and estuary.
Thus, some of the continuing problems of the estuary will also be biological
ones and the problems of salmon will continue to revolve around dissolved
oxygen, metabolic energy, and many associated factors to be described in
this report.


                         Training Aspects

      An  important  part of  this project which will not be published in
any scientific journal is the  training of new scientists in environmental
science.   Taking the  laboratory into the field with minimum non-scientific
crew required that  graduate students participate  in fishing, cooking,
boat-handling, proposal and report  writing, purchasing of supplies
 (including engine parts,  food  and chemicals), planning experiments, and
arranging commuting schedules  using car, boat,  ferry, and floatplane.
And  in  the case of  our research  in  fish physiology, there were usually
no readily available  research  tools which could be applied directly to
our problems, so new methods had  to be devised  and tested before some of
the experiments could be completed.  In some  cases, new  equipment also
had to  be designed, built,  and its  operational  characteristics measured.
An example of this  was our respirometer and swimming  chamber for adult
salmon (described in Section IV).

      The most noteworthy aspect  of the present program  is the wide  scope
of the  training provided for graduate students.   After several years
participation in one or more of the programs  aboard the  floating laboratory,
a newly-graduated  Ph.D. is capable of independent investigation  in a wide
variety of research lines concerning aquatic biology.  The students  are
certainly not limited to working on problems  concerning  salmonid physiology.
 Further, they learn to work independently, as part of a  team, and cooperatively
with other investigators and officials  from city, state, and federal agencies,
and to  communicate  with the general public and the news  media.   The  result
of this wide scope  involvement has been,  internally,  an  excellent esprit
de corps among the  project staff at all levels resulting in  a high work

-------
output, and, externally, an excellent ability to compete for ]obs.  The
first Ph.D. to graduate from the program is presently with the Biology
Department at Marquette University in Milwaukee.  Two Master's degrees
have been completed in the program, and four additional Ph.D. degrees are
expected in the next one or two years, all having been partially supported
by FWPCA funds.  Regardless of where their careers take them.^these
graduates will always be promoters of environmental conservation.

-------
                            SECTION  IV


                       MATERIALS AND METHODS

      This section describes a number of general methods which are basic
to most of the individual sections of the report.  In addition, each
section will contain further description of its own unique methods.


        Designing, Building and Using a Floating Laboratory

      The basic facility needed was  a means of performing physiological
experiments on salmon during most stages of their life cycle.  The
primary alternatives were shoreside  laboratories with sea-water systems,
a portable laboratory, and a new laboratory at the existing inland site
with recirculating sea-water systems where the various environments
would be simulated.  Eventually the choice narrowed to a non-propelled
barge because of the maximum space and stability that it would provide
for a minimal cost of construction and operation.  Such a vessel could anchor
in bays close to the marine migration routes of salmon.  Freshwater stages
of several salmonids were available, along with freshwater moorage for the
barge, at the University of Washington campus on the Lake Washington Ship
Canal.

      Eventually  a small  salvage, surplus, and marine construction company
was found that  had a  surplus  hull at the right price and would undertake
the construction  as well.   Plans were drawn up to suit the available hull,
a portion of a  WW II  LSM (Figs.  3,  4,  5, and 6).  Living quarters for
eight persons,  a  bunkroom for additional persons, a wet laboratory, a
chemical laboratory,  instrument rooms,  storage and refrigeration compart-
ments,  a machine  shop,  and engine room with three diesel-electric
generators  - 2-10 kw  and 1-10 kw -  were provided.  The barge was designed
to  support  a crew and staff of 12 persons  for  eight weeks  at a remote
research  station  completely independent of shoreside  services.

       The basic problems that necessitated the construction  of the floating
laboratory  were largely solved.  Experiments were performed  under  environ-
mental conditions that  would have been very difficult to simulate  ashore.
Usually pitch or  roll exceeded 2-3° only from  vessel wakes.   When  experi-
 encing vessel  motion,  we soon learned to weigh chemicals only during
 certain portions  of the barge's swinging around the  anchor,  to limit water
 depth in  aquaria, and to mount pressure transducers  to  avoid motion and
 engine vibration.  Transporting people to the  laboratory instead  of the
 fish proved advantageous.  Float plane charter flights  were  scheduled  between
 one and three times weekly, making  long distance commuting practical.
 Efficiency and productivity were high  because  the  scientific staff
 enjoyed between two and seven days  of  endeavor away from telephones,
 committee meetings, and the stress  of  urban life.   Even when the
 barge was anchored in the waters of metropolitan Seattle, where commuting

-------
                                     3
00
                                                          STARBOARD
                       1.  Crane
                       2,  Portholes
                       3.  Entrance hatch to wet laboratory
                       4.  Engine exhaust manifolds
5.  Exhaust vent
6.  Entrance hatch to engine room
7.  Entrance hatch to galley
t.  Anchor winch
                                                            PORT
                           Fig.  3.  Drawings of port and starboard sections of R.V. Kumtuks.

-------
     BOW
                  STERN
1.  Anchor winch
2.  Porthole
3.  Paint locker
4.  SCUBA diver's locker
5.  Entrance to wet laboratory
            Fig.  4.  Drawings of bow and stern sections of R.V.  Kumtuks.

-------
                                        Upper  deck
                                           STARBOARD
i
             OHatch
  Forward
    deck
  Anchor
State- *
j room J-^
1 \
1
State- J
] room x<
1


State-
S room

/State-
room



L
Head
{
Head
Head
^
Laundry



f*lr\om 1 nH


° ° H
Wet Lnb


**
V
Galley Eating Area ^
1




_ ^-^_^____ij^^»j


^•••l^H
Radio

-r->—
r^

•— «
-*^
- o-
-o-
~\s~\
<
Small
Insttu
room
-i — »•


\
Instrument
room

i '



: + :
z ci
-.81
>*. r



• g*3 ^ • -+-£^5

kJ
O
*-
V)
—
^
0
CO
O
•••
             O Hatch
                       •Gas tank

                       ^Entry

                          Aft
                         deck
                                                                                 .:/Entry
                     Entry
  Entry      Entry
Engine room
                      ^Entry


                    O -Cargo boom
                   O Hatch
                                    ss
                                    B
                                             PORT
     Fig.   5.   Upper deck facilities of R.V. Kumtuks.

-------
                                     Lower  deck
                                       STARBOARD
       r-i
       L.J
Storage
      O
                                         Sea
               Sleeping

               quarters
Classroom
                          — 3 -
                   Storage
                               Storage
                              chest
                               I
Lathe
                               ^7 Pump
             Pump
                                                            Compressor
                       Storage
                                  Storage
                                                      Work

                                                      bench
                                            -Q.-
                                                  Engine room
                                               Generator
                                      Pumps         rn

                                      Generators
                         Sea  chest  —  —     '—
                                                                                             to
                                                                                             H
                                                                                             M
                            Storage
                                         PORT
                        Fig.  6. Lower deck facilities  of R.V. Kumtuks.

-------
by car was practical, working for significant lengths of time - two days
or longer - was most effective.  Isolation from urban stress was nearly
as good in the city as in rural areas, except for theft of deck equipment.

      Towing the barge to the research site proved convenient and economi-
cal: it has a light displacement by barge loading standards - about 300
dead weight tons when completely loaded with fuel and water.  It was towed
at 5-1/2 or 6 knots by a 350-hp tug and at about 9 knots by a 1,000-hp tug
and was moved at short distances under calm conditions with our 18-ft,
155 hp inboard-outboard boat.  With the barge skegs under the aft rake,
there was no problem of veer from side to side during towing.  A 750-lb
Danforth anchor proved secure in winds of over to knots; thus the lack of
propulsion engines for maintaining position during bad weather was not a
worry, at least not after we had successfully ridden out the first storm
and become confident of the vessel's capabilities.  We later weathered
a tO-knot blow with a 90-ft seiner and a 40-ft troller moored to the barge.

      Operating the Kumtuks at a research station involves a combination of
shipboard and shoreside practices.  Once anchored on station, the docks
and small boats are lowered and assembled behind the barge.  All personnel,
including the scientific crew, usually help with the chores.  Next the
flexible suction line for the aquarium water supply is lowered, primed, and
the wet laboratory activated.  Masking tape is removed from cupboard doors
and drawers in the chemical laboratories and instrument rooms (having been
applied for the trip) and contents unpacked.  Breakage during towing, even
with only minimal packing,  is not a problem.  Twice we successfully carried
out experiments while the Kumtuks was being towed from Puget Sound through
the locks into freshwater.  Preparations for departure from a research
station are  the reverse of the above.

       It was originally thought that the scientific  staff could contribute
to engine room operations, especially when only one or two members of the
scientific staff were aboard.  However,  the scientific staff were generally
too engrossed in their work to perform regular engine checks and too valuable
to be spending the time necessary for oil changes and other matters of
routine engine maintenance.   Conversely, once the staff had finished their
activities in the late evening and most of the heavier intermittent electrical
load was unnecessary, everyone could go to bed with one engine running
unattended until morning.  In several cases, our chief marine engineer
detected minor problems that  a mechanically less experienced person would
not have noted and thereby probably prevented major difficulties.

      The power system, while generally satisfactory, has  provided some
problems.  Electric heat was  installed, partly because its loading
characteristics were supposed to minimize voltage changes  when large
electric motors were started.  In spite of the heating ballast, voltages
momentarily dropped from 120  to 100 v, and some scientific instruments
were affected.  After a number of possibilities were  tried, a 10-kw
generator was installed to supply power only to the scientific instruments.
                                12

-------
Although 10-kw are more than would ever be needed for instruments, this
large a generator was installed so that it could carry the entire shipload
on warm nights and use less fuel than the larger engines.  It also serves
as an emergency generator.

      A primary requisite of any physiology laboratory is an adequate,
dependable supply of healthy animals.  When studying wild animals, one
must catch and maintain them in good condition.  Purse seines and traps
have proved to be the least injurious or stressful to salmon.  Whenever
possible fish were obtained in collaboration with other projects, partly
for reciprocal sharing of biological and physiological information about
the fish in question and partly because experience demonstrated that
professional fishermen catch more fish per unit of effort than physiol-
ogists.  When the staff had to conduct the fishing, an 18-ft inboard-outboard
boat with a 30-ft x 600-ft hand-operated seine was used.  Fish were trans-
ported in the same boat, in a flow-through live-tank.  With the net aboard
and live-tank filled the safe loading limit for the boat was exceeded, so
it was used mostly for seining or transporting, but not for both together.
The same boat with a towing post installed amidship also served as seine
skiff for a chartered seiner and for towing sections of our docks lashed
together as an improvised barge.

      Fish were   held  in a variety of containers, including small-sized
confinement chambers submerged  in a shallow water table, 100-gal aquaria,
4-ft- and 6-ft-diameter fiberglass tanks,  8-ft-diaraeter plastic swimming
pools, and 15-ft square pens made of nylon mesh.  The latter were supported
 inside of the U-shaped docks and servedto  hold fish safe  in case of pump
or power failure, although  pump failure was unlikely because any of several
pumps can serve  in place  of the laboratory pump during an emergency.   We
 found that  any confinement  was  stressful to salmon; the  smaller the container
 in proportion to the size of the fish, the greater was the degree of  stress.
 The  wide range of container sizes, therefore, proved advantageous for
handling many sizes of salmon.

       While one  reason for the  inexpensive operation of  the  Kumtuks has been
 the  lack of certification requirements and subsequent requirements for a
 three-shift licensed crew,  the  Coast Guard provided a  courtesy inspection
 and  made recommendations, so that the vessel  and its equipment could  meet
 all  standard  safety requirements.   A number of short cuts were taken  in the
 construction  and operation of the vessel, but safety has never been a matter
 for compromise.

       The  costs  of operation are difficult to determine exactly.   Fixed  annual
 costs  are about  $20,000, and 20 weeks of independent operation cost about
 $300/week,  so that 120 days of charter at $250/day would pay crew salary,
 operation,  and maintenance for a year.  Food  costs were not included in  these
 estimates,  because they vary widely, depending on numbers of people  and  food
 preferences.   Also not included are the depreciation on the original  capital
 investment of $160,000 and the cost of scientific and other non-integral
 equipment.
                                 13

-------
    A Modified Version of the Blazka Respironveter and Exercise

                       Chamber of Large Fish


Design

      A basic problem of studying salmon, especially adults, was that
they swim almost continuously.  Valid physiological measurements,
therefore, should be taken from swimming fish.  Respirometers are
devices which make such things possible and we adapted one to our
needs.

      Our version (Fig. 7) was made with two plexiglass (acrylic) tubes
12 inches (30.5 cm) and 8 inches  (20.3 cm) in diameter with walls 1/4 inch
(6.6 mm) thick.  The smaller tube is loosely centered inside the larger
tube at four vanes at  each end.  A removable set of vanes inside the
smaller  tube  at the upstream end controls direction of flow.  Flanges
were machined  from flat plexiglass plate and glued to the outer tube for
attachment of  the end  blocks and  for mounting of the tubes on a plywood
base.  The end blocks  were laminated from lumberyard hardwood, turned on
a wood lathe,  and fiberglassed for waterproofing.  The shape shown for the
end blocks has proved  sufficiently satisfactory so that we plan to have
them duplicated  in cast  aluminum.  The plywood base  is supported on an
axle and  a framework  that allow the whole respiroraeter to be tilted^for
insertion of fish  into the respirometer while it  is  partly filled with
water.  Partly filling the respirometer in the tilted position minimizes
the amount of time that  the  fish  is out of water.  A 3/u-hp motor with
a variable-speed magnetic clutch  drives a jet outboard impeller mounted
on a stainless steel driveshaft.  The driveshaft  passes through the end
block in a  standard, graphited packing gland.  The jet outboard  impeller
causes less  spiral movement of the water than a propeller or  centrifugal
 imoeller  would, and producesaflow having very low turbulence  and velocities
up to 131 cm/sec.  When  the experimental fish fills  a significant portion
of the cross-sectional area of the  chamber,  the  effective velocity is
 considerably greater, depending on  the size  of the fish.  Water enters the
respiroroeter at the  bottom and leaves from a large diameter,  low-turbulence
 standpipe at the top.  Tubes,  wires, and other  attachments  to the fish
 also exit there.


 Operation

       An anesthetized fish is put into the partly-filled respirometer through
 the end opposite the fcnpeller.  Any tubes, wires, or other attachments are
 pulled through the standpipe and a hole in the inner tube with a Booked
 wire.  The inner vanes are inserted, the end block is secured with wing
 nuts,  and the respirometer leveled.  The impeller is rotated while the
 respirometer completes filling so that the water movement provides a
 respiratory current for the fish and induces recovery from the plane III
  (respiratory arrest) level of anesthesia used during surgery.  Between 2

-------
Packing
Gland
                                                          End  block
                                                         (removeable)
                 Axle for tilting  respirometer
Mounting  platform
Fig.  7.   Diagram  of modified Blazka respirometer used for investigating
         physiological responses of salmon swimming in water of  low
         dissolved oxygen.

-------
and 24 hr of recovery in the respirometer are allowed before an experiment
is begun, the length of time depending on the experimental requirements
(Fig. 8).

      The reverse procedure is followed for removal of the fish from the
respirometer.  The respirometer is tilted, the water level  lowered, and
the end block removed.  Either the fish is pulled out by its tubes (after
being disconnected from the recorder, etc.) or the respirometer is tilted
down again and the remaining water with the fish poured into a large
container.

      Controlling the position of the fish in the respirometer without
interfering with free swimming is a continuing problem.  Several respiro-
meters use screens or grids downstream from the fish to keep the fish out
of the pump and provide electrification of a grid to stimulate swimming.
However, we wished to minimize frictional losses and electrolytic products
in sea water are toxic; therefore, screens were not included in the design.
We had already found that small adult salmon gave no overt response when
towed by a cannula anchored in their nasal cartilage.  Preliminary trials
in this respirometer showed that most fish will swim either when the water
velocity increases or when their tail touches the rotating impeller.  How-
ever, larger fish may break, pull out, or damage most of the tubing and
routine attachments.  Thus we inserted a heavy nylon cord through the
muscles under the anterior edge of the fish's dorsal fin - the approximate
node of its swimming oscillations and also its approximate center of gravity.
We call this cord a "tether" or "towline."

      In use, the towline is led out of the standpipe and tied with minimal
slack while the fish recovers from surgery or previous periods of exercise.
Swimming is stimulated by increasing the water velocity and giving the fish
more slack in the towline.  Brightly illuminating the downstream half of
the respirometer and darkening the upstream half help the fish to
learn to hold position in the swimming chamber.  The towline system has been
used successfully on salmonids ranging in weight from 0.3 to 10 kg for up to
2 weeks.  The towline does not seem to cause necrosis or promote infection
at the puncture site, even in the presence of the fungus Saprolegna else-
where on the fish.  Fish become accustomed to the towline usually within
minutes.  Because water flow produced by the impeller is sufficient
to meet the fish's respiratory needs, fish have an extremely low level of
ventilatory activity while they are being towed.
                Anesthesia, Multiparameter Sampling, and Surgery

Problems of Sampling Live  Swimming Fish

      Repeated sampling of blood, urine, and inspired and expired water is
necessary for evaluating immediate physiological responses of a free
swimming fish to environmental changes.  Surgical installation of catheters
or tubes is presently the  most direct method of access to blood vessels,
                                 16

-------


Fig.   8.     Adult male coho salmon swimming in respirometer after surgical im-
        plantation of various cannulae and catheters.


-------
urine bladder, and gill regions.  A method of sampling pre-gill water^
was developed by Saunders (1962), and members of our project have modi-
fied and developed methods for continuous blood sampling (Smith and
Bell, 1964), urine sampling (Miles, 1967, 1969; Mearns et al., in
preparation), and post-gill (expired water) sampling (Davis and Watters,
1970).  Simultaneous application of all the techniques have proved feasible
and successful during the present study.  With two or three technicians,
we have been able to monitor all samples every ten or fifteen minutes
while the fish is swimming.


Problems of Preparing Fish for Physiological Monitoring

      A salmon completely prepared and rested for physiological monitoring
of environmental responses has unavoidably been exposed to a series of prior
"handling" stresses.  These include initial capture, confinements in a
holding pen or tank, netting, anesthesia, surgery, and acclimation to the
swimming chamber.  Our  experience during the 1968 field season has shown
that the stresses of anesthesia and surgery in particular may prove lethal
to salmon in  the low oxygen environment.  Although indicative of a lowered
stamina for these particular fish, it nevertheless posed problems for
further physiological studies.  Our decision was to separate the stresses
of capture, netting, and anesthesia-surgery by rest periods of 12 to 24
hours  for each fish.  In the 1969 studies, only fish held on board the
Kumtuks for two days were used for surgery.  A fish was then netted from
the holding tank or pen and placed in a dark chamber for 12 to 24 hours
 (overnight) to recover  from the netting itself.  Sufficient anesthetic was
then  infused  into the dark chamber to tranquilize the rested fish in 3 to
4 minutes.  The relaxed fish was removed and placed on the surgical^table
with  adequate irrigation of aerated water and anesthetic over the gills
 (Smith and Bell, 1967). Catheters and tubes were installed at this time
 (Fig.  9) and  the fish was brought to nearly complete recovery before being
placed in the respirometer  (stress tunnel).  Finally, the fish was
allowed to recover in the tunnel 12 to 20 hours  (overnight) prior to the
swimming performance and  physiological studies.  This approach  (separating
handling stresses) has  not only proved feasible, but highly successful
in insuring the validity  of conducting physiological studies on wild fish.
It has also given  us new  insight into the effect of various environments on
the ability of fish to  overcome handling stresses.
                                  18

-------
• o
         Fig.  9.   Installation of catheter behind  the  gill  in  adult  male  coho  salmon.
                Buccal catheter is visible arising  from the  snout  of  the  fish.
                in the salmon's mouth  supplies  oxygenated water and anesthetic.

-------
                            SECTION V
               STRESS DURING OUTMIGRATION OF SMOLTS


      Upon reaching a certain size characteristic for each species,
juvenile Pacific salmon which had been working to stay in their natal
stream or lake decisively turn downstream and seek the strongest river
currents to aid them in their pell-mell dash to the sea.  Their
plunge into salt water puts many strains on their physiological
systems — new environment, unfamiliar food, unexpected dangers.
This section deals with our attempts to identify which of these new
problems are most significant to outmigrant salmon.


        Ionic Regulation in Migrating Juvenile Coho Salmon


Introduction

      The purpose of this study was twofold.  The first was to define the
concentrations of sodium, potassium, calcium, magnesium, chloride, and water
in blood plasma in juvenile coho salmon residing in freshwater.  All meas-
urements were made for each fish to determine normal values and the
relationships among these parameters in the individual fish.  The second
purpose was to learn what changes in these constituents occur after entry
into  sea water.
Materials  and Methods

       Coho salmon, Oncorhynchus kisutch, residing in Big Beef Creek, Kitsap
County,  Washington, were used throughout the study.  Big Beef Creek dis-
charges  into the Hood  Canal near Seabeck.  The facilities of the Big Beef
Creek  Field Station of the College of Fisheries, University of Washington,
were used  in the field work.  The station is located on the creek about 1/4
mile from  its mouth.

       Collections of non-migratory fish in the winter and early spring
(November  12, 1966 through February 18, 1967) were taken by means of a
15-ft  braided nylon seine.  The sample on March 22, 1967 was taken from
fish found in a box trap set below a pipe draining a pond near the station.
The fish caught in the box below the pond may have been stressed by the^
high velocity of water flowing through the box during a freshet.  The fish
collected  thereafter  (through May 10, 1967) were downstream migrants caught
in a floating-box fyke net located 1200 ft from the stream mouth.  The
migrants moved  mainly  at night, and the samples for this study were removed
from the floating box  in the morning after a night of collection.  The
floating box was covered and the fish were not crowded; abnormal^stress
was improbable. The fish were removed from the traps with a brail fitted
                                  20

-------
with 1/4-inch braided nylon net and taking care to avoid loss of scales.
The fish were then placed in a bucket containing a solution of MS 222
(Tricaine  Methanesulfonate) in a concentration such that they reached
respiratory arrest in about five minutes.  The blood sampling was
completed within 20 minutes after the fish were placed in the anesthetic
solution.

      Thirty-four coho caught in the fyke net at section 1200 on Big Beef
Creek were put in four 2-1/2-gal buckets of freshwater and were transported
5 miles to Seabeck, Washington, where 29 of them were placed in a holding
pen floating in sea water (25.5 ppt, measured with a refractometer). The
holding pen was a 3-ft cube constructed with 1/2-inch galvanized hardware
cloth.  The five coho that were not placed in the holding pen were
anesthetized and taken back to the Big Beef Field Station for blood
sampling.  A sample of five fish was dipped    from the holding pen
without deliberate selection every 6 hours for the succeeding 36 hours.
The fish were anesthetized as described above, and blood sampling was
conducted within 15 minutes after removal from the holding pen.

      Wet weight was taken on a balance to the nearest tenth of a gram^and
total length was measured on a wet measuring board to the nearest milli-^
meter.  The dried  caudal peduncle, wrapped in a tissue to prevent contami-
nation, was severed with either large  surgical scissors or a scalpel.
Blood was  collected   into microhematocrit tubes coated with ammonium heparin
as anticoagulant.  The total handling  time from removal of the fish  from
the anesthetic solution to  the completion of the  blood collection was
usually less  than 45 sec.   The blood samples in the heparinized tubes were
 centrifuged at 13,500 x g  for  5 minutes, and hematocrit was determined  using
 a Spiracrit microhematocrit reading device.  Then the microhematocrit tubes
 were broken immediately above  the layer of leucocytes  so  as to separate the
 cells and plasma.   A 20-microliter aliquot of the plasma  was  removed and
 run into a 1-ml volumetric flask containing  0.980 ml of deionized water and
 was thoroughly mixed by repeated inversions  of the flask.  When the per-
 centage of total dissolved solids in plasma was determined,  a small,
 hand-held refractometer ("TS" Meter, American Optical Co.)  was used.
 Chloride ion was measured with a Buchler-Cotlove chloridimeter, an elec-   ^
 tronic potentiometric titrator, operated in the low range.   The concentration
 of the metal ions was measured with a Perkin-EHner, Model 290 flame ab-
 sorption spectrophotometer.  The blank used was deionized water and the
 standard was a solution containing 0.4 mEq/1 Na, 0.3 mEq/1 K, 0.3 mEq/1
 Ca, and 0.1 mEq/1 Mg in deionized water.  One-tenth ml of the diluted
 plasma was further diluted to one ml for the sodium determination.


 Results
       Throughout the experiment (from November 12, 1966 to May 11, 1967),
 length, weight, and hematocrit of the fish in samples taken in Big Beef
 Creek were measured.  There was a total of 103 observations.  Measurement
                                   21

-------
of plasma ion concentrations was begun on April 6, 1967 and measurement of
total dissolved solids was begun on April 12, and both were continued for
the remainder of the experiment.  The means, variances, and ranges of
these physical and blood parameters for freshwater and sea water observa-
tions are presented in Tables in the appendix.

Discussion

      Premigratory Blood Characteristics.  Hematocrit was measured in
juvenile coho salmon residing in freshwater during the winter and spring
months.  There was considerable variation in hematocrit with time which
might be explained by the variation in water temperature (Fig. 10).
Snieszko (1960) reported that the hematocrit values of fish are sensitive
to temperature and especially sensitive to the amount of dissolved oxygen.
The changes in stream temperature may be seen to correspond with the
changes in hematocrit with two exceptions.  This correlation is probably
due to the higher dissolved oxygen concentration and lower   metabolic rate
of the fish with lower stream temperatures.  With a lower metabolic rate,
the fish would require less oxygen delivery to the tissues while at the
same time the environmental concentrations of oxygen would be higher.  Both
of these conditions are compatible with a hypothesis that fish have fewer
circulating red cells, as indicated by lower hematocrit, at lower tempera-
tures than at higher temperatures.

      During the period between April 12 and May 10, there was no statis-
tically detectable change in the concentration of dissolved solids
(Appendix Table 3).  This indicates that there was no major change in the
equilibrium between excretion of water by the kidney and uptake of water
across the gills.  Houston (1959) reported that plasma water and chloride
concentrations varied inversely with weight in premigratory development of
steelhead trout.  In the present study, these parameters were found to be
directly correlated with weight (Table 1).  There were no decreases in
chloride levels as have been described from previous studies (Kubo, 1955;
Houston, 1959), but compared to previously reported chloride values, those
reported in this study are somewhat low.  It is possible that a decrease in
chloride concentration occurred before the sampling began.

      A correlation matrix (Table 1) of parameters from all of the fish
sampled from April 12 through May 10 was prepared without regard to sample
date.  Hematocrit was correlated with both length and weight, but this
correlation was probably of secondary nature since growth and stream
temperature both increased with time during the period of observation.
The increase in length and weight was due to growth, and the increase in
hematocrit was due in part to the rise in temperature.  Total solids also
were positively correlated with length, weight, and hematocrit.  Since
there was no corresponding correlation of the ion concentrations with
these factors, the correlation was probably due to the changes in the
protein fraction of the total dissolved solids, which may have been a
function of growth.  The levels of the predominantly extracellular ions
sodium, calcium, and chloride were mutually related as were the intra-
cellular ions potassium and magnesium.
                                 22

-------
       7c
 o
50
                            Hematocrit  vs.  Temperature
fO
CO
     37.5
                  J-jHem.

                  o—o Tern p.
      2.5
°F
55
                                                             45
            Nov.     Dec.    Jan.     Feb.    Mar.   April    May
                                                             35
                  Fig. 10.  Seasonal changes in hematocrit and stream temperature.

-------
                   Table 1. Correlation matrix of physical and blood parameters of
                             freshwater residents (April 12 through May  10,  l*t>f)
Hemato- Total
Length Weight crit Solids
Length 1.000 0.972** 0.702** 0.465**
Weight 1*000 0.675** 0.488**
Hematoerit 1-000 0.557*
M Total solids i-000
Sodium Ion
Potassium Ion

Calcium Ion

Magnesium Ion

Chloride Ion
JL «f» T^ MfT
Na K Ca ug
0.183 -0.135 0.343* 0.125
0.193 -0.172 0.313* 0.150
0.052 -0.113 0.303* 0.355*
0.016 -0.147 0.310* 0.236
1.000 -0.352* 0.494** -0.079
1.000 -0.202 0.512**
1.000 0.177

1.000



Cl"
_— — — — — —
0.300*
0.333*
0.083
0.024
0.596**
-0,224
0.235

0.075

1.000

 * Correlation significant at the 95% level.

** Correlation significant at the 99% level.

-------
      The physiological and behavioral changes occurring during the parr-
smolt transformation and migration are incompletely understood.  In the
period of these observations, there were no dramatic changes in the ionic
constituents (Figs.11,12,13, and 1*0 of the plasma.   Conte et al. (1966)
found that coho throughout winter and spring were equally able to survive
transfer to sea water and presumably were capable of migrating.  Baggerman
(1960), however, reported a salinity preference of coho for sea water only
in late April and May, and a preference for freshwater in periods preceding
and following this time.  Conte et al. (1966) concluded that the ability
to survive in sea water was independent of the parr-smolt transformation.
The data gathered in the present work showed no relation between pre-
migratory plasma ion concentrations and the parr-smolt transformation.


      Adaptation to Sea Water.  In this experiment, the juvenile coho were
able to complete the adjustive phase referred to in earlier literature
(Black, 1951; Fontaine and Koch, 1950; Keys, 1933) in about 36 hours.
Conte et al. (1966) found this period to be about 36 to 40 hours for coho.
Conte and Wagner (1965) found the period of adjustment for rainbow trout
to be 60 to 100 hours, and Houston (1959) found it to be HO to 70 hours in
50-per-cent sea water for fish of the same species.  Black (1951) and Houston
(1957) reported a period of 36 hours for chum fry.  The adaptation to sea
water in the present study was characterized by increased concentrations of
plasma sodium, chloride, magnesium, and during one six-hour period, an
increase in the concentration of potassium.

      The percentage of packed erythrocytes rose during the first 18 hours
of exposure of the fish to sea water (Fig. 15A).  A rise in hematocrit can
be accounted for by an increase in the size of individual erythrocytes, an
increase in the number of erythrocytes, or a decrease in the plasma volume.
Since the fish were osmotically losing water through the gill epithelium to
the environment, the plasma must have become temporarily hyperosmotic to the
erythrocytes and they must have tended to lose fluid rather than to gain it.
It is also highly unlikely, although unproven, that the hemopoeitic capa-
bilities of the fish were such that they could produce approximately a
25-per-cent increase in the number of circulating erythrocytes in a period
of 18 hours.  Therefore, it seems that the increase in hematocrit was due to
a decrease in plasma volume.  If this hypothesis is accepted, the hematocrit
values obtained in this experiment can be used as measures of plasma volume.

      In freshwater, the juvenile salmon is hyperosmotic to its environment,
resulting in an inflow of water through the gills, which is excreted by the
kidney as a relatively large volume of dilute urine.  Upon entering sea
water, the fish is in a hypertonic environment, and water is lost by
diffusion through the gills while the kidney continues production of a
smaller volume of dilute urine.  Holmes (1961) reported that urine flow
declined in rainbow trout  (Salmo gairdneri) from 75-90 ml/kg/day in fresh-
water to 0.5-1.0 ml/kg/day in sea water.  A diagrammatic account is given
of the changes in water flux at the three major exchange sites  - gill,
                                  25

-------
ro
          50-
          40-
30

6.0
      ^4.75-
          3.5-
                               Hematocrit
                       Total  Dissolved Solids
               Apr.6  Apr. 12  Apr.20 Apr. 28  May 4   May 10
                 Fig. 11.  Observations of hematocrit and total dissolved solids
                        in migrating juvenile echo salmon.

-------
10
135

130
        l«j
           100-
                               Plasma  Sodium
       T	T

Plasma Chloride
                 Apr. 6  Apr. 12  Apr. 20 Apr. 28   May4  May 10

               Fig. 12. Observations of sodium and chloride concentrations in the blood
                     plasma of migrating juvenile coho salmon.

-------
to
00
           8.5
           7.0J
5.5
1.5-
          0.5
                               Plasma Calcium
Plasma Magnesium
                Apr.6    Apr.12  Apr.20  Apr.28 May 4   May 10
               Fig. 13. Observations of calcium and magnesium concentrations in the blood
                     plasma of migrating juvenile coho salmon.

-------
10
            16-
                              Plasma  Potassium
         \
            8-
            0
                Apr.6  Apr.12  Apr. 20  Apr. 28  May4   May 10
                 Fig. m. Observations of potassium concentration in the blood plasma
                       of migrating juvenile coho salmon.

-------
co
o
       55
       45-
35


6.0-
       4.5-
       3.0
       F.W.
                                 Hematocrit
  	1	1	r~

  Total  Dissolved  Solids
6        12      18      24

    Hours in Sea water
                                                          30
36
           Fig. 15. Changes in the heroatocrit and percentage of total dissolved solids in the

                  blood plasma of juvenile coho salmon with adaptation to sea water.

-------
kidney, and gut - in Fig. 16 from the data and theories of other workers
and the data obtained in this study.

      Upon entry of the juvenile coho salmon into sea water, there is an
influx of sodium and chloride across the gills.  This influx, combined with
the loss of plasma water, results in increased concentrations of these ions
(Fig. 17) during the first 6 hours.  It appears that then the gills begin
to actively transport these ions from plasma to the sea water.  When con-
tinued dehydration stimulates ingestion of sea water, the fish again must
cope with increased plasma levels of these ions as they are passively
absorbed from the gut with the water.

      Some workers have observed decreases in the sodium-chloride ratio
during adaptation to sea water in several salmonid species (Gordon, 1959;
Houston, 1957; Parry, 1966).  Such decreases indicate a loss of some other
anion, most likely bicarbonate.  This is consistent with Busnel's (1943)
observation of a decrease in the pH of the blood of rainbow trout after
their transfer from freshwater to sea water.  In the present study, a
decline in the mean sodium-chloride ratio from 1.31 to 1.23 was observed
during the 36-hour experimental period, but the variance of each mean was
high and rendered the differences statistically insignificant.  Decreases
in pH have been observed to cause decrease in membrane permeability and
therefore may aid passively in the adaptation to sea water (Houston, 1964).

      The potassium movements observed in this study were probably passive.
There was a movement of potassium from the tissue to the extracellular fluid
(Fig. 18) along with the dehydration of the tissue early in the adjustive
phase, but this movement reversed direction (Houston, 1959) as the potassium
level reached equilibrium with the concentration in the environment.  Since
the concentration of potassium in sea water was 7.36 mEq/1, it is unlikely
that there ever was a strong gradient of this ion between the environment
and the plasma.

      The concentration of calcium in the plasma remained stable  (Fig. 19A)
throughout the experimental period until the thirtieth hour.  Houston (1959)
observed that the plasma level of calcium remained rather constant along with
rising tissue concentrations of this ion.  Calcium ion may contribute to the
decrease in surface permeability and reduce the rate of ion flux  across the
gills and skin.  The drop in the calcium level in this experiment at the
thirty-sixth hour was probably associated with initiation of divalent ion
control by the hindgut.

      The concentration of magnesium in the sea water used in the experiment
was  39.6 mEq/1.  This concentration provides a substantial gradient for
passive magnesium transport across the gills.  The data (Fig. 19B) show
a decreasing rate of the influx of magnesium into the plasma, probably as a
result of decreasing membrane permeability, and then a decrease in plasma
concentration when active transport of divalent ions begins in the hindgut.
                                  31

-------
0
to
Efflux



Stasis




Influx
                V
                                                                                 ............  Kidney


                                                                                 	Gill

                                                                                 ——•— •—  Tissue

                                                                                 	Intestine
       x  fresh   y^
       x  '        x*ST'
          water
                                 0
                                                      seawater
                                                  tissue v/ater loss
                                                                     tissue water replaced
                                                                     seowater  ingestion
                                                                                     excretion  of divolentjons
                                                                                             \
                                                                              30
                  6           12          18         24

                                 Hours  in  Seawater

Fig. 16.  Theoretical flow rates of plasma water in juvenile coho salmon during

         the adjustive phase.
36

-------
w
CO
       170
    \
140

!45
       105
F.W.
                                Plasma  Sodium
               Plasma  Chloride
6        12       18      24

   Hours  in Seawater
                                                          30
                                                             36
                 Fig. 17.  Changes in concentrations of sodium and chloride in the blood
                        plasma of juvenile coho salmon with adaptation to sea water.

-------
      15.0
s?  \
        0
              FW.
                             Plasma  Potassium
6        12       18      24
   Hours  in  Seawater
30
36
                Fig. 18. Changes in concentration of potassium in the blood plasma of
                       juvenile echo salmon with adaptation to sea water.

-------
oo
tn
         8
     \

     £f 61
          -
   ki
       2.5-
       0.5-
             F.W.
                             Plasma  Calcium
Plasma Magnesium
      12      18      24

     Hours In  Seawater
30
36
               Fig. 19. Changes in concentrations of calcium and magnesium in the blood

                      plasma of juvenile echo salmon during adaptation to sea water.
                                                                          B

-------
      Relationship of theSea Water Experiment to the Ecological
Situation^In behavioral experiments conducted by Houston (1957),     ^
smoits moved into sea water (21.83 ppt) about 2 hours after they were given
the opportunity to do so.  Thus, the fish apparently spend little time in
the estuarine environment, perhaps only the time it takes to follow the
increasing salinity gradient into full strength sea water.  In a stream
that empties directly into sea water, such as Big Beef Creek, the fish
could make the transition in a matter of minutes.  Therefore, the results
obtained by transferring smelts directly from freshwater to sea water very
closely approximate the response to osmotic stress in wild fish migrating
at will.

      Lipid Composition and Heroatology of Juvenile Chinook Salmon

      (Oncorhynchus tshawytscha) Before and After Migrating Through

                       The Duwamish Estuary

Introduction

      Chinook salmon (Oncorhynchus tshawytscha) reared at Soos Creek
Hatchery near Auburn, Washington, encounter problems of feeding in their
unfamiliar environment following release and downstream migration. ^The
young salmon were reared in a hatchery environment and fed a commercial
pelleted diet.  They were liberated when they reached approximately 50
fish per pound (9 g each).  Following release from the hatchery, the
young salmon were free to migrate down the Green River into the estuary
 (Duwamish Waterway) and then into Elliott Bay, Washington.  The young fish
may remain and forage in Elliott Bay for a temporary period prior to their
seaward migration through Puget  Sound and the Strait of Juan de Fuca into
the Pacific Ocean.  While retained  in the hatchery environment, the young
fish were fed a pelleted diet periodically, and hence were accustomed to
the feeding schedule of the hatchery attendants.  In the environment of
river and estuary, the young fish had to seek and catch food.  The Green
River, at the time of the salmon's  release from the hatchery, offered few
food items.  The Duwamish Waterway  was a polluted area near Seattle Harbor,
containing reduced oxygen levels, and receiving chemical, oil, and domestic
wastes.   The estuary probably contained a minimal food supply for the
young  fish.

       Young  salmon were captured by tow net at specific locations along ^
 their migration  route from the  hatchery to Elliott Bay to study  changes in
 their lipid  composition,  specific fatty acid  composition, and hematology.
 To compare these changes  quantitatively, a control sample of fish was taken
 from the hatchery at the  time of release.  Additional fish were  taken^from
 the estuarine environment and placed aboard the R.V. Kumtuks located  in
 the Duwamish Waterway, and fed  a commercial diet.  Quantitative  analyses
 of fork length,  body weight, and total lipid  and fatty acid concentrations
 were performed to assess  the quality and amounts  of  food being ingested,
 and to evaluate  the influence of diet  upon  the lipid components  of the
 fish.
                                  36

-------
Materials and Methods

      Sampling of the Fish.  Downstream-migrating chinook, released from
the hatchery in late May, were captured by tow net in the Duwamish Water-
way (Fig. 20).  The fish were taken from the tow net and immediately
packed in ice for transportation to the research laboratory at the Uni-
versity of Washington.  Some of the captured salmon were utilized for
hematological experiments, and others were transported live to the
R.V. Kumtuks for mortality studies and studies by other individuals in
related areas of physiological work.

      Lipid Analysis.  Individual samples of the young salmon were homo-
genized in a mixture of chloroform and methanol for lipid extraction
(Bligh and Dyer, 1959).  Total lipid analyses were taken to determine the
amount of lipid in the entire salmon, and this was related to the fish's
fork length and weight.  Methylation of the total fatty acids was accom-
plished prior to gas chromatographic analysis of the individual fatty
acids present.  Supportive thin-layer chromatography and gas-liquid
chromatography were used to verify the identification of individual fatty
acids.  The fatty acids were detected by a Model 5750 research gas chromat-
ograph manufactured by Hewlett-Packard.  Detection of the fatty acids
present was made at 190 C, employing a flow rate of 60 mm/min of helium
gas in a column containing 15 per cent ethylene glycol succinate bound to
chromasorb-p  (60 - 80 mesh).

      Identification of the Fatty Acids.  Comparison of known fatty acid
standards and related material from other salmon lipids was used to identify
the fatty acids present in the lipids of the young salmon.  Procedures
employing retention time and supportive work with thin-layer chromatography
have been described earlier by Saddler et al.  (1966).

      Hematology.  Juvenile chinook salmon from the hatchery at the University
of Washington were cultured on Abernathy dry pellet  in freshwater and sea
water.   Fish  from these two environments were  compared to juvenile chinook ^
salmon cultured concurrently at  Soos Creek Hatchery on Oregon moist pellet,
and with the  same stock of salmon following their release and subsequent
capture  by  tow netting in Elliott Bay.  A portion of the  fish captured in
Elliott  Bay were cultured for 12 days in sea water on the R.V. Kumtuks on
Abernathy dry pellets.

      Hematological analyses included measurements of packed cell volume
 (PCV  or  hematocrit) by the microhematocrit technique, hemoglobin  concen-
tration  by  the cyanmethemoglobin method, mean  corpuscular hemoglobin  concen-
tration  (MCHC) by standard calculations  (Wintrobe, 1967), and total dissolved
 solids with a Goldberg refractometer.  Fork length and body weight were  also
      "^Obtained from R. V. Moore Company, La Conner, Washington.
                                   37

-------
                                      Eos!

                              West   Wolcrwol[
                            Woterwoy        T
Fig.  20.   The Duwamish Estuary and Green-Duwamish River.

                                 38

-------
determined for each fish.  Chinook salmon sampled at Soos Creek Hatchery
as well as Soos Creek chincok captured in Elliott Bay contained insufficient
volumes of blood to permit measurement of all hetnatological variables in
each fish.  The values for salmon sampled at Soos Creek Hatchery represent
pooled observations.  Insufficient sample sizes did not permit complete
evaluations of blood in Chinook captured in Elliott Bay.
Results
      Chinook salmon reared at Soos Creek Hatchery and those cultured in
these experiments were fed either Oregon taoist pellets or Abernathy dry
pellets.  Table 2 gives the percentage composition of the basic ingredients

     Table 2.  Percentage composition of basic ingredients in Oregon
               moist pellet and Abernathy dry pellet diets.  Oil content
               was derived for both diets from fish, soybean, and
               cottonseed oils
             Crude     Crude     Crude     Carbohydrate,     Water
             protein,  fat,      fiber,
  Diet          %       %          %             %             %

Oregon noist   35.0     5.0        4.0           -             35.0
 pellets

Abernathy
dry pellets    43.0    10.5         -            26.0          8.0
 for the two diets.  The oil  source  is  listed as  crude fat and contained fish
 oil, soybean oil, and  cottonseed  oil.   The plant oils contributed greater
 amounts of linoleic acid  (18:2) than is usually  found in aquatic marine
 food items utilized by salmon (Gruger   et al., 1964).  The two diets
 contained twenty-eight major fatty  acids.  These fatty acids  contained from
 8 to 24 carbon atoms and  from 0 to  6 double bonds.   Six of the twenty-
 eight fatty acids were selected for detailed examination because of their
 quantitative and physiologic importance:  palmitic  (16:0), oleic (18:1),
 linoleic  (18:2), arachidonic (20:4), eicosapentaenoic (20:5), and
 docosahexaenoic  (22:6) acids. In addition, the  two diets contained the
 same fatty acids found in hatchery  salmon and in native salmon inhabiting
 the Puget Sound area.  Major differences in the  two diets existed in  the
 percentages of linoleic acid (18:2) and the major polyunsaturated fatty
 acids containing five  and six double bonds.  Percentage relationships for
 fatty acid contents sometimes are misleading and difficult to evaluate
 quantitatively.  Basically,  each  fatty acid is relative to the total
                                  39

-------
number present and an increase or decrease in percentage may mislead the
reader.  Table 3 gives concentrations (mg) and proportions of the
individual fatty acids in three-gram samples of the Oregon moist pellet
and Abernathy dry pellet diets.  The information in these tables indicates
the amount of lipid available to the fish following consumption.

      The juvenile chinook salmon released from the hatchery frequently
remained in freshwater near the hatchery for seven to ten days before
migrating downstream into the estuary.  Tables 4 and 5 give the
relative percentages of the individual fatty acids for salmon taken
directly from the hatchery and for salmon that were retained in freshwater
and fed Abernathy pellets.  Major changes during this time included a
10-mm increase in length, and a 33 per cent increase in weight from an
average of 3.8 g to 5.7 g.  The total lipid percentage for the salmon fed
Abernathy pellets was 3.6 per cent, while those from the hatchery was only
2.6 per cent.  Major changes in fatty acids included an approximately
two-fold increase in the amount of 18:2 and a major reduction in the amount
of polyunsaturated fatty acids for the salmon fed Abernathy pellets.
Concentrations  (mg) of saturated and monounsaturated fatty acids increased
in hatchery -reared fish and in hatchery fish fed Abernathy pellets in
freshwater.  Linoleic acid increased from 13 mg to 51 mg, while at the same
time, concentrations of eicosapentaenoic and docosahexaenoic acids remained
approximately the same.  Tables 6 and 7 give the comparisons for the weight
relationships of the major fatty acids present.
       Chinook salmon migrating through the Duwamish River into the      ^
 Waterway, near the entrance of Elliott Bay, showed significant changes  in
 the percentages of specific fatty acids.  Linoleic acid accounted for 6.0
 per cent of the total  fatty acids present.  This constituted a marked reduc-
 tion relative to hatchery fish which contained 14.6 per cent linoleic acid,
 and hatchery fish fed  Abernathy pellets which contained 29.7 per cent.
 Tables 8 and 9 present the percentage and weight relationships for
 juvenile chinook salmon captured in the West waterway of the Duwamish
 River, where the proportion of docosahexaenoic acid increased 26 per cent.
 However, the concentration (mg) of this acid  (17.7 mg) was very similar to
 the concentrations found in chinook salmon reared on Oregon moist pellet
 and residing in the environments studied.  Juvenile salmon migrating into
 the West Waterway were captured and retained on the research vessel Kumtuks
 and fed Abernathy pellets. These fish showed a reversal in the original
 fatty acid pattern found in salmon cultured in freshwater, namely,  a sharp
 increase in the percentage of linoleic acid and a decrease in polyunsaturated
 compounds.  Tables 10 and 11  give the relative percentage and the
 weight relationships for the  fatty acids obtained from these fish while
 on the R. V. Kumtuks.

       Hematology.  Soos Creek Hatchery salmon were  smaller  in  size, and gave
 lower readings for packed cell volume, hemoglobin,  and total dissolved  solids
 than salmon cultured on Abernathy diet in  either freshwater or sea  water.
 Following release of the hatchery salmon into the Green River,  and  upon their
 stibsequent capture and sampling in the estuary,  there were  further  declines

-------
       Table 3.  Fatty acid contents of Oregon moist and Abernathy dry pellet
                 diets expressed in per cent and mg weight
Fatty Acid
Fatty Acid
8:
10:0
12:0
14:0
14:1
15:0
15:1
16:0
16:1
16:2
17:1
18:0
18:1
18:2
18:3
18:4
20:1
20:2
20:3
20:4
20:5
22:1
22:2
22:3
22:4
22:5
22:6
24:1
Content in
Oregon moist
pellet,*
%
0.00
0.03
0.06
3.35
0.21
0.34
0.02
16.31
4.48
0.49
0.34
3.23
18.28
20.69
0.12
2.50
7.21
0.07
0.01
0.39
13.79
0.15
0.05
0.06
0.07
0.35
7.12
0.09
Content in
Oregon moist
pellet,*
mg
0.00
0.06
0.13
7.82
0.59
0.79
0.05
38.04
10.45
1.13
0.79
7.54
42.64
48.27
0.27
5.84
15.83
0.17
0.01
0.90
32.16
0.34
0.11
0.14
0.15
0.83
16.60
0.22
Content in
Abernathy
dry pellet,*
%
0.00
0.00
0.12
1.46
0.09
0.15
0.01
12.45
2.13
0.19
0.09
3.20
19.18
40.78
6.90
3.82
0.01
0.02
0.02
0.08
6.21
0.04
0.09
0.02
0.01
0.14
2.58
0.13
Content in
Abernathy
dry pellet,*
mg
C.OO
0.00
0.32
3.84
0.25
0.40
0.02
32.70
5.60
0.49
0.23
8.40
50.40
107.16
18.14
10.04
0.08
0.05
0.06
0.21
16.31
0.10
0.24
0.64
0.24
0.38
6.78
0.35
*Based upon a 3-g sample of test diet.
                                      41

-------
Table 4.  Proportions of the 28 fatty acids found in Soos Creek
          Hatchery juvenile chinook salmon fed Oregon moist pellet
Average percentage contribution of individual fatty acids
Number
of
carbons
8
10
12
14
15
16
17
18
20
22
24
Number of double bonds
0123456
0
0
0.05
2.16 0.09
0.28 0.02
18.20 3.53 0.64
0.20
5.09 18.64 14.63 0.21 1.50
3.88 0.26 0.14 1.10 6.27
0.09 0.10 0.12 0.38 0.57 21.16
0.29
                   Average  weight =  3.80 g
                   Average  length = 71.6  mm
                   Average  lipid  =  2.6  %
                              42

-------
Table 5.   Percentage fatty acid composition of juvenile, hatchery-
           reared Chinook salmon retained in freshwater and fed
           Abernathy pellets.
Average
Number
of
carbons
8
10
12
14
15
16
17
18
20
22
24
percentage

0
0
0
0.07
1.85
0.23
15.71

contribution of individual fatty acids
Number of double bonds
123456



0.09
0.20
2.78 0.41
0.18
4.67 19.12 29.72 0.63 3.93



3.73 0.45 0.54 0.74 3.03
0.70 0.06 0.07 0.19 0.80 9.77
0.42

-------
Table 6.  Concentrations (in mg) of 28 fatty acids found in juvenile
          chinook salmon cultured at Soos Creek Hatchery on Oregon
          moist pellets
Average weight (mg) of individual fatty acids
Number
of
carbons
8
10
12
14
15
16
17
18
20
22
24
Number of double bonds
0123456
0
0
0.04
1.86 0.08
0.20 0.02
14.80 3.09 0.52
0.18
4.11 15.88 12.98 0.16 1.35
3.45 0.23 0.11 0.85 5.37
0.08 0.09 0.12 0.34 0.52 16.78
0.23
                  Average weight =  3.80 g
                  Average length = 71.6 mm
                  Average lipid  =  2.6 %
                             44

-------
Table 7.   Concentrations  (in mg) of 28 fatty  acids found in juvenile
           chinook salmon  retained  in freshwater and fed Abernathy
           dry pellet  diet
Average concentrations (in mg) of individual fatty acids
Number
of
carbons
8
10
12
14
15
16
17
18
20
22
24
Number of double bonds
0123456
0
0
0.13
3.24 0.16
0.40 0.04
27.35 4.90 0.72
0.31
8.04 33.09 51.46 1.07 6.82
6.48 0.79 0.90 1.27 5.15
1.20 0.10 0.12 0.34 1.28 16.47
0.68
                   Average weight =  5.68 g
                   Average length =81.8  mm
                   Average lipid  =  3.6  %

-------
Table 8.   Percentage fatty acid composition of 28 fatty acids found
           in juvenile chinook salmon that had been released from
           Soos Creek Hatchery and captured in the West Waterway of
           the Green-Duwamish river estuary
Average
Number
of
carbons
8
10
12
14
15
16
17
18
20
22
24
percentage

0
0
0
0.23
1.58 0
0.28 0
16.24 4
0
5.99 21
2
0
0
contribution of individual fatty acids
Number of double bonds
123456



.16
.06
.13 0.82 0.50
.31
.54 5.97 0.37 0.50
.57 0.24 0.48 2.50 5.96
.10 0.12 0.23 0.38 1.32 26.37
.57
                   Average weight =  3.58 g
                   Average length = 72.2 mm
                   Average lipid  =  2.3  %
                              46

-------
Table 9.   Fatty acid concentrations of juvenile chinook salmon that
           were released from Soos Creek Hatchery and captured in the
           West Waterway of the Green-Duwamish river estuary
Average weight (rag) of individual fatty acids
Number
of
carbons
8
10
12
14
15
16
17
18
20
22
24
Number of double bonds
0123456
0
0
0.15
1.27 0.14
0.21 0.04
11.29 3.32 0.59
0.39
4.07 15.43 4.50 0.29 0.40
2.31 0.18 0.43 1.64 4.40
0.09 0.09 0.17 0.21 0.97 17.72
0.47
                    Average weight =  3.58 g
                    Average length =72.2  mm
                    Average lipid  =  2.3  %
                               47

-------
Table 10. Percentage composition of 28 fatty acids of juvenile
          chinook salmon that had been released from Soos Creek
          Hatchery, captured in the estuary, and retained and
          cultured aboard the R.V. Kumtuks on Abernathy pellets

Average percentage contribution of individual fatty acids
Number
of
carbons
8
10
12
14
15
16
17
18
20
22
24
Number of double bonds
0123456
0
0
0.07
1.56 0.06
0.19 0.03
16.35 2.29 0.45
0.14
5.57 17.70 20.42 0.50 2.21
2.19 0.35 0.53 1.39 5.35
0.07 0.13 0.15 0.21 0.33 20.50
0.34
                   Average weight  =   6.52  g
                   Average length  =76.4   mm
                   Average lipid   =   2.2   %
                             48

-------
Table 11.  Concentrations of 28 fatty acids of juvenile chinook
           salmon that had been released from Soos Creek Hatchery,
           captured in the estuary, and retained and cultured
           aboard the R.V. Kumtuks on Abernathy pellets
Average weight (mg) of individual fatty acids
Number
of
carbons
8
10
12
14
15
16
17
18
20
22
24
Number of double bonds
0123456
0
0
0.08
2.25 0.10
0.27 0.03
20.40 3.29 0.57
0.18
6.55 23.48 28.48 0.69 3.13
4.63 0.49 0.71 1.52 7.06
0.08 0.09 0.15 0.21 0.38 19.45
0.31
                             49

-------
in blood constituents.  Maintenance of these salmon, captured in the Green-
Duwamish estuary, on an Abernathy dry pellet diet for 12 days restored the
PCV 22 per cent (Table 12).


Discussion

      The interaction between the feeding adjustments and behavioral adjust-
ments of juvenile salmon that encounter an alien environment after having
been released from a hatchery is not known.  During the time spent in the
hatchery the young salmon were fed periodically and showed behavioral
patterns similar to the feeding schedule.  Upon their release into the river,
active predation was a necessity.  Stomach contents taken from the migrating
juvenile Chinook salmon included items usually ingested by salmon, but in
addition, some nondigestible items such as pieces of wood and fir
needles.  This could possibly suggest a lack of adequate food items or
changes in the feeding behavior of the migrating fish.

      Tables 13 and 14 give the average per cent contribution of major
fatty acids and the weight values for six major fatty acids of the
diets and feeding environments utilized in this study.  The six fatty
acids listed in the table often account for as much as 80 per cent of
the total fatty acids present in the diet and in the young salmon.

      Studies by Mead et al. (1960) on the biogenesis of polyunsaturated acids
in Tilapia mossambica employed radioactive tracers.  Fish were found to syn-
thesize large amounts of saturated and monounsaturated fatty acids.  However,
polyunsaturated fatty acids showed the least activity and there was question
as to whether or not the essential polyunsaturated fatty acids could be
metabolized in sufficient concentration by the fish.  Thus, eicosapentaenoic
and docosahexaenoic acids, which frequently accounted for as much as 40 per
cent of the total fatty acids in fish inhabiting the estuary, need to be
supplied in the diet.  These two compounds were present in both diets,
with the Oregon moist pellets containing approximately twice the amount
contained in the Abernathy diet.  The weight in milligrams for these two
fatty acids showed a similar relationship for the two diets.  Bioenergetic
studies on the swimming performance of salmon by Krueger et al. (1968)
found that juvenile coho salmon swimming over twenty-four-hr periods
preferentially utilized palmitic and oleic acids for energy.  These results
indicated preferential use of these compounds.  Essential polyunsaturated
fatty acids are conserved and utilized for cell structure requirements in
the growing salmon (Gruger, 1964).

      Different species of wild juvenile salmon taken from streams in Western
Washington have been found to contain between one and two per cent linoleic
acid (Saddler and Koski, in preparation).  It is not known what effect the large
percentages of linoleic acid retained by the juvenile chinook salmon fed
artificial diets may have on their survival, growth, general nutrition, and
migration following their release from the hatchery.  In approximately seven
to ten days, the time estimated for migration from the hatchery into the
West Waterway of the Duwamish River, the percentage of linoleic acid
                                 50

-------
Table 12.  Hematological characteristics of juvenile Chinook salmon cultured on different diets
           and residing in different environments
Group
Soos Creek Chinook Salmon*
at hatchery (May 19, 1969 )*
University of Washington
Salmon Hatchery, Chinook
Salmon (June 4, 1969)**
Soos Creek Chinook Cultured
R/V Kumtuks on Abernathy
pellet diet (June 4, 1969)
Soos Creek Chinook captured
by tovmetting in Elliott
Bay (June 1, 1969)
Soos Creek Chinook captured
by townetting on June 4 and
fed Abernathy diet for 12
days (June 12, 1969)
Fork
length ,
cm
7.40
±0.85
8.04
±0.13

9.63
±0.25

7.25


7.56
±0.20


Wet
weight ,
g
4.44
±0.12
5.49
±0.32

9.38
±0.90

3.75


3.86
±0.48


PCV,
%
33.27
±0.59
39.13
±1.35

39.7
±1.19

25.00


32.00



HB,
g-%
6.1846
±0.18
6.93
±0.30

8.56
±0.31

	


9.03
±0.77


MCHC , TDS ,
% %
20.48 5.11
±0.16
17.74 6.17
±0.90 ±0.15

21.60 4.93
±0.72 ±0.10

4.60


28.21 4.4



    ^Cultured on Oregon moist pellet.

   **Cultured on Abernathy dry pellets.

-------
en
10
           Table 13.   Percentage composition of the six major fatty acids in juvenile Chinook salmon
                       cultured on different diets and residing in different environments
Diets and environments
Estuary-captured
Hatchery, Diet I*
Estuary-retained, Diet II**
Diet I*
Freshwater-retained, Diet II**
Diet II**
16:0,
%
16.24
18.20
16.35
16.31
15.71
12.t5
18:1,
%
21.54
18.64
17.70
18.28
19.12
19.18
18:2,
%
5.97
14.63
20.42
20.69
29.72
40.78
20:4,
%
2.50
1.10
1.39
0.39
0.74
0.08
20:5,
%
2.96
6.27
5.35
13.79
3.03
6.21
22:6,
%
26.37
21.16
20.50
7.12
9.77
2.58
           *Diet I - Oregon moist pellets,

          **Diet II - Abernathy diet.

-------
tn
CO
           Table 14.   concentrations (in mg) of the six major fatty acids in  juvenile  chinook  salmon
                       cultured on different diets and residing in different environments

Diets and environments
Estuary-captured
Hatchery, Diet I*
Estuary-retained, Diet II**
Diet I*
Freshwater-retained, Diet II***
Diet II**
16:0,
mg
11.29
14.80
20.40
28.04
27.35
32.70
18:1,
mg
15.43
15.88
23.48
42.64
33.09
50.40
18:2,
mg
4.50
12.98
28.48
48.27
51.46
107,16
20:4,
mg
1.64
0.84
1.52
0.90
1.27
0.21
20:5,
mg
4.40
5.37
7.06
32.16
5.15
16.31
22:6,
mg
17.72
16.78
19.45
16.60
16.47
6.78
             *0regon moist  pellet.

            **Abemathy dry pellet.

-------
decreased from 20.4 to 6.0 per cent of the total.  In contrast, salmon fed
the Abernathy diet possessed the greatest amount of linoleic acid (29.7
per cent).  Metabolically, fish cannot convert linoleic acid into the
longer-chained polyunsaturated fatty acids, 20:5 and 22:6.  Compounds
related to linoleic acid, namely 20:2, 20:3, and 20:4, do not increase
or change in the same proportion as the dramatic percentage increase of 18:2
in the fish studied.  It is possible that migrating salmon utilize linoleic
acid for energy during migration.  Saddler and Cardwell (in preparation).
found that linoleic acid was the most extensively utilized fatty acid in
juvenile pink salmon (Qncorhynchus gorbuscha) that had been captured in
sea water near Neah Bay, Washington, tagged with the Dennison internal
anchor tag (Dell, 1968), and held for 33 days.  However, the fate of 18:2
in the metabolic pattern of the young salmon cannot be concluded from
these studies.

      Soos Creek salmon were suffering from a low-grade infection of
bacterial gill disease (Myxobacteria spp.), which probably accounted for
the anemia that prevailed at the time of their release.  During the
juvenile salmon's 25-km migration to the estuary, there was further
hematological deterioration.  This condition could have resulted
collectively from the bacterial infection, the limited quantity of food
available in the river and estuary, and reduced food consumption by the
salmon as they changed to an actively foraging type of behavior.  An
inadequate food supply is known to produce erythropoietic depression
(Zanjani et al., 1969) and plasma protein diminution (Lysak and
Wojcik, I960).  Decreased numbers of red blood cells would contribute
substantially to deterioration of the salmon's physiological condition by
limiting the oxygen supply to the tissues.  This in turn would reduce the
fish's stamina for burst and sustained swimming activity, implicating that
these juvenile fish would be limited in their capabilities to escape
predators and capture food organisms relative to juvenile salmon possessing
a healthy hemogram.
                               54

-------
                            SECTION VI
        NATURAL STRESS DURING SPAWNING MIGRATION OF ADULTS


      The migration problems of outmigrant juveniles and  inmigrant  adult
salmon differ in several respects.   The juveniles descend into sea water
with considerable speed because they combine the river velocity with that
of their own swimming.  Even if lost, delayed or hindered in their journey,
as long as they are not killed outright enroute, they are likely to end
up in the estuary, having been minimally influenced by environmental degra-
dation along the way.  Adults on the other hand may enter several estuaries,
and once committed to a particular one, stay in the lower reaches of the
river mouth for several weeks.  Our impression was that they would venture
as far into a degraded environment as their physiological capabilities
would allow, being subjected to prolonged periods of exposure to low DO,
toxicants, and whatever else mankind had to offer them.  The waiting
problem was further complicated by their cessation of feeding - any serious
delay or excessive use of stored energy could cause failure to reach the
spawning grounds or cause the eggs to be of such poor quality as to effec-
tively produce the same result.

      This section of the report assesses the biological  significance of
several estuarine problems encountered by adult salmon in the perspective
of the fact that spawning Pacific salmon eventually deteriorate and die
after spawning anyway.


   Normal Hematological Variations During the Spawning Migration
                         of  Chinook  Salmon

      Concurrent with studies on the effects of low environmental dissolved
oxygen on the physiology of  chinook  and coho salmon, an  investigation was
undertaken to evaluate the normal hematological variations  in chinook
salmon following their arrival  in the Duwamish-Green River  estuary and  sub-
sequent  25-km spawning migration to  Soos Creek  Hatchery,  near Auburn, Wash-
ington.  Additional observations of  blood constituents were made on sub-
adult coho and chinook salmon and on adult  sockeye salmon (0_. nerka)
captured at the entrance to  the Strait of Juan  de Fuca.   The basic problem
was  to distinguish between the  effects of low DO  or  increased residence
time in  the estuary  from the physiological  change which  normally
accompanies the salmon's undisturbed spawning migration.

       The hematological  variables  examined  for  all groups of fish  were
 PCV (hematocrit),  TDS (total dissolved solids), and TPP  (total  plasma
 protein).   Five plasma protein fractions,   presumably  fibrinogen,
       Fractions tentatively identified in salmon plasma using differential
 precipitation with sodium sulfate.
                                  55

-------
beta globulins, alpha globulins, albumin, and pre-albumin, were examined
in sexually nature chinook salmon using electrophoresis and cellulose
acetate support media.

      Blood samples collected from estuarine adult male and female
chinook salmon revealed a sexually homogeneous and above-normal hemogram.
The prespawning salmon in the estuary and at the hatchery were notably
polycythemic relative to subadult salmon entering the Strait of Juan de
Fuca (Tables 15 and 16).  Evidently there was increased erythrocyte
production during the final stages of maturation.  The above-average
red blood cell content (polycythemia) and the elevated plasma protein
content of the adult sockeye sampled in the ocean was a reflection of the
advanced maturity of these fish.  After spawning, however, there was a
rapid decline in hematocrit, which was more acute in female than male fish.
Mature female salmon, in general possess fewer erythrocytes than males
during and after upstream migration.  Apparently the functional deteriora-
tion so characteristic of sexually mature salmon does not include the
erythropoietic mechanisms until, or after, spawning.  It is unlikely that
the number of erythrocytes is the physiological factor limiting the ad-
justments of these salmon to hypoxia.  Total dissolved solids and total
plasma protein related well to changes in hematocrit.  Commonly these
two variables are significantly correlated (P = 0.05).  The plasma
protein values in estuarine chinook and prespawning male chinook were
higher than readings obtained from sub-adult and juvenile saloon, and
indicate a condition of hyperproteinemia.  The diminution in protein con-
centration in the terminal stages of the salmon's life cycle is caused
directly by starvation, although an increase in the volume of the extra-
vascular space after the fish had entered freshwater would also have initially
increased the progressive reduction in protein.  The declines were more
marked in female fish, presumably because ovarian development required
more energy and proteinaceous materials than the developing germinal
epithelium of males (Tables 15 and 16).  Plasma proteins and the ability
of the blood to attract metabolites from the tissues (oncotic pressure)
are interrelated, with reductions in TPP contributing to the tissue edema
characteristic of salmon migrating upstream.

      The plasma protein composition of upstream migrating salmon deviated
from patterns commonly obtained from immature fish.  There was a decrease
in alhtimtn and concurrently increased globulins, notably the alpha fraction
and one of the least electrophoretically mobile beta globulins (Fig. 21).
Relative to TPP, all plasma protein fractions were significantly reduced.
Some of the beta globulins (the beta-2 fraction) possess antibodies
                                  56

-------
en
          Table 15*   Hematological characteristics of sub-adult  chinook and  coho  salmon, and adult
                      sockeye salmon captured at  the  entrance  to  the  Strait of Juan  de Fuca.  Values
                      are given  as  means ±  standard errors.
Group
Adult sockeye
Adult sockeye
Sub-adult coho

Sub -adult coho

Adult chinook

Fork
length ,
Location Sex n cm
Ocean1 Male 4 61.38
±0.78
Ocean Female 3 59.70
±2.14
Ocean Male 3 51.80
±1.91
Ocean Female 2 59.10
±3.11
Ocean Male 2 46.70
±4.77
Weight ,
g
2,573.75
± 283.97
2,657.00
± 470.89
1,571.75
± 149.73
2,201.00
± 154.86
1,332.50
± 369.47
PCV,
%
54.50
±2.11
53.17
±0.55
46.25
±3.70
60.75
±0.53
41.75
±3.71
TDS,
0,
•0
9.96
±0.32
11.73
±1.02
5.88
±0.59
8.73
±0.30
5.25
±0.32
TPP,
%
6.94
±0.15
8.82
±1.79
4.64
±0.73
6.43
±0.34
3.58
±0.04
                 Entrance of Strait of Juan de Fuca.

-------
in
oo
            Table 16.  Hematological variations in adult Green-Duwamish River chinook salmon at
                       various stages of maturation.   Values are given as means +_ standard errors,
Sample
group
Estuarine
adult1
Uns pawned
adult male
Unspawned
adult female
Spawned-out
adult male
Spawned-out
adult female
Location
Duwamish
Waterway
Soos Creek
Hatchery
Soos Creek
Hatchery
Soos Creek
Hatchery
Soos Creek
Hatchery
Fork
length ,
n cm
5 73.40
±5.12
5 84.00
±4.80
5 83.90
±1.95
5 80.42
±5.42
4 81.25
±1.45
Wet
weight ,
gm
5,830.00
±1,107.90
7,360.00
±1,263.10
7,646.00
± 759.20
6,358.30
±1,020.10
6,455.00
± 960.40
PCV,
%
—
51.26
±4.82
44.76
±2.59
38.00
±6.86
38.68
±5.32
IDS,
%
7.64
±0.37
6.72
±0.60
5.86
±0.40
4.14
±0.35
2.98
±0.53
TPP,
gm-%
9.48
±0.69
5.24
±0.70
4.20
±0.35
3.25
±0.39
2.37
±0.20
                  ^Sample group sexually intermixed.

-------
       Ill
                                                                      III
   t.
                                                                 Ill
p.   21   Differences in the electix>phoretic composition of the plasma proteins
          in Chinook salmon at various stages of their life cycle:  juvenile (A),
          estuarine adult male (B), adult female with severe myxobacterial infec-
          tion and hypoproteinernia (C), pre-spawning adult male (D), and post-
          spavminp; adult male (E).  Protein fractions corresponding to those
          discussed in text include beta globulins (I), alpha globulins (II), and
          albumin (III).
                                   59

-------
similar to mammalian gamma globulins.  Although increased relative to
the albumins, the depressed total globulin concentrations in spawning
salmon  imply  a diminished resistance to stress and infection.  Albumin
declines are characteristic of many pathologies and protein malnutrition,
and presumedly these small molecular weight proteins function indirectly
in gluconeogenesis and furnish amino acid substrates for wound repair.
                                 60

-------
            Renal Function in Migrating Adult Coho Salmon


Introduction

      The present study was undertaken to try to define the normal changes
in kidney function that occur when an adult coho salmon enters fresh-
water on the spawning migration.  These normal changes can then be
compared, in later work, with the changes observed in salmon migrating
through polluted estuaries.  These basic data are necessary for the
future evaluation of the effects of water quality upon physiological
functions of fish.  A secondary purpose of the work was to evaluate
the use of  p-aminohippuric acid (PAH) in the measurement of renal
plasma flow of the coho salmon.


Methods and Materials
      Location and Collection of Animals.  Coho salmon, Oncorhynchus
kisutch, captured while migrating into Big Beef Creek, Kitsap County
(Washington), were used in the study.  An upstream trap, a facility of
the Big Beef Field Station of the College of Fisheries, University of
Washington, was used to capture the migrating adult fish.  These fish
were typically about 65-70 cm in length and 3.0-3.3 kg in weight.  The
fish were removed from the trap in the tidal estuary and transported by
boat to the R. V. Kumtuks, anchored in Seabeck Bay. They were then placed
in a 5 x 5 x 3 meter holding pen in sea water for at least one week prior
to experimentation.  Fish used only in freshwater were in freshwater for
at least one week prior to experimentation.

      Preparation of an Experimental Animal.  The fish was netted from the
holding pen and placed directly into sea water containing the anesthetic
MS-222.  When it reached plane III anesthesia C"No respiratory activity.
The fish may be easily revived by removing it to untreated water."
(Klontz, 1964)], it was placed on an operating table (Smith and Bell,
1967) where the gills were continuously irrigated with salt water or
freshwater, as appropriate, which contained MS-222.  A catheter, made from
PE 160 (polyethylene, I.D. = 1.1U mm)  tubing (Clay-Adams) which was
shaped by gentle heating, was inserted into the bladder.  This catheter was
anchored with three stitches through the base of the anal fin; an additional
stitch was placed on the dorsal midline posterior to the dorsal fin.

      Two holes were then made in the snout of the fish using a large bore
hypodermic-needle.   Each hole was lined with a short piece of PE 200
tubing (I.D. = mo mm),.that was heat flared at one end and inserted from
the inside of the buccal cavity to resist being pulled out.  A long (1.5
meter) piece of PE 60 (I.D. = 0.76 mm) tubing (heat flared at one end) was
pulled through one liner from the inside until flush with the palate.  At
this time, a stitch of braided silk suture was placed through the skin of
the palate on the midline just anterior to the gill arches (Smith and
Bell, 1967).
                                 61

-------
      A 16 gauge cannulating syringe  (Aloe Co.) filled with Cortland saline
(Wolf, 1963) was inserted into the dorsal aorta.  The syringe and needle
were withdrawn leaving the short plastic sleeve into which a PE 60 tube
filled with Cortland saline was quickly inserted to minimize blood loss.
The tube was then secured to the palate with  the silk suture previously
placed there and pushed through the other snout liner.  On the outside of
the snout, a tie was placed around each of the liners and pulled tight enough
to secure the buccal and dorsal aorta cannulae without obstructing them.
A restraining "tether" was tied through the epaxial musculature just
anterior to the dorsal fin.  The length of time the fish were on the
operating table varied from 8 to 12 minutes.

      After preparation, the animal was brought to plane II anesthesia
(opercular and fin movement) and placed in an exercise chamber (Smith
and Newcomb, 1970) to recover.  The dorsal aorta cannula  (filled with
Cortland saline), and the buccal cannula were connected to differential
pressure transducers  (Sanborn Model 267B) which were attached to a Brush
amplifier and recorder system.  Heart rate, blood pressure, and breathing
rate were recorded.  Urine  from the catheter  was collected in a fraction
collector (Buchler) at intervals dependent on the urine flow rate.

      Insulin and  PAH.    C Insulin and H PAH  (p-aminohippuric acid) were
injected as a single dose  (Table 1).   The  H  PAH was mixed with unlabeled
PAH to  reduce the specific  activity.

      Collection  and  Processing of Blood  Samples.  Blood was withdrawn
from the dorsal aorta cannula at irregular intervals with a 1 ml syringe.
After filling the syringe,  2 microhematocrit  tubes were filled directly
from the cannula.  One milliliter  of  Cortland saline  (Wolfe, 1963) was then
injected through  the  cannula to replace the sample volume and maintain blood
volume. The microhematocrit tubes were then  centrifuged at 12,000 RPM
(r = 9  cm)  for five minutes.  After centrifugation, the hematocrit (packed
cell volume) was  determined and  the plasma  supernatant was taken for
measurement of per cent  total plasma  solids.  The per cent of total solids
was measured with a hand-held refractometer (Bausch and Lombe "TS Meter").

      The 1 ml sample was  then  centrifuged at 12,000  (r = 9 cm) for five
minutes and the plasma decanted.   A 200 yliter  aliquot was dissolved
in 15 ml of "cocktail"  in  a standard  scintillation vial.  The "cocktail"
used was made by  dissolving *K69  g POP (2,  5-Diphenyloxazole),0.469 g
POPOP  (1, «*-bis-[2-(4-Methyl -5-Phenyloxayolyl)D), and 62.5 ml "Biosolve"
(Beckman BBS-3)  in reagent grade  toluene  and making up to one liter.  A
100 pliter  aliquot of plasma was  put  dropwise while stirring on a vortex
mixer into  0.900  ml of a solution  containing  5% (w/v) trichloroacetic
acid, 5%  (w/v)  HC1, and  1% (w/v)  lanthanum (Willis, 1960 and 1961).
This mixture was  centrifuged to separate the precipitated protein.  The
supernatant solution was aspirated directly into an atomic absorption
spectrophotometer (Perkin-Elmer Model 290)  for calcium analysis, and
appropriate dilutions of the supernatant solution were used for the
analysis of sodium, potassium, and magnesium by use of the same spectro-
photometer.  A 50 uliter aliquot of plasma was  used for determination
                                  62

-------
of chloride ion by use of a potentimetric titrater (Buchler-Cotlove
Chloridimeter).

      Plasma  Ultrafiltration.   Dialysis tubing was soaked in distilled water
until soft and then  folded to  make a U-shaped tube.  An aliquot of plasma
was placed in the tube and the tops of the  tube tied together with heavy
thread (Toribara  et  al., 1957).  This bag,  containing the plasma, was then
placed in a screw-top centrifuge tube; the  top of this tube was screwed
down over the trailing threads, thus suspending the bag in the tube.  The
tube was then centrifuged  for 12 hours at 2,000 RPM (r = 13 cm).  At the
end of that time, the filtrate was removed from the bottom of the centrifuge
tube and frozen  for later  analysis.

      Collection and Processing of Urine Samples.  Urine samples were
collected by  an  automatic  fraction collector at intervals determined by^
the urine flow rate and collecting tube size. The volume of the collection,
if less than  5 ml was measured with a pipette of appropriate size.  If the
sample was larger than 5 ml, it was measured with a 10 ml graduated
cylinder.  A  2 ml aliquot was frozen for later analysis.  The same set of
analyses was  done on the urine samples; similar methods  (omitting the
protein precipitation step) as used  in the plasma analysis described
previously were employed.

      Analysis of Urinary Precipitate.  After aliquots were removed  for ion
analysis, five urine samples  containing precipitate were combined in a  15
ml centrifuge tube  and  centrifuged for 30 minutes  at  3,600 RPM  (r =  13  cm).
The  supernatant was decanted,  the precipitate was  washed and centrifuged
two  additional times and dried at 105% CC.   A sample  of  the  precipitate
was  pressed  into a  potassium  bromide block and its infrared spectrum
analyzed  with a  Perkin-Elmer  Infrared Spectrophotometer.

       Calculations. The catheter dead space was corrected^by the method
of Hickman (l968a). The symbols in Table  17 will be used in the equations
 for the calculation of  the renal parameters.


 Results

       The results  from  this study are extensive and will^be published in
 full by H. M. Miles in  Comparative Biochemistry and Physiology sometime
 in 1971. That  which follows is excerpted from his Ph.D. thesis and
 includes the most significant results.  Individual fish are designated
 by letters and  data from each fish is presented in tables in the
 appendix.

       Urine Flow Rate.  After the fish were transferred to  freshwater,
  the urine rate exhibits a five- to tenfold  increase  over the _ salt water
  urine rate with a  highly variable freshwater urine rate. This high
  variability persisted  even after considerable time  in freshwater.   In
  fish 0 the  injection of 1 rag adrenaline at  3000 minutes after the
                                   63

-------
Table 17. Symbols used in the equations for the calculation of the renal

          paraneters (Koch, 1965)
Parameter
Urine flow rate
Glomerular filtration rate
Renal plasma flow rate
Excretion rate of Cl~
Plasma concentration of Na
Filtered load of K+

Proportion protein bound ion
Symbol
V
u
V
g
V
P
QC1
[Na]p
•
Kg
k
Units
ml/(kg x hr)
ml/ (kg x hr)
ml/(kg x hr)
liequiv/(kg x hr)
mequiv/1
yequiv/(kg x hr)

diirensionless
           Glonerular filtration rate.
                              _   [Inulin]uxVu


                            g ~    [Inulin]
           PAH clearance.
                            PAH
[PAH]
           Filtration fraction.
 given).
                                   PAH



           Excretion rate of Na  , K  , Ca   , Hg  , and Cl  (Example Na
                              u
                                       [Ma]  x V
                                           u    u

-------
         Filtered load of Na+, K+, Ca+t, Hg+*, and Cl" (Example K*


given).
                        Qv  =  V   x _  _
                         Kg    g     P



          Total load of Na+, K*.  Ca++, Hg++,  and Cl" based on PAH clearance



(Exairple Ca+  given).


                         6     = V,, „ x [Ca3
                         ^Ca     PAH       p
                            P




          Clearance ratio of water
                             VPAH



          Clearance ratio of Na+, K*. Ca**, Mg**,  and Cl"  (Example Mg**


 given).
                           s
           Filtration ratio of water.




                               •

                               V
                               _u

                               •

                               V
                                g




           Filtration  ratio of Ma*,  K+,  Ca++ ,  Mg**, and Cl"  (Example  Cl'



  given).
                                 g
                                    65

-------
experimental period resulted in a depression of the urine rate from
around 3.6 ml/(kg x hr) to about 1.8 ml/(kg x hr) that persisted for about
600 minutes when the urine rate returned to normal, if not slightly
elevated levels.  When all samples are pooled (Table 18) the mean urine
flow in salt water is 0.406 ml/(kg x hr) while in freshwater the mean
urine flow is 1.65 ml/(kg x hr).

      Glomerular Filtration Rate.  The glomerular filtration rate (GFR)
for the pooled samples "(Table  18) taken from fish held in salt water
is 1.48 ml/(kg x hr) and in the  samples taken while the fish were in
freshwater, the mean GFR was 9.06 ml/(kg x hr).  Although this increase
was slightly less than the increase in urine flow, a correlation at the
1 per cent level (r=0.769, df=266) exists  between the GFR and the urine
rate for the pooled freshwater samples.  After the injection of 1 mg
adrenalin in fish 0, th« GFR showed a depression from about 8 ml/(kg x hr)
to about 5 ml/(kg x hr).  The  mean filtration ratio of water (Table 18),
or the ratio of the rate of filtration of  water through the glomerulus to
the rate of excretion of water in the urine, changed from 0.380 in salt
water to 0.525  in freshwater.  This indicates that about 15 per cent^more
water was reabsorbed from the  glomerular filtrate in sea water than in
freshwater.  This 15 per cent  increase does not account for all of the
observed increase in urine rate, yet the filtration ratio of water is
correlated  (r=0.564, df=266) at  the 1 per  cent level with urine rate in
the pooled  samples from fish held  in freshwater.  This shows that
reabsorption rate does play a  significant  part in the rate of urine
production.

       PAH  Clearance.  The clearance of PAH, while usually being greater
than  the GFR,  varied with and  was  correlated  (r=0.564, df=266) at the 1 per
cent  level with the GFR.   In fish  0 after  injection of 1 mg adrenaline, the
PAH clearance  also varied with the GFR.  For the pooled samples (Table 18),
PAH clearance  increased from a mean of 2.86 ml/(kg x hr) in salt water to
a mean of  13.3 ml/(kg x hr)  in freshwater.

       Magnesium Clearance.  The  magnesium  clearance, calculated from taking
the mean values in  salt water  for urine  flow rate, plasma magnesium
concentration, and urine magnesium concentration for fish H (Appendix),^
is 79.6 ml/(kg x hr).  The magnesium clearance value for fish L (Appendix),
similarly  calculated,  is 4.56  ml/(kg x hr).

       Urine Ion Concentrations.   There was considerable variation among
the ion concentrations of the  individual fish.  The most dramatic change
common to  all  of them was the  greatly decreased concentrations of magnesium
and chloride ions in the urine.   Some details  for  individual fish are pre-
sented in the  appendix tables  and  are expected to  be published in full by
Miles  in 1971.

       Urine Ion Excretion Rates.  The excretion rates of  ions are dependent
on both the urine flow and the concentration  of ions  in the urine.  It can
be seen in Appendix B and in Table 18 that the excretion rates of sodium,
                                  66

-------
Table  18. Means, standard deviations (S.D.), and sample sizes (n),  pooled for renal parameters
           in all salt water fish and in all freshwater fish
Salt water
Parameter
Flow rates (ml/(kg x hr))
Urine rate
Glorr.erular filtration rate
PAH clearance
Urine ion concentrations
(mequiv/1)
Sodium
Potassium
Calcium
Magnesium
Chloride
Plasma ion concentrations
(rcequiv/1)
Sodium
Potassium
Calcium
Magnesium
Chloride
Excretion rates
(pequiv/(kg x hr))
Sodium
Potassium
Mean

0.106
l.»*8
2.86


55.5
2.48
8.21
160
133


181
2.72
2. 45
3.35
156


16.9
0.863
S.D.

0.217
0.723
1.41


50. 0
1.33
5.34
74.6
68.5


2.30
0.134
1.43
3.33
2.24


11.3
0.370
n

19
14
14


19
19
19
19
19


16
16
16
16
16


19
19
Freshwater
Mean

4.65
9.06
13.3


12.3
1.69
2.93
10.2
13.2


157
2.53
2.38
2.01
133


68.1
7.05
S.D.

3.66
4.70
8.04


12.1
1.88
1.72
35.4
23.8


8.75
0.293
0.904
0.859
9.81


126
7.08
n

309
268
268


273
275
275
275
301


282
282
282
282
289


273
275

-------
0>
00
      Table  18.  Means, standard deviations (S.D.), and sample sizes (n), pooled for renal parameters
                 ,in all salt water fish and in all freshwater fish - Continued
Salt water
Parameter
Calcium
Magnesium
Chloride
Mean
2.85
72.4
61.0
S.D.
1.33
52.7
47. 0
n
19
19
19
Freshwater
Mean
12.2
14.1
60.7
S.D;
9.13
20.9
115
n
275
275
301
Filtered ion load
  (uequiv/(kg x hr)>

  Sodium
  Potassium
  Calcium
  Magnesium
  Chloride
227
3.62
1.41
5.29
224
112
1.81
1.37
6.53
112
14
14
14
14
14
1200
20.2
6.62
14.3
1180
631
10.3
4.02
10.7
662
262
262
262
262
268
      Ion load based
        on PAH clearance
        (pequiv/(kg  x hr))

        Sodium
        Potassium
        Calcium
        Magnesium
        Chloride

      Clearance ratios based
        on PAH clearance
        (dimensionless)

        Water
        Sodium
517
7.78
5.90
7.51
445
251
3.79
2.02
4.95
220
14
14
14
14
14
2100
33.9
30.3
25.9
1740
1380
22.4
20.1
15.7
971
262
262
262
262
268
                                        0.154
                                        0.0524
0.0191
0.0467
14
14
0.405
0.0347
0.314  258
0.0454 245

-------
      Table   18.  Means,  standard deviations  (S.D.),  and sample sizes (n),  pooled  for renal parameters

                   in all  salt water fish and  in  all freshwater  fish  - Continued
o>
to
Salt water
Parameter
Potassium
Calcium
Magnesium
Chloride
Filtration ratios
( dimensionless )
Water
Sodium
Potassium
Calcium
Magnesium
Chloride
Filtration fraction
(dimensionless)
Kematocrit (%)
Plasma total solids (%)
Blood pressure (mm Hg)
Dorsal aorta pulse
pressure (mm Hg)
Heart rate (beats/min)
Mean
0.145
0.556
17.1
0.126


0.380
0.0876
0.297
4.66
78.5
0.413
0.682
20.5
3.35
28.8
3.80
59.4
S.D.
0.0839
0.198
12.6
0.0747


0.209
0.0279
0.0852
2.90
61.6
0.309
0.565
1.02
0.710
7.09
0.519
9.. 35
n
14
14
14
14


14
14
14
14
14
14
14
16
16
15
15
19
Freshwater
Mean
0.227
0.504
1.45
0.0378


0.525
0.0501
0.337
2.23
4.96
0.0538
0.765
14.8
3.42
26.7
5.07
52.4
S.D.-
0.170
0.310
4.77
0.552


0.343
0.0583
0.200
1.24
22.2
0.0929
0.262
6.47
0.817
8.06
1.79
8.59
n
247
247
247
263


268
245
247
247
247
264
268
299
299
253
253
253

-------
potassium, and calcium actually increase when the fish enters freshwater.
The excretion rates of magnesium and chloride ions, however, decrease with
entry into freshwater.  While there are several significant correlations
among the ion excretion rates in both salt water and freshwater (Tables 19
and 20), the closest correlation in salt water is that between magnesium
and chloride and the closest correlation in freshwater is between sodium
and chloride.

      Protein Binding of Plasma Ions.  Plasma was subjected to pressure
filtration by centrifugation through dialysis membrane.  Analysis of the
filtrate from each sample so treated yielded the percentages of protein
bound ions shown in Table 21.
   Table 21.  Percentages of protein bound  sodium, potassium, calcium,
              magnesium, and chloride  in blood  plasma  from fish M
    Ion                                        Per cent  bound
    Sodium                                         15.0
    Potassium                                      11.0
    Calcium                                        69.0
    Magnesium                                      27.0
    Chloride                                        3.1
All of the calculations of  filtered ion load utilized the percentages
listed in Table 21.

      Filtered Ion Load.  The  filtered ion load is dependent on  the
glomerular filtration rate  and the concentration of unbound ions in the
blood plasma.  The means  for filtered ion load are presented in  Appendix Table
B-6.  In the pooled  freshwater sample significant correlations are found
between the per cent plasma total solids and the filtered load of sodium,
calcium, and magnesium  (Table  22).
                                  70

-------
Table 19. Correlation coefficient matrix with sample sizes of sodium, potassium, calcium, magnesium,
          and chloride excretion rates for the pooled freshwater sampler,

Sodium

Potassium

Calcium

Mangesium

Chloride

Sodixim Potassium Calcium
1.00 0.543-"* 0.653-"
(273) (273) (273)
1.00 0. 5 46 "•••'••
(275) (275)
1.00
(275)




Magnesium
0.0255
(273)
-0.1'* 3*
(275)
0.489-*
(275)
1.00
(273)


Chlori
0.968*
(272)
0.500*
(274)
0.681*
(274)
0.137*
(274)
1.00
(301)
de
,

i;

ft





     '"Correlation significant at the 5% level.

    '•'•""Correlation significant at the 1% level.

-------
Table 20. Correlation coefficient matrix (n=19) of sodium, potassium, calcium, magnesium, and
          chloride excretion rates for the pooled salt water samples
Sodium Potassium Calcium Magnesium Chloride
Sodium
Potassium
Calcium
Magnesium
Chloride
1.00 0.614** 0.320 -0.477'"' -0.480'''
1.00 0.614** 0.226 0.242
1.00 0.283 0.269
1.00 0.996'*'*
1.00
     '•'•'Correlation significant at the 5% level.

    ""'Correlation significant at the 1% level.

-------
     Table  22.   Correlation  coefficients  (r) and sample size (n) for
                correlations between plasma total solids and the
                filtered load of  sodium,  potassium, calcium, magnesium,
                and chloride
     Ion
Sodium
Potassium
Calcium
Magnesium
Chloride
0.130*
0.0775
0.218**
0.226**
0.199**
262
262
262
262
268
   Correlation significant at the 5% level.
  **Correlation significant at the 1% level.

      Ion Load and Clearance Ratios Based on PAH Clearance.  The mean
results from the calculation of ion load and clearance ratios from the
PAH clearance are presented in Appendix Table B-7 and Table 18.  The clearance
ratio is the ratio of the rate of excretion of a substance to the rate^
at which it is presented to the kidney by the renal circulation.  A priori,
this ratio could not exceed a value of 1.00.  However, the mean clearance
ratio of magnesium (Table 18) in salt water is 17.1 and in freshwater is
1.45, both ratios being greater than 1.00.

      Ion Load and Clearance Ratios Based on Magnesium Clearance.  The
mean magnesium clearance of 79.6 ml/(kg x hr) was used as an estimate of
renal plasma flow in the calculation of the total ion load and clearance
ratios presented in Table 23.  The mean plasma ion concentrations and
excretion rates from Table 18 were used in these calculations.

      Filtration Ratios.  The filtration ratio is the ratio of the rate
of excretion of a substance to its rate of filtration.  These values are
reported in the appendix tables.  If the filtration ratio  is greater than
1.0, active secretion of that substance into the tubule lumen is indicated.
A filtration ratio of less than 1.0 indicates active reabsorption from
the glomerular filtrate into the blood.  The mean filtration ratios for
the pooled freshwater and salt water samples  (Table 18) reveal that except
for calcium and magnesium, there  is a  net reabsorption of  water and ions
by the tubule.

      Filtration Fraction.  The filtration  fractions, the  ratios of the
glomerular filtration rate to the total renal plasma flow, reported in
the Appendix and Table  18 are based on the  PAH clearance as  a measure
                                  73

-------
Table 23.  Total ion load and clearance ratios based on the magnesium clearance of fish H in
           salt water and calculated from the mean plasma ion concentrations and excretion
           rates of the cooled samoles in salt water and freshwater5'
Ion
Sodium
Potassium
Calcium
Magnesium
Chloride
Salt
Total load
uequiv/(kg x hr)
14,400
217
195
267
12,400
water
No units
clearance ratio
0.00117
0.00399
0.0146
0.272
0.00491
Fres
Total load
uequiv/(kg x hr)
12,500
201
189
160
10,600
hwater
No units
clearance ratio
0.00545
0.0350
0.0644
0.0381
0.00573
  -Figures  reliable to 3 significant figures only.

-------
 of total renal  plasma flow.   If these values are calculated using the
 high renal  plasma  flow indicated by  the magnesium clearance of fish
 H  (79.6 ml/(kg  x hr)) and the mean values  for GFR for the pooled salt
 water   ^ and  freshwater samples  (Table 18), filtration fractions of
 0.0186 in salt  water  and 0.114 in freshwater are obtained.

      Blood Parameters.   In  general, the hematocrit and per cent plasma
 total solids decreased with  introduction to freshwater, the blood pressure
 remained the same  or  decreased slightly, and the heart rate remained
 relatively  constant.   The dorsal aortic pulse pressure increased.  This
 indicated the occurrence of  a dilation of  the branchial capillary beds
 with introduction  into freshwater.

      Urinary Precipitate.   The infrared spectrum for the purified materi-
 al obtained from this precipitate was comparable with that for components
 of human urinary calculi. The precipitate in the fish urine appears to
 consist chiefly of the mineral brushite CaHPO   • 3H 0.


 Discussion

      These observations have demonstrated some of the changes in the
 renal function  of  salmon when they enter freshwater during the final
 portion of  their spawning migration.  In general, in the salt water envi-
 ronment , the salmon kidney has an osmoregulatory function involving
 excretion of magnesium and sulfate ions (Hickman, 1968c_) and conserva-
 tion of water.  In freshwater, the kidney  excretes large amounts of
 water while conserving salts.

      Maintenance  of  Internal Homeostasis.  When fish enter the freshwater
 environment  from the  salt-water environment, there is a decrease in the
 concentrations  of  the  plasma  ions.   Green  (1904) observed that the
 freezing point  depression was greater in the blood from the Chinook
 salmon (Oncorhynchus  tschawytscha) taken in salt water (-0.762° C) than
the mean of  those  taken  from  the salmon taken in freshwater (-0.613° C).
This was probably  the  first published observation on the osmoregulatory
physiology of the  genus  of Pacific salmon  (Oncorhynchus).  Since that
time there have been no  detailed observations comparing the composition
of the plasma of adult salmonids in both salt water and freshwater.  In
the present work, there  was observed a definite decrease in plasma ion
concentration, except  in the case of potassium and calcium.  There seem
to be two homeostatic levels  for ion concentrations, one in salt water
and one in freshwater, and both are equally functional.

      The excretion of magnesium, which is one of the major roles of the
kidney in salt water,  is  an extremely important function.  It has been
found (Engbaek,  1952;  Del Castillo and Engbaek, 1954) that magnesium in
concentrations greater than 10 mequiv/1 will produce a neuromuscular
                                 75

-------
block.  The kidney, the primary excretory site for magnesium, excretes
most of this cation that is absorbed by the intestine (Hickman, 1968c).
It thus plays a critical role.                                      ~~

      Precipitation of magnesium and calcium salts in the tubular lumen
is an efficient mode of excretion.  After crystalization, these salts
would therefore no longer participate in the osmotic gradient from the
peri tubular blood to the lumen of the tubule.

      Other reports of crystalline material are by Pitts (1934), and by
     Lin and Ennis (1934) who reported MgHPO  • 3H 0 in marine fish
urine.  Hickman (1962b) reports CaHPO  in the urine of the southern
Grafflin and Ennis (1934) who reported MgHPO  •  3H 0 in marine fish
urine.  Hickman (1962b_) reports CaHPO^ in the urine of the southern
flounder.  The precipitate found in the present work on adult coho salmon
was CaHPO  •  3H.O.

      In his  work with southern flounder in salt water, Hickman (1968b)
found that this fish actively secreted magnesium and calcium into the""
tubule and actively reabsorbed sodium, potassium, and chloride.  The
same relations were found in the present work.
                                 76

-------
                            SECTION VII


            EFFECTS OF MAN-HADE STRESSES ON ADULT SALMON


            Stress During the Migration of Adult Salmon


      We assume that the high seas migration of salmon is relatively un-
stressful.  However, as these salmon approach the estuary of their natal
stream, a number of major physiological changes occur in response to envi-
ronmental and internal cues.  The rates of swimming and general metabolism
increase (Royce, Smith, and Hartt, 1968; Brett, 1965).  Feeding ceases
prior to entry into the river, but the exact timing of this appears to be
unknown.  The scales are absorbed, tne skin thickens about threefold, and
mucous production increases.  The kidney, gills, and gut prepare to
change from their marine functions of excreting both monovalent and divalent
ions and conserving water to excreting excess water and conserving mono-
valent ions as the fish enters freshwater (Miles, 1969).  The blood levels
of the adrenocorticosteroid hormones increase about fivefold (Fagerlund,
1967), and in response many of the blood constituents such as blood cells
and plasma proteins decline and change dramatically by the time the fish
has completed spawning (Cardwell, 1968).  Resistance to disease also dimin-
ishes markedly (Wedemeyer, In press).  Dramatic changes take place in pro-
tein and lipid metabolism (Idler and Bitners, 1959).  The fish completes
spawning with its lipid reserves exhausted and much of its muscle tissues
replaced by water.  These changes in salmon are intriguingly similar to
degenerative diseases like Gushing's syndrome in humans, and may be
reversible in the salmon if food is given after spawning (McBride et al.,
1963).  We define all such changes as natural stress responses.

      Man-made stressers which are not immediately lethal to the fish
evoke many of the same responses as the natural stressers.  Therefore,
the problem of quantifying responses to man-made stressers is one of dis-
tinguishing between degrees of responses rather than between types of
responses.  Studying degrees of responses is a much more difficult kind
of research than studying types of responses.  It is vitally important in
several ways to understand the fish's adjustments to reduced water quality.
First, salmon are not feeding in the estuary or river and therefore cannot
replace lost or wasted energy.  If the man-made stresser requires too
much energy in addition to that normally needed for migration, the fish
will die prior to reaching the spawning grounds.  Second, since the man-
made stressers are likely to be cumulative, the two together can be fatal,
although either one alone might not.  Third, the fish have a limited ability
to respond to stressing agents owing to chronic elevation of circulating
corticosteroids (Fagerlund, 1967).  Most salmon die from disease after
spawning, but not because they can no longer meet their basic physiological
requirements.  Studies by McBride et al. (1963) on mature sockeye salmon,
kept alive after spawning, showed that the primary factors causing death
were vitamin deficiencies and disease, not functional insufficiencies.
                                 77

-------
To understand the full effects of stressing agents on salmon, one must
understand a whole spectrum of man-made and natural stressers and the
degree of response by the fish.
   Effects of Low Levels of Dissolved Oxygen on Chinook and Coho

                  Salmon in the Duwamish Estuary

General Considerations

      Before beginning a description of our experiments, it is useful to
point out that salmon have alternative courses of action available for
coping with the problems of low DO.  Each of these alternatives involves
a compromise.  Each action taken by the fish to remedy its difficulties in
maintaining adequate oxygen consumption under conditions of decreased
oxygen availability produces at least one new difficulty.  Thus, the pro-
blems salmon encounter in water of low DO are difficult to predict because
there are a large number of considerations and alternatives, some of which
may be suitable at one DO level and not at another.  The experiments
described below will illustrate some of the ways in which salmon select
these alternatives as best they can.


Materials and Methods
      For these particular experiments, a series of four fish was put in
the swimming chamber:  1) no catheterizations and no prolonged anesthesia;
2) no catheterizations but with prolonged anesthesia; 3) dorsal aorta
and urethra catheters; and 4-) pre- and post-gill catheterizations.  The
fish were rested in a  chamber after capture, anesthetized, catheterized
using an operating table for fish, placed in a swimming chamber, and
rested overnight to recover from anesthesia and surgery.  The turnover of
in situ estuarine water (salinity 21-25 °/oo and temperature 12.0-13.5 C)
in the swimming chamber was 10 1/min.  For the first hour the water was
aerated, giving DO's ranging from 5.5 to 6.5 ppm.   The aeration was stopped
during the second hour so that the DO decreased to the range of H.O to 5.0
ppm.  The current in the chamber was set to provide a moderate swimming
velocity.  At the end  of the two hour period, the  current was reduced.
The fish's recovery could then be followed with the fish resting in aerated
water.

      The series of four fish were repeated three  times using different
fish in ambient estuary water having a moderately  low DO and then repeated
one more time in the sea water of Elliott Bay (about two miles away from
the first site), where DO was saturated and no pollution was obvious.  In
the latter case, water flow through the chamber was reduced during the
second hour so that the fish's own oxygen consumption produced DO levels
                                 78

-------
in the chamber comparable to those we observed in the estuary.   As an
additional control for the coho salmon which we caught in the estuary
and whose swimming might have been affected by pollution agents other
than low DO, we also brought coho salmon from Hood Canal where pollution
contamination was very unlikely and tested them at the Elliott Bay site
on the same schedule as the other salmon.
Results
      Swimming Stamina.  Although it was not part of our experimental
design to test swimming stamina per se, some comments on swimming stamina
are warranted from our data.  Swimming stamina may be some kind of inte-
grated indicator for the status of a fish's energy transfer systems, but
high speed swimming until fatigued - the usual definition of stamina -
probably does not occur in nature.  Just as with people, most fish will
not work to complete exhaustion unless forced to do so by unusual circum-
stances.  However, some of the fish we tested did become fatigued at our
relatively moderate test velocity of 56 cm/sec within the two-hour swimmini
period of the experiment.  Data concerning the environmental oxygen levels
and swimming endurance are shown in Table 24.  Fish exposed to 4.0 - 4.5
mg/liter fatigued in the test period.
     Table  24.  Relation between  dissolved  oxygen concentrations
                and fatigue  in  swimming  adult  coho  salmon-1-»


                                First  hour             Second hour
Number of fish
Fatigued
0
Not
fatigued
3
Fatigued
4
Not
fatigued
4
Dissolved oxygen     mean      -            5.8          4.3         4.8

Concentration mg/1   range     -         5.0-6.6      4.0-4.5      4.5-5.0
     """Environmental temperature  range was  12-13.5  °C.
     2
      Salmon were  swum  at  56  cm/sec.
                                  79

-------
       Ammonia concentration at low DO levels may have some effect on
 swimming  stamina since all four of the fish which fatigued had DO levels
 below  4.5 mg/liter and ammonia concentrations above 0.65 ppm, while the
 four that did not become fatigued in the same test series had DO levels
 above  4.5 mg/liter and ammonia concentrations of less than 0.65 ppm.
 Some synergistic effects between ammonia and oxygen appear possible and
 will be discussed later under ammonia excretion.

       Respiratory Changes.  The three respiratory variables which we
 measured  - oxygen consumption, ventilation volume, and extraction
 coefficient - are summarized in Fig. 22.  During the first hour of swim-
 ming in high DO, the consumption remained nearly constant and dropped
 slightly  at the end of the hour.  This drop was probably real and could
 have been caused by a decrease in the excitement of the new swimming
 task being performed.  Similar observations have been made by J. R.
 Brett  (personal communication).  A similar change was seen in ventilation
 volume, probably for the same reason.  The extraction coefficient was
 commonly the complement of the ventilation volume.  As the level of
 dissolved oxygen was decreased, the oxygen consumption and extraction
 coefficient decreased by about one -third almost immediately and then slowly
 recovered.  From the oxygen metabolism data alone, it appeared that the
 fish were recovering from the initial shock of the dissolved oxygen
 drop.

      Two observations seem especially noteworthy.  First, the decreased
 oxygen consumption occurred during a period of constant or slightly-
 increased activity.   The decreased consumption must therefore represent
 an oxygen debt and lactate accumulation.  Second, the ventilation volume
 increased rather slowly as compared to the consumption and extraction.   This
 indicates, we  believe, that an increase in ventilation volume is more
 "expensive" physiologically than going into oxygen debt, and that an
 increase in ventilation volume occurs only when the low DO persists long
 enough that the  lactate accumulation begins to become a problem.  In our
 swimming chamber,  the fish did not have the option of reducing its level
of activity prior  to fatigue as a means of reducing its lactate.

      Changes  in Levels of Lactate in the Blood.  We have been successful
 in completely  catheterizing relatively few individual fish.   Therefore,
we will present  information from individual fish, since each proved to  be
different and  each illustrates different points.  In Fig. 23, fish 1C
began  swimming with  a moderately-high blood lactate level, but improved
 it slightly by the end of the first hour.  Upon entry into water of low DO,
however, the lactate increased rapidly until the fish became fatigued just
before the end of  the second hour.  The recovery period after fatigue was
normal and uneventful, declining to the normal 5-20 mg% level of a typical
rested fish.

      In fish  2C (Fig.  23), the blood lactate was normal at  the  start of
 the swimming,  but  rose during swimming, showing a possible tendency to
                                 80

-------
    5.5-

I5
    4.5-
Ambient oxygen  concentration
|l50-

Oxygen  consumption
      7-

      6-

      5-
Ventilation  volume
      18-

      15-

      12-
Extraction  coefficent
              15          45          I--I5         1:45

                   Time   from   starting  activity

        Fie  22. Oxygen consumption, extraction coefficient, and ventilation
               volume with change in environmental oxygen concentration in
               swimming coho salmon.  Temperatures ranged from 12 to 15 °C,
               and salinities from 20 to 28 ppt.
                           81

-------
75-
50-
25-
^75-
t>>
^50-
|25-
^
^75-
|so-
*25-
75-
50-
25-
COHO 1C
a h 
-------
stabilize toward the end of the first hour.  The adjustments in lactate
levels in both this fish and in fish 1C probably correspond to the slight
decrease in oxygen consumption as seen in Fig. 22.

      Fish 3C and HC (Fig. 23) illustrate the problems of controlling
the behavior of the fish.  In fish 3C the sharp peak in lactate concentra-
tion between 105 and 150 minutes resulted from violent, nonswimming
activity.  Thus, the fish indicated that it would have preferred to turn
downstream and remove itself from that environment rather than continue to
swim upstream.  Low blood lactate concentrations in fish 4C resulted from
the fish's refusal to swim or exert itself in any manner.  These two fish
also illustrate the degree to which normal values can be influenced by
erratic behavior.

      We believe that an average response to our swimming schedule is
represented by this figure (Fig. 24).  In adequate DO there was a minimal
resting level of lactate which rose slightly during any  increase in activity.
Once some critical point was reached  in  either decreased DO or increased
activity, the blood lactate level increased rapidly until the fish became
fatigued and stopped swimming, or until  it reached the end of the test
period and was allowed  to rest.

      Figures 25 and 26 illustrate  the direct application of lactate data
to water quality criteria.  In Fig.  25,  lactate  levels in the blood were
determined at 15 min. intervals  while the  fish  swam at a constant velocity
in decreasing concentrations  of  dissolved  oxygen.  Although there was
considerable individual variation,  the general  pattern seemed to be a rel-
 atively constant  level of blood lactate until  the DO concentration decreased
below 5 mg/1.  Then blood lactate increased rapidly and  fatigue occurred
shortly thereafter as the DO  continued to  decrease.

      Two significant interpretations can  be  made from this figure.  First,
coho salmon experience  a range of DO levels within which they  can adjust
their respiratory  functions to meet a large  fraction  of  their  energy needs
-  i.e., they do not go  into oxygen  debt  by producing  much  lactate.   Second,
they reach a critical DO level at which  the respiratory  adjustments are
insufficient to meet their needs and they  produce lactate  very rapidly and
soon fatigue.  For the Duwamish Waterway  at ambient temperatures and  a
swimming rate which we  assumed to be typical, the critical  DO  level was
just below  5 mg/1.

       Some possible generalizations on the lactate-DO data of Fig.  25  are
shown  in Fig. 26.   This figure  suggests  that  the rate of lactate  production
is a  function of  DO.  As DO decreased, lactate production  increased  so that
the sum of  oxygen  consumption and  lactate  production  was approximately
constant.   Eventually,  fatigue is  virtually  certain  if the lactate  is  high
enough  or the DO  is low enough.   Fish also can have  high lactate  levels
 in normal DO  due  to  excessive activity produced by handling,  chasing,  or
                                  83

-------
          80n
00
•F
                      0
                     Fig. 24,
       60            120

       TIME  (MINUTES)
Temporal variations in blood lactate concentrations
in three coho salmon swimming at 56 cm/sec in aerated
(100% saturation = "a") and hypoxic (50% saturation
= b) waters.
240

-------
   70 -
   60 -
o
o
 E

UJ
   40 -
-» 30 1
O
O
     o-
     o
A Resting

O Swimming

• Fatigue
      4.0     4.5    5.0     5.5     6.0     6.5     7.0
                   DISSOLVED  OXYGEN   mg/l
              —r~
              7.5
       Fig. 25.  Relation between blood  lactate, dissolved  oxygen
                and duration of swimming in four coho salmon
                from the Duwamish Waterway, 3 to 4 kg adult fish.
                Samples taken every 15  min proceeding from right
                to left.  Coho 1C, dotted line; coho 2C,  solid
                line, coho 3C, dashed line, and coho HC,  broken
                line.
                                85

-------
  80 -
 £

o
\
070^
  60 -
UJ
H50H



^ 40 H


Q
EC
  20-


   10-
                                                                     600
                                                                         -

                                                                          . ,.
                                                                         .

                                                                     •500^
                                                                         o
                                                                     •400 jZ
                                                                         a.
                                                                         CO
                                                                     300 g

                                                                         ! '
                                                                    -200
                                                                         x
                                                                         o
                                                                    -100
                 -p-    -r-
                 45678
                       DISSOLVED OXYGEN
                                                      10
                                                                                      to
                                                                                      00
                                                                             «
                                                                             ....
                                                                             ' i


-------
 other strenuous activity.  These latter fish would not be expected to fatigue,
 The relation between oxygen consumption and DO is partly based on our data
 and partly on data from Salvelinus fontinalis (Fry, 1957).

       Excretion of Lactate.  The first evidence that lactate can appear in
 the urine of salmonids was produced by Hunn (1969).  The first evidence
 that blood lactate levels may increase in proportion to sub-maximal levels
 of activity was just presented.  The two facts together raise the question
 of how much lactate (and therefore energy) might a salmon lose if subjected
 to increased blood lactate while swimming in chronically low DO.

       We have some experiments in progress at the time of writing which may
 answer that question.  In a coho salmon whose blood lactate concentration
 ranged between 10 and 15 mg%, 30-45 gm/kg/hr were excreted in the urine.
 If we extrapolate to a blood level of 45 mg% and 135 gmAg/hr urinary
 excretion, a one-kg fish would lose 3.2 mg/day or 97.2 mg/month, a month
 not being an unreasonable period of time for the fish to wait in the  estuary
 before ascending the stream.

       Whether this energy leak is significant to the fish's energy budget
 or not is impossible to say now, because we do not know how crucial the
 last few milligrams of energy stores may be to the fish when it is on the
 spawning grounds.   Also, there probably are other routes of lactate loss,
 such as through the gills.   This point was suggested by an experiment in
 which a coho salmon had a very large dose of lactate injected directly into
 the blood stream (concentration rose to 938 mg% - far beyond physiological
 levels),  but only  57% of it was recovered in the urine.

       Ammonia Excretion. Ammonia is the primary end product of nitrogenous
 metabolism in teleost fish  and excretion occurs primarily through  the  gills.
 Since  the  gills  are very difficult to isolate, we monitored ammonia excre-
 tion only  as  the differential in ammonia content between the water entering
 and leaving the  swimming chamber.

       Data from  ten fish indicate  two patterns of ammonia excretion (Fig. 27).
 The rate of ammonia excretion increased during activity (in  water with
 adequate oxygen).   Fish  that  did not fatigue during the second hour (low DO)
 characteristically  showed a depressed rate  of ammonia  excretion during
 exposure to the  low DO.  Fish that did fatigue during  the  low  DO period
 had rates of ammonia excretion (dotted line)  which remained  constant or
 continued to increase until fatigue  occurred.   It  is also worthwhile to
 note that fish which became fatigued were exposed to low DO which was
 about  0.5 mg/liter  lower than those  which did not become fatigued, and
 that the nonfatigued fish elevated their rates of ammonia excretion again
when adequate DO was restored.   Both patterns of ammonia excretion were
accompanied by the typical pattern of increased lactate levels as shown
 in the upper part of Fig. 27.
                                 87

-------

    40-

8  20-
     0

15-

10-
     0-
                Blood  \oc\a\e
          Ammonia excretion
                                           ..— fatigue


    rest-4«	swim 56cm/sec—4«	rest—
    	High do—H*—Low do   »|«   -High do
              0     30    60    90    120   150    180
 Fig. 27.      Upper graph indicates average changes in blood lactate
              levels of adult coho salmon (Oncorhynchus kisutch)
              contacting a low dissolved oxygen (50% saturation)
              while swimming at 56 cm/sec.  Lower graph indicates
              variations in total ammonia excretion in swimming
              adult coho salmon subjected to various levels of
              dissolved oxygen.
                            88

-------
     Rate of Urine Production.  Kidney function in marine salmon is
concerned primarily with the excretion of divalent ions, particularly mag-
nesium, and organic acids including, as we have shown, lactic acid.  The
major excretory load of sodium, ammonium, and chloride ions is excreted by
cells in the gills, so that urine volume in marine salmon is only about
one-eighth of that in freshwater salmon (Hickman and Trump, 1969; Miles,
1969).  However, any major decrease in the rate of kidney function could
cause problems through the toxicity of the accumulated magnesium ions in
marine salmon or accumulated water in freshwater salmon.

     Our present data is sufficient only to say that there appeared to
be a decrease in urine production when the fish was subjected to low levels
of DO.  We plan additional research in this area, but cannot now say how
important this effect of  low  DO may be.  However, if excretory mechanisms
are impaired by low  DO, then  toxic  levels of  various blood constituents
may be reached.
                                  89

-------
                              SECTION VIII
                             ACKNOWLEDGMENTS

     Some of the personnel contributing to this project were partly
supported by a contract, No. 14-17-0007-1114, from the Bureau of
Commercial Fisheries (now National Marine Fisheries Service) for
study of problems encountered while tagging juvenile Pacific salmon
at sea.  A number of the physiological methods devised during the
1966-1970 period proved equally valuable for studying the stress of
either pollution or tagging and, therefore, the costs of developing
these methods were distributed between the two agencies.

     Mr. Alan Mearns was supported during part of his participation
in this project by FWQA Training Grant No. 5T1-WP-175 as well as
directly by this project.
                                    90

-------
                            SECTION IX


                            REFERENCES


Baggerman, B.  1960.  Salinity preference, thyroid activity and the
    seaward migration of four species of Pacific salmon (Oncorhynchus).
    J. Fish. Res. Bd. Canada 17(3):295-322.

Black, V. S.  1951.  Changes in body chloride, density, and water
    content of chum (Oncorhynchus keta) and coho (£. kisutch) salmon fry
    when transferred from fresh water to sea water.  J. Fish. Res. Bd.
    Canada 8(3):164-177.

Bligh, E. G., and W. J. Dyer.  1959.  A rapid method of total lipid
    extraction and purification.  Canad. J. Biochem. Physiol. 37:911-917.

Brett, J. R.  1965.  The swimming energetics of salmon.  Sci. Amer.
    8:80-85.

Busnel, R. G.  1943.  Recherches de physiologie appliquees a la pisci-
    culture:  a propos  de la migration de  la truite arc-en-ciel.  Bull.
    Francais Pisciculture,  No. 128, p. 108-117.

Cardwell, R. D.   1968.  Qualitative and quantitative changes in plasma
    proteins of Pacific salmon resulting  from natural  and  induced stress.
    M.S. Thesis,  Univ.  Washington, Seattle.  94 p.

Conte, F. P., and H. H. Wagner.   1965.  Development of osmotic and  ionic
    regulation  in juvenile  steelhead  trout Salmo  gairdneri.  Comp.
    Biochem. Physiol.  14:603-620.

Conte, F. P., H.  H.  Wagner, J.  Fessler, and C. Gnose.   1966.  Develop-
    ment  of osmotic and ionic regulation  in juvenile coho  salmon
    Oncorhynchus  kisutch.   Comp.  Biochem.  Physiol.  18:1-15.

Davis, J.  C.,  and K. Watters.   1970.   Evaluation  of opercular catheteri-
    zation  as  a method for sampling water expired by fish.   J.  Fish.
    Res.  Bd.  Canada 27(9):1627-1635.

 Del Castillo,  J., and  L.  Engebaek.  1954.  The nature  of the neuro-
    muscular block produced by magnesium.  J. Physiol. 124:370-384.

 Dell, M.  B.  1968.  A new fish tag and rapid, cartridge-fed applicator.
    Trans.  Amer.  Fish. Soc. 97:57-59.

 Engbaek, L.  1952.  The pharmacological actions  of magnesium ion with
     particular reference to the neuromuscular and the  cardiovascular
     systems.  Pharmacol.  Rev. 4:396-414.
                                 91

-------
Fagerlund, U. H. H.  1967.  Plasma cortisol concentrations in relation
    to stress in adult sockeye salmon during the freshwater stage of
    their life cycle.  Gen. Comp. Endocrinol. 8:197-207.

Fontaine, H., and H. Koch.  1950.  Les variations d'euryhalinitie et
    d'osmoregulation ches les poissons.  J. Physiol. (Paris) 42:287-318.

Fry, F. E. J.  1957.  Respiration.  Jfa M. E. Brown [ed.], The physiology
    of fishes, Vol. 1.  Academic Press, New York.

Gordon, M. S.  1959.  Ionic regulation in the brown trout (Salroo trutta
    L.).  J. Exp. Biol. 36:227-252.

Grafflin, A. L., and D. Ennis.  1934.  The effect of blockage of the
    gastro-intestinal tract upon urine formation in a marine teleost,
    Myoxocephalus octodecimspinosus.  J. Cell. Comp. Physiol. 4:283-296.

Greene, C. H.  1904.  Physiological studies of the Chinook salmon.
    Bull. Bur. Fish. 24:429-456.

Gruger, E. H., Jr., R. W. Nelson, and H. E. Standsby.  1964.  Fatty acid
    composition of oils from 21 species of marine fish, freshwater fish
    and shellfish.  J. Amer. Oil Chem. Soc. 41:662-667.

Hickman, C. P., Jr.  1968a.  Glomerular filtration and urine flow in the
    euryhaline southern flounder, Paralichthys lethostigma, in seawater.
    Canad. J. Zool. 46:427-437.

Hickman, C. P., Jr.  1968b_.  Urine composition and kidney tubular
    function in southern flounder, Paralichthys lethostigma, in seawater.
    Canad. J. Zool. 46:439-455.

Hickman, C. P., Jr.  1968c_.  Ingest ion, intestinal absorption, and elim-
    ination of seawater and salts in the southern flounder, Paralichthys
    lethostigma.  Canad. J. Zool. 46:457-466.

Hickman, C. P., Jr., and B. F. Trump.  1969.  The kidney, p. 91-239.
    In W. S. Hoar and D. J. Randall [eds.], Fish physiology, Vol. 1.
    Academic Press, New York.

Holmes, R. H.  1961.  Kidney function in migrating salmonids.  Rep.
    Challenger Soc. 3:23.

Holmes, W. N., and I. M. Stainer.  1966.  Studies on the renal excretion
    of electrolytes by the trout  (Salmo gairdneri).  J. Exp. Biol.
    44:33-46.

Houston, A. H.  1957.  Responses of juvenile chum, pink, and coho salmon
    to sharp sea water gradients.  Canad. J. Zool. 35:371-383.

Houston, A. H.  1959.  Osmoregulatory adaptation of steelhead trout
    (Salmo gairdneri Richardson) to sea water.  Canad. J. Zool.
    37:729-748.
                                 92

-------
Houston, A. H.  1964.  On passive features in the osmoregulatory adapta-
    tion of anadromous salmonids to sea water.  J. Fish. Res. Bd. Canada
    21(6)-.1535-1538.

iunn, J. u.   1969.  Chemical composition of rainbow trout urine follow-
    ing acute hypoxic stress.  Trans. Amer. Fish. Soc. 98:20-22.

Idler, D. R., and I. Bitners.  1959.  Biochemical studies on sockeye
    salmon during spawning migration.  V.  Cholesterol, fat, protein,
    and water in the body of standard fish.  J. Fish. Res. Bd. Canada
    16:235-241.

Keys, A. B.   1933.  The mechanism of adaptation of varying salinity in
    the common  eel  and the general problem of osmotic regulation in
    fishes.   Proc.  Roy. Soc. B,  112:184-199.

Klontz, G. H.  1964.  Anesthesia of  fishes.   In_ Proceedings  of the Sym-
    posium on Experimental Animal Anesthesiology, Brooks  Air Force Base,
    14-16  December, 1964.   [Mimeographed.]

Koch, A.   1965.  The kidney, p.  843-870.   In T.  C.  Ruch and  H. D. Patton
    [eds.],  Physiology  and biophysics.   W. B.  Saunders, Philadelphia.

Krueger, H.  M., J.  B. Saddler, G. A. Chapman, I.  J.  Tinsley, and R.  R.
    Lowry.   1968.   Bioenergetics, exercise,  and fatty acids  in fish.
    Amer.  Zool. 8:119-129.

Kubo,  T.   1955.  Changes of some characteristics of blood of sraolts  of
    Oncorhynchus masou during seaward migration.  Bull. Fac. Fish.
    Hokkaido Univ.  6:201-207.

 Lysak,  A., and K. Wojcik.  I960.  Electrophoretic investigations on  the
    blood of carp fed with food containing various protein amounts.
     Acto Hydrobiol. 2:49-61.

 McBride, J.   R. , U. H. M. Fagerlund, H. Smith, and N. Tomlinson.   1963.
     Resumption of feeding by and  survival of adult sockeye salmon
     (Oncorhynchus nerka) following advanced gonad development.  J.  Fish.
     Res. Bd. Canada 20:95-100.

 Mead, J. F., M. Kayama, and R. Reiser.  I960.  Biogenesis of polyun-
     saturated  acid in fish.  J. Amer. Oil Chem. Soc. 37:438-440.

 Mearns, A. J., L. S. Smith, and H. M. Miles.  [In preparation.]  A_tech-,
     nique for  sampling urine in  free-swimming salmon with observations
     of urogenital anatomy variation in salmonids.

 Miles  H. M.  1967.  Ionic regulation in migrating juvenile coho salmon,
     Oncorhynchus kisutch.  M.S.  Thesis, Univ. Washington, Seattle.  35 p.

 Miles, H. M.  1969.  Renal  function in migrating adult coho salmon.
     Ph.D. Thesis,  Univ. Washington, Seattle.  80 p.
                                  93

-------
Parry, G.  1966.  Osmotic adaptation in fishes.  Biol. Rev. 41:392-444.

Pitts, R. F.  1934.  Urinary composition in marine fish.  J. Cell. Comp.
    Physiol. 4:389-395.

Royce, W. F., L. S. Smith, and A. C. Hartt.  1968.  Models of oceanic
    migrations of Pacific salmon and comments on guidance mechanisms.
    U.S. Fish Wildl. Serv., Fish. Bull. 66:441-462.

Saddler, J.  B., and R. D. Cardwell.  [In preparation.]  The effect of
    tagging  upon the fatty acid metabolism of juvenile pink salmon
    (Oncorhynchus gorbuscha).  Submitted to Comp. Biochem. Physiol.

Saddler, J.  B., and K Koski.  [In preparation.]  Fatty acid alterations
    during the early development and migration of Pacific salmon.
    Submitted to Lipids.

Saddler, J.  B., R. R. Lowry, H. M. Krueger, and I. J. Tinsley.  1966.
    Distribution and identification of the fatty acids from the coho
    salmon.  Oncorhynchus kisutch (Walbaum).  J. Araer. Oil Chem.  Soc.
    43:321-324.

Salo,  E. 0.  1969.  Final report for the period June  1, 1965 - September
    30,  1968; Estuarine Ecology research report.  Univ. Washington,
    Coll. Fish., Seattle.  80 p.  [Processed.]

Saunders, R. L.  1962.  The irrigation of  the  gills of  fishes.  II.
    Efficiency of oxygen uptake in relation to respiratory  flow activity
    and  concentrations of oxygen and carbon dioxide.  Canad. J. Zool.
    40:817-862.

Smith, L. S., and G. R. Bell.  1964.  A technique for prolonged blood
    sampling in free swimming salmon.  J.  Fish. Res.  Bd.  Canada 23:
    711-717.

Smith, L. S.,  and G. R. Bell.  1967.  Anesthetic  and  surgical  techniques
    for Pacific salmon.  J. Fish. Res. Bd. Canada 24:1579-1588.

Smith, L. S.,  and T. W. Newcomb.   1970.  A modified version of the Blazka
    respirometcr and exercise chamber  for  large  fish.  J.  Fish. Res. Bd.
    Canada  27(7):1321-1324.

Snieszko, S. F.  1960.   Microhematocrit as a  tool in  fishery research
    and management.  U.S.  Fish Wildl.  Serv.,  Spec. Sci.  Rep. Fish. 341.
    15 p.

Toribara, T. Y., A.  R.  Terepka,  and P. A.  Dewey.   1957.   The ultra-
    filtrable calcium of human serum.   I.  Ultrafiltration methods and
    normal  values.   J.  Clin.  Invest.   36:738-748.

Wedemeyer,  G.   [In press.]  The  role of  stress in the disease  resistance
    of fishes.   Amer.  Fish.  Soc.,  Spec.  Publ.
                                   94

-------
Willis, J. B.  1960.  Determination of calcium in blood serum by atomic
    absorption spectroscopy.  Nature 186:249-250.

Willis, J. B.  1961.  Determination of calcium and magnesium in urine
    by atomic absorption spectroscopy.  Anal. Chem. 33:556-559.

Wintrobe, M.  1967.  Clinical hematology.  Lea and Febriger, Baltimore.

Wolf, K.  1963.  Physiological salines for freshwater teleosts.  Proer.
    Fish-Cult. 25:135-140.

Zanjani, E. D., Man-Lira Yu, A. Perlmutter, and A. S. Gordon.  1969.
    Humoral factors influencing erythropoiesis in the fish (blue gourami
    Trichogaster trichopterus).  Blood 33:573-581.
                               95

-------
                              SECTION X


         APPENDIX A - LIST OF PUBLICATIONS FROM THE PROJECT

Published Papers

   Miles, H. M., and L. S. Smith.  1968.  Ionic regulation in migrating
       juvenile coho salmon, Oncorhynchus kisutch.  Comp.  Biochem.
       Physiol.  26:381-398.

   Smith, L. S.  1970.  Building and operating a floating laboratory.
       Lab. Practice 19(7):709-712.

   Smith, L. S., and T. W. Newcomb.  1970.  A modified version of the
       Blazka respirometer and exercise chamber for large fish.  J.
       Fish. Res. Bd. Canada 27:1321-132U.


Completed Theses

   Cardwell, Rick D.  1968.  Hematologic responses of Pacific salmon to
       acute  and chronic stress.  M.S. Thesis, Univ. Washington, Seattle.
       9H p.

   Miles, H. M.  1967.  Ionic regulation in migrating juvenile coho
       salmon, Oncorhynchus kisutch.  M.S. Thesis, Univ. Washington,
       Seattle.  35 p.

   Miles, H. M.  1969.  Renal function in migrating adult coho salmon.
       Ph.D. Thesis, Univ. Washington, Seattle.   80 p.


Papers in Press (as of February, 1971)

   Cardwell, R. D., and L. S. Smith.  Hematological manifestations of
       a marine bacterial infection  upon juvenile chinook salmon
       (Oncorhynchus tshawytscha).   Progr. Fish-Cult.

   Miles, H. M.  Renal function  in migrating coho salmon.  Comp.
       Biochem. Physiol.

   Smith et al. Physiological changes experienced by Pacific salmon
       migrating through  a polluted  urban estuary.  "FAO Technical
       Conference  on  Marine Pollution and its  Effects on Living
       Resources and  Fishing."
                                   96

-------
Papers Submitted or in Preparation (as of February, 1971)

   Mearns, A. J.  Lactic acid regulation in salmonid fishes.  Ph.D.
       Thesis, in preparation.  (This thesis will eventually become
       2 or 3 papers relating lactate metabolism to swimming stamina
       and low DO.)

   Saddler, J. B.s R. D. Cardwell, and L. S. Smith.  Lipid composition
       and hematology of juvenile chinook salmon (Oncorhynchus
       tshawytscha) cultured on different diets and residing in
       different environments.  (Submitted to Lipids.)

   Saddler, J. B., and P. R. Dorn.  Comparative fatty acid changes
       during rapid growth of trout in salt water ponds.  (In
       preparation.)

   Saddler, J. B., and K V. Koski.  Fatty acid alterations during the
       early development and migration of Pacific salmon.  (Submitted
       to Lipids.)
                                  97

-------
                         SECTION  X


             APPENDIX B - SUPPLEMENTARY  TABLES
                                                              Page No.
Table B-l.  Means, variances, and ranges for length,
            weight, hematocrit, and ion concentrations
            in samples of coho salmon resident in fresh-
            water 	      99

Table B-2.  Results of one-way analysis of variance in
            length, weight, hematocrit, and ion concentra-
            tionsbetween samples of juvenile coho salmon
            resident in freshwater  	     100

Table B-3.  Means, variances, and ranges for length, weight,
            hematocrit, and ion concentrations in all coho
            salmon samples taken during sea water adapta-
            tion  	     101

Table B-4.  Results of one-way analysis of variance in
            length, weight, hematocrit, and ion concentra-
            tions between samples of juvenile coho salmon
            during adaptation to sea water  	     102

Table B-5.  Means, standard deviations (S.D.), and sample
            sizes (n) for renal parameters in salt water
            and freshwater for fish H   	     103

Table B-6.  Means, standard deviations (S.D.), and sample
            sizes (n) for renal parameters in salt water
            and freshwater for fish L   	     106

Table B-7.  Means, standard deviations (S.D.), and sample
            sizes (n) for renal parameters in freshwater
            for fishes N and 0	     109

Table B-8.  Means, standard deviations (S.D.), and sample
            sizes (n) for renal parameters in freshwater
            for fish P	     112
                              98

-------
        Appendix Table B-l.   Means,  variances,  and ranges  for length, weight, hematocrit, and ion concentrations
                             in samples of coho salmon resident  in  freshwater
Sampling
period
Nov. 12, 1966 to
May 11, 1957
(N=103)

Apr. 6, 1967 to
10 May 11, 1967
(N=55)






Parameter
Total length (mm)
Wet weight (g)
Hematocrit (%)
Total length (mm)
Wet weight (g)
Hematocrit (%)
Sodium (mEq/1)
Potassium (mEq/1)
Calcium (mEq/1)
Magnesium (mEq/1)
Chloride (mEq/1)
Mean
104.21
9.87
37.77
114.73
12.49
39.33
146.7
8.58
6.73
1.08
117.33
Standard
deviation
19.22
6.22
5.52
19.63
7.43
5.75
8.24
3.69
0.83
0.20
7.66
Standard
error of
mean
1.89
0.61
0.54
2.65
1.00
0.77
1.11
0.50
0.11
0.03
1.03
Maximum
170
34.24
53.0
170
34.24
53.0
168
19.01
8.85
1.55
132.57
Minimum
72
3.14
27.0
86
4.29
28.5
130
1.80
4.95
0.65
90.85
Range
98
31.1
26. C
84
29.95
24.5
38
17.21
3.9
0.9
41.72
Apr. 12, 1967 to
Kay 11, 1967       Total solids (%)
(N=45)
4.77
0.66
                        0.10
                          6.6
3.1
3.5

-------
Appendix Table B-2. Results of one-way analysis of variance in length, weight, hematocrit, and ion
                   concentrations between samples of juvenile  coho  salmon resident in freshwater
Parameter
Total length
Wet weight
Kenatocrit
0 Total solids
Sodium
Potassium
Calcium
Magnesium
Chloride

Source
Between samples
Within sample
Betweeen samples
Within sample
Between samples
Within sample
Between samples
Within sample
Between samples
Within sample
Between samples
Within sample
Between samples
Within sample
Between samples
Within sample
Between samples
Within sample
Sum of
squares
13393.0085
7407.8991
1905.7651
1071.4912
898.8094
884.2639
3.6510
15.7290
871.8420
2793.5737
120.7501
614.0101
17.2715
20.2751
0.7526
1.3840
195.0506
2971.8135
Degrees of
freedom
5
49
5
49
5
49
4
40
5
49
5
49
5
49
5
49
5
49
Mean
square
2678.6017
151.1816
381.1530
21.8672
179.7619
18.0462
0.9127
0.3932
174.3684
57.0117
24.1500
12.5308
3.4538
0.4138
0.1505
0.0282
39.0101
60.6493
F
ratio Significance
17.7178 **
17.4304 **
9.9612 **
2.3212 N.S.
3.0585 *
1.9273 N.S.
8.3482 >•»•
5.3292 **
0.6432 N.S.


-------
       Appendix Table B-3.   Means,  variances,  and ranges  for length, weight, hematocrit, and ion concentrations
                            in all  coho  salmon samples^ taken during sea water adaptation.  (N=29)
Parameter
Total length (mm)
Vfet weight (g)
Hematocrit (%)
Total solids (%)
£ Sodium (mEq/1)
Potassium (mEq/1)
Calcium (mEq/1)
Magnesium (mEq/1)
Chloride (mEq/1)
Mean
120.48
12.69
45.29
4.86
158.3
7.19
6.38
2.17
126.6
Standard
deviation
8.41
2.78
5.26
0.67
5.89
2.99
0.78
0.95
7.43
Standard
error of
mean
1.56
0.52
0.98
0.12
1.09
0.55
0.14
0.18
1.38
Maximum
144
21.06
55.5
5.9
170
13.35
7.5
4.2
143.68
Minimum
101
7.55
33.5
3.2
149
3.15
4.5
0.95
111.6
Range
43
13.51
22.0
2.7
21.0
10.2
3.0
3.25
32.08
     "^A sample of five fish was taken every 6 hours over a period of 36 hours after introduction of the fish
into sea water.

-------
      Appendix Table B-4.   Results of one-way analysis of variance in length, weight, hematocrit, and ion concen-

                           trations between  samples of juvenile coho salmon during adaptation to sea water
o
to
Parameter
Total length (mm)
Wet weight (g)
Hematocrit (%)
Total Solids (%)
Sodium (mEq/1)
Potassium (mEq/1)
Calcium (mEq/1)
Xagnesium (mEq/1)
Chloride (mEq/1)
Source
Between samples
Within sample:
Between samples
Within sample
Between samples
Within sample
Between samples
Within sample
Between samples
Within sample
Between samples
Within sample
Between samples
Within sample
Between samples
Within sample
Between samples
Within sample
Sum of
squares
161.4206
2205.51498
18.7746
218.6759
521.6125
416.3875
5.2076
12.0900
859.6794
658.8299
94.1381
174.9645
11.4946
7.3055
18.8704
13.7711
1134.1743
1438.3055
Degrees of
freedom
6
27
6
27
6
27
6
27
6
27
6
27
6
27
6
27
6
27
Mean
square
26.9034
81.6870
3.1291
8.0991
86.9354
15.4218
0.8679
0.4478
143.2799
24.7715
15.6897
6.4802
1.9158
0.2706
3.1451
0.5100
189.0291
53.2706
F
ratio Significance
0.3293 N.S.
0.3864 N.S.
5.6372 **
1.9383 N.S.
5.7841 **
2.4212 N.S.
7.0804 **
6.1663 **
3.5485 -"

-------
            Appendix Table B-5.
                         Means, standard deviations (S.D.), and sample  sizes  (n) for renal parameters
                         in salt water and freshwater for fish H
Flow rates (ml/(kg x hr))
Urine rate
Glomerular filtration rate
PAH clearance

0.487
1.05
3.52

0.216
0.443
1.36

13
9
9

1.81
2.57
4.37

1.14
1.05
0.902

37
25
25
o
CO
Urine ion concentrations
   (raequiv/1)

  Sodium
  Potassium
  Calcium
  Magnesium
  Chloride

Plasma ion concentrations
   (ir.squiv/1)

  Sodium
  Potassium
  Calcium
  Magnesium
  Chloride

Excretion rates
   (pequiv/(kg x hr})

  Sodium
  Potassium
  Calcium
  Magnesium
  Chloride
23.6
1.62
5.71
208
177
7.55
0.115
0.911
19.1
11.4
13
13
13
13
13
16.5
1.47
3.96
70.5
37.8
7.37
0.517
3.12
97.6
55.5
25
25
25
25
36
181
2.70
1.53
1.27
155
2.81
0.155
0.281
0.179
1.95
11
11
11
11
11
154
2.36
1.53
0.944
134
4.62
0.375
0.273
0.144
5.06
34
34
34
34
34
11.0
0.791
2.72
100
85.6
4.34
0.370
1:0?
39.4
35.3
13
13
13
13
13
22.1
1.75
3.10
35.9
33.8
20.3
1.15
0.967
50.9
29.8
25
25
25
25
36

-------
       Appendix Table B-5,
                    Means, standard deviations (S.D.),  and sample sizes  (n) for renal parameters
                    in salt water and freshwater for fish H -  Continued
o
£
Parameter
Salt water Freshwater
Mean S.D. n Mean S.D. n
Filtered ion load
   (uequiv/(kg x hr))

  Sodium
  Potassium
  Calcium
  Magnesium
  Chloride

Ion load based on
  PAH clearance
   (yequiv/(kg x hr))

  Sodium
  Potassium
  Calcium
  Magnesium
  Chloride

Clearance ratios based
   on PAH clearance
   (dimensionless)

  Water
  Sodium
  Potassium
  Calcium
  Magnesium
  Chloride
162
2.54
0.466
0.944
158.
70.4
1.09
0.191
0.286
68.2
9
9
9
9
9
336
5.24
1.16
1.72
330
132
1.71
0.448
0.787
130
25
25
25
25
25
                                                0.155
                                                0.0208
                                                0.0913
                                                0.598
                                               26.0
                                                0.178
70.4
1.09
0.191
0.286
68.2
251
3.92
3.68
1.16
215
0.0159
0.0070
0.013S
0.0818
3.22
0.0199
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
637
9.53
5.03
4.34
546
251
3.92
3.68
1.16
215
9
9
9
9
9
677
10.5
6.63
3.94
534
139
2.69
1.86
0.817
119
25
25
25
25
25
0.307
0.0294
0.164
0.466
9.89
0.0539
132
1.71
0.448
0.787
130
139
2.69
1.86
0.817
119
0.207
0.0236
0.110
0.104
12.5
0.0515
25
25
25
25
25
25
25
25
25
25
25
24
24
24
24
25

-------
          Appendix Table B-5.  Means, standard deviations  (S.D.), and sample sizes  (n)  for renal parameters

                               in salt water and freshwater for  fish H  -  Continued
o
tn
Salt water
Parameter
Filtration Ratios
( dimensionless )
Water
Sodium
Potassium
Calcium
Magnesium
Chloride
Filtration fraction
( dimensionless )
Hematocrit <%)
Plasma total solids (%)
Blood pressure (mm Hg)
Dorsal aorta pulse
pressure (mm Hg)
Heart rate (beats/min)
Mean


0.526
0.0828
0.348
6.60
121
0.628
0.298
21.1
2.88
23.5
3.71
54.1
S.D.


0.0584
0.0304
0.0428
1.25
19.0
0.090
0.0543
0.485
0.0745
2.01
0.367
5.65
n


9
9
9
9
9
9
9
11
11
9
9
13
Freshwater
Mean


0.478
0.0557
0.301
3.12
42.7
0.149
0.587
16.9
2.54
23.0
4.89
35.6
S.D.


0.166
0.0251
0.0978
1.60
60.1
0.196
0.226
0.850
0.0524
3.97
1.91
7.55
n


25
24
24
24
24
25
25
34
34
34
34
34

-------
     Appendix Table B-6.  Means, standard deviations (S.D.), and sample sizes (n) for renal parameters in

                          salt water and freshwater for fish L
o

-------
o
-J
       Appendix Table B-6.
                     Means, standard deviations (S.D.),  and  sample sizes (n) for renal parameters in
                     salt water and freshwater for fish  L  -  Continued
Parameter
Calcium
Magnesium
Chloride

Mean
3.11
12.6
7.90
Salt water
S.D.
1.87
3.43
1.55

n
6
6
6

Mean
20.1
19.1
155
Freshwater
S.D.
9.63
15.4
167

n
94
94
96
Filtered ion load
   (pequiv/(kg x hr))

  Sodium
  Potassium
  Calcium
  Magnesium
  Chloride
345
5.55
3.12
13.1
344
62.2
0.990
0.618
4.46
62.3
5
5
5
5
5
1620
25.8
9.17
24.7
1680
700
11.1
3.98
10.4
733
96
96
96
96
96
       Ion load based on
          PAH clearance
          (yequiv/(kg x hr))

         Sodium
         Potassium
         Calcium
         Magnesium
         Chloride

       Clearance ratios based
          on PAH clearance
          (dimensionless)

         Water
         Sodium
302
4.64
7.47
13.2
264
39.8
0.610
1.07
3.67
34.9
5
5
5
5
5
2090
31.7
32.5
36.6
1900
898
13.5
14.4
14.6
832
96
96
96
96
96
                                        0.153
                                        0.109
0..0260
0.0263
5
5
0.645
0.0627
0.390
0.0616
96
93

-------
Appendix Table B-6.  Means, standard deviations (S.D.), and sample sizes (n)  for renal parameters in
                     salt water and freshwater for fish L - Continued
Salt water
Parameter
Potassium
Calcium
Magnesium
o
Chloride
Filtration ratios
(dimension less)
Water
Sodium
Potassium
Calcium
Magnesium
Chloride
Filtration fraction
( dimensionless )
Hematocrit (%)
Plasma total solids (%)
Blood pressure (mm Hg)
Dorsal aorta pulse
pressure (mm Hg)
Heart rate (beats/rain)
Mean
0.242
0.479
1.13
0.0316


0.116
0.0962
0.205
1.16
1.17
0.0240

1.37
19.2
4.36
36.8

3.94
71.0
S.D.
0.0662
0.321
0.506
0.0053


0.0286
0.0231
0.0597
0.694
0.496
0.0035

0.321
0.154
O.CS18
2.44

0.707
1.79
n
5
5
5
5


5
5
5
5
5
5

5
5
5
6

6
6
Mean
0.276
0.655
0.569
0.0754


0.730
0.0809
0.347
2.35
0.315
0.0874

0.927
16.5
3.65
18.3

3.05
54.4
Freshwater
S.D.
0.172
0.272
0.425
0.0707


0.486
0.0823
0.214
1.06
0.546
0.0888

0.134
1.12
0.350
7.35

1.21
5.06
n
94
94
94
94


96
93
94
94
94
94

96
100
100
51

51
51

-------
Appendix Table B-7.   Means, standard deviations (S.D.), and sample sizes (n) for renal parameters in
                     freshwater for fishes M and 0
Parameter
Flow rates (ml/(kg x hr))
Urine rate
Glomerular filtration rate
PAH clearance
Urine ion concentrations
( mequiv/i)
Sodium
Potassium
Calcium
Magnesium
Chloride
Plasma ion concentrations
Sodium
Potassium
Calcium
Xapnesium
o
Chloride
Excretion rates
(uequiv/(kg x hr))
Sodium
Potassium


Mean

3.
8.
13,


9.
2.
3.
6.
9.

152
2.
_J_ ^
1.
131


25.
6.

38
88
0


82
13
92
89
02


52
67
80



7
66
Fish
S.

1
2
5


10
1
1
15

2
0
0
0
2


16
5
M
D.

.57
.95
.41


.0
.74
.94
.4
. 0

.63
.149
.523
.555
.29


.4
.76

n

79
77
77


69
69
69
69
77

72
72
72
72
79


69
69


Mean

3
8
24


10
2
2
1
2

172
2
2
1
115


39
7

.86
.04
.4


.6
.13
.63
.13
.31


.97
.78
.39



.2
.90
Fish
S

1
1
8


3
0
0
0
1

4
0
0
0
3


17
3
0
.D.

.30
.95
.57


.83
.652
.854
.539
.34

.27
.199
.922
.184
.24


.3
.02

n

44
41
41


43
43
43
43
43

44
44
44-
44
44


43
43

-------
        Appendix  Table  B-7.
Means, standard deviations (S.D.), and sample sizes (n) for renal parameters in
freshwater for fishes M and 0 - Continued
O
Fish M
Parameter
Calcium
Magnesium
Chloride
Filtered ion load
(uequiv/(kg x hr) )
Sodium
Potassium
Calcium
Magnesium,
Chloride
Ion load based
on PAH clearance
(yequiv/(kg x hr))
Sodium
Potassium
Calcium

Chloride
Clearance ratios based
on PAH clearance
(dimensionless )
Water
Sodium
Mean
11
12
18


1130
19
4
11
1130



1880
31
19
21
1710



0
0
.5
.3
.3



.6
.45
. 0




.4
.9
.2




.257
.0166
S.D.
5.14
12.7
8.92


384
5.93
1.90
3.82
380



784
13.7
8.08
7.7*
723



0.0849
0.0150
n
69
69
77


71
71
71
71
77



71
71
71
71
77



77
64
Mean
9.72
3.85
7.75


1180
21.3
6.65
8.03
895



4210
71.0
61.3
33.2
2790



0.179
0.0109
Fish
S
3
0
3


282
5
2
2
221



1510
21
19
11
948



0
0
0
.D..
.85
.910
.59



.76
.87
.12




.3
.5
.2




.0948
.0080

n
43
43
43


41
41
41
41
41



41
41
41
41
41



41
40

-------
Appendix Table B-7.  Means, standard deviations (S.D.), and sample sizes (n) for renal parameters in
                     freshwater for fishes M and 0 - Continued





Parameter
Potassium
Calcium
Magnesium
Chloride

Fis
Mean
0
0
0
0
.232
.638
.853
.0144
>h M

S.D.
0
0
1
0
.218
.289
.14
.0154

n
64
64
64
76


Mean
0
0
0
0
.124
.178
.130
.0032
Fish
o
0
0
0
0
0
. JJ.
.0636
.0967
.0609
.0019

n
40
40
40
39
Filtration ratios





( dirr.ensionless )
Water
Sodium
Potassium
Calcium
Hagr.csium
Chloride

0
0
0
2
0

.365
.0249
oo £
• o O*.)
.73
.43
.0215

0
0
0
0
1
0

.111
.0187
.266
.972
.74
.0298

77
64
64
64
64
76

0
0
0
1
0
0

.476
.0328
.386
.69
.516
.0088

0
0
0
0
0
0

.0928
.0117
.103
.379
.179
.0044
Filtration fraction

u
(dimensionless)
ematocrit (%)
Plasma total solids (%)
3
lood pressure (mm. Hg)
0
4
2
29
.719
.35
.84
.7
0
0
0
3
.151
.800
.124
.41
77
72
72
77
0
16
2
36
.369
.9
.95
.6
0
0
0
o
\J
Dorsal aorta oulse

•JJ
pressure (mm Hg)
eart rate (beats/rcin)
4
53
.89
.8
0
4
.819
'.92
77
77
7
56
.63
.1
0
5
.162
.865
.0382
.83

.651
.83

41
40
40
40
40
40

41
44
44
43

43
43

-------
Agjendix Table  B-8.   Means,  standard deviations  (S.D.), and  sample  sizes  (n)  for renal parameters in
                     freshwater  for fish  P
Parameter
Flow rates (ml/(kg x hr))
Urine rate
Glomerular filtration rate
PAH clearance
Urine ion concentrations
(mequiv/1)
Sodium
Potassium
Calcium
Magnesium
Chloride
Plasma ion concentrations
(mequiv/1)
Sodium
Potassium
Calcium
Magnesium
Chloride
Excretion rates
(yequiv/(kg x hr))
Sodium
Potassium

Mean

2.74
6.47
6.07


5.83
1.97
1.54
1.57
3.54


146
2.48
4.10
1.65
132


15.0
5.56
Fish P
S.D.

1.59
2.77
2.55


2.76
3.83
0.866
1.45
1.55


2.31
0.222
0.159
0.0322
1.51


8.89
11.2

n

49
29
29


43
44
44
44
49


32
32
32
32
32


43
44

-------
Appendix Table B-8.
Means, standard deviations  (S.D.), and sample sizes (n) for renal parameters in
freshwater for fish P -  Continued
Parameter
Calcium
Magnesium
Chloride

Mean
4.39
4.05
8.62
Fish P
S.D.
14.62
4.06
5.02

n
44
44
49
 Filtered ion  load
   (yequiv/(kg x hr))

   Sodium
   Potassium
   Calcium
   Magnesium
   Chloride

 Ion  load based
   on PAH clearance
   (uequiv/1)

   Sodium
   Potassium
   Calcium
   Magnesium
   Chloride

 Clearance ratios based
    on PAH clearance
    (dimensionless)

   Water
   Sodium
                   803
                   883
                    15.1
                    24.7
                    10.1
                   802
                     0.408
                     0.0195
342
372
  7.23
 10.4
  4.29
336
  0..111
  0.0071
29
14.3
8.15
7.84
827
6.79
3.52
3.40
354
29
29
29
29
29
29
29
29
29
29
24

-------
Appendix Table B-8.  Means, standard deviations (S.D.), and sample sizes (n) for renal parameters in
                     freshwater for fish P - Continued
Parameter
Potassium
Calcium
Magnesium
Chloride
Filtration ratios
(dirnensionless)
Water
Sodium
Potassium
Calcium
Magnesium
Chloride
Filtration fraction
( dimension less )
Kematocrit (%)
Plasma total solids (%)
Blood pressure (ran Hg)
Dorsal aorta pulse
pressure . (mm Kg)
Heart rate (beats/min)

Mean
0.252
0.157
0.336
0.0098


0.335
0.0212
0.265
0.479
0.431
0.0095

1.06
22.7
4.86
24.5

5.31
56.4
Fish P
S.D.
0.0798
0.111
0.279
0.0031


0.101
0.0072
0.0821
0.361
0.350
0.0029

0.0780
5.39
0.621
7.12

1.04
4.29

n
25
25
25
29


29
24
25
25
25
29

29
49
49
48

48
48

-------
 BIBLIOGRAPHIC:
    L.  S. Smith, J. B. Saddler, R. C. Cardwell,
 A.  J.  Meeras, H. M. Miles, T. W. Nevoorab, and K.
 C.  Watters.  Fisheries Heswrcts seen
Included decreased swlMilng stamina and respir-
atory efficiency, decreased oxygen consumption
and increased laetate,  decreased urine flow
ACCESSION NO.
   KEY WORDS:
Fish Physiology
Fish Migration
Environmental Effects
Pacific Salmon
Oxygen Sag

-------
are! anmonia excretion, especially In the pres-
ence of environmental amaonia.  Longer tem
disruptions in heMatology and lipld netabolis*
»ere seen.  Most of the effects occurred at DO
concentrations just below 5 mg/liter, except
for synerglstio effects between ammonia and
lev DO at sonewhat higher concentrations.

   This report wes submitted In fulfillment of
Grant No. 18050 EBK under the sponsorship of  the
Federal Water duality Administration.
and amnonla  excretion, especially in the pres-
ence  of  environmental ammonia.  Longer term
disruptions  in hematoi ogy and lipld metabolism
were  seen.  Most of the effects occurred at DO
concentrations just below 5 rag/liter, except
for synergistie effects between ammonia and
low DO et somewhat higher concentrations.

   This  report was submitted in fulfillment of
Grant No. 18050 EB1C under the sponsorship of the
Federal  Water fiuality Administration.
 and  aomonia  excretion, especially in th« pres-
 ence of environmental ammonia.  Longer ter«
 disruptions  in henatology and lipid raet»bolism
 were seen.   Most  of the  effects occurred at DO
 concentrations Just below 5 ng/llter, except
 for  synerglstie effects  between anaonia and
 low  DO at •onewnat higher concentrations.

   This report was submitted in fulfillment of
 Grant No. 18050 EBK under the sponsorship of the
 Federal Water fiuallty Administration.

-------
    Accession Number
 w
  Subject Field & Group

    VI G

Group 21, 28
                                          SELECTED WATER RESOURCES ABSTRACTS
                                                 INPUT TRANSACTION  FORM
    Organization

        Fisheries Research Institute,  University  of Washington
        Seattle, Washington  98105
    Title
        "Responses of Teleost  Fish  to  Environmental Stress" (Final Report, FWQA
        Grant No. 18050 EBK.)
10

Authors)
L. S. Smith, J. B.
R. C. Cardwell, A.
H. M. Miles, T. W.
and K. C. Watters.
Saddler ,
J. Mearns^
Newconib ,
16

21

Project Designation
Note
22
    Citation
23
Descriptors (Starred First)

Fish Migration
Fish Physiology
Anadroroous  Fish
Salmon
Environmental Effects
                                        Water Pollution Effects
                                        Oxygen Sag
                                        Oxygen Requirements
                                        Animal Metabolism
 25
Identifiers (Starred First)
   *
Aquatic Environments
Fish  Migration
Salmonids	
27
    /Abstract
     A  floating laboratory was built for conducting multiparameter physiological
     studies  on salmon in marine, estuarine, and fresh waters.  New  methods were
     developed using a swimming chamber-respirometer for adult  salmon.   Normal values
     were  measured for a variety of physiological  functions,  then  repeated on  salmon
     migrating through an urban estaary characterized by sewage pollution  and  low DO.
     Effects  seen included decreased swimming stamina and respiratory  efficiency,
     decreased oxygen consumption and increased lactate, decreased urine flow  and
     ammonia excretion, especially in the presence of environmental  ammonia.   Longer
     term  disruptions in hematology and lipid metabolism were seen.  Most  of the
     effects  occurred at DO concentrations just below 5 mg/liter,  except for^syner-
     gistic effects between ammonia and low DO at  somewhat  higher  concentrations.

     This  report was submitted in fulfillment of Grant No.  18050 EBK under
     sponsorship of the Federal Water Quality Administration.
Abstractor
          L. S.  Smith
                              /n.s 11' tut ion
                                  University of Washington
 YVRM02 (REV JULY 1969)
 W R SI C
                        SEND W'TH COPY OF DOCUMENT, TO: WATER RESOURCES SCIENTIFIC 1NFOF
                        StNO, w. I H         --^       j.s. DEPARTMENT OF THE INTERIOR
                                                  WASHINGTON. D. C. 2024C

-------