x°/EPA
            United States
            Environmental Protection
            Agency
            Environmental Research
            Laboratory
            Corvalhs OR 97330
EPA-600 '3-78-090
September 1978
            Research and Development
EFFECTS OF SEVERAL
METALS ON SMOLTING
OF COHO SALMON

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                RESEARCH  REPORTING SERIES

Research reports of the Office of Research and Development, U.S. Environmental
Protection Agency,  have been grouped into nine series. These nine broad cate-
gories were established to facilitate further development and application of en-
vironmental technology. Elimination of traditional  grouping  was  consciously
planned to foster technology transfer and a maximum interface in related fields.
The nine series are:

      1    Environmental Health Effects Research
      2.   Environmental Protection Technology
      3.   Ecological Research
      4   Environmental Monitoring
      5   Socioeconomic Environmental Studies
      6.  Scientific and Technical Assessment Reports (STAR)
      7.  Interagency Energy-Environment Research and Development
      8.  "Special" Reports
      9.  Miscellaneous Reports

 This report has been assigned to the ECOLOGICAL RESEARCH series. This series
 describes research on the effects of pollution on humans, plant and animal spe-
 cies, and materials. Problems are assessed for their long- and short-term influ-
 ences.  Investigations include formation, transport, and  pathway studies to deter-
 mine the fate of pollutants and their effects. This work provides the technical basis
 for setting standards to minimize undesirable changes  in living organisms in the
 aquatic, terrestrial, and atmospheric environments.
This document is available to the public through the National Technical Informa-
tion Service, Springfield, Virginia 22161.

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                                              EPA-600/3-78-090
                                              September 1978
EFFECTS OF SEVERAL METALS ON SMOLTING OF COHO SALMON
                        by

                  Harold W.  Lorz
                 Ronald H. Williams
                 Charles A.  Fustish
         Research and Development Section
      Oregon Department of Fish  and Wildlife
              Corval1 is, Oregon  97331
                   Grant #R-80^283
                    Project Officer

                    Gary A. Chapman
            Western Fish Toxicology Station
      Corval1 is Environmental  Research Laboratory
                Corval1 is, Oregon 97330
      CORVALLIS ENVIRONMENTAL RESEARCH LABORATORY
          OFFICE OF RESEARCH AND DEVELOPMENT
         U. S. ENVIRONMENTAL PROTECTION AGENCY
                CORVALLIS, OREGON 97330

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                                 DISCLAIMER

     This report has been reviewed by the Corvallis Environmental Research
Laboratory, U. S. Environmental Protection Agency, and approved for publi-
cation.  Approval does not signify that the contents necessarily reflect the
views and policies of the Environmental Protection Agency, nor does mention
of trade names of commercial products constitute endorsement or recommen-
dation for use.
                                       i i

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                                    FOREWORD

     Effective regulatory and enforcement actions by the Environmental
Protection Agency would be virtually impossible without sound scientific
data on pollutants and their impact on environmental stability and human
health.  Responsibility for building this data base has been assigned to
EPA's Office of Research and Development and its 15 major field install-
ations, one of which is the Corvallis Environmental Research Laboratory
(CERL)-

     The primary mission of the Corvallis Laboratory is research on the
effects of environmental pollutants on terrestrial, freshwater, and marine
ecosystems; the behavior, effects and control  of pollutants in lake systems;
and the development of predictive models on the movement of pollutants in
the biosphere.

     This report describes a potentially adverse effect of pollutants on
fish such as salmon which must migrate from freshwater to seawater, and
demonstrates that under certain conditions exposure to sublethal  levels of
pollutants can result in mortality when fish subsequently enter seawater.
Laboratory test methods are described which should detect this effect in
screening tests and advance knowledge on the effects of pollutants on
aquatic ecosystems.
                                                  A.  F-  Bartsch
                                                  Director,  CERL
                                       i i i

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                                    ABSTRACT

     The 96-h LC50 value for cadmium  (CdC^) for yearling coho salmon
 COncorhynchus kisutch) was 10.4 pg/liter in January but appeared to have
 increased  in smelting fish in May.  The 96-h LC50 of mercury  (HgC^) for
 yearling coho salmon was 240 yg/liter.  All static bioassay tests were con-
 ducted at  10°C  in water with alkalinity and hardness ranging  from 70-83
 mg/liter and 85~93 mg/liter as CaCOj,  respectively.

     Exposure of yearling coho salmon  for <_144 h to sublethal levels of Cd,
 Ni  (NiCl2), Cr  (l^C^O^) or Zn (ZnCl^)  in freshwater had no apparent affect
on  the (Na, K)-st imulated ATPase activity of the gill.  Fish  exposed to
concentrations >4 pg/ liter Cd died  in  a dose dependent manner following
transfer to seawater.  Coho salmon  given a 5~day rest  (non-toxicant exposure
 in freshwater)  between Cd exposure  and transfer to seawater exhibited
seawater survival comparable to control fish.  Few deaths occurred following
transfer of the Ni , Cr or Zn exposed fish into seawater.
     Yearling coho salmon exposed to £_4.5 yg/liter Cd for  144 h did not
migrate as well as the control  fish following  release into a natural stream.
However,  fish chronically exposed to <4.5 ug/liter Cd (47  and 70 days)
migrated  as  well  or  occasionally better  than control fish  following their
release.

      The  (Na, K)-st imulated  ATPase activity of the gills of coho salmon
chronically  exposed  to Cd or Zn was suppressed and the downstream migration
was  reduced  when  10  yg/liter Cu was added with the Cd or Zn.  Deaths also
occurred  when Cd-Cu  or Zn-Cu exposed fish were challenged with seawater.
Neither Cd nor Zn appeared to affect feeding activity, growth or survival
during exposures of  70 and 27 days respectively; however,  the addition of 10
pg/liter  Cu  for only 6 days  to  Zn exposed fish significantly reduced
condition factor.

     Histological examination of liver,  kidney and gill  tissue of coho
salmon exposed to Cd or Zn (singly or  in combination with  Cu) showed no
aberrations  in these organs.  Cadmium was found to accumulate in the gills,
liver and kidney, but only minor amounts occurred in muscle tissue.

     This report was submitted  as partial fulfillment of Grant R-804283 by
the Oregon Department of Fish and Wildlife under the partial sponsorship of
the U. S.  Environmental  Protection Agency.  This report covers the period
January 5, 1976 to December  20, 1976.
                                       IV

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                                CONTENTS

                                                                   Page

DISCLAIMER	    ii
FOREWORD	   i i i
ABSTRACT	    iv
FIGURES	     v
TABLES	    i x
ACKNOWLEDGMENTS	     xi

     I   CONCLUSIONS	     1
    II   RECOMMENDATIONS	     3
   I I I   INTRODUCTION	     4
    IV  METHODS	     5
          EXPERIMENTAL FISH	     5
          EXPOSURE TO TOXI CANT	     5
          TOLERANCE TO SEAWATER	     8
          GILL ATPASE ACTIVITY	     8
          DOWNSTREAM MIGRATION	     8
          OSMOTIC AND IONIC REGULATION	     8
          ASSESSMENT OF COEFFICIENT OF CONDITION AND GROWTH	     9
          HISTOLOGICAL EXAMINATION AND TISSUE CADMIUM LEVELS	     9
          WATER QUALITY	     9
     V  RESULTS AND DISCUSSION	    11
          ACUTE LETHAL I TY TESTS	    11
               Cadmium	    11
               Chromium	    14
               Nickel	    14
               Mercury	    14
               Zinc	    15
          EFFECT OF METAL EXPOSURE ON GROWTH AND SURVIVAL	    16
               Cadmium	    16
               Zinc	    16
          EFFECTS OF METAL EXPOSURE ON GILL ATPASE ACTIVITY AND
          SURVIVAL IN SEAWATER	    17
               Cadmium	    17
               Zinc	    24
               Chromium and Nickel	    26
               Mercury	    27
          EFFECT OF METAL EXPOSURE ON PLASMA OSMOTIC PROPERTIES...    28
          EFFECT OF METAL EXPOSURE ON CORTICOSTEROID RESPONSE	    28
          EFFECT OF METAL EXPOSURE ON DOWNSTREAM MIGRATION	    30
               Cadmium	    30
               Zinc	    34
    IV  DISCUSSION	    37

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                                                                    Page
REFERENCES
APPENDICES
     APPENDIX
     APPENDIX
              II
     APPENDIX III
             RECOMMENDED SEAWATER-ENTRY TEST PROCEDURE
             PARTIAL CHARACTERIZATION OF GILL (NA, K)-
             STIMULATED, OUABAIN SENSITIVE ADENOSINE
             TRIPHOSPHATASE FROM COHO SALMON, Oncorhynchus
             kisutch
             . HISTOLOGICAL EFFECTS OF SEVERAL METALS  IN
              SELECTED TISSUES OF YEARLING COHO SALMON
                                                                     60
APPENDIX IV.  CADMIUM UPTAKE AND ACCUMULATION IN COHO SALMON.
                                                                     74
                                                                     79

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                                 FIGURES

Number                                                              Page

  1   Diagrammatic sketch of flow-through diluter	    6

  2   Exposure tanks with diluter in background	    7

  3   Mortality of yearling coho salmon exposed to various cadmium
     concentrations	   12

  k   Median mortality-time of yearling coho salmon exposed to
     solutions of cadmium	   13

  5   Effect of zinc, copper and zinc-copper mixtures on
     coefficient of condition of yearling coho salmon	   17

  6   Percent survival of yearlng coho salmon exposed to cadmium
     in freshwater (static) and subsequent survival  upon transfer
     to seawater	   18

  7   Percent survival of yearling coho salmon exposed to cadmium in
     freshwater (flow-through) and subsequent survival  upon
     transfer to seawater	   19

  8   Percent survival of yearling coho salmon exposed to cadmium in
     freshwater (static) and subsequent survival  upon transfer to
     seawater	   23

  9   Gill  (Na, K)-stimulated ATPase activity of yearling coho
     salmon during exposure to cadmium, zinc and cadmium-copper
     mixture	   2k

 10   Percent survival of yearling coho salmon exposed to inorganic
     mercury (HgC^) in freshwater (static) and subsequent
     survival upon transfer to seawater	   27

 11   Effect of cadmium exposure in freshwater on the osmolality
     of plasma of coho salmon	   29

 12   Effect of cadmium exposure on the chloride ion  concentration
     of plasma of coho salmon	   29
                                      VI I

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Number                                                             Page

 13  Percent downstream migration of yearling coho salmon
     previously exposed to cadmium  in  freshwater and released
     April  l*t, 1976	  32

 ]k  Percent downstream migration of yearling coho salmon
     previously exposed to cadmium, copper  and  cadmium-copper
     mixtures  in  freshwater  and  released  May 5,  1976	  33

 15  Percent downstream migration of yearling coho salmon
     previously exposed to zinc, copper and zinc-copper
     mixtures  in  freshwater  and  released  May 26,  1976	  35

 16  Percent migration of yearling  coho salmon  previously
     exposed to zinc, copper and zinc-copper mixtures  in
     freshwater and  released June 9, 1976	:	  36
                            APPENDIX  FIGURES

Number                                                              Page

   1   Relationship  of  gill  (Na,  K)-stimulated  ATPase  activity
      of coho salmon;  A.  pH,  B.  tissue concentration,  C.  ouabain
      concentrat ion,  D.  d i sodi urn ATP	   67

   2  Relationship  of gill  (Na,  K)-stimulated  ATPase  activity
      of coho salmon:  A.  MgCK,  B.  (Na + K)  concentration,
      C. ratio of NaCl/KCl, D.  KC1	   68

   3  Relationship  between Nad  concentration  and  gill  (Na,  K)-
      stimulated ATPase  activity	   69

   k  Gill  (Na,  K)-stimulated ATPase activity	   71
                                        VI II

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                                 TABLES

Number                                              .              Page

  1   CHEMICAL AND PHYSICAL CHARACTERISTICS  OF TEST  WATER  (STATIC
     AND FLOWING-WATER SYSTEMS)	   10

  2   SURVIVAL OF YEARLING COHO SALMON EXPOSED TO  MERCURIC
     CHLORIDE IN FRESHWATER FOR ]kk h (APRIL  20-26,  1976)	   15

  3   SURVIVAL OF YEARLING COHO SALMON WITH  EITHER REST  PERIODS OR
     IMMEDIATE TRANSFER TO SEAWATER FOLLOWING EXPOSURE  TO  CADMIUM
     FOR 1 kk h	   20

  k   SURVIVAL OF YEARLING COHO SALMON EXPOSED TO  CADMIUM  IN
     FRESHWATER AND THEIR SUBSEQUENT SURVIVAL AFTER TRANSFER
     TO SEAWATER	   21

  5   SURVIVAL OF YEARLING COHO SALMON EXPOSED TO  CADMIUM OR
     A CADMIUM-COPPER MIXTURE IN FRESHWATER AND THEIR SUBSEQUENT
     SURVIVAL AFTER TRANSFER TO SEAWATER	   22

  6   SURVIVAL OF YEARLING COHO SALMON EXPOSED TO  ZINC OR A
     ZINC-COPPER MIXTURE IN FRESHWATER AND  THEIR  SUBSEQUENT
     SURVIVAL AFTER TRANSFER TO SEAWATER	   25

  7   SURVIVAL OF YEARLING COHO SALMON EXPOSED TO  CHROMIUM
     OR NICKEL IN FRESHWATER AND THEIR SUBSEQUENT SURVIVAL
     AFTER TRANSFER TO SEAWATER	   26

  8   PERCENT MIGRATION THROUGH JULY 6, 1976 OF YEARLING COHO
     SALMON RELEASED INTO A SMALL COASTAL STREAM  FOLLOWING ACUTE
     OR CHRONI C METAL EXPOSURE	   31
                                     IX

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                             APPENDIX TABLES

Number                                                              Page
  1   MEASURED CONCENTRATIONS OF CADMIUM AND COPPER DURING
     CHRONIC EXPOSURE TESTS
  2  MEASURED CONCENTRATIONS OF ZINC AND COPPER DURING CHRONIC
     EXPOSURE TESTS .................................................   50

  3  MEASURED CONCENTRATIONS OF THE METALS; CHROMIUM, NICKEL,
     AND MERCURY DURING ACUTE EXPOSURE TESTS ........................   51

  k  MERCURY CONTENT OF SELECTED TISSUES OF COHO SALMON EXPOSED
     TO VARIOUS CONCENTRATIONS OF MERCURIC CHLORIDE .................   52

  5  EFFECTS OF CADMIUM OR CADMIUM-COPPER EXPOSURE ON AVERAGE
     LENGTH, WEIGHT, AND CONDITION FACTOR OF COHO SALMON ............   53

  6  EFFECTS OF ZINC-COPPER EXPOSURE ON AVERAGE LENGTH, WEIGHT,
     AND CONDITION FACTOR OF COHO SALMON ............................   5^
  7  GILL  (NA, K) -STIMULATED ATPASE ACTIVITY OF COHO SALMON
     EXPOSED TO CADM I UM FOR 1 Mt h ...................................  55

  8  GILL  (NA, K)-STIMULATED ATPASE ACTIVITY OF COHO SALMON
     CHRONICALLY EXPOSED TO CADMIUM OR CADMIUM-COPPER MIXTURE ......   56

  9  GILL  (NA, K)-STIMULATED ATPASE ACTIVITY OF COHO SALMON
     CHRONICALLY EXPOSED TO ZINC AND ZINC-COPPER MIXTURES, WITH
     SUBSEQUENT SALTWATER CHALLENGE AND FRESHWATER RECOVERY ........   57

  10  GILL  (NA, K)-STIMULATED ATPASE ACTIVITY OF COHO SALMON
     EXPOSED TO MERCURY FOR iMt h ..................................   58
  11  PLASMA  SERUM OSMOLARITY AND PLASMA CHLORIDE LEVEL OF COHO
     SALMON  EXPOSED TO CADM I UM .....................................   59

  12  PERCENT MIGRATION (TO JULY 6,  1976) OF YEARLING COHO SALMON
     RELEASED  INTO A SMALL COASTAL  STREAM FOLLOWING ACUTE AND
     CHRON I C EXPOSURE TO SEVERAL METALS ............................   60

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                              ACKNOWLEDGMENTS

     This investigation was supported in part by the U.  S. Environmental
Protection Agency, Research Grant R-804283, and was funded through the
Corvallis Environmental Research Laboratory, Corvallis,  Oregon.  Many
contributed to this study and their assistance is gratefully acknowledged:
Dr. G. A. Chapman, Project Officer, Western Fish Toxicology Station (WFTS),
EPA provided technical assistance and made arrangements  with CERL for
chemical water analyses. Mr. Joel McCrady (chemist, WFTS)  carried out the
analyses for the metals cadmium, copper, zinc, chromium  and nickel in water.
Dr. D. R. Buhler, Agriculture Chemistry, OSU, provided equipment and
technical assistance in the analysis of mercury.  Mr.  B. Doerge and Mrs. S.
Glenn assisted in the laboratory and literature search.

     The authors gratefully acknowledge Drs. G. A. Chapman, R.  D. Ewing,
A. V. Nebeker, C. B. Schreck, and H. H. Wagner who provided constructive
criticism on the manuscript.
                                      xi

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                                  SECTION I

                                  CONCLUSIONS

1.  Cadmium was the most toxic of the metals tested to yearling coho salmon;
    96-h LC50 of 10.4 yg/liter in January.

2.  Few deaths occurred when yearling coho salmon previously exposed to 4 yg/liter Cd died in a  dose-dependent manner following
    transfer to seawater.

3.  Coho salmon yearlings given a 5~day rest (non-toxicant exposure period)
    between Cd exposure and transfer to seawater exhibited survival com-
    parable to the control  groups when challenged with seawater.

4.  Yearling coho salmon previously exposed  to salts of nickel  or chromium
    (<5 mg/liter) survived  the seawater challenge, whereas fish exposed to
    mercury (50-300 yg/liter)  died in a dose-dependent manner upon transfer
    to seawater.

5.  Gill (Na, K)-stimulated ATPase activity  in yearling coho salmon was
    not affected by cadmium, chromium, nickel  or zinc salts.
6.  Yearling coho salmon exposed to f_4.5 yg/liter Cd for 144 h did not
    migrate as well as control  fish following a release into a natural
    stream.  However, fish chronically exposed to Cd (47 to 70 days)  and
    Zn (27 days), respectively, showed acclimation to the toxicants and
    exhibited migration similar to the controls.

7.  The (Na, K)-stimulated ATPase activity of the gills of chronically
    exposed yearling coho salmon was suppressed and downstream migration of
    the released fish was reduced when 10 yg/liter Cu was combined with the
    Cd or Zn.  Low levels of mortality also occurred when the Cd-Cu or Zn-
    Cu exposed fish were challenged with seawater.

8.  Neither Cd nor Zn exposures appeared to affect feeding, growth or
    survival during exposures of 70 and 27 days respectively; however,
    addition of 10 yg/liter Cu for only 6 days to the Zn-exposed fish signi
    ficantly reduced their condition factor.

9-  No histological changes were noted in examination of liver,
    kidney, or gill tissues of yearling coho salmon chronically exposed to
    Cd or Zn either singly or in combination with Cu.

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10.   Exposure of yearling coho salmon to Cd in freshwater did not affect
     their ability to maintain normal blood plasma osmotic pressure and
     chloride ion concentration.

11.   Copper produced a marked dose-dependent cortisol  stress response.
     Cadmium, even at concentrations that produced death, did not elicit a
     plasma cortisol stress response in yearling coho salmon.  Cadmium
     exposed fish were capable of a stress response as noted by handling or
     seawater challenge tests.

12.   Cadmium was found to accumulate in the gills, liver, and kidneys of
     yearling coho salmon but only a minor amount was found in the muscle
     tissue.  The bioaccumulation appeared to be dependent both on duration
     of exposure and exposure concentration.

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                                  SECTION II

                                RECOMMENDATIONS

1.   The techniques and methodology developed in this and the prior study
     (Lorz and McPherson 1977) are recommended for the determination of
     pollutant effects on anadromous fishes.  The Seawater Entry Test
     (Appendix 1) is a quick method for determining if pollutants in fresh-
     water could be potentially harmful to the downstream migration and
     subsequent seawater life history phases.

2.   Prudence in applying the results of this study directly and quantita-
     tively to field situations is recommended since other factors such as
     acclimation, water quality, presence of other chemicals, disease,
     organic content of the water, etc., could alter the reported responses
     of yearling coho salmon to a given toxicant concentration.

3.   Additional  research should be conducted to determine if the inter-
     ference observed with smelting of anadromous salmonids by sublethal
     levels of metals or their combination (Cu,  Cd-Cu, Zn-Cu) is a general
     phenomenon.  Research on affects of toxicant exposure to earlier life
     stages and subsequent effects on smolting are also needed.

k.   Another potential area of research would be to determine if application
     of toxicants to a natural stream and its fish population would produce
     results similar to those noted in the laboratory study.

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                                 SECTION I I I

                                INTRODUCTION

     Potential metal contamination of waterways may occur from industrial,
thermal-electric power generation, agricultural, municipal, mining and
natural sources.  Considerable research has been done to determine safe
limits of various metals for survival and growth of juvenile salmonids in
freshwater, but there is little information on effects of metals affecting
migration and survival of fish in seawater.   The need for this information
is recognized in the Pacific Northwest where large populations of anadromous
salmonids are a valuable commercial and sport fishery resource.

     Studies carried out in Eastern Canada indicated that copper-zinc mining
pollution interrupted the upstream migration of adult Atlantic salmon, Salmo
salar, and caused considerable numbers to return downstream (Sprague, El son
and Saunders 1965, Saunders and Sprague 1967).  Laboratory studies by
Sprague (l96Aa, 1964b) and Sprague and Ramsay (1965) determined not only the
lethal levels of copper and zinc,  (singly and in combination)  but also the
concentrations that produced avoidance behavior in juvenile Atlantic salmon.
From the laboratory data, Sprague, Elson and Saunders (1965) postulated that
the combined affect of severe pollution in most years, plus the necessity of
a 3 year juvenile residency, resulted  in lowered salmon smolt production in
the Miramichi River.  Similarly, Sb'dergren (1976) suggested the decline of
the Atlantic salmon population from the Ricklea, a small salmon river in
northern Sweden, was due to heavy metal pollution of the stream's ecosystem.

     This report  is the second of a three part study by the Oregon Depart-
ment of Fish and Wildlife and funded  in part by the Environmental Protection
Agency.  In the previous study, Lorz and McPherson  (1977) noted deleterious
effects of copper exposure  (sublethal  levels in freshwater) on yearling coho
salmon survival when transferred to seawater, a decrease in the  (Na, K)-
stimulated ATPase activity of the gill, and a reduced migratory tendency
when copper-exposed fish were placed  into a natural stream.  The present
study  examined similar parameters  (tolerance to seawater,  (Na, K)-stimulated
ATPase activity,  and migratory disposition) following freshwater exposure to
the metals cadmium, zinc, and combinations of cadmium-copper and zinc-
copper.  In addition, tolerance of yearling coho salmon to seawater was
determined after  exposure to chromium, nickel, and mercury salts.

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                                  SECTION IV

                                    METHODS

EXPERIMENTAL FISH

     All experiments were conducted on coho salmon between ages of 12 and 17
months.  Coho salmon of the 197^ year class^ from Fall Creek Salmon Hatchery
(Alsea River, Oregon) were used.  Fish were hatched and reared from fertil-
ized eggs brought into the laboratory under conditions similar to those
reported by Lorz and McPherson (1977).

EXPOSURE TO TOXICANT

     Yearling coho salmon were exposed to cadmium (CdCl2'2.5 H£0) for
periods ranging from 6 to 70 days, zinc (ZnCl2)  for up to 28 days, mixtures
of cadmium-copper for 6 days, and mixtures of zinc-copper for up to 21 days.
Five 96-h LC50 static tests were conducted in 0.61 m diameter fiberglass
tanks with four metallic compounds: two tests were run with cadmium (Cd)
and one test each was run with chromium (KoCroOy) , mercury (HgC^), and
nickel (NiCl2*6H20) •  In the static tests (l^-n exposure) the water was
continuously aerated, and 85% of the 120 liters  of test water was exchanged
once each day.  Toxicant solutions were mixed in a separate container prior
to introductions to the tanks.  Total metal  concentrations of daily water
samples were assayed (by WFTS) by flame or flameless atomic absorption
spectrophotometry and there was no detectable loss of metal from the water
during any 2^-h period.  Results of the AA analysis taken during the con-
tinuous exposure indicated that the mean weekly  measured concentration
approximated the nominal concentrations (Table A-l to 3).  Water temperature
was maintained at 10 + 1°C in all static tests.
     All exposures of >}kk h were conducted in a flowing water system.  This
system consisted of a gravity flow diluter (Fig. 1) that delivered 12 L/min
to each of ten 1.5^ m diameter fiberglass tanks (five concentrations repli-
cated, Fig. 2).  The background concentration of cadmium and zinc in the
well water was <1 yg/L and <^ yg/L, respectively.   A volume of 1000 L was
maintained in each of the tanks, and 35% of the water was replaced every
3-7 h.  Submersible pumps were used in each tank to provide additional
current, aeration and mixing.
 Year class refers to year of spawning.

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        in  Water inlets with                                                         .,  ,.
        IJJ  */"'<''/ />*^
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Figure 2.  Exposure tanks with diluter in background.
     Smaller groups of fish were exposed periodically to the flowing toxi-
cant in 0.61 m diameter fiberglass tanks.   Water flow (5 L/min;  95% replace-
ment every 1.5 h) to these 120-liter tanks was provided by a siphon from
mid-depth in the 1.5^ m chronic exposure tank, with no difference in water
quality between tanks.  All tanks were covered to prevent loss of fish.

     Starting in April 1976, 10 yg/L Cu was added for 6 days (with a Mariotte
bottle) to four of the tanks of fish that  were receiving Cd and  one control
tank.  Similarly, four of the zinc exposure tanks received 10 yg/L Cu for up
to 21 days in May and June 1976.

     Cadmium and zinc concentrations chosen for chronic study were deter-
mined from prior static toxicity tests.  Total metal  concentration was
analyzed at least once per week from each  exposure tank (from a  composite
water sample) by atomic absorption spectrophotometry.  Water and toxicant
flows in the diluter were checked at least once daily and only occasional
minor adjustments of flow were required.

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TOLERANCE TO SEAWATER

     Ten to 20 fish from each exposure  tank  (containing concentrations of
Cd, Zn, Cd-Cu, Zn-Cu or well water) were  periodically  tested  for tolerance
to seawater and percent survival  provided  a  presumptive measure of osmoreg-
ulatory ability.   Fish were  transferred directly  from  the  exposure tanks to
0.61 m diameter fiberglass tanks  containing  120 L of natural  seawater and
held for at least  10 days.   Eighty-five percent of the 120 L  was exchanged
daily  and the  seawater was continuously aerated.   Water was maintained at a
salinity of 30 +_ 0.5 °/oo and a  temperature  of  10 +_ 1°C.   Procedure  for a
seawater challenge test that can  be used  to  survey the possible detrimental
effect of any  chemical  (in freshwater)  is  given  in Appendix  I.

GILL ATPASE ACTIVITY

      (Na, K)-stimulated ATPase activity was  measured by the whole gill
method (Johnson et al.  1977).  Conditions  for optimal  enzyme  activity were
determined  from appropriate  characterization curves (Appendix II).   In
addition,  (Na,  K)-stimulated ATPase  in  microsomes isolated from gill fila-
ments  was assayed  by  the  procedures of  Zaugg and  McLain  (1970).  The
 (Na,  K)-stimulated ATPase activity was  measured on four to six fish  per
metal  concentration.   The whole  homogenate assay  procedure was a more
convenient  assay  because  it  required  no centrifugation and thus allowed for
a  larger  number of samples  to  be run  as well as allowing  freezer storage of
the  samples for up to 2 weeks  prior  to  analysis.

DOWNSTREAM  MIGRATION

      The  effect of exposure  to metals (Cd, Zn,  Cd-Cu and  Zn-Cu) on the coho
salmon smelt's migratory  disposition  was  assessed by  releasing marked con-
trol  and  metal-exposed fish  into Crooked  Creek,  a tributary  of the North
Fork of the Alsea River and  monitoring  their migration to a  trap 6.4 km
downstream.   The  trap was built  into  a  permanent  weir  and was checked daily
for  the first 10  days following  release.   On the  day prior to release, 50 to
 100  fish  from each exposure  tank were anesthetized in  MS-222, weighed,
measured, and identified  by  freeze branding  and  a fin  clip.   To  insure that
all  fish  in the migration studies had attained  the smolt  size no fish smaller
than  11  cm  were  released.  Seaward migration  of wild coho  salmon juveniles
began  at  the  trap in  late April,  peaked in late May and ceased by  late June.
Releases  of metal-exposed and  control fish were made between  April 14 and
June  9>  1976.   Trapping was  terminated  July  6,  1976 almost one month after
the  last  release.   The stream  was electrofished on July 6 and 7, 1976 from
above  the  release points  down  to the  weir, in an  attempt  to  collect  fish
that  had  not  migrated.

OSMOTIC AND IONIC REGULATION

      Changes  in osmolality and  chloride ion  (Cl~) concentration  in blood
plasma were monitored by  sacrificing  groups  of  yearling fish  at various
times  after the  initiation of  toxicant  exposure.   The  methods and equipment
used  in  the analysis  were outlined by Lorz and  McPherson  (1977)-


                                        8

-------
ASSESSMENT OF COEFFICIENT OF CONDITION AND GROWTH

     Each month, approximately ^40 fish were randomly selected from each
exposure tank after a 24-h starvation period, anesthetized, weighed, and
measured.  Individual fish were weighed to 0.1 g and fork length was deter-
mined to 0.1  cm.  The coefficient of condition was determined for each fish
in the sample using the formula K = 100 W/L^, where W denotes weight in
grams, and L denotes fork length in centimeters (Hoar 1939)-

HISTOLOGICAL EXAMINATION AND TISSUE CADMIUM LEVELS

     Histological sections of several  tissues were prepared from fish
previously exposed to metals and an assessement of histopathological effects
was made (Appendix III).  Similarly, levels of cadmium and mercury accumu-
lation in various fish organs were determined (Appendix IV, Table A-4).

WATER QUALITY

     The source of water for the study was wells located approximately  one
mile east of the Willamette River, Linn County, Oregon and the chemical
characteristics were measured by Corvallis Environmental Research Laboratory
(CERL) (Lorz and McPherson 1977).  Alkalinity, hardness, dissolved oxygen,
pH, and ammonia in the exposure tanks  were measured routinely at the labora-
tory and these chemical  characteristics are presented in Table 1.  Water
temperatures were monitored continuously in both the static and flow-through
systems with recording thermographs.

-------
TABLE 1.  CHEMICAL AND PHYSICAL CHARACTERISTICS OF TEST WATER
          (STATIC AND FLOWING-WATER SYSTEMS).

Charac-
istic
Alkal inity
Hardness
Dissolved
oxygen
Ammon i a
PH
Unit
mg/L as
CaCO^
mg/L
mg/L
mg/L
NH3-N

Year
1975
1976
1975
1976
1975 static
1975 flowing
1976 static
1976 flowing
1975
1976
1975 static
1975 flowing
1976 static
1976 flowing
Mean + SD
72 + 3
76 + 4
93 + 3
90 + 3
9.8 + 0.3
8.7 + 0.9
9-9 + 0.9
9-5 + 0.8
0.39 + 0.18
0.15 + 0.07
7.30
7.26
7.59
7.03
Range
66 - 81
70 - 83
84 - 98
85 - 93
9.4 - 10.1
6.2 - 10.9
7.8 - 11.1
6.6 - 10.6
0.12-0.57
0.08-0.21
6.81-7.54
7.07-7.54
7.00-7.82
6.48-7-37
n
49
13
32
13
8
52
34
48
7
8
6
42
16
7
                               10

-------
                                  SECTION V

                            RESULTS AND DISCUSSION

     From January to July 1976, five static toxicity tests using four metals
were completed.  These tests included acute lethality determinations,
effects on gill (Na, K)-stimulated ATPase activity, and effects on the
ability of juvenile coho salmon to adapt to seawater following exposure to
metals in freshwater.  Additionally, 10 tests of tolerance to seawater were
completed using yearling coho salmon previously exposed to either Cd or Zn
in the flowing freshwater toxicant system.  Included in these seawater
tolerance tests were groups of Cd exposed fish that had received a fresh-
water recovery period prior to the seawater and also groups of yearling coho
salmon exposed to mixtures of Cd-Cu or Zn-Cu.  During the 1976 migratory
period four releases of yearling coho salmon were made into a small  coastal
stream and the downstream movement of migrants monitored.

ACUTE LETHALITY TESTS

Cadmium

     In January-February 1976, the 96-h LC50 was about 10.4 yg/L Cd; but in
May it appeared to be considerably higher as only a 10% mortality was noted
at a concentration of 10.3 pg/L Cd (Fig. 3)-  This decrease in sensitivity
may have been related to increasing size and age of the coho salmon.  Unlike
the results with Cu (Lorz and McPherson 1977) the parr-smolt transformation
did not appear to cause the coho salmon to be more sensitive as smolts to
Cd.  The reason for lack of 100% deaths at the 30 yg/L group in the  January
test is unknown, however Chapman, (personal communication) noted the
occasional similar occurrence of incomplete kill at high concentration in
his studies.  When the "incipient lethal level" (ILL) was plotted following
the technique of Sprague (I964a), the ILL for Cd was about 9-0 ug/L  (Fig.
k).  A sharp differentiation between lethal and non-lethal concentrations
for Cd occurred, and the line relating concentration to survival time breaks
and runs nearly parallel to the time axis (Fig. k).  The longest LT  50 (time
to 50% mortality)  in any test was 138 h for one test tank.  Generally the LT
50 for Cd occurred from 82 to 130 h (Fig. k).  Time to 50% death of  coho
salmon was considerably greater with Cd than previous studies with Cu or
Zn (Lorz and McPherson 1977).
                                      11

-------
  99.99n
<
O
CD
o
a:
CL
a:
o
LU
o
cr
LU
Q.
 99-1



 95

 90




 70


 50



 30




 10


  5
0.2
                                      •  JAN. 21-27, 1976 (STATIC)

                                      o  FEB. 6-12, 1976 (FLOWING)

                                      •  MAY 19-25, 1976 (STATIC)

                                      X  ABERRANT VALUES
                              6     10      20      40

             CONCENTRATION  OF CADMIUM
  Figure 3.  Mortality of yearling coho salmon exposed to various cadmium

  concentrations (line fitted by eye with aberrant values excluded).
                               12

-------
iooo:
2 600-
>: 400-
or 200-
§ 150-
0 '00:
in 80
g 60-
^ 40-
*~
20

S$ 44 4&


(44«/
J
(30%;


^So ..
'^L
^>S3 O
Jan 21-27/76 Feb 6-12/76 ^
(nominal concentration)
0-3 »-4
O-6 +-8
0-9
A-12 A-12
0-15
0-30
                            23    579      20    40

                          CONCENTRATION  OF CADMIUM
Figure 4.  Median mortality-time of yearling  coho salmon  exposed  to  solutions
of cadmium.


     Our 96-h LC50 values for Cd (10.4 yg/L)  for  yearling coho  salmon are
higher than those reported by Chapman (1975)  for  coho  salmon of the  same
stock (2.7 pg/L for 6 mo parr).   Our water alkalinity  and hardness were
approximately four times greater than those reported  in Chapman's study.
Our LC50 values are similar to that reported  by Ball  (196?) f°r rainbow
trout, Salmo gairdneri (15 yg/L) in water with a  hardness of 290 mg/L as
CaCOo.  Other researchers have recorded LC50  concentrations of  400-8000
yg/liter Cd for various warm water and spiny-rayed fish  (Chapman  1973).  As
noted by other researchers, despite the high  toxicity  of  cadmium, the re-
sponse of the fish is initially very slow, even at high Cd-concentrations.
Sauter et al. (1976)  found brook trout fry survival  (in soft water)  through
30 days post hatch was not affected by exposures  to Cd concentrations as
high as 47 yg/L,  but total length of fry (at these concentrations)  was
significantly reduced.  However survival  was  significantly reduced during
the 60-day exposure to test concentrations of cadmium  >6  yg/L.  In hard
water, Sauter et al.  (1976) found brook trout fry survival  significantly
reduced during 60 days of exposure to 21  and  12 yg/L Cd,  indicating  a cumulative
effect during the 30-60 day exposure period.
                                      13

-------
Chromi urn

     No coho salmon mortalities occurred during our chromium toxicity test,
even at 5 mg/L, a concentration 50 times the recommended acceptable concen-
tration (0.10 mg/L, U.S.E.P.A. 1976).  Benoit  (1976) noted that the 96-h
LC50 values of hexavalent chromium for rainbow trout and brook trout were 69
and 59 mg/L Cr, respectively and believed the difference between species was
due to difference in age of fish used.  Warm water fish species also are
relatively  insensitive to hexavalent chromium as noted by their 2k- and 96-h
LC50 concentrations of 40-280 and 20-130 mg/L chromium, respectively (Trama
and Benoit  I960, Pickering and Henderson 1966, Ruesink and Smith 1975).

     Holland et al. (I960) found that trivalent chromium was more toxic to
coho salmon than hexavalent chromium, although chromium sulfate (trivalent)
precipitated to form non-ionized chromous carbonate at concentrations
<50 mg/L.  Mills (1975) noted very steep mortality curves for trivalent Cr
with yearling coho salmon in seawater.  Although the' above cited authors
note significant mortalities to juvenile-yearling salmonids  (LC50's from 30-
70 mg/L), Olson and Foster (1956) noted that survival and growth of alevin
chinook salmon and rainbow trout were affected by concentrations as low as
0.02 mg/L Cr (hexavalent) during A months of exposure and this agreed
closely to  the findings of Benoit (1976) who used rainbow and brook trout.
Strik et al. (1975) noted that 25 percent of the rainbow trout exposed to 10
mg/L Cr  (hexavalent) for  15 days showed decreased activity and food intake.
They also observed swollen blood vessels in the intestine and evidence of
liver damage and impaired gill and kidney function.  Buhler et al.  (1977)
reported on tissue accumulation and effects of hexavalent chromium on
enzmatic activity  in two  strains of rainbow trout. Benoit (1976) suggests
an application factor  (MATC/96-h LC50) for brook trout and Cr would be
between 0.003 and 0.006.  However, Sauter et al. (1976) suggest that the
current  recommendations for chromium  (100 yg/L, U.S.E.P.A. 1976) are not
stringent enough, and  the maximum permissible  concentration of chromium in
the aquatic environment should be reduced.

Nickel
      Doudoroff and  Katz  (1953)  noted  that  toxic  concentrations of nickel
 ranged  from  1.0 mg/L  to  10 mg/L depending  on  fish  species, pH, exposure
 time, synergism,  etc.  Pickering (197*0  found  the  96-h  LC50 of nickel for
 fathead minnows was 32 mg/L  Ni.   However,  he  noted  that a nickel concen-
 tration of 0.73 mg/L  caused  a  significant  reduction  (following chronic
 exposure)  in both the number of eggs  per spawning  and the hatchability of
 the eggs.  Rehwoldt et al.  (1971,  1972)  noted  that  Ni was less toxic
 (96-h LC50 6.2-46.2 mg/L)  than copper or zinc  to several fish species from
 the Hudson River.   In our  study no mortalities occurred in yearling coho
 salmon  following  144-h exposure to Ni  even at  concentrations of 5 mg/L.

 Mercury

     The estimated  96-h  LC50 for coho salmon  yearlings  exposed to a series
 of different inorganic mercury (HgC^) concentrations was 2AO yg/L Hg

-------
 (Table 2).  Similar values were reported for juvenile  rainbow trout by
MacLeod and Pessah (1973) who found 96-h LC50 values for HgCl2 of 220, 280,
and 400 yg/L, at 20, 10 and 5°C, respectively.

            TABLE 2.   SURVIVAL OF YEARLING COHO SALMON EXPOSED TO
                      MERCURIC CHLORIDE IN FRESHWATER FOR 144 h
                      (APRIL 20-26, 1976).

Nominal concentration                        Percent survival
    yg/L Hg	 (144-h exposure)"3
0
30
50
100
200
300
400
500
100
100
100
100
61.9
4.8
0
0
(20)
(19)
(22)
(20
(21)
(21)
(20)
(21)
 Number of fish exposed in parentheses.

     Due to microbial action, inorganic mercury is known to change chemical
form when released into the natural aquatic environment.  It has been shown
that microorganisms present in bottom sediments, fish intestine and the
external mucus of fish synthesized methylmercury from inorganic mercury
(jernelov and Lann, 1970-  Fish can absorb both inorganic and organic forms
of mercury, although metabolic pathways, accumulation and excretion rates
are quite different (Jensen and Jernelov 1969, Jernelov and Lann 1971,
Hannerz 1968).  Buhler et al. (1973) reported that 60-80% of the total
mercury present in freshwater and estuarine fish is in the methylmercury
form.  The 96-h LC50 concentration of methylmercury for juvenile coho salmon
was found to be 38.9 yg/L (McPherson 1973),  which is six times more toxic
than the HgCl2 used in this study.

     Tissue analysis for Hg showed a typical  pattern  of absorption-accumu-
lation as described by Jernelov and Lann (1971).  Our analysis revealed
highest total  Hg concentrations occurred in the gills, while kidney and
liver tissue contained lesser but significant amounts.  Very small  amounts
of total Hg were found in the muscles and gonads (Table A-4) .

Zinc

     No acute lethality tests were carried out for zinc this year,  however,
tests carried out in 1974 (Lorz and McPherson 1977) gave 96-h LC50 values of
4600 yg/L Zn for yearling coho salmon.   There was  also an indication that
fish became increasing sensitive to Zn  during smolting.  Zitko and Carson
(1977) noted that the incipient lethal  level  (ILL)  of zinc to juvenile
Atlantic salmon (in freshwater with a hardness of  14  mg/L) varied from 150
to 1000 yg/L as a function of season and developmental stage of the fish.

                                      15

-------
They noted that the  ILL  increased from 500 to 1000 yg/L Zn during the first
year but reduced to  150  yg/L Zn the following spring.  Zitko and Carson
suggest that the most sensitive stage in the Altantic salmon's life history,
as evidenced by decreased  ILL, coincides with and is probably related to the
pari—smolt transformation.

     Herbert and Wakeford  (1964) noted that the resistance of yearling
rainbow trout and Atlantic salmon smolts to zinc sulphate increased with
salinities up to 30-40%  seawater, in which these species could withstand
concentrations of zinc sulphate 13 times greater than those in freshwater
during 48 h of exposure.   If the salinity was increased to 72% seawater then
a reduction in tolerance to the zinc salts occurred.  Salmon were more
susceptible to zinc  poisoning than trout in freshwater and at all salinities
tested.

EFFECT OF METAL EXPOSURE ON GROWTH AND SURVIVAL

Cadmium

     Coho salmon in  the  higher concentrations of Cd did not appear to feed
quite as actively as the controls.  However, even yearling coho salmon held
under control conditions for 70 days starting in late February 1976 experi-
enced little additional  growth, and the Cd concentrations tested did not
appear to reduce growth  (Table A-5)•

     Yearling coho salmon subjected to 10 yg/L Cu for the last 5 days of 68-
day exposure to Cd showed no apparent ill effects on growth or coefficient
of condition (Table A-5) as had been noted previously with coho salmon (Lorz
and McPherson 1977) or goldfish and carp (Ozaki  et al. 1970)  exposed to just
Cu.  Almost no mortality occurred during 68 days of exposure to Cd although
accumulation of Cd occurred in some tissues (Appendix IV).

Zinc

     Yearling coho salmon were exposed to Zn (ZnC^) for 27 days in May and
June with half of the tanks also receiving 10 yg/L Cu for the last 20 days.
No mortalities occurred during the exposure.  Coefficient of condition was
significantly lower  in groups that received only Cu or Zn-Cu mixtures as
compared to the controls or the Zn-only exposed fish (Fig. 5,  Table A-6).
Coho salmon receiving Cu or Zn-Cu exposures did not feed as readily as
controls or Zn-only  groups but did feed better than coho salmon exposed to
higher Cu concentrations in a previous study (Lorz and McPherson 1977).  The
effect of Cu and Zn-Cu mixtures in lowering the coefficient of condition was
evident after only 6 days of Cu exposure (Table A-6).  It appears that Cu
may not only reduce  the  feeding response, but also may increase the metabolic
rate when combined with  Zn, causing a reduction in condition factor.
                                      16

-------
•z.
o
1 	
1^
Q
Z
0
O
u_
o
I-
z
UJ
o
COEFFI


1.06-
1.05-
1.04-
1.03-
1.02-
i.oi-
1.00-
0.99-
0.98-
0.97-
0.96-
0.95-
y
X
0.90



I —




:
                                                  Q  \*2n for 27 days

                                                  H*Zn only for 7 days and
                                                      Zn~Cu  for 20days(IO>tg/LCu3
                                                   I»95% C.I. on mean
CONTROL  4OO      800     1600

          ZINC  CONCENTRATION
                                                   2400
Figure 5-  Effect of zinc,  copper and zinc-copper  mixtures on  coefficient
of condition of yearling coho salmon.
EFFECTS OF METAL EXPOSURE ON GILL ATPASE ACTIVITY  AND  SURVIVAL  IN SEAWATER

Cadmium

     Exposure of coho salmon during January  and  February  to  concentrations
of Cd >k yg/L usually caused death of at least 20% of  the test  fish during
the toxicant exposure.  When the survivors of these 1^4-h static and  flow-
through tests were transferred directly to seawater (30 °/oo),  at least 60%
of the transferred fish died (Fig. 6 and 7).  Deaths normally occurred
within the first 72 h following transfer to  seawater;  this result contrasts
with the effects observed for Cu exposed fish where deaths occurred predomi-
nantly after 72 h in seawater (Lorz and McPherson  1976).
                                      17

-------
oo
       IOOI-X


        90


        80
     <  70


     >  60


     (/>  50
   40
        30
LJ
O
     rr
     LU
     CL 20
         10
         —X-
—X —X	X	
          Ojug/L
          3;ug/L

         30pg/L

         6;ug/L J
                    X	X	X
                        Ojjg/L
                        3;ug/L
                                                                             6/jg/L
               FW        SW
         Jon 21 -> Jan 27
                          Feb 6,1976
                        	i     I
               24       72

                   Freshwater
                             120
                     24
96
168
240
                                                        Sea water
                                 EXPOSURE   TIME  (hours)
        Figure 6.  Percent survival of yearling coho salmon exposed to cadmium in freshwater (static) and
        subsequent survival upon transfer to seawater.

-------



<
CE
5
— »
PERCEIv

100
90
8O
70
60
50
40
30
20
10
     -X	X
                                                         Oftg/L Zttg/L
                                                                        a
             FW      SW        i
       Feb 6 —>Feb 12 —>. Feb 24,1976
                                          '•^12/ug/L
                                                                      -V-1-
                                                                       »«5OQ
                                                                        288
24      72       120   0   24           96           168
    Freshwater                          Seawater

              EXPOSURE  TIME  (hours)
Figure 7-   Percent  survival of yearling coho salmon exposed to cadmium  in
freshwater (flow-through) and subsequent survival upon transfer to seawater.


     Yearling coho  salmon given a 5~day rest (non-toxicant exposure)  following
Cd exposure did  not die when transferred to seawater, whereas, similar
groups transferred  directly showed considerable mortality (Table 3).   It
appeared that during the rest period the coho salmon were able to adjust
their body burden of the metal and thus avoid the immediate detrimental
effects observed in seawater.
                                      19

-------
        TABLE 3-  SURVIVAL OF YEARLING COHO SALMON WITH EITHER
                  REST OR IMMEDIATE TRANSFER TO SEAWATER
                  FOLLOWING EXPOSURE TO CADMIUM FOR 144 h.

Nominal                 Percent               Percent                  Percent
concentration       survival in FW         survival in FW          survival in SW
(yg/L)	(144-h exposure)	(120-h "rest")	(280-h exposure)

A. Direct transfer to seawater

   0  (56)a                100                                          100
   2  (32)                 100                                          100
   4  (55)                  98.2                                        93.3
   8  (82)                  52.4                                        10
  12  (82)                  40.2                                         0

B. Five days rest In freshwater prior to seawater exposure

   0  (19)                                       100                    100
   4  (26)                                       100                    100
   8  (18)                                       100                    100
  12  ( 9)                                        88.9                  100
aNumber of fish exposed in parentheses.

      Deaths of fish  chronically  exposed  to Cd and then transferred to sea-
water occurred only  in the groups exposed to 4.5 yg/L Cd.  Coho salmon
chronically exposed  to Cd did  not exhibit greater mortality  in seawater than
those acutely exposed  (Tables  k  and  5).  Thus,  there appeared to be no
cumulative effect of chronic exposure  to Cd either during exposure to the
toxicant or upon subsequent transfer to  seawater.  These results were con-
siderably different  from those observed  during  chronic Cu exposure, where
many  yearling coho salmon died during  exposures to 20 and 30 yg/L Cu and also
upon  subsequent exposure to seawater (Lorz and  McPherson 197&)-

      Following 1660  h of Cd exposure,  144 h of  Cu exposure at 10 yg/L, or
combined exposure to both metals fish  transferred to seawater incurred 13-
43% mortality  in the groups receiving  Cu only or the Cd-Cu mixture, while the
4.5 yg/L Cd-only exposed fish  exhibited  <7% mortality  (Table 5).

      The yearling coho salmon  exposed  to Cd  in  May  (Fig. 8) were more
tolerant to both Cd  and seawater than  were groups challenged in January (Fig.
6).   The deaths that did occur in seawater following Cd exposure were dose
dependent.  The  improved survival  in May may have been related to the
increased age and size of fish tested  or to physiological changes that occur
during the parr-smolt  transformation.   In a previous study,  Lorz and
McPherson  (1977) noted an  increased  tolerance to seawater but a decreased
tolerance to Cu during the parr-smolt  transformation.
                                        20

-------
       TABLE 4.  SURVIVAL OF YEARLING COHO SALMON EXPOSED TO
                 CADMIUM  IN FRESHWATER AND THEIR SUBSEQUENT
                 SURVIVAL AFTER TRANSFER TO SEAWATER.
Nominal con-
centration
(yg/L Cd)
A.





B.



C.





Feb. 26, 1976-Mar. 19,
0
0.75
1.5
3.0
4.5
Mar- 12-18, 1976 (6-day
0 (21)*
3.0 (36)
4.5 (37)
Feb. 26, 1976 - April 9
0
0.75
1.5
3.0
4.5
Percent sur-
vival in
freshwater
1976 (22-day exposure)
100a
100
100
100
99-3
exposure)
100
100
100
, 1976 (43-day exposure)
100
100
100
100
99.2
Percent sur-
vival in
seawater

100
100
100
100
95-1

100
96.7
96.7

100
100
100
100
95.0

(4l)Jb/c
(42)
(42)
(41)
(41)

(I6)2)'d
(30)
(30)

(31)h'e
(42)
(42)
(45)
(40)
Originally placed 300-325 fish into each of 10 tanks.
Number of fish exposed in parentheses.
23 days exposure in SW.
11 days exposure in SW.
10 days exposure in SW.
                                      21

-------
TABLE 5
                  SURVIVAL OF YEARLING COHO SALMON EXPOSED TO CADMIUM
                  OR A CADMIUM-COPPER MIXTURE IN FRESHWATER AND THEIR
                  SUBSEQUENT SURVIVAL AFTER TRANSFER TO SEAWATER.
Nominal con-
centration
(yg/L Cd)
Percent sur-
vival in
freshwater
ATPase
activity3
(freshwater)
Percent sur-
vival in
seawater
A. April 28, 1976 - May 4, 1976 (6-day exposure)
3.0
4.5
100
100
                                                           100
                                                            93.5
                                                                         (3D
B. Feb. 26, 1976 - May 6, 1976  (70-day exposure)
0
0.75
1.5
3.0
100d
100
100
100
 99-2
                                          3.75
                                          4.80
                                                                   100   (4l)e
                                                                   100   (44)
                                                                   100   (45)
                                                                   100   (43)
                                                                    97.7 (44)
C. Feb. 26,  1976 - May 5,  1976  (63-day of Cd; last 6 days Cd + Cu)
0

0.75
1.5
3.0
4.5


Cd +
Cd +
Cd +
Cd +

10
10
10
10
10

Cu
Cu
Cu
Cu
Cu
100
100
100
100
100
99.2
3
2



1
.75
.0



.70
100
82.
57.
75.
87.
70.

9
1
6
2
0
(41F
(41)
(42)
(41)
(39)
(50)
a(Na, K)-stimulated ATPase activity  (\imoles ATP hydrolyzed/mg protein/h) .
 Number  of fish  exposed in parentheses.
°13 days exposure in SW.
 Originally placed 300-325 fish  into each of 10 tanks.
e!2 days exposure in SW.
 14 days exposure in SW.
                                      22

-------
    100

     90

     80
 _)

 >  7°
 £  6°
 ^
 CO  50

 H-  40
 z
 UJ
 o  3°
 a:
 S  20
     10
	X
                                                  X 0>ug/L
                                                   4;ug/L
                                                   10 jug/L
      FW       SW      ,
May !9->May 25—>  June 7,1976
          24       72      120   0 24     96    168   240    312

               Freshwater                 Seawater
                       EXPOSURE   TIME   (hours)

Figure 8.  Percent survival  of yearling coho salmon exposed  to cadmium in
freshwater (static) and subsequent  survival  upon transfer  to seawater.
     Fish exposed to 8 and 12 yg/L Cd in early February had  mortalities  of
48 and 60% respectively in the toxicant and subsequent  mortalities  of  90 and
100% respectively when transferred to seawater,  (Table  3), however,  only the
12 yg/L Cd fish had (Na,  K)-stimulated ATPase activities significantly lower
than the control  fish (Table A~7) .  Since Cd-exposed  fish died  quickly when
transferred to seawater,  perhaps important metabolic  systems other  than
osmoregulation were being affected.   In coho salmon yearlings previously
exposed to copper in freshwater, mortalities in  seawater usually  did not
occur until the third or  fourth day after transfer (Lorz and McPherson
1977)-  Osmoregulatory effects due to copper (increased osmolality  of  plasma
following transfer to seawater) took 12-36 h to  develop and  about 200  h  to
return to normal.

     The (Na, K)-stimulated ATPase activity peaked in mid-April and remained
high through May in both  the control fish and yearling  coho  salmon  chroni-
cally exposed to Cd since February (Fig. 9a).  There  was no  significant
difference in specific activity in the groups that received  4.5 yg/L Cd  as
compared to controls.  The group of fish exposed to  10  yg/L  Cu  for  6 days
and the group exposed to  Cd-Cu mixture exhibited significantly  lower ATPase
specific activity than the controls or the 4.5 yg/L Cd  groups (Fig. 9b,
                                     23

-------
Table A-8).  The whole  g.    and  microsomal  methods  of gill  preparation
showed similar patterns of  ATPase activity  (Table A-8).

c
> 8
r—
O Cn
.C
6.0
4.0

2.0

A.
T>

i ~~~~--4
i
~T
^~JL^y' '''
'"11 [

I j-

• Control
, A 4.5ng /I
j * (144-1,
- 2,400^1!
j (336-5
r\4
i

               Feb   I   Mar   I   Apr
                     May  1    Jun
              5.O

              4.01

              3.0

              2.O
B
                                  4.5)i9/LCd (l,68Oh)
                                 Control
                                  lOjifl/LCu (|68h)
                                  4.5(ig/U Cd + IO
                                     Cu
                   31     5   10  15  2O  25  3O    5

               Mar                Apr             May


 Figure 9-   Gill  (Na,  K)-stimulated ATPase activity of yearling coho salmon
 during exposure  to cadmium, zinc and cadmium-copper mixture.
 Zjnc

      Fish chronically exposed to Zn for 13 and 28 days and then challenged
 with  seawater had no mortalities in seawater (Table 6).  However, most of
 the groups that received the Zn-Cu mixture (13 days) exhibited some mor-
 tal 1ity when exposed to seawater, but when the Zn-Cu groups were exposed for
 a  longer period (28 days)  and then challenged with seawater only minimal
 mortality occurred (Table 6).  The Na, K)-stimulated ATPase activity of
 yearling coho salmon exposed to Zn showed no significant difference from
 control fish while the groups receiving Cu or Zn-Cu exhibited significantly
 lower ATPase activity than the controls (Table 6, Table A-9).  Considering
 that  both the ATPase activity and coefficient of condition of the Zn-Cu
 exposed fish (Table 6, Fig. 5) following 28 days of exposure are signifi-
 cantly lower than controls, it is difficult to explain why so few fish
 died  during the second seawater challenge (Table 6).

-------
            TABLE 6.  SURVIVAL OF YEARLING COHO SALMON EXPOSED TO
                      ZINC OR A ZINC-COPPER MIXTURE IN FRESHWATER
                      AND THEIR SUBSEQUENT SURVIVAL AFTER TRANSFER
                      TO SEAWATER.
Nominal con-
concentration
yg/L Zn
Percent sur-
vival in
freshwater
ATPase
activi tya
(freshwater)
Percent survival
in seawater
(310-h exposure)
A. Zinc (315 h May 12-26 S-June 7, 1976).
B.
       0
     400
     800
    1600
    2400
                         100
                         100
                         100
                          99-4
                          99-9
               4.09
               4.09
Zinc and copper (171  h Zn + 144 h Zn + Cu)
     400 Zn +
     800 Zn +
    1600 Zn +
    2400 Zn +
           10 Cu
           10 Cu
           10 Cu
           10 Cu
           10 Cu
100
100
100
100
100
                                           2.55
1.42
C. Zinc (675 h May 12-June 9, 1976 bi June 22, 1976)
                                SW
       0
     400
     800
    1600
    2400
                         100
                         100
                         100
                          99-4
                          99.0
               3.3
               4.1
D. Zinc and copper (171 h Zn + 504 h Zn + Cu)
                100
                100
                100
                100
                100
     (20)b
     (20)
     (21)
     (21)
     (21)
100  (20)
 90  (20)
100  (20)
 90  (20)
 77.8(18)
                100
                100
                100
                100
                100
     (39)
     (21)
     (40)
     (40)
     (40)
10 yg/1 Cu
400 Zn + 10 Cu
800 Zn + 10 Cu
1600 Zn + 10 Cu
2400 Zn + 10 Cu
100
100
100
100
100
1.6



1.5
100
100
95.1
100
100
(40)
(20)
(41)
(40)
(39)
 (Na, K)-stimulated ATPase activity (\imoles ATP hydrolyzed/mg protein/h)-
^Number of fish in parentheses.
                                      25

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Chromium and Nickel

     Little mortality occurred during the seawater challenge of yearling
coho salmon previously exposed to chromium or nickel for 144 h in freshwater
(Table 7).  The maximum  level of Cr tested (5 mg/L) was 50 times the
acceptable concentration  (100 yg/L Cr - U.S.E.P.A. 1976).  Thus, the water
quality recommended for  Cr appears adequate for coho salmon entering sea-
water.  Sauter et al. (1976), however, recommended the maximum permissible
concentration of chromium in the aquatic environment should be reduced.
Similarly, the effect of  interaction of metals or the effects of chronic
exposure of these metals on the salmonids migratory disposition or subse-
quent tolerance to seawater is not known.

        TABLE 7.  SURVIVAL OF YEARLING COHO SALMON EXPOSED TO
                  CHROMIUM OR NICKEL IN FRESHWATER AND THEIR
                  SUBSEQUENT SURVIVAL AFTER TRANSFER TO SEAWATER.

Nominal con-                      Percent survival
centration                        in freshwater               Percent survival
mg/L	(l44-h exposure)	in seawater	

A. Chromium (March 9~15  S- -March 25, 1976)

   0                                   100                        100   (20)a
   0.03                                100                        100   (19)
   0.05                                100                        100   (20)
   0.08                                100                        100   (20)
   0.10                                100                        100   (20)
   0.30                                100                        100   (20)
   0.50                                100                        100   (20)
   3.0*                                100                        100   (11)
   5.0b                                100                         91-7 (12)
 B.  Nickel  (April  2-8^ -April  19,  1976)
0
0.25
0.50
0.75
1.0
2.5
4.0
5.0
100
100
100
100
100
100
100
100
100
100
100
95
100
100
100
100
(20)
(20)
(20)
(20)
(20)
(21)
(20)
(20)
 aNumber of fish exposed in parentheses
 Exposed to chromium for 96  hours prior  to  transfer to seawater.
                                      26

-------
Mercury

     Coho salmon yearlings previously exposed to mercuric chloride for ]kk h
were transferred to seawater and their survival  monitored.   Deaths occurred
in all concentrations >50 yg/L Hg in a dose dependent  manner (Fig. 10).   The
mortalities in seawater occurred within the first 72-h exposure,  which is
similar to the results observed for Cd-exposed fish  (Fig.  7  and 8),  but
dissimilar to that noted for Cu-exposed fish (Lorz and McPherson  1976).   The
(Na, K)-stimulated ATPase activity of coho salmon exposed  to Hg  (^50 yg/L)
was suppressed but the decrease was not statistically  significant (Table  A-
10).
  ioo L__ x—x—x	x	x
                                     •X	X	X	X	X	X	X	X	HQftg/L
                                      ,	A	A	A	A	A	A- -A	A30 ««'«-
                                     -                         50/jg/L

                                          .—A-—A---A—&—A—A—A
                                                             lOO/ug/t
                                                FW        SW
                                         April 20 —> April 26 —> May 6,1976
                                                        200 ug / L
                                                                    24O
                 72      120  0   24           96          168

              Freshwater                        Seawater
                       EXPOSURE   TIME   (hours)

Fig. 10.   Percent survival  of  yearling coho salmon exposed to  inorganic
mercury (HgC^)  in freshwater  (static)  and subsequent  survival upon
transfer to seawater.
                                      27

-------
EFFECT OF METAL EXPOSURE ON PLASMA OSMOTIC PROPERTIES

     Exposure of yearling coho salmon to Cd  in freshwater did not appear to
affect their ability to maintain either normal osmolality or chloride concen-
trations of blood plasma although a statistically non-significant decrease
in osmolality and chloride  ion concentration was noted following 96-h expo-
sure to 12 yg/L Cd  (Figs. 11 and 12 Table A-ll).  Larsson (1975) noted that
Cd had only slight  effects  on the major plasma electrolytes, sodium and
chloride,  in flounders but  found the levels of other plasma  ions (K, Ca and
Mg) were remarkably changed after Cd exposure.  The osmolality of the plasma
of fish exposed to  Zn, or 10 yg/L Cu did not differ statistically from
controls.  Similarly, Skidmore  (1970) found  that the osmotic concentration
and the concentrations of Na, K, Ca, Mg and  Zn  in blood were largely unaffected
in rainbow trout  immobilized by zinc sulfate.  Coho that had received the
Zn-Cu  (2400 yg/L  Zn + 10 yg/L Cu) mixture for 480 h exhibited reduced osmolality
(Fig.  11).  The lack of effect of Cd on plasma osmolality and plasma chloride
is in  contrast to the changes  in blood parameters noted  in coho, brook trout
and brown  bullhead  exposed  to sublethal copper concentrations (Christensen
et al.  1972, Lorz and McPherson  1977, McKim  et al.  1970).  However, McCarty
and Houston  (1976)  found found  that goldfish exposed to  sublethal levels of
cadmium  (44.5 and 380 yg/L  for a period of 25 days  showed significant
changes  in plasma chloride.  Goldfish held  in 44.5  yg/L  had  apparently
compensated  for most of the initial cadmium  effect  after 50  days exposure
while  those  exposed to 380  yg/L  exhibited significant deviations in plasma
sodium and chloride levels.

EFFECT OF  METAL EXPOSURE ON CORTICOSTEROID RESPONSE

     To  determine the feasibility of using plasma cortisol levels as an
indicator  of heavy  metal toxicity,  yearling  coho salmon  were exposed to Cu
and Cd and the cortisol  level of the plasma  measured.  The work was carried
out cooperatively with Dr.  C.  B. Schreck, Oregon Cooperative Fishery Research
Unit,  Oregon State  University.   The basic findings  of the study were as
follows: Cu  produced a marked,  dose-dependent cortisol stress response, but
Cd did not elicit a cortisol elevation, even in moribund fish.  Handling and
confinement  produced similar corttsol elevation  in  controls  and Cu or Cd
treated  fish, but Cu exposure  reduced the salmon's  ability to survive the
stress of  handling  and confinement  (Schreck  and Lorz 1978).  Two groups of
coho  salmon  were  treated to determine the potential additive or synergistic
effects  of low  levels of Cu and  Cd.  Both groups received Cu at 10 yg/L as
CuCl£  on April 29,  1976.  One group, however, had been exposed continuously
since  February 26,  1976  to  4.5  yg/L Cd, and  thus received both Cu and Cd
simultaneously.   Following  96  h  of  Cu exposure, serum was collected from 10
fish  for each treatment.  The mixture of the two metals  did  not produce an
elevation  in cortisol  [x =  46.6  +9-1  (SE)];  their  cortisol  level was
similar to that of  fish  exposed  to  10 yg/L Cu only  [x =  51.5 +_ 8.6  (SE)].

      Groups  of coho salmon  exposed  to Hg as  HgCl2 for 120 h  were sampled for
cortisol.  Although the data were not conclusive,  it appeared that the Hg
caused an  elevation in plasma cortisol  levels;  mean titers  (+_ SE) were
37.4 + 5-9 for the  control  and  66.9 + 9-0, 51.0 +  14.1,  and  F5.5 + 6.7 for


                                        28

-------
 E
 O
 3310
 Q_
 u_ 290
 O

 t 270
    250
O

O
                                                 l2»ig/LCd
                                                !
              24
                         72
    20
   168
                                                           i Control
                                                                    . Zn (48Oh)
                                                           $IOyg/L Cu(360h)
                           2,4OOng/LZn(l20n)+
                           Zn-Cu(36Oh)
 Figure  11.  Effect of cadmium exposure  in freshwater on the plasma  osmolality
 of coho salmon.  Mean +_ SE (effect  of zinc, copper and zinc-copper  exposure
 on plasma osmolality).  n = 5-
\
cr
UJ
(/) I3O
_l
°- no
z
„ 90^
O
    70
           24
                      72
120
168
                                                           2.400 ng/LZn (48Oh)
                                                           ontrol
                                                           2,400ug/LZn{l2Oh)t
                                                            Zn-Cu(360 h)
                       EXPOSURE TIME (h)

 Figure 12.  Effect of cadmium exposure  in freshwater on the chloride  ion
 concentration of plasma of coho salmon.  Mean +_ SE (effect of zinc, copper,
 and  zinc-copper exposure on chloride  ion concentration),   n = 5.
                                   29

-------
the 30, 50, and 100 yg/L Hg treatments, respectively.  The mean level
produced by the 100 yg/L Hg was not only higher than the control groups, but
also higher than any mean of a control found in the other experiments. We
concluded  (Schreck and Lorz 1978) that cortisol and other characteristics of
the general adaptation syndrome of stress should not be universally applied
as indicators of heavy metal stress.

EFFECT OF METAL EXPOSURE ON DOWNSTREAM MIGRATION

     The proportion of released test  fish that migrated downstream in
Crooked Creek was almost always greatest for control fish (Table 8), but
there was  not a dose-dependent response as noted for Cu exposed fish  (Lorz
and McPherson 1977)-   In the fish chronically exposed to Cd or Zn some
groups migrated better than the controls but not in a consistent manner
(Table 8).  Most downstream migration occurred within the first 10 days of
release and often the major portion was completed  in 5 days (Table A-12).

     A 7 km section of the stream was electrofished from above the release
sites  to the weir in an attempt to collect non-migrants.  Only two yearling
coho that  had been released earlier were found, although native cutthroat
and rainbow of comparable size to the released fish were caught.  There were
only two small areas in the stream where our electrofishing gear appeared
inadequate because of  the depth of water.  Thus, fish that had not migrated
by early July apparently died as a  result of predators, stress, natural
causes or  other  indirect effects of the toxicant exposure.

Cadmium

     The first release of Cd-exposed  fish was made in mid-April.  The
controls of the acutely exposed  fish  migrated  best, although  there was only
1% difference  in  rates of migration of  the five release groups  (Fig.  13A).
The chronically exposed fish  (47 days)  did not migrate as well as the
acutely exposed groups but  there was  no suggestion of a dose  response  (Fig.
13B).   It  appeared that Cd  had  little effect on migratory disposition.

     A second  release  of  15 groups  of yearling coho salmon  (5 groups exposed
for 6  days; 5  groups exposed  for 67 days; and  5 groups exposed  to Cd  for 67
days plus  10 yg/L Cu for  the  last 5 days prior to  release) was made  into
Crooked Creek  in  early May.   There  appeared  to be  little effect of Cd  on the
downstream migration of either  acutely  or chronically exposed fish, although
the former groups exhibited better  migration  (Fig. 14, Table  8).   In  the
acutely exposed  groups the  controls exhibited  the  best migration and
although there was no  overall dose-response  exhibited, the 4.5 yg/L group
showed 16% less migratory  tendency  than the  controls  (Fig.  14A).  The
chronically exposed  groups  that  received Cu  for 5  days  in addition to  the Cd
showed a decrease  in their  migratory  disposition  (Table 8,  Fig. 14B).  The
Cd-Cu  mixture  appeared to act synergistical1y, as  migration was inhibited to
a greater  extent  than  noted for  fish  exposed to Cu alone  (Fig.  14B).
                                        30

-------
        TABLE 8.   PERCENT MIGRATION THROUGH JULY 6,  1976 OF YEARLING
                  COHO SALMON RELEASED INTO A SMALL  COASTAL STREAM
                  FOLLOWING ACUTE OR CHRONIC METAL EXPOSURE.
Exposure and
nominal con-
centration
yg/L
A. Chronic exposure*3
Control
10 Cu
0.75 Cd
0.75 Cd + 10 Cu
1.5 Cd
1.5 Cd + 10 Cu
3.0 Cd
3.0 Cd + 10 Cu
4.5 Cd
4.5 Cd + 10 Cu
B. Acute exposure (144 h)
Control
0.75 Cd
1.5 Cd
3.0 Cd
4.5 Cd

C. Chronic exposure0
Control
10 Cu
400 Zn
400 Zn + 10 Cu
800 Zn
800 Zn + 10 Cu
1600 Zn
1600 Zn + 10 Cu
2400 Zn
2400 Zn + 10 Cu
D. Acute exposure (144 h)
Control
10 Cu
400 Zn + 10 Cu
800 Zn + 10 Cu
1600 Zn + 10 Cu
2400 Zn
2400 Zn + 10 Cu
Percent
4/14/763

60.2

68.7

62.4

65.9

59.4


80.2
77.8
73-5
73-5
73.7
5/26/76a

80.2
63.7
75.9
52.5
71-0
47.0
82.0
34.0
66.0
37.7

No release
it
ii
11
ii
ii
ii
Migration
5/5/76*

67.0
62.3
69-9
47.0
55.6
51.4
69.3
56.7
66.3
54.8

85.1
71.8
67.6
80.7
69-0
6/9/76a

71.2
65.4
73-1
58.1
67.5
61.9
67.6
63.5
74.5
64.5

81.5
66.1
72.0
67.2
53.1
62.1
58.3
aDate of release.
 Exposed to Cd since Feb.  26,  1976.
 Exposed to Zn since May 10, 1976
                                     31

-------
o
Q
                                                          x Control
                                                          n 0.75*jg/ICd
                                                          Q'4.5 /ig/ICd
                                                          -1.5 /jg/ICd
                                                          ^3.0 jug/ICd
                 J	I
IK,    I	1	I	I	I
    80-

                  ,-°'
                '!>—t—A-	" s'/'-s
              o//^-«—^-		-"-'	-^  C°""°T
                                                           4.5 pg/ICd
               B
                                 10
             35
45
55
                   DAYS    POST    RELEASE

  Figure 13-   Percent downstream migration of yearling coho salmon previously
  exposed to  cadmium in freshwater and released April  14, 1976.  A. represents
  9^-102 fish per group exposed for  144 h.  B. represents 180-192 fish per
  group exposed for 47 days.
                                 32

-------
                                                     « Control

                                                     a 3.O/ug/l Cd
                                                     A 0.75/jg/ICd
                                                     fl 1.5
                                                       4.5
                                                             Cd
                                                       Control
                                                       4.5pg/l Cd
                                                      x 10 >ig/icu

                                                             Cd + lOug/ICu
                                     25
35
45
55
              DAYS   POST   RELEASE
Figure 14.   Percent downstream migration of yearling coho salmon previously
exposed to  cadmium, copper,  and cadmium-copper mixtures  in freshwater and
released May 5, 1976.  A. represents  6?-88 fish per group exposed for
]kk h to cadmium.   B. represents 100-114 fish per group  exposed to cadmium
(67 days),  copper (5 days),  or cadmium-copper mixture (62 days Cd + 5
days Cd-Cu).

-------
Zi nc

     A release made  in late May of  10 groups of yearling coho salmon showed
that movement of the groups exposed to Zn was similar to that of the
controls, except in  the highest Zn  group where fewer migrants were observed
(Fig. 15, Table 8).  In the group exposed to Cu and the groups exposed to a
Zn-Cu mixture fewer  migrants were observed than in the controls or the
corresponding Zn (only) groups  (Fig.  15, Table 8).  The reduced movement of
the groups exposed to the Zn-Cu mixture again showed the apparent synergistic
action of the combined metals as well as indications of a dose-dependent
response.

     Another release of yearling coho salmon exposed to Zn, Cu, or Zn-Cu
mixtures was made  in early June.   In  the acutely exposed groups  (6 days) the
controls showed the  best migration  while those receiving 2400 yg/L Zn or
10 yg/L Cu migrated  at a lesser  intensity  (Table 8, Fig. 16A).  The groups
receiving the highest Zn-Cu mixtures  (1600 and 2400 yg/L Zn" + 10 yg/L Cu)
migrated with considerably less  intensity  than controls  (Fig. 16A), and this
was similar  to the migration  patterns exhibited in the earlier release  (Fig.
15)-   In the chronically exposed groups  (27 days), controls were intermediate
in their movement  compared to the  four Zn  groups  (Table 8,  Fig.  16B).  The
fish from the highest Zn concentrations which had previously exhibited  less
movement than controls  (Fig.  15) exhibited slightly better  migration than
the controls  in the  June release.   Whether the yearling coho salmon had
acclimated during  the extended  exposure or whether the downstream movement
was complicated by  the  lower  June  streamflow  is not known.  The groups  that
received only Cu or  the Zn-Cu mixture migrated better than  comparable groups
released  in  late May although their migration was still 6 to 13% less than
the controls or corresponding groups  exposed only to Zn  (Fig. 16B).
Similarly, the groups that received the  longer exposure to  Zn or Zn-Cu
mixture  (27  days)  appeared to have acclimated and responded with greater
survival  in  the seawater challenge test  than  the groups that had been exposed
to Zn  for  13 days  plus Cu  for the  last 6 days  (Table 6).  Sprague  (1970)
reviewed several studies that reported acclimation to zinc.  Lloyd  (I960)
found  increased survival times  for trout following two weeks of acclimation,
however  Sprague  (1968a) found that the avoidance threshold, a sublethal
response of  rainbow  trout, was  not influenced by acclimation.  Chapman
 (1978) found  that  chinook  salmon fry  acclimated to Cu and Zn (but not to Cd)
survived concentrations of Cu and  Zn  which were lethal to non-acclimated
salmon.  However,  he found there was  an optimal concentration for Cu accli-
mation  (9~16 yg/L).   In avoidance  studies  Chapman  (1978) noted that 80% of
the control  fish  (steelhead  trout  fry) avoided Cu at 10 or  20 yg/L, but none
of the acclimated  fish avoided  any tested  concentration, even ones that was
lethal.  Thus, Chapman  stated,  "the development of acclimation may be a
mixed  blessing;  it  allows  existence in a marginal environment but  interferes
with behavioral defense mechanisms."

-------
V-O
vn
        80

     O
UJ
a:
     c/)
        so
        50
        40
        30
     O
        20
         10
                             o	
                                                                     •o 1600 pg/I Zn
                                                                     -x Control
                                          —/.	.	v  SOOjjg/l Zn

                                  	/,	A.	A 2400«g/IZn
                                      a	_	a
                                                                                Zn»
                                                                                              Cu
                                                                         •A 2400fig/I Zn+ 10^g/ICu
                                                                         -• 1600/jg/IZnflO/jg/ICu
                                          LJV
                                    8
                                     10
15
20
25
30
35
                               DAYS   POST   RELEASE
   Figure 15-  Percent downstream migration of yearling coho salmon previously exposed to zinc, copper, and
   zinc-copper mixtures in freshwater and released May 26, 1976.  Each line represents a release of 100-106
   fish previously exposed for; a) 13 days to zinc, b) 6 days to Cu, and c) 7 days to Zn + 6 days Zn-Cu.

-------
 901-
                                                     x  Control
                                                             Zn
                                               45
55
               DAYS    POST   RELEASE
Figare 16.  Percent migration of yearling coho salmon previously exposed to
zinc, copper and zinc-copper mixtures in freshwater and released June 9,
1976.  A.  represents 50-65 fish exposed  for 1^4 h to zinc, copper, or zinc-
copper mixture.  B. represents releases  of 102-110 fish exposed for; a)
27 days to Zn, b)  20 days to Cu, and c)  7 days to Zn + 20 days Zn-Cu.
                              36

-------
                               SECTION VI

                               DISCUSSION

     As mentioned in the introduction, considerable laboratory research has
been done to determine water quality criteria for cadmium, copper, and zinc
based on survival and growth of juvenile salmon ids in freshwater (Chapman
1973, 1975, Christensen 1975, Hodson and Sprague 1975, Lloyd I960, McKim and
Benoit 1971, 197*», Sauter et al. 1976, Sinley et al.  1971*, Sprague \S6ka,
and Sprague and Ramsey 1965).  However, only a few field studies are avail-
able where contaminants in the natural environment have been related to
effects on fish populations  (Geckler et al 1976, Goettl et al. 1973, Grande
1967, Herbert et al. 1965, Nehring and Goettl 197^, Sodergren 1976, and
Sprague et al. 1965).  There are also several laboratory studies available
where researchers working with mixtures of metals have described their
findings.  Lloyd (I960 noted that copper and zinc were additive in their
toxicities, in acute threshold mortality tests, in both hard and soft water.
Sprague and Ramsay (1965) also observed summation of copper and zinc toxi-
city to Atlantic salmon in soft water whereas; Anderson (1973) working with
guppies (Poecilia reticulata) described his results of copper and zinc
mixtures as "supra-additive" while mixtures of copper and nickel were
concentration additive.  Brown  (1968), Brown et al. (1969), and Brown and
Dalton (1970) described a method of predicting toxicities of mixtures of
substances based on 48-h LC50 determinations and assuming direct summation.
Their 48-h LC50 values were  thought to provide a "reasonable estimate" of
acute threshold concentrations.  However, Eaton (1973) studied the effects
of chronic toxicity of cadmium, copper and zinc mixtures to fathead minnows
(Pimephales promalas Rafinesque),  and he concluded that the toxicity of the
trimetal mixture could not have been predicted closely from the results of
single-toxicant chronic tests by assuming they were totally additive in
their toxic units.  Eaton noted (acute tests) that it took considerably less
metal in combination to obtain a threshold value and therefore suggested
that some toxicity-increasing interaction was occurring. Similarly, Eisler
and Gardner (1973) working with the estuarine teleost (Fundulus heteroclitus)
noted that mixtures of Cu and Zn (96-h bioassay studies) produced more
deaths than expected on the  basis of toxicities of individual components.
If concentrations of Cd not  ordinarily lethal were added,  they exerted a
negative effect on survival  of fish previously intoxicated by salts of Cu,
Zn, or both.  Spehar et al.  (1978)  found that when flagfish (Jordanella
floridae) were exposed to sublethal levels of cadmium and zinc that the
action of the toxicants was  not additive, but a joint action was indicated.
Effects on survival showed that the toxicity to cadmium and zinc mixtures
was little if any greater than the toxicity of zinc alone.
                                       37

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     The early research studies have served to determine the relative sen-
sitivities of aquatic organisms to various metals or their mixtures and the
effects of water quality parameters such as alkalinity, hardness, pH, and
dissolved oxygen concentration.  However,  in the last  10 years considerable
progress has been made toward determining  the relationship of toxicity of
heavy metals to their aqueous chemistry:i.e., toxicity of concentrations of
ionic and molecular forms  in solution, physiological mechanism involved, and
application of the results to field situations.  Andrew  (1976) reviewed some
of the  literature that demonstrated chelation of copper by organic reagents
effectively reduced its toxicity and summarized EPA's  recent experiments
that attempted to clarify  the relationship between copper toxicity and water
chemistry.  Sprague (1968b) found  that use of the trisodium salt of NTA
(nitrilotriacetate) increased the  tolerance of Atlantic salmon to copper up
to 33  times the lethal threshold.  Similarly, Biesinger et al. (197*0 noted
that chelates of copper or zinc with NTA are relatively non-toxic to Daphnia
magna.  Wilson  (1972) observed  that addition of pulp mill spent  sulphite
liquor, allowed Atlantic salmon parr to  survive at copper- concentrations
considerably greater  than  their 96-h LC50  values.  Wilson found  that
chelation  of copper by sulphite  liquor greatly  increased  the difficulty  in
developing a simple predictive  model for copper toxicity  in receiving
waters.   Similarly, Zitko  et al.  (1973)  noted that the addition  of humic
acid to solutions of  copper or  zinc  increased the  incipient lethal level
of copper  by binding  cupric  ions  but did not affect  the  ILL of zinc to
juvenile  Atlantic salmon.  Chynoweth et  al.  (1976) noted  that bound copper
was  less  toxic  than unbound copper to  guppies  (Lebistes reticulatus) when
several organic compounds  were  added to  copper  bioassays.  An  inverse
relationship was observed  between  the  degree of copper binding and copper
toxicity.   Similarly, Brown et  al.  (197*0  noted that  the  toxicity of a  given
total  concentration of copper  to  rainbow trout was  reduced with  additions of
sewage effluent, an amino  acid,  humic  substances, or  suspended organic
matter- These  authors concluded that data  from  toxicity  tests with copper  in
which  natural  surface waters are  used  for  dilution purposes cannot define
the  true  toxicity of  copper or  have  application to other  natural waters
except when  the concentration  of  the toxic chemical  species  is known.

      Stiff (1971),  using  equilibrium equations  investigated the  formation of
complex carbonate  species  in copper-bicarbonate solutions and  suggested  that
the  difference in  toxicity of  copper to  fish with  differences  in water
hardness  might  be dependent  upon  carbonate complexation.  Stiff  proposed
that the  free  cupric  ion  was  the  primary toxic  form.   This conclusion  is
supported by  Pagenkopf et  al.  (197*0 who examined  published copper toxicity
data,  developed an  equilibrium model,  amd  postulated  that CuOH+  might also
be  involved  in  the  toxicity.   Chapman  and  McCrady  (1977)  using a computer
program computed  the  free copper  concentrations for  a  variety of alka-
 linities  and  pH values and obtained  results  similar  to Stiffs',  however  they
suggested that  pH may be  an  important  factor  in copper toxicity  in addition
to  its usual  association  with  alkalinity.

      Hara et  al.  (1976)  noted  that, "chemosensory  receptors are  immediate
detectors of  environmental chemical  stimuli.  Environmental changes are
detected  by  transducer elements of the receptors and  information is sub-
sequently carried along  nerve  fibers to  various  integrating centers of  the

                                       38

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body. Olfaction plays an important role in the survival of fish.   It
mediates such diverse phenomena as feeding, recognition of prey and pre-
dator, sexual and social behavior, orientation, and migration.  Since the
olfactory membranes are not protected by external barriers, man-made
alterations  in water quality could easily  interfer with their functioning,
the consequences being a breakdown in communication among fish and the
environment." Earlier, Hara (1972) found that pretreatment of the nasal
cavity of sockeye or coho salmon with solutions of HgCl2 or CuSOi|  for as
little as 10 seconds completely blocked the olfactory response.  However,
the effect was reversible.  Hara et al. (1976) continued the studies and
found that the olfactory responses to a standard stimulant in rainbow trout
could be depressed by mercury and copper at sublethal concentrations.  The
lowest concentration of mercury and copper to cause appreciable effects
within 2 h  was estimated at 100 and 8 ug/L, respectively.  The authors
speculated that these metals may possibly  interfere with the perception of
the normal homestream odors that guide salmon to their spawning tributary.
Exposure of 0.5-5-0 mg/L Cu for 6-24 h permanently damaged the olfactory
mucosa of some estuarine fishes (Gardner and LaRoche 1973), and Hara et al.
(1976) suggest that persistent binding of  heavy metals to receptor sites at
sublethal concentrations may also result in irreversible damage to the
receptor cells.  Voyer et al. (1975) noted tissue damage in nasal  passages
and buccal cavities of mummichogs exposed  to 28 mg/L Cd for 6.5 hours.

     The insecticides, DDT and Sumithion (fenitrothion) have been  shown to
modify salmonid physiology, behavior and learning ability following exposure
to sublethal concentrations (Anderson 1971, Bull and Mclnerney 197**, Hatfield
and Anderson 1972, Symons 1973, and Warner et al. 1966).  Similarly, studies
on sublethal effects of metal pollutants have elicited changes in  the
physiology and behavior of fishes (Kania and O'Hara 197**, Kleerekoper 1973,
and Seller et al . 1975)-  Recently, Sullivan et al. (1978) discussed changes
in prey-fish behavior resulting in selective predation of stressed prey at
concentrations of the toxicant well below  lethal levels.  Sullivan et al.
(1978) were able to demonstrate increased  prey vulnerability of the fathead
minnow (Pimephales promelas) exposed to acute (2k h) and subacute  (21 day)
sublethal cadmium levels prior to interacting with largemouth bass
(Micropterus salmoides).  Prey exposed to  cadmium displayed altered behavior
patterns including abnormal schooling behavior at concentrations of cadmium
previously suggested to be safe for fathead minnows (Pickering and Gast
1972).  Sullivan et al. recommend that behavioral studies be an integral
part of future bioassay studies where water quality criteria are being
determined.  Similarly, Solbe and Flook (1975) observed a behavioral change
in the stone loach, Noemacheilus barbatulus (L) , the loss of instinct to
hide  in daylight, following exposure to cadmium.  They suggested this behavioral
change made  the stone loach more vulneralbe to predation and thus may explain
the stone loach's absence from parts of the River Tean.

      In a recent study. Weltering et al. (1978) found that predator-prey
interactions of largemouth bass and the mosquitofish (Gambusia affinis) were
altered depending on the density of the prey species and ammonia concentration.
In the study both species were simultaneously and continuously exposed to


                                      39

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the toxicant (ammonia) for the duration of the experiment.  Weltering et al,
found that the largemouth bass were more sensitive than the mosquitofish to
ammonia and at high ammonia and high prey densities the harassment of the
predator by the prey was generally lethal to the bass.

     From this brief review of the literature and the results obtained in
our studies it is apparent that additional research is necessary to deter-
mine whether metal exposures  in the early life stages or during smelting in
salmonids could result in permanent damage to the population.  In our
studies we observed reduced seawater tolerance and migratory response in
some metal  intoxicated fish,  however, we do not know  if the effects would
result  in lowered adult returns.

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     T. P. Wuite.  1975.  Toxicity of chromium (VI) in fish, with special
     reference to organoweights, liver, and plasma enzyme activities, blood
     parameters and histological alterations,  pp.31-42.  In: Koeman, J.  H.
     and Strik, J. J. T. W. A. eds. Sublethal effects of toxic chemicals  on
     aquatic animals.  Elsevier Publ. Co. New York.

Sullivan, J. F.,  G. J. Atchison, D. J. Kolar, and A.  W. Mclntosh.  1978.
     Changes in the predator-prey behavior of fathead minnow (Pimephales
     promelas) and largemouth bass (Micropterus salmoides)  caused by
     cadmium.  J. Fish. Res. Board Can. 35(4) :446-451 .

Symons, P. E. K.   1973-  Behavior of young Atlantic salmon (Salmo salar)
     exposed to or force-fed fenitrothion, an organophosphate insecticide.
     J. Fish. Res. Board Can. 30(5):651-655-

Trama, F- B., and R. J. Benoit.  I960.  Toxicity of hexavalent chromium to
     bluegills.  J. Water Pollut. Control. Fed. 32:868-877.

U. S. Environmental Protection Agency.  1976.  Quality criteria for water.
     U. S. Environ. Prot. Agency Washington D.C.  256 pp.

Voyer, R. A., P.  P. Yevich, and C. A. Barszcz.   1975.  Histological  and
     toxicological responses of the mummichog Fundulus hetercelitis (L.)
     to combinations of levels of cadmium and dissolved oxygen in a
     freshwater.   Water Research 9:1069-1074.

Warner, R. E., K. K. Peterson, and L. Borgman.   1966.  Behavioral patho-
     logy in fish: A quantitative study of sublethal  pesticide toxication.
     J. Appl. Ecol. (Suppl.) 3:223-247.

Weltering, D. M., J. L. Hedtke, and L. J. Weber.   1978.  Predator-prey
     interactions of fishes under the influence of ammonia.   Trans.  Am.
     Fish. Soc. 107 (3)-.500-504.

Wilson, R. C. H.   1972.  Prediction of copper toxicity fn receiving waters.
     J. Fish.Res. Board Can. 29(10):1500-1502.

Zaugg, W. S. and  L. R. McLain.  1970.  Adenosinetriphosphatase activity in
     gills of salmonids: seasonal variations and salt water  influence in  coho
     salmon, Oncorhynchus kisutch.  Comp. Biochem.  Physiol.  35:587-596.

                                     47

-------
Zitko, V., and W. G. Carson.  1977-  Seasonal and developmental  variation  in
     the lethality of zinc to juvenile Atlantic salmon (Salmo salar).   J.
     Fish. Res. Board Can. 3MD : 139-141.

Zitko, V., W. V. Carson and W. G. Carson.  1973.  Prediction of  incipient
     lethal levels of copper to juvenile Atlantic salmon in the  presence of
     humic acid by cupric electrode.  Bull. Environ. Contam. Toxicol.  10(5):
     265-271.

-------
         TABLE A-l.  MEASURED CONCENTRATIONS OF CADMIUM AND COPPER
                     DURING EXPOSURE TESTS.
Tank Nominal concentration
No. yg/L
Cadmium exposure
E- 1
E- 2
E- 3
E- 4
E- 5
E- 6
E- 7
E- 8
E- 9
E-10
Copper exposure
E- 1
E- 2
E- 4
E- 6
E- 8
E-10
(Feb. 27-May 6, 1976)
0
0
1.5
1.5
3.0
3-0
0.75
0.75
4.5
4.5
(April 30-May 6, 1976)
0
10
10
10
10
10
Measured concentration ug/L
Mean ± SDa Range
n = 10
0.09 + 0.19
0.02 + 0.35
1.41 + 0.15
1.41 + 0.10
2.73 + 0.21
2.69 + 0.17
0.91 + 0.38
0.78 + 0.24
3.90 + 0.31
4.05 ± 0.39
n = 1
1.7*
29. 3C
17.0
13.1
10.4
10.5

0
0 -
1.2 -
1.3 -
2.3 -
2.4 -
0.6 -
0.6 -
3.6 -
3.6 -








1.6
1.0
1.7
1.6
3-0
2.9
1.6
1.4
4.5
4.9







 Results of weekly composite water samples.
^Results of a 6-day composite water sample.
°Suspected contamination of sample container.
                                      49

-------
 TABLE A-2.  MEASURED CONCENTRATIONS OF ZINC AND COPPER
             DURING  CHRONIC  EXPOSURE TESTS.
Tank
No.
Nominal concentration
vg/L
Zinc exposure (May 12-June 9, 1976) n
E- 1
E- 2
E- 3
E- 4
E- 5
E- 6
E- 7
E- 8
E- 9
E-10
Copper
E-1,3,
E- 2
E- 4
E- 6
E- 8
E-10
0
0
800
800
1600
1600
400
400
2400
2400
exposure (May 19-June 9, 1976)
5,7,9 0
10
10
10
10
10
Measured
Mean +
» 4
4.8 +
7 +
698 +
721 +
1384 +
1391 +
354 +
359 +
2027 +
2087 +
n - 3
2.5 +
13-2 +
12.4 +
12.3 +
12.5 +
12.1 +
concentrat
SDa

5.1
6.5
67.0
26.1
95.1
96.0
18.0
16.3
202.9
187-1

1.2
3.1
2.1
1.0
1.1
0.6
ion yg/L
Range

0
0
613 -
687 -
1273 -
1285 -
331 -
340
1737 -
1810

1.1
10.9
10.1
11.5 -
11-3 -
11.4 -

12
13
768
746
1482
1507
369
374
2180
2210

4.5
16.8
14.3
13.4
13.4
12.6
of weekly composite water samples.
                             50

-------
             TABLE A-3.  MEASURED CONCENTRATIONS OF THE METALS;
                         CHROMIUM, NICKEL, AND MERCURY DURING
                         ACUTE EXPOSURE TESTS.
Tank
no.
Chromium (March 9~15
21, 31
22, 23
2k, 25
26, 27
28
32, 33
34, 35
36, 37
38
Nickel (April 2-8, 1
21, 22
23, 2k
25, 26
27, 35
31, 32
33, 3k
36, 37
28, 38
Mercury (April 20-26
21 , 22
23, 2k
25, 26
27, 28
31, 32
33, 34
35, 36
37, 38
Nominal concentration
yg/L
, 1976) n = 6
0
30
100
80
5,000
500
300
50
3,000
976) n = 6
0
1 ,000
500
750
250
5,000
4,000
2,500
, 1976) n =* 6
50
400
0
300
500
30
100
200
Measured concentration pg/L
Mean + SDa

12 +
32 +
113 +
95 +
-
543 +
349 +
62 +
-

0
1118 +
530 +
828 +
285 +
5275 +
4145 +
2610 +

28 +
317 +
0
284 +
500 +
22 +
67 +
193 +

0
14
14
13

48
73
10



76
87
34
40
413
97
65

24^
47

53
50
8
12
10
aResults of daily grab water samples.
 Due to excessive storage time these values may not be accurate.
                                     51

-------
         TABLE  A-4.   MERCURY CONTENT OF SELECTED  TISSUES  OF  COHO
                     SALMON EXPOSED TO VARIOUS  CONCENTRATIONS  OF
                     MERCURIC CHLORIDE.
Nominal concen-
tration
yg/L
300
300
300
300
400
400
400
400
400
500
500
500
500
Exposure time
(h)
96
96
96
96
48
48
48
48
48
24
24
24
24
Tissue
Gill
Liver
Kidney
Muscle
Gill
Liver
Kidney
Muscle
Testes
Gill
Liver
Kidney
Muscle
ng Hg/mg tissue3
(wet weight)
37-6
8.0
22.9
0.8
43.4
5.3
17.0
0.8
0
62.8
4.5
15.6
0.3
One fish per concentration analyzed with Coleman Mercury Analyzer.
                                     52

-------
          TABLE  A-5. EFFECTS OF CADMIUM OR CADMIUM-COPPER EXPOSURE  ON  AVERAGE  LENGTH,  WEIGHT,
                      AND CONDITION FACTOR OF COHO SALMON.
Date Days
of of
sample exposure
1/13/76 0
2/24/76 0
" 0
" 0
" 0
11 0
" 0
11 0
11 0
" 0
11 0
vn 4/14/76 48
48
48
48
48
48
48
48
48
48
5/4/76 68
68
68
68
68
68
68
68
68
68
Nominal concentration
yg/L
Cd Cu
0
0
0
0.75
0.75
1.5
1.5
3.0
3-0
4.5
4.5
0
0
0.75
0.75
1.5
1.5
3.0
3-0
4.5
4.5
0
0
0.75
0.75
1.5
1.5
3.0
3-0
4.5
4.5
_
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
0
10
0
10
0
10
0
10
0
10
Fork
length
cm +_ SE
14.7 + 0.14
15-3 + 0.19
15.4 + 0.24
15-2 + 0.21
15-0 + 0.19
15.2 + 0.23
15-5 + 0.23
14.8 + 0.20
15.1 + 0.18
15-1 + 0.23
15-7 + 0.22
15.8 + 0.24
16.8 + 0.27
16.1 + 0.29
15-4 + 0.22
16.3 + 0.28
16.2 + 0.23
15.6 + 0.22
16.3 + 0.24
15.6 + 0.26
16.3 + 0.28
16.5 + 0.26
16.2 + 0.34
16.2 + 0.28
16.6 + 0.22
16.2 + 0.25
16.9 + 0.29
16.1 + 0.26
16.5 + 0.23
15.7 + 0.24
16.3 + 0.24
_ — -_ 	 , 	 . 	 . 	 _ —
Weight
g + SE
40.8 + 1.25
41.8 + 1.58
42.1 + 1.90
40.9 + 1-76
39.2 + 1.53
41.4 + 2.00
43.4 + 2.00
37.3 + 1.62
40.7 + 1-46
40.7 + 1.86
45.2 + 1.99
42.7 + 2.17
50.3 + 2.54
45.5 + 2.68
39.0 + 1.77
46.8 + 2.67
45.6 + 2.05
40.5 + 1.87
47.3 + 2.11
40.7 + 2.22
46.9 +2.56
46.2 + 2.23
44.5 + 3-36
43-9 + 2.39
46.3 + 1-99
43.4 + 2.22
50.2 + 2.97
43.4 + 2.30
45.8 + 2.11
39.4 + 1.92
44.0 + 2.15
Condi tion
factor3
KFL + SE
1.232 + 0.005^
1.141 + 0.009C
1.124 + 0.008C
.139 + 0.007
.134 + 0.007
.141 + 0.010
.149 + 0.010
.132 + o.oio
1 .151 + 0.008
1.141 + 0.007
1.143 + 0.003
1 .043 + 0.009
1.025 + 0.011
1.056 + 0.012
1.057 + 0.018
1.049 + 0.012C
.039 + 0.011
.028 + 0.009
.064 + 0.017
.044 + 0.014
1.039 + 0.012
0.997 + 0.009
0.989 + 0.013d
0.993 + 0.011
0.988 + 0.013d
0.988 + 0.013
0.995 + 0.012d
1.011 + 0.011
0.988 + 0.008d
0.985 + 0.009
0.988 + 0.008d
aSample size 40 except where noted, "Sample size 199,  °Sample size 39,  °5  days  of copper  exposure.

-------
VJl
•t-
                     TABLE A-6.   EFFECTS  OF ZINC-COPPER EXPOSURE ON AVERAGE LENGTH, WEIGHT,

                                  AND  CONDITION  FACTOR OF COHO SALMON.
Date Days of
of exposure
sample Zn Cu
5/25/76 13
13
13
13
13
13
13
13
13
13
6/8/76 27
27
27
27
27
27
27
27
27
27
0
6
0
6
0
6
0
6
0
0
0
20
0
20
0
20
0
20
0
20
Nominal concen- Fork
tration yg/L length3
Zn Cu cm + SE
0
0
400
400
800
800
1600
1600
2400
2400
0
0
400
400
800
800
1600
1600
2400
2400
0
10
0
10
0
10
0
10
0
10
0
10
0
10
0
10
0
10
0
10
19.1 + 0.25
17.6 + 0.27
18.6 + 0.30
18.0 + 0.29
18.2 + 0.24
17-3 + 0.14
18.1 + 0.28
17-7 + 0.21
17-9 + 0.22
17-2 +_ 0.24
18.8 + 0.25
18.6 + 0.29
19-2 + 0.32
18.1 + 0.24
18.9 + 0.36
17.7 + 0.26
17-9 + 0.28
17-8 + 0.28
17.8 + 0.26
17-9 + 0.24
Weight5
g + SE
70.1 + 2.90
55,3 + 3-07
67.6 + 3.39
59.8 + 3.39
62.4 + 2.50
51.6 + 1.33
61.8 + 2.96
55-7 + 2.37
57-4 + 2.07
50.4 +_ 2.18
69.2 + 2.93
64.3 + 3.45
75.8 + 4.17
58.6 + 2.72
73.1 + 4.39
54.5 + 2.56
60.3 + 3.03
56.2 + 2.84 '
57.6 + 2.55
55-9 + 2.49
Cond i t ion
factor3
KFL + SE
0.989 + 0.007
0.973 + 0.009
1.027 + 0.011
0.984 + 0.008
1.018 + 0.009
0.990 + 0.009
1.017 + 0.008
0.984 + 0.009
0.990 + 0.010
0.968 + 0.007
1.027 + 0.010
0.965 + o.oio
1.036 + 0.009
0.961 + o.oio
1.025 + 0.010
0.966 + 0.010
1 .019 + 0.009
0.958 + 0.008
0.999 + 0.009
0.948 + 0.007
95% C.I.
on mean KFL
0.9749-1.0031

1.0058-1.0492

0.9998-1.0362

1.008-1.0332

0.9698-1.0102

1 .0068-1.0472

1.0178-1.0542

1.0048-1.0452

1.0008-1.0372

0.9808-1.0233

     Sample size equal to 40.

-------
          TABLE A-7-  GILL (NA,K)-STIMULATED ATPASE ACTIVITY
                      OF COHO SALMON EXPOSED TO CADMIUM FOR
                      144 h.
Nominal con-
centration
0
8
12
Sample
date
2/12/76
2/12/76
2/12/76
Specific activity
n
6
6
4
Mean +
2.49 +
2.32 +
1.89 +
SD
0.39
0.54
0.23
35% C.I.
2.09-2.90
1.75-2.89
1.50-2.26
 0                 3/22/76         5            1-90  +_   0.78     0.93-2.87
 3                 3/22/76         6            1.85  +   0.75     1.06-2.64
4.5                3/22/76         7            2.53  +   1.10     1.51-3.55
                                   55

-------
       TABLE A-8.  GILL (NA, K)-STIMULATED ATPASE ACTIVITY OF COHO SALMON CHRONICALLY
                   EXPOSED TO CADMIUM OR CADMIUM-COPPER MIXTURE.  WHOLE GILL TECHNIQUE
                   USED EXCEPT WHERE NOTED.
Nominal con-
centration
ug/L
0
4.5
0
4.5
0
4.5
0
4.5
0
4.5
0
4.5
0/10
4.5/10
0
4.5
0/10
4.5/10
0
4.5
0/10
4.5/10
Exposure
(h)
Cd
816
816
816
816
1152
1152
1344
1344
1344
1344
1608
1608
1608
1608
1680
1680
1680
1680
1680
1680
1680
1680
time
Cu
0
0
0
0
0
0
0
0
0
0
0
0
96
96
0
0
168
168
0
0
168
168
Sample
date
3/31/76
3/31/76
3/31/76
3/31/76
4/14/76
4/14/76
4/22/76
4/22/76
4/22/76
4/22/76
5/3/76
5/3/76
5/3/76
5/3/76
5/6/76
5/6/76
5/6/76
5/6/76
5/6/76
5/6/76
5/6/76
5/6/76
n
6
6
6
6
15
15
6
6
6
6
5
6
6
6
6
6
6
6
6
6
6
6
ATPase specific
Means +_ SD
2.5 + 0.6
2.6 + 0.7
49.4 + 20.3
55.3 + 20.6
2.3 + 0.9
3.1 + 0.4
4.5 + 1.0
4.2 + 1.3
100 + 21.5
101.1 + 25.9
2.8 + 1.1
4.1 + 0.9
1.8 + 0.5
2.3 + 0.6
3-8 + 1.6
4.8 + 1.1
2.0 + 0.3
1.7 + 0.4
29.6 + n.o
37.0 + 4.6
15.4 + 2.0
16.9 + 4.2
activity
95% C.I.
1.8-3.1
1.9-3.3
26-72a
34-77a
1.8-2.8
2.6-3.6
3.5-5.6
2.9-5.6
77-123*
74-128a
1.4-4.1
3.2-5.0
1.2-2.5
1.7-3.0
2.1-5.4
3.7-6.0
1.7-2.3
1.3-2.3
l8-4ia
32-42a
12-17. 5a
12. 5-21. 2a
aMicrosomal preparation.

-------
VJl
       TABLE A-9.   GILL (NA,  K)-STIMULATED ATPASE ACTIVITY OF COHO SALMON CHRONICALLY EXPOSED
                   TO ZINC AND ZINC-COPPER MIXTURE,  WITH SUBSEQUENT SALTWATER CHALLENGE AND
                   FRESHWATER RECOVERY.   WHOLE GILL  TECHNIQUE USED EXCEPT WHERE NOTED.
Nominal con-
centration
0
2400
10
2400; 10
0
2400
10
2400; 10
0
2400
10
2400; 10

0
2400
10
2400; 10
0
10
2400; 10
Exposure
(h)
Zn
0
336
0
336
528
528
0
624
528
528
0
624
Zn; Cu; SW
0 0 288
675 0 288
0 500 288
675 500 288
0 500 0
0 500 0
675 500 0
time
Cu
0
0
168
168
0
0
456
456
0
0
456
456
FW
0
0
0
0
288
288
288
Sample
date
5/26/76
5/26/76
5/26/76
5/26/76
6/3/76
6/3/76
6/7/76
6/7/76
6/3/76
6/3/76
6/7/76
6/7/76

6/21/76
6/21/76
6/21/76
6/21/76
6/21/76
6/21/76
6/21/76
ATPase specific activity
n
6
6
6
6
6
6
6
6
6
6
6
6

10
10
10
10



Mean
4
4
2
1
3
4
1
1
41
41
17
12

4
4
4
4
2
1
1
.09
.09
.56
.42
.27
.08
.55
.52
.10
.10
.60
.88

.20
.70
.61
.49
.40
.86
.44
+_ SD
+ 0.
+ 1.
+ 0.
+ 0.
+ 0.
+ 0.
+ 0.
± °-
+ 15.
+ 9.
+ 6.
± ]-

+ 0.
+ 0.
+ 0.
± °-
+ 0.
+ 0.
+ 0.
7
4
6
3
7
9
5
4
4
6
6
3

6
3
6
9
4
9
3
35% C.I.
3.
2.
1 .
1.
2.
3.
1.
1.
26.
31.
10.
11.

3-
4.
4.
3.
1.
0.
1.
3
6
9
1
5
1
0
0
0
6
7
5

8
5
2
8
9
7
0
- 4.8
- 5.5
- 3-2
- 1.7
- 4.0
- 5.1
- 2.1
- 1-9
-56. 2a
-51. 8a
-24. 5a
-14. 3a

- 4.6
- 4.9
- 5.0
- 5.1
- 2.9
- 2.9
- 1.8
      Microsomal preparation.

-------
             TABLE A-10.  GILL (NA, K)-STIMULATED ATPASE ACTIVITY
                          OF COHO SALMON EXPOSED TO MERCURY FOR
                          ]kk h.

Nominal
concentration                                       Specific activity
   yg/L                           n               Mean + SD        35% C.I

     0                            3                5.7 + 0.7      3.9-7.4

    50                            5                3-3 + 0.9      2.2-k.k

   100                            3                4.5 + 2.0      0.5-9.5
                                       58

-------
                       TABLE A-11.   PLASMA SERUM  OSMOLARITY  AND  PLASMA  CHLORIDE  LEVEL
                                    OF COHO SALMON  EXPOSED TO  CADMIUM.
ui
Nominal con-
centration
(pg/L)
Control




8





12





Exposure time
(h)
6
24
48
96
168
6
12
24
48
96
168
6
12
24
48
96
168
x osmolari ty +_ SD;
(mOsm)
301.
295.
273.
300.
302.
292.
302.
288.
294.
304.
298.
275.
314.
295.
276.
280.
277.
0
8
7
5
4
4
2
2
9
0
2
0
0
0
5
0
3
+
+
+"
+"
+"
+
+"
+
+
"+
T
+
+"
+"
+
+"
+
14.3,
19.8,
15.9,
12.4,
3.4,
4.28,
6.34,
7.23,
10.72,
6.10,
3.88,
25.2
11.2,
28.7,
12.8,
8.4,
16.5,
6
8
7
5
1
1
2
3
3
2
1
11
5
12
5
3
7
SE
.4
.9
.1
.6
.9
.9
.0
.2
.4
.7
• 7
.3
.0
.8
.7
.8
.4
x chlorinity + SD, SE
(m Eq/1 iter)
112
118
114
113
114
111
112
115
114
114
115
113
117
121
117
111
105
.0
.8
.3
.7
.1
.0
.9
.3
.4
.4
.0
.1
.8
.5
.2
.3
.2
+ 6.6,
+ 10.1,
+ 7.5,
+ 5.7,
+ 1.4,
+ 6.4,
+ 5.1,
+ 2.9,
+ 6.5,
+ 2.5,
± 3.4,
+ 10.9,
+ 5.5,
+ 10.1,
+ 15.8,
+ 12.9,
+ 9.6,
2.9
4.5
3.3
2.9
0.6
2.8
1.6
1.3
2.9
1.1
1.5
4.8
2.5
4.5
7.0
5.7
4.3

-------
                TABLE A-12.   PERCENT  MIGRATION  (TO  JULY  6,  1976)  OF YEARLING  COHO
                              SALMON RELEASED  INTO A SMALL COASTAL STREAM  FOLLOWING
                              ACUTE AND  CHRONIC  EXPOSURE  TO  SEVERAL METALS.
Nominal con-
centration
yg/L


(1-5)
Percent migration
(Days post release)
(6-10) (11-20) (21+) (1-5) (6-10)
A. Chronic exposure
0 Cd
10 Cu
0.75 Cd
0.75 Cd + 10 Cu
1.5 Cd
1.5 Cd + 10 Cu
3.0 Cd
3.0 Cd +
4.5 Cd
4.5 Cd +
58.1
67.7
60.7
63-2
59
68
61
64
Apri
.1
.2
.8
.3
1 14, 1976a
(11-20)
(21+)
May 5, 1976*
60.2 60.2
68.7 68.7
62.4 62.4
65.9 65.9
10 Cu


56.7
58
• 9
59.4 59.
4
10 Cu
B. Acute exposure
0 Cd
0.75 Cd
1.5 Cd
3.0 Cd
4.5 Cd





(144 h)c






78.7
75.8
73-5
73.5
73.7

80
76
-

""
C. Chronic exposure

0
10 Cu
400 Zn
400 Zn
800 Zn
800 Zn
1600 Zn
1600 Zn
2400 Zn
2400 Zn




+ 10

+ 10

+ 10

+ 10
D. Acute exposure
0
10 Cu
400 Zn
800 Zn
1600 Zn
2400 Zn
2400 Zn


+ 10
+ 10
+ 10

+ 10




Cu

Cu

Cu

Cu
(144 h)*


Cu
Cu
Cu

Cu
(1-5)
75.5
51.9
72.2
48.8
64.0
37.0
79-0
27.2
59.2
27-3









.9
.8



May
(6-10) (1
79
63
75
52
71
47
82
33
65
37








.2
• 7
.9
.5
.0
.0
.0
.0
.0
.7









80.
77.8 77.
73.
73.
73-
26, 1976e

9
8
5
5
7

1-20) (21+)
80.2 80.
63.
75.
52.
71.
47.
82.
34.0 34.
66.0 66.
37.








2
7
9
5
0
0
0
0
0
7








65.1
59.4
65.0
42.0
47.8
44.9
67-5
47-1
61.4,
43.2

85.1
71.8
64.7
78.4
67-9

(1-5)
64.4
51.9
64.1
51.4
59-3
50.4
57.8
50.5
68.6
56.4

76.9
62.9
66.0
55.7
40.6
51.7
45.0
68.0
43.0
48.6
49.5
49.2
-
4g.O

_
-
-
79.5
—
June 9,
(6-10)
69.2
62.5
67-9
56.8
63.8
59-0
64.7
60.7
69.6
64.5

81.5
64.5
72.0
67.2
53-1
58.6
53.3
66.0
60.4
69-9
45.0
53-0
50.5
68.4
54.8
65.3
50.0

_
-
67.6
80.7
69.0
]376f
(11-20)
71.2
63-5
71.8
58.1
66.7
61.9
66.7
63.5
73-5
64.5

81.5
66.1
72.0
67.2
53-1
62.1
58.3
67.0
62.3
69-9
47-0
55.6
51.4
69-3
56.7
66.3
54.8

85. ld
71.8
67.6
80.7
69.0

(21+)
71.2
65.4
73.1
58.1
67.5
61.9
67.6
63-5
74.5
64.5








 Release of 180-192 fish per group (47 days Cd).
^Release of 100-114 fish per group (67-days Cd; 5-days Cu; 62 days Cd + 5 days Cd-Cu).
°Release of 94-102 fish per group (6-days Cd).
^Release of 67-88 fish per group (6-days Cd).
eRelease of 100-106 fish per group (13-days Zn; 6-days Cu; 7 days Zn + 6 days Zn-Cu).
fRelease of 102-110 fish per group (27-days Zn; 20 days Cu; 7 days Zn + 20 days Zn-Cu).
^Release of 50-65 fish per group exposed for 6 days.
                                                  60

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APPENDIX I.  RECOMMENDED SEAWATER-ENTRY TEST PROCEDURE

     Sublethal exposure to toxic chemicals can produce aberrations in physio-
logy which diminish the organism's ability to grow, reproduce, or otherwise
adequately perform its overall biological function.  Detection of these
aberrations usually requires the use of testing procedures beyond those
necessary to produce routine acute toxicity data.  Juvenile anadromous
salmonids undergo a series of physiological changes, collectively known as
smelting, which enable and cause the fish to migrate from freshwater to the
ocean.  Toxic chemicals are capable of disrupting normal  smelting by inter-
fering with osmoregulatory processes or downstream migration (Lorz and
McPherson 1976, 1977, Bouck and Johnson MS).  Procedures  which detect
interferences in smolting have been developed using enzyme assays, field
migration studies, or blood sodium levels, but these procedures are often
complex, limited to specific toxicants, or insufficiently documented for use
in routine screening methods for toxic wastes or chemicals.  One test proce-
dure which lends itself to routine toxicity screening methods is "the
seawater-entry test" (SET).  This procedure involves a routine freshwater
acute toxicity test followed by immediate transfer of survivors to 30 °/oo
seawater and holding for one week.  Interference with smolting is frequently
manifested in osmotically related mortality of toxicant-exposed fish following
transfer to seawater-  The SET is recommended for use with all chemicals or
wastes likely to be discharged into rivers or estuaries having populations
of anadromous fish.  Like any toxicity test, SET provides an index of
probable environmental damage and an effect level for use in risk assessment.

Test Procedure

     The acute toxicity test comprising the first phase of SET should be
conducted according to the procedures outlined in Methods for Acute Toxicity
Tests with Fish, Macroinvertebrates, and Amphibians (EPA, 1975).  It is re-
commended that SET be conducted with either coho salmon (Oncorhynchus
kisutch), Atlantic salmon (Salmo salar), or steelhead trout (anadromous Salmo
gairdneri}.  Smolting characteristics of these species are such that smolts
are normally larger than 7 cm in length; however, the sole criterion recom-
mended for suitability of test fish for SET is a greater  than 90% survival
of control fish in seawater.

     At the termination of the 96-h acute toxicity test in freshwater the
survivors are randomly split into two groups of equal numbers, with one
group transferred into 30 °/oo seawater and the other into normal (non-
toxicant containing) freshwater.  This procedure enables  the investigator to
separate seawater-related, toxicant-induced mortalities from direct residual
effects of the toxicant exposure.  Both freshwater and seawater temperatures
should be  12°C.

     Fish loading for the holding period following the acute toxicity test
should not exceed 5 g/L.  Since no toxicant is involved in this latter
phase, static tests and aeration of the water are acceptable.  The dissolved
                                      61

-------
oxygen concentration should not fall below 75% saturation and concentrations
of unionized ammonia should not exceed 20 yg/L.  The test organisms should
not be fed throughout the test.

     A salinity of 30 °/oo  is recommended.  This salinity allows the use of
near-shore supplies of natural seawater which usually do not achieve the
approximately 35 °/oo salinity of full strength seawater.  Dilution of more
saline seawater to 30 °/oo  is achieved with any good quality freshwater
suitable for acute toxicity testing.  Artificial seawater is acceptable as a
substitute for natural seawater and can be reconstituted with the composition
given  in Table 1.

     The salinity must be adjusted  to 30 +_ 0.5 °/oo and the pH of the seawater
measured.  Dissolved oxygen should  be determined daily in each test tank
during the second phase of  static tests to assure that aeration  is sufficient.

     The overall duration of  the test is  11 days, with an initial 96-h
freshwater  acute toxicity  phase followed immediately by a 7~day seawater
exposure.

     The number of dead organisms in each test chamber must be counted at
least  every 24 hours after  the beginning of the test and dead organisms
should be  removed as soon as  they are observed.

     Following a freshwater acute toxicity test only one or two  toxicant
concentrations may have enough survivors  for  the SET; if these concentrations
result  in  mortality of seawater-exposed fish,  it is necessary to repeat the
test procedure starting with  a second acute toxicity test in freshwater
using  lower concentrations  of the toxicant.
                                       62

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                TABLE 1.  RECOMMENDED PROCEDURE FOR PREPARING
                          RECONSTITUTED SEAWATER3

Add the following technical grade chemicals  and waterc to make one liter
of seawater.  It is important that the listed order of ingredient additions be
followed and that each  ingredient be dissolved prior to adding the next one;
this is best accomplished by constant mixing in the course of preparation.

 1. Add 890 ml of tap water to a suitable container

 2. Add 2.85 mg               NaF

 3. Add 19-5 mg               SrCl2-6H20

 k. Add 30.0 mg               H3B03

 5. Add 100.0 mg              KBr

 6. Add 699.0 mg              KC1

 7. Add 1.1 g                 CaCl2-2H20

 8. Add 3-99 g                Na2SOi,

 9. Add 5.0 g                 MgCl2-6H20

10. Add 23.48 g               NaCl

11. Add 19-5 mg               Na2Si0.^9^0

12. Add 0.6 mg                ethylenediaminetetraacetate (EDTA)  tetrasodium salt

13. Add 199.5 mg              NaHC03 • The pH should be 8.0 + 0.2

14. Fill to one-liter mark, the salinity should be 30 +_ 0.5 °/oo.  If a lower
    test salinity is desired, dilute with additional tap water.

fFrom Zaroogian et al. 1969 and LaRoche et al. 1970.
 Reagent grades of EDTA are used because of unavailability of technical grades.
°Good practice to determine chemical characteristics of water being used
 in making seawater.
                                     63

-------
                                 REFERENCES

Bouck, G. R. and D. A. Johnson. (1977 MS).  Inhibition of saltwater tolerance
     in coho salmon (Oncorhynchus kisutch) by disease treatment.
     National Fisheries Research Center - Seattle, Washington.  12 pp.

Committee on methods for toxicity tests with aquatic organisms.  1975.
     Methods for acute toxicity tests with fish, macroinvertebrates, and
     amphibians.  Ecol. Res. Ser. EPA-660/3-75-009.  U. S. Environ. Prot.
     Agency.  Corvallis, Oregon.  61 pp.

LaRoche, G., R. Eisler and C. M. Tarzwell.  1970.  Bioassay procedure for
     oil and oil dispersant toxicity evaluation.  J. Wat. Pollut. Cont. Fed.
     4(11) :1982-1989-

Lorz, H. W. and B. P- McPherson.  1976.   Effects of copper or zinc  in fresh
     water on the adaptation to sea water and ATPase activity, and the
     effects of copper on migratory disposition of coho salmon  (Oncorhynchus
     kisutch).  J. Fish. Res. Board Can.  33(9):2023-2030.

Lorz, H. W. and B. P. McPherson.  1977-   Effects of copper and zinc on
     smoltication of coho salmon.   Environmental Protection Agency.
     Corvallis, Oregon EPA 600/3-77-032.  69 pp.

Zaroogian,  G. E., G. Pesch and G. Morrison.  1969-  Formulation of an arti-
     ficial sea water media suitable for  oyster larvae development.  Am.
     Zool.  3:}}kk.

-------
APPENDIX II.  PARTIAL CHARACTERIZATION OF GILL (NA, K)-STIMULATED, OUABAIN
SENSITIVE ADENOSINE TRIPHOSPHATASE FROM COHO SALMON, Oncorhynchus kisutch.
                             INTRODUCTION

     An enzyme system composed of a (Na, K)-stimulated, ouabain sensitive
adenosine triphosphatase has been shown to be a fundamental mechanism for
active ion transport across cell membranes (Maetz 1969, Post et al. I960,
Skou I960, 1972).  Numerous investigators have found that the gill (Na, K) -
stimulated ATPase system plays an important role in saltwater adaptation of
euryhaline fishes (Epstein et al. 1967, Jampol and Epstein 1970, Kamiya and
Utida 1968, 1969, Sargent and Thomson 197*0-  Parr-smolt transformation and
migratory behavior of several salmonid fishes have been shown to correlate
with an increase in gill (Na, K)-stimulated ATPase (Giles and Vanstone 1976,
Lorz 1974, Zaugg and McLain 1970, 1972, Zaugg and Wagner 1973).

     Changes in enzyme activities of vertebrates have been reported due to
exposures of heavy metals.  Donaldson et al.  (1971) found inhibition of (Na,
K)-stimulated ATPase in rat brain preparations by several metals.  Activities
of five liver enzymes of a cyprinodont fish changed either by exposing the
liver homogenate, or the fish itself to salts of various metals  (Jackim et
al. 1970).  Lorz and McPherson  (1976) were able to detect gill  (Na, K)-ATPase
inhibition in coho salmon exposed to sublethal levels of copper.  Consequ-
ently, during the present study periodic monitoring of gill  (Na, K)-stimulated
ATPase activities was undertaken to reveal any changes attributable to heavy
metal exposure.  Significant change in this biochemical mechanism  in an
anadromous fish may disturb the normal migratory behavior or the ability of
the fish to adapt to a hypertonic environment.

     In past studies at this laboratory, an enzyme assay was used for gill
microsomal (Na, K)-ATPase which involved detergent extraction and centri-
fugation as portions of a lengthy procedure (Zaugg and McLain 1970).  The
complexity of the procedure limited the number of fish sampled to the extent
that statistical analysis of the data was difficult.   In addition, these
procedures were never characterized sufficiently to reveal the extent of
variability of enzyme activity  if chemical components of the assay were
varied.  An assay method was developed to determine (Na, K)-stimulated,
ouabain-inhibitable ATPase activity quantitatively in whole homogenates of
gill filaments  (Ewing and Johnson 1976).  All centrifugation and detergent
treatments were eliminated.  The reduction in analytical time, as well as
the versatility of the whole gill homogenate  technique, allows the investigator
the advantage of a larger number of samples and field or laboratory appli-
cation.  Johnson et al. (1977) described in detail a characterization of
spring chinook salmon, whole gill (Na, K)-stimulated ATPase.  Several of
their observations were assumed to be consistent in the coho salmon enzyme
system, and therefore, were not investigated  in this characterization.
These  mcluded  ion requirements, stability upon storage with 2-mercaptoethanol,
fractionation with and without deoxycholate,  and reaction time.
                                       65

-------
                                    METHODS

     Coho salmon, Oncorhynchus kisutch, from the Fall Creek Hatchery, Oregon,
were reared for approximately 14 months in well water at  12°C.  Fish were
fed at recommended levels  (ODFW-hatchery procedures) with Oregon Moist
Pellet (Bioproducts,  Inc.).  Fish chosen for analysis were pre-smolts and
approximately  15 cm  in  length.

     Procedures for  enzyme analysis followed those described by Ewing and
Johnson  (1976) with  a few changes  in concentrations of components.  Gill
filaments were homogenzied  in a Potter-Elvehjem homogenizer containing a
solution of 0.1 M  imidazole  buffer, 20 mM Na£EDTA, 0.3 M  sucrose, and 10 mM
2-mercaptoethanol. adusted  to pH 1.2.  Fifteen mg wet weight of gill filament
per mL of homogenizing  media (HMM) was used  routinely. To initiate the re-
action,  0.1 mL of  the homogenate was added to 0.4 mL of a reaction mixture
containing; 100 mM  imidazole buffer, 10 mM Na2ATP, 20 mM  MgCl2, 90 mM KCL,
and 270  mM NaCl , adjusted  to pH 7.2. Incubation  was-carried out at 37°C
 (without shaking)  for 20 min.  The  reaction  was stopped with 3.0 mL of 1.66
N  H2S02j  and liberated phosphate was measured by the method of  Ernster et al.
 (1950).  Protein was measured by a  modification of the method  of Lowry et
al.  (1951).   (Na,  K)-stimulated activity was calculated as the difference
between  rates  of  inorganic  phosphate liberated  in the presence or absence of
0.5 mM ouabain and  divided  by the  protein value.


                                    RESULTS

     Whole gill  homogenates  of coho salmon may  contain numerous enzymes
 capable  of hydrolyzing  ATP.  The activities  of  these enzymes can be stimulated
 by Mg2+, Na+,  K+,  possibly Ca   and other  ions, while significant  inhibition
 of some  activity  occurs in  the presence of ouabain.  The  (Na,  K)-stimulated
 ouabain  sensitive  portion  is the component  responsible for the (Na, K)  ion
 transfer phenomenon (E.G.  3.7-6-2)  and  is of interest here.  The ouabain
 insensitive portion appears to  represent a complex of activities and was not
 considered of primary importance  in this study.

 PROPERTIES OF OUABAIN SENSITIVE ATPASE

     The optimal  pH for the reaction was 7-3, with  sharp  declines  in activity
 on either  side of this  value (Fig.  1A).

      Effects  of  tissue  concentration and the chelators EDTA or EGTA  in the
 HMM, were  tested  for their influence on activity.   Figure IB shows a definite
 suppression of activity by EGTA, while  in  the  presence of EDTA the activity
 was  proportional  to the tissue  concentration.   A  tissue concentration of 15
 mg/mL  was  considered adequate.  Although  inhibition  of the enzyme  has been
 reported by  concentrations of EDTA in  excess of 1 mM (Wallick  et al.  1973),
 this  problem  didn't appear in  our  assays.  The  20 mM of MgC^  in our  reaction
 mixture  may have alleviated the  inhibitory effect of EDTA.
                                       66

-------
3.9i-
                                                                   — EDTA 20 m M

                                                                   — EGTA 10 m M
                                                             10       is      20
                                                            Tissue Concentration
                                                        tissue/ml. homogenizing soln.)
              .2     .3     .4     .5

       Ouobain Concentration, mM
 5     10     15     20    25
Nag ATP Concentration, mM
 Figure 1.  Relationship of gill  (Na, K)-stlmulated ATPase  activity of  coho
 salmon to: A. pH  of reaction mixture; B. tissue concentration and the
 chelators EDTA  and EGTA; C. ouabain concentration; D. disodium ATP concen-
 tration.
                                       67

-------
                10    20    30    4O
               MgCI  Concentration, m M
                                       so
        2.5 -
CO
                           120       ISO       240
                           240       ISO       120
                              Concentration, m M
270
 90
300
60
360
 0
                                                                          D
                              100     200     300     400     500
                                      {Na + K) Concentration, mM
                                                                                                                     600
40    60     80     100    120
       KCI Concentration,
140
      160
            180
       Figure  2.   Relatinship of gill  (Na, K)-stimulated ATPase activity of coho  salmon to: A.  magnesium
       chloride concentration; B. combined concentrations of  NaCl and  KCI;  C. ratios  of NaCl/KCl  in reaction
       mixture; D. potassium chloride  concentration.

-------
     The inhibitory effect of ouabain is shown in Fig. 1C.  Maximal  inhibi-
tion occurred at a concentration of 0.1 mM, while half maximal inhibition
occurred at 0.025 mM.  Disodium ATP concentrations were varied (Fig. ID) and
maximal reaction rates were observed at 10 mM ATP in the presence of 20 mM
MgCl2, 270 mM NaCl , and 90 mM KC1.  Optimal activity occurred at MgCl2
concentration of 20 mM in the presence of 10 mM Na2ATP (Fig.2A).

     The activity of the ouabain sensitive enzyme complex was not only de-
pendent upon the concentrations of Na+ and K+ but also to the ratio of these
ions and their combined ionic strength in the reaction mixture.  Four tests
were performed to define the influence of these variables in promoting
enzyme activity.  Optimal activities occurred when the ionic strength was
between 360-450 mM (Fig. 2B).  Using 360 mM (Na+K) ionic strength as our
standard, the Na+:K+ ratio was varied.  Although a complex relationship
exists between the ratios and enzyme activity (Fig.ZC), a 3:1 ratio  (270 mM
Na:90 mM K.) gave consistently maximal activities.  In the presence of 20  mM
MgCl2 and 270 mM NaCl maximal activity occurred at a KC1  concentration of
approximately 60 mM (Fig. 2D).   The sodium concentration curve was more
complex but revealed an optimal activity at 270 mM NaCl (Fig. 3).
                      4.0!
                   o>
                 ^ Q.  3.0
                 O £

                   ^2.0
                 UJ £
                 CO
                 0:$  i.o
                 I- °
                 5 £
                           36 72 108  180    270   360

                              NaCl CONCENTRATION, mM
Figure 3-  Relationship between NaCl concentrations and gill (Na, K)-stimulated
ATPase activity.
                                      69

-------
                                  DISCUSSION

     An analytical procedure utilizing a whole gill homogenate of coho salmon
(O. kisutch), required reaction conditions different from those previously
reported for microsomal isolates.  Whereas Jorgensen and Skou (1971) found
maximum activation of  (Na, K)-ATPase by addition of deoxycholate (DOC) to
microsomal preparations of rabbit kidney, a loss of activity in whole gill
homogenates of chinook salmon  (O. tshawytscha) occurred from exposure to DOC
(Johnson et al. 1977).  Although the effects of DOC on whole gill homogenates
from coho salmon were not specifically tested it was assumed that a reduction
in enzyme activity would occur similar to that found in chinook salmon.
Other conditions which differed from various commonly used  (Na, K)-ATPase
analyses were: ionic strength; Na:K ratio; Mg2+; and ATP concentrations.
Reasons for the differences are not known.

     Since  the subcellular units in the homogenate were not fractionated,
sedimentation occurred upon standing.  Considerable variation in the assay
resulted  if thorough homogenization and resuspension of all particles was not
accompli shed.

      It appeared  that  the enzyme activity determined by ouabain  inhibition
was the best method  in which to calculate the (Na, K)-stimulated ATPase
activity  using the whole gill  homogenate assay.  Tests conducted during this
study with  0-age  class coho salmon parr reveal an  inhibition of total ATPase
activity  (Mg   only  stimulated), by the addition of extraneous NaCl and KC1
to the  reaction mixture.   If further testing substantiates  this observation,
it will have definite  implications on  the manner  in which the Na+,  K+-
transport enzyme  activity  is calculated.  At this  time we believe that
determination of  the Na+,  K+-transport enzyme activity  in coho salmon  is best
calculated  by the difference between reactions  involving: substrate + enzyme
+  (Mg2+,  Na+, K+) and  substrate + enzyme +  (Mg2+,  Na+,  K+,  ouabain).

      In any given population of fish,  the  (Na,  K)-stimulated ATPase activi-
ties  are  quite variable  between  individuals.  This  is  illustrated often by
large  standard deviations  in specific  activities  (Fig.  k).  The coefficients
of variation  (C.V.)  were calculated for all assays  run on control fish for
the period  February  12 to  June 21,  1976.  Using these  C.V.  a brief  comparison
was made  between  the two assay methods used at our  laboratory; a) gill
microsomal  technique (Zaugg and McLain  1970), and  b) whole  gill  homogenate
technique (Ewing  and Johnson  1976).  The  results  (Table  l),  indicated
slightly  greater  average C.V.  occurred with the microsomal  preparation.
However,  the C.V. experienced  in our whole  gill homogenate  assays of coho
salmon, were approximately 12% higher  than  those  of  14  spring chinook  salmon
assayed by  the same  procedure  (Johnson et al.  1977).   Differences are
probably  inherent in the variation of  enzyme activities  between  individuals,
and not due solely  to  the  technique used.

      Several  investigators have  found  seasonal  variations  in  (Na, K)-ATPase
using microsomal  preparations  of coho  salmon gill  (Giles  and Vanstone  1976,
Zaugg and McLain  1972).  We felt  it would be of  interest  to determine  if  this
                                        70

-------
seasonal variation occurred when whole gill preparations were used.  Using
coho salmon of the 1975 brood, 30 fish from "stock" tanks were sampled
monthly.  The whole gill homogenates were analyzed for  (Na, K)-ATPase as
described.  Figure 4 shows the seasonal fluctuation in  the  (Na, K)-ATPase
activities.  A distinct rise  in activity occurred  in the spring (February
thru May) of their second year of life.  This rise in activity coincided with
migratory movements we have observed in both wild and experimental groups
from this stock.  Zaugg and McLain  (1972) found a similar rise in activities
of yearling coho salmon, while Giles and Vanstone  (1976) reported increased
activities occurred in mid-winter and decreased activities  in the spring.
 >£   8
-------
            TABLE 1.  COEFFICIENTS OF VARIATION OF MEAN SPECIFIC
                      ACTIVITIES OF GILL (NA, K) ATPASE USING TWO
                      DIFFERENT ASSAY TECHNIQUES.

                                        No. of    Total No.   Mean
Preparation _ assays    of fish _ C.V.   Range
Microsomal (coho)                         k         2k         3k%  22%-k\

Whole gill homogenate  (coho)             11         69         27%

Whole gill homogenate  (spring chinook)3   1         ]k        14.5%


a Data from Johnson et al. 1976.
                                        72

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                                  REFERENCES

Donaldson, J., T.St-Pierre, J. Minwich, and A. Barbeau.  1971.  Seizures in
     rats associated with divalent action inhibition of Na+-K+-ATPase.  Can.
     J. Biochem.  49:1217-1224.

Epstein, F. H.-, A. J. Katz, and G. E. Pickford.  1967-  Sodium and potassium-
     activated adenosine tr iphosphatase of gills: role in adaptation of
     teleosts to sea water.  Science. 156:1245-1247.

Ernster, L. R. , R. Svetterstrom and 0. Lindberg.  1950.  A method for the
     determination of tracer phosphate in biological material.  Acta. Chem.
     Scand.
Ewing, R. D. and S. L. Johnson.  1976.  A simplified procedure for analysis
     of  (Na+K) activated ATPase.  Oregon Dept. of Fish and Wildlife;
     Information Rept. Series, Fisheries No 76~3-

Giles, M. A. and W. E. Vanstone.  1976.  Changes in ouaba in-sensitive
     adenosine tr iphosphatase activity in gills of coho salmon (Oncorhynchus
     kisutch) during parr-smolt transformation.  J. Fish. Res. Board Can.
     33:54-62.

Jackim, E., J. M. Hamlin, and S. Sonis.  1970.  Effects of metal  poisoning
     on 5 liver enzymes  in the killifish (Fundulus heteroclitus) .   J. Fish.
     Res. Board Can. 27:383-390.

Jampol , L. M. and F. H.  Epstein.  1970.  Sodium-potassium-activated adenosine
     tr iphosphatase and osmotic regulation by fishes.  Am. J. Physiol.
     218(2):607-611.

Johnson, S. L., R. D. Ewing and J. A. Lichatowich.   1977-  Characterization
     of gill (Na+K)-act ivated adenosine tr iphosphatase from Chinook salmon,
     Oncorhynchus tshawytscha.  J. Exp. Zool .  199:345~354.

Jorgensen, P- L. and J.  C. Skou.  1971-  Purification and characterization
     of  (Na+ + K+)-ATPase.  Biochem. Biophys.  Acta. 233:366-380.

Kamiya, M. and S. Utida.  1968.  Changes in activity of sodium-potassium
     activated adenosinetr iphosphatase in gills during adaptation  of the
     Japanese eel to sea water-  Comp. Biochem. Physiol.  26:675-685.

_ .  1969.  Sodium-potassium activated  adenos inetriphosphatase activity
     in gills of fresh-water, marine and euryhaline teleosts.  Comp. Biochem.
     Physiol.  31:671-674.

Lorz, H. W.  1974.  The development of the parr-smolt transformation in
     anadromous salmonids in relation to some  environmental conditions.
     Progress Rept. AFS Project 20-6.  Oregon  Wildlife Commission.  12 pp.
                                       73

-------
Lorz, H. W. and B. P. McPherson.  1976.  Effects of copper or zinc in fresh-
     water on the adaptation to seawater and ATPase activity, and the effects
     of copper on migratory disposition of coho salmon (Oncorhynchus kisutch),
     J. Fish. Res. Board Can. 33(9):2023-2030.

Maetz, J.  1969.  Seawater teleosts:evidence for a sodium-potassium exchange
     in the branchial sodium-excreting pump.  Science 166:613-615-

Post, R. L., C. R. Merritt, C.  R. Kinsolving, and C. D. Albright.  I960.
     Membrane adenosine triphosphatase as a participant in the active
     transport of sodium and potassium in human erythrocyte.  J. Biol.
     Chem. 235:1796-1802.

Sargent, J. R. and A. J. Thomson.   1974.  The nature and properties of the
     inducible sodium-plus-potassium  ion-dependent adenosine triphosphatase
     in the gills of eels  (Anguilla anguilla) adapted to fresh water and
     sea water.   Biochem.  J. 144:69~75.

Skou, J. C.  I960.   Further  investigations on a Mg^+-Na+ activated ATPase,
     possibly related to the active transport of Na+ and K+ across the
     nerve membrane.  Biochem.  Biophys. ACTA 42:6-23-

Skou, J. C.  1972.   Preparations  from mammalian brain and kidney of the
     enzyme system  involved  in  active transport of Na+ and K+.  Biochem.
     Biophys.  ACTA  58:314-325.

Wai lick, E. T., J. C. Allen, and  A. Schwartz.   1973-  Differential effects
     of metal chelators on Na+,  K+-ATPase activity.  Arch. Biochem. Biophy.
      158:149-153.

Zaugg,  W.  S. and  L.  R. McLain.   1970.  Adenosine triphosphatase activity
      in gills of  salmonids:  seasonal  variations and salt water  influence
     on coho salmon, Oncorhynchus kisutch.  Comp. Biochem. Physiol. 35:
     587-596.

Zaugg,  W.  S. and  L.  R. McLain.   1972.  Changes  in gill adenosine-triphos-
     hatase activity associated with  parr-smolt transformation  in steelhead
      trout,  coho, and  spring chinook  salmon.  J. Fish. Res.  Board Can.
     29:167-171.

Zaugg,  W.  S. and  H.  H. Wagner.   1973.  Gill ATPase activity  related to
     pan—smolt  transformation  and  migration  in steelhead trout  (Salmo
     gairdneri):Influence  of photoperiod and  temperature.  Comp.  Biochem.
      Physiol.  45(B):955-965.

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APPENDIX III.  HISTOLOGICAL EFFECTS OF SEVERAL METALS IN SELECTED TISSUES OF
YEARLING COHO SALMON.

                                INTRODUCTION

     In an attempt to explain observed differences in migratory behavior,
(Na, K)-stimulated ATPase levels and saltwater tolerance,  histological
sections were prepared from selected tissues of fish exposed to several
metals.

                                   METHODS

     Five exposed fish and 5 control fish were taken at  each sampling.   The
following metals, exposure periods and concentrations were used:

     Cadmium

     12 yg/L for 48 h
     12 yg/L for 120 h
      3 yg/L for 21 days
    4.5 yg/L for 70 days
    4.5 pg/L for 70 d + 10 yg/L copper for the last 6 days

     Copper

     10 yg/L for 6 days
     10 yg/L for 20 days (3 fish)

     Mercury

     500 yg/L for 24 h

     Zinc

     2400 yg/L for 28 days
     2400 yg/L for 28 days + 10 yg/L copper for the last 20 days.

     Fixing, mounting and staining methods recommended by  Galigher and
Kozloff (1971) were followed.  Gills,  liver, gonads,  head  kidney,  and a
portion of the middle kidney were taken from each fish.   Tissues  were  im-
bedded in paraplast on an Autotechnicon and 10 micrometers (ym)  sections were
cut.   Mayers' egg albumen was used as an affixative. Slides were stained
with Harris1 hematoxylin with eosin as a counterstain.  Sections  were mounted
from the beginning, middle and end of each band of parafin.
                                       75

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                           RESULTS AND DISCUSSION

     Tissues from fish exposed to metals were compared to control fish to
determine differences in organ and cell structure, staining properties, and
the consideration of the nucleus.  No apparent histological aberrations
resulted from the metal exposures (Table 1).  Specific data for tissues
examined are given below.

     Gills  No major structural damage from any of the metals or combination
of metals tested.  Post-mortem decomposition destroyed the gills of fish
exposed to mercury.

     Gonads  No necrosis was observed that could be attributable to metals.
There was some discoloration and tissue necrosis  in ovaries that came  into
contact with bile fluid.  Due to the prevalence of the burst gall bladders
in control fish, we assumed that the necrosis observed were not caused by
the metals but by of the bile.  The caustic action of bile has been shown to
cause destruction of liver tissue (Hendricks et al. 19/6).  Sangalang and
O'Halloran  (1973) found that the testes of mature  brook trout when exposed
to 25 yg/L Cd for 24 h showed extensive hemorrhagic necrosis and disintegration
of lobule-boundary cells.  In a second experiment with fish exposed for a
maximum of 30 days to  10 yg/L Cd similar injury to the testes was noted but
the damage was not as extensive.  In our experiments fish were sexually
immature, our concentrations of Cd <2.5 times Sangalang and O'Halloran's
experiment.  This may explain the lack of histopathological effects.

     Kidneys  No necrosis was noted that could be  directly attributable to
heavy metal  intoxication.  Differences between samples and controls were
suspect,  because of  fixation procedure.  The kidney should be, but was not
excised prior to fixation.   If formalin  is used as a fixative, it should be
fresh and changed every  two weeks during the preservation  period.  Formalin
degrades  naturally to  formic acid which  is caustic and could cause decom-
position  of  fixed tissues.

     Liver   Liver sections showed two  forms of necrosis, but neither one
could be  attributed  to metal  intoxication.  Sections showed subcapsilar
necrosis  where gall  bladders became  ruptured or appeared to leak. Secondly,
fish used  in  static  bioassays were not fed and did not have as many glycogen
deposits  in  liver cells  as did the well-fed chronically tested fish.

     The  levels  of metals  used  in these  studies were too  low or  the exposure
too  short  to  produce detectable  tissue necrosis.   Voyer et al.  (1975)  found
no histopathology  in the (_freshwater)  mummichog  (Fundulus  heteroclitus)
exposed  to  cadmium at  concentrations of  3 mg/L and suggest this  level  may
have been  too  low  to stimulate morphological change.   Other authors also
have presented evidence  that  histopathological changes occur only when the
fish have been exposed  to  metals  above a minimal  threshold concentration:
Lloyd  (I960)  found gill  damage  in  rainbow  trout subjected  to 20  mg/L Zn for
2.5  h but absent in  specimens  exposed  to 3 mg/L Zn, a  sublethal  concentration,
for  2 days;  similarly  no histopathology was apparent  in mummichogs exposed
to 5 mg/L Cd  for one year, although  intestinal  lesions and pathological


                                       76

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TABLE 1.  SUMMARY OF HEAVY METALS HISTOPATHOLOGY.
Nominal con-
centration
Metal pg/L
Cd 12



12



3



4.5 + 10 yg/L Cu


Cu 10



Hg 500


Zn 2400


Zn 2400 + 10 yg/L Cu


Sample
N
5
5
5
5
5
5
5
5
5
5
5
5
3
3
3
8
8
8
5
3
3
3
3
3
3
3
3
3
Exposure
period
48 h
48 h
48 h
48 h
120 h
120 h
120 h
120 h
21 days
21 days
21 days
21 days
70 days + Cu
last 6 days

6 days
20 days
20 days
20 days
24 h
24 h
24 h
28 days
28 days
28 days
28 days
+ Cu for
last 20 days
Tissue
gills
1 iver
gonads
kidney
gills
liver
gonads
kidney
gills
1 iver
gonads
kidney
gills
1 iver
kidney
gills
liver
kidney
gonads
gills
1 iver
kidney
gills
1 iver
kidney
gills
1 iver
kidney
Histopathology
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
Post-mortem decomp
None
None
None
None
None
None
None
None

-------
changes of the kidney and gills developed within 1 h at 50 mg/L (Gardner and
Yevich 1970).  Skidmore and Tovell  (1972) studied the histological changes
that occurred in gill tissue of rainbow trout following exposure to kO mg/L
Zn.   They found the epithelium covering the secondary lamellae lifted away
from the pillar cell system, thus  increasing the diffusion distance from
water to blood.  Eisler and Gardner  (1973) observed renal pathology in the
estuarine mummichog subjected to either 1 or 10 mg/L Cd in combination with
copper; lesions were always present  in fish exposed to 10 mg/L Cd, but were
sometimes missing  in fish exposed  to  1 mg/L Cd.  All of Eisler and Gardner's
(1973) metal concentrations that showed histopathological damage also caused
some level of mortality during the exposure period.  Newman and MacLean
(197^) noted histopathological effects in cunner, Tauggolabrus adspersus,
following exposure to cadmium concentrations of A8 mg/L for 96 h but noted
few changes  in fish  receiving lesser  concentrations.  Similarly, Gardner and
LaRoche  (1973) found no visible evidence of hepatic alteration in the
mummichog or Atlantic silverside  (Menidia menidia) exposed to copper for up
to 21 days.  Some  lesions were present in some mummichog receiving paired
injections of  100  mg/L copper, and the liver was marked by focal areas of
necrosis  in  these  cases.  Gardner  and LaRoche suggest that liver cell
functions could  be affected  long  before a cellular alteration can be identi-
fied with a  light  microscope.

     Thus,  from  the tissues  sampled it appears that the mortalities or be-
havioral  changes observed following exposure to several metals were probably
due  to  biochemical rather than tissue lesions.
                                       78

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                                  REFERENCES
Eisler, R. and G. R. Gardner.  1973-  Acute toxicology to an estuarine
     teleost of mixtures of cadmium, copper and zinc salts.  J. Fish. Biol.
     5:131-142.

Galigher, A. E. and E. N. Kozloff.  1971.  Essentials of practical microtech-
     nique.  Lea and Febiger, Philadelphia, Penn.  531 pp-

Gardner, G. R. and G. LaRoche.  1973-  Copper induced lesions in estuarine
     teleosts.  J. Fish. Res. Board Can. 30(3):363-368.

Gardner, G. R. and P. P. Yevich.  1970.  Histological and hematological
     responses of an estuarine teleost to cadmium.  J. Fish. Res.  Board
     Can. 27(12):2185-2196.

Hendricks, J. D., L. G. Hunter and J. H. Wales.  1976.  Postmortem bile
     damage to rainbow trout  (Salnto gairnderi) livers.  J. Fish. Res.
     Board Can. 33(11):26l3"26l6.

Lloyd, R.  I960. The toxicity of zinc sulphate to rainbow trout.  Ann. Appl.
     Biol. 48(0:84-94.

Newman, M. W. and S. A. MacLean.  1974.  Physiological response of the cunner,
     Tautogolabrus adspersus  to cadmium.  VI Histopathology.  NOAA Technical
     Rept.  NMFS SSRF-681:27~33-

Sangalang, G. B. and M. J.  O'Halloran.  1973-  Adverse effects of cadmium
     on brook trout testis and on in Vitro testicular androgen synthesis.
     Biology of Reproduction.  9=394-403-

Skidmore, J. F., and P. W.  A. Tovell.  1972.  Toxic effects of zinc
     sulphate on the gills of rainbow trout.  Water Research. 6:217-230.

Voyer, R. A., P. P. Yevich and C. A. Barszcz.  1975-  Histological and toxico-
     logical responses of the mummichog Fundulus heteroclitus (L.) to
     combinations of levels of cadmium and dissolved oxygen in a freshwater.
     Water Research.  9:1069-1074.
                                      79

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APPENDIX  IV: CADMIUM UPTAKE AND ACCUMULATION  IN COHO SALMON.

                                 INTRODUCTION

     The  phenomenon of accumulation of  toxic  substances by aquatic organisms
is of concern not only to  fisheries biologists, but also  to the general
public as users and consumers of  these  organisms.   It has been shown that
metals can accumulate  in aquatic  organisms exposed  to sublethal doses and  in
fact can  become more concentrated in  the  tissues of the animal than  in the
environment  (Clearly and Coleman  197^,  Hannerz  1968, Mount and Stephan
1967)-  This poses  two potential  problems, deleterious effects upon  the
individual or  its offspring via  gradual accumulation of metal  in  the tissue,
and trophic  level bioconcentration of the toxicant.

     The  primary objective of this study  was  to  investigate cadmium  accumu-
lation from  the water  in tissues  of Cd  exposed  fish.

                                    METHODS

     Fish were killed  at the end  of the chronic Cd  exposure test  from the
control and  4.5 vg/L  (1685 h) and from  8  and  12 yg/L Cd concentrations after
~\kk h.  Fish were killed by a sharp blow  on  the head and  frozen for  approxi-
mately 2-3 months prior  to processing.

     Tissue  was dissected, weighed and  dried  for 2k h at  90°C.  Dry  weight
was taken and  10-200 mg  of the  tissue was placed  in glass Kimax 20 mL test
tube with a  teflon  lined cap.   Digestion  procedures were  modified from
Adrian  (1971).  Since  only milligram  portions were  used,  the  acid-mix volume
was  1.0 mL.  Heating was done  in a Gilson water bath at 80°C  for  8 h.

     After  cooling,  the  volume  was brought up to  10 mL by the addition of
glass distilled water.   Analysis of total cadmium was conducted by flameless
atomic absorption  spectrophotometry  (Perkin-Elmer 305B) with  a Hollow Graphite
Atomizer  2000.

                            RESULTS AND DISCUSSION

     Concentrations of Cd  found in  tissue of yearling coho  salmon are  listed
 in Table  1.  Values obtained  for control  fish tissues are similar to values
 in fish  from natural waters  (Lovett et al. 1972,  Lucas et al. 1970)  and
 salmonids from Oregon  hatcheries (McCrady, WFTS personal  communication).
The distribution  of Cd in  various tissues follows a typical pattern  with  the
 kidney containing the  highest  level  in both  control and Cd  exposed fish.
 Concentrations of Cd in  the  kidney have been reported  to  increase with
 increasing  exposure times  (Kumada et  al.  1973,  Smith et al.  1976).   This
 phenomenon  does  not appear to  be universal.   Eaton  (197^) found no  increase
 in Cd  concentration in hepatic  or renal tissue with longer  periods of
 exposure.  Similarly,  Mount  and Stephan  (1967)  found no direct correlation
 between  exposure  tfme  and  absolute liver Cd  concentration in  the  bluegill
 (Lepom±s  macroch±rus).  Rehwoldt and  Karixian-Teherani  (1976) studied  the
                                       80

-------
uptake -,nd effect of Cd in the zebrafish  (Brachydanio rerio) and found that
there was a period of rapid accumulation of Cd followed by a period of
reduced uptake which gradually reached a plateau.

     In our study, a general trend of increasing tissue Cd concentrations
was seen between control fish and those exposed to cadmium chloride.  The
trend is well illustrated in the chronically exposed group where the accumu-
lation is of the following order: liver 2.2x; kidney l8x; and gill  43x the
level measured from control fish (Table 1).  This trend in Cd accumulation
is typical of fishes and has been shown to occur in other species as well
(Clearly and Coleman 197^, Eisler 1971, Eisler and Gardner 1973, Kumada et
al. 1973, Lander and Jernelov 1969, Mount and Stephen 1967, Rowe and Massaro
197*0-  It is apparent that the  internal organs begin accumulating Cd
within 24 h at exposures of 8 ug/L Cd, and in all concentrations (4, 8, 12
yg/L) within 96 h.  Muscle tissues did not accumulate noticeable amounts of
Cd in the acute exposure tests.

     Kumada et al. (1973) found  that juvenile rainbow trout accumulated the
highest levels of Cd in the kidney with a steady-state level being reached
in 10-20 weeks after which the concentration was essentially constant.
They also found that the liver,  but not the kidney of rainbow trout showed a
reduction in Cd (in Cd-free water) following a 10-week exposure of Cd.
Analysis of gill tissue prior to exposure to the clean water revealed a con-
siderable accumulation of Cd.  The hypothesis was made that the gills were
the site of absorption of Cd from the water and the metal was excreted
through the kidneys.
                                       81

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                  TABLE  1.   CONCENTRATIONS  OF  CADMIUM  IN VARIOUS TISSUES OF  COHO  SALMON  SMOLTS
                            EXPOSED  TO  LETHAL  AND  SUBLETHAL  CONCENTRATIONS OF  CADMIUM  CHLORIDE.
      Type  of  exposure
        Concentration of cadmium yg/L
Liver	Kidney	G i 11	Muscle
                                      Ovary
oo
NJ
      Control  group with
      7  days  10  yg/L  Cu

      4.5  yg/L Cd with
      7  days  10  yg/L  Cu
      Approximate LC50 value
      ^Lethal dose >LC50 value
                                           Chronic exposure 2/26-5/7/76
  0.24
0.62
0.18
  0.52        11.2          7.8

  Acute exposures  6/21-7/2/76
                                        0.70*
Control group -
Cd 4 yg/L
Cd 4 yg/L
Cd 8 yg/L
Cd 8 yg/L
Cd 12 yg/L
* n » 1
** n » 2
*** n = 3
2k h
2k h
96 h
24 ha
96 ha
96 bb



3
k
6
6
6 .
6



0.16
-
0.11*
0.26
0.40
0.68



0.46
-
0.80***
1.0
0.78
1.78



0.26
0.44
1.15
0.43
0.70
0.87**



-
0.09
0.02
<0.01
0.02
~



0.48*
-
0.13*
0 . 09***
0.09***
0.35**




-------
                                 REFERENCES
Adrian, W. J.  1971.  A new wet digestion method for biological material
     utilizing pressure.  Atomic Absorption Newsletter, 10:97.

Cleary, J. E. and R. L. Coleman. 1974.  Cadmium toxicity and biocon-
     centration in largemouth bass and bluegill.  Bull. Environ. Contam.
     Toxicol.  11:145-151.

Eaton, J. G.  1974.  Chronic cadmium toxicity to the bluegill  (Lepomis
     macrochirus Rafinesque).  Trans. Am. Fish. Soc. 103(4):729-735.

Eisler, R.  1971.  Cadmium poisoning in Fundulus heteroclitus  (Pisces;
     Cyprinodontidae), and other marine organisms.  J. Fish. Res. Board
     Can. 28:1225-1234.

Eisler, R. and G. R. Gardner.  1973-  Acute toxicology to an estuarine
     teleost of mixtures of cadmium, copper and zinc salts.  J. Fish.
     Bfol. 5:131-142.

Hannerz, L.  1968.  Experimental investigations on the accumulation of
     mercury in water organisms.   Inst. Freshwater Res.
     Drottnfngholm.Rept.  48:120-176.

Kumada, H,, S. Kimura, M. Yokote and Y. Matida.  1973-  Acute and chronic
     toxTcity, uptake and retension of cadmium  in freshwater organisms.
     Bull. Freshwater Fish. Res. Lab.  (Tokyo) 22(2):157-164.

Lander, L. and A. Jernelov.  1969-  Cadmium in aquatic systems; in Metals
     and Ecology Symposium.  Ecological Research Committee Bull. No. 5.
     Swedish Natl. Scf. Res. Council; Stockholm.

Lovett, R. J., W. H. Gutenmann, I. S. Pakkala, W. D. Youngs, D. J. Lisk,
     G. E. Burdick and E. J. Harris.   1972.  A survey of the total cadmium
     content of 406 fish from 49 New York State fresh waters.  J. Fish. Res.
     Board Can. 29(91:1283-1290.

Lucas, H. F- Jr., D. N. Edgington, and P. J. Colby.  1970.  Concentrations
     of trace elements in Great Lakes fishes.  J. Fish. Res. Board Can.
     27(41:677-684.

McCrady, J.  1976.  Personal communication.  Analyzed fish from Marion Forks
     Salmon Hatchery, Oregon and the Western Fish Toxicology Station (EPA)
     Corval1ts, Oregon.

Mount, D.  \. and C. E. Stephan.  1967-  A method for detecting cadmium
     poisoning fn fish.  J. WiIdlife Manage. 31:168-172.

Rehwoldt, R. and D. KarIxian-Teherani.  1976.  Uptake and effect of
     cadmTum on zebrafish.  Bull.  Environ. Contam. Toxicol. 15(4):442-446.


                                      83

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Rowe, D. W. and E. J. Massaro.  1974.  Cadmium uptake and time dependent
     alterations in tissue levels of the white catfish Jctalurus catus
     (Pisces:Ictaluridae).  Bull. Environ. Contam. Toxicol.  11:244-249-

Smith, B. P., E. Hejtmancik and B. J. Camp. 1976.   Acute effects of cadmium
     on Jctalurus punctatus (catfish).  Bull. Environ. Contam. Toxicol.
     15(3):271-277-

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                                   TECHNICAL REPORT DATA
                            (Please read Instructions on the reverse before completing)
1. REPORT NO.
   EPA-600/3-78-090
                              2.
                                                           3. RECIPIENT'S ACCESSION NO.
4. TITLE AND SUBTITLE
 Effects of Several  Metals on Smolting  of Coho Salmon.
             5. REPORT DATE
               September 1978
                                                           6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)

 Harold W. Lorz,  Ronald H. Williams  and  Charles A.
 Fustish.
                                                           8. PERFORMING ORGANIZATION REPORT NO
9. PERFORMING ORGANIZATION NAME AND ADDRESS
 Oregon Dept  of Fish and Wildlife
 Research  Laboratory
 28655 Highway  34  Corvallis, OR 97330
             10. PROGRAM ELEMENT NO.

                 1BA608
             11. CONTRACT/GRANT NO.
                                                               R-804283
12. SPONSORING AGENCY NAME AND ADDRESS
 Con/all is Environmental  Research Laboratory
 Office of Research  and Development
 U.S. Environmental  Protection Agency
 Corvallis, Oregon 97330
             13. TYPE OF REPORT AND PERIOD COVERED
              Final 1-5-76  to 12-20-76
             14. SPONSORING AGENCY CODE
                EPA/600/02
15. SUPPLEMENTARY NOTES
 Report contains   94  references and a recommended seawater-entry test procedure.
16. ABSTRACT
    Exposure to sublethal  levels of copper  in  freshwater reduces Na,K-activated  gill
 ATP'ase in coho salmon  and results in latent  effects such as poor migration  and poor
 survival in seawater  (EPA-660/3-75-009).   Similar tests with cadmium, chromium, mercur
 nickle and zinc indicated that only mercury produced similar latent effects.  Addition;
 tests with cadmium  and  zinc plus 10 ug/liter  of  copper produced latent effects
 considerably more severe  than those produced  by  10 ug Cu/liter by itself.  Histologica
 examination of liver, kidney and gill showed  no  effect of cadmium or zinc, either
 singly or in combination  with copper.  Cadmium was found to accumulate in gill, liver
 and kidney, but only minor amounts occurred in muscle tissue.  A seawater-entry test
 (SET) is recommended for  detecting latent  effects of toxicants on salmonid smolts.
17.
                                KEY WORDS AND DOCUMENT ANALYSIS
                  DESCRIPTOPS
                                              b.IDENTIFIERS/OPEN ENDED TERMS
                          c.  COSATI Field/Group
 Salmon, Copper, Zinc, Cadmium,  Nickle,
 Chromium, Mercury
  Smolting,
  Migration,
  Osmoregulation
06/A,F,T
13. DISTRIBUTION 57 ATfcMENT
                                              19. SECURITY CLASS (This Report)
                                               unclassified
                           21. NO. OF PAGES

                              98
       Release to Public
20 EtCUHITY CLASS (This page I

 unclassified
                                                                         22. PRICE
EPA Form 2220-1 (9-73)
                                            85
                                                            a U.S. GOVERNMENT PRINTING OFFICE: 1978—798-081/5

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