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
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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.
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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
-------
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
-------
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
-------
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
-------
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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Anderson, P- D. 1973- An approach to the study of multiple toxicity through
the derivation and use of quantal response curves. Ph.D. Thesis,
Oregon State University. Corvallis, Oregon. 80 pp.
Andrew, R. W. 1976. Toxicity relationships to copper forms in natural
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Konasewich. eds. Toxicity to biota of metal forms in natural water.
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Buhler, D. R. , R. M. Stokes and R. S. Caldwell. 1977- Tissue accumulation
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Heavy Metals in Environment. Water Resources Research Institute,
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-------
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43
-------
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44
-------
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45
-------
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46
-------
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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
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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
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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
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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
-------
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
-------
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
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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.
-------
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
-------
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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