EPA-600/3-76-050

May 1976

MORTALITY, SALTWATER ADAPTATION AND REPRODUCTION

OF FISH DURING GAS SUPERSATURATION

Gerald R. Bouck

Allen V. Nebeker

Donald G. Stevens

Western Fish Toxicology Station*

Environmental Research Laboratory-Duluth Corvallis, Oregon 97330

(*Western Fish Toxicology Station is now attached

to the Corvallis Environmental Research Laboratory, Corvallis, Oregon 97330)

U.S. ENVIRONMENTAL PROTECTION AGENCY

OFFICE OF RESEARCH AND DEVELOPMENT

ENVIRONMENTAL RESEARCH LABORATORY

DULUTH, MINNESOTA 55804
EPA-600/3-76-050

May 1976

MORTALITY, SALTWATER ADAPTATION AND REPRODUCTION

OF FISH DURING GAS SUPERSATURATION

Gerald R. Bouck

Allen V. Nebeker

Donald G. Stevens

Western Fish Toxicology Station*

Environmental Research Laboratory-Duluth Corvallis, Oregon 97330

(*Western Fish Toxicology Station is now attached

to the Corvallis Environmental Research Laboratory, Corvallis, Oregon 97330)

U.S. ENVIRONMENTAL PROTECTION AGENCY

OFFICE OF RESEARCH AND DEVELOPMENT

ENVIRONMENTAL RESEARCH LABORATORY

DULUTH, MINNESOTA 55804
DISCLAIMER

This report has been reviewed by the Environmental Research

Laboratory-Duluth, U.S. Environmental Protection Agency, and approved for publication. Mention of trade names or commercial products does

not constitute endorsement or recommendation for use.

ii
ABSTRACT

Tests were conducted using continuous exposure in shallow water

at levels of total dissolved gas pressure ranging from 110-140% of

barometric pressure (hyperbaric pressure = 103-410 g/cm2). Both times

to 20% and to median mortality were determined on several life stages of Pacific sa1monids (Oncorhynchus and Salmo) and largemouth bass

(Micropterus sa1moides). Mean times to 20% mortality at 115% total

gas saturation were 309~ 154~ and 125 hours for adults, smo1ts and

parr. At 120% saturation mean times to 20% mortality were 48, 41,

and 53 hours for adults, smolts~ and parr. At 125% saturation, mean

times to 20% mortality decreased to l8~ 17~ and 24 hours for adults,

smolts, and parr. Factors which influence time to death included

genera, life stage, acclimation temperature~ activity level, sex,

and body size. Mortality curves were typically skewed to the right. Gross pathology of gas bubble disease was described relative to these

experiments. High gas levels that killed 50% of three species of

salmon smo1ts had no apparent effect on the ability of survivors to

tolerate an immediate transfer into seawater (30 ppt C1). Long-term (3-month) continuous exposure of adult spring chinook salmon to 110%

saturation had no readily apparent adverse impact on the fertilization

and hatching of their eggs.

iii
Section

I

II

III

IV

CONTENTS

Abstract

List of Figures

List of Tables

Acknowledgments

INTRODUCTION

CONCLUSIONS AND RECOMMENDATIONS

METHODS AND MATERIALS

Supersaturation Exposure Facilities

Wa ter Qua1i ty

Biological Methods

Design of Experiments

Statistical Procedures

RESULTS

General Tolerances ~f Sa1monids and Non-Sa1monids

Sample Responses to Supersaturation

Pathobio10gy and Times to Mortality

Tolerance to 130% Saturation

Tolerance to 125% Saturation

To1erance to 120% Saturation

To1erance to 115% Saturation

Tolerance to 110% Saturation

Estimated Times to 20 Percent Mortality

Rank Order of Tolerance to Supersaturation

v

Page

iii

vii

viii

ix

1

4

6

6

6

15

15

18

19

19

19

26

26

30

30

31

32

32

35
Page

Factors Affecting Mortality by Supersaturation 35

Test Temperature 35

Swimming Activity 39

Sex 39

Life Stage 42

Body Size and Condition Coefficient 42

Other Factors Influencing Tolerance to 45

Supersaturation

Tolerance to Seawater After Exposure to 45

Supersaturated Water

Effects of Chronic Supersaturation on Reproduction 47

V

VI

DISCUSSION

REFERENCES

vi

48

52
FIGURES

No.

1 Diagram of facilities for exposing fish to 7

supersaturated water

2 Supersaturation exposure facilities 8

3 Diagram of modified Weiss Saturometer 14

4 Time to death of fasting winter steel head trout parr 23

(Salmo gairdneri) at 125% saturation and 10 C in water

65 cm deep

5 Mortality response of rainbow trout (Salmo gairdneri) 24

to 120% saturation

6 Means and 90% confidence belts of times to 20% mortality 33

at various life stages of salmonids and levels of super- saturation

7 Effects of test temperature on the time to median 37

mortality from supersaturation

8 Effects of swimming activity on the mortality of 40

largemouth bass (Micropterus salmoides) in water

supersaturated to 140%

9 Effect of percent saturation and sex on mortality of 41

adult coho salmon (Oncorhynchus kisutch) at 10 C in

tanks'65 cm deep

10 Effect of percent saturation and life stage on mortality 43

of coho salmon (Oncorhynchus kisutch) at 10 C in tanks

65 cm deep

11 Cumulative mortality of salmonid smolts during exposure 46

to 120% air supersaturation and immediate transition

to 30 0/00 seawater

vii
TABLES

No. Page

1 Examples of natural and man-made sources of 2

supersaturated water

2 A summary of chemical and physical parameters in the 10

water supply of Western Fish Toxicology Station 1972-1973

3 Nominal and measured test levels of total gas saturation 12

4 Conversion table and equivalent units for expressing 16

gas saturation data

S Fish species tested 17

6 Summary statistics of time to death at three levels of 20

supersaturation by twenty population samples of rainbow

trout, Pacific salmon, and largemouth bass

7 Hours of exposure to reach 20% mortality among fishes 27

exposed to supersaturation in shallow water at 10 C

8 Estimated hours to median mortality (ETSO) among fishes 29

exposed to supersaturation in shallow water at 10 C

9 Statistical analysis of pooled observations on hours to 34

20% mortality among three life stages of five species of

salmonids

10 Rank order of tolerance between fish tested at 120% total 36

gas saturation (203 g/cm2 hyperbaric)

11 Summary of the relationship between acclimation temperature 38

and time to median mortality in supersaturated waters

12 Correlation analyses of time to death at various levels 44

of supersaturation to body length, weight, and condition

coefficient (K) for adult spring chinook (Oncorhynchus

tshawytscha)

viii
ACKNOWLEDGEMENTS

We gratefully acknolwedge the assistance of the following

agencies: Fish Commission of Oregon, Wildlife Commission of

Oregon, National Marine Fisheries Service, U.S. Army Corps of

Engineers, and the Columbia River Basin Interagency Fisheries

Technical Advisory Committee.

ix
SECTION I

INTRODUCTION

Scientists have studied supersaturation and its pathologic result,

Gas Bubble Disease (GBD), for over 100 years with recurrent cycles of

interest. Within the last decade interest has been renewed because

man's activities have supersaturated large volumes of surface water,

making them potentially lethal to fish (Ebel, 1969; Bouck, 1972).

Causes of excess dissolved gas pressure (Table 1) include operations

at hydroelectric or other dams, heating of water, and eutrophication (photosynthesis). Since most means of diminishing supersaturation could involve large financial costs, there must be a firm data base

for establishing construction priorities or the necessary levels of

water treatment and its achievable benefits.

An additional impetus for renewed research has been the develop~

ment of improved techniques such as gas partition chromatography and

the Weiss Saturometer which permit rapid analysis of total and

individual dissolved gases. One might now resolve long standing questions regarding the biological impact of individual gases and

easily monitor supersaturation in the aquatic environment.

The condition causing gas bubble disease has several names including supersaturation, air supersaturation. and nitrogen supersaturation. The

term "air supersaturation" (Table 1) is used here because it correctly implies the typical problem. namely that the sum of the pressures of

the dissolved components of air exceeds the combined compensatory

pressures of the atmosphere, water. and tissues. Boyle~s Law prescribes that gases will not cavitate regardless of how supersaturated a given

gas may be, unless the total dissolved gas pressure (tension) exceeds

the compensatory pressures.

The primary goal of this study was to obtain data on the acute

lethality of supersaturated water to Pacific salmonids and a predator

species (largemouth bass). These data were needed to evaluate what

criterion of saturation (relative to barometric pressure!!) would

adequately protect fish and aquatic invertebrates. Shallow water and

continuous exposure were selected for test conditions because they

!I Dissolved gas(es) levels conventionally are described as a percentage of some reference value, typically barometric pressure which varies

continuously due to weather and between locations due to altitude.

Continued use of this convention adds a potentially significant error

to the data. This is because the relative value of 110% saturation can

represent a (surface) uncompensated absolute force ranging from about

77.2 Kdynes at 7200 ft. above sea level to about 101.2 Kdgnes at sea

level. However, abandoning this convention might seriously confuse

engineers and biologists who are not associated with this research.

The conversion of relative saturation levels to absolute physical units

is presented in Table 4.

1
TABLE 1. EXAMPLES OF NATURAL AND MAN-MADE SOURCES OF SUPERSATURATED WATER

Type No. I

AIR ENTRAINMENT

Type No. II

THERMAL Type No. IIIORGANIC

Heating water in fish culture

stations to achieve optimal growth.

l. Cold water recharging an l. Eutrophic levels of

aquifer or lake, then photosynthesis in

geotherma1ly warmed, then conjunction with high returned to surface. levels of temperature and solar radiation.

2. Hot weather, often preceded by cold rain.

l. Heating water to cool steam- 1. Same as above except may electric stations or other occur in polluted water

industrial processes. to a much greater degree.

2. Turbulence in high velocity streams.

A. Natural 1. Water falls with deep plunge basins.

B. Man-made 1. Flood gates or other

spillways at dams which

entrain air bubbles and

carry them to depths.

2. Injection of air to prevent 2.

"water hammer" in turbines,

sluiceways, or to reaerate

reservoir water.

I:oV

3. Venturi action at pipe joints, or pumps sucking air.
represented the maximum environmental stress, eliminated significant hydrostatic compensation, and allowed a more direct comparison of

tolerances between species. Collateral goals were to identify factors

which might diminish tolerance to gas bubble disease, to determine

residual effects on salt water adaptation, and to estimate if reproduc- tion was readily and adversely affected by supersaturation.

The literature on Gas Bubble Disease has been reviewed recently by Harvey (1975), Wolke, Bouck and Stroud (1975), Bouck (1973), Weitkamp and Katz (1973), and Rucker (1972). These publications contain

additional resource material and more detailed descriptions of Gas

Bubble Disease.

3
SECTION II

CONCLUSIONS AND RECOMMENDATIONS

1. Some mortality occurred among sensitive fish at dissolved gas levels of 110-115% of barometric pressure when they were restricted

to shallow water. These and higher gas levels may be safe for wild

fish if they sound to compensatory depths.

2. The mean hours to 20% mortality and two-tailed 90% confidence

limits are:

Salmonids*

Adul ts

Smolts

Juveniles

115%

309 (± 114)

154**

125 (t 122)

Supersaturation Level

120% 125%

48 (:!: 8.7) 18.3 (:t 3.2)

41 (:tlO.l) 17 .2 (± 5.2)

53.5 (± 23.8) 23.6 (:t 4.4)

*(10 C, shallow water) **(= single sample only)

3. Response (time to death) curves are skewed to the right and contain

at least three phases which include: (1) a sublethal period; (2) a

period of log-linear mortality; and (3) a period of protracted survival.

These skewed curves indicate that: (1) as much as 75% of a laboratory population of fish can be killed in about half the time required to kill

the total population; (2) probably few factors influence the mortality; and (3) the median is probably the best measure of central tendency in

these data.

4. Times to death varied among the tests, genera and species in some

cases. Variability was greater in long-term exposure tests than in

short, acute exposures.

5. Several additional factors were significantly correlated with time

to death. These included life stage, physiological condition, body size, activity, behavior, and water temperature.

6. Largemouth bass survived prolonged exposure at 120% of barometric

pressure (203 g/cm2 hyperbaric) which killed salmon and trout. This

level caused external signs of gas bubble disease on bass but did not

prevent them from preying on young salmonids. Thus supersaturation may

exert ecological succession pressure against salmonids and favor bass.

4
7. The external lesions of gas bubble disease such as dermal emphysema

or exophthalmus varied between species and became most severe when

conditions were marginally lethal; such conditions allowed the longest survival, hence the longest period for the pathologic process to develop. In acutely lethal cases with short exposure time, early mortality frequently showed no external signs of gas bubble disease. Therefore,

the presence of air emboli in the blood along with identification of

supersaturated water must be used to confirm acutely lethal gas bubble

disease.

8. Supersaturation levels which killed 50% of the smo1ts in a species did not prevent the survivors from tolerating a direct transfer to 30 0/00

seawater where they survived for five days.

9. Appearance, fertility and hatching of eggs were not adversely affected in spring chinook which had been held at 110% for three months

(10 C, 65 cm of water) and artifica11y spawned.

10. Actual conditions encountered by fish in supersaturated water

should be clearly defined, such as intermittent exposure and water depth.

11. A zero supersaturation limit is recommended for sa1monid fish

hatcheries; any supersaturation typically indicates a more basic problem that is capable of reaching harmful levels especially when the water is

heated or pumped.

5
SECTION III

METHODS AND MATERIALS

SUPERSATURATION EXPOSURE FACILITIES

Each exposure chamber operated independently and consisted of

a large fiberglass tank about 3.75 m in diameter by 1 meter deep. A frame supported rubber-coated nylon sides (1.2 m high) and 2.5 cm

mesh netting over the top. These tanks were located outdoors, but

insulated with 5 cm of urethane foam and located under a roof to

shield them from direct sunlight (Figures 1 and 2).

Well water was aerated to near saturation before being pumped

at 1900 kilo 9ynes/cm2 through each exposure system. A small amount

of this water was recycled to the aeration tank to promote aeration.

Compressed air was injected at a constant rate into the pressurized

water immediately prior to passing thru pipe ells (Figure 1) where

turbulence promoted the solution of the gases. The water passed into a retention tank and the undissolved air was removed by drawing air off the top; water was drawn off the bottom and delivered to the

test tank. The level of total dissolved gas pressure (supersaturation)

was proportional to the volume of air being injected into the system. As tested, this system could deliver 75 llmin of water with a total

dissolved gas pressure easily as high as 150 percent of barometric

pressure. Water was introduced into the exposure chamber via a

subsurface port to reduce gas losses to the air. Valves were omitted

wherever possible to avoid sharp drops in pressure which remove gases from solution.

Additional exposure chambers were located within a circular cage made of wood, coated with epoxy paint, and covered with nylon bobinet

(Figure 2 G). Each cage, about 1.3 m in diameter and 1 mhigh, was

equipped with hinged doors and sub-divided into quadrants making four

additional exposure chambers. The cage assembly sat over and was

held in place by the tank stand-pipe. Water currents within the cage promoted good mixing of water, but velocities were slower in the cage than in the open tank.

WATER QUALITY

Water for these experiments was drawn from a well located adjacent

to the Willamette River. Water from this source has been used for fish

culture for five years, and its general characteristics are listed in

Table 2. Hardness and alkalinity were typically low (20 ppm as CaC03)

and ranged between pH 7.0-7.4 after aeration. Water temperature was

kept at 10 C except where it was altered as an experimental variable.

6
FIGURE 1. DIAGR4M OF FACILITIES FOR EXPOSING FISH TO SUPERSATURATED WATER.

SUPERSATURATION GENERATOR

air line

-

Heater ..-Air Escape Vent

" '. " t·

" .,." ......

.' . ,* '.~:. , '.

.:: ,'-"',. '. "

'., .~".: " '~.. • t •••• :.,

r;:::.==" Exposure

Tank

Retention

Tank

-

Water

Flow Meter

1urbulence Loop ( air T water)

-

water

--

If)

If)

(1)

l...

Q.

... E .- 0 <tu

Well

Water

Float._H -.l~_

Valve ~~Jl::~~
A--Test tanKS with roof to shield out

direct sunlight.

B--Aeration tank with incoming water

supply and continuous temperature

control system.

C--Test tank with saturometer in

preparation for gas analysis.

Figure 2. Supersaturation exposure facilities

8
f--Air and/or compressed gas dellvery

system of supersaturation generator.

D (left), E (right)--Supersaturation generator system (see Figure 1).

G--Four-chambered nylon bobinet cages

within the test (exposure) tanks.

Young sockeye salmon can be seen in

the chambers.

Figure 2 (continued). Supersaturation exposure facilities

9
TABLE 2. A SUMt4ARY OF CHEMICAL AND PHYSICAL PARM1ETERS IN THE WATER

SUPPLY OF WESTERN FISH TOXICOLOGY STATION 1972-l973!!.

ParameterY Min Max Mean ParameterY Min Max Mean

Temperature (OC) 7.0 18.2 13. 1

pH 6.70 7.00 6.80

QUANTITY (mg/1, ppm) QUANTITY (~g/l, ppb)

Dissolved Oxygen 2.8 9.0 4.9 Heavy Metals

Total A1 ka1 inity 18 26 22 Cadmium 0.0 0.3 0.1

Total Hardness 21 25 23 Chromium 1.2 5.0 1.3

Nitrites <.001 <.001 <.001 Cobalt 0.5 3.0 1.4

Nitrates .138 .330 .176 Copper 1.0 9.0 3.5

Ammonia <.001 .048 .014 Iron 55 360 110

Calcium 5.8 13.7 7.3 Lead 3.0 5.0 4.7

Magnesium 1.4 2.4 1.7 Magnesium 1.0 22.0 2.9

Sodium 4.1 5.4 4.6 Nickel 1.0 4.0 1.9

Potassium 0.48 0.70 0.57 Zinc 1.0 24.0 6.4

Su1 fate 2.0 4.0 2.9 Mercury <0.5 2.0 0.7

Dissolved Solids 34 68 52

Suspended Solids <1.0 4.0 1.7

Chloride 3.0 9.0 6. 1

1I Unpublished Data. Donald F. Samuelson

Y Number of samples for all parameters ranged between 23-26.

Maximum values occurred during winter months (Dec-Feb) when

saturated ground water dominated the river water supplying

our well .

10
Water quality in the experimental and control tanks was monitored

continuously for temperature; usually once a day for pH, alkalinity and

oxygen; and several times each day for total dissolved gas pressure. Chemical analyses were done according to "Standard Methods for the

Examination of Water and Wastewater" (Anon., 1971).

Total dissolved gas pressure (TDGP) was measured with a dissolved

gas tensionmeter!l (Table 3). Fickeisen, Schneider and Montgomery

(1974) reported the accuracy of this instrument to be comparable to

the results obtained by partition chromatography.

Figure 3 depicts a dissolved gas tensionometer consisting of

four components: (l) a molecular sieve which excludes liquid water but

allows gases to reach pressure equilibrium with their dissolved phases

(400 ft. coil of 0.025" 0.0. X 0.012" 1.0. dimethyl silicone rubber

tube); (2) a gauge for measuring gas pressure or tension; (3) nylon tubing (gas tight) to connect components (1) and (2); and (4) a framework

to support and protect the rubber tubing. The sensor is placed under

water, and the gas pressure in the tubing equilibrates with the gas

pressures in the water, including water vapor pressure. The gauge directly measures the resulting total gas pressure, indicating a

hyperbaric or positive pressure in supersaturated water and a hypo- baric or negative pressure in water that is not fully saturated.

The measurement of dissolved gas pressure is based on the principle

that the membrane is selectively permeable to gases but not to liquids.

A dissolved gas crosses the membrane and in due time reaches a pressure equilibrium with its dissolved gas phase. The gauge sums the pressures of the individual gases including that of water vapor, but always

relative to ambient barometric pressure. Therefore, this instrument

measures the total pressure difference (~P) between barometric pressure and the sum of dissolved gas pressures including water vapor pressure.

The relative total dissolved saturation is calculated by the

formula:

BP + ~P - VP

BP

X 100, where

BP = barometric pressure in mmHg; ~p =differential gas pressure in mmHg (from saturometer);

VP = vapor pressure of water in mmHg.

Thus "percent saturation" is a term which relates to the local

barometric pressure, which in turn changes with altitude and weather

conditions. In our case, barometric pressure averaged about 755 mmHg

!I Developed by Dr. Ray Weiss, Scripts Institute of Oceanography,

LaJolla, California, and initially called a "saturometer".

11
TABLE 3. NOMINAL AND MEASURED TEST LEVELS OF TOTAL GAS SUPERSATURATION.

Nomi na 1 Measured Tota 1 Gas Saturation (%)

Test Test Total Gas

No. Chamber Saturation (%) N Mean Range

1 1 125 16 125.2 123 . 6 - 127.6

2 1 120 59 120.1 116.6 - 123.7

3 1 125 31 125.5 122.7 - 127.8

4 1 120 44 120.2 11 5. 2 - 123.5

5 1 115 42 115.6 114.0 - 125.0

6 1 125 43 125.0 123.5 - 127.7

2 120 53 120.1 118. 2 - 122. 2

7 1 120 37 120.3 118.3 - 122.6

2 115 39 114.9 113.0 - 116.3

8 1 125 45 124.7 123.2 - 126.3

9 1 125 43 124.8 122.6 - 126.3

2 120 42 120.1 118.7 - 121.0

10 1 140 20 139.3 137.2 - 141.0

2 130 30 129.7 128.2 - 130.8

11 1 120 24 120.6 117 . 7 - 122. 0

3 115 34 114.7 112.6 - 116.5

12 2 140 8 140.6 139.9 - 141.0

13 1 140 16 140.7 138.4 - 142.4

2 130 22 130.1 127.5 - 133.4

14 1 135 7 136.0 133.3 - 138.5

15 1 140 10 141 .2 138. 9 - 143. 2

16 1 120 10 120.0 117.4 - 121.1

2 115 21 115.2 113.5 - 116.9

3 110 19 109.8 109. 1 - 11 O. 6

17 1 120 7 119.8 11 9. 1 - 120 . 5

12
TABLE 3. NOMIr~L AND MEASURED TEST LEVELS OF TOTAL GAS SUPERSATURATION (CONTINUED).

Nominal Measured Total Gas Saturation (%)

Test Test Total Gas

No. Chamber Sa tura ti on (%) N Mean Range

18 1 125 7 125.5 123 . 9 - 127.2

19 1 130 17 130.4 126.4 - 133.0

2 125 17 125.0 123 . 3 - 127.2

3 120 43 119.8 115.1 - 123.4

20 1 120 35 120.2 117.1 - 121.9

2 115 59 115.0 111.9 - 117.3

3 110 157 109.8 107 . 0 - 116. 5

13
Handle .......

SATUROMETER

....-Nylon Tubing

Pressure

Guage

---- Gas Permeable Membrane

(Dimethylsilicone tubing)

i-HfIIIlt-- Protective Support

Figure 3. Diagram of modified Weiss Saturometer.

14
and varied typically by less than 20 mmHg (2.6% of average). However

a given "total" saturation value does not imply that each component

gas was saturated to that level. For example! a total saturation of

110% can consist entirely of high N2 levels and low 02 levels or vice

versa.

Since the causative agent of gas bubble disease is related (in

part) to the dissolved gas pressure above barometric levels (Bouck!

1973; D'Aoust and Smith! 1974), this is called the ~p or hyperbaric

pressure, and it can be measured both accurately and directly with the

saturometer. The ~p is the uncompensated hyperbaric pressure that

provides the driving force to form emboli and emphysema by overcoming the compression forces in blood and other tissues. Hyperbaric pressure

may prove to be a more universal and determinative parameter than

"percent saturation II because it is independent of elevation, barometric

pressure, salinity, or other influences. Table 4 lists equivalent values for percent saturation, g/cm2, mmHg, and p.s.i.

BIOLOGICAL METHODS

Fish for these experiments were obtained from several sources

listed in Table 5. All juvenile fish were hatchery reared, mainly at

this laboratory, but some adult fish were returnees to hatcheries.

Largemouth bass, sockeye, and possibly spring chinook salmon were wild

fish. Fish usually were placed into the exposure tank and allowed to

adjust to their surroundings for at least 48 hours prior to the onset

of supersaturation. Food was routinely withheld during each experiment! but some exceptions are so noted. Adult fish were tested in the open

tanks! but smaller fish were usually tested in the bobinet cages (Figure 2).

Common and scientific names of fishes conform to that of Bailey!

et ~., (1970).

Adult salmon were spawned artificially by the "dry" method after they

ripened sexually (Leitriz, 1959). Eggs were incubated in tray type incubators. Dead and infertile eggs were removed after pigment was

evident in the embryonic eye and again after hatching was complete. Temperatures varied seasonally during incubation from 10 C down to

about 6 C.

DESIGN OF EXPERIMENTS

The three objectives were to: (1) determine and compare the exposure times (in hours) which killed 50% of the specimens at given test

conditions; (2) determine the effects of supersaturation on saltwater

adaptation; and (3) determine the effect of long-term exposure to low

15
TABLE 4. CONVERSION TABLE AND EQUIVALENT UNITS FOR

EXPRESSING SATURATION DATA

Percent Hyperbaric Pressure

saturation

g/cm211 1b/in2at 755 mmHg Torr or mmHg (psi) Bars.Y

100 0 0 0.00 0

105 51.4 37.8 0.75 0.05

110 102.6 75.5 1.47 0.10

115 153.9 113.2 2.21 0.15

120 205.3 157.0 2.94 0.20

125 256.7 188.8 3.68 0.25

130 307.9 226.5 4.41 0.30

135 359.2 264.2 5.15 0.36

140 410.6 302.0 5.88 0.40

145 461.9 350.0 6.62 0.46

150 513.6 377 .5 7.35 0.50

1J To convert g/cm2 to dynes, multiply by 980 cm/sec2 (which is the

approximate acceleration of gravity)

Bars are one of two pressure units recommended for use by the

Society for Experimental Biology and Medicine (1972):

1 bar = 105 Pascals =14.5 lbs/in2 = 0.987 atmospheres

16
TABLE 5. FISH SPECIES TESTED

Species

Coho Salmon

(Oncorhynchus kisutch)

Sockeye Salmon

(Oncorhynchus nerka)

Chinook Salmon

spring migrating variety

(Oncorhynchus tshawytscha)

Steel head Trout

Salmo gairdneri)

summer race

winter race

Rainbow Trout

(Salmo gairdneri)

Suckers

(Catostomus ~.)

Largemouth Bass

(Micropterus salmoides)

Test No.

6,7

6,7

6,7

6,7

4,5,7

16,17,18

1,2,3,4,5

20

16

19,20

8

16

1,2,5

7,8

16

11

9

3,4,5,9

20

9,10,12,13,14,15

9,10,12,13,14,15

Life Stage

parr*

post smolts*

jacks

adults

parr*

smolts*

adults

parr*

smolts*

adults

parr

smo 1ts

adults

parr*

smolts*

adults

parr*

yearlings*

adults

juveniles

adults

Source

Oregon Fish Commission Hatchery Fall Creek, Lincoln Co., Oregon

Bonneville Hatchery - Ore. Fish Comm.

Columbia River, Bonneville Ladder

Oregon Fish Commission, Willamette

Hatchery, Oakridge, Oregon

Columbia River Bonneville Ladder

Washougal River Hatchery, Washington Game Commission

Columbia River, Bonneville Ladder

Oregon Wildlife Commission Hatchery N. Fk. Alsea R., Benton Co., Oregon

Oregon Game Commission, Roaring River Hatchery, Linn Co., Oregon

Columbia River at Bonneville

Ponds, Harrisburg, Lane Co., Oregon

* Reared from eggs at WFTS.
level supersaturation on maturation and fertilization of sex products and subsequent hatching success. Collateral objectives included the

identification of factors influencing mortality and observations of

,the pathobiology of gas bubble disease.

Fish were assigned randomly to tanks, and a fish was judged to

be dead when it no longer moved or ventilated. Eight levels of

saturation were selected for testing: 100 (control), 110, 115, 120,

125, 130, 135, and 140% of barometric pressure (hence total gas

saturation). Circumstances never permitted the concurrent testing at

every level, but a control group and at least one previously tested

level were repeated in a series. Replicates or re-runs were conducted

whenever the circumstances permitted, and several life stages or species

were tested concurrently in most cases. The fish were observed

frequently, and times to mortality were plotted on log probit paper for

visual estimation of the time to median mortality (ET50). Raw data

were plotted and used to estimate the relationship between various levels

of supersaturation and respective times to 20% and median mortality.

For the latter purpose all sa1monid data were pooled for a given life

stage.

To study tolerance to the combined stresses of supersaturation

followed by saltwater stress, smolts of three species were exposed to

three levels of supersaturation for four days at 10 C or until 50%

mortality occurred. Immediately thereafter, the survivors were placed in 20 liter jars containing seawater (30 0/00) and equipped with an

air supply and refrigeration to maintain 10 C ± 1°. Survival was

noted at the end of a five-day period.

The effects of sublethal supersaturation upon sexual maturation,

fertility, and hatching were studied using adult spring chinook trapped in the Columbia River. These were held at 110% from April 23, 1973

until August 3 (103 days), then maintained at normal gas levels for

about three weeks before the fish were artificially spawned.

The general effects of temperature on mortality were studied at

10, 15, and 18 C. The fish were adjusted at a rate 3 C/day until they

reached the test temperature. Fish were maintained at the test temperature for at least two days prior to initiating supersaturation.

STATISTICAL PROCEDURES

Fish were assigned to treatments using prepared random number cards.

All statistical procedures conform to those of Snedecor and Cochran

(1973). Statistical significance was assigned whenever the probability

of obtaining a given result by chance alone was <.10.

18
SECTION IV

RESULTS

GENERAL TOLERANCES OF SALMONIDS AND NON-SALMON IDS

Preliminary concurrent exposure of several representative species,

conducted at 130-150% saturation (10 C), established that salmonid

species (Salmo and Oncorhynchus) were highly susceptible to gas bubble

disease. Shiners (Notro~ ) and crappies (Pomoxis ~.) also died

rapidly under those conditions. Bluegill (Lepomis macrochirus),

squawfish (Ptychocheilus oregonesis), and warmouth (Lepomis gUlosus)

were intermediate in tolerance to bullhead (Ictalurus ~~), largemouth

bass (Micropteru~ salmoides), and carp (Cyprinus carpio) which were

extremely hardy under those test conditions. These results reveal

differences in tolerance between genera and demonstrate that salmonids

were both highly sensitive and easy to use in these tests.

SAMPLE RESPONSES TO SUPERSATURATION

A general estimation of population response (mortality) to supersaturation is essential to understanding the overall problem. Specific

information on this subject has not been found in the literature: hence,

we analyzed the data from twenty bioassays (each used either 10 or 20

fish per bioassay) and estimated the response curve. Individual times

to death from experiments with 100% mortality constituted the raw data.

Various measures of central tendency or dispersion were estimated for

population parameters listed in Table 6.

As a preliminary step, pooled times to death data for all life

stages were tested by analysis of variance, and this revealed that

the samples were significantly heterogeneous at the 10% level for a given

level of saturation. Subsequently the data were pooled only within a

given life stage because gross differences in tolerance were apparent between life stages.

Curves plotted for cumulative mortality and rate of death (Figures 4 and 5) show three phases of population response to supersaturation. The initial phase consists of a sublethal morbid period during which

emboli form and related dysfunctions approach critical limits for fish

life. The second phase is a period of extensive mortality which appears

linear with the 10910 of exposure time. The third phase did not occur

in all samples nor at all saturation levels, although its intermittent

occurrence indicates likelihood if larger samples or more replicates

were tested for a longer time. During the third phase the more tolerant

fish died at a greatly reduced rate, typically over an extended period

of time. This protracted survival period gave many fish samples a

definite and significant skewness that rejects the hypothesis of random

normal distribution. Possibly a fourth phase may exist consisting of

post-exposure mortality or recovery from all effects that carried over

after returning to normal saturation levels.

19
TABLE 6. SUMMARY STATISTICS OF TIME TO DEATH AT THREE LEVELS OF SUPERSATURATION BY TWENTY

POPULATION SAMPLES OF RAINBOW TROUT, PACIFIC SALMON, AND LARGEMOUTH BASS.

Number 90% Coefficient Coefficient

in Range Average Belt r~edian Standard of of

Sample (hours) (hours) for ].l (hours) Variation Skewness

10C, 130% Supersaturation

1. Spring chinook salmon (Oncorhynchus tshawytscha) (XFL =81.0cm; XWt =7.738kg) adult female, migrating

a. Time to dei.1th 11 7-12 8. +0.929 8.5 1.700 19.0 +0. 60

b. Log 10 0.845-1.0792 0.943 +0.044 0.954 0.OB2 8.7 +0.419

2. Spring chinook salmon 60cm; XWt = 5.618g)

adult male, migrating

a. Time to death 9 7-11 .440 +0.701 10 1.130 12.0 1.

b. Log 10 0.845-1.041 0.972 +0.034 1.00 0.056 .7 .497

10C, 125% Supersaturation; 10C

3. Rainbow trout 15.2cm; XWt =42.2g)

parr

. Time to death 20 20-48 30. 150 +3.276 27 8.474 28.1 1.115

b. 1. 301-1. 681 1.4645 +0.044 1.431 0.114 7. 0.874

4. Rainboltl trout =28.18cm; - 259.39)

yearling

a. Time to death 17-45 33.70 +5.556 31 9.696 28.8 O.

b. Log 10 .230-1.653 1. 1510 +0.0792 1.4914 0.1385 12.0 7.

5. Winter stee1head trout (Salmo gairdneri) (XFL =7.8cm; XWt =5.6g)

parr

a. Time to death 20 10-224 66.55 +24.34 35 62.966 94.6 1.360

b. Log 10 1.000-2.3502 1.6519 -+0.966 1.5524 0.150 24.4 0.559

6. Sockeye salmon (Oncorhynchus nerka) (XFL =10.2cm; XWt =12.0g)

parr

a. Time to death 20 13-189 62.150 +18.566 40.0 48.022 77.3 1.509

b. Log10 1.0128-2.2765 1.6551 -+0.1420 1. 6021 0.367 22.2 0.615

7. Spring chinook salmon (Oncorhynchus tshawytscha) (XFL =79.6cm; XWt = 5.816kg)

adult females

a. Time to death 16 12-22 17.875 +1.357 18 3.095 17.3 0.605

b. L0910 1.0792-1.3424 1.2456 +0.0346 1.2553 0.0798 6.4 0.810

8. Coho salmon (Oncorhynchus kisutch) (XFL =20.4cm; XWt =87.5g)

post smolts

a. Time to death 20 6-21 12.500 +1. 559 12.0 4.032 32.2 0.372

b. Log 10 0.7782-1. 3222 1.0578 +0.0556 1.0792 0.1468 13.8 0.437
TABLE 6. SUMMARY STATISTICS OF TIME TO DEATH AT THREE LEVELS OF SUPERSATURATION BY TWENTY

POPULATION SAMPLES OF RAINBOW TROUT, PACIFIC SALMON, AND LARGEMOUTH BASS (CONTINUED).

Number 90% Coefficient Coefficient

in Range Average Belt t"ledi an Standard of of

~ampl e (hours) (hours) (hours) Variation Skewness

9. Coho salmon (Oncorhynchus 40. XWt = 795g) jacks (precocious)

a. Time to death 20 12-21 7.200 .276 19 3.3015 19.2 1.635

b. L0910 1. 0792-1. 3222 1.2275 .0054 1. 2788 0.0865 7.0 1.780

10. Coho salmon = 71.1em; XWt = 4.173kg) adult female,

a. Time to 10 15-28 20.900 .902 21 3.2812 15.7 0.091

b. Log l0 1.1761-1. 4472 1.3153 .0392 1.3222 0.0683 5.2 0.301

11. Coho salmon (Oncorhynchus = 66.8cm; XWt 3.678kg) adult male, ripe

a. Time to death 10 12-42 23.2 161 20 8.904 38.38 1.078

b. Log l0 1.0792-1.6232 1.3403 .1867 1.3010 0.1520 11.34 0.015

[)..:;

I-'

12. Rainbow trout 15.3cm; = 43.9kg)

parr

a. Time to death 20 32-193 70.650 6.403 51 42.475 60.1 1.388

b. 0 1.5051-2.2856 1.7950 .0802 1.7076 0.2078 11. 6 1.262

13. Winter steelhead trout 69.2cm; = 3. 563k9)

adult females, gravid

a. Time to death 10 59-164 104.50 .316 92 33.323 31.9 1.107

b. Log 10 1.7709-2.2148 1.9987 .0797 1.9638 0.1377 6.8 0.1377

14. Winter steel head trout (XFL = 70.2cm; XWt = 3.336kg)

adult males, ripe

a. Time to death 10 57-144 90.30 +16.599 79 28.6397 31.72 1.1837

b. Logl0 1.7559-2.1584 1.9370 -+0.0766 1.8973 0.1324 6.83 0.8998

15. Coho salmon (Oncorhynchus kisutch) = 73.0cm; XWt =4.372kg)

adult female. migrating, gravid

a. Time to death 10 31-64 45.700 +5.178 45 8.9324 19.5 0.235

b. .Log lO 1.4914-1.8062 1. 6525 !:0.0488 1.6532 0.0843 5.1 0.025

16. Coho salmon (Oncorhanchus kisutch) (XFL = 70.4cm; XWt =4.080kg)

adult female. gravi

a. Time to death 10 42-110 58.80 +11. 348 51 19.577 33.2 1.1950

b. Logl0 1.6232-2.0414 1.7526 -+0.0693 1.7076 0.1197 6.8 1.1288
TABLE 6. SUMMARY STATISTICS OF TIME TO DEATH AT THREE LEVELS OF SUPERSATURATION BY TWENTY

POPULATION SAMPLES OF RAINBOW TROUT, PACIFIC SALMON, AND LARGEMOUTH BASS (CONTINUED).

CoefficientCoefficient

Median Standard of of

(hours) Deviation Variation Skewness

54.44 +16.735 51

1.6676 -+0.1450 1.7076

45.45 +4.782 45

1.6424 :0.0454 1.6532

0.337

0.452

0.109

0.274

27.2

7.2

56.2

15.9

12.369

0.1175

30.623

0.2653

90%

Belt

for j.l

Number

in Range Average

Sample (hours) (hours)

(XFL = 66.7cm; XWt = 5.208kg)

9 22-99

1.3424-1.9956

= 22.4cm; XWt - 163.4g) 20 25-69

1.3979-1.8388

17. Spring chinook salmon (Oncorhynchus ~~~~)

adult female, migrating

a. Time to death

b. Log10

20C, 130% Supersaturation

18. Largemouth bass l.r!!..:~~~ salmoides)

a. Time to death

b. Logl0

19. Largemouth bass, adults (XFL =23.1; XWt =187.5g)

a. Time to death 20

b. Log10

20. largemouth bass. adults (XFL = 24.0cm; XWt = 209.6g)

a. Time to death 20

b. Logl0

10-22

1.000-1.3424

11-19

1.0414-1.2788

16.550 :1.3924 16 3.349 20.2

1.2098 ~0.0381 1.2041 0.0924 7.6

12.45 to.8551 11 2.2117 17.8

1.0895 :0.0696 1.0414 0.0696 6.4

0.493

0.183

1.966

2.074
MEDIAN AVERAGE

100 t t CUMULATIVE MORTALITY 20

90 ')

"0 I I

80 I I a - I I 15 a::

70 w - Cl. ...J ,I 60 . I >-

a:: I ..... 0

5°1 I I 10 ...J

a 40 I \ a::

w / \ Coefficient of skewness = 1.36 0

I:\:l

c:" 30 / \ ...J 5 ..... ::> 20 / "- z

:E / "- w

::> ......... ____ MORTALITY PER DAY u

10 I a:: u ----------------- lLJ U Cl. <! 0

10 20 30 40 50 60 70 80 90 100

DAYS OF EXPOSURE

Figure 4. Time to death of winter steelhead trout parr (Salmo gairdneri)

at 125% supersaturation and 10C in water 65 em deep.
140 160 180 200

PHASE:m:

(PROTRACTED SURVIVAL)

MORTALITY

100 120

HOURS

•••

80

••

60

•

•

•

•

•

•

•

•

•

•

••:-- RATE OF

40

MEDIAN

•

•••

• •

• • •

•

•

•

•

•

•

•

•

•

•

•

•

•

•

•

•

•

•

20

I

PHASE-I I PHASE-II I

I

MORBIDITY II I LINEAR )1 (MORTALITY •

I MEAN

+

o

~40

-

....

:) 20

:::>

a 0 -+--__----__----r-----,r----__- __--....--~..;;...;...;;;...;;..;;.;;..;;;.;.;;~.;;;..;;;.;;...;;;..;

o

>-100

....

-

--'

80

0:::

o

60

Figure 5. Mortality response of rainbow trout (Salmo

gairdner9 to 120% supersaturation.
Skewness was prominent (Table 6) whenever phase III mortality occurred and relatively high whenever the mortality data were plotted

as hours to death. Although a log transformation of the data generally diminshed skewness, it still was a prominent factor approaching a high level of statistical significance (Snedecor and Cochran, 1973). Its

occurrence at high and low levels of supersaturation indicates that the

shortest time to mortality is more sharply defined than the maximum

time to death. Further, skewness usually indicates that a relatively small number of factors are influencing the resulting population

response, and this may be true for the fish's tolerance to supersaturation.

However, skewness varied between tests and, in the case of adult female

coho salmon (at 120% supersaturation), the coefficient of skewness

increased from 0.235 in late September to 1.195 in late October.

Possibly this reflects the impact of selection via prespawning mortality prior to capture but, whatever the cause, it influenced the response

curve. Higher levels of skewness were more associated with low levels

of saturation, male fish, and immature juvenile fish than their counterparts.

Central tendency was examined by comparing means, median, and

modal values. Confidence limits can be derived for means, a valuable

advantage when statistically comparing different populations or developing population models. However, mean time to death had required a 100% kill

which neither occurred at 115% supersaturation nor, in many cases, at

120% even after prolonged exposure. This was due in part to the skewed

population response described above. Thus, the median time to death

typically occurred before the more protracted mean time to death.

Median time to death, therefore, not only permits more tests to be

conducted in any given period, but also is an appropriate measure of

central tendency in these tests. However, applying confidence limits to

it is difficult.

Table 6 lists mean times to death varied among levels of supersaturation, species, and life stages. The width of the 90% (two-tailed)

confidence estimate of the population mean (~) at 120% saturation varied

from a low of about ± 6 hours for juvenile rainbow trout to a high of

± 19 hours for adult female steelhead (winter). Confidence belts were

generally smaller at higher saturations, and the previous results would

predict extremely broad confidence belts at low levels of supersaturation.

Variability was a function, in part, of the supersaturation level,

hence coefficients of variation (C.V.) were inversely proportional to

the supersaturation level. At 120% saturation C.V. values reached a

high of 60. However, variability usually diminished a great deal when

the data were expressed at 16910 hours to death, and this was the case

at each saturation level. This emphasizes the need for a log transformation of exposure times in studying gas bubble disease, as well as

toxicants.

25
Similar to the coefficients of variation, standard deviations

were inversely proportional to the saturation level. Frequently the

standard deviations approached or exceeded 35% of the means; however,

these were much smaller when the data had been transformed into 10910

values.

PATHOBIOLOGY AND TIMES TO MORTALITY

Tolerance to 130% Saturation

Spring chinook salmon and largemouth bass were the only species

tested at this high level of saturation. The times required to reach

20% mortality and median mortality at 130% are listed in Tables 7 and 8.

Adult spring chinook began dying within about seven hours, reaching

median mortality in about 8.5 hours and total mortality within 12 hours.

From these data continuous exposure to 130% apparently can kill salmonid

fish in a relatively short time provided that the fish remains in shallow

water. However, if a fish dives to a depth of 3 meters, the hydrostatic

pressure of water would prevent bubbles from forming.

Adult spring chinook at 130% of air saturation indicated approaching

mortality by their behavior. After 4 hours of exposure, morbid chinook

would swim aimlessly, unresponsive to external stimuli. Coughing and

shaking occurred frequently, usually followed by bursts of violent but

generally aimless swimming. Necropsy found few external signs of gas

bubble disease, but emboli usually were present in the blood vessels

of the gills causing discoloration. Amassive air-embolus filled the

intravascular space between the capillary beds of the gills and the heart.

Air emboli could be seen in the intercostal arteries and in the dorsal

aorta. After median mortality had been reached, small emphysema (bubbles)

were evident in some fins of some fish, but usually not seen in the tail

of adult spring chinook. Most adult spring chinook salmon sank at death

and did not float during the short period between death and necropsy. These results were reported in greater detail by Stroud, Bouck and

Nebeker (1975).

Largemouth bass, lethargic at 130% saturation and 10 C, died only

after exposures 10 times longer than those lethal to adult spring chinook

salmon. The longer survival of bass probably accounts for more fully

developed and more frequent incidence of external signs of gas bubble

disease in these fish. After two days of exposure, all the bass were

still alive, but they contained sufficient emphysema to change portions

of their normally transparent fins to a itish translucent color.

Emphysema usually was not observed under the skin or scales of bass.

Necropsy revealed that portions of the gills which extended to the heart

were filled with air at pressure .sufficient to distend the myocardium.

largemouth bass floated prior to and after death from gas bubble disease,

possibly because their swim bladder has no opening to their esophagus for volitional release of excess air. This resulted in tympanites of

the swim bladder.

26
TABLE 7. HOURS OF EXPOSURE TO REACH 20% MORTALITY AMONG FISHES EXPOSED

TO SUPERSATURATION IN SHALLOW WATER AT lOGC.

%saturation

130 125 120 115 110

Coho salmon

parr 37,22 30, 73, >336

134

smo1ts

held over smolts 8, 12 30, 23, 120, 303

41,30

jacks 14 49, 40 336

adult females (gravid) 19 38.5, 43 122

adult males (ripe) 19 56, 40 207

Spring chinook salmon

parr

smolts 30 >268 1440

adult females, migrating 7 14 30, 30 430

adult males, migrating 8 15 50, 22 498

Sockeye salmon

parr 20 36, 45 49, 167

smolts 11, 19, 35, 27, >268 >268

16 50, 58

adults, migrating <18 60 140, 650

Stee1head Trout, Summer

parr

smolts 14, 14, >268 >268

29

adults, migrating 25 72 140

Steel head Trout, Winter

parr 19, 38, 40, 25 160

20

smo1ts 46 154 >268

adu1 t females, (gravi d ) 68 332

adult males (rioe) 68 263

Rainbow Trout

parr 23, 24 45

yearlings 23, 218 79

27
TABLE 7. HOURS OF EXPOSURE TO REACH 20% MORTALITY AMONG FISHES EXPOSED

TO SUPERSATURATION IN SHALLOW WATER AT 10°C.

%saturation

Largemouth bass

juveniles

adults

Suckers (Catostomus sp.)

adults

130

115

65

125

-0-

142

20

226

115 110

Tests were conducted with a maximum attainable depth of 65 cm, and 10 fish or

more per test.

28
TABLE 8. ESTIMATED HOURS TO MEDIAN MORTALITY (ETSO) AMONG FISHES EXPOSED TO SUPERSATURATION

IN SHALLOW WATER AT looe.

SPECIES 130 125

Gas levels (% saturation)

120 115 110

Coho salmon (Oncorhynchus kisutch)

a. parr b. smolts

c. held-over smolts

d. precocious males (jacks)

e. adult females

f. adult males

Spring chinook salmon (Oncorhynchus tshawytscha)

a. parr b. smolts

c. adult females, migrating d. adult males, migrating

Sockeye salmon (Oncorhynchus nerka)

a. parr b. smolts

c. adults

Steelhead Trout, Summer (Salmo gairdneri)

a. parr b. smolts

c. adults, migratin~

Stcelhead Trout, Winter (Salmo gairdneri)

a. parr b. smolts

c. adult females, gravid d. adult males

Rainbow Trout (Salmo gairdneri)

a. parr b. yearlings Largemouth Bass (Micropterus salmoides)

a. juveniles b. adults

Suckers (Catostomus sp.)

a. adul ts

8. 1

8.9

175

130

70, 44

11 ,13

17

20

20

17.5

16.0

40

20,23,22

<18

19,18

33

38,70,35

25

31

>240

>240

280 ,120 ,1 50 :

I

44,34,40

60,60,50

44,52

80,71

50

45,60

65,43

70,95

60,54,60

83

52

108

90,75

>48

92

80

56

42

>240

>240

>216

>336

>240

270,>336

>336

180

265

>268

540

500

100,183

>268

>168

>268

>168

270

>268

>336

>336

140

>268,>1440

>1440

>l440

>268
Tolerance to 1 Saturation

A 125% saturation was ethal to all the sa monid fish in six days under these test conditions, but l~~r,omr~!lth bass and suckers were not

killed. Median mortality was reac an overall average of 28 hours.

Time to first mortal ty ra rs r hold-over smoltsl! to

25 hours for adul t ter s 1, ica ly occurred after 12

hours of exposure in this shallow wa nce. Times to median

mortality ranged 1 hours ts to 70 hours

for juvenile coho sal to hours for bass (Table 8).

Tolerance differed e sexes of a given species at

this level, but differe t i uen showed marked differences in tolerance, e.g., co e Median survival times

were about twice as 1 juveniles of nter steel head as for summer

steelhead, indicating tolerance exist due to racial

stocks. However, men 1 error account for these differences

because variability samet mes exc

At least 90% of th s survived 125% saturation

for 10 days, but they 1 ex sive areas of emphysema in their

fins. At this time juvenile salmon were added to the tanks, whereupon

the bass caught a ate is 1 to concl sion that the bass

were still sufficiently to sus n ir predatory inclinations.

Meekin and Turner (1974 tha bl 1 gas bubble disease

inhibited predation by squawfish (Ptychocheilu~ oregonensis).

External signs f

at 125% than at 130%

gas had longer to act

minimal external signs

were more readily mottled by emboli a

under the skin. Eye i

because exposure time

the exception and devel

formed sooner and more

Tolerance to 120% --'-'----'-'----'-'--

Time to median

coho smolts to as

bass died at 120%

parr was in 16 hou

to four days before

l! Smolts not libera

of testing in early

generally don't survi

nanor~lly more evident

onger and therefore the

li es at 125% had

icallyexternal signs

gns include gills the lateral line, and

mal at 125%, possibly

a rainbow trout were

dermal emphysema, which

test fish.

hours for hold-over

rr, but no largemouth

mortality to steelhead nOlnny'~lly survived three

in tanks until the time

do not exist in nature and
Differences in tolerance to gas bubble disease were evident at

120% between adult male and female coho~ gravid coho females were

less tolerant among coho and possibly this also was the case for gravid

sockeye salmon. A sex-related difference in tolerance was not noted

in adult spring chinook, possibly because they were not gravid. Adult

winter steel head died sooner than adult summer stee1head, again possibly

because the winter fish were ready to spawn.

Most of the previously described signs of gas bubble disease were

observed at 120% saturation where longer survival, hence longer exposure

time, increased the severity of the lesions. Eye damage became evident

at this level of supersaturation, but it occurred in probably less than

10% of the fish and primarily among the various racial stocks of Sa1mo

gairdneri. The most frequently observed eye involvement was exophtha1mia

from retrobu1bu1ar emphysema. This caused the eyeball to protrude well

beyond the orbit and may have diminished the functional status of the

eye. Another form of eye involvement consisted of air bubbles in the

aqueous humor and in the retina causing distorted optics, retinal detachment, and hemorrhage. The latter lesions are considered a serious problem that can lead to blindness, inability to spawn, and death.

Another significant result of gas bubble disease was the development

of petechial and ecchymotic hemorrhages in various locations including

cutaneous and intra-muscular regions. Presumably these hemorrhages occurred in the surviving fish, but their impact and extent are unknown.

Also, adult salmon with gas bubble disease frequently developed fungal

infections which spread rapidly across their bodies.

Tolerance to 115% Saturation

Prolonged exposure was required to reach median mortality (Table 8)

at 115% total gas saturation. Total mortality never occurred at this

level during the test period (up to 30 days), hence the average 10g10

time to death could not be computed. The most sensitive fishes were

sockeye parr (ET50 in 100 hours) and adult female coho (ET50 in 180

hours), and the least sensitive salmonids were juvenile coho salmon

(0 mortality in 336 hours). Intermediate tolerance was indicated by

juvenile steel head (ET50 in 270 hours) and adult spring chinook (ET50

in 526 hours). Sex related differences were more pronounced at this level.

Symptoms of gas bubble disease were most developed at 115% mainly

due to the prolonged exposure to supersaturation. Exophtha1mia became

even more exaggerated and frequent but still involved probably less

than 10% of the animals. Emphysema were abundant in the skin and fins

of essentially all the fish. Fungal infections became very frequent

and covered areas of ulceration. Petechial hemorrhage of hyperemia often gave the skin a reddish color.

31
Muscular emphysema were noticed in adult spring chinook after two

weeks of exposure at 115%. The emphysema grew in size as exposure time

increased to three weeks until slices of the muscle were so riddled with

holes that they resembled red "swiss cheese." The size of these

emphysema, described in greater detail by Stroud and Nebeker (1975),

reached to slightly greater than 1 cm in diameter.

Tolerance to 110% Saturation

Only a few tests were conducted at 110% total gas saturation and

were generally tolerated by the test fish. One chronic exposure was

conducted on spring chinook salmon adults and smolts. During a 3.5

month period, 10% of the adults died of gas bUbble disease and secondary complications. Similarly, 20% of the spring chinook salmon smolts died

in two months at 110%. In this case about 30% of the smo1ts had

exophtha1mia and at least 50% had other external signs of gas bubble

disease such as emphysema in the skin or fins. Perhaps emphysema would have been discovered in even more fish had we inspected them via

ultrasonic sound reflection (Mackay and Rubissow, 1971). Necropsy revealed that most of the dead smo1ts had emboli in the blood.

Food pellets were offered to the spring chinook smo1ts to prevent

starvation during the long exposure period. Those smolts with

exophtha1mia attempted to and could usually feed, but their actions

suggested impaired vision and, in some cases, blindness.

ESTIMATED TIMES TO 20% MORTALITY (THRESHOLD TOLERANCE)

We established the time to 20% mortality as the threshold for

mortality. Based on the previously described skewed mortality curves,

additional exposure will probably produce disproportionally greater mortality when the 20% mortality level is achieved. Conversely, remaining well below this exposure time would probably prevent losses due to

gas bubble disease. Therefore we estimated the true population mean

(p) from the observed times to 20% mortality (x) by determining the 90%

(two-tailed) confidence limits of the sample mean (x). Although the

curves are skewed, the data were pooled from all sa1monid species of a

given life stage because we have not yet proven that these species were

significantly different, especially in nractica1 terms.

The 90% confidence limits for average time to 20% mortality are

listed in Table 9 and shown in Figure 6. At the highest stress level

(125% saturation), one can readily predict within narrow limits the

probable time to 20% mortality for a given life stage. Smolts and adults

required about 3/4 of a day and parr required nearly a full day at 125%

saturation to reach 20% mortality. As the stress level diminished to

120% of saturation, time to 20% mortality approached two days of exposure

32
350

300

>-

.....

-...J

250

f:r

o

fI!. 200

o

(\J

::>

(/) 100

X

LLJ

(/) 50

f:r

::>

o

J:

110 /15 120 125

PERCENTAGE SATURATION

Figure 6. Means and 90% confidence belts of times to

20% mortality at various life stages of

salmonids and levels of supersaturation.

33
TABLE 9. STATISTICAL.AMALYSIS OF POOLED OBSERVATIONS ON HOURS TO

20% MORTAbITY AMONG THREE LIFE STAGES OF FIVE SPECIES OF

SALMONIDS ..l1

LIFE STAGE

Adults Smolts Parr

125%

mean hours 18.33 17.17 23.63

90% limits of mean :3.19 :5.24 :4.41

range 14-25 11-29 14-38

n 6 6 11

120%

mean hours

90% limits of mean

range

n

115%

mean hours

90% limits of mean

range

n

48.12

~8.69

22-72

12

309.11

±114.28

122-650

9

41.00

:10.06

27-58

6

154

1

53.50

±23.83

25-134

8

125.30

±122.25

49-67

3

!I Included spring chinook salmon, coho salmon, sockeye salmon,

summer steel head trout, winter steel head trout, and rainbow trout.

34
and the confidence belt ranged in width from 8 hours to 24 hours. At

the lowest stress level that routinely produced mortality (115% saturation),

mean times to 20% mortality increased to about 13 days (t 5 days) of

exposure for adults and about 5 days (t 5 days). Stress levels at

110% saturation did not routinely produce mortality.

Sa1monid fish must typically spend several days at a maximum depth of less than 60 cm if their populations are to experience as much as

20% mortality at saturation levels of 115%. However, caution is urged because the confidence limits are approximately 100% of the mean, hence

the response is relatively unpredictable at low stress levels. Evidently several factors influence tolerance at low levels of saturation, and

some of these are indicated in the sub-section on factors affecting mortality by supersaturation.

RANK ORDER OF TOLERANCE TO SUPERSATURATION

The life stages of the various test species were placed in a rank

order beginning with the least tolerant and proceeding to the most

tolerant (Table 10). The data base was constructed from the e~timated time to

median mortality at 120% total gas saturation (203 g/cm2 hyperbaric). The authors selected this saturation level because the resulting mortality

was sufficiently protracted to permit the expression of inherent

differences while generally excluding the effects of disease and malnutrition

FACTORS AFFECTING MORTALITY BY SUPERSATURATION (GAS BUBBLE DISEASE)

Test Temperature

Mortality at comparable saturation levels was determined at

temperatures between 10 C and 20 C. Sockeye salmon and largemouth bass

(both adults and juveniles) reveal markedly different response patterns

to supersaturation when tested at different acclimation temperatures (Figure 7 and Table 11). Adult sockeye were considerably more tolerant

to 120% and 125% saturations when they were acclimated slowly from 10 C

and tested at 18 C. But sockeye salmon and rainbow trout parr showed

a variable response; increased test temperatures increased tolerance

in one case and decreased tolerance in two cases. Both juvenile and

adult largemouth bass showed a decrease in tolerance with increased

test temperature, possibly because they were stressed by temperature acclimation and by supersaturation.

Obviously, test temperature has an influence on tolerance to

supersaturation, but resulting variability creates uncertainty in

interpreting the impact of acclimation time, temperature preference,

or both. For example, adult sockeye had been acclimated to their

35
TABLE 10. RANK ORDER OF TOLERANCE BETWEEN FISH TElTED AT 120% TOTAL

GAS SATURATION (203 g/cm2 hyperbaric). -I

Species

Held-over coho smo1ts

Yearling Rainbow Trout

Adult female coho

Spring chinook smo1ts

Summer steel head smo1ts

Adult female spring chinook

Adult male spring chinook

Rainbow trout parr

Precocious male coho (jacks)

Sockeye smo1ts

Adult male coho

Adult male winter stee1head

Sockeye parr

Winter steel head parr

Adult sockeye

Adult female stee1head

Adult summer steel head

Coho parr

Largemouth bass

ET 2/

50

39.3

42.0

48.0

50.0

52.0

52.5

54.0

56.0

56.6

58.0

75.0

80.0

82.5

82.5

83.0

92.0

108.0

183.0

>240.0

To1er~nce 3/

ratlo -

1.00

1.06

1.22

1.27

1.32

1.34

1.37

1.42

1.44

1.48

1.90

2.04

2.10

2.10

2.11

2.34

2.75

4.66

>6.00

1/

- Tests were conducted at lOo e with a maximum water depth of 65cm.

2/

- Times to median mortality averaged from Table 8.

y

Tolerance ratio = IIX II time to median mortality

smallest time to median mortality

36
20

, P

/ / /

-u / I

- / / J

a:: / J / I

::> 15 ..... / « I /

a:: / II

0- / 1/

I, >168 w / t- Il/ /

o\~ / /

/

01 I /

~~ 0\0/ I 0/ ~~7 / ~,

10 - '"

0 50 100

HOURS TO MEDIAN MORTALITY

e--... adult sockeye (Oncorhynchus nerka)

A---6 juvenile sockeye

0--0 juvenile largemouth bass

0--0 adult largemouth bass (Micropterus sQlmoides)

0---0 rainbow trout (Salmo gairdneri)

Figure 7. Effects of test temperature on the time to

median mortality from supersaturation.

37
TABLE 11. SUMMARY OF THE RELATIONSHIP BETWEEN ACCLIMATION TEMPERATURE

AND TIME TO MEDIAN MORTALITY IN SUPERSATURATED WATERS.

Hours to median mortality at temperature

10 C 15 C 18 C

Species and Saturation level

1. Sockeye salmon (Oncorhynchus nerka)

a. adults (1) 125%

(2) 120%

b. parr (1) 125%

(2) 120%

2. Rainbow trout (Salmo gairdneri)

a. parr 125%

120%

3. Largemouth bass (Micropterus salmoides)

a. adults (1) 140%

(2) 130%

b. juveniles (1) 140%

(2) 130%

38

18

83

38

58

29

46

32

130

45

175

22

21

46

168

16

75

1a

92

12

42

10

60
test temperatures for a t

showed increased tolerance

temperatures. This was a so

and rainbow trout whi h were

the other salmonid fish i

temperature acclimation uveni

showed decreased tolerance

testing. These fish

h gher acclimation

one p of juvenile sockeye

to warm water. All of

ttle or no time for

a rainbow trout) and they

on.

ponds on November 27, 1972,

December 4, 1972. The bass

3. Then the fish were warmed

ust for an additional 2.5 days n~nrOr1l1re was repeated several times

cold to warm temperatures , but their response

ds which experienced l~n"'O\l<'~, bass prefer 'warm water

si y the observed differences

5S and salmon are based on hO~I.IOl,n warm water fishes and cold water

Bass had been collec by selnlng

and some fish were tested at 10 C

were kept at about 10 C until rch

to 20 C in about 4 days a all

prior to beginning the test.

in the testing of the 55.

Whether the relati y rapid c

had influenced the tolerance

conforms to the pattern es

minimal temperature accl;

and were more active at

in tolerance to gas bu

more fundamental diT+,,~on~<,c

fishes.

Based upon these data, it

tolerance to supersaturation.

to clarify the role of temperature.

temperature can influence

wo k will be needed

Swimming Activi_t,l

The effects of swimming ac

salmon and bass in supersatura

are shown in Figure 8. s

very little current 0

bass that were placed concurren

(higher velocity) too les

sockeye died at about the same

tank. Increased mortality res

tests. Those individuals c

out of the water usually di

quiet.

on juvenile sockeye

r largemouth bass

tank center experienced

26 hours. But

ion of the tank

mortality. Juvenile

r location in the

was seen in other

y ac ve nd often jumped

e which remained relatively

Sex

In some circumstances differences in lerance to supersaturation

varied between sexes for adult fish (i re 9). lerance was different

between males and females when (1) te rtality was low enough

39
100

80 15 C

SWIMMING

>- 60

t-

.-J

a::

0 40

0

\-

Z

W

U 20

a::

w

GO

16 18 20 22 24 26

TIME TO DEATH (hours)

Figure 8. Effects of swimming activity on the mortality of

largemouth bass (Micropterus salmoide in water

supersaturated to 140%.
100 ,. ,.' ,.. 90 ,..

"

>-

females ,.,."males males 80

l-- 1250/0 - 70 jj -.J

;I'

'" 60 ,.... 0: " ,. 0 50 ......

40 .......,---,20%

I- _.....

..... z 30 ,AII"' .....

w ,. ,. u 20 '" _.#/1" 0: -- w - 10 a..

10 20 30 40 50 60 70 80 90 100

TIME (hours)

Figure 9. Effect of percent saturation and sex on mortality

of adult coho salmon (Oncorhynchus kisutch) at lOC

in tanks 65 cm deep.
to permit its expression, and (2) if significant collateral differences

also existed. For example, female and male salmonids both died very rapidly at or above saturation levels of 125% and there was no apparent difference in tolerance. Also, no apparent difference was evident at

120% or lower when male and female fish experienced comparable conditions,

i.e. migrating spring chinook salmon. However, when the males and

females were not in comparable condition, the result was a difference

in tolerance to supersaturation. For example, male and female winter

steel head showed a difference in tolerance. but this was probably due

to fighting among the males which weakened them. Female coho salmon

were less tolerant than males and we speculate that this difference was

due to the weakened condition of the gravid females (Figure 10).

Largemouth bass did not die except at high levels of supersaturation which may have precluded the expression of sexual differences. No

differences were noted in the rate or total mortality between male and

female largemouth bass tested at 130% saturation and 20 C.

Life Stage

Figure 10 shows the concurrent times to mortality among juveniles

(parr), post smolts, jacks and adult coho salmon. Median mortality

was reached first by adults. then by jacks. and last by parr at 120%

saturation. At 115% saturation proportionally fewer adults were killed

and essentially no juveniles were killed.

Body Size and Condition Coefficient

Body size and/or condition had a significant influence on time to

death in some instances. Correlation analyses were conducted for 10910

hours to death on body weight. body length. and condition coefficient

(K) for adult coho. The only significant correlation at 125% saturation

was between the length of female coho and 10910 hours to death

(r = 0.93); larger fish took longer to die. These fish were essentially gravid and ready to spawn. Juveniles were not tested.

Adult spring chinook had a significant correlation between 10910

time to death and body weight (Table 12). While these results have

statictical significance and indicate that larger adult salmon tend to

survive longer in supersaturated w2ter. the overall impact seems

relatively small and questionable. For example, Meekin and Turner (1974)

determined that the size of juvenile Pacific salmon was inversely related

to survival in gas supersaturated water; large juveniles died sooner

than their smaller siblings. Perhaps fat content influences this as

indicated by Boycott and Damant (1908) and Gersh, Hawkinson, and Rathbun

(1944).

42
adult mares smolts

1200/0 1200/0

~adults juveniles

1200/0 125%

"__ ::0 ~-- __ -0

L__~~~=======:=::=:==7~'jaCks 12_~ ,,---

-.-juveniles

1200/0

\ J V

smolts jacks adults

125% 125% 1250/0

100

90

80 - 70

60

50

..... 40

30

20

l1J

a.. 10

20 40 60 80 fOO 120

TIME (hours)

140 160 lao 200

Figure 10. Effect of percent saturation and life stage on

mortality of coho salmon (Oncorhynchus kisutch)

at 10e tanks 65 cm deep.
TABLE 12. CORRELATION ANALYSES OF TIME TO DEATH AT VARIOUS LEVELS OF

SUPERSATURATION TO BODY LENGTH, WEIGHT, AND CONDITION

COEFFICIENT (K) FOR ADULT SPRING CHINOOK (Oncorhynchus tshawytscha).

Body Body Condition

Total Gas Length weirt Coefficient

Saturation Sex (cm) (9 (K)

r =+0.653* +0.679* +0.278

Males (9) a = -41.18 -22117 +1.163

b = +121.74 +29370 +0.017

130%

r =+0.480 +0.349 -0.307

Females (11) a =+15.823 -6056 +1.672

b =+70.628 +14928 -0.036

r = -0.674 -0.820 +0.946*

Males (5) a =+87.384 +4563 -3.745

b = -13.838 -1184 +0.007

125%

r = +0.225 +0.137 -0.167

Females (15) a =+37.143 +1221 . +1.207

b = +34.320 +13593 -0.009

r =+0.978* +0.813* -0.690*

Males (9) a = -130.82 -12818 +5.813

b = 111.92 10825 -2.416

120%

r =+0.970* +.723* +0.688*

Females (8) a = -90.52 -3294 +1.137

b =81.03 +6267 +0.085

r = -0.642 -0.624 +0.047

Males (7) a =+177.9 25421 +1.236

b = -61.09 -17713 +0.059

120%

r = -0.650* +0.659* ...0.252

Females (9) a = -12.132 -14240 +1.568

b =+54.793 +12712 -0.152

115% Females (7) r =+0.837 +0.487 +0.520

* Significant at 90% level.

44
Other Factors Influencing Tolerance to Supersaturation

At least two other factors may influence tolerance to gas bubble

disease based on our observations. The first is the effect of small

but definite increases in dissolved gas pressure induced by solar

radiation and fluctuations in the control system. Slight increases

in saturation seemed to cause increases in mortality in critically morbid fish; conversely, small decreases in saturation caused decreases

in mortality. Possibly the same effect was caused by changes in depth which would alter the amount of hydrostatic pressure to compensate the emboli or emphysema.

A second important factor that is difficult to quantitate is

behavior. The investigators spent many hours watching the test animals

and agree that behavior can have significant impact upon the results.

For example, adult sockeye salmon are schooling fish and tend to

pursue a common mode or level of activity. Other fish such as adult

steelhead tend to be very individualistic and their results seem to

be more variable. Adult chinook tended to remain at maximum depth in

the tanks; if this is the case in nature, the investigators speculate that these fish would probably find ample compensation by hydrostatic

pressure, if available.

TOLERANCE TO SEAWATER AFTER EXPOSURE TO SUPERSATURATED WATER

Three species of anadromous salmonids were tested for tolerance

to seawater (30 ppt Cl) after exposure to three levels of super~

saturation (110, 115, and 120%).

Exposure to 120% continued for about 47 hours when the following levels of mortality had been reached: winter steelhead = 15%; sockeye salmon =45%; spring chinook salmon 60%; and summer steel head = 70%

(Figure 11). Immediately thereafter the fish were transferred to air

equilibrated seawater and no further mortality occurred during a

subsequent 96 hour period that could be related to supersaturation or

seawater.

Other groups of smolts were placed in 115% saturation water for

268 hours. During this time 10% of the sockeye smolts and 20% of the

winter steel head smolts died of gas bubble disease. None of the summer

steel head or spring chinook smolts died during the 268 hour exposure

to 115% saturation. However, the survivors of each species had some

emphysema in the skin and fins and a few had protruding eyes from

emphysema around or behind the tye. Signs of gas bubble disease were

most prevalent and severe among the winter and summer steel head. Yet

these fish survived direct transfer to 30 0/00 seawater and continued

to survive for 124 hours when the experiment was ended.

45
100

:r: 90

(/)

u. 80

o

(\J

u. 70

o

>- 60 I-

...J

50

0:::

0 40

30

20

lJJ

U

0:: 10

FRESH WATER AT

120cro OF AIR

SATURATION (10°C)

TRANSFERRED ABRUPTLY TO

30% SEAWATER (10°C)

SUMMER STEELHEAD I(S. oirdneri)

SPRING CHINOOK I(O. tshaw tseho)

I

SOCKEYE ( O. nerko)

WINTER STEELHEAD I (S. oirdneri)

END OF

TEST

•

10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170

HOURS

I NO MORTALITY IN CONTROL GROUPS

Figure 11. Cumulative mortality of salmonid smolts during exposure

to 120% air supersaturation and immediate transition to

300 /00 seawater.
No smolts died that were exposed to 110% saturation for 268

hours followed by abrupt transfer to 30 0/00 seawater.

These results confirm our previous pilot studies on coho salmon

smolts and indicate no latent mortality to smolts from gas bubble

disease after they have entered seawater. Essentially identical

results were reported by Dawley, Monk, Schiewe, and Ossiander (1975).

EFFECTS OF CHRONIC SUPERSATURATION ON REPRODUCTION

The effects of supersaturation on reproduction were investigated only at 110% saturation because higher levels of supersaturation produced extensive mortality of adults in shallow tanks. The exposure of 20 adult spring chinook began April 30, 1973. One female developed partial paralysis early in July and was sacrificed and examined about

three weeks later. Another fish died at about this time when dissolved

gas levels jumped unexpectedly to 116%, but this fish had a heavy infection in the gills by fungus and parasitic copepods. The test

exposure ended August 3 when it became apparent that further mortality would result if we did not treat for the infective fungus and copepods. Thereafter no mortality occurred in either group until spawning operations began. Apparently the infections had weakened both the control and test

fish to the extent that heavy mortality occurred each time the fish were

handled and inspected for sexual maturity.

Experimental male spring chinook salmon ripened readily and their

semen was normal in appearance and consistency. Likewise, the eggs of

all experimental females developed to typical size and were normal in

color and general appearnace. However, prespawning mortality from

handling the fish eliminated four of the six females in the experimental lot; extensive mortality also occurred in the control lot.

Two experimental females survived the stress of supersaturation and subsequent prespawning handling; these ripened and were spawned artificially. Their eggs were typical in all respects and approxi- mately 90% of these eggs hatched. This is well within normal limits

and was comparable to the control fish. The results of this limited

sampling revealed no evidence of adverse effects to the sex products

or to the ability of the resulting embryos to hatch.

47
SECTION V

DISCUSSION

Perhaps some readers may want to extrapolate beyond these data

to natural circumstances in a river, reservoir, or lake. We urge

extreme caution in such endeavors because the research design did not

permit the testing of remedial behavioral responses such as avoidance,

sounding, or other aspects resulting in less than continuous exposure

to supersaturation stress.

Behavioral responses are important because they are known to

influence survival in supersaturated water. Dawley, et~l., (1975),

reported that juvenile fall chinook sustained less than ~mortality

after 60 days of exposure at 120% supersaturation when a maximum depth of 2.5 mwas permitted; greater than 95% mortality occurred in the same

period when siblings were exposed to 120% supersaturation in water 0.25

mdeep. Meekin and Turner (1974) reported similar results for juvenile fall chinook salmon, juvenile coho salmon, and squawfish, but juvenile steel head trout did not survive quite as well.

In 5-hour tests Meekin and Turner (1974) reported that juvenile chinook salmon avoided supersaturated water and congregated in the

control water. Juvenile coho salmon showed no preference during this

short period, which may in part, be related to their great tolerance

for supersaturation. Juvenile sockeye salmon consistently sound to

maximum depth when placed in supersaturated water hence actively avoid

gas bubble disease (Wesley J. Ebel, personal communication). Thus

avoidance responses could exert an extremely important influence upon the potential lethality of supersaturation in a river.

Tolerance to gas bubble disease appears to involve different

biological factors at high versus low levels of supersaturation. In

acutely lethal conditions, emboli form in the blood and cause rapid death. Thus survival at acutely lethal conditions may be influenced

heavily by the ability of individual fish to tolerate changes in

vascular dynamics. But survival at long term sub-acute exposures may be more dependent upon complex alterations of physiological functions

such as immune responses, infectious agents, or adaptive behavior.

Several other factors were identified that influenced tolerance

to supersaturation, including temperature, activity, life stage, and

body size. One of the most important was the difference in tolerance

between fish families, and to a lesser extent, tolerance varied between

species or races. Another obvious difference was that trout and salmon

which are phyostomous were generally more sensitive at 10 C than bass

48
which are physoc11stous. One hypothesis to explain this difference

is based on the observation that adult salmon expell gas frequently from their swimbladder and sink when dead. Conversely, bass became

bloated with considerable pressure in their gas bladder and usually floated before death. Possibly the tolerance of bass to supersaturation

was promoted in part, by a concurrent and compensatory increase of

intrabody pressure. Evidence of high intrabody pressure was indicated

in necropsy examinations. When punctured, the heart and swimbladder

emitted an audible release. High intrabody pressure would keep gases in solution within vital organs of the area adjacent to the body cavity and thus protect the fish. However, a sudden release of gas (from the swimbladder of salmon) would tend to sharply diminish intrabody pressure and promote cavitation of gas or the growth of accumulated

embol i.

Apparent differences in tolerance to supersaturation were related

to genetic stocks, sex, and life stage, but the attendant variation

renders it impossible to determine appropriate significance. ~ex was

a factor in two cases, but only when the respective species approached spawning condition. Adult male winter steel head were in spawning condition when tested and fought territorial battles in the tanks which

may have promoted emboli formation and death or decreased resistance.

Whatever the reasons, male steel head died much sooner than the females.

Adult summer steel head in spawning condition did not fight and,

although the sample size was small, there was no readily apparent difference in tolerance between males and females. The other case of

sex-related tolerance occurred with adult coho salmon, where gravid females died more rapidly than ripe males. Although the evidence is

not clear, indications are that poor physiological condition is a

greater influence on tolerance than sex pea se. Physiolggical condition may well be the cause of observe dlfferences in tolerance

to supersaturation between different life stages.

Osmotic and ionic regulation in seawater provides great stress

to smolts of Pacific salmon and steelhead. If these functions are.

impaired to any great extent, the fish generally die within a few

days. In this study the survivors of lethal supersaturation apparently suffered no significant impairment of osmotic and ionic re9ulation

because they survived direct transfer to seawater (30 0/00) and no

mortality resulted within a five-day period thereafter. This result

was confirmed by Dawley, et ~., (1975), and by Bouckll.

Gas composition in these experiments was not determined beyond the oxygen level which was typically high. High oxygen-nitrogen ratios

have been shown by Rucker (1974) to prolong survival. Apparently helium

11 Bouck, G. R. 1972. Unpublished data on coho salmon smolts.

49
has a similar effect because the decompression period of skin divers

can be reduced 50% when half of the nitrogen in the breathing air is

replaced with helium (Workman, 1963). Gas composition was not controlled

in this experiment and its variability may have influenced the resulting times to death.

Residual effects on sex products were not readily apparent among Columbia River spring chinook salmon which had been held for 3.S

months in shallow water saturated to 110% of barometric pressure. This is more than twice the exposure period that these fish would

experience in passing through the Columbia River. The resulting hyper- baric pressure is approximately equivalent to what a chinook would

experience at a depth of about 2 meters in water with 130% saturation.

Natural spawning was not possible in this research, but the sex products had a good appearance when spawned artificially, and both fertilization

and hatching were within normal limits.

Variability was relatively large for the observed times to median

mortality; this was true even within a life stage of a given race of

a given species. The ETSO value varied by 100% in some cases. If one

adds to this the potential influence of depth, swimming activity, and

acclimation temperature, it is then easy to hypothesize that data from

most salmonids can be treated as if they were from the same population without regard to race or species. Thus it appears that the sum of the

factors causing increased or decreased tolerance to supersaturation tend

to overwhelm the influence of a single factor except when it achieves

critical importance on a given occasion.

The interpretation of these data relative to the Columbia and Snake

Rivers requires the consideration of many factors. Wild fish live in

a dynamic circumstance and are free to alter their depth which, in turn,

alters their compensation by hydrostatic pressure. Likewise, in nature

intermittent exposure is probably the rule thus permitting higher tolerance than indicated by these data. Also, the potential exposure time of fish must be considered. For adult salmon in the Columbia River

typical passage time between the ocean and safe tributary streams is

probably three weeks or less.

There is no question that the three species of Pacific salmon,

racial variants of steel head trout, and even bass, can get into

serious trouble if they remain in shallow water at high saturation

levels for over a day. This has considerable significance in fish

hatcheries and laboratories where exposure to supersaturation may be

for a longer period and in shallow water. What must be determined is

the extent that these data apply to a large and complex river ecosystem.

Supersaturation appears to be exerting ecological selection

pressure in the Columbia River Basin (Ebel, et al., 1974), but its

direction and final result are not evident yet.--Thus far, most

50
researchers have investigated only the detrimental effects of super- saturation, describing supersaturation only in terms of morbidity and

mortality. But broader ecological effects are potentially possible and remain undescribed. For example, a low level of supersaturation (or intense supersaturation at certain times of the year) might be an

effective tool in eradicating rough fish populations. From this

aspect supersaturation has the distinct advantage of leaving no

residue, harmful or otherwise. Likewise, if supersaturation kills

bass fry and inhibits predation by squawfish, the net result could

benefit salmon. Further, if supersaturation selectively eliminates

small adults from breeding populations, the net result might be

larger salmon therefore better adapted to the Columbia River. This

is also suggested by the studies of Cramer (1974).

While a supersaturation level of 110% was not acutely lethal,

it did produce low level mortality and signs of ill health in adult

and juvenile spring chinook salmon during a 3.5 month laboratory

exposure in shallow water conditions. Such a prolonged exposure (especially in shallow water) would be unlikely for wild chinook,

but may occur in fish culture stations or possibly in the outfalls of

heated effluents. The latter could be particularly important when

seasonal temperature cycles dropped beyond the preferred temperatures of local fish species. In such cases, fish congregating in the warmer

water might die or be adversely affected by gas bubble disease.

Therefore, a saturation level of 110% might be acceptable for a river,

but may not be acceptable for heated effluents.

51
SECTION VI

REFERENCES

Anon. 1971. Standard Methods for the Examination of Water, Sewage and Industrial Wastes. 13th Edition. Amer. Public Health

Assoc., Inc., NY 1350 pp.

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54
TECHNICAL REPORT DATA

(Please read Ins.tructions on the reverse before completing)

1. REPORT NO. 12. 3. RECIPIENT'S ACCESSIOr+NO.

EPA-600/3-76-050

4. TITLE AND SUBTITLE 5. REPORT DATE

May 1976 (Issuing Date)

MORTALITY, SALTWATER ADAPTATION AND REPRODUCTION 6. PERFORMING ORGANIZATION CODE

Of FISH DURING GAS SUPERSATURATION

7. AUTHOR(S) 8. PERFORMING ORGANIZATION REPORT NO.

Gerald R. Bouck, Allen V. Nebeker, and Donald "

~1. Stevens

9. PERFORMING ORGANIZATION NAME AND ADDRESS 10. PROGRAM ELEMENT NO.

Western Fish Toxicology Station* lBA608

US EnviroTh~ental Protection Agency 11. CONTRACT/GRANT NO.

1350 SE Goodnight Ave

Corvallis, Oregon 97330 -- 12. SPONSORING AGENCY NAME AND ADDRESS 13. TYPE OF REPORT AND PERIOD COVERED

Environmental Research Laboratory Final 1970-1972

Office of Research and Development 14. SPONSORING AGE'NCY CODE

U.S. Environmental Protection Agency EPA-ORD Duluth, Minnesota 55804

15. SUPPLEMENTARY NOTES

*Now attached to the Corvallis Environmental Research Laboratory, Corvallis, Oregon

16. ABSTRACT

Tests were conducted using continuous exposure in shallol.\, water at levels of total

dissolved gas pressure ranging from 110_1LOo~ of barometric pressure (hyperbaric

pressure ': 103-410 g/cm2). Both times to 20% and to median mortality were determined

on several life stages of Pacific salmonids (Oncorhynchus and Salmo) and Largemotlth

bass (Micropterus salmoides). Hean times to 20% mortality at 115~;' total gas saturation were 309, 154 and 125 hours for adults, smol t s and parr. At 120~~ saturat ion

mean times to 20% mortality were 48, 41 and <:;1

-I ~~' hours for adul ts, smolts and parr.

At 125 % saturati on, mean times to 20% mortality decreased to 18, 17, and 24 hours

for adults, smolts, and parr. Factors which infl uenced time to death inc1 uded

genera, life stage, ace1imat ion temperature, activity level, sex and body size.

Mortality curves were typically skewed to the right. Gross pathology of gas hubble

disease was described relative to these experiments. High gas levels that killed

50% of three species of salmon smolts had no apparent effect on the ability of the

survivors to tolerate an immediate transfer into seawater (30 ppt cl). Lonp-te>t"fTl

(3-month) continuous exposure of adult spring chinook sal mon to 110'70 saturation

had no readily apparent adverse impact on the fertil ization and hatching of their

eggs.

17. KEY WORDS AND OOCUMENT ANALYSIS

a. DESCRIPTORS b.IDENTIFIERS/OPEN ENDED TERMS C. COSATI Field/Group

----- temperature, animal physiology, salmon, gas bubble dic:ease 06/c/f

trout, enVironments, eye diseases, gas 07/b

dynamics, pathophysilogy, water pollution, 13/ ilk

fresh water, supersaturation

18. DISTRIBUTION STATEMENT 19. SECURITY CLASS (This Report) 21. NO. OF PAGES

UNCLASSIFIED 65

Unlimited release 20. SECU RITY CLASS (This page) 22. PRICE

UNCLASSIFIED

EPA Form 2220·1 (9·73) 55

n u. S. GOVERNMENT PRINTING OFFICE: 1976-657-695/5424 Reg No.5-II

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