SUBLETHAL EFFECTS OF AMMONIA
AND CAB‘MIUM ON GROWTH
0F GREEN SUNFISH

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This is to certify that the
thesis entitled
SUBIETHAL EFFECTS OF AMMONIA AND
CADIVEEUM ON GROWTH OF GREEN SUNFISH

presented by

David John Jude

has been accepted towards fulfillment
of the requirements for

iPh.D 4degree in Fisheries 8c Wildlife

2/22 flaw

Major professor

1.

Date __September 5, 1913

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SUBLETHAL EFFECTS OF AMMONIA AND CADMIUM
ON GROWTH OF GREEN SUNFISH

BY

David John Jude

AN ABSTRACT OF A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Fisheries and Wildlife

1973

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ABSTRACT

SUBLETHAL EFFECTS OF AMMONIA AND CADMIUM

ON GROWTH OF GREEN SUNFISH
BY

David John Jude

Nine experiments were initiated to evaluate effects of
ammonia and cadmium on growth and food consumption of green
and pumpkinseed sunfish. RNA-DNA ratios were determined for
fish from selected experiments and the cadmium content of
the whole body, gills and liver of all fish exposed to
cadmium was also obtained. Comparative LCSO values also
were determined. In all experiments eight fish per treat-
ment were exposed to toxicant in individual cells within an

aquarium using a flow-through system. Food (Gambusia) was

 

available continuously to all fish and amounts eaten were
monitored daily.

Results of the 40-day experiment exposing green sunfish
to 6 ppm ammonia as N at three different temperatures showed
that decreased food consumption and growth of exposed fish
was directly proportional to the temperature.

Pumpkinseeds and green sunfish showed an initial
decline in feeding and growth when exposed to concentra-
tions of ammonia greater than 2 ppm at one temperature.

Fish exposed to 2 ppm grew considerably larger than controls
and exhibited the highest RNA-DNA ratio. The magnitude and

long-term detrimental effects on growth increased and were

r‘ :reater :

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David John JUde
of greater duration the higher the concentration of
ammonia. Fish exposed to higher concentrations exhibited
an acclimation phenomenon whereby feeding eventually was
re-established at a rate comparable to that of controls.

An LCSO value for green sunfish exposed to ammonia
was 33 ppm as N, while for pumpkinseeds the LC50 was 9.4
ppm as N. For green sunfish exposed to cadmium the LCSO
value was 20.5 ppm Cd.

Green sunfish exposed to 3, 7 and 15 ppm Cd exhibited
reduced food intake and growth. Sunfish exposed to lower
concentrations of cadmium (0.23-2.48 ppm) also grew at
rates lower than control fish. Growth of fish exposed to
0.05 ppm, however, exceeded that of control fish. Fish
exposed for 20 days to 1 ppm Cd at three different tempera-
tures appeared to be unaffected at cold and medium tempera—
tures when contrasted with controls. Fish exposed to 30 C
and 1 ppm Cd exhibited decreased food intake and growth as
well as highest mortality.

Short-term exposure of sunfish to high concentrations
of cadmium (S-SO ppm) and subsequent growth in uncontamin-
ated water indicated that cadmium was detrimental to growth
of exposed fish. Food conversion ratios were variable and
no consistent trends were apparent.

Whole-body and gill cadmium content on a wet-weight

basis for control fish was about 1 ppm. Whole-body

castration
was prop-o:
tration ir.
in 12 days
.3133 Cd ac:
posed to a
UFtake did

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David John JUde

measurements were the most consistent values obtained

among whole body, gill and liver, while liver usually
contained the highest concentration. Dead fish accumulated
more cadmium than corresponding live fish at the same con-
centration. Uptake by fish exposed to 3, 7 and 15 ppm Cd
was proportionally greater at 15 ppm. A threshold concen—
tration in whole-body cadmium content above which fish died
in 12 days was found at about 20 ppm. Fish exposed to 0.05
ppm Cd accumulated in 20 days as much cadmium as fish ex-
posed to almost 2 ppm. Effects of temperature on cadmium
uptake did not appear dramatic as fish exposed to 1 ppm Cd
accumulated similar levels at all temperatures. Cadmium
elimination after exposure to high concentrations for short
periods was complete by 60 days.

An STC (Stimulation Threshold Concentration) is pro-
posed as a more reliable and useful approach for replacing
LC50 data and in some cases for preliminary determination
of MATC (MaXimum Acceptable Toxicant Concentration) values.
The STC is defined as that concentration of a compound which
over a long period of time promotes growth greater than
controls. The STC value was used to calculate an applica-
tion factor for green sunfish exposed to cadmium which

agreed well with literature values.

SUBLETHAL EFFECTS OF AMMONIA AND CADMIUM
ON GROWTH OF GREEN SUNFISH

BY
David John Jude

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Fisheries and Wildlife

1973

One!

this wcr}:

is ACKNOWLEDGMENTS

One of the most difficult tasks in the completion of
this work is the accurate portrayal of the way in which the
suggestions, help and support of my friends affected the
shape and direction of my research. I owe a debt of thanks
to all these people for the time and effort expended in my
behalf which has ultimately culminated in these pages.

I first want to thank Suzann Pyzik for the sacrifices
and time spent in helping me with the capture and feeding of
fish as well as recording of the large amounts of data
connected with each experiment. She also helped with some
of the water chemistries, did key punching, edited the
thesis and typed many of the rough drafts and all of the
tables. For belief in my ideas and the many other intangibles
extended during times of stress, I can only remain indebted.

To Joseph Ervin, I want to extend my gratitude, for the
many times he solved problems I could not, for the many times
he helped me collect fish on short notice, for the many times
he assisted in recording data, for times he acted as a
listening ear, and for the times he gave me encouragement.

I want to acknowledge the academic stimulation, the
guidance and many suggestions given me by Dr. H. E. Johnson

who first started me thinking about the effects of stress on

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animals and who introduced me to the "Stress of Life". He
was instrumental along with my major professor in obtaining

a National Institute of Health Biomedical Sciences Support
grant for which I am grateful. He critically read the thesis
a number of times in its earlier versions and gave many
invaluable criticisms.

My major professor, Dr. N. R. Kevern, was always avail-
able for discussing problems and shouldered the difficult
task of reading the first and later drafts of the thesis.

I want particularly to thank him for the financial support
he provided me, both in obtaining the National Defense
Education Act Fellowship and in finding means to support my
unplanned equipment needs and computer time. For financial
assistance I also want to acknowledge support from the
Michigan State University Agricultural Experiment Station.

I want to thank the other members of my committee for
their time in trying to make this a better thesis. My appre-
ciation goes to Dr. R. E. Monroe for his understanding, to
Dr. J. R. Hoffert for his physiological acumen and to Dr.

P. O. Fromm for his critical review of my thesis and for
the wisdom of his comments.

I wish to also express my gratitude to Jake Eckenrode
for help with seining, for the pesticide analyses and for
the very helpful suggestions in the design of the flow-
through apparatus. Hugh Wright also assisted in the design

and building of that apparatus. Other people who helped me

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with the seining and sometimes recording of data were Art
Talsma, Frank Tesar, Tim Ervin, Jim Waybrandt, Robert Hite
and Johnathan Livingston Janssen. Dean Eyman and Al McIntosh
gave many valuable suggestions, helped with seining and
tutored me in the use of the atomic absorption unit. I also
received help with the spectrophotometer from Charles Annett
and Dennis Tierney.

Dr. F. D'Itri gave me the benefit of his chemical
knowledge in the design of the cadmium recovery system.
Dr. W. Conley and particularly Dr. C. E. Cress took time to
explain both the meaning and uses of the statistical tests
as well as computer programs to calculate them. To Jackie
Church for the seemingly endless sheets of data she so
cheerfully and speedily punched for me, I owe many days of
saved time. Mr. Goodsel of Lansing Vector Control provided
Gambusia and information on ponds where they could be
located. I want to thank Harold Bergman for many lively
discussions and many useful suggestions. Thanks also are due
my typist, Jean Fickes, for her speedy and efficient render-
ing of pages of scribbling and type into final form. Lastly
I wish to acknowledge the support and patience of my family,
who helped me in times of financial need, and who had faith

in things I wanted to do and who helped me accomplish them.

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TABLE OF

LIST OF TABLES. . . . .

LIST OF FIGURES . . . .

CONTENTS

 

INTRODUCTION. . . . . .
MATERIALS AND METHODS .

Bioassay Apparatus
Fi Sh O O O O O O 0

Fish Tissue Digestions

RNA/DNA Procedures

Chemicals and Chemical

RESULTS AND DISCUSSION.

Experiment 6—F .
Experiment 7-F .
Experiment lO-F.
Experiment ll-F.
Experiment 12-F.
Experiment 13-F.
Experiment lO-S.

SUMMATION DISCUSSION. .

Met
Computational and Statistical P o e

Common Responses to Stress

RIVA-DNA Data. 0 o o

Cadmium Uptake Dat .
Discussion of the Toxic Action

Ammonia and Cadmium.
Application Factors.

SWRY O C O O O O O 0

Experiment 6-F . .
Experiment 7-F . .

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Page

vii

10
ll
12
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16
37
45
57
79
96
123

142
142
147
148

155
160

164

164
164

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TABLE OF CONTENTS--C0ntinued Page

Experiment lO-F. . . . . . . . . . . . . . . 165
Experiment ll-F. . . . . . . . . . . . . . . 166
Experiment 12-F. . . . . . . . . . . . . . . 166
Experiment 13- F. . . . . . . . . . . . . . . 166
Experiment 10- S. . . . . . . . . . . . . . 167
Cadmium Uptake Data. . . . . . . . . . . . . 168

LITERATURE CITED. . . . . . . . . . . . . . . . . 170

APPENDIX. C O O O O O O O O O O O O O O O O O O O 177

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TABLE

1A.

18.

LIST OF TABLES

Chemical characteristics of water used in
the continuous-flow experiment 6-F. (N is
the number of samples used in determinations;
X is the mean with one standard error
enclosed in parentheses; C = Cold, M =
Medium, H = Hot, S = Stressor; t = less

than 0.01 ppm). . . . . . . . . . . . . . .

Chemical characteristics of water used in
the continuous-flow experiment 6—F. (N

is the number of samples used in determina-
tions; X is the mean with one standard
error enclosed in parentheses; C = Cold,

M = Medium, H = Hot, S = Stressor; t =

less than 0.01 ppm) . . . . . . . . . . . .

Chemical characteristics of water used in
the continuous-flow experiment 6-F. (N is
the number of samples used in determinations;
X is the mean with one standard error
enclosed in parentheses; C = Cold, M =
Medium, H = Hot, S = Stressor; t = less

than 0.01 ppm). . . . . . . . . . . . . . .

The analysis of variance table for the
effects of threestEmperatures and two
levels of ammonia (stress) over four
periods on the percent weight gain of
green sunfish. A log (percent weight

gain + 20) transformation was used. . . . .

Time and weight response of green sunfish
that died after continuous exposure to
three different temperatures and ammonia
concentrations. . . . . . . . . . . . . . .

The analysis of variance table for the
effects of three temperatures and two
levels of ammonia stress over four periods
on the consumption of Gambusia by green
sunfish . . . . . . . . . . . . . . . . . .

 

vii

Page

17

18

19

23

25

29

LIST OF TA

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LIST OF TABLES--Continued

TABLE
5.

10.

ll.

12.

The analysis of variance table for the
effects of three temperatures and two
levels of ammonia (stress) over four
periods on the food conversion ratios

of green sunfish. . . . . . . . . . . . . .

The analysis of variance table for the
effects of three temperatures and two
levels of ammonia (stress) over four
periods on the RNA-DNA ratios of green
sunfish . . . . . . . . . . . . . . . . . .

Chemical characteristics of water used

in the continuous—flow experiment 7—F.

(N is the number_of samples used in
determinations; X is the mean with one
standard error enclosed in parentheses;

t = less than 0.01 ppm) . . . . . . . . . .

Mean weight gain and food conversion

ratio of fish exposed to ammonia.

(Standard error is given in paren—

theses following the mean). . . . . . . . .

Mean concentrations of RNA, DNA and the RNA-
DNA ratio of pumpkinseed sunfish before

and after 30 days exposure to ammonia.

(DFFT is dry fat-free tissue from the

dorsal muscle excluding skin. Standard
error is enclosed in parentheses) . . . . .

Chemical characteristics of water used in
the continuous-flow experiment lO-F.

(N is the number of samples used in deter-
minations; X is the mean with one standard
error enclosed in parentheses). . . . . . .

The analysis of variance table for the

effects of seven concentrations of ammonia
(stress) over six periods on the amount of
Gambusia consumed by green sunfish. . . . .

The analysis of variance table for the
effects of seven concentrations of

ammonia (stress) over six periods on

the growth of green sunfish . . . . . . . .

viii

Page

32

36

38

43

44

46

50

54

LIST OF TA

TABLE

l3.

17.

13A.

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LIST OF TABLES--Continued

TABLE

13.

14.

15.

16.

17.

18A.

18B.

18C.

19.

Death and weight response of fish exposed
to various concentrations of ammonia at
15.8 C. O O O O O O O O O O O O O O C O O O

The analysis of variance table for the
effects of seven concentrations of ammonia
(stress) over six periods on the food
conversion ratios of green sunfish. . . . .

Chemical characteristics of water used in
the continuous—flow experiment ll-F. (N

is the number of samples used in determina-
tions; X is the mean with one standard
error enclosed in parentheses; N.D. means
not—detectable, less than 0.01 ppm) . . . .

The analysis of variance table for the
effects of four concentrations of cadmium
(stress) over three periods on the amount
of Gambusia consumed by green sunfish . . .

The analysis of variance table for the
effects of four concentrations of cadmium
(stress) over three periods on the food
conversion ratios of green sunfish. . . . .

Whole-body cadmium concentration on a wet-
weight basis of green sunfish from experi-
ment ll-F. (N is the number of fish used
in analysis; one standard error is given
in parentheses after the mean). . . . . . .

Cadmium concentration on a wet-weight
basis in gills of green sunfish from
experiment ll-F. (N is the number of

fish used in analysis; one standard error
is given in parentheses after the mean) . .

Cadmium concentration on a wet-weight
basis in the liver of green sunfish from
experiment ll-F. (N is the number of

fish used in analysis; one standard error
is given in parentheses after the mean) . .

Chemical characteristics of water used in
the continuous-flow experiment 12-F. (N
is number of samples used in determina-
tions; Y'is the mean with one standard
error enclosed in parentheses; N.D. means
not-detectable, less than 0.01 ppm) . . . .

ix

Page

56

6O

62

66

74

76

78

80

v
a

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LIST

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30.

LIST OF TABLES--Continued

TABLE

25B.

25C.

27.

28.

29.

30.

Chemical characteristics of water used

in the continuous—flow experiment 13-F.

(N is the number of samples used in deter—
minations; X is the mean with one standard
error enclosed in parentheses; C = Cold;

M = Medium; H = Hot; S = Stressor; N.D. =

Not Detectable, less than 0.01 ppm). . . .

Chemical characteristics of water used

in the continuous-flow experiment 13-F.

(N is the number of samples used in deter-
minations; X is the mean with one standard
error enclosed in parentheses; C = Cold,

M = Medium; H = Hot; S = Stressor; N.D. =

Not Detectable, less than 0.01 ppm). . . .

The analysis of variance table for the
effects of three temperatures and two
levels of cadmium stress over four periods
on the consumption of Gambusia by green
sunfish. . . . . . . . . . . . . . . . . .

The analysis of variance table for the
effects of three temperatures and two
levels of cadmium stress on the food
conversion ratios of green sunfish . . . .

Mean concentrations of RNA, DNA and the
RNA-DNA ratios of green sunfish from
experiment 13-F before and during con-
tinuous exposure to 1 ppm cadmium at
three different temperatures for 20 days.
DFFT is dry, fat-free tissue from the
dorsal muscle excluding skin. Standard
error is enclosed in parenthesis . . . . .

The analysis of variance table for the
effects of three temperatures and two
levels of cadmium stress over four
periods on the RNA—DNA ratios of green
sunfish. . . . . . . . . . . . . . . . . .

The analysis of variance table for the
effects of three temperatures and two
levels of cadmium stress over four periods
on the whole-body burden of cadmium in
green sunfish. . . . . . . . . . . . . . .

xi

Page

100

101

105

112

113

114

118

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LIST OF TABLES--Continued
TABLE Page

31. The analysis of variance table for the
effects of three temperatures and two
levels of cadmium stress over four
periods on green sunfish cadmium uptake
in the gill. . . . . . . . . . . . . . . . . 122

32. Concentration of cadmium on a wet-weight
basis in the livers of fish exposed
continuously to 1 ppm Cd at three temper—
atures for 20 days. (Samples size was
four live fish unless otherwise indicated;
standard error is enclosed in paren-
theses). . . . . . . . . . . . . . . . . . . 124

33. The analysis of variance table for the
effects of three temperatures and two
levels of cadmium stress over four periods
on green sunfish cadmium uptake in the
liver. . . . . . . . . . . . . . . . . . . . 125

34. The mean and standard error of chemical
parameters, except pH, found for the water
used in experiment lO-S. (Concentrations
of cadmium given are for the COntinuous-
flow portion of the experiment; water
chemistry data are for the static
part). . . . . . . . . . . . . . . . . . . .. 126

35. Time of death and final weight of fish
exposed for short periods of time to
various concentrations of cadmium in
experiment lO-S. . . . . . . . . . . . . . . 128

36A. Final concentration (wet-weight basis)
of cadmium in pooled samples of fish 60
days after short exposures to various
continuous-flow concentrations of cadmium.
(Standard errors are enclosed in paren-
theses). Comparative data are given
in Table 36B . . . . . . . . . . . . . . . . 139

368. Some comparative data on whole—body
burdens of cadmium in various fishes from
the present study and from other sources.
(N.D. means less than 0.01 ppm). . . . . . . 140

xii

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LIST OF TABLES—-Continued

TABLE

37.

D.

E.

A summary of studies showing some values
of application factors (Mount and Stephan,
1967b) as well as corresponding MATC

(Maximum Acceptable Toxicant Concentration)

and the 96-h]: LCSO VBlUES. o o o a o o o 0

APPENDIX

A summary of water quality characteristics

of filtered tap water from the Limnological

Research Building. Samples were collected
on June 2, 1970 and June 1, 1971 . . .....

A summary of pertinent information on the
method and location of capture and health
of sunfish collected for bioassay purposes

Pesticide residue analysis for five green
sunfish collected for bioassay purposes
from a Williamston pond. Fish from this
collection were used in experiment 6-F . .

Gambusia pond locations. . . . . . . . . .

Mean concentrations of RNA, DNA and the
RNA-DNA ratios of green sunfish from
experiment 6-F before and during contin—
uous exposure to various concentrations

of ammonia at three different temperatures
for 40 days. DFFT is dry fat-free tissue
from the dorsal muscle excluding skin.
Standard error is enclosed in parentheses.

A summary of pertinent data for the LC50
determination on pumkinseeds (4.46 i

0.31 g) in experiment 8-F. Sample size
for ammonia was four, and for all other
determinations was one. (Standard error
is enclosed in parentheses; t 2 less than
0.01 ppm). . . . . . . . . . . . . . . . .

A summary of pertinent data for the LCSO
determination on green sunfish (8.39 i
1.37 g) in experiment 9-F. Sample size
for ammonia was seven, for dissolved
oxygen and pH two, and for alkalinity and
hardness one. Standard error is enclosed
in parentheses; range is given for pH.

(t = less than 0.01 ppm) . . . . . . . . .

xiii

Page

162

178

179

183
184

185

186

187

Ei.

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6 SC]; SCICl SC1C1 SC2C1
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LIST OF TABLES-~Continued

TABLE

H.

Standard errors for the mean weight
changes of green sunfish exposed to
various concentrations of ammonia in
experiment lO-F. . . . . . . . . . . . .

Standard errors for the mean weight
changes of green sunfish exposed to
various concentrations of cadmium in
experiment ll-F. (N.D. means less than
0.01 ppm Cd) . . . . . . . . . . . . . .

Standard errors for the mean weight
changes of green sunfish exposed to
various concentrations of cadmium in
experiment 12-F. (N.D. means less than
0.01 ppm Cd) . . . . . . . . . . . . . .

Standard errors for the mean weight
changes of green sunfish exposed for

15 min to various concentrations of
cadmium in experiment lO-S. (N.D. means
less than 0.01 ppm). . . . . . . . . . .

Standard errors for the mean weight
changes of green sunfish exposed for

1 hr to various concentrations of
cadmium in experiment lO-S. (N.D. means
less than 0.01 ppm). . . . . . . . . . .

Standard errors for the mean weight
changes of green sunfish exposed for

24 hrs to various concentrations of
cadmium in experiment lO-S. (N.D. means
less than 0.01 ppm). . . . . . . . . . .

xiv

Page

188

189

190

191

192

193

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FIGURE

1.

LIST OF FIGURES
Page

Schematic diagram showing the main

features of the bioassay apparatus used

to deliver continuous-flow concentrations

of toxicants to twelve large aquaria

containing individual cells for eight

fish per aquarium . . . . . . . . . . . . . 7

The ratio of the initial weight of

green sunfish to their final weight

under three different temperatures and
concentrations of ammonia. Each point
represents the mean of four observations

except as noted by a number in parentheses.

One standard error is given as a dark

vertical bar on only one side of the

point for clarity . . . . . . . . . . . . . 22

Rate of consumption of Gambusia in mg

per hr for green sunfish exposed contin—

uously to three different temperatures

and concentrations of ammonia. Each

point represents from sixteen to four

fish depending on deaths. Mean standard

error is given for each concentration . . . 28

Food conversion ratios of fish exposed
continuously to three different tempera-

tures and concentrations of ammonia. Each
point represents the mean of four fish

except where otherwise noted by a number

in parentheses. One standard error is

shown by a vertical bar . . . . . . . . . . 31

Average RNA-DNA ratio of green sunfish

exposed continuously to three different
temperatures and concentrations of

ammonia. Each point represents the

mean of four fish with one standard

error given as a dark vertical bar on

only one side of the point for clarity. . . 35

 

E.
c.

a r"
[Ti‘I
bfiuyt

 

C

RhLLttrCinVC

.c ‘\ 1“ .II I‘

10,

11.

2 LIST OF

FIGURE

6.

8.

10.

11.

FIGURES--Continued
Page

Rate of consumption of Gambusia in mg per

hr by pumpkinseeds exposed continuously to

seven concentrations of ammonia. Each

point represents the mean of eight fish.

Mean standard error is given for each
concentration . . . . . . . ... . . . . . . 41

 

Rate of consumption of Gambusia in mg per

hr by green sunfish before and after con—
tinuous exposure to different concentra-

tions of ammonia. Each point represents

the mean of eight fish unless deaths

reduced that number. Mean standard error

for each concentration is given as a dark
vertical bar. Period of stress is indi-

cated by the stippled area. . . . . . . . . 49

Mean weight of green sunfish before,

during (stippled area) and after contin-

uous exposure to ammonia. Each point

represents the mean of eight fish except

where deaths decreased this number. See
Appendix Table H for standard errors. . . . 53

Food conversion ratio is shown before

(day 0-4), during (day 4-28, stippled

area) and after (day 28-36) continuous

exposure to seven different ammonia con-
centrations. Each point represents the

mean of eight fish, except where deaths
decreased this number. Mean standard

error is shown with dark vertical bars. . . 59

Rate of consumption of Gambusia in mg per

hr by green sunfish 96 hrs before and

during 384 hrs of continuous exposure to

seven different concentrations of cadmium.

Each point represents eight fish except

where deaths reduced this number. Mean

standard error is given for each concen-

tration as a dark vertical bar. . . . . . . 65

 

Mean weight of green sunfish 4 days before

and during 16 days of continuous exposure

to seven different concentrations of

cadmium. Each point represents the mean

of eight fish except where deaths

decreased that number. See Appendix

Table I for standard errors . . . . . . . . 68

xvi

 

 

iT‘CFF

«.4
«H.

-vnvv

0-2:—

LIST OF FIGURES—-Continued

FIGURE

12.

13.

14.

15.

16.

Food conversion ratios of fish exposed
for 16 days (after a 4-day acclimation
period) to various high concentrations
of cadmium. Each point represents the
mean of eight fish except where deaths
reduced that number. Mean standard
error for each concentration is shown

by a dark vertical bar. . . . . . . . . .

Rate of consumption of Gambusia in mg per
hr 96 hrs before and during 480 hrs of
continuous exposure to seven different
concentrations of cadmium. Each point
represents eight fish except where

deaths reduced this number. Mean
standard error is given for each
concentration . . . . . . . . . . . . . .

 

Mean weight of green sunfish 4 days be-
fore and during 20 days of continuous
exposure to various concentrations of
cadmium. Each point represents the

mean of eight fish except where deaths
decreased this number. See Appendix
Table J for standard errors . . . . . . .

Food conversion ratios of green sunfish
before and during continous exposure to
different concentrations of cadmium.

Each point represents the mean of eight
fish except where deaths reduced this
number. Mean standard error for each
concentration is shown by a dark
vertical bar. . . . . . . . . . . . . . .

Rate of consumption of Gambusia in mg per
hr for green sunfish 96 hrs before and
during 480 hrs of continuous exposure to
1 ppm Cd at three different temperatures.
Each point represents eight fish except
where deaths reduced this number. Mean
standard error is given for each temper-

 

ature and cadmium concentration . ... . .,

xvii

Page

71

C
(.0

86

90

104

18.

19.

21.

 

LIST OF FIGURES--Continued

FIGURE

17.

18.

19.

20.

21.

Page

Mean weight of green sunfish (the
points) and the regression line of the
mean weight against time for three
different temperatures and two levels
of cadmium stress. The number of fish
comprising each mean is shown in paren-
theses. One standard error is shown
with a dark vertical bar on only one
side of the point for clarity;

standard errors less than 0.5 are

not shown . . . . . . . . . . . . . . . . . 108

Food conversion ratios of fish exposed

(after a 4-day acclimation period) for

20 days (stippled area) to 1 ppm Cd at

three different temperatures. Number

of fish used is given in parentheses.

One standard error is shown with a

dark vertical bar on only one side

of the point for clarity. . . . . . . . . . 111

Whole-body concentration of cadmium on

a wet-weight basis in surviVing green

sunfish before and during continuous

exposure to 1 ppm Cd at three different
temperatures. Each point represents

four fish except as otherwise noted by

the number in parentheses. One standard

error is given by a dark vertical bar on

only one side of the point for clarity. . . 117

Cadmium uptake on a wet—weight basis by

the gills of surviving green sunfish

before and during continuous exposure

to 1 ppm Cd at three different tempera-

tures. Each point represents four fish

except as otherwise noted by the number

in parentheses. One standard error is

given by a dark vertical bar on only one

side of the point for clarity . . . . . . . 121

Mean weight of green sunfish before (3

days) and 60 days after 15 min, 1 hr

and 24 hrs of continuous exposure to

various concentrations of cadmium. Each

point represents the mean of 10 fish

unless deaths decreased this number.

Standard errors are found in Appendix

Tables K, L, M. . . . . . . . . . . . . . . 130

xviii

.44
5/5

23.

LIST OF FIGURES--Continued

FIGURE

22.

23.

24.

Page

Food conversion ratio of green sunfish

over 4 or 8-day intervals. Fish were

exposed for 15 min to various concentra-

tions of cadmium. Each point represents

the mean of 10 fish except where deaths

reduced this number . . . . . . . . . . . . 133

Food conversion ratio of green sunfish

'over 4 or 8-day intervals. Fish were

exposed for 1 hr to various concentra—

tions of cadmium. Each point represents

the mean of 10 fish except where deaths

reduced this number . . . . . . . . . . . . 135

Food conversion ratio of green sunfish

over 4 or 8-day intervals. Fish were

exposed 24 hrs to various concentrations

of cadmium. Each point represents the

mean of 10 fish except where deaths

reduced this number . . . . . . . . . . . . 137

xix

Staff: 2. n;

““8 ‘4

 

 

INTRODUCTION

One of the central themes in pollution biology is
the determination of "no-effect" levels of various com-
pounds, important for setting "safe" water quality
standards for aquatic life. To answer this question,
scientists have used the traditional bioassay approach
of subjecting a number of organisms to various levels of
a substance, using death as a criterion of effect. The
TLm (medium tolerance limit) or more currently LCSO value
(median lethal concentration--that concentration of a sub-
stance just killing 50% of the test organisms) is used to
evaluate the toxicity of the substance with some safety
or application factor applied to arrive at a "safe" level.
Other techniques for detecting a "no-effect" level have
focused on cellular damage to gills (Mount and Stephan,
1967), uptake and threshold concentrations of toxicants in
the whole body, gills and blood (Eisler, 1971; Eisler,
,g§,§l., 1972; Hogan and Roelofs, 1971; Mount, 1964; Eisler
and Weinstein, 1967; Lane and Livingston, l970),organ-body
ratios (Robinson, gt_al,, 1960), reproductive success (Mc-
Kim and Benoit, 1971; Pickering and Gast, 1972), effects on

liver mitochondria (Hiltibran, 1971), on urine composition

2
(Hunn, 1969), on low mobility serum proteins (Bouck and
Ball, 1965; Fujiya, 1961), on cell strains (Rachlin and
Perlmutter, 1968), and on osmolality (Lewis and Lewis,
1971). Goodyear (1972) has suggested evaluation of a
toxicant by recording the behavior of prey fish exposed
to the toxicant and placed in a situation with predators
where any reduced ability in their avoidance response is
fatal. The concept of an animal's "scope for activity"
as outlined by Fry (1947) is also a useful principle for
evaluating the metabolic cost of a stressor on an organism.
Brown (1967) suggested that fish populations can generally
exist where the sum of the fraction of the 48-hour LC50
of all soluble poisons does not exceed 0.3-0.4. Use of
LC10 or LC90 is also prOposed (Sprague, 1969).

In my research, I was interested in "stress" and its
effect on fish, particularly as it was related to tempera-
ture--of concern because of the increasing use of thermal
nuclear power and the need it creates for disposal of waste
heat. My intent in this research was to help give a more
refined answer to the overall question of "safe" levels
and to specifically gain more information on the growth
response of sunfish to ammonia and cadmium. Thus my objec-
tives were: 1) to develOp a faster and more accurate method
for determining a "no-effect" concentration; 2) to eval-
uate cadmium uptake in the whole body, gills and liver of

fish as a valid biopsy technique; and 3) to obtain

information on rate and levels of uptake under different

temperature and cadmium regimes.

3

To accomplish these objectives nine experiments were
conducted, five involving ammonia and four involving cad~
mium as the toxicant. Of the five ammonia experiments
three involved green sunfish and two involved pumpkinseeds.
An LC50 value and growth response of pumpkinseeds and
green sunfish under ammonia stress accounted for four of
the experiments. The other ammonia experiment was a
temperature (3) by stress (2) factorial allowing evaluation
of the effect of 6 ppm ammonia on growth of green sunfish
at three different temperatures. The four experiments with
cadmium used green sunfish, all of which were analyzed for
cadmium content. These experiments established an LCSO
value, furnished information about effects of low levels
of cadmium on fish growth, and evaluated the effect of 1
ppm Cd on growth of green sunfish at three different
temperatures. One static bioassay was designed to deter-
mine post-treatment effects on green sunfish exposed con—
tinuously to a series of cadmium concentrations for
three different lengths of time.

Growth of exposed fish was used to determine if a
substance had a detrimental effect since growth is one of
the best indications of health in an animal and is easily
measured. In bioassays, I devised a method to insure
that feeding conditions would be as natural as possible
and would provide for measurement of amount of food eaten
over a given interval. This was accomplished by placing

Gambusia, a readily available and uniform source of food,

to varic
determir:

toxicant

 

4

in aquaria with predator fish (sunfish). Food consumption,
growth and food conversion efficiency of sunfish exposed

to various toxicants and temperature regimes was then
determined. "Safe" levels and chronic effects of the

toxicants were then judged on the basis of these studies.

H‘vuc—vu '

similar

providei

 

MATERIALS AND METHODS

Bioassay Apparatus

Bioassays were conducted in a flow-through system
similar to that of Chadwick and Brocksen (1969) which
provided for three different water temperatures and con-
trolled delivery of toxicants (Figure 1). One variation
utilizing only seven aquaria and no temperature control
was employed to give seven treatments at one temperature.
When needed, water was heated by stainless-steel immersion
heaters and controlled by thermoregulators placed in the
hot-water head tank. Water flowed from head tanks to mix-
ing boxes where toxicants were introduced from a Mariotte
bottle and mixed with dilution water. Flow rates to the
test tanks were controlled by varying the angles of glass
drip tubes inserted in the delivery boxes.

Test tanks were twelve 70-1 (20 gal) glass aquaria
equipped with overflow tubes, plexiglass covers, and
aerated by pumps and air stones. Each tank was partitioned
into eight chambers (about 12 x 12 cm) with fiberglass
screen to provide individual fish chambers. Two-in
sections of 2-in diameter polyvinyl chloride (PVC) pipe
were placed in each chamber to provide cover for fish.
Water used (see Appendix Table A) was East Lansing tap

water entering through iron pipes and then passing through

 

 

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8

a carbon filter which was periodically backwashed. Water
was then fed through PVC pipes to the two head tanks.
Flow rate to aquaria for ammonia and cadmium experiments
was 300 ml/min and 200 ml/min respectively, with 90%
replacement times (Sprague, 1969) of 5 and 7.2 hours.

Light was provided by overhead mercury-vapor lamps.
Photoperiod was manually controlled with approximately 8
hours of darkness and 16 hours of light daily.

The static bioassay apparatus consisted of seventeen
20-1 (10 gal) aquaria each fitted with glass covers and
aerated. Aquaria were located on both shelves of a two—
shelf structure and treatments were randomly assigned to
aquaria. Aquaria on the top shelf were consistently warmer
‘by about 1-2 C. A number of 2-in sections of l%—in diam-
eter PVC pipe as well as empty brown jars were placed in
each tank for cover. About 22 1 of carbon-filtered
tap water were used with 10 fish per tank. Recommended
fish weight to water ratios of 1 g/l were sometimes exceed-
ed (American Public Health Association, 1965). Lighting

was similar to that of the flow-through system.

Fish

Green sunfish (Lepomis cyanellus) were used in all
experiments except experiment 7-F and 8-F in which pumpkin-
seed sunfish (Lepomis gibbosus) were used (see Appendix
Table B, C). Fish were collected from the Lansing area

using seines and electroshocking. Once at the lab, fish

with _'I_"
allevi.
experi:
fish we
Tempere
0f abet
tempera
around
60 Cm)
Af
to the J
baldnCe
minimal

anesthe-

 

 

9
were held in rectangular (860 l) or circular (1,680 l)
tanks for a minimum of 2 weeks but usually much longer
before they were used in an experiment. Depending on the
experiment, stock fish were fed with either ground-up fish

or small—sized trout chow, which fish readily accepted

 

after a few days acclimation. Problems with Trichodina,
a protozoan parasite, and some "popeye" were noted. Fish
with Trichodina were eliminated and additional aeration
alleviated "popeye" problems. For the two temperature
experiments three groups of randomly selected green sun-
fish were acclimated to test temperatures for 10 days.
Temperatures were raised from a holding-tank temperature
of about 12 C (cold temperature) to around 20 C (medium
temperature) and 28 C (hot temperature) at the rate of
around 2 C per day in separate holding tanks (240 cm x
60 cm) equipped with thermoregulators.

After anesthetization weights of fish were determined
to the nearest 0.01 9 using MS-222 and a top—loading
balance. Fish were anesthetized only to the degree that
minimal movement still occurred and no ill effects due to
anesthetization were noticed. Fish were randomly assigned
to treatments from a group of fish previously selected for
size and health.

LC50 values were obtained using graphical interpola-
tion and methodology of the American Public Health Associa-
tion (1965). Criteria for death were cessation of movement

and no response after stimulation with a glass rod.

Th
sunfish

from pc

through
Each fi

dead, r«

r‘ :
.IEt {TILE

rinsed W

removed ‘

 

10
The prey species used in all experiments as food for

sunfish was the mosquito fish, Gambusia affinis, obtained
from ponds (Appendix Table D) in the Lansing area.

Gambusia were sorted according to size with average weight
obtained by weighing a number of anesthetized fish after
blotting. Gambusia were placed into each cell of the flow-
through bioassay system which contained green sunfish.

Each fish cell was usually checked daily and the number

dead, regurgitated and live Gambusia remaining was recorded.

Fish Tissue Digestions

Fish exposed to cadmium were analyzed separately to
determine rate and amount of cadmium absorbed. Fish were
rinsed with tap water and then gill arches and liver were
removed, weighed and placed into separate beakers. The
remaining carcass, sometimes minus a strip of muscle from
the upper right dorsal area (for RNA—DNA analysis), was
also placed into a beaker. A boiling stone and nitric
acid were then added. In the case of gills and liver,
10-20 ml of 4 N nitric acid was added and for the carcass,
about 5 ml of concentrated nitric acid per g of fish
was used. These beakers, covered with watch glasses, were
then placed on a hot plate and allowed to boil until almost
dry. If the fluid was reasonably clear 5 ml of 4 N nitric
acid followed by distilled water was added and the contents
then filtered through number 3 Whatman filter paper. Final
volume of fluid used for cadmium analysis for gills and liver

was 25 or 50 ml and for the carcass 50 or 100 ml was used.

Samples
5347:7185
with ea
amount

was 10c

PJLAV/fih}
\u‘

 

11
A number of these Whatman filters were analyzed for cadmium

content. No cadmium was found on seven filters when the
concentration of cadmium in the fish was less than 0.10
ppm Cd. When concentrations of cadmium in the fish were
greater then 0.10 ppm it was found that an average of
3 i 1.29 percent of the cadmium found in the fish was pre-
sent on the filter. No adjustment was made for this find-
ing. Unused filters were digested and no cadmium was ever
detected in these samples.

Twenty samples of ground fish and beef heart of
about 10 g each were "spiked" with 0.0025 mg Cd (8
samples), 0.010 mg Cd (8 samples), and 0.050 mg Cd (4
samples). A control sample was determined concurrently
with each group and background levels subtracted from the

amount found in spiked samples. The mean percent recovery

was 100.25 1 7.23.

RNAJDNA Procedures

The procedure for determining RNA-DNA ratios was that
of Bulow (1970). A description of technique and necessary
steps for analysis also appears in Haines (1969). Fish
were removed from the freezer and then a longitudinal
muscle strip along and just below the dorsal fin was
excised and the skin removed. This flesh was then defatted
using a chloroform ethanol—mixture and a 50 mg sample
weighed. To calculate mg of DNA and RNA, the ratio of dry
fat-free weight to wet weight was needed, which for flesh

samples (0.72 to 3.32 g) from ten green sunfish was

g of DFE

were ca:

 

was WEE;

were fr

4‘1“ “1":
“‘dSVQ i \

to Stan

 

12
0.1749 1 0.002. Flesh samples were then digested with

trichloracetic acid and treated after immersion in a hot-
water bath with orcinol for RNA and diphenylamine for DNA.
Final concentrations are reported as mg of RNA or DNA per
g of DFFT (dry fat-free tissue). RNA-DNA ratios also

were calculated. Final concentration of the color reaction“
was measured using a Beckman DK2A spectrophotometer. Fish
were frozen whole and stored sometimes up to 6 months

before analysis.

Chemicals and Chemical Methods

Routine water chemistry tests (hardness, alkalinity,
dissolved oxygen and nitrates) were determined according
to Standard Methods (American Public Health Association,
1965). A pH meter was used for measuring pH, and tempera-
ture was determined with a glass mercury thermometer.

Ammonia was added to bioassay containers as ammonium
chloride and analyzed using direct nesslerization (American
Public Health Association, 1965) with a Klett-Summerson
colorimeter. Concentrations of duplicate samples were
similar using direct nesslerization and distillation with
nesslerization. Concentrations of ammonia are reported
ppm as N (Nitrogen). In the text ammonia refers to all
forms of ammonia (NH3, NH4+), while un-ionized ammonia
(NH3) will be specified when NH3 is discussed. Concentra-

tions of un-ionized ammonia (NH3) were calculated using

Table 14 in Spotte (1970).

Concert.
basis.

aciditi-

 

13

Cadmium was added as cadmium chloride and measured
on a Jarrell-Ash Atomic Absorption spectrophotometer
Model 800. Samples were run at 2360 Angstroms in the
range 2 ppm to 0.02 ppm, the maximum sensitivity range.
Concentrations were recorded as ppm Cd on a wet-weight
basis. A number of 1 ppm cadmium standards of different
acidities all had similar peak heights when measured on
the spectrOphotometer. Standards were compared with
those from the Crops and Soils Science Department, Michi-
gan State University and found in good agreement.

Water samples for chemical analyses were removed from
the closest, right-most cell of the aquarium after temper-
ature, ammonia and dissolved oxygen checks from the front,
middle and back revealed complete mixing occurred in the
entire aquarium. Samples for water chemistry were
collected periodically when time for analysis was avail-
able. Samples for toxicant determinations were taken ar—
bitrarily and attempts were made to get a sample at least
once per day and sometimes more often. Ammonia samples
were untreated but refrigerated immediately (3.5C). Sam-
ples in cold storage analyzed after a period of time for
ammonia were not different from samples analyzed immediate-
ly. Cadmium water samples were treated with 3 ml nitric
acid per 1 of water. In cadmium experiments glassware
and bottles were washed between uses in dichromate—sulfuric
acid cleaning solution (American Public Health Association,

1965).

 

of freed
tor each
at least
each ca]
Mee
ratiOs ‘
iays fo:
S‘l‘fled.
int"31”Va.
nonld r

ing One

l4
Computational and Statistical Procedures

The CDC 3600 and 6500 computers were used for data
analysis. Special programs were written for some calcula-
tions, while the statistical Stat Pac from the Michigan
State Computer Library was used for analysis of variance
and linear regression. When analysis of variance was
calculated, missing data, because of dead fish, were re-
placed with the mean of that particular cell. One degree
of freedom was subtracted from error degrees of freedom
tor each time this was done. Attempts were made to verify
at least some data on cards with the original data from
each calculation.

Mean growth, food consumption and food conversion
ratios were determined over an interval of time, usually 4
jays for weight, and about 1 day for amount of food con-
sumed. If a fish died over half-way through one of these
intervals, the convention was used that its contribution
would remain for that interval, but for none of the succeed-
ing ones. If a fish died before it was half-way through an
interval, its contribution to the mean for that interval
was drOpped. In all graphs depicting amount of Gambusia
eaten and food conversion ratios, points were plotted mid-
way through the interval over which the particular parameter
was calculated.

The food conversion ratio for each fish was calculated
by dividing the net weight gain for a given time interval

by the weight of Gambusia eaten over that same interval.

The mea:
rather ‘
some of
certage:
intervai
tion of
weight
since e:
occurre.
Stl
root of
“Fan st:
all Sta;
line 0};
errors .
errOrS I
mean as

(

 

L
‘0 SavJ

in the

15

The mean for the entire group was then obtained. Ratios
rather than efficiency values were calculated to avoid
some of the statistical problems associated with per-
centages. In cases where one fish in the group in a given
interval did not eat, its ratio was excluded from calcula-
tion of the mean. Any fish which registered a negative
weight gain was given a food conversion ratio of zero,
since extreme variation in mean food conversion ratios
occurred if it was not treated as zero.

Standard error (S.E.) was calculated as the square
root of the variance (52) divided by N. In some cases, a
mean standard error was calculated which is the average of
all standard errors for each of the means shown for one
line on a graph. This method was used only when standard
errors were small and uniform across time. If standard
errors were not small and uniform but correlated with the
mean as occurred with mean weight of fish, standard
errors were shown for each mean using vertical, half bars
(to save space) or individual standard errors were placed

in the appendix.

to detei
green 5;
temperai
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RESULTS AND DISCUSSION

Experiment 6-F

Experiment 6—F was designed as a 3 x 2 x 4 factorial
to determine the RNA-DNA ratio and growth response of
green sunfish continuously exposed to three different
temperatures and concentrations of ammonia over four 10-
day periods. The experiment lasted 40 days from November
10 to December 21, 1970. Four fish from each of three
temperatures were sampled initially for RNA-DNA ratios.
Thereafter, every 10 days four fish from each of the six
treatment combinations were randomly selected from the
two replicate aquaria. Three sets of four aquaria were
maintained at mean temperatures of 13 C (cold), 22 C
(medium) and 28 C (hot) (Table 1A, 1B, 1C). It was desired
to maintain one concentration of ammonia in all aquaria
receiving toxicant for each of the three temperatures so
chronic effects of temperatures on ammonia toxicity could
be evaluated. From pilot experiments, 6 ppm ammonia as
N was selected as a nominal concentration which would not
kill any fish. However, because ammonia was oxidized to
nitrates proportionately more with increasing temperatures
(Table 1A, 13, 1C) a uniform nominal concentration of
ammonia was not maintained over the three test temperatures.

At the cold temperature 5.5 ppm ammonia as N was found,

16

Table 1A .

H

 

Treatment
E“.

m as N
Uri-ionized
PM as N

pH

Teaperatur,

DiSSOIVe d
We" (pp:

A“Kalini ty
‘3 C ac

Ear11118 38
a! Cac

 

5C3 (Ppm a

 

17

Table 1A. Chemical characteristics of water used in the
continuous flow experiment 6-F. (N_is the number
of samples used in determinations; X is the mean
with one standard error enclosed in parentheses:
C - Colds M I Medium: H - Hots S = StressorI t -

loss than 0.01 ppm).

 

Aquarium Number

 

1 11 3 5
Treatment C C CS CS
NH N 3 3 17 19
ppg as N X 0.13 0.12 5.48 5.53
(0.06) (0.04) (0.15) (0.19)
Un-ionizsd NH3 t t 0.08 0.08
ppm as N
pH N 6 6 6 7
Range 7.68” 7073- 7058- 7052-
7.82 7.89 7.79 7.80
Temperature N 30 30 30 30
X 12.8 13.4 13.1 13.3
(0.1) (0.2) (0.2) (0.2)
Dissolved N 5 5 5 5
Oxygen (ppm) X 8.1 8.2 6.4 6.6
(0.1) (0.3) (0.5) (0.4)
Alkalinity N 4 4 4 4
ppm as 0:003 X 342 346 5 342
(2) (1) (3 (3)
Hardness N_ B E 2
ppm as CaCO3 X 3 6 3 7 3 6 9
(5) (7) (9) (8)
N03 (ppm as N) ,N - - l l
X 0.8 1.0

 

Table 17

H

 

Treatme
M

ppm as

L731-i0r1i
From as

DH

Temper;
'«

:138011
~Xygen

Alkali:
PM as

18

Table 18. Chemical characteristics of water used in the
continuous flow experiment 6-F. (N_is the number
of samples used in determinations; X is the mean
with one standard error enclosed in parentheses;

 

 

 

C I Colds M = Medium: H I Hot; S = Stressor; t =
less than 0.01 ppm).
Aquarium Number
6 7 2 10
Treatment M M MS MS
NH“ N 2 3 18 19
ppm as N X 0.15 0.12 4.04 4.20
(0.05) (0.04) (0.20) (0.17)
Un-ionized NH3 t t 0.16 0.16
ppm as N
pH N 6 6 6 6
Range 7095- 7075- 7062- 7059-
8.06 8.00 8.12 7.95
Temperature 3 30 30 30 30
(C) X 20.8 21.5 20.0 21.3
(0.2) (0.2) (0.2) (0.2)
Dissolved N 5 5 6 5
Oxygen (ppm) X 7.3 7.1 5.2 5.3
(0.2) (0.3) (0.6) (0.7)
Alkalinity N 4 a 4 3
ppm as CaCO3 X 345 3 9 333 326
(1) (4) (3) (1)
Hardness N 3 B 3 3
ppm as CaCO3 X 352 3 3 347 2 6
(u) (10 (5) (5)
N03 (ppm as N) N - - 1 l
X 1.0 1.6

 

Table 1C .

 

 

 

Treament

rain
ppm as N

(n-ionize
m as N

pH

Temperatu
(C)

318301Ved|

Alkalinit
PM as C 8

 

HardREBB

N03 (Ppm

 

19

Table 10. Chemical characteristics of water used in the
continuous flow experiment 6-F. (N_is the number
of samples used in determinations: X is the mean
with one standard error enclosed in parentheses:
C I Cold; M I Medium: H I Hot: S = Stressors t I
less than 0.01 ppm).

 

 

Aquarium Number

 

4 9 8 12
Treatment H H HS HS
NH N , 2 2 18 19
pp3 as N X 0.08 0.09 1.49 1.69
(0.08) (0.01) (0.13) (0.16)
Un-ionized NH3 0.01 0.01 0.11 0.12
ppm as N
pH N 7 6 7 6
Range 7.80- 8.05- 7.56- 7.63-
8.22 8.20 8.20 8.25
Temperature N 30 30 30 30
(C) X 28.0 28.7 27.9 27.5
(0.2) (0.2) (0. ) (0.2)
gissolved ) N 65 65 “5 “6
xygen ppm X l .5 .7 .
(0.4) (0.3) (o. ) (0.6)
Alkalinity E 4 3 3 3
ppm as CaCO3 X 347 350 314 307
(4) (3) (4) (3)
Hardness N’ 3
ppm as Ca.CO3 X 352 334 337 338
(6) (6) (5) (6)
N0 (ppm as N) ‘3 - - 1 l
3 x 3.0 4.0

 

while 4
hot ten

was res

each te
control
atures ,
ture, t
oxygen

T‘

,—

in weifi
Collecq

weeks

{1'

would i

 

20

while 4 and 1.5 ppm ammonia as N were found at medium and
hot temperatures respectively. In addition, temperature
was responsible for the reduction of dissolved oxygen in
the controls from 8 ppm at cold temperature to 7 and 6
ppm for medium and hot temperatures respectively, while
dissolved oxygen in all aquaria receiving toxicant at
each temperature was about 2 ppm less than corresponding
controls, 6, 5, and 4 ppm for cold, medium and hot temper-
atures, respectively. Effects of ammonia across tempera-
ture, therefore, may be partially confounded by dissolved
oxygen concentration.

The 108 green sunfish used in this experiment ranged
in weight from 3.97 to 15.79 grams (see Appendix Table E,
Collection 4, 5 and 6). Fish were not fed during the three
weeks before the start of the experiment so RNAkDNA ratios
would be at a minimum.

Growth of green sunfish, shown as percent increase
over the initial weight (because of the large variability
in size of fish used in the experiment), was depressed at
cold temperatures (13.2 C) and almost linear at medium
temperatures (20.9 C) (Figure 2). At high temperatures
(28 C) the greatest increase in growth occurred with a
gradual tapering-off after day 20 to values comparable
to those exhibited by fish exposed at medium temperatures.
The analysis of variance (Table 2) showed that all main
effects, temperature (3 levels--13, 20, 28 C), stress

(2 levels-—nomina1 concentrations of 0 and 6 ppm ammonia

Figure 2.

21

The ratio of the initial weight of green
sunfish to final weight under three
different'temperatures and concentrations
of ammonia. Each point represents the
mean of four observations except as noted
by a number in parentheses. One standard
error is given as a dark vertical bar on
only one side of the point for clarity.

 

 

maxph
th
.1)
(F3
G__
X——4
1'21
3)
)
1
8
d
a
6
'3
E
/
(LL//
r:
C

T

22

 

 

 

 

 

 

 

 

 

 

 

4" EXPERIMENT 6-F 1
PERCENT GPOWTH 0F GREEN SUNFISH
UNDER AMMONIA STRESS
T (3)).
G———D (3.20. CONTROL
.A——A ”.20 5.50 PPM N / .
o——o 20.9c CONTROL // !
x———x 20.90 4.:2 PPM N /
v___q ze.oc coNTRoL /
...—.... ze.oc Leo PPM N 77/
3* ~
/
/
/
/ .1
p ...“.
/ , (3)/1‘
/ /
8 1' / /
H /
"2 - /
It)
2 A/ l
g /
a / /
E— ’ /
z‘ / / (3)
m /
/ , /
o. / /
. / /
.. / /
I/ / T
o J 1 L I
o-uo 20-30 50-40

l0-20
INTERVAL IN DAY.

Table 2 0

Source

 

Temperatu
Stress

T x S
Period

T x P

S x P

T X S x It
Error

Total

 

23

Table 2. The analysis of variance table for the effects of
three temperatures and two levels of ammonia
(stress) over four periods on the percent weight

gain of green sunfish.
+ 20) transformation was used.

A log (percent weight gain

 

 

 

Sum of Degrees of Mean

Source Squares Freedom Square F Statistic
Temperature 4.95 2 2.48 54.23**
Stress 0.84 1 0.84 l8.38**
T x S 0.22 2 0.11 2.43
Period 3.53 3 1.18 25.80**
T x P 0.29 6 0.05 1.07
S x P 0.13 3 0.04 0.97
T x S x P 0.29 6 0.05 1.04
Error 3.29 72 0.05
Total 13.55 95

 

330.01 significance level.

underl;
these :
an ave:
across
fish.

expose'
at the
at the
Growth

to or T

 

 

 

24
as N), and period (four periods of 10 days each) were

significant. Since standard errors were postively corre-
lated with mean percent increase, a log (percent increase
plus 20) transformation was used to better satisfy the
underlying assumption of homogeneity of variances. Thus
these data indicate among other things, that ammonia had
an average, additive detrimental effect on exposed fish
across temperatures and time when compared with control
fish. In all cases except for hot temperature, fish
exposed to ammonia exhibited growth less than controls

at the same temperature. Some evidence of acclimation

at the highest temperatures existed, since after day 20,
growth increase of ammonia-exposed fish was at least equal
to or higher than that of controls.

Fish exposed to ammonia were more excitable than
control fish, and those that died followed a consistent
behavior pattern. Usually they stopped feeding, became
listless and would, in cases where the aerator stream of
bubbles was in their cell, maintain themselves in that
stream. Prey Gambusia harassed moribund sunfish by nipping
and biting at fin rays. In an effort to escape their
cell, some ammonia-exposed sunfish (and a few controls)
experienced very severe abrasions of their lower mandibles.
The extent to which this affected their resistance to
ammonia is unknown. Many fish that died in stressor tanks,
however, possessed split lower mandibles. No control fish

died, while six in the stressor tanks succumbed (Table 3).

H

77
89
21

13

25

Table 3. Time and weight response of green sunfish that died
after continuous exposure to three different temper-
atures and ammonia concentrations.

 

 

 

Time of Initial Final
Fish Death Type of Weight Weight
No. (hrs) Stress (g) (g)
80 249 medium stressor 9.63 8.98
60 364 hot stressor 4.82 4.90
77 393 medium stressor 7.15 7.70
89 460 hot stressor 11.21 9.94
21 496 cold stressor 7.72 7.63

13 678 medium stressor 8.69 11.29

 

26

One fish died at cold temperature, three at medium tempera—
ture and two died at the highest temperature.

The depressing effect of ammonia on food consumption
under different temperatures was also shown by computing
the mg of Gambusia eaten on an hourly basis (Figure 3).
The depressing effect of ammonia on food consumption was
minimal at cold temperature, but larger differences were
found at higher temperature. These trends were highly
significant, as borne out by the analysis of variance
(Table 4) which showed that all main effects, temperature,
stress and period were significant. Amount of food con-
sumed was directly proportional to temperature. The
effect of stress in depressing food consumption was great-
est at highest temperature. At the low temperature a small
but consistently lower amount of food was eaten by exposed
fish when compared with controls. The significant effect
of time is related to growing fish consuming more and more
food the larger they become.

Food conversion ratios, net weight gain divided by
weight of food eaten, offered no consistent trends (Figure
4). In all cases except one, however, ratios for stressed
fish were lower than values found for control fish. The
analysis of variance showed that ammonia stress did not
have a significant effect on food conversion ratios of all
stressed fish when compared with all control fish over
temperature and time (Table 5). However, there was a

significant effect of temperature when averaged over time

Figure 3.

27

Rate of consumption of Gambusia in mg per
hr for green sunfish exposed continuously
to three different temperatures and con-
centrations of ammonia. Each point repre-
sents from sixteen to four fish depending
on deaths. Mean standard error is given
for each concentration.

W0

50

m0

MG W EATEN/HOUR

jvrfi
‘

DO

V fir fi—ij—‘

A;

 

v—v—vfivffv
._
l.-

I

+7—v V'fi V ' V

firi 7'17

. ,2»

if

i

v

 

IOO .

50.

EWINENT 6-F

l3.2 C

+——+ CONTROL -

A 5.50 PPM NN4 As N

9 ONE FISH DEATH 8.6. 1' £
- +

 

A

 

 

IOO .

M6 W EATEN/HOUR

 

 

 

 

 

 

0L 28 '0 c ' 8 ' T+- ' + co'NTROL' ' ' '
IOO ' 4 A LOO RPN NM A8 N
8.6. +1
~ Ill
w. ‘
O , . , . .
0 200 400 000 soo nooo

Table

H

501

Tempe}

Stres

29

Table 4. The analysis of variance table for the effects of
three temperatures and two levels of ammonia
(stress) over four periods on the consumption of
Gambusia by green sunfish.

 

Sum of Degrees of Mean

Source Squares Freedom Square F Statistic
Temperature 1793.99 2 896.99 65.6l**
Stress 180.62 1 180.62 13.18“
T x S 83.11 2 41.56 3.04
Period 190.26 3 63.42 4.63**
T x P 27.06 6 4.51 0.33
S x P 49.35 3 16.45 1.20
T x S x P 87.30 6 14.55 1.06
Error 931.06 a68 13.69
Total 3342.77 95

 

270.01 significance level.
4 degrees of freedom were subtracted for dead fish.

 

Figure 4.

30

Food conversion ratios of fish exposed
continuously to three different temper—
atures and concentrations of ammonia.
Each point represents the mean of four
fish except where otherwise noted by a
number in parentheses. One standard
error is shown by a vertical bar.

FOOD CONVERSION RATIO X 0.0l

rev—H

40[

30’

'T‘*

10

'7‘"

20

10

A)

30>

20

("7

10

v
_

 

 

o
o
I

 

FOOD CONVERSION RATIO X 0.0I

40

30

30

20

10

3'1

132 C EXPERIMENT 6-_F_
NET NT. GAIN/W EATEN

4—1- CONTROL
A- —A 5.50 ppm N

 

 

 

 

1 1 1 L
20.9 C
_ +——+ (DNTROL
9" ‘4 M12 PPM N J_/‘r
INK, , .3.
F \ //
/
/
/
r- /
/
/
l J l J
28.0 C +——+ CONTROL

A——A 1.59 ppm N

 

 

 

T /
/
20 w( 3_,__ — -—. _— _— N
//
l 1 I L
0-10 10-20 20-30 30-40

Ingpvu IN DAYS

Table 50

 

Sourcq

Temperab
Stress

T x 8
Period
T x P

S x P

T x S x
Error
Total

N
a’o-Ol s

degm'
fish,

 

 

32

 

 

 

Table 5. The analysis of variance table for the effects of
three temperatures and two levels of ammonia
(stress) over four periods on the food conversion
ratios of green sunfish.
Sum of Degrees of Mean
Source Squares Freedom Square F Statistic
Temperature 0.065988 2 0.032994 5.30**
Stress 0.023937 1 0.023937 3.84
T x S 0.024409 2 0.012204 1.96
Period 0.090232 3 0.030077 4.83**
T x P 0.064991 6 0.010832 1.74
S x P 0.037245 3 0.012415 1.99
T x S x P 0.063560 6 0.010593 1.70
Error 0.411670 a66 0.006231
Total 0.782032 95

 

:*0.01 significance level.

6 degrees of freedom were subtracted for dead and non-feeding

fish.

 

51

,MJ

)1

(I)

{1)

r1

33

and stress. The respective means were 0.34, 0.38 and
0.40 for fish at cold, medium and hot temperatures
respectively, indicating that fish became progressively
more efficient in their food conversion with increasing
temperature. In addition, there was an unexpected
significant effect of time on food conversion ratios as
fish became progressively more efficient in their utiliza-
tion of Gambusia. The means for the first through fourth
lO-day period were 0.35, 0.36, 0.39 and 0.43.

RNA-DNA ratios of fish at all temperatures started
low (around 10) then increased to levels between 20 and 30
for the remaining 40 days (Figure 5, Appendix Table E).
The analysis of variance revealed that temperature, stress
and period were all significant (Table 6). The means
pooled over temperature indicated that fish at the cold
temperature had a higher mean RNA-DNA ratio (27.21) than at
either medium temperature (19.14) or hot temperature (23.14).
The effect of ammonia stress considered across time and
temperature showed a depressing effect on RNA-DNA ratios
when the control value (25.62) was contrasted with that of
ammonia-exposed fish (20.71). This reduction in RNA-DNA
ratios indicates that, by some mechanism, RNA synthesis by
stressed fish was depressed. Considering mean RNA-DNA ratios
at each temperature over time indicated the difference be—
tween stressed and control fish was greatest for fish at cold
temperatures (around 8), small for fish at medium temperatures

(1.6) and somewhat larger for fish at high temperatures.

Figure 5.

34

Average RNA-DNA ratio of green sunfish
exposed continuously to three different
temperatures and concentrations of ammonia.
Each point represents the mean of four fish
with one standard error given as a dark
vertical bar on only one side of the point
for clarity.

w

M

N

30‘

 

10

RNA/DNA RATIO

40

30

20’

10

'0
O

. n
o

35

13.2 C

// —__—
zr~-~ /
// 1’

/
/

b

_,___,. comm. EXPERIMENT 6-F
A— -a 5.50 PPM N

 

 

 

1 1 J
20.9 C +—+ CONTROL
AF- —A 4.12 ppm N
. / \
// \\
 , / ""1—
1 1 1 1

 

 

 

10 ~0——+ CONTROL
A- —A 1-59 PPM N
am 1 1 1 L-
0 10 20 30 40

DAYS

Table 6. Th

’01
(s
of
Source

Temperature

Stress

S x '1‘

Period

T x P

S x P

T X 3 x P

Error

Total

W

36

Table 6. The analysis of variance table for the effects of
three temperatures and two levels of ammonia
(stress) over four periods on the RNA-DNA ratios

of green sunfish.

 

 

 

Sum of Degrees of Mean

Source Squares Freedom Square F Statistic
Temperature 1040.97 2 520.48 9.l9**
Stress 577.02 1 577.02 10.19”
S x T 169.98 2 84.99 1.50
Period 949.26 3 316.42 5.59“”
T x P 419.32 6 69.89 1.23
S x P 67.98 3 22.66 0.39
T x S x P 311.03 6 51.84 0.92
Error 4078.78 72 56.65
Total 7614.34 95

 

730.01 significance level.

(around 5).
ETTA-DNA ra'
that ratio;
declined c:

leveled of

Exoerinert

Pumpk
was a 30-c‘
11,1971. I
of ammonia
thatresul:
green SUnf
"Pumpkinsel
distributel
more food
(Appendix
from 4.13
temperatu:
tratiOns
a flow rat
a 6—day so
day wither
with fOOd

Sixty
were Saar:

initial Pd

and end 0:

 

37

(around 5). A consideration of the effect of time on the
RNA-DNA ratios across temperatures and stress revealed
that ratios were highest after the first 10 days (27),
declined considerably in the second 10 days (18), then

leveled off for the remaining periods at around 23.

Experiment 7-F

Pumpkinseed sunfish were used in experiment 7-F which
was a 30-day experiment conducted from March 11 to April
11, 1971. The experiment was done to evaluate the effect
of ammonia on growth of a different species of sunfish so
thatrresults could be compared with those obtained using
green sunfish. unfortunately, as learned later, 13 of the
"pumpkinseeds" were green x pumpkinseed hybrids, randomly
Idistributed among tanks. Data showed these hybrids consumed
more food than pumpkinseeds. Fish were seined from a pond
(Appendix Table B, Collection 4, 5, 6) and ranged in weight
from 4.13 to 9.22 9. Fish were exposed for 24 days to a
temperature of 10.1 C (Table 7) and seven nominal concen-
trations of ammonia (0, 2, 6, 8, 10, 12, 14 ppm as N) at
a flow rate of 300 ml/min per tank. Pumpkinseeds were given
a 6-day acclimation period in the seven tanks, the first
day without Gambusia present, and from the second day on
with food present.

Sixty—four fish were used in this experiment; eight
were sacrificed on the first day of the experiment for

initial RNA-DNA ratios. Fish were weighed at the beginning

and end of the experiment. Thus initial growth response

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Similar tr

ment, but I

39

and any acclimation trends may be masked by not having
measured weights during the experiment. The standard 96-
hr LCSO determination was also performed on a group of
these fish (Experiment 8-F). This was done so that: 1)
the prior standard method results could be compared with
results from my chronic tests and 2) so that application
factors could be calculated. This 96-hr LC50 value was
found for pumpkinseeds (no hybrids) to be 9.4 ppm as N
(Appendix Table F).

Patterns of food consumption (Figure 6) indicated that
all concentrations of ammonia above 5.72 ppm caused fish
to stop feeding when the toxicant was introduced. Some
regurgitation of previously eaten food also occurred. The
higher the concentration of ammonia the less the amount of
food that was eaten and the longer the time before increased
consumption occurred. A one-way analysis of variance showed
there was a significant difference among treatments in the
amount of Gambusia eaten (calculated F = 2.45, tabular F,
0.05 level (6, 56) : 2.34). However, Dunnett's 2-tailed
test (Steel and Torrie, 1960) to compare the control with
all treatments failed to show any differences. This is
undoubtedly related to the great variability in the growth
and feeding response of sunfish among treatments which
decreased the precision of these measurements. Food con-
sumption by fish exposed to 2.13 ppm ammonia as N was
similar to that of controls in the early part of the experi-

ment, but throughout the remainder of the experiment amounts

Figure 6.

40

Rate of consumption of Gambusia in mg per
hr by pumpkinseeds exposed continuously to
seven concentrations of ammonia. Each
point represents the mean of eight fish.
Mean standard error is given for each
concentration.

30

«HOUR
o

O

20

 

”3 W EATEN
O

 

 

 

 

l——

305'

...—O

20

IO-

/'&40UR
0
#;TV

 

41

EXPERIMENT 7-F
I O. I C

S.E.

+——-+CONTROL EA

M N
e——-—-e 2. i .
0—0 5332'); 1;“ MNN i I i

TOX ICANT

l/ INTRODUCED
x. a.

 

 

20

“3 W EATEN
o

O

 

 

 

 

 

eaten were
addition fi
eventually
eater. by CC
Mean w
weight) of
angested f
of ammonia
weight than
control (0.
(Calculated
from WEight
tiOns Of an

81 thOUgh nc

 

g.
‘lSh, was

fish. ‘

Food c
diVided by
0.25, the c
signifiCam
ted p = 0..

mdiCated 1

42

eaten were greater than those eaten by control fish. In
addition fish exposed to the other ammonia concentrations
eventually ate amounts of Gambusia comparable to amounts
eaten by controls.

Mean weight gain (initial weight divided by final
weight) of exposed fish was a variable measurement that
suggested fish exposed to the three highest concentrations
of ammonia (9, 11 and 14 ppm) gained considerably less
weight than controls (Table 8). Mean weight gain for the
control (0.14), however, was not statistically different
(calculated F = 0.83, tabular F, 0.05 level (6, 56) = 2.34)
from weight gains made by fish exposed to other concentra-
tions of ammonia. Weight gain by fish exposed to 2 ppm,
although not significantly higher than that of control
fish, was the highest mean weight gain of all groups of
fish.

Food conversion ratios (net weight gain of a fish
divided by weight of Gambusia eaten) ranged from 0.18 to
0.25, the control value being highest (Table 8). No
significant difference was found among treatments (calcula-
ted F = 0.12, tabular F, 0.05 level (6, 56) = 2.34), which
indicated that food conversion efficiency was unaffected by
ammonia stress. However, variation and measurement of
weights only at the beginning and end of the experiment
would tend to mask any trends which occurred during the
initial exposure and acclimation process.

The RNA-DNA ratios (Table 9) more closely reflected

trends established by the food consumption curves (Figure 4).

Table 80 ME
0]

\L

Aquarius C1

No.

W

43

Table 8. Mean weight gain and food conversion ratio of fish
(Standard error is given in
parentheses after the mean).

exposed to ammonia.

 

 

 

aMean Food
Aquarium Cone. of NH“ No. of Mean Weight Conversion
No. (ppm as N) Fish Gain Ratio
1 Control 8 0.1453(0.0565) 0.2514(0.0858)
8 2.13 8 0.1638(0.0369) 0.2092(0.0354)
3 5.72 8 o.1532(o.0304) 0.2318(0.0353)
10 7.88 8 0.1562(o.0532) 0.2193(0.0631)
5 9.16 8 0.0864(0.0354) 0.2087(0.0?73)
2 11.27 8 0.0536(0.0277) 0.1871(0.0899)
12 13.59 8 0.1229(o.0339) 0.2211(0.0550)

 

uNigetive weight gains were treated a570.

 

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44

 

 

 

 

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Aao.av ~n.oa Aa.av m.m~ as.o~v ~.mn~ m s~.HH N
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Initially
30 days cc
analysis J
level (6,
the RNA-DI
Dunnett's
exposed t
higher R‘.’
trol fist.

2 ppm am

—-‘———

produce c

Equ

reSPOTse

 

0f ammonj
grewth 0‘
COnducteC
56 grEen
nOH‘inal E

25 Ppm a:

 

Sunfish .

45
Initially the RNA-DNA ratio was low (5.26), while after
30 days control fish had a mean ratio of 11.50. An
analysis of variance (calculated F = 2.68, tabular F, 0.05
level (6, 56) = 2.34) showed a significant difference among
the RNA-DNA ratios of fish from the various treatments.
Dunnett's test (Steel and Torrie, 1960) showed that fish
exposed to 2 ppm ammonia as N possessed a significantly
higher RNA-DNA ratio (17.82) when contrasted with the con-
trol fish ratio (11.50). It is clear that fish exposed to
2 ppm ammonia were definitely stimulated to eat more and

produce correspondingly more RNA.

Experiment lO-F

Experiment lO-F was performed to determine the growth
response of green sunfish exposed to seven concentrations
of ammonia. Also investigated was the post-treatment
growth of fish in toxicant-free water. The experiment was
conducted from May 12 to June 17, 1971 using seven tanks,
56 green sunfish (Appendix Table E, Collection 8) and
nominal ammonia concentrations of 0, 2, 5, 10, 15, 20 and
25 ppm ammonia as N (Table 10). In this experiment all
sunfish were placed in aquaria and fed Gambusia for 4 days
before introduction of toxicant. Ammonia was then intro-
duced for 20 days and then removed. Fish were then fed for
8 additional days in toxicant-free water. Fish were
weighed at the beginning and every 4 days during the exper-

iment to monitor changes in weight and food conversion

 

 

#09832 ESHHOSU<

 

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46

 

 

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. ca 0.3.09

ratios.
to each
To
value an
one was
value we
l’ne LCSO
tions an
Table 6)
for blue
using ta
a static
Scheier
6-0 ppm
large fi
The
OUS cone
was firs
PrOHOunC
Of aml'nOn
respOHS e
deCrQBSe
ammonia
than COn
reflEc;Ce
effects

highly S

47

ratios. Mean water temperature was 15.8 C and flow rate
to each tank was 300 ml/min.

To compare chronic results with the standard LCSO
value and with the LCso value obtained for pumpkinseeds,
one was determined for green sunfish. In addition this
value was needed for calculation of an application factor.
The LC50 value was determined under continuous-flow condi-
tions and found to be 33 ppm of ammonia as N (Appendix
Table G). Other values reported by McKee and WOlf (1963)
for bluegills included a 48-hr LC50 of 15 ppm as N at 20 C
using tap water and a static system and 18.5 ppm as N for
a static system using reoxygenated water. Cairns and
Scheier (1959) gave a 96-hr LC50 value for bluegills of
6.0 ppm as N for small fish and 7.7 ppm ammonia as N for
large fish (14 cm) under static test conditions.

The number of Gambusia eaten by fish exposed to vari-
ous concentrations of ammonia declined when the toxicant
was first introduced (Figure 7). The decline was most
pronounced and lasted longer at the highest concentrations
of ammonia tested, while fish at 1.93 ppm showed no initial
response and those at 4.84 ppm exhibited a short-term
decrease in feeding. Thereafter, fish exposed to 1.93 ppm
ammonia consumed on the average consistently more food
than control fish. The analysis of variance (Table 11)
reflected the variable nature of the response as main
effects and the ammonia stress x period interaction were

highly significant. The significant interaction indicates

Figure 7.

48

Rate of consumption of Gambusia in mg per
hr by green sunfish before and after con-
tinuous exposure to different concentra-
tions of ammonia. Each point represents
the mean of eight fish unless deaths
reduced that number. Mean standard error
for each concentration is given as a dark
vertical bar. Period of stress is indi-
cated by the stippled area.

75

50

2&

“3 W CATCH/HOUR

a-
O

N
(D

 

O

50

25

49

+-—§- CONTROL
H |.93 PPM N
H 4.84 PPM N

 

ONE FISH DEATH

3

 

 

 

CONTROL
.02 PPM N

20
...... 24.03 PPM N /

:3

 

.1
E

 

 

"F EXPERIMENT lO-F

 

 

Table 11.

Source

Stress
Period
S x P
Error

Total

*0.61 Big!

“‘11 degreeg'

 

50

Table 11. The analysis of variance table for the effects of
seven concentrations of ammonia (stress) over six
periods on the amount of Qggbggia consumed by
green sunfish.

 

 

 

Sum of Degrees of Mean
Source Squares Freedom Square F Statistic
Stress 3H7.23 6 57.87 “0.69““
Period 158.22 5 31.6h 22.2u**
S x P 73.09 30 2.h4 1.71“
Error 359.82 a253 1.42
Total 938.36 335

 

ԤO.UI significance level.

“#1 degrees of freedom were subtracted for dead fish.

that the
not cons
that fis
considerI
fish ap;l
ing rate:
consumpt
especiall
mmmtcl
controlsl
Thel
was appé
After a:
had mea:
erEnce k
to 4.84
than COr
4‘84 Ppn
particul
of Varie
time, bL
Signific
caution
the larc

 

51

that the feeding response caused by the toxicant was
not consistent over time, supporting the view (Figure 7)
that fish were initially stressed to the point that a
considerable decline in feeding occurred. Later these
fish apparently acclimated to the ammonia stress as feed-
ing rate became similar to that of control fish. Food
consumption appeared to increase after toxicant removal
especially in the two highest concentrations, where
amount of food eaten was comparable to that eaten by
controls.

The lowest concentration of ammonia (1.93 ppm as N)
was apparently stimulatory to fish growth (Figure 8).
After about 12 days, fish exposed to this concentration
had mean weights greater than control fish. This diff—
erence became greater as time progressed. Fish exposed
to 4.84 ppm of ammonia exhibited growth slightly lower
than controls. At concentrations of ammonia greater than
4.84 ppm ammonia, growth was much less than for controls,
particularly for 20 and 24 ppm ammonia as N. The analysis
of variance (Table 12) showed a significant effect of
time, but more importantly that ammonia stress had a
significant effect on the growth of these fish. Some
caution in interpretation must be exercised, because of
the large number of dead fish, but it is still clear that
stress adversely affected sunfish growth. Dunnett's

procedure was applied to the mean weight averaged over

time and it was found that growth of fish exposed to 20

 

Figure 8.

52

Mean weight of green sunfish before,
during (stippled area) and after contin-
uous exposure to ammonia. Each point
represents the mean of eight fish except
where deaths decreased this number. See
Appendix Table H for standard errors.

26

22

18

14

10
26

22

22.

18.

 

14.

 

1%

CL' m

WEIGHT IN GRAMS

26

22

18-

14

T

53

EXPERIMENT lO-F /

GROWTH OF GREEN SUNFISH X
UNDER AMMONlA STRESS

    
 

15 8 C ’ ONE FISH DEATH

..... CONTROL
"""" A'— 1093 PP“ N
...... Dung 4.84 Pm N

 

 

 

 

 

 

 

 

10d-
26 *3; 1 1
15-8 C +——+ CONTROL
22 ~ A—-—A 8.67 ppm N ,x
G----0 13.23 ppm N
13 b //
on"
I a ”/"
14L -—’ ”/
10 _
L L
26 .
+—+CONTROL
22 . A——420.02 ppm N

 

 
 

0----O 24.03 PPM N

 

 

 

Table 1 2 I

Source

Stress
Period
3 x P
Error
Total

31:3.01 Big]

degree,

 

 

54

Table 12. The analysis of variance table for the effects of
seven concentrations of ammonia (stress) over six
periods on the growth of green sunfish.

 

 

 

Sum of Degrees of Mean
Source Squares Freedom Square F Statistic
Stress 979.52 6 163.25 32.33’“
Period 1269.35 5 253.87 50.77**
S x P 123.89 30 4.13 0.82
Error 1281.67 a254 5.05
Total 365“.“3 335

 

“0.01 afganicance—ievelf L

340 degrees of freedom were subtracted for dead fish.

and 24 PP
ively) wa
(16.06 g)

More
although
(Table 13
experimen
at 24 ppn
nents bef
experimen
ammonia a
than fish

Remc
on fish 9;
the Same
observed
13‘23 Pp?
grew at I
removal c

30mg
indicateCl

greater 4.

 

COnsidErE

after f1.

 

lnCreasi‘

55

and 24 ppm of ammonia as N (12.80 and 11.81 g respect—
ively) was significantly lower than growth of controls
(16.06 9).

More deaths occurred at higher concentrations,
although one control fish and one at 1.93 ppm died
(Table 13). Three succumbed at 13.23 ppm early in the
experiment (190 hrs); then later two died at 20 and one
at 24 ppm as N. Fish usually lost weight in other experi-
ments before death, while only four of nine did in this
experiment. Fish exposed to higher concentrations of
ammonia also had a greater incidence of mandible abrasions
than fish at lower concentrations.

Removal of toxicant appeared to have no great effect
on fish growth, as most fish continued to grow at about
the same rate and exhibited the same trends of growth
observed during exposure (Figure 8). Fish at 8.67 and
13.23 ppm appeared to be somewhat of an exception, as they
grew at rates comparable to controls in the 8 days after
removal of toxicant.

Some acclimation by fish under ammonia stress was
indicated by growth data (Figure 8). For those treatments
greater than 4.84 ppm as N, mean fish weight declined
considerably after introduction of the toxicant, indicating
in some cases a mean weight loss of about 1 g per fish
over the first 4 days after toxicant introduction. There-
after fish exposed to 8 and 13 ppm ammonia grew at an

increasing rate while growth of fish exposed to 20 and 24

Table 13 .

\\

Fish No.

35
37
10
59
89
9h

75

 

 

56

Table 13. Death and weight response of fish exposed to
various concentrations of ammonia at 15.8 C.

 

 

 

Time of Type of Initial Final
Death Stress Weight Weight

Fish No. (hrs) (ppm N) (S) (8)
34 190 13.23 11.50 11.79
35 190 13.23 10.24 10.53
37 190 13.23 11.99 11.49
10 190 8.67 12.00 11.10
59 191 1.93 10.95 12.04
89 264 24.03 10.24 9.69
94 288 24.03 13.38 12.81
1 356 Control 12.31 14.75
75 524 20.02 10.02 10.59

 

ppm rem;
in growi

Foc
first 4
centrat:
initial
these f:
exposed
reactio:
elabora‘
sion ra+
ratio 0;
able frJ
0f Vari.
iOd int:

that the

 

Consist!
the ini

0f the .

349%

Ex
SeDtemb.
as the

record

57
ppm remained the same for 12 days before greater increases
in growth occurred.

Food conversion ratios showed an initial decline the
first 4 days after toxicant introduction by fish from con-
centrations greater than 4.84 ppm (Figure 9). During the
initial 4-day period, non-feeding and weight loss for
these fish occurred. Apparently stressed fish (those
exposed to ammonia) utilized energy for the "stress
reaction" (Selye, 1956), thereby resulting in less tissue
elaboration (growth) and consequently, lower food conver—
sion ratios. After the first 4 days, the food conversion
ratio of all stressed fish became essentially indistinguish-
able from control fish values (0.30-0.40). The analysis
of variance for these data indicated that the stress x per-
iod interaction was significant (Table 14), which shows
that the effect of ammonia on food conversion was not
consistent over time. The reason for this interaction,
the initial decline in food conversion after introduction

of the toxicant, has been discussed.

Experiment ll-F

Experiment ll-F, performed between August 18 and
September 7, 1971, was the first experiment using cadmium
as the stressor. Objectives of the experiment were: 1) to
record the growth response of green sunfish exposed to
relatively high concentrations of cadmium; 2) to determine
at what concentrations sub-lethal tests should be conducted;

3) to obtain an LCSO value for green sunfish; 4) to

Figure 9.

58

Food conversion ratio is shown before
(day 0-4), during (day 4-28, stippled
area) and after (day 28—36) continuous
exposure to ammonia concentrations.

Each point represents the mean of eight
fish, except where deaths decreased this
number. Mean standard error is shown
with dark vertical bars.

X OJDI
.
0

FOOD CONVERSION RATIO

SOr

u
0

N
O

...:
O

O

    
   

 

EXPERIENT lO-F

 

 

59

 

CONTROL 352?

A——A8.67 ppmN +40
0 ----- 013.23 ppmN "I

 

 

4-0 0-12 12-16 1.6-20 20-24 24-28 20-32 32-36

INTIIVIL IN DAYS

Table 14 .

w
”

Source

Stress
Period
3 x P
Error

Total

 

3.0.01 31
“1 degr

 

6O

 

 

 

Table 14. The analysis of variance table for the effects of
seven concentrations of ammonia (stress) over six
periods on the food conversion ratios of green
sunfish.

Sum of Degrees of Mean

Source Squares Freedom Square F Statistic

Stress 0.0772 6 0.0129 0.79

Period 0.8299 4 0.2075 12.16"

S x P 1.0191 24 0.0425 2.49**

Error 4.0964 3241 0.0170

Total 6.0226 279

 

:i0.01 significance level.
44 degrees of freedom were subtracted for dead fish.

 

deternul
and who
biopsy

weight

Collect
concent
ppm--se
of 4 da
mean wa
at the
after.
to the
able, a
more si

affects

 

water g
eSCape
abrasec
Entire

in
uouS-E:
Picker:

“50 v;

Chemis
p.
exp0811.

Cadmiu;

61
determine the rates of cadmium uptake in the gills, liver
and whole body; and 5) to evaluate the use of chemical
biopsy techniques. Fish used in this study ranged in
weight from 7.20 to 10.81 9 (see Appendix Table E,
Collection 9, 10). They were subjected to seven nominal
concentrations of cadmium (O, 5, 10, 20, 30, 40 and 50
ppm--see Table 15) for 16 days after a pre—stress period
of 4 days. Flow rate to each tank was 200 ml/min and
mean water temperature was 19.9 C. Sunfish were weighed
at the beginning of the experiment and every 4 days there—
after. Gambusia were present at all times. Fish exposed
to the higher concentrations of cadmium were very excit-
able, and handling stress during weighing undoubtedly was
more significant for these fish than for controls. Fish
affected by cadmium remained quietly at the surface of the
water for long periods of time, or darted around trying to
escape their respective cells. Many fish had severely-
abrased lower mandibles and mucus secretions over their
entire body, particularly on the eyes, mouth and fins.

The LCSO value derived for green sunfish under contin-
uous-flow conditions of this experiment was 20.5 ppm Cd.
Pickering and Henderson (1966) found a considerably higher
LCSO value, 66 ppm, which was obtained under similar water
chemistry conditions but using a static system.

Food consumption by fish at all levels of cadmium
exposure was lower than that of controls; the higher the

cadmium concentration, the lower the amount of food

 

 

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63

consumed (Figure 10). Fish exposed to the two lowest
concentrations of cadmium (3 and 7 ppm) showed an initial
decline in feeding rate from about 30 mg Gambusia/hr to
values around 5 mg/hr when the toxicant was first intro-
duced. Feeding resumed at values around 30 mg/hr in the
next 20—30 hrs and remained at this level for the remainder
of the test, while control fish consumed food at a rate of
40—50 mg/hr. The analysis of variance confirmed the detri-
mental effect of the toxicant as the main effects, both
stress and period, were highly significant (Table 16).
Dunnett's test (Steel and Torrie, 1960) showed that the
average rate of Gambusia consumption by control fish over
all periods (4.18 mg/hr) was significantly different from
consumption rates of 2.11, 1.77 and 0.79 mg/hr exhibited
by fish at 3, 7 and 15 ppm Cd respectively.

Cadmium had a detrimental effect on growth rate of
green sunfish at all concentrations (Figure 11). Amount
of growth depression as well as the pattern of death was
dose-dependent. At 51 ppm Cd all fish died in less than
24 hrs, while all fish at 35 ppm and 27 ppm Cd were dead
in 4 days. Two died at 15 ppm, while none died in the
control or at 7 and 3 ppm Cd during the l6-day period.
Regression equations were calculated for controls and
fish at 3, 7 and 15 ppm Cd relating mean growth and time.
Only control fish exhibited a significant linear trend
(F = 46.36, 0.05 level, 30 d.f.) with the regression

equation being: Y (weight) = 0.30 X (time) + 8.47. The

 

Figure 10.

64

Rate of consumption of Gambusia in mg

per hr by green sunfish 96 hrs before

and during 384 hrs of continuous exposure
to concentrations of cadmium. Each point
represents eight fish except where deaths
reduced this number. Mean standard error
is given for each concentration as a dark
vertical bar.

65

 
   
   

I00
EXPERIMENT ll-F
'0 19.9 C New
3.2:.

  
 
 

Taxman + n

5 0 , INTRODUCED II

 

+——+ CONTROL

5
H 15.44 PPM co
H2163 PPM 00 +317 r
TOXICANT I ' /
FIITRODUCED /

HOUR
a
O

6

 

:Arsu
3

O

O

 

 

 

4O

20

 

 

 

 

66

 

 

 

Table 16. The analysis of variance table for the effects of
four concentrations of cadmium (stress) over three
periods on the amount of Qggpggi; consumed by
green sunfish.

Sum of Degrees of Mean

Source Squares Freedom Square F Statistic

Stress 145.95 3 48.65 29.85**

Period 14.70 2 7.35 4.51“

S x P 8.86 6 1.48 0.90

Error 130.37 aso 1.63

TOtll 299087 95

 

I0.05 significance level.
**0.01 significance level.

an

degrees of freedom were subtracted for dead fish.

67

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69

equation explained 61% of the variation relating growth of
the control fish to time. The regression coefficient of
the control was compared with the regression coefficient
of all stressed fish. Because of the extreme variability
in fish weights, no significant differences were detected
between the control and any of the three treatments. This
is a good example of the type of phenomenon which occurs
in nature and in many laboratory tests. The stressor
significantly affects a fairly large number of fish which
soon die and others grow very little or lose weight.
Others, which are genetically suited, are able to resist
the stressor and even grow well. This leads to a large
difference between individuals among stressed fish. Con-
trol fish, not subjected to such a drastic stress, are not
selectively pushed to their genetic limits. When these
factors are Operating, statistical tests using an average
variance to test effects can be inaccurate.

During the first 4-day pre-stress period fish possessed
food conversion ratios very close to 0.40 (Figure 12).
After introduction of toxicant, conversion ratios for fish
under cadmium stress, when contrasted with controls, were
considerably lower for the three remaining intervals. It
appears that all concentrations of cadmium between 3 and 35
ppm were detrimental to fish since energy which normally
would go into elaboration of tissue for new growth was
diverted elsewhere or some detrimental effect on biochem-

ical systems was occurring. This judgment was confirmed

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IO‘O X OIL'U NOISIIANOO 000!

O
M

O
1'

on

72

by the analysis of variance (Table 17) which showed a
significant main effect (F = 24.75), and a Significant
interaction of stress with period (F = 4.56). The inter-
action effect was probably due to the initial decline in
food conversion ratios by exposed fish while control
conversion ratios remained high. The increase exhibited
in the last period by fish exposed to 3 ppm Cd was also
a factor.

Eight fish were randomly selected from the 72 fish
used in this experiment and analyzed for cadmium at the
beginning of the experiment and eight were analyzed at the
end of 4 days of no stress (two control tanks were maintained
for this reason). Then all 56 experimental fish were
analyzed at the end of the experiment. Fish that died at
15 ppm Cd contained 5-8 times the amount of cadmium found
in live fish at that concentration (Table 18A). Therefore
dead fish were not pooled with live fish, but tabulated
separately. Initially fish contained whole-body burdens
of 0.68 ppm Cd and after 4 days of feeding on Gambusia in
toxicant-free water, whole—body burdens increased to 0.98
ppm. The only reason which can be advanced for this
increase is that Gambusia eaten contained 0.50 1 0.18 ppm
Cd (wet-weight basis), and that a considerable amount Of
this food was eaten, up to 2.5 g. The cadmium concentra-
tion in Gambusia was obtained from determinations on 3
separate samples from different times of the year weighing

16, 18 and 4 g respectively. After 16 days live fish

 

 

 

Table 17. The analysis of variance table for the effects of
four concentrations of cadmium (stress) over three
periods on the food conversion ratios of green
sunfish.

Sum of Degrees of Mean

Source Squares Freedom Square F Statistic

StrCUB 0 e 8081 0 e 269“ 24 e 75"

Period 0.0150 0.0075 0.69

S x P 0.2979 6 0.0496 4.56*’

Error 0 .8706 380 0 . 0109

Total 1.9900 95

 

“0.01 signifiEance level. ,
degrees of freedom were subtracted for dead fish.

at.

74

Table 18A. Whole-body cadmium concentration on a wet-weight
basis of green sunfish from experiment ll-F. (N is
the number of fish used in analysis: one standard
error is given in parentheses after the mean).

 

 

 

 

 

Treatment Live Fish Dead Fish

Day Ppm Cd N Ppm Cd N Ppm Cd

0 Control 8 0.68(0.07) - -

4 Control 8 0.98(0.13) - -

16 Control 8 0.82(0.09) - -

16 3.83(0.18) 8 3.99(o.30) - -

16 7.95(0.23) 8 4.77(0.38) - -

16 15.44(0.39) 6 21.52(3.75) 2 114.07(0.93)
16 27.63(0.99) - - 8 74.43(9.36)
16 35.92(0.70) - - 8 104.74(14.86)
16 51.51(2.26) - - 8 148.78(9-35)

 

75

exposed to 0, 3, 7 and 15 ppm Cd exhibited a linear
relationship when the log of the cadmium concentration
in the fish plus one (Y) was regressed against the cadmium
concentration in the water (X). The equation describing
this relationship (Y = 0.065 X + 0.316) explained 89% of
the variation involved in measuring these variables. Fish
exposed to 15 ppm accumulated proportionately more cadmium
than fish at 3 and 7 ppm Cd. Apparently mechanisms that
prevent excessive cadmium uptake such as mucus secretions,
metaloproteins (Wisniewska, gt 21., 1970) and other unknown
means become saturated. Saturation permits cadmium build-
up which apparently reaches a lethal limit in green sunfish
exposed for 16 days to 15 ppm, since two fish at this level
and all fish exposed to higher concentrations died. The
higher accumulation of cadmium by dead fish when compared
with live fish at the same concentration cannot be attribu-
ted wholly to surface adsorbtion on mucus since all fish
were washed thoroughly under tap water. Dead fish exposed
to higher concentrations of cadmium generally accumulated
increasing amounts of cadmium, the higher the concentra-
tion to which they were exposed. It would have been
informative to have analyzed fish from these higher concen-
trations of cadmium before they died.

A similar pattern of cadmium uptake was exhibited
by the gills from these fish, although gill cadmium content
was more variable (Table 18B). Cadmium content in the gills

increased from 0.46 on the first day of the experiment to

76

Table 188. Cadmium concentration on a wet-weight basis in
gills of green sunfish from experiment ll-F. (N is
the number of fish used in analysis: one standard
error is given in parentheses after the mean).

 

 

 

 

 

Treatment Live Fish Dead Fish

Day Ppm Cd N Ppm Cd N Ppm Cd

0 Control 8 0.46(0.15) - -

4 Control 8 0.99(0.28) - -

16 Control 8 0.62(0.16) - -

16 3.83(0.18) 8 3.22(0.uu) - -

16 7.95(o.23) 8 3.uo(o.68) - -

16 15.44(0.39) 6 21.uu(4.uu) 2 l68.79(4.05)
16 27.63(0.99) - - 8 108.86(14.52)
16 35.92(0.79) - - 8 170.27(22.70)
16 51.51(2.26) - - 8 242.18(18.02)

 

77
0.99 ppm Cd on the fourth day. After 16 days of exposure
a similar, though less exact pattern of cadmium uptake was
shown by the gills as was seen in whole-body uptake. The
equation explaining 74% of the variation observed in
measuring cadmium uptake by the gills was: Y (log of the
cadmium content in the gills + 1) = 0.066 X (cadmium
content in the water) + 0.222. The concentration of
cadmium in the gills of dead fish was higher than concentra-
tions found in the whole body of these fish.

Cadmium content measurements of fish livers were the
most variable among structures measured (Table 18C).
Cadmium levels also increased in the liver from 0.35 ppm on
day 0 to 1.12 ppm on day 4. After 16 days exposure the
same type of relationship between cadmium in water and in
tissue was observed. Only 60% of the variation was
explained by the regression equation: Y = 0.086 X + 0.153.
There was a very high accumulation by livers of live fish
exposed to 15 ppm, twice as high (42 ppm) as concentrations
found in livers of dead fish at higher concentrations.

After fish exposed to 50 ppm Cd had succumbed, eight
large green sunfish (71.40 i 9.16 g) were added to this
tank. Most fish died within 24 hrs, a little sooner than
did smaller fish at that concentration. Gills were removed
from the large green sunfish after death and analyzed for
cadmium content. Concentration of cadmium in these fish
gills was 182.19 1 20.95 ppm Cd which was less than that
in gills of smaller fish (242.18 1 18.02 ppm Cd--Table 18E)

exposed to 50 ppm Cd.

78

 

 

 

 

 

Table 180. Cadmium concentration on a wet-weight basis in the
liver of green sunfish from experiment ll-F. (N is
3?:£82313.“ifihpfif-Sfitii‘ffififii:’.§2°.2§§3‘?”°

Treatment Live Fish Dead Fish

Day Ppm Cd N Ppm Cd N Ppm Cd

0 Control 8 0.35(0.35) - -
4 Control 8 1.12(0.35) - -

16 Control 8 0.41(o.20) - -

16 3.83(0.18) 8 4.27(0.66) - -

16 7.95(0.23) 8 7.2u(3.00) - -

16 15.uu(o.39) 6 42.81(9.09) 2 60.48(11.40)

16 27.63(0.99) - - 8 33-53(3-05)

16 35.92(o.70) - - 8 37.17(4.01)

16 51.51(2.26) - - 8 39.03(1.78)

 

79

Experiment 12-F

In this experiment, an attempt was made to maintain
green sunfish at low concentrations of cadmium which are
more likely to be found in waters as a result of man's
activities. Growth, food consumption and RNA-DNA ratios
were monitored under the low cadmium levels as was whole
body, gill and liver cadmium uptake. The 72 green sunfish
used ranged in weight from 5.67 to 7.47 g (Appendix Table
B, Collection 9, 10). From this group of fish, eight were
randomly assigned to each of nine different treatments,
the first group of eight being sacrificed on day 0 to
determine initial cadmium levels and RNA-DNA ratios. The
second group was one of two control groups which was sacri-
ficed on day 4 to determine what effect the 4-day acclima-
tion period had on cadmium uptake and RNA-DNA ratios. The
remaining seven groups made up the experimental groups of
one control and six treatments exposed to 0.05, 0.23, 0.32,
1.31, 1.93 and 2.48 ppm Cd (Table 19). The experiment
was started on September 10, 1971 and continued for 24 days
including a 4—day period of acclimation to experimental
conditions, then 20 days of stress with cadmium introduction.
Food (Gambusia) was unlimited for sunfish throughout the
experiment. Fish were exposed continuously at a flow rate
of 200 ml/min and a mean temperature of 18.6 C and were
weighed every 4 days to monitor growth.

Except for the 0.05 ppm treatment, fish at all concen-

trations of cadmium ate amounts of Gambusia comparable to

 

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81

or less than that eaten by controls (Figure 13). Greatest
depression of feeding and highest mortality occurred at
1.31 ppm Cd where three fish died toward the latter part of
the experiment. One fish succumbed at 1.93 ppm Cd while
none died at 2.48 ppm. Analysis of variance showed that
cadmium stress and period had a significant effect on the
amount of Gambusia eaten by these fish (Table 20). To sort
out differences among treatments Dunnett's test (Steel and
Torrie, 1960) was used, but it failed to show any treatments
different from the control. It was noted, however, that
fish exposed to 0.05 ppm Cd consumed the greatest amount of
Gambusia (a mean of 1.67 g) as compared with the control
value of 1.48 g. Fish exposed to higher concentrations of
cadmium consumed amounts ranging from 0.87 to 1.21 9.

After about 200 hrs exposure to 0.05 ppm Cd fish began
to eat at an increasingly higher rate when compared with
control fish. It appeared that this concentration definitely
stimulated growth and may be an important threshold value of
significance in setting safe water quality standards. Some
signs of acclimation toward the end of the experiment were
shown by fish exposed to higher concentrations. A longer
study would have better documented this phase of the chronic
toxicity response. 2

Growth of green sunfish was depressed at all concentra-
tions of cadmium, except at 0.05 ppm Cd (Figure 14). The
analysis of variance (Table 21) showed that both main effects

(cadmium stress and period) had a significant effect on

Figure 13.

82

Rate of consumption of Gambusia in mg per
hr 96 hrs before and during 480 hrs of
continuous exposure to seven different
concentrations of cadmium. Each point
represents eight fish except where deaths
reduced this number. Mean standard erIxDI?
is given for each concentration.

83

30 r ‘
"- EXPERIMENT 12-F -+—-+ CONTROL

o-—-oo.05 ppm CO A
O—eo.23 ppm on o :4

ONE FISH ,x/
DEATH

  
  
   

 

 

 

‘ AN 5
\ x—-x 0.32 ppm CD + A
* 1 H 1.31 ppm CD I il

20 1 13‘. \

 

MG 5mg EATEN PER HOUR
O
T
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r
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X

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L. v—V1.93ppMCO *V0

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\ . /
20 I" ; E: \x ’3;\ /
. “‘\ \ 4' f \ r /
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x‘1'-‘ \ \ _. r1 , ' ",/
. . +7] . ‘_ . .1. /
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0 100 200 300 400 500
HOURS

84

Table 20. The analysis of variance table for the effects of
seven concentrations of cedmium (stress) over five
periods on the amount of Gglbugie consumed by
green sunfish.

 

 

 

Sum of Degrees of Mean
Source Squares Freedom Squere F Stetistic
Stress 16.65 6 2.77 4.86”
Period 16.99 h 4.25 7.4uee
S x P 1#.OO 24 0.58
Error 135.81 5‘238 0.57
Total 183.u5 279

 

{30.01 significance level. _
7 degrees of freedom were subtracted for dead fish.

85

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87

 

 

 

Table 21. The analysis of variance table for the effects of
seven concentrations of cadmium (stress) over five
periods on the mean weight gains of green sunfish.

Sum of Degrees of Mean

Source Squares Freedom Square F Statistic

Stress 32.71 6 5.45 7.68**

Period 19.28 h “.82 6.79"

S x P 7.6“ 24 0.32 O.b5

Error 168.88 238 0.71

228.51 279

Total

 

8‘i5.01 significance level.
7 degrees of freedom were subtracted for dead fish.

88

growth of these fish. Dunnett's test (Steel and Torrie,
1960) showed that fish exposed to 0.23 and 1.31 ppm Cd
possessed mean weights significantly less than the control.
Fish exposed to 0.05 ppm Cd had the highest mean weight, but
it was not statistically higher than the control weight. The
stimulatory effect of 0.05 ppm Cd clearly shown by food con—
sumption (Figure 13) appeared somewhat later in the growth
response (Figure 14). The greater rate of growth of these
fish, when compared with controls, started after 3 days
exposure. By the end of the experiment mean weight of these
fish was greater than that of controls.

Food conversion ratios (Figure 15) were extremely varia-
ble, but the analysis of variance showed that both stress and
period had a significant effect (Table 22). The magnitude
of the F value showed time to be the greater of the two
effects. Examination of the tables of means averaged over
time showed that fish at 0.05 ppm had the highest food con—
version ratio (O.25), while values from the other cadmium-
exposed fish except one, were lower than the control value
(0.20). None were significantly different (Dunnett's test)
from control values. Fish exposed to 0.05 ppm Cd having
the highest food conversion ratio suggests that the greater
growth exhibited by these fish is due both to greater intake
of food and a better efficiency of conversion when compared
with controls. There was no consistent effect of period on

the mean ratio considered over all treatments.

u I, ‘- M

r

‘m

Figure 15.

89

Food conversion ratios of green sunfish
before and during continuous exposure to
different concentrations of cadmium.

Each point represents the mean of eight
fish except where deaths reduced this
number. Mean standard error for each
concentration is shown by a dark vertical
bar.

40

30

20

10

+1“; /
30* *——-X 0.32 pm CD 'll ,

FOOD CONVERSION RATIO x 0.0:
3
/

90

[ EXPERIMENT 12-F MEAiN ,5-33-
18.5 C NET WEIGHT GAIN/ » I I I

GAMBUS‘IA EATEN

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‘~~. , //\ /
L _....4' ~ _. / ,
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+——+ CONTROL
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9—00.23 PPM CD

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18.6 C MEAN s E.
4————+ CONTROL

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.

+—-+ CONTROL
V——-v 1,93 ppM CD MEAN S.E.

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H 2.48 PPM CD Y' o

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INTERVAL IN DAYS

91

 

 

 

Table 22. The analysis of variance table for the effects of
seven concentrations of cadmium (stress) over five
periods on the food conversion ratios of green
sunfish.

Sum of Degrees of Mean

Source Squares Freedom Square F Statistic

Stress 0.2944 6 0.0491 2.27*

Period 1.1238 4 0.2810 13.01**

S x P 0.6674 24 0.0278 1.29

Error 5.0338 a233 0.0216

Total 7.1195 279

 

*0.05 significance level.
;*Q.Ol significance level.
12 degrees of freedom were subtracted for dead fish.

92

The mean RNA-DNA ratio for this group of fish started
at 16.97 and increased to 19.28 at the end of the first 4
days before cadmium introduction (Table 23). After the next
20 days fish from the control tank possessed a mean RNA—DNA
ratio of 22.54. Cadmium-stressed fish had ratios ranging
Lrom 21.87 (1.31 ppm Cd) to 77.51 (1.93 ppm Cd) with the
0.05 ppm-exposed fish having a ratio of 26.29. There was no
significant difference among treatment means (calculated F =
0.34, tabular F, 0.05 level, (6, 49) : 2.37) after 20 days.
Thus the RNA-DNA data were unable to demonstrate some of the
results already shown. For example, one would have expected
the 0.05 ppm-treated group to have a considerably higher
ratio. This might have been shown if samples could have
been taken midway through the experiment.

Whole—body burdens were measured initially at 0.95 ppm
Cd and then decreased to 0.55 ppm after 4 days of feeding be-
fore cadmium was introduced (Table 24A). Apparently differ-
ences in background levels and perhaps water characteristics
were responsible for this loss. In experiment ll-F this
change was just the opposite. Control fish in experiment
lZ—F after 20 more days contained 0.65 ppm Cd, comparable to
levels found after 4 days (0.55 ppm), so apparently equilib-
rium was reached at least by 4 days and maintained near that
for the next 20 days. Fish exposed to 0.05 ppm Cd contained
three times as much cadmium (1.84 ppm) as control fish (0.05
ppm), and even greater levels than found in fish exposed to

0.23 and 0.32 ppm. It is because of this unique behavior of

93

 

 

 

 

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94

Table 24A. Whole-body cadmium concentration on a wet-weight
basis of green sunfish from experiment 12-F. (N is
the number of fish used in analysis: one standard
error is given in parentheses after the mean).

 

 

 

 

 

Treatment Live Fish Dead Fish
Day Ppm Cd N Ppm Cd N Ppm Cd

0 Control 8 0.95(0.16) - -

4 Control 8 0.55(0.08) - -
24 Control 8 0.65(0.07) - -
Zn 0.05 8 1.84(0.21) - ‘
24 0.23 8 1.21(O.15) - -
24 0.32 8 1.28(o.10) - -
20 1.31 5 2.12(o.23) 3 5.34(2.u7)
24 1.93 7 2.l4(0.27) l 3.73
20 2.48 8 2.82(0.36) - -

 

95
fish exposed to 0.05 ppm in accumulating cadmium that
regression equations relating cadmium in the water to cad-
mium uptake by fish failed to explain large amounts of the
variability, even though significant linear trends were
shown by analysis of variance (significant effect due to
regression). The linear trend was highly significant for
whole-body burdens when the amount of cadmium in the fish
and that to which they were exposed was examined (calculated
F = 40.24, tabular F, 0.05 level, (6, 45) = 2.37). This
trend was also significant for the cadmium in gills and liver.
The regression equation for whole-body concentration relating
Y (log of the cadmium concentration in the body + l) to X
(cadmium concentration in the water) was Y = 0.097 X + 0.325

with an r2

(coefficient of determination) equal to 0.46,
while for gill and liver the r2 was 0.10 and 0.38 respectively.
Since green sunfish exposed to 0.05 ppm Cd were stimulated to
eat more Gambusia and grow more they may have ingested more
cadmium via this route than by water alone. Chadwick and
Brocksen (1969) found that fish exposed to 0.5 ppb dieldrin
and fed worms containing known amounts of dieldrin, did not
accumulate more dieldrin than fish exposed to the same con-
centration but fed uncontaminated worms. Undoubtedly
Gambusia eaten by sunfish will contain different amounts of
cadmium depending on the length of exposure and concentration
of cadmium in the experimental aquarium. Future experiments
should expose fish with and without food at the same concen-

tration of cadmium to clarify which route of heavy metal

uptake is most important.

96

The pattern of cadmium uptake by green sunfish gills
paralleled that of whole-body burdens (Table 24B). Initially
fish gills contained a mean concentration of 1.42 ppm which
decreased to 0.23 ppm after 4 days. Control fish contained
0.49 ppm Cd after the next 20 days at the end of the experi—
ment. Gills of fish exposed to 0.05 ppm accumulated almost
as much (2.08 ppm) as gills of fish exposed to 1.31 ppm Cd
(2.38 ppm). Livers were the most variable of the structures
measured but the same trends as found in the whole body and

gills were apparent (Table 24C).

Experiment 13-F

This experiment was designed to evaluate effects of 1
ppm Cd on fish exposed to three different temperatures, cold
(17.5 C), medium (23.9 C) and hot (30.0 C). The experiment
was conducted for 24 days from October 20 to November 13,
1971. Four fish from each of the six treatments were
sampled 2, 6, 12 and 20 days after toxicant introduction to
evaluate changes in growth, food consumption, RNA-DNA ratios
and cadmium uptake in the whole body, gills and liver. Two
duplicate aquaria containing eight fish each were maintained
at each of the six treatment combinations (3 temperatures
and 2 levels of stress). No undesirable water chemistry
changes were experienced (Table 25A, 258, 25C) among treat-
ments or between treatments and the control, as was a factor
in experiments with ammonia. The 108 fish used in this
experiment (see Appendix Table B, Collection 12, 13) ranged

in weight from 5.17 to 9.10 g and were assigned randomly to

Table 243.

97

Cadmium concentration on a wet-weight basis in

gills of green sunfish from experiment 12-F. (N is
the number of fish used in analysis; one standard
error is given in parentheses after the mean).

 

 

Live Fish

 

 

 

Treatment Dead Fish
Day Ppm Cd N Ppm Cd N Ppm Cd

Control 8 l.42(0.50) - '-
4 Control 8 0.23(0.16) ‘- -
24 Control 8 0.49(0.15) -' -
24 0.05 8 2.08(0.34) - -
2a 0.23 8 1.64(0.49) - -
24 0.32 8 1.96(0.63) -' -—
24 1.31 5 2.38(0.86) 3 1.07(0.58)
24 1.93 7 1.93(o.49) 1 2.75
24 2.48 8 2.55(0.21) —' '-

 

98

Table 240. Cadmium concentration on a wet-weight basis in the
liver of green sunfish from experiment 12-F. (N is
the number of fish used in analysis: one standard
error is given in parentheses after the mean).

 

 

 

 

 

Treatment Live Fish Dead Fish
Day Ppm Cd N Ppm Cd N Ppm Cd
0 Control 8 0.98(0.98) - -
14 Control 8 1.l7(0.80) ‘ ‘
24 Control 8 0.51(0.34) "' "
24 0.05 8 2.41(0.84) - "'
24 0.23 8 l.57(0.85) - -
24 0.32 8 0.31(0.31) - -
24 1.31 5 1.63(1.63) 3 9.84(5.22)
24 1.93 7 3.87(1.09) l 3.89
24 2.48 8 5.08(2.12) -> -

 

99

Table 25A. Chemical characteristics of water used in the
continuous-flow experiment l3-F. (E is the number
of samples used in determinations: X is the mean
with one standard error enclosed in parentheses;

C = Cold; M = Medium: = Hot; S = Stressor; N.D.
= non-detectable, less than 0.01 ppm).

 

Aquarium Number

 

1 11 3 5

Treatment 0 C CS 03

Cadmium N. 4 4 l6 16
ppm as Cd X N.D. N.D. 0.99 0.94
(0.02) (0.02)

pH N 2 2 2 2
Range 7077- 707""- 7075' 7078'

7.81 7.93 7.88 7.90

Temperature N 20 20 20 20
(C) x 17.3 17.8 17.4 17.6
(0.2) (0.2) (0.2) (0.2)

Dissolved N 2 2 2 2
Oxygen (ppm) X 8.4 7.4 7.8 8.4
(0.6) (0) (0.1) (0.1)

Alkalinity N 2 2 2 2
ppm as CaCO3 X 342 34? 344 338
(0) (3 (0) ( )

Hardness fl 2 2 2 2
ppm as CaCO3 X 325 332 332 330
(5) (o) (4) (2)

 

100

Table 258. Chemical characteristics of water used in the
continuous-flow experiment l3-F. (fl is the number
of samples used in determinations: X is the mean
with one standard error enclosed in parentheses;

C a Cold: M a Medium: H a Hot; S = Stressor; N.D.
= non-detectable, less than 0.01 ppm).

 

 

Aquarium Number

 

6 7 2 10

Treatment M M MS MS

Cadmium N 4 4 16 16
ppm as Cd X N.D. N.D. 0. 92 0. 98
(o. 02) (0. 02)

pH N 2 2 2 2
Range 7.92- 7.98- 7.74- 7. 81-

8.00 8.20 7.91 7. 91

Temperature 5 20 20 20 20
(c) x 23.9 24. 0 23.4 24.4
(0.2) (0. 2) (0.1) (0.1)

Dissolvfd ) N 26 26 2 24

Oxygen ppm X 7. 7. 7.1 7.
(0) (0.1) (0) (0.2)

Alkalinity N 2 2 2 2
ppm as CaCO3 X 343 336 339 344
(1) (2) (9) (0)

Hardness N 2 2 2 2
ppm as CaCO3 X 331 327 329 330
(1) (1) (3) (2)

 

a”. ....”a. “flog;—
; V.

101

Table 250. Chemical characteristics of water used in the
continuous-flow experiment 13-F. (N is the number
of samples used in determinations; X is the mean
with one standard error enclosed in parentheses;

C = Cold: M = Medium. H a Hot; S = Stressor; N.D.
' a non-detectable. less than 0.01 ppm).

 

 

Aquarium Number

 

4 9 8 12

Treatment H 11 HS HS

Cadmium N 4 4 16 16
ppm as Cd X N.D. N.D. 1.00 1.01
(0.02) (0.02)

pH N 2 2 2 2
Range 8.01- 8.01- 8.1 - 8.02-

8.22 8.11 8.2 8.09

Temperature N 20 20 20 20
(C) X 29.6 29.6 30.4 29.4
(0.1) (0.1) (0.1) (0.1)

Dissolved N 2 2 2 2
Oxygen (ppm) X 7.2 6.8 7.0 6.6
(o) (0) (0.1) (0.1)

Alkalinity N 2 2 2 2
ppm as CaCO3 X 338 344 344 346
(2) (2) (2) (2)

Hardness N_ 2 2 2 2
ppm as CaCO3 X 327 332 332 331
(1) (o) (o) (1)

 

102

the 12 aquaria of the experimental design. Before beginning
the experiment 12 green sunfish were initially sacrificed,
four from each temperature, to provide initial cadmium
levels and RNA-DNA ratios. Fish were placed in the tanks
and allowed to feed for 4 days prior to toxicant introduction.
The first fish samples taken on day 2 after toxicant intro-
duction represent accumulated changes from the first 4 days
of pre-stress as well as the first 2 days of toxicant intro-
duction. Each treatment combination started intially with
16 fish, and decreased by four at each sampling date so values
for growth, food consumption and food conversion ratios were
determined with reduced sample size as time progressed.

Examination of the amount of Gambusia eaten (Figure 16)
revealed that 1 ppm Cd depressed amounts eaten by fish at all
temperatures except 23.9 C, when contrasted with respective
controls. Decreased consumption appeared greatest at the
highest temperature where the only mortality in the experi-
ment was recorded. The analysis of variance (Table 26) indi—
cated that all main effects and interactions except the stress
x period interaction were highly significant. Considering the
magnitude of the F values and the table of means, it is
probable that most of the interaction comes from two consider-
ations. Stressed fish at medium temperatures consumed on the
average more Gambusia (5.28 9) than controls (4.67 g) at that
temperature, whereas cadmium-exposed fish at other tempera-
tures consistently consumed less than controls. The other

consideration is that the effect of temperature (averaged

 

Figure 16.

103

Rate of consumption of Gambusia in mg
per hr for green sunfish 96 hrs before
and during 480 hrs of continuous
exposure to 1 ppm Cd at three different
temperatures. Each point represents
eight fish except where deaths reduced
this number. Mean standard error is
given for each temperature and cadmium
concentration.

104

 

 

 

 

 

 

' EXPERIMENT l3-F
? ONE FISH DEATH
175 C +——+ CONTROL MEAN 5.1:.
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105

Table 26. The analysis of variance table for the effects of
three temperatures and two levels of cadmium
(stress) over four periOds on the consumption of

Gambusia by green sunfish.

 

 

 

Sum of Degrees of Mean

Source Squares Freedom Square F Statistic
Temperature 241.61 2 120.81 62.89**
Stress 17.47 1 17.47 9.09**
T x S 48.55 2 24.27 12.63**
Period 42.03 3 14.01 7.29**
T x P 45.19 6 7.53 3.92**
S x P 1.20 3 0.40 0.21
T x S x P 50.42 6 8.40 4.37**
Error 134.46 a70 1.92
Total 580.93 95

 

230.01 significanCe level:-

2 degrees of freedom were subtracted for dead fish.

106

over stress) on Gambusia consumption was not consistent
over each of the four periods. Fish at cold temperatures
ate less and less over time, while consumption at medium
and hot temperatures fluctuated randomly between periods.

Four deaths were recorded among fish exposed to
cadmium at high temperatures. These deaths can probably be
attributed to an interaction of cadmium with temperature.
However, one of the 16 control fish at hot temperatures
also died toward the end of the experiment.

Fish grew least at 17.5 C (Figure 17). Growth of
fish at 23.9 C was greater than that observed at cold tempera-
tures, while fish at 30‘C grew slightly larger than fish at
23.9 C. Regression equations for control and stressed fish
at each temperature were calculated and regression coeffi-
cients compared to determine if cadmium stress had a signif-
icant effect on growth. No significant differences between
control and stressed fish were found at any temperature,
the calculated F value being very low in every case. Fish
exposed at 30 C and 1 ppm Cd were detrimentally affected
since four of the 16 fish died during the 20-day period.
Thus only two fish were available for the regression
analyses for two sampling dates. This small sample size as
well as the greater number of samples toward the beginning
of the experiment were sufficient to prevent the regression
equations from showing a significant difference in growth

between control and exposed fish at hot temperatures.

 

ij‘. ..r-l v ' V

Figure 17.

107

Mean weight of green sunfish (the points)
and the regression line of the mean weight
against time for three different temper-
atures and two levels of.cadmium stress.
The number of fish comprising each mean is
shown with a dark vertical bar on only one
side of the point for clarity; standard
errors less than 0.5 are not shown.

19

15

...;
UI

WEIGHT 3N GRAKS
O

15

108

f EXPERIMENT l3-F

GROWTH OF GREEN SUNEiSH
‘ " ‘ A—A CONTROL

' UNDER CADMIUM STRESS - 9-19 1 ppm CO

.. 1 ONE FISH DEATH
TOXICANT

 

10L} 17 ' 5 C INTRODUCED
(8) J1"
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109

Food conversion ratios (Figure 18) reflected the same
trends as those found for growth (Figure 17). The analysis
of variance again showed a significant temperature x stress
interaction (Table 27), due mainly to fish exposed to cad-
mium at medium temperatures possessing average food conversion
ratios of 0.38 compared with the control value of 0.32. This
trend is opposite to that exhibited by fish at cold and hot
temperatures, where cadmium-exposed fish had consistently
lower food conversion ratios than control fish. Food con-
version ratios over time (the significant effect of period)
declined consistently from 0.40 to 0.24. It was concluded
that cadmium had an adverse effect on energy utilization at
cold and probably hot temperatures, but energy utilization was
not affected at medium.temperatures.

RNA-DNA ratios were variable among individuals, and no
obvious differences were apparent between control and cadmium-
exposed fish, with the exception of fish at high temperatures
(Table 28). Ratios appeared to increase to a maxima in fish
at the three temperatures, the time to reach it being
inversely related to temperature. Maxima were reached on
day 16 for fish at cold temperature, on day 10 for fish at
medium temperature and on day 6 for fish at hot temperature.
Analysis of these data (Table 29) showed that stress did not
have a significant effect on RNA-DNA ratios. Temperature and
a temperature x period interaction were found to be signifi-
cant. The temperature x period interaction is related to the

previously discussed fact that RNA-DNA ratios appeared to

 

Figure 18.

110

Food conversion ratios of fish exposed
(after a 4—day acclimation period) for
20 days (stippled area) to 1 ppm Cd at
three different temperatures. Number of
fish used is given in parentheses. One
standard error is shown with a dark
vertical bar on only one side of the
point for clarity.

lll

5” EXPERIMENT 13- F

,0 L NET NEIOHT CAIN/W EATEN

H CONTROL
G--‘9 1 PPM CD

1

   
   

30*

201'

 

10'

 

 

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FOOD CONVERSION RATIO X 0.0|
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0 4 8 12 16 20 24

112

Table 27. The analysis of variance table for the effects of
three temperatures and two levels of cadmium
(stress) over four periods on the food conversion
ratios of green sunfish.

 

 

 

Sum of Degrees of Mean

Source Squares Freedom Square F Statistic
Temperature 0.3054 2 0.1527 9.15**
Stress 0.0667 1 0.0667 4.00*
T x S 0.1352 2 0.0676 4.05*
Period 0.4556 3 0.1519 9.10**
T x P 0.0646 6 0.0108 0.64
S x P 0.0425 3 0.0142 0.85
T x S x P 0.0892 6 0.0149 0.89
Error 1.1351 868 0.01669
Total 2.2943 95

 

30.05 significance level.
3*0.01 significance level.
4 degrees of freedom were subtracted for dead fish.

2113

 

 

 

Table 284 iMean concentrations of RNA, DNA and the RNA-DNA ratios of green
sunfish from experiment 13-F before and during continuous
exposure to 1 ppm cadmium at three different temperatures for
20 days. DFFT is dry fat-free tissue from the dorsal muscle
excluding skin. Standard error is enclosed in parentheses.

Day pg DNA per pg RNA per RNA‘DNA
100 mg DFFT 100 mg DFFT Ratio
Cold 0 19.4(1.6) 302.1(29.2) 16.00(2.58)
6 l9.9(2.2) 502.5(71.2) 26.62(4.68)
10 23.6(2.8) 506.7(35.5) 22.34(3.60)
16 l7.3(2.3) S70.1(44.6) 28.84(1.48)
24 17.3(1.0) 506.7(37.9) 28.84(1.48)
Cold 0 - — -
Stressor 6 23.6(2.5) 481.0(48.4) 21.09(3.59)
10 17.8(3.9) 411.2(65.2) 27.65(9.43)
16 19.4(2.0) 655.1(32.S) 34.28(1.75)
24 18.8(2.1) 473.1(34.6) 26.14(3.3S)
Medium 0 25.2(2.3) 316.3(52.0) 12.88(2.52)
6 26.2(S.0) 581.1(85.9) 25.78(7.3S)
10 22.0(3.6) 760 0(169.S) 38.82(11.30)
16 22.6(1.8) 485.2(21.2) 22.36(2.62)
24 25.2(2.3) 509.3(45.1) 21.18(2.27)
Medium 0 - - -
Stressor 6 24.7(2.9) S62.8(163.0) 21.94(3.87)
10 26.2(2.6) 760.0(202 0) 3l.80(10.50)
16 18.9(l.9) 434.8(40.1) 23.28(1.56)
24 15.7(l.4) 388.1(4l.2) 25.82(4.98)
Hot 0 22.0(1.0) 317.3(10.2) 14.44(.69)
6 22.0(2.2) 688.1(90.6) 33.44(7.04)
10 24.7(1.8) 360.9(34.5) 14.59(.73)
16 21.5(2.3) 461.6(30.6) 23.00(4.09)
24 19.4(3.7) 366.1(62.9) 22.18(6.86)
Hot 0 - - -
Stressor 6 23.6(3.S) 503.5(49.3) 21.S6(1.11)
10 23.1(1.5) 448.4(4S.3) 20.05(1.73)
16 24.1(2.8) 421.2(54.2)_ l7.94(3.07)
24 15.7(3.1) 263.3(101.1) l7.31(4.87)

 

114

Table 29. The analysis of variance table for the effects of
three temperatures and two levels of cadmium
(stress) over four periods on the RNA-DNA ratios
of green sunfish. -

 

 

 

Sum of Degrees of Mean
Source Squares Freedom Square F Statistic

Temperature 729.18 2 364.59 3.31*
Stress 98.48 1 98.48 0.89
T x S 52.49 2 26.24 0.24
Period 82.02 3 27.34 0.25
T x P 1745.60 6 290.93 2.64*
S x P 227.99 3 75.99 0.69
T x S x P 367.74 6 61.29 0.56
Error 7933.54 72 110.19

Total 11237.03 95

 

h0.05 significance level.

115

increase to a maxima in fish, the time to reach it being
dependent on temperature. '

Whole-body cadmium uptake by green sunfish was not as
greatly affected by temperature as expected (Figure 19).
This might be partially related to the fact that dissolved
oxygen concentrations (Table 25) were similar in all tanks,
so that decreased oxygen at higher temperatures and concomi—
tant cadmium uptake due to increased ventilation rates did
not occur to any significant degree. Such a correlation
between respiration and uptake was found by Murphy and
Murphy (1971). Lloyd (1961) also demonstrated that the
toxicity of many compounds to fish increased almost equally
when oxygen was reduced by the same amount. He concluded
that increased toxicity was due to increased respiratory
irrigation bringing more of the toxicants to gill surfaces.
After 20 days exposure control fish at all temperatures con—
tained about 1 ppm Cd. Cadmium-exposed fish at cold tempera-
tures accumulated 2 ppm or doubled their concentration over
control levels after 2 days exposure to 1 ppm Cd and then
remained at about 1.5 ppm for the remaining times. Exposed
fish at medium and hot temperatures accumulated increasing
amounts of cadmium the longer they were exposed to the
stressor with no equilibrium values reached after 20 days.
Analysis of variance for these data showed a highly signifi-
cant effect of cadmium stress (F = 156.54) on cadmium uptake
by these fish (Table 30). However, there was a significant

temperature x stress as well as a temperature x period

 

Figure 19.

116

Whole-body concentration of cadmium on a
wet—weight basis in surviving green sun—
fish before and during continuous exposure
to 1 ppm Cd at three different tempera-
tures. Each point represents four fish
except as otherwise noted by the number in
parentheses. One standard error is given
by a dark vertical bar on only one side of
the point for clarity.

1

0

PPM CD - WET WEIGHT BASIS
|.—I N

117

r EXPER|MENT I3-F CADMIUM UPTAKE BY

‘TOXlCANT GREEN SUNFISH (NNOLE BODY)
INTRODUCED

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DAYS

118

Table 30. The analysis of variance table for the effects of
three temperatures and two levels of cadmium
(stress) over four periods on the whole body burden
of cadmium in green sunfish.

 

 

 

Sum of Degrees of Mean

Source Squares Freedom Square F Statistic
Temperature 5.06 2 2.53 11.48**
Stress 34.45 1 34.45 156.54**
T x S 2.41 2 1.20 5.47**
Period 2.63 3 0.88 3.98*
T x P 3.12 6 0.52 2.36“
S x P 0.37 3 0.12 0.56
T x S x P 2.42 6 0.40 1.83
Error 14.97 a68 0.22
Total 65.41 95

‘0:05 significance level.
3*0.01 significance level.
4 degrees of freedom were subtracted for dead fish.

119
interaction. Examination of the table of means for the
effect of stress (averaged over period) at each of the temp-
eratures showed a trend toward increasing amounts of cadmium
in both control and stressed fish the higher the temperature.
Some overlap at the highest temperature was observed, however,
undoubtedly contributing to the significant interactive
effect. The other interaction of temperature x period
averaged over stress showed that fish at cold temperatures
initially accumulated the highest levels (1.40 ppm Cd) but
thereafter lost cadmium. This trend was reversed for fish
at the higher temperatures, thus giving a significant inter-
action.

Patterns of accumulation in the gills of green sunfish
at these three temperatures were similar, although more
variable, than trends established for whole—body burdens
(Figure 20). The analysis of variance (Table 31) indicated
that only stress had a significant effect on cadmium uptake.
Since there was no significant effect of time, it can be con—
cluded that the concentration of cadmium in fish gills reached
an equilibrium level at least over the first 20 days of
exposure to cadmium. Data of Mount and Stephan (1967a),
however, showed that bluegill whole-body levels reached an
equilibrium concentration only after 30 to 60 days, and Eaton
(personal communication) reported values in bluegills exposed
at lower concentrations for 11 months that were much higher
than I observed. In addition, the analysis of variance
indicated that accumulation of cadmium by gills was the same

at all temperatures.

 

Figure 20.

120

Cadmium uptake on a wet—weight basis by
the gills of surviving green sunfish
before and during continuous exposure

to 1 ppm Cd at three different temper-
atures. Each point represents four fish
except as otherwise noted by the number
in parentheses. One standard error is
given by a dark vertical bar on only one
side of the point for clarity.

121

3" EXPERIMENT l3-F
' H CONTROL

CADMIUM UPTAKE BY e-—
GREEN SUNFISH GILLS v *9 1 PPM CD

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122

Table 31. The analysis of variance table for the effects of
three temperatures and two levels of cadmium
(stress) over four periods on green sunfish cadmium
uptake in the gill.

 

 

 

Sum of Degrees of Mean

Source Squares Freedom Square F Statistic
Temperature 1.78 2 0.89 0.71
Stress 40.35 1 40.35 32.04**
T x S 2.90 2 1.45 1.15
Period 1.16 3 0.39 0.31
T x P 2.21 6 0.37 0.29
S x P 1.38 3 0.46 0.37
T x S x P 4.55 6 0.76 0.60
Error 85.62 a68 1.25
Total 139.96 95

 

Ti0.01 significance leve1.#—
4 degrees of freedom were subtracted for dead fish.

123

Cadmium accumulation in the livers of fish followed the
same trends established for whole-body and gill cadmium
uptake (Table 32). All main effects and a stress x period
interaction were significant (analysis of variance-—Tah1e 33).
For the liver, cadmium uptake apparently was affected by
temperature. The stress x period interaction showed that
the effect of cadmium stress on uptake by the liver was not

consistent across time.

Experiment lO-S

The possibility of fish being exposed to high concentra-
tions of cadmium for short periods of time in a river situa-
tion prompted an investigation of the effects such a tempor-
ary exposure would have on fish. An experiment was designed
involving 17 static aquaria with 10 green sunfish per aquarium.
Three sets of fish were exposed in the flow—through apparatus.
The first set of fish was exposed for 24 hrs and included the
control and 10 fish each at 5, 10, 20 and 30 ppm Cd (Table
34), as higher concentrations were shown to kill all fish in
24 hrs. The second set of fish was exposed for 1 hr to 5,
10, 20, 30, 40 and 50 ppm Cd (Table 34), and the third set
of fish was exposed for 15 min to the same six concentra-
tions as above. These fish ranged from 4.51 to 5.50 9 (see
Appendix Table B, Collection 12, 13) and were exposed in
order: 24 hrs, then 1 hr and lastly 15 min. Immediately
after exposure fish were weighed and transferred to the 17
static aquaria containing around 22 l (10 gal) of water and

an unlimited supply of Gambusia. Fish were placed in the

 

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125

Table 33. The analysis of variance table for the effects of
three temperatures and two levels of cadmium
(stress) over four periods on green sunfish cadmium
uptake in the liver.

 

 

 

Sum of Degrees of Mean

Source Squares Freedom Square F Statistic
Temperature 61.11 2 30.55 5.56**
Stress 71.14 1 71.14 12.94**
T x S 15.82 2 7.91 1.44
Period 60.59 3 20.20 3.67*
T x P 55.10 6 9.18 1.67
S x P 46.91 3 15.63 2.84*
T x S x P 33.27 6 5.55 1.01
Error 373.81 ass 5.49

Total 717.73 95

‘0.05 significance level.
;*0.0l significance level.
4 degrees of freedom were subtracted for dead fish.

 

126

 

 

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.sm capes

127

static aquaria and fed for 3 days prior to cadmium exposure
in the flow-through apparatus. The experiment was conducted
for 63 days, including the first 3 days prior to toxicant
treatment, and 60 days of post-treatment feeding in the 17
cadmium-free aquaria. The experiment extended from
September 4 to November 4, 1971. Water in the static aquaria
was changed every 4 days for the first 28 days, then changed
every 8 days for the remaining time. Mean water temperature
for these aquaria varied between 20.5 and 21.7 C while mean
dissolved oxygen concentrations ranged from 6.6 to 8.4 ppm
(Table 34).

Mortality during exposure to cadmium was only recorded
at the highest concentration during the 24 hr exposure, when
seven of ten individuals exposed to 30 ppm Cd died (Table 35).
Post-exposure mortality followed no consistent trends,
although six of the ten deaths were from exposure at the three
highest concentrations of 30, 40 and 50 ppm Cd (see Table 35).

Growth of fish from all treatments was essentially indis-
tinguishable from the control through day 40 (Figure 21).
Thereafter some differences became apparent. Fish exposed
for 15 min to the six concentrations of cadmium exhibited
growth curves which were all lower than the control, except
fish exposed to 5 ppm Cd. The difference in mean weight
between the control and stressed fish from the 15 min expo-
sure ranged from 1 to 5 g on day 63. Fish exposed to 5 ppm
Cd in both the 15 min exposure and 1 hr CXposure grew at

rates similar to or greater than control rates. For the 1

128

Table 35. Time of death and final weight of fish exposed for
short periods of time to various concentrations of
cadmium in experiment lO-S.

 

 

 

 

Type of Stress Final
Aquarium Time of Exposure Weight
No. Death (Days) Time (ppm Cd) (g)
2 l 24 hr 30 4.67
1 24 hr 30 5.07
1 24 hr 30 .10
1 24 hr 30 .14
1 24 hr 30 4.21
1 24 hr 30 4.66
1 24 hr 30 5037
4 12 15 min 40 5.90
18 15 min 10 5.44
7 18 15 min 50 4.18
18 15 min 50 5.11
1 22 24 hr 10 5.13
10 24 1 hr 30 5.23
11 25 15 min 5 4.66
1 31 24 hr 10 5.23
7 48 15 min 50 6.17

52 15 min 40 4.31

 

 

Figure 21.

129

Mean weight of green sunfish before

(3 days) and 60 days after 15 min, 1 hr
and 24 hrs of continuous exposure to
various concentrations of cadmium. Each
point represents the mean of 10 fish
unless deaths decreased this number.
Standard errors are found in Appendix
Table K, L, M.

HEIGHT IN GRAMS

ab

12

10

12

H
O

a.

12

10

p

p

T

 

130

"EXPERIMENT Io-s

GROWTH OF GREEN SUNFISH AFTER ’ ,2.

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H 5 PPM CD 15 MIN EXPOSURE TO CADMIUM

H10 PPM CD

    

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21.0 C
L 1 l L 1 J
’ GROWTH OF GREEN SUNFISH AFTER
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H CONTROL GROWTH OF GREEN SUNFISH AFTER ,5?
- +——I- 5 PPM co 24 HR EXPOSURE TO CADMIUM ,,
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131
hr exposure growth curves were similar to those observed for
fish exposed for 15 min. All concentrations of cadmium
except 5 ppm inhibited growth to some degree when contrasted
with the control. Fish exposed for 24 hrs exhibited a clearly
interpretable relationship. Cadmium at 5, 10, 20 and 30 ppm
decreased growth when compared with the control. This became
apparent after day 40 when control fish grew at a greater
rate than any treated fish. This judgment was not confirmed
by statistical determinations. Variability as discussed
previously was Operating as the range in fish weights became
extreme with time, this same type of phenomenon being observed
by Brown (1946) and commented on extensively in Brown (1957,
p. 372) as the hierarchy effect. She suggested the size-
hierarchy effect was related to the order of dominance, with
the largest, dominant fish growing fastest. Carline (1968)
as cited in Warren (1972) also found a hierarchial effect
among coho salmon. Although food was unlimited to all fish,
dominant Group I fish prevented fish in subordinate groups
from obtaining all the food they would otherwise have con-
sumed. This effect was greatest with this static test (lO-S)
since individual fish were not separated as in flow-through
tests.

Food conversion ratios calculated for all treatments
followed an irregular, but similar pattern (Figures 22, 23,
24). Initially, because fish were not fed the 24 hrs during
treatment of the first group, and because of the stress

associated with exposure to cadmium, most fish recorded a

 

Figure 22.

132

Food conversion ratio of green sunfish
over 4 or 8-day intervals. Fish were
exposed for 15 min to various concen-
trations of cadmium. Each point repre-
sents the mean of 10 fish except where
deaths reduced this number.

133

50' EXPERIMENT '0-3 NET wr GAIN/GAMBUSIA EATEN

15 MIN EXPOSURE
. TO CADMIUM +—+ CONTROL

H 5 PPM CD \
H10 PPM CO

’°+ $ A
1‘ .
20L ‘ “‘ :.
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30 "I "I o

 

 

Figure 23.

134

Food conversion ratio of green sunfish
over 4 or 8-day intervals. Fish were
exposed for 1 hr to various concentra—
tions of cadmium. Each point represents
the mean of 10 fish except where deaths
reduced this number.

X 0.0l

FOOD CONVERSION RATio

‘0

30

20

10

40

5“ EXPERIMENT Io-s

135

NET W'! GAIN/CAMBUSIA EATEN

1 HOUR EXPOSURE +——i~ CONTROL "——‘_

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H 10 PPM CD

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136

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138

weight loss even though considerable amounts of Gambusia

 

were eaten prior to that time. Thus all values for food
conversion ratio were zero. Thereafter considerable varia-
tion was found in the ratios, but no large differences or
trends were noted between control values and those of the

16 treatments. Thus short-term exposures to cadmium apparent-
ly did not affect food conversion efficiency in the days

after exposure.

To determine if fish retained cadmium after exposure,
cadmium whole-body burdens were determined for a pooled
sample of all live fish remaining in each treatment tank
after the experiment was ended (Table 36A). It was not
known how much cadmium was taken up by fish from each treat-
ment during exposure to cadmium and before introduction to
clean water, but it was suspected in some cases to be con—
siderable. For example, live fish from experiment ll-F
exposed to 15 ppm Cd for 12 days contained 22 ppm (Table 18).
Dead fish exposed to 15-50 ppm Cd contained from 74 to 148
ppm Cd in their bodies. Results of experiment lO-S indicated
there was no significant long-term storage of cadmium by these
fish as all values (Table 36A) were less than 1 ppm which
was the amount found in control fish from all previous exper-
iments. Comparative data also support this contention
(Table 368).

In conjunction with this experiment, I became interested
in cadmium uptake in natural populations which may be exposed

to cadmium. Therefore 12 green sunfish (14.65 i 4.15 g) were

139

 

 

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scum NOCNHH mCOHud> CH ssHsuNo mo mCocusn avopnOHonz CO «paw o>Hpauanoo oaom .m n OHpae

141
collected on June 15, 1972 below the East Lansing Waste-

Water Treatment Plant outfall assuming that this area was
most likely to be contaminated by cadmium. No analysis

of the water was made. The mean whole-body cadmium content
of collected fish was low, 0.16 i 0.06 ppm Cd on a wet-
weight basis, which was the lowest concentration of cadmium

found among the sets of control fish collected from ponds.

SUMMATION DISCUSSION

Common Responses to Stress

In almost all experiments conducted a number of recur-
ring salient features became obvious. When first exposed to
higher concentrations of the toxicant, fish ceased feeding
and even regurgitated food. Decreased feeding resulted in
decreased growth of fish in succeeding periods. With ammonia
as the toxicant, fish eventually adapted and after a period
of time began to feed at a rate similar to that of controls.
Even if fish adapted to the stress, as in the case of ammonia,
their initial loss of weight was slowly if ever regained dur-
ing the course of the experiment. Lloyd and Orr (1969) have
also documented an acclimation response by rainbow trout
exposed to ammonia. For cadmium at high concentrations, an
initial decline in feeding by exposed fish was observed,
but recovery and adaptation were small or nonexistent.

Among low concentrations Of cadmium and ammonia, one
concentration stimulated food Consumption so that growth of
these fish exceeded that of controls. Smyth (1967) discussed
this phenomenon at length, referring to it as the "peck-of—
dirt maxim" and as "sufficient but not overwhelming challenge."
He discussed radiation data which showed that mice exposed to
low levels Of x—rays lived longer than controls. The suffi-
cient challenge phenomenon in the medical field is named the

142

143

Arndt-Schulz law which is the basis of all chemotherapy.
Smyth maintained that small, nonspecific responses measured
in chronic toxicity studies are readjustments or adaptations
to a non-lethal stress and show the well-being of an animal
which is healthy enough to maintain homeostasis. He also
stated that among animals exposed to various concentrations
of a compound some responses will be indistinguishable from
control responses. Between these and higher concentrations
will be a narrow range in which exposed animals will perform
better than controls, while concentrations above those in
this range will cause dose-related injuries. This phenomenon
was recorded in experiments lO—F and 7-F (ammonia) and in
experiment 12-F (cadmium). A number of toxological studies
in the literature also have noted such a response, but
either failed to recognize it as a beneficial effect or
merely alluded to that possibility. McKim and Benoit (1971)
for example, found that low concentrations of copper caused
greater growth in brook trout when compared with controls.
Pickering and Gast (1972) found that egg production by fat-
head minnows under some concentrations of cadmium was twice
that Of controls, and suggest this may be an example of
"sufficient Challenge". Pickering (1968) Observed that low
concentrations Of zinc promoted growth of bluegills, while
Cope, g£_§;, (1970) found that growth of bluegills exposed
to 2,4-D when compared with controls was highest at the low-
est concentrations. Cairns, gghgl, (1967), in another
example, found that guppies grew better than controls when

exposed to low levels Of dieldrin.

144

I was consistently able to show a Stimulation Threshold
Concentration (STC), defined as that concentration of a
compound which over a long period of time promotes growth
greater than controls. For ammonia this level was 2 ppm as
N, while for cadmium it was 0.05 ppm Cd. This concept could
be important in establishing water quality criteria for fish.
Mount and Stephan (1967b) have proposed use of an LFPI
(Laboratory Fish Production Index) which involves continuous
exposure of fish (usually fatheads) to various concentrations
of toxicant through at least one generation. A "no-effect"
level is then obtained by comparing one or more of the toxi—
cant levels with the control and determining by mortality,
survival and growth of eggs, larvae and adults, which con-
centrations are "safe". My experiments are more complicated
than EPA bioassays in one respect, since they require procure-
ment and care of a large number of prey. However these exper—
iments were considerably shorter than the 1 year or 1 life
cycle required for some of the Environmental Protection
Laboratory assays (McKim and Benoit, 1971; Pickering and
Gast, 1972; Brungs, 1971; Mount and Stephan, 1967b). Ammonia
(2 ppm) stimulated growth of pumpkinseeds after 4 days and
growth of green sunfish after 10 days, while 0.05 ppm Cd

stimulated Gambusia consumption by green sunfish after 10

 

days exposure.’ With some further confirmatory experiments,
the STC might be used as a preliminary "safe" level in
setting water quality criteria. Although it requires more

time, the STC also would be a more apprOpriate method for

145

determination of the toxicity of many compounds now being
judged solely with LC50 data using death as the criterion.
Preliminary data for bluegills exposed to cadmium (John
Eaton, personal communication) are available to compare with
results of my experiments on green sunfish. Eaton exposed
groups of 18 fish for 11 months to 0.03, 0.08, 0.24, 0.74
and 2.14 ppm Cd. All fish died in the 0.74 and 2.14 ppm
tanks, while 16, 9 and 0 fish died at 0.24, 0.08 and 0.03
ppm, respectively. He estimated a "Safe" concentration to
be 0.03 ppm Cd on the basis of the above mortality data,
spawning success and survival of larvae. Schweiger (1957),
as cited in McKee and Wolf (1963), found that 0.03 ppm Cd
was not harmful to one and two-year old tench, carp, rainbow
trout and char, nor to the crustacea, worms and insect larvae
on which they fed. Thus, depending on how the STC is regard-
ed, a "safe" level for green sunfish in my experiments could
be set at 0.05 ppm Cd if the stimulation of growth is viewed
as beneficial, or at less than 0.05 ppm Cd if the effect is
regarded as abnormal. For ammonia, I would set a "safe"
level around 2 ppm as N for green and pumpkinseed sunfish.

McKim, 22.§l° (1970) have also advocated use of a short-
cut method to estimate the "safe" concentration. They found
that after exposure of brook trout to COpper for 6, 21 and
337 days, blood characteristics indicNted fish were under
stress during the first 21 days of exposure, but that an
accommodation or adaptation seemed to occur subsequently

for all blood factors except one. This same pattern was

146

established for bluegills and perch. After comparing the
concentration at which short-term "transient" effects
(6-37 days) occurred with the concentration of copper at
which long-term effects after 337 days were noted, they
concluded that blood measurements made after short exposure
to a toxicant can be indicative of harmful concentrations
which would occur after long—term exposure. Pickering and
Gast (1972) advocated use of acute toxicity studies on the
most sensitive life stage as a good indication of the chronic
"safe" concentration. For fathead minnows, this was the
developing embryo.

I believe that the STC is not a harmful concentration
but that it definitely should not be exceeded, as Eaton
found a small difference between a "safe" cadmium level
(0.03 ppm) and one that can be injurious (0.08 ppm). Another
point to consider with the STC is that uptake of cadmium
by fish exposed to low concentrations is usually considerably
higher than that found in controls. For my experiments,
fish exposed to 0.05 ppm accumulated 1.84 ppm Cd after 20
days of active feeding, while control fish contained 0.65
ppm Cd. In conflict with this belief is Eisler (1971), who
required that cadmium levels of fish from his "no-effect"
concentration be similar to that found in controls. He
found 0.1 ppm Cd was a "safe" concentration for mummichogs
using that criterion. However he did not feed his fish
during this exposure, a somewhat unrealistic procedure since

wild fish would probably feed if not under severe stress and

147

if food was available. The heavy metal accumulation
problem should be checked by monitoring fish for cadmium
uptake over a longer time period to see that accumulation
under low levels of exposure does not increase above lethal

levels.

RNA-DNA Data

Use of RNA-DNA data for detecting sublethal effects of
toxicants was found to have limited worth from reSponses I
measured. Closer monitoring of daily changes due to the
toxicant were more clearly shown with food consumption curves
and to some extent by growth curves. In addition, the tech-
nique was time-consuming and variability in populations of
fish was so extreme that only large differences were detected.
Ratios were determined on the fish from two ammonia experi-
ments and two cadmium experiments. In only one experiment,
7-F (ammonia), was there a significant difference shown with
the RNA-DNA data but not with other data, such as growth or
food consumption. In the other ammonia experiment, there
was a significant depressant effect of ammonia on ratios of
exposed fish when compared with controls. No significant
differences were found among the RNA—DNA ratios of fish from
either of the cadmium experiments (lZ-F and 13-F) despite the
fact that food consumption, growth and mortality data
indicated a detrimental effect of the toxicant. It appears
that RNA-DNA data would be useful in experiments where

either a greater number of samples were used or where less

148

variability existed in experimental fish. RNA-DNA ratios
may be useful in detecting a short—term response before
growth has occurred, since in the early days of toxicant
introduction the ratios did monitor growth fairly well. My
data as well as those of Bulow (1970) have shown that values
tended to reach asymptotic levels which then changed little
over time. This asymptotic level was reached earlier the
higher the temperature in experiment l3-F (Table 28).
Another advantage of this technique which may have applica-
tion to toxicity research is that measurement of ratios in

a field situation could be used to assess growth rate occur-
ring at a given time once base levels were established.
Measurement of the ratios from a number of fish from the
field could then be compared with the levels established for
slow-growing and fast-growing fish, or fish from above and

below a point of toxicant introduction could be compared.

Cadmium Uptake Data

Dead fish accumulated considerably more cadmium than
live fish at the same concentration so that dead fish were
always kept separate from live fish in data analysis. This
effect was also noted by Eisler (1971), while Mount and
Stephan (1967a) proposed this difference as a means for
detecting cadmium poisoning in fish. Higher concentrations
of cadmium in dead fish when compared with live fish at the
same concentration were probably due to absorption from the

test medium and breakdown of detoxification mechanisms which

149
would mask any threshold concentration these fish may
have reached before death. Gills of dead fish usually had
the highest concentrations of cadmium among all structures
studied, presumably because gills secrete large amounts of
mucus, which complexes cadmium. It has also been shown that
fish increase their respiratory ventilation rate during
stress (Chiszare, gt al., 1972), which should also increase
cadmium uptake. Eisler (1971) devised an experiment to
determine how much cadmium dead and live fish could take up
and eliminate. He found that dead mummichogs accumulated 53
times as much cadmium in 24 hrs as live fish and 89 times as
much as live fish exposed for 48 hrs. He found average loss
of whole-body cadmium 24 hrs after treatment by fish surviv—
ing exposure for 24 and 48 hrs was 40 and 12%, respectively,
while dead fish lost 48 and 36% after transfer to clean
water. His data seemed to indicate that a considerable
amount of passive diffusion was involved in uptake and elim—
ination processes.

To judge if abnormal accumulation of cadmium has occurred
in fish obtained from a fish kill or a field monitoring
situation, background levels are necessary. For green sun—
fish, concentrations in the whole body of control fish were
consistently the least variable of the three structures
measured. Control fish contained about 1 ppm Cd or less on
a wet-weight basis for the 19 sets of determinations of fish
(sample size from 4 to 8)., For the gills this value was

slightly more variable, but still about 1 ppm Cd. In the

150

liver mean values were the most variable, between 0 and

4.79 ppm Cd. Data from experiment 10-5 and those of Lucas,
‘gt,al. (1970); Hesse and Evans (1972); Mathis and Cummings
(1971); Uthe and Bligh (1971) and Lovett, gt a1. (1970) seem
to indicate that exposure of fish to abnormal concentrations
of cadmium should be suspected if whole-body burdens exceed
much over 2 ppm Cd.

Among structures measured, both in control and exposed
fish, liver cadmium concentrations were usually higher than
those in the gill and whole body. This result was also
reported by Mount and Stephan (1967a), who analyzed a number
of other structures (bone, muscle, gut, spleen and kidney)
and found that only gill and liver showed dose-related uptake.
Buildup of cadmium in the liver of bluegills exposed for 11
months to 0.03 to 0.24 ppm was between 218 and 524 ppm Cd
on a wet-weight basis (data of John Eaton, personal communica—
tion). The highest levels of cadmium I found in fish exposed
to low levels of cadmium for 20 days (Table 24C) was 5 ppm.
Fish exposed to 15 ppm for 16 days accumulated an average
of 42 ppm in the liver (Table 18). Mount and Stephan found
that the liver took up negligible quantities of cadmium when
fish were exposed to lethal concentrations for short periods.
However fish that died in this study which were exposed at 30—
50 ppm Cd had livers with mean cadmium levels ranging from
33 to 39 ppm.

Good agreement was found among experiments in the con-

centration of cadmium found in fish exposed to comparable

151
concentrations. For example, whole—body burden of cadmium
for fish in experiment ll-F exposed to 3.83 ppm Cd at 19.9
C was 3.99 ppm Cd. Gills contained 3.22, while liver con-
tained 4.27 ppm Cd. These values compared well with fish
exposed in experiment 12-F at a cadmium concentration of 2.48
ppm in the water and a temperature of 18.6 C. Whole-body
burdens were 2.82 ppm, gill levels were 2.55 ppm and liver
values were 5.08 ppm Cd.

It is postulated that uptake under exposure to high con—
centrations of cadmium reached a threshold concentration,
above which fish died (Table 18). For experiment ll—F this
value was about 20 ppm for accumulated cadmium in whole body
and gills; for the liver the value was 40 ppm. Thus it can be
stated that any fish which has accumulated levels of cadmium
comparable to threshold levels (20 ppm in the whole body) was
probably exposed to high concentrations of cadmium and is in
danger of death. Mount and Stephan (1967a) reported that an
equilibrium concentration of cadmium was found in bluegill
gill and liver samples between 30 and 60 days. They also
suggested a threshold value was apparently reached for cadmium
in the gill and that death occurs when this value is exceeded.
Eisler (1971) found a threshold concentration of cadmium in
the body of mummichogs in excess of 86 mg Cd/kg ash (dry—
weight basis).

Fish in all my experiments were given an unlimited supply
of food. While this approximates natural environments in

that fish must actively seek their prey, in some cases this

152
approach does not reflect the natural situation, since prey
are hardly ever unlimited in nature. Thus fish exposed to
toxicants in my experiments were able to resist adverse
effects of the toxicant more vigorously than would fish fed
on a limited diet. This type of experiment has been docu-
mented by Chapman (1965), as cited in Warren (1971). Chapman
found that cichlids exposed to potassium pentachlorophenate
in low concentrations and given unlimited food behaved exactly
as did some fish in my experiments. Exposed fish initially
consumed less, but then began to consume considerably more
until their growth was equal to or surpassed growth of
controls. A group of these same fish exposed at the same
concentrations, but given a limited food supply, were not
able to grow as fast as control fish. This was explained by
the mechanism of toxic action of the chemical, which uncouples
oxidative phosphorylation leading to a decrease in the
efficiency with which energy is utilized to maintain life
processes. Thus exposed fish, by consuming more food, com-
pensated for decreased efficiency of energy utilization and
attained the size of those not poisoned, while stressed fish
fed limited amounts of food could not compensate. In my
studies on ammonia (experiment 6-F) feed conversion efficiency
was generally lower for exposed fish at all three temperatures
when compared with controls. No difference was found among
food conversion ratios in experiment 7-F, while in lO-F only
an initially low value was recorded, but thereafter exposed

fish exhibited the same food conversion efficiency as

153

controls. For cadmium, fish exposed to 3, 7 and 15 ppm

in ll-F possessed conversion efficiencies less than control
values, while in 12-F there was little difference between

the control and exposed fish. In the factorial experiment
13—F, there was a definite depression of food conversion
efficiency of the exposed fish at cold and hot temperatures
when compared with controls. Thus it appears that the mechan—
ism discussed above (Chapman, 1965) was not occurring with my
fish, since fish exhibiting low efficiencies seldom consumed
enough to grow as well as controls. Conversely, those fish
that were stimulated to consume more than controls possessed
food conversion efficiencies comparable to or greater than
control fish efficiencies.

Temperature did not have a significant effect on cadmium
uptake (Figure 18, 19, Table 32) even though it was definitely
established that fish at higher temperatures ate and grew more
than fish at lower temperatures. Failure to observe increased
cadmium uptake in fish at higher temperatures could be rela—
ted to the higher rate of metabolism causing detoxification
and elimination mechanisms to be considerably greater and
thus off-set increased uptake. In addition dissolved oxygen
levels were comparable among treatments which would also tend
to equalize uptake rates, since ventilation rates would not
be increased because of lower dissolved oxygen at higher
temperatures. In view of the increased possibilities of
additional heating of our waters, the interaction of temper-
ature with uptake relationships of heavy metals certainly

deserves more investigation.

154

One aspect investigated was whether short exposures of
fish to high concentrations of cadmium exerted an effect on
fish at some time after exposure (experiment lO-S). Gardner
and Yevich (1970) eXpressed concern over post-exposure
effects and Bonnell, 23 a1. (1960) noted that cadmium—caused
anomalies became progressively greater even though exposure
had ceased. In another study Knoll and Fromm (1960) found
rainbow trout exposed to chromium were found after return to
non-toxic water to have selectively retained chromium in the
kidney and spleen while other major sites such as gills and
liver showed a rapid decline. In experiment lO-S, approxi-
mately 6 percent of the fish surviving the initial exposures
died during the 60 days following exposure to various concen-
trations of cadmium. Whole-body cadmium content of these
fish (about 0.6 ppm Cd) after 60 days was comparable to that
of control fish in my other experiments and to concentrations
found in fish from a number of surveys in Michigan, the
Great Lakes, New York and Canada. Thus cadmium was eliminated
by fish that were transferred to uncontaminated water. In a
study similar to mine, Eisler (1971) found that mummichogs
experienced post-treatment mortality which was a direct
function of initial cadmium concentration and exposure period.
Final cadmium concentrations in the groups of fish were still
proportional to initial exposures although considerably

lower.

155
Discussion of the Toxic Action of Ammonia and Cadmium

The main equations governing the behavior of ammonia in

water are: NH

+ H O -* NH + + 0H-

3 2 *’ 4 4

Thus ammonia (NH3) upon dissolution in water forms ammonium

OH-‘z‘NH

hydroxide which in turn dissociates to form an ammonium ion
plus a hydroxide ion. The toxic component (NH3) is thus a
function of pH, the higher the pH the more toxic the solution.
Downing and Merkens (1955) stated that reduction in pH of
water from 8.0 to 7.0 resulted in a tenfold decrease in the
concentration of un-ionized ammonia. Spotte (1970) reported
that only NH3 can cross tissue barriers, which means that
diffusion of ammonia will occur in the direction of lower pH
(greater number of hydrogen ions). Fromm and Gillette (1968)
stated that the free base (NH3) is able to diffuse across all
membranes easily because of its lipid solubility and lack of
charge, whereas the ammonium ion (NH4+) penetrates membranes
less readily because it is hydrated, charged and has low-
lipid solubility. Brockway (1950) found that ammonia excre-
tion increased with increased activity, with a rise in water
temperature and after feeding. He noted that fish lost the
ability to use oxygen when the concentration of ammonia in
water increased, since blood carbon dioxide increased about
15% causing hemoglobin to take up considerably less oxygen.
Lloyd and Herbert (1960) have shown for salmonids that the
concentration of ammonia at the gill surface is the critical
factor affecting ammonia toxicity. Carbon dioxide excreted

by fish causes a decrease in pH at the gill surface which

156

reduces the amount of un—ionized ammonia at the gill. Thus
depression of the amount of un-ionized ammonia at the gill is
greatest when the carbon dioxide content of the water is low—
est. They also stated that increased toxicity of NH3 would
occur under low dissolved oxygen conditions because decreased
oxygen results in decreased carbon dioxide excretion at the
gills. With decreased carbon dioxide, pH would increase at
the gills, resulting in an increase in the toxicity of
ammonia present, because more would be in the un-ionized
state. This was documented by Downing and Merkens (1955),
who showed increased ammonia toxicity under low dissolved
oxygen tensions.

Fromm and Gillette (1968) found a direct, linear correla-
tion between water NH3 and blood NH3, the blood NH3 being
higher than water NH3. Since blood NH3 concentration always
exceeded the water NH3 level, the increases in blood NH3 were
attributed to inhibition of NH3 excretion rather than inward
transfer of NH3 against a concentration gradient. They also
found that with an increase in water NH3 total nitrogen (and
ammonia) excretion decreased. They further suggested that
increased toxicity of ammonia at higher temperatures is due
to increased metabolism which causes greater production of
internal ammonia (NH3).

Burrows (1964) found that concentrations of un-ionized
ammonia (NH3) as low as 0.006 ppm as N could cause extensive
hyperplasia in the gill epithelium of continuously-exposed

Chinook salmon fingerlings. These fingerlings could tolerate

157
levels of ammonium hydroxide as great as 0.7 ppm for 1 hr
per day without apparent effect, while exposure periods
greater than 12 hrs per day at levels of 0.1 ppm or greater
caused reduced growth rate. Hyperplasia was thought to cause
salmonids to become susceptible to gill disease. Continuous
exposure to 0.1 ppm caused reduced stamina and disease
resistance. Kawamoto (1961) showed a reduced growth rate of
carp exposed to 0.3 ppm ammonium chloride for 3 months.
Spotte (1970) reported that another study exposing fish to
sublethal concentrations of ammonia found gill hyperplasia,
congestion of mucus cells in the skin, abnormal concentration
of blood corpuscles in the epidermis, congested blood vessels
and inflammation of the liver. Ball (1967) found the lethal
threshold (0.3-0.4 ppm as N-nun-ionized ammonia) did not
differ between trout and coarse fish, but more trout than
coarse fish died in the early part of the test.

The toxic action of ammonia can be understood by consid-
ering the role of ammonia in fish metabolism. Nitrogen waste
is usually eliminated as ammonia from blood at the gills by
passive diffusion. Under "normal" conditions urea production
is a minor component in nitrogen waste removal. Fromm and
Gillette (1968) showed that ammonia concentration in trout
blood almost doubled when fish were exposed to high ambient
concentrations of ammonia. At some critical level of blood
ammonia, they noted, a fish must either decrease its sensi-
tivity to ammonia or convert ammonia to a less toxic com-

pound. Further work of Olson and Fromm (1971) showed that

158

trout exposed to high external ammonia levels decreased
ammonia excretion and total nitrogen excretion. Urea
excretion increased slightly for trout, while goldfish
significantly increased urea production in a very short time.
The route of urea production was suggested to be from ammonia
through purine synthesis and catabolism. It is thus suggested
that green sunfish probably adapted to external ammonia levels
by a gradual shift to urea production. Green sunfish did not
succumb at relatively high concentrations of ammonia (25
ppm as N or about 0.5 ppm un—ionized ammonia). In addition
green sunfish grew and consumed food at rates near those of
controls after a certain acclimation period which suggests
this sunfish may have the capacity to rapidly initiate
mechanisms for ammonia detoxification and urea production.

The mechanism of toxic action of cadmium is not known
and may well be different for different conditions of expo-
sure. Eisler (1971), for example, reported that with brief
exposure to high concentrations of cadmium, the gill appeared
to be the primary site of damage, but prolonged exposure to
low concentrations of cadmium affected the intestine, kidney
and possibly other tissues not analyzed. This relation
was generally borne out in literature surveys. Eaton
(personal communication) found highest accumulation in the
liver of bluegills (218-524 ppm Cd on a wet-weight basis)
exposed continuously for 11 months to low concentrations of
cadmium (0.03-0.24 ppm). High levels were also found in

kidney, liver and caecum. Mount and Stephan (1967a) reported

159
that bluegills contained more cadmium in the liver (500 ug/g
of tissue-~dry weight) than gills (130 ug/g of tissue—~dry
weight) of fish exposed over 30, 60 and 90 days. Acute
exposures that resulted in death of fish showed gills to
contain high concentrations of cadmium while the liver con—
tained minimal levels of cadmium.

The fact that gills are severely affected at high con—
centrations was certainly confirmed by the response of fish
in my study. Fish exposed to 30-50 ppm Cd prior to and
after death were covered with excessive mucus secretion, and
considerable fusing of gill lamellae was noted. Gardner and
Yevich (1970) also noted hypertrophy of gill filaments and
hyperplasia of the epithelial surface of the respiratory
lamellae in fish exposed to 50 ppm Cd. Schweiger (1957), as
cited in Eisler (1971), noted that high concentrations of
cadmium were found to cauterize gill lamellae of several
freshwater fishes. Eisler (1971) found the same high rate
of accumulation in gills of adult tautog, a marine fish.

Another finding by Lewis and Lewis (1971) may also be
related to the toxic action of heavy metals. They found
that fish exposed to copper and zinc exhibited a reduction
in blood-serum osmolality which could result in mortality.
The osmotic drOp was principally related to damage of the
head and gill region. They found sodium chloride added to
water containing copper and zinc prevented distress symptoms
and mortality usually associated with exposure to these

heavy metals. Thus they concluded that heavy metals affected

160
the gill area and caused a drop in the salt concentration
of the blood. Another important mechanism explaining the
toxic action of heavy metals is thought to be poisoning of
enzyme systems (McKee and Wolf, 1963). Metals are readily
chelated by organic molecules and cadmium is listed as one
of the metals which could combine with the cell membrane
and affect permeability. Thus transport of sodium, potas-
sium, chloride ions or organic molecules could be affected.
Rupture of these membranes was also listed as a possible
effect. Hiltibran (1971) investigated effects of cadmium
and zinc on energy production as indicated by changes in
oxygen and phosphorus metabolism in bluegill liver mito—
chondria. He found that cadmium and zinc can disrupt energy
production through inhibition of oxygen uptake within the
cells and that this disruption can occur at relatively low
levels of cadmium and be of such severity as to cause death

of fishes, particularly bluegills.

Application Factors

The concept of the LFPI (Laboratory Fish Production
Index) proposed by Mount and Stephan (1967b) involves deter-
mination of a "safe" level or MATC (Maximum Acceptable
Toxicant Concentration). The MATC is the concentration of
toxicant which is judged safe by considering the mortality
and growth of the adults, and eggs and fry derived from them
after long-term exposure (a year or one life cycle) to the

toxicant. The application factor is then derived by dividing

161

the MATC by the 96—hr LCSO‘ These values have been derived
for a number of studies, mainly by workers from the Environ-
mental Protection Agency (Table 37). Two things should be
noted with these data. First, Pickering and Gast (1972) also
gave the lethal threshold concentration value, which is
another value usually equal to the 96—hr LCSO value. It is
defined as that concentration of toxicant found when the
toxicity curve becomes essentially parallel to the time axis.
Using this value in deriving the application factor gives

a value of 0.08—0.13, which is different by a factor of 10
from the tabular value of the application factor (Table 37).
Secondly, for my data the STC (Stimulation Threshold Concen-
tration) was used in determining the MATC. Considering that
the four studies involving cadmium reported here involved
such obvious differences in water conditions as freshwater
versus saltwater, of different species of fish, and of
different water chemistry parameters, agreement among these
four values is very good.

For the ammonia experiments the MATC for both pumpkin-
seeds and green sunfish was judged to be 2 ppm as N. The
LCSO value for pumpkinseeds was 9.4 ppm as N at a temperature
of 12.0 C (see Appendix Table F); for green sunfish the LC50
value was 33 ppm ammonia as N at a temperature of 12.5 C
(see Appendix Table G). Thus application factors for pump-
kinseeds and green sunfish are 0.21 and 0.06 respectively.
Lloyd and Orr (1969) gave an application factor of 0.12 for

trout. The difference between the larger application factors

 

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163

for ammonia as compared with cadmium derives from the
apparent reduced toxicity of ammonia on a relative scale
compared with cadmium. Fish appeared to acclimate readily

to ammonia, while no such acclimation to cadmium was observed.
In addition, cadmium-exposed fish experienced a depression

in feeding rate which remained throughout exposure.

These data and comparative literature data give
considerably more support for advocating the STC (Stimula—
tion Threshold Concentration) as a useful parameter in
toxicity research. The STC, derived using food consumption
and growth as the criteria, is suggested as a more meaningful
parameter from which accurate water quality standards can
be judged and should provide a more productive approach for

evaluating potentially toxic substances.

SUMMARY

Experiment 6-F

Experiment 6-F, performed to evaluate the interaction
of three temperatures with one level of ammonia, was not
completed as designed, because decreased levels of ammonia
and dissolved oxygen were observed at higher temperatures
and confounded results. Temperature exerted a major effect
on fish, the higher the temperature the greater the growth.
In all cases except the latter 10 days at hot temperature,
growth of ammonia-exposed fish was less than that of corres-
ponding controls. The greatest difference occurred at

medium temperature. Amount of Gambusia eaten reflected the

 

same trends as observed with growth data. Food conversion
ratios were variable and in all cases except one were
lower than control values. RNA-DNA ratios, like efficien-
cies were generally lower for stressed fish, particularly
for fish at cold temperature, when contrasted with corres-
ponding control values. A tendency for values to level

off was noted for all treatments.

Experiment 7—F

Pumpkinseed sunfish exposed to concentrations of
ammonia 5.72 ppm and greater were stressed to the point
that feeding was temporarily halted, which affected their
growth and possibly their conversion efficiency.

164

165

When compared with controls, fish exposed to 2 ppm
ammonia as N were stimulated to eat more food which resulted
in these fish possessing a significantly higher RNA—DNA
ratio. Fish at all other concentrations exhibited an
acclimation phenomenon whereby after the initial decrease
in food consumption (and also growth), feeding eventually
was established at a rate comparable to controls. The
recovery was sufficient enough so that values for mean
weight gain and food conversion efficiency, although undoubt-
edly lower after introduction of toxicant, were not dis-
tinguishable from control values at the end of the

experiment.

Experiment lO-F

Fish exposed to ammonia concentrations greater than
1.93 ppm reacted initially by regurgitation of previously
eaten food and cessation of feeding. Fish exposed to 1.93
ppm ammonia as N were stimulated to eat and grow more than
control fish. The long-term detrimental effects on growth
increased and were of greater duration the higher the con-
centration of ammonia. Acclimation was observed only in
the fast recovery Of exposed fish (increased feeding and
growth after a severe decline) in the second and succeeding
4-day periods after initial introduction of toxicant. Food
conversion ratios of fish exposed to 8 ppm ammonia as N and
greater were lower than those of controls during the first
4—day period. Thereafter ratios of all groups of fish were

similar.

166

Experiment ll-F

Results of experiment ll-F showed that concentrations
of cadmium greater than 27 ppm killed all fish within 4
days (LC50 value was 20.5 ppm Cd). At concentrations of
3, 7 and 15 ppm Cd, growth, food consumption and food con-
version ratios were detrimentally affected, since feeding
continued, but little weight increase occurred. These
concentrations of cadmium are to be considered unfavorable
for green sunfish growth, and perhaps eventually would

cause death of the fish.

Experiment 12-F
Green sunfish exposed to low concentrations of cadmium

(0.23 to 2.48 ppm) consumed lesser amounts of Gambusia and

 

grew at a rate lower than controls. Fish exposed to 0.05
ppm, however, were stimulated by this lower level of cadmium,
as amounts of Gambusia eaten increased over that eaten by
controls, and mean weight consistently increased until it
was greater than that of controls at the last sampling
period. Food conversion ratios were slightly depressed in
the first 4 days after toxicant introduction, but thereafter
were indistinguishable from control values. RNA-DNA ratios
increased from initial levels, but no significant differ-

ences were found among treatments after 20 days of exposure.

Experiment l3-F
Evaluation of the effect of 1 ppm Cd on growth and

survival of green sunfish at three different temperatures

167

was based on growth, feeding, food conversion ratios and
RNA—DNA ratios of these fish. At the cold temperature 1
ppm Cd appeared to have little effect on growth and food
consumption. Efficiency of food conversion was depressed
when contrasted with control efficiencies. Cadmium uptake
was double that of controls; however no deaths at cold
temperatures occurred during the duration of the study

(20 days).

At medium temperatures growth, food consumption and
efficiency of stressed fish were indistinguishable from
respective values of control fish. Cadmium uptake was
twice that of control fish.

Fish exposed at hot temperatures were detrimentally
affected by the combination of cadmium and high temperature.
Food consumption of stressed fish was considerably lower
than amounts eaten by control fish, as was growth rate. In
addition four of the 16 fish exposed at 1 ppm Cd and hot

temperature died, as did one control fish.

Experiment lO-S

This experiment demonstrated that short-term exposure
to various high concentrations of cadmium was detrimental
to subsequent growth of cadmium-exposed fish when compared
with control fish. Post-treatment growth of fish exposed
to six concentrations of cadmium for 15 min and for 1 hr
was less, but not significantly, than growth of controls

for almost all treatment combinations. Growth of fish

168
after 24 hrs exposure to 5, 10, 20 and 30 ppm Cd was
severely depressed over that of control fish. Food con-
version efficiencies were extremely variable and differences

among treatments were not consistent.

Cadmium Uptake Data

1. An equation (Y = 0.065 x + 0.316) with an r2 = 0.89
adequately described the uptake curve relating the indepen-
dent variable X (cadmium concentration in the water up to
15 ppm Cd) to the dependent variable Y (log of the cadmium
concentration in the fish + 1).

2. Fish exposed to 3, 7 and 15 ppm Cd accumulated
cadmium in a geometric manner, with highest water concen-
trations causing proportionately greater uptake than the
two lower water concentrations of cadmium. Concentrations
of cadmium greater than 15 ppm killed all fish in 16 days.

3. Whole body, gills and livers of dead fish contained
considerably more cadmium than corresponding live fish at
the same concentrations.

4. Livers of live fish consistently contained highest
levels of cadmium, while for dead fish gills usually con—
tained the highest concentration of cadmium among whole
body, gills and liver.

5. Whole-body burdens of control fish contained a mean
of about 1 ppm Cd on a wet-weight basis.

6. Among structures measured for cadmium content,

whole-body burdens were the most stable and least variable.

169

7. A threshold concentration in whole-body cadmium
content above which green sunfish died was found at about
20 ppm.

8. Fish exposed to the STC (Stimulation Threshold
Concentration) of cadmium (0.05 ppm) for 20 days contained
as much cadmium as fish exposed to almost 2 ppm for the
same time. Higher uptake by fish exposed at 0.05 ppm is
thought to be due to greater food consumption by these fish.

9. Fish exposed at three different temperatures (17.5,
23.9, 30.0 C) to 1 ppm Cd contained similar levels of cad-
mium about twice that of control fish.

10. Cadmium elimination after exposure to high con-
centrations for short periods was complete after 60 days
and probably much sooner.

Stimulation of growth was observed for one low concen-
tration of ammonia (2 ppm) and one low concentration of
cadmium (0.05 ppm). This response, termed the STC
(Stimulation Threshold Concentration), is proposed as a
more accurate method than use of LC50 data and as a shorter
method for obtaining tentative MATC (Maximum Acceptable
Threshold Concentration) values. Application factors for
this and three other studies were compared and found in

good agreement.

LITERATURE CITED

170

LITERATURE CITED

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Ball, I. R. 1967. The relative susceptibilities of some
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Bonnell, J. A., J. H. Ross, and E. King. 1960. Renal lesions
in experimental cadmium poisoning. Brit. J. Ind. Med.
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Bouck, G. R., and R. C. Ball. 1965. Influence of a diurnal

oxygen pulse on fish serum proteins. Trans. Amer. Fish.
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Brockway, D. 1950. Metabolic products and their effects.
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Brown, M. E. 1946. The growth of brown trout (Salmo trutta
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Brown, M. E. 1957. The Physiology of Fishes. Vol. I.
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Brown, V. M. 1969. The prediction of the acute toxicity of
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March 27,28,29, 1969.

Brungs, W. 1969. Chronic toxicity of zinc to the fathead

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Brungs, W. A. 1971. Chronic effects Of low dissolved oxygen
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Brungs, W. A. 1971. Chronic effects of constant elevated
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Bulow, F. 1970. RNA-DNA ratios as indicators of recent
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Burrows, R. E. 1964. Effects of accumulated excretory
products on hatchery-reared salmonids. U. S. Bur.
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Cairns, J. Jr., N. Foster, and J. Loos. 1967. Effects of
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Cairns, J., and A. Scheier. 1959. The relationship of
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chemicals. Proc. 13th Indus. Waste Conf. Purdue
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Carlson, A. 1972. Effects of long-term exposure to carbaryl
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Chadwick, G. G., and R. W. Brocksen. 1969. Accumulation of
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J. Wildl. Mgmto 33:693-698.

Chiszar, D., M. Moody, and J. T. Windell. 1972. Failure of
bluegill sunfish, Lepomis machochirus, to habituate to
handling. J. Fish. Res. Ed. Canada 29:576-578.

Cope, C. B., E. Wood, and G. Wallen. 1970. Some chronic
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Downing, D., and J. Merkins. 1955. The influence of dissol-
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Eaton,J. 1972. Personal communication. National Water
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Eisler, RN 1971. Cadmium poisoning in Fundulus heteroclitus
(Pisces: Cyprinodontidae) and other marine organisms.
J. Fish. Res. Ed. Canada 28:1225-1234.

Eisler, R., and M. P. Weinstein. 1967. Changes in metal
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Eisler, R., G. Zaroogian, and R. Hennekey. 1972. Cadmium
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Fromm, P., and J. R. Gillette. 1968. Effect of ambient
ammonia on nitrogen excretion of rainbow trout

(Salmo gairdneri). Comp. Biochem. Physiol. 26:887-896.

Fujiya, M. 1961. Use of electrophoretic serum separation
in fish studies. J. Wat. Poll. Cont. Fed. 33:250-257.

Gardner, G. R., and P. P. Yevich. 1970. Histological and
hematological responses of an estuarine telost to
cadmium. J. Fish Res. Bd. Canada 27:2185-2196.

Goodyear, C. P. 1972. A simple technique for detecting
effects of toxicants or other stresses on a predator-
prey interaction. Trans. Amer. Fish. Soc. 101:367-369.

Haines, T. A. 1971. Effects of nutrient enrichment and a
rough fish population (carp) on a game fish population
(smallmouth bass). Ph. D. thesis Michigan State
University, 96 pp.

Hesse, J. L., and E. D. Evans. 1972. Heavy metals in surface
waters, sediments and fish in Michigan. Mich. Wat.
Resour. Commis., Bur. Wat Mgmt., Dept. Nat. Resour.

Hiltibran, R. 1971. Effects of cadmium, zinc, manganese,
and calcium on oxygen and phosphate metabolism of blue-
gill liver mitochondria. J. Wat. Pol. Cont. Fed.
43:818-823.

Hogan, R. L., and E. W. Roelofs. 1971. Concentrations of
dieldrin in the blood and brain of the green sunfish,
Lepomis cyanellus, at death. J. Fish. Res. Bd. Canada
28:610-612.

Hunn, J. 1969. Chemical composition of rainbow trout urine
following acute hypoxic stress. Trans. Amer. Fish. Soc.
98:20-22.

Kawamoto, N. Y. 1961. Influence of excretory substances of
fishes on their own growth. Prog. Fish-Cult. 23:70-75.

Knoll, J., and P. O. Fromm. 1960. Accumulation and elimina-
tion of hexavalent chromium in rainbow trout. Physiol.
2001. 33:1-8.

Lane, C., and R. Livingston. 1970. Some acute and chronic
effects of dieldrin on the sailfin Molly, Poecilia
latipinna. Trans. Amer. Fish. Soc. 99:489-495.

 

174

Lewis, S., and W. Lewis. 1971. The effect of zinc and
copper on the osmolality of blood serum of the channel
catfish, Ictalurus punctatus Rafinesque, and golden
Shiner, Notemigonus crysoleucas Mitchell. Trans. Amer.
Fish. Soc. 100:639-643.

 

 

Lloyd, R. 1961. Effect of dissolved oxygen concentrations
on the toxicity of several poisons to rainbow trout
(Salmo gairdnerii Richardson). J. Exp. Biol. 38:447—
455.

Lloyd, R., and D. W. M. Herbert. 1960. The influence of
carbon dioxide on the toxicity of un—ionized ammonia to
rainbow trout (Salmo gairdnerii Richardson). Ann. Appl.
Biol. 48:399-404.

 

Lloyd, R., and L. Orr. 1969. The diuretic response by rain-
bow trout to sub-lethal concentrations of ammonia.
Water Research. 3:335-344.

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

Lucas, H. F., Jr., D. N. Edgington, and P. J. Colby. 1970.
Concentrations of trace elements in Great Lakes fishes.
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Mathis, B. J. 1972. Personal communication. Bradley
University. Peoria, Illinois.

Mathis, B. J., and T. F. Cummings. 1971. Distribution of
selected metals in bottom sediments, water, clams,
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River. WRC research report no. 14. University of
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McKee, J. E., and H. W. Wolf. 1963. Water quality criteria.
The resources agency of California. 2nd ed. St. Wat.
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McKim, J. M., G. M. Christensen, and E. P. Hunt. 1970.
Changes in the blood of brook trout (Salvelinus
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175

Mount, D. 1964. An autopsy technique for zinc-caused fish
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Mount, D. I. 1968. Chronic toxicity of copper to fathead
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ing cadmium poisoning in fish. Jour. of Wildl. Mgmt.
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Mount, D. I., and C. E. Stephan. 1967b. A method for
establishing acceptable toxicant limits for fish-
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Murphy, P. G., and J. V. Murphy. 1971. Correlations
between respiration and direct uptake of DDT in
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Toxicol. 4:581-588.

Olson, K. R., and P. O. Fromm. 1971. Excretion of urea by
two teleosts exposed to different concentrations of
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Pickering, O. H. 1968. Some effects of dissolved oxygen
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gill, Lepomis machochirus, Raf. Water Research
2:187—194.

Pickering, Q. H., and M. H. Gast. 1972. Acute and chronic
toxicity of cadmium to the fathead minnow (Pimephales
promelas). J. Fish. Res. Bd. Canada 29:1099-1106.

 

Pickering, Q. H., and C. Henderson. 1966. The acute
toxicity of some heavy metals to different species
of warm water fishes. Air Wat. Poll. Int. J. 10:453-
463.

Rachlin, J. W., and A. Perlmutter. 1968. Fish cells in

culture for study of aquatic toxicants. Wat. Res.
2:409-414.

Robinson, P. L., C. G. Wilber, and J. Hunn. 1960. Organ-
body weight relationship in the toadfish, Opsanus tau.

 

Smyth, H. F. 1967. Sufficient challenge. Food Cosmet.
Toxicol. 5:51-58.

176

Spotte, S. H. 1970. Fish and Invertebrate Culture,
Water Management in Closed Systems. Wiley-Interscience,
a Division of John Wiley and Sons Inc. New York, 145 pp.

Sprague, J. B. 1969. Measurement of pollutant toxicity to
fish. Water Research. 3:793-821.

Steel, R. G. D., and J. H. Torrie. 1960. Principles and
Procedures of Statistics. McGraw—Hill Book Co., Inc.,
New York, N. Y. 481 pp.

Uthe, J. F., and E. G. Bligh. 1971. Preliminary survey of
heavy metal contamination of Canadian freshwater fish.

Warren, C. E. 1971. Biology and Water Pollution Control.
W. B. Sauders Co., Philadelphia 434 pp.

Wisniewska, J. M., B. Trojanowska, J. Piotrowski, and M.
Jakubowski. 1970. Binding of mercury in the rat
kidney by metallothionein. Tox. and App. Pharm. 16:
754-763.

 

APPENDIX

177

 

178

Table A. A summary Of water quality Characteristics Of
filtered tap water from the Limnological Research
Building. Samples were collected on June 2, 1970
and June 1, 1971.

 

 

 

Parameter Concentration
Alkalinity ppm as CaCO3 292
Hardness ppm as CaCO3 328
PH 7.8
Ammonia ppm as N 0.03
Chloride ppm as Cl 5.2
Nitrate ppm as N 0.01
Phosphorus (total) ppm as P 0.85
Sulfate ppm as S04 6.0
Cadmium ppm as Cd <0.01
Zinc ppm as Zn (0.015
Copper ppm as Cu <0.01
Iron ppm as Fe <0.lO
Lead ppm as Pb (0.10

Conductivity (mho) 682

 

179

 

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183

 

 

 

Table 0. Pesticide residue analysis for five green sunfish
collected for bioassay purposes from a Williamston
pond. Fish from this collection were used in
experiment 6-F.

Concentration in ppb
Fish weight (g) Sex DDE DDD DDT Dieldrin
6.8000

(composite sam 1e
of three fish?

9.3385

51.18 11.86 34.11 #.07

52.63 12.69 23.63 2.57
43.57 9.84 25.00 2.07

ZZW’SS

 

184

Table D. Gangugig pond locations.

 

 

Collection Site

Location

 

# ponds located on the Michigan State
campus north of the Grand Trunk Railroad
and east of Farm Lane

1 pond at the north-east corner of
Waverly Golf Course, intersection of
Waverly Road and Saginaw in Lansing

Ponds in Grand Woods Park in Lansing
1 pond located north and east of the

intersection of Hulett Road and Jolly
Road in Meridian Township

3 large concrete ponds located at the
Water Research Laboratory on campus

 

185

Table EL Mean concentrations of RNA, DNA and the RNA’DNA ratios of green
sunfish from experiment 6-F before and during continuous
exposure to various concentrations of ammonia at three different
temperatures for 40 days. DFFT is dry fat-free tissue from the
dorsal muscle excluding skin. Standard error is enclosed in

 

 

 

parentheses.
Day pg DNA per fig RNA per RNA-DNA
100 mg DFFT 100 mg DFFT Ratio
Cold 0 22.6(4.8) 223.4(42.4) 12.69(5.14)
10 15.7(2.6) 530.8(47.6) 35.97(S.31)
20 20.5(l.0) 539.7(84.0) 27.74(5.15)
3O 15.7(2.S) 565.9(56.5) 36.09(3.68)
40 16.8(1.S) 414.9(14.0) 25.40(2.13)
Cold 0 - - -
Stressor 10 15.7(1.8) 335 2(29.1) 21.62(2.88)
20 19.9(3.3) 378.7(22.7) 19.87(2.04)
30 l7.8(1.8) 402.3(100.l) 25.37(8.13)
40 l7.3(2.9) 415.4(53.3) 25.58(4.86)
Medium 0 24.7(2.9) 186.2(15.0) 8.10(1.64)
10 24.1(5.5) 509.3(91.7) 23.76(5.68)
20 23.6(2.2) 367.2(19.4) 15.84(1.65)
30 18.9(4.1) 358.2(23.0) 22.12(5.04)
40 l6.3(1.3) 287.4(10.7) 18.l9(l.87)
Medium 0 - - -
Stressor 10 20.5(2.2) 472.6(77.7) 22.68(2.46)
20 19.4(1.3) 312.1(60.4) 15.81(2.51)
30 21.5(4.2) 331.0(54.9) 16.60(3.40)
4o 15.7(3.3) 265.4(11.4) 18.12(2.16)
Hot 0 l9.9(2.0) 216.6(32.3) 10.66(.95)
10 16.8(3.1) 490.4(65.7) 30.45(2.54)
20 19.4(1.6) 295.8(S.1) 15.58(1.05)
30 16.3(6.5) 285.8(19.2) 24.10(5.26)
40 8.4(1.S) 260.1(17.0) 32.13(3.83)
Hot 0 - - -
Stressor lO l7.9(l.4) 481.5(23.8) 27.47(2.28)
20 16.8(.9) 246.0(33.4) 14.64(2.01)
30 13.1(1.8) 248.6(23.1) 19.47(l.98) .

40 11.0(2.0) 226.6(10.0) 21.30(2.99)

 

 

186

Table F. A summary of pertinent data for the L050 determination
on pumpkinseeds (4.h6 t 0.31 g) in experiment 8-F.
Sample size for ammonia was four, and for all other
determinations was one. (Standard error is enclosed
in parentheses: t = less than 0.01 ppm).

 

 

Aquarium Number

 

1 8 3 10 5 2 12
% survival
after 96 100 100 no 20 o o 0
hrs
Ammonia 0.08 “.02 11.30 14.23 18.07 24.95 28.10
(ppm as N) (0.03) (0.14) (0.21) (0.21) (0.58) (1.53) (0.82)
Un-ionized NH3 t 0.10 0.35 0.21 0.56 0.60 0.53
(ppm as N)
Dissolved 8.7 8.4 8.3 8.3 8.0 8.5 8.3

Oxygen (ppm)
pH 7.76 7.88 7.94 7.55 7.90 7.83 7.70

Tamperature 11.7 12.1 11.9 12.3 12.0 12.0 12.2

 

187

Table G. A summary of pertinent data for the L050 determination
on green sunfish (8.39 t 1.37 g) in experiment 9-F.
Sample size for ammonia was seven. for dissolved
oxygen and pH two, and for alkalinity and hardness
one. Standard error is enclosed in parentheses: range
is given for pH. (t = less than 0.01 ppm).

 

 

Aquarium Number

 

1 8 3 10 5 2 12
% survival
after 96 ~100 100 80 80 0 0 0
hrs
Ammonia 0.11 8.81 26.69 30.89 35.84 47.30 5h.40
(ppm as N) (0.01) (0.26) (0.38) (0 82) (1.62) (2.35) (2.00)

Un-ionized NH3 t 0.21 0.82 0.7“ 1.36 1.19 0.96
(ppm as N)
Dissolved 8.6 8.0 8. 8.0 8.“ 8.1 8.5

7
Oxygen (ppm) (0.2) (0.2) (0.u) (0.3) (0.2) (0.3) (0.3)

PH 7082' 7080' 7093- 7076- 7095- 7076' 7072-
7-8“ 7.90 8.00 7.79 7.97 7.79 7.76

Temperature 12.0 12.h 12.2 12.6 12.2 12.2 12.

0 (0.1) (0.2) (0.1) (0.2) (0.2) (0.1) (0.3

Alkalinity 336 328 336 336 332 338 326
ppm as CaCO3

Hardness 340 336 340 332 340 336 336
ppm as CaCO3

 

 

188

 

 

 

Table H. Standard errors for the mean weight changes of
green sunfish exposed to various concentrations
of ammonia in experiment lo-F.

Time Treatment (ppm NHu as N)
(Days) 0.15 1.93 4.84 8.67 13.23 20.02 24.03
0.3? 0.38 0.40 0.47 0.44 0.26 0.53
4 0.48 0.60 0.48 0.57 0.53 0.38 0.52
0.43 0.66 0.60 0.60 0.53 0.34 0.49
12 0.52 0.67 0.66 0.84 0.83 0.03 0.04
16 0.56 0.87 0.87 0.89 1.08 0.57 0.3?
20 0.78 0.95 0.99 1.03 1.18 0.79 0.39
24 0.94 1.02 1.15 1.10 1.38 1.10 0.42
28 1.10 1.12 1.36 1.21 1.21 1.18 0.53
32 1.10 1.30 1.47 1.38 1.57 1.62 0.74
36 1.17 1.65 1.64 1.50 1.3? 2.05 1.02

 

189

 

 

 

Table I. Standard errors for the mean weight changes of
green sunfish exposed to various concentrations of
cadmium in experiment ll-F. (N.D. means less than
0.01 ppm Cd).

Time Treatment (ppm Cd)
(Days) N.D. 3.83 7.95 15.44 27.63 35.92 51.15
0.29 0.35 0.40 0.26 0.41 0.43 0.48
4 0.31 0.48 0.41 0.39 0.49 0.45 0.58
8 0.42 0.56 0.47 0.33 0.64 0.42 ——
12 0.45 0.74 0.51 0.25 -— — .—

 

190

 

 

 

Table J. Standard errors for the mean weight changes of
green sunfish exposed to various concentrations
of cadmium in ex eriment lZ-F. (N.D. means less
than 0.01 ppm Cdg.

Time Treatment (ppm Cd)
(Days) N.D. 0.05 0.23 0.32 1.31 1.93 2.48
0.17 0.16 0.17 0.12 0.17 0.12 0.12
4 0.32 0.17 0.20 0.35 0.31 0.12 0.24
8 0.24 0.18 0.25 0.38 0.33 0.13 0.26
12 0.29 0.20 0.32 0.39 0.33 0.14 0.33
16 0.31 0.21 0.33 0.40 0.32 0.15 0.37
20 0.37 0.26 0.32 0.42 0.31 0.18 0.38
24 0.48 0.36 0.36 0.52 0.46 0.29 0.43

 

191

 

 

 

Table K. Standard errors for the mean weight changes of
green sunfish exposed for 15 min to various
?§?8‘3“$§§§§°3‘§s‘s’f.§2§’“3‘f‘3112.33“?”iment 1”"

Time Ppm Cd - 15 Min Exposure
(Days) N.D. 5 10 20 30 40 50
0 0.10 0.11 0.05 0.08 0.09 0.10 0.09
3 0.10 0.10 0.06 0.09 0.09 0.11 0.09
7 0.17 0.18 0.10 0.14 0.20 0.17 0.12
11 0.16 0.32 0.20 0.16 0.26 0.25 0.21
15 0.19 0.39 0.24 0.19 0.33 0.28 0.33
19 0.31 0.44 0.29 0.24 0.41 0.45 0.42
23 0.36 0.57 0.34 0.28 0.46 0.49 0.53
27 0.40 0.57 0.37 0.29 0.50 0.52 0.56
31 0.46 0.67 0.43 0.33 0.56 0.58 0.60
39 0.61 0.81 0.49 0.37 0.66 0.68 0.80
47 0.94 1.10 0.53 0.51 0.88 0.83 1.12
55 1.28 1.52 0.61 0.73 1.18 1.07 1.49
63 1.58 2.14 0.78 1.04 1.44 1.49 1.89

 

192

 

 

 

Table L. Standard errors for the mean weight changes of
green sunfish exposed for 1 hr to various con-
gzgggafiggstfiincgdgiugpin experiment lO-S. (N.D.

Time Ppm Cd - 1 Hr Exposure
(Days) N.D. 5 10 20 30 40 50
0 0.10 0.06 0.09 0.10 0.07 0.09 0.09
3 0.10 0.06 0.08 0.08 0.07 0.05 0.08
7 0.17 0.16 0.11 0.09 0.17 0.12 0.16
11 0.16 0.24 0.19 0.17 0.23 0.17 0.22
15 0.19 0.28 0.22 0.24 0.34 0.17 0.21
19 0.31 0.34 0.37 0.29 0.43 0.31 0.24
23 0.36 0.38 0.43 0.32 0.48 0.39 0.31
27 0.40 0.44 0.46 0.36 0.54 0.47 0.33
31 0.46 0.50 0.51 0.36 0.63 0.62 0.37
39 0.61 0.63 0.64 0.40 0.70 0.80 0.42
47 0.94 0.79 0.88 0.53 0.89 1.09 0.52
55 1.28 1.09 1.09 0.62 1.30 1.26 0.64
63 1.58 1.35 1.36 0.66 1.61 1.68 0.75

 

193

Table M. Standard errors for the mean weight changes of
green sunfish exposed for 24 hrs to various
concentrations of cadmium in experiment lO-S.
(N.D. means less than 0.01 ppm).

 

 

 

Time Ppm Cd - 24 Hr Exposure

(Days) N.D. 5 10 20 30
0 0.10 0.09 0.07 0.11 0.11
0.10 0.11 0.06 0.11 0.17
7 0.17 0.16 0.11 0.18 0.77
11 0.16 0.21 0.16 0.30 1.03
15 0.19 0.26 0.19 0.43 1.25
19 0.31 0.30 0.27 0.59 1.51
23 0.36 0.34 0.27 0.67 1.65
27 0.40 0.41 0.33 0.72 1.71
31 0.46 0.43 0.41 0.81 1.78
39 0.61 0.51 0.47 0.99 2.01
47 0.94 0.63 0.65 1.23 2.11
55 1.28 0.87 0.93 1.60 2.52
63 1.58 1.21 1.24 1.77 3.11

 

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