WATER POLLUTION CONTROL RESEARCH SERIES
18050 DST 12/70
TOXIC ACTION OF
WATER SOLUBLE POLLUTANTS
ON FRESHWATER FISH
ENVIRONMENTAL PROTECTION AGENCY • WATER QUALITY OFFICE
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WATER POLLUTION CONTROL RESEARCH SERIES
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TOXIC ACTION OF WATER SOLUBLE POLLUTANTS
ON FRESHWATER FISH
Dr. Paul 0. Fromm, Professor
Department of Physiology, Michigan State University
East Lansing, Michigan U8823
for the
Water Quality Office
ENVIRONMENTAL PROTECTION AGENCY
Grant Number 18050 DST
December, 1970
For sale by the Superintendent of Documents, U.S. Government Printing Office, Washington, D.C. 20402 - Price to cento
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ABSTRACT
In studies of the effect of stress on trou^we found that exposure to
Cr and to forced exercise caused a transient rise in plasma cortisol.
Fasted fish treated with exogenous cortisol for 1 week had higher
liver glycogen stores and excreted more waste nitrogen than controls,
whereas data from fed animals were similar to control data. Ammo-
nia appears to kill fish by preventing excretion of ammonia and not by
interfering with gill respiratory exchange or inhibition of oxygen
transport by hemoglobin. Under comparable conditions goldfish
excrete much more urea than trout, and the excretion rates are much
more responsive to ambient ammonia in goldfish. Hyperexcitability
observed in ammonia -exposed trout was not noticeable in the more
resistant goldfish. Greater histopathology was seen in trout gills
than in goldfish gills exposed to the same or greater ammonia levels.
In studies using individual perfused gill arches of rainbow trout we
found that: (a) the routes for blood flow are as described in previous
studies using non-perfused gills; (b) significant changes in filamental
and lamellar flow patterns appear to be controlled adrenergically,
probably by vasoconstriction and vasodilation of sinus vessels and
lamellar arterioles; (c) when perfused with Ringer solution there was
a small but significant loss of sodium into the bath solution, whereas
perfusion with sodium poor Ringer solutions always resulted in a net
uptake of sodium; (d) perfusion fluid sodium and epinephrine appear
to control sodium uptake by the gill; (e) uptake dependent on ATP
energy produced aerobically was generally independent of the rate and
pattern of fluid flow through the gill; (f) transfer of dieldrin into the
vascular system occurred only when plasma protein, or more probably
plasma lipoprotein was present in the perfusion fluid; (g) short-term
exposure to dieldrin, rotenone, malathion and MS-222 reduced perfu-
sion flow rate through isolated gills; exposure to 1 mg/L methoxychlor
was without effect. Decrease in flow rate correlated well with
increased lamellar perfusion.
This report was submitted in fulfillment of Grant No. 18050 DST under
partial sponsorship of the Federal Water Quality Administration.
Keywords: Fish, stress, chromium, insecticides, gill, sodium
transport, gill blood flow, ammonia toxicity, nitrogen excretion.
111
*rairibow trout, SaJmo gairdneri
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CONTENTS
Section Page
I INTRODUCTION 1
II RESPONSE OF INTERRENAL GLAND
OF RAINBOW TROUT TO STRESS 3
III EFFECT OF AMMONIA ON TROUT
AND GOLDFISH
IV STUDIES USING ISOLATED-PERFUSED
RAINBOW TROUT GILLS 23
V ACKNOWLEDGMENTS 47
VI REFERENCES CITED 49
VII PUBLICATIONS 53
VIII PERSONNEL 55
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FIGURES
No. Page
1 Total ammonia in trout blood as related to total
ammonia in the water 11
2 Non-ionic ammonia in trout blood as related to
non -ionic ammonia in the water 12
3 Photomicrograph of gill lamellae from trout
exposed to low ambient ammonia for 8 weeks 18
4 Photomicrograph of gill lamellae from trout
exposed to 5 yg ammonia/L for 8 weeks 18
5 Greater magnification of gill lamellae from
trout exposed to 5 yg ammonia/L for 8 weeks 20
6 Greater magnification of gill lamellae from
trout exposed to 5 yg ammonia/L for 8 weeks 20
7 Constant pressure perfusion apparatus 25
8 Circulation in gill filaments and lamellae of
trout 27
9 Circulation in gill lamellae of trout 28
10 Rate of sodium uptake by gills as a function of
the sodium concentration of the perfusion fluid 33
11 Typical gas chromatography tracings of solu-
tions analyzed in experiments with dieldrin 39
VI
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TABLES
No. Page
I Plasma cortisol levels in fish exposed to
forced exercise for 2 and 4 hours 4
II Effects of exogenous cortisol on nitrogen
excretion, liver glycogen and plasma
glucose of fasted trout 5
III Effects of exogenous cortisol on nitrogen
excretion, liver glycogen and plasma
glucose of fed trout 6
IV Excretory nitrogen components from trout
subjected to various levels of ambient
ammonia 14
V Effect of acclimation on urea excretion by
goldfish 16
VI Sodium uptake as affected by variable sodium
concentrations, inhibitors and vasoactive
agents 31
VII Factors affecting rate of fluid flow through
isolated trout gills 34
VIII Factors affecting pattern of fluid flow through
isolated trout gills 36
IX Summary of information on uptake of dieldrin
by isolated trout gills 41
X Acute effect of various chemicals on rate of
fluid flow through isolated -perfused gills
of trout 42
Vll
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No. Page
XI Acute effect of various chemicals on pattern
of blood flow through isolated-perfused gills
of trout 43
Vlll
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SECTION I
INTRODUCTION
The research conducted during the past five years represents an
extension of earlier work (EF 00162) which was concentrated pri-
marily on an investigation of the toxicity of chromium to fish. Al-
though much of the work reported below has been published, few
reprints are now available; hence the research is discussed giving
sufficient details for understanding.
Except for the few experiments with goldfish, all fish used in the
experiments discussed below were rainbow trout (Salmo gairdneri)
supplied to us by the Michigan Department of Natural Resources
hatcheries at Harrietta or Grayling, Michigan. All arrangements for
supplying fish were made with Dr. L. N. Allison, Fish Pathologist,
who also offered advice on problems relating to husbandry and dis -
eases of hatchery fish. All fish were held in the laboratory in
300 liter tanks lined with fiberglass for at least one week prior to
use. They were fed commercial trout pellets during the phase of
acclimation to laboratory conditions. Treatment and feeding of fish
varied with each experiment and are indicated below.
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SECTION II
RESPONSE OF THE INTERRENAL GLAND
OF RAINBOW TROUT TO STRESS
A series of experiments were undertaken to determine whether
chronic exposure to low levels of noxious stimuli will cause an
increase in plasma levels of glucocorticoids in rainbow trout. We
also investigated some of the metabolic effects of artificially ele -
vated levels of glucocorticoids. At the outset of these investigations
it was considered that the cortisol assay procedure of Guillemin
et al. (1959) was the method of choice. Since that time others have
shown that abbreviated procedures such as the one used can lead to
erroneous results. It is known that the principal steroid elaborated
by the interrenal of rainbow trout is cortisol, but there are fluorogens
other than cortisol also present such as 20 3 dihydrocortisone. Most
of the plasma samples had been processed before we realized that
in order to state categorically that the changes in fluorescence of
plasma that we measured were due to cortisol alone, we should have
isolated cortisol (chromatography) and then measured it fluorometri-
cally. Our strongest argument for stating that the changes we
observed were due to changes in plasma cortisol comes from the fact
that when cortisol pellets were implanted in fish we were able to
determine a significant elevation in the fluorescence of the plasma
extracts.
Chromium stress: Two groups of fish were held in water containing
0. 02 and 0.20 mg Cr/L; controls were held in aged tap water. At
these levels radiochromium was found to accumulate steadily in rain-
bow trout throughout a 28-day experimental period (Fromm and
Stokes, 1962). Fish exposed to 0.2 mg Cr/L for 1 week had plasma
cortisol levels nearly twice those of controls (54. 3 vs. 30. 5 ygm/
100 ml). Those fish exposed to the lower concentration also had sig-
nificantly elevated plasma cortisol. After exposures of 2 and 3 weeks
the Cr-treated fish had plasma levels essentially similar to those of
controls. Fish exposed to 20 mg Cr/L for 3 days showed plasma
cortisol levels of 56. 8 yg/100 ml compared to 37. 8 for controls.
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Fish exposed for 6 and 7 days showed no increase in blood levels over
controls; and those exposed 10 days had somewhat elevated plasma
cortisol, but they were not statistically higher than values for controls.
Effect of exercise: Fish were given two daily half-hour periods of
forced exercise for one week and two weeks in separate experiments.
The speed of rotation of the circular tank used was regulated so that
the fish were swimming as rapidly as they were willing to do for the
half-hour period.
Table I
Plasma cortisol levels in fish exposed to forced exercise
for 2 or 4 hours. Blood samples taken immediately after
cessation of exercise.
Duration
(hours)
Control
2
4
Number
of data
5
5
5
Plasma cortisol
(yg/100 ml)*
54. 4 ± 3.5
61. 0± 2.3 (p> 0.05)
64.2 ± 4. 3 (p< 0.05)
* mean± S.E.
When the blood samples were taken 24 hours after cessation of exer-
cise the exercised fish had cortisol levels slightly lower than those
for controls. In a second series of experiments fish were forced to
swim for 2 and 4 hours and blood samples were taken immediately
after capture. Plasma cortisol levels in fish exercised for 2 hours
were somewhat elevated above those for control fish, and the eleva -
tion after 4 hours of exercise was statistically significant.
Metabolic effects of exogenous cortisol: To study the effects of ele-
vated serum cortisol, exogenous cortisol in cholesterol pellets was
administered to fish by intraperitoneal implantation. Pellets of
cholesterol only were implanted in controls. Some characteristic
effects of elevated levels of adrenal cortex activity in mammals include
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an increase in liver glycogen, an elevation in plasma glucose, and an
increase in the utilization of amino acids for energy metabolism and
gluconeogenesis, with a resulting increase in nitrogen excretion.
These metabolic parameters were measured in fish that received
intraperitonally implanted pellets containing cortisol.
Table E
Effects of 1 -week exposure to exogenous cortisol on Nitrogen Excre
tion, Liver Glycogen and Plasma Glucose of fasted rainbow trout.
Parameter
Control (6)*
Experimental (8)*
Plasma cortisol
(ug/lOOml)
Liver glycogen
(g/100 g tissue)
Nitrogen excretion
(mg N/kg/day)
Plasma glucose
(mg/100 ml)
48.7 ±4.5
0.59± 0.16
219 ± 29
86.3 ± 11.5
125.7 ± 16.0
1.17± 0.12
306 ± 61
74.1 ± 11.0
* all data: Mean ± S. E.; number of data in parentheses.
In fasted experimental fish the glycogen content of the liver and values
for nitrogen excretion were higher than those for controls. Plasma
glucose in treated fish were similar to values for controls. A similar
experiment was conducted with fish that were fed at a rate of 2% of
body weight per day during the period between implanting of pellets
and sampling. Neither liver glycogen levels nor nitrogen excretion
was elevated in the cortisol-treated fish which had been fed.
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Table HI
Effect of 1 -week exposure to exogenous cortisol on Nitrogen Excretion
and Liver Glycogen of fed rainbow trout.
Parameter
Control (7)*
Experimental (6)*
Liver glycogen
(g/100 g tissue)
Nitrogen excretion
(mg N/kg/day)
3.63± 0.69
348 ± 77
3.55± 0.56
326 ± 12
* all data: Mean ± S. E. ; number of data in parentheses.
Discussion and summary: Exposure to chromium resulted in a tran-
sient elevation in the concentration of fluorogenic materials in the
plasma of rainbow trout. The response of fish to 20 mg Cr/L, a
concentration approaching the 48 -hour median tolerance limit for the
species, caused a response that was of a much lower magnitude than
that observed in spawning Pacific salmon, which, according to
Robertson and co-workers, causes detrimental degenerative secondary
responses. Results somewhat similar to ours were obtained by
McKim (1966), who measured output of urinary 17 hydroxycortico -
steroids (17-OHCS) metabolites in rainbow trout exposed to a continu-
ous stress of sublethal environmental levels of the detergent, alkyl
benezene sulfonate (ABS). Excretion rates of 17-OHCS of experi-
mental fish increased about twofold in the first 24 hours of exposure
but, in all cases, decreased to only slightly above (at 7 mg ABS/L)
or equal to (at 3 and 5 mg ABS/L) the rates of control fish at the end
of 7 days. There is reasonable doubt as to whether the untreated
controls for the chromium study represented unstressed fish, since
there was some elevation of cortisol levels during the 3-week experi-
mental period and we have no plausible explanation of the increase
observed. Variations of inter renal activity may have been influenced
by the social interaction of fishes, although no observations of this
interaction were made in the present study. Erickson (1967) found
that green sunfish in small aquaria established a social hierarchy
and that fish of lower social rank, presumably the more stressed
individuals, had significantly greater volumes of interrenal tissue.
He found that the quantity of interrenal tissue present was negatively
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correlated with the number of aggressive actions that the fish
initiated. During the course of our study we noted considerable vari-
ability in the values for plasma cortisol in controls that may reflect
a possible seasonal variation in the activity of the interrenal tissue of
trout and/or variations in the rate of clearance. There is evidence
that the responsiveness to ACTH of the adrenal cortex of rats and
humans is decreased by high circulating corticoid levels in the blood.
Hane et al. (1966) injected mammalian ACTH and caused a fourfold
increase in plasma 17-OHCS in nonspawning Pacific salmon captured
at sea and similar results in sexually undeveloped fish captured at the
beginning of their spawning migration. Responsiveness to ACTH
decreased as the fish approached sexual maturity, coincident with
increased plasma 17-OHCS, and little response was seen in spawned
fish whose plasma 17-OHCS levels were greatly elevated. Any
seasonal variation in plasma cortisol levels of untreated fish is of
particular significance to anyone wishing to use this parameter as an
indicator of stress. Direct comparisons of values obtained at dif-
ferent times of the year would be valid only if completely adequate
control groups could be used. Short-term stress has consistently
resulted in elevated circulating glucocorticoid levels in fish in our
experiments and those of others (Hatey, 1958; McKim, 1966; Fager-
lund, 1967). There is evidence from McKim1 s ABS data and from our
chromium experiments that the magnitude of the response can be
expected to vary with the magnitude of the noxious stress. This re-
sponse might be useful for evluating stress in short-term studies.
The apparent important role of elevated circulating corticosteroids,
whether the result of increased activity of the interrenal gland or
impaired clearance, in bringing about the degenerative changes
observed in spawning salmon has been given strong support by the
successful reproduction of most of these changes by the administra
tion of exogenous cortisol to immature rainbow trout (Robertson
et al., 1963). The high corticosteroid levels found in migrating
Pacific salmon may be an unusual phenomenon associated with the
simultaneous demands of long -sustained exertion of migration and of
maturation of a large mass of gonadal tissue and gametes. Our
results, as well as those of McKim (1966) and Fagerlund (1967), sup-
port the idea that the response of the interrenal gland of fish to chronic
sublethal stress does not play a major role in the genesis of adverse
reactions that may be caused by these stressors. As noted above,
analysis of the response of this tissue during short-term studies of
stress may be quite valuable.
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SECTION in
EFFECT OF AMMONIA ON TROUT AND GOLDFISH
Effect of ammonia on trout: Deamination of amino acids by the liver,
metabolic activity of nerve and muscle tissue, as well as activity of
enzymes contained in the flora of the gut on substrates derived from
the diet and the blood, all lead to the production of ammonia. Am-
monia is quite toxic to most organisms, and it must be either con-
tinually eliminated or converted to less toxic compounds to prevent
any buildup to harmful concentrations within the body.. When the pH
of an aqueous solution of ammonia is increased, the amount of non-
ionized ammonia is increased. The free base (NHg) is able to diffuse
across cell membranes easily because of its lipid solubility and lack
of charge, whereas the ammonium ion penetrates membranes less
readily because it is hydrated, charged and has a low lipid solubility.
This being the case, one would expect that the pH of an ammonia
solution would have a great effect on the toxicity of the solution. The
toxicological actions of ammonia on fish are not completely known.
Burrows (1964) has observed extensive proliferation and consolidation
of gill lamellae of salmonids exposed to ammonia and similar observa -
tions have been reported by Reichenbach-Klinke (1967). These affected
fish were quite susceptible to gill disease, and Burrows regards the
ammonia irritation as a precursor to the disease. The consolidation
of lamellae reduces the surface area of the gills and thereby reduces
the ability of the fish to liberate COg and to absorb oxygen. Brockway
(1950) has correlated an increase in ambient ammonia with a reduction
in oxygen level of the blood and suggested that ammonia affects the
oxygen transport ability of fish blood. If ammonia is to act internally,
it appears probably that blood levels of ammonia should increase in
fish concurrent with an increase in ambient ammonia. Any change in
blood ammonia under these conditions could be due to either inhibition
of excretion or inward diffusion of ammonia. The aims of the experi-
ments described below were to investigate changes that occur in blood
ammonia levels and in ammonia excretory rates when fish are exposed
to increased concentrations of ambient ammonia. Data for daily
excretion of total nitrogen were obtained to see if any change in
excretion rate or form of nitrogenous waste occurred in fish exposed
9
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to different concentrations of ambient ammonia. We also investigated
the effect of ammonia on the oxygen carrying ability of hemoglobin.
When rainbow trout were exposed to increased ambient concentrations
of total ammonia, the levels of total ammonia in the blood increased.
Slight differences in the pH of the water samples was noted which
altered the proportion of NHg and NH, in samples of equal total
ammonia concentration. When blood ammonia, total and unionized,
was plotted versus ambient ammonia (total and unionized, respective-
ly), both graphs indicated a direct linear correlation between blood
ammonia and water ammonia. Equations for the slopes determined
by the method of least squares are given in Figures 1 and 2. In all
cases the concentration of total and non-ionic ammonia in the blood
was higher than their respective concentrations in the water from
which the fish were taken.
No measure of the external action of ammonia on trout was made in
our experiments, but it was certainly obvious that fish placed in the
high concentrations of ammonia were hyperexcitable. Wuhrmann &
Woker (1948) and McCay & Vars (1950) reported similar observations.
We have no information on the production of ammonia by neural tissue
or the effects of ammonia on the excitability and/or metabolism of
neural tissue of fish. In mammals there are indications that the
formation of ammonia by nerve tissue is due to reactions involving
proteins and nucleoproteins; however, details concerning the nature
of its precursors and the mechanism of its liberation are lacking.
The main stream of ammonia disposal is the combination of one
molecule of ammonia with a-ketoglutarate to form L-glutamate.
Then a second molecule of ammonia combines with glutamate, with
the utilization of an ATP, to form glutamine, a compound which
traverses the blood-brain barrier more easily than glutamate. An
auxiliary mechanism utilizing transamination exists. L-glutamate,
pyruvic and oxalacetic acids are involved, with the resultant forma-
tion of alanine and asparate. When the ammonia concentration is
increased, it appears to stimulate glycolysis in neural tissue. The
end-result of excessive ammonia production is an accumulation of
pyruvate and lactate brought about by stimulation of the glycolytic
pathway and concurrent suppression of citric acid cycle activity due
to a diversion of a -ketoglutarate and oxalacetate into increased amino
acid production. Although the ammonia binding mechanisms may give
rise to many of the effects associated with increased production of
ammonia, Weil-Malherbe (1962) concludes that they are necessary
in order to prevent accumulation of free ammonia, which would be
even more detrimental to the organism.
10
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100
* 80
60
HJ
8 40
20
Y = 38.85 + 4.025 X
Sy.x " 10'5
2 4 6 8 10 12 14
Water Ammonia (HH, + NEL+)
3 4
Figure 1. Total ammonia concentration in the blood as a function of
total ammonia concentration in the water. The solid line was drawn
using the method of least-squares. Ninety-five per cent confidence
limits for points on this line are indicated by the dotted lines.
11
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1.2-
1.0-
ffl
c 0.8-
3*
H
0.6-
O
0.4-
0.2-
X
•x
X
X
X
X
• •
• •
X
X
X
X
X
X
X
X
X
X
X
X
•• X
X
V
Y - 0.630 + 675 X
y.x
or o oj O O 0^ i.o
Unionized Ammonia in Water (HH,)
Figure 2. Non-ionic ammonia concentration in the blood as a function
of non-ionic ammonia concentration in the water. The solid line was
drawn using the method of least-squares. Ninety-five per cent con-
fidence limits for points on this line are indicated by the dotted lines.
12
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Ammonia in the water appears to be toxic to trout by suppressing the
liberation of ammonia at the gill surface. Since the blood ammonia
level always exceeded that in the environment from which the fish
were taken, the source of the blood ammonia must have been endoge -
nous. Thus the rate of metabolic production and/or release of
ammonia into the blood exceeded the combined rates of excretion and
detoxification in these fish. Evidence for detoxification of ammonia
was obtained by experiments in which the rate of total waste nitrogen
and ammonia nitrogen excretion was determined for fish exposed to
various concentrations of ambient ammonia. As the water ammonia
level increased, total nitrogen excretion decreased. Ammonia ex-
cretion decreased also and the decrease in ammonia excretion could
account for about 48% of the decrease in total nitrogen excretion up
to an ambient concentration of 5 yg/ml. At an ambient concentration
of 8 yg/ml the amount of ammonia excreted was greatly reduced but
excretion of total nitrogen remained quite high, indicating that the
reduction in ammonia excretion was to some extent compensated for
by increased excretion of some other nitrogenous compound.
Brockway (1950) reported that when the ammonia in water increased
to about 1 mg/L the oxygen content of trout blood decreased to approxi-
mately one-seventh of its normal value, and the carbon dioxide content
increased about 15%. One might argue that the external action of
ammonia on the gill epithelium could affect respiratory exchange and
give rise to the alterations noted in the blood gases. In experiments
(unpublished) we found that rainbow trout exposed to 1. 6-4. 3 mg NH3/L
for 24 hours had rates of oxygen consumption which ranged from 53
to 106 per cent above the normal resting level. In these fish the
exchange of respiratory gases was not impaired by ammonia, although
the rates of oxygen usage and carbon dioxide production could have
given rise to alterations in the blood gas content similar to those
reported by Brockway.
To test the effect of ammonia on the ability of hemoglobin to combine
with oxygen in vitro, the following procedures were followed. Whole
blood, obtained from untreated fish, was centrifuged and the plasma
removed. To a volume of cells an equal volume of ammonia-free
Ringer solution was added, and to aliquots of this mixture equal
volumes of Ringer solution which contained known quantities of ammo -
nia were added. These RBC-Ringer mixtures, which had hematocrit
values of about 25 per cent, were then aerated by bubbling air through
them for a minimum of 5 minutes immediately prior to the analyses
given below. During all of the procedures the temperature of the
samples was maintained at 12°C, and the pH of the final mixtures
13
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Table IV
Excretory nitrogen components from rainbow trout subjected to various levels of ambient ammonia.
Values for nitrogen excretion are as yg N/gm of fish/day. The per cent N accounted for is:
excreted ammonia N + urea N + protein N X 100/Total N excreted.
Mean ambient
ammonia
(ug/ml)
2.39± 0.09*
4. 58 ± 0.17
6.05± 0.13
8.30± 0.21
n
19
12
10
17
Ammonia
N
excretion
86 ± 7.8
63 ± 7.2
35 ± 4.5
24± 6.9
Urea
N
excretion
39± 7.6
28± 5.8
30 ± 4. 9
30± 3.4
Protein
N
excretion
35 ± 4.9
44 ± 9.0
24 ± 4. 3
38 ± 4.2
Total
N
excretion
161 ± 15.9
151 ± 19.9
91 ± 11.0
103 ± 10.1
Per cent N
accounted
for
99
89
97
89
* Standard error of mean.
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was about 7. 4. After aeration, aliquots were taken for determination
of oxygen content using the Natelson Microgasometer and hemoglobin
content by the acid hematin method (B & L Spectronic 20 Clinical
Technique Manual). It was found that ammonia in concentrations up
to 10 yg/ml had no significant effect on the ability of trout hemoglobin
to combine with oxygen in vitro.
In general summary our results indicate that ammonia in water (1 ppm
as NH3> is toxic to trout because of its effect of preventing the excre-
tion of normal amounts of ammonia. The toxic action most probably
is at the cellular level, and the nervous system appears to be affected
earliest. Ammonia at the levels studied does not appear to kill fish
by preventing exchange of the respiratory gases at the gill surface or
by inhibiting transport of oxygen in combination with hemoglobin.
In another series of experiments teleosts which inhabit distinctly
different environments were studied to determine if any species
variability to ammonia toxicity exists. Rainbow trout, which require
relatively clean water, and goldfish (Carassius carassius), which have
a tolerance for stagnant water, were used.
Trout subjected to increased ambient ammonia with no previous
acclimation period showed a decrease in total nitrogen excreted
(Table IV) and a concomitant decrease in waste nitrogen excreted as
ammonia. Except for a slight increase in urea excretion at low
ambient ammonia, urea and protein nitrogen excretion rates showed
no change as ambient ammonia increased. Ninety-four per cent of
the total nitrogen excreted by trout was as ammonia, urea and pro-
tein nitrogen. Two groups of trout were then acclimated to high
(5 yg/ml) and low (0. 5 y g/ml) ambient ammonia and then subjected
to about 3 yg/ml while their urea excretion rate for 24 hours was
determined. Those exposed to high ammonia actually excreted slightly
less urea than those exposed to low ammonia, which was just the
opposite of what was expected.
In studies with goldfish, determinations of urea excretion rates were
made, but excretion of total waste nitrogen was not investigated.
Fish were acclimated to low and high ambient ammonia, and then the
rate of urea excretion was determined at the concentrations as indi-
cated in Table V. It is apparent from the data that pre -conditioning
or acclimation to high ammonia has little or no effect on urea excre-
tion. Urea excretion rate is dependent on the ambient ammonia levels
during the collection period and is independent of any acclimation
15
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concentration or duration of acclimation. The goldfish appear to
respond to a change in ambient ammonia almost instantaneously.
Table V
Urea excretion during 24 hours at various levels of ambient ammonia
by goldfish which had been acclimated to either low (0. 5 u g/ml) or
high (5.0 to 25.0 yg/ml) ambient ammonia.
Days
fish
acclimated
26
46
56
26
20
30
Acclimation
concentration
y g ammonia/ml
0.5
0.5
0.5
5.0
25.0
25.0
n
7
3
7
11
4
9
Ambient
concentration
during
experiment ,
yg ammonia /ml
2.37 ± 0.23*
0.75± 0.03
0.08± 0.01
2.22 ± 0.14
0.68± 0.11
0.10± 0.01
Urea
excretion
yg N/gm
of
fish/day
134 ± 31
52 ± 7
27 ± 3
128 ± 21
22 ± 10
28 ± 2
* Standard error of mean.
Trout placed in water containing more than 3 yg ammonia/ml became
hyper excitable. Any disturbance of the tank or movements above the
tank visible to the fish resulted in disoriented escape attempts which
sent the fish crashing into the sides of the tank. If these fish were
then placed in water containing no ammonia, they appeared to return
to normal, i.e., were no longer hyper excitable. The highest am -
monia concentration (about 8 yg/ml) to which trout were exposed
caused about 50 per cent mortality within 24 hours. There was a
decrease in mortality with the corresponding decrease of ambient
ammonia concentrations during the 24 hour period. The onset of
16
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death was characterized by violent thrashing movements which were
functionless as propulsive swimming movements. Trout used in the
high ammonia acclimation experiments were also hyperexcitable
initially; however, after about two days they appeared to calm down,
and after the third day they showed no signs of elevated excitability.
Conversely, goldfish did not appear to be bothered at all by ammonia
as high as 25 yg/ml, some eight times greater than the concentration
which affected the trout. When the goldfish were placed in 40 yg
ammonia/ml, about 10 per cent died in 24 hours. The onset of death
was characterized by a gradual cessation of swimming movements,
during which time the fish slowly settled to the bottom of the aquarium.
After one or two hours nearly all of the dying fish curled laterally in
the form of a "U" and opercular movements dropped considerably. If
left in the ammonia solution, death soon followed. Three fish near
death were removed from the ammonia water and placed in ammonia -
free water; two of the fish lived for several days before dying, and
the third completely recovered. Although severe, the effects of
ammonia on fish apparently are, to some extent, reversible.
The results of the experiments with trout and goldfish can be briefly
summarized as follows:
When rainbow trout were subjected to increasing ambient ammonia
concentrations at 13°C, the total nitrogen excreted decreased, which
is reflective of a decreased excretion rate of ammonia. Except for
an initial increase of urea excretion at very low ambient ammonia
levels, urea- and protein-N excretion rates remained constant at the
other levels tested. An average of 94 per cent of the total nitrogen
excreted by trout consists of ammonia, urea and protein nitrogen.
Rainbow trout acclimated to elevated ammonia levels showed no
increase in urea excretion over that of controls acclimated to low
ammonia.
When goldfish were exposed to increased ammonia levels at 20-23°C,
the rate of urea excretion increased. Acclimation of goldfish to dif-
ferent elevated ammonia levels for variable periods of time show
that the change in rate of urea excretion was dependent solely on the
ambient ammonia level during the experimental period and was un-
affected by prior treatment. The ability of goldfish to change their
urea excretion rate concomitant with a change in ambient ammonia
appears to be either instantaneous or with a time course so short
that any lag time is insignificant in 24 hours.
17
-------
Four photomicrographs of gill lamellae from rainbow trout appear on
the following pages. The sections were cut at 8 microns and stained
with hematoxylin and eosin. Descriptions of these figures are as
follows:
Figure 3. Photomicrograph of gill lamellae from rainbow trout
exposed to low ambient ammonia (< 0. 5 yg/ml) for 8 weeks. Gills
exposed to this very low level of ammonia have long slender lamellae
which exhibit no significant pathology. (X 133)
Figure 4. Photomicrograph of gill lamellae from rainbow trout
exposed to 5 yg ammonia/ml for 8 weeks. The lamellae are shorter
and thicker than those seen in Figure 3, and they have bulbous ends.
Some consolidation of lamellae was also noted in fish exposed to high
ammonia, 5 yg/ml. (X 133)
18
-------
Figure 4
19
-------
Figures 5 and 6. Higher magnification (X 532) photomicrographs of
lamellae from trout exposed to 5 yg ammonia/ml for 8 weeks. Two
types of pathology can be recognized. Many filaments show a rather
limited hyperplasia (Figure 5) which is accompanied by the appearance
of cells containing large vacuoles whose contents stain positive for
protein. Other lamellae (Figure 6) show a definite hyperplasia of the
epithelial layer, as is evident by an increase in the number of cell
nuclei.
20
-------
Figure 5
Figure 6
21
-------
With regard to species variability between rainbow trout and goldfish,
it can be noted that quantitatively goldfish excrete much more urea
than trout, and urea excretion rates are much more responsive to
ambient ammonia levels in goldfish. Goldfish are able to survive
(0% mortality) ammonia levels more than three times greater than
that which is lethal to trout. Hyperexcitability due to ammonia
exposure which was observed in trout was not noticeable in goldfish.
There appeared to be greater histopathology of the gills of trout
exposed to ammonia than to the gills of goldfish exposed to the same
or greater ammonia concentrations. Enzyme studies by other investi-
gators support the hypothesis that purine catabolism and not ornithine
cycle activity is probably the main metabolic pathway for urea synthe-
sis at the rates found in goldfish and trout.
22
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SECTION IV
STUDIES USING
ISOLATED-PERFUSED RAINBOW TROUT GILLS
The gill surfaces used in respiratory exchange by most aquatic
animals such as fish are maximally exposed to water borne or water
soluble pollutants. It has been recognized for years that heavy metal
ions have an effect on gills, but much of the research accomplished to
date has been biased towards the determination of lethal concentra-
tions for a particular pollutant. Of all organs present in fish, we
figure that the gills receive the greatest initial insult when these
animals are placed in polluted waters. A toxic agent may act exter-
nally on the gills causing, for example, erosion or precipitation of
gill mucus; or it may act internally, bringing about its deleterious
effect by interfering with metabolic cycles. We originally planned to
perfuse the whole branchial apparatus of trout, measure selected
physiological parameters and then test the effect of certain chemicals
on this preparation. We finally gravitated to the use of a single gill
arch in order to enhance our chances of doing more quantitative
measurements. Early studies indicated that the pattern of blood flow
through isolated gills was a significant variable with which we had to
contend. Experiments using the isolated gill technique have produced
some interesting results and the technique, slightly modified, is cur-
rently being used in new experiments in our laboratory.
The initial aim of this perfusion study was to obtain data for "normal"
or control level of sodium transport by gill epithelial cells. The
gills were bathed with a 1 per cent Ringer solution and perfused with
100 per cent Ringer solution. Under these conditions no measurable
uptake of sodium occurred. We then lowered the internal sodium
concentration by a partial substitution of sodium chloride with choline
chloride. It had previously been suggested that acetylcholine caused
a shift in the blood flow pattern through the filaments of teleosts, thus
it was necessary to investigate the flow patterns in trout gills in order
to ascertain whether or not choline chloride affected flow. To do this
we perfused gills with Ringer solutions containing India ink along
23
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with the material to be tested. Histological preparations of these
gills were examined.
Gill blood flow: Rather extensive studies were made of the patterns
of blood flow through filaments and lamellae of isolated -perfused
rainbow trout gills. To do these experiments we used the constant -
pressure perfusion apparatus shown diagrammatically in Figure 7.
Prior to cannulation, the dorsal and ventral ends of the gill were
trimmed of excess bone and tissues, exposing the efferent and afferent
branchial arteries. The valve on the perfusion apparatus was opened
and a steady, rapid flow of fluid was established. The afferent cannula
was then inserted into the afferent branchial artery and tied in place
with No. 30 cotton thread. The ligature was tied around the arch
rather than directly around the artery, thus preventing loss of fluid
due to leakage at the cut end of the arch. The efferent artery was
cannulated in the same manner, and the gill was immersed in 50 ml
of 1 per cent Ringer solution. The placement of the ligatures was
such that approximately 15 per cent of the filaments were outside of
each ligature, and thus only about 70 per cent of the filaments on a
given arch were perfused. Gills were first perfused with Ringer
solution containing the test substance (control, 10~^M epinephrine,
10~5 acetylcholine). These perfusions were carried out with the free
end of the efferent cannula at approximately the same height as the
gill and were continued until no more blood could be seen entering the
collecting tube. The volume of perfusate collected during this period
was never less than 100 yL. At the end of the initial perfusion, the
free end of the efferent cannula was raised to a height of 20 cm above
the gill and was inserted into another 100-yL pipette. The valve on
the perfusion apparatus was then turned and India ink, which contained
the test substance in the concentrations as noted above, was allowed
to flow through the gill until at least 100 yL of ink solution had been
collected from the efferent cannula. The ink used was Pelikan Bio-
logical India Ink (John Henschel Co., New York). According to
Peterson et al. (1965), this ink contains 10 per cent carbon with a
particle size of 0. 02 -0. 03 y , 4. 3 per cent fish glue, 1. 0 per cent
phenol, and none of the shellac or ammonia normally found in other
ink preparations.
Additional ligatures which had been placed around the ends of gill
arches were tightened as soon as the cannulas were removed at the
end of the perfusion period, thus minimizing loss of ink from the
branchial vasculature during subsequent processing. Gills were
placed in Dietrich' s fixative (10 parts formalin, 20 parts 95% ethanol,
2 parts glacial acetic acid, and 59 parts water) for 12 hours, then
24
-------
Figure 7. Constant pressure perfusion apparatus: s, syringe;
v, valve; pe, polyethylene tubing; c, cannula.
25
-------
dehydrated, infiltrated with and embedded in Paraplast (A. H. Thomas,
Co., Philadelphia) using standard procedures. Sections of gills 8y
thick were mounted on glass microscope slides and stained using
standard procedures of Masson' s trichrom stain. Only gills from
which there was no visible leakage of India ink during perfusion were
prepared for histological examination. In fish, blood flows from the
ventral aorta into afferent branchial arteries, thence to afferent fila-
mental arteries, through lamellae, collects in efferent filamental
arteries, then flows into efferent branchial arteries and into the dorsal
aorta. Filamental and lamellar circulation were studied in detail, for
it is in these parts where exchange of materials between the blood
and the environmental water takes place. The India ink distribution
in acetylcholine -treated gills was essentially identical to that seen in
untreated controls. In both cases the ink was concentrated in the
filamental sinus and collateral vessels, with very little ink appearing
in the lamellar lacunae or marginal channel (see Figures 8 and 9). In
the epinephrine-treated gills the ink was concentrated in the lamellae,
with little or no ink in the filamental sinus and collateral vessels. In
gills perfused with India ink made up in "choline Ringer solution, " the
distribution of ink resembled much more closely that seen in
acetylcholine-treated and control gills than it did that in epinephrine -
treated gills. The distribution of ink in gills perfused with "choline
Ringer solution" containing lO'^M atropine, however, was more
similar to the pattern seen in epinephrine-treated gills than to that
seen in acetylcholine-treated and control gills. Histological cross -
sections of the afferent-lamellar and efferent-lamellar arterioles
revealed that both of these vessels contain muscular elements within
their walls. The presence of contractile elements in the walls of the
efferent-sinus vessels has not been clearly demonstrated histologically,
but neither the dimension of the vessels themselves nor the thickness
of the vessel walls precludes such a possibility. It was also observed
that the rate of fluid flow through the gill was strongly affected by the
nature of the perfusing fluid. The addition of epinephrine to the fluid
caused a 177 per cent increase in flow rate and perfusion with "choline
Ringer solution" resulted in a 66 per cent reduction in flow rate.
Addition of atropine to the "choline Ringer solution" resulted in a flow
rate which was not significantly different from the control rate. In
their studies of the pattern of blood flow through the gill, Steen &
Kruysse (1964) placed freshly excised gill filaments into physiological
salt solutions on a microscope slide. They then placed a cover-slip
over the filament and observed the patterns of fluid flow when pres -
sure was applied to the cover-slip. Using this method, they found that
in untreated gills blood flowed, often simultaneously, between the
afferent and the efferent filamental arteries by way of the (a) lamellae,
26
-------
Figure 8. The patterns of fluid flow through the teleost gill with
arrows indicating the direction of flow: eb, efferent branchial
artery; ab, afferent branchial artery; bl, branchial lymphatic vessel;
si, s^nus-lymphatic vessel; as, afferent sinus vessel; fl, filamental
lymphatic vessel; af, afferent filamental artery; me, marginal chan-
nel of the lamella; al, afferent-lamellar arteriole; 11, lamellar
lacumae; el, efferent lamellar arteriole; ef, efferent filamental
artery; es, efferent sinus vessel; fs, filamental sinus.
27
-------
el
Figure 9 Diagrammatic representation of a cross -section of a gill
filament: cs, cartilaginous support; rbc, red blood cell; ec, efferent
collateral vessel; 1, lamella; pc, pillar cell; ac, afferent collateral.
All other abbreviations are as in Figure 8.
28
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(b) filamental sinus, and (c) direct connection between the afferent
and efferent filamental arteries at the tip of the filament. When
acetylcholine was added to the salt solution bathing a filament, they
found that the blood flowed through the filamental sinus and around the
tip of the filament. Addition of adrenalin, on the other hand, caused
all of the blood to flow through the lamellae. The absence of a normal,
unidirectional, afferent-to-efferent pressure gradient in their experi-
ments makes it difficult to determine the true physiological significance
of the flow patterns described. In our study, using approximately
normal afferent-to-efferent pressure gradients, and with the acetyl-
choline or epinephrine in the perfusing fluid rather than applied to the
outside of the gill, the results we obtained were similar to those of
Steen & Kruysse (1964). The effects of epinephrine and acetylcholine,
however, were not as absolute as those reported by these investigators.
For example, perfusion with India ink containing acetylcholine did not
completely eliminate the flow of fluid through the lamellae, and per-
fusion with epinephrine did not result in 100 per cent lamellar flow.
The effect of atropine indicates that there may be some tonic cho-
linergic regulation of gill blood flow. Since we observed only small
differences between the distribution of India ink in control and
acetylcholine -treated gills, we believe that the pattern and/or regula-
tion of blood flow through teleost gills is primarily under adrenergic
control. In rainbow trout the pattern of blood flow through the gill
seems to vary from "purely cholinergic" flow (exclusively through
the filamental sinus) to "purely adrenergic" flow (exclusively through
the lamellae). The site of control of gill blood flow pattern probably
involves some combination of the lamellar arterioles and the sinus
vessels. Acetylcholine, which not only causes blood to flow through
the filamental sinus but also decreases the flow rate (Ostlund &
FSnge, 1962), probably acts solely to cause vasoconstriction of the
lamellar arterioles. Epinephrine gives rise to greater blood flow
through the high-resistance lamellar circulation and increases
lamellar flow rate. Thus epinephrine must cause both vasoconstric-
tion of the sinus vessels and vasodilation of the lamellar arterioles.
Sodium uptake by isolated -perfused gills of rainbow trout: As indi-
cated above, the initial aim of the perfusion studies was to obtain
information on the level or rate of sodium transport by gill epithelial
cells. In any analysis of the net uptake or excretion of ions by the
gill, four major factors must be taken into account. They are:
(1) the mechanism by which the ions are moved, e.g., active trans-
port, exchange diffusion, etc., (2) the surface area across which
exchange can occur, (3) the duration of exposure between the vascular
fluid (or perfusion fluid) and the fluid bathing the gill, and (4) the
29
-------
concentration gradient against which the ions must move. Experiments
designed to examine the net sodium flux across isolated-perfused
trout gills with respect to each of these parameters. Some informa-
tion on the mechanisms involved was obtained by examining the effects
of metabolic inhibitors on net flux of sodium. The effective surface
area available for exchange was studied by histological examination
of gills which had been perfused with India ink containing various
vasoactive agents (as noted above), and the rate of fluid flow through
the branchial vasculature was used as a measure of the duration of
exposure between the perfusion fluid and the water. The concentra-
tion gradients for sodium across the gills were determined in all
experiments and were kept relatively constant during any single ex-
periment. The perfusion system used was similar to that described
above. Gills were isolated and perfused with Ringer solution to clear
them of blood. Following this initial perfusion, a typical experiment
was conducted as follows. The free end of the outflow tube was raised
to a height of approximately 20 cm above the gill in order to establish
a normal afferent-to-efferent pressure gradient. The gill was then
placed in 50 ml of fresh 1 per cent Ringer solution and a clean 100 y L
pipette was placed over the free end of the outflow tube. A stop watch
was started and the time required to collect 100 y L of fluid was
recorded. Fifty-microliter aliquots of the stock perfusion fluid and
the fluid collected after passing through the gill were taken for sodium
analyses to provide control data. The valve on the perfusion apparatus
was then turned to allow the experimental perfusion fluid to flow
through the gill. A 100 yL sample of the fluid flowing from the
efferent cannula was collected and discarded. The gill was then
placed in 50 ml of fresh 1 per cent Ringer solution, a clean 100 yL
pipette was placed over the free end of the outflow tube and the time
required for the collection of 100 yL of fluid was determined and
recorded, Aliquots of the experimental perfusion fluid and the fluid
collected after passing through the gill were again taken for sodium
analyses. The sodium concentration of the bath solution was monitored
to check for leaks of perfusion fluid from the gills. If the bath con-
centration increased by more than 1. 5 m-equiv. Na /L, all data for
that experiment were discarded. Sodium was measured using a
Coleman Model 21 flame photometer and a Coleman Model 22 Galv-o-
meter. All dilutions were made with 0. 02% Sterox SE in distilled
water. Sodium concentration was read as per cent transmission and
converted to m-equiv. Na /L using a standard curve. As a constant
check on the accuracy of the readings, the solutions were read in the
following order: bland, 150 m-equiv. /L standard, bland, sample,
bland, etc. The accuracy with which the sodium concentration of a
given sample could be determined was ±1.0 m-equiv. Na /L. When
30
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Table VI
Sodium uptake as affected by variable sodium concentrations,
inhibitors, and vasoactive agents. The rates of sodium uptake are
given for gills perfused with experimental solutions containing dif-
ferent sodium concentrations, inhibitors, and vasoactive agents.
Solution
100% Ringer
Choline Ringer 1
Choline Ringer 2
Choline Ringer 3
Choline Ringer 4
Choline Ringer 4 +
10 "3M A tr opine
Choline Ringer +
10 ~5M Epinephrine
Choline Ringer 4 +
10-4M Ouabain
Choline Ringer 4 +
10 ~*M Cyanide
Choline Ringer 4 +
10 ~*M lodoacetate
100% Ringer +
10 ~**M Epinephrine
concentration
in
perfusion
fluid
mEq/L± S. E.
156.2 ±0.5
139,2 ± 0.4
124. 0± 0.6
116.1 ± 1. 1
83.9 ±0.0
81. 4± 0.9
81.2 ± 0.8
86. 3± 1.3
84. 9 ± 1.1
83. 3 ± 1.2
150. 4 ± 1.8
Rate of
Na+ uptake
in
pEq/min ± S. E,
-0.026± 0.014
0.068± 0.025
0.142 ± 0.040
0. 122 ± 0. 040
0.189± 0. 110
0.223 ± 0.070
1.154± 0.182
0.034± 0.017
0.086± 0.033
0.201 ± 0.064
0.388± 0.137
31
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isolated trout gills were perfused with 100 per cent Ringer solution,
there was a small but significant loss of sodium to the bathing
medium; however, in all cases where the perfusion fluid had concen-
trations of sodium less than that of normal plasma or when epinephrine
was added to the perfusion fluid, sodium uptake was significantly
greater than zero (Table VI). When the sodium concentration of the
perfusion fluid was reduced to 139, 123, 115 and 84 m-equiv. Na2/L
(choline Ringer 1 -4) by replacing sodium chloride with choline chloride,
it was found that the rate of sodium uptake was inversely related to
the sodium concentration of the perfusion fluid (Figure 10). The
internal sodium concentration at which no net uptake or loss of sodium
would be expected to occur (152 m-equiv. /L) is very close to the
normal plasma sodium concentration (155. 4 ± 1.2 m-equiv. /L) which
was found in the rainbow trout studied. Addition of atropine (10~^M)
to the perfusion fluid did not result in any significant change in the
rate of sodium uptake by gills perfused with choline Ringer 4. The
most rapid uptake of sodium occurred when gills were perfused with
choline Ringer 4 which contained 10~5M epinephrine. The rates of
sodium uptake by gills perfused with choline Ringer 4 containing
10 M ouabain or cyanide were 86 and 65 per cent lower, respectively,
than the rates of uptake by gills perfused with choline Ringer 4 which
contained no inhibitor. The differences were significant at the P =
0. 025 and P less than 0. 10 levels, respectively. In a similar experi-
ment it was found that iodoacetate had no significant effect on the rate
of sodium transport by isolated gills. The various perfusion solutions
used in this study not only affected the rate of sodium uptake by the
gills, but also altered the rates and patterns of fluid flow through the
gills. These data are summarized in Table VII. When the experi-
mental perfusion solution used was the same as the control solution
(100 per cent Ringer), there was a significant increase in flow rate.
When epinephrine was added to the choline Ringer 4 and 100 per cent
Ringer perfusion fluids, the flow increased by 48 and 177 per cent
of the control values, respectively. Flow rates for all of the other
perfusions were significantly less than control rates, except that no
significant change in flow rate occurred when gills were perfused
with choline Ringer 2 or choline Ringer 4 containing atropine.
General discussion and summary of research on uptake of sodium by
trout gills: When isolated gills were perfused with Ringer solution
which had a sodium concentration equal to or above that found in
normal trout plasma, we observed a slight but significant loss of
sodium to the bath solution. When the sodium concentration of the
perfusion fluid was below that of normal trout plasma, we were able
to measure a net transfer of sodium from the bath into the vascular
32
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.to
.IB
.10
RATE
OP
H.»
UPTAKE
.08
aoo
-JOB
•0 MO NO ItO BO MO
Mo' CONCENTRATION M PERPUSMN FLUB taw^/l)
«0
Figure 10. The rate of sodium uptake by gills as a function of the
sodium concentration of the perfusion fluid.
33
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Table VII
Factors affecting rate of fluid flow through isolated trout gills. The
rates of fluid flow through gills during control (100% Ringer) per-
fusions are given along with the per cent changes in flow rates during
perfusion with experimental solutions containing different sodium
concentrations, inhibitors, and vasoactive agents.
Solution
Initial rate of
fluid How
yL/min± S.E.
(100% Ringer
solution)
Per cent change
in flow rate
(experimental
perfusion)
100% Ringer
Choline Ringer 1
Choline Ringer 2
Choline Ringer 3
Choline Ringer 4
Choline Ringer 4 +
10~3M Atropine
Choline Ringer 4 +
10 ~5M Epinephrine
Choline Ringer 4 +
10~4M Ouabain
Choline Ringer 4 +
10"% Cyanide
Choline Ringer 4 +
10~TVI lodoacetate
100% Ringer +
10~5M Epinephrine
24.7 ±2.6
21. 9± 2.7
17.2 ±2.5
17.8± 2.6
22.2 ±2.2
33.3 ±3.9
34. 5 ± 6.4
15. 6 ± 4.4
19.5± 2.5
21.6± 3.8
22.7 ±3.7
+ 17.2 ±2.6
- 39.4± 11.9
- 43.2 ±7.6
- 33.7± 10.1
- 65.7 ± 7.3
- 1.9± 14.0
+ 48.8± 15.2
- 66.4± 12.4
- 74.1 ± 4.8
- 31. 6± 15.1
+177. 6 ± 40.3
34
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system of the gills. This net inward transfer of sodium was against
concentration gradients as high as 100:1, and the rate of uptake varied
inversely with the sodium concentration of the perfusion fluid. Thus
it appears that the gills themselves are able to regulate the net sodium
flux between the vascular system and the fluid bathing the gill and that
this autoregulatory action is dependent upon the concentration of
sodium in the perfusion fluid. The inhibition of sodium uptake by
ouabain and cyanide indicated that sodium uptake by isolated, perfused
rainbow trout gills is an ATP-dependent process and that much of the
ATP used is derived from oxidative metabolism. The complete lack
of inhibition by iodoacetate suggests that glycolysis per se is not
required for sodium uptake by the gill. These results, along with
those obtained by Kamiya (1967) using salt-water-adapted eels, sug-
gest that glycolysis may be generally unimportant as a metabolic
pathway in the gills of fish. The data and observations recorded in
Table VIII indicate that the rate of sodium uptake is not primarily
dependent upon either flow rate or flow pattern through the gill. Two
factors which do appear to affect the rate of net sodium movement
across the gill are the sodium concentration of the perfusion fluid and
epinephrine.
Deviations of the internal or vascular sodium concentration from
normal plasma levels may alter the rate and direction of net sodium
movement by either of two ways. Decreased sodium may directly
stimulate and increased sodium may directly inhibit the sodium
pumping mechanism present in gill epithelial cells. Alternatively,
the sodium pump may be continuously active at a level or rate which
just balances the normal sodium loss from the fish, thus a decrease
in the internal sodium concentration would decrease the passive loss
of sodium across the gill and result in a net uptake of sodium. The
converse would be true when the internal sodium concentration is
above the normal level. The fact that gills perfused with 100 per cent
Ringer solution containing 10 M epinephrine took up sodium at the
rate of 0. 388 y-equiv. /min as opposed to a net loss of 0. 026 y -equiv. /
min in the absence of epinephrine indicates that epinephrine has a
direct stimulatory effect on the sodium pumping mechanism. This
direct action of epinephrine was also evident when the sodium uptake
rate by gills perfused with choline Ringer 4 containing epinephrine
was compared with that by gills perfused with choline Ringer 4 alone.
It is of interest to note that the rate of sodium uptake by gills per-
fused with choline Ringer 4 containing epinephrine is much greater
than the rate of sodium uptake by either gills perfused with 100 per
cent Ringer containing epinephrine or by those perfused with choline
Ringer 4 alone. The fact that the combined effects of low sodium
35
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Table VIII
Factors affecting pattern of fluid flow through isolated trout gills.
This table gives the rates of sodium uptake, the per cent changes in
the rates of fluid flow, and the patterns of fluid flow through gills
perfused with experimental solutions containing different sodium con
centrations, inhibitors, and vasoactive agents.
Perfusion fluid
100% Ringer
Choline Ringer 1
Choline Ringer 2
Choline Ringer 3
Choline Ringer 4
Choline Ringer 4
containing atropine
Choline Ringer 4
containing epinephrine
Choline Ringer 4
containing ouabain
Choline Ringer 4
containing cyanide
Choline Ringer 4
containing iodoacetate
100% Ringer
containing epinephrine
Rate of
sodium uptake
(yEquiv. /min)
-0.026
+0.068
+0. 122
+0.125
+0.189
+0.223
+1.154
+0.034
+0.086
+0.201
+0.388
Per cent
change in
flow rate
+ 17.2
- 39.5
- 43.2
- 33.7
- 65.7
- 1.9
+ 48.8
- 66.4
- 74.1
- 31.6
+177.6
Flow pattern
filamental sinus
filamental sinus
filamental sinus
filamental sinus
filamental sinus
lamellar
lamellar
filamental sinus
filamental sinus
filamental sinus
lamellar
36
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concentration and epinephrine on sodium uptake are more than
additive has been interpreted to mean that these two stimuli act at
different levels on the sodium uptake mechanism. For example,
there may be two separate sodium pumping mechanisms, one which
transports sodium into the filamental sinus which is most responsive
to low internal sodium, and another which transports sodium into the
lamellae and is most responsive to epinephrine. The similarity of
the sodium uptake rates during perfusions with choline Ringer 4 and
choline Ringer 4 containing 10 "^M atropine indicates that there is no
cholinergic inhibitory mechanism acting to oppose the stimulatory
effect of epinephrine. In summary, the experiments reported above
strongly indicate that the uptake of sodium by isolated-perfused rain-
bow trout gills is an oxidative metabolism-dependent, ATP-dependent
system which is controlled by the sodium concentration of the perfusion
fluid and by epinephrine, and which is generally independent of the rate
and pattern of fluid flow through the gill.
Uptake of dieldrin by isolated perfused gills of rainbow trout: During
the course of our experiments with isolated-perfused gills, the
Fisheries and Wildlife Department at Michigan State University
allowed us the use of a gas chromatograph on a limited time basis.
We chose to work with the insecticide dieldrin in order to take advan-
tage of the experience and advice offered by Dr. H. Johnson and
coworkers in that department. Dieldrin, a cyclodiene, is a widely
used pesticide with a relatively long residual life in the environment.
Aquatic organisms such as fish may assimilate dieldrin from water
through the gill membranes or skin or via the alimentary tract.
Holden (1962, 1965), Premdas and Anderson (1963), Crosby and
Tucker (1966), and Lenon (1968) have all suggested that uptake of DDT
and related insecticides including dieldrin occurs primarily by way of
the gills. No specific data on gill uptake were presented by these
workers, but the extremely rapid uptake and dispersal of DDT in fish
led them to conclude that the gills are the chief port of entry and the
blood is the chief means of transport of assimilated insecticides. The
purpose of our experiments was to investigate the transfer of dieldrin
from the environmental water into the vascular system of isolated-
perfused gills of rainbow trout.
Gill arches were surgically removed and attached to a gill perfusion
apparatus as described above. The arches were perfused initially
with Ringer solution containing glucose for about 15 mins to clear the
filaments of blood cells, and during this time the gills were bathed
with the nutrient Ringer solution. Dieldrin was dissolved in ethanol
(10 mg/ml) and bath solutions containing approximately 1000 ug/L
37
-------
were prepared by adding 0.1 ml of dieldrin stock solution to 1 liter of
dechlorinated tap water. Bath solutions were prepared at least 24 hours
prior to use. About 80 ml were then put into a glass beaker and stirred
continuously with a teflon-coated stirring bar. Analyses of samples
indicated that during the 9 -min equilibration period a variable amount
of dieldrin was volatilized or adsorbed on the beaker walls so that the
initial concentrations to which the gill arches were exposed varied
from one experiment to another. All experiments were performed in
a walk-in refrigerator at 13°C.
To closely mimic normal physiological conditions the initial perfusion
fluid used was heparinized plasma. Since it was difficult to obtain
sufficient volumes of plasma from a single fish for perfusion and we
disliked the idea of using pooled samples from several donor fish, a
fish Ringer solution containing glucose was substituted as the perfusion
fluid. When no dieldrin appeared in the perfusate of Ringer-perfused
gills, we used TC-199 instead of the Ringer solution. The synthetic
tissue culture medium (TC-199) is a product of Difco Co., Detroit,
Michigan, which contains salts, vitamins and several amino acids but
no protein.
In a typical experiment a 1 ml aliquot of the dieldrin bath was taken
for analysis at the time the gills were placed in the solution, and a
second aliquot was taken after 1 ml of perfusate had been collected
from the gill. A 1 ml aliquot of the perfusion fluid was also taken for
analysis. For determinations of the amount of dieldrin adsorbed on
or bound to gill tissues, gills were removed from the bath, flushed
with Ringer solution for 3 -5 min, weighed, and then digested with
20 per cent alcoholic KOH for 1 hr at 76°C. For analytical consistency
all solutions analyzed were subjected to the same digestion procedure
as the gill tissues even though not all of the samples contained protein
material. After digestion all samples were individually extracted with
petroleum ether from which water was removed using anhydrous sodium
sulfate. The dieldrin content of the final solution was determined by
gas chroma tog raphy. In our initial analyses ether extracts were run
through a Florisil column to eliminate co-extractives, and many of
the smaller peaks in the chromatogram were eliminated. This pro-
cedure was deemed unnecessary and was discarded in the studies
reported here. The chromatograph used was an Aerograph HY-FL
model 600-C (Wilkens Inst. & Res. Inc.) with a proportional isothermal
temperature control (Model 328) and an electron capture detector.
The 5' X |" pyrex glass column was packed with 3 per cent QF-1 on
100/120 mesh Gas-Chrom Q. Column temperature was 180°C and
gas (nitrogen) flow 40 ml/min.
38
-------
EXP I
WINGER)
EXP 2
(TC-199)
EXP 3
(PLASMA)
CO
CO
EXP 4
/PLASMA\
^ SUPER I
Figure 11. Gas chromatography tracings of (A) perfusion fluid, (B) perfusate, and (C) perfusate to
which a known amount of dieldrin was added (spiked), excepting that experiment 3C is spiked per-
fusion fluid. In experiments 1, 2 and 3 the gills were perfused with Ringer solution, TC-199, and
rainbow trout plasma respectively. Tracings for experiment 4A and B are of the supernatant solu-
tions which remained after plasma samples had been treated with barium hydroxide -zinc sulfate
and centrifuged to remove protein. Experiment 4C is a chromatogram for spiked supernatant solu-
tion. Dieldrin peaks are indicated with solid black arrows.
-------
Results: Typical gas chromatography tracings of the solutions analyzed
in the experiments (Figure 11) show that no change occurred either in
Ringer solution (experiment 1A, B) or in TC-199 (experiment 2A, B)
during passage through isolated gills. That none of the peaks which
appear in the tracings of the experimental solutions represented
dieldrin is shown by tracings of spiked samples (experiment 1C and
2C). When isolated gills perfused with plasma were placed in a bath
of tap water containing dieldrin, the insecticide was found in the plasma
perfusate (experiment 3B). Tracings of plasma perfusate from gills
which were not exposed to dieldrin were identical to those for non-
perfused plasma (experiment 3A), whereas samples of spiked non-
perfused plasma (experiment 3C) were identical to those for plasma
from dieldrin-exposed gills (experiment 3B). Although the tracings
are not shown, spiking of plasma from dieldrin-exposed gills caused
an increase in size or height of the dieldrin peak and confirmed the
original presence of dieldrin in these samples.
The proteins in two samples of plasma perfusate from gills exposed
to dieldrin and in a sample of nonperfused plasma were preciptated
using barium hydroxide and zinc sulfate (Somogyi procedure). Gas
chroma tog raphic tracings of the supernatant fluid from these samples
(Figure 11, experiment 4A, B) indicate that the dieldrin present in the
plasma samples was preciptated along with the plasma proteins.
Large amounts of dieldrin were found adsorbed on or bound to perfused
gills (Table IX) with relatively greater quantities being found associated
with plasma-perfused gills than those perfused with Ringer solution.
The dieldrin concentration in the plasma perfusate was uniformly less
than that in the solution which bathed the gills; and over the range of
concentrations tested, the amount of dieldrin transferred across
isolated gills appeared to be dependent upon the concentration of the
insecticide in the bath.
Discussion and summary: We have clearly demonstrated that dieldrin
can be transferred from environmental water into the vascular system
of isolated perfused gills of rainbow trout. This transfer occurred
only when protein, or more probably lipoprotein, was present in the
perfusion fluid. Since the dieldrin concentration in the plasma per-
fusate from gills was always less than that of the bath fluid, a gradient
for inward diffusion existed and there is no need to postulate any other
mechanism for inward movement of the insecticide by the gill. The
greater quantities of dieldrin found in gills perfused with plasma than
in those perfused with Ringer solution may indicate either a greater
entry of dieldrin into epithelial cells of gills perfused with plasma and
a consequent greater internal binding of the insecticide or inadequate
40
-------
flushing of the gill lamellae prior to analyses. As the plasma used had
been obtained by bleeding the fish from which the gills were taken, it
is probable that epinephrine was released into the blood during the
blood letting procedure. Epinephrine causes high blood flow into
lamellae of perfused gills and it may be that the lamellae were not
flushed out by the Ringer solution, which contained no epinephrine.
Table IX
Summary of information on amount of dieldrin bound to perfused gills
and effect of concentration of dieldrin in bath on dieldrin transfer by
gills. Concentration of dieldrin is given as parts per billion (yg/L).
Per fusion
fluid
Ringer
Ringer
TC-199
TC-199
Plasma
Plasma
Number
of
perfusions
2
8
2
2
2
9
Concentration
of dieldrin
in bath
none
119-339
none
289
none
169-515
Dieldrin
bound
to gill
no
a
yes
b
b
no
yes
Concentration
of dieldrin
in perfusate
none
none
none
none
none
64-220
But much less than that found in plasma perfused gills.
Not analyzed for dieldrin.
Lipoprotein is present in mammalian blood, and evidence has been
accumulating that much of the lipid material in human plasma is
combined with protein. This may also be true for fish plasma. We
suggest that dieldrin- and other related insecticides diffuse through
41
-------
gills of fishes and are dissolved in the lipid portion of plasma lipo-
protein, in which form they are transported to and become dissolved
primarily in the lipid portion of the various tissues. Since the
insecticide is much more soluble in lipid than in water, the tissue
concentrations can attain levels far above that of the environmental
water independent of any active transport mechanism. Little is known
about the pathways used for the excretion of dieldrin, but since release
is rapid (Gakstatter and Weiss, 1967; Lenon, 1968) it appears unlikely
that excretion of most of dieldrin is dependent upon lipid turnover in
the tissues.
Effects of some insecticides and MS-222 on isolated-perfused gills of
trout: Gills of rainbow trout were prepared for perfusion as previously
described. The gills were initially bathed with 1 per cent Ringer
solution and perfused with 100 per cent Ringer to clear them of blood.
Gills were then placed in a fresh 1 per cent Ringer bath and perfused
with Ringer solution in which choline chloride was substituted for NaCl
so that the solution contained only 80 per cent of the normal amount of
sodium (control). After control flow rates were determined, they
were placed in a fresh 1 per cent Ringer bath which contained the test
chemical and perfused with the low sodium Ringer solution (experi-
mental). To determine the pattern of gill blood flow under the different
experimental conditions, gills were perfused with an India ink solution
and then prepared for histological examination as described above. No
gills exposed to malathion were perfused with India ink.
Table X
Acute effect of various chemicals on rate of fluid flow through
isolated-perfused gills of rainbow trout. Flow rates are in
for the control and experimental periods as described in the text
Conditions
Untreated gills
Dieldrin (1 ppm)
Methoxychlor (1 ppm)
Rotenone (1 ppm)
MS -222 (100 ppm)
Malathion (5 ppm)
n
11
10
6
6
10
6
Control
37
34
33
41
38
35
Experimental
34
27
30
25
28
28
Per cent
change
10
21
8
39
24
20
42
-------
Date for mean flow rates in VL/min during the control and experimental
periods are presented in Table X. In each case the mean flow rate
was lower during the experimental than during the preceding control
period. All differences excepting those for untreated gills and those
exposed to methoxychlor were statistically significant (p < 0. 10).
With respect to patterns of blood flow through gills, histological
sections of gill filaments were examined and the number of lamellae
which contained ink were noted. For example, some 2348 sections
of lamellae from one gill were observed, and 1865 contained ink;
thus, 79 per cent of the lamellae for that particular gill exhibited
lamellar flow. The other data presented in Table XI were obtained
in the same manner.
Table XI
Acute effect of various chemicals on pattern of blood flow through
isolated-perfused gills of rainbow trout. Data represents per cent
of lamellae perfused with India ink and. values were obtained as
described in the text.
Control
79
70
92
81
Rotenone
92
94
82
82
92
98
Methoxychlor
61
81
85
95
Dieldrin
94
97
93
MS -222
83
90
98
X
80
90
80
94
90
Statistics: Rotenone > Control, p = 0.057; Dieldrin > Control,
p = 0. 028; Methoxychlor = Control, p > 0. 343; MS-222 = Control,
p= 0.114.
43
-------
All chemical compounds tested except methoxychlor had an acute effect
on isolated gills and reduced the flow of perfusion fluid through them.
In this same series of experiments we measured the sodium content
(flame photometry) of the afferent and efferent perfusion fluids and,
using appropriate calculations, we were able to detect changes in
inward sodium transport per unit of time. In many cases the dif-
ferences in the concentration of sodium in the afferent and efferent
solutions were so small as to be within the range of accuracy of the
analytical procedure employed. To determine the effect of the test
chemicals on sodium transport by isolated gills, it will be necessary
to use radiosodium, and we plan to carry out experiments of this
nature in our laboratory.
It should be emphasized that the decreases in flow rates occurred at
constant perfusion pressure, which means that resistance to fluid flow
through gills was increased. Blood flow from afferent to efferent
filamental vessels in fish gills is via the filamental sinus, apical and
lamellar lacunae pathways. It has been assumed that the presence of
pillar cells in lamellar lacunae make this a high resistance pathway
for fluid flow. Smooth muscle, capable of controlling blood flow
patterns, has been found in the walls of the afferent and efferent
vessels of both the filamental sinus and the secondary lamellae.
Although increased lamellar perfusion and decreased flow rates
generally go hand-in-hand, the addition of epinephrine to the perfusion
fluid was shown to give rise to both an increase in lamellar perfusion
and an increase in the overall flow rate through the gill, as noted
above. Thus the substances tested may have exerted their effects at
one or more of the various control sites in the afferent and efferent
branchial vasculature.
The decreased flow of fluid through gills treated with rotenone and
dieldrin appears to result from a shift of flow into the high resistance
lamellar route. Flow rates for methoxychlor treated gills were not
significantly different from those which occurred in controls and both
groups had similar percentages of lamellar perfusion. The absence
of a significant change in the pattern of ink flow through MS-222 treated
gills may have been due to a change in the resistance of the other
blood flow pathways, or the data may represent an artifact due to the
small number of observations and the variability of the gills examined.
In summary; Isolated gills of rainbow trout were perfused with a
sodium deficient Ringer solution in the presence of various pesticides
and MS-222 and flow rates were determined. This was followed by
44
-------
perfusion with India ink and preparation for histological determination
of fluid flow patterns through the branchial vasculature. It was found
that short-term (acute) exposure to dieldrin, rotenone, malathion
and MS-222 resulted in a statistically significant reduction in perfusion
flow rate through the isolated gills. Exposure of gills to 1 mg/L
methoxychlor was without effect. Results of India ink perfusions
indicated that decrease in rate of fluid flow through the gills corre-
lated well with increased lamellar perfusion.
45
-------
SECTION V
ACKNOWLEDGME NTS
This research was carried out in the Comparative Physiology Labo-
ratory, Department of Physiology, Michigan State University, East
Lansing, Michigan 48823.
The research was supported in part by the Michigan Agricultural
Experiment Station. The rainbow trout used in the experiments were
obtained from the Michigan Department of Natural Resources through
the cooperation of Dr. L. N. Allison, Fish Pathologist, Grayling
Research Station, Grayling, Michigan.
The gas chromatograph used in experiments with dieldrin was made
available through the courtesy of Dr. Howard Johnson, Department
of Fisheries and Wildlife, Michigan State University.
47
-------
SECTION VI
REFERENCES CITED
Burrows, R. E. (1964) Effects of accumulated excretory products
on hatchery -reared salmonids. U.S. Dept. of Interior,
Bureau of Sport Fisheries & Wildlife Research Report 66:
1-11.
Brockway, D. R. (1950) Metabolic products and their effects. Progr.
Fish-Cult. 12: 127-129.
Crosby, D. G., and Tucker, R. K. (1966) Toxicity of aquatic herbi -
cides to Daphnia magna. Science 154: 289-291.
Erickson, J. G. (1967) Social hierarchy, territoriality and stress
reaction in sunfish. Physiol. Zool. 40: 40-48.
Fagerlund, U. H. M. (1967) Plasma cortisol concentration in rela-
tion to stress in adult sockeye salmon during the freshwater
stage of their life cycle. Gen. Comp. Endocrinol. 8: 197-
207.
Fromm, P. O., and Stokes, R. M. (1962) Assimilation and metabo-
lism of chromium by trout. J. Water Pollution Control
Federation 24:1151-1155.
Gakstatter, J. H. . and Weiss, C. M. (1967) The elimination of
DDT-C , dieldrin-C14 and lindane-C14 from fish following
a single sublethal exposure in aquaria. Trans. Am. Fish.
Soc. 96: 301-307.
Guillemin, R., Clayton, G. W., Lipscomb, H. S., and Smith, J. D.
(1959) Fluorometric measurement of rat plasma and adrenal
corticosterone concentration. J. Lab. Clin. Med. 53: 830-
832.
49
-------
Hane, S. , Robertson, O. H., Wexler, B. C., and Krupp, M. A.
(1966) Adrenocortical response to stress and ACTH in
Pacific salmon (Oncorhynchus tschawtscha) and rainbow
trout (Salmo gairdnerii) at successive stages in the sexual
cycle. Endocrinology 68: 791-800.
Hatey, J. (1958) Influence de 1'agitation motrice sur la teneur du
plasma en 17-hydroxycorticosteroids d'un teleosteen; la
carp (Cyprinus carpio L.). Compt. Rend. 246: 1088-1091.
Holden, A. V. (1962) A study of adsorption of 14C-labelled DDT from
water by fish. Ann. Appl. Biol. 50: 467-477.
Holden, A. V. (1965) Contamination of fresh water by persistent
insecticides and their effects on fish. Ann. Appl. Biol. 55:
332-335.
Kamiya, M. (1967) Changes in ion and water transport in isolated
gills of the cultured eel during the course of salt adaptation.
Annot. Zool. Jap. 40: 123-139.
Lenon, H. L. (MS, 1968) Translocations and storage equilibria
involving sublethal levels of dieldrin in aquatic ecosystems.
Ph.D. Thesis, Michigan State University, Dept. of Fisheries
and Wildlife, East Lansing, Mich. 85 p.
McCay, C. M., and Vars, H. M. (1950) Studies upon fish blood and
its relation to water pollution. In A biological survey of the
St. Lawrence watershed, Suppl. to Twentieth Ann. Rep.,
N.Y. State Cons. Dept., 230-231.
McKim, J. M., III. (1966) Stress hormone metabolites and their
fluctuations in the urine of rainbow trout (Salmo gairdnerii)
under the influence of various sub-lethal stressors. Ph.D.
thesis, U. of Michigan, Ann Arbor, Mich.
Ostlund, E., and Fange, R. (1962) Vasodilation by adrenaline and
noradrenaline, and the effects of some other substances on
perfused fish gills. Comp. Biochem. Physiol. 21: 415-424.
Peterson, R. A., Ringer, R. K., Telzloff, M. J., and Lucas, A. M.
(1965) Ink perfusion for displaying capillaries in the chicken.
Stain Technol. 40: 351-356.
50
-------
Premdas, F. H., and Anderson, J. M. (1963) The uptake and
detoxification of C14-labelled DDT in Atlantic salmon,
Salmo salar. J. Fish. Res. Bd. Canada 20: 827-837.
Reichenbach-Klinke Von H. H. (1967) Untersuchungen iiber die
Einwirkung des Ammoniakgehalts auf den Fischorganismus.
Arch. Fischereiwiss. 17: 122-132.
Robertson, O. H., Hane, S., Wexler, B. C., and Rinfret, A. P.
(1963) The effect of hydrocortisone on immature rainbow
trout (Salmo gairdnerii). Gen. Comp. Endocrinol. 3: 422-
436.
Steen, J. B., and Kruysse, A. (1964) The respiratory function of
teleostean gills. Comp. Biochem. Physiol. 12: 127-142.
Weil-Malherbe, H. (1962) Ammonia metabolism in the brain. In
Neurochemistry (Edited by Elliot, Page and Quastel),
pp. 321-329.
Wuhrmann Von K., and Woker, H. (1948) Betrage zur toxikologie
der fische. II. Experimentelle untersuchungen iiber die
Ammoniak- und blausaure-vergiftung. Schweiz. Z. Hydrol.
11: 210-244.
51
-------
SECTION VII
PUBLICATIONS
Hill, C. W., and P. O. Fromm. (1968) Response of interrenal
gland of rainbow trout (Salmo gairdneri) to stress. Gen.
Comp. Endocrinol. 11: 69-77"
Fromm, P. O., and J. R. Gillette. (1968) Effect of ambient ammonia
levels on blood ammonia and ammonia excretion by trout.
Comp. Biochem. Physiol. 26: 887-896.
Fromm, P. O. (1968) Some quantitative aspects of ion regulation in
teleosts. Comp. Biochem. Physiol. 27: 865-869.
Richards, B. D., and P. O. Fromm. (1969) Patterns of blood flow
through filaments and lamellae of isolated -perfused rainbow
trout (Salmo gairdneri) gills. Comp. Biochem. Physiol. 29:
1063-1070.
Fromm, P. O., and R. C. Hunter. (1969) Uptake of dieldrin by
isolated-perfused gills of rainbow trout. J. Fish. Res. Bd.
Canada 26: 1939-1942.
Richards, B. D., and P. O. Fromm. (1970) Sodium uptake by
isolated-perfused gills of rainbow trout (Salmo gairdneri).
Comp. Biochem. Physiol. 33: 303-310.
Fromm, P. O., Richards, B. D., and R. C. Hunter. (1970) Effects
of some insecticides and MS-222 on isolated-perfused gills
of trout. Accepted for publication in the Progressive Fish-
Culturist, Sept. 1970.
53
-------
SECTION VIII
PERSONNEL
Staffing: All persons, other than the Principal Investigator and
R. C. Hunter, who were formerly or are now employed on this grant
project have been graduate students in the Department of Physiology
at Michigan State University. Appointments were usually on a one -
half time basis and were entitled "Special Graduate Research Assis-
tant. " Appointees, along with a brief biographical sketch, are listed
in chronological order of employment. Dates of employment are in
parentheses.
Janet Gillette, B. S. Biology, Bridgewater State College; M. S.
Physiology, Michigan State University, 1967. Currently employed
as an instructor in Biological Science at University of Pittsburgh at
Greensburg, Pennsylvania, and is working toward Ph. D. at University
of Pittsburgh. (6-66 to 9-67)
C. Hill, B.S. Chemistry-Physics, Wisconsin State College, Superior;
B. S. and M. S. Fish and Wildlife Management, Montana State Uni-
versity; Ph.D. Physiology, Michigan State University, 1967. He is
currently employed as Assistant Professor, Department of Biology,
California State College, Long Beach, California. (6-66 to 7-67)
Mack Holt, B. S. Biology, Ft. Valley State College; withdrew from
graduate program in Physiology at Michigan State University, 2-15-68.
Current address is unknown. (9-67 to 2-68)
Wayne Price, B.S. Biological Sciences, Michigan State University;
withdrew from Master1 s program in Physiology at Michigan State
University, December 1968. Currently in U. S. Armed Forces.
(3-68 to 12-68)
B. D. Richards, B.S. Zoology, University of Michigan; M. S. Biology,
Florida State University; Ph.D. Physiology, Michigan State Univer-
sity, 1969. Currently employed as Assistant Professor, Department
of Biological Sciences, Illinois State University, Normal, 111. (9-67
to 6-68)
55
-------
R. C'. Hunter. H. S. Physiology, Michigan State University; M. B. A.
Michigan State University, 1969. Currently employed by Pfizer Drug
Company, New York, New York. Mr. Hunter was a U.S. Navy
veturan, a fornn-r corpsman, and was hired on an hourly basis as a
technician. (6-69 to 12-69)
Konneth Olson, B. S, Biochemistry, University of Wisconsin at
LaCrosse; M. S. Physiology, Michigan State University, 1970. Cur-
rently a graduate student in Physiology at Michigan State University.
(6-69 to present)
R. I.. Walker, B. S. Biological Sciences, Alma College. Currently
a graduate student in Physiology at Michigan State University. (6-70
to present)
56
-------
\um6«r
5ubf«r>
Ftfldtt Group
SELECTED WATER RESOURCES ABSTRACT!
INPUT TRANSACTION FORM
Michigan State University, Department of Physiology, E. Lansing, Mi. 48823
Toxic action of water soluble pollutants on freshwater fish
• A Aulhaiff)
Dr. Paul 0. Promm
11
i • i t
16
Date
12-5-70
Pro/«cf Numbw
18050DST
J2jP""
56
i
21
. c Contract .Vumber
Note
22 Ctltlian
"23}
D»«cr(pfor« (Slarrtd Flnl)
Fish*, stress*, chromium, Insecticides, ammonia, sodium transport
gill blood flow, nitrogen, fish physiology, nitrogen excretion*
Pish" stress, nitrogen excretion, fish physiology
27
Over a five year period experiments on rainbow trout Indicated that exposure
to chromium ant to forced exercise caused a transient increase in plasma
cortisol. Exposure to ammonia (a) caused a decrease in the rate of total
nitrogen excretion and in ammonia excretion (b) caused some histopathologl-
cal changes in trout gills but oxygen transport by hemoglobin was unaffected
(c) caused a very slight increase in urea excretion by trout but a vary
significant rise in goldfish. Hyperexcitability observed in ammonia-exposed
trout was not noticeable in the more resistant goldfish. Ammonia may kill
fish by prevention of excretion of normal amounts of endogenous ammonia.
Experiments with isolated-perfused gills of trout have shown among other
things that (a) gill blood flow patterns are significantly affected by epi-
nephrine (b) when perfused with Ringer solution there was a small but signi-
ficant loss of sodium into the bath solution, whereas perfusion with sodium-
poor Ringer solutions always resulted in a net uptake of sodium (c) perfusion
fluid sodium and epinephrlne appear to control sodium uptake by the gill (4)
transfer of dieldrin into the vascular system occurred only when plasma
protein, or more probably plasma lipoprotein was present in the perfusion
fluid (a) short term exposure to dieldrin, rotenone, malathlon and MS-222
reduced perfusion flow rate through isolated gills but exposure to 1 rag/L
methoxychlor was without effect. Decrease in flow rate correlated well
with lamellar perfusion.
Intlllulion
Mich. State University
NM|t«i miv OCT. mi)
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------- |