Ecological Research Series
TOXIC EFFECT OF WATER SOLUBLE POLLUTANTS
ON FRESHWATER FISH
Environmental Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Duluth, Minnesota 55804
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RESEARCH REPORTING SERIES
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EPA-600/3-77-057
May 1977
TOXIC EFFECT OF WATER SOLUBLE POLLUTANTS
ON FRESHWATER FISH
by
Paul 0. Fromm
Department of Physiology
Michigan State University
East Lansing, Michigan 48824
Grant Number R-801034
Project officer
James M. McKim
Environmental Research Laboratory-Duluth
Duluth, Minnesota 55804
ENVIRONMENTAL RESEARCH LABORATORY-DULUTH
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
DULUTH, MINNESOTA 55804
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DISCLAIMER
This report has been reviewed by the Environmental Research Labor-
atory-Duluth, U.S. Environmental Protection Agency, and approved for
publication. Approval does not signify that the contents necessarily
reflect the views and policies of the U.S. Environmental Protection
Agency, nor does mention of trade names or commercial products consti
tute endorsement or recommendation for use.
ii
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FOREWORD
Our nation's freshwaters are vital for all animals and plants, yet our
diverse uses of water — for recreation, food, energy, transportation, and
industry — physically and chemically alter lakes, rivers, and streams. Such
alterations threaten terrestrial organisms, as well as those living in water.
The Environmental Research Laboratory in Duluth, Minnesota develops methods,
conducts laboratory and field studies, and extrapolates research findings
—to determine how physical and chemical pollution affects aquatic
life,
—to assess the effects of ecosystems on pollutants,
—to predict effects of pollutants on large lakes through use of
models, and
—to measure bioaccumulation of pollutants in aquatic organisms that
are consumed by other animals, including man.
While the results of this project do not provide "numbers" for direct
agency use, the study is no less important to EPA. Only by understanding
modes of action and physiological pathways can aquatic toxicologists predict
effects of pollutants without long and expensive testing. Otherwise our
data produced are applicable only to the conditions studied.
This report moves us another step closer to better predictions of
toxicity.
Donald I. Mount, Ph.D.
Director
Environmental Research Laboratory
Duluth, Minnesota
ill
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ABSTRACT
Studies of the effect of inorganic and organic mercury on trout indicated
that uptake was primarily via the gills in non-feeding fish. Organic, mercury
entered fish at a faster rate than inorganic mercury. Exposure of trout to
10 yg Hg/1 (methyl forir.) had no effect on the gill oxygen consumption measured
in vitro or on the plasma electrolytes. The hematocrit index increased
significantly. Studies of the metabolism of iron hy normal and iron deficient
trout (made deficient by bleeding) indicated that the liver, spleen, and head
kidney are the major iron storage organs. Liver iron was reduced by bleeding
whereas splenic iron was unaffected. In iron deficient fish more radioiron
appeared in erythrocytes than in normal controls. Studies of isolated-perfused
gills revealed the presence of both a and 3 adrenergic receptors and the data
obtained indicate the functional surface area of trout gills can be regulated
by changes in perfusion pathway through the gills. Use of perfused gills
appears to be a very sensitive model to detect deleterious action of pollutants
on fish. Evaluation of heat exchange in perfused gills indicates that the
presence of epinephrine increased the transfer maximum of the gill but they
were unaffected by the administration of acetylcholine. Analysis of a simple
model indicated that the gills may account for as much as 60% of the total
heat exchange by trout.
iv
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CONTENTS
Foreword iii
Abstract „. •, iv
Figures , , , vi
Tables „ „ viii
Acknowledgments ix
1. Introduction 1
2. Conclusions 2
3. Recommenda t ions...., »... 4
4. Effects of methyl mercuric chloride and mercuric chloride on
rainbow trout 5
A study of uptake pathways into whole fish 5
Uptake of mercury by trout erythrocytes in vitro 7
Mercury uptake and ion distribution in gills of
rainbow trout „ 10
Effect of methyl mercury on gill metabolism and blood
parameters of rainbow trout 12
Effect of mercury exposure on rainbow trout gill
ultrastructure •. 14
5. Iron metabolism „ 17
Metabolism of iron by normal and iron deficient
rainbow trout 17
6. Respiratory physiology „ 24
Control mechanisms for regulating the functional
surface area of fish gills „ 24
The role of fish gills in heat exchange 32
The vascularity of trout gills 44
References „ „ 48
Publications 51
Index 52
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FIGURES
Number Page
1 Percent uptake of methyl mercury or divalent mercury by
red blood cells as a function of time 9
2 Three component exponential curve of iron clearance from
plasma of rainbow trout following i.p. injection of ->9pe 19
3 Specific activity of iron in tissues of trout following
intraperitoneal injection of ^'Fe 21
4 Specific activity of iron in tissues of iron deficient
trout following intraper itoneal injection of -*'Fe 21
5 Diagram of gill perfusion apparatus 26
6 Results from a typical epinephrine experiment 28
7 The effect of epinephrine on ^C-urea influx 29
8 The effect of adrenergic blockade on epinephrine induced
C-urea influx 29
9 The effect of acetylcholine on ^C-urea influx 30
10 Typical effect of acetylcholine on perfusion pressure 31
11 Typical effect of norepinephrine on perfusion pressure
in ;'he absence and presence of adrenergic blockers 32
12 Diagram of apparatus used to perfuse isolated heads of
rainbow trout 35
13 Experimental data for heat transfer plotted as a function of _1
ventilatory flow for one isolated head perfused at 20 ml min 36
14 The effects of alterations in perfusion flow and heart rate
on heat exchange from perfused head of rainbow trout 37
15 The effects of vasoactive agents on heat exchange from
perfused head of rainbow trout 38
vi
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FIGURES (continued)
Number Page
16 Diagram of model used to evaluate gill heat exchange in
terms of whole body heat exchange 41
17 Graph of percent of total heat flux from the model gill
with increasing perfusion flows 42
18 Model prediction of 7oQtot; as a function of ventilatory flow
(VK) using experimental data at perfusion flows of 16 and
20 ml rain'1 43
19 Microfil cast of a single pair of gill filaments from
rainbow trout , 43
vii
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TABLES
Number page
1 The 24-hr uptake of mercury by trout exposed to 275 ng/liter
mercury as either C^HgCl or HgCl2 6
2 Values for some blood parameters of control and mercury
exposed (10 ng Hg/liter) rainbow trout 13
3 In vitro oxygen consumption of gill tissue from control
and mercury exposed rainbow trout in 100% and 107,
phosphate buffered saline (PT>G) 14
4 Calculated values for the iron content of tissues of a
hatchery-reared lOOg rainbow trout 18
5 Hematocrit, hemoglobin, MCHC, and percent reticulocytes of
blood samples from control and experimental fish 22
6 Heat transfer (liA) of isolated-perfused second gill arches
of rainbow trout 36
7 Heat transfer for isolated-perfused head of rainbow trout
during control periods 36
v±ii
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ACKNOWLEDGMENTS
The research work reported herein was carried out in the Comparative
Physiology Laboratories, Department of Physiology, Michigan State Univer-
sity, East Lansing, Michigan 48824.
The research was supported in part by the Michigan Agricultural Experiment
Station, Project 122 and by the Institute of Water Research, Michigan State
University as OWRR Project No. A-064-MICH. Some of the rainbow trout used
in the experiments were obtained from the Michigan Department of Natural
Resources, Grayling Research Station, Grayling, Michigan. We are also in-
debted to Drs. W. Willford and R.E. Reinert, Great Lakes Fishery Laboratory,
Bureau of Sport Fisheries and Wildlife, Ann Arbor, Michigan for their sug-
gestions and generous supply of mercury exposed rainbow trout.
I am particularly indebted to Dr. J.R. Hoffert and Mrs. Esther Brenke not
only for their continued interest in this project but for their work in
preparing and photographing the many illustrations. I also thank Dr. R.
Pax, Wm. Jackson, G. Eldred, T. Snow and J. Ubels for their help with
various technical aspects of the research and for many lively discussions
of the project.
Needless to say, this research work would not have been completed without
the aid of the support personnel listed below:
Principal Investigator; Paul 0. Fromm, B.S. Zoology, M.S. Physiology,
Ph.D. Physiology all from Univ. of Illinois, Urbana, 111. Professor,
Department of Physiology, Michigan State University, East Lansing, Mi.
Support personnel: All persons other than R.L. Bergman who worked on or
were 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 Assistant1. A brief biographical sketch is given with each
person listed.
R.L. Walker, B.S. Biology, Alma College, M.S. Physiology, Ph.D. Physiology,
Michigan State University. Currently an Instructor, Department of Biology,
Univ. of Calgary, Calgary, Alberta, Canada.
W.F. Jackson, B.S. Zoology, M.S. Physiology, Michigan State University.
Currently a graduate student in Dept. of Physiology, Michigan State
University.
ix
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D.V. O'Connor, B.S. Biology, Oakland Univ., M.S. Physiology, Michigan
State University. Currently employed as a biologist for the U.S. Co?-ps
of Engineers, Baltimore, Md.
P.R. Sorenson, B.S. Zoology, Michigan State Uni.vcisity. Currently a
graduate student, Dept. of Physiology, Michigan State University.
K.R. Olson? B.S. Chemistry & Zoology, LaCrosse State University, M.S.
Physiology, Ph.D. Physiology, Michigan State University. Currently Asst.
Prof. Indiana Univ. School of Medicine, U. of Notre Dame, Notre Dame, Ind,
H..L. Bergman, B.A. Biology, M.S. Biology, Eastern Michigan University,
Ph.D. Fisheries Biology, Michigan State University. Currently Asst. Prof.
Dept. of Zoology and Physiology, Univ. of Wyoming, Laramie, Wyo.
* received no research assistant stipend, however, funds from Grant
R-801034 were used to support research project.
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SECTION 1
INTRODUCTION
The research work supported in part by Environmental Protection
Agency Grant No. R-801034 was conducted over a period of approximately
three calendar years. There were essentially three general research areas,
all of which involved laboratory experiments with rainbow trout. All
projects were carried out in the Comparative Physiology Laboratory, Depart-
ment of Physiology, Michigan State University, East Lansing, Michigan. The
results of most of the research work have previously been published and the
reader should consult the respective publications for a more detailed dis-
cussion than that which appears below. The information on the effects of
exposure to mercurials on the ultrastructure of gills is being prepared
for publication and the report of heat flux across the gills has been
accepted for publication in the Journal of Comparative Physiology,
The experimental animals used in all experiments discussed herein
were hatchery-reared rainbow trout (Salmo gairdneri). Fish were obtained
from the Michigan Department of Natural Resources hatchery at Grayling,
Michigan and a few wen supplied by the Great Lakes Fishery Laboratory,
Bureau of Sport Fisheries and Wildlife at Ann Arbor, Michigan. The most
recent experiments have been conducted with fish purchased from Midwest
Fish Farming Enterprises, Inc., Harrison, Michigan. During the period of
acclimation to laboratory conditions fish were kept in large (300 liter)
fiberglass tanks in flowing dechlorinated tap water at about 12° C under
a controlled photoperiod of 16 hours light per day. In our holding
facilities the fish were fed commercial trout food, either EWOS or Purina
Chow pellets. Subsequent treatment and feeding of fish varied with each
experiment and are so indicated below.
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SECTION 2
CONCLUSIONS
Twenty-four hour uptake rate of either 203Hgd2 or CH3203Hj',Cl by
rainbow trout was not affected by esophageal ligation. Uptake of these
two mercurials in non-feeding trout appears to be by way of the gills.
Methyl mercury enters the fish at a faster rate than the inorganic form
and anomalous tissue distribution of these two mercurials suggests that
inorganic mercury does not require methylation prior to entry into the
fish. In vitro experiments using radioactive mercurials demonstrated
high affinity of methyl mercury for red cells, up to 90% was bound to red
cells in 40 minutes. Only 9% of inorganic mercury was taken up by red
cells, but, this percentage was increased up to 65% if the cells were
washed and suspended in Ringer solution prior to incubation with mercury.
Mercury was found in gills of rainbow trout which had been exposed
to inorganic mercury but not in those exposed to methyl mercury. No
specific site for mercury uptake was identified and it is suggested that
inorganic mercury enters the gill across the general lamellar surface.
High concentrations were found associated with the gill cartilage. Since
little ion diffusion occurs during tissue preparation, localization and/
or identification of tissues can be accomplished by scans for various
elements: sodium, potassium chlorine and sulfur. The technique is not
suitable for identification of highly volatile compounds such as methyl
mercury due to the necessity of subjecting tissues to high vacuum condi-
tions, however, electron probe analyses may prove to be useful in studies
of active ion transport systems in gill tissue and in investigations of
the effects of heavy metal pollutants on fishes.
Exposure of fish to methyl mercury for 12 weeks had no effect on the
oxygen consumption of gill tissue or on plasma electrolyte concentration
but the hematocrit index increased significantly.
In studies of iron metabolism by control and iron deficient (bled)
trout we found that within 24 hours after injection most radioiron was
absorbed from the peritoneal cavity and that clearance of iron from the
plasma is a 3 component exponential process. The erythropoietic response
to bleeding is slow. The incorporation of radioiron into red cells
probably represents (a) incorporation of iron for hemoglobin formation
and (b) exchange of iron between cytopiasmic non-heme stores and the
plasma. The liver, spleen and head kidney are the main iron storage
organs in trout and bleeding significantly reduced the liver iron content
but had little effect on splenic iron. Iron metabolism in rainbow trout
appeared to be a closed, recycling system as essentially no radioiron
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was excreted in feces and urine.
The relative functional surface area of isolated-perfused gills was
evaluated by measuring the influx of radiocarbon labelled urea, a passively
diffusing molecule. The catecholamines, norepinephrine and epinephrine,
increase functional gill surface area and decrease overall branchial vas-
cular resistance. Surface area and resistance effects of adrenergic agon-
ists and blocking agents demonstrated the presence of both « and 3 adren-
ergic receptors in rainbow trout gills. Stimulation of a adrenergic re-
ceptors increased both functional surface area and branchial vascular
resistance, while P> adrenergic receptor stimulation increased functional
surface area but decreased branchial vascular resistance. Acetylcholine
decreased functional gill surface area and increased overall branchial
vascular resistance. The data presented strongly indicate that the func-
tional surface area of rainbow trout gills can be regulated by changing
perfusion pathway with adjustments in the relative vascular resistance
across the different pathways.
Casts of the gill vasculature of rainbow trout perfused with Microfil
were examined visually. The most conspicuous feature seen was enlargements,
called blebs, of the afferent filamental vessels that were located in the
outermost region of the interfilamental septa. Several lamellae were
supplied by profusely branched afferent lamellar veseels, whereas most
lamellae were individually connected to the efferent filamental vessel.
Vessels contained in the septal area between filaments were also visualized
as well as vessels that ran parallel to the long axis of the filaments.
Heat exchange was evaluated in isolated-perfused second gill arches
and the intact branchial basket of rainbow trout. The presence of ICT-'M
epinephrine in the perfusion solution increased the transfer maximum of the
gill suggesting a change in perfusion pathway and/or vascular dimensions.
Changes in perfusion flow altered heat exchange by the gill which is again
due in part to changes in vascular dimensions. A model for the evaluation
of the gills in relation to whole body heat exchange indicated that in the
range of perfusion flows from 4 to 20 ml min~l the gill may account for as
much as 30 to 607» of the total heat exchange in the animal.
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SECTION 3
RECOMMENDATIONS
1. The electron microprobe should be satisfactory for use in investi-
gations of active ion transport systems in gill tissues and in research
on the effects of heavy metal ions (pollutants) on fishes. The technique
is not suitable for identification of highly volatile compounds such as
methyl mercury due to the necessity of subjecting tissues to high vacuum
cond it ions.
2. Incorporation of radioiron into erythrocytes is recommended as a
very sensitive parameter for study of the effects of environmental pollu-
tants on fish. The study of any one parameter, however, would probably
provide insufficient information on which a meaningful i'ia«nosis could be
base.', thus it is suggested that detc-rmin.it >'o:is nf blood hemoglobin and
erythrocyte volume (hematocrit) as well as reticulocyte counts be made
concurrently with experiments using radioiron.
3. It is highly recommended that the isolated-perfused gill pre-
paration be further developed and standardized for use as an assay pro-
cedure to determine the effects of water soluble pollutants on fish.
Using this preparation one could quantitatively describe the effects of
pollutants on gills in terms of changes in gill permeability to water,
gill ion transport and possible biotransformation of pollutants by pul-
monary (gill) metabolism.
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SECTION 4
EFFECTS OF METHYL MERCURIC CHLORIDE AND MERCURIC
CHLORIDE ON RAINBOW TROUT
A STUDY OF UPTAKE PATHWAYS INTO WHOLE FISH
Uptake of mercury by fish can occur via three possible routes, the gas-
trointestinal (GI) tract, the skin, or the gills. The importance of the GI
tract in mercury uptake has been demonstrated by Hannerz (1968), Backstrom
(1969), Miettinen et_ _al. (1969, 1970), Miettinen (1970), and others. Al-
though orally administered mercury was often regurgitated in their experi-
ments, the general consensus was that mercury, especially methyl mercury, is
absorbed through the GI pathway. In nonfeeding, freshwater teleosts, which
also accumulate high tissue levels of mercury, the amount of mercury absorbed
via the GI tract may be limited by a low drinking rate (around 13 yliters/hr
per 100 g for a freshwater adapted eel (Maetz and Skadhauge, 1968). However,
if exposure to mercury stimulates drinking rates, the GI tract pathway could
play a significant role in mercury uptake. To our knowledge there have been
no efforts to delineate the importance of each of these pathways in nonfeed-
ing teleosts.
Methods
In order to determine the significance of the GI tract in mercury uptake
by nonfeeding trout, the GI pathway was eliminated in one group of fish by
esophageal ligation. The fish were divided into three groups: control, sham
ligated, and ligated. The purpose of the sham ligation procedure was to de-
termine if any mercury was taken up through the body wall incision. Eight
fish from each group were placed into 100 liters of aged tap water than con-
tained 275 ng/liter Hg as either 2°3HgCl2 or CH3203HgCl. After 24-hr expo-
sure fish were randomly removed and blood samples taken from the caudal vein.
They were then killed and tissue samples from the gills, liver, kidney, heart,
skeletal muscle, stomach and intestine were removed and placed into preweighed
vials. Tissue activity was determined with a 2-inch thallium activated sodi-
um iodide crystal well detector and Nuclear Chicago analyzer/sealer. Tissue
dry weights were also determined. Analysis of the data was done on a Control
Data Corp. 6500 computer. One way analysis of variance was used for all sta-
tistical comparisons.
Results and Discussion
Uptake of divalent mercuric ions, as well as methyl mercuric ions, occurs
primarily by way of the gills. Ligation of the esophagus has no apparent ef-
fect on 24-hr accumulation of either form of mercury by any tissue with the
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exception of gills from fish exposed to inorganic mercury. In this instance
gills from the sham-operated fish had the highest mercury concentration.
There was a noticeable trend, although not statistically significant, toward
increased tissue mercury concentrations for sham-operated fish in both experi-
ments. The explanation for this is unknown but it possibly could be related
to variations in surgical trauma. In both methyl and inorganic mercury-ex-
posed fish, dry weight to wet weight ratios remained constant for all tissues
except liver and kidney. Even though there was an increase in hydration of
liver and kidney tissue the change was no greater than 1.5% and probably does
not reflect gross inward water flux which would be needed to significantly
affect tissue mercury concentrations.
Methyl mercury was taken up by all tissues at a much faster rate than
inorganic mercury over the 24-hr period and the large concentration differen-
ces in gill tissue observed between the two compounds (28 vs. 405 ng Hg/g dry
weight) can be explained only by faster penetration of the methylated form.
Mucus which continually coats the gill epithelium can act as an ion binding
resin to trap mercury and prevent its access to the tissue. Since ambient
concentrations of both mercurials were identical for the experiments and as-
suming both ion species were trapped or precipitated by the mucus, the con-
centration of inorganic mercury at the gill should be no less than one half
that of the organic form. This is based on the assumption that one sulfhy-
dryl (SH) binding site is occupied by each monovalent methyl mercuric ion and
a maximum of two SH sites by each divalent inorganic mercury cation.
TABLE 1
THE 24-HR UPTAKE OF MERCURY BY TROUT EXPOSED TO 275 NG/LITER MERCURY
AS EITHER CH3HgCl or HgCl2 (n = 8)
Tissue
Gill
Liver
Stomach
Intestine
Heart
Kidney
Ku s c 1 e
Blood
••— ' '• • 1 • •! « •• --•- ••-• — .U - -
Tissue activity
CH3HgCl
404.6
29.3
9.6
15.9
30.7
92.1
2.4
81.8
(ng Hg/g dry vt . )
HgCl2
28.1
2.6
2.3
12.3
2.6
4.6
0.2
15.4
Ratio
Me t hy 1 Hg : i nor na n i c Us;
14.4
11.3
3.5
1.3
11.8
19.8
15.4
5.3
A ratio of gill methyl mercury concentration to gill inorganic mercury
concentration (Table 1) of 14.4 indicates that the increased uptake of the
methylated form must reflect either entry of mercury into the gill tissue
where additional binding sites are available or increased mucus secretion
which binds the methylated form on the exterior of the gill. Observations of
fish from this and numerous other experiments have shown that methyl mercury
does not stimulate greater mucus secretion than inorganic mercury and, in
fact, the opposite is often the case. The increased penetration of methyl
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mercury into gill tissue may simply reflect the greater lipid solubility of
this compound over that of inorganic mercury. This explanation would hold if
mercury gains entry into gill epithelial cells across a lipid-containing cell
membrane and not via water-filled pores. On the other hand the bivalency of
inorganic mercury may promote tighter binding to mucoproteins thereby re-
stricting its diffusion into gill tissue.
Comparison of the distribution of the two mercury compounds within a tis-
sue demonstrates preferential accumulation of mercury based on its chemical
form. Ratios of methyl mercury to inorganic mercury in various tissues after
a 24-hr exposure are given in Table 1. The magnitude of these ratios is par-
tially attributable to increased uptake of methyl mercury by the gill, how-
ever, all the tissues should exhibit approximately the same ratios if there
is no selectivity by the tissues for specific forms of mercury. The differ-
ences in these ratios show that tissues such as the kidney, muscle, gill,
heart and liver preferentially accumulate the methylated form and blood, sto-
mach and intestine do not. Low ratios for the latter three tissues could be
due to selective accumulation of the inorganic form which is masked by higher
uptake rates of the organic compound. Using radioautography Halbhuber et al.
(1970) have demonstrated selective accumulation of radiomercury by the Paneth
cells in the intestine of the rat and guinea pig. It was postulated that one
function of these cells is to transfer divalent heavy metal ions from the
body fluids into the lumen of the gut. Similarly Paneth cells in the trout
gut might sequester divalent mercury but not methylater forms.
Backstrom (1969) administered methyl and inorganic mercury intravenously
to speckled trout. After 24 hr the distribution patterns of methyl mercury
were similar to our data for rainbow trout with the exception of the low val-
ues for the gills. He reported highest inorganic mercury concentrations in
the kidney; values for blood and gill were about 50% lower. These small dis-
crepancies in distribution when compared to data presented here are probably
due to the mode of administration of the mercurial. The tissue mercury dis-
tributions reported by Hannerz (1968) for cod exposed for 24 hr to waterborne
mercurials are similar to those found in the present study. He postulated
gill uptake as the principal mode of entry of mercury into cod and pike.
The wide variation in organic/inorganic tissue concentration ratios that
we noted supports the idea that inorganic mercury can cross the gill epithe-
lium as divalent mercury ions or as some complex without prior methylation.
The present experiments have eliminated the GI tract as a major pathway for
mercury uptake in nonfeeding trout and it is widely accepted that the skin is
relatively impermeable (Prosser, 1973, pp. 48).
UPTAKE OF MERCURY BY TROUT ERYTHROCYTES _IN VITRO
Methods
Heparinized whole blood or washed cells were placed in 25-ml stoppered
Erlenmeyer flasks. The mercury solution was added to the blood at time zero
and the flasks stirred at 16 rpm with a multipurpose rotator. At timed inter-
vals 0.2 ml of blood was removed and placed in a 0.4 ml polyethylene
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m
icrocentrifuge tube. Packed red cells were then resuspended in Ringer solu-
tion, centrifuged and the subsequent supernatant was placed in a third micro-
centrifuge tube. Hematocrits were determined in duplicate for all blood sam-
ples incubated. The concentration of mercury from the packed red cells, plas-
ma and wash was determined by scintillation counting. Percent uptake of mer-
cury into the cellular fraction was standardized by correction for equal vol-
umes of red cells and plasma. All glassware used was coated with silicone to
minimize absorption of the mercurial onto the glass.
Results and Discussion
The percent uptake of both mercurials into erythrocytes as a function of
time is plotted in Figure 1. With the exception of methyl mercury incubated
with whole blood, equilibrium was reached in less than 10 minutes. Uptake of
methyl mercury did not reach equilibrium when incubated with whole blood until
after 30 to 40 minutes. Analysis of this reaction revealed a two-component
uptake system, the first having a half time of under 1 min, the second about
11 min. (Uptake values of less than 1 min are only estimates due to the in-
herent time lag incurred during blood centrifugation and separation.) This
can be explained by initial binding of a small percentage of mercury to plas-
ma proteins or thiols, the remainder being taken up by the red cells as a
function of the free or nonplasma bound mercury. After the red cells have re-
moved most of the unbound mercury from the plasma a new equilibrium (slow com-
ponent) is established that is dependent on the dissociation of bound mercury
from 'plasma sites' prior to red cell uptake. This postulate is further sup-
ported by the mode of uptake of methyl mercury into red blood cells suspended
in Ringer solution. In this instance the uptake rate is so fast that only
one component can be determined. The increased G^Rg"*" binding at equilibrium
by washed cells in Ringer solution compared to cells in plasma (95% vs. 90%)
is probably indicative of some irreversible mercury binding to plasma protein.
Failure of the washed cells to bind 100% of the mercury could be due to slight
hemolysis coupled with some mercury binding by the incubating media or absorp-
tion on glassware.
Inorganic mercury incubated with whole blood binds almost exclusively to
the plasma fraction; less than 9% was taken up by red cells after 1440 min
(not shown in Figure 1). Washed cells suspended in Ringer solution take up
9-10 times more inorganic mercury than cells do in plasma. Removal of plasma
proteins undoubtedly is the critical factor. There is also a significant
difference in percent uptake by red cells that is dependent on the suspending
media; cells suspended in Abbott Ringer take up less Kg"1""*" than cells suspen-
ded in phosphate buffered Ringer solution. Since the only difference is the
presence of magnesium and the buffering capacity of the PER either the magne-
sium or pH or perhaps both factors must enhance equilibrium binding by red
cells.
In other experiments it was shown that uptake of either methyl or inor-
ganic mercury was, in all instances, independent of the length of the incuba-
tion period which preceded addition of mercury to the cell suspension. Thus,
secretion of a chelator by trout red cells is not a factor in the failure of
these cells (especially washed cells) to bind all mercury present. As
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indicated in Figure 1 less than 100% of the available methyl or inorganic
mercury was taken up by red cells in these experiments.
LJ
IOO-,
90-
80-
70-
o
03 60-
cr
40-
30-
20-
10-
10
30
60
-/A
180
TIME (MIN)
Figure 1. Percent uptake of methyl mercury or divalent mercury by red blood
cells as a function of time. A = CI^HgCl incubated in whole blood (15);
HgCl2 in whole blood (6); A = CI^HgCl incubated with washed cells suspended
in Abbott Ringer (4); = HgCl2 incubated with washed cells suspended in
Abbott Ringer (4); ° = HgCl2 incubated with washed cells suspended in Phos-
phate Buffered Ringer (4). Exposure concentrations approximately 0.0133 to-
tal ppm CH-jHgCl and 0.054 ppm total HgCl2- Number of observations (N). Stan-
dard errors are not shown on the graph as they were smaller than the diameter
of the data points.
-------
It appears that saturation of trout erythrocytes with mercurials must in-
volve either (1) some group(s) such as SH, amono, or other which are respon-
sible for cell membrane integrity and are not saturated at the time of hemol-
ysis or (2) a disruption of ionic balance and subsequent osmotic hemolysis
caused by mercury levels below those required for total cell saturation. SH-
containing compounds such as albumin, cysteine and reduced glutathione failed
to remove either inorganic or organic mercury previously bound to red cells,
which suggests strong mercury-red cell binding.
MERCURY UPTAKE AND ION DISTRIBUTION IN GILLS OF RAINBOW TROUT
Efforts to localize and/or characterize the pathway of uptake of mercury
in gills of trout using radioautographic techniques were largely unsuccess-
ful. Autographs of gills of fish exposed to either methyl or inorganic mer-
cury exhibited a diffuse grain pattern which precluded identification of the
uptake site. Lack of grain density in areas containing many chloride cells
probably indicates that these cells are not involved in the uptake of either
mercurial. The thinness of lamellae and the small interlamellar distances
prevented localization of preferential mercury binding sites. Dense grain
patterns due to 203jjg-H- were -found associated with the filamental cartilage.
Our second attempt to localize the gill pathway for mercury uptake was done
with the electron microprobe. The following is a report of these experi-
ments.
Methods
Fish were exposed to the mercurials which were added to water in 100-
liter tanks. The concentrations used were 0.25 and 0.05 ppm Hg for inorganic
and organic mercury, respectively. After 23-hr exposure a second amount of
mercury equal to the first was added to the tanks and fish were removed 1 hr
later and killed. The second pair of gill arches were removed from mercury-
exposed fish within 15 sec after the fish was killed. The arches were quick
frozen in isopentane which has been cooled to near -160° C with liquid nitro-
gen. Details of mounting, embedding, sectioning and preparation of sections
for use in the ARL Microprobe are given by Olson and Fromm, 1973. Tissue
scans and image scan micrographs were recorded on Polaroid 200 black and
white film and the line scans were recorded on a Hewlett Packard x-y recorder.
Low peak to background rations during mercury analysis made the tissue scan
procedure difficult to interpret, however, line scan procedures proved ade-
quate for mercury identification. Line and tissue scans were also used for
detection of other elements as both an aid in tissue orientation and a check
on diffusional ion movements which might have occurred during sample prepara-
tion. The 0.5 y diameter of the primary electron beam permits fairly high
resolution in the localization of ions within a given area especially with
the line scan procedure. All analytical data obtained were qualitative or
only semiquantitative as the refinements and sophistication required for quan-
titative determinations were beyond the scope of this preliminary study.
Results and Discussion
For the line scans as the beam entered the area known to contain large
numbers of chloride cells, concentrations of all ions except mercury
10
-------
increased. Interlamellar areas contained definite mercury peaks and the lo-
cation of lamellae was marked by increases in the concentrations of Na, K, Ca
and Cl. Mercury content appeared to be inversely related to concentrations
of the other elements and it did not increase until the beam entered the
space normally filled by environmental water. This confirms results of radio-
autographic studies and supports the concept that inorganic mercury is not
selectively taken up by chloride cells nor concentrated by them in 24 hr. It
should be stated that we were unable to detect mercury in gills from tout ex-
posed to methyl mercury even though our previous study showed that fish ex-
posed to methyl mercuric chloride contained 10 times more mercury than those
fish exposed to mercuric chloride. The inability to detect mercury after
methyl mercury exposure was probably due to the loss of this element during
tissue preparation. Crystals of methyl mercury will sublime at room tempera-
ture, therefore, mercury could be lost during freeze drying, carbon evapora-
tion or in the vacuum volumn of the microprobe. All of these procedures re-
quire a high vacuum.
The mercury peaks observed were due in large part to mercury which was
added 1 hr prior to sampling and demonstrated the effectiveness of the inter-
lamellar mucus to trap ions. Presumably mercury is contained in the mucus
from which it can either enter the gill or be eliminated with the sloughed off
mucus. McKone e^ al. (1971) exposed goldfish to 0.25 ppm HgCl2 for 3 hr and
found that 79.3% of the mercury in excised gills was removed by washing with
80% ethanol. They concluded that this fraction represented mercury trapped
by the mucus coating the gill, however, they did not examine gill tissues af-
ter the ethanol wash to verify that only mucus was removed. Gills are very
fragile and it is quite possible that some epithelium was also removed in the
wash process.
Tissue scans of other material showed that mercury concentrations were
highest in cartilage and were correlated with high calcium, sulfur and phos-
phorus levels. Microprobe (and radioautographic) studies have clearly indi-
cated that there is no preferential accumulation of mercury in lamellae. It
appears that mercury can enter gills through the general lamellar surface,
thus active uptake is probably of little consequence in mercury influx. Once
in the filament, much inorganic mercury is accumulated by cartilagenous tis-
sue. Other gill tissues exhibit no preferential uptake of mercury with re-
spect to a given cell type, i.e. epithelial cells, pillar cells, or chloride
cells.
Preparation of tissues for electron microprobe analysis avoids the use
of various aqueous solvents as are commonly employed in routine analytical
histochemistry. Thus, localization of many ions (e.g. small molecular weight
monovalents) with the probe probably reflects a fairly true tissue distribu-
tion of these ions. We suggest that electron probe analyses should prove
beneficial in a study of ion distribution in chloride cells of fish exposed
to various salinities or to pharmacological agents. Further information on
the ion transfer role of mucus, which covers the surface of chloride cells
from marine species, could be gained by studies utilizing the electron
microprobe.
11
-------
EFFECT OF METHYL MERCURY ON GILL METABOLISM AND BLOOD PARAMETERS OF TROUT
The gills of freshwater teleosts function as the primary site for the
active absorption of ions from the external media and for the exchange of
respiratory gases. Large amounts of water must be passed over the gill sur-
faces to meet the oxygen demands of the fish consequently when fish are in
contaminated waters their gills can be exposed to large amounts of water sol-
uble pollutants. The experiments discussed below were performed to determine
if methyl mercury has any effect on the metabolism (oxygen consumption) or
physiological function (plasma electrolyte regulation) of the gill.
Methods
The experimental design involved exposing a group of starved fish to 10
yg Hg/liter administered as methyl mercuric chloride while another group of
starved fish served as controls. The mercury was administered to the experi-
mental fish using a gravity feed system in which the flow rate of the concen-
trated mercury solution into the tank was correlated to the flow of water
through the tank to maintain a concentration of 10 yg Hg/liter. At the end
of 4, 8 and 12 weeks the fish were killed and determinations of the follow-
ing parameters were made: hematocrit, oxygen consumption of gill tissue,
protein content of gill sample and plasma electrolyte concentrations (Na+,
K , Cl~, Mg++ and Ca~H"). Plasma sodium and potassium were determined on a
Beckman flame photometer, magnesium and calcium were determined on a Perkin
Elmer atomic absorption spectrophotometer and chloride was measured with a
Buchler chloridometer. All plasma electrolyte determinations were done in
duplicate. Samples of gill filaments from the second and third branchial
arches and were suspended in 3 ml of 10% and 100% phosphate buffered saline
(PBS). The oxygen consumption of the filaments was then measured polarograph-
ically at 12° C with a YSI biological oxygen monitor. Calculation of oxygen
consumption was based on the solubility of oxygen in 10% and 100% PBS at 12°
C and the percent of initial oxygen consumed during a 10 minute period. Fol-
lowing oxygen consumption measurements the 3 ml samples of PBS containing the
gill filaments was sonified to remove as much tissue as possible from the
cartilaginous skeleton and the protein content of the mixture was determined.
Results and Discussion
Results from this investigation (Table 2) indicate that up to 12 weeks
exposure to methyl mercuric chloride (10 yg Hg/liter) does not significantly
affect the in vitro metabolism of the gill or the concentration of plasma
electrolytes in rainbow trout. There was a significant increase in the he-
matocrit of experimental fish after 12 weeks exposure. Webb (1966) pointed
out that many mercurials are known to cause hemolysis of RBC's which would
tend to cause a decrease in the hematocrit index, not an increase as we found
in our experiments. Possibly methyl mercury directly stimulated erythropoi-
esis causing an increase in hematocrit of trout or the fish may have over-
compensated erythropoietically in response to RBC loss resulting from expo-
sure to mercury.
12
-------
TABLE 2
VALUES FOR SOME BLOOD PARAMETERS OF CONTROL AND MERCURY
EXPOSED (10 NG Hg/LITER) RAINBOW TROUT
Blood
Parameter
Plasma Na+
(meq/L)
Control
Hg-treated
Plasma K1"
(meq/L)
Control
Hg-treated
Plasma Cl"
(meq/L)
Control
Hg-treated
Plasma Ca~*~*"
(meq/L)
Control
Hg-treated
i i
Plasma Kg"1"1"
(meq/L)
Control
Hg-treated
Hematocrlt
(7. RBC)
Control
Hg-treated
Mean±S.E.(N)
Length of
4
140. 08±1. 35(12)
142.58±1.93(12)
2.74±0.17(12)
2.61±0.13(12)
123. 36±0. 95(12)
123. 27±2. 07(12)
4.36±0.28(12)
3.97±0.25(12)
1.82±0. 05(12)
1.66±0.09(12)
19. 44±1. 28(12)
18.00±0.88(12)
Exposure (Weeks)
8
142.68±3.48( 8)
149. 20±1. 83(12)
1.36±0.22( 8)
1.42±0.19(12)
122.02±5.13( 8)
128. 73±1. 71(12)
4.00±0.15( 8)
4.15±0.26(12)
1.96±0.04( 8)
2.02±0.06(12)
19.59±2.63( 8)
21. 52±1. 38(12)
12
139. 37±3. 13(8)
133.75±7.66(8)
1.70±0.27(8)
l.77±0.36(8)
121. 75±4. 00(8)
110.85±11.14(8)
3.76±0.15(8)
3.73±0.25(8)
2.15±0.09(8)
1.92±0.23(8)
21.31±1.46(8)
30.34±2.40(8)
Although there were no significant differences in plasma electrolyte
levels for paired comparisons between control and mercury treated fish after
4, 8 and 12 weeks exposure, over the 12 week period several of the electro-
lytes did vary considerably. Plasma sodium for controls remained fairly con-
stant at about 140 meq/liter while those for experimental fish varied between
133 and 149 meq/liter. This greater variability in the experimental fish may
represent some effect of methyl mercury on the active transport of sodium by
gills but further work is needed to substantiate this hypothesis. No plaus-
ible explanation can be offered for the increase in plasma magnesium levels
of control fish after 12 weeks exposure.
Gill samples from control and mercury exposed fish measured in 100% PBS
showed a significantly higher rate of oxygen consumption than samples mea-
sured in 10% PBS (Table 3). This could be the result of the sudden transi-
tion of gill tissue from fresh water (5-10 mOsm) to 100% PBS (285-290 mOsm)
which represents both an osmotic shock and a probable increased salt load for
the tissue. Chloride cells present in the gill filaments could conceivably
have responded with an increased rate of active transport to maintain osmo-
tic equilibrium and thus account for at least some of the difference in oxy-
gen consumption. It is interesting to note that the decrease in oxygen con-
sumption for control and mercury treated fish in 10% PBS occurred during the
same period of time that a decrease in plasma potassium concentration was
13
-------
seen. It would appear that these changes represent some form of accommoda-
tion to the effects of starvation since they were observed in both control
and experimental fish.
TABLE 3
IN VITRO OXYGEN CONSUMPTION OF GILL TISSUE FROM CONTROL AND
MERCURY EXPOSED (10 pG Hg/LITER) RAINBOW TROUT IN 100% AND
10% PHOSPHATE BUFFERED SALINE (PBS)
Data expressed as pi 02 consumed/hr/mg protein.
Sample
100% PBS
Control
Hg-treated
10% PBS
Control
Hg-treated
MeaniS.E.
(N)
17.
18.
14.
13.
4
35±0
53±1
13±0
13±0
Length
.74(12)
.37(12)
.78(12)
.64(12)
of Exposure (Weeks)
15.
14.
8.
8.
8
56±0
48±1
74±0
64±0
.98( 8)
•15(12)
.53( 8)
.50(12)
16
17
9
9
12
.47±2
.27±1
.78±0
-57±0
.07(8)
.15(8)
.77(8)
.63(8)
The data presented above seems to indicate that longterm exposure to
methyl mercuric chloride does not alter the metabolism of the gill or affect
its role in plasma electrolyte regulation. Such a generalization may at best
be premature. McKim et al. (1970) reported significant changes in seven blood
parameters of brook trout after exposure to three different concentrations of
copper for periods of 6 and 21 days, however, when the experiment was exten-
ded to 337 days five of the seven parameters were similar to control values.
It is possible that methyl mercury exerted a similar transient effect on the
fish in this study and remained undetected or possibly a longer period of ex-
posure is necessary before significant changes in oxygen consumption or plas-
ma electrolyte concentrations occur. The need for longer term studies with
shorter time intervals between samplings is evident and would do much to fur-
ther our understanding of the physiological effects of mercurials on fish.
EFFECT OF MERCURY EXPOSURE ON RAINBOW TROUT GILL ULTRASTRUCTURE
Several investigators using light microscopy and standard histological
techniques have described the gross histopathological or structural changes
in the gill epithelium after exposure of fish to mercurials. Schweiger (1957)
found that fish exposed to 0.03 mg Hg/liter survived longer than seven days
and considered this concentration harmless. At higher concentrations the sur-
vival time was progressively shortened and massive damage to the respiratory
epithelium was observed. Below is a description of some morphological
changes that occurred in the ultrastructure of gills of rainbow trout which
were exposed to methyl mercuric chloride and mercuric chloride. Using light
microscopy, no significant histopathological changes were noted in these
gills when compared to untreated controls.
14
-------
Methods
Rainbow trout which had been exposed to monomethyl mercury for 4 weeks
or longer were obtained from the Great Lakes Fisheries Laboratory, Bureau of
Sport Fisheries and Wildlife, Ann Arbor, Michigan. These fish ranged from 35
to 60 g and were exposed to an average concentration of 200 ng Hg/liter (as
CH3HgCl) at 5, 10 and 15° C in a constant flow bioassay apparatus. Gill sam-
ples from these fish were collected and fixed at Ann Arbor. For transmission
electron microscopy (TEM) individual gill filaments were dissected from sup-
porting tissue of the second gill arch of a freshly killed fish, fixed in 4%
glutaraldehyde buffered with S«5rensen buffer and post fixed in 1% osmium tet-
roxide. The tissues were then dehydrated in graded concentrations of ethanol
or acetone and embedded in Spurrs embedding media. Thin sections were
stained in uranyl acetate and lead citrate and were examined with a Philips
EM 300 electron microscope. For scanning electron microscopy (SEM) the
second gill arch (contralateral) side was removed, rinsed with tap water and
fixed in 50% glutaraldehyde. After six rinses of 30 minutes each in S^rensen
buffer the tissues were either frozen in isopentane cooled with liquid nitro-
gen and subsequently lyophilized or they were dehydrated with ethanol and
amylacetate and dried by the critical point method. Dried filaments were
separated from the supporting cartilage, attached to coverslips and coated
with carbon and gold palladium. The tissues were then examined with an AMR
Model 900 scanning electron microscope.
Results and Discussion
For detailed descriptions of normal rainbow trout gill ultrastructure
the reader should consult Olson and Fromm (1973) for SEM work and Morgan and
Tovell (1973) for TEM observations. Investigations with the SEM indicated
that a 24 hr exposure to 0.05 mg/liter methyl mercury produced some slight
disarrangement of the microridges present on the outer surface of lamellar
epithelial cells although junctions between epithelial cells appeared essen-
tially normal. This same exposure resulted in what is believed to be an in-
crease in the degenerative process in chloride cells which, in SEM micro-
graphs is typified by loss of surface microvilli. There was also an increase
in the number of degenerating chloride cells in gills of fish exposed to 200
ng/liter methyl mercury for 4 to 8 weeks. Exposure to HgCl2 produced some-
what similar changes in the surface features of chloride cells and also led
to the appearance of many smooth non-ridged lamellar epithelial cells.
Observations with the TEM revealed that exposure to 0.05 mg/liter methyl
mercury had relatively little effect on the appearance of the epithelial mi-
croridges which appear more like villi in TEM micrographs. Some vacuolation
of chloride cells was apparent but many of these cells appeared normal. A 4
week exposure to 200 ng/liter methyl mercury produced red cell vacuolation
and slight loss of epithelial cell ridge integrity. The vascular space was
often filled with debris, possibly the contents of ruptured red cells. De~
generating or abnormal chloride cells appeared to be present in greater num-
bers but normal chloride cells were also present. Extensive vacuolation of
chloride cells was noted in fish exposed to 200 ng/liter methyl mercury for
8 weeks. This vacuolation extended throughout the cell and appeared to be
15
-------
associated with the smooth endoplasmic reticulum (SER) of these cells. In
addition to their becoming vacuolated these cells were often detached from
subepithelial structures. Many red cells in fish exposed to this concentra-
tion appeared to be normal but vacuolation was not uncommon. Chloride cells
in gills of fish exposed for 5 days to 0.25 mg/liter inorganic mercury and
those exposed to lower concentrations for longer periods of time showed ex-
tensive vacuolation and loss of surface microvilli. Small vacuoles with a
low electron density were found near the apical area of the chloride cells,
whereas, large (up to 0.5 um diameter) vacuoles were located in the basal
portion of the cell and were more electron dense. This is in contrast to
methyl mercury exposed chloride cells in which the small vacuoles were pres-
ent throughout all but the extreme apical portion of the cell. This differ-
ence may reflect different penetration rates of these two mercurials or dif-
ferent biochemical interactions. Cross sections through the smooth areas on
the lamellar epithelial surface revealed that these areas were the outer or
apical surface of large cells which appear to push apart and replace regular
epithelial cells on the lamellar surface. These smooth cells are somewhat
larger and less electron dense than normal epithelial cells; they contain a
relatively large nucleus, numerous mitochondria and cytoplasmic vacuoles.
Except for the absence of a slightly ridged epithelial surface, these cells
are similar to the 'new' type of epithelial cell seen in methyl mercury ex-
posed fish.
16
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SECTION 5
IRON METABOLISM
METABOLISM OF IRON BY NORMAL AND IRON DEFICIENT RAINBOW TROUT
Iron is an essential element involved in the physiological functions of
oxygen transport and cellular respiration. An enormous volume of literature
has resulted from measurements made on human subjects and rats which deals
with iron absorption, the mode of iron transport via the plasma, and the
utilization and conservation of iron. There is, however, relatively little
information concerning iron metabolism in lower vertebrates. The study
reported here was designed to increase our knowledge of the metabolism of
iron in salmonidae. The objectives were: (1) to study iron distribution and
utilization in normal control and iron deficient trout over a 30 day period
following an intraperitoneal (i.p.) injection of 59Fe, (2) to investigate the
absorption of iron into red blood cells and (3) to evaluate the relative im-
portance of various iron storage pools in providing iron to be used during
time of increased erythropoietic activity by head kidney tissue.
Methods
One group of fish were made iron deficient by removal of 40% of the
blood volume in four bleedings of 10% of the blood volume over a seven day
period with 48 hours between bleedings. On the day of the last bleeding
both experimental and control fish received an i.p. injection of 1 yCi-^FeCl-,/
100 g fish in 0.9% saline. Six experimental and six control fish were sacri-
ficed 1, 2, 4, 8, 11, 16, 23, and 30 days following 59Fe injection. Blood
samples were taken via the hemal arch and fish were killed with a sharp blow
to the head. Tissue samples were taken and weighed using a Roller-Smith
Balance. Intestinal contents were tare weighed on a Mettler Model B5 Balance.
Tissues and intestinal contents were stored at 0°C in glass culture tubes
until analyzed for total iron and/or 59pe content.
The percentage of pack red blood cells was determined by the micro-
hematocrit method and hemoglobin by the cyanmethemoglobin method using Hycel
(Houston, Texas) standards and reagents. Reticulocyte counts were made on
blood smears prepared by supra-vital staining. After staining a total of
1000 cells, both immature and mature red cells, were counted using an ocular
grid. The number of reticulocytes was expressed as a percentage of this
population. Plasma iron was determined colorimetrically and expressed as
ug Fe/100 ml plasma (yg%). Plasma from any hemolyzed samples was discarded.
The plasma total iron binding capacity (TIBC) was calculated from the plasma
iron content and the unsaturated iron binding capacity (UIBC). Tissue iron
was measured colorimetrically after wet-ashing in sulfuric-nitric acid
(5:1). Total blood volume and tissue blood volume was determined using 51(]r-
labelled RBCs. The tissue blood volumes were used to correct tissue 59pe
activity and total iron concentration for the blood iron content. To evaluate
17
-------
absorption of radioiron by the intestine the method of De Benedetto and
Farmanfarmaian (1975) was used to measure renal excretion of radioiron the
urinary bladders of trout were cannulated and urine collected in a manner
described by Froiran (1963).
Results and Discussion
With respect to normal control rainbow trout the data presented in Table
4 indicate that the spleen has the highest iron concentration but the liver
due to its mass, represents the prime storage area for iron in trout. Tissue
iron concentration and/or total iron content corrected for blood iron content
remained constant over the 30 day study period. Plasma contained very small
amounts of iron but plasma does contain transferrin, the iron transport pro-
tein which shuttles iron back and forth between the various iron pools, thus
it plays an essential role in the metabolism of iron by trout.
TABLE 4
CALCULATED VALUES FOR THE IRON CONTENT OF TISSUES OF A
HATCHERY-REARED 100 G RAINBOW TROUT
Tissue yg Fe/g (X + S.E.) yg Fe Total
Liver
Spleen
Head kidney
Caeca
Intestine
Plasma
168
381
122
36
34
0.55
± 13
± 46
± 11
± 3
± 3
± 0.03
185.0
81.2
24.3
45.3
14.3
1.2
The clearance of iron from the plasma of rainbow trout following i.p.
injection of 59Fe was determined. When plotted as log concentration against
time, radioiron content of the plasma presents the clearance curve seen in
Figure 2 upper left. The curve is composed of three exponential components
G!» C? anc* C3» similar to those characterized for human plasma iron clearance.
The equation for the line is given at the top of Figure 2 where: Ct = fraction
of the initial dose of 59Fe present in the plasma at time t; Co^, Co2, 003 = y-
intercepts for components 1, 2 and 3 expressed as decimal equivalents of
initial dose; k]_, k2, k3 = rate constants for components 1, 2, and 3; e =
base of natural logarithms and t = time in days.
The first component (Cj) with a half time (Tl/2) of 5.98 hours represents
the flux of radioiron-labelled transferrin from the plasma into the tissue
extravascular spaces. The second component with a half-time of 1.16 days
represents the return of 59pe-transferrin to the plasma pool, a process which
is responsible for the change in the slope of the clearance curve between
days 2 and 8 following i.p. injection of radioiron. The attainment of
equilibrium between radioiron in the plasma pool and the erythropoietic and
storage labile iron pools occurs after day 8 and is represented by the third
component of the plasma clearance curve (tl/2 = 39/38 days). As indicated
above fish were made iron deficient by removal of 40% of their blood volume.
No fish died as a result of the bleeding. Recovery from hemorrhagic shock was
18
-------
100
<>3
k3-.OI76
11/2 -39.38 DAYS
.25 .50 .75 1.0
DAY
.01
345 67
DAY
Figure 2. Three component exponential curve of iron clearance from plasma
of rainbow trout following i.p. injection of -'%e. Each component is
Ri-fiphed separately to show its y-intercept (Co), Tl/2 and slope (k) . De-
finitions of the symbols used in the equation are Given in the text.
-------
rapid and fish were actively swimming within minutes after bleeding. Some
fish gupled air at the surface but the majority exhibited normal activity.
There was very little change in total iron concentration in the various
tissues except the liver during the 30 day period following the final bleed-
ing and averages for the different tissues did not differ from those for
controls. The iron conent of the liver dropped significantly after day 11
and was significantly below that of controls. The plasma clearance curve
for iron deficient fish was similar to that shown in Figure 2. The Tl/2
for the GI component appeared to be shorter than the control but statistically
there was no difference between the two curves.
The movement of 59pe to and from the extravascular spaces of tissues is
indicated by plots of data in Figures 3 and 4 for controls and iron defici-
ent fish respectively. It is evident that 59pe activity of tissue is in
equilibrium with the iron pool of the plasma some 8 days after administration
of radioiron. The clearance of ^Fe from spleen, head kidney and cecal
tissue was rapid and muscle (not shown) was similar in that respect. The
specific activity of muscle iron was the lowest of any tissue and only 3 to
5% of the initial injected activity remained in the total muscle mass 16
days after injection (controls). In contrast, an average of 15% of the
initial injected radioactivity remained in the liver of controls over the
30 day experimental period and the specific activity of iron in the liver was
higher than that for any tissue from day 4 through day 30 (Figure 3). This
indicates that a significant amount of 59pe was deposited and/or stored in
the liver and remained there throughout the 30 day study period. In iron
deficient fish the initial increase in liver radioiron between days 1 and
4 is similar to that seen in controls but thereafter the 59pe activity
decreased steadily in the liver of bled fish whereas it remained unchanged
in controls. There is some indication that the head kidney of bled fish is
more active in sequestering ^9pe than those from untreated controls. Mean
values for specific activity ( and % of initial dose given) are higher in
the experimental fish head kidney than in the controls but since these values
were not statistically different we are unable to state that bleeding had
any verifiable effect on head kidney tissue. Thus the experiments with
iron deficient trout tend to indicate that the liver contains the most
important labile iron storage pool which can be utilized for the accelerated
erythropoietic process. We found that the spleen iron stores were not
significantly reduced following bleeding thus this storage pool apparently
is non-responsive to acute induced iron deficiency.
Between 70 and 80% of the injected dose of radioiron was found in the
red blood cells of bled fish by day 16. The difference in red cell iron
uptake between the bled and control groups was statistically significantly
different. The peak erythrocyte radioiron content coincided with the in-
crease in reticulocyte population (Table 5). Following day 16 there was
a decline in red blood cell radioactivity similar to that seen in control
fish. In cells from control fish initial uptake of 59pe was rapid and
reached a maximum of 55 to 60% on the initial injected activity by day 11.
20
-------
90n
CONTROL
® LIVER
A SPLEEN
23 HEAD KIDNEY
O CAECA
I 2 4
DAY
".Figure 3. Specific activity of iron in tissues of trout following.
intraperitoneal injection of -^Fe.
150
EXPERIMENTAL
© LIVER
A SPLEEN
0 HEAD KIDNEY
O CAECA
DAY
Figure 4. Specific activity of iron in tissues of iron deficient
trout following intraperitoiieal injection of ^Fe.
21
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TABLE 5
HEMATOCRIT, HEMOGLOBIN, MCHC, AND PERCENT RETICULOCYTES OF
BLOOD SAMPLES FROM CONTROL AND EXPERIMENTAL FISH.
INCLUDED ARE MEAN ± S.E. (N).
Days
after
inj_. j
1-30
1
2
4
8
11
16
23
30
Group
Control
Exp.
Exp.
Exp.
Exp.
Exp.
Exp.
Exp.
Exp.
Hematocrit
37.7 + 1.1(48)
16.3 ± 2.2 (6)*
14.2 ± 1.9 (6)*
19.8 ± 2.0 (6)*
26.3 ± 2.3 (6)*
24.7 ± 1.8 (6)*
31.7 ± 1.0 (6)*
33.5 ± 2.7 (6)
35.2 ± 3.8 (5)
Hemoglobin
(gram%)
7.9 ± 0.2(48)
3.5 ± 0.4 (6)*
2.9 ± 0.4 (6)*
3.2 ± 0.3 (6)*
4.7 ± 0.5 (6)*
3.9 ± 0.3 (6)*
5.1 ± 0.1 (6)*
5.9 ± 0.6 (6)*
5.8 ± 0.7 (5)*
MCHC
20.8
21.3
20.1
16.2
17.7
15.9
16.0
17.7
16.5
Reticuloytes
_ _(%)
2.75 ± 0.3(28)
6.98 ± 0.8 (5)*
5.00 ± 1.5 (2)
11.07 ± 5.5 (5)
17.65 ± 6.7 (5)*
10.62 ± 2.1 (6)*
5.07 ± 2.1 (6)*
5.00 ± 0.9 (2)
* Significantly differnet from controls at p = 0.05
As shown in Table 5, the hematocrit index was lowest 2 days after the
last bleeding but thereafter the red cell volume gradually increased and was
back to the control level by day 23. Although the red cell volume was
normal by day 23 the hemoglobin concentration remained below normal during
the 30 day study period. The man corpuscular hemoblobin concentration
(MCHC) decreased from about 21% to 15-16% and remained below the normal
level throughout the remainder of the 30 day experimental period. Appar-
ently the red cell volume was restored to normal by the release of young
cells, which lacked functional hemoglobin, into the circulating blood. Even
though the accuracy of reticulocyte counting is poor there was some evidence
of an increase in the immature red cell population by day 11 to replace those
cells lost via bleeding. It is apparent that the processes involved in
hemoglobin formation are significantly slower than those involved in the
formation and/or release of red blood cells from the erythropoietic head
kidney.
The total iron binding capacity (TIBC) of the plasma, which is an in-
direct indicator of the transferrin concentration, was measured. There was
a noticeable and significant increase in the TIBC following bleeding, however,
there was also an increase in the TIBC for controls during the same period.
This nullified any attempt to relate the increase to induced iron deficiency.
The TIBC values for trout plasma (400-700 yg%) are higher than values re-
ported for humans (250-350 pg%) and for the tench (250 yg) reported by
Hevesy et^ al. (1964). The values for trout blood were reproducible with the
method used and human plasma tested in the exact same manner yielded values
within the accepted range.
The results of the absorption studies indicated that there was a direct
correlation between the amount of iron injected into the gut segment and the
22
-------
amount recovered in the tissues of the trout. The dose given was insuffi-
cient to exceed the absorptive capacity of the gut, thus it was not possible
to establish a miniumum daily requirement for iron by trout based upon in-
testinal absorption. Possibly some iron enters trout by absorption across
the gill and uptake by this pathway has yet to be investigated. However, it
seem unlikely that absorption of iron by the gill would be of any great
importance since we have shown that iron is readily absorbed via the gut.
Following i.p. injection, very little radioiron is lost from trout via the
feces. The radioiron in fecal material taken from the intestine of fish
used in this study was initially as high as 4% of the injected dose, but
by day 2 it was barely detectable and less than 0.08% was found infecal
material after day 8.
To check iron loss in urine a group of trout which had been on the same
feeding regimen as controls were cannulated (urinary bladder cannulation)
and urine was collected over a 4 day period following i.p. injection of
No detectable quantity of radioiron was found in the urine. Thus the data
from experiments with the gut and kidney indicate that the iron cycle in
fish is a closed recycling system with very little iron input or excretion
and in this respect is similar to the iron cycle in humans.
Summary
Iron metabolism in normal and iron deficient rainbow trout was studied
after intraperitoneal injection of 59]7e<, in both groups most of the 59pe
was absorbed from the peritoneal cavity within 24 hours and equilibrium be-
tween the plasma 59pe pool and that of the tissues and attained 8 days after
i.p. injection. Liver iron, the main storage pool in trout, was reduced from
the control level of 185 yg/g to below 100 Mg'g 16 days after bleeding,
whereas, splenic iron stores were unaffected. In iron deficient fish the
RBC 59Fe content increased to 70-80% of the injected dose by day 16 com-
pared to 50% in controls. This was attributed to the difference in reticu-
locyte count which was 10-20% for the bled fish and 2-3% in controls. Some
iron accumulated by erythrocytes is temporarily stores as non-heme iron by
these cells. An average of 15% of the i.p. injected 59Fe was taken up by
hepatic tissue of control fish and remained there throughout the 30 day
study. In iron deficient trout liver radioiron was reduced from a high of
15% on day 2 to less than 1% of the initial dose by day 16 post bleeding.
There was essentially no detectable loss of 59Fe in the urine or feces of
either normal control or iron deficient fish.
23
-------
SECTION 6
RESPIRATORY PHYSIOLOGY
CONTROL MECHANISMS FOR REGULATING THE FUNCTIONAL SURFACE AREA OF FISH GILLS
Research on the effects of water borne pollutants is gradually moving
from the realm of determination of LD-50 concentrations or TLm values into
that of clinical determinations of the cause of death of test animals.
Information on the toxicological and/or pharmacological action of pollutants
obtained from experiments with fish may prove to be very beneficial in as-
sessing the effect(s) these materials may have on mammals, including man.
During the past several years we have been forced to interpret data on the
toxic action of materials on fish in light of information from experiments
with rats or other 'higher' experimental vertebrates. With the advent of
many suitable micro-procedures which can be used to obtain physiological and
biochemical data and with more sophisticated chemical and physical ana-
lytical systems available we are in a position to extend our collection of
information on the toxic action of pollutants on fish.
Ventilation of the gills of fish maintains at the gill surface the con-
centration of any pollutant that is in the aquatic environment. It follows
that the study of gill structure and function can contribute valuable in-
sight into the action of pollutants. Suspended solids and sulfite liquor
from paper-mill wastes, ions of heavy metal salts from mining industries,
detergents, phenols and a variety of other discharges from agricultural
practices and industrial processes as well as specific fish poisons, e.g.,
rotenone, are known to affect the gills of fishes.
Thickening of the gill epithelium is one of the first changes noticeable
in fish that are exposed to heavy metal salts, detergents and phenols.
Epithelial thickening is usually followed by fusion of adjacent secondary
lamellae and detachment of the epithelium from the basement membrane of
the gill filaments and their secondary lamellae. Changes in the gill
epithelium during poisoning by detergents are accompanied by vasodilation
of the gill arterioles and blood spaces of the secondary lamellae, producing
extensive vascular stasis or hematomos. Heavy metal ions are known to coag-
ulate mucus secreted on the gills and the gills of chronically exposed fish
have a thinner epithelium covering the lamellae than those acutely exposed.
These changes occur to varying degrees in different parts of the gill so
that diagnosis must be based on a substantial sample of filaments from
different parts of gills.
It is important to note that even though histological changes found
after exposure to various toxic materials have been described in detail,
they do not necessarily indicate the preceise cause of death. Heavy metals
24
-------
may react with enzymes in sill epithelial cells causing formation of lyso-
somes, vesicles and vacuoles in the gill. Damage to the epithelium may
affect gas exchange, extrarenal excretion or gill ion exchange although os-
motic or ionic imbalance has not been found to be the precise cause of death
after exposure, to zinc or mercury. It is probable that in many cases where
fish are exposed to toxic materials, death is due to hypoxia resulting from
the detachment of the gill epithelium from the vascular bed of the lamellae
causing a lengthening of the water-blood pathway. Under these conditions a
significant decrease in the diffusing capacity of the gills would result and
even greatly increased gill ventilation would be unable to meet the oxygen
requirements of the fish. Water borne toxic materials might also have
serious detrimental effects on blood flow through the gills so that even with
adequate external ventilation the internal perfusion might not be adequate
for transport of oxygen from the gills to the various body tissue where it
can be utilized.
It is apparent from the above discussion that more detailed experiments
involving use of the isolated gill preparation are in order. From experi-
ments of this type information should be obtained relating to critical or
detrimental effects that water soluble pollutants, or changes in certain
water quality criteria, might have on freshwater fish. The objectives of
the experiments discussed below were to (1) modify published isolated-
perfused gill techniques to permit longer experiments under conditions re-
sembling, as closely as possible, those found in vivo, and (2) to add to
our existing knowledge of the physiology of the fish gill so that the
preparation could be utilized for studies of the effects of pollutants on
gills. With respect to the latter we wished to confrim or deny regulation
of functional surface area of the teleost gill and determine the nature of
physiological control mechanisms which would be responsible for regulating
functional surface area. For these experiments the influx of -^C-urea was
used as a relative measure of gill functional surface area. Urea is not
metabolized by rainbow trout gill tissue and is not known to be actively
transported by teleost gills. The method is based on the assumption that
diffusional influx of l^C-urea is limited principally by the extent of
secondary lamellae perfusion, i.e., the functional surface area of the gill
available for diffusional influx of the marker.
Methods
Previously described methods for isolated gill perfusion studies were
modified by using a peristaltic pump to deliver perfusion solutions and by
collecting perfusate samples in a fraction collector (Figure 5). Two in
dependent perfusion channels were used to simulataneously perfuse the second
pair of gill arches from each fish. All experiments were conducted in a
cold room at 11 ± 1°C. The peristaltic pump aspirated perfusion solutions
from polyethylene bottles and delivered them through PE 60 polyethylene
tubing and the afferent cannulae to the gills. Perfusate leaving the gills
passed through efferent cannulae and PE 60 tubing to photoelectric drop
counters above the fraction collector. The channel B drop counter signal
drove the fraction collector, which was set to turn after a pre-selected
number of drops had been collected. Fraction collector turns along with
the individual drop signals from the channel A drop counter were recorded
25
-------
Figure 5. Diagram of gill perfusion apparatus. Solid lines represent
perfusion channels and connections; dashed lines represent electronic
connections. A and B gill arches in A and B perfusion channels; D
photoelectric drop counters; FC fraction collector; FCD fraction col-
lector drop count accumulator; G Grass polygraph; PC pressure cali-
bration manometer; PP peristaltic pump; PS perfusion solutions; R
polygraph recording; S 4-way stopcocks; T pressure transducers.
on a Grass 5jy polygraph. To monitor perfusion pressures, 't' connectors
between the pump and gill arches (10 cm from the afferent cannula tips) were
connected to Statham P23^c pressure transducers. A calibration manometer was
also connected through valves and PE 60 tubing to the transducers. The can-
nula tips, manometer base, and transducers were all positioned in the same
horizontal plane to facilitate accurate calibration and measurement of
pressures. Output from each transducer was recorded continuously on the
Grass 5j) polygraph.
The gill cannulation procedure has been described in detail (Bergman
et al. 1974). After cannulation, the pump output to each arch was increased
to 0.5 ml/min and held constant throughout the remainder of each experiment.
Since only about 70% of the filaments were perfused in these gills the
0.5 rnl/min flow rate was consistent with the expected single arch flow rate
(0.75 ml/min, assuming equally divided flow to all eight gill arches) cal-
culated from cardiac output estimates for resting 200-400 g rainbow trout.
Drop size calibration for the photoelectric drop counters permitted calcula-
tion of flow rate through each gill arch. In preparations where severe leak-
age developed the measured flow rate fell below pump output and data from
these experiments were discarded. Pressures usually ranged within the limits
reported for ventral aortic blood pressure in rainbow trout. To mimic
26
-------
systemic resistance, efferent pressure was set at 15 mm Hg by elevating the
efferent tube outflow 20 cm above each gill arch.
Perfusion solutions consisted of vasoactive drugs or hormones added to
Ringer solution. At 11°C the pH of this solution was 7.5 which compares
closely to mixed venous-arterial blood pH for the same species and temperature
in this laboratory. However, this value is lower than the arterial blood pH
of 7.9 reported at 10.5°C by Randall and Cameron (1973) for rainbow trout.
Before vasoactive agents were added, the perfusion solution was vacuum fil-
tered through a 0.22 ym millipore filter. The solution was then vigorously
shaken to assure atmospheric equilibration.
After cannulation, gill arches were perfused for an equilibration period
of about one hour before experiments were begun. During this time each gill
arch was perfused with the same solution that was to be used in that arch
during the initial period of the experiment. After the initial period, drug
or hormone concentrations were increased in 10-fold steps in the experimental
arch while they were either not added or held constant in the control arches.
At the end of a few experiments under each protocol the vasoactive perfusion
pressure and l^C-urea flux to control values. Starting with the initial
period, perfusate fractions of equal volume were collected directly into
vials for determination of -^C-urea activity in a liquid scintillation
counter. The l^C-urea counts from the initial experimental period for the
arch. The sample activities from the initial period were averaged for each
gill arch, and the average was defined at 100% of initial activity. The
activities of samples collected during the remainder of the experiment were
then converted to a percentage of this mean initial activity. To demonstrate
qualitative pressure responses to the drug and hormone treatments, typical
perfusion pressure records were selected to represent each type of experiment.
Where quantitative expressions of pressure responses were appropriate, the
data were treated in the same way as the l^C-urea data. All statistics were
calculated with log transformed data, and the means of 95% confidence limits
were used for all comparisons and Tukey's w-procedure was used for multiple
comparisons among means.
Results and Discussion
The influx of -^C-urea into isolated-perfused gills was altered
markedly by the vasoactive drugs and hormones used in this study. The
results of a typical epinephrine (EPI) experiment are depicted in Figure 6.
In the figure the open circles = control arch fractions and x = experimental
arch fractions. Vertical dashed lines delineate experimental periods with
EPI concentrations in the experimental arch shown above. EPI concentration
in the control was 10"7M throughout. The solid horizontal line represents
the control initial activity for both gill arches. Typically, the percent
of initial activity increased (or decreased depending upon the agent used)
toward some new steady-state value in response to step-wise changes in the
vasoactive drug concentration. For all experiments following one protocol,
steady-state percent of initial activity values are tabulated for the drug
concentrations used.
27
-------
TOO
600
b 500
*20O
100
lO'7 M
EPINEPHRINE CONCS.
IO'6 M IF* M IO'7 M
,*>"*''
•ffir—
0 1.0
L5 2-0 2.5 3-0 3.5
POST CANNULATION TIME (MRS)
Figure 6. Results from a typical epinephrine experiment. See text for
details.
The results from epinephrine (EPI) experiments (Figures 7 and 8) were
qualitatively the same as those found for norepinephrine (NEPI), although
a narrower concentration of EPI was used. EPI, like NEPI, is a mixed agonist
which can stimulate both a and 3 adrenergic receptors. Because -^C-urea in-
flux data were less variable in the EPI experiments than in NEPI experiments,
more clear-cut differences were evident when the EPI effect was blocked with
phenoxybenzamine (POB) and propronolol (PROP) as noted in Figure 8 below.
The unblocked 10~5M EPI induced l^C-urea influx is significantly greater
that seen in the control or blocked gills. * Influx in the single block
experiments (a or 3) was still significantly higher than in the controls,
and double blockade (a and £) of the EPI effect was necessary to reduce in-
flux to the control value.
Acetylcholine (ACH) significantly reduced ^C-urea influx when perfused
alone (Figure 9) or when perfused along with 10~5M EPI. The reduction was
greater in experiments where ACH was perfused without the EPI. Stimulation
of a and/or 8 adrenergic receptors with catecholamines or pharmacological
drugs in this study produced marked and usually significant Increases in
14c-urea influx, whereas ACH significantly reduced influx of the marker.
The most tenable explanation for the observed changes in l^C-urea influx and
branchial vascular resistance is that alterations in the internal perfusion
pathway resulted in changes in functional surface area of these gills. It
could be aruged that the vasoactive agents may have affected the permeability
of the gills to l^C-urea but it is doubtful that the changes in permeability
could account ror the effects these agents had on branchial vascular resistance
(Figures 10 and 11). This is especially true in the case where, with opposite
effects on resistance, stimulation of either a or 3 adrenergic receptors
caused an increase in 14C-urea influx. We conclude that these vasoactive
28
-------
TOO
t 600
o 900
3 400
300
£00
IOO
1 CONTROL EPI
[~~3 EXPERIMENTAL EPI
£
i?
f
icr6 to-3
EPINEPHHINE CONCS. (MOLES/LITER)
Figure 7. The effect of epinephrine on l^C-urea influx. During the initial
period (not shown) both control and experimental gills were perfused with
10~^M epinephrine. The mean, 957» confidence interval, and N value are
shown for each control and treatment from successive experimental periods.
Significance for paired comparisons; *0.01 P 0.05; **0.001 P 0.01;
*** 0.001.
7OO,
£ soo
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J 400
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OL EPI ^ W BLOCKADE
EXPERIMENTAL EPIS /3 BLOCKADE
^ Ota/3 BLOCKADE
EPINEPHRINE CONCS. (MOLES/LITER)
Figure 8. The effect of adrenergic blockade on epinephrine induced l*C-
urea influx. During the initial period (not shown) all arches were per-
fused with 10~7M EPI. In blocked arches, the blocking drug(s) was per-
fused throughout the entire experiment. All values are from the same ex-
perimental period, a block = 1Q-5M POB; 3 block = 10~5M PROP. The means,
957» confidence interval and N values are shown. The means listed below
which are not underlined by the same line are significantly different (0.05)
Control a + 8 a B Exp.
EPI
72
block
100
block
187
block
189
EPI
564
29
-------
**
140-1
120
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1 CONTROL (RINGERS ONLY)
EXPERIMENTAL ACH
10"" 10*' 1C"
ACETYLCKOLINE CONCS. (MOLES/LITER)
Figure 9. The effect of acetylcholine on l^C-urea influx. During the
initial period (not shown) both control and experimental gills were perfused
with Ringer solution only.
agents had little effect on permeability and that the observed changes in
14C-urea uptake reflect changes in the relative functional surface area of
the gills. For any series of experiments with a vasoactive agents, the
pressure effect was qualitatively consistent, but the sensitivity of different
preparations to a drug or hormone varied. This may have resulted from.
differences in prevailing vascular tqne.
Perfusion pressure was reduced by NEPI (Figure 10 ), although in a few
experiments a transient pressure increase was seen first (Figure 10B). At
1Q-5M NEPI the average percent of initial pressure (88%) was significantly
lower than in the 10~9M NEPI controls. Although not statistically signifi-
cant, reductions in perfusion pressure were observed in some preparations
with NEPI concentrations as low as 10~8M. Figure IOC, D and E show the typi-
cal pressure responses when adrenergic blockers were perfused along with NEPI
just as in the unblocked gills. After 8 blockade, however, a pressure in-
crease was usually unmasked and when both a and $ blockers were sued no pres-
sure change resulted from the increased NEPI concentration. Qualitative
pressure responses in gills perfused with EPI alone or with adrenergic block-
ing agents were identical to those seen in the NEPI experiments, hence these
data are not presented. When the ACH content of the perfusion solution was
increased from 10~Jl to 10~7M a slight but significant increase, in perfusion
pressure was noted (Figure 11A). Another ten-fold increase to 10~°M ACH
resulted in a very significant pressure increase (Figure 11 B). The effects
of four different drugs on the increased perfusion pressure induced with
10-6M ACH were also noted. The muscarinic and nicotinic (ganglionic) block-
ers, atropine and hemamethonium respectively, both eliminated most of the
pressor effect of ACH. The a adrenertic block POP completely eliminated
the pressure increase whil phentolamine, also an a adrenergic blocker, had
little effect.
30
-------
E
o
CO
£ 60r
o. 30«-
I
30L i , ,
1 I MIN
Figure 10. Typical effect of norepinephrine on perfusion pressure in the
absence and presence of adrenergic blockers. Arrows indicate where NEPI con-
centration was increased from 10~6 to 10~5M. (A) and (B) NEPI alone; (C) a
blockard of NEPI with 10~5M FOB; (D) 3 blockade of NEPI with 1CT5M PROP; (E)
a and 3 blockade of NEPI with 10~5M FOB and 10~5M PROP.
Summary
The physiological viability of the perfused gills in this study was
probably due partly to the use of filtered perfusion fluid (Rankin and Maetz,
1971) as well as pulsatile pump-perfusion, which appeared qualitatively
superior to perfusion with a hydrostatic head (Richards and Fromm, 1969;
Rankin and Maetz, 1971; Randell e£ al. 1972; Wood, 1974) or a constant pres-
sure syringe pump (Shuttlcworth, 1972). The appearance of the gill arches
usually remained good during and after perfusion except that secreted mucus
accumulated on the surface of a few gills. Although no edema was evident,
colloid osmotic, pressure was low in our perfusion fluids and some edema could
have developed. However, branchial vascular resistance remained constant and
vasoactive agent effects were reproducible for as long as 12 hours. With
flow rates which approximated the expected single arch flow rate in intact,
resting rainbow trout (Stevens and Randall, 1967a), the perfusion pressures
in these experiments were generally within the range of ventral aortic blood
31
-------
en
I60F
SOL
LU
CC.
tO
Ul
a:
o.
! MIN
Figure HA and B. Typical effect of acetylcholine on perfusion pressure.
Arrows indicate where ACH concentration was increased. (A) ACH increase
from 10~8 to 10~7M; (B) ACH increase from 10~7 to 10~6M.
pressure reported for this species (Stevens and Randall, 1967b). Pressures
in excess of the normal range were observed only in the gills perfused with
10~% ACH. Significant effects on resistaace and 1/lC-urea uptake were seen
at NEPI and EPI concentrations which were comparable to the maximum plasma
catecholamine levels reported for distrubed rainbow trout (Nakano and
Tomlinson, 1967). However, the adrenergic agonist concentrations which
produced threshold effects on resistance were sometimes 100-fold higher than
previously reported for the same drugs and hormones in isolated teleost gill
preparations (Rankin and Maetz, 1971; Wood, 1974). The reasons for this are
probably related to differences in pH, temperature, perfusion pressure and
composition of the perfusion fluids.
The physiological control mechanisms responsible for adjusting the
resistance to flow in teleost gills are probably both hormonal and neural in
nature. The catecholamine-induced decrease in branchial vascular resistance
increase in functional surface area seen in these experiments agree with pub-
lished observations on the resistance (Keys and Bateman, 1932; Ostlund and
Fange, 1962; Reite, 1969) and blood pathway effects of catecholamines (Steen
and Kruysse, 1964; Richards and Fromm, 1969). These findings, combined with
the observation that plasma catecholamine concentrations are elevated during
exercise (Nakano and Tomlison, 1967), strongly support hormonal control of
gill blood flow and functional gill surface area. Wood (1974) has proven
the involvement of 3^ adrenergic receptors in this catecholamine effect. Al-
though the critical catecholamine potency-series experiments have not yet
been reported as proof, evidence in this and other studies suggest a ad-
renergic receptor involvement in the catecholamine effect as well.
THE ROLE OF FISH GILLS IN HEAT EXCHANGE
Another series of experiments dealing with heat exchange of fishes were
conducted using isolated-perfused gills as well as isolated-perfused heads
of rainbow trout. Investigations dealing with rates of thermal exchange in
32
-------
fish have been few in number. Stevens and Fry (1970, 1974) have reported
values for a coefficient of temperature exchange in a variety of species,
however, there appear to be no data directly in terms of heat exchange by
a teleost. Fry (1967) speculated that the gills play a major role in overall
thermal exchange but pertinent experiments to substantiate this hypothesis
are lacking.
In attempting to quantitate heat exchange by the gills of rainbow trout
a generalized equation for a tubular heat exchanger was adopted (Kay, 1963):
Q - hAAln (1)
Where Q_= total heat flux in cal min"1; h = heat transfer factor in cal
min~-'-0C cm" ; A = surface area in cm^ and AT^n = logarithmic mean temperature
gradient in °C. The value of h will be determined by three factors: (1)
convective transfer to the inner wall of the vasculature, (2) conduction
through the vessel wall and (3) convective transfer from the gill surface to
the environment. The surface area will be determined by the mean length and
mean diameter of the vasculature. Because we were unable to accurately de-
termine a value for surface area (A), Equation 1 was solved for the quantity
hA following determinations of Q and T^n using Equations 2 and 3 respectively
(Kay, 1963):
Q - f Cpp(Ti-T0) (2)
Tln Tj - TQ
. Ti - To
TO - Tb (3)
Where f = perfusion flow rate in ml min ; Cp = specific heat of perfusion
fluid (= 0.962 cal gm-l'C"1 for 0.1% NaCl, CRC Handbook); p = density of
perfusion fluid flowing into the gills, out of the gills, and of the bath
respectively. Equation 3 represents the mean temperature gradient for a
tube of uniform dimensions and may not be rigorous for the case of the
branching vasculature. However, this equation provides the best means of
assessing the mean gradient driving heat across the gill. Also, with uni-
directional ventilation, the increase in temperature of the ventilatory
stream as it passes the gills may be accounted for an equation similar to
(3), but makes an insignificant contribution to the results reported here.
The experiments performed were designed to provide information on (1)
the magnitude of heat exchange occurring in isolated-perfused gills of
rainbow trout, (2) the effect of changes in vasculature gemoetry (i.e.,
changes in functional surface area) on heat exchange and (3) the effect of
ventilation on heat exchange. Finally, a simple model is evaluated in an
attempt to relate gill heat exchange to the whole body thermal response.
Methods
The second gill arches from 200-400 gm rainbow trout (Salmo gairdneri)
were prepared for cannulation as outlined by Bergman et^ a±. (1974). Inflow,
outflow and bath temperatures were measured (+ 0.05°C) with thermistors
(32A7, Victory Engr.,) using a continuous balancing Wheatstone bridge re-
33
-------
corder. Inflow and outflow thermistors were inserted in the cannulas to
within 1 cm of the supportive branchial bar of each gill arch. Heat ex-
change was determined in gills perfused at 0.5 ml min~l by a Ringer solution
alone or Ringer solution which contained 10~^M epinephrine (EPI). With
T± held constant at 10 + 1°C, data were obtained and Equation 1 solved for
hA at steady state values for Tfo of 2, 5, 8, 13, 16, and 19°C. Statistical
treatment of the data was performed using the Student's _t_ test for sample
means.
For whole head preparations fish were decapitated just posterior to
the opercula and the intact branchial basket was allowed to clear of blood as
described by Bergman et al. (1974). A cannula with a thermistor probe
threaded through the lumen to the cannula tip was inserted into the ventral
aorta and secured with a ligature around the bulbus arteriosus. A similar
cannula was placed in the dorsal aorta and secured by a ligature around the
dorsal aorta and spinal column. A third ligature was placed around the cut
end of the esophagus. The head was transferred to a temperature regulated
water bath and a ventilatory flow (Vg) was diverted into the mouth via a
flowmeter and varied between 40 and 130 ml rain"1 (Figure 12). Perfusion
solutions were delivered by a positive displacement type pump with an in-
dependently adjustable stroke volume and rate. Perfusion pressure was
monitored continuously with mean perfusion pressure of all control experi-
thermistor for a reliable measure of To precluded the use of an outflow
resistance resulting in larger than normal perfusion pulse pressures. Iso-
lated heads were perfused with four different solutions: Ringer solution
alone and Ringer solution which contained 10~5M EPI, 10~8M or 10~7M ACH.
Heat exchange was determined in the steady state with an inflow temperature
(Ti) of 12 + 1°C and bath temperature (Tt,) of 8 + 0.1°C. The thermistor
probes were calibrated against a differential thermometer and temperatures
recorded + 0.01°C. All experiments were preceded by a 30 min equilibration
period. Changes in drug concentration were followed by a 30 min equilibration
period and changes in perfusion flow were followed by a 15 min equilibration
period.
Results and Discussion
In the isolated-perfused heads, the influence of ventilatory flow
on heat transfer was evaluated and is shown for a representative experiment
in Figure 13. Values for hA at Vg below 400 ml min~l were not determined
because of marked variations in hA due to (1) extraneous heat picked up by
the fluid passing through the flowmeter which was in contact with ambient
temperature (20°C) and (2) possible mixing of the low temperature bath with
the warmer ventilatory fluid by back flow into the opercular chamber at
low Vg. Despite these experimental limitations, two constraints may be
placed on the values of hA with increasing Vg. Since Vg influences only the
convective heat loss from the gill surface, as Vg increases a point is reach-
ed where heat transfer is limited solely by the transfer of heat to the inner
vessel wall and the conduction of heat through the vessel wall and hA becomes
independent of Vg. This constraint represents0an asymptotic maximum of hA
(hAmax). In a similar manner, for 0 ml min"1 Vg there will ideally be no
heat lost from the system in steady state and hA must be zero. We chose
to use a rectangular hyperbolic function to predict hA for a given Vg:
34
-------
FLOWMETER
TEMPERATURE
REGULATED
WATER
BATH
Figure 12. Diagram of apparatus used to perfuse isolated heads of rainbow
trout. See text for details.
hA = hA V
max g
V - Vasym
O
(4)
The term -Vasym also represents the value of V necessary to reach one-half
of hA (Vn c ) and Equation 4 becomes:
max 005 max M
hA = hA V
max g
Va
(5)
0.5 max
From equation 5, the transfer maximum (hA ) and ventilatory flow at one-
half the transfer maximum (Vn _ ) were obtained from a linear regression
u•j max
of hA -1 on V -1. Changes in hA and V_ _ were evaluated using a 2
way analysis of variance and when appropriate"a §?udent-Newman-Keuls test
for significance group means (Sokal and Rohlf, 1969).
Values for heat transfer (hA) In the isolated-perfused gills are shown in
Table 6. Perfusion with ICT^M EPI significantly increased hA. There were
no uniform or systemic alterations In hA or perfusion pressure associated
with changes in bath temperatures.
35
-------
hA
cal/sec/°C
0.4 J
0.3-
0.2-
0.1-
max
- 25.59 cal
VQ.5 max " 205 ml nln
-1
400
—, 1
800 1200
ml/min
Figure 13. Experimental data for heat transfer plotted as a function of
ventilatory flow (Vg) for one isolated head perfused at 20 ml min"1. hAmax
and VQ.Smax were determined by a double reciprocal linear regression and the
solid line represents the theoretical curve.
TABLE 6
HEAT TRANSFER (hA) OF ISOLATED-PERFUSED SECOND GILL ARCHES
OF RAINBOW TROUT. PERFUSION RATE - 0.5 ML MIN
_P_e_rfusion Fluid
Heat transfer (hA)cal min"
Ringer solution
10~->M EPI in Ringer solution
0.774 + 0.024*
0.858 + 0.024*+
*X + SE; + p less than 0.05
Values of hAmax and V0.5jnax at perfusion flow rates of 16 and 20 ml
min"1 for the initial control periods of all experimental groups are summarized
in Table 7. The effects of altering perfusion flow are shown in Figure 14.
TABLE 7
HEAT TRANSFER FOR ISOLATED-PERFUSED HEAD OF RAINBOW TROUT
DURING CONTROL PERIODS. VALUES (X + SE, n)
ARE GIVEN FOR FLOW RATES OF 16 and 20 ML MIN"1.
Perfusion flow rate
16 ml min"1 20 ml min'1
(cal min"1^"1)
0.5max
21.27 + 0.57 (21)
144 + 17 (21)
24.79 + 0.77 (21)
183 + 23 (21)
36
-------
Increasing the perfusion flow increased
Also, at a given perfusion flow rate,
a 20% change in stroke volume.
but had no effect on V
and Vo.5max were unaffected by
The effects of drug infusions are shown in Figure 14 (below) for a
perfusion flow of 20 ml min*"1. Each experimental group was followed
through control, drug infusion and post-control periods. During
each period hA was determined for perfusion flows of 16 and 20 ml min~ at
a stroke rate of 50 min"1. VQ.Smax was unaltered for any of the experiment-
al treatments. With infusion of 1Q-5M EPI hA^x was significantly increased
above the values obtained during the pre- and post-infusion control periods,
whereas, perfusion of solutions containing 10~8M and 10" 'M acetylcholine
had no effect on hA^x- These results are qualitatively the same at both
perfusion flow rates. When perfusion flow was increased from 16 to 20 ml
min"1 during constant infusion of either EPI or ACH the change in hA^x was
identical to that seen in the control experiments (Figure 15).
'man
col/»K/-C
20 ml/mln
50
0.5-
0.4-
0.3-
0.2-
0.1-
t
T
JL
5
5
5
S
1
fft
I
i
4
JL
5
I
5
.
4
6
1
!!
i!:i
6
|
6
1P<0.05
mi/milt 1 1 Ring
300-
200-
ICO-
,-5.
T
I
9
11
9
5
|
9
^2 IOJMEpi
I
1
1
&
n
4
i
5
H] IO"8M Ach
{H IO"7M Ach
I
,s;
:-
I
4
flJlT
ji!»l
« 6 C
ijijii
Figure 14_ The effects of vasoactive agents on hAmax and VQ.Smax
(X + SE) of the perfused head at a perfusion flow of 20 ml min~l. + signif-
icant change in hAmax during 10~5M EPI from pre- and post-infusion control
periods.
37
-------
cal/sec/eC
t P<0.05
ml/min
0.5-
0.4-
0.3-
0.2-
0.1-
t
_ T
4
4
4
4
50 40 60 40 beat
16 20 ml
T
4
T
4
T T
4
4
-200
- 100
/
min 50 40 60 40
•nin 16 20
Figure 15. The effects of alterations in perfusion flow and heart rate
on hAmax on the left and Vo.5max on the right (X + SE) for the isolated-
perfused head (n = 4) . + significant change in hA^x at perfusion flow of
20 ml min"1 over 16 ml min"1.
In interpreting changes in heat transfer (hA) following experimental
manipulations, the basic determinants of h and A must be considered. These
determinants are not independent. A change in vascular dimensions ^will
alter not only the length and diameter of the vessels, but wall thickness
and perfusate velocity such that the value of both h and A are affected.
The effects of ventilatory flow on external convection will be determined
by the velocity of the stream as well as its relation to the path of in-
ternal perfusion.
The use of values for maximal heat transfer (hA^x) and ventilatory
flow at one-half hAmax (Vo.5max) allows some of these factors to be evaluat-
ed independently. An increase in Vo.5max without a change in hAmax would
result in a decrease in hA at any given $g. Assuming the internal dimensions
and hence internal convection and conduction are stable during the determi-
nation of hA^x and V0.5max' the only alteration which would account for the
increase in V0.5max would be a decrease in convective loss from the gill
surface. A decrease in external convective loss can be brought about by
two means: (1) a conformational change of the gill in the ventilatory
stream or (2) a change in the mean ventilatory velocity over the perfused
lamellae. If a gross conformational change of the gill has not occurred,
the patterns of ventilatory velocity over the gill should be unaltered. The
increased VQ.Smax would then indicate a change in perfusion pathway away
38
-------
from the better ventilated secondary lamellae with a resultant increase in
ventilation-perfusion inequality. In contrast to Vo.5max» hAmax provides
a VK independent measurement of heat transfer and should be altered only by
changes in internal thermal convection from the perfusate to the vessel wall
and thermal conduction through the vessel wall. By evaluating the two para-
meters of the hyperbolic function relating hA to Vg, independent changes in
either V0 5raax or hAmax wil1 provide information regarding alterations in
(1) the ventilation-per fusion relationship of the gill (hAmax) . Simultaneous
changes in hAmax and Vo.5max would indicate some indeterminate combination
of these factors.
The theoretical determinants of hA^x could be correlated to experimental
observations by noting the effects of increasing perfusion flow. The in-
creased flow and resultant increase in perfusion pressure may cause an in-
crease in mean perfusate velocity and/or distension of the vasculature.
These changes alter the internal convection and conduction processes and
result in a significant increase in hAmax in response to the increased per-
fusion flow (Figure 14). We were unable to detect any significant alter-
ation in VQ_5max following our experimental manipulations. This may be due
in part to a basic insensitivity of V0.5max to detect ventilation-perfusion
changes. However, the importance of ventilation-perfusion matching cannot
be overemphasized when evaluating exchange processes in any respiratory
organ.
Holeton and Randall (1967) reported that trout exposed to a hypoxic
enviroment exhibit a marked bradycardia with no alteration in cardiac out-
put. In this case, the animal should attempt to maximize oxygen transport
by minimizing ventilation-perfusion inequalities. The evidence for a cap-
activie 'bleb' in the afferent filamental vessels (Fromm, 1975 and see
below) gave rise to speculation concerning the frequency dependence of the
distensible gill vasculature. If the gill vessels could exhibit a frequency
dependent nature to allow for passive changes in perfusion pathways in
response to changes in heart rate, the hypoxic bradycardia could give rise
to a passive redistribution of perfusion to aid in oxygen transport. Thi
is apparently not the case since, as shown in Figure 14, a 30° change in
heart rate (40 to 60 min"-1-) did not alter the Vo.5max or hA^y of the gills.
However, the diffusion distance and membrane permeability characteristics
than heat, thus there are many changes that may be occurring in vascular
flow and the lamellar membrane that will alter oxygen uptake, but have an
insignificant effect on thermal exchange. If a passive vascular response
to changes in heart rate occurs, this could be coupled with an active mech-
anism(s) to efficiently regulate ventilation-perfusion ratios.
Along with the possibility changes in the gill vasculature, there have
been several investigations dealing with active regulation of the gill
vasculature (Bergman e£ al. , 1974; Steen and Kruysse, 1964). Bergman
et al. , (1974) concluded that EPI increased functional surface area for
39
-------
uptake and that it was decreased by acetylcholine. The primary
changes in these experiments were attributed to perfusion pathway changes
which should be reflected by alterations in the hA^x value for heat ex-
change. As shown in Table 6 and Figure 15, 10-% EPI significantly in-
creased hA in the isolated perfused gill and hAmax in the isolated perfused
head. This increase is not as dramatic as that seen by Bergman et_ ja. (1974),
and is indicative of the relative sensitivities of the two markers used. In
the brachial basket the major change in vascular dimensions is presumed to
take place at the secondary lamellae which would greatly alter the l^C-urea
uptake and also change heat transfer. However, the remainder of the large
vessels in the gill are readily exchanging heat but not ^C-urea indicating
there is a greater effective surface area for thermal exchange than for mo-
lecular exchange. This could explain why there was a much smaller increase
in total heat flux (13%) than total 14C-urea flux (400%, Bergman*£t al.,
1974) for the same perfusion pathway change. Acetylcholine at concentrations
of 10~8M and 10-?M did not significantly alter hA^x- There are several pos-
sible reasons for this lack of supportive evidence of ACH modulation of the
exchange process: (1) the relative sensitivities of a molecular versus ther-
mal diffusion phenomena to small changes in vascular geometry, (2) the ini-
tial control state of the gill vasculature and (3) the possibility of a dif-
ferential response by the gill arches to ACH that would mask overall changes
in the brachial basket but not in the isolated gill arch. This final point
has received little attention and the redistribution of perfusion pathways
at the gill arch level as well as the lamellar level should be considered for
all vasoactive agents.
Aside from the basic quantitation of heat exchange by the gill, a sim-
ple model (see Figure 16) was constructed to determine the relative contri-
bution of the gill to whole body heat exchange. The body of the fish con-
sists of a cylinder with thermal conductivity (k) of 1.4 x 10~J cal°cnr* (k
for water, Crc Handbook) and an internal core of 0.5 cm radius maintained at
some temperature (T^ above ambient (Tb). Blood is pumped through an ex-
changer and returned to the body at T0. To simplify the model it was as-
sumed that: (1) there is no convective mixing in the body, (2) there is no
interference with thermal conduction from the body due to the flow of blood
through the gills, and (3) there is no heat conducted from the ends of the
cylinder. Thus, the total heat flux (Qtot) will be the sum of the convective
loss from the gill (Qcv) and the conductive loss from the body (Qcd)•
Qtot - Qcv + Qcd (5)
Qcd can be determined using Equation 6 (Bennett and Meyers, 1962).
Qcd = k 2 lATi/(ln r2/ri) (6)
Where 1 = length of cylinder; TI - internal radius of cylinder; r2 - external
radius of cylinder and T± = T1 - Tb. Qcv can be determined by using Equation
2. The solid lines in Figure 17 represent a plot of the percent of total
heat flux (% Qtot) exchanged via the model gill as a function of perfusion
flow when the % equilibration (1 - To/Ti)100 is held at 100% and 50%. The
maintenance of a constant % equilibration in the face of increasing perfusion
flow will probably not occur and the actual model response should tend to
40
-------
Figure 16. Diagram of model used to evaluate gill heat exchange in terms of
whole body heat exchange, r^ = 0.5 cm, r<^ = 2.0 cm. Other terms as given
in the text.
flatten as the heat delivered by the perfusion fluid exceeds the exchange
capability of the gills.
f C
Experimental data were evaluated in a similar manner. Since Q
pP
cv
~ T0) (Equation 2) and Qcv = hAATln (Equation 1) if we express To
as a fraction, X, of T-^ then:
f Cpp (Tj - T0) = hA T± -
In Ti - Tb
- Tb
and:
In
- Tb
XT± - Tb
hA (T± -
- XT±)
Subtracting Tb from all temperatures, i.e., measuring all temperatures rela-
tive to Tb:
41
-------
In
(Ti - Tb)
X(T± - Tb)
hA
f C
PP
Solving for X:
hA
X = e f C
PP
- hA
and:
% equilibration = (1-e
) 100
Using this value of X:
and:
f C
PP
Qcv = f Cp
_ f Cn (1 - X)
(7)
(1 - X) + k 2 ir 1 100 (8)
In
100
__ _. Equilibration
T T
8 12 16
Cardiac Output ml/min
Figure 17. Graph of percent of total heat flux (% Qtot) from the model gill
with increasing perfusion flows. Solid lines indicate the model response at
a constant % equilibration, 50 and 100%. Dotted line indicates model re-
sponse using experimental data.
42
-------
For the specific model of Figure 16, Equation 8 was solved using experi-
mental data. In Figure 17 the data point at 0.5 ml min~l is the value ob-
tained for a single gill arch (Table 6) and the point at 4 ml min~l is extrap-
olated assuming 8 independent gill arches all exchanging at the same rate as
the single arch perfused at 0.5 ml min~l. The points at perfusion flows of
16 and 20 ml min~l were calculated using the hA^jj values given in Table 7.
The dotted line connecting these data points lies between the 50 and 100%
equilibration curves for the idealized model and % Qtot is leveling off with
higher flow rates.
The model may also be used to determine the influence of gentilation on
% Qtot exchanged at the gills. Values of hA as a function of Vg were deter-
mined using Equation 5 and substituting for hA^.,,. and V0.5 max at perfusion
flows of 16 and 20 ml min~l. Values for % Qtot were obtained by solving Equa-
tion 7 as a function of V and using Equation 8 to determine the % QtOf
marked effect on % Qtot as Vg is increased is shown in Figure 18. Several
cautions should be placed on the interpretation of the model responses.
%Q
tot
lOO-i
20ml/min
16 ml/min
400
1 T
800 . 1200
V ml/min
1600
o
Figure 18. Model prediction of % Qtot as a function of ventilatory flow (Vg)
using experimental data at perfusion flows of 16 and 20 ml min~l.
First, we are only able to estimate the effect of perfusion flow on the max-
imal rate of heat transfer by the gills, the In vivo state will lie at some
point below the dotted curve of Figure 17. Second, we are only able to esti-
mate the effect of a continuous ventilatory stream at perfusion flows of 16
and 20 ml min~l. Data for Intact animals may be at some point below the
43
-------
curves of Figure 18 due to lower cardiac outputs and the complicating factor
of a phasic respiratory flow. Finally, the model of the fish body as being
a purely conductive cylinder will be complicated by the convective mixing of
the blood. This factor will tend to raise QC(j and lower all the values of
gill heat transfer when expressed as % Qtot. The model is primarily illus-
trative of the maximum relative contribution the gills may make to whole body
heat exchange. The actual contribution to heat exchange by the gills awaits
further investigation.
Summary
The heat exchange has been evaluated in isolated-perfused sec-
ond gill arches and the intact branchial basket. The presence of 10~5M epi-
nephrine increases the transfer maximum of the gill suggesting a change in
perfusion pathway and/or vascular dimensions. Changes in perfusion flow al-
ter heat exchange by the gill which is again due in part to changes in vas-
cular dimensions. A model for the evaluation of the gills in relation to
whole body heat exchange indicates that in the range of perfusion flows from
4 to 20 ml min~l the gill may account for as much as 30 to 60% of the total
heat exchange in the animal.
THE VASCULARITY OF TROUT GILLS
The final project which received financial support from Grant R-801034
was work which was an outgrowth of studies on circulation in trout gills in-
jected with latex and examined after acid digestion (Richards and Fromm,
1969). More recently Microfil, a vulcanizing liquid silicone compound that
flows through vessels of capillary size much more readily than latex, became
available. The following is a summary of observations made on gill circula-
tion in specimens infused with this compound.
Methods
Rainbow trout were anesthetized with MS-222 and placed ventral side up
in a V-shaped trough, and an incision was made to expose the heart and bul-
bus arteriosus. Freshly mixed Microfil (2.5 ml MV 112 white, 2.0 ml MV di-
luent, and 0.135 ml curing agent)(Canton Bio-Medical Products, Inc., Boulder,
Colo.) was drawn into a 5 ml syringe and infused via a polyethylene cannula
(PE 90) that had been inserted and secured into the ventral aorta. Gentle
pressure on the syringe plunger was applied manually and excessive disten-
tion of the ventral aorta avoided by extending the infusion over a period of
12-15 min. The infusion pressure was not recorded. About 5 ml Microfil was
injected into fish weighing around 200 g, a much larger volume than that re-
quired to fill the gill vasculature. Significant amounts passed through the
gills and appeared in vessels of the musculature and the splanchnic area.
After injection, the specimens were stored in a refrigerator overnight to
allow complete polymerization of the Microfil. They were then cleared in
methyl salicylate (Merck), and selected casts with adhering cleared tissue
were submerged in methyl, salicylate and photographed (see Figure 19).
-------
Figure 19. Microfil cast of a single pair of gill filaments from rainbow
trout. AG, afferent gill vessel; AF, afferent filamental vessel; EG, effer-
ent gill vessels; EF, efferent filamental vessel; B, afferent filamental
blebs; SL, secondary lamellae. Calibration bar = 1 mm.
45
-------
Results and Discussion
The trout circulatory system from the ventricle to the secondary lamel-
lae fit the classical description except that the efferent gill vessel (EG)
was found to leave the arch a short distance below the upper limit of the
gill, leaving a cul-de-sac extension to drain blood from vessels of the more
dorsally located filaments. Many vessels that arise from the base of the
afferent filamental or from the afferent gill vessel were observed in the in-
terfilamental septal area. Vessels on either side of the septum were seen to
loop toward the base of the filament and then leave the septal area and run
parallel to the afferent filamental vessel. Although it is not as clearly
visible, a second somewhat larger vessel, distal to the bleb, also ran par-
allel to the afferent filamental vessel toward the tip of the filament. It
appeared as if this vessel might provide cross connections between afferent
lamellar vessels at the point where they empty into the space surrounding
the pillar cells. Another septal vessel of smaller diameter than the effer-
ent filamental vessels was found in several but not all gill arches. This
vessel was located in the basal portion of the septal area and ran parallel
to the afferent gill blood vessel. Several additional short intraseptal vas-
cular connections between successive pairs of filaments on a gill arch were
also noted.
Perhaps the most conspicuous feature of the gill casts was the presence
of blebs. These aneurysm-like enlargements of the afferent filamental ves-
sels were located in the most distal part of the septal area, roughly one-
third of the distance from the base to the tips of filaments. The casts ap-
peared as if those on opposing filaments would make contact with one another
when fully distended but no vascular connections between blebs were found.
It appears presumptive that the blood vessels in the interfilamental
septal area and those that run parallel to the afferent filamental vessels
are not involved in gas exchange. The function of the filamental blebs, on
the other hand, is even more speculative.
In these experiments we were aware that perfusion pressure during cast-
ing was rather critical, especially with respect to flow into the central
filament space between afferent and efferent filamental vessels. It was not
practical to record perfusion pressure but care was taken to avoid excessive
distension of the ventral aorta during infusion. There was no evidence of
the application of excessive pressure, e.g., rupture of gill vessels; thus,
we believe that the casts represent fairly accurately the size and shape of
vessels and blebs in trout gills.
Using a dissecting microscope and intense direct illumination we have
observed a turbulent, pulsatile flow of blood into the bleb area of rainbow
trout gills in situ and the frequency of this flow into the blebs was found
to correlate perfectly with a simultaneous recording (EGG) of heart rate.
Also when a large nerve carrying fibers to an isolated-perfused gill arch
was stimulated, adduction of the filaments occurred, accompanied by an in-
crease of about 15% in the perfusion pressure. When stimulation was exten-
ded for as long as 60 sec the increased pressure rapidly subsided, lasting
46
-------
only about 15-20 sec. It is tempting to speculate that contraction of the
adductor muscles may have increased tissue pressure surrounding the blebs
and facilitated their emptying. If rhythmical isotonic or isometric con-
traction of these muscles occurred synchronously with heart beat, the pump-
ing effect would be analogous to that of gill hearts that aid in forcing
blood through the gill vasculature. Unfortunately, this idea is the obser-
vation by Bijtel (1949) that adduction movement of the filaments (which might
not occur with isometric contraction of adductor muscles) appears sporadi-
cally and then only in connection with coughing movements.
47
-------
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Randall, D.J. and J.N. Cameron. 1973. Respiratory control of arterial pH
as temperature changes in rainbow trout Salmo gairdneri. Am. J. Physiol.
225:997-1002.
49
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Rankin, J.C. and J. Maetz. 1971. A perfused teleostean gill preparation:
Vascular actions of neurohypophysial hormones and catecholamines. J. Endocr.
51:621-635.
Reite, O.B. 1969. The evolution of vascular smooth muscle responses to hista-
mine and 5-hydroxytryptamine. I. Occurrence of stimulatory actions in fish.
Acta Physiol. Scand. 75:221-239.
Richards, B.D. and P.O. Fromm. 1969. Patterns of blood flow through fila-
ments and lamellae of isolated-perfused rainbow trout (Salmo gairdneri) gills.
Comp. Biochem. Physiol. 29:1063-1071.
Schweiger, G. 1957. Die Toxikologische Einwirkung von Schwermetallizalzen
auf Fische und Fischnahrtiere. Arch. f. Fishchereiwiss. 8:54-77.
Shuttleworth, T.J. 1972. A new isolated perfused gill preparation for the
study of the mechanisms of ionic regulation in teleosts. Comp. Biochem.
Physiol. 43A:59-64.
Sokal, R.R. and F.J. Rolf. 1969. Biometry. W.H. Freeman Co., San Francisco.
776 p.
Steen, J.B. and A. Kruysse. 1964. The respiratory function of teleostean
gills. Comp. Biochem. Physiol. 12:127-142.
Stevens, E.D. and F.E.J. Fry. 1970. The rate of thermal exchange in a tele-
ost Tilapia mossambica. Can. J. Zool. 48:221-226.
Stevens, E.D. and D.J. Randall. 1967a. Changes of gas concentrations in
blood and water during moderate swimming activity in rainbow trout. J. Exp.
Biol. 46:329-337.
and . 1967b. Changes in blood pressure, heart rate
and breathing rate during moderate swimming activity in rainbow trout. J.
Exp. Biol. 46:307-315.
Wood, C.M. 1974. A critical examination of the physical and adrenergic fac-
tors affecting blood flow through the gills of the rainbow trout. J. Exp.
Biol. 60:24-265.
50
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PUBLICATIONS
Olson, K.R., H.L. Bergman, and P.O. Fromm. 1973. Uptake of methyl mercuric
chloride and mercuric chloride by trout: A study of uptake pathways into
the whole animal and uptake by erythrocytes in vitro. J. Fish. Res. Bd.
Canada 30:1293-1299.
Olson, K.R. and P.O. Fromm. 1973. Mercury uptake and ion distribution in
gills of rainbow trout (Salmo gairdneri): Tissue scans with an electron
microprobe. J. Fish. Res. Bd. Canada 30:1575-1578.
Olson, K.R. and P.O. Fromm. 1973. A scanning electron microscopic study of
secondary lamellae and chloride cells of rainbow trout (Salmo gairdneri) Z.
Zellforsch. 143:439-449.
O'Connor, D.V. and P.O. Fromm. 1974. The effect of methyl mercury on gill
metabolism and blood parameters of rainbow trout. Bull. Env. Cont. Tox. 13:
406-411.
Bergman, H.L., K.R. Olson, and P.O. Fromm. 1974. The effects of vasoactive
agents on the functional surface area of isolated-perfused gills of rainbow
trout. J. Comp. Physiol. 94:267-286.
Fromm, P.O. 1974. Circulation in trout gills: presence of "blebs" in af-
ferent filamental vessels. J. Fish. Res. Bd. Canada 31:1793-1796.
Walker, R.L. and P.O. Fromm. 1976. Metabolism of iron by normal and iron
deficient rainbow trout. Comp. Biochem. Physiol. 55A:311-318.
Sorenson, P.R. and P.O. Fromm. 1977. Heat transfer characteristics of iso-
lated-perfused gills of rainbow trout. J. Comp. Physiol. 8112:345-357.
51
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INDEX
Acetylcholine, effect on
permeability, 28
perfusion pressure, 28
heat exchange, 37
Acknowledgments, ix
blood parameters, 13, 22
14C-Urea, 24 et seq.
electron microscopy, 14
electron microprobe, 10
EPA 2220-1 form, 53
Epinephrine, effect on
heat exchange, 35
permeability 27, 28, 30
experimental animals, 1, 10, 12
gills
circulation in, 44, 46
functional surface area, 25, 31
ion distribution in, 11
oxygen consumption, 12
gill cannulation, 26
gill perfusion, 25, 27
heat exchange in fish, 33
hematological parameters, 22
holding facilities, 1
Introduction, 1
iron
uptake by fish, 13 et seq.
tissue content, 18
deficiency, 17
metabolism of, 23
mercury
distribution in tissues, 6, 11
uptake into fish, 6
methyl form, 6
inorganic, 7
uptake by RBC's, 8
uptake by gills, 10
mercury, effect on
metabolism, 14
blood parameters, 13
gill ultrastructure, 15
Microfil, 44
nor-epinephrine, 28
Personnel, ix
phenoxybenzamine, 28
plasma
electrolytes, 13
clearance of ^Fe, 13
propranolol, 28
Publications, 51
References, 48, 49, 50
statistics, 27
tissue, fixation of, 15
whole head cannulation, 34
52
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPORT NO.
EPA-6QQ/3-77-Q57
3. RECIPIENT'S ACCESSION-NO.
4. TITLE AND SUBTITLE
TOXIC EFFECT OF WATER SOLUBLE POLLUTANTS ON
FRESHWATER FISH
5. REPORT DATE
May 1977 issuing date
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
Paul 0. Fromm
8. PERFORMING ORGANIZATION REPORT NO
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Department of Physiology
Michigan State University
East Lansing, Michigan 48824
10. PROGRAM ELEMENT NO.
11. CONTRACT/GRANT NO.
Grant No. R-801034
12. SPONSORING AGENCY NAME AND ADDRESS
Environmental Research Laboratory - Duluth, MN
Office of Research and Development
U.S. Environmental Protection Agency
Duluth. Minnesota 55804
13. TYPE OF REPORT AND PERIOD COVERED
Final 3-1-72 through 6-3Q-JZ5.
14. SPONSORING AGENCY CODE
EPA/600/03
15. SUPPLEMENTARY NOTES
16. ABSTRACT
Studies of the effect of inorganic and organic mercury on trout indicated that uptake
was primarily via the gills in non-feeding fish. Organic mercury entered fish at a
faster rate than inorganic mercury. Exposure of trout to 10 yg Hg/1 (methyl form)
haa no effect on the gill oxygen consumption measured in vitro or on the plasma
electrolytes. The hematocrit index increased significantly. Studies of the
metabolism of iron by normal and iron deficient trout (made deficient by bleeding)
indicated that the liver, spleen, and head kidney are the major iron storage organs.
Liver iron was reduced by bleeding whereas splenic iron was unaffected. In iron
deficient fish more radioiron appeared in erythrocytes than in normal controls.
Studies of isolated-perfused gills revealed the presence of both a and gadrenergic
receptors and the data obtained indicate the functional surface area of trout gills
can be regulated by changes in perfusion pathway through the gills. Use of perfused
gills appears to be a very sensitive model to detect deleterious action of pollutants
on fish. Evaluation of heat exchange in perfused gills indicates that the presence
of epinephrine increased the transfer maximum of the gill but they were unaffected by
the administration of acetylcholine. Analysis of a simple model indicated that the
gills may account for as much as 60% of the total heat exchanged by trout.
7.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.lDENTIFIERS/OPEN ENDED TERMS C. COSATI Field/Group
Trout
Mercury
Iron
Metabolism
Blood Analysis
Perfused fish gills
Iron metabolism
06A
060
06T
8. DISTRIBUTION STATEMENT
RELEASE TO PUBLIC
19. SECURITY CLASS (ThisReport)
UNCLASSIFIED
21. NO. OF PAGES
63
20. SECURITY CLASS (Thispagef
UNCLASSIFIED
22. PRICE
EPA Form 2220-1 (9-73)
53
ft U.S. GOVERNMENT PRINTING OFFICE: 1977-767-066/6474
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