UPTAKE, DISTRfiBUTlON AND

EXCRETION 05 ms fiAD-lONUCLED-E I”
BY RAENBOW mow:
(SALMO GARDNER!)

 

 

Thesis {or “19 Degree of pk. D.
MECHEGAN STATE UNIVERSITY
Joseph Bruce Hunn

1963

1

THESIS

This is to certify that the

thesis entitled

Uptake, Distribution and Excretion
of the Radionuclide 1131 by
Rainbow Trout (Salmo gairdneri)

presented by

Joseph Bruce Hunn

has been accepted towards fulfillment
of the requirements for

.__P_ll:2:_ degree in M08?

”fix/J @ (7%

Major professor

 

Date JuneJ27, 1963

0-169

 

 

LIBRARY

Michigan State

University

 

ABSTRACT

UPTAKE, DISTRIBUTION AND EXCRETION OF THE

RADIONUCLIDE 1131 BY RAINBOW

TROUT (SALMO GAIRDNERI)
by Joseph Bruce Hunn

Rainbow trout (Salmo gairdneri) were exposed to various

solutions (distilled water, tap water, 0.6% NaCl and 66%
. . . . 131

Ringer's) containing carrier free I for 24 hours. At the
end of the exposure period,the fish were anesthetized with
MS-222 and samples of blood, heart, gill, lower jaw (thyroid),
liver, spleen, caeca, head kidney, kidney,intestine, stomach,
muscle and eye were taken. The relative accumulation and

distribution of 1131 in these tissues was determined.

131 . . . . .

The uptake of I by the gills of trout is inhibited
by NaSCN, reduced by chloride, inversely related to the
calcium level of the medium and is not affected per se by
osmotic gradient or thiouracil.

131 . . . .

I in the blood of the trout is more than 90% ionic
and its biological half-life at 14°C is 1.7 days.

Iodine storage capacity of trout tissues was determined

using an equilibrium technique. Results indicate that muscle

may contain 30% of the total body iodine.

Joseph B. Hunn

Iodine is lost from the body of trout via the gills and
urine. Fecal loss was not measured. About four times as
131 . . . . .
much I is lost Via the urine as Via the gills. Measure-
l3l .
ments of the renal clearance of I and urine to plasma
. . . . 131 . .
concentration ratios indicate that I is filtered and

reabsorbed and that I- clearance is greater than Cl-

clearance.

UPTAKE, DISTRIBUTION AND EXCRETION OF THE
131
RADIONUCLIDE I BY RAINBOW

TROUT (SALMO GAIRDNERI)

BY

Joseph Bruce Hunn

A THESIS

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

DOCTOR OF PHILOSOPHY

Department of Physiology and Pharmacology

1963

ACKNOWLEDGEMENTS

The author wishes to express his gratitude to Dr. Paul
O. Fromm for his patience, understanding and insight in
helping bring this study to fruition. He also wishes to
thank Dr. E. P. Reineke for his many helpful suggestions.
Appreciation is also expressed to H. Kaczkofsky for his
technical assistance.

The author is indebted to the National Institutes of
Health for a predoctoral research fellowship (March, 1962
to March, 1963) and to the Department of Physiology and
Pharmacology for assistantships which made this study

possible.

ii

TABLE OF CONTENTS

INTRODUCTION . . . . . . . . . . . . . . . . . . .
HISTORICAL SURVEY . . . . . . . . . . . . . . . .
Iodine

Ion Regulation in Fresh-water Fish
Role of the Gill in Ion Regulation
Role of Endocrine Glands

Biology of Iodine in Fish

MATERIALS AND METHODS . . .

Experimental Animal

Uptake and Distribution Studies

Injection Studies 131

Effect of NaSCN and Thiouracil in I Uptake
Excretion Studies

Binding of 1131 in the Blood

RESULTS . . . . . . . . . . . . . . .

Uptake of 1131 from Media of Different
Osmolarities and Ionic Makeup

Distribution of 1131 in the Blood

Uptake of 1131 and the Effect of Known
Inhibitors of Iodine Uptake Mechanisms

Iodine Levels in the Tissues of Rainbow Trout
Having an Adequate Level of Iodine Available
to Them

Excretion of 1131 by Rainbow Trout

DISCUSSION 0 O O O O O O O O O O O O O O O O O O 0
SUMMARY AND CONCLUSIONS . . . . . . . . . . . . .

REFERENCES CITED . . . . . . . . . . . . . . . . .

iii

to

<30qu

21
22
23
25
25
29
31
32
37
37
41
50
58
74

76

APPENDICES

Tissue IM Values for 24 Hour Studies and
24-29 Day Study . . . . . . . . . . . . . . .
. 131 . .
Uptake of Injected Doses of I With Fish
Injected Under MS-222 Anesthesia . . . . .

Uptake of 1131 by Goldfish and Rainbow Trout .

Turnover of I.P. Injected Radioactive
Thyroxine in Rainbow Trout . . . . . . . . . .

Method for the Determination of Plasma

Protein-bound Iodine and Total Iodine Content
of the Lower Jaw (Thyroid) . . . . . . . . . .
Procedure for Paper Chromatography . . . . . .

Ringer's Solutions for Fish . . . . . . . . .

References Pertaining to Iodine in Fish . . .

iv

Page

83

89

91

95

101
105
107

109

Table

10.

11.

12.

13.

LIST OF TABLES

Page
. 131
Effect of Osmolarity on I Uptake by
Rainbow Trout . . . . . . . . . . . . . . . . 33
131 .
Uptake of I as Affected by the CalCium
and Chloride Level of the Surrounding Medium . 34
. . . 131 . .
Distribution of I in the Blood FolloWing
24 Hour Uptake . . . . . . . . . . . . . . . . 38
. . 131
Influence of NaSCN and ThiouraCil on I
Uptake by Rainbow Trout . . . . . . . . . . . 40
Influence of NaSCN on 24 Hour Uptake of an
Injected Dose of 1131 in Rainbow Trout . . . . 42
Comparison of the Iodine Content of Thyroid
and Muscle in Rainbow Trout . . . . . . . . . 43
Blood Iodine Values in "Normal" Trout vs.
Trout in Iodine Equilibrium . . . . . . . . . 44
Estimated Iodine Storage Capacity of Rainbow
Trout Tissues . . . . . . . . . . . . . . . . 45
Body Weight—Organ Weight Ratios in Rainbow
Trout ... . . . . . . . . . . . . . . . . . . 46
. ' i
Estimated Maximum Iodine Content of a
Theoretical 100 Gram Rainbow Trout . . . . . . 47
. 131 .
Whole Body Excretion of I FolloWing 24
Hour Exposure to 1131 . . . . . . . . . . . . 51
. . . 131 T
Blood Distribution of I and M'Values from
Trout at the End of Whole Body Excretion
Study . . . . . . . . . . . . . . . . . . . . 51
Turnover of 1131 in the Blood of 1131
Injected Rainbow Trout . . . . . . . . . . . . 52

Table

14.

15.

Page

Excretion of 1131 by Rainbow Trout Following

Exposure to 1131 in Distilled Water . . . . . 55
, 131 . .

Excretion of I by Rainbow Trout FolloWing

I.P. Injection of I 31 . . . . . . . . . . . . 56

APPENDIX TABLES

Page
131 .
Uptake of I from Tap Water by Rainbow
Trout . . . . . . . . . . . . . . . . . . . . 84
131 . .
Uptake of I from Distilled Water by
Rainbow Trout . . . . . . . . . . . . . . . . 85
131 .
Uptake of I from 0.6% NaCl by Rainbow
Trout . . . . . . . . . . . . . . . . . . . . 86
131 .
Uptake of I from 0.66% Ringer's by
Rainbow Trout . . . . . . . . . . . . . . . . 87
Iodine Equilibrium Study 25-29 Day fifValues . 88
131 . . .
Turnover of I in Rainbow Trout Follow1ng
an I.P. Injection Under MS-222 Anesthesia . . 90
131 . .
Uptake of I by Goldfish and Rainbow Trout . 94
Turnover of an I.P. Injected Dose of
Radioactive Thyroxine in Rainbow Trout . . . . 99

Distribution of Radioactivity in the Blood
Following an I.P. Injection of 1131 Thyroxine. 100

Whole Body Excretion Following Injection of
IlBl-Thyroxine . . . . . . . . . . . . . . . . 100

vi

LIST OF FIGURES

Figure Page
1. Urine Collection Apparatus . . . . . . . . . . 26

2. Flow-by Apparatus for Whole Body Excretion

Study . . . . . . . . . . . . . . . . . . . . 28
_ 131 . .
3. Half-life of Non-bound I in Rainbow Trout
Blood . . . . . . . . . . . . . . . 53
APPENDIX FIGURES
Figure Page
D-l. Turnover of Radioactivity Associated with
the Protein-bound Fraction of the Blood
and the RBC's of 1131-Thyroxine Injected
Rainbow Trout . . . . . . . . . . . . . . . . 97
131 .
D-2. Turnover of Non—bound I in the Blood of
113l—Thyroxine Injected Rainbow Trout . . . . 98

vii

INT RODUCT I ON

The nuclear age has brought many advances in science
and technology but has also introduced a number of perplex-
ing problems not the least of which is the problem of
radioactive waste disposal and its control. Atomic power
plants located adjacent to large bodies of potable fresh
water, as in Michigan, pose a problem as a potential source
of radionuclides which might contaminate this aquatic
habitat. Whether or not these radionuclides become an
actual danger to aquatic life depends upon the type and
quantity of radionuclide(s) released. Our knowledge of the
biological role of many radionuclides is rather meager.
Additional knowledge is, however, vital in determining the
quantities of isotope(s) that may be released into a body
of water without having potentially harmful effects on
aquatic life.

The present study was undertaken to broaden our knowl-
edge about the iodine metabolism in fishes and the possible
involvement of 1131 in the life of aquatic animals. Rain-
bow trout (Salmo gairdneri) were chosen as the experimental

animal because (1) of my interest in fish, (2) they could be

obtained gratis from the Michigan Department of Conservation,

(3) adequate holding facilities for cold water fish were
available and (4) these fish inhabit the Great Lakes and
many adjacent bodies of water.

The problem investigated covered three general areas:
(1) the direct uptake of iodine from the habitat by fish,
(2) the distribution and storage capacity for iodine in
fish tissues and (3) the rate of turnover of iodine in the

fish.

HISTORICAL SURVEY

Iodine

Iodine was discovered by Courtois in 1811 and proven
one of the four halogen elements by Gay-Lussac. Shortly
thereafter Prout in England introduced iodine as a treatment
for goitre.

Early reports of iodine being present in fish or fish
products were by Jonas (1838) in herring, Gmelin (1839) in
cod liver oil, and Girardin and Preisser (1842) in the liver
of the ray. In 1850 Raboudin published an analytic method
for the determination of iodine which was to become the
basis for the extensive work of Chatin (1850-1876). Chatin
improved the method and was able to detect as little as
0.1 gamma of iodine. This investigator was the first to
associate the occurrence of goitre and cretinism with
geographical areas having insufficient iodine in the water,
soil, air and foodstuffs; however, his idea was severely
criticized by his fellow scientists (Elmer, 1938).

In 1896 Baumann showed conclusively that iodine is found
in the thyroid and that it was strongly linked to protein,
yet, it was not until 1914 that Kendall extracted and cry-

stallized thyroxine. Kendall and Osterberg (1919) concluded

from analytical data thatthyroxine was a tryptophan deriva-
tive, a claim disproved by Harington and Barger (1927) who

showed by direct synthesis that thyroxine was a derivative

of tyrosine (Elmer, 1938).

Baumann's work stimulated research on the biochemistry
of iodine and the use of iodine in treatment of goitre lead
to an extensive search for dietary sources of this material.
Many reports contain information on the occurrence of iodine
in fish or fish products in addition to a wealth of infor-
mation on the iodine content of plants and other animals.

References to this work are given in appendix G.

Ion Regulation ig_Fresh Water Fish

 

In aquatic animals hyperosmotic regulation, that is,
maintaining the body fluids at a higher osmotic concentra-
tion than the surrounding environment, is characterized by
processes for the uptake of ions and for the expulsion of
water. The total energy required for the maintenance of a
hyperosmotic state in higher animals, is utilized almost
exclusively for the active transport of ions. The energy is
consumed either entirely at the external surface in direct
contact with the surrounding water or shared between this

surface and tubule cells of the excretory organ. Where the

osmotic work is shared, the proportioning of work between
the surface membranes and the excretory organ depends upon
both the relative permeability of the body surface to water
and salts, and on the efficiency of the excretory tubule
cells in removing ions from the urine (Shaw, 1960).

One of the first studies on the uptake of ions by fish
from the surrounding medium was done indirectly by Marine
and Lenhardt (1910 a, b) in their study of goitre prevention
in brook trout. They found that adding I- to the water
which flowed past the fish resulted in a reduction of the
goitrous condition of the fish. Later Krogh (1937b, 1938,
1939) was able to show uptake of C1_ and Br- but not of I-
by goldfish. He concluded that I- penetrates the gills of
the goldfish very slowly by diffusion. Further work on
anion uptake will be discussed later.

The absorption of an anion (C1- or Br_) by the gills of
fish need not be accompanied by any absorption of cation,
however, when a cation is absorbed simultaneously with the
anion, the rate is higher and the total quantity of anion
absorbed is definitely larger (Krogh, 1939). This uptake
of anion is against a rather large concentration gradient
especially in the case of the fresh water fish and the actual

mechanism for this uptake is not known.

Ion movement against a concentration gradient may be
due to active transport, an energy-requiring process, facili-
tated diffusion Which requires no direct expenditure of
energy or differential permeability. Solvent drag may also
play a role in ion movement.

The role of the gastro-intestinal tract in the uptake
of ions in fresh water fish is rather a question mark. Some
authors believe that aquatic animals get most of their salt
requirement from food (Wikgren, 1953). Apparently little
water is drunk by fresh water fish and it is assumed that
the uptake of ions from this source is minimal.

The urine of fresh water fish is hypoosmotic to the
blood but always contains some salts. Calculations of Krogh's
(1939) data give urinary chloride loss for rainbow trout
ranging from a minimum of 0.11 mEq/Kg/day to a maximum of
1.25 mEq/Kg/day. For the goldfish, urinary chloride loss
ranges from a minimum of 0.10 mEq/Kg/day to a maximum of
0.41 mEq/Kg/day. Wikgren (1953) gives as the urinary Cl-
loss 0.25, 0.12 and 0.10 mEq/Kg/day for the lamprey, salmon
and carp respectively. Fromm (1963) finds that the rainbow
trout has an average urinary chloride of 10.2 i SE .9 mEq/l
and a urine flow of 101 1 SE 8 ml/Kg/day, thus a urinary

chloride loss of about 1 mEq/Kg/day. The loss of chloride

via the integument is probably equal to or less than the
urinary loss. Krogh (1939) gives the rate of loss in trout
as 0.1 to 0.2 uM/cmz/hr and Wikgren (1953) reported the
level of chloride loss in the goldfish as 0.07 and 0.05
uM/cmz/hr as computed from Krogh's (1937b) and Smith's
(1929) data respectively.

Loss of ions across the gill is difficult to assess.
Krogh (1937), in studies of Cl- loss via the gills of gold—
fish in distilled water, found that "the loss of Cl- from
the head is very small because it is compensated by absorp-
tion." Because ion movement out of fish (outflux) and
uptake (influx) may be operating simultaneously, most studies
have measured the net movement of ions. With the develop-
ment of the double isotope technique, flux rates can now
be measured in both directions simultaneously.

In freshwater fishes fecal loss of absorbable salts is
probably minimal; however, no specific data appear to be

available on this subject.

Role 9f_the Gill in.Ion Regulation

 

Since the description of the so-called chloride cells
in eel gills by Keys and Willmer (1932), much circumstantial

evidence has been brought forth to support their role in

salt regulation. The exact significance of these cells in
fresh water fish is still in doubt. Krogh (1939) expressed
his thoughts on it by writing "the alternative that the
whole of the respiratory epithelium might be also secretory
appears to me more likely . . . ." Doyle and Gorecki (1961)
after studying gills by use of the electron microscope came
to the conclusion that the location in the epithelium and
the interspecific distribution of the "chloride cells" casts
doubt on their supposed function as the principal site of
chloride exchange. Fleming and Kamemoto (1963) report
histochemical and isotopic evidence which strongly indicates
the so-called chloride cells are not the site of Na exchange
in the gills of Fundulus.

The efficiency of the uptake mechanism of the gills can
be seen in fish that are starving. In this case the net
movement inward through the gill must equal integumentary
and urinary losses (rv70 mg/Kg/day in trout) if ionic balance
is to be maintained. If the C1- content of Michigan trout
waters as determined by Urshel and Hooper (1961) is typical
(1-30 mg/l) then the gill must be extremely efficient in
extracting the C1_. Such efficiency speaks strongly in
favor of many cells rather than a few specialized cells being
involved in maintaining the proper ion level of the blood

(4,000 - 5,000 mg c1"/1).

Role gf_Endocrine Glands

 

The material available on the role of the endocrines
in ionic regulation in fish is limited and confusing. The
role of the thyroid gland in osmoregulation has been reviewed
by Fontaine (1956) and Pickford and Atz (1957). Although
much of the work published in this area is contradictory
some general conclusions can be drawn. First, stimulation
of the thyroid in decreasing salinity and inhibition in the
reverse situation is a transitory response in marine fish.
Secondly, there is an increase in thyroid activity in fish
migrating from fresh water to salt water. This is paralleled
by the development of the so-called chloride cells in the
gills. The development of the chloride cells, the increase
in thyroid activity and other physiological changes represent
a phase in the maturing process which enables the fish to
continue its life cycle in a marine habitat. Thirdly, the
promotion of salinity tolerance by thyroxine may be related
to the release of growth hormone or the synergism of growth
hormone action by thyroxine. Gorbman and Bern (1962) in
summarizing the role of the thyroid in osmoregulation state
that if thyroid hormones are involved in osmoregulation they
may only modify the actions of the more potent adrenal

corticosteroids.

10

The work of Smith (1956) indicates that growth hormone
may play a major role in osmoregulation. Other indirect
evidence supports his contention. At sexual maturation the
salinity tolerance of trout decreases; a significant increase
in salinity tolerance has been shown to occur simultaneously
with periods of increased growth rate in the spring and fall.
And lastly, the increase in salinity tolerance can be evoked
with physiologic levels of growth hormone.

The evidence to support the idea that the corticosteroids
are involved in ionic regulation is meager. Sexton (1955)
working with goldfish found that DCA slightly inhibited the
uptake of radioactive sodium through the gills but didn't
effect sodium loss. Holmes (1959) likewise found that DCA
suppressed sodium uptake but he noted an increase in total
sodium output from saline-loaded trout after injection with
DCA or hydrocortisone. His inability to collect urine via
cannulation casts doubt on values presented for renal excre-
tion of sodium. Also, values for the extra renal excretion
of sodium using data from cannulated fish can not be con-
sidered as reflecting the "normal" excretory pattern.

Edelman §£_§1, (1960) were unable to show any change in the

response of euryhaline and stenohaline marine species to

immersion in freshwater following injection with

ll

hydrocortisone. The fact that glucocorticoids may affect
ion regulation in terms of suppressed sodium uptake is of
interest. There is good evidence that teleost interrenal
tissue increases its activity in response to the demands of
physical activity both under experimental conditions and
during migration. Here, the corticoid role may be that of
stimulating gluconeogenesis to satisfy metabolic needs
attendant on increased energy output. Any osmoregulatory
action is probably subsidiary to its metabolic role.

The influence of aldosterone on ion regulation has only
recently been investigated. Edelman _t__l, (1960) could
find no change in response following injection of aldosterone.
Bentley and Follet (1962) have demonstrated that aldosterone
reduces the net loss of Na in the lamprey. Chester Jones
_t_§l, (1962) indicate that aldosterone inhibits the uptake
of sodium in the eel adapted to fresh water but does not
affect the renal excretion of sodium. In some experiments
the gill inhibition was not recordable until 8 hours had
passed. This suggests the possibility that the steroid does
not act directly on the gill uptake mechanism. Failure of
the kidney to respond to aldosterone treatment by increasing

sodium reabsorption may be due to its lack of capacity to

respond. That is to say, probably the kidney was functioning

12

to its maximal capacity for sodium retention without, or
prior to, the aldosterone treatment. Holmes and Butler
(1963) show aldosterone can significantly reduce plasma
sodium in rainbow trout but the route of loss of this sodium
was not ascertained. Even though aldosterone has been
identified in the plasma of all groups of fishes examined
exclusive of the cyclostomes, no clear picture of its func-
tion in fish has been elucidated. Its presence in the blood
does suggest that it may serve some regulatory function
possibly, as in higher vertebrates, in the regulation of Na
and K at the kidney level.

Work on the neurohypophyseal hormones in fish has seen
two phases: (1) isolation and identification and (2)
experimentation on the possible role of these hormones in
ion regulation. The early work on the role of the neuro-
hypophyseal hormones is reviewed by Fontaine (1956), and
Pickford and Atz (1957). Work in the 1930's showed that
pituitary extracts and pituitrin caused neither a decrease
in urine volume nor an increase in body weight in fresh-
water fishes. Fontaine and co-workers were unable to show
any measurable weight gain in fish following injection of
pituitary extracts. Sexton (1955) found that pitressin

increased urine flow in the goldfish and significantly

13

reduced the loss of Na via the gills; however, there was

no change in the rate of Na uptake. Since 12 of 16 of his
experimental fish died within one day following pitressin
injections, the results on the urine flow probably indicate
a stress reaction. Maetz and Juien (1961) have shown that
neurohypophyseal extracts of Carassius as well as oxytocin
stimulate the influx of Na across the gills of Carassius
while the outflux of Na remains constant. Bentley and
Follett (1962) show that oxytocin, arginine vasotocin and
pitressin are all effective in promoting a net Na loss in
the lamprey. Whole mammalian neurohypophyseal extract also
increased net Na loss. Meier and Fleming (1962) have
recorded a number of effects of pitocin and pitressin on
water and Na movements in the killfish. Pitocin affected
water influx and outflux while comparable doses of pitressin
had no effect. Both pitocin and pitressin stimulated Na
uptake from tap water. The renal loss of Na was affected
by neither pitocin nor pitressin while the gut loss showed
a five-fold increase. By tying off anus, renal papilla or
both, the amount of Na loss via various routes was estimated;
gill 65%, kidney 25% and gut 10%. Also survival time of
fish transferred from tap water to high concentrations of

NaCl was increased by pitocin but not by pitressin.

14

The idea that the gut in fish may play a significant
role in ion regulation has been largely ignored. The indi-
cation of a five—fold increase in loss of Na by the gut under
the influence of posterior pituitary is of interest especi-
ally since it is known that these hormones exert an effect
on the contraction and tonus of the intestines of several
teleosts.

The study of neurosecretory cells in relation to ion
regulation has provided circumstantial evidence suggesting
hypothalamic control and/or involvement. Fontaine (1956)
states that the results obtained in fish are comparable to
those described for mammals and amphibians as a result of
experimental changes in their osmotic equilibrium. It
therefore seems logical to presume these neurosecretory
products are similar in function but the intermediary stages
through which they act in osmoregulation are still to be
identified. Carlson and Holmes (1962) have been able to
show an increase in pituitary and hypothalamic neurosecre-
tions in rainbow trout subjected to handling. Transfer of
the fresh water fish to 60% sea water was accompanied by a
decline in pituitary anti-diuretic material and an increase
in oxytocic activity. The disappearance or accumulation of

neurosecretory material has been used as circumstantial

15

evidence indicating an increase or decrease in the rate of
release of this material. These observations give little
indication as to the rate of synthesis or utilization of the
material by the body tissues. The measurements of this and
other parameters (blood level of hormones, urine flow, etc.)
would do much to integrate the picture of ion regulation in
fishes.

The caudal neurosecretory system of fish was first
described anatomically by Weber in 1827 as a warty appendix
at the end of the spinal cord of the carp. In the 1920's
Speidel confirmed the existence of Dahlgren cells in the
posterior part of the spinal cord of skates and other fishes.
From his cytological studies it became evident that a
morphologically discrete material was elaborated by these
cells. Work by Enami and coaworkers reviewed in 1959 sug-
gests that the urohypophysis (a shortened term for caudal
neurosecretory system) has much the same structure as the
posterior pituitary. The experimental analysis of the uro-
hypophysis was carried out by subjecting fish (loach
Misgurnus) to osmotic stress via salt loading and by surgical
removal of the gland followed by injection of various frac-
tions of the gland. After salt loading, the urohypophysis

exhibited increased secretory activity. Repeated saline

16

injections caused complete loss of secretory activity of

the cells. Following surgical removal of the urohypophysis,
fish were adapted to 50% sea water or fresh water. The fish
were then injected with urohypophyseal extracts. Those in
sea water decreased in total sodium content while those in
fresh water increased in sodium content. This suggests a
possible relation between the urohypophysis and certain
target organs responsible for sodium regulation. The exact
role of this gland is yet to be clearly experimentally

defined.

Biology Q§_Iodine in_§i§h_

Marine and Lenhardt's (1910 a,b) reduction of goitre
in brook trout by adding Lugol's Solution to the water was
the first study which indicated the ability of fish to take
iodine directly from water. Krogh (1938) states the fol—
lowing about experiments using goldfish: "A single experi-
ment with 0.1 NaI shows a slight absorption of 0.007 qu in
three hours, but in other experiments with NaI no change
whatever could be detected and the gills are no doubt prac-
tically impermeable to I-.” Earlier Keys (1937) had sug-

gested the same thing about eel gills. All recent i vivo

 

data on uptake come from studies in which the iodine was

17

injected (see Leloup and Fontaine, 1960). The only ig_yit£9_
data available is the study by Maqsood, Reineke and Fromm
(1961).

The routes and rate of excretion of iodine in fish have
been little studied. Bieter (1933) reports work by E. K.
Marshall, Jr., in which toadfish were injected with NaI and
loss via the gill and kidney measured. At the end of 23
hours the gill loss was 74 and 92% respectively for the two
fish used while the urinary loss was 0.67% and 1.1%. The
toadfish is a marine, aglomerular fish. Hickman (1959)
concludes from his 1131 injection studies of the starry
flounder that "it is evident that a very large proportion
of the excreted I131, perhaps as much as 80%, is removed
extrarenally in both salt and fresh-water flounder."

The distribution of iodine within the various tissues
of the body, with the exception of the thyroid and blood,
has received little attention. In the appendix are listed
a number of references on the iodine content of fish. Un—
fortunately few of these papers contain any breakdown as to
organ content. Robertson and Chaney (1953) have reviewed
the few data available and contributed data on the iodine
content of muscle, thyroid, eggs, testes, liver and other

organs in Michigan rainbow trout and California steelhead.

18

Two things stand out in this study; one, that there is a
large capacity for iodine in the tissue of rainbow trout and
two, that the development of the eggs in the female places

a great demand on the body's iodine supply. Leloup and
Fontaine (1960) have shown the concentration of iodine in
the ovaries of salmon and trout to be about 10 times that of
the plasma.

The first work on fish blood iodine is that of Leloup
(1949) in which he studied 11 species of marine fish. The
total iodine ranged from 12.2 to 91.5 09%, the Somogyi pre-
cipitable iodine 5.1 to 35.7 ug% and the butanol extractable
iodine (BEI) l to 19.2 ug%. Fontaine and Leloup (1950),
studying salmon and shad at the start of their reproductive
migration, found total serum iodine values of 77 - 231 ug%
in salmon and 345 - 574 09% in the shad. Robertson and
Chaney (1953) give average blood serum values for wild rain-
bow trout in Michigan ranging from 2.7 - 8.1 09% whereas
hatchery trout had a mean value of 15.5 ug%. They also
report an average blood serum iodide of 49 ug% for sea run
steelhead in California. Hickman (1962) has suggested that
PBI in fish is not a reliable index of circulating thyroid
hormone and that only BEI will measure this adequately. His

data show that in the starry flounder serum PBI and BEI

1!

19

values are not well correlated. Leloup and Fontaine (1960)
had previously shown that 1131 will bind to rainbow trout

and salmon plasma proteins ig_yitgg, Data on the inorganic
iodine in the blood of Whitefish in fresh water, Hickman
(1962), suggest the Whitefish has the ability to take up

and retain relatively large amounts of iodine. Hidkman
(personal communication) has stated that the Whitefish is
peculiar in this respect as the other species of fish in the
same body of water do not exhibit high serum inorganic iodine
levels.

The first measurement of the iodine content of fish
(elasmobranch) thyroids was recorded by Cameron (1913).
Burwash (1929) measured the iodine content in two species
of elasmobranchs and one teleost. The more recent work is
reviewed by Leloup and Fontaine (1960).

The advent of radioactive iodine in the late 1930's has
lead to a tremendous amount of work on the physiology of
fish thyroid. Labeled iodine has been used to help identify

T ).

the various compounds in the thyroid (MIT, DIT, T3, 4

Various studies on uptake of injected doses have been used
in an attempt to evaluate thyroid function. Estimations
of output rates of thyroid hormone have been made using

various methods involving labeled iodine (Hoffert and Fromm,

20

1959; Leloup and Fontaine, 1960). As yet no single method
has been put forth which will adequately measure thyroid
function in fish. This is partially due to the fact that
the exact parameter(s) affected by the thyroid in fish have

yet to be elucidated.

MATERIALS AND METHODS

Experimental Animal

Rainbow trout (Salmo qairdneri) used in this study were
obtained gratis from the Wolf Lake State Fish Hatchery
courtesy of the Michigan Department of Conservation. These
fish were transported via aerated tanks to East Lansing
where they were kept in a temperature controlled room. The
water used in the static tanks was aged, aerated tap water.
The water temperature in the room was 12 i 2°C. The water
in the tanks is changed every third day.

The fish were fed trout pellets (Formula 2-B Trout Food
made by Zeigler Brothers Feed Mills, Inc., Gardner, Penna.).
Trout newly arrived from the hatchery must be acclimated
about a week before they start feeding. The feeding regime
here differs from the hatchery in that no liver is fed and
feeding is every other day.

Trout used in the experiments were approximately of the
same body weight for any one experiment. Few of the animals
were sexually mature enough to determine the sex without the

aid of histologic sections.

21

22

Uptake and Distribution Studies
Fish were exposed to various solutions (distilled water,
. . . . 131
tap water, 0.6% NaCl and 66% Ringer's) containing I
for 24 hours. Two liters of media were placed in covered
plastic containers (4 3/4 x 6 3/4 x 10 3/4) with constant

. . 0 .
aeration. The room was maintained at 13 i l C With constant

illumination. The 1131 solution was added to the media

5 — 10 minutes before the fish were put in. The fish were
not acclimated to the various media before being placed into
them. Upon termination of the experiment, the fish were
anesthetized with MS-222 (Tricaine Methanesulphonate, Sandoz)
and samples of blood, heart, gill, lower jaw, liver, spleen,
caeca, head kidney, kidney, intestine, stomach, muscle and
eye were taken. Blood samples taken using the method of
Schiffman (1959) varied from 0.3 to 1.0 ml per fish. After
removal the tissues were rinsed three times in fish Ringer's,
blotted dry and weighed to the nearest milligram on a Roller-
Smith balance. The heart samples included the conus; liver
samples included the gallbladder and contents; the gill sam-
ple was taken from the upper half of the gills to exclude
thyroid tissue; the lower jaw is that piece of tissue from

the lst to the 4th gill arch cleaned of gill and muscle; the

stomach sample was taken from the upper stomach while the

23

intestine sample was the whole lower gut (rectum); the
muscle sample was taken on the right side of the fish im-
mediately under the adipose fin and the eye sample was the
entire right eye. Upon being weighed the samples were
placed in five dram plastic vials and wet-ashed with nitric
acid overnight. A ten ml media sample was also placed into
a similiar vial. »After dilution to the counting volume,
all samples were counted using a two-inch thallium treated
NaI well scintillation detector, Model PHA-ICA pulse height
analyzer, and Model DS-lA decade scaler used as a slave
scaler. Counting was done at the 5% level of error or less

(according to the method of Calvin, 1949).

Injection Studies

Three different experiments were carried out using
. . 131 ,
injected doses of I . A 24 hour uptake experiment was

. . . . . 131 131

made injecting unanesthetized trout With I and I plus
20 mg NaSCN. These trout (5 per group) were placed into 40
liter aquaria containing 36 liters of aged tap water after
injection. The room conditions were as previously described.
After 24 hours water, blood, gill and lower jaw samples were

taken and treated as in the distribution studies.

A second injection study was made as above except that

24

no NaSCN was injected and water from each aquarium was cir-
culated through a separate charcoal filter. A sample of
five fish was taken at 24, 72 and 120 hours after injection.
The blood samples were centrifuged at 2500 rpm for 20 min-
utes and the plasma drawn off. The plasma was then preci-
pitated using ZnSO4 and NaOH and washed twice with 10 ml of
glass distilled water. The precipitate and all washes were
counted as well as the aquaria water and lower jaw.

An iodine equilibrium study was made by placing 9 trout
in an 80 liter aquarium containing 10 ug of 1127 per liter
of aged tap water and injecting them daily with a constant
volume of a decaying I131 solution (start .1 ml = 0.50 uc
I131). The water was changed three times a week and the 1127
renewed each time. These fish were starved except for one
feeding on day 15. The fish were sacrificed as follows; 1
on day 25, 2 on day 26, 2 on day 27, 2 on day 28, and 2 on
day 29. The fish were anesthetized with MS—222 a blood
sample drawn and all other tissues taken and prepared as in
distribution studies except the lower jaw. Plasma protein
precipitates and the lower jaws were prepared for iodine
determination (for method see appendix E). After the iodine

determination, aliquots from the precipitates and lower jaws

were counted along with the other tissues.

25

Effect 9§_NaSCN and Thiouracil QQ_I131 Uptake

Fish were exposed to three levels of NaSCN 0.1%, 0.01%
and 0.001% made up in distilled water. The procedure was
the same as in the distribution studies except that only
water, blood, gill and lower jaw samples were taken. The
other groups were injected and placed into distilled water
containing 1131. One group of fish was injected with 10 mg
NaSCN per fish while the second group was injected with

10 mg thiouracil per fish. Sampling of tissue was as above.

Excretion Studies

. 1 . . .
Excretion of 113 was studied in two groups of fishes.

The first group was exposed to 1131 in distilled water for
24 hours. At the end of the exposure period the fish were
anesthetized with MS-222 and a cannula (Intramedic poly-
ethylene tubing PE 60) was inserted into the urinary bladder.
The cannula had a flared tip and could be inserted under a
slight pressure. Once the cannula was in place, a suture
was made through the body wall and the cannula anchored.

The cotton thread was then wrapped around the base of the
cannula a number of times and again tied. The fish was then

placed into the urine collecting apparatus (see fig. 1) head

first. The body was held in place by damp cotton. Distilled

26

Figure 1

Urine Collection Apparatus

 

 

 

of
Water

 

. .-%/-
Inflow 2; fl

 

Urine
Collection
in
Graduated
Cylinder

Ion Exchange Resin

 

Outflow
of
Water

27

water, pumped over the head of the fish, flowed out of the
side arm into two columns in series containing 10 gm of ion
exchange resin IRA 400. The cannula delivered the urine
into a 10 m1 graduated cylinder. At the end of the urine
collection period, the fish was again anesthetized with
MS-222, a blood sample drawn and the lower jaw taken out
and weighed. The blood was then centrifuged and the plasma
drawn off. The freezing point depression of the plasma was
determined with a Model C Fiske Osmometer using a small
sample probe. The plasma sample was then readied for
counting as well as the lower jaw and the exposure media.
The second group was injected ip with an 1131 solution and
placed into tap water for 24 hours. The remainder of the
procedure is as for the first group except that tap water
was used.

Whole body excretion was studied using the apparatus
pictured in fig. 2. Fish were exposed for 24 hours in
distilled water containing 1131 as previously described.
After 24 hours they were anesthetized with MS-222 and placed
into the tube heading into the flow. Distilled water was
then circulated past the fish at about 150 ml/min. The
water then flowed through two tubes in series containing

30 gm of IRA 400. The tubes were packed in such a manner

28

Figure 2

Flow-by Apparatus for Whole Body Excretion Study

 

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29

as to hold the ion exchange resin in place between two plugs
of glass wool. One tube was placed in a lead shield so that
the resin would be immediately under the scintillation
detector. The scintillatiOn detector was a one-inch thal-
lium treated NaI crystal. The scintillation tube was
powered by a Model 1620 count Rate Meter (Nuclear-Chicago)
and the activity was recorded via a Model AW Esterline-Angus
Graphic Ammeter. The activity was recorded over a 24 hour
period. Upon termination, the fish was anesthetized with
MS-222 and the blood and lower jaw sampled. Samples of the
exposure media, blood and lower jaw were then counted in

the well counting apparatus as previously described.

 

 

Binding Qf_1131 in.the Blood
Since the amount of binding of 1131 in the blood will
affect the distribution and the excretion of 1131,it was

thought necessary to determine the amount of free ionic 1131

in the blood. Fish were exposed as described in the uptake
and distribution section. Blood samples were drawn, cen-
trifuged and the RBC's and plasma separated. The plasma
was then precipitated with TCA or ZnSO4 and NaOH or was run

through a column containing two cc of IRA 400. The relative

activity of each fraction was then determined. Fish were

30

also injected with I131 and samples of the plasma chroma-
tographed on one-inch Whatman 3 MM paper in an ammonia-
collidine system (see appendix F). After drying, the strips
were placed on Kodak no-screen medical X-ray film and the
film exposed for 48 hours. At that time the film was devel-
oped. Upon drying the film was placed on an X—ray viewer
and the spots marked on the strips. The strips were then
cut up according to the activity pattern and placed into
plastic vials for counting. The relative activities of the

various portions of the strip were then determined.

RESULTS

Other than the work of Marine and Lenhardt (1910 a,b),
there are no data showing uptake of iodine from water by
fish. The only comprehensive data on the uptake of iodide
by fish tissues is the in vitro study of Maqsood, Reineke
and Fromm (1961). Therefore it was decided that the I131

uptake data be expressed in a form comparable to that of the

 

in vitro study. The 24 hour a values were calculated as
follows: cpm per unit wet weight of tissue divided by the
cpm per unit of medium. The blood fi'values were derived by
dividing the cpm per ml of blood by the cpm per m1 of ex-
posure media. All other fi'values were calculated using
blood as the medium.

The tissue/blood ratio of iodine has been used to
demonstrate the iodine concentrating ability of the thyroid
gland in many animals. The discrete nature of the thyroidal
tissue in most vertebrates makes exact weighing of the gland
possible. In the trout, as in most fishes, the thyroid
tissue is diffuse, with the greatest concentration of thy-
roidal tissue being in the lower jaw region. The use of the

lower jaw region in the determination of thyroidal iodine

uptake resulted in very low values due to the presence of

31

32

much extraneous tissue. Quiring (1950, 1962) has estimated
that the thyroidal tissue of rainbow trout ranges from 0.60
to 6.07 mg/100 gm in fish weighing from 2,750 grams to 42.8
grams. If this estimate had been used then the thyroidal
fi'values, assuming the portion of the lower jaw removed
contained all the thyroid tissue, would have been in the
thousands. In order to keep the values comparable to the
in.yitrg study, all thyroidal 2'values were calculated using

M

the weight of the trimmed lower jaws.
131 . .
Uptake Qf_I from Media 9§_Different
Osmolarities and Ionic Makeup
In general the water surrounding the fresh-water fish
is of low osmolarity compared to the body fluids of fish
and the diffusion gradient for salt is from the fish to the
surrounding medium. Therefore the water gradient is from
the surrounding medium to the fish and the salt gradient
from the fish to the medium.
. . . 131
The media, in which the trout were exposed to I ,
ranged from a distilled water hypoosmotic to the trout's
"normal" habitat (tap water) to 1.27% NaCl Which is hyper—
osmotic both to the normal habitat and the fish (see Table l).

The ionic make-up of the media was such that the con-

centration of all ions was less than the concentration in

33

TABLE 1

EFFECT OF OSMOLARITY ON I131

BY RAINBOW TROUT

 

24 hour g‘values

 

 

. Osmolarity .
Media milliosmoles Blood Therid
Medium Blood
/K9
i Q i SE i 1 SE
Distilled 6* 6.34 i 1.12 1.49 i .19
water
n = 10
Tap water 8** 1.59 i .28 2.03 i .43
n = 10
0.6% NaC1 192 1.40 i .31 2.75 i .41
n = 10
66%
Ringer's*** 212 0.50 i .06 1.30 i .06
n = 10
1.27%,NaC1 401 0.79 i .13 4.29 i .84

n = 8

 

*ll mOS/kg after 24 hours
**10 mOS/Kg after 24 hours

***Ringer'S after Schiffman (see appendix G)

Note: Osmolarity of rainbow trout plasma normally ranges
from 280 to 300 in m03.

34

TABLE 2

UPTAKE OF I131 AS AFFECTED BY THE CALCIUM AND

CHLORIDE LEVEL OF THE SURROUNDING MEDIUM

 

 

c1" Ca++
. Blood
Medium n content content EEEIEE'
mEq/1* mEq/1*
Distilled water 10 - - 6.34 i 1.12**
Tap water 10 <1 2.6 1.59 i .28
0.6%1NaC1 10 102 - 1.40 i .31
66% Ringer's 10 114 8.6 0.50 i .06
1.27%1NaC1 8 217 - 0.79 t .13

 

*Normal plasma calcium (total) in rainbow trout is 5.3
mEq/l; chloride 136 mEq/l.

**Standard error of the mean.

35

the body fluids of the trout with two exceptions: (l) in
1.27% NaCl the fish were in a "positive" sodium and Chloride
balance and (2) in Ringer's solution the calcium concen-
tration was greater than that of the blood.

The relative uptake from the various media and tissue
distribution of 1131 are presented in Tables A 1—4. A one-
way analysis of variance indicates that there is no statis—
tically significant difference between the fi-values for the
four treatments in the case of the spleen, caeca, kidney,
intestine and muscle. In fish exposed in distilled water,
the heart and head kidney 2"'values are significantly higher

M

than those of fish exposed in the other media, while the 3'

values for gill, lower jaw, liver, stomach and eye are
significantly higher in fish exposed in 0.6% NaCl.

I131

The relative uptake of by the trout is reflected

in the blood fi'values. It is apparent from the data pre-
sented in Table 1 that the osmotic gradient per se does not
greatly affect the uptake.

Data in Table 2 indicate that Ca++ is involved in the
uptake pattern. The highest uptake is noted in fish exposed
to 1131 in distilled water. The uptake of 1131 from tap

, , ++ -
water which contained 2.6 mEq Ca /1 and almost no C1 was

statistically significantly less. There is no difference

36

in the uptake of 1131 between trout in tap water and in

0.6% NaCl despite the fact there is a 100 fold increase in
the amount of a similar halide (C1-) in the medium. A
significant reduction from "normal" in uptake is noted in
trout exposed in Ringer's solution. It is unlikely that the
slight increase in either Cl- content or osmolarity in the
Ringer's solution could alone bring about such a change.
Davson and Daneilli (1952) have shown that the presence of
Ca++ and Ca++ plus K+ inhibit movements of certain ions
across certain biological membranes. Therefore the presence
of a high level of Ca++ and some K+ seems the more likely
reason for the reduced uptake in Ringer's solution. The
reason for the reduced uptake in 1.27% NaCl is unclear.
However two significant changes have occurred: (1) the
medium is hypertonic to the fish and (2) the chloride dif-
fusion gradient has been reversed.

In a normal Michigan trout habitat.the level of Ca++ in
the water may be a definite factor influencing the uptake
of available I- from the water by the fish. The low level

of C1_ (1 mEq/1 or less) probably rules out Cl— as a signif-

icant influence in I- uptake.

37

Movement of ions into and out of the blood is affected
by the amount of binding of these ions to plasma proteins
and/or red blood cells.

. . . . 131 . ,

The relative distribution of I in the blood follOWing
a 24 hour uptake period is listed in Table 3. Paper

. . 131
chromatography of in_v1tro I -tagged plasma and of plasma

from Il3I-injected fish also showed some 94-97% of the 1131

to be in the ionic form. Twelve hour dialysis of Il3l-tagged

plasma at 3°C showed 1-5% of the I131 to be non-dialyzable.
The plasma (0.5 - 1.0 ml) was dialyzed against a liter of
fish Ringer's (Stokes, 1963; see appendix G).
It appears that the role of binding of 1131 is minimal
131

in controlling the movement of I into or out of the

vascular compartment of trout.

Uptake Qf_Il31 and the Effect 9§_Known

Inhibitors gf_Iodine Uptake Mechanisms
, 131 . .

The high uptake of I from distilled water by trout

suggests that an active mechanism may be involved. To test
. . 131 .

this hypotheSis, the uptake of I was measured in the
presence of a known competitive inhibitor of the thyroidal
iodide trapping mechanism, NaSCN. Since it is possible that

uptake could involve organic binding, fish were also treated

38

TABLE 3
131
DISTRIBUTION OF I IN THE BLOOD
FOLLOWING 24 HOUR UPTAKE

Distribution of Activity as % of Total Blood Activity

 

Treatment IRA 400

Fish no. RBC PBI Inorganic
l 2.2 3.3 94.5
2 1.4 0.6 97.9
3 1.7 0.02 98.3

Treatment TCA Precipitation
1 3.2 1.6 95.1
2 . . 95.8
3 . 1.6 94.9
4 2. 97.1
5 . . 96.0

Treatment ZnSO4 - NaOH Precipitation
1 97.5
2 0.6 . 98.8
3 . . 98.8

 

39

with thiouracil to prevent organic binding of the iodide.

Trout were exposed to I131 in distilled water which
also contained three levels of NaSCN. From the results
(Table 4) it is evident that thiocyanate competitively in-
hibited the uptake of 1131 at the gill level.

Trout weighing from 64 to 124 grams were injected i.p.
with either 10 mg of NaSCN or thiouracil and placed into
distilled water containing 1131. Thiouracil had to be in-
jected as it will not stay in solution at pH's conducive to
fish life. At the same dose level, NaSCN is much more ef-
fective in reducing 1131 uptake in the gill and thyroidal
regions than is thiouracil. Since thiouracil did not
suppress uptakeuuzis possible that the dose was too small
or that organic binding of I' is not necessary for the
transfer of I' across the gill. The elevated lower jaw i-
value for these fish suggests that thiouracil had little or
no effect in 24 hours which is consistent with the supposi-
tion that the overall iodination process is slow in the
thyroids of fishes.

NaSCN is known to inhibit the uptake of I_ by the thyroid
but it is also thought to have the ability to discharge the

I" from the inorganic iodine pool of the_thyroid. The

mechanism for the latter action is not known. NaSCN is also

40

TABLE 4

131

INFLUENCE OF NaSCN AND THIOURACIL ON I
UPTAKE BY RAINBOW TROUT

Medium and

24 hourg‘fi values

 

 

 

n Blood Thyroid
treatment Medium Blood
9 SE 2 t SE

Nandi & Bern 2 or 4 1.537 t 0.82*
Nandi & Bern
0.2 mg/ml
NaSCN 2 or 4 0.673 t .033*
Nandi & Bern
.02 mg/ml
thiouracil 2 or 4 1.612 t .065*
0.I% NaSCN
in distilled water 7 0.13 i .03 0.13 i .08
0.01%1NaSCN
in distilled water 8 0.48 i .04 0.41 i .01
0.001% NaSCN
in distilled water 8 1.12 i .15 0.94 i .15
Distilled water
10mg NaSCN
injected per fish 6 1.30 i .62 0.46 i .02
Distilled water
10mg thiouracil
injected per fish 7 6.72 i 3.24 2.57 i .94

 

*;n_vitro study of Magsood, Reineke and

Fromm (1961)

41

known to increase the excretion of I“ from the body of mam-
mals. It was of interest to determine whether or not NaSCN
would affect the loss of 1131 from trout. In Table 5 are
recorded the effects of an injected dose of NaSCN on the
uptake by several tissues of an i.p. injected dose of 1131
(2.26 lie/fish) . The aquarium water (36 liters) of the con-
trol fish contained 21.9% of the total of the injected
doses while that of the NaSCN treated fish contained 69.7%
after 24 hours. This greater loss may be due to reduced
uptake as well as increased excretion. The blood § values
indicate that there was still a fairly large diffusion
gradient between the blood and the water (NaSCN fi'66.36 vs
control fi'l,027.97) at the end of 24 hours. The route of
loss was not determined but the lower gill values in the
NaSCN-treated fish may indicate an increased loss via this
route.
Iodine Levels in the Tissues of
Rainbow Trout Having an Adequate
Level of Iodide Available to Them

The amount of iodine in fish is dependent upon the
availability of iodine in the food and in the water surround-

ing the fish. The study of Robertson and Chaney (1953) in-

dicates a large difference existed between the iodine content

42

TABLE 5

INFLUENCE OF NaSCN ON 24 HOUR UPTAKE OF AN
INJECTED DOSE OF 1131

IN RAINBOW TROUT

% Injected Dose per Gram Tissue

 

CONTROL

Fish no. Bodygxeight Blood Gill Lower Jaw
1 156.7 2.74 0.56 7.82
2 110.7 2.60 0.67 11.42
3 89.1 5.10 0.85 16.25
4 159.1 2.36 0.50 8.48
5 132.7 2.70 0.54 12.02
2' 129.7 3.10 0.62 11.20
SE i ~51 i .06 i 1.50

20 mg NaSCN per Fish

1 98.1 0.72 0.26 0.37
2 163.3 1.14 0.47 0.50
3 149.6 0.68 0.31 0.31
4 172.6 0.46 0.22 0.34
5 154.4 0.19 0.06 0.05
2' 147.6 0.64 0.26 0.31
SE i 0.16 i 0.07 i 0.07

 

43

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44

TABLE 7
BLOOD IODINE VALUES IN "NORMAL" TROUT VS.
TROUT IN IODINE "EQUILIBRIUM"

 

 

 

 

"Norma1"* Equilibrium study
PBI Inorganic PBI*** Inorganic**
119% 99% 99% 99%
3.5 10 45.1 470
10.1 5 53.8 301
8.0 2 61.6 276
6.2 0 65.0 325
4.7 0 72.2 216
7.9 3 70.6 451
6.7 0 67.8 233
5.2 0 37.5 335
5.7 0 47.0 184
3.5 0

 

*Determinations by Albert L. Chaney Chemical Laboratory,
Glendale,

California.

**Calcu1ated values.

***Determined by alkaline fusion method

(see Appendix E).

45

TABLE 8

ESTIMATED IODINE STORAGE CAPACITY OF
RAINBOW TROUT TISSUES

 

 

Tissue ug/lOO gm
2 t SE

plasma, Inorganic 392 i 50
Plasma, P.B. 58 i 4
Heart 63 i 9
Gill 57 i 5
Lower Jaw 4,641 i552
Liver 118 t 19
Spleen 52 i 10
Caeca 48 i 5
Head Kidney 108 i 14
Kidney 117 i 19
Lower Gut 61 i 8
Stomach 52 i 6
Muscle 15 t 2

H-
N

Eye 43

 

46

TABLE 9

BODY WEIGHT-ORGAN WEIGHT RATIOS
IN RAINBOW TROUT

Orqan Weight

 

 

Body Weight X 100
2 i SE
Body Weight 169.2 i 14.1
Heart 0.14 t .01
Gill 1.59 i .05
Lower Jaw 0.27 t .02
Liver 0.95 i .04
Gall Bladder 0.03 i .01
Spleen 0.14 i .01
Caeca 1.34 t .07
Head Kidney 0.14 i .01
Kidney 0.64 t .04
Stomach 1.62 i .09
Mid-gut 0.47 i .03
Lower-gut 0.52 i .03
Eye 0.54 i .02
Lens 0.039 t .001

 

47

TABLE 10

ESTIMATED MAXIMUM IODINE CONTENT OF A THEORETICAL
100 GRAM RAINBOW TROUT

 

 

Tissue 38:33: aIOO ng 09 I tofaIfI
Plasma Iodide 1.80 392 7.06 19.91
PBI 0.04 58 0.02 0.06
Heart 0.14 63 0.09 0.25
Gill 1.59 57 0.91 2.57
Lower Jaw 0.27 4,641 12.53 35.34
Liver 0.95 118 1.12 3.16
Spleen 0.14 52 0.07 0.20
Caeca 1.34 48 0.64 1.80
Head kidney 0.14 108 0.15 0.42
Kidney 0.64 117 0.75 2.12
Lower Gut 0.52 61 0.32 0.90
Stomach 1.62 52 0.84 2.37
Muscle 70.00* 15 10.50 29.61
Eyes 1.08 43 0.46 1.30

 

*From Robertson and Chaney (1953).

48

of sea-run rainbow trout and Michigan wild trout. They
ascribed this to the difference in availability of iodine
in the environment. It was of interest to find out whether
or not rainbow trout given sufficient iodide in the water,
could take up and maintain levels equal to those of trout
in an iodine-rich environment. The role of diet was ruled

out by fasting the fish.

127 used in the water was 10 09/1 which is

The level of I
about 3-100 times the reported levels of iodine in Michigan
waters (Eldridge, 1924) and about one-fifth the iodine level
of sea water.

The radioactivity of the tissues listed in Table A-1 was
determined as well as the iodine content of the lower jaws
and plasma protein precipitates. Using these data the radio-
activity (3:) of the tissues was determined. The iodine
measurement of the PBI and lower jaw allowed the calculation
of the specific activity of iodine (uc/ug) in these tissues.
Assuming the trout were in iodine equilibrium, the iodine
content of the remaining tissues was calculated using radio-
activity of the tissue in the terms of uc/gm divided by the
sp. act. of the PBI (uc/ug I). The specific activity (uc/

ug I) of the PBI of each fish was used for the tissues of

the fish. The blood PBI was used instead of thyroidal iodine

49

because it was thought that this iodine would exchange much
more readily with blood inorganic iodide and more closely
resemble the other body tissues in this respect. As stated
earlier Hickman and other workers have shown that PBI is made
up of protein-bound thyroxine as well as a much larger amount
of non—thyroxine iodine. The actual amount of thyroxine
(BEI) could not be determined because the blood samples were
too small to do both PBI and BEI determinations. If one
assumes that the sea-run trout sampled by Robertson and
Chaney (1953) were in iodine equilibrium, the iodine values
from those fish should coincide with the values obtained in
the present study. Table 6 shows that the thyroidal iodine
values do indeed coincide.

Table 8 contains the estimated content of iodine in the
tissues of the rainbow trout studied. Note that the calcu-
lated iodine content of the muscle is the same as that com-
puted from RObertson and Chaney's (1953) data (see Table 6).
Using the data from Tables 8 and 9, the maximum iodine content
of the tissues of theoretical 100 gram trout have been calcu-
lated (see Table 10).

Assuming the trout in this study were in iodine equilib-
rium, it is of interest to note that the tissue E'values,

Table A-5, are about the same as the 24 hour fi'values for

50

trout in tap water with the exception of the lower jaw.
This suggests that the values for relative distribution at
the end of 24 hours is indicative of the iodine distribu-

tion pattern at equilibrium.

. 131 .
Excretion 9f_I by_Rainbow Trout

 

Net uptake of iodide results when the rate of uptake
exceeds the rate of loss. Almost nothing is known of the
routes of iodide loss or their relative importance in fishes.
Marshall's work suggests that the gill is the most important
route of iodine loss in an aglomerular fish. No data are
available on glomerular fish such as the trout.

The rate of loss of iodide from the whole fish was studied
using the apparatus shown in fig. 2. Trout used in this
study were exposed to I131 in distilled water for uptake.
Distilled water was then used as the circulating medium
because (1) its composition could be controlled, (2) it would
provide a maximum diffusion gradient for the iodide and (3)
by probably creating a diuresis would give the maximum urinary
rate of loss. This measured rate of loss then represents the
maximum rate at which trout would lose iodide. The data from

this experiment are listed in Table 11. Fish No. 1 was over

anesthetized with MS-222 and used as a control measuring loss

WHOLE BODY EXCRETION OF I

51

TABLE 1 1

FOLLOWING
24 HOUR EXPOSURE TO 1131

 

T l‘(hours)

 

 

Fish no. a* bx rxy 2
1** 3.49412 -.00833 .99 37.5
2 4.29648 -.01286 .99 24.06
3 4.69671 -.00733 .99 41.25
4 3.98372 -.01832 .99 16.0
5 3.90703 -.01797 .99 18.3

*Log cpm = a + bx
**Over anesthetized
TABLE 1 2
131

BLOOD DISTRIBUTION OF I

T
AND M VALUES FROM TROUT

AT THE END OF WHOLE BODY EXCRETION STUDY

 

 

 

 

 

 

Fish % total 1131 fitvalues
no. RBC PBI Ionic Blood* Lower Jaw
Medium Blood
1 _ _ _ _ _
2 5.31 0.38 94.31 20.36 2.57
3 1.21 1.50 97.19 37.24 2.82
4 0.70 2.13 97.17 12.58 5.69
5 1.03 1.23 97.74 13.15 5.63
*Exposure medium contained from 1.6 - 2.1 uc of I131 per

two liters of medium.

TURNOVER OF I

52

TABLE 13

131 131

IN THE BLOOD OF I

INJECTED RAINBOW TROUT

 

 

 

24 hour 72 hour 120 hour
56* 2* 55*
% dose per gram plasma 4.53 2.35 0.91
% dose PBI per gram
plasma precipitated .037 .016 .026
% dose as non-bound
per gram plasma 4.49 2.33 0.88
131 .
% I protein bound 0.83 0.70 2.90
% dose per gram
lower jaw 7.87 18.94 31.79
131

N°nigiund plasma I 4,681.40 2,761.80 837.76

I in water

T

Lower Jaw —' 1.82 7.89 36.36

M

 

53

Figure 3

Half-life of Non-bound 1131 in Rainbow Trout Blood

% Dose Non-bound 1131 per Gram of Plasma

 

 

log % dose = 0.8286 + (-.1773)x

 

l _l g
2 3 4

Days After 1131 Injection

 

54

by diffusion only. Data Show that three out of four "normal"
fish lost iodine at a rate greater than that by diffusion
loss alone (fish no. 1). The blood fi'values (Table 12) sug-
gest, however, that the fish has a rather efficient mechanism
for retaining iodide even under conditions calculated to
bring about the maximum rate of loss.

Since the blood fi'values remained high following maxi-
mum whole body excretion, it was decided that a study be

made of the biological half-life of 1131 in the blood.

Fifteen trout were injected with 3.87 uc 1131 each and placed
into three aquaria (5 in each). Samples of five fish were
taken at 24, 72 and 120 hours. Data from this experiment
are presented in Table 13 and fig. 3.

The apparatus shown in fig. 1 was constructed so that
the ions added to water passing over the gill area would not
be available for reabsorption and the iodide loss via gill
area could be separated from that lost via the kidney. The
gut loss was not measured. Data on fish exposed to distilled
water and tap water are presented in Tables 14 and 15. The
urine flow of the trout (m1/Kg/24 hr.) in both studies is
high compared to the normal values for rainbow trout given

by Krogh (1939) 60-106 ml/kg/day, Holmes (1961) 75-90 ml/Kg/

day and Fromm (1963) 101 i SE 8 ml/Kg/day. This could be

55

TABLE 14

EXCRETION OF 1131 BY RAINBOW TROUT FOLLOWING

EXPOSURE TO 1131 IN DISTILLED WATER*

 

Fish 1 2 3 4 5

 

Urine

Urine Volumes

Collection period hr 2.0 2.0 1.25 1.5 2.0
Urine volume ml 1.4 1.9 1.3 1.5 1.9
ml/Kg/hr 5.4 9.3 6.7 8.8 7.5
ml/Kg/24 hr 129.6 232.2 160.8 211.2 190
Clearance
Osmotic
Urine mOS/Kg 61 40 34 29 48
Plasma mOs/Kg - 296 279 235 292
Clearance ml/hr - .13 .12 .12 .16
I131
Urine/plasma ratio .30 .07 .27 .34 .10
Clearance ml/hr .22 .06 .17 .25 .10
Urine/gill ratio 3.92 2.74 6.29 3.05 3.61

 

*Distilled water was also the circulating medium used during
the excretion study.

56

TABLE 15

EXCRETION OF I131 BY RAINBOW TROUT FOLLOWING

I.P. INJECTION OF 1131*

 

 

 

Fish 1 2 3 4
HEADS.
Collection period 2 hr 2 hr 2 hr 2 hr
Urine volume ml 1.5 1.3 1.2 1.2
ml/Kg/hr 7.4 6.3 6.4 8.0
m1/Kg/24 hr 176.4 151.2 153.6 192.0
Clearance
Osmotic
Urine mOS/Kg 51 38 81 36
Plasma mOs/Kg 302 282 288 299
Clearance ml/hr .13 .09 .17 .07
I131
Urine/plasma ratio .08 .09 .10 .07
Clearance ml/hr .04 .06 .06 .04
Urine/gill ratio 4.0 4.5 - 2.66

 

*After injection the trout were placed into four liters of
tap water for a 24 hour uptake. The medium circulated
over the gill area during the excretion study was also
aged tap water.

57

due to the anesthetic, a "handling diuresis," or in the case
of the fish exposed to circulating distilled water, an in—
creased uptake of water. Fish tested in distilled water show
a higher g'ratio and clearance than do those fish tested in
tap water. This reduced kidney reabsorption of iodide may
be due to the diuretic state of the animals.

The urine to gill ratios indicate that the urinary loss
is about four times that of the gill diffusion loss. Under
normal conditions where the net movement of iodide may be

inward across the gills, the urine would be the major route

of excretion.

DISCUSSION

Fresh—water fish may Obtain salt from food consumed or by
direct uptake via the gills. In starving fish ionic balance
or stability is maintained by active uptake of ions by the
gills. If most salt necessary for the maintenance of
homeostasis is supplied via the gut, it is most probable
that the fine control of ionic homeostatic levels is carried
out by the gill uptake mechanism. Regulation of the gill
mechanism may be assumed to be under the control of hormonal
substances present in the blood. The stimuli responsible
for altering the circulating levels of these postulated
hormonal substances are unknown. It would appear likely,
however, that these stimuli arise from changes in the rela-
tive ionic composition or osmotic concentration of the blood
or environmental water.

The very nature of the uptake mechanism may impose a
differential rate on the uptake of various ions available in
the habitat, and needless to say, the ionic composition of
the habitat water will affect their availability to the fish.
The ionic composition may influence ion uptake by affecting
the gill epithelium directly. The effect of Ca++ on 1131

. . . ++ .
uptake by trout is one example. This effect of Ca in the

58

59

medium appears to be similar to that observed by Phillips
_£_§1, (1955, 1958) who reported that the uptake of certain
ions was related inversely to the level of Ca++ in the
medium. Calcium in the habitat may be used by fish as a
source for body Ca++ and it can alter the permeability of
cell membranes. Increased permeability due to low Ca++ may
lead to an increased water influx across the gill which
would in turn increase the Urine volume and loss of ions.
The decrease in ion levels plus the lowered osmotic concen-
tration could trigger a mechanism which increases the rate
of ion uptake by the gill.

A high level of Ca++ at the gill surface by decreasing
permeability probably inhibits movement of ions across the
epithelium, a phenomenon observed repeatedly with other
biological membranes. By reversing the Ca++ gradient (out-
ward to inward) the rate of movement of Ca++ into the trout
may increase thereby eliminating certain routes of entry
for other ions through the gill epithelium.

Anion uptake has been extensively studied using Cl-.
Krogh (1937b) was able to show that a number of fresh-water
fish could take C1- or Br- directly from the surrounding
medium if they had previously been "washed out" by exposure

to distilled water. He was unable to show the uptake of I-

60

or NO3-. Meyer (1948) using goldfish partially covered with
a rubber bag found that fish initially lost Cl- via the
gills after transfer from holding tanks to battery jars.
Following this initial "shock" phase, they took up C1_. By
injecting distilled water, he was able to Show that dilution
of body fluids retarded Cl- excretion while the uptake re-
mained the same. Salt loading reduced uptake while the
excretion remained unaffected. Wikgren (1953) confirmed the
observations of Meyer (1948) that fish need not be "washed
out" in distilled water before active uptake is initiated.
He did not, however, find the so-called "shock" effect ob-
served by Meyer (1948). The author also noted that when
Petromyzon were placed into distilled water, the C1- level
increased rapidly but then dropped back to a low level which
was maintained throughout the experiment; however, the con-
ductivity of the medium continued to increase. This indi-
cates a fine degree of control over the Cl- handling mechan—
isms in the face of a large diffusion gradient. Jorgensen
and Rosenkilde (1956) have shown that Cl_ can be taken up
directly from tap water by goldfish. In these fish the
excretion or retention of 150 to 238 qu Cl- was followed

by retention or excretion of one ml of water. They suggest

that the osmoregulatory mechanisms in the goldfish are

61

probably sensitive to variations as small as i 3% in the
osmotic pressure of the body fluids when such change occurs
in 24 hours or less.

There are no data which suggest a possible competitive
effect between the various halides in relation to the ion
uptake mechanism of the gill. Krogh (1937b) stated that
the fish gill cannot distinguish Br- from C1-. Data pre-
sented were on the uptake of 1131 by trout from solutions
containing various levels of Cl- Show no competitive effect
of Cl- on I- uptake. A hundred fold increase in C1. (tap
water to 0.6% NaCl) did not significantly change the uptake

I131

of . Doubling the C1- content 0.6% NaCl to 1.27% NaCl)

reduced the 1131 uptake but it should be noted that the Cl-
diffusion gradient reversed with the two solutions. Whether
the reduction in I- uptake was purely a C1- effect is doubtful.
The effect of metabolic inhibitors on gill uptake has
received little attention. Meyer (1952) has shown that a
mercuric ion at a concentration of 10-5 M completely inhibits
Na+ uptake and increases Na+ gill loss 10 fold in goldfish.
Sexton and Russell (1955) suggested this inhibition is due
to a reduction in gill succinic dehydrogenase activity.

Data reported herein clearly demonstrate that NaSCN can

inhibit iodide uptake by the gills of rainbow trout. A

62

graded response, in terms of blood fi'values, can be seen in
relation to the three levels of NaSCN tested. It should be
noted that the thyroidal fi'values also showed possible ef-
fects of NaSCN indicating it passed through the gill into
the blood stream. Injected doses of NaSCN were also effec—
tive in reducing gill uptake.

The injection of thiouracil, a compound known to block
the iodination of tyrosine showed that organic binding is
probably not necessary for the movement of iodide through
the gill. Carrier mechanisms have been postulated for cations
but few have been mentioned in relation to anions.

The role of the diet in supplying iodide and other salts
to fresh-water fish has not been evaluated. There are
virtually no data in the literature on the iodine content
of freshawater plants or animals. Just what role diet plays
in supplying iodide to fresh-water fish remains to be
elucidated.

Blood iodine levels are governed by the amount of iodine
taken into the body, the exchange of iodine between the
blood and the tissues and the amount excreted. The blood
iodine picture in fishes is quite different from that of

mammals. In mammals most iodine in the blood is protein-

bound and in the form of iodothyronines with little inorganic

63

iodine. In fishes the converse is true especially if there
is adequate iodine in the environment. Leloup (1949), in
studying the blood iodine of marine fish, found that the BEI
ranged from 2-56% of the total iodine and PBI 7-89% of the total.
The BEI comprised 13-74% of the PBI iodine. Similar blood
iodine values have been reported for the salmon and shad
(Fontaine and Leloup, 1950). Robertson and Chaney (1953)
found that about 50% of the total blood iodine was PBI in
California steelhead trout. Hickman (1962) reported that

in the starry flounder the PBI and inorganic iodide conform
to seasonal variations in the environmental level of iodine.
The BEI remained constant except for the month of July and
represented only a small portion of the total blood iodine.
He has also Shown a direct correlation between the inor-
ganic iodide levels and the PBI levels whereas the level of
BEI remained constant and hence exhibits no correlation with
PBI or inorganic iodide levels.

The present study supports the fact that most of the
iodine in the blood of fish is in the inorganic form. From
94-99% of the 1131 in the blood of the trout following a
24 uptake period (Table 3) is in the non-bound form. Data
presented in Table 13 Show a similar distribution pattern

131

in the blood of i.p. injected I . Tong t al, (1961) have

64

shown that 88.4% of plasma iodine is iodide in the hagfish
four days after injection of 400 uc of 1131.

The amount of blood iodide found in or attached to the
red blood cells of fish varies from species to species.
Leloup and Fontaine (1960) have expressed the distribution
of iodide between the plasma and the RBC's as H/P ratios
(the ratio of 1131 per gram of RBC's to the 1131 per gram
of plasma). Data on mammals and birds show a range of H/P
ratios of 0.40 to 0.60. The H/P ratios for teleosts may
be similar (eel, carp, conger, sea perch) or lower (Sal-
monidae, shad, mullet). The minimal value found by Leloup
and Fontaine (1960) was 0.098 in salmon. The present study
(using data from the uptake studies) indicates that the
average H/P ratios for rainbow trout is 0.05 (range 0.01 —
0.16). This is about 1/4 the value reported for rainbow
trout by Leloup and Fontaine (1960). Their values are from
an ig_yit£9_study.

The amount of non—hormonal iodine bound to plasma pro-
teins in fish blood may exceed 50% of the PBI. Fontaine and
Leloup (1960) have shown that 1131 will combine with salmon
and trout plasma proteins in_yitr9, The present study sug-

gests that ig_vitro binding is similar to that which occurs

in_vivo during 24 hours following injection.

 

65

Data from the uptake studies (Table 3) also suggests
that some binding takes place as it is improbable that any
radioactive T4 is released into the circulation in so short

t al, (1959) reported in studies on intra-

a time. Berg
thyroidal iodine (1131) metabolism in fish that labeled
thyroxine is often slow to appear and is present in only
trace amounts or only in certain seasons. Leloup and Fon-
taine (1960) report that chromatographic studies on thyroid
hyrolyzates show that inorganic iodine often exists in
significant amounts and may persist for a long time. Tong
_t_§l, (1961) report that labeled thyroxine was first
detectable in hagfish plasma 6 days after I131 injection.
Fontaine and Fontaine (1962) report that in non-migratory
rainbow trout the pituitary thyrotropic stock is nil or very
low.

The distribution of iodine (1131) in the tissues of the
trout as related to the blood (E) is similar to that of
mammals (Wallace and Brodie, 1937) except for muscle. In
the data presented for rainbow trout the thyroid is the only
tissue which concentrates iodine over the blood level.‘ An
iodide pump is known to exist in the stomach of tadpoles
(Lipner and Hazen, 1962) and in higher vertebrates (Brown-

Grant, 1961). No data were obtained on the stomach contents

66

of trout to confirm the presence Of an iodide pump. As
previously mentioned, the ovary is also capable of concen-
trating iodine.

Data on the iodine content of various fish are few.
Information as to the environmental level of iodine as well
as other pertinent data are often not included in the reports
of these values. Robertson and Chaney (1953) reviewed the
literature and have contributed data on the iodine content
of the blood, muscle, thyroid, eggs, testes and liver of wild
Michigan rainbow trout and California steelhead trout. Leloup
and Fontaine (1960) have published values for plasma iodine
for cylostomes, selachians and teleosts. Hickman (1962) gives
values for thyroidal and serum iodine in the starry flounder,
rainbow trout and Whitefish. In the present study the levels
of plasma iodine in hatchery trout maintained as previously
described were measured. Lower jaw and plasma PBI iodine
levels were also determined in hatchery trout exposed to
water containing 10 ug per liter of 1127. These data and
those of Robertson and Chaney (1953) indicate that there is
a large storage capacity for iodine. The tissues of hatchery
trout exposed to 10 ug I-/1 contained some 16 times more
muscle iodide and 30 times more thyroidal iodine than the

same tissues from wild Michigan trout.

67

The Observation (Robertson and Chaney, 1953) that the
ovary sequesters large amounts of iodine into the developing
eggs should be of interest to trout culturists in a low
environmental iodine area such as the Great Lakes. Addi-
tion of iodine to the diet during the time when the fish
are sexually ripening would probably give rise to a healthier
brood stock because the iodine demand of the thyroid and
ovary could be adequately met. Survival of the developing
embryos and fry might also be enhanced by having adequate
iodine stores.

Contamination of waters in the Great Lakes region with
sufficient radioactive iodine could have detrimental effects
both on aquatic animals and man. Since fish can take up
iodine from the water and concentrate it in the developing
eggs, sufficiently high levels of radioactive iodine could
bring about genetic damage in addition to possible damage
to the developing embryOnic tissue, especially thyroid.

The human population could be affected by drinking con-
taminated water or consuming radioactivity contained in sport
and/or commercial fish. As stated previously trout muscle
has a large capacity for storage of iodine and may contain
as much as 30% of the total body iodine.

Iodide may be lost from the body of a trout: (1) via

68

diffusion across the gills, (2) diffusion from the general
body surface, (3) the feces, (4) urine and (5) laying of
eggs by the female.

Whole body loss was studied using a flow-by system to
prevent reabsorption of the iodide from the surrounding
medium. The T i'values for whole body loss indicate that
the amount of 1131 being lost per hour is reduced by one—
half in an average of 24.9 hours.

The whole body loss in terms of percent dose of I131
lost into the medium surrounding the fish following an i.p.
injection has been studied by a few workers. Leloup (1952)
studied two species of marine teleosts and found that Muge
lost 24.7% in 24 hours and 45.3% in 72 hours; Congre lost
7% in 24 hours. Chavin (1956) reported that the fresh—water
goldfish lose 65% of an injected dose of 1131 into the
aquarium (tap water) in 24 hours. Leloup and Fontaine
(1960) have reviewed the literature and compiled a table of
data on the rate of excretion of 1131 by fresh-water teleosts.
Their data indicate that rainbow trout kept at 20°C lose
33.5% of an injected dose of 1131 in 24 hours. In the
course of the present study one experiment in which five

131

unanesthetized trout were injected with I (see Table 5)

showed that the aquarium water contained 21.9% of the total

69

injected I131 24 hours after injection. The second experi-
ment involved a series of 10 trout which were anesthetized
with MS-222, injected with I131 (average 1.31 uc per fish,
see appendix B) and placed into plastic containers holding
four liters of tap water at 14°C. After 24 hours, the tap
water contained an average of 41.0% of the injected dose
(range 25.5 - 62.4%). The action of MS-222 on ion regula-
tion in fish is not known. A recent note by Collins and
Hulsey (1963) indicates that 0.5 percent NaCl solution with
a concentration of 1 gram of MS-222 to two gallons of solu-
tion provides a greater survival rate for transport of
threadfin shad than a solution of MS-222 without the salt.
It is of interest to note that compounds known to
inhibit the normal iodine metabolism of the thyroid also
effect the loss of I131 from the bodies of fishes. Leloup
(1952) has shown that marine teleost Muge kept in sea water
containing thiourea (l g/liter) from 8—21 days lost 55.8 -
58.7% of injected doses of 1131 in 24 hours whereas untreated
controls lost 24.7% in 24 hours. The Congre injected daily
with thiouracil (30 mg/kilogram) for 12 days lost 61.4%
of injected 1131 in 24 hours versus 7% in the control. In

the present study five rainbow trout which were injected

with 20 mg NaSCN/fish concomitant with an injected dose of

70

I131 lost 69.7% of the total dose in 24 hours (control =

21.9%). With the blocking of the thyroidal uptake, it might
be expected that the blood level of 1131 would rise but
suprisingly it did not. Leloup (1952) found that in Muge
treated with thiourea the blood level of 1131 (% dose per
gram of blood) actually was lower than that of the control:
8 hours post-injection control 3.21%; thiourea 1.68%; 72 hours
post-injection control 2.38%; thiourea .74 - .94%. In the
present study similar results were obtained for rainbow
trout treated with NaSCN; the % dose per gram of blood for
controls averaged 3.1% versus 0.64% for the NaSCN treated
trout. The increased rate of loss of I131 as reflected by
the blood levels may have occurred at the renal or gill
level. Bricker andiflad (1955) have shown that SCN- does

not appreciably alter the I13l/inulin clearance ratios in

humans. It did, however, markedly increase the plasma 1131
levels in hyperthyroid patients and caused a moderate rise
in euthyroid patients.

The data in Table 5 indicate that there is a 50% decrease
. . 131 .
in the gill I content in the NaSCN treated as compared
to the controls. Whether this means a greater rate of

outflux via the gills or a reduced uptake with a ”normal"

rate of outflux is not known.

71

An examination of the literature shows that almost
nothing is known about the normal rate of turnover of I131
in the blood of fresh-water fish. Hickman (1962) studied
fresh-water starry flounder and found that the T %'for blood
1131 was 202.6 hours and 279.4 hours for two flounder as
calculated from data obtained 30 and 70 hours after injec—
tion. For rainbow trout blood (fig. 3) the T é'for the non-
bound 1131 is 1.7 days.

As stated previously Marshall's work on the excretion
of iodide by the toadfish is the only published data on
actual measurements of the routes and relative rates of
excretion of iodine by fish. No data on iodide excretion
are available on fresh—water fish having glomerular kidneys.
The present study is the first to shed some light on this
unknown area. With the same reasoning which has been used
with mammalian data,the U/P ratios of 1131 indicate that
iodide is filtered and mostly reabsorbed. Reabsorption of
iodide is known to take place primarily in the proximal
segment of the tubules in the kidneys of dog and man. The
rainbow trout kidney has a proximal segment but no distal
segment. Bricker and Hlad (1955), Giebisch §§_§l, (1956)
and Williamson t al, (1962) have shown that the reabsorp-

tion of iodide in mammals is a passive diffusion process.

72

Due to the fact that only one parameter, the U/P ratio of
131 . . . .
I , was measured in the trout, no definite conclus10n can
be reached as to whether or not the reabsorption is an active
or passive process.
131
Renal clearance values of I for the trout have been
expressed in ml per hour rather than the usual ml per minute.
In man the normal renal clearance of iodide is 30 to 40 ml
of plasma per minute when the glomerular filtration rate is
normal. For trout in tap water the average renal clearance

of 1131 is 0.05 ml per hour, measured 24 hours after injec-

tion of I131. The average 1131 clearance for trout exposed
to distilled water is three times that of trout in tap water.
The work of Giebisch _t.§l, (1956) shows that in the dog
renal I131 clearance may increase five-fold during a mannitol
induced diuresis. Trout exposed to distilled water exhibit
a higher rate of urine flow than those exposed in tap water
(181.0 t SE 16.9 vs. 168.3 t SE 9.7 ml/Kg/day). It is, how-
ever, improbable that this difference in urine flow could
account for the three-fold difference in the clearance values
between the two groups of trout.

In trout as well as in mammals the Cl- clearance is less

than the iodide clearance (C1- 6.8 ml/Kg/day as determined

by Fromm (1963) vs. I_ 12.6 ml/Kg/day in the tap water trout).

73

Inulin clearance in rainbow trout at 100 is 169 i 11.8 ml/

Kg/day as measured by Holmes and McBean (1963).

The outflux of iodide from the gills of fresh-water fish
was measured simultaneously with the urinary loss. These
measurements indicate that 3.8 times as much iodide is lost
via the kidneys as by the gills per unit of time. Whether
this ratio is constant or changes depending on iodide levels
in the blood is not known. The fact that there is an out-
flux across the gills as well as a kidney loss helps to
explain why fresh-water fish in a goitre area have little
or no ionic iodine in the blood. But as has been previously
shown, if adequate iodide is present in the habitat, fast-
ing trout will take up and retain large amounts of inorganic

iodide free in the blood.

SUMMARY AND CONCLUSI CNS

1. Rainbow trout can accumulate 1131 directly from the

medium which surrounds them. This uptake is not affected

per se by the osmotic gradient between the environment and

the fish but is inversely related to the calcium level of

the medium.

2. The gill uptake of 1131 is inhibited by NaSCN. Injected
thiouracil does not appear to have any effect on uptake via
this pathway.

3.' The relative distribution of iodine in trout tissues
(tissue vs. blood concentration) is similar to that which
occurs in mammals with the exception of musCle which is lower
than in mammals.

4. In rainbow trout having adequate iodine available to

them, more than 80% of the blood iodine is in the ionic form
in contrast to the mammals where 90% or more is protein-bound.
The protein—bound iodine of the blood is made up of hormonal
iodine from the thyroid and by direct binding of iodide to
plasma proteins which can take place both ig_yiyg_and in yitgg,
5. The biological half-life of non-bound 1131 in the blood of

rainbow trout at 14°C is 1.7 day.

74

75

6. The retention of injected 1131 is significantly reduced
by NaSCN given intraperitoneally. Reduced gill levels of
1131 suggest a possible increased loss via this route as the
result of NaSCN treatment.
. 131 .
7. Kidney loss of I was found to be about four times
that of the gill when both were measured simultaneously.
. 131 .
8. Kidney clearance of I has been measured. As in mam-
mals, I- clearance is greater than that of Cl-.
9. The urine/plasma ratios for I131 indicate that 1131 is
filtered and mostly reabsorbed by the kidney.
10. Comparison of the iodine content of wild trout and trout

in iodine "equilibrium" reveals that there is a large iodine

storage capacity in trout tissues.

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8O

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APPENDIX A

Tissue fi'Values For 24 Hour

Studies and 24—29 Day Study

83

84

 

TABLE A-l
131
UPTAKE OF I FROM TAP WATER BY RAINBOW TROUT
T_ _ cpm/unit tissue
24 Hour M — cpm/Unit medium Values

Each value is the average of 10 observations

 

 

Tissue X i S.E.
Blood* 1.59 i .28
Heart .06 i .03
Gill .25 i .02
Lower Jaw 2.03 i .43
Liver .45 i .05
Spleen 14 i .06
Caeca 20 i .05
Head Kidney .18 i .07
Kidney .29 i .03
Intestine .17 i .04
Stomach .18 i .02
Muscle .02 i .01
Eye .14 i .02

 

*Average level of activity in exposure medium = 1.57 uc/liter

85

 

TABLE A-2
131
UPTAKE OF I FROM DISTILLED HOH BY RAINBOW TROUT
24 Hour —' = cpm/unit tissue Values
M cpm/unit medium

Each value is the average of 10 observations

 

 

Tissue X i S.E.
Blood* 6.34 i 1.12
Heart .32 i .03
Gill .23 i .03
Lower Jaw 1.49 i .19
Liver .36 i .07
Spleen .17 i .02
Caeca 13 i .01
Head Kidney .35 i .06
Kidney 30 i .04
Intestine .15 i .03
Stomach .20 i .02
Muscle .06 i .01
Eye .19 i 08

 

*Average level of activity in exposure medium = 2.38 UC/liter

86

TABLE A-3

UPTAKE OF 1131 FROM .6% NaCl BY RAINBOW TROUT

25 Hour %' = cpm/unit tissue Values
cpm/unit medium

 

Each value is the average of 10 observations

 

+

 

Tissue X - S.E.
Blood* 1.40 i .31
Heart .20 i .05
Gill .56 i .08
Lower Jaw 2.75 i .41
Liver 1.07 i .10
Spleen .12 i .04
Caeca .23 i .04
Head Kidney .28 i .05
Kidney .37 i .06
Intestine .19 i .03
Stomach .27 i .02
Muscle .04 i .01
Eye .21 i .02

 

*Average level of activity in exposure medium = 1.20 HC/liter

UPTAKE OF I

131

T _
24 Hour M. —

87

TABLE A—4

cpm/unit tissue

 

cpm/unit medium

Values

Each value is the average of 10 observations

FROM 66% RINGER'S BY RAINBOW TROUT

 

l+

 

Tissue X S. E.
Blood* .50 i .06
Heart 0
Gill .43 i .06
Lower Jaw 1.30 i .06
Liver .40 i .07
Spleen .03 i .03
Caeca .18 i .02
Head Kidney .03 i .03
Kidney .23 i .06
Intestine .09 i .03
Stomach .19 i .01
Muscle .02 i .02
Eye .14 i .01

 

*Average level of activity in the exposure medium

liter

1.63 uc/

88

TABLE A-5

IODINE EQUILIBRIUM STUDY

 

 

25-29 Day fi' Values
Tissue* K i .E.
Heart 0.16 i .01
Gill 0.16 i .01
Lower Jaw 18.39 i .49
Liver 0.32 i .06
Spleen 0.13 i .02
Caeca 0.13 i .01
Head Kidney 0.27 i .01
Kidney 0.29 i .02
Lower Gut 0.16 i .01
Stomach 0.13 i .01
Muscle 0.04 i .01
Eye 0.11 i .01

 

APPENDIX B
. 131 .
Uptake of Injected Doses of I With

Fish Injected Under MS—222 Anesthesia

89

9O

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mo. gm.m mo.m mm.o Nh.m mm H
Hm.H o.av mm.ma om.m no.om M
n.a mm.mm em.ma mm.m o.moa OH
o.H mo.mm hm.mm ov.oa o.mb so
m.H hm.mm mo.m mn.m H.00H m
v.H H¢.m© H®.> om.v ¢.mm so
v.H mm.mm mh.oa om.m n.Hm w
m.a OH.N¢ go.¢a mm.h m.moa em
S.H o¢.mm mm.¢m hm.m N.mm o
m.o oa.mv N¢.ha m¢.HH o.moa rm
o.H oo.a¢ m¢.m mm.m m.>oa m
N.H mo.m¢ om.w mo.o N.oh H
o: mnwufia w Emum Hum Emnm madam
mmop pmpomncH Hmum3 mos 3MU H0304 Umwwm DEUAOB moom .Os amen

 

HMHH mo moon UmuommcH mo RH

 

BDOfiB 30mZH¢m ZH

Hma

fiHmmmBmmZ< NNNIWZ MMDZD
ZOHBUHbZH .m.H Zfi OZHSquom

H m0 mm>OszB

Hlm mqm<fi

APPENDIX C
Uptake of I131 by Goldfish

and Rainbow Trout

91

92

Uptake pf_1131 py_Goldfish and Rainbow Trout

Twenty-five goldfish (Carassius) purchased from a local
store were placed into a 40 liter aquarium of aged tap water
and acclimated to a temperature of 14°C for a two week
period. During this time they were fed trout pellets. At
the end of the acclimation period, the goldfish were placed
into an aquarium containing 30 liters of aged tap water to
which had been added 68.8 uc of carrier free 1131. Twenty-
five trout were also placed into another aquarium set up the
same as that for the goldfish. During the course of the
experiment the fish were not fed nor the water changed.

A sample of five fish was taken from each aquarium at
24, 48, 72, 96, and 120 hours. Each fish was damped dry
and weighed to the nearest 0.1 of a gram. The goldfish were
wet ashed in 30 ml of concentrated nitric acid. Due to the
size variation in the trout, they were wet ashed in various
volumes (30 to 100 m1) of concentrated nitric acid.

Ten ml aliquots of the aquarium water and fish digests
were counted as described in the materials and methods
section. All counts were corrected back to zero time, the
beginning of the experiment.

It was thought necessary to determine the availability

of the added 1131. This was accomplished by running aquarium

93

water through 2 cc of ion exchange resin IRA 400. The count
of the aquarium water before and after passing through the

. . 131 . . .
reSin was used to estimate the percent I in the ionic

form.

94

TABLE C-l

UPTAKE OF 1131 BY GOLDFISH AND RAINBOW TROUT

FROM TAP WATER AT 14°C

 

 

Exposure No. Bgdy wt. gpm/gm %.ionic
time Fish of X and X and 1131
fish range range in water
24 hours* Goldfish 5 1.7 gm 4,308 100
0.8-2.7 3,507-5,535
Rainbow 5 13.4 gm 563 100
trout 6.8-18.8 226—807
48 hours Goldfish 5 1.5 gm 2,692 96
0.9-2.4 2,325-3,090
Rainbow 5 17.4 gm 495 96
trout 7.8-23.7 367-638
72 hours Goldfish 5 2.9 gm 4,357 100
1.4-4.0 1,119-12,664
Rainbow 5 13.0 gm 604 96
trout 4.3-25.2 475-837
96 hours Goldfish 5 2.4 gm 5,038 96
1.2-3.5 2,816-7,172
Rainbow 5 18.4 gm 516 87
trout 15.5-22.2 491-562
120 hours Goldfish 5 1.4 gm 6,511 100
1.0-2.4 l,503-18,242
Rainbow 5 9.9 gm 535 89
trout 8.2-12.2 459-597

 

*Level of radioactivity in water containing the goldfish was
467 cpm/m1, rainbow trout water 462 cpm/m1.

APPENDIX D

Turnover of i.p. Injected Radioactive

Thyroxine in Rainbow Trout

95

96

Turnover p§_i.p. Injected Radioactive
Thyroxine ip_RainbOW Trout

 

 

 

 

Nine unanesthetized rainbow trout ranging from 59.5 grams
to 106.4 grams were injected i.p. with 0.21 ug (7.5 uc) of
radioactive thyroxine and each placed into a plastic container
holding two liters of aged tap water at 14°C. Every 24 hours
a sample of two fish was taken. At that time a 10 ml aliquot
of the tap water was taken from each container and the re-
maining fish were placed into two liters of uncontaminated
aged tap water.

The fish to be sampled were anesthetized with MS-222 and
a blood sample taken. The lower jaw was also removed, trimmed
and weighed. The blood was centrifuged and the RBC's, plasma
supernatant, plasma precipitates and lower jaw prepared for

counting.

Figure D-l

Turnover of Radioactivity Associated with the
Protein-bound Fraction of the Blood and
131 . .
the RBC's of I -Thyrox1ne Injected

Rainbow Trout

% Dose per Gram

0.1'
0.09:

0.08
0.074
0.06

 

 

N|I-'

H

‘0
0———O Protein-bound
A Fraction
A---A Red Blood Cells
(RBC's)

 

I I I I \ I

 

1 2 3 4 5
Days After Il3I-Thyroxine Injection

 

98

Figure D-2
131 .
Turnover of Non-bound I in the Blood of

IlBl-Thyroxine Injected Rainbow Trout

 

 

 

 

r
\
\
an
S
v.
a
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0 098765 2 l
2 100000 0 0

mEmmHm SOHO Hum

Hma

H UCDOQIGOZ meQ X.

Days After Il3l-Thyroxine Injection

99

.snae Hmooa>accs now one cornea nsoaum>uonno Hem.

 

I mm.m mm.m mm.~ os.a 3th nosoe
ca.m mm.g mm.~ Hm.H He.s smug nod onoo R
I mo. mo. mm. mm. mason samuoum mammam
ca. me. no. es. am. so and whom x
I ea. mm. gm. mm.o usmumcuwmom .umm mEmDHQ
No. me. mm. em. so.m some nod onoo R
I mo. so. mo. mm. 0mm scum
Ho. me. so. co. ca. nod onoc R

 

muooz oma mHoOS om whoon NB mason we undo: em

 

«BDOMB SOmZHdm 2H mZHXOMMEB
m>HBU<OHQ<m ho mmOQ QmBUmeH .m.H Z4 m0 mm>OZMDB

HIQ mam<8

100

TABLE D-2

DISTRIBUTION OF RADIOACTIVITY IN THE BLOOD FOLLOWING

AN I.P. INJECTION OF 113l-THYROXINE

 

Time after

% of total activity in blood

 

 

 

 

 

injection RBC Protein-bound Non-bound
24 hours 4.01 15.53 80.46
12.19 39.93 47.90
48 hours 5.72 21.09 73.19
5.49 27.19 67.32
72 hours 4.68 18.85 76.47
5.37 25.60 69.03
96 hours 0.96 6.86 92.18
5.58 35.14 59.28
120 hours 0.69 13.99 85.32
TABLE D-3
WHOLE BODY EXCRETION FOLLOWING INJECTION
OF 1131-THYROXINE
. No. of X‘% dose AcgumulatIVe
Time fish excreted x,% dose
per 24 hours excreted
24 hours 9 19.17 19.17
48 hours 7 15.28 35.22
72 hours 5 13.22 47.28
96 hours 3 10.58 61.60
120 hours 1 4.45 84.04

 

APPENDIX E

Method for the Determination of Plasma Protein—bound
Iodine and Total Iodine Content

of the Lower Jaw (Thyroid)

101

102

Method for the Determination p£_Plasma
Protein-bound Iodine and Total Iodine
Content pf the Lower Jaw (Thyroid)
Preparation PBI

To 1 ml of plasma add 1 cc of zinc sulfate (10%), 1 cc
of sodium hydroxide (0.5 N) and 7 cc of glass distilled water,
mix and let stand for 10 minutes. The mixture is then cen-
trifuged and the supernatant saved or discarded as deemed
necessary. The precipitate is then resuspended in 10 cc of
glass distilled water and the procedure repeated twice. The
precipitate is then transferred into a nickel crucible con-

taining 1 cc of 4 N sodium carbonate.

Lower Jaw

The lower jaw was cut from the anesthetized trout,
trimmed and weighed to the nearest mg on a Roller-Smith
torsion balance. Upon being weighed the lower jaw was placed
in a nickel crucible containing 2 cc of 4 N sodium carbonate

and 1 cc of glass distilled water.

Common Procedure
The nickel crucibles were then placed into a drying
oven at 90-95°C to dry over night. The dried samples were

ashed for about two hours at 600 i 10°C in an electric muffle

103

furnace and then allowed to cool. The ash was dissolved in

2 cc of 2 N hydrochloric acid plus 2 cc of 7 N sulfuric acid.
The PBI samples were made up to a total volume of 14 cc and
the lower jaw samples to 25 cc with glass distilled water.
Contents were thoroughly mixed and transferred to a pyrex
centrifuge tube. The insoluble carbonaceous material was
allowed to settle and when necessary removed by centrifugation.

Two 5 m1 aliquots (PBI) and two 0.1 m1 aliquots (lower
jaw), for duplicate analyses, were pipetted from the centri-
fuge tubes into round bottomed optically calibrated color-
metric tubes. These tubes contained 0.5 ml of arsenious
acid and were made up to volume 5.5 ml with glass distilled
water. The tubes were then mixed well and placed into a
water bath accurately regulated at 27 t 0.20C for 15 minutes
incubation.

0.5 ml of ceric ammonium sulfate was added successively
to each tube at 60 second intervals using a "blow-out"
pipette and the tubes were thoroughly mixed and returned to
the water bath. Exactly 15 minutes after addition of ceric
ammonium sulfate the % transmittance was read in a Coleman
Model 11 Spectrophotometer using a No. PC—4 blue filter and
a wave length of 430 millimicrons. The initial setting of

the instrument (10% T) was made up against distilled water.

104

A reagent blank was prepared exactly as described above except
that the sample was omitted.

The reading of the reagent blank was subtracted from
the reading of the iodine standard or reading of unknown
samples. The concentration of iodine was read directly
from a standard curve plotted on arithmetic graph paper. The
total iodine was then calculated using the necessary correc-
tions for dilutions.

All determinations were performed in duplicate. A con-
trol standard of known concentration of iodine (Hycel's
Standard Iodine Reagent) and a reagent blank were included

with each series of samples.

Reference

Barker, S. R., M. G. HUmphrey and M. H. Soley, 1951. The
clinical determination of protein—bound iodine. J. Clin.
Invest., 30:52-62.

APPENDIX F

Procedure for Paper Chromatography

105

106
Procedure for Paper Chromatography

Paper

Paper strips (Whatman 3 MM) one inch by 12 7/8 inches
were cut. The origin was marked 2.5 inches from one end.
The origin end was tapered, starting 1/2 inch below the

origin, to 1/4 inch.

Technique for Applying Solutions

The paper strips were placed between clean glass plates
with the origin extending about one inch from the plates.
The solutions were applied at the origin with lambda
pipettes. The paper was then placed under a heat lamp to
dry. The dried strips were afixed to pins and placed on a
suspension rack. The rack was then placed into a steam hood
and subject to steaming for 15-25 minutes. After steaming
the rack was placed into a developing jar containing col-
lidine-HzO-ammonia. The strips were developed in this sys-
tem for 18 hours, after which they were removed and the

solvent fronts marked. After drying the strips were placed

on X-ray film for a 48 hour exposure.

Developing Reagent

Collidine 125 ml
H20 44 m1
NH4OH beaker of concentrated NH4OH is placed in

chamber

APPENDIX G

Ringer's Solutions for Fish

107

108

Ringer's Solutions for Fish

 

Schiffman's

58.5 grams NaCl
3.8 grams KCl
4.8 grams CaC12
10 liters distilled water

Stokes'
7.37 grams NaCl
.31 grams KCl
.37 grams MgSO4 - 7H20
.14 grams CaC12
1 liter distilled water
References

Schiffman, R. H. 1961. A perfusion study of the movement of
strontium across the gills of rainbow trout (Salmo
gairdnerii). Biol. Bull.,120(l):110-1l7.

Stokes, R. M. 1963. Glucose absorption and metabolism by
the gut of rainbow trout, Salmo gairdneri. Ph.D.
thesis, Michigan State University.

APPENDIX H

References Pertaining to Iodine in Fish

109

110

References Pertaining pp_Iodine 1p.Fish

 

Bourcet, P. 1899. Recherché' et dosage colormetrique de
petites quantities d'iode dons les matiéres organiques.
Comp. rend. Acad. Sci., 128:1120-1122.

Cameron, A. T. 1913. Note on the iodine content of fish-
thyroids. Biochem. J., 7:466-70.

Cameron, A. T. 1913-1914. The iodine content of the thy-
roid and of some bronchial cleft organs. J. Biol. Chem.,
16:465-473.

Cameron, A. T. 1914. Contributions to the biochemistry of
iodine. I. The distribution of iodine in plant and
animal tissues. J. Biol. Chem., 18:355-380.

Cameron, A. T. 1915. Contribution to the biochemistry of
iodine. II. The distribution of iodine in plant and
animal tissues. J. Biol. Chem., 23:1-39.

Closs, K. 1931. Uber das Vorkommen des Jods in Meer und
in Meeres organismen. Arch. Math og Naturvidensk.,
40:1-150. Cited from Biol. Abstr., 7(3):5433 (1933).

Coulson, E. J. 1935. Iodine content of American fishery
products. U. S. Bur. Fisheries, Invest. Rept. 1, No.
27, 7 pp.

Forbes, E. B. and F. M. Beegle. 1916. The iodine content
of foods. J. Med. Res., 34:445-58. Cited from Chem.

Abstr., 10:2773 (1916).

Jarvis, N. D., R. W. Clough and E. D. Clark. 1926. Iodine
content of the Pacific coast salmon. Univ. of Washing—
ton Publ. Fisheries, l(6):109-l40.

Jarvis, N. D. 1928. Iodine content of Pacific coast sea-
foods. Univ. of Washington Publ. in Fisheries 1:239—250.

Kupzis, J. 1932. Die Jodfrage in Lettland imzusammen
hange mit dem Kropfe. Zeitschr. Hyg. u. Infektienskrankh
11 : 51-573. Cited from Biol. Abstr., 10(3):5496 (1936).

° I

111

Lunde, G. 1928a. The geochemistry and biochemistry of
iodine with particular regard to Norwegian goiter pro-
phylaxis. Wiener klin. Wochschr., 41:15. Cited from
Chem. Abstr., 22:1370 (1928).

Lunde, G. 1928b. Occurrence and distribution of iodine in
fish and in fish products. Teknisk Ukeblad, 75:201-
203. Cited from Chem. Abstr., 25:1292 (1931); 23:2193
(1929).

Lunde, G. 1928c. Biochemistry and geochemistry of iodine.
Etiology and prophylaxis of endemic goiter. Northwest
Med., 27:412. Cited from Chem. Abstr., 23:401 (1929).

Lunde, G. and K. Closs. 1928. Iodine compounds in fish
products. Tids. Kem. Bergvesen, 8:33-34. Cited from
Chem. Abstr., 23:1443 (1929).

Lunde, G., K. Closs, H. Hauland and S. O. Madsen. 1928.
The iodine content of Norwegian fish and fish products.
Aars beretning vedkommende Norges Fisherier No. IV:
3-41. Cited from Chem. Abstr., 23:1964 (1929).

Lunde, G. and J. Bee. 1928. The iodine content in species
of fish from the east and west coasts of U.S.A. Pre-
liminary communication. Tids. Kemi og Bervesen, 8:
139-40. Cited from Chem. Abstr., 23:1939 (1929).

Lunde, G. 1929. The geochemistry of iodine and its cir-
culation in nature. Chem. Rev., 6:45-61.

Lunde, G., J. BOe and K. Closs. 1930. Iodine content of
American marine animals. J. Conseil. Intern. Explora-
tion Mer., 5:216-25.

McClendon, J. F. 1927. The distribution of iodine with
special reference to goiter. Physiol. Rev., 7(2):
189-258.

Nilson, J. W. and E. J. Coulson. 1939. Iodine content of
fillets of fish (marine) and canned salmon. U.S. Dept.
of Com., Bureau of Fisheries Inv. Rept. NO. 41, pp.
1-7.

112

Ripperton, J. C. 1935. Iodine content of some Hawaiian
food products. Hawaii Agr. Expt. Sta. Ann. Rept.
1934:27. Cited from Chem. Abstr., 29:4840 (1935).

Roman, W. 1930. Eine vergleichende Ubersicht fiber den
Jodgehalt von Merr-und Sfisswasserfischen. Ein beitrag
zur kroptprohylaxe. Miteil. Deutsch. Seefischerei-Ver.
46:552-555. Cited from Biol. Abstr. 6(5):13030 (1932).

Rudolph, W. 1947. Iodine and vitamins from the sea. Med.
Monatsschr., 1:276-78. Cited from Chem. Abstr., 44:
7459 (1950).

Tressler, D. K. and A. W. wells. 1924. Iodine content of
sea foods. U. S. Bur. Fisheries Doc. No. 967, 12 pp.

Wille, O. 1929. Uber den Jodgehalt deutscher Seefische
und Seefisch produkte. Mitteil. Deutsch. Seefischeri-
Ver., 45:418-419. Cited from Biol. Abstr., 6(4):9744
(1932).

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