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ACCUMULATION AND ELIMINATION OF HEXAVALENT CHROMIUN

IN RAINBOW TROUT

By

Jack Knoll

AN ABSTRACT

Submitted to the School of Science and Arts of
Michigan State University of Agriculture and Applied
Science in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Physiology and PharmacOIOgy

1959

——

Approved L21 0‘ FAWV

ARSTRACT

Studies of fish toxicity resulting from inorganic metals
and their salts have been confined largely to the determina-
tion of lethal doses. Little investigational work has been
conducted on the effects of sub-lethal levels of such materi-
als. In order to investigate the effects of sub-lethal levels
of hexavalent chromium, rainbow trout were exposed to a solu-
tion of KZCrOu containing 2.5 mg. Cr/l., part of which was
in the form of Crsl. Representative tissues were removed;
their chromium content was determined by radiological meth—
ods. These studies revealed that hexavalent chromium was
accumulated in quantities proportionally exceeding the
aquarium water in spleen, liver, posterior gut, pyloric caeca
and kidney. With the exception of the spleen, these organs
were all capable of excretion, thus it was hypothesized that
accumulation in these tissues might be correlated with ex-
cretion.

In another study, fish were exposed to 2.5 mg. Cr/l.
for 12 days; then returned to clean water from which h or 5
fish were removed every 5 days. These fresh water elimina-
tion studies showed a rapid decrease of chromium from the
blood, liver, stomach, pyloric caeca and posterior gut.

These studies also indicated a retention of chromium in the

spleen and kidney.

11

Three fish, given radiochromate via stomach tube, were
returned to clean water for one day. Tissues from these
fish were examined for Cr51, indicating no appreciable chro-
mium content in organs other than the stomach, pyloric caeca
and posterior gut. E50phageally occluded fish exhibited an
accumulation of chromium similar to that in nnn—occluded
fish, when both were exposed to 2.5 mg. Cr/l. for 1 day. As
a result of these observations, it was hypothesized that
the major route of entry of hexavalent chromium was via
the gills. Except in one fish, the blood concentration of
chromium never surpassed that of the surrounding water,
tending to indicate that hexavalent chromium probably crossed
the gill membrane as a result of simple diffusion. A very
low rate of increase in chromium concentration was noticed
in blood, indicating either that the chromium combined into
a complex when it entered the blood, or that nucleated fish
erythrocytes behaved differently than do mammalian red blood
cells.

On the basis of a rapid accumulation of chromium by the
pyloric caeca, during the first few days of exposure, fol-
lowed by a diminution of the rate of accumulation, it was
hypothesized that the pyloric caeca, along with bile and
kidneys, were a possible route of chromium excretion.

Based on information obtained, fish, caught in chromium
contaminated streams not exceeding a concentration of 2.5 mg.

Cr/l. of water, would probably not contain appreciable amounts

of chromium in muscle tissue.

iii

ACCUMULATION AND ELIMINATION OF HEXAVALENT CHROMIUM

IN RAINBOW TROUT

BY

JACK KNOLL

A THESIS

Submitted to the School of Science and Arts of
Michigan State University of Agriculture and Applied
Science in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Physiology and Pharmacology

1959

ACKNOWLEDGMENTS

Gratitude is expressed to Dr. P. 0. Fromm for his
continual guidance and interest.

In addition the writer is indebted to the Michigan
Department of Conservation for providing a continuous
supply of uniformly sized fish and to the United States
Public Health Service for providing funds in support of

this 5 tUdYe

To Ella and Bob

vi

INTRODUCTION 0 O O O O O O O O O O O O O O 0

REVIEW

I.

II.

III.

METHOD S

I.

II.

III.

IV.

RESULTS

I.

TABLE OF CONTENTS

OF LITERATURE O O O O O O O O O O O O O

Chromium Toxicity Studies . . . . . .
Tissue Uptake of Chromium . . . . . .

Accumulation of Fission Products in
Fish TisSU.S e e e e e e e e e e e e

Accumulation of Hexavalent Chromium .
Procurement and holding facilities .
Experimental aquaria . . . . . .
Tissues studied . . . . . . .
Preparation of tissue . . . . .
Counting equipment . . . . . . . . .

Feasibility of counting whole tissues

Expression of data . . . . . .

Elimination of Hexavalent Chromium upon

Return of the Fish to Fresh Water

Administration of Hexavalent Chromium
Via StomaCh TUbe e e e e e e

Hexavalent Chromium Concentrations in
with Esophageal Occlusions . . . .

Accumulation of Hexavalent Chromium

vii

Page

10
19
19
19
20
21
22

23

27

27

28

Page

Blood . . . . . . . . . . . . . . . . . . . 29
Spleen . . . . . . . . . . . . . . . . . . . 29
Liver . . . . . . . . . . . . . . . . . . . 30
Posterior portion of gut . . . . . . . . . . 30
Pyloric caeca . . . . . . . . . . . . . . . 31
Stomach . . . . . . . . . . . . . . . . . . 31
Kidney . . . . . . . . . . . . . . . . . . . 31
Muscle . . . . . . . . . . . . . . . . . . . 36

II. Elimination of Hexavalent Chromium from

Tissues upon Return of the Fish to Fresh
“'ater e e e e e e e e e e

e e e e e e e e e e 36

III. Elimination of Hexavalent Chromium

Administered via Stomach Tube . . . . . . . . 37
IV. Hexavalent Chromium Concentration in Fish
with Esophageal Occlusions . . . . . . . . . 37
DISCUSSION 0 O O O O O O O O O O O C O O O O O C O O 0 no
I. Possible Entry Routes of Hexavalent Chromium ho
II. Transport of Hexavalent Chromium in the Fish #1
III. Hexavalent Chromium Accumulation in Individual
organs 0 O O C Q C O O O O O O C O O O O C O 1‘1
IV. Excretion of Hexavalent Chromium . . . . . . #3
SUMMARY AND CONCLUSIONS . . . . . . . . . . . . . . . “6
REFERENCES CITED 0 O O O O O O O O O O O O O O O O O O “8
APPENDIXES e e e e e e e 0 e e e e e e e e e e e e e e 52
A. Accumulation of Chromium in ug./g. Tissue
With Time e e e e e e e e e e e e e e e e e e 53
B. Elimination of Chromium with Time Following
Return of Fish to Fresh Water after 12 Days
Exposure to 2.5 ug. Chromium . . . . . . . . 62

viii

Distribution Studies of Radiochromate
Administered to Rainbow Trout via
Stomach Tube and Distribution Studies

of Rainbow Trout Subjected to Esophageal
Occlusion . . . . .

Statistical Formulas. .

ix

Page

67
69

TABLE

LIST OF FIGURES

Absor tio f
P “Sf

Using a Cr Source .

Absorption ff
Using a Cr5

Source .
Chromium Accumulation
Chromium Accumulation

Chromium Accumulation

Chromium Accumulation
POSterior Gut e e e e

Chromium Accumulation
Pyloric Caeca . . . .

Chromium Accumulation

Chromium Accumulation

and

and

and

and

and

and

Elimination

Elimination

Elimination

Elimination

Elimination

Elimination

Elimination

0.32 Mev Gamma Energy by Water

0 O O O

0.32 Mev Gamma Energy by Lead

Blood
Spleen

Liver

Stomach

Kidney

Two Fish with Esophageal Occlusions Compared
with Mean 2b Hour Values for Normal Fish . . .

LIST OF TABLES

Per Cent of Total Activity Remaining in
Chromate Exposed Fish after Return to

Clean Water e e e e e

25

26

32

33

33

3h
3h

35

38

Page

37

INTRODUCTION

Although there is no known function of chromium in
animals, it is present in trace amounts (Grushko, l9h8; Van
der Walt and Van der Merwe, 1938; Koch et a1., 1956; Udy,
1956). Gray and Sterling (1950) reported mean values of
chromium in human blood to be 20 ug. per cent for packed
red blood cells and 1% ug. per cent for plasma. Similar
data for other animals has not been found in the literature.

Chromium, atomic number 2h, atomic weight 52.01, occurs
in group VI of the Periodic Table, and although there are
four naturally occurring isot0pes (Cr50, Cr52, Cr53, Cr5u),
Cr52 is by far the most abundant. The following table
presents a brief outline of some of the characteristics of

the element (Hopkins, l9h2).

 

 

Valence Class Reaction Reprezzgfiative Nizzig
2 Chromous Basic CrCl2 Cation
3 Chromic Weakly Basic CrClB Cation
3 Chromite Weakly Acidic NaCrO2 Anion
6 Chromate Acidic NazCrOu Anion
6 Dichromate Acidic NazCr207 Anion

Generally speaking, hexavalent chromium exists in water
in true solution, regardless of H+ concentration or the

presence of other ions. Trivalent chromium exists in water

1

in solution, as a complex, or as a colloidal or flocculent
precipitate depending on the pH and dissolved substances.

Chromium compounds containing Cr51 have been used
clinically in recent years. Beierwaltes et a1. (1957) have
reviewed procedures preposed for a reticulo-endothelial test,
diagnostic test for hemolytic anemias, measurement of red
cell volume and half-life, location and quantitation of
intestinal bleeding and measurement of plasma volume, all of
which utilize Cr51 in one form or another.

Gray and Sterling (1950), in a major contribution to
chromium literature, showed that mammalian erythrocytes have
a marked affinity for anionic chromate. They hypothesized
that hexavalent chromium was bound or modified after gaining
entry into the red cell. This binding was shown to be
essentially irreversible. In addition, they reported that
trivalent chromium was bound by albumin and this binding
was also essentially irreversible.

Collins (1958), studying chromium excretion in the dog,
ascertained that the major excretory route for intravenously in-
jected chromium was via the urine. After a single intravenous
injection, renal clearances of CrCl3 decreased exponentially
with time. Measurement of dialyzable Cr-51 in plasma showed
that the per cent of chromium not bound to plasma proteins
also decreased exponentially with time. Glomerular filtra-
tion and tubular reabsorption were two mechanisms involved

in the renal handling of unbound chromium. Renal function

was not impaired over a large range of plasma chromium
concentrations. In vivo reduction of hexavalent chromium
occurred. It was indicated that chromium was excreted at
least in part in organic combination.

Chromium is of significance as a pollutant to streams,
lakes and potable water supplies due to waste disposal by
industry. Cr51 in low amounts is also present in the
effluent of nuclear reactors along with Nag“, Cusu, P32,
A376 and rare earths (Davis and Foster, 1958).

Studies of metal toxicity have been confined largely
to the determination of lethal doses, with little investi-
gational work being conducted on the effects of sub-lethal
levels of a toxic substance.

In an effort to gain knowledge of possible routes of
entry, methods of transport and routes of excretion in
rainbow trout, a study of hexavalent chromium distribution
patterns was undertaken, using 2.5 mg. Cr/l. Because

chromium might be present in the environment of fish, a

study of this type was felt to have value.

REVIEW OF LITERATURE

I. Chromium Toxicity Studies

Many papers dealing with the toxicity of both trivalent
and hexavalent chromium have been reviewed by Doudoroff and
Katz (1953). To date, most of the information has pertained
to the determination of acutely toxic levels! Rushton (1922)
observed that a 6 hour exposure to 100 mg./l. of chr2°7
proved fatal to trout within 12 hours after their return to
fresh water. Oshima (1931) stated that the survival time of
young eels in a solution of KCr(SOu)2 averaged 18.7 hours at
a concentration of 5.2 mg. Cr/l. Anderson (l9hh) found that
KZCrOu immobilized Daphnia magga in 16 hours at a concen-
tration of less than 0.6 mg./1. The toxicity of KZCrOu and
K20r207 to yearling rainbow trout was evaluated by Grindley
(1946). He concluded the lower limits of toxicity were
slightly below 20 mg. Cr/l. Abegg (1950) studied the effects
on bluegills of NaZCrOQ and NaZCr207. He expressed the 2h
hour TLm (median tolerance limit) as 930 mg. of NaZCrOh/l.
and 788 mg. of Na20r207/1. Abegg also reported a slight
but significant increase in the fluid content of muscle and
a decrease in the blood specific gravity caused by exposure

to NaZCr LeClerc and Devlaninck (1950), using trivalent

2O7.

chromium in the sulphate form, determined the minimum fatal

concentration for a 6 hour exposure period for minnows in
distilled water to be near to mg. Cr/l.

Fromm and Schiffman (1958) determined the TLm for
largemouth bass to be 195 mg. Cr/l. Under the conditions
described, exposure to 9b mg. Cr/l. caused, initially, a
slight but not highly significant increase in the oxygen
consumption of bass, followed by a gradual decline to 27
per cent below normal after 68 hours exposure. Exposure to
chromium caused severe pathological changes in the intestine
immediately posterior to the pyloric caeca that in all
probability completely destroyed its digestive function.
The normal respiratory rhythm of the bass was not observably
altered following exposure to chromium; however, coughing
movements which occurred at fairly regular intervals were
observed. No significant histological change in the gills
of fish exposed to chromium were noted. In concentrations
that were not acutely lethal, chromium apparently did not
kill fish by interfering with the respiratory function of
the gill.

Schiffman (1957) reported a 2h hour TLm for rainbow
trout as being 100 mg. Cr/l. when using KZCrOu. Tracer
experiments indicated that the spleen and gall bladder,
including bile, accumulated chromium above the level of a
section of the caudal peduncle, whereas there was no evi-
dence that kidney or liver did. Weights of kidney, liver,

spleen and total body water did not change when the fish

were exposed to KZCrOu. The hematocrits of both splenec-
temized and intact fish were significantly higher than
control fish in tap water. This rise in hematocrit was due
to an increase in cell number and cell volume. There was

no evidence that the plasma or blood volume increased or
decreased in fish exposed to KZCrOu. The presence of spleen
was not necessary to increase the hematocrit under chromium
stress. The hematocrit reached its maximum value at a
concentration between 2 and h mg. Cr/l. Data indicated that
the spleen may act as a limited buffer mechanism to sudden
hemoconcentration.

Olson and Foster (1956a), studying the effects of low
concentrations of NaZCr207 on young Chinook salmon and
rainbow trout, found that the eggs would hatch when placed
in the highest concentration used (0.18 mg. Cr/l.). However,
survival of the young salmon and trout was affected at 0.08
mg. Cr/l., and growth was possibly retarded in the low con-
centration of 0.013 mg. Cr/l. They reported the expected
weight gain with time demonstrated a diminution as the
chromium was increased. In a following study, Olson and
Foster (1956b), reported further information regarding the
toxicity of hexavalent chromium to Chinook salmon. Chronic
release of dichromate was slightly more toxic to aquatic
life than the same quantity released intermittently. The
dichromate appeared to have a greater toxic effect at h1°F.

than at 50°F. However, no simple correlation between

temperature and toxicity was indicated. Davis and Foster
(1958) conducted detailed studies of radioactivity uptake by
different tissues of Columbia River fish, in relation to ef—
fluent from the Hanford, Washington reactor. Fish maintained
in effluent from the Hanford reactors concentrated Raga in
tissues about 130 fold. Direct absorption of other dominant
isotopes in the effluent, Cr51, CuGu, P32, A576 and rare
earths, appeared to be inconsequential. The contribution
of food chains to the concentration of radioisot0pes in
aquatic animals was apparent. Fish collected down-river
from the reactors were approximately 100 times as radio-
active as laboratory fish that were exposed to equivalent
mixtures of the effluent, but fed uncontaminated food.
Koenuma (1956) found that a 5x10-“ M. sea water solu-
tion of uranyl nitrate inhibited the formation of the
fertilization membrane in Urechis eggs and led to polyspermy.
These findings indicated that in determining the effects of
toxic materials more than lethal effects should be considered.
The hardness of the experimental water used also plays
an important part in determining lethal doses of some metals.
Cairns (1957), using bluegills exposed to zinc, noted a 96
hour TLm to be 2.86 mg. Zn/l. at 18°C. and 1.93 mg. Zn/l.
at 30°C. in soft water. In hard water, it was 10.13 mg.
Zn/l. at 18°C. and 10.15 mg. Zn/l. at 30°C. This would
indicate that temperature as well as hardness is an impor-

tant factor in toxicity studies.

II. Tissue Uptake of Chromium

Visek et al. (1953), investigating the metabolism of
chromium compounds, employed 200 rats sacrificed at intervals
of up to h5 days after injection. They also correlated the
electrophoretic behavior of each chromium compound present
in serum with its tissue distribution. Ninety per cent of
the injected NaCr-Slo2 was taken up by organs of the reticulo-
endothelial system. The electrophoretic pattern showed it
to be colloidal in serum. The liver and spleen retained 33
and 50 per cent respectively of their h day activity at the
end of h2 days, whereas the lung showed only 10 per cent of
its h day activity. After Cr51013 injection, the activity
was greatest in the liver, spleen and bone marrow, a reflec-
tion of reticulo-endothelial uptake. When NaCr5102 was
given, uptake of radioactivity by these tissues was somewhat
less. The activity, once deposited, was removed very slowly.
The concentration in the liver at 45 days was 35 Per cent
of its maximum value observed at 2h hours. All other tissues
were practically constant throughout the #5 day period with
the exception of blood and lungs. The blood retained practi-
cally no activity after the seventh day and the lungs at
U5 days retained about 15 per cent of their maximum value,
which was observed at 6 hours following injection. The
spleen gained activity over the h2 day period. Orally

administered Cr51C13 was almost totally excreted in the

feces at the end of h days. Less than 0.5 per cent of the
dose was absorbed from the G.I. tract. Chromic chloride
behaved as a colloid under these conditions. The electro-
phoretic pattern indicated that it was protein bound. Sodium
chromate showed a major uptake by the liver, but less than
that observed for Cr51013 or NaCrSIOZ. Twenty-five per cent
of the activity was observed in the liver 30 minutes after
injection, while less than 1 per cent was present at the end
of #2 days. Here again, the spleen seemed to gain activity
with time. Skeletal tissues showed relatively small con-
centrations of chromium. The blood at 21 days had a concen-
tration of approximately 1/5 the 30 minute value, while at

h2 days it was practically zero. Crslcl3 in acetate (pH 5.5)
and citrate (pH h) buffers followed the same general pattern
as non-buffered Cr51013. There was however, a much larger
urinary excretion when buffered Cr51C13 solutions were ad-
ministered intravenously. The behavior of Nazcrslou was
"probably ionic” under these conditions. The electrophoretic
pattern indicated that it was ionic in serum and bound by
hemoglobin in cells.

MacKenzie (1957) fed trivalent and hexavalent chromium
to rats in drinking water for a period of one year. The
chromium concentration in the water varied from 1 to 25 mg.
Cr/l. as chromate ion. He reported no effect on water in-
take, food consumption or weight gain during the period of

exposure. Also, no significant histological changes were

10

observed in the blood or tissues examined. There was an
abrupt rise in retention in kidney, liver, femur and spleen
at water concentrations above 5 mg. Cr/l. as chromate ion.
Starved rats administered radiochromate via stomach tube
absorbed approximately 5.5 per cent of the dose, while non-
starved animals absorbed approximately 2.5 per cent. Blood,
stomach, intestine, liver, kidney, urine and feces were
analyzed. The highest activity in the blood occurred at 6
hours or earlier, whereas, the highest activity in the liver
and kidney occurred at 2h hours. In a similar experiment,
blood samples were taken h hours after administering NaZCrOu
or CrC13 via stomach tube. The starved animals receiving
NaZCrOh solution absorbed the most chromium, while the non-
starved group given CrCl3 absorbed the least. There was
considerable reduction of the chromate ion in the digestive
tract. After injecting NaZCrOh and CrCl3 directly into the
intestine, much higher blood values were obtained than in

the previous experiment, indicating that most of the chromate

reduction occurred in the stomach.

III. Accumulation of Fission Products in Fish Tissues

Numerous studies have been conducted on uptake of nuclear
fission products by fish. Boroughs et al. (1956a) conducted
distribution studies of Sr89 in black skipjack, yellowfin
tuna, and dolphins. They also conducted studies on the dis-

tribution of Yt91 in the marine fish Tilapia mossambica

11

(Boroughs et al., 1956b). Their most recent paper (Boroughs
et al., 1957) discussed the uptake of Cau5 from artificial
sea water containing the same calcium concentration as normal
sea water. It was shown that marine fish can take up calcium
directly from sea water and also that marine fishes will dis-
criminate against strontium in favor of calcium. Rosenthal
(1956) reported a considerable retention of Cal"5 from water
by guppies. Chipman (1956) placed pieces of skin separating
two sea waters, one of which contained Sr89, C3137 or Ru106.
The diffusion of these isotopes through the tissues under the
skin of iced fish was observed after a number of days contact
with the skin surface. Cesium entered the fish through the
skin very readily. After h days contact of the skin with the
radioactive solutions, the activity from cesium of the deep
muscle tissue was equal to that on the skin surface. On the
other hand, after 8 days, the activity of the deep tissue

was only A per cent of the skin surface activity for Sr89

and l per cent for Ru106. Chipman et a1. (1957) reported
studies of metabolism of Zn65 by marine fishes. Zn65 was
pipetted directly into the stomach of croakers, then they
were placed in fresh sea water. Zn65 rapidly entered the
blood stream and accumulated in the kidney, spleen and liver.
There appeared to be a rapid loss from the internal organs
and a slow and long continued accumulation, although still
only a small amount of the administered dose, in the bone

and muscle tissues. Welander (1957), studying radioactivity

12

in reef fishes of Belle Island, Eniwetok Atoll, collected
693 specimens. Skin, muscle, bone, liver and viscera were
removed and examined for radioactivity. The greatest per-
gram concentration of radioactive materials occurred in the
alimentary tract with the liver, skin, bone and muscle having
successively lesser amounts.

Following the hydrOgen bomb tests at Bikini Atoll in
the spring of l95h, radioactive fish began appearing on the
Japanese markets. On March 1, l95h, the first of these fish
arrived on the Fukuryu Maru No. 5, the ship on which the
”Bikini ash" had fallen. On May 31, 195h, the Japanese
Ministry of Health and Welfare established an arbitrary
maximum permissible contamination of 100 c.p.m. when measured
with a G-M tube placed 10 cm. from the skin of the fish.
Fishing vessels returning from the general Bikini area were
compelled to put in at certain harbors where the entire catch
was subjected to scrutiny with a G-M counter. Following
this decree, a great deal of scientific research was con-
ducted to test the validity of the 100 c.p.m. maximum per-
missible level. This and other investigations by Japanese
investigators noted below are presented in the two volume
issue of the Japan Society for the Promotion of Science
entitled "Research in the Effects and Influences of the
Nuclear Bomb Test Explosions", 1956.

Saiki et a1. (1956) reported radioactivity distributions

in the tissues of various marine fish that were collected

13

from Japanese fish markets. The most radioactive portions
were liver, kidney, spleen, pyloric caeca, stomach content,
stomach and rectum. The radioactivity of muscles was re-
ported to be low. Amano et a1. (1956) reported similar re“
sults on studies made on fresh caught Pacific fishes taken by
the government research vessel, R. V. Shunkotsu Varu. His
group also attempted to chemically analyze the contaminants

by separating them into five fractions following the proce-
dures of qualitative chemical analysis. They stated that

the major contaminant in white muscle was Zn65. Kawabata
(1956), calculating decay rates and absorption curves, along
with chemical fractionation, determined the principal con-
taminant in white muscle to be Zn65 with other unidentified
isotOpes. Nagasawa et a1. (1956) reported some of the unknown
activity in white muscle to be due to Sr90.

Yoshii (1956) conducted electron diffraction studies on the
first vertebrae of Katsuwonus vagans. Only the patterns of
Ca3(POu)2 and CaCO3 were indicated. This same investigator
conducted radioautographic studies that led to the following
conclusions:

Stomach wall: In the tunic mucosa, radioactive sub-
stances are distributed from the mid-portion to the basal
membrane, around lymphatic cavities and the vessels.

Intestinal wall: Radioactive substances are strongly

localized in the walls of vessels and lymphatic cavities of

the mucous membrane. They were also distributed along the

1h

margins of muscle fibers.

Muscle: Radioactive substances were distributed along
the margin of muscle fibers and the muscle bundles, and
clearly on the dark zone of the striated muscles.

Skin: Most of the activity was concentrated in the
connective tissue of the epithelium.

Testis: The activity was uniformly distributed around
the circumference of the spermatocyte.

Gill: Activity was located on the gill filaments.

Liver: Radioactive substances were abundantly distri-
buted in the liver cell strings.

Atlas: The deposition of radioactive substances was
marked inside of the atlas and on the spinal cord.

Saeki and Sano (1956) placed goldfish in solutions of "Bi-
kini ash". Tissue examination showed the gill, fins, and diges—
tive tract, with contents, to have an activity exceeding that
of the water in which the fish were placed. They reported
skin and muscle as showing no activity. In fresh water
experiments on fish previously exposed to the radioactive
solution, it was reported that goldfish did not lose the
absorbed radioactivity any faster than could be attributed
to decay alone.

Hiyama et a1. (1956), in laboratory studies, demonstrated
that fresh water fish absorbed fission products faster than did
salt water species. Inorganic contaminants placed directly

in the stomach were almost unabsorbed, while contaminated

15

food was taken up more readily. (Note the food was con-
taminated Artemia).

Suyehiro et a1. (1956) placed Japanese killfish in 10%
physiological saline solution containing Sr90. They reported
that the uptake of this isotope was in preportion to the

strength of the Sr90

solution. Goldfish were placed into
water containing 10,000 c.p.m./l. Whole fish counts indi-
cated an increasing accumulation trend till near the tenth
day at which time the absorption rate tapered off. They
also showed a correlation between the count of gills, liver
and serum. In the carp, eel, goldfish, Labyrinth fish and
conger eel, there was little difference in the distribution
of radioactive material.

Ichikawa and Hiyama (1956), studying strontium, reported
that a great part of the absorbed isotope was deposited on
hard tissues such as scales, vertebrae, fin and gill, be-
having in a similar manner to calcium.

Tomiyama et al. (1956a) conducted experiments to deter-
mine the route of calcium uptake from the surrounding water
medium. Upon placing goldfish in water containing Ca”5 and
sacrificing, followed by counting separate tissues, it was
noted that the gill exhibited the greatest activity, followed
by the scales. The next series of experiments consisted of
placing goldfish in small glass chambers. A rubber diaphragm

isolated the anterior portion of the chamber and the fish

from the posterior portion. The anterior portion of the

16

fish was then exposed to a concentration of 125 mg. Ca/l.
The posterior portion was exposed to well water with no Can5
ion. After an 18 hour exposure, the fish was removed and
another placed in the apparatus, with the posterior portion
being exposed to Cans. It was demonstrated that when the
anterior portion only was exposed to calcium, a high uptake
took place while very little uptake was observed when the
posterior portion only of the fish was exposed. This group
postulated that the gill is the most likely path for accumu-
lation of calcium. Absorption of Can5 via the water medium
versus that taken up through food by goldfish was also com-
pared by this group (Tomiyama et al., 1956b). Irrespective
of different routes for Can5 uptake, the deposition pattern
was similar except in the intestine. This same group,
Tomiyama et al. (1956c), studying marine fishes, noted a
high concentration of Ca“5 in the intestine, suggesting an
uptake of radiocalcium by the intestine as well as the gill.
The route of absorption of 5r9° was studied by placing carp
in a diaphragm type of apparatus to separate the anterior
from the posterior end of the fish and by occluding the
esophagus (Tomiyama et al., 1956d). It was demonstrated that
Sr90 was absorbed primarily through the gill, to a small ex—
tent through the skin, and not absorbed in a measureable
amount through the digestive tract. Except for the muscle
and gall bladder, activity was detected in tissues studied

after as little as 30 minutes exposure. The deposition

17
pattern was similar to that of Cans. Sr9O administered
intramuscularly (Tomiyama et al., l956e) showed the same
distribution as Sr9o taken up from the surrounding water.
This group, Tomiyama et a1. (l956f), also reported that
PBZOA ion was readily absorbed by carp from the surrounding
water. The absorbed P32 was distributed in the gill, caudal
fin and scales similarly to Sr90 and CauS. In contrast to
5r9°, the blood, heart, liver and kidney were found to be
high in P32. The deposition pattern of P32 administered
intramuscularly to carp was similar to the absorption of the
isotOpe from the surrounding water (Tomiyama et al., l956g).

Hibiya and Yagi (1956) exposed fish eggs of different
Species in various stages of development to different con-
centrations of ash taken from the deck of the Fukuryu-Maru.
Japanese killfish eggs were killed by placing them into a
concentration of ash that counted 62 c.p.m./cc.

Mikami et al. (1956) studied the effects of different
concentrations of the radioactivity found in rainwater on the

hatching success of zebra fish. When the activity was less

than b.2 x 10‘“ uc./1., the activity found in the original

rainwater, survival was not affected, but the mortality
increased considerably when the medium exceeded 21 x 10'“
uc./l. Total mortality occurred when the concentration
reached 150 to 210 x 10'“ uc./l.

Suyehiro and Hibiya (1956) injected Sr9O into goldfish

and then removed three fish, for blood study, after varying

18

periods in fresh water. Blood was removed from the caudal

artery. Slides were prepared using a variety of stains. A

count was then made of all cells observed. A ratio obtained

by dividing the number of erythrocytes into the number of

leucocytes decreased with time after the first two days of

injection, however, the ratios exhibited considerable vari-

ability.

METHODS

I. Accumulations‘gf Hexavalent Chromium

 

Procurement and Holding Facilities

Rainbow trout, §Elfl£ gairgflggii, obtained from the
Michigan Department of Conservation Wolf Lake Hatchery, were
used in this study. The fish were transported to the labor-
atory, in one hundred lot quantities in two double thickness
plastic bags, each containing 50 fish.

Fish were held in four 100 liter glass aquaria under
constant illumination at 1&0 i 2°C. in a constant temperature
room. No more than 35 fish were maintained in each aquarium.
Aged tap water with the following properties was used:
total hardness (Schwarzenback titration) 33h mg. of CaCO3
per liter and pH of 8.5 to 8.8. The fish in each holding
aquarium were transferred into clean, aged water once every
three days. This coupled with aeration via a stone air-
breaker and filtration of the water through a gravel, glass
wool, charcoal filter made it possible to maintain the fish
in a healthy condition. Fish in the holding aquaria were
fed dried trout pellets, also obtained from the Michigan
Department of Conservation. One half hour after feeding,

excreta and uneaten pellets were siphoned off the bottOm of

the holding aquaria.

19

20

Experimental Aquaria

The experimental aquaria were kept under the same
conditions as the holding facilities. The water volume in
each experimental aquarium was established and maintained
at no liters. These tanks were aerated and filtered through
a gravel, glass wool, gravel complex. Schiffman (1957)
determined that such a system would not remove dissolved
chromium or hardness from the aquarium water.

The fish were exposed to approximately 2.5 mg./1. of
chromium in the form of KZCrOh. Schiffman (1957) reported
a rise in the hematocrit of fish exposed to potassium chromate
solutions. He stated that ”The hematocrit reached its maxi-
mum rise in value at a point between 2 and u mg. Cr/l." The
2.5 mg. Cr/l. concentration was believed to represent the
near maximum tolerable level in which fish could live for
extended periods of time.

A standard solution of KZCrOh containing 10 mg. Cr/ml.
was used in preparing the experimental aquaria. One milli-
51

curie of Cr was also added to the ho liters of water in

each aquarium. This isotope, supplied by the National
Laboratory, Oak Ridge, Tennessee in the form of CrCl3, was
converted to hexavalent NaZCrOu before adding to the aquaria.
A sample of the aquaria water was then analyzed chemically
by the Saltzman method in order to determine the chromium
content more exactly. Both ash and non-ash procedures were

run periodically in an effort to detect any valence change

21

of the chromium in the water, and data indicated that no
change occurred.

Ten fish were placed in each experimental aquarium.
These fish were removed at the end of the desired exposure
period, the aquarium was allowed to stand for at least 2h
hours, then 10 more fish were placed in the aquarium to be
exposed for a subsequent period. Three aquaria were used
in this manner. Fish were not fed while in the experimental
aquaria; for this reason the study was to be conducted either
until maximum uptake was established or starvation threatened
to be an influencing factor in the results. The decision
against feeding was based on the observation that feeding
trout pellets tended to cloud the water. An increased
biological oxygen demand (B.O.D.) coupled with decomposition

of uneaten pellets or excreta could add to the toxicity of

the water.

[issues Studied
A preliminary study was conducted in an effort to
establish chromium concentrating organs. Fish were exposed
to hexavalent chromium containing Cr51, and all accessible
organs were removed, weighed and counted. The tissues
studied included brain, gonads, urinary bladder, air bladder,
heart, kidney, fin, liver, gill, eye, muscle, skin, stomach,

pyloric caeca, posterior gut with contents, posterior gut

washed, spleen, gall bladder with bile, blood, urine and

22

bone. The kidney, liver, blood, stomach, pyloric caeca,

and posterior gut warranted further study as possible chro-
mium concentrating organs. The posterior gut with contents
was weighed and counted in the final experiment. Although
initially the spleen showed little activity, Schiffman (1957)
reported it as exhibiting a concentrating ability for chro-
mium and it was retained for this reason. The gall bladder
was not counted, since the rate of bile flow would have to
be determined in order to quantitate any such information.
Although it showed no significant activity, muscle tissue
was counted routinely for purposes of comparison. A thorough

study was made of muscle because of its use as a food source.

Preparation of Tissue

After exposure to chromium, the fish were removed
singly, washed in running tap water and anesthesized with
MS-222 (300 mg./l.) (tricaine methane sulfonate - Sandoz).
When fully immobilized, the fish were blotted dry with a
towel, weighed and measured. Blood was removed by heart
puncture using a heparinized syringe fitted with a 27 gauge
needle. The blood was expressed into an aluminum planchet
and weighed. The remainder of the tissues were then removed
in the following order: spleen, liver, gut, pyloric caeca,
stomach, kidney and muscle. The total extraction process
involved approximately 30 minutes per fish. With the ex-

ception of blood, all tissues were weighed using a Roller-

Smith balance having a maximum capacity of 500 mg. A11
weights refer to wet weights. After weighing, the tissues
were placed in previously unused aluminum planchets. All
samples were then dusted lightly with pyrethrum, as a deter-

rent to ants, and allowed to air dry before counting.

Counting Equipment

The counting equipment consisted of the following
components: a two inch thallium treated sodium iodide
scintillation detector assembly, model D55, housed in a
lead castle, model 3036, a radiation analyzer, model 1810,
and a model 183 scaling unit, all manufactured by the Nuclear
Instrument and Chemical Corporation of Chicago, Illinois.
Use of the radiation analyzer enabled differentiation against
all energy levels except that to which it was set. This
instrument, with the window width set at 5 volts, was ad—
justed to produce a maximum count from a Cr51 standard. The
efficiency of the counting equipment was maintained at a
constant level during the total counting period. Using the
Cr51 standard as a source, the equipment was first adjusted
for peak counting efficiency. The standard was counted
twice daily during the entire counting period. If any
significant loss in efficiency occurred, the equipment was

readjusted. All tissues were counted at constant geometry.

Feasibility_of Counting Whole Tissues

Chromium-51 is a low energy gamma emitter (Comar, 1955).

2h

As a result, the possibility of counted activity being
affected by mass differences in the samples was investigated.
Absorption of Cr51 gamma rays by water and lead was studied.
In the study of absorption by water, a tall form, 100 m1.
glass beaker was suspended by the flared lip from the top
shelf of the lead castle. The Cr51 standard was placed
underneath the beaker with a lead collimator placed between
and a count was made. The beaker was then removed, weighed
and 10 m1. of distilled water were pipetted into it; it was
reweighed and counted again. This procedure was continued
in 10 ml. steps until 100 ml. of water had been used as an
absorber. The average inside diameter of the beaker was
determined and the results were expressed graphically in
Figure l, as the leg of activity versus absorber thickness
in g./cm.2 of water. Absorption of gamma radiation by lead
was studied. Varying thicknesses of the absorber material
were placed between the Cr51 sample and the scintillator
tube. These data were expressed graphically in Figure 2,
as the log of activity versus absorber density in g./cm.2.
Absorption of radiation by water is near that of tissue
For the gamma energy of chromium, 0.32 Mev (Nine and Brownell,
1956). As a result, there would be little loss by counting
whole tissue of the size extracted in this study. Four

‘hundred mg./cm.2

of water absorbed approximately 3 per cent
of the activity. This weight corresponded to some of the

larger samples counted during the course of experimentation.

Counts per Minute

25

Figure 1. Absorption of 0.32 Nev gamma energy by water
using a Cr source.

 

3000 -

2000 r'

1000

900
800

"141 ‘r‘

700

600!—

500 P

/

U00:—

300 L—

200L-

d; = 7.1 ge/C'nez

 

 

J J l l l_ J L_
a 6 8 lo

Grams/cm.2 of Water

L

 

Counts per Minute

26

Figure 2. Absorption of 0.32 Mev gamma energy by lead
using a Cr source.

 

2000-

1000'.

80°F.

600+-

hoo #-

200-

100%-

60-

now-

20L- dl = 1.92 g./cm.2

 

l l J l I l l I l l

 

 

’4 6 8 10
Grams/cm.2 of Lead

N

27

Expression of Data

 

For standardization purposes, all data were expressed
as pg. Cr/g. tissue. This was accomplished by counting a
measured sample of the experimental aquarium water in ap-
proximately the same geometry as the tissues were counted.
With the chromium content of the water having been previously
determined, it was possible to express the water activity as

being equivalent to a particular concentration of chromium.

_ Activity_of_fl29_minus background
Activity Of 1 ”3’ Cr _ “Th1. H20 counted)_Tpg. Cr/ml.)
The time of counting was recorded for each sample and

proper decay corrections were introduced into all calcula-

tions.

II. Eliminatigp of Hexavglent Chromium

from Tissues upon Return of the Fish to Fresh Wateg
General procedures were the same, for this experiment,

as were outlined in the chromium accumulation study. Fish

were exposed to 2.5 mg./1. of KZCr510u for 12 days, after
which time they were returned to non-chromated water. Four

or five fish were sacrificed every 5 days during a 25 day

period. This study was conducted to determine whether or
not hexavalent chromium, once accumulated, could be elimi-
nated readily by various organs. The removal and counting
techniques were the same as those followed in the accumula-

tion study.

28

III. Administration of Hexavalent Chromium
Via Stomach Tube
Three fish were given, via stomach tube, .1 m1. of a
solution containing 2.5 mg. KZCrSIOu/l. They were then
returned to normal aquarium water for one day, after which
time they were sacrificed. Tissues were removed and counted
following procedures described previously.

IV. Hexavalent Chromium Concentrations in Fish
with EsoPhageal Occlusions

 

Small corks were adapted, for esophageal occlusion, by
changing their shapes and attaching 2 small barbs to prevent
their expulsion by the fish. With the aid of forceps, the
cork was placed into the esOphagus of an anesthetized fish.
The fish was then placed into an aquarium containing 2.5 mg.
Cr/l. for 1 day. Five fish were prepared in this manner.
After 1 day of exposure, the fish were sacrificed. The
procedures followed in the removal and counting of tissues

were the same as those followed in the previous studies.

RESULTS

I. Accumulations of Hexavalent Chromium

Vean concentrations of chromium, in pg. Cr/g. tissue,
were plotted versus exposure time in days. Open circles of
Figures 3 through 9 represent these mean values. When
amenable, linear regression lines V = a + bX, with the
corresponding .95 two-tailed 100 P6 confidence belts for the
amount of chromium present in a fish after X days of exposure,
were plotted. In Appendix A are found the data from which

this series of graphs was constructed.

Blood (Figure 2)

Statistical treatment of the 85 individual observations
on blood indicated the possibility of a linear regression
during the time of study. The slope, 0.05, was signifi-
cantly different from zero at the .95 limit of confidence
(r = 0.58). With the exception of 1 fish, the chromium
concentration in blood, although exhibiting considerable

variability, never exceeded the concentration of the sur-

rounding water.

Spleen (Figure Q)
The spleen exhibited an increased accumulation of chro-

mium with time. This increase followed a linear regression

29

30

pattern, the slepe of which was significantly different from
zero, at the .95 level of confidence (b = 0.19, r = 0.77)-
Due to a very low RS/RB ratio (sample activity/background
activity), mean values for the first h days, although in-
cluded in Figure h, were not statistically significant at a
5 per cent level of counting error. As a result, these
particular values should not be considered as significant.
The concentration of chromium in the spleen, as indicated

by the mean regression line, exceeded that of the water

medium at 15 days.

Liver (Figure 5)

Chromium concentration in the liver increased with time.
The average concentration, as indicated by the mean regression
line V = -0.33 + 0.h2X, surpassed the average concentration
of the aquarium water by the seventh day. Observations on
the seventeenth and twenty-fourth days exhibited considerable

variability.

Epsterior Portion of Gutg(Figure 6)

Chromium levels in the posterior gut rose rapidly during
the first A days to an estimated level of 6 pg./g., after
which they tended to taper off somewhat. This was plotted
as a linear regression although, by inspection, a curving

line, guided by the plotted mean values, might also have

applied.

31

gyloric Caeca (Figure 7)

This organ accumulated chromium rapidly, reaching a
concentration above that of the aquarium water in slightly
over 2 days time. This rapid uptake continued until about
the tenth day, when the accumulation reached a value of
approximately 12.5 pg. Cr/g. tissue. After 10 days exposure,
the rate of accumulation leveled off considerably, reaching
a value near 15.5 NC. at 2h days. This information was not

plotted as a linear regression line, but rather as a tapering

curve in Figure 7.

Stomach (Figure 8)

The stomach also exhibited a linear type of concentrating
ability for chromium. The concentration of chromium in the
stomach surpassed that of the aquarium water after 11 days
exposure. The mean regression line derived from the formula

A
‘Y = 0.2“ + 0.20X is shown in Figure 8 (r = 0.78).

_§idney (Figure 9)

By the fifth day of exposure, the chromium level in the
kidney exceeded that of the surrounding water. From the
standpoint of radioactive analyses, kidney tissues were ideal
in that they coupled a relatively high degree of activity with
a very uniform degree of geometry. These organs presented
the perplexing problem of a high degree of variability with
time, coupled with relatively low standard errors for the

daily mean values. Consequently, no fitted line was attempted.

32

Figure 3. Chromium Accumulation and Elimination.

 

pg. Cr/g. Tissue

 

 

 

 

 

Blood
K,
Y = .53 + .05X
20’
16-
12’
8-
u-
ég";’r\ """ 6 """"""
‘ T l
Accumulation 1q 1% 2a 25 39 32
Elimination 0 5 10 15 20 25

Time in Days

Figure h. Chromium Accumulation and Elimination.

Spleen

 

= -e20 + .19X

N
O

%

pg. Cr/g. Tissue
H H
m to ox
T l I

c-
I

-"
.0"
a

 

   

 

 

Accumulation lo. 15 20 2; 30 3;

Elimination o 3 lb 15 ab 25
Time in Days

33

Figure 5. Chromium Accumulation and Elimination.

 

   

   

 

  

 

 

 

Liver
9 = -.33 + .uzx
20*
e
5’: w
n16 "x,
d ”’
e ,,-'
“.012!- I””,
7? ’ <>
0
C 8—
w I
1 ."'
h...
J 1.x" 1 1L“
Accumulation 10. 1% 20. 2% 30. 32
Elimination 0 5 10 15 20 25

Time in Days

Figure 6. Chromium Accumulation and Elimination.

Posterior Gut

 

 

 

 

A a
Y = 2.71 + .70x ,I’
20F I,”’o
e ,I” O
a
0316? I’,’
a ,’
F ,’ e ,
h ’I’ 1”,
o ,’
e 8*- v"
m I!
n . . . ’l”
ab -/’
o "I
O 1 x” 1 1 l 1. ______ _____
Accumulation 10| 15. 20' 25' 30. 35.
Elimination O 5 10 15 20 25

 

3b

 

 

 

 

 

 

 

 

 

Figure 7. Chromium Accumulation and Elimination.
Pyloric Caeca
20-—
3
nl6-
n
vi
(.4
élzt-
\
a
o
O 8—
no
:1
1,-
I L I I (
Accumulation 10. 137 20
Elimination o g 1b 13 2b 23
Time in Days
Figure 8. Chromium Accumulation and Elimination.
Stomach
? = .2u + .20x
20—-
3
316-
w
9
$12-
\
h
o
O 8—
M
a
u
Accumulation 1% 15 20 25 30 35
Elimination 0 5 1b 13 25 £5

Time in Days

Figure 9. Chromium Accumulation and Elimination.
Kidney
20- e
3
nl6— .
n
d
E qP
012 .—
'10
\
2;
e 8'— I
to e e
1 e
L”. 1“ .
. . . Ah
I I _Ii I 213__5L_5I_
Accumulation 10, 15I 2% I q 5.
Elimination o 5 10 15 20 25

35

 

 

 

 

Time in Days

 

36

Muscle

It was concluded on the basis of 86 individual samples
that muscle did not accumulate chromium in appreciable
amounts during the course of this study. Due to a rather
consistent count slightly above background, the concentration
of chromium in ug./g. muscle was calculated. These data are
presented in Appendix A, however, it should be stated that
they are of questionable value, except to indicate that
chromium does not accumulate in muscle to any appreciable
extent.

II. Elimination of Hexavalent Chromium

from Tissues upon Return of the Fish tg_Fresh Water

These data, along with the associated standard errors,
are presented graphically in Figures 3 through 9 as shaded
circles. Table 1, in which the per cent of graph estimated
initial values is expressed, summarizes these results. The
elimination of chromium was rapid in most tissues, the
spleen and kidney being notable exceptions. The former lost
little, if any, of its chromium content, even after the fish
had been in fresh water for 25 days. Although the kidney
retained only 50 per cent of its estimated initial activity
at the end of 25 days, a smooth curve drawn through the mean
values tended to become asymptotic, starting at 20 days, to

the Y axis near a value of 5 ug. Cr/g. kidney.

37

TABLE 1

PER CENT OF TOTAL ACTIVITY REMAINING IN CURONATE
EXPOSED FISH AFTER RETURN TO CLEAN WATER

 

 

 

 

 

Days ORGAN

in __

Fresh , POST. PYLORIC ,

Water BLOOD SPLEEE LIXER GUT CAECA STOMACH KIDiEY
5 as 88 no M9 3h 80 102
10 28 39 29 11 32 uz 73
15 37 99 38 3 2 22 60
20 M3 136 17 3 3 20 51
25 25 95 10 2 1 5 5o

 

III. Elimination of Hexavalent Chromium
Administered via Stomach Tube

Over one~half of the dose administered via stomach tube
was eliminated within a 2h hour period, with no appreciable
concentrations noted in any organs other than those of the
digestive system. The greatest per cent administered dose
of chromium, with the exception of the digestive system, was
found in the liver (0.05 i 0.02 per cent). This was followed
by the kidney (0.0a t 0.01 per cent) and blood (0.03 + 0.01

per cent). The pertinent data are presented in Appendix C.

IV. Hexavalent Chromium Concentrations
in Fish with Esophageal Occlusions

Although 5 fish were treated, the occlusion proved
satisfactory in only 2. The 2 successful fish are graphed

individually in Figure 10. The greatest activity was noted

38

Figure 10. Two Fish with E30phagea1 Occlusion Compared
with Mean 2h Hour Values for Normal Fish.

Blood‘

Spleen

Esophageal occluded Liver

fish exposed to 2.5
mg. Cr/l. as KZCrOu.

Stomach
Kidney

Blood
Spleen
Liver
Gut

Esophageal occluded
fish exposed to 2.5
mg. Cr/le as chrouo

Caeca
Stomach

Kidney

Blood
Spleen

Liver

Gut
Pyloric
Caeca

Stomach
Kidney

Mean values of non-
occluded fish exposed
to 2.5 mg. Cr/l. as
K2Cr0u.

Gut
Pyloric‘
Caeca.

 

 

Pyloric‘

 

 

 

 

 

 

 

 

 

I f’ 1 1 y ’
.5 1.0 1.5 2.0 2.5
ug. Cr/g. Tissue

39

in the kidney, followed by the liver. The gut, pyloric
caeca and blood were all approximately the same value.
These results compared favorably with normal fish exposed

to hexavalent chromium over the same period of time.

DISCUSSION

I. Possible Entry Routes of Hexavalent Chromium

In fish, three avenues of entry are available to hexa—
valent chromium: absorption through the skin, ingestion into
the stomach, or entry across the gill membrane.

The possibility of chromium gaining entrance via the
skin would tend to be discredited, due to a consistently low
muscle concentration of chromium, even when samples were
taken adjacent to the skin surface.

Radiochromate, placed directly into the stomach, failed
to be absorbed in significant amounts, thus tending to dis-
credit the theory of uptake via the digestive system. Another
factor Opposing uptake via the digestive system is the theory
that fresh water fishes do not drink appreciable amounts of
water, there being a considerable osmotic force tending to
drive water into the animal (Smith, 1951).

Tissue distributions attained by the esophageal occluded
fish compared favorably with those of normal fish exposed to
hexavalent chromium. With these observations in mind, it is
postulated that the gill is the most likely path for the en-
trance of hexavalent chromium. 'Tomiyama et a1. (1956) re-
ported Caus uptake in goldfish, and Sr90 uptake in carp, as

having taken place via the gills.

ho

U1

II. Transport of Hexavalent Chromium in the Fish

 

As previously mentioned, chromium levels in the blood
rarely exceeded, and were for the most part, considerably
lower than the level of the surrounding water. As a result
of this concentration gradient, entry of chromium into the
fish would be possible by diffusion across the gill membrane.
The linear accumulation patterns with time would also support
this supposition.

Gray and Sterling (1950), after injecting anionic Cr51
into dOgs, noted that approximately 85 per cent of the acti-
vity was concentrated in the erythrocytes within 1 day. The
lack of a substantial increase of activity with time, in fish,
indicated either that chromium was tied up in a complex, and
thus was not free to bind, or that the nucleated fish ery-
throcytes behaved differently than non-nucleated mammalian
red blood cells. This latter point will be explored, by
further research, in the near future.

Regardless of the method, it may be postulated that
chromium is transported by the blood, from which it is re—
moved by certain organs in the digestive, excretory and

reticulo-endothelial systems.
III. Hexavalent Chromium Accumulation in Individual Organs

Fresh water elimination studies showed that blood, liver,

stomach, pyloric caeca and posterior gut lost activity rapidly.

#2

This would tend to indicate that hexavalent chromium did

not bind to blood in appreciable amounts, that the liver
could excrete chromium via bile, and that the stomach, pyloric
caeca and gut could secrete chromium into the lumen, or that
it was already in the lumen. It was noted that there was a
substantial bile flow, along with a full gall bladder, in
most of the fish dissected. Bile probably carries chromium
into the lumen of the digestive system, to be eliminated via
the anal route. The possibility of some chromium elimination
via the gill also exists when the fish is placed in fresh
water.

Kidney and spleen did not lose activity rapidly, upon
return of the fish to fresh water. The spleen exhibited no
significant decrease, even after the fish had been in fresh
water for 25 days. Visek et a1. (1953) reported an increase
in spleen activity in rats with time, after a single injec-
tion of NaZCrSIOh. Expressing their values as per cent of
dose per gram of fresh tissue, they reported 0.91 I 0.16 per
cent at h days after injection, and “.8 I 1.6 per cent at
hz days after injection. This accumulation could be due
possibly to phagocytosis where the chromium was then bound
in the macrOphages. The significant linear regression for
blood, even though the slope was only +0.05 (r = 0.58, N =
85), indicates the probability of some erythrocytic binding
of chromium. The delay in splenic uptake could well be ex-

plained by phagocytosis of aged or defective erythrocytes.

43

Kidney retention was an estimated 50 per cent of the
12 day accumulated value, after 25 days in fresh water, with
indications of little further diminution. Collins (1958),
studying excretion in the dog, postulated that glomerular
filtration and tubular reabsorption were two methods involved
in the renal handling of unbound chromium. If these methods
applied to fish having glomerular kidneys, there is a possi-
bility that bound chromium in the blood is bound to a small
enough molecule, so as to pass into the renal tubule through
Bowman's capsule, thence excreted. If these bonds were bro-
ken, however, then tubular reabsorption could return the free
chromium into the blood stream. The irreversible binding in
the kidney could be due to this free chromium entering the
kidney cells and being unable to leave. The very slow in-
crease in blood activity may be similarly explained by re-
lease of chromium from the kidney. Comar (1955) reported
the kidney to be the critical organ for Cr51, viz, that organ
which receives radiation damage that results in the greatest
insult to the total body. Visek et al. (1953) did not report

as high a chromium retention, in the kidneys of rats, as

observed in this study.

IV. Excretiog of Hexavalent Chgomium

In addition to the possibility of gill excretion of
chromium by fish in fresh water, three possible routes of

excretion exist: the bile, the kidney, and direct excretion

hh

into the lumen of the digestive system. Without collection
of bile, urine, and feces, these routes could not be quanti-
tated. The role of bile and kidney have been previously
discussed.

The fact that the pyloric caeca did not appear to follow
a linear regression pattern in their accumulation of chromium,
focused attention on this organ as a possible source of
excretion of chromium from the body. The estimated slope of
accumulation during the first 8 days was approximately +1.5.
During the first few days, this organ was accumulating chro-
mium at a more rapid rate, gram for gram, than any other
tissue. The tapering of concentration increase with time,
when plotted as mean values of ug. Cr/g. pyloric caeca
versus the 10g of time, produced points that were capable of
being graphed as a straight line. A plot of the reciprocal
of time versus log of mean concentrations produced points,
that with the exception of those for the first 3 days, could
be connected into a straight line, producing a value of 21
ug./g. pyloric caeca when extrapolated to infinity. The
half value of 10.5 mg. was attained at near 8 days. Although
the possibility of a limiting mechanism in the pyloric caeca
exists in reference to chromium, it would seem more plausible
that this organ is behaving in an excretory manner. It would
appear that the legarithmic increase with time represents
chrOmium deposited in the lumina of the various blind pouches

of the pyloric caeca. From here, the chromium could move out

“5

into the main lumen, then into the gut, thus accounting for
the lack of further increases. If the above hypothesis were
true, the 510pe of the first few days should represent the
actual excretory rate of the pyloric caeca. Fromm and
Schiffman (1958) also reported a high chromium concentration
in this area, resulting in severe pathological changes in
the intestine immediately posterior to the pyloric caeca.

The question of chromium secretion directly into the
lower gut must also be considered. If it were excreting
chromium into the lumen, the gut would be expected to exhibit
higher values of chromium than the pyloric caeca. An exami-
‘nation of the concentrations showed no statistical difference
laetween the two, although the gut mean values did exceed

those of the'pyloric caeca somewhat in 6 out of 9 cases.

SUVVARY AND CONCLUSIONS

1. Hexavalent chromium was accumulated in quantities
exceeding the aquarium water in spleen, liver, posterior gut,
pyloric caeca, stomach and kidney. With the exception of the
spleen, these organs are all capable of excretion; thus it
is hypothesized that accumulation in these tissues may be
correlated with excretion.

2. In the rainbow trout, the major route of entry, into
the fish, of hexavalent chromium, is probably via the gills.
Fish with esophageal occlusions demonstrated accumulations of
chromium similar to those of normal fish in chromated water.
Hexavalent chromium given to fish via stomach tube was not
significantly accumulated by tissues other than the digestive
system in 2b hours.

3. Hexavalent chromium probably crosses the gill mem-
brane as a result of simple diffusion. Except for one fish,
the blood concentration of chromium never surpassed that of
the surrounding water.

h. Either this chromium combines into a complex when it
enters the blood, or nucleated fish erythrocytes behave dif—
ferently than do mammalian red blood cells. A very low rate
of increase in concentration was noticed in blood. If ery-
throcytic binding were taking place, a high rate of increase

in activity would be observed.

h6

U7

5. Fresh water elimination studies showed a rapid
decrease of chromium from the blood, liver, stomach, pyloric
caeca and posterior gut. These studies also indicated a
retention of chromium in the spleen and kidney.

6. Possible sites of excretion of chromium from the
body are liver (bile), kidney and pyloric caeca.

7. Based on information obtained, fish caught in
chromium contaminated streams, not exceeding a concentration
of 2.5 ug. Cr/ml. of water, would probably not contain

appreciable amounts of chromium in muscle tissue.

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U9

Chipman, w. A. 1957. Quarterly Report, January-March.
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Hopkins, 8. S. l9h2. General Chemistry for Colleges.

New
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50

Ichikawa, R., and Y. Hiyama. 1956. Deposition of Radio-
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Kawabata, T. 1956. Radiochemical Studies of Contaminated
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KOCh, He Jo, Jre, Ee Re Smith, Ne Fe Shimp, and Jo Connor.
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51

Olson, P. A., and R. F. Foster. l956b. Further Studies on
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52

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pp. 917-936-

 

APPENDIXES

5’4

APPENDIX A

ACCUMULATION OF CWRONIUM

IN ug./g. TISSUE WITH TIME

55

56

ACCUVULATION 0F cwnoxlux WITH TIME
FOLLOWING EXPOSURE T0 2.5 mg. Cr/l.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

BLOOD
EXPOSURE TIME IN DAYS
1 2 3 u 5.2 8.7 lb 17 23.8
0.26 0.93 0.25 0.65 0.82 1.h5 0.48 1.u2 1.7a
g 0.59 1.uu 0.51 1.29 1.52 0.81 0.66 1.85 1.u5
g 0.20 0.72 0.66 0.73 1.08 1.13 1.25 1.81 2.u2
H 0.36 1.21 0.18 0.29 1.06 1.70 1.u1 1.91 1.31
D 0.76 1.5a 0.07 0.1a 0.69 1.01 0.53 2.02 1.97
g 0.62 2.16 0.21 0.29 0.73 0.57 1.05 0.96
ea 0.58 1.2a 0.25 0.37 1.hh 3.28 0.70 2.47
5 0.5M 0.39 0.12 0.33 0.86 0.96 0.80 2.17
. 0.2u 0.5a 0.36 0.52 1.0a 1.u3 0.06
an 0.21 0.26 0.87 0.97 1.38
1 0.91
O.u8
1.98
1.19
Y’ o.uu 1.12 0.29 0.55 1.02 1.28 0.87 1.80 1.81
5 £0.20 £0.56 $0.17 10.35 £0.30 £0.70 io.u0 30.22 10.60
-Std.
Error £0.06 £0.19 $0.05 10.11 20.09 10.19 10.13 20.10 30.21
Coeff.
EEF- 95 50 59 6h 29 55 #6 12 33

 

Data for plotting linear regression line

and .95 confidence interval:
A
Y = e53 + .OSX
r = .58
N = 85

syex = 05317

ACCTNULATION
FOLLOWING EXPOSURE T0 2.5 mg. Cr/l.

0F CHROH 1U“.-

SPLEEN

W ITH T IKE

57

 

 

 

 

 

 

 

EXPOSURE TIME IN DAYS
1 2 3 0 5.2 8.7 10 17 23.8
5 0.30 0.03 0.03 0.30 0.79 0.70 0.13 1.30 5.03
g 0.31 0.30 0.06 0.06 0.90 2.02 1.26 6.68 2.13
a 0.06 0.02 0.30 0.99 0.67 0.56 0.37 2.91 3.73
m 0.71 0.89 0.03 0.25 0.81 0.91 2.28 3.02 5.03
g 0.03 0.00 0.67 0.21 1.07 0.69 3.86 5.96
St 0.06 0.02 0.69 2.30 2.50 3.53
5 0.51 0.22 1.10 1.80 3.51
. 0.02 0.21 1.33 2.29 0.95
m 0.99 0.86 1.32 6.81
1 0.35 1.70
0.21
5.19
1.28
I 0.30 0.29 0.19 0.16 0.83 1.06 2.02 3.63 0.20
s 0.00 10.86 $0.26 10.30 i0.30 $1.30 $2.00 i2.00 11.37
Std.
3§rror $0.01 $0.27 £0.08 $0.15 i0.09 $0.35 $0.63 £0.89 $0.56
(3 ff.
1!::. 12 296 137 188 36 89 83 55 32

 

 

Data for plotting linear regression line
.95 confidence interval:

and

A

Y =

r:

\' —.
. 1‘

S

Y'X

‘e20 +
.77
80

= 1.06

.19X

ACCUVULAIION OF CHROVIPU wITU TIVE

 

 

 

 

 

 

 

 

FOLLO”ING EXPOSURE T0 2.5 mg. Cr/l.
LIVER
ExPOSURE TIME IN DAYS
1 2 3 0 5.2 8.7 10 17 23.8
0.10 0.25 0.30 0.93 1.59 0.28 2.39 3.02 17.98
L. 0e20 Oegu 0e63 0e27 1e52 1.148 Zesu 17e12 2.1“}
g 0.16 0.90 0.68 0.76 1.38 10.79 2.50 9.20 5.65
'H 0.20 1.09 1075 2.07 1.11 1e18 12e33 IOeOl‘ 10e69
H 0.65 1.51 0.20 0.20 0.57 1.93 2.26 10.18 9.90
.g 0.06 1.27 0.60 1.15 0.68 1.30 12.90 3.76
7: 0.00 0.25 0.69 0.36 3.79 2.60 3.19 6.50
C) Oe29 Oe29 0e32 Oe51 1e28 1e80 3e39 8e33
. 0.15 0.16 0.12 0.69 0.82 5.53 3.98
m 0.32 0.03 0.26 1.10 2.55 12.62
1 1.01
1.76
7.08
3.99
E' 0.29 0.70 0.56 0.81 1.02 3.00 5.82 9.91 8.16
s :0.17 10.09 10.07 10.60 11.19 12.80 30.70 15.00 10.80
- d.
::ror t0.05 $0.15 io.15 10.19 $0.38 £0.75 $1.09 12.20 $1.70
0 rt.
V::. 59 70 80 70 80 82 01 50 59

 

 

Data for plotting linear regression line

and .95 confidence interval:
A
r — .72
‘ = 86

ACCUKULATIOY

OF OUR 07-7111”

0' 'T‘T'
..\ I ‘ A

TIVE

FOLLOWING EYPOSURE To 2.5 mg. Cr/l.

POSTERIOR GUT

 

 

 

 

 

 

 

 

EXPOSURE TITE IN DAYS
9 1 2 3 0 5.2 8.7 10 17 23.8
g
0.18 0.52 1.33 7.81 10.00 20.27 8.76 5.65 27.01
S 1.00 1.20 16.55 0.93 5.33 16.89 0.85 19.70 5.03
g 0.52 0.90 9.53 20.08 5.80 7.76 8.18 26.02 6.58
3 2.20 2.92 3.28 0.99 0.09 7.18 22.99 28.86 17.51
m 1.91 1.80 1.95 7.03 0.08 6.07 5.83 17.07 25.62
g 0.56 3.17 3.17 0.67 2.86 9.61 17.18 5.98
. 0.60 1.98 11.77 1.26 13.35 0.96 9.33 10.65
m 0.30 1.25 2.02 0.80 0.37 7.76 9.78 22.01
7: 3.60 5.27 1.72 3.81 3.97 12.30 12.66
0 0.21 0.02 1.67 3.25 23.71 29.23
. 6.90
g 11.56
17.80
13.79
i' 1.16 1.95 5.30 5.95 5.38 12.19 12.88 19.55 18.10
5 £1.29 £1.09 £5.30 16.90 13.90 £6.30 $7.90 $9.00 $9.10
5 d.
E:ror £0.01 £0.07 $1.69 12.18 11.23 11.68 £2.50 $0.02 13.22
c ff.
E;;. 111 20 100 116 72 52 61 06 50

 

 

Data for plotting linear regression line

and

A

Y = 2.71 + .70K

.61

r:

N = 86

.95 confidence interval:

60

ACCUMULATION OF CHROMIUM WITH TIME
FOLLOWING EYPOSURE 70 2.5 mg. Cr/l.

PYLOR IC CAECA

 

 

 

 

 

 

 

 

EXPOSURE TIME IN DAYS
I
§ 1 1 2 3 0 5.2 8.7 10 17 23.8
3 ‘0.12 0.35 2.01 9.02 11.16 17.62 12.90 6.20 23.10
0 0.60 1.56 16.22 0.80 7.51 16.50 0.57 17.81 0.70
g 0.25 2.59 10.22 6.52 9.52 13.72 15.50 19.30 13.10
c 2.25 3.63 3.53 2.72 6.63 0.26 27.26 21.05 15.02
'; 2.36 2.01 3.20 1.00 2.57 11.28 5.26 11.05 16.51
S 0.62 3.01 10.07 8.82 0.18 12.02 13.90 10.70
é 1.55 2.11 13.79 1.83 17.97 15.06 11.50 18.31
is 0.97 0.90 3.35 0.90 0.38 13.98 13.30 20.31
5 1.01 0.07 0.98 0.80 7.62 11.28 13.09
0.28 1.20 1.57 5.56 7.58 17.16
B 3.07
a 8.86
12.80
13.07
E' 1.05 2.23 6.93 0.60 7.50 11.51 13.09 15.25 15.27
5 £0.82 $1.23 £5.96 £3.00 £5.20 £0.20 £6.30 £6.30 $5.80
d.
giro, 0.26 $0.39 $1.88 £0.95 £1.60 £1.12 $1.99 $2.83 12.05
c ff.
v::, 78 17 70 65 69 36 07 01 38

 

 

ACCUMULATION OF CHROMIUU WITH TIDE
POLLOYIXG EXPOSURE TC 2.5 mg. Cr/l.

 

 

 

 

 

 

 

 

 

 

 

 

STOMACH
EXPOSURE TIME IN DAYS
1 2 3 0 5.2 8.7 10 17 23.8
.C
3 0.10 0.25 0.33 1.00 0.97 2.37 0.29 2.32 5.57 3
s 0.08 0.60 1.10 0.20 0.79 2.00 2.17 2.76 2.36 ;
3 0.23 0.61 0.99 1.31 1.07 1.11 2.66 2.63 0.29 -
n 0.89 0.96 1.01 0.66 1.00 1.02 8.51 2.76 5.18
- 0.58 0.60 0.91 0.05 1.09 1.30 2.67 3.03 5.55
if 0.35 0.55 1.00 0.62 1.32 2.23 3.32 0.06
g; 0.51 0.35 0.07 0.01 1.73 1.68 6.08 0.20
0.26 0.35 0.00 0.97 1.02 2.00 1.91 0.66
g 0.57 0.63 1.10 1.02 1.19 2.00 2.89
a 0.16 1.21 0.38 1.02 2.02 9.21
1.00
1.65
2.65
1.81
f’ 0.02 0.62 0.77 0.81 1.26 1.81 0.01 2.70 0.53
5 £0.23 £0.28 £0.35 £0.00 £0.30 £0.08 £2.60 £0.30 £1.00
2:33, £0.07 £0.09 £0.11 £0.13 £0.09 £0.13 £0.82 £0.13 £0.35
Sgiff. 55 1“ 95 09 20 27 59 11 22

 

 

Data for plotting linear regression line
and .95 confidence interval:

A

Y = .20 + .20x
r = .78

N = 86

s - 1.12

ACCUMULATION

I

E

EXPOSURE TIME IN DAYS

OF CHROMIUM

KIDNEY

WITH TIME
FOLLOWING EXPOSURE T0 2.5 mg. Cr/l.

62

 

 

 

 

 

 

Ear.

 

1 2 3 0_‘ 5.2 8.7 10 17 23:§
3 0.68 -1.58 1.07 1.59 2.69 8.00 3.85 15.56 20.35
g 2.03 8.98 9.59 1.81 5.67 5.56 11.83 27.55 15.83
a 1.09 7.35 6.01 3.01 2.57 5.02 0.00 15.12 22.36
x 3.33 7.78 3.25 2.10 0.72 6.29 11.20 16.59 19.08
g 0.81 11.07 0.52 0.89 2.17 3.82 3.67 25.10 16.01
~2 3.70 8.60 1.20 2.10 2.23 7.02 9.73 10.87
5 0.00 2.01 0.82 2.06 6.80 7.56 0.93 13.52
. 2.88 1.35 0.57 1.36 3.53 3.15 0.02 15.08
M 1.00 0.99 1.00 2.60 2.69 7.10 6.21
1 3.32 2.12 0.81 5.13 6.08 7.50
2.77
6.68
13.02
8.33
f 2.73 5.23 2.53 2.27 3.67 6.50 6.60 19.98 16.79
3 £1.00 £3.78 £3.08 £1.20 £1.50 £1.65 £3.00 £5.90 £3.70
3:33, £0.00 £1.20 £0.97 £0.38 £0.07 £0.00 £0.95 £2.60 £1.31
C°eff' 51 23 122 53 01 25 05 30 22

 

ACCU‘-’ULAT ION

OF CW? 03! IUM

wITR TIME
FOLLOWING EXPOSURE T0 2.5 mg. Cr/l.

C\
I.)

 

 

 

 

 

 

 

 

 

 

RUSCLEt
EXPOSURE TIME IN DAYS
1 2 3 0 5.2 8.7 10 17 23.8
C) - " 0.09 " 0e13 Gel-8 "' Gel“v 0.10
'3 - 0e01 OJIO Oe19 0.1“ 0e06 0.09 0.514 OeZO
n - 0.08 0.06 0.03 0.08 0.15 0.00 0.25 0.00
g - 0.09 0.70 - 0.06 0.06 0.13 0.17 0.08
. £0.07 0.02 - - 0.28 0.21 0.17 0.37 0.37
RD 0e10 0e03 0.08 - 0.09 0.20 0e28 Oe19
‘: 0.06 - - - 0.08 0.57 0.20 0.18
0 0.01 - - - - 0.10 - -
t 0.09 - " - " 0.02 0e01
g - 0.00 - 0.06 0.17 0.10
0.21
0.33
f 0.03 0.03 0.13 0.03 0.10 0.16 0.10 0.29 0.10
3 £0.03 £0.01 £0.22 £0.01 £0.10 £0.10 £0.10 £0.15 -
Std.
Error £0.01 - £0.07 - £0.03 £0.03 £0.03 £0.07 -
Coeff.
Var. 107 - 169 - 71 63 71 51 -
*Due to low counting ratios, none of the above values are

statistically significant.

FIE;

. .‘ .' _ _ A

V?" _.,. ,3

we;

APPENDIX B

ELIMINATION OF CHROMIUM WITH TIME FOLLOWING RETURN OF FISH

TO FRESH WATER AFTER 1?. DAYS EXPOSURE T0 2.5 mg. Cr/l.

ELIEINATIOY

OF CUPOWIUM WITH TIME FOLLOWING RETURN OF FISH

C1

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

T0 FRESH WATER AFTER 12 DAYS EXPOSURE T0 2.5 mg. Cr/l.
BLOOD
DAYS IN FRESH WATER

g 0* 5 10 15 20 25
\

{38 1.2 0.63 0.22 0.53 0.50 0.26

,3 0.03 0.30 0.26 0.00 0.36
0L0 0.06 0.02 0.72 0.50 0.28

3 0.36 0.25 0.60 0.31

0.05
I 0.55 0.33 0.00 0.52 0.30
5 10.1111 550.10 $0.23 $0.10 -
Std.
Error $0.19 t0e06 t0e12 t0.05 -
Coeff.
SPLEEN
DAYS IN FRESH WATER

& 0* 5 10 15 20 25
\C

£38 2.1 1.96 1.01 1.23 1.20 1.17

,3 3.18 0.78 2.63 0.03 0.23
Mu: 1.96 0.26 2.91 3.37 5.82

3 0.83 1.53 2.21 0.73

1.33

Y’ 1.85 0.82 2.08 2.80 1.99
5 £0.88 £0.58 £0.82 £1.00 £2.58
Stde + + + 1 4'
Error -0.39 -0.33 -0.01 -0.70 -1.29
Coeff.
Var. “8 71 39 50 130

 

 

*Estimated Initial Value

ELIMINATION OF CHROMIUM WITH TIME FOLLOWING RETURN OF FISH
TO FRESH WATER AFTER 12 DAYS EXPOSURE T0 2.5 mg. 0r/1.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

LIVER
DAYS IN FRESH WATER
é 0* 5 10 15 20 25
\
5 3 0.7 1.50 1.35 0.23 0.86 0.58
.3 2.11 1.99 1.12 0.90 0.52
Up! 1063 0060 1.19 0.88 0.32
3 2.36 0.60 0.55 0.50
1.90
f’ 1.90 1.35 1.79 0.80 0.ua
Std.
Error 20.16 :0.u5 £0.83 £0.08 $0.05
Coeff.
Var. 18 92 21 21
POSTERIOR GUT
DAYS IN FRESH wATER
8 0* 5 10 15 20 25
7:»
o g 11.1 9.75 1.58 0.36 1.11 0.15
. 3.11 1.2a 0.h0 0.09 0.16
g 3.h7 0.72 0.21 0.18 0.28
8.22 0.h6 0.11 0.50
2.71
i’ 5.u5 1.18 0.36 0.37 0.27
3 £3.28 t1.u3 £0.21 :0.u9 £0.17
Std.
Error :1.u7 £0.83 £0.10 £0.20 £0.08
Coeff.
Var. 60 120 58 132 63

 

 

*Estimated Initial Value

67

ELIMINATION OF CHROMIUM WITH TIME FOLLOWING RETURN OF FISH

TO FRESH WATER AFTER 12 DAYS EXPOSURE T0 2.5 mg. Cr/l.

PYLORIC CAECA

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

t“
“ DAYS IN FRESH WATER
O
25: L‘ 0* 5 10 15 20 25
\O .
5 o 13.0 7.29 0.80 0.3u 1.06 0.02
.2 2.72 6.53 0.20 0.21 0.10
g: 5.91 1.68 0.110 0.12 0.16
a 2.86 0.39 0.29 0.23
9 0.02
I 0.56 0.35 0.33 0.u1 0.10
3 £1.99 £5.59 £0.19 £0.h3 £0.10
2:33, £0.89 £3.23 £0.09 £0.21 £0.05
0 . ,
vziff “a 129 58 105 71
STOHAOH
DAYS IN FRESH WATER
t5; 0* 5 10 15 20 25
E:
o a 2.6 3.89 0.90 0.55 0.63 0.21
.3 0.77 1.96 0.62 0.30 0.17
3" 3.85 0.91 0.75 0.u1 0.16
1.10 0.30 1.0a 0.57
0.81
i’ 2.08 1.09 0.56 0.53 0.10 ~
5 £1.63 £1.30 £0.30 £0.32 £0.20
"STE” 1...”... ‘ #‘
Error- i0073 10.75 £0017 30.16 1.0010
c ff.
v::. 78 119 61 60 103

 

 

*Estimated Initial Value

ELIMIYATION

OF CHROMIU“ WITH TIME FOLLOWING RETURN
TO FRESH WATER AFTER 12 DAYS EXPOSURE T0 2.5 mg. Cr/l.

OF FISH

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

KIDNEY
-mfllpfl. .4 __ 4—— i r
DAYS IN FRESH WATER
g 0* 5 10 15 20 25
~\>.
€33 10.6 6.58 7.72 6.01 10.67 5.07
0 12.90 7.10 10.02 5.15 3.01
.33: 12.36 8.53 5.31 0.65 0.93
n 8.07 3.65 0.99 7.06
10.07
§' 10.80 7.78 6.35 5.36 5.32
5 £3.25 £9.02 £2.89 £3.99 £1.67
Std.
Error £1.05 £5.21 £1.00 £1.99 £0.83
Coeff.
12r.. 30 115 06 70 31
EUSCLE
¥_fi DAYS IN FRESH WATER
Q l 0* 5 10 15 20 25
\0
£33 - 0.02 0.00 0.17 0.65 0.03
.3 0003 0003 " 0.10 0033
ME 0005 0003 " 0.18 008,-"
3 0.01 0.01 - -
i’ 0.02 0.03 0.05 0.23 0.30
3 £0.02 - $0.08 10028 $0039
Std' 1 £0.01 - £0.00 £0.10 £0.19
Error __
C°eff’ 100 - 160 122 130
Var.

 

 

*Estimated Initial Value

 

APP END IX C

D ISTRIBUTION STUDIES OF RADIOCHROMATE ADMINISTERED T0
RAINBOW TROUT VIA STOP-I CH TUBE AND DISTRIBUTION STUDIES
OF ’T’YYTZCBTIU‘? I); RATN'SOW TROUT SUB ECTED TO ESOPHAGEAL

C", CLUS IOV .

u\
\C)

.F-‘Iu

Ila.

<11!

DISTR [BUTTON STUDIES OF RADIOCHROMATE ADE-EINISTERED

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

T0 RAINRON TROUT VIA STONAOH TUBE
“.3 ORGAN
O h
‘,3 LOOD L Y L POST. PYLORIO 0v I . M
E.,: 8 SP EEN IVER GUT CAECA ST “A01 KIDNEY .USCLE
O-H O
05"? 0.02 - 0.06 19.00 6.60 10.57 0.05 0.01 "“ =:,.
T
g E 0.00 0.01 0.07 36.03 10.06 1.33 0.00 - 3
c.§ 0.02 - 0.03 15.66 15.66 21.97 0.02 0.02 *
i 0.03 0.01 0.05 23.56 10.91 11.29 0.00 0.01 g
5 £0.01 £0.01 £0.02£10.95 £0.55 £10.30 £0.01 £0.01
Std. _‘-’_H ‘I’ .
Error - .'--:- -t0001 16.33 12063 t5.98 - - Eur . 2|
Coeff.
Var, 33 50 '40 [£7 ’4'2 92 25 100
DISTRIBUTION STUDIES OF CHROMIUM IN RAINBOW TROUT
SUHJEOTED TO ESOPHAGEAL OCCLUSION
5 ORGAN
7:5 TLOO L POST’ PYLORIC OHAO IDN x1~ CL
C 3 3 D SPLEEN IVER GUT CAECA ST H K .EY .US E
‘30 0033 0009 0050 0032 0.32 0.12 1.1L9 -
3. 1 0.28 " 0.36 0028 0031‘ 0.11 1013 0001

 

APPENDIX D

STATISTICAL FORMULAS

71

 

 

72

STATISTICAL FORVULAS*

Sasic Conversion Formulas:
x = X - I
2
2x2 FXZJEX)
- __?r__.
2
2_-2 (EY)
2" ’2" T—

} xy 2 XY - firmné Y)

Linear Regression Formulas:

4
II

a + bX (Equation for a line)

b = 2 xy (SlOpe of a line)

I

a=;Y;b2X

5 xy
W2 XZHE Y2)

Linear Regression Confidence Limit Formulas:

 

(correlation coefficient)

 

 

 

 

 

Upper two-tailed 100 P% confidence
belt for amount of chromium present

2 in a fish after X days of exposure.

A
Y - SY t1+p = Lower two-tailed 100 PT confidence
‘5“ belt for amount of chromium present

in a fish after X days of exPosure.

*Statistical procedures were suggested by Dr. P. J. Clark,
ZOOlOgy Dept., M.S.U.

11

S]
I".