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Toxicity for cats of methylmercury in contaminated fish from Swedish lakes and of methylmercury hydroxide added to fish
RESEARCH 5, 425-442
ENVIROSMENTAL
Toxicity
for
Swedish
lakes
L. ALBANUS, G.
Cats and
of
(1972)
Methylmercury of Methylmercury
L. FRANKENBERC, NORDBERG,
M.
in Contaminated Hydroxide
Fish Added
from to Fish
C. GRANT, U. VON HAARTMAN, A. JERNEL~~, A. SCHULTZ, AND S. SKERFVING~
RYDXLV,
Research Institute of National Defense, S-172 04 Sundbyberg, Department of Pathology, Karolinska Hospital, S-104 01 Stockholm, Water and Air Pollution Research Laboratory, S-114 28 Stockholm, Department of Environmental Hygiene, Karolinska Institute, S-104 01 Stockholm, Department of Occupational Medicine, University Hospital, S-221 85 Lund, Food Research Department, National Food Administration, S-104 01 Stockholm, and Department of Hygiene, UniveTsit!/ of Lund, S-220 02 Lund, Sweden Received
March
9. 1972
To assess the toxicity of methylmercury accumulated in fish, five cats (Group 1)‘ were fed a homogenate of pike from a mercury-contaminated lake. Five other cats (Group 2) were fed a homogenate of pike from a “noncontaminated” lake, but with methylmercury hydroxide added to the homogenate. The methylmercury level in both homogenates was about 6 mg Hg/kg and the exposure of the cats about 0.45 mg Hg/kg body weight/day. Control cats (Group 3) were fed solely on noncontaminated pike. The exposure period to clinical onset was 60-83 days. The overall clinical and neuropathological patterns were consistent with methylmercury poisoning and similar for Groups 1 and 2. More than 90% of the mercury was absorbed of which at least 20% was incorporated in the hair. Mercury levels in the brain constituted about 1% of the total body burden (fur excluded). Total mercury levels in brain, liver, kidney, muscle, blood cells, and plasma averaged at termination 18, 39, 31, 27, 55, and 0.7 @g/g, respectively, with only minor differences between the two exposed groups. In brain and muscle about 100% of the mercury was recovered as methylmercury, while in kidney and liver the corresponding figures were 62 and 80%. The results demonstrate that toxicity studies with simple methylmercury salts give results comparable with those obtained with methylmercury “naturally” accumulated in fish. INTRODUCTION
The basis for evaluating maximum acceptable exposure to methylmercury is. epidemiological data from human beings E mainly after poisoning by methylmercury-containing fish and shellfish in Japan together with some cases of industrial exposure to methylmercury salts-and experimental data obtained by the administration of methylmercury salts to human beings and animals (cf. Swedish Commission Report, 1971). Toxicologically, methylmercury as it is present in fish has been assumed to be equivalent to methylmercury salts. This study is an 1 The ordinator Medicine,
paper is submitted as part of doctoral thesis by S. Skerfving, for this project. Reprint requests to Dr. S. Skerfving, Department University Hospital, 5-221 85 Lund, Sweden. 425
Copyright All rights
@ 1972 by of reproduction
Academic in any
Press, Inc. form reserved.
who
served as coof &cupationa].
ALBANUS
ET
AL.
TOXICITY
OF
MERCURY
IN
FISH
FOR
CATS
427
attempt to test the validity of this assumption by comparing the toxicity for cats of methylmercury in fish from a heavily contaminated Swedish lake and a methylmercury salt added to noncontaminated fish. MATERIALS
Labeled Methylmercury
AND METHODS
Hydroxide
[203Hg]Methylmercury hydroxide was obtained from mercury( ‘O”Hg) oxide by an isotope-exchange procedure (Berlin, 1963) and purified by Ostlund’s method, step 1 (1969). The purity of the Iabeled methylmercury hydroxide was checked by thin-layer chromatography (Westbii, 1966) and by the distribution of radioactivity, at repeated shakings, between benzene phases and 2 N hydrochloric acid phases. The content of inorganic mercury in the solutions used for preparation of fish homogenates was less than 1%of the total mercury. For specific activity see below. Fish Homogenate 1. “Contaminated” pike (Esox Zucius) were caught in 1969 in a Iake system (DelHngersHn) in Sweden. A paper mill had discharged phenylmercury acetate into the lakes between the 1940s and 1966.?The fish was frozen and shipped whole to the laboratory. Head, gills, fins, abdominal organs, and kidneys were removed. The fish was homogenized in a mincer and blended in a dough blender at about 4°C. A trace amount of [20”Hg]methylmercury hydroxide in water solution and with a specific activity of 0.15 Ci/g was sprayed as an aerosol over the homogenate during blending. Loss of radioactive material during the spraying procedure was negligible. The amount of methylmercury added corresponded to 0.6% of the mercury present in the homogenate. The radioactivity of the homogenate was 5.2 ,&i/kg at the start of the experiment, and the coefficient of variation between different samples 9%. Samples of about 100 gs were taken from different parts of the homogenate and after renewed homogenization were analyzed for total mercury and methylmercury (Table 1) and for energy and nutrients (Table 2). The homogenate was frozen in 2-kg blocks until fed to the cats. Homogenates 2 and 3. “Noncontaminated” pike were caught in lake Storhjalmaren in central Sweden. The noncontaminated fish was prepared in the same way as the contaminated fish. One half of this batch was sprayed with [20”Hg]methylmercury hydroxide having a specific activity of 0.92 mCi/g and formed Homogenate 2 (5.2 ,&i/kg). The mercury level obtained was close to that of Homogenate 1 (Table 1). The other half was frozen without further treatment and formed Homogenate 3, the control diet (Table 1). ’ Microbial transformationhas been assumedto explain the accumulationof mercury fish as methylmercury (Jensenand Jernebv,
1969).
in
428
ALBANUS
ET
TABLE LEVELS
OF ENERQY
AND NUTRIENTS
(standard methods of analvsis. National
AL.
2 IN HOMOGENATE OF PIKF. Swedish Food AdministrationY
Per kg Physiological energy, MJ (kcal) Wat,er, g Protein, g Fat, g Calcium, g Phosphorus, g Iron, mg Vitamin A, IU of which Vitamin AZ, IU Biologically active carotine, mg Thiamin, mg
3.5 (840) 780 180 9b 7.2 6.4 8 4000 2000
0 Supplemented with retinol palmitate, 1400 IU., Vitamin A, calciferol, 280 IU, vitamin D, tocopheryl acetate, 4.3 mg, thiamin chloride, 0.6 mg, riboflavin, 0.3 mg, pyridoxin chloride, 0.3 mg, and pantothenol, 2.8 mg, per cat per day (cf. Scott, 1964). Each cat was given 50 mg iron as a sorb&o1 citric acid complex intramuscularly at the beginning of the experiment,. Once weekly each cat was given an additional supplement of 100 mg tocopheryl acetate. * No peroxides detected.
ExpeTimenta~ Animals Fifteen European short-haired cats, bred and raised under standard conditions at the Research Institute of National Defence, 11 months old and weighing 2.33.9 kg at the beginning of the experiment, were divided into three experimental groups, each containing three females and two males. The animals were kept in separate cages at night and in two big common cages with outdoor runs during daytime, one for each sex. Feeding The cats were fed the fish homogenates as their only food together with water. A weighed portion of frozen homogenate was placed in the cage of each cat in the evening. The portions were adjusted to be consumed completely. If any remnants were left, they were weighed in the morning. All cats were fed Homogenate 3, the control diet, for an initial period of 55 days. Then Group 1 was fed Homogenate 1, Group 2 Homogenate 2, and five control cats (Group 3) Homogenate 3. Each cat was supplied daily with a combined vitamin solution (Table 2). Examination and Sampling The cats were observed daily and weighed twice a week. A modified McGrath ( 1960) system was applied to each cat twice a week to test the common reflexes. A standard protocol specifying 22 items was used to check systematically the general behavior, carriage, and activity of the cats, their movements, their ability to climb and jump up as well as down, the righting and placing reflexes, position and movements of the head and ears, spontaneous eye movements and the fixing
TOXICITY
OF MERCURY
IN
FISH
FOR
CATS
429
reflex,. the blink reflex, and the pupillary response to light. The cats were checked by two persons familiar with the animals. Once deviations from the normal were observed for any cat it was examined daily. Every 2 weeks each cat was filmed while performing standard movements-getting up, walking, jumping, and a rough test of the peripheral visual fields. Whole-body radioactivity measurements were performed once a week. Venous blood (about 3 ml) was taken once every 2 weeks and immediately prior to killing. BIood cells were separated from plasma by centrifugation for I.5 min at 12,000 X g in an Eppendorf microcentrifuge 3200. Autopsy and Pathological Examination The cats were killed when they showed definite signs of poisoning, i.e., convulsions. A modified Cammermeyer technique (1960) was used. With the cats in barbiturate anesthesia the aorta caudal to the origin of the renal arteries and the Vena cava caudalis were cannulated. After retrograde flushing through the aorta with 2-3 liters of heparinized 5.6% gum. arabic. in 0.9% NaCl solution at a pressure of 200-250 mm Hg, neutral formol-calcium solution under the same pressure was connected to the system and the fixative (2-3 liters) was then perfused through the cats until the efflux consisted purely of fixative. The cats maintained cardiac and respiratory movements until actua1 fixation was begun, about 20 min after the first abdominal incision and 2-3 min before completing perfusion. Samples of tissues (see Tables 5 and 7) were taken for immediate radioactivity measurements. In a separate experiment, it had been checked that perfusion did not significantly alter the level of radioactivity. Samples of liver, kidney, and skeletal muscle were frozen for subsequent mercury analyses. The cats were skinned and the pelt cleaned from fat and connective tissue. Radioactivity was determined in the whole pelt, and after clipping, in the skin and in the total amount of hair. Blocks of tongue, pharynx, larynx, thyroid and parathyroid, trachea, lung, thymus, myocardium, liver, pancreas, spleen, adrenal, kidney, stomach at several levels, duodenum, jejunum, ileum, cecum, colon, mesentery with lymph nodes, skeleta1 muscle, rib, and femoral bone marrow were taken for microscopical examination. The gonads were fixed in weak Bouin’s solution. All tissues were stained initially with azure A eosinate at pH 6.5. Frozen sections of myocardium, liver, kidney, and adrenal were stained with oil red 0. The skinned skull and intact spine were Ieft intact in neutral FormoI-calcium fixative for a week. There was no detectable loss of mercury to the neutral fixative. The brachial plexus with the median nerve and the (nonperfused) sciatic nerves were dissected free, straightened, and allowed to dry slightly on pieces of cardboard before being immersed in formol-calcium. After fixation, blocks of spinal cord together with adjacent nerve roots and dorsal root ganglia were taken at the levels of the cervical and lumbar intumescentia. The perfused eyes were removed from the orbit and postfixed in weak Bouin’s solution before trimming and sectioning. Radioactivity of the whole brain was measured after fixation and removal from
430
ALBANUS
ET
AL.
the skull. The brain was then divided in the midline. The left half was analyzed for total mercury and methyhnercury. The right half was sectioned transversely at 2-mm intervals from the olfactory lobe to the medulla oblongata in a Plexiglas miter box and marked so that the resulting microscopical sections represented even coronal steps and that corresponding sections from different brains could easily be compared, Brain and other tissues fixed in formol-calcium were prepared for microscopy by washing in running water overnight and subsequent dehydration in increasing concentrations of ethanol at intervals of at least 24 hr. The tissues were cleared in methylbenzoate, washed quickly in xylol, and embedded in Fibrowax under vacuum. The brains were sectioned primarily at 8 pm and stained with azure A eosinate at pH 6.5 and with Nissl’s cresyl violet. Other tissues were sectioned at 4-5 pm. Radioactivity
Measurements
The radioactivity in samples of fish homogenates, blood cells, plasma, and organs was measured by gamma scintillation counting against standards of the same geometry in a 3 x l-in. NaI well-type crystal connected to a three-channel gamma-spectrometer with autogamma unit (Model 3375, Packard, Downers Grove, IL). The efficiency of the measurements was about 40%. The statistical counting error was always 1% or less. The coefficient of variation between two measurements of the same sample on different occasions was 6%. The amounts of mercury were calculated from the specific activity in the homogenates (including methylmercury originally present and added). The set-up for whole-body counting is shown in Fig. 1. The detector was a 3 X l-in. well-type crystal shielded by lead, connected with the gamma-spectrometer described above. The counting efficiency was 0.03%. The amount of mercury was calculated by comparison with a standard consisting of the body of a cat, which had received an intravenous injection of 20 mg mercury as [20SHg]methylmercury hydroxide (a total of 18 &i) and had been frozen in a suitable crouching position. Each cat was monitored at least twice on each counting occasion. The coefficient of variation for 10 repeated countings of the same cat was 12%. The statistical counting error was always 1% or less. Whole brains, whole pelts, skins, and fur clippings were measured against standards of the same geometry in a large volume detector consisting of a cylindrical tank (8 x 16 in.) containing liquid scintillator and equipped with six 3-in. photomultiplier tubes. This equipment, giving essentially 4 r geometry, was connected to a gamma-spectrometer (Model 3375, Packard). The efficiency was about I4%, the coefficient of variation at duplicate measurements was 7% and the statistical counting error always 1% or less. Analyses
of Total Mercury
and Methylmercury
All glass was first cleaned by ordinary laboratory procedures, then by shaking with 1 M ammonium hydroxide solution, and then by deionized water. Plastic vials used did not contaminate water kept in them for 1 month. Samples of fish homogenate, brain, liver, kidney, and muscle were homogenized
TOXICITY
OF
MERCURY
IN
FISH
I 250
FOR
4:31
CATS
LEXIQLASS YLINDER
!
---LEAD
SHIELD
--.DETECTOA
FIG. 1. Set-up in millimeters.
used
for
measurement
of whole-body
radioactivity.
Figures
denote
distances
under cooling in an Ultra-Turrax homogenizer (Janke and Kunked, Staufen, W. Germany). Fractions of the samples of brain, liver, kidney, and muscle were freeze-dried (Kylteknik AB, Huddinge, Sweden). No detectable losses of radioactivity occurred during the drying procedure. The samples were kept frozen until analyzed. Total mercury level was determined by neutron activation analysis (Sjijstrand, 1964) and atomic absorption spectrometry (Schiitz, 1969) on dried samples. Methylmercury analyses of fresh fish homogenates were made by the method of West% (1967 and 1968) and Westi% and Rydglv ( 1971), of fresh brain b!; Westiiij (1970), and of liver, kidney, and muscle by Westi% (1967 and 1968). The samples analyzed represented 0.1-0.5 g wet weight. At least duplicate samples were analyzed for total mercury by atomic absorption spectrometry and for methylmercury. In the range 20-30 pg Hg/kg the coefficient of variation of 16 duplicate analyses of fresh tissue for methylmercury was 9%. The corresponding figure for analyses of total mercury by atomic absorption in nine duplicate
432
ALBANUS
ET AL.
analyses of dried samples in the range 60-80 pg Hg/g was 5%. In both cases possible lack of homogeneity of the samples is included in the error. RESULTS
Clinical
Signs
Fish consumption and mercury intake calculated as an average for the whole exposure period have been summarized in Table 3. One of the original control cats (No. 34) refused to eat. It was taken out of the experiment after 21 days and killed for examination. Since the prime object of this study was to compare Group 1 and Group 2, we shall confine ourselves here to a general description of the clinical observations. A more detailed analysis of the clinical signs in relation to pathological lesions and mercury levels in the individual cats is to be published separately. All cats in Groups 1 and 2 developed convulsions. For nine of the 10 cats the first convulsions occurred between Days 60 and 73 and for one (No. 13 in Group l), on Day 83. There was no significant difference between the two groups as to the length of the exposure period to the onset of convulsions. Changes in behavior and movements were noticed in seven cats prior to the onset of convulsions, four in Group 1 and three in Group 2. With one exception the changes were first observed 4-11 days before the convulsions. For one cat (No. 13 in Group 1) th ere was from about Day 50 a long period with low food consumption, declining body weight, and ataxia of steadily increasing severity until the onset of convulsions on Day 83 (Fig. 2), The observed behavioral and locomotory signs were wariness, aggressiveness, affectionateness, and a stiff, waddling gait, particularly of the hindlegs, progressing to distinct ataxia. No definite evidence of visual impairment could be detected, at least as long as the cats could be adequately examined i.e., up to onset of convulsions. The convulsions, whether or not preceded by behavioral and locomotory signs, followed a common pattern. Brief tonic spasms were followed by clonic spasms involving the limbs, trunk, neck, and head and were accompanied by profuse :salivation, dilatation of the pupils, and piloerection on the tail but not by urination or defecation. After 10 set to 2 min the convulsions gradually subsided. Once convulsions began, their frequency was roughly proportional to the duration and severity of the preceding clinical signs. Reviews of the films made during the exposure period did not result in the ,detection of any signs not noted earlier.
FISH
CONSUMPTION
AND
Intake Fish, kg/day/cat mg Hg/kg body
MFJTHYLMERCURY WHOLE EXPOSURE
TABLE 3 INTAKE CALCULATED AS AN AVERAGE PERIOD (MEAN f SEM)
Group
weight/day
0.28 0.45
1
+ 0.04 + 0.03
Group 0.31 0.47
2
+ 0.02 f 0.02
FOR TEE
Group 0.34 0.017
3
l 0.03 f 0.004
TOXICITY
OF MERCURY
IN
FISH
FOR
433
CATS
There were minor differences between different cats in the clinical signs, but variation within each group was as great as between groups. None of the controls (Group 3) showed any neurological signs. In all animals of Groups 1 and 2 there was a decrease of body weight during the last days of exposure (Fig. 2). In some there was a leveling off of body weight or a decrease before any neurological signs had been noted, in some cats even 1 month before other signs occurred. The total amount of mercury as methylmercury ingested when convulsions occurred was 30 ? 1 mg per kg body weight (mean -C SEM) for Group 1 and 35 t 2 for Group 2. Pathological
Findings
The pathological findings have been briefly reported (Grant, 1972) and, together with the clinical findings, will be described in detail elsewhere. The relevant changes were limited to the central and peripheral nervous systems. On the whole, there was no essential qualitative or quantitative differences between the cats of Groups 1 and 2. The quantitative differences between cats within BODY WEIGHT kg
“1
t 13P
GROUP 1
GROUP 2
1310 GROUP 3
0
20
LO ’
60
90
100
DAYS OF EXPOSURE
FIG. period. number
2. Body weight of the cats on control homogenate for 55 days and Onset of neurological signs (ataxia, convulsions, or both) is indicated and sex of the animals are indicated.
during exposure by arrows. The
434
ALBANJS
ET
AL.
the same group were greater than those between the groups. No relevant changes were encountered in the nervous system of the control cats (Group 3). The basic pathological pattern consisted of degenerative changes in the granular layer of parts of the cerebellar cortex, the peripheral nerves and their dorsal roots, and in the cerebral cortex. In the cerebellum, degenerative changes were extensive and remarkably uniform both within and between the exposed groups. Degeneration within the granular layer of the cortex followed the known pattern for methylmercury intoxication (Swedish Commission Report, Chap. 8, 1971). The topographica distribution was highly selective with preferential involvement of the midline and basal areas-the lingula, nodulus, and uvula together with the basal medial areas of the anterior, medial, and ansiform lobes. In the peripheral nerves and their dorsal roots degenerative changes involved segments of nerve fibers scattered among intact fibers. While quantitatively relatively slight, the pattern of degeneration was the usual one of swelling and fragmentation of the axon and the myelin sheath. There were no obvious group differences. In the cerebral cortex, the damage involved scattered small foci in practically all cortical regions except the tips of the frontal lobes, the olfactory cortex, and the pyriform lobe. The focal cortical damage involved particularly the deeper TOTAL BODY BURDEN w
Hg
100 -I
* = GROUP 1 (MEAN -
AND RANGE)
DAYS
FIG. 3. Whole-body retention From Day 64 on cats were killed
of mercury as calculated from as they developed convulsions.
OF EXPOSURE
radioactivity
measurements.
TOXICITY
OF MERCURY
IN
FISH
FOR
CATS
435
layer in lamina II and lamina III. Neuron changes were predominantly of Nissl’s “ischaemic” type with shrinkage, loss of cytoplasmic detail with increased affinity The most impressive change in the for basic stains, and nuclear pyknosis. damaged areas, however, was microgliosis with formation of rod cells. Perivascular cuffs of lymphocytes were often encountered in or near the damaged areas. It must be emphasized that the cerebral cortical damage involved numerous small areas scattered apparently at random throughout most of the cortical areas. There were wider individual differences between cats within the same group in this respect than between the groups. Analyses
of Total Mercury
and Methylmercury
The accumulation of whole-body radioactivity was parallel in the two experimental groups (Fig. 3). The considerable range of whole-body radioactivity of the animals in Group 1 early in the experiment was due to one cat (No. 13), which consumed less homogenate than the others. Groups 1 and 2, respectively, retained within the body or in the fur, 96 2 2 (mean + SEM) and 93 I 2% of the dose administered during the first week. The accumulation of mercury in blood cells and plasma was similar in Groups 1 and 2 (Table 4). Th e average blood cell levels increased 50-70 times and the plasma levels about 40 times, the ratio blood cell/plasma being about 55 at the start of exposure and about 75 at the end. There was a good agreement between radioactivity measurements and neutron activation analyses. In the controls (Group 3) the blood cell 1eve 1 rose about three times and the plasma level about two times, indicating that the exposure from the control homogenate exceeded that of the diet prior to the experiment. With the three methods used (atomic absorption, neutron activation, and radioactivity) analyses of total mercury in brain, liver, kidney, and muscle showed a general agreement, although there were some systematic differences in both experimental groups (Table 5). As an overall average for Groups 1 and 2 the atomic absorption and radioactivity measurements gave levels corresponding to 113 + 2 (mean & SEM) and 111 + 3% of those obtained by neutron activation. Both values differ significantly from 100% (P < 0.001). The radioactivity measurements do not include the mercury present at the start of exposure. This, however, constitutes only a small fraction of the total mercury. In liver. kidney, and muscle there were no conclusive differences between mercury levels in Groups 1 and 2 (Table 5). This was true for both total mercury and methylmercury. However, the brain concentrations differed slightly but were statistically significantly in regard to total mercury and methylmercury levels as well as specific activity of ‘““Hg (in Table 5 relation between chemical determinations and mercury levels based on radioactivity). The methylmercury levels obtained in Groups 1 and 2 made up 106 + 3 (mean + SEM) % of the total mercury (mean of atomic absorption, neutron activation. and radioactivity analyses) in muscle, 80 +- 3% in liver, and 62 + 3% in kidney (Table 6). In brain the methylmercury percentage was lower in Group 1 than in Group 2 (91 +: 4 vs 116 + 6%). The methylmercury percentage based on both groups was I.04 + 5%.
0.60
3
f 0.15
f 0.22
+ 0.16
blood cells
Day
before
f SEM)
0.022
0.021
0.018
b&g
onset
Group
radioactivity
than
0.004
0.004
64-86,
*
f
& 0.003
plasma
OF TOTAL MERCURY AND RADIOllCTIVITY
in Group 1 were killed on Days four values. three values. activation analyses were higher
1.1
2
0 The animals * Based upon c Based upon d The neutron
0.79
rglg
-
(MEAN
1
Group
LEVELS
71-80,
measurements
2 on Days
zk 1.8) * 0.28
(P
and
< 0.05).
Group
3 on Days
77-94.
+ 5.3) $- 0.20
(54 1.7
blood cells
(41 f 2.0) 1.1 f 0.03
dg
(26 1.4
cells
56 + 1.6b (55 f 1.9) 56 f 4.8
blood
52-53
39 zk 3.7 (42 + 4.2) 39 I!I 3.9
pg/g
of exposure
ACTIVATION
27 + 1.7 (27 + 1.9) 24 + 1.6
24-25
Days
TABLE 4 IN BLOOD CELLS AND PLASMA. NEUTRON MEASUREMENTS (WITHIN PARENTHESES)
(0.60 0.038
0.76 (0.73 0.71
fig/g
At autopsy@
ANALYSIS,
+_ 0.034) f 0.008
f 0.047 + 0.026) f 0.049d
plasma
F
fl
$ s
TOXICITY
OF MERCURY
IN
FISH
FOR
437
CATS
I
N
z E R
’
I
I
438
ALBANUS
METHYLMERCURY IN PERCENTAGE ATOMIC ABSORPTION, NEUTRON Group 1 2 3
ET
AL.
TABLE 6 (MEAN + SEM) OF TOTAL MERCURY (AVERAGE ACTIVATION, AND RaDIOACTIVITY MEASUREMENTS)
Brain”
Liveti
Kidney*
91 f 3.8 116 + 5.7 73 f 2.F
78 Tk 6.6 81 f 0.5 52 f 8.4
OF
Muscle
62 f 6.4 62 &- 3.1 42 + 2.2
a Group 2 < Group 1 (P < 0.01); Group 2 (P < 0.05) and Group different from 100%. b All groups statistically different from 100% (P < 0.001). c Based upon three values.
105 + 4.1 108 k 4.6 102 + 6.4c
3 (P < 0.001)
st,atistically
Total mercury levels, as calculated from radioactivity measurements on myocardium, spleen (free of blood), and the gastrointestinal tract (Table 7) were comparable to those in the brain, but lower than in liver, kidney, and muscle. At the end of the experiment more than half of the total dose of mercury was retained in the body (Table 8) and about half of the retained amount was present in the fur. Less than 1% of the total body burden (fur excluded) was present in the brain. DISCUSSION
This study demonstrated that (1) cats fed methylmercury-containing fish from a contaminated lake (Group 1) developed clinical signs and pathological lesions consistent with poisoning with a simple methylmercury salt (Group 2)) and that (2) the toxicity and mercury distribution were similar in the two groups. Thus,
TOTAL
MERCURY
TABLE LEVELS IN ORGANS FROM RADIOACTIVITY
Organ Myocardium Thymus Pancreas Spleen Ovary Testis Duodenumc Jejunumc Ileumc Colonc Cecumc Bile a Based upon three values. b Group 2 < Group 1 (P < 0.05). c Washed free of contents.
7 (MEAN + SEM) MEASUREMENTS
Group (/*g/g) 19 f 9.6 + 6.7 + 23 f 16 + 8.6; 17 + 17 * 16 -t 17 f 17 * 4.4;
I
1.1 1.5a 1.0” l.O* 3.0” 10 1.5 1.9 1.5 0.68 1.7 4.9
AS CALCULATED
Group b&d
2
20 * 7.8 f 7.5 + 17 f 13 + 7.8; 19 + 15 f 16 +16 k 20 f 8.5 +-
0.49 0.78 0.87 1.V 0.580 7.3 1.9 2.0 0.86 1.5 1.9 1.5
TOXICITY
OF MERCURY
IN
FISH
FOR
439
CATS
TABLE 8 MERCURY
IN
WHOLE
BODY,
I~ADIOACTIVITY
PELT,
FUR,
AND
MEASUREMENTS
BRAIN
(MEAN
AT
TERMINATION
+
SEM) OF
(koup Total dose administered (TD), mg Hg Whole body burden (WB), yQ of TI) Pelt, yfi of WB Fur, pg Q/g Fur (F), yOof WB Whole body without fur (WB-F), s, of TI1 Brain, TC,of (WB-F)
104
+
1 14
77 +_ 5.6 54 + 6.2 310
*
28
27 + e5.6 55 + 5.4 0.81
+ 0.08
9s
CALCULATED
FROM
EXPOSURE
Group 2 132
+ 8.0
64 62 3x0 34 42
* + + + f
3.5 3.4 45 2.4 3.3
*
0.05
0.88
the use of methylmercury salts appears to be generally relevant in assessing the toxicity of methylmercury in fish. In a collaboratory study, Albanus et ai. (1970) fed two cats homogenate of contaminated pike (exposure 0.4 mg mercury per kg body weight per day as methylmercury). Since no methylmercury compound, radioactive or inactive, was added to the homogenate, these cats constitute a suitable “inactive” comparison group for the cats in Groups 1 and 2. Total intake of methylmercury to onset of convulsions (26 and 30 mg mercury per kg body weight; 70 and 80 days), total mercury levels in the brain ( 19 pg/ g ) , and clinical and neuropathological patterns were fully comparable with the results for the cats in Groups 1 and 2. The hematological and blood chemistry studies on the “inactive” cats as well as their concurrent dietary controls gave essentially normal results. The methylmercury which had accumulated in the pike fed to the cats in Group 1 had been synthesized, probably by microbial activity (Jensen and Jerneliiv, 1969), from an arylmercury compound (phenylmercury acetate) discharged as waste into the water. The major epidemics of methylmercury poisoning among Japanese through consumption of fish and shellfish have been attributed to the discharge of methylmercury as such and its accumulation in food chains (Niigata Report, 1967; Minamata Report, 1968). The admittedly incomplete data available for cats fed shellfish from Minamata (Kai, 1963; Yamashita, 1964; Kitamura, 1968; Takeuchi, 1968) and data on cats fed methylmercury proteinate (Pekkanen, 1969; Rissanen, 1969)) are compatible with the observations made in this study. In the general context of methylmercury poisoning, the cats again provided evidence for primary neurotoxicity. The pattern of cerebellar damage fits in with what has been described by Takeuchi (1968) for cats. The topographical distribution of damage observed in the present study has not been pointed out previously, presumably because the method of examination used here permits a more systematic evaluation. In earlier studies examination of peripheral nerves and their dorsal roots has apparently not been carried out, or at best reported ambiguously, for cats as well as other species, including human beings, with few exceptions (cf. Swedish Commission Report, Chap. 8, 1971). When clearly reported, however, the damage observed fits in with what has been seen in the cats in this study, The damage in the cerebral cortex, however, poses problems
440
ALBANUS
ET
AL.
of interpretation. The nature of the cortical damage in the cats deviates from what would be expected from the general description by Takeuchi (1968) butpresumably because of inexact translation-is not necessarily incompatible with his description. The pattern seen in our cats deviates in details of neuron change and glia reaction from what we are familiar with in squirrel monkeys, rats, and human beings (Nordberg et al., 1971; Swedish Commission Report, Chap. 8, 1971; Grant, 1972) but is somewhat similar to what Hunter et al., 1940) observed in the cerebral cortex of a rhesus monkey. There is a possibility that the cerebral cortical changes in our cats are a result of hypoxia during convulsions, The lesions of thiamine deficiency encephalopathy (cf. Jubb et al., 1956) were sought for but not present. The exposure in this study, 0.35-0.55 mg mercury as methylmercury per kg body weight per day, well exceeds the minimum necessary to poison cats. This is shown by the rapid accumulation of mercury in blood cells and whole body without any indication of a leveling off of the accumulation curves. The distribution of mercury in cats exposed to methylmercury differs from that in other species. The erythrocyte/plasma ratio is considerably higher than in squirrel monkeys (Nordberg et al., 1971; Berlin et al., 1972), and man (Swedish Commission Report, 1971) while the fraction of the total body burden in the brain is considerably lower (Berlin et al., 1972; Aberg et al., 1969). In cats exposed to methylmercury, and in rats (Gage, 1964; Norseth and Clarkson, 1971) and mice (Norseth, 1971) a considerable fraction of the mercury in the kidney and liver is not present as methylmercury, indicating a breakage of the carbonmercury bond. The working hypothesis when planning the experiment was that metabolic differences between methylmercury as methylmercury hydroxide added to fish and methylmercury incorporated in fish protein in viva would show up as a difference in mercury accumulation between the two groups and possibly as a difference between the groups in specific activity in the organs. In liver, kidney, and muscle no such differences were observed. In the brain, however, the atomic absorption analyses showed a minor (1%) but significantly higher (P < 0.05) average total mercury Ievel in Group 1 than in Group 2. On the other hand the brain methylmercury level in Group 1 was significantly lower than in Group 2 (P < 0.05). The specific activity was lower in the brains of Group 1 than in those of Group 2 (P < 0.01). It cannot be excluded that these minor differences indicate a slight difference in metabolism, although the possibility of analytical errors must be kept in mind. These differences, if real, are of little toxicological significance. From the available data, it appears that, regardless of the mercury compound entering water, methylmercury will appear in fish, and that all mercurycontaining fish, including tuna caught at sea (Smith et al., 1971)) constitute the same toxicological problem. ACKNOWLEDGMENTS The Swedish Medical
investigation was supported Environmental Protection Research Council.
by Board
Grant and
7-45/68 b from partly by Grant
the Research Board 13X-2081 from the
of the Swedish
TOXICITY
OF MERCURY
IN FISH
FOR
CATS
441
REFERENCES (1969). MetabABERG, B., EKMAN, L., FALK, R., GREITZ, U., PERSSON, G., AND SNIHS, J.-O. olism of methyl mercury (“‘Hg) compounds in man, excretion and distribution. Arclr. Environ. Health 19, 478484. ALHANUS. L., FRANKENBERG, L., AND SUNWALL, A. ( 1970). Mercury poisoning in cat, fed Swedish fresh-water fish. Report C 1373-79, Research Institute of National Defense, Dept. 1, S-172 04 Sundbyberg 4, Sweden. Mimeographed (in Swedish). BERLIN, M. ( 1963). Renal uptake, retention and excretion of mercury. II. A study in the rabbit during infusion of methyland phenylmercuric compounds. Arch. En&on. Health 6, 610-616. BERLIN, M., NORDBERZ, G., AND HELLBERG, J. The uptake and distribution of methylmercury in the brain of Saimiri sciureu.s in relation to behavioral and morphological changes. In “Mercury, Mercurials and Mercaptans” (M. W. Miller and T. W. Clarkson, eds.) (From the 4th Rochester Conference on Environmental Toxicity, June 17-19, 1971) Thomas, Springfield, IL (in press 1972). CAMMERMEYER, J. (1960). A critique of neuronal hyperchromatosis. J. Neuropathol. 19. 141-142. GAGE, J. C. ( 1964). Distribution and excretion of methyl and phenyl mercury salts. Brit. 1. Ind. Med. 21, 197-202. GRAST, C. A., Pathology of experimental methyl mercury intoxication: Some problems of exposure and response. In “Mercury, Mercurials and Mercaptans” (M. W. Miller and T. W. Clarkson, eds.) (From the 4th Rochester Conference on Environmental Toxicity. June 17-19, 1971) Thomas, Springfield, IL (in press 1972). HUSTER, D., BOMFOAD, R. R., AND RUSSEL, D. S. ( 1940). Poisoning by methyl mercury compounds. f. Neural. Neurosurg. Psychiat. 17, 235-241. JENSEN, S., AND JERNEL~V, A. ( 1969). Biological methylation of mercury in aquatic organisms. Nature 223, 753-754. ( 1956). Thiamine deficiency encephalJUBB, K. V., SAUNDERS, L. Z., AND COATES, H. V. opathy in cats. J. Comp. Pathol. 66, 217-227. KAI, F. (1963). Metabolism of mercury compounds in animals administered with the shellfish from Minamata bay and methylmercuric compounds. .I. Kztrnumoto Med. Sot. 37. 673-700. (English abstr.) KITAMURA, S. (1968). Determination on mercury content in bodies of inhabitants, cats. fishes and shells in Minamata district and the mud of Minamata bay. In “Minamata Disease” (M. Kutsuna, ed.), pp. 257-266, Study Group of Minamata Disease, Kumamoto Ilniversity, Japan. MCGRATH, J. T. ( 1960). Neurologic examination of the dog with clinicopathological observations. 2nd ed. Lea & Febiger, Philadelphia. MINAMATA REPORT ( 1968). In “Minamata Disease” ( M. Kutsuna, ed. ), Study group ot Minamata Disease, Kumamoto University, Japan. NII~ATA REPORT. ( 1967). Report on the cases of mercury poisoning in Niigata, Ministry ot Health and Welfare, Tokyo. Mimeographed (in Japanese). NOHDBERG, G., BERLIN, M., AND GRANT, C. A. ( 1971) . Methylmercury in the monkeyautoradiographical distribution and neurotoxicity. Proc. 16th Int. Congr. Occup. Health pp. 234-237, Tokyo. NORSETH, T. ( 1971). Biotransformation of methyl mercuric salts in the mouse studied 1,) specific determination of inorganic mercury. Acta Pharmucol. 29, 375-384. NORSETH, T., AND CLARKSON, T. W. ( 1970). Studies on the biotransformation of ““‘Hglabeled methyl mercury chloride in rats. Arch. Environ. Health 21, 717-727. OSTLUND, K. ( 1969). Studies on the metabolism of methyl mercury and dimethyl mercury in mice. Acta Pharmacol. 27, Suppl. 1. ( 1969). Clinical and neurological findings in cats poisoned by methylmercru-y. PEKKAXEN, T. In “Swedish-Finnish Mercury Symposium” (J. K. Miettinen, ed.), pp. 28-34, Helsinki. ( Mimeographed in Swedish). RISSANEN, K. ( 1969). Retention and distribution of mercury in cat. In “Swedish-Finni&
442
ALBANUS
ET AL.
Mercury Symposium” (J. K. Miettinen, ea.), pp. 21-27, Helsinki. Mimeographed (in Swedish). SCH~TZ, A. ( 1969). A method for determination of submicrogram quantities of mercury in blood, urine and other biological material. Report 691020 from Department of Occupationd Medicine Uniuersity Hospital, S-221 85 Lund, Sweden. Mimeographed (in Swedish). SCOTT, P. P. ( 1964). Nutritional requirements and deficiences. In “Feline Medicine and Surgery” (E. J. Catcott, ea.), pp. 60-70, Amer. Veterinary Publications, St. Barbara, CA. SMITH, J. C., CLARKSON, T. W., GREENWOOD, M., VON BURG, R., AND LASELLE, E. ( 1971). The form of mercury in North American fish. Fed. Proc. 30, 221 ( Abstr. ). SJ~STRAND, B. ( 1964). Simultaneous determination of mercury and arsenic in biological and organic materials by activation analysis. And. Chem. 36, 814419. SWEDISH COMMISSION REPORT ( 1971). “Methyl mercury in fish. A Toxicologic-Epidemiologic Evaluation of Risks. Report from an Expert Group” (S. Skerfving, ea.). No&, Hyg. Tskr. Suppl. 4, pp. 364. (Swedish edition, 1970.) SWEDISH WATER AND AIR POLLUTION RESEARCH LABORATORY ( 1968). Gas chromatographic determination of methylmercury. Stockholm, Sweden. Mimeographed (in Swedish). TAKEUCHI, T. ( 1968). Pathology of Minamata disease. In “Minamata Disease” (M. Kutsuna, ed. ), pp. 141-228, Study Group of Minamata Disease, Kumanoto University, Japan. WEST%, G. (1966). Determination of methylmercury compounds in foodstuffs: I. Methylmercury compounds in fish, identification and determination. Acta Chem. Scand. 20, 21312137. WEST~%, G. ( 1967). Determination of methylmercury compounds in foodstuffs: II. Determination of methylmercury in fish, egg, meat, and liver. Acta Chem. Scud 21, 1790-1800. WEST%, G. ( 1968). Determination of methylmercury salts in various kinds of biological material. Acta Chem. Scud. 22, 2277-2280. WEST&, G. ( 1970). Mercury and methylmercury levels in pig’s brain. Vdr fiida 22, 147. (English abstr.) WEST%, G., AND RYDHLV, M. ( 1971). Methylmercury levels in fish caught March, 1968April, 1971. V&r fiidu 23, 179318. (English abstr.) YAMASHITA, M. (1964). Distribution in organs and excretion of mercury on experimental organic mercury compounds poisoning. J. jap. Sac. Intern. Med. 53, 529538. ( English abstr. )
ENVIROSMENTAL
Toxicity
for
Swedish
lakes
L. ALBANUS, G.
Cats and
of
(1972)
Methylmercury of Methylmercury
L. FRANKENBERC, NORDBERG,
M.
in Contaminated Hydroxide
Fish Added
from to Fish
C. GRANT, U. VON HAARTMAN, A. JERNEL~~, A. SCHULTZ, AND S. SKERFVING~
RYDXLV,
Research Institute of National Defense, S-172 04 Sundbyberg, Department of Pathology, Karolinska Hospital, S-104 01 Stockholm, Water and Air Pollution Research Laboratory, S-114 28 Stockholm, Department of Environmental Hygiene, Karolinska Institute, S-104 01 Stockholm, Department of Occupational Medicine, University Hospital, S-221 85 Lund, Food Research Department, National Food Administration, S-104 01 Stockholm, and Department of Hygiene, UniveTsit!/ of Lund, S-220 02 Lund, Sweden Received
March
9. 1972
To assess the toxicity of methylmercury accumulated in fish, five cats (Group 1)‘ were fed a homogenate of pike from a mercury-contaminated lake. Five other cats (Group 2) were fed a homogenate of pike from a “noncontaminated” lake, but with methylmercury hydroxide added to the homogenate. The methylmercury level in both homogenates was about 6 mg Hg/kg and the exposure of the cats about 0.45 mg Hg/kg body weight/day. Control cats (Group 3) were fed solely on noncontaminated pike. The exposure period to clinical onset was 60-83 days. The overall clinical and neuropathological patterns were consistent with methylmercury poisoning and similar for Groups 1 and 2. More than 90% of the mercury was absorbed of which at least 20% was incorporated in the hair. Mercury levels in the brain constituted about 1% of the total body burden (fur excluded). Total mercury levels in brain, liver, kidney, muscle, blood cells, and plasma averaged at termination 18, 39, 31, 27, 55, and 0.7 @g/g, respectively, with only minor differences between the two exposed groups. In brain and muscle about 100% of the mercury was recovered as methylmercury, while in kidney and liver the corresponding figures were 62 and 80%. The results demonstrate that toxicity studies with simple methylmercury salts give results comparable with those obtained with methylmercury “naturally” accumulated in fish. INTRODUCTION
The basis for evaluating maximum acceptable exposure to methylmercury is. epidemiological data from human beings E mainly after poisoning by methylmercury-containing fish and shellfish in Japan together with some cases of industrial exposure to methylmercury salts-and experimental data obtained by the administration of methylmercury salts to human beings and animals (cf. Swedish Commission Report, 1971). Toxicologically, methylmercury as it is present in fish has been assumed to be equivalent to methylmercury salts. This study is an 1 The ordinator Medicine,
paper is submitted as part of doctoral thesis by S. Skerfving, for this project. Reprint requests to Dr. S. Skerfving, Department University Hospital, 5-221 85 Lund, Sweden. 425
Copyright All rights
@ 1972 by of reproduction
Academic in any
Press, Inc. form reserved.
who
served as coof &cupationa].
ALBANUS
ET
AL.
TOXICITY
OF
MERCURY
IN
FISH
FOR
CATS
427
attempt to test the validity of this assumption by comparing the toxicity for cats of methylmercury in fish from a heavily contaminated Swedish lake and a methylmercury salt added to noncontaminated fish. MATERIALS
Labeled Methylmercury
AND METHODS
Hydroxide
[203Hg]Methylmercury hydroxide was obtained from mercury( ‘O”Hg) oxide by an isotope-exchange procedure (Berlin, 1963) and purified by Ostlund’s method, step 1 (1969). The purity of the Iabeled methylmercury hydroxide was checked by thin-layer chromatography (Westbii, 1966) and by the distribution of radioactivity, at repeated shakings, between benzene phases and 2 N hydrochloric acid phases. The content of inorganic mercury in the solutions used for preparation of fish homogenates was less than 1%of the total mercury. For specific activity see below. Fish Homogenate 1. “Contaminated” pike (Esox Zucius) were caught in 1969 in a Iake system (DelHngersHn) in Sweden. A paper mill had discharged phenylmercury acetate into the lakes between the 1940s and 1966.?The fish was frozen and shipped whole to the laboratory. Head, gills, fins, abdominal organs, and kidneys were removed. The fish was homogenized in a mincer and blended in a dough blender at about 4°C. A trace amount of [20”Hg]methylmercury hydroxide in water solution and with a specific activity of 0.15 Ci/g was sprayed as an aerosol over the homogenate during blending. Loss of radioactive material during the spraying procedure was negligible. The amount of methylmercury added corresponded to 0.6% of the mercury present in the homogenate. The radioactivity of the homogenate was 5.2 ,&i/kg at the start of the experiment, and the coefficient of variation between different samples 9%. Samples of about 100 gs were taken from different parts of the homogenate and after renewed homogenization were analyzed for total mercury and methylmercury (Table 1) and for energy and nutrients (Table 2). The homogenate was frozen in 2-kg blocks until fed to the cats. Homogenates 2 and 3. “Noncontaminated” pike were caught in lake Storhjalmaren in central Sweden. The noncontaminated fish was prepared in the same way as the contaminated fish. One half of this batch was sprayed with [20”Hg]methylmercury hydroxide having a specific activity of 0.92 mCi/g and formed Homogenate 2 (5.2 ,&i/kg). The mercury level obtained was close to that of Homogenate 1 (Table 1). The other half was frozen without further treatment and formed Homogenate 3, the control diet (Table 1). ’ Microbial transformationhas been assumedto explain the accumulationof mercury fish as methylmercury (Jensenand Jernebv,
1969).
in
428
ALBANUS
ET
TABLE LEVELS
OF ENERQY
AND NUTRIENTS
(standard methods of analvsis. National
AL.
2 IN HOMOGENATE OF PIKF. Swedish Food AdministrationY
Per kg Physiological energy, MJ (kcal) Wat,er, g Protein, g Fat, g Calcium, g Phosphorus, g Iron, mg Vitamin A, IU of which Vitamin AZ, IU Biologically active carotine, mg Thiamin, mg
3.5 (840) 780 180 9b 7.2 6.4 8 4000 2000
0 Supplemented with retinol palmitate, 1400 IU., Vitamin A, calciferol, 280 IU, vitamin D, tocopheryl acetate, 4.3 mg, thiamin chloride, 0.6 mg, riboflavin, 0.3 mg, pyridoxin chloride, 0.3 mg, and pantothenol, 2.8 mg, per cat per day (cf. Scott, 1964). Each cat was given 50 mg iron as a sorb&o1 citric acid complex intramuscularly at the beginning of the experiment,. Once weekly each cat was given an additional supplement of 100 mg tocopheryl acetate. * No peroxides detected.
ExpeTimenta~ Animals Fifteen European short-haired cats, bred and raised under standard conditions at the Research Institute of National Defence, 11 months old and weighing 2.33.9 kg at the beginning of the experiment, were divided into three experimental groups, each containing three females and two males. The animals were kept in separate cages at night and in two big common cages with outdoor runs during daytime, one for each sex. Feeding The cats were fed the fish homogenates as their only food together with water. A weighed portion of frozen homogenate was placed in the cage of each cat in the evening. The portions were adjusted to be consumed completely. If any remnants were left, they were weighed in the morning. All cats were fed Homogenate 3, the control diet, for an initial period of 55 days. Then Group 1 was fed Homogenate 1, Group 2 Homogenate 2, and five control cats (Group 3) Homogenate 3. Each cat was supplied daily with a combined vitamin solution (Table 2). Examination and Sampling The cats were observed daily and weighed twice a week. A modified McGrath ( 1960) system was applied to each cat twice a week to test the common reflexes. A standard protocol specifying 22 items was used to check systematically the general behavior, carriage, and activity of the cats, their movements, their ability to climb and jump up as well as down, the righting and placing reflexes, position and movements of the head and ears, spontaneous eye movements and the fixing
TOXICITY
OF MERCURY
IN
FISH
FOR
CATS
429
reflex,. the blink reflex, and the pupillary response to light. The cats were checked by two persons familiar with the animals. Once deviations from the normal were observed for any cat it was examined daily. Every 2 weeks each cat was filmed while performing standard movements-getting up, walking, jumping, and a rough test of the peripheral visual fields. Whole-body radioactivity measurements were performed once a week. Venous blood (about 3 ml) was taken once every 2 weeks and immediately prior to killing. BIood cells were separated from plasma by centrifugation for I.5 min at 12,000 X g in an Eppendorf microcentrifuge 3200. Autopsy and Pathological Examination The cats were killed when they showed definite signs of poisoning, i.e., convulsions. A modified Cammermeyer technique (1960) was used. With the cats in barbiturate anesthesia the aorta caudal to the origin of the renal arteries and the Vena cava caudalis were cannulated. After retrograde flushing through the aorta with 2-3 liters of heparinized 5.6% gum. arabic. in 0.9% NaCl solution at a pressure of 200-250 mm Hg, neutral formol-calcium solution under the same pressure was connected to the system and the fixative (2-3 liters) was then perfused through the cats until the efflux consisted purely of fixative. The cats maintained cardiac and respiratory movements until actua1 fixation was begun, about 20 min after the first abdominal incision and 2-3 min before completing perfusion. Samples of tissues (see Tables 5 and 7) were taken for immediate radioactivity measurements. In a separate experiment, it had been checked that perfusion did not significantly alter the level of radioactivity. Samples of liver, kidney, and skeletal muscle were frozen for subsequent mercury analyses. The cats were skinned and the pelt cleaned from fat and connective tissue. Radioactivity was determined in the whole pelt, and after clipping, in the skin and in the total amount of hair. Blocks of tongue, pharynx, larynx, thyroid and parathyroid, trachea, lung, thymus, myocardium, liver, pancreas, spleen, adrenal, kidney, stomach at several levels, duodenum, jejunum, ileum, cecum, colon, mesentery with lymph nodes, skeleta1 muscle, rib, and femoral bone marrow were taken for microscopical examination. The gonads were fixed in weak Bouin’s solution. All tissues were stained initially with azure A eosinate at pH 6.5. Frozen sections of myocardium, liver, kidney, and adrenal were stained with oil red 0. The skinned skull and intact spine were Ieft intact in neutral FormoI-calcium fixative for a week. There was no detectable loss of mercury to the neutral fixative. The brachial plexus with the median nerve and the (nonperfused) sciatic nerves were dissected free, straightened, and allowed to dry slightly on pieces of cardboard before being immersed in formol-calcium. After fixation, blocks of spinal cord together with adjacent nerve roots and dorsal root ganglia were taken at the levels of the cervical and lumbar intumescentia. The perfused eyes were removed from the orbit and postfixed in weak Bouin’s solution before trimming and sectioning. Radioactivity of the whole brain was measured after fixation and removal from
430
ALBANUS
ET
AL.
the skull. The brain was then divided in the midline. The left half was analyzed for total mercury and methyhnercury. The right half was sectioned transversely at 2-mm intervals from the olfactory lobe to the medulla oblongata in a Plexiglas miter box and marked so that the resulting microscopical sections represented even coronal steps and that corresponding sections from different brains could easily be compared, Brain and other tissues fixed in formol-calcium were prepared for microscopy by washing in running water overnight and subsequent dehydration in increasing concentrations of ethanol at intervals of at least 24 hr. The tissues were cleared in methylbenzoate, washed quickly in xylol, and embedded in Fibrowax under vacuum. The brains were sectioned primarily at 8 pm and stained with azure A eosinate at pH 6.5 and with Nissl’s cresyl violet. Other tissues were sectioned at 4-5 pm. Radioactivity
Measurements
The radioactivity in samples of fish homogenates, blood cells, plasma, and organs was measured by gamma scintillation counting against standards of the same geometry in a 3 x l-in. NaI well-type crystal connected to a three-channel gamma-spectrometer with autogamma unit (Model 3375, Packard, Downers Grove, IL). The efficiency of the measurements was about 40%. The statistical counting error was always 1% or less. The coefficient of variation between two measurements of the same sample on different occasions was 6%. The amounts of mercury were calculated from the specific activity in the homogenates (including methylmercury originally present and added). The set-up for whole-body counting is shown in Fig. 1. The detector was a 3 X l-in. well-type crystal shielded by lead, connected with the gamma-spectrometer described above. The counting efficiency was 0.03%. The amount of mercury was calculated by comparison with a standard consisting of the body of a cat, which had received an intravenous injection of 20 mg mercury as [20SHg]methylmercury hydroxide (a total of 18 &i) and had been frozen in a suitable crouching position. Each cat was monitored at least twice on each counting occasion. The coefficient of variation for 10 repeated countings of the same cat was 12%. The statistical counting error was always 1% or less. Whole brains, whole pelts, skins, and fur clippings were measured against standards of the same geometry in a large volume detector consisting of a cylindrical tank (8 x 16 in.) containing liquid scintillator and equipped with six 3-in. photomultiplier tubes. This equipment, giving essentially 4 r geometry, was connected to a gamma-spectrometer (Model 3375, Packard). The efficiency was about I4%, the coefficient of variation at duplicate measurements was 7% and the statistical counting error always 1% or less. Analyses
of Total Mercury
and Methylmercury
All glass was first cleaned by ordinary laboratory procedures, then by shaking with 1 M ammonium hydroxide solution, and then by deionized water. Plastic vials used did not contaminate water kept in them for 1 month. Samples of fish homogenate, brain, liver, kidney, and muscle were homogenized
TOXICITY
OF
MERCURY
IN
FISH
I 250
FOR
4:31
CATS
LEXIQLASS YLINDER
!
---LEAD
SHIELD
--.DETECTOA
FIG. 1. Set-up in millimeters.
used
for
measurement
of whole-body
radioactivity.
Figures
denote
distances
under cooling in an Ultra-Turrax homogenizer (Janke and Kunked, Staufen, W. Germany). Fractions of the samples of brain, liver, kidney, and muscle were freeze-dried (Kylteknik AB, Huddinge, Sweden). No detectable losses of radioactivity occurred during the drying procedure. The samples were kept frozen until analyzed. Total mercury level was determined by neutron activation analysis (Sjijstrand, 1964) and atomic absorption spectrometry (Schiitz, 1969) on dried samples. Methylmercury analyses of fresh fish homogenates were made by the method of West% (1967 and 1968) and Westi% and Rydglv ( 1971), of fresh brain b!; Westiiij (1970), and of liver, kidney, and muscle by Westi% (1967 and 1968). The samples analyzed represented 0.1-0.5 g wet weight. At least duplicate samples were analyzed for total mercury by atomic absorption spectrometry and for methylmercury. In the range 20-30 pg Hg/kg the coefficient of variation of 16 duplicate analyses of fresh tissue for methylmercury was 9%. The corresponding figure for analyses of total mercury by atomic absorption in nine duplicate
432
ALBANUS
ET AL.
analyses of dried samples in the range 60-80 pg Hg/g was 5%. In both cases possible lack of homogeneity of the samples is included in the error. RESULTS
Clinical
Signs
Fish consumption and mercury intake calculated as an average for the whole exposure period have been summarized in Table 3. One of the original control cats (No. 34) refused to eat. It was taken out of the experiment after 21 days and killed for examination. Since the prime object of this study was to compare Group 1 and Group 2, we shall confine ourselves here to a general description of the clinical observations. A more detailed analysis of the clinical signs in relation to pathological lesions and mercury levels in the individual cats is to be published separately. All cats in Groups 1 and 2 developed convulsions. For nine of the 10 cats the first convulsions occurred between Days 60 and 73 and for one (No. 13 in Group l), on Day 83. There was no significant difference between the two groups as to the length of the exposure period to the onset of convulsions. Changes in behavior and movements were noticed in seven cats prior to the onset of convulsions, four in Group 1 and three in Group 2. With one exception the changes were first observed 4-11 days before the convulsions. For one cat (No. 13 in Group 1) th ere was from about Day 50 a long period with low food consumption, declining body weight, and ataxia of steadily increasing severity until the onset of convulsions on Day 83 (Fig. 2), The observed behavioral and locomotory signs were wariness, aggressiveness, affectionateness, and a stiff, waddling gait, particularly of the hindlegs, progressing to distinct ataxia. No definite evidence of visual impairment could be detected, at least as long as the cats could be adequately examined i.e., up to onset of convulsions. The convulsions, whether or not preceded by behavioral and locomotory signs, followed a common pattern. Brief tonic spasms were followed by clonic spasms involving the limbs, trunk, neck, and head and were accompanied by profuse :salivation, dilatation of the pupils, and piloerection on the tail but not by urination or defecation. After 10 set to 2 min the convulsions gradually subsided. Once convulsions began, their frequency was roughly proportional to the duration and severity of the preceding clinical signs. Reviews of the films made during the exposure period did not result in the ,detection of any signs not noted earlier.
FISH
CONSUMPTION
AND
Intake Fish, kg/day/cat mg Hg/kg body
MFJTHYLMERCURY WHOLE EXPOSURE
TABLE 3 INTAKE CALCULATED AS AN AVERAGE PERIOD (MEAN f SEM)
Group
weight/day
0.28 0.45
1
+ 0.04 + 0.03
Group 0.31 0.47
2
+ 0.02 f 0.02
FOR TEE
Group 0.34 0.017
3
l 0.03 f 0.004
TOXICITY
OF MERCURY
IN
FISH
FOR
433
CATS
There were minor differences between different cats in the clinical signs, but variation within each group was as great as between groups. None of the controls (Group 3) showed any neurological signs. In all animals of Groups 1 and 2 there was a decrease of body weight during the last days of exposure (Fig. 2). In some there was a leveling off of body weight or a decrease before any neurological signs had been noted, in some cats even 1 month before other signs occurred. The total amount of mercury as methylmercury ingested when convulsions occurred was 30 ? 1 mg per kg body weight (mean -C SEM) for Group 1 and 35 t 2 for Group 2. Pathological
Findings
The pathological findings have been briefly reported (Grant, 1972) and, together with the clinical findings, will be described in detail elsewhere. The relevant changes were limited to the central and peripheral nervous systems. On the whole, there was no essential qualitative or quantitative differences between the cats of Groups 1 and 2. The quantitative differences between cats within BODY WEIGHT kg
“1
t 13P
GROUP 1
GROUP 2
1310 GROUP 3
0
20
LO ’
60
90
100
DAYS OF EXPOSURE
FIG. period. number
2. Body weight of the cats on control homogenate for 55 days and Onset of neurological signs (ataxia, convulsions, or both) is indicated and sex of the animals are indicated.
during exposure by arrows. The
434
ALBANJS
ET
AL.
the same group were greater than those between the groups. No relevant changes were encountered in the nervous system of the control cats (Group 3). The basic pathological pattern consisted of degenerative changes in the granular layer of parts of the cerebellar cortex, the peripheral nerves and their dorsal roots, and in the cerebral cortex. In the cerebellum, degenerative changes were extensive and remarkably uniform both within and between the exposed groups. Degeneration within the granular layer of the cortex followed the known pattern for methylmercury intoxication (Swedish Commission Report, Chap. 8, 1971). The topographica distribution was highly selective with preferential involvement of the midline and basal areas-the lingula, nodulus, and uvula together with the basal medial areas of the anterior, medial, and ansiform lobes. In the peripheral nerves and their dorsal roots degenerative changes involved segments of nerve fibers scattered among intact fibers. While quantitatively relatively slight, the pattern of degeneration was the usual one of swelling and fragmentation of the axon and the myelin sheath. There were no obvious group differences. In the cerebral cortex, the damage involved scattered small foci in practically all cortical regions except the tips of the frontal lobes, the olfactory cortex, and the pyriform lobe. The focal cortical damage involved particularly the deeper TOTAL BODY BURDEN w
Hg
100 -I
* = GROUP 1 (MEAN -
AND RANGE)
DAYS
FIG. 3. Whole-body retention From Day 64 on cats were killed
of mercury as calculated from as they developed convulsions.
OF EXPOSURE
radioactivity
measurements.
TOXICITY
OF MERCURY
IN
FISH
FOR
CATS
435
layer in lamina II and lamina III. Neuron changes were predominantly of Nissl’s “ischaemic” type with shrinkage, loss of cytoplasmic detail with increased affinity The most impressive change in the for basic stains, and nuclear pyknosis. damaged areas, however, was microgliosis with formation of rod cells. Perivascular cuffs of lymphocytes were often encountered in or near the damaged areas. It must be emphasized that the cerebral cortical damage involved numerous small areas scattered apparently at random throughout most of the cortical areas. There were wider individual differences between cats within the same group in this respect than between the groups. Analyses
of Total Mercury
and Methylmercury
The accumulation of whole-body radioactivity was parallel in the two experimental groups (Fig. 3). The considerable range of whole-body radioactivity of the animals in Group 1 early in the experiment was due to one cat (No. 13), which consumed less homogenate than the others. Groups 1 and 2, respectively, retained within the body or in the fur, 96 2 2 (mean + SEM) and 93 I 2% of the dose administered during the first week. The accumulation of mercury in blood cells and plasma was similar in Groups 1 and 2 (Table 4). Th e average blood cell levels increased 50-70 times and the plasma levels about 40 times, the ratio blood cell/plasma being about 55 at the start of exposure and about 75 at the end. There was a good agreement between radioactivity measurements and neutron activation analyses. In the controls (Group 3) the blood cell 1eve 1 rose about three times and the plasma level about two times, indicating that the exposure from the control homogenate exceeded that of the diet prior to the experiment. With the three methods used (atomic absorption, neutron activation, and radioactivity) analyses of total mercury in brain, liver, kidney, and muscle showed a general agreement, although there were some systematic differences in both experimental groups (Table 5). As an overall average for Groups 1 and 2 the atomic absorption and radioactivity measurements gave levels corresponding to 113 + 2 (mean & SEM) and 111 + 3% of those obtained by neutron activation. Both values differ significantly from 100% (P < 0.001). The radioactivity measurements do not include the mercury present at the start of exposure. This, however, constitutes only a small fraction of the total mercury. In liver. kidney, and muscle there were no conclusive differences between mercury levels in Groups 1 and 2 (Table 5). This was true for both total mercury and methylmercury. However, the brain concentrations differed slightly but were statistically significantly in regard to total mercury and methylmercury levels as well as specific activity of ‘““Hg (in Table 5 relation between chemical determinations and mercury levels based on radioactivity). The methylmercury levels obtained in Groups 1 and 2 made up 106 + 3 (mean + SEM) % of the total mercury (mean of atomic absorption, neutron activation. and radioactivity analyses) in muscle, 80 +- 3% in liver, and 62 + 3% in kidney (Table 6). In brain the methylmercury percentage was lower in Group 1 than in Group 2 (91 +: 4 vs 116 + 6%). The methylmercury percentage based on both groups was I.04 + 5%.
0.60
3
f 0.15
f 0.22
+ 0.16
blood cells
Day
before
f SEM)
0.022
0.021
0.018
b&g
onset
Group
radioactivity
than
0.004
0.004
64-86,
*
f
& 0.003
plasma
OF TOTAL MERCURY AND RADIOllCTIVITY
in Group 1 were killed on Days four values. three values. activation analyses were higher
1.1
2
0 The animals * Based upon c Based upon d The neutron
0.79
rglg
-
(MEAN
1
Group
LEVELS
71-80,
measurements
2 on Days
zk 1.8) * 0.28
(P
and
< 0.05).
Group
3 on Days
77-94.
+ 5.3) $- 0.20
(54 1.7
blood cells
(41 f 2.0) 1.1 f 0.03
dg
(26 1.4
cells
56 + 1.6b (55 f 1.9) 56 f 4.8
blood
52-53
39 zk 3.7 (42 + 4.2) 39 I!I 3.9
pg/g
of exposure
ACTIVATION
27 + 1.7 (27 + 1.9) 24 + 1.6
24-25
Days
TABLE 4 IN BLOOD CELLS AND PLASMA. NEUTRON MEASUREMENTS (WITHIN PARENTHESES)
(0.60 0.038
0.76 (0.73 0.71
fig/g
At autopsy@
ANALYSIS,
+_ 0.034) f 0.008
f 0.047 + 0.026) f 0.049d
plasma
F
fl
$ s
TOXICITY
OF MERCURY
IN
FISH
FOR
437
CATS
I
N
z E R
’
I
I
438
ALBANUS
METHYLMERCURY IN PERCENTAGE ATOMIC ABSORPTION, NEUTRON Group 1 2 3
ET
AL.
TABLE 6 (MEAN + SEM) OF TOTAL MERCURY (AVERAGE ACTIVATION, AND RaDIOACTIVITY MEASUREMENTS)
Brain”
Liveti
Kidney*
91 f 3.8 116 + 5.7 73 f 2.F
78 Tk 6.6 81 f 0.5 52 f 8.4
OF
Muscle
62 f 6.4 62 &- 3.1 42 + 2.2
a Group 2 < Group 1 (P < 0.01); Group 2 (P < 0.05) and Group different from 100%. b All groups statistically different from 100% (P < 0.001). c Based upon three values.
105 + 4.1 108 k 4.6 102 + 6.4c
3 (P < 0.001)
st,atistically
Total mercury levels, as calculated from radioactivity measurements on myocardium, spleen (free of blood), and the gastrointestinal tract (Table 7) were comparable to those in the brain, but lower than in liver, kidney, and muscle. At the end of the experiment more than half of the total dose of mercury was retained in the body (Table 8) and about half of the retained amount was present in the fur. Less than 1% of the total body burden (fur excluded) was present in the brain. DISCUSSION
This study demonstrated that (1) cats fed methylmercury-containing fish from a contaminated lake (Group 1) developed clinical signs and pathological lesions consistent with poisoning with a simple methylmercury salt (Group 2)) and that (2) the toxicity and mercury distribution were similar in the two groups. Thus,
TOTAL
MERCURY
TABLE LEVELS IN ORGANS FROM RADIOACTIVITY
Organ Myocardium Thymus Pancreas Spleen Ovary Testis Duodenumc Jejunumc Ileumc Colonc Cecumc Bile a Based upon three values. b Group 2 < Group 1 (P < 0.05). c Washed free of contents.
7 (MEAN + SEM) MEASUREMENTS
Group (/*g/g) 19 f 9.6 + 6.7 + 23 f 16 + 8.6; 17 + 17 * 16 -t 17 f 17 * 4.4;
I
1.1 1.5a 1.0” l.O* 3.0” 10 1.5 1.9 1.5 0.68 1.7 4.9
AS CALCULATED
Group b&d
2
20 * 7.8 f 7.5 + 17 f 13 + 7.8; 19 + 15 f 16 +16 k 20 f 8.5 +-
0.49 0.78 0.87 1.V 0.580 7.3 1.9 2.0 0.86 1.5 1.9 1.5
TOXICITY
OF MERCURY
IN
FISH
FOR
439
CATS
TABLE 8 MERCURY
IN
WHOLE
BODY,
I~ADIOACTIVITY
PELT,
FUR,
AND
MEASUREMENTS
BRAIN
(MEAN
AT
TERMINATION
+
SEM) OF
(koup Total dose administered (TD), mg Hg Whole body burden (WB), yQ of TI) Pelt, yfi of WB Fur, pg Q/g Fur (F), yOof WB Whole body without fur (WB-F), s, of TI1 Brain, TC,of (WB-F)
104
+
1 14
77 +_ 5.6 54 + 6.2 310
*
28
27 + e5.6 55 + 5.4 0.81
+ 0.08
9s
CALCULATED
FROM
EXPOSURE
Group 2 132
+ 8.0
64 62 3x0 34 42
* + + + f
3.5 3.4 45 2.4 3.3
*
0.05
0.88
the use of methylmercury salts appears to be generally relevant in assessing the toxicity of methylmercury in fish. In a collaboratory study, Albanus et ai. (1970) fed two cats homogenate of contaminated pike (exposure 0.4 mg mercury per kg body weight per day as methylmercury). Since no methylmercury compound, radioactive or inactive, was added to the homogenate, these cats constitute a suitable “inactive” comparison group for the cats in Groups 1 and 2. Total intake of methylmercury to onset of convulsions (26 and 30 mg mercury per kg body weight; 70 and 80 days), total mercury levels in the brain ( 19 pg/ g ) , and clinical and neuropathological patterns were fully comparable with the results for the cats in Groups 1 and 2. The hematological and blood chemistry studies on the “inactive” cats as well as their concurrent dietary controls gave essentially normal results. The methylmercury which had accumulated in the pike fed to the cats in Group 1 had been synthesized, probably by microbial activity (Jensen and Jerneliiv, 1969), from an arylmercury compound (phenylmercury acetate) discharged as waste into the water. The major epidemics of methylmercury poisoning among Japanese through consumption of fish and shellfish have been attributed to the discharge of methylmercury as such and its accumulation in food chains (Niigata Report, 1967; Minamata Report, 1968). The admittedly incomplete data available for cats fed shellfish from Minamata (Kai, 1963; Yamashita, 1964; Kitamura, 1968; Takeuchi, 1968) and data on cats fed methylmercury proteinate (Pekkanen, 1969; Rissanen, 1969)) are compatible with the observations made in this study. In the general context of methylmercury poisoning, the cats again provided evidence for primary neurotoxicity. The pattern of cerebellar damage fits in with what has been described by Takeuchi (1968) for cats. The topographical distribution of damage observed in the present study has not been pointed out previously, presumably because the method of examination used here permits a more systematic evaluation. In earlier studies examination of peripheral nerves and their dorsal roots has apparently not been carried out, or at best reported ambiguously, for cats as well as other species, including human beings, with few exceptions (cf. Swedish Commission Report, Chap. 8, 1971). When clearly reported, however, the damage observed fits in with what has been seen in the cats in this study, The damage in the cerebral cortex, however, poses problems
440
ALBANUS
ET
AL.
of interpretation. The nature of the cortical damage in the cats deviates from what would be expected from the general description by Takeuchi (1968) butpresumably because of inexact translation-is not necessarily incompatible with his description. The pattern seen in our cats deviates in details of neuron change and glia reaction from what we are familiar with in squirrel monkeys, rats, and human beings (Nordberg et al., 1971; Swedish Commission Report, Chap. 8, 1971; Grant, 1972) but is somewhat similar to what Hunter et al., 1940) observed in the cerebral cortex of a rhesus monkey. There is a possibility that the cerebral cortical changes in our cats are a result of hypoxia during convulsions, The lesions of thiamine deficiency encephalopathy (cf. Jubb et al., 1956) were sought for but not present. The exposure in this study, 0.35-0.55 mg mercury as methylmercury per kg body weight per day, well exceeds the minimum necessary to poison cats. This is shown by the rapid accumulation of mercury in blood cells and whole body without any indication of a leveling off of the accumulation curves. The distribution of mercury in cats exposed to methylmercury differs from that in other species. The erythrocyte/plasma ratio is considerably higher than in squirrel monkeys (Nordberg et al., 1971; Berlin et al., 1972), and man (Swedish Commission Report, 1971) while the fraction of the total body burden in the brain is considerably lower (Berlin et al., 1972; Aberg et al., 1969). In cats exposed to methylmercury, and in rats (Gage, 1964; Norseth and Clarkson, 1971) and mice (Norseth, 1971) a considerable fraction of the mercury in the kidney and liver is not present as methylmercury, indicating a breakage of the carbonmercury bond. The working hypothesis when planning the experiment was that metabolic differences between methylmercury as methylmercury hydroxide added to fish and methylmercury incorporated in fish protein in viva would show up as a difference in mercury accumulation between the two groups and possibly as a difference between the groups in specific activity in the organs. In liver, kidney, and muscle no such differences were observed. In the brain, however, the atomic absorption analyses showed a minor (1%) but significantly higher (P < 0.05) average total mercury Ievel in Group 1 than in Group 2. On the other hand the brain methylmercury level in Group 1 was significantly lower than in Group 2 (P < 0.05). The specific activity was lower in the brains of Group 1 than in those of Group 2 (P < 0.01). It cannot be excluded that these minor differences indicate a slight difference in metabolism, although the possibility of analytical errors must be kept in mind. These differences, if real, are of little toxicological significance. From the available data, it appears that, regardless of the mercury compound entering water, methylmercury will appear in fish, and that all mercurycontaining fish, including tuna caught at sea (Smith et al., 1971)) constitute the same toxicological problem. ACKNOWLEDGMENTS The Swedish Medical
investigation was supported Environmental Protection Research Council.
by Board
Grant and
7-45/68 b from partly by Grant
the Research Board 13X-2081 from the
of the Swedish
TOXICITY
OF MERCURY
IN FISH
FOR
CATS
441
REFERENCES (1969). MetabABERG, B., EKMAN, L., FALK, R., GREITZ, U., PERSSON, G., AND SNIHS, J.-O. olism of methyl mercury (“‘Hg) compounds in man, excretion and distribution. Arclr. Environ. Health 19, 478484. ALHANUS. L., FRANKENBERG, L., AND SUNWALL, A. ( 1970). Mercury poisoning in cat, fed Swedish fresh-water fish. Report C 1373-79, Research Institute of National Defense, Dept. 1, S-172 04 Sundbyberg 4, Sweden. Mimeographed (in Swedish). BERLIN, M. ( 1963). Renal uptake, retention and excretion of mercury. II. A study in the rabbit during infusion of methyland phenylmercuric compounds. Arch. En&on. Health 6, 610-616. BERLIN, M., NORDBERZ, G., AND HELLBERG, J. The uptake and distribution of methylmercury in the brain of Saimiri sciureu.s in relation to behavioral and morphological changes. In “Mercury, Mercurials and Mercaptans” (M. W. Miller and T. W. Clarkson, eds.) (From the 4th Rochester Conference on Environmental Toxicity, June 17-19, 1971) Thomas, Springfield, IL (in press 1972). CAMMERMEYER, J. (1960). A critique of neuronal hyperchromatosis. J. Neuropathol. 19. 141-142. GAGE, J. C. ( 1964). Distribution and excretion of methyl and phenyl mercury salts. Brit. 1. Ind. Med. 21, 197-202. GRAST, C. A., Pathology of experimental methyl mercury intoxication: Some problems of exposure and response. In “Mercury, Mercurials and Mercaptans” (M. W. Miller and T. W. Clarkson, eds.) (From the 4th Rochester Conference on Environmental Toxicity. June 17-19, 1971) Thomas, Springfield, IL (in press 1972). HUSTER, D., BOMFOAD, R. R., AND RUSSEL, D. S. ( 1940). Poisoning by methyl mercury compounds. f. Neural. Neurosurg. Psychiat. 17, 235-241. JENSEN, S., AND JERNEL~V, A. ( 1969). Biological methylation of mercury in aquatic organisms. Nature 223, 753-754. ( 1956). Thiamine deficiency encephalJUBB, K. V., SAUNDERS, L. Z., AND COATES, H. V. opathy in cats. J. Comp. Pathol. 66, 217-227. KAI, F. (1963). Metabolism of mercury compounds in animals administered with the shellfish from Minamata bay and methylmercuric compounds. .I. Kztrnumoto Med. Sot. 37. 673-700. (English abstr.) KITAMURA, S. (1968). Determination on mercury content in bodies of inhabitants, cats. fishes and shells in Minamata district and the mud of Minamata bay. In “Minamata Disease” (M. Kutsuna, ed.), pp. 257-266, Study Group of Minamata Disease, Kumamoto Ilniversity, Japan. MCGRATH, J. T. ( 1960). Neurologic examination of the dog with clinicopathological observations. 2nd ed. Lea & Febiger, Philadelphia. MINAMATA REPORT ( 1968). In “Minamata Disease” ( M. Kutsuna, ed. ), Study group ot Minamata Disease, Kumamoto University, Japan. NII~ATA REPORT. ( 1967). Report on the cases of mercury poisoning in Niigata, Ministry ot Health and Welfare, Tokyo. Mimeographed (in Japanese). NOHDBERG, G., BERLIN, M., AND GRANT, C. A. ( 1971) . Methylmercury in the monkeyautoradiographical distribution and neurotoxicity. Proc. 16th Int. Congr. Occup. Health pp. 234-237, Tokyo. NORSETH, T. ( 1971). Biotransformation of methyl mercuric salts in the mouse studied 1,) specific determination of inorganic mercury. Acta Pharmucol. 29, 375-384. NORSETH, T., AND CLARKSON, T. W. ( 1970). Studies on the biotransformation of ““‘Hglabeled methyl mercury chloride in rats. Arch. Environ. Health 21, 717-727. OSTLUND, K. ( 1969). Studies on the metabolism of methyl mercury and dimethyl mercury in mice. Acta Pharmacol. 27, Suppl. 1. ( 1969). Clinical and neurological findings in cats poisoned by methylmercru-y. PEKKAXEN, T. In “Swedish-Finnish Mercury Symposium” (J. K. Miettinen, ed.), pp. 28-34, Helsinki. ( Mimeographed in Swedish). RISSANEN, K. ( 1969). Retention and distribution of mercury in cat. In “Swedish-Finni&
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Mercury Symposium” (J. K. Miettinen, ea.), pp. 21-27, Helsinki. Mimeographed (in Swedish). SCH~TZ, A. ( 1969). A method for determination of submicrogram quantities of mercury in blood, urine and other biological material. Report 691020 from Department of Occupationd Medicine Uniuersity Hospital, S-221 85 Lund, Sweden. Mimeographed (in Swedish). SCOTT, P. P. ( 1964). Nutritional requirements and deficiences. In “Feline Medicine and Surgery” (E. J. Catcott, ea.), pp. 60-70, Amer. Veterinary Publications, St. Barbara, CA. SMITH, J. C., CLARKSON, T. W., GREENWOOD, M., VON BURG, R., AND LASELLE, E. ( 1971). The form of mercury in North American fish. Fed. Proc. 30, 221 ( Abstr. ). SJ~STRAND, B. ( 1964). Simultaneous determination of mercury and arsenic in biological and organic materials by activation analysis. And. Chem. 36, 814419. SWEDISH COMMISSION REPORT ( 1971). “Methyl mercury in fish. A Toxicologic-Epidemiologic Evaluation of Risks. Report from an Expert Group” (S. Skerfving, ea.). No&, Hyg. Tskr. Suppl. 4, pp. 364. (Swedish edition, 1970.) SWEDISH WATER AND AIR POLLUTION RESEARCH LABORATORY ( 1968). Gas chromatographic determination of methylmercury. Stockholm, Sweden. Mimeographed (in Swedish). TAKEUCHI, T. ( 1968). Pathology of Minamata disease. In “Minamata Disease” (M. Kutsuna, ed. ), pp. 141-228, Study Group of Minamata Disease, Kumanoto University, Japan. WEST%, G. (1966). Determination of methylmercury compounds in foodstuffs: I. Methylmercury compounds in fish, identification and determination. Acta Chem. Scand. 20, 21312137. WEST~%, G. ( 1967). Determination of methylmercury compounds in foodstuffs: II. Determination of methylmercury in fish, egg, meat, and liver. Acta Chem. Scud 21, 1790-1800. WEST%, G. ( 1968). Determination of methylmercury salts in various kinds of biological material. Acta Chem. Scud. 22, 2277-2280. WEST&, G. ( 1970). Mercury and methylmercury levels in pig’s brain. Vdr fiida 22, 147. (English abstr.) WEST%, G., AND RYDHLV, M. ( 1971). Methylmercury levels in fish caught March, 1968April, 1971. V&r fiidu 23, 179318. (English abstr.) YAMASHITA, M. (1964). Distribution in organs and excretion of mercury on experimental organic mercury compounds poisoning. J. jap. Sac. Intern. Med. 53, 529538. ( English abstr. )