Aquatic Toxicology 50 (2000) 287 – 299
www.elsevier.com/locate/aquatox
Pathologic alterations in adult rainbow trout, Oncorhynchus
mykiss, exposed to dietary
2,3,7,8-tetrachlorodibenzo-p-dioxin
Gail L. Walter a,1, Paul D. Jones c,d,*, John P. Giesy b,c,d
a
Department of Pathology, College of Veterinary Medicine, Michigan State Uni6ersity, East Lansing, MI 48824, USA
b
Department of Zoology, Michigan State Uni6ersity, East Lansing, MI 48824, USA
c
Institute of En6ironmental Toxicology, Michigan State Uni6ersity, East Lansing, MI 48824, USA
d
Food Safety and Toxicology Building, National Food Safety and Toxicology Center, Michigan State Uni6ersity, East Lansing,
MI 48824, USA
Received 29 June 1999; received in revised form 31 January 2000; accepted 2 February 2000
Abstract
Adult female rainbow trout (Oncorhynchus mykiss) fed [3H]2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) impregnated diet at 0, 1.8, 18 or 90 ng/kg food for up to 320 days were evaluated by clinical pathology, gross pathology and
histopathology procedures. Gross pathological changes were limited to a slight increase in the incidence of lesions of
the caudal fins in the treated groups. Mixed mononuclear inflammatory infiltrates were present in multiple organs of
fish from control and treatment groups, but did not appear to be treatment related. Lesions associated with exposure
to TCDD were observed histologically in liver and spleen after 100 and 250 + days of exposure. The livers of
TCDD-treated fish contained less hepatocellular glycogen, more mitotic figures, greater anisokaryosis, anisocytosis,
nuclear chromatin clumping and margination. Prominent nucleoli were directly proportional to TCDD dose.
Hepatocellular changes in fish exposed to TCDD also included single cell necrosis and clear cytoplasmic vacuoles
consistent with lipid. Some fish from all TCD exposed groups had lower lymphoid density compared to controls at
all time intervals. Fish exposed to the highest TCDD dose had decreased peripheral leukocyte counts after 50 and 100
days. The lowest observable adverse effect level (LOAEL) for these effects was 5.69 ngTCDD/kg in diet and 0.90 ng
TCDD/kg liver. © 2000 Elsevier Science B.V. All rights reserved.
Keywords: Dioxin; Fish; Accumulation; Pathology; Histopathology; Toxicity
1. Introduction
* Corresponding author. Tel.: +1-517-4326333; fax: +1527-4322310.
1
Present address, 325 Grandview Ave., Kalamazoo, MI
49001, USA.
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is
the by-product of industrial processes and pyrolytic reactions (Harrad and Jones, 1992; Rappe
and Kjeller, 1994; Brzuzy and Hites, 1996).
0166-445X/00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved.
PII: S 0 1 6 6 - 4 4 5 X ( 0 0 ) 0 0 0 9 5 - 3
288
G.L. Walter et al. / Aquatic Toxicology 50 (2000) 287–299
TCDD has been shown to accumulate into the
tissues of fish (Schmieder et al., 1995; Niimi, 1996;
Delorme et al., 1998; Johnson et al., 1998; Tietge
et al., 1998). Some fish species, particularly
salmonids, are sensitive to the effects of TCDD
(Walker and Peterson, 1994; Peterson et al., 1993;
Newsted et al., 1995). While a number of chemicals
structurally similar to TCDD can cause adverse
effects in fish through the same mode of action
(Hanberg et al., 1990; Walker and Peterson, 1991;
Newsted et al., 1995), TCDD is the most potent of
the class of polyhalogenated, diaromatic hydrocarbons (PHDHs; Giesy and Kannan, 1998). The
toxicity of complex mixtures of PHDH is often
expressed as TCDD equivalents (TEQs; Safe,
1987). TCDD equivalency factors have been
derived for fish (Van den Berg et al., 1998) so that
the toxic potency of complex mixtures of Ah-receptor (Ah-R) active compounds to fish can be calculated. However, to be able to interpret the potential
effects of these complex mixtures on fish a dose-response relationship including a threshold for effect
is needed. Previously, the toxicity of TCDD to fish
has been investigated in short-term studies at
exposure concentrations greater than those found
in the environment. Furthermore, the vector of
exposure was generally via ‘intra peritoneal’ injection, single gavage dose or from aqueous media
containing solvent carriers (Spitsbergen et al.,
1991). Alternatively eggs have been exposed to
concentrations of TCDD in water above the compounds water solubility or TCDD was injected into
eggs (Walker and Peterson, 1991). While these
studies established that TCDD is extremely toxic to
fish, no reference doses were available.
The research presented here was conducted as a
definitive study of the long-term effects of small
concentrations of TCDD in the diet of rainbow
trout. The study was designed to investigate the
effects of concentrations of TCDD in the diet that
were similar to those currently in the environment
or that once occurred in the environment. Furthermore, the greatest concentration of TCDD used in
the current study was less than the total concentration of TEQs often measured in fish from the North
American Great Lakes (Jones et al., 1993; Giesy et
al., 1999). The vector and rate of exposure were
selected to allow an estimation of ecological effects
of TCDD on a salmonid fish at environmentally
relevant concentrations. In addition, the pathological responses are catalogued such that they can be
used as a functional measure of TCDD exposure
under field conditions. This paper presents only the
pathological findings, detailed descriptions of the
accumulation disposition and effects on reproduction and survival can be found elsewhere (Giesy et
al., 2000; Jones et al., 2000).
2. Materials and methods
Adult (age class II; 350 g), female rainbow trout
(Oncorhynchus mykiss) of the spring-spawning
Shasta strain were collected from the rearing ponds
at the Stoney Creek Trout farm (Grant, MI). Fish
were sorted by sex and held at the Michigan State
University Aquaculture facility until exposure began in March of 1991. Fish were acclimated for 60
days in the exposure tanks before the commencement of exposure.
Tritium labeled TCDD was synthesized and
purified at the Pesticide Research Center, Michigan
State University. The radiochemical purity (\
99.9%) and specific activity were confirmed by gas
chromatography-mass spectrometry (GC-MS) and
liquid scintillation counting. Concentrations of
TCDD in food and selected fish tissues were
confirmed by GC-MS and ELISA (Enzyme linked
immunosorbent assay). Food was spiked with both
3
H-labeled TCDD and non-labeled TCDD such
that, while the TCDD dose varied, the TCDD
specific activities (DPM/pg TCDD; DPM= disintegrations per minute) were also varied so that the
radiometric dose remained constant. In this way
fish were exposed to the same dose of radiation over
the course of the study (Table 1). Control fish were
not exposed to 3H.
Fish were exposed in 1700 l, flow-through
tanks. The flow rate was 71.5 l/h resulting in
approximately two turnovers of the water per
day. Temperature was maintained at 12°C and
photoperiod was adjusted weekly to match ambient outside conditions. Tanks were situated in a
negative pressure facility with three levels of containment for water and one for air. Control fish
were held in the same facility with the same
G.L. Walter et al. / Aquatic Toxicology 50 (2000) 287–299
containment but in an adjacent room to prevent
TCDD carry over between tanks.
Fish were fed Silver Cup Fish Feed (Murray
Elevators, Murray, UT) with or without tritiumlabeled TCDD for up to 320 days. Stock solutions
of TCDD were prepared in acetone and applied
to the food. The acetone was allowed to evaporate. TCDD-spiked food containing 5.4 54 or 270
ng TCDD/kg was fed every third day while no
spiked food was fed on the other 2 days. This
resulted in average daily dietary concentrations of
0, 1.8, 18–90 ng TCDD/kg moist weight of food
(Table 1). The food contained a background concentration of 24 ng/kg TCDD-EQ as determined
by the H4IIE bioassay (Sanderson et al., 1996) of
which less than 0.2 ng/kg was 2,3,7,8-TCDD. The
other compounds that contributed to the Ah-R
activity were unknown, but could have been polychlorinated biphenyls (PCBs), polycyclic aromatic
hydrocarbons or short-lived ‘natural’ inducers.
The quantity of food fed was adjusted throughout
the experiment to maintain a constant ration of
1.5% of body weight per day.
The experiment was initiated with 35 females in
each of the four exposure groups (three TCDD
exposure concentrations and one control). Two to
four fish from each of the three treatment and
control groups, were evaluated for clinical pathology alterations and gross lesions after 50, 100,
150, or 200 days of exposure. Additional fish were
collected and evaluated at various time points at
the time of spawning from day 255 to 320, and
data from these fish have been grouped together
as a 250+-day treatment group.
289
Fish were anesthetized by submersion in MS222 (tricaine methane sulfonate). Blood was collected by venipuncture of the caudal vein and
placed on ice. Samples were collected in EDTA
(ethylene diamine tetraacetic acid) for complete
blood counts and without anticoagulant for
serum chemistry evaluation. Clinical pathology
tests were performed on the same day as blood
collection. A 1:200 dilution in modified Dacie’s
solution was used to determine leukocyte counts
by previously described methods (Blaxhall and
Daisley, 1973; Campbell, 1988b). Erythrocyte
counts and hemoglobin determinations were done
by voltage impedance (Counter ZBI) by previously described methods (Campbell, 1988a). Leucocytes were classified by microscopic evaluation
of Wright’s stained blood smears. Serum from
two fish was typically pooled to provide adequate
sample volume for analysis. Serum chemistry
evaluation included sodium, potassium, total carbon dioxide, anion gap, iron, albumen, alkaline
phosphatase, amylase, total bilirubin, urea nitrogen, calcium, cholesterol, creatine kinase, creatinine, gamma glutamyl transferase, glucose,
magnesium, phosphorus, sorbitol dehydrogenase,
aspartate aminotranserase, alanine aminotransferase, total protein and osmolality. Tests were
performed on a tandem access analyzer (Abbott
Spectrum) using reagents supplied by the manufacturer. Tests were not modified from standard
methodologies used for mammals. Enzyme analysis was performed at 37°C. Osmolality was determined by freezing point depression.
Table 1
Concentrations of 2,3,7,8-TCDD, TCDD-EQs and radiometric doses in food
Dose group
TCDD in food (ng/kg)
Specific activity (DPM/pg TCDD)
Radiometric dose (DPM/g food)
Bioassay (ng TCDD/kg food)b
TCDD only dose
a
Control
B0.2
Naa
Na
24
0
ng TCDD/kg diet
1.8
18
90
5.4
541
974
28.9
4.9
54
57.0
1026
46.9
22.9
270
10.5
949
154
129.8
Na, Not applicable.
Background concentrations of dioxin like activity were measured using the H4IIE bioassay. The presence of this activity is
discussed in Section 2.
b
G.L. Walter et al. / Aquatic Toxicology 50 (2000) 287–299
290
but one fish and samples of ovary were collected
from most fish. Following routine processing and
paraffin embedding, 6-mm sections were prepared
and stained with hematoxylin and eosin. Periodic
Acid Schiff (PAS) staining was also performed on
some sections. Incidence and severity of lesions
were enumerated and the values for treatment
groups were compared to controls at each time
interval.
3. Results
Fig. 1. Accumulation of TCDD in the livers of rainbow trout
exposed to TCDD for up to 200 days. Values are mean of each
treatment group; solid =1.8 ng TCDD/kg; dashed = 18 ng
TCDD/kg; dotted 90 ng TCDD/kg. Error bars indicate one
standard error of the mean. As the control group was not
exposed to 3H-TCDD no values are available.
Anaesthetized fish were killed by concussion
followed by cervical spinal cord transection. Liver
and ovaries were removed and weighed, gross
lesions were noted and samples for histopathological evaluation were taken within 5 min of death
and placed in 10% neutral buffered formalin.
Samples of liver, midsection kidney, hematopoietic tissue, gill, and stomach were collected from
each fish. Samples of spleen were obtained in all
There were no statistically significant differences in growth among treatment groups after 200
days of exposure. TCDD was rapidly accumulated in the livers of TCDD-treated fish and
reached a steady state after 50 days of exposure
(Fig. 1 and Table 2). There was an apparent but
non-significant decrease in liver TCDD concentration in all TCDD-treatment groups at 150 days.
However liver concentrations increased again between 150 and 200 days to reach levels equivalent
to those observed at 100 days of exposure. This
decrease may represent a mobilization and transfer of lipid and associated TCDD to the developing gonads. Relatively uniform increases in the
ovary/somatic index were noted over the entire
exposure period (results not shown) so alterations
in TCDD distribution would indicate a specific
phase of egg development. Details about tissue
accumulation and distribution of TCDD in this
experiment are provided elsewhere (Giesy et al.,
2000; Jones et al., 2000).
Table 2
Concentrations of 2,3,7,8-TCDD (pg/g wet weight) accumulated in rainbow trout livers during the 200-day feeding perioda
Interval (days)
Control
ng TCDD/kg diet
1.8
Nab
Na
Na
Na
50
100
150
200
a
b
Values are mean (S.D.).
Na, not applicable.
0.255 (0.06)
0.312 (0.051)
0.218 (0.083)
–
18
1.65 (0.57)
2.875 (0.72)
1.717 (0.56)
2.85 (0.639)
90
–
12.92 (2.75)
9.933 (0.528)
16.242 (2.21)
G.L. Walter et al. / Aquatic Toxicology 50 (2000) 287–299
3.1. Gross pathology
The only consistent gross external lesion was
necrosis of the caudal and occasionally anal fins.
The incidence of fin lesions was slightly greater in
treated groups than in the controls at the same
time intervals (Table 3) but this response was not
dose dependent. Lesions were characterized histopathologically as epithelial erosion with mild subjacent fibrosis and occasional necrosis. The degree
of severity of lesions was not dose dependent
suggesting that these lesions were probably due to
confinement in the tanks.
3.2. Clinical pathology
No TCDD-related alterations in any serum
chemistry parameters or in erythrocyte parameters were observed at any exposure time (results
not shown). The total number of leukocytes was
decreased in the 90-ng TCDD/kg treatment dose
group fish when compared to controls for samples
although there was not a clear dose-response relationship across dose groups.
3.3. Histopathology
Inflammation was frequently present and often
involved the peritoneal serosal or capsular surfaces of the stomach, spleen and liver, the submucosa, muscularis and subserosa of the stomach,
the ovary, and the parenchyma of the liver. Inflammatory infiltrates generally consisted of mixed
mononuclear cells (lymphocytes, plasma cells,
macrophages). Occasionally fibroblasts and neovascularization were increased on the stromal surTable 3
Incidence (lesions/number of fish examined) of fin lesions
(gross) in rainbow trout exposed to TCDD
Exposure time (days)
Control
ng TCDD/kg diet
1.8
50
100
150
200
1/3
0/4
2/4
0/2
0/4
4/4
2/4
1/6
18
0/4
3/4
3/3
1/3
90
2/4
2/3
2/4
0/4
291
faces. These changes were not dose-related and
occurred with similar frequency among all treatment groups. Specific etiologic agent(s) were not
identified. Other organ-specific changes are addressed individually.
3.4. Li6er
There were dose-related hepatocellular changes
in all TCDD treated groups but not in untreated
fish (Table 4 and Fig. 2). The livers of control fish
were characterized by uniform small hepatocytes
with small, oval monomorphic nuclei, which had
evenly dispersed finely granular chromatin and
rare mitotic figures. In the livers of fish exposed to
18 or 90 ng TCDD/kg exposed fish, there were
increased mitotic figures and nuclear changes, including variably enlarged nuclei (anisokaryosis),
prominent nucleoli, marginated and/or coarsely
clumped chromatin and vesiculation (Fig. 3). Anisocytosis due to increased hepatocellular size was
also observed in 18 and 90 ng/kg treatment
groups when compared to controls. Cells with
yellow–brown, slightly refractile cytoplasmic pigment, suggestive of bile, were noted in the 18- and
90-ng/kg TCDD treatment groups at 200 and
250+ days (data not shown). Individual cell necrosis was occasionally observed in the livers of
fish exposed to 18 or 90 ng TCDD/kg (Table 4).
Dose-dependent decreases in hepatocellular glycogen were also observed in all TCDD-treated
groups. Hepatocytes of control fish contained
moderate to abundant glycogen in irregular, fibrillar intracytoplasmic vacuoles which were strongly
PAS positive (Fig. 3). Hepatocellular vacuolization was less intense and cytoplasm was more
densely eosinophilic in TCDD-exposed fish (Fig.
3). There was little PAS stain affinity in TCDDtreated fish. Sharply-outlined, PAS negative, clear
vacuoles, consistent with lipid, were present in
some fish in the 18- and 90-ng TCDD/kg treatment groups but were not seen in untreated fish.
There were periportal, mixed mononuclear inflammatory infiltrates and capsulitis/peritonitis in
fish from control and treatment groups; therefore,
these were not considered to be treatment related
(Fig. 4). Thus the lowest observable adverse effect
level (LOAEL) for these effects was 5.69 ng
G.L. Walter et al. / Aquatic Toxicology 50 (2000) 287–299
292
Table 4
Incidence of liver lesions (lesions/number of fish examined) in rainbow trout exposed to TCDD
Parameter
Exposure time
(days)
Control
ng TCDD/kg diet
1.8
18
90
Glycogen decreased
100
150
200
250+
0/3
0/4
1/4
0/2
2/4
1/4
2/4
0/6
1/4
4/4
0/4
2/4
3/4
2/3
2/3
4/5
Lipid-like vacuoles
100
150
200
250+
0/3
0/4
0/4
0/2
1/4
2/4
0/4
0/6
1/4
3/4
0/4
0/4
2/4
2/3
0/3
1/5
0/3
0/4
1/4 (1/10 hpf)a
0/4
0/4
0/4
1/4 (2/10 hpf)a
2/4 (1.5/10 hpf)a
3/4 (2/10 hpf)a
3/4 (1.7/10 hpf)a
0/4
3/4 (5/10 hpf)a
2/3 (3/10 hpf)a
2/3 (5/10 hpf)a
0/5
Increased mitotic figures
100
150
200
250
Nuclear or cellular change
100
150
200
250+
0/3
0/4
0/4
0/2
0/4
0/4
3/4
3/6
1/4
4/4
1/4
2/4
4/4
3/3
2/3
4/5
Individual cell necrosis
100
150
200
250+
0/3
0/4
0/4
0/2
0/4
0/4
0/4
0/6
0/4
0/4
0/4
2/4
1/4
0/3
1/3
0/5
Peritonitis liver
100
150
200
250+
0/3
1/4
1/4
0/2
1/4
0/4
0/4
0/6
0/4
0/4
1/4
0/4
1/4
0/3
0/3
0/5
Inflammatory infiltrates
100
150
200
250+
1/3
1/4
3/4
0/2
1/4
1/4
1/4
0/6
0/4
1/4
1/4
2/4
1/4
2/3
2/3
1/5
Inflammation/peritonitis
abdominal, not liver
100
3/3
2/4
1/4
4/4
150
200
250+
4/4
3/4
1/2
1/4
0/4
3/6
1/4
2/4
1/4
0/3
1/3
2/5
a
Mean number of mitotic figures per number of high power fields (hpf).
TCDD/kg in diet (geometric mean of 1.8 and 18 ng
TCDD/kg) and 0.90 ng TCDD/kg liver (geometric
mean of 0.285 and 2.85 ng TCDD/kg) based on
measured liver concentrations at 200 days.
3.5. Spleen
There was a decrease in lymphoid density in all
fish exposed to TCDD-treatments at all time
points. Occasionally the lymphoid tissue of untreated fish was scant but was never as little as the
TCDD-treated fish (Fig. 5). This lesser density
was usually associated with an apparent increase
in stromal and vascular structures and often
vascular and/or sinus congestion. Occasional foci
of individual cell necrosis and hemosiderosis were
also observed in the 18- and 90-ng TCDD/kg
treatment groups. Some fish in the TCDD treat-
G.L. Walter et al. / Aquatic Toxicology 50 (2000) 287–299
293
ment groups were also characterized by multiple
foci with clusters of small endothelial-lined vascular structures, which contained small amounts of
intraluminal eosinophilic fibrillar material or erythrocytes (Fig. 6). These foci were often adjacent
to large or moderately-sized blood vessels.
Inflammation occurred with similar frequency
in treatment and control groups and was not
considered to be treatment related. Etiologic
agent(s) were not identified. The inflammation
was characterized by the infiltration of mononuclear cells into the splenic capsule with neovascularization and fibrous adhesions in some fish.
Extension of this reaction through the capsule
into the subcapsular parenchyma was uncommon.
3.6. Stomach
No dose-related toxicological effects on the
stomach were observed. The most frequently observed gastric lesions included chronic inflammation of the serosa with lymphocyte, mononuclear
cell, and occasional plasma cell infiltration. This
often involved the neural, perineural and perivascular tissues with variable amounts of capsular
thickening due to fibrovascular proliferation. In
Fig. 3. (A, B) Liver from an untreated fish (panel A) from a
fish fed 90 ng TCDD/kg food for 100 days (Panel B). There is
abundant cytoplasmic glycogen, which appears as an empty
space within the hepatocytes (straight arrow Panel A). Nuclei
are uniform in size with fine granular chromatin (curved
arrow, panel A). There is decreased hepatocellular glycogen,
slight variation in nuclear size and chromatin, and increased
numbers of mitotic figures (curved arrows, Panel B). Scale bar
on both panels represents 25 mm.
Fig. 2. Occurrence of histopathological lesions in the livers of
trout exposed to TCDD for up to 250 + days.
many samples, this reaction extended to the
mesentery, and in a few cases extended into the
muscularis. This inflammation was observed in
294
G.L. Walter et al. / Aquatic Toxicology 50 (2000) 287–299
untreated as well as all TCDD-treated groups.
Infectious agents were not identified and the etiology could not be determined. Multifocal mineralization, usually involving the submucosa and
Fig. 4. (A, B) Liver from a fish fed 90 ng TCDD/kg food for
50 days. There is prominent hepatocellular anisocytosis and
anisokaryosis. Note variation in nuclear chromatin patterns
and sharply outline vacuoles consistent with lipid (open arrowhead, Panel B). Infiltrates of lymphocytes (small arrow, Panel
B) are present in the portal areas. Bile duct (curved arrow,
Panel B), artery (large arrow), vein (solid arrowhead, Panel B).
Scale bar on panel A is 25 mm. Scale bar on panel B is 50 mm.
Fig. 5. (A, B) Spleen from an unexposed fish (Panel A) after
100 days and from a fish fed 90 ng TCDD/kg food for 150
days (Panel B). In the untreated fish there is a mixed population of lymphoid cells (arrow, Panel A), stromal cells (open
arrowhead), erythrocytes (solid arrowhead), and occasional
melanomacrophages (curved arrow, Panel A). In the exposed
fish there are increased numbers of melanomacrophages
(curved arrows, Panel B), stromal cells (open arrowhead), and
erythrocytes (solid arrowhead). Lymphoid cells (arrow, Panel
B) are decreased in number. Scale bar on panel A is 50 mm.
Scale bar on panel B is 25 mm.
G.L. Walter et al. / Aquatic Toxicology 50 (2000) 287–299
occasionally the subserosa and/or muscularis, often between inner and outer muscle layers, was
noted in all treatment groups. In most cases,
granulomatous inflammatory infiltrates were associated with the mineralized foci. Similar foci of
295
inflammation also occurred without evidence of
mineralization. Less common lesions included
lymphocytic or lymphohistocytic infiltration of
the cardiac glands with loss or effacement of
glandular epithelium. Vacuolization, clefting and
individual cell degeneration of the submucosal
basilar zone was noted in many stomach specimens but was considered to be an artifact of
sample preparation.
3.7. Peritoneum
Peritonitis occurred in all fish from control and
TCDD-treated groups with similar frequency and
was defined as the presence of inflammatory cell
infiltrates or fibrous to fibrovascular adhesions on
the serosal surfaces of one or more abdominal
organs or in the mesentery. Inflammatory cells in
all cases were primarily macrophages with fewer
numbers of lymphocytes and plasma cells, with
variable numbers of fibroblasts and fibrocytes.
Etiologic agents were not identified.
3.8. Gill
Gill lesions were infrequent, mild in severity
and occurred randomly between groups. Lesions
most frequently noted were focal chronic granulomas, occasional blunting and fusion of secondary
lamellae or lymphocytic infiltrates. Infectious
agents were not identified.
3.9. Hematopoietic tissue
Fig. 6. (A, B) Spleen from a fish fed 90 ng TCDD/kg food for
250 days. There are multiple foci of endothelial-lined vascular
structures (curved arrows, Panel A). Lymphoid density is
decreased. Erythrocytes (straight arrow, Panel B) and
melanomacrophages (open arrowheads, Panel B) are present.
Scale bar on panel A is 50 mm. Scale bar on Panel B is 25 mm.
Variation in numbers of melanomacrophages
was observed but could not be associated with
any dose group or time interval. Despite consistent differences between total numbers of erythrocytes or leukocytes in fish from the control and
90-ng TCDD/kg dose group fish, histopathological alterations in hematopoietic tissue were rarely
observed. Plasma cell infiltrates with adjacent foci
of hemosiderosis were present in one control fish
at 250 + exposure, and non-suppurative neuritis/
ganglioneuritis was present in one fish fed 18 ng
TCDD/kg in the diet for 150 days.
296
G.L. Walter et al. / Aquatic Toxicology 50 (2000) 287–299
3.10. Kidney
4. Discussion
Renal lesions were infrequent. Variations in the
number of melanomacrophages were noted but
were not treatment or dose dependent. Intensely
eosinophilic droplets were observed in the renal
tubular epithelium of a majority of the fish and
were not unique to any particular treatment
group.
Liver vacuolization has been reported to be
variable in salmonids with glycogen deposits causing variable distention of hepatocellular cytoplasm (Yasutake and Wales, 1983). Two patterns
of hepatocellular vacuolization were noted in the
present study. Indistinct fibrillar vacuolization
with varying prominence was associated with PAS
positive stain reaction and was considered to be
consistent with glycogen. This vacuolization pattern was most consistently observed in untreated
fish and fish fed 1.8 ng TCDD/kg. Clear, sharplyoutlined hepatocellular vacuolization was observed in the livers of some fish fed 18 and 90 ng
TCDD/kg. The localization, appearance and lack
of affinity for PAS suggest that the material was
lipid. Although these patterns of hepatocellular
vacuolization were not exclusive to specific treatment or control groups the trend suggests that, in
this study, vacuolar patterns were altered by administration of TCDD. Other treatment-related
hepatic alterations were consistently found and
included a variation in nuclear size and chromatin
pattern, mild variation in hepatocyte size and/or
increased numbers of mitotic figures. These results
are consistent with two effects associated with
TCDD toxicity in mammals, wasting syndrome
(Rozman, 1984) and fatty liver.
The hepatocellular effects of TCDD exposure
on fish have been documented in previous studies
(Fisk et al., 1997; Tietge et al., 1998). However,
unlike our study, previous studies exposed eggs,
fry or juvenile fish to waterborne TCDD or intraperitoneal (i.p.) injection. Sinusoidal dilatation,
hepatocellular swelling and necrosis, nuclear chromatin margination, decreased glycogen and cytoplasmic inclusions were observed following
exposure of eggs, yolk sac fry and juvenile rainbow or lake trout to varying concentrations of
TCDD in water for 48–96 h (Helder, 1981; Spitsbergen et al., 1991). Juvenile rainbow trout exposed to a single i.p. injection of TCDD in doses
from 0.1 to 125 mg/kg (Spitsbergen et al., 1988)
exhibited hepatic lesions characterized by bile
duct hyperplasia and the presence of intracytoplasmic eosinophilic inclusions. The incidence and
severity of both of these lesions varied among
3.11. O6ary
Ovaries generally contained eggs in varying
stages of development (primary and secondary
oocytes). The proportion of primary to secondary
oocytes varied among fish but was not associated
with any dose group, time interval or other histopathological alteration. Occasionally, degeneration of oocytes was observed and, in some cases,
was associated with mononuclear inflammation.
In some, but not all cases, peritonitis and ovarian
inflammation were both present. One fish from
the 90-ng TCDD/kg treatment group (150 days
exposure) contained both ovarian and testicular
tissue. This fish had ambiguous gonads on gross
examination.
3.12. Other tissues
Thymic and thyroid tissues were not obtained
from all fish; therefore definitive conclusions cannot be made. However, no apparent treatment-related alterations were observed. Thyroid follicles
varied in both size and height of follicular epithelium. Lymphocytic neuritis and perineuritis was
noted in pancreatic tissue of one fish exposed to
18 ng TCDD/kg after 100 days exposure. Adenomatous hyperplasia with multifocal interstitial
mineralized concretions of the Corpuscle of Stannius was noted in one fish exposed to 1.8 ng
TCDD/kg at 200 days exposure. Cardiac lesions
were limited to mixed mononuclear inflammatory
infiltrates on the serosal (pericardial) surface of
one control fish at 150 days and within the adjacent pericardial adipose tissue of one 18-ng
TCDD/kg treatment group fish after 200 days
exposure. Histologic alterations were not identified in interrenal, chromaffin, or ocular tissues.
G.L. Walter et al. / Aquatic Toxicology 50 (2000) 287–299
strains. The absence of bile duct hyperplasia and
hepatocellular eosinophilic inclusions in this study
may have been due to a number of factors, including strain of trout, lesser doses, age of fish at
the time of study, route of administration or
possible repair of lesions before the first histopathologic evaluation at day 100. Six weeks following a single i.p. injection of either 0.06 or 3.06
mg TCDD/kg, livers of juvenile rainbow trout
contained less glycogen compared to unexposed
fish (van der Weiden et al., 1992). Livers of the
exposed fish also exhibited hepatocellular
swelling, hydropic degeneration, vacuolization,
single cell necrosis and occasional focal necrosis
with mononuclear inflammation. Twelve weeks
after the single i.p. injection liver glycogen content
had increased, and there was an increased number
of mitotic figures in the 3.06-mg/kg dose group
(van der Weiden et al., 1992). In the current
study, mild mononuclear inflammatory infiltrates
were present in the liver to a similar degree in all
treatment groups. A number of fish exposed to 18
and 90 ng TCDD/kg had livers which contained
cells with yellow–brown slightly refractile granules in the cytoplasm. These granules were suggestive of bile, although staining techniques to rule
out iron and hemosiderin were not conducted.
Dietary exposure of fingerling rainbow trout to
494 ng/kg TCDD for 91 days did not result in any
detectable gross lesions, although histologic examination was not performed (Kleeman et al., 1986).
This exposure was of shorter duration than our
study, but to a greater TCDD concentration than
even the greatest TCDD concentration in our
study.
In the present study, multifocal gastric mineralization with associated granulomatous inflammation was observed in some fish from every group,
including controls. The stomach alterations observed are most consistent with mineralized foci
of inflammation defined as visceral granulomas
(Ferguson, 1988; Roberts, 1989) and were not
considered to be related to TCDD exposure.
Dose-related gastric lesions noted in previous
studies of trout include cardiac gland necrosis
(Helder, 1981), degeneration of enterocytes (Spitsbergen et al., 1991) and atrophy or hyperplasia of
serous gastric glands which varied between hatchery strains (Spitsbergen et al., 1988).
297
Lymphoid involution of the thymus and spleen
and hypocellularity of the hematopoietic tissue
have been reported following i.p. injection of juvenile rainbow trout with 10 mg TCDD/kg (Spitsbergen et al., 1988). Thymic tissue was collected
and examined microscopically from a number of
the fish in the present study. The amount of
lymphoid tissue varied among fish but this was
attributed, in most cases, to sampling variation.
Due to the size and location, piscine thymic tissue
is a difficult organ to consistently sample and
evaluate, especially in adults. Thymic tissues were
difficult to identify and age-related thymic involution had most likely already occurred in these fish.
Increases in numbers of melanomacrophages,
lymphoid depletion, and increased splenic erythrocytes have previously been reported following
a single i.p. injection of 0.27–2.93 mg TCDD/kg
in juvenile mirror carp (Cyrinus carpio; van der
Weiden et al., 1993). Melanomacrophages are
considered to be repositories of end products of
cell breakdown, such as phospholipids, erythrocytes, particulate matter and antigens (Ferguson,
1988). In the spleens of TCDD-exposed fish, there
was evidence of increased degeneration of
lymphocytes, and possibly erythrocytes which
would lead to an increased need for functional
macrophages to phagocytoze cellular debris. This
could account for both increased numbers of
melanomacrophages and macrophages with yellow–brown intracytoplasmic globular material,
interpreted as hemosiderin.
The presence of multiple foci of endotheliallined vascular structures were present in a few
TCDD-exposed fish at the 250+ time period.
The significance of this observation is unknown,
although it is possible that these structures might
represent vascular or lymphatic channels, which
formed or opened secondary to congestion or
impaired circulation.
In the present study, decreases in splenic
lymphoid density observed in TCDD-treated fish
were accompanied by prominent splenic stroma,
sinuses, vasculature and blood content. However,
this may have been due to the presence of fewer
lymphoid cells. The somewhat variable nature of
the lymphoid depletion and lack of histopathologic changes in hematopoietic tissue observed in
298
G.L. Walter et al. / Aquatic Toxicology 50 (2000) 287–299
this study, as compared to more severe lesions
observed in previous studies, may be due to differences in strain and age of fish, dose and route of
administration of TCDD, duration of study and
sampling times. Unlike mammalian bone marrow,
hematopoietic tissue of fish has no bony spicules
or adipocytes with which to judge cellularity by
comparing the proportion of cells to fat. This lack
of stromal support also causes fish hematopoietic
tissue to collapse when sectioned, which can mask
mild to moderate changes in cellularity. Subtle
changes may be more easily distinguished in plastic-embedded thin sections (Spitsbergen et al.,
1988), than in paraffin-embedded specimens as
done in this study.
Acknowledgements
This research was supported by a grant from
the Biology Panel of the Exploratory Research
Program on the US-EPA (R814850). The assistance of R. Crawford, L. Williams, D. Tillitt, J.
Newsted, K. Henkel, and W. Lawrence are
greatly appreciated. Dr S. Sleight of the Department of Pathology reviewed all of the histopathology conclusions.
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