WATER POLLUTION CONTROL RESEARCH SERIES
18050 DXJ 05/71
Histochemical and Cytophotometric
Assay of Acid Stress in
Freshwater Fish
ENVIRONMENTAL PROTECTION AGENCY • RESEARCH AND MONITORING
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WATER POLLUTION CONTROL RESEARCH SERIES
The Water Pollution Control Research Series describes the results
and progress in the control and abatement of pollution in our
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should be directed to the Chief, Publications Branch, Research
Information Division, Research and Monitoring, Environmental
Protection Agency, Washington, D.C. 20460.
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HISTOCHEMICAL AND CYTOPHOTOMETRIC ASSAY OF ACID STRESS
IN FRESHWATER FISH
by
The Pennsylvania State University
Department of Biology
University Park, Pennsylvania 16802
for the
ENVIRONMENTAL PROTECTION AGENCY
Grant Number DXJ 18050
May 1971
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ii
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ABSTRACT
The feasibility of using histochemical and histopathological changes in
brook trout, longnose dace and fathead minnows as bioindicators of acid
pollution was investigated. Laboratory studies entailed using a gravity
flow diluter system. Field studies involved using net traps in polluted
streams. Exposure durations were 4-5 days and 28-30 days. Histo-
chemical and cytophotometric analyses were made of gills, Stannius
corpuscle, blood, spleen, kidney and liver.
The primary mode of acid toxicant action is gill damage which results in
impaired respiratory, excretory and liver functions. Short term indices
of acid stress include: colloidal iron and PAS staining of gills and
renal Stannius corpuscles. A useful bioindicator of prolonged acid
exposure is decreased azure B-RNA staining of liver cells; this assesses
the extent of liver impairment and reflects a reduced tolerance of fish
to other toxicants.
Sublethal levels of acidity are not cumulative. However, pH levels of
about 5.0 should be considered hazardous since they prove toxic to
breeding fish having increased oxygen needs and also reduce the liver's
ability to detoxify noxious substances present in acid polluted waters.
This report was submitted in fulfillment of Grant No. 18050 DXJ under
partial sponsorship of the Water Quality Office, Environmental Protection
Agency.
111
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CONTENTS
Section Page
I CONCLUSIONS 1
II RECOMMENDATIONS 3
III HISTOCHEMICAL ANALYSES OF GILLS AND STANNIUS CORPUSCLES
IN ACID EXPOSED BROOK TROUT 5
IV ERYTHROCYTIC INDICES OF STRESS IN ACID EXPOSED BROOK
TROUT 21
V HEMAL CHANGES IN ACID EXPOSED FATHEAD MINNOWS AND
LONGNOSE DACE 49
VI HEPATIC INDICES OF STRESS IN ACID EXPOSED BROOK TROUT . 65
VII ACKNOWLEDGMENTS 83
VIII REFERENCES 85
IX PUBLICATIONS 91
X GLOSSARY 93
XI PERSONNEL 95
XII APPENDIX 97
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FIGURES
Figure Page
1. DNA distribution patterns for gill supporting cells
of brook trout exposed to acid water for acute (4-5
day) and subacute (28 day) periods 11
2. Periodic acid Schiff (PAS) distribution patterns for
Stannius corpuscle cells of brook trout exposed to acid
water for acute (4-5 day) and subacute (28 day) periods . . 13
3. DNA distribution patterns for Stannius corpuscle cells
of brook trout exposed to_ acid water for acute (4-5 day)
and subacute (28 day) periods 15
4. Total cellular RNA distribution patterns for Stannius
corpuscle cells of brook trout exposed to acid water for
acute (4-5 day) and subacute (28 day) periods 16
5. Hematocrit, hemoglobin, peripheral RBC count and venous
blood p02 values in nonbreeding trout exposed to acid for
five days (pH 4.0-3.5) 25
Total blood volume determined with radioactive iodinated
serum albumin (RI131SA) in nonbreeding trout exposed to
acid for five days (pH 4.0-3.5) 26
7. Photomicrographs of immature erythroid cells from a
renal tissue imprint. P - proerythroblast, B - basophilic
erythroblast, PC - polychromatophilic erythroblast,
R - reticulocyte (Giemsa stain, 1440X) 27
8. Feulgen-DNA distribution patterns for a mixed population
of erythroid cells from renal and splenic imprints of
nonbreeding trout exposed to acid for five days (pH 4.0-
3.5) 29
9. Total cellular RNA content (+S.E.) of erythroid maturation
stages in renal and splenic imprints of nonbreeding trout
•exposed to acid for five days (pH 4.0-3.5) 30
10. Radioactive iron incorporation in RBC of non-breeding,
acute acid exposed trout (pH 4.0-3.5 x 5d) 31
11. Hematocrit, hemoglobin and venous blood p02 values in
nonbreeding trout exposed to acid (pH 4.9) for 26-28
days 32
VI
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Figure Page
12. Feulgen-DNA analyses of erythroid nuclei in renal
and splenic imprints of subacute acid exposed trout
during nonbreeding season (pH 4.9 x 26-28d) 34
13. Total cellular RNA content (±S.E.) of erythroid
maturation stages in renal and splenic imprints
of subacute acid exposed trout during nonbreeding
season (pH 4.9 x 26-28d) 35
14. Hematocrit, hemoglobin and peripheral RBC count
in reproductively active trout exposed to acid
(pH 4.9) for 26-28 days 36
15. Total cellular RNA content (±S.E.) of erythroid
maturation stages in renal and splenic imprints
of reproductively active, subacute acid exposed
trout (pH 4.9 x 26-28d) 37
16. Total blood volume determined with RI131SA in
reproductively active, subacute acid exposed
trout (pH 4.9 x 26-28d) 38
17. Feulgen-DNA measurements of erythroid blast nuclei
in renal and splenic imprints of reproductively
active trout exposed to acid (pH 4.9) for 26-28 days ... 39
18. Radioactive iron incorporation in RBC of repro-
ductively active, subacute acid exposed trout
(pH 4.9 x 26-28d) 40
19. Hematocrit, hemoglobin and venous blood p02 values
in subacute acid exposed trout immediately
following breeding season (pH 4.9 x 26-28d) 41
20. Hematocrit of nonbreeding trout exposed to acid
(pH 4.8) for two days' in the field 42
21. Feulgen-DNA distribution patterns for a mixed
population of erythroid cells from renal and
splenic imprints of nonbreeding, acute acid
exposed trout in the field (pH 4.8 x 2d) 43
22. Histograms representing Feulgen DNA values for
splenic lymphocytes of longnose dace exposed to
acid water for subacute (28 day) period 55
vii
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Figure Page
23. Histograms representing Feulgen DNA values for
splenic lymphocytes of fathead minnows exposed
to acid water for acute (1 day) period in two
acid streams 57
24. Histograms representing Feulgen DNA values for
splenic lymphocytes of longnose dace exposed
to acid water for acute (1 day) period in two
acid streams 58
25. Histograms representing nuclear histone values for
splenic lymphocytes of longnose dace exposed to
acid water for acute (1 day) period in two acid
streams 59
26. Histograms representing Feulgen DNA values for
splenic lymphocytes of fathead minnows exposed
to a graded severity of pH for a chronic (7 months)
period 60
27. Spectral absorption curves of Feulgen-DNA and fast
green histone complexes in trout liver nuclei, 67
28. Spectral absorption curves of FDNB-azure B and
FDNB-Sakaguchi complexes in trout liver nuclei 69
29. Frequency distribution profiles of Feulgen-DNA
and fast green histone stained liver nuclei of
acid exposed trout 71
30. Effects of acid exposure on RNA, (lysine + tyrosine)/
arginine ratio, lysine + tyrosine, and arginine
levels in trout liver nuclei 73
31. Microdensitometer tracings of electrophoresed
histone fractions from control and acid exposed
trout liver 75
32. Schematic outline of hemal, erythrocytic and
hepatic responses in fish exposed to acid polluted
water 82
Vlll
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TABLES
Table Page
1. Histochemistry of hatchery reared brook trout gill
following acute (4-5 day) and subacute (28 day)
exposure to acid water 6
2. Histochemistry of hatchery reared brook trout Stannius
corpuscle following acute (4-5 day) and subacute
(28 day) exposure to acid water 6
3. Histochemistry of gill mucopolysaccharides in
fathead minnows exposed to graded severities of
pH for seven months 7
4. Chemical basis for histochemical and histological
stains employed 8
5. Mucous cell changes in acid exposed fish 9
6. Summary table of major findings 19
7. Differential counts in blood smears from non-
breeding brook trout exposed to acid water for
five days (acute exposure) and 26-28 days
(subacute exposure) 26
8. Maturation sequence of trout erythrocytes in
renal and splenic hemopoietic tissue 27
9. Differential red blood cell counts in renal and
splenic touch preparations from nonbreeding brook
trout exposed to acid water for five days and
26-28 days 28
10. Differential counts in blood smears from repro-
ductively active brook trout exposed to acid
water for 26-28 days 37
11. Differential red blood cell counts in renal and
splenic touch preparations from reproductively
active brook trout exposed to acid water for
26-28 days 38
IX
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Table Page
12. Stream conditions during field studies of acute
acid exposed fish ..................... 52
13. White blood cell differential counts of control
and acid exposed longnose dace after 28 day
exposure ......................... 54
14. White blood cell differential counts of control
and acid exposed fathead minnows during field
studies of acute exposure ................. 56
15. White blood cell differential counts of control
and acid exposed fathead minnows after a 7 month
exposure ......................... 59
16. Cytophotometric measures of RNA, lysine + tyrosine
and arginine in liver nuclei of acid exposed trout .... 72
17- Relative numbers of pink, green and purple staining
nuclei in acid exposed trout liver (eosin-fast
green stain) ....................... 74
18. Comparison of extinction ratios, Ee^o/Esoo, of
eosin-fast green stained histone fractions from
trout liver extracts ................... 76
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SECTION I
CONCLUSIONS
(A) General Conclusions (relating to utility of histochemical methodology)
1. A number of histochemical methods, primarily developed and used
for diagnosis of human disease states, can be used to assess
toxicant effects in fish of sublethal levels of water acidity
under laboratory and field conditions.
2. Analytical histochemical techniques, which involve use of cyto-
photometry (microscopic microchemistry), enable the detection of
tissue chemical changes in the picogram (10~^2g) range with a
routine accuracy of ±2%. Thus, for microscopic analysis of organic
constituents in tissue cells, the detection limit, sensitivity and
accuracy of cytophotometry approach theoretical limits imposed by
light microscopy -
3. Analytical histochemistry is a simpler and more versatile tool
than most physical analytical procedures, in that — purified
samples are not required, minimal amounts of tissue (<1.0mg)
are needed and routine processing, microtomy and staining methods
are generally involved. Instrumentation is also not costly;
microspectrophotometer components can be purchased for less than
$5,000 if a laboratory is already equipped with a research microscope.
4. Use of a battery of several histochemical tests is preferable to a
single test in establishing safe versus harmful levels of stream
pollutants since measures based on combined tests enable one to
differentiate between acclimative and debilitative responses.
(B) Specific Conclusions (regarding acid induced histochemical changes in
fish)
5. Histochemical changes in mucopolysaccharides and nucleic acids of
the trout gill and renal Stannius corpuscle cells are useful
short-term (l-7d) indices of sublethal levels of water acidity.
These are transient changes and are not evidenced in 28 day
exposed fish.
6. Ribonucleic acid levels in certain renal blood cell precursors
are depressed during acute exposure and elevated during prolonged
(28 d) exposure of trout to sublethal levels of acidity and can
thus serve as a useful index of acid pollution in both acute and
subacute exposure test situations.
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7. Hematocrit (or hemoglobin) measures, leucocyte differential counts
and total serum lipid levels are generally more useful supplemental
indices of hemal responses to sublethal levels of acid exposure
than biochemical measures of blood pC^j radioiron incorporation,
radioiodinated serum albumin, electrophoretic histone analyses or
chromatographic analyses of serum fatty acids.
8. A given level of water acidity is much more toxic under field
conditions (i.e. in presence of other stream contaminants) than
under controlled laboratory conditions.
9. Reproductively active trout are less tolerant to acid water than
nonbreeding trout.
10. Brook trout, fathead minnows and longnose dace exhibit the same
basic pattern of acid induced histochemical and histological
responses in gills, hemopoietic organs and circulatory elements.
11. Liver function is impaired in acute and subacute acid exposed
trout as reflected in a decreased azure B-RNA content of liver
cell nuclei. Since a major function of the liver is removal of
blood toxins any overall decrease in liver metabolism would
reduce the tolerance of fish to toxicants present in acid
polluted streams.
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SECTION II
RECOMMENDATIONS
Recommendation 1. Since standards for water quality must ultimately
be based on biological as well as physical and chemical criteria, more
emphasis should be placed on developing sensitive bioassays of water
pollution. Because many histochemical methods are extremely sensitive
and quantifiable, greater use of cytophotometry as a bioassay approach
is recommended. Metal ion toxication should be especially amenable to
bioassay using histochemical techniques since, unlike acid pollution,
effects of heavy metals tend to be cumulative.
Recommendation 2. Careful consideration should be given to concentrating
attention on using fish tissue changes as biological indicators of water
pollution. Fish constitute a valuable resource from the conservational,
recreational and economic standpoint. Of equal importance, since fish
and man exhibit remarkable similarities in metabolic regulatory mecha-
nisms at the cellular level, pollutants causing histopathology in fish
should be regarded as potential health hazards.
Recommendation 3. Special study is required of what constitute "norms"
for various biochemical, physiological and histological parameters in
various fish species. Present information is deficient with respect to
normal levels of tissue constituents as well as microscopic and histo-
chemical appearance of healthy fish. Of particular interest to fish
pathologists and physiologists would be data on hemal, endocrine and
excretory tissue parameters.
Recommendation 4. There is an urgent need for intensified research on
toxicant effects of mixtures of water contaminants since the development
of adequate standards of water quality ultimately will require detailed
information on the mode of entry, mechanism of action, cumulative
effects, etc., of a specified toxicant in the absence and presence of
other toxicants.
Recommendation 5. Serious consideration needs to be given to devising a
general procedure for interlaboratory exchange of tissue specimens for
analysis, as well as of material which has been histochemically analyzed,
to speed up standardization of various types of analytical procedures on
a nationwide basis. Implementation of such an exchange could be
initiated by setting up a repository at one of the Federal Water Quality
Laboratories.
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SECTION III
HISTOCHEMICAL ANALYSES OF GILLS AND STANNIUS
CORPUSCLES IN ACID EXPOSED BROOK TROUT
Preliminary work in our laboratory showed that acute exposure of trout to
severe acidity (pH 3.9) disrupts the cellular integrity of gill lamellae
(Plonka and Neff, 1969) and also results in depressed body sodium levels
(Packer and Dunson, 1970). Since both gills and renal Stannius cor-
puscles are involved in ion and water regulation (Conte, 1969; Fontaine,
1964), a series of experiments were undertaken to investigate the nature
of histochemical responses in these organs in fish subjected to sublethal
levels of acidity for short (4-5 day) and relatively long (28 day) dura-
tions of exposure. The specific aims of this phase of work were: (1) to
analyze histochemical changes in gills and Stannius corpuscles with
methods selected to provide measures of cellular constituents involved in
regulation of metabolism (i.e., nuclear DNA, RNA, and histone content) as
well as measures of cytoplasmic endproducts of synthesis (mucin, PAS posi-
tive material or nonspecific protein content); (2) to determine which
tests show promise as potential short-term bioassays for toxicant effects
of sublethal levels of water acidity; (3) to compare histochemical
effects of subacute (28 day) versus acute (4-5 day) exposures to sub-
lethal levels of acidity on designated parameters; and (4) to elucidate
the physiological basis of histochemical response patterns in terms of
possible long-term effects on the functional capacity of the gills and
Stannius corpuscle.
Methods. Tissue specimens came from three sources: hatchery reared brook
trout (Salvelinus fontinalis), obtained from the Pleasant Gap Fish
Research Station, Pleasant Gap, Pennsylvania; wild brook trout, caught in
Tomtit Run, Clearfield County, Pennsylvania; and fathead minnows
(Pimephales) (fixed in 4% formalin, preserved in 70% ethanol) obtained
from the National Water Quality Laboratory, Duluth, Minnesota. Prior to
experimentation, trout were acclimated to laboratory conditions for one
to two weeks in 150 gallon tanks at 12 C under constant aeration. At
the end of each test run, all fish were sacrificed by a sharp blow on the
head, weighed (±0.1 gm), measured from head to caudal fin (±0.5 gm), dis-
sected, and sexed. Hatchery trout varied in length from 17-20 cm and
weighed 75-120 gins with the exception of one group of larger fish used
for an acute study (21-25 cm; 170-225 gms). Wild trout varied in length
from 11-18 cm and weighed 10-60 gms.
Acute toxicity tests (4-5 days) were conducted in two systems. In one, a
group of 50 control and 50 experimental hatchery reared trout were main-
tained in a static system. The water was not changed during the 4-5 day
period, although it was constantly aerated, filtered and kept at 12 C.
Acidity was maintained by addition of concentrated HC1 (specific gravity
1.84; 37% by weight) to establish a pH of 4.0 ± 0.2. Control tanks were
maintained at a pH of 7.0 ± 0.5. In the second system, 70 wild brook
trout were exposed to varying levels of acidity in a gravity flow
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Table 1 Histochemistry of hatchery reared brook trout gill following acute (4-5 day)
and subacute (28 day) exposure to acid watei:
~ " No. of analyses
Treatment Staining reactions (No. of fish)
— Contr~ " 1. Colloidal iron, PAS, alcian blue and 300(100)
PH"7.0-7.5 for 5 days mucicarmine for mucopolysaccharides
50 ^".olorl'davs 2. Feulgen cytophotometry for DNA 200(20)
3. Eosin-fast green, alkaline fast
green and eosin Y for histones
4.
20 Control
pH 7.0 for 28 days 1.
2.
Gomori ' s
and eosin
As above
As above
trichrome and hematoxylin
for histological detail
20(10)
100(50)
120(40)
200(20)
20 Acid exposed,
pH 4.8 for 28 days 3. As above
4. As above 20<1°>
40(20)
aThe number of analyses refers to the number of tissue sections prepared and examined
microscopically. Where cytophotometry was performed the number of analyses represents the
number of individual cellular measures.
Table 2. Histochemistry of hatchery reared brook trout Stannius corpuscle following acute
(4-5 day) and subacute (28 day) exposure to acid water
N Treatment Staining reactions
50 Control, 1. Colloidal iron, alcian blue and
pH 7.0-7.5 for 5 days mucicarmine for mucopolysaccharides
2. Periodic acid Schiff for aldehydes
(cytophotometry)
50 Acid exposed, 3. Feulgen cytophotometry for DNA
pH 4.0 for 5 days
4. Azure B cytophotometry for RNA
5. Eosin-fast green, alkaline fast
green and eosin Y for histones
6. Sudan black B for lipid
7. Gomori 's trichrome and hematoxylin
and eosin for histological detail
20 Control
pH 7.0 for 28 days 1. As above
2. As above
3. As above
20 Acid exposed, 4] As above
pH 4.8 for 28 days .
1 5. As above
6 . As above
7 . As above
No. of analyses3
(No. of fish)
300(100)
200(50)
200(20)
180(20)
20(10)
20(10)
20(10)
100(50)
120(40)
200(20)
200(20)
150(20)
40(20)
20(10)
40(20)
40(20)
aThe number of analyses refers Co the number of tissue sections prepared and examined
microscopically. Where cytophotometry was performed the number of analyses represents the
number of individual cellular measures.
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proportional diluter system (Mount and Brungs, 1967) with aquaria main-
tained at pH's of 4.1 ± 0.4, 4.7 + 0.5, 5.1 ± 0.4, 6.6 + 0.3, and
7.1 ± 0.2. Temperature was maintained at 12 C using an automatic cool-
ing unit and pH monitored by a multichannel recorder system.
Tables 1 and 2 list various histochemical tests performed on gill and
Stannius corpuscle tissue from acute acid exposed hatchery trout and
wild trout exposed to graded levels of acidity.
Subacute toxicity studies (28 day) on a group of 53 hatchery trout were
all conducted in a gravity flow diluter system. The pH's of the three
experimental tanks were 6.3 ± 0.1, 5.9 ± 0.4, and 4.8 ± 0.2, and the pH
of the control tank was 7.0 ± 0.2.
Chronic exposure data were based on tissue from fathead minnows exposed
to graded severities of acid for seven months (in a gravity flow diluter)
at the Duluth Water Quality Laboratory (Table 3). These fish were fixed
in formalin (4%) and preserved in 70% ethanol before shipment by air
express to our laboratory.
Table 3. Histochemistry of gill mucopolysaccharides in fathead
minnows exposed Co graded severicies of pH for seven
monchs
SCaining reaccions No. of analyses
N TreatmenC for all groups (No. of fish)
20 ConCrols 1. Colloidal iron, alcian 134(67)
(two groups at blue and mucicarmine for
pH 7.0 and 7.A) mucopolysaccharides
A7 Experimentals 2. Hemacoxylin and eosin 67(67)
(five groups ac for hisCological detail
pH 6.5, 6.0, 5.5,
5.0, and 4.7)
The number of analyses refers Co the number of tissue sections
prepared and examined microscopically. Where cyCophoComeCry was
performed Che number of analyses represenCs Che number of individual
cellular measures.
Portions of gills and kidney (with Stannius corpuscles) were fixed in 4%
neutral buffered formalin for 24 hours, dehydrated, cleared, paraffin
embedded and sectioned at 6y. Touch preparations of Stannius corpuscles
were also made, some of which were fixed in CHoOH while others were
stained immediately for lipids. Formalin fixed touch preparations were
used for azure B-RNA staining. Sections of Stannius corpuscles and
gills were analyzed for DNA using the Feulgen reaction, basic fuchsin,
CI 42500 and for mucopolysaccharides using periodic acid Schiff's (PAS)
reagent (Humason, 1967). Touch preparations of Stannius corpuscles were
treated with DNAase (Worthington Corp.) and stained by azure B for RNA.
Cytophotometric measurements were made of DNA, RNA and PAS positive
material using a single beam microspectro^hotometer using the two wave-
length method of cytophotometry described bv Patau (1952) and Ornstein
(1952) . For F-DNA and PAS the wavelengths chosen were 560 nm and 495 nm;
for azure B-RNA the wavelengths selected were 560 nm and 510 nm.
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Xonhomogeneous areas (i.e., whole nuclei or entire cells) were measured
by centering the nucleus in the photometric field, excluding as much
surrounding light as possible.
Qualitative histochemical analysis of histones in gills and Stannius
corpuscles were made using eosin Y-fast green (eosin Y CI 45380, fast
green FCF CI 42053) (Bloch, 1966), alkaline fast green (Humason, 1967)
and eosin Y (Bloch and Hew, 1966). Unfixed Stannius corpuscle touch
preparations were also stained with Sudan black B, CI 26150 (Humason,
1967) for lip ids. In addition, three mucopolysaccharide stains, col-
loidal iron, alcian blue, CI 74240 (Mowry, 1963) and mucicarmine,
CI 75470 (Humason, 1967) were used for studies of mucin content in gill
and selected Stannius corpuscle sections. For routine microscopic
analyses, henatoxylin and eosin and Gomori's trichrome (chromotrope
CI 16570; fast green FCF CI 42053) were employed (Humason, 1967) (Table 4)
Table ^. Chemical basis for histochemical and histological stains
employed
Tissue substrate
nested
Chemical groups
in cells
involved in staining
Reaction or stain
employed
Nuclear proteins
(histones)
Cytoplasmic lipids
Mucopolys accharides
Mu capolysaccharides
Mucopolysaccharides
Compounds containing
free aldehyde groups
Acidophilic and baso-
philic const!tuents
Acidophilic and baso-
philic constituents
aldehydes
phosphates
basic amino acids
lipids
acidic groups of
mucopolysaccharides
carboxyl groups
and sulfates
unknown
aldehydes
anionic and cationic
components
anionic and cationic
components
Feulgen reaction
(Schiff reagent)
basophilia
(azure B and DNAase)
acidophilia
(eosin-fast green)
mutual solubility of
fat and dye
(Sudan black B)
(colloidal iron)
chelate with metal ion
subsequently reduced
by K ferrocyanide
basophilia
(alcian blue)
mucicarmine affinity
PAS reaction
(Schiff reagent)
hematoxylln and
eosin
Gomori'<= trichrome
A preliminary aspect of work involved a comparative evaluation of acid
induced histochemical changes in gills and Stannius corpuscles of wild
and hatchery reared trout. That differences between wild and hatchery
bred trout exist in size, physical state, behavioral activity, and
tolerance to water toxicants is well recognized. However, not unexpect-
edly, there were no differences in the histological or histochemical
appearance of tissues or organs (including the gills and Stannius cor-
puscles) between fish obtained from the hatchery and those collected in
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field streams. Moreover, no differences were found in the type or
intensity of acute acid induced histochemical changes which could be
related to size, weight, sex or collection site of the fish used.
Therefore, the following data are presented without reference to the
source of fish.
General behavioral observations were made on fish exposed to acid water
(pH 4.0-4.85). Both acute (4-5 day) and subacute (28 day) exposed trout
exhibited a noticeable decrease in activity- Fish in acidic water ate
poorly, whereas controls consumed 4-5 fish pellets per day. Wild trout,
which were initially highly colored, showed progressive loss of colora-
tion in all gradations of acidity (pH 4.5, 5.0, 5.5, 6.0) but blanching
was most marked at low pH's (4.5, 5.0). Both subacute and acute acid
exposed fish surfaced more than controls and gulped air through their
mouths. In extremely low pH water (below 4.0) trout secreted copious
amounts of mucus such that a mucous film became visible over their whole
body, especially in the gill area. Fish in this state showed a loss of
equilibrium, often swimming with their ventral sides up. Acute and sub-
acute acid exposed trout also exhibited hyperventilation as reflected in
an increased rate of opercular movements. No attempt was made to under-
take a systematic study of behavioral changes. The intent was simply to
confirm that exposure of trout to moderate and high levels of acidity
did evoke external behavioral symptoms of respiratory distress. It was
established, however, that behavioral reactions were most pronounced in
the first four days and tended to diminish with continued exposure. The
average mortality rate of subacute exposed trout maintained in control
(pH 7.0) and moderately acid (pH 5.5, 6.0) aquaria was 10% whereas
mortality at lower pH levels (pH 5.0, 4.5) was 30%.
Table 5. Mucous cell changes in acid exposed fish
N
50
50
5
5
20
20
Presence of
Treatment mucous cells
Hatchery trout, control , +
pH 7.0-7.5 for 4-5 days
Hatchery trout, acute exposed, +++ to
pH 3.7-4.0 for 4-5 days 1 1 II
Wild trout, control, +
pH 7.0-7.5 for 4-5 days
Wild trout, acute exposed, +-H- to
pH 4.0-4.5 for 4-5 days -H-H-
Hatchery trout, control, +
pH 7.0-7.5 for 28 days
Hatchery trout, subacute exposed, ++
pH 4.0-4.5 for 28 days
Location of
mucous cells
tip of filaments
base of 1 ame 1 1 ae
tip of filaments
along length of
lamellae
tip of filaments
most at lamellar
base, some along
side
along lamellae
tip of filaments
between lamellae
tip of filaments
at lamellar base
tip of filaments
along lamellae
Extracellular Filament
material clubbing
to +
•H-+ to -t-H- to
MM Mil
+ +
-t-H- +++
to +
++ ++
67 Fathead minnows, chronic exposed, +• at lamellar base
all gradations,
pH 4.5-7.0 for 7 months
aThese observations were made from cross sections of whole fish; each filament was therefore
cut and it was not possible to examine the tips of the filaments for mucous cells.
-------
Microscopic analyses of gill changes in acute and subacute acid exposed^
brook trout are summarized in Table 5. A marked increase in the number
of gill mucous epithelial cells was evidenced in acute acid exposed
trout as well as in unattached epithelial cells, extracellular material
and mucin so that interlamellar spaces were almostly completely obscured.
In subacute acid exposure studies, trout exhibited a moderate increase in
lamellar mucous cells and accumulation of extracellular materials, but
the spaces between lamellae were not completely obliterated. Moreover,
the distribution of mucous cells along the lamellae differed in control
and subacute or acute acid exposed fish. Mucous cells of controls were
almost exclusively limited to the base of each lamella whereas they were
found along the entire lamellar length in acid exposed fish. Chronic
exposed fathead minnows exhibited none of the changes observed in trout.
There was no difference in relative numbers of lamellar mucous cells
between acid exposed fish and controls and there was no evidence of
increased cellular and extracellular material between lamellae.
In brief, the major findings in this phase of work can be summarized as
follows. First, acute acid exposure elicited a marked increase in muco-
polysaccharide staining of gill epithelial cells. This was more pro-
nounced at the lowest pH levels (pH 4.0-5.0). Furthermore, both hatchery
and wild trout exhibited the same intensity of mucification and extent of
histopathology (interlamellar cell debris). Second, there appears to be
a diminution in this response with increased duration of exposure. After
28 days of continuous exposure in low pH aquaria (pH 4.0-4.5), only a
moderate increase in mucopolysaccharide staining was evidenced in trout
gills while subacute exposures to moderate levels of acidity elicited
little or no response. Significantly, there was no difference in the
histochemical appearance of gills from fathead minnows exposed to pH
gradations ranging from 4.5 to 7.0 after seven months of exposure.
Cytophotometric analysis of DNA in gill cells. Data summarized in
Figure 1 represent a total of 400 individual measurements of nuclear
Feulgen DNA (F-DNA). Determinations were made on gill supporting cells
which are located at the lamellar base and can be distinguished from
other gill epithelial cells by their oval shape and large vesicular
nuclei. Because the increase in interlamellar material begins at the
lamellar base and supporting cells are consistently found in conjunction
with lamellar "clubbing," it was reasoned that nuclei of supporting
cells might reflect either signs of damage (karyolysis) or changes in
mitotic (i.e., proliferative) activity.
F-DNA data are conventionally presented as frequency histograms because
this facilitates the detection of changes in the proportion of nuclei
which exhibit either chromatin loss (reduced F-DNA content) or mitotic
activity (tetraploid F-DNA level). To facilitate comparison of the expo-
sure groups, dotted lines were included on histograms to designate the
2C DNA interval; the 2C level represents the nonreplicating diploid con-
tent of DNA. To insure that the value assigned as the 2C level of DNA
truly represented the diploid amount of DNA, F-DNA content was also mea-
sured in 50 sperm cells from these fish. As expected, the mean DNA con-
tent for the sperm cells was one-half that of the somatic cells.
10
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Figure 1.
ACUTE EXPOSURE
CONTROL GROUP
(PH 7.0-7.5)
ACUTE EXPOSURE
EXPERIMENTAL GROUP
(PH4.0)
20^
© ! © ! ©
| j ^ SUBACUTE EXPOSURE
CONTROL GROUP
(PH 7.0)
40 n
20-1
B
10
SUBACUTE EXPOSURE
EXPERIMENTAL GROUP
(PH4.8)
12
AMOUNT OF FEULGEN-DNA IN ARBITRARY UNITS
DNA distribution patterns for gill supporting cells of
brook trout exposed to acid water for acute (4-5 day)
and subacute periods (28 day). Numbers in circled
inserts refer to percentage of cells between and on
each side of dotted division lines.
As is evident from the data presented, there are few nuclei with 4C
amounts of DNA, indicating that neither acute nor subacute acid exposure
causes any stimulation in mitotic activity of supporting cell elements.
Moreover, there is no appreciable increase in nuclei with very low DNA
levels, indicating the absence of nuclear damage in acid exposed gills.
Thus, supporting gill lamellar cells are not the major source of cellular
debris which comprises the interlamellar coagulum present in acute acid
exposed gills.
11
-------
Histochemical analysis of basic nuclear proteins in gill cells. Another
useful parameter of nuclear function is the histone or basic protein
content. Relative changes in histones can be analyzed qualitatively or
semiquantitatively using the eosin-fast green method providing there is
assurance that one can differentially stain nuclei which exhibit prefer-
ential staining for eosin as opposed to fast green. Analyses of eosin-
fast green stained tissue sections from control trout showed this to be
the case. However, all epithelial cell nuclei (in both gills and
Stannius corpuslces) proved to stain green whereas erythrocyte nuclei
in adjacent blood vessels were stained red with eosin. When tissue sec-
tions from untreated trout were stained with either eosin Y or fast
green, epithelial cell nuclei of gills and Stannius corpuscle showed an
affinity for both dyes, staining red in the first instance and green in
the second.
Analyses of sections from acid exposed trout revealed that neither the
level of acidity nor duration of exposure had any effect on the intensity
or color of gill cell nuclei or Stannius corpuscle nuclei with eosin-fast
green staining. All nuclei were green in color and cells of acid exposed
fish showed comparable staining based on visual examination of the
stained specimens. No attempt was made to measure dye concentration
cytophotometrically since considerable nonspecific staining occurred. For
example, when control slides were run (i.e., using acetic anhydride to
acetylate histones or IN HC1 hydrolysis to remove histones), both eosin
Y and fast green resulted in some nonspecific staining of nonhistone
cellular constituents.
In summary, this phase of the study demonstrated the following: First,
eosin-fast green staining has potential value in differentiating between
functionally distinct cell types in trout tissue. Second, there is no
evidence that acid exposure causes any alteration in the relative amounts
of eosinophilic or fast green staining nuclear components in either gill
epithelial or Stannius corpuscle cells.
Histological analyses of the Stannius corpuscle. Grossly, Stannius cor-
puscles appear as whitish globular structures embedded in the middle
third of the kidney. These vary in size from less than one to five
millimeters in diameter, depending somewhat on the size of the fish. The
number of Stannius corpuscles per trout range from 4 to 10; most trout
had 3-4 large (3-4 mm) and 1-2 small (less than one mm) corpuscles; how-
ever, in some, no large and 8-10 small corpuscles were found. Thus, it
would be very difficult to measure changes in the total mass of glandular
elements (e.g., hypotrophic or hypertrophic responses) on a tissue area
or on a weight basis.
Histologically, the Stannius corpuscle is an encapsulated organ consisting
of several lobules separated by strands of connective tissue. Each lobule
contains groups of columnar cells circularly arranged around a central
lumen. The large round basophilic nuclei, which are hasally located,
exhibit marked heterochromaticity (i.e., chromatin appears in the form of
strands). Portions of cells abutting the lumen are agranular and periodic
12
-------
acid Schiff (PAS) negative whereas basal portions are intensely colored
when stained with PAS. No cytomorphological differences were noted
between acid exposed and control trout corpuscles following staining
with H and E or Gomori's trichrome stains. However, cursory examination
of these tissues following staining with PAS indicated greater staining
in acute acid exposed trout as compared to controls. As a consequence,
PAS affinity was selected to assess possible effects of acute and sub-
acute acid exposure on secretory function of the Stannius corpuscle.
4Ch
20-
ACUTE EXPOSURE
CONTROL GROUP
(PH 7.0 -7.5)
ACUTE EXPOSURE
EXPERIMENTAL GROUP
(PH4.0)
SUBACUTE EXPOSURE
CONTROL GROUP
(PH7.0)
40n
20-
SUBACUTE EXPOSURE
EXPERIMENTAL GROUP
(PH4.8)
2 4 6 8 ID 12 14 16 18
AMOUNT OF PAS POSITIVE MATERIAL IN ARBITRARY UNITS
Figure 2. Periodic acid Schiff (PAS) distribution patterns for
Stannius corpuscle cells of brook trout exposed to
acid water for acute (4-5 day) and subacute (28 day)
periods.
13
-------
Cytophotometric analyses of PAS staining in. Stannius corpuscles.
Although PAS is a nonspecific stain (i.e., mucopolysaccharides, glycogen,
glycolipids and some other compound lipids are PAS-positive), it can be
used to measure relative changes in secretory material on an individual
cell basis. Although PAS staining is not stoichiometric, one can assign
relative numerical values to cells exhibiting decreased or increased
staining affinity. As with the F-DNA data, results were expressed in
the form of histogram profiles to facilitate comparison of the various
experimental groups. From data summarized in Figure 2, it is apparent
that Stannius corpuscle cells from trout exposed to acute acid exhibited
a pronounced increase in the number of cells containing more PAS-positive
material. On the other hand, no increase in relative numbers of cells
exhibiting high affinity for PAS was evidenced in Stannius corpuscles of
the subacute exposure group.
Several histochemical tests were employed for analysis of lipids and
nuclear proteins in the Stannius corpuscle. Sudan black B showed that
lipid in the Stannius corpuscle is particularly concentrated in cell
membranes. There was no discernible difference in the intensity of
sudanophilia between control and acute acid exposed trout. As indicated
previously, basic protein stains (eosin-fast green, eosin Y, and fast
green) showed no differences in the pattern of nuclear staining in acid
exposed and untreated trout Stannius corpuscles.
Cytophotometric analysis of DNA in Stannius corpuscle nuclei. The pro-
nounced increase in PAS staining of the Stannius corpuscle in acute acid
exposed trout prompted inquiry into the possibility that histochemical
changes might also be evidenced in the nucleic acids, DNA and RNA. Cyto-
photometric analyses of F-DNA were undertaken since it was reasoned that
an acid induced increase in chromatin dispersion might eventuate in a
lowered resistance to acid hydrolysis and a consequent increase in the
net yield of aldehydes from some nuclei. One would thus expect higher
F-DNA values to be associated with chromatin dispersion.
Figure 3 represents a total of 400 individual measures of nuclear F-DNA
in acute and subacute exposed trout and their corresponding controls.
As with the F-DNA data of gill epithelial nuclei, the 2 C or diploid
segment of the histogram is shown between dotted lines and indicates
that the majority of cells in acid exposed and untreated groups contain
50-80 arbitrary units (A.U.) of F-DNA. It is noteworthy, however, that
in both acute and subacute groups there is a shift in the number of
nuclei exhibiting high F-DNA values (i.e., ca_. 80 A.U.) and a propor-
tional decrease in nuclei with low amounts of F-DNA (ca. 40 A.U.)- Very
few nuclei are in the premitotic stage (ca.. 140 A.U.)~ The number of
nuclei showing higher F-DNA levels in experimental groups proved to be
sufficiently great to be reflected in an increase in the average F-DNA
content of the entire population of nuclei (62-72 A.U. for the acute
group and 59-73 A.U. for the subacute group).
14
-------
Ul
o
z
U-
o
IT
UI
CO
ACUTE EXPOSURE
CONTROL GROUP
(PH 7.0-7.5)
ACUTE EXPOSURE
EXPERIMENTAL GROUP
(PH4.0)
SUBACUTE EXPOSURE
CONTROL GROUP
(PH7.0)
SUBACUTE EXPOSURE
EXPERIMENTAL GROUP
(PH4.8)
2 4 6 B 10 IE 14 16 18
AMOUNT OF FEULGEN-DNA IN ARBITRARY UNITS
Figure 3. DNA distribution patterns for Stannius corpuscle cells
of brook trout exposed to acid water for acute (4-5 day)
and subacute (28 day) periods. Numbers in circled
inserts refer to percentage of cells between and on
each side of dotted division lines.
In brief, these data indicate that both acute and subacute acid exposure
results in an increase in F-DNA content in a considerable number of
Stannius corpuscle nuclei. The increase, measured on an individual
nuclear basis proved to be of sufficient magnitude to be reflected in
a greater mean F-DNA value of the nuclear cell population. Significantly,
the mean F-DNA contents of the two acid exposed groups were of comparable
magnitude.
15
-------
Cytophotometric analyses of total cellular RNA in Stannius corpuscles.
Having established that acid exposure results in alteration in the
physical chemical state of chromatin as reflected in increased F-DNA
values, the next step was to determine if these were associated with
changes in total cellular RNA content. Total cellular RNA is a useful
and sensitive index of metabolic activity since RNA content generally
parallels synthesis of cytoplasmic protein constituents. Thus, on the
basis of the observed PAS and F-DNA responses, one would expect highest
levels of cellular RNA in Stannius corpuscles of acute exposed trout.
This proved to be the case.
40-1
20-
40, © I ©
20-
40-,
20-
ACUTE EXPOSURE
CONTROL GROUP
(PH7.0 -7.5)
= 40
ACUTE EXPOSURE
EXPERIMENTAL GROUP
(PH4.0)
N = 4-0
SUBACUTE EXPOSURE
CONTROL GROUP
(pH 7.0)
N =50
1
1
1
1
r-r
1
SU8ACI
EXPER
(pH4.
N = 50
— , 1
2 4 6 8 10 12
AMOUNT OF AZURE B - RNA IN ARBITRARY UNITS
Figure i. Total cellular RNA distribution patterns for Stannius
corpuscle cells of brook trout exposed to acid water for
acute (i-5 day) and subacute (28 day) periods. Numbers
in circled inserts refer to percentage of cells between
and on each side of dotted division lines.
16
-------
Data summarized in Figure 4 represent a total of 180 measures made on
individual Stannius corpuscle cells from acute and subacute acid exposed
trout and corresponding control groups. The data are presented as per-
centages of cells containing different amounts of RNA to facilitate
comparison with the F-DNA histogram data. As is evident from these data,
the average content of RNA is significantly greater (45 ± 2 A.U.) in
Stannius corpuscles of acute acid exposed trout than that of controls
(38 ± 2 A.U.). On the other hand, subacute exposure to acid did not
affect the RNA content of Stannius corpuscle cells. In fact, the average
content of the subacute exposed group (41 ± 2 A.U.) was not significantly
different from either of the two control groups. Therefore, the F-DNA
increase noted in subacute exposure was not paralleled by an increase in
cellular RNA content.
Supplemental biochemical analyses. Several biochemical measures were run
to determine whether the observed his tochemical changes were sufficiently
great to be amenable to chemical analysis and to obtain some clues
regarding the general type of secretory constituents present in the
Stannius corpuscle. In one experiment, lipids were extracted from fresh,
unfixed aliquots of Stannius corpuscles and the extract plated on thin
layer chromatographic plates. It was determined that approximately 20 mg
of tissue from 2-6 fish (number of fish dependent on their size) would
yield 3-4 mg of lipid which was sufficient for thin layer chromatographic
analysis of neutral and'polar lipids. Acute acid exposed fish showed an
increase in phospholipid content. This correlated well with the finding
that the corpuscles from acid exposed fish contained a greater amount of
PAS positive material since phospholipids are PAS positive. Acid expo-
sure had no effect on the number and, presumably, the types of lipids
present in the Stannius corpuscle as gauged by comparison of their
mobility with known standards.
Electrophoretic analysis of gill histones was also undertaken to deter-
mine if different histone fractions were separable in gill tissue and
to possibly gain insight into the nature of gill mucification. Histones
were extracted from acute, subacute and control fish gills. Six histone
fractions were identified by comparison with known standards (Worthington
Biochemical Corp.). There were no visually discernible differences
between acute, subacute, or control animals, in either number of distri-
bution of bands. This supports the histochemical findings which indi-
cated a lack of histone histopathology in response to either acute or
subacute exposure to acid water.
Discussion and Summary. This report! is concerned in part with the feasi-
bility of using histopathological and histochemical changes to detect
sublethal effects of acidity on selected tissues of fish. Histopathology,
defined as the microscopic appearance of aberrant or diseased tissue,
remains the classical form of diagnosing disease states, metabolic dis-
orders and toxicant effects in vertebrate species. It also often provides
one with insight into possible mechanisms involved in the induction of
various types of disorders. More recently, quantitative histochemical
17
-------
methods have been successfully used to detect signs of incipient damage,
long before cytopathological changes are microscopically evident. For
example, changes in DNA, RNA, and histones, coupled with other histo-
chemical techniques have proven especially useful in analyses of tissue
dysfunction in many animal species (Wied, 1966; Wied and Bahr, 1970).
Because of the chemical and structural similarity of the basic tissue
types (hemopoietic, connective, epithelial, muscle, nerve) in fish and
mammals, it was reasoned that selected histochemical methods, limited
largely in the past to the study of mammalian tissues, should work
equally well for analyses of fish tissue. Preliminary work, together
with the present study, showed this to be the case. The Feulgen reac-
tion for DNA, azure B staining for RNA, mucopolysaccharide stains such
as colloidal iron, alcian blue, and the periodic acid Schiff reaction
give identical responses in fish and mammalian tissues. In fact,
greater staining differences are often evidenced between organs and
tissue types in an individual animal (fish or mammal) than one finds
when comparisons are made of staining affinities of specified organs
between animal species. This is not too surprising since the specific
function of specialized cell types is often closely related to its
chemical make-up. It is noteworthy that although the application of
histochemistry and experimental histopathology in mammals is well
established, very little is known about the histochemistry of fish
tissues. In view of this, it is felt that one of the important contri-
butions of the present study is the demonstration that a number of
histochemical methods developed primarily for use with mammalian tissue
can be applied to the analysis of fish tissues. Furthermore, since two
of the staining reactions (i.e., Feulgen, azure B) have a known chemical
basis and are thus amenable to quantitation, these can serve as excel-
lent indices of cytopathology such as might be induced by acid exposure.
Gills were selected as one of the tissues to be analyzed because pre-
liminary work (Plonka and Neff, 1969) established that histopathological
changes in gill epithelial cells are elicited by short exposure of trout
to severe acid water (pH 3.9). For example, histological examination of
gill sections from acid treated trout revealed increased mucin produc-
tion, disruption of lamellar integrity, "clubbing" of lamellae and the
occlusion of interlamellar spaces by a coagulum of cellular debris. On
the basis of microscopic study, however, it was not possible to ascertain
the mechanism of pathological change, i.e., the source of the cellular
debris nor the extent to which remaining supporting epithelial cells
were affected by acid treatment.
The Stannius corpuscle was selected for histochemical analysis since
some recent work in our laboratory revealed that shifts in electrolyte
and water balance appear to be associated with acid exposure (Packer and
Dunson, 1970; Vaala and Mitchell, 1970). Since Hanke and Chester-Jones
(1966) and Chester-Jones et al. (1966) have shown that one of the major
functions of the Stannius corpuscle is ion regulation, it was felt that
acid induced metabolic or secretory changes in epithelial cells compris-
ing this endocrine organ should be manifested in their histochemical
staining reactions.
18
-------
Although the scope of this paper is restricted to analysis of these two
tissues, the work described actually forms an integral portion of a
broader project concerned with "screening" histochetnical responses of
tissues representing most of the vital organ systems in the trout. The
underlying rationale for this coordinated approach which involved the
participation of a number of graduate students working on different
tissues was to check for the possible existence of a "toxic response
syndrome," adopting the terminology proposed by Warner (1966). There is
no doubt that the physiological significance of histochemical responses
can best be interpreted in light of alterations in other body systems.
Thus, one of the goals of the study was to determine whether histo-
chemical changes in the gill and Stannius corpuscle are indications of
his topatho logy or signs of acclimation to acid exposure.
Table 6. Summary table of major findings
Type of acid exposure
Acute (4-5 day)
High
Subacute (28 day)
Moderate High
acidity" acidityb acidity3 acidity
A. Gross responses
2. hyperventilation , loss of equilibrium, surfacing t
3. loss of coloration t
B. Gill changes (histological , histochemical and biochemical)
1. increase in number of raucous cells t
2. presence of interlamellar debris t
3. mucopolysaccharide staining of gill cells and inter-
lamellar area (colloidal iron , alcian blue ,
mucicarmine) t
4. F-DNA of epithelial nuclei
5. basic nuclear proteins of epithelial cells (eosin-fast
green, alkaline fast green, eosin Y staining)
6 . electrophoretic analyses of histones
C. Stannius corpuscle changes
1. PAS staining ft
2. F-DNA of Stannius corpuscle cells
3. neutral lipids
4. polar lipids
5. RNA of Stannius corpuscle cells
6. basic nuclear protein (eosin-fast green, eosin Y,
alkaline fast green)
aModerate exposure pH 5.0-6.5 - no change
^Severe exposure pH 4.0-5.0 t moderate increase
•f 4
TT
tt
t
tt
tt
tt
t
tt
t
t
t
t
t tt
t
t
t
t
t slight increase
tt marked increase
A synopsis of the overall pattern of cytological and histochemical
changes as a function of the severity of acidity and exposure duration
is presented in Table 6 where relative intensities of changes are indi-
cated by direction and width of arrows. Two conclusions are supported
by these data: First, it is evident that acute exposure to moderate
and high levels of acidity results in increase in mucopolysaccharide
content of gill cells and increased PAS staining of Stannius corpuscle
cells. Second, in gill cells the intensity of the histochemical
response is much greater in trout exposed to high levels of acidity
under acute conditions than in moderate acid exposed groups. In con-
trast, PAS staining of the Stannius corpuscle was equally intense in
both moderate and high acid exposed trout. No indication was found of
19
-------
a progressive decline in the functional capacity of these organs follow
ing prolonged exposure to moderate and high severities of acidified
water as measured by differences in RNA and his tone levels.
The above findings indicate that one of the major aims formulated at the
onset of this investigation was accomplished, i.e. demonstrating that
histochemical tests have potential value as physiological or toxicant
bioassays. Of equal importance, such tests convincingly show that
acidity, per se, has no direct toxic action on internal vital organs
since all of the observed changes occur as a result of respiratory and
circulatory impairments. The observation that F-DNA histogram profiles
of supporting cells in trout gills are identical in control, acute, and
subacute acid exposed animals signifies that exposure to sublethal
levels of acidity does not damage gill supporting cells. Therefore, one
can also conclude that interlamellar debris seen in acute acid exposed
fish probably originates from sloughed mucous cells rather than support-
ing epithelial cells.
On the other hand, F-DNA profiles of Stannius corpuscle nuclei of acute
acid exposed trout revealed the presence of considerably more nuclei
with higher than average amounts of F-DNA when compared to controls; so
much so, that the average F-DNA level was significantly higher in acute
acid exposed nuclear populations relative to the control nuclei.
Stannius corpuscle cells of acute acid exposed fish also showed an
increase in azure B-RNA when compared to controls. Actively synthesiz-
ing cells would be expected to show an increase in DNA, RNA, and cellular
anabolites. The combined DNA and RNA data, therefore, indicate an
increase in metabolic activity of Stannius corpuscle cells in acute acid-
exposed trout. This probably represents an acute stress reaction to acid
since F-DNA levels remain high in Stannius corpuscles from 28 day exposed
fish whereas total cellular RNA content returned to control levels.
Alterations in total cellular RNA are useful indicators of protein
synthesis since all functional types of RNA are directly concerned with
the mechanism whereby information stored in chromosomal DNA is made
operative at the sites of protein synthesis (polyribosomes). Signifi-
cantly, one finds highest levels of RNA in growing cells or when their
synthetic machinery has been triggered into action by some exogenous or
endogenous stimulus. Conversely, resting cells or cells in a steady
state (homeostatic equilibrium) contain moderate to low levels of cyto-
plasmic RNA. In view of these considerations, presence of increased
RNA during acute acid exposure, but not following subacute acid expo-
sure, can be explained in one of two ways. One possibility is that fish
are stressed to near their tolerance limit. A more probable explanation
is that stressed fish achieve a new level of homeostasis, at least in
respect to this particular gland. Data on cytoplasmic PAS content and
nuclear histones support this interpretation since increased cytoplasmic
content of PAS was not detected in subacute exposed fish and histone
staining did not indicate a cessation or inhibition of metabolic activity.
20
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SECTION IV
ERYTHROCYTIC INDICES OF STRESS IN
ACID EXPOSED BROOK TROUT
Most workers favor the idea that fish exposed to acid pollution experi-
ence a hypoxemia and tissue anoxia due to impaired oxygen transport
across the gill respiratory epithelial surface (Carpenter, 1927) since
acid stressed fish often exhibit a thick mucus-like coagulum in the
interlamellar spaces of gill filaments (Westfall, 1945; Plonka and Neff,
1969). However, many fish can withstand a considerable extent of gill
mucification and apparently recover with no grossly detectable evidence
of disability except for a marked loss in body coloration. To date,
little attention has been focused on possible cumulative or long term
consequences of acid-induced hypoxemia in fish subjected to sublethal
levels of acidity. In homeotherms, hypoxia initiates erythropoiesis
through release of an erythropoietic stimulating factor (Jacobson and
Doyle, 1962) . Thus, it was decided to investigate the nature of hemo-
poietic responses in trout subjected to near toxic levels (ca. pH 4.0-
3.5) of acidity for a short term (five day) and to sublethal levels
(pH 4.9) for a longer (26-28 day) duration of exposure. The specific
aims of this phase of work were:
1. to characterize the peripheral hemal response during acute and
subacute acid exposure by monitoring changes in selected blood
parameters such as hematocrit, hemoglobin content, differential
counts, and blood Po9;
2. to determine if acute and subacute exposure to acid stimulates
hemopoiesis as reflected in the cytochemical Feulgen-DNA pro-
file of immature erythroid cells;
3. to investigate the influence of acute and subacute exposure to
acid on the metabolism of hemopoietic cells in renal and splenic
tissue as reflected in RNA content; and
4. to determine if the pattern of acid induced erythrocytic changes
is the same in nonbreeding and reproductively active fish.
Methods. Two hundred and seventy hatchery-reared brook trout,
Salvelinus fontinalis, (Research Station of the Pennsylvania Fish
Commission, Pleasant Gap, Pennsylvania) 12-18 months old, weighing
130-170 g and 20-25 cm in length were used. Prior to experimentation,
fish were acclimated to laboratory conditions for at least one week in
150 gallon holding tanks at 12 ± 1 C under constant aeration.
Acute toxicity studies (five days) were conducted on nonbreeding trout
(spring-summer months) in a static system where water was not changed
during the exposure period. Controls were maintained at pH 7.0-7.3 ± 0.1.
21
-------
The procedure of acute acid exposure involved the addition every eight
hours of 2-3 ml of concentrated HC1 (specific gravity 1.84; 37% by
weight). Within one day, pH dropped from 7.0 to 5.0 ± 0.2; on days
2, 3 and 4 water was maintained at pH 4.0 ± 0.2; and on day 5, pH
dropped to 3.5 ± 0.1.
Subacute toxicity studies (26-28 days) were conducted on nonbreeding
trout (spring-summer months) and reproductively active trout (fall).
In addition, one study was done immediately after the breeding season
(winter). These experiments were conducted using a gravity flow
proportional diluter system (Mount and Brungs, 1967). Controls were
maintained at pH 7.0 ± 0.1 and experimental at pH 4.9 ± 0.1. A stock
solution of 9.5 N HC1 was used in this system.
Field studies were also conducted on hatchery trout placed in net traps
in a stream, Upper Three Runs in Clearfield County, Pennsylvania. This
stream conveniently provided water plateaus of different pH's. Controls
were subjected to a pH 6.4 ± 0.2 while experimentals were exposed to
pH 4.8 ± 0.2. Exposure time was approximately 24-48 hours, after which
fish were transported to the laboratory for analysis.
Several peripheral blood measurements were made to determine if there
were any erythrocytic adjustments associated with acute and subacute
acid exposure. Blood collection was performed by severance of the caudal
peduncle or cardiac puncture using heparin, ethylenediaminetetraacetic
acid (EDTA), or sodium citrate as anticoagulants according to dictates
of specific tests used. Blood taken by puncture of the heart ventricle
was employed for hematocrit measurements using heparinized glass capil-
lary tubes. Hemoglobin determinations were based on blood obtained by
severance of the caudal peduncle using the cyanmethemoglobin method
(U.S. Armed Forces Medical Journal. 1954. 5:696). Differential counts
of neutrophils, lymphocytes and monocytes were made on dried blood
smears. WBC/RBC and reticulocyte/RBC ratios were also calculated.
Counts of immature erythroid cells per 1000 mature RBC were made on
renal and splenic touch preparations stained with Giemsa (Humason, 1967).
On several occasions, when enough blood was available, checks were made
on the total RBC count using a standard RBC diluting pipette and
Hendrick's diluting solution (Hesser, 1960).
Supplemental measures of peripheral blood characteristics included: (a)
blood P02 determinations on cardiac blood using a Copenhagen radiometer
oxygen electrode setup (Vaala and Mitchell, 1970); (b) blood volume
measures using radioiodinated serum albumen (Rll31sA, Squibb); (c) incor-
poration of radioiron (Fe59 citrate, Squibb) into hemoglobin; (d) poly-
acrylamide gel electrophoresis of hemoglobin using the method of Moss
and Ingram (1968); and (e) electrophoretic analysis of total histones in
erythrocytes following the method of Bonner et al. (1968).
For cyto chemical analyses of nucleic acids in erythropoietic tissue of
trout (kidney and spleen), groups of at least six fish each were acid
exposed for five and 26-28 days. Each experimental set had its own
22
-------
control group of fish and microscopic slides representing each animal were
used for DNA and RNA analyses. Following exposure, fish were sacrificed,
splenic and renal touch imprints were air dried and fixed for five
minutes in absolute methanol. Individual slides from each animal were
stained for morphological analysis with Giemsa (Humason, 1967), for DNA
by the Feulgen procedure (Deitch, 1968), and for RNA by azure B (Flax
and Himes, 1952). To minimize error, control and experimental slides
for each set of experiments were stained simultaneously- Optimum hydro-
lysis time for Feulgen staining was determined to be 60 minutes with
methanol fixed tissue when using 5N HC1 at 20 C. Since azure B stains
both RNA and DNA, azure B-RNA was measured on sections pretreated with
electrophoretically purified, RNAase free, bovine pancreas DNAase
(Worthington Corp.). In addition, one slide was always stained with
Schiff's reagent. This yielded a negative Feulgen reaction, and served
as a control for azure B staining. Two Feulgen-DNA controls were always
run, a non-hydrolyzed control and a control in which DNA was extracted,
both giving negative Schiff reactions.
A single beam Leitz microspectrophotometer was used for cytophotometric
measurements of immature erythroid cells. Nuclei ranging from 4.3y to
8.4y in diameter represented a graded series of red cell nuclei which
were individually measured for Feulgen-DNA content. The nuclear sizes
were chosen to represent various erythroblast maturation stages, baso-
philic erythroblast to reticulocyte. Fixed numbers of each cell type
were selected for measurement, to insure that histograms were not influ-
enced by population shifts in developmental stages. Total cellular RNA
(azure B) measurements were made on whole cells with nuclear diameters
ranging from 4.3y to 10.5y which represented cells in erythroid matura-
tion from proerythroblast to reticulocyte. Specific erythropoietic
cells were identified on the basis of nuclear morphology, nuclear area
and cytoplasmic tinctorial properties.
The two wavelength method of microspectrophotometry described by Patau
(1952) and Ornstein (1952) was used to avoid measuring errors caused by
heterogeneous distribution of stained material. Wavelengths (\2 and ^i)
employed for Feulgen-DNA were 550 nm and 500 nm; corresponding wave-
lengths for azure B-RNA were 595 nm and 515 nm.
General observations. During acute exposure (five day) of trout to near
toxic levels of acidity (pH ca. 4.0), the most marked symptoms of respir-
atory distress were evidenced when the pH was brought down from 5.0 to
4.0 on the second day of exposure. At this time a noticeable decrease
in activity was observed, the rate and depth of ventilation increased,
surfacing was often noted, and fish ate poorly. Copious amounts of
mucus were secreted such that a film became visible over the entire skin
surface, especially in the gill area. These symptoms persisted from day
two to five at which time the pH was lowered to 3.5. At a pH of 3.5
trout showed a loss of equilibrium, swimming with their ventral sides up
and if left at this level of acidity died within several hours. Trout
collected during the nonbreeding season and used in sub acute exposure
(28 day) experiments at sublethal levels of acidity (pH 4.9) exhibited
23
-------
the same svmptoms of respiratory distress as were evidenced in acute
exposure studies. Stress reactions were first noted within 24-36 hours
after introduction into acidified water and persisted for 8-10 days; by
the end of the second week, hyperventilation was no longer evidenced
nor -as there anv mucus secretion present, suggesting the trout were no
longer hvpoxemic'. Significantly, the mortality of nonbreeding trout at
a pll of 4.9 was 10% during the 28 day exposure interval. In contrast,
in subsequent experiments with reproductively active trout exposed to
sublethal levels of acidity for 23 days, the mortality rate was extremely
high, 75%. Interestingly, most of the reproductively active^trout died
within the first 8-10 days of exposure and showed no overt signs of
acclimation to acidic conditions. No attempt was made to undertake a^
systematic study of behavioral response. The main intent was to confirm
that moderate to high levels of acidic exposure did evoke external
behavioral symptoms of respiratory distress. Tissue analyses were
limited to surviving fish.
Lrythrocytic responses in nonbreeding trout following acute (five day)
exposure to acidity. In both short term (five day) and longer term (28
day; exposure studies, measures were usually made of three basic periph-
eral blood characteristics (hematocrit, hemoglobin and PQ^ levels) and
three related hemopoietic tissue parameters (DNA content, RNA content
and differential counts of erythroblast cells in renal and splenic tissue)
To facilitate presentation, results in this and the succeeding subsec-
tion are presented in terms of basic and supplemental measures pertaining
to (a) peripheral hemal response patterns and (b) hemopoietic tissue
changes in acute and subacute exposed trout.
a. Peripheral hemal responses. Data on hematocrit, hemoglobin, blood
PQ? levels and peripheral red blood cell count changes in acute
acid exposed trout are summarized in Figure 5 with supplemental
measures of blood volume and differential white counts presented
in Figure 6 and Table 7, respectively. Briefly, the major findings
were as follows. After five days of exposure to near toxic levels
of acidity, trout exhibited a hematocrit increase of about 17% and
a hemoglobin increase of 29%. Despite these compensatory hemal
changes, acid exposed trout remained hypoxemic since the PQO level
was approximately 20% lower in the exposure group relative to
controls.
Differential counts of Giemsa stained smears were limited to lympho-
cytes (which comprise about 90% of the leucocytes), monocytes and
polymorphs. Basophils and eosinophils were rarely found in smear
preparations of either control or experimental trout. Percentage
of lymphocytes was lower whereas that of polymorphs was higher in
acute acid exposed trout (Table 7). The WBC/RBC ratio was lower in
acid exposed trout. Examination of Giemsa stained peripheral
smears revealed an exceptionally high percentage of reticulocytes
in both acid exposed (10.1 ± .5%) and control (10.0 ± .5%) trout.
24
-------
3-
N = 20
10
20
30
40
HEMATOCRIT(volume percent ± S.E.)
NONBREEDING,ACUTE
EXPOSURE,LAB. TEST
[3 CONTROL ( pH 7.0)
0 ACID (pH 4.0 - 3.5)
N • no. of trout
5 10
HEMOGLOBIN (gm/100 ml blood ± S.E.)
N"8
'////////////////////A P<-OOI
.4 .8 1.2
PERIPHERAL RBC COUNTS
( x IOe/mm3 blood ± S.E .)
N = 14
+
N»I4
'///// ///////////-\- P < .0 1
i i
5 10
PO ( m m H g
Figure 5. Hematocrit
blood P
I 1
15 20
±S.E.)
, hemoglobin, peripheral RBC count and venous
values in nonbreeding trout exposed to acid
for five days (pH li.O-3.5).
b. Hemopoietic changes. Microscopic examination of splenic and renal
imprints revealed no cytomorphological differences in blast cell
types between controls and acid exposed trout. Four maturation
stages were readily identifiable in Giemsa stained preparations
when nuclear s±ze, chromatin dispersion and cytoplasmic basophilia
were used to characterize cell types (Table 8, Figure 7). As
maturation proceeds from proerythrob last to the mature erythrocyte,
there is a progressive reduction in nuclear size, an increase in
compaction or condensation of nuclear chromatin and increased
acidophilia of the cytoplasm. The stem cell, or lymphoid hemo-
blast, was not characterized in this study because of controversial
literature regarding its identity. It was felt that the four
stages - proerythroblast, basophilic erythroblast, polychromato-
philic erythroblast, and reticulocyte - represented an identifiable
25
-------
N-M7
p <-001
i 2 3 *
TOTAL BLOOD VOLUME
(ml/100 gm body wt I S.E.)
NONBREEDING, ACUTE
EXPOSURE,LAB. TEST
[^CONTROL ( pH 7.0)
^ ACID (pH 4.0 - 3.5)
N = no. of trout
Figure 6. Total blood volume determined with radioactive iodinated
serum albumin (RI SA) in nonbreeding trout exposed to
acid for five days (pH 14.0-3.5).
Table 7. Differential counts in blood smears from nonbreeding brook trout exposed to acid water for five
days (acute exposure) and 26-28 days (subacute exposure).a
% PMN
% Lymphocyte
Monocyte
WBC/KBC
Blood Acute exposure
control (H=10)
pH - 7.0
experimental (N=10)
pH U.0-3.5
Subacute exposure
control (11=8)
pH - 7.0
experimental (11=8)
pH It. 9
It.
18.
It,
3.
110.7
.6+3.7*
,01 .6
.7± .8
90.
71.
93.
93
,0± 8.
,lt± 6.
.1± 5
,6
.9*
.5
.0111.0
5,
10,
2
3
,9+1. It
,0il.8t
.9± .3
.31 .8
2
,71.
1.9±.
1
1
. U+
.lt±
>
,3t
.1
.1
Average values ( S.E.) are based on 10 differential counts per trout or approximately 100 differential
counts per group. Asterisks designate significant differences at the 5? level of confidence using the
i-test; t indicates p < .10, N = number of trout.
26
-------
I
Figure 7. Photomicrographs of immature erythroid cells from a renal
tissue imprint. P proerythroblast, B basophilic
erythroblast, PC - polychromatophilic erythroblast,
R - reticulocyte (Giemsa stain,
Table 8. Maturation sequence of trout erythrocytes in renal and
splenic hemopoietic tissue.
(1)
(2)
(3)
(ft)
(5)
Maturation Kuclear
Stages Diameter
proerythroblast (9.5-10.5y)
+
basophilic (7.3- 8. liy)
erythroblast
polychromatophilic (6.0- 6.8p)
*
reticulocyte (It. 3- 5.5p)
1
erythrocyte (3x5p)
Chromatin
dispersed
moderately
condensed
condensed
highly
condensed
highly
condensed
Cytoplasm
moderately
basophilic
intensely
basophilic
basophilic (with
faint aci do-
phi li a)
acidophilic (with
faint basophilia)
acidophilic
(no basophilia)
27
-------
maturation sequence which could be counted and analyzed histochem-
ically on an individual cell basis. The total number of immature
cells per 1000 RBC in renal and splenic tissue from exposed trout
was not significantly different from control values. However, in
kidney tissue there was a significant reduction in the percentage
of basophilic erythroblasts with a relative increase in reticulo-
cytes in exposed trout (Table 9). Few mitotic figures were found
in either control or acid exposed renal or splenic specimens.
Table 9. Differential red blood cell counts in renal and splenic touch preparations from nonbreeding brook
trout exposed to acid water for five days and 26-28 days.a
Tissue and
treatment
groups
Renal :
Acute exposure
control (11=10)
pH - 7.0
experimental (H=10)
pH - U. 0-3. 5
Subocute exposure
control (»-=8)
pH 7.0
experimental (8=8)
pH - It. 9
Splenic:
Acute exposure
control (H=ll)
pH - 7.0
experimental (H=ll)
pH - It. 0-3. 5
Subacute exposure
control (li=8)
pH - 7.0
experimental (11=8)
pH - It. 9
Ho. of immature
RBC/1000 mature
RBC
170 ±13
152 ±16
138 +13
60*±10
180 ±15
213 ±20
60 ± 5
kk"± 2
I
J.8.8±2.
% of each type RBC precursor ± S.E.
II III
3
l6.lt+7.8
12.1t±3.
10.0±1.
1.7±0.
1.5±0.
6.6±1.
11.3±1*.
6
6
5
9
&
5
21
It
17
21
11
13
33.
9.
.7 ±
.6»±
.U +
.6 +
.7 ±
.6 +
"t.7
1.9
5.0
5.0
7.2
5.1
.3 ±11.6
.3*+
2.2
2U
25
17
30.
18,
23.
15.
27.
.7
.6
,lt
±8.8
±6.5
±1*.3
.0*±5.0
.3
,lt
.1
2
±l*.l*
±6.1
±5.0
±6.8
3l*
53
52
38
68
61
1*5.
52.
IV
.8 +lt.7
.l**+6.5
.8 +7.2
.1* ±8.3+
.3 ±6.6
.5 ±7.0
.0 ±6.6
,2 ±6.8
Complete count data (with standard errors) in Table 6 (Appendix). Asterisks indicate significance at the
5i level of confidence using t-test; t indicates p < .10; N = number of trout.
'Types of precursors: I proerythrsblast, II basophilic erythroblast, III polychromatophilic
erythroblast and IV - reticulocyte
Feulgen DNA analyses of blast cell nuclei revealed very few baso-
philic erythroblasts containing 4C amounts of F-DNA where C is
defined as the haploid amount of DNA. When these data were plotted
in the form of frequency histograms, with F-DNA levels expressed in
arbitrary units (A.U.) on the abscissa and number of cells in dif-
ferent maturational stages on the ordinate, no differences were
found between acid exposed and control trout tissue (Figure 8). Most
cells contained a diploid (2C) amount of F-DNA. Only 7-12% of total
blast cells in renal and splenic imprints showed interclass (3C)
levels of F-DNA. It was found that late maturational stages (poly-
chromatophilic blasts and reticulocytes) with condensed nuclei have
a lower F-DNA content than the earlier basophilic erythroblast
stage which contains dispersed chromatin.
28
-------
NONBREEDING CONTROL
(N = IO), pH 7,0 x 5d
80r
60-
40-
20
u
D
Z
U.
o
(E
10 12
NONBREEDING ACID
(N = 10), pH 4.0 -3.5 x 5d
80r
60-
40
20
RENAL
n = 160
\ll
10 12
14
4 6 8 10 12 14 " 4 6 8 10 12 14
RELATIVE AMOUNT OF DNA IN ARBITRARY UNITS (A.U.)
Figure 8- Feulgen-DWA distribution patterns for a mixed population of
erythroid cells from renal and splenic imprints of non-
breeding trout exposed to acid for five days (pH ^.0-3.5).
Dotted lines indicate the 2C category. Numbers in circles
indicate percentages of cells to the right of the 2C
category. Maturation stages: I(basophilic blast),
IKpolychromatophilic blast) and III (reticulocyte) are
hatched, solid and open bar graphs, respectively.
Total cellular RNA content generally parallels the rate of synthesis
of cytoplasmic protein and therefore can be used as a sensitive
index of metabolic activity. For example, early stages of matura-
tion in hemopoietic tissue of control and exposed fish often show
much higher total cellular RNA levels than are found in polychroma-
tophilic erythroblasts or reticulocytes. Cytophotometric analyses
of azure B-RNA levels in cells representing stages II, III and IV
of the erythroid maturation sequence revealed a 21% decrease in RNA
content of basophilic erythroblasts and a 10% decrease in total
cellular RNA of polychromatophilic erythroblasts in renal tissue of
acid exposed trout as compared to controls (Figure 9). However, the
RNA content in renal proerythroblasts and reticulocytes did not
29
-------
appear to be affected by acute acid exposure Splenic proerythro-
blasts showed slightly lower RNA levels in acid exP°**f "out
(p < .05) than in controls but there were no differences in RNA
levels in the spleen in maturation stages II, III and IV.
50r
w 30- jS ^^
*' TA*^'^
~ 20-
- 10-
LJ III
1-
z
o
o
z
a:
NONBREEOING, ACUTE
EXPOSURE, LAB. TEST
o o CONTROL (N "71 ,pH 7.0
• • ACID lN-8),pH 4.0-3.5
Eoch point is overog* of 25 RNA
analyses
30r
30
20
10
m
nr
PRO- BASOPHILIC POLYCHRO-
MATOPHILIC
ERYTHROBLAST STAGES
RETICULO-
CYTE
Figure 9. Total cellular RMA content (±S.E.) of erythrold maturation
stages in renal and splenic imprints of nonbreeding trout
exposed to acid for five days (pH 14.0-3.5).
One additional observation should be noted. Renal proerythroblasts
of both control and acid exposed fish had lower RNA levels than
stages II to IV. This was not evidenced in splenic proerythro-
blasts nor in renal or splenic tissue of controls or acid exposed
trout used in subsequent longer term exposure (see Figures 9,
13, 15).
59
Supplemental measurements. Fe incorporation, which provides an
estimation of the rate of iron incorporation into hemoglobin, did
not differ in exposed fish from controls when both sets of values
were calculated on the basis of an assumed standard blood volume.
Fe59 uptake in control fish was 25 ± 2% and in experimental it
was 27 ± 2%. However, when actual blood volume in each group of
animals was calculated, exposed trout showed an increased rate of
30
-------
iron uptake (p < .001) after five days of acute exposure (Figure 10)
The same results were obtained whether radioiron uptake data were
calculated on the basis of initial, final or mean body weight.
Thus, for uniformity all data were recorded on the basis of final
body weight.
N = 9
N = 7
10 20 30 40 50
PERCENT FE59 INCORPORATION IN R8C (± S.E.)
NONBREEDING, ACUTE
EXPOSURE,LAB. TEST
O CONTROL (pH 7.0)
0 ACID (pH 4.0 - 3.5)
(corrected for blood vol.)
| ACID (pH 4.0 - 3.5)
N = no. of trout
Figure 10. Radioactive iron incorporation in RBC of nonbreeding
acute acid exposed trout (pH ^.0-3.5 x 5d).
Polyacrylamide electrophoretic analyses of hemoglobin in 16 blood
samples from acute acid exposed and control trout revealed no dif-
ference in the pattern of electrophoretic mobility or the relative
proportions of bands. Five bands were visually identified and
scanned. Polyacrylamide electrophoresis of the same 16 blood
samples for total his tones yield two major fast moving histone
fractions and two to three minor components with lower electro-
phoretic mobilities. Based on previous work in our laboratory
using fish liver (Crissman and Therrien, 1970), three major frac-
tions were tentatively identified as an arginine rich and two
slightly lysine rich histones, with the three minor fractions
representing an arginine rich and two lysine rich histones. In
the acute exposure experiment, only two major fractions were
resolved in both controls and experimental and the minor arginine
rich histone fraction was absent in experimental fish blood but
present in controls. No attempt was made to quantify these data
since pure histone preparations were not available for use as
reference standards.
31
-------
Erythrocytic responses in nonbreeding trout following subacute (28 days)
exposure to acidity.
a. Peripheral hemal response. Measurements of hematocrit, hemoglobin
and blood PQ2 are summarized in Figure 11. As in the five day
exposed fish, hematocrit and hemoglobin levels were elevated in
28 day exposed fish as compared to controls. However, unlike the
acute exposure group, trout were no longer hypoxemic. Determination
of blood oxygen levels revealed that the PQ~ of cardiac blood sam-
ples was the same (11-12 mm Hg) in both control and 28 day exposed
trout.
N = 10
V///////////77////A- p<-°>
10 20 30 40 50
HEMATOCRIT (volume percent ± S.E.)
N'9
N • 8
246 8 10
HEMOGLOBIN (gm/100 ml blood + S.E.)
N • 8
N • 6
5 10 15
P0 (mm Hg± S.E. )
20
25
NONBREEDING. SUBACUTE
EXPOSURE, LAB. TEST
(_J CONTROL ( pH
7.0)
0 ACID (pH 4.9)
N
= no.
of trout
Figure 11. Hematocrit, hemoglobin and venous blood P values in
nonbreeding trout exposed to acid {pH li.g) for 26-28 days.
32
-------
Differential counts of WBC also revealed a restoration of relative
percentages of lymphocytes, polymorphs and monocytes to control
levels in subacute exposed fish (Table 7) . Similarly, the leuco-
cyte to erythrocyte ratio was no longer depressed in subacute
exposed trout as was the case in acute exposed fish. Reticulocyte
counts (per 100 RBC) were also comparable in subacute exposed
(1.7 ± .5) and control (1.1 + .5) trout.
Hemopoietic changes. Differential counts of immature erythroid
cells in Giemsa stained renal tissue indicated that the maintenance
of an elevated hematocrit was related to a depletion of immature
cells in 28 day exposed trout. There was more than a 50% reduction
in the immature to erythrocyte cell ratio in subacute exposed fish.
The control was 138 immature cells (stages I-IV) per 1000 erythro -
cytes; that for exposed trout was 60 per 1000 RBC. Percentages of
maturation stages I to IV in unexposed trout renal tissue were 12,
18, 17 and 53%, respectively; corresponding values for subacute
exposed trout were 10, 22, 30 and 38%. Thus, there was a relative
drop in reticulocytes (p < .10) with a proportional increase in
polychromatophilic erythroblasts (p < .05) in renal tissue of 28
day exposed trout.
Similar analyses of differential counts in spleen cells again
revealed a lower total number of immature to mature RBC ratio in 28
day exposed trout as compared to controls. Only the percentage of
maturation stage II, however, was reduced in the acid exposed group.
It was further noted that the immature to mature RBC ratio in acute
exposure controls in splenic tissue was three fold greater than that
of controls in the 28 day exposure experiment, suggesting an atypic-
ally high level of erythropoiesis in the former group of control
fish used in acute exposure studies.
Cytophotometric measurements of nuclear DNA made on blast cells in
renal and splenic hematopoietic tissue of subacute exposed trout
and corresponding controls (Figure 12) revealed little ploidy
(1-11% of total cells measured) which was restricted to basophilic
erythroblasts. There was no apparent stimulation of mitotic activ-
ity in subacute exposed fish as evidenced by lack of increased
ploidy in blast cells.
Total cellular RNA was measured cytophotometric ally in maturing
blast cells from renal and splenic tissue imprints of subacute
exposed and control fish. The proerythroblast stage in renal
tissue of exposed trout as compared to controls revealed a 32%
increase in total cellular RNA (Figure 13). This was not evident
in proerythroblast cells from splenic tissue. However, immature
erythroid cells from both hemopoietic tissue imprints showed an
initially high amount of RNA which progressively decreased as blast
cells differentiated into erythroblasts.
33
-------
50
40 -
30
20
liJ
_1
o
z
NONBREEDING CONTROL
( N = 8), pH 7.0 » 28 d
Tr4
8 10 12 14
v/1-
4 6 8 IO 12
NONBREEOING ACID
( N = 8), pH 4.9 x 28d
6 8 10 12 14 " 4 6 8 IO 12
RELATIVE AMOUNT OF ONA IN ARBITRARY UNITS (A.U.)
Figure 1£, Feulgen-DNA analyses of erythroid nuclei in renal and
splenic imprints of subacute acid expi.sed trout during
nonbreeding season (pH 'i.9 x 26-28d). Dotted lines
indicate the 2C category. Numbers in circles indicate
percentages of cells to the right of the 2C category.
Maturation stages I, II and III are designated by
hatched, solid and open bar graphs.
Supplemental measurements. Polyacrylamide electrophoretic analyses
of blood histone and hemoglobin were again run on blood samples
from eight control and eight subacute exposed trout. No differences
were found in banding patterns of hemoglobin as evidenced visually
and from microdensitometer scans of gels.
34
-------
60
50
40
30
Lli
-------
conclusion are presented in Figures 14 to 18. Again to facilitate pre-
sentation, the major results are summarized below in terms of selected
parameters measured.
a. Peripheral hemal response. Hematocrit and hemoglobin levels were
found to be considerably higher in reproductively active control
trout than in corresponding nonbreeding controls (Figure 14). In
fact, the average hematocrit (33 ± 1.3) and hemoglobin (7.6 ± 0.4)
values are comparable to those obtained in nonbreeding trout
exposed to acid water for 28 days (Figure 11).
N = 30
N = 8
'/////////////^A— p<°5
10 20 30 40
HEMATOCRIT (volume percent ± S.E.)
N = 24
2468
HEMOGLOBIN (gm/100 ml blood ± S.E.)
N * 19
N =8
REPROD. ACTIVE,SUBACUTE
EXPOSURE.LAB. TEST
Q CONTROL (pH 7.0)
[2 ACID (pH 4.9)
• NONBREEDING
CONTROLS ( pH 7.0)
N= no. of trout
.4
.8
1.2 1.6
PERIPHERAL RBC COUNTS
( K I0s/mm3 bloodi S.E.)
Figure 14. Hematocrit, hemoglobin and peripheral RBC count in
reproductively active trout exposed to acid (pH h.9
for 26-28 days.
Unlike nonbreeding trout, where these indices increased by acid
exposure, reproductively active trout had a lower hematocrit, hemo-
globin and peripheral count after 28 days of exposure. Corres-
pondingly, the supplemental measurement of total blood volume
(Figure 16) showed a drop of 11% in subacute exposed fish during
36
-------
reproductively active months. Differential WBC counts of blood
smears revealed a higher percentage of polymorphs but lymphocyte
and monocyte percentages did not change in acid exposed fish
(Table 10). PQ was not measured due to the limited number of
surviving trout, i.e. mortality was 75%.
100
80
60
in
+1
40
20
REPROD, ACTIVE,SUBACUTE
EXPOSURE,LAB. TEST
.o o CONTROL (N=7),pH 7.0
. » ACID (N = 8), pH 4.9
Each point is average of 25 RNA
analyses
UJ
(J
50
40
30
20
10
[SPLENIC!
n
m
TSL
PRO- 8ASOPHILIC POLYCHRO-
MATOPHILIC
ERYTHROBLAST STAGES
RETICULO-
CYTE
Figure 15. Total cellular RNA content (±S.E.) of erythroid maturation
stages in renal and splenic imprints of reproductively
active, subacute acid exposed trout (pH U.9 x 26-28d).
Table 10. Differential counts in blood smears from reproductively active brook trout exposed to acid water
for 26-28 days,a
Tissue
Blood
Treatment
Subacute exposure
control (H=6)
pH 7.0
experimental (N=7)
pH It. 9
% PMN % Lymphocyte
5.U+1.5 93,2-8.8
10.011.3* 88.1t±6.9
% Monocyte
l,lt±0.1
1.6+O.U
WBC/RBC
1,2±0,1
3.0+0,2*
aAverage values (±S,E.) are based on 10 differential counts per trout or approximately 100 differential
counts per group. Asterisks designate significant differences at the 5$ level of confidence using the
t-test. N = number of trout.
37
-------
N« 7
N -8
P<-05
1.0 2.0 3.0 4.0
TOTAL BLOOD VOLUME (ml/100 gm body wt ± S.E.)
REPROD. ACTIVE,SUBACUTE
EXPOSURE, LAB. TEST
Q CONTROL (pH 7.0)
(2 ACID (pH 4.9)
N • no. of trout
Figure 16. Total blood volume determined with HI SA In
reproductively active, subacute acid exposed trout
(pH 14.9 x 26-28d).
Table 11. Differential red blood cell counts in renal and splenic touch preparations from reproductively
active brook trout exposed to acid water for 26-28 days.a
Tissue and Ho. of immature
treatment RBC/1000 mature
groups RBC
Renal:
Subacute exposure
control (N=6) 2l»9 ±20
pH 7.0
experimental (N=7) 714"+ 5
pH It. 9
Splenic:
Subacute exposure
control (N=6) 57 ± It
pH - 7.0
experimental (N=7) It9 ± 3+
pH 1. 9
% of each type
I II
9.2±1.6 13.3±lt.8
9-5±1.3 16.2±1.3
ll(.l±3.5 12.3±5.2
22.lt±li.0 10.3±6.1
RBC precursor ± S.E.
Ill
12.0 ±2.8
29.7»±6.7
17.5 ±3.5
20.lt ±6.1
IV
65.5 ±10.0
ltU.6t±10.8
56.1 ±10.5
1(6.9 ± 6.1
Complete count data (with standard errors) in Table 7 (Appendix). Asterisks indicate significance at the
5% level of confidence using t-test; + indicates p < .10; N - number of trout.
Types of precursors: I - proerythroblast, II basophilic erythroblast, III - polychromatophilic
erythroblast and IV - reticulocyte.
38
-------
b. Hemopoietic response. Measures of immature RBC to mature RBC numbers
in renal tissue indicated this ratio was much higher in breeding con-
trols than in nonbreeding controls (see Tables 9 and 11), whereas
splenic values were comparable in both control groups. This again
agrees with the interpretation that during the reproductively active
period there is marked augmentation of erythropoiesis. Total cellu-
lar RNA content of proerythroblasts was also greater in renal and
splenic tissue imprints from reproductively active controls than in
nonbreeding controls (see Figures 13 and 15).
Immature erythroid differential counts summarized in Table 11 on
renal imprints from sub acute exposed trout during the reproductively
active months indicated a significantly lower number of immature
RBC/1000 mature RBC. In splenic tissue the number of immature RBC
to 1000 mature RBC also dropped in exposed trout compared to controls.
70
6O
50
40
30
20
10
o
3
Z
u_
O
-------
Peulgen-DNA measurements on maturing blast nuclei in renal and
splenic hemopoietic tissue are summarized in Figure 17. There was
no proliferative response of blast cells in reproductively active,
exposed trout nor any evidence of incipient cellular damage. Azure
B-RNA cytochemical measurements (Figure 15) revealed an initially
high amount of total cellular RNA in proery throb lasts from renal
and splenic imprints of exposed trout. RNA levels markedly
declined as blast cells matured. In renal hemopoietic tissue of
exposed fish, total cellular RNA showed a pronounced rise of 115%
in basophilic erythroblasts and 88% in polychromatophilic erythro-
blasts. Even polychromatophilic erythroblasts from splenic imprints
revealed a rise of 32% in total cellular RNA.
Uptake of Fe59 in erythrocytes of trout exposed to acid during repro-
ductively active months dropped by 23%. When the percentage of Fe^
incorporation was corrected for blood volume, the decrease in uptake
was 33% in exposed trout (Figure 18). Electrophoretic analyses of
extracted hemoglobin revealed no differences between experimentals
and controls.
N = 7
V///////////////77-
N - 6
10 20 30 40 50
PERCENT FE" INCORPORATION IN RBC (± S.E.)
60
REPROD. ACTIVE,SUBACUTE
EXPOSURE,LAS. TEST
Q CONTROL ( pH 7O)
[2 ACID (pH 4.9)
(corrected for blood vol.)
| ACID (pH 4.9 )
N = no. of trout
Figure 18. Radioactive iron incorporation in RBC of reproductively
active, subacute acid exposed trout (pH h.g x 26-28d).
40
-------
Hemal responses iu postbreeding trout exposed to acidity for 28 days.
Checks were made immediately postbreeding to ascertain the nature of
peripheral hemal reactions to acid exposure when the reproductive tract
was involuted, i.e. when metabolic activity associated with reproduction
is again at an ebb and therefore oxygen requirements begin to approach
those of the nonbreeding or reproductively quiescent trout. In brief,
this group of trout presumably would be less hypoxemic than reproduc-
tive^ active fish during acid exposure. This proved to be the case.
Mortality was again about 10% corresponding to nonbreeding trout. Hema-
tocrit and hemoglobin content as well as the blood P0? levels approxi-
mated those of nonbreeding controls (Figure 19). Significantly, 28 day
acid exposed trout in the postbreeding period showed no overt behavioral
symptoms of respiratory distress and blood PQ- levels were slightly
higher than corresponding controls.
N = 7
10 20 30 40
HEMATOCRIT (volume percent ± S.E.)
N = 7
N = 10
POSTBREEDING.SUB ACUTE
EXPOSURE,LAS. TEST
QCONTROL (PH 7.0)
0ACID (pH 4.9)
• NONBREEDING CONTROLS
(pH 7.0)
N = no. of trout
5 10
HEMOGLOBIN (gm/100 ml blood±S.E.)
N = 7
Y////////////////A-
10
15
20
P0 (mm Hg ± S.E.)
Figure 19. Hematocrit, hemoglobin and venous blood P values in
subacute acid exposed trout immediately following
breeding season (pH 1|.9 * 26-28d).
41
-------
Field studies of nonbreeding trout placed in acid streams. Considerable
time and effort were expended in attempts to validate laboratory findings
under field situations, i.e. in streams containing levels of acidity
comparable to those employed in the laboratory but stemming from acid
mine drainage. A number of stream sites were used and several hundred
fish introduced after acclimating them to nonacid segments of an acid
polluted stream. None of the acid exposed fish survived longer than
one week at pH 4.8 ± 0.2. This precluded making hemal and hemopoietic
measurements following 28 day intervals of exposure. Measurements of
peripheral parameters following acute exposure were also limited due to
the high incidence of mortality. Hematocrits from surviving exposed
trout were elevated compared to controls (Figure 20). Blast cells showed
no cytomorphological changes. F-DNA profiles of the immature erythroid
cells revealed increased proliferation in both renal and splenic imprints
from both controls and experiments (Figure 21), i.e. 40-46% of the total
blast cells measured fell into interclass or 4C amounts of DNA compared
to previous F-DNA profiles (see Figures 8, 12, 17), where 1-16% of the
total cells measured fell to the right of the 2C category. It was felt
that this stimulation in blast cells in both controls and experiments
under stream conditions is related to factors other than acid exposure.
Thus, field studies revealed that toxicants or unknown factors other than
acidity aggravated the acid induced hypoxemia observed in the laboratory
during the acute phase of exposure.
N • 9
N - 10
P< .05
10 20 30 tO
HEMATOCRIT (volume percent ± S.E.)
I
50
NONBREEDING ACUTE
EXPOSURE,FIELD TEST
QCONTROL (pH 6.4)
£2 ACID (pH 4.8)
N " no. of trout
Figure 20. Hematocrit of nonbreeding trout exposed to acid (pH 14.6}
for two days in the field.
42
-------
4Or
NOM8REEDING CONTROL
( N=6),pH 6.4 x 2d
SPLENIC
1 n = 100
"4 6 8 10 12 14 16 18 "468 10 12 14 16
RELATIVE AMOUNT OF DMA IN ARBITRARY UNITS (A.U.)
Figure 21. Feulgen- DNA distribution patterns for a mixed population
of erythroid cells from renal and splenic imprints of non-
breeding, acute acid exposed trout in the field
(pH 1|.8 x 2d). Dotted lines indicate the 2C category.
Numbers in circles indicate percentages of cells to the
right of the 2C category. Maturation stages I, II and III
are designated by hatched, solid and open bar graphs.
Discussion. A major contribution of this phase of work is that it eluci-
dates certain aspects of the erythropoietic process following short and
long term exposure of nonbreeding brook trout to sublethal levels of
acidity. Specifically, peripheral blood measurements indicated compensa-
tory hemal changes suggesting increased mobilization of blood stores and
a hemoconcentration in both short and long term exposure. However, RNA
cytochemical measurements of blast cell populations indicated that meta-
bolic aspects of hemopoiesis were not the same in short and long term
acid exposed trout. Acute exposure caused a reduction in metabolic
activity (depressed RNA levels) while subacute exposure stimulated regu-
latory aspects of erythroid maturation. When reproductively active trout
are exposed to acid, compensatory hemal responses are unable to meet
higher oxygen demands stemming from increased energy expenditure (asso-
ciated with reproductive changes) and decreased oxygen transport across
the gill epithelium. As a consequence, there is a breakdown in peripheral
adaptive blood responses, despite an exaggerated increase in RNA levels
in the early blast cells of hemopoietic tissue.
43
-------
It is noteworthy that although the hematocrit is elevated during acute
acid exposure, trout remain hypoxemic, i.e. blood ?Q2 levels remain
sufficiently low to elicit behavioral symptoms of respiratory distress.
Thus, it is not surprising to find that ^productively active trout
exhibit a lower tolerance to acid' by virtue of their higher oxygen
requirements. Nonbreeding trout require at least two weeks before they
lose overt symptoms of oxygen lack. It is probable that this coincides^
with the time when PQ levels return to normalcy and that this is attri
butable to increased Tiemopoietic activity. Support for this is
derived from the observation that 28 day exposed trout have normal Po2
levels and also show signs of increased metabolic activity in hemopoietic
sites. This pattern of response to hypoxia, consisting of hemoconcentra-
tion during the acute phase followed by active hemopoiesis, is basically
the same as that of warm blooded vertebrates.
Hematological findings in subacute exposure experiments indicate that the
ultimate physiological basis for acclimation is an increased availability
of oxygen effected through an increased hematocrit. During the onset of
acid exposure, most of the observed increase in RBC mass comes from
splenic and renal stores (Vaala and Mitchell, 1970). Other workers (Hall
_et_aL., 1926; Phillips, 1947; Prosser _et_ aL., 1957; Ostroumova, 1970)
support the idea that rapid changes in RBC mass can be achieved in fish
through mobilization of erythrocyte reserves in the spleen. However, this
is probably a temporary expedient preceding the establishment of a higher
level of hemopoiesis with longer term exposure to acidity. The most
plausible explanation for the observed increase in circulating RBC mass
in 28 day exposed trout is an increased rate of maturation and turnover
of erythroid precursor cells. The basis for postulating a stepped up
rate of erythroid maturation in 28 day exposed trout stems from observed
changes in numbers of immature erythroid cells in kidney hemopoietic
tissue. A 50% decrease was found in the ratio of blast cells to mature
RBC in kidney of acid exposed fish, suggesting a faster production and
output of erythrocytes into the circulation.
In keeping with the hypothesis that stimulation of regulatory aspects of
cellular metabolism is associated with the erythroid maturation process
in acid exposed trout, one would anticipate increased RNA levels in early
blast cells. This proved to be the case. Total cellular RNA was higher
in proerythroblasts in renal tissue of acid exposed trout indicating a
stimulatory effect in the maturational activity of early blast cells.'
Azure B-RNA profiles also revealed higher total cellular RNA in early
maturation with subsequent stages showing a progressive decline in RNA.
This RNA pattern, seen in both splenic and renal imprints, has been fairly
well established from studies on many types of vertebrates (Cameron and
Prescott, 1963; Grasso and Woodward, 1966, 1967; Torelli e_t al., 1964;
Storti and Torelli, 1965; lorio, 1969). In short, one aspect~of accli-
mation is undoubtedly stimulation of the erythron, a term which sometimes
is used to describe the functional hemopoietic unit, i.e. population of
cells delegated with the function of RBC production. In other words,
there is an apparent increase in rate of maturation of immature RBC in
subacute exposed trout.
44
-------
Although one would expect an increased blast cell proliferation in acid
exposed trout to account for the increased hematocrit, very few mitotic
figures were seen in either renal or splenic tissue. Furthermore, DNA
profiles of maturation stages indicated few cells in the premitotic or
mitotic state (e.g., with 3-4C amounts of F-DNA). These observations
indicate that the S-G2 phase in teleost erythroid blast cells must be
extremely short and, therefore, even with a high proliferation rate very
vew cells contain 4C amounts of DNA at any one time. Further investiga-
tion is needed to document this, perhaps utilizing combined techniques
of autoradiography and incorporation of labelled thymidine into DNA.
Two major conclusions (based on hematocrit and hemopoietic changes) can
be drawn regarding the nature of the erythrocytic response during acid
exposure. Simply restated, these are as follows: (1) the acute phase
of acid exposure is characterized by an increased mobilization of erythro-
cytic stores with no augmentation of hemopoiesis and (2) following longer
term exposure there is an amelioration of hypoxemia attributable to
increased hemopoiesis.
59
Measures of several related blood characteristics (e.g. Fe uptake, his-
tone profiles, blast cell counts), as well as data on mortality and hemal
response patterns of reproductively active and field exposed trout, pro-
vide added support to these conclusions. However, none of these supple-
mental measures were considered very useful in terms of providing new
information or clarifying the physiological basis or pattern of erythro-
cyte adjustments during acid exposure. Some of the observed changes,
notably in Fe^9 uptake, in differential counts and radioactive iodinated
serum albumin index (RISA) of blood volume,are difficult to interpret in
the absence of information on normal levels of blood parameters being
measured in teleosts.
59
For example, Fe incorporation (calculated on a percentage basis) was
observed to be identical in short term acid exposed and control trout.
This lends support to the observation that metabolic aspects of hemo-
poiesis and maturation of red blood cells are not enhanced but actually
may be depressed during the acute phase of exposure. However, if one
calculates Fe^9 incorporation on a blood volume basis, the "apparent"
rate of iron uptake, and hence rate of hemoglobin synthesis, would appear
to be extremely high. The reason for this is that the use of RISA for
calculating blood volume yielded erroneously high values in acute exposed
trout probably because rate of dilution of the tracer compound was much
higher (as a consequence of hemoconcentration) in acid exposed than in^g
control trout. Thus, although the 24 hour interval recommended for Fe
uptake (Hevesy et al., 1964) and the 15 minute time span used for RISA
dilution (established in preliminary studies) might be valid for trout
not undergoing volume changes in blood to cell compartments, these are
probably not the optimum times one should use in situations where hemo-
concentration is going on. In brief, it is felt that these two tests as
used have questionable validity in instances where one group is exhibit-
ing hemoconcentration.
45
-------
Differential count data of immature erythroid cells proved to be quite
variable and therefore was not considered as valid as the immature to
mature RBC ratio since the percentages of cells in differential counts
are always relative. It is felt that asynchronous division might also
cause some of the observed inconsistency in percentages of maturation
stages in different groups of trout. The leucocyte differential counts
on blood smears revealed a drop in lymphocytes with a rise in polymorphs
in acute exposed fish with a return to normalcy in subacute exposed, non-
breeding trout suggesting that the former group of trout is more stressed.
This same type of shift was observed to occur in minnows following acute
acid exposure (Dolsky, 1971). A similar reaction was seen by Ostroumova
(1970) in trout exposed to increased salinity. These changes are diffi-
cult to explain since the function of leucocytes in fish has not been
clarified.
It was interesting to note that hematocrit and hemoglobin values from
controls collected during the spawning season, a period of high metabolic
activity and presumably increased oxygen demand, are comparable to hema-
tocrit and hemoglobin content seen in nonbreeding subacute exposed trout.
In addition, a considerably higher ratio of proerythroblast to reticulo-
cyte RNA content was evident in reproductively active controls compared
to nonbreeding subacute controls. This further substantiates involvement
of hemopoietic tissue cells which exhibited an equally high enhancement
of metabolic activity in nonbreeding long term exposed trout. The latter
trout, however, could acclimate since they presumably had a lower overall
tissue requirement for oxygen compared to reproductively active trout.
Exposed reproductively active trout, however, showed a failure of compen-
satory hemal adjustments and a high mortality. Symptoms of respiratory
distress persisted throughout the exposure period suggesting trout were
unable to acclimate. Peripheral measurements (hematocrit, hemoglobin
content and peripheral count) all decreased. Correspondingly, blood
volume was significantly below control values and radioiron incorporation
was depressed. This collapse of the hemal compensatory response is sug-
gestive of ineffective erythropoiesis. Kinetic models of erythropoiesis
from Lajtha (1965) and Stohlman et_ a^. (1962) indicate that death of a
certain population of cells, i.e. cessation of mitosis and differentia-
tion, results in ineffective erythropoiesis.
As in nonbreeding trout, DNA cytophotometric profiles of renal and splenic
tissue of reproductively active trout also showed no increase in numbers
of proliferating blast cells. However, a pronounced increase in total
cellular RNA was evidenced in basophilic and polychromatophilic erythro-
blasts. This might reflect an attempt to restore the depressed erythro-
pbietic line and suggests that the erythropoietic stimulus is operating,
but some other mechanism must be malfunctioning. Significantly, even
proerythroblasts in splenic (an accessory hemopoietic organ in trout)
tissue imprints revealed a higher RNA content.
46
-------
Studies similar to those described above were conducted under field condi-
tions which involved the presence of water toxicants, as well as a low pH.
All trout died within one week of exposure at pH levels which were not
lethal under laboratory conditions (pH 4.8). Undoubtedly, additional
environmental stresses somehow lessen the ability of fish to acclimate
under field situations. Thus, projection of laboratory methodology to
field situations must be regarded with caution. A pH of 5.0 may be
tolerable under laboratory conditions in nonbreeding season, but in the
field, where unknown toxicants may be present, standards must be based
on more than one water quality criterion for maintenance of fish
populations.
Summary. Four groups of hatchery-reared brook trout were used. One group
consisted of nonbreeding trout exposed to near toxic levels of acidity for
five days where pH was brought from 7.0 to 5.0 on day one, maintained at
4.0 for three days, and lowered to 3.5 on the fifth day of exposure. The
second consisted of nonbreeding trout continuousJ.y exposed to sublethal
levels of acidity (pH 4.9) for 26-28 days. A third group was composed of
reproductively active trout exposed for 28 days at a pH of 4.9. The final
group was composed of nonbreeding trout exposed to acidity under field
conditions.
Hemal changes associated with acid induced hypoxemia were assessed using
peripheral blood measures of hematocrit, hemoglobin content, blood P()9>
and differential counts with supplementary measures of peripheral RBC
counts and blood volume. The hemopoietic response pattern was character-
ized through Feulgen-DNA and azure B-RNA quantitative cytophotometry of
developing blast cells in renal and splenic tissue imprints in addition
to blast cell to mature RBC ratios in imprints. Supplemental information
on hemoglobin and blood histones was obtained using polyacrylamide gel
electrophoresis and determination of Fe^" uptake by erythrocytes.
The major results are as follows: (1) Acute exposure to near toxic levels
of acidity evoked behavioral symptoms of respiratory stress and hypoxemia
(low blood PQ?) • There was an increase in hematocrit and hemoglobin, but
no evidence of increased hemopoietic activity in blast cell elements of
spleen and kidney. Instead, total cellular RNA content in maturing
erythroid cells in kidney was depressed in basophilic and polychromato-
philic erythroblasts. (2) Following 28 days of exposure to a pH of 4.9,
nonbreeding trout exhibited symptoms of respiratory distress only during
the first 8-10 days of exposure and not during the last 10-28 days.
After 28 days of exposure the hematocrit and hemoglobin were elevated and
blood Pg9 levels were normal. Feulgen-DNA data again failed to reveal
any increased proliferative activity; however, there was a definite
increase in total cellular RNA in kidney proerythroblast cells. (3)
Reproductively active trout had a very high mortality rate (75%) as com-
pared to nonbreeding (10%) trout when subjected to a pH of 4.9. Further-
more, the surviving trout exhibited signs of respiratory distress
throughout the 28 day exposure period. Peripheral blood measurements in
these fish indicated failure of hemal compensatory mechanisms, i.e. the
hematocrit was depressed relative to both nonbreeding and reproductively
47
-------
active controls. On the other hand, basophilic and polychromatophilic
erythroblasts in the kidney and spleen showed very high levels of total
cellular KNA indicating a very high rate of metabolic activity in these
hemopoietic sites. (4) None of the nonbreeding trout exposed to acidity
under field conditions survived longer than one week in water of pH 4.8.
Hematocrits were elevated in surviving exposed trout. F-DNA profiles
revealed increased proliferation in both renal and splenic imprints from
both control and experimental fish. (5) Results of Fe-*" uptake, histone
profiles, blast cell counts, and RISA estimates of blood volume generally
provided additional support for the conclusions drawn from hematocrit and
hemopoietic nucleic acid data. However, it was felt these supplemental
measures did not add any appreciable information relating to physiological
mechanisms involved in acid induced erythrocytic changes.
The significance of these results is that they characterize the erythro-
cytic response in brook trout during short and long term acidic exposure
in terms of hemal and hemopoietic parameters. From this it is concluded
that the most useful bioindicators of acid induced erythrocytic changes
in peripheral blood are hematocrit (or hemoglobin) coupled with blood PQ
measurements and related bioassays of hemopoietic tissue are azure
B-RNA profiles of renal blast cells and the immature to mature RBC ratio
in the kidney.
48
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SECTION V
HEMAL CHANGES IN ACID EXPOSED FATHEAD MINNOWS
AND LONGNOSE DACE
This subproject deals with the influence of acidified water on white
blood cells of two species of fish, fathead minnows and longnose dace,
which were exposed to sublethal levels of low pH water for acute, sub-
acute, and chronic exposure periods. Also, histological and his to-
chemical analyses of the spleen were performed to determine the extent
of hemopoietic involvement during acid exposure. One objective of this
work was to explore the possibility that splenic and hemal responses in
small fish might be used as bioindicators of sublethal levels of acid
contaminants in water.
No firm cytological or physiological basis is available for establishing
a uniform or standardized nomenclature for specific types of leucocytes
in teleost blood. As a consequence, considerable confusion exists regard-
ing the identification of various white blood cells. In this study, the
terminology for teleost blood proposed by Jackowska (1956) and Weinreb
(1963) is adhered to since these workers have documented their reports
with photomicrographs of the major cellular elements described.
Examination of smears of the peripheral blood of minnows and dace,
treated with Wright's stain, reveals the following mature white cell
types: lymphocytes, monocytes, thrombocytes, neutrophils, eosinophils
and basophils. Basophils and eosinophils are very rare in the circula-
tion of minnows, dace and other teleosts under both normal and adverse
conditions. When seen, these cells are similar in appearance to their
mammalian counterparts and are easily recognizable by virtue of their
prominent cytoplasm!c granules. Lymphocytes are the most prevalent white
cells in dace and minnows as in other teleosts where the reported number
is as high as 20 per 300 erythrocytes (Weinreb, 1959). They average six
micra in diameter, with size and shape varying according to the amount of
cytoplasm present. The large, rounded nucleus occupies most of the cell
and contains distinct dark and light areas of chromatin. Larger lympho-
cytes tend to have more eccentric nuclei and a more granulated cytoplasm.
There is no reliable cytological basis for differentiating between mono-
cytes and Large lymphocytes; hence, monocytes were not considered as a
distinct cell type in the present study.
Although neutrophils are the most numerous of the granulocytes in a whole
blood smear, they only number about two neutrophils per 300 erythrocytes
(Weinreb, 1959). Their average size is 11 micra in diameter and they are
round to oval in shape. The eccentric nucleus may be found in variable
forms: round, indented, kidney shaped, or bilobed, as in higher verte-
brates. The slightly acidophilic cytoplasm contains very fine acidophilic
granules.
49
-------
Neutrophils have been termed "scavengers of the blood stream," in teleosts
as in mammals, for they exhibit great phagocytic activity and are drawn to
areas of infection for this function. Neutrophils have been found in
great abundance in the circulation of trout during acute inflammatory
reactions (Weinreb, 1958). In view of these considerations, particular
attention was focused on acid induced changes in circulating lymphocytes
and neutrophils and relating these to possible alterations in Feulgen DNA
and histone staining of spleen lymphocytes.
Methods. Fathead minnows (Pimephales promelas) and longnose dace
(Rhinicthyes cataractae) were obtained by electro-fishing from Spring
Creek, Centre County, or by net at the Pennsylvania Fish Hatchery, Benner
Springs, Pennsylvania. The minnows weighed 1.9-4.8 g and varied in
length from 4-7 cm. Dace weighed 8.4-11.5 g and varied in length from
7.5-10.5 cm. Tissues from fathead minnows exposed to graded severities
of acidity for seven months were sent from research workers at the
Duluth Water Quality Control Laboratory for processing and analysis in
our laboratory.
In preliminary studies, several hundred fathead minnows and several
hundred longnose dace were subjected to graded severities of pH to estab-
lish tolerance limits and mortality rates of the two fish species under
controlled laboratory conditions. A continuous flow system, similar to
that described by Mount and Brungs (1967) was employed to maintain a
graded series of acidity in seven different tanks. Hydrochloric acid
was used. The pH was continuously monitored by a multichannel recorder
system. The water was constantly aerated, filtered and kept at 12 C
using an automatic cooling unit. Minnows or dace were kept in mesh
baskets to separate them from larger trout that were present in tanks.
Fish were fed once a day in the form of dry pellets (Strike Fish Feed,
Agway, Inc.). To standardize the water coming into the gravity flow
diluter system it was passed through an ion exchange tank and a charcoal
filter (Culligan filtration cylinders). This provided water which was
free of Ca, Fe, and Cl and had a pH range of 7.5-7.8. However, since
fish will not survive in the absence of any Ca, enough was added to
aquaria in the form of CaCl2 twice a day to maintain the Ca level at
about 4-10 p.p.m. Water samples were taken from each tank and an ion
analysis was performed by mass spectrometry (Appendix). Other analyses
included measures of total acidity, total alkalinity, conductivity, and
dissolved oxygen following procedures and recommendations outlined in
"Standard Methods" published by APHA, AWWA, and WPCF. Fish behavioral
changes and mortality were recorded at each pH level, and hematocrit
determinations were performed on the blood of fathead minnows.
Subacute exposure studies (28 day) on a group of 49 field captured long-
nose dace were conducted in a gravity flow diluter system as described
above. The pH's of the four experimental tanks were 5.89 ± .68, 6.33 ±
.39, 6.73 ± .20, and 6.98 ± .14; the pH of the control tank was 7.1 ± .10.
Blood smears were made from the fish at each pH for white blood cell dif-
ferential counts. Splenic tissue was also taken from these fish for rou-
tine histological studies and cytophotometric analyses of Feulgen-DNA
(F-DNA) in lymphocytes.
50
-------
Two streams of known acidity (ca. pH 4.9 and 5.3) were chosen for acute
exposure studies of longnose dace and fathead minnows in natural water-
ways': Black Moshannon Creek in Centre and Clearfield Counties; Upper
Three Runs in Clearfield County (Appendix). These streams are small
tributaries of the West Branch of the Susquehanna River and contain soft
acid water from this sandstone area. Natural fish populations occur in
the upper regions of each stream, while in the lox^er regions tributaries
of acid mine water from strip mining operations enter the streams causing
a decrease in pH levels. Control sites were established above the acid
water tributaries on each stream and test sites were established below
the entry of the acid mine water tributaries. Another control base used
mainly for acclimation purposes was established on Spring Creek, an
alkaline limestone stream (pH 7.8). About 50 fathead minnows and 20
longnose dace were placed on different occasions in each live trap at
the selected sites. Traps were made of nylon net suspended from metal
frames, and anchored to the bottom of the stream with rocks. Fish were
temperature acclimated at the control site on Spring Creek, and pH accli-
mated before placement in the test areas with as little handling as
possible to reduce stress. Holding pens were examined on a 6-12 hour
basis. Water samples were taken from all sites and water analyses per-
formed as described previously (Appendix). A representative number of
fish was returned to the laboratory for analysis. Blood smears were
taken for leucocyte differential counts, and the viscera of fathead min-
nows and the spleen of longnose dace were processed for histological and
histochemical studies. The number of fish, exposure periods, and water
conditions are found in Table 12.
Tissues from fathead minnows exposed for seven months to graded severities
of acid in a gravity flow dilution system at the Duluth Water Quality
Laboratory were shipped to our laboratory after being fixed in 4% formalin
for 24 hours and preserved in 70% ethanol. Prior to tissue processing,
while fish were cut dorsal-ventrally into four pieces; these were then
dehydrated, cleared, embedded in paraffin and sectioned at six micra.
Histological staining and analysis of F-DNA were performed on only those
body sections containing the spleen. Blood smears of representative fish
from each pH treatment group were also obtained for white blood cell
differential counts.
Blood was obtained by severing the caudal peduncle of both dace and
minnows and smears stained with Wright's stain. Lymphocytes, large
lymphocytes (including monocytes), eosinophils, neutrophils and basophils
were counted following the procedure outlined by Hessor (1960). Hemato-
crit measurements were performed only on fathead minnows exposed to a pH
of 7.1 and 6.3 for 14 days, plus those exposed to a pH of 7.1 and 4.0-4.5
for 5.5 hours. Blood for hematocrits was taken from the caudal peduncle
using heparinized capillary tubes.
Routine histological stains (hematoxylin and eosin) were used for locali-
zation of spleen and lymphocytes found in the spleen, and alternate
slides were Feulgen stained for DNA. Optimum hydrolysis time was deter-
mined to be 60 minutes using 5N HC1 and 20 C as outlined by Dietch (1968).
51
-------
Control slides were prepared by hydrolyzing spleen sections in 5% trich-
loroacetic acid at 100 C for 15 minutes to remove all nucleic acid and
stained simultaneously with the slides to be used for F-DNA analyses. The
two wavelength method of microspectrophotometry (Ornstein, 1952; Patau,
1952) was used to calculate relative amounts of nuclear DNA on an indi-
vidual cell basis. Two histochemical techniques were employed for nuclear
histones: the picric acid-eosin Y method (Bloch and Hew, 1960), and the
alkaline fast green method (Alfert and Geschwind, 1953) . The latter stain
was subsequently used for cytophotometric analysis of nuclear his tone
content. The reason for this was that control slides stained with eosin Y
showed very bright non-specific staining after treatment with 5N HC1,
IN HC1, 0.2N HC1, and trypsinization to remove the histones. On the other
hand, control slides stained with alkaline fast green showed little non-
specific staining after removal of histones using the method of Swift
(1964). The procedure for removing histones simply consists of placing
blood smears in 0.2N HC1 for two hours before fixation in 4% formalin.
Formalin fixation apparently hinders HC1 solubilization of histones. Again,
an absorption curve for alkaline fast green was established and the two
wavelength method of microspectrophotometry used for nuclear histone
quantitation. For Feulgen-DNA the wavelengths chosen were 565 millimicra
and 496 millimicra; for alkaline fast green the wavelengths selected were
635 millimicra and 590 millimicra.
Table 12. Stream conditions during field studies of
acute acid exposed fish
Fathead minnows
June 1-2, 1970
pH
Temperature
Exposure time
No. of fish
June 3-4, 1970
pH
Temperature
Exposure time
No. of fish
Upper Three Runs
Control area Test area
6.4 - 5.8 4.8
48 F - 55 F 55 F
136.5 hrs. 20 hrs.
5 5
6.4 - 5.9 4.9
48 F - 54 F 54 F
184.5 hrs. 17.5 hrs.
5 5
Longnose dace
Black rtoshannon Creek
Control area Test area
6.4 - 5.9 5.2
53 F - 62 F 62 F
137.5 hrs. 21 hrs.
5 5
6.4 - 6.2 5.3
53 F - 59 F 61 F
186.5 hrs. 13.5 hrs.
5 5
Control Area Upper Three Runs
Spring Creek Above acid Acid Branch
pH
Temperature
Exposure time
No. of fish
Survival
7.8 6.
64 F 60
collection site 210
10 10
10 10
0 4.5
F 60 F
hrs. 12 hrs.
10
0
52
-------
Preliminary work. The pilot studies conducted on several hundred longnose
dace and fathead minnows were designed primarily to determine approximate
tolerance limits of these species to acid water, and to determine which of
these two species was best suited for studies of subacute (28 day) expo-
sures to graded severities of sublethal levels of acidity. Briefly, the
major findings were as follows:
1. Water with a pH of 3.0-4.0 was extremely toxic to both species. All
fish died rapidly, in less than two hours, and exhibited massive
hemorrhaging at the base of pectoral fins, mouth and gill regions.
This was presumed to be indicative of contact chemical injury to
these vital regions. Epidermal mucification was also very pro-
nounced over the entire body surface, including the pectoral, dorsal
and tail fins. A pH range of 4.0-4.5 proved equally toxic since all
fish died within the first 24 hours. Maximum survival time was 5.5
hours. Again copious skin mucification was evidenced as was massive
hemorrhaging. The major behavioral response at these low pH levels
(3.0-4.5) was a marked decrease in locomotor activity and an increase
in opercular movement. The fish did not exhibit surfacing reactions
or gasping responses observed in larger trout exposed to similar
conditions.
2. At pH 5.0-5.8 mortality of the fish was still high, ca. 75%. On one
occasion all minnows died at this pH, while on another" occasion 57%
of the dace died at this pH. However, some fish survived up to 28
days. None of the survivors exhibited contact chemical injury which
resulted in hemorrhage. Mucification was extensive in all fish but
transient (3-4 days) in those fish which did not die in a period of
one to four weeks.
3. During the course of the above studies a number of fathead minnows
obtained from the hatchery exhibited a high incidence of skin fungus
and some also developed symptoms of Whirling disease or Popeye even
in control tanks (pH 7.0). None of the field-captured dace exhibited
these symptoms. As a consequence, dace were used for 28 day exposure
studies.
4. No differences were found in the hematocrit of control and acid
exposed fish. It was noted, however, that the mean hematocrit of
fish captured in the fall tended to be slightly higher than that
of summer-captured specimens.
5. In summary, the pilot studies established that the pH range of 5.0-
5.8 is close to the tolerance limit for longnose dace, and a pH of
6.0 could be considered sublethal over a 28 day exposure interval.
Effect of 28 days exposure to graded severities of acidity on leucocyte
counts and F-DNA content of spleen lymphocytes in dace. One of the most
striking changes observed in the blood of acid exposed dace was a shift
in the differential leucocyte count. The percentage of lymphocytes
dropped from a mean of 91.3 to 52.0%, while that of neutrophils increased
53
-------
from 8.6 to 47.6% (Table 13). Since the responsive cell types in all
experimental groups were lymphocytes and neutrophils, the standard error
and level of significance are given only for these cell types. Increased
acidity brought decreased in the percentage of leucocytes regardless of
the time of year or the sex of the animals. Neutrophils in acid-exposed
dace smears increased in number to such an extent that in one microscopic
field five to six neutrophils could be counted (at a magnification of
900x), while corresponding areas of control smears normally had zero to
one neutrophils.
Table 13. White blood cell differencial counts of control and
acid exposed longnose dace after a 28 day exposure.
% Lymphocytes P* % Neutrophils P*
pH
No
pH
No
pH
No
PH
No.
PH
No.
•= 7
. of
- 7.
. of
- 6.
. of
6.
, of
- 5,
of
.1
fish
.0 +
fish
.7 +
fish
.3 +
fish
,9 + .
fish
= 8
.1
= 7
.2
= 6
,4
= 6
.7
= 5
91
s .
91
a .
90
s .
94
s .
52,
s .
.3 + 9.
e. = 3
.7 + 7.
<=. = 2
.1 +• 7.
e. - 2
.2 + 4.
e. •- I
.0 + 11
e. = 5
6
.6
4
.8
6
.9
2
.7
.7
.2
8.6 + 8
s . e . -•
7.6 + 5
s . e.
9.7 + 7
S . e . =
5.8 + 3
a . e .
0.001 46.6 -t-
s . e . =
.8
3.
.2
2.
.0
2.
.8
1.
10.
1,
4
0
9
6
7 0.001
5
Level of significance.
Data summarized in each histogram in Figure 22 represent 50 individual
measurements of nuclear F-DNA in a population of splenic lymphocytes from
each acid exposure group of dace. It was reasoned that the observed
decrease in circulating lymphocytes in acid exposed dace might reflect a
reduction in splenic lymphopoiesis or increased rate of destruction. With
decreased mitotic activity one would expect to find fewer lymphocytes in
the S phase of DNA synthesis (i.e. fewer cells with DNA levels exceeding
2C); increased numbers of degenerating cells on the other hand would lead
to a greater number of cells with F-DNA content below the 2C level.
Histograms are conventionally used to present F-DNA data on cell popula-
tions comprising specific tissues. The profile of such histograms serves
as a useful index of changes in proportions of nuclei exhibiting chromatin
loss (DNA values less than the 2C level) or increased mitotic activity
(DNA content greater than the 2C level). To facilitate comparison of
exposure groups, dotted lines were included on the histograms to designate
the diploid 2C or non-replicated DNA segment of the lymphocyte nuclear
population. To insure that the value assigned as the 2C level of DNA
truly represented the diploid amount of DNA, F-DNA content was also mea-
sured in 50 sperm cells from these fish. As expected, the mean DNA
content for the sperm cells was one half that of the somatic cells.
54
-------
LONG NOSE DACE 1
28 DAY EXPOSURE
pH - 6.89 ± 0,68
pH-6.33 ±0.39
n-50
pH = 6.73 ±0.20
pH = 6.98 ± 0.14
n-50
pH = 7.1
n = 50
RELATIVE AMOUNT OF FEULGEN - DNA (A U I
SPLEEN
Figure 22. Histograms representing Feulgen DNA values for splenic
lymphocytes of longnose dace exposed to acid water for
subacute (28 day) period. Dotted lines or the histo-
grams designate the 2C interval of F-DNA
As is evident from the data presented, there was no increase in nuclei
with less than 2C amounts of DNA in splenic lymphocytes of acid exposed
dace; this indicated that the rate of pyknosis was comparable in acid
exposed and untreated groups. No differences were observed in the histo-
logical appearance of lymphocytes or in the extent of yellow pigment
deposits in the spleen of acid exposed dace; this is also indicative of
the absence of any increased lymphocytic or erythrocytic breakdown.
There was also no indication that acid exposure caused any augmentation
in mitotic activity. Both control and experimental spleens exhibited
few if any nuclei which fell into the 4C (replicated) F-DNA category.
Field experiments on fathead minnows and longnose dace. Acute acid expo-
sure of fathead minnows in stream traps provided as pronounced a change
in the white blood cell differential as was seen in the laboratory sub-
acute acid exposed dace. After exposure for 20 hours, fish from two
acidic streams (pH 5.2 and 4.8) showed a reduction in lymphocytes from
93.4% to 58.8%, and 91.3% to 77.6%. Circulating neutrophils, on the
55
-------
other hand, increased from 6.6% to 41.4% and 8.3% to 24.0% (Table 14). A
repeat test of fathead minnows in these two streams for 17 hours showed a
percentage reduction in lymphocytes from 87.6 to 79.0 and 97.0 to 85.8.
The percentage of neutrophils increased from 8.5 to 20.8 and 2.0 to 13.6
(Table 14).
Table 14. White blood cell differential counts of control and
acid exposed fathead minnows during field studies of
acute exposure.
Treatment % Lymphocytes P* % Neutrophils P*
Upper Three Runs 93.4 + 5.8 6.6 + 5.2
pH = 6.4 5.8 s. =. - 2.6 ». =. = 2.3
No. of fish 5
Upper Three Runs 58.8 + 14.3 0.01 41.4 + 5.6 0.001
pH - 4.8 =.. e. = 6.4 s. e. = 8.3
No. of fish 5
Black Moshannon 91.3+11.7 8.2+9.9
pH - 6.4 =,. e. = 5.9 s. e. = 4.9
No. of fish = 4
Black Moshannon 77.6+10.4 0.1 24.0+10.2 0.05
pH » 5.2 ». =. = 4.7 a. =. ' 4.6
No. of fish - 5
Upper Three Runs 87.6 + 10,3
pH = 6 .4 =.=.=4.6
No. of fish 5
Upper Three runs 79.0 + 4.4 20.8 + 4.0 0.001
pH = 4.9 =.=.=1.9 s. e. = 1.8
No. of fish 5
Black Moshannon 97,0+2,3 0.1 2.0+1.6 0.1
pH • 6.4 =, =. = 1,1 s. eT 0.7
No. of fish - 5
Black Moshannon 85.8 + 13.1 13.6 + 11.3
pH » 5.3 =. B.~= 5.8 ». =.~= 5.0
No. of fish = 5
it
Level of significance
Splenic lymphocyte F-DNA data from acid exposed minnows were again sum-
marized in the form of histograms with each histogram representing 100
nuclear measurements for designated exposure groups (Figure 23). As can
be seen from these data, 20 hours of acid exposure had no effect on F-DNA
distribution pattern of lymphocyte nuclei, indicating no alteration of
the rate of lymphopoiesis of lymphocytolysis.
Longnose dace were also subjected to acute acid exposure In selected acid
streams. Figure 24 consists of three histograms each representing 100
individual measurements of nuclear F-DNA. For these measures, a con-
certed effort was made to select lymphocytes which contained a larger
nucleus; as a consequence, the F-DNA data are representative of a nuclear
population of larger, more immature lymphocytes. This explains why the
shape of the histogram is not the same as that obtained for fathead min-
now splenic lymphocytes. It was felt that earlier stages in lymphocyte
maturation might be more sensitive to acid exposure than the more mature,
56
-------
smaller lymphocyte. However, as with minnows, no differences were found
in the distributional pattern of F-DNA in lymphocyte nuclei of acid
exposed and control dace.
RELATIVE AMOUNT OF FEULGEN - DMA (A.U.I
(A U. = ARBITRARY UNITS)
BLACK MOSHANNON CREEK
CONTROL pH = 6.4 - 5.9
n = 100
BLACK MOSHANNON CREEK
ACID BRANCH
pH - 5.2
n- 100
UPPER THREE RUNS
CONTROL
pH - 6.4 - 5.8
n -69
UPPER THREE RUNS
ACID BRANCH
pH = 4.8
n = 69
I D LARGE LYMPHOCYTES
1 ] SMALL LYMPHOCYTES
Figure 23. Histograms representing Feulgen DNA values for splenic
lymphocytes of fathead minnows exposed to acid water for
acute (1 day) period in two acid streams. Dotted lines
on the histograms designate the 2C interval of F-DNA.
57
-------
LONG NOSE DACE
ACUTE EXPOSURE
BENNER SPRINGS
pH = 7.B
n = 100
n
UPPER THREE RUNS
pH = 6.0
n= 100
UPPER THREE RUNS
pH-4.5
n« 100
RELATIVE AMOUNT OF FEULGEN - DNA IA U 1
(A U = AHBITflAflY UNITS)
Figure 24. Histograms representing Feulgen DNA values for splenic
lymphocytes of longnose dace exposed to acid water for
acute (1 day) period in two acid streams. Dotted lines
on the histograms designate the 2C interval of F-DNA.
Cytophotometric analyses of nuclear histones also showed that acid expo-
sure has no quantitative effect on nuclear histones in dace splenic
lymphocytes. The histograms in Figure 25 represent a total of 300 indi-
vidual measurements of nuclear histones stained by the alkaline fast
green method.
Hematological effects of chronic (seven month) exposure of fathead
minnows to graded severities of acidity. During the course of this study,
differential counts of blood smears and F-DNA analyses of splenic lympho-
cytes were also made on tissues from fathead minnows subjected to a seven
month acid exposure to graded severities of pH (5.0 ± .1, 5.5 ± .1,
6.0 ± .1, 6.5 ± .1, 7.0 ± .1, 7.4 ± .1). Differential counts of blood
smears showed that chronic exposure had no effect on the leucocyte count
of minnows exposed to any degree of acidity. Blood from these fish-
showed a mean range of 85.8 to 91.9% lymphocytes and a mean range of 7.0
to 14.0% neutrophils over the entire pH range involved (Table 15). Blood
smears stained with Wright's stain also showed no cytomorphological dif-
ferences which could be attributed to acid exposure.
58
-------
LONG NOSE DACE
ACUTE EXPOSURE
BENNER SPRINGS
pH-7.8
n-93
UPPER THREE RUNS
n-92
r-l
UPPER THREE RUNS
pH = 4.5
RELATIVE AMOUNT OF NUCLEAR HISTONE IA.U 1
IA.U. ARBITRARY UNITS!
Figure 25. Histograms representing nuclear histone values for splenic
lymphocytes of longnose dace exposed to acid water for acute
(1 day) period in two acid streams. Dotted lines indicate
the nonreplicating Interval of nuclear histones.
Table 15. White blood cell differential counts of control and
acid exposed fathead minnows after a 7 months
exposure.
Lymphocytes
Neutrophlls
pH - 7.4 + .1
No. of fish - 8
90.5 + 5.3
s. e. = 1.9
9.5 + 4.6
s. «=. = 1.6
pH = 7.0 + .1
No. of fish = 8
91.9 + 8.0
s. e. = 2.8
7.9+ 6.6
s. e. = 2.3
pH = 6.5 + .1
No. of fish - 8
90.6 + 6.0
s. c. - 2.1
9.3 + 5.4
s. e. = 1.9
pH - 6.0 + .1
No. of fish - 8
85.8 + 9.0
b. e. =• 3.2
14.5 + 8.5
s. e. ' 3.0
pH = 5.5 + .1
No. of fish = 8
90.0 + 7.9
s. e. = 2.8
10.0 + 7.4
s. e. = 2.6
pH - 5.0 + .1
No. of fish = 8
90.3 + 4,9
s. e, = 1.7
9.9 + 4.5
s. &. = 1.6
59
-------
FATHEAD MINNOWS
7 MONTH EXPOSURE
Ur-riTl PI I h-F^ r
pH " 6.5 ± 0.1
n-60
pH = 6.0 ± 0.1
n-60
pH = 5.5 t 0.1
n*60
pH = 5.0 ± 0.1
n-60
pH " a.7 i 0.1
RELATIVE AMOUNT OF FEULGEN - DNA (A U I
IA U - ARBITRARY UNITS)
PI LARGE LYMPHOCYTES
I I SMALL LYMPHOCYTES
Figure 26. Histograms representing Feulgen DNA values for splenic
lymphocytes of fathead minnows exposed to a graded
severity of pH for a chronic (7 months) period. Dotted
lines designate the 2C interval of F-DNA.
Results of F-DNA studies are presented in Figure 26. Each, histogram
represents 60 measurements of nuclear F-DNA from lymphocytes of desig-
nated exposure groups. Again, as in acute and subacute exposure studies,
no changes were noted in the distributional pattern of F-DNA staining in
splenic lymphocytes of acid exposed fish. In short, F-DNA studies indi-
cate that neither one day nor seven months acid exposure has an effect on
the physical chemical state of the chromatin of lymphocytes as reflected
in their Feulgen staining affinity.
Discussion. One conclusion supported by the study is that short term
exposure of minnows and dace to sublethal levels of acidity evokes circu-
lating leucocytic changes. The functional significance of lymphopenia
and neutrophilia observed in acute (ca. 24 hours) and subacute (28 day)
exposed fish is difficult to explain since the role of leucocytes in fish
has not been clarified. A shift in the lymphocyterneutrophil ratio is
often witnessed at the time of spawning, and in cases of parasitism and
infection. Since control fish did not exhibit a lymphopenia, one can
60
-------
conclude that the leucocyte changes are a consequence of acid exposure.
Although we have no information regarding the physiological basis for the
observed leucocytic responses, it is quite likely that a disturbance in
acid-base balance and electrolyte pattern is involved in triggering hemal
changes. Among the factors which would tend to promote acidosis and an
ion imbalance in acid exposed fish are hyperventilation, disruption or
injury to the gill epithelium (Plonka, 1971) and renal changes. Packer
and Dunson (1970) found that trout exposed to waters of pH less than five
showed a decrease in blood pH from 7.39 to 6.97. According to A. V.
Vasil'ef (1948) when the pH of mammalian blood changes, shifts in leuco-
cyte counts are observed, with a decrease in lymphocytes occurring during
acidosis. Ostroumova (1970) reported similar shifts in leucocytes in
trout exposed to waters of increasing salinity which, he felt, caused a
disturbance in the blood pH.
Minnows exposed to low pH for seven months did not show such leucocyte
shifts. It can be assumed that these fish have acclimated. Thus, it
appears that in fish, as in mammals, the early or acute stages of a toxic
response syndrome are followed by a recovery phase which is characterized
by a homeostatic readjustment of physiological parameters and an asso-
ciated abatement or return to "normalcy" of many tissue characteristics.
In related work with acid exposed trout, it was shown that histochemical
changes in the gill and Stannius corpuscle, which are evoked during
acute exposure, disappear after one month exposure (see Section III).
Significantly, even behavioral symptoms of respiratory distress and skin
mucification are transient responses, indicating fish can become accli-
mated to acid levels which are close to the toxic limit for a given
species. Such studies emphasize the importance of using short term tissue
responses as bioindicators of water toxicants rather than using tissue
indices based on relatively long exposure intervals.
A few workers have studied changes in blood characteristics of fish
exposed to sublethal levels of metal ion toxicants (Fujiya, 1964;
Schiffman and Fromm, 1959). McKim e_t al_. (1970) found that many physio-
logical blood changes of trout exposed to copper were transient in nature,
disappearing after extended exposure. Acid exposed trout (Vaala, 1971)
often exhibit initial changes in peripheral blood parameters which disap-
pear after a subacute exposure (see Section IV). The present study shows
that for acid exposed minnows and dace, changes in white blood cell dif-
ferential counts are a useful index of acidity if employed in acute
exposure situations, but not in subacute (28 day) or chronic exposure
(seven months).
Another important aspect of this work was the study of the role of the
spleen with regard to changes in leucocyte counts of peripheral blood.
The spleen is the major site of white blood cell production in the fish
and varies in importance as a hemopoetic site from species to species.
It was thought that acid induced lymphopenia and neutrophilia might be
secondary consequences of an alteration in splenic hemopoiesis. His to-
logical and histochemical studies showed that was not the case. Spleen
tissue of acid exposed fish appeared the same as that of controls when
61
-------
examined microscopically following routine hematoxylin-eosin staining.
For example, there was no cytological evidence of pyknosis or degeneration
in lymphocytes or any indication of increased mitotic activity in neutro-
philic elements.
In this study, F-DNA and nuclear histone analyses failed to show an
increased rate of lymphocytolysis in the spleen of acid exposed dace and
minnows after 17 hours or seven months, indicating that the potential
for hemopoiesis is not impaired by exposure of fish to low pH water.
Fujiya (1961) exposed Sparus macrocephalus to pulp mill waste for 24
hours and also found no evidence of histopathology in the spleen.
It is quite possible that during acid exposure there is an increased pro-
duction of leucocytes in both fathead minnows and dace. Consistent with
this interpretation is our finding that the number of leucocytes per 1000
erythrocytes increases from 9.0 ± 1 in control field specimens to 30.0 ± 2
under experimental field conditions. However, differential cell counts
also demonstrated that there was a decrease in the percentage of lympho-
cytes concurrent with an increase in the number of neutrophils. Since
differential counts are based on percentages of specific cell types per
100 leucocytes counted, a reduction in splenic output of lymphocytes
which normally comprise about 80-90% of the circulating leucocytes would
result in a relative increase in neutrophils. Weinreb et_ ^L^ (1969) has
recently postulated that an increase in the number of neutrophils may be
due to cellular transformations or differentiations occurring in the
peripheral blood. This is considered unlikely since it has not been
established that leucocytes can differentiate from one stem line to
another either in the peripheral circulation of mammals or poikilothermic
vertebrates.
Previous work in this laboratory (Plonka, 1971; Vaala, 1971) and else-
where (Westfall, 1945) has shown that one of the major physiological
consequences of acid exposure is impaired respiratory function and a
hypoxemia (Westfall, 1945; Plonka, 1971; Vaala, 1971). It has also been
shown that in some species this is followed by an increased hematocrit,
which possibly stems from a combination of factors such as hemoconcen-
tration, increased erythropoiesis and mobilization of erythrocytes from
hemopoietic stores. Increased mobilization of blood stores from the
spleen has been cited by Ostromouva (1970) and Vaala (1971) to account
for the increased red blood cell volume and increased hematocrits of
stressed trout.
In conclusion, it should be stated that the lymphopenia and neutrophilia
witnessed in acid exposed fish may very well be nonspecific stress
responses of the fish since similar responses are known to occur in rela-
tion to breeding and parasitism. Results of a recently completed study
(Vaala, 1971) indicate that trout exposed to low pH for an acute period
show a lymphopenia and neutrophilia, whereas a 28 day acid exposure shows
return of the leucocyte count to normal (see Section IV) . In support of
this are the results of our study of chronic acid exposed fish which
exhibited a readjustment of percentages of leucocytes to the normal level
62
-------
after a seven month exposure. Therefore, this leucocyte shift must be
used in conjunction with other parameters, such as histochemical changes
in the gill and Stannius corpuscles of acid exposed fish, in order to
establish indices of acid water pollution.
Summary. The major findings of this subproject can be summarized as
follows:
1. Under both laboratory and field conditions the pH tolerance limit
for both minnows and dace is approximately pH 6. Maximum survival
time at a pH less than 5.0 is 5.5 hours. Workers at the Duluth
Water Quality Laboratory provided us with several groups of fathead
minnows that reportedly survived for seven months at pH's 4.7, 5.0,
and 5.5. Blood and spleens from these fish were analyzed and con-
stituted our "chronic" exposure group.
2. Both species exhibit behavioral symptoms of stress and contact
chemical injury after acute exposure to high levels of acidity (pH
4.0, 5.0). These symptoms include decreased locomotor activity,
increased secretory activity of mucous cells, and massive hemor-
rhaging at the base of fins, mouth and gills.
3. Subacute exposure of dace to sub lethal levels of acidity (pH 5.8 to
7.0) and acute exposure of fathead minnows to acidity under field
conditions evokes a shift in the leucocyte differential counts
resulting in a lymphopenia and a neutrophilia.
4. However, chronic exposure of fathead minnows to graded levels of pH
(7.4, 7.0, 6.5, 6.0, 5.5, 5.0, 4.7) showed no relationship between
the differential count and severity of acidity. Percentages of
lymphocytes and neutrophils are identical in both control and acid
exposed groups.
5. No histopathological changes are found in the spleen of dace or fat--
head minnows following acute, subacute or chronic exposure to
acidified water. Histochemical analyses of both Feulgen-DNA and
nuclear his tone content of splenic lymphocyte also indicate an
absence of any increase in mitotic or degenerative changes in these
hemopoietic cells.
6. Changes in leucocyte counts of the peripheral blood is a useful short
term index of acid contamination in water.
63
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SECTION VI
HEPATIC INDICES OF STRESS IN ACID EXPOSED BROOK TROUT
Previous studies showed that acute exposure of brook trout to low pH
water evokes a toxic response syndrome which involves behavioral
symptoms of respiratory distress, increased secretory activity of gill
mucous and Stannius corpuscle epithelial cells (Section III), decreased
efficiency of respiratory gas exchange and hypoxemia (Section IV).
However, with prolonged exposure, fish exhibit adaptive responses as
evidenced by increased hemopoietic activity, normal p02 levels and
normal behavioral activity (Section IV). It was felt that acclimation
of fish to oxygen lack must necessitate compensatory adjustments in
tissue metabolism. In this respect, the liver, as a key organ in
metabolic regulation should reveal, at the cellular level, alterations
in DNA, RNA and histone staining which might be used to assess the
metabolic state of the fish during long term exposure to low pH water.
Many studies attest to the involvement of DNA in the regulation of cell
and tissue metabolism. Recent studies by Garcia (1967, 1969a, 1969b)
and others (Noeske, 1971) have shown that small but measurable differ-
ences in nuclear Feulgen-DNA (F-DNA) staining occur in cells exhibiting
different degrees of chromatin dispersion, with dispersed chromatin
exhibiting slightly higher F-DNA contents than compact chromatin.
Furthermore, biochemical studies by Littau et_ al_. (1964) have shown
that dispersed chromatin provides a more efficient template for RNA
synthesis than compact chromatin. One can postulate therefore that
more dye binding sites are available in dispersed chromatin of metabol-
ically active cells; such cells should exhibit a greater Feulgen staining
affinity than metabolically inactive cells. Another study (Bloch, 1966)
indicates that changes in histone composition can also be correlated
with functional changes in cells.
The purpose of this project is to determine whether acute (3-5d) and
subacute (28d) exposure of brook trout to acid water results in detect-
able changes in Feulgen-DNA (F-DNA), Azure B - RNA, fast green-histones
(FG-histones) or histone fractions in liver parenchymal cells and to
assess the utility of such changes in detecting metabolic impairment
of liver tissue. One reason for undertaking this work was to evaluate
the potential of using liver histochemical changes as rapid and sensi-
tive bioassay methods for acid contaminated water.
Methods: Two hundred and fifty seven brook trout, Salvelinus fontinalis,
(Research Station of the Pennsylvania Fish Commission, Pleasant Gap,
Penna.), 12-16 months in age, weighing 50-80 g and measuring 15-18 cm
in length were used. Prior to experimentation, fish were acclimated to
laboratory conditions for one to two weeks in 150 gallon holding tanks
at 12 ± 1 C under constant aeration. Fish were fed dry pellets (Strike
Fish Feed, Agway, Inc.) once daily.
65
-------
Acute toxicity studies (five days) were conducted in a static system
where water was not changed during the exposure period. Forty control
(pH 7.0-7.3 ±0.1) and sixty experimental fish were used. Acidity of
experimental tanks was maintained at a pH of 4.0 ± 0.2 using HC1. On
day 5, the pH was dropped to 3.5 ± 0.1. An additional 16 control and
16 acid exposed trout were also used in tracer studies dealing with
effects of acute acid exposure on liver histones and liver lipids.
Subacute exposure studies (28-30 days) were conducted using a gravity-
flow proportional diluter system (Mount and Brungs, 1967) to maintain
a constant flow of graded levels of acidity into a series of five
aquaria. The pH's of the five tanks (continuously monitored 6.6 ± 0.3
and 7.1 ± 0.2. Temperature was maintained at 12 C by an automatic
cooling unit. Water samples were taken from each tank and ion analyses
performed by mass spectrometry. Other analyses included measures of
total acidity, total alkalinity, conductivity and dissolved oxygen
following procedures and recommendations outlined in "Standard Methods"
published by APHA, AWWA and WPCF in 1965. A total of 73 fish were used.
Several acute exposure experiments were also conducted under field
conditions. Hatchery trout were placed in net traps in a stream, Upper
Three Runs in Clearfield County, Pennsylvania. Natural fish populations
occur only in upper regions of this stream, since in the lower region a
tributary of mine acid water from a strip mining operation drains into
the stream. A control site was established above the entry of the acid
mine water tributary. Twenty two control fish were subjected to a pH
6.4 ± .2 while 30 experimental fish were exposed to pH 4.8 ± .2 under
field conditions. Exposure time was approximately 24-48 hr, after which
fish were transported to the laboratory for analysis.
Livers were excised from experimental and control fish and small portions
fixed in phosphate buffered 4 per cent formaldehyde (pH 7.0) for 24 hr,
dehydrated, embedded in paraffin and sectioned at 6 microns. Other
portions, used in biochemical studies, were frozen and stored at -20 C.
Individual slides from each animal were stained with hematoxylin and
eosin for morphological analysis (Humason, 1967), by the Feulgen proce-
dure (Deitch, 1968) for DNA and for nuclear histones by the alkaline
fast green procedure (Alfert and Geschwind, 1953). Control and experi-
mental sections were stained simultaneously to minimize error. Optimum
hydrolysis time for Feulgen staining was determined to be 45 min with
formalin fixed liver using 5N HC1 at 20 C. Two Feulgen-DNA control
slides were always run, a nonhydrolysed control and a control in which
DNA was previously extracted by hydrolysis in 5 per cent trichloroacetic
acid at 95 C for 15 min. Both types of controls gave negative Schiff
reactions. Histones were extracted from some tissue sections by treat-
ment in 1 N HC1 for 3 hr at 20 C. These control slides showed no
alkaline fast-green staining. Spectral absorption curves of F-DNA and
fast green histones in liver nuclei are shown in Figure 27.
66
-------
.25
.20
en
.10
Q.
O .05
0
560
450 500 550 600 650
WAVELENGTH (nm)
700
>
j-
cn
UJ
Q
O
I-
Q_
O
.4-
.3-
.2-
.1 -
0
FAST GREEN HISTONE|
635
_L
450 500 550 600 650
WAVELENGTH (nm)
700
Figure 27. Spectral absorption curves of Feulgen-JNA and fast green
histone complexes in trout liver nuclei.
The eosin-fast green technique was also used to differentiate arginine-
rich and lysine-rich histone types on the basis of nuclear staining
(Bloch, 1966). The staining reaction is semiquantitative since analysis
is based on counts of the nuclei exhibiting pink, green or purple
staining.
Two quantitative histochemical procedures were employed, each of which
requires sequential staining of tissue with two different dyes. Using
cytophotometry, quantitative comparisons of two chemical components can
67
-------
be made in the same cell. One procedure used FDNB (fluorodinitrobenzene)
(Bloch and Brack, 1964) and azure B staining (Vaughn, 1966). Under
proper conditions the overall staining reaction is specific for (lysine +
tyrosine) and RNA. The metachromatic stain, azure B, stains both DNA
and RNA so that pretreatment of tissue with deoxyribonuclease is required
for RNA determinations. After deoxyribonculease treatment, control
slides give a negative Feulgen reaction. Since tyrosine accounts for
less than 10 percent of FDNB staining (Therrien, 1967), intensity of
tissue staining is largely a reflection of lysine content. The relative
lysine/RNA ratios in nuclei are computed using the formula E4QQ(lysine)/
E560(RNA) where E = extinction and subscripts refer to wavelengths used
for obtaining extinction values. Since cytoplasmic lysine and RNA also
contribute to the staining intensity of each stain, the following
formula is used to obtain corrected nuclear extinction values:
En = Es - (1 - p/d) Ec, where En = corrected nuclear extinction, Es =
uncorrected nuclear extinction, p is the mean optical path, d = section
thickness and Ec is the extinction of cytoplasm adjacent to the nucleus
(Swift and Rasch, 1956). Cytophotometric analyses were performed using
the plug method of cytophotometry (Swift, 1950). Nuclear lysine or RNA
content can be obtained in arbitrary photometric units (A.U.) using the
formula, M = 2/3r2En, where M = relative content, En = nuclear extinction
and r = radius of the sphere (Swift, 1950). For spherical nuclei, the
formula is M = r2En.
The second double staining technique involves initial staining of tissue
with FDNB followed by staining using the Sakaguchi reaction (Bloch, 1966).
These stains are specific for (lysine + tyrosine) and arginine, respec-
tively, and may be used to obtain molar ratios of these components in
nuclei using the formula: moles (lysine + tyrosine)/arginine = 1.14
[E^Q0(FDNB)/E520(Sakaguchi)] -0.08 (Bloch, unpublished). Correction
for cytoplasmic staining and cytophotometry involved the same procedures
described earlier. Wavelengths used for determining extinctions were
400 nm for FDNB and 520 nm for the Sakaguchi stain. Spectral absorption
curves for the two double staining procedures are shown in Figure 28.
Cytophotometric measurements of parenchymal liver nuclei were made with
a single beam microspectrophotometer using the two wavelength method
(Patau, 1952, Ornstein, 1952) for nuclear Feulgen-DNA and fast green
histone. The standard plug method (Swift, 1950) was also employed in
some cases. For Feulgen-DNA analyses, the wavelengths chosen were 560 nm
and 495 nm; for fast green, the wavelengths selected were 640 nm and
675 nm.
Supplemental measurements included: (a) electrophoretic analyses of
histones, (b) histone acetylation measures, (c) lipid chromatography and
(d) qualitative histochemical study of lipids and glycogen. Procedures
for these are briefly given below.
Liver histones were extracted as described by Bonner et_ al. (1958) and
protein content estimated by the procedure of Lowry et_ al. (1951) .
68
-------
45O 5OO 550
WAVELENGTH (nm)
600
FDNB-
AZURE B
0
"400 450 500 550 600 650 700
WAVELENGTH (nm)
Figure 28. Spectral absorption curv&s of FDNB-azure B and
FDNB-Sakaguchi complexes in trout liver nuclei.
Histone samples were dissolved in 10 M urea to a final concentration of
1 Ug/yl. Polyacrylamide gel electrophoresis was used to identify on each
gel. After electrophoresis, gels were stained for 18 hr in a 0.5 percent
solution of 7 percent acetic acid. Some gels were stained with eosin-fast
green (0.1 percent eosin, 0.1 percent fast green) in 4 percent formalin
(pH 8.1) for 18 hr at 4 C and destained in 4 percent formalin. Micro-
densitometer tracings of stained gels were made using a Gilford densito-
meter. Gels stained with amido black were scanned at a speed of 1 cm gel/
min at a wavelength of 660 nm, with a full scale deflection of 2.5 O.D.
Eosin-fast green stained gels were scanned at 500 nm (eosin) and
rescanned at 640 nm (fast green).
69
-------
Eight control and eight experimental fish were injected intracardially
with 5 yC of 1£*C-acetate (sodium salt, specific activity 20 mc/mM) in
0.1 ml of fish Ringer's solution. Fish were subjected to acute acid
exposure and sacrificed at 30 min and 1 hr intervals. Liver lipids
were extracted from one gram samples of liver by method of Folch et_ al.
(1957) and dissolved in 5 ml of 1:1 chloroform-methanol. Aliquots (500
of each sample were added to 10 ml scintillation fluid and samples
counted in a Packard liquid scintillation counter for 10 min. Thin
layer chromatography (silica gel) was used to fractionate liver lipids
(100 yl) into polar or neutral fractions. Plates were stained with
iodine vapors and lipid fractions located and scraped into vials con-
taining 10 ml of scintillation fluid; counts were made for 10 min
intervals.
Two semiquantitative histochemical stains, Sudan Black B and the periodic
acid Schiff (PAS) reaction (Humason, 1967) were used for staining lipids
and glycogen, respectively. Glycogen content was assessed by visual
comparison of diastase treated and nondiastase treated tissue sections
following PAS staining.
Cytophotometric analyses of DNA and histones in liver nuclei of acid
exposed trout. Data on Feulgen DNA (F-DNA) and fast green histone
(FG-histone) content of liver nuclei are presented in the form of
frequency histograms in Figure 29. These data represent 600 individual
measurements (100 per histogram) of liver parenchymal nuclei. No attempt
was made to analyze other cell types in liver tissue such as connective
tissue cells or vascular elements. Comparison of both the DNA and
histone profiles reveals no differences in average contents of F-DNA and
FG-histone between control and experimental groups. Neither acute nor
subacute exposure results in any detectable shift in the number of nuclei
with F-DNA or FG-histone values which deviated markedly from control
levels. This indicates that acid exposure of trout does not materially
affect liver cells with respect to their nuclear affinity for either the
Feulgen stain or the fast green stain.
The unimodal F-DNA histograms of control as well as experimental fish
indicate most cells contain 2 C (unreplicated diploid) amounts of DNA.
The absence of a 4 C category indicates that few nuclei in fish liver
exhibit premitotic activity. The data thus reveal that neither acute nor
subacute exposure stimulates mitotic activity or induces cell damage in
trout liver. It is noteworthy that profiles of F-DNA and FG-histone
histograms are strikingly similar indicating that in brook trout, as in
other vertebrate species, the DNA and histone levels parallel one another.
This suggests a close functional relationship between DNA and histones.
Cytophotometric analyses of RNA, (lysine + tyrosine) , and arginine in
liver nuclei of acid exposed trout. Since no detectable changes were
found in total F-DNA and FG-histone, studies were designed to clarify
the role of RNA and histones in liver nuclei of acid exposed trout.
Lysine-rich histone fractions are known to inhibit DNA-dependent RNA
synthesis. On the basis of this, it was postulated that if acid
70
-------
30
20
10
X =5.33+ .06
DNA
CONTROL
J1!
X = 6.27+ .05
HIS TONE
_ 30
o
20
o
tr
I0
5.35+.04
FIVE DAY EXPOSED
DNA
6.23+.06
HISTONE
L*., 1.4 ill
THIRTY DAY EXPOSED
5.30±.05 X = 6.25 + .07
DNA
10-
HISTONE
345678 456789
Relative Amount of DNA or Histone in Arbitrary Units (A.U.)
Figure 29. Frequency distribution profiles of Feulgen-DNA and fast
green histone stained liver nuclei of acid exposed trout.
exposure impairs or depresses liver metabolism, this should be reflected
in changes in levels of nuclear RNA, possible accompanied by changes in
the two major amino acid residues of histones, lysine and arginine. Cyto-
photometry of liver sections stained by the FDNB-azure B and FDNB-
sakaguchi reactions supported this postulate.
The RNA data and the (lysine + tyrosine)/arginine molar ratios are pre-
sented in Table 16 while the comparison of both sets of data is seen in
Figure 30. It is evident that both acute and subacute exposure of trout
results in a decrease in nuclear RNA with a concomitant increase in the
(lysine + tyrosine)/arginine molar ratio. Nuclear RNA decreases by 43
71
-------
Table 16. Cytophotoraetric measures of RNA, lysine + tyrosine and arginine in liver nuclei of acid
exposed brook trout*
Control Trout
Acid Exposed Trout
RNA (A.U
(lysine
(lysine
lysine +
arginine;
.)
+ tyrosine) /RjNA
+ tyrosine) /arginine
tyrosine (A. LJ . )
(A.U.)
2.3 ± .12
1.9 + .07
2.5 ± .08
2.3 ± .08
1.1 ± .03
(40)
(40)
(40)
(40)
(40)
5 day
1.3 + .08
2.7 ± .11
5.0 + .12
3.8 ± .12
0.8 + .03
28-30 day
(40)
(40)
(40)
(40)
(40)
1.2 ±
2.9 ±
4.1 ±
3.0 +
0.8 ±
.08
.12
.11
.11
.03
(40)
(40)
(40)
(40)
(40)
*number of nuclei measured are in parenthesis; (lysine + tyrosine)/RNA is dimensionless being equal
to the extinction at 400 nm over that at 560 nm; the (lysine + tyrosine)/arginine ratio is in moles;
A.U. arbitrary photometric units. All experimental values differ from controls at the one percent
level of confidence (using Students t test).
per cent while the (lysine + tyrosine)/arginine ratio doubles after acute
acid exposure. The nuclear RNA level remains depressed in liver tissue
of 28 d exposed trout and the (lysine + tyrosine)/arginine ratio doubles
after acute acid exposure. The nuclear RNA level remains depressed in
liver tissue of 28 d exposed trout and the (lysine + tyrosine)/arginine
ratio remains 64 percent above control levels. These findings indicate
that curtailment of RNA synthesis in liver nuclei is a prompt and
sustained response to acid exposure. Furthermore, the decrease in
nuclear RNA is accompanied by alterations in relative amounts of the two
major basic amino acid residues present in histones.
To determine what factors contribute to the observed increase in the
(lysine + tyrosine)/arginine molar ratios, measurements were then made
of double stained nuclei. This permitted direct comparison of the content
of (lysine + tyrosine) with that of arginine in a given nucleus. These
data also appear in Table 16 and Figure 30. It is evident that increased
(lysine + tyrosine)/arginine ratios in both experimental groups are due
to an increased (lysine + tyrosine) content and a concomitant decrease in
arginine content of liver nuclei.
In brief, the above data indicate that acid exposure of trout results in
decreased nuclear RNA levels with corresponding increased (lysine +
tyrosine) content and decreased arginine content in liver nuclei. Although
the (lysine + tyrosine) data do not reflect the nuclear lysine-rich
histone levels per se_, the fact that much of the nuclear lysine is a
component of histone and that tyrosine accounts for less than 10 per cent
of this fraction suggest that the increased (lysine + tyrosine) values are
largely due to increased lysine-rich histone levels.
72
-------
V)
2
13 2
cc
cc
t I
m
cc
RNA
en
UJ
control five day thirty day
4
5
s'~* O
co
I-
E
D 2
cc
cc
t 1
CD
CC
i^m« -T- ~
-
-
-
"i
^
/
X^
' \
*-,
w
,:
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-
-
£
-
2.0
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control five day thirty day
Figure 30. Effects of acid exposure on RNA, (lysine + tyrosine)/
arginine ratio, lysine + tyrosine, and arginine levels
in trout liver nuclei.
Cytochemical analyses of nuclear histones in liver nuclei of acid
exposed trout. Having established that changes in protein lysine and
arginine occur in liver nuclei during acid exposure, it was decided to
employ the eosin-fast green stain which, in many instances, has proven
useful for differentiating lysine rich and arginine rich nuclear histones.
Nuclei containing an abundance of lysine rich histones and/or exhibiting
a decrease in RNA synthesis often show a preferential affinity for
eosin. Increased eosinophilia, however, was not evidenced micro-
scopically in intact nuclei of acid exposed trout tissue despite a
subsequent finding that electrophoresed histone extracts from experi-
mental trout livers were more eosinophilic than controls.
Microscopic analysis of eosin-fast green stained liver tissue from acid
73
-------
exposed and control trout revealed three spectrally distinct cell 'types
which could be differentiated on the basis of their nuclear affinities
for eosin and fast green. Thus, in fish liver, as in mammalian species,
there are at least three histochemically distinct cell types. Both
acid exposed and control fish liver tissue exhibited an equal abundance
of both pink and green nuclei. Only a small number of nuclei stained
purple (Table 17).
Table 17. Relative numbers of pink, green and purple staining nuclei in acid exposed trout liver
(eosin-fast green stain)
Exposure
Controls
Acute (5 d)
Subacute (30 d)
n*
15
10
10
Mean %
Pink
Nuclei ± S.E.
31.8 ± 1.6
32.6 + 1.7
31.5 ± 1.4
Mean 7.
Green
Nuclei ± S.E.
60
60
61.
.7 + 1.9
.2 ± 1.4
.5 ± 2.0
Mean %
Purple
Nuclei ± S.E.
7.5 + 0.4
7.2 ± 0.5
7.0 + 1.1
*n refers to the number of trout; at least 100 nuclei were counted per liver section.
Supplemental measurements. Electrophoretic studies of liver histones
were conducted to determine if qualitative or quantitative changes occur
in various histone fractions during acid exposure. It was found that the
histones in control fish liver can be resolved into six major bands which
correspond in number and electrophoretic mobility to calf thymus histones
when examined with the gel system used. Visual inspection as well as
microdensitometer tracings of the gels reveals the absence of one arginine
rich band and a quantitative increase in a slower moving lysine rich band
in gels of both the acute exposed and subacute exposed trout (Figure 31).
It appears there is a relative increase in the lysine rich fraction at
the expense of the arginine rich fraction, with no marked change in
total histone. These results support cytophotometric data which indicated
that acid exposure resulted in increased lysine and decreased arginine.
All six electrophoretically separated histone fractions of control trout
liver stain well with either eosin or fast green when used independently;
however, all fractions stain somewhat more intensely after fast green
staining than after eosin staining. Eosin stained histone fractions
show no absorbance at 640 nm whereas the- absorbance of fast green stained
histone fractions at 500 nm is negligible. For this reason, the E61tQ(fast
green)/E5QQ(eosin) can be used as an index of preferential staining
affinity in gels. However, gels must be stained with eosin-fast green
in a formalin solution as bands diffuse from the gels in the absence of
fixative. Eosin-fast green stained histone fractions exhibit colorations
ranging from purple to violet depending on the content of each stain. No
74
-------
bands stain pure pink or green.
cn
LLJ
Q
o
I-
QL
O
l.0r CONTROL TROUT
AR,
.5
J
LR,
1.0
.5
AR,
FIVE DAY EXPOSED
J
1.0
.5
THIRTY DAY EXPOSED
LR,
Figure 31. Ilicrodensitometer tracings of electroplioresed historic
fractions from control and acid exposed trout liver.
Histone gels of acute acid exposed and control trout were stained with
eosin-fast green, scanned at 500 nm (eosin), rescanned at 640 nm (fast
green) and the E61f0/E500 ratios computed (Table 18). It was found
that the histone bands show a greater affinity for fast green than
75
-------
Table 18. Comparison of extinction ratios, Ee^o/^soo' °^ various
eosin-fast green stained histone fractions from trout
liver extracts.
Histone Fraction Control Acute acid exposed
Arginine rich, (AR,) 5.4 4.6
Slightly lysine rich; (SLR,) 6.3 5.4
Slightly lysine rich2 (SLR2) 8.9 5.2
Arginine rich2 (AR2) 4.2 absent
Lysine rich, (LR,) 5.7 7.3
Lysine rich2 (LR2) 7.0 5.5
*extinctions (E) were measured at 640 nm and 500 nm for fast green and
eosin, respectively; fractions obtained by polyacrilamide gel electro-
phoresis.
eosin; it was also found that certain histone bands are more eosinophilic
than other bands. Furthermore, although three resolvable histone fractions
(ARj, SLRj, SLR2) appear unaltered with respect to electrophoretic
ability, all three fractions exhibit a greater affinity for eosin in
acid exposed trout. These data support the hypothesis that some
eosinophilic component, detectable in both isolated histone fractions
and tissue sections, functionally inhibits RNA transcription.
Since previous studies involving mammalian liver have shown that increased
acetylation of histones accompanies increased RNA synthesis, another
exploratory study dealt with histone acetylation in the liver of acid
exposed brook trout. It was found that histone acetylation also occurs
in fish liver as in mammalian species; no changes were noted in the
extent or rate of histone acetylation in trout after acute exposure for
30 min and one hr.
An exploratory study employing radioactive acetate as a lipid precursor
was also conducted to shed light on lipid synthesis in liver during acute
acid exposure. Results showed that incorporation of 1'tC-acetate into
lipids occurs in livers of both experimental and control fish. A time
lag in release of lipids from the liver was noted in experimental fish.
Some differences were also noted in lipids synthesized and released by
the liver; control lipids were predominantly free fatty acids and
triglycerides while experimental lipids were mainly phospholipids and
to a lesser extent free fatty acids and triglycerides.
Since both lipid content and glycogen content are often used as parameters
of the functional state of liver, a number of liver tissue specimens,
from control and acid exposed fish were stained with Sudan Black B for
lipids and the PAS reaction for glycogen. Positive staining was obtained
76
-------
with each procedure. No visually detectable changes in Sudan Black B
staining were evidenced after either acute or subacute exposure. A
slight decrease in PAS staining was noted after acute acid exposure;
however, no differences between control and experimental fish were
discernible after subacute exposure.
Discussion. One contribution of this subproject is that it demonstrates
that acid exposure impairs liver function in brook trout and that the
site of impairment is in the nuclear machinery involved in regulation of
synthetic processes. Specifically, in acid exposed trout nuclear
regulatory mechanisms responsible for initiating and controlling protein
synthesis are inhibited or possibly impaired. This is reflected in a
decreased content of nuclear RNA which could eventuate in a decreased
potential for synthesis of cytoplasmic protein constituents by liver
tissue. Data obtained from electrophoretic analyses of nuclear histones
further suggested that the possible agent effecting the observed decrease
in nuclear RNA may be a lysine rich histone. Lysine rich histones are
known to be involved in suppression of DNA-dependent RNA synthesis
(Huang et al. 1964) and such histones were found to be increased in
liver nuclei of acid exposed trout.
An equally important aspect of this study concerns the physiological
significance of these types of liver alterations. There is no question
that any inhibition of RNA synthesis at the nuclear level would probably
result in a reduction in most, if not all, aspects of liver function
(i.e. oxidative metabolism of nutrients, inactivation of toxins, energy
stores, etc.). Under such circumstances one would anticipate a reduced
tolerance of trout to other toxicants or to environmental stresses
which would call for increased levels of liver metabolism.
Related findings in our laboratory support this interpretation. For
example, with both brook trout and longnose dace, we observed that levels
of acidity which are sublethal under laboratory conditions are toxic
under field situations where acid water contains appreciable concentra-
tions of metal ion toxicants. It was also found that levels of acidity
which nonbreeding trout tolerate under laboratory conditions prove to be
very toxic to reproductively active trout which presumably have much
higher energy requirements (Section IV).
In another respect, however, a reduction in liver metabolism could
temporarily increase tolerance of fish to hypoxemia during the acute
phase of acid exposure. Any reduction in oxidative processes such as
those involved in hepatic regulation of carbohydrate, protein and fat
metabolism would reduce overall bodily needs for oxygen at a critical
time when trout suffer from oxygen lack stemming from acid induced gill
damage.
The fact that the decreased RNA values after subacute exposure are not
significantly different from those obtained after acute exposure indicate
that the decrease in RNA occurs early and is a sustained response to
77
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acid exposure. Since nuclear RNA content is considered a reflection of
cytoplasmic protein synthesis, it would follow that a curtailment of
protein synthesis is among the first manifestations of liver dysfunction
following acid exposure. A reduction in nuclear regulatory aspects of
protein synthesis in liver cells would not necessarily result in reduced
synthesis of cytoplasmic structural proteins since the cells are not
damaged but metabolically depressed. Thus, the fact that the histological
appearance of liver cells is the same in acid exposed and control trout
does not signify these cells have comparable levels of metabolic activity.
The absence of any evidence of nuclear or cytoplasmic pathology simply
indicates that there is no cell deterioration associated with either
acute or subacute exposure to acidified water.
Quantitative analyses of Feulgen DNA (F-DNA) and fast green histone
(FG-histone) content confirmed that acid exposure of trout does not cause
any nuclear damage (DNA loss) nor change the rate of mitotic activity in
liver cells. Comparison of the F-DNA or FG-histone in each fish group
reveal similar profiles which probably reflects the existence of a close
functional relationship between DNA and histones in fish liver. Bloch and
Godman (1955) were among the first to demonstrate that an increase in
F-DNA occurs coincidentally with an increase in FG-histone in mitotic
cells of mammalian liver. They concluded that the synthesis of DNA and
histones proportionally parallel each other.
Quantitative analyses of F-DNA were initiated to investigate the function-
al relationship between DNA and RNA. On the basis of earlier work
performed in this laboratory (Pearson, 1970) it was anticipated that a
negative shift of small magnitude might be seen in F-DNA corresponding to
the decrease in nuclear RNA noted in experimental fish. Such a shift
would represent a qualitative rather than a quantitative change in DNA
and would reflect the inaccessibility of some portion of DNA for Feulgen
dye binding due to compaction or coiling of that portion of the DNA.
This rationale stems from a number of recent studies showing that the
synthesis of RNA is directly proportional to the availability of sites on
DNA. Of importance is the fact that the Feulgen stain in many instances
seems to bind somewhat proportionally, but not exclusively, to the same
sites which are available for templating RNA. It is generally agreed
that only 5 per cent of the genome serves as template for RNA synthesis.
The anticipated changes in F-DNA values, due to the unavailability of
binding sites, would thus be small. In view of these considerations, it
is not too surprising to find that Feulgen dye binding was not altered in
acid exposed trout liver tissue.
Although no change in total histone content was noted, quantitative histo-
chemical changes in nuclear (lysine + tyrosine) and arginine were found
associated with decreased nuclear RNA levels in experimental fish. It
was shown by electrophoretic studies of acid-extracted histones that these
changes could be attributed to an increase in lysine rich and a decrease
in arginine rich histones. It should be further noted that the increase
in lysine rich histone occurs at the expense of the arginine rich histone
78
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fraction which probably explains why the amount of total histone (i.e.,
FG-histone) remains unaltered. These data lend indirect support to the
contention that lysine rich histones are effective inhibitors of DNA-
dependent RNA synthesis (Huang et. al., 1964).
The utility of eosin-fast green staining technique for evaluating func-
tional metabolic changes in the rat liver was established in a previous
study (Crissman, 1968). It was found that liver nuclei bordering the
portal vein exhibit an increase in eosinophilia following exposure of
rats to oxygen lack. Failure to demonstrate this in liver cells of
acid exposed trout might be due to the fact that fish liver is not
organized into small lobules as are found in mammalian liver where
certain cells are much less oxygenated than others.
Staining of histone fractions in polyacrylamide gels with eosin-fast
green may prove to be a useful application of the staining method.
Histone fractions varied in their affinities for eosin and fast green
as reflected by the E61)0/E5QO ratios obtained by spectrophotometric
analyses. Claypool and Bloch (1967) have shown that with a decreased
RNA synthesis there is a decreased E6it0/E500 ratio in nuclei stained
with eosin-fast green. In the present study the E /E500 ratio was
lower in all three histone fractions of experimental fish when compared
to controls although no other observable differences were noted.
Further work would be needed, however, to establish that the acquisition
of eosin in histone fractions, as reflected in a decreased E6tt0/E500
ratio, could be used as an index of depressed RNA synthesis.
At present, there is good evidence that acetylation of histones repre-
sents one of the earliest chemical events in the process of gene acti-
vation for RNA synthesis. This is based mainly on studies of rat liver
preparations. The role of histone acetylation in fish liver is not
known; however, the data in this study show that histone acetylation
probably occurs in fish liver in much the same manner as in rat liver.
Acetate is rapidly incorporated into histones at a constant rate over a
one hr interval. No differences in the acetylation patterns were noted
between experimental and control fish indicating that histone acetyl-
ation in trout liver is not altered during the first hr of acute acid
exposure.
Data obtained on lipid synthesis demonstrate that acetate is rapidly
incorporated into fish liver lipids. Both control and experimental fish
were found to synthesize and release lipids such as fatty acids and tri-
glycerides which are generally used as energy sources. These exploratory
tests merely indicate that the liver is not totally incapacitated with
respect to lipid synthesis during early periods of acid exposure.
Supplemental histochemical analyses of glycogen and lipid content in
fish liver again attest to the value of histochemical methodology in
79
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detecting metabolic alterations. These studies show that energy stores
in the form of glycogen and lipids are not markedly depleted in liver
during acid exposure. This probably reflects an overall reduction in
utilization of energy by bodily tissues. Again this provides evidence
that liver cells are not damaged but simply metabolically inhibited
during acid exposure.
Summary. Three groups of hatchery-reared "brook trout (Salvelinus
fontinalis) were exposed to different levels of acidity over various
time periods. One group of trout was exposed to near toxic levels of
acidity for 5 d where pH was maintained at 4.0 for 4 d and lowered to
3.5 on day five (acute exposure). The second group of trout was
continuously exposed to sublethal levels of acidity (pH 4.7) for 28-30 d
(subacute exposure). The final group of hatchery trout was exposed to
acidity under field conditions (pH 4.8 for 24-48 hr). Control groups
were maintained in each case.
A portion of liver from each fish was fixed in neutral formalin, paraffin
embedded, sectioned at 6 microns and stained by the Feulgen or alkaline
fast green method for DNA and histone analyses. Some tissue sections
were double-stained for (lysine + tyrosine) and for arginine, by the
fluorodinitrobenzene (FDNB) and the Sakaguchi reactions,. Other tissues
were double stained for (lysine + tyrosine) and for RNA by the FDNB and
the azure B methods. Sections stained with azure B were pretreated with
deoxyribonuclease. Eosin-fast green staining for differentiating nuclear
histone types was also employed.
Cytophotometric analyses were performed on liver parenchymal nuclei from
each stained group. These studies indicate that no quantitative change
occurs in total Feulgen-DNA or fast green-histone after acid exposure.
However, analyses of nuclear REA and two amino acid residues (lysine and
arginine) of histones revealed alterations in these components after both
acute and subacute exposure of trout. The data showed an increase in
the nuclear (lysine + tyrosine)/arginine molar ratio associated with a
decrease in nuclear RNA. Since lysine rich histones are effective
inhibitors of DNA dependent RNA synthesis, one would suspect a decrease
in RNA synthesis concomitant with an increase (lysine + tyrosine)
content. This proved to be the case.
Data obtained from electrophoresis of histones extracts substantiate
the histochemical data. These studies show a quantitative increase in
lysine rich histone with a proportional decrease in arginine rich histone
in acid exposed trout.
Spectrophotometric analyses of eosin-fast green stained nuclei reveal a
peak at 620 nm in eosinophilic liver nuclei of control and experimental
fish which is not observed in eosinophilic rat liver nuclei. It was
speculated that the 620 nm peak reflects some differences in molecular
configuration of fish liver histone as compared to rat liver. No signifi-
cant differences were noted with respect to the number of eosinophilic
80
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nuclei in acid exposed trout or control trout.
Supplemental information on liver lipid and glycogen levels was obtained
following semiquantitative histochemical staining by the Sudan Black B
or the periodic acid-Schiff method, respectively. These studies reveal
that the levels of these metabolites in liver are not markedly changed
after acid exposure. Supplemental data obtained from ^C-acetate
tracer studies showed no alterations in the pattern of histone acetylation
or lipid synthesis during the first hr of acute acid exposure. However,
insufficient data were obtained to draw conclusions regarding rates of
utilization of these liver metabolites during acid exposure. The data
did, however, reveal that acid exposure of trout does not totally
incapacitate metabolic mechanisms in liver.
It was suggested that decreased liver function may be a contributing
factor responsible for the reduced tolerance of trout to acid polluted
streams.
A schematic summary which related acid induced liver changes to other
tissue responses described in previous subsections III, IV and Vi is
shown in Figure 32. Included in this figure are some of the histo-
chemical tests which can serve as indices of acid stress.
81
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GILL MUCIFICATION
• colloidal iron
• PAS
PRIMARY
EFFECTS
- IMPAIRED EXCRETION,CIRCULATORY FLOW
HYPOXEMIA
Oxidative
meto bolism
depressed in all
tissues
SECONDARY TARGETS
KIDNEY 8 SPLEEN
STANNIUS CORPUSCLE
ERYTHROPOIETIC
TISSUE
- LEUCOPOIETIC
TISSUE
NATURE OF RESPONSE
• histochemicol indices
increased secretion / water
and ion regulation altered
• PAS
mobilization of RBC/increased
hematocrit
• F-DNA + azure B-RNA
non specific stress reaction,
neutrophilia 8 lymphopenia
• Giemsa
LIVER
NUCLEI OF
PARENCHYMAL CELLS
=>
— impaired metabolism/reduced
tolerance to toxicants
• ozure B-RNA
SUMMARY OF ACID INDUCED HISTOCHEMICAL RESPONSES
Figure 12 . SL he FT. j tic outline of hemal, erythrocytic and hepatic responses
in fish exposed Co acid polluted water
82
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SECTION VII
ACKNOWLEDGMENTS
A large number of faculty and students in the Department of Biology
have contributed to this project. The grant director and author
of this report was Dr. Adam Anthony, Professor of Zoology and Chair-
man of the Physiology Program. Project coinvestigators were
Drs. E. L. Cooper, C. D. Therrien, R. B. Mitchell and W. H. Neff.
This work was carried out in the Physiology and Histochemistry
Laboratories, Department of Biology, The Pennsylvania State University,
University Park, Pennsylvania 16802.
The research was supported in part by Federal Water Pollution Control
Administration grant USDI 18050 DXJ and also The Pennsylvania Agricul-
tural Experiment Station.
We wish to acknowledge the contributions of Dr. Robert L. Butler of
the Pennsylvania Cooperative Fisheries Unit and Dr. Arthur Bradford,
Chief Fisheries Division of the Pennsylvania Fish Commission in the
procurement of fish and apparatus needed for maintenance of fish in
the laboratory. We also wish to thank Dr. Quenton Pickering and
Dr. William A. Brungs of the Newtown Laboratory at Cincinnati, Ohio
for helping us with setting up the gravity flow diluter system used
in our work.
Particular appreciation is extended to Dr. James M. McKim III of FWPCA
National Water Quality Laboratory in Duluth, Minnesota, who acted as
Project Officer of this grant and arranged for the shipment of fish
tissue specimens from research projects dealing with chronic exposures
to toxicants at the Duluth and Newtown FWPCA laboratories.
83
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SECTION VIII
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of desoxyribose nucleic acid (DNA) in cells of the same tissue and
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93-100.
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blood of brook trout (Salvelinus fontinalis) after short-term
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1883-1889.
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87
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Mowry, R. W. 1963. The special value of methods that color both
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Jerusalem.
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Schiffman, R. A. and P. 0. Froram. 1959. Chromium-induced changes in
the blood of rainbow trout, Salmo gairdneri. Sewage Ind. Wastes.
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light. In_ G. Oster and A. W. Pollister (eds.), Physical techniques
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ribonucleic acid and basic protein in the plasmodial slime mold
Physarum pusillum. Mycologia 59:757-766.
Vaala, S. S. 1970. Blood p02 changes in acid exposed brook trout.
Pa. Acad. Sci. 44:in press.
Vaala, S. S. 1971. Erythrocytic indices of stress in brook trout
exposed to sub lethal levels of acidity. Ph.D. Thesis, The
Pennsylvania State University. 65pp.
Vaala, S. S. and R. B. Mitchell. 1970. Blood p02 changes in acid
exposed brook trout. Pa. Acad. Sci. 44: in press.
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(Hematology of agriculture animals). Moskva, Sel'khozgiz.
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to the fate of displaced histones following histone transition
in rat spermiogenesis. J. Cell. Biol. 31:247-278.
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contaminants., Bull. World Health Organ. 36:181-207.
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the rainbow trout. Salmo_ gairdneri irideus. I. Hematology:
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the rainbow, trout , Salmo gairdneri irideus. II. Effects of
induced inflammation and cortisone treatment on the digestive
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Weinreb, E. L. 1963. Studies on the fine structure of teleost blood
cells. I. Peripheral blood. Anat. Rec. 147:219-238.
Weinreb, E. L. and S. Weinreb. 1969. A study of experimentally
induced endocytosis in a teleost. I. Light microscopy of peripheral
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26:283-287.
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cytochemistry. Vol. II, Academic Press, New York. 551 pp.
90
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SECTION IX
PUBLICATIONS
Master of Science Theses
Plonka, A. C. 1969. Mucopolysaccharide histochemistry of gill epithelial
secretions in brook trout (Salvelinus fontinalis) exposed to acidic
conditions, The Pennsylvania State University. 30p.
Weyandt, T. B. 1970. Gas chromatographic analysis of muscle and liver
lipids of brook, brown and rainbow trout, The Pennsylvania State
University. 41p.
Dively, J. L. 1970. Gas chromatographic analysis of muscle lipids in
normal and acid exposed brook trout, The Pennsylvania State
University. 31p.
Trusal, L. R. 1970. Feulgen-DNA cytophotometry of adipose cells,
erythrocytes, fibroblasts and spermatogenic cells of brook trout,
* The Pennsylvania State University. 50p.
Mudge, J. E. 1970. Histology and DNA cytophotometry of the pancreas
in acid exposed brook trout, The Pennsylvania State University.
48p.
Dolsky, M. F. 1971. Hemal changes in acid exposed fathead minnows
and longnose dace, The Pennsylvania State University.
Ph.D. Dissertations
Plonka, A. C. 1971. Histochemical and histological analyses of gills
and Stannius corpuscles in acid exposed brook trout, The
Pennsylvania State University. 68p.
Vaala, S. S. 1971. Erythrocytic indices of stress in brook trout
exposed to sublethal levels of acidity, The Pennsylvania State
University. 92p.
Crissman, H. A. 1971. Cytochemistry of DNA, RNA and histones in liver
nuclei of acid exposed brook trout, The Pennsylvania State
University. 73p.
91
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Papers
Crissman, H. A. and C. D. Therrien. 1969. Cytochemical analysis of
liver histones in acid exposed brook trout, Salvelinus fontinalis.
Proc. Pa. Acad. Sci. 43:49-52.
Plonka, A. C. and W. H. Neff. 1969. Mucopolysaccharide histochemistry
of gill epithelial secretions in brook trout exposed to acid pH.
Proc. Pa. Acad. Sci. 43:53-55.
Vaala, S. S., R. B. Mitchell .and A. Anthony. 1969. Cytophotometric
studies of DNA in circulating erythrocytes of brook trout exposed
to acid pH. Proc. Pa. Acad. Sci. 43:191-194.
Crissman, H. A. and C. D. Therrien. 1970. Cytochemical and biochemical
analyses of DNA and histones in liver parenchymal nuclei of acid
exposed brook trout. Proc. Pa. Acad. Sci. 44:
Mudge, J. E. and W. H. Neff. 1970. Microscopic anatomy of the pancreas
of the brook trout. Proc. Pa. Acad. Sci. 44:
Vaala, S. S. and R. B. Mitchell. 1970. Blood PQ changes in acid
exposed brook trout. Proc. Pa. Acad. Sci. 44:
Neff, W. H., L. R. Trusal and A. Anthony. 1970. Feulgen-DNA cyto-
photometry of erythrocytes, fibroblasts and adipose cells of
brook trout. Anat. Rec. 166(2):354.
Plonka, A. C. R. B. Mitchell and A. Anthony. 1971. Quantitative
cytophotometry of PAS positive material in Stannius corpuscles of
brook trout exposed to acidic water. Proc. Pa. Acad. Sci. 45:
in press.
Dively, J. L. , G. Porter and A. Anthony. 1971. Fatty acid composition
of muscle lipids of normal and acid exposed brook trout. Proc.
Pa. Acad. Sci. 45: in.press.
Vaala, S. S., R. B. Mitchell and A. Anthony. 1971. Erythrocytic
changes associated with subacute acid exposure of brook trout.
Proc. Pa. Acad. Sci. 45: in press.
92
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SECTION X
GLOSSARY
acidophilia - cytoplasmic affinity for acid dyes.
AU - arbitrary units. The total amount of absorbing material in a
nucleus or cell is generally expressed in arbitrary photometric
units and is a function of the measured optical density (extinction),
the specimen area and the extinction coefficient of the dye used.
azure B-RNA - complex of ribonucleic acid stained with azure B; nuclear
and cytoplasmic RNA can be measured cytophotometrically following
azure B staining provided DNA is first enzymatically removed
using DNAase.
basophilia - nuclear and cytoplasmic affinity for basic dyes. The
substance largely responsible for basophilic;staining in the
cytoplasm is ribonucleoprotein.
compact or condensed chromatin - refers to those segments of chromosomes
that are tightly coiled in the interphase nucleus.;' are visible
as chromatin particles with light microscopy; also referred to as
heterochromatin although this term has cdrtain connotations to
cytogeneticists which make use .of the prefixes hetero- and eu-
less suitable for describing chromatin stainability or its
appearance.
dispersed chromatin - refers to chromatin of th'ose segments of chromo-
somes that are extended and -with light microscopy are invisible
in the interphase nucleus; also referred to as diffuse chromatin
or euchromatin by histochemists.
DNAase - desoxyribonuclease, a purified enzyme available commercially
and used for preparing control slides in F-DNA analyses and in
cytophotometric analysis of RNA.
F-DNA - Feulgen stained desoxyribonucleic acid complex. The Feulgen
reaction utilizes the Schiff reaction for aldehydes.but involves
the use of mild hydrolysis (under standard conditions). This
liberates aldehydes from the D-2-deoxyribose of DNA but not those
from the ribose of RNA nor any other sugars.
H and E - hematoxylin (a basic dye) plus eosin (an acid dye) staining
represents the most common combination of stains used for routine
microscopic analyses of tissues.
93
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hemopoiesis - formation of white blood cell or leucoid elements
(leucopoiesis) and red blood cell or erythroid elements
(erythropoiesis). In teleosts, erythropoiesis occurs primarily
in renal hemopoietic tissue and leucopoiesis in the spleen
although both organs manufacture WBC and RBC.
histones - relatively simple basic proteins. These are the most
abundant and most important proteins in chromosomes. Histone
levels show a quantitative correlation with DNA content in many
cell types.
microspectrophotometry - application of spectroscopic methods for the
quantitative analysis of chemical constituents in tissue cells.
Synonyms used include: cytophotometry, microphotometry and
analytical microscopic histochemistry.
PAS - the periodic acid Schiff method is a two step histochemical
procedure for mucopolysaccharides, first utilizing periodic acid
to release aldehydes from glucose residues of carbohydrates and
then treating sections with Schiff reagent (leucofuchsin) which
reacts with the aldehydes to form a purple dye (basic fuchsin).
RNAase - ribonuclease, a purified enzyme available commercially and
used for preparing control slides in RNA analyses.
S-G2 stage - the DNA duplication or synthesis (S) stage and post-
duplication (62) stage of interphase represent the intervals of
the mitotic cycle where one would expect nuclei to contain inter-
class (3C) and tetraploid (4C) contents of F-DNA.
94
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SECTION XI
PERSONNEL
Staffing: Most of the work described was performed by graduate students
supervised by the following staff members in the Department of Biology.
Dr. Adam Anthony, Professor of Zoology and Principle Investigator;
Dr. E. L. Cooper, Professor of Zoology, Coinvestigator; Dr. C. D. Therrien,
Associate Professor of Biology, Coinvestigator; Dr. R. B. Mitchell,
Assistant Professor of Biology and Dr. W. H. Neff, Assistant Professor
of Biology. Mrs. Athleen J. Stere and Charles Wagner were employed on
a half time basis as Research Assistant and Fisheries Research Technician,
respectively. Brief biographical sketches of graduate students, most
of whom held traineeships which permitted them to participate in this
research as part of their graduate training are given below with their
interval of association with this project in parenthesis.
Harry A. Crissman, M.S., 1968, Ph.D., 1971, The Pennsylvania State
University. He held an NDEA fellowship from 9-68 to 8-71. Currently
has accepted a research position at the Los Alamos Radiation Laboratory,
New Mexico effective August, 1971. (3-68 to 5-71).
James L. Dively, M.S. 1970 in Physiology, The Pennsylvania State University.
Currently holds an NDEA fellowship and is a doctoral candidate in
Physiology. (3-68 to 5-71).
Mary F. Dolsky, M.S. 1971 in Physiology, The Pennsylvania State University.
Was awarded an NSF traineeship which she has resigned to enable her to
accept a FWPCA research fellowship effective September, 1971. (3-69 to
5-71).
James E. Mudge, M.Ed, 1969; M.S...1970, The Pennsylvania State University.
Currently a doctoral candidate in Physiology and holds a teaching
assistantship in biology. (10-69 to 5-71).
Andria C. Plonka, M.S., 1969; Ph.D. 1971, The 'Pennsylvania State
University (3-68 to 3-71).
Lynn R. Trusal, M.Ed., 197o', M.S., 1970, The Pennsylvania State
University. Was admitted as doctoral candidate in Physiology and was
drafted. Currently a 2nd lieutenant teaching physiology at the Medical
Field Service School, Fort Sam Houston, Texas. (9-68 to 8-70).
Sharon S. Vaala. M.S. 1968, Ph.D. 1971, The Pennsylvania State
University. Held an NSF traineeship 1967^-1971. Currently has accepted
a position,in the Department of Anatomy, University of Rochester
Medical School, Rochester, New York. (3-68 to 5-71).
95
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Timothy B. Weyandt, M.S. 1970, The Pennsylvania State University. Held
a NASA traineeship 1969-1970. Admitted as doctoral candidate and drafted
Currently teaching in Medical Field Service School, Fort Sam Houston,
Texas. (9-68 to 9-70).
96
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SECTION XII
APPENDIX
A. Microspectrophotometric Procedures 99
Figure 1. Single Beam Microspectrophotometers Used for
Analytical His tochemical Analyses 98
Figure 2. Two Beam Recording Microspectrophotometer Used for
Validation of Analytical Procedures 100
Figure 3. Typical Hydrolysis Curve of Feulgen Stained
Erythrocytes (used to establish optimal HC1
hydrolysis time prior to staining) 102
Figure 4. Typical Absorption Spectrum of Feulgen Stained
Erythrocytes (used for predetermining appropriate
wavelengths to correct for nonhomogeneous distri-
bution of dye complex in measured specimen area) . 102
Figure 5. Fortran Program Used for Computing Feulgen-DNA
Content by the Two Wavelength Method of
Microspectrophotometry 103
Figure 6. Typical Absorption Spectra of Fish Liver Nuclei
Stained by the FDNB-Sakaguchi and FDNB-azure B
Methods 104
Figure 7. Typical Absorption Spectrum of Alkaline Fast Green
Stained Erythrocytes 105
B. Data Relating to Water Quality Measures and Exploratory
Biochemical Tests 106
Table 1. Ion Analysis of Water Used in the Gravity Flow
Diluter Systems 107
Table 2. Ion Analysis of Streams Used in Acute Acid
Exposure Field Studies 107
Table 3. Water Quality in Black Moshannon Creek 29 April
1970 and During Trial Run Period 108
Table 4. Water Quality in Upper Three Runs 29 April 1970
and During Trial Run Period 108
Figure 8. Map of Streams Used in Field Studies 109
Figure 9. Hemoglobin Microdensitometric Tracings (scan speed -
1 cm/min, 540 my) of Polyacrylamide Gels from
Nonbreeding, Acute Acid Exposed Trout 110
Figure 10. Total Blood Histone Microdensitometric Tracings
(scan speed - 1 cm/min, 660 my) from Polyacrylamide
Gels from Nonbreeding, Acid Exposed Trout Ill
Figure 11. Total Blood Histone Microdensitometric Tracings
(scan speed - 1 cm/min, 660 my) of Polyacrylamide
Gels from Nonbreeding, Acid Exposed Trout 112
Figure 12. Mount-Brungs type gravity flow diluter system . . . 113
97
-------
Figure 1. Single Beam Microspectrophotometers used for Analytical
Histochemical Analyses.
98
-------
APPENDIX
A. MICROSPECTROPHOTOMETRIC PROCEDURES
Cytophotometric measurements were made with two specially constructed
microspectrophotometers (Figure 1), each consisting of the following
basic components: (1) Photovolt power supply and meter; (2) Leitz
or Zeiss monochromator and tungsten light source; (3) Leitz Ortholux
microscope with 20x, 0.4 N.A. quartz objective used as a condenser and
95x, 1.32 N.A. objective lens; and (5) photodetector RCA 1P21 photo-
multiplier. Validation checks of cytophotometric methodology were
also performed using a custom built, two beam, UV-visible recording
microspectrophotometer (Figure 2).
Relative amounts of nuclear DMA in various cell types were determined
using the two wavelength method of microspectrophotometry. The two
wavelength method was introduced simultaneously by Ornstein (1952)
and Patau (1952) as an application of the Beer-Lambert Law. The
procedure corrects for distributional error for a wide variety of
chromophore distributions and therefore is useful when the material
being measured is irregular or heterogeneous.
Objects in a photometric field are measured at two wavelengths pre-
determined 'to give an extinction ratio of 2:1 for the chromophore under
investigation. Appropriate mathematical manipulation of transmittance
divides the field into absorbing molecules and nonabsorbing components.
An estimate of absorbing molecules depends upon a linear relation between
extinction and concentration of dye molecules. The procedure involves
selection of two wavelengths (Xx and X2) from an absorption curve of a
homogeneous area such that the extinction at one wavelength (E^ equals
half the extinction (E2) at the other to give 2 Ej = E2. Under these
conditions, El = log IO/IS at X1, and E2 = log I0/IS at X2. Io represents
intensity of background or incident light, while Is is the intensity of
the incident beam after being transmitted through a photometric field
containing the specimen. For Feulgen-DNA cytophotometry, one has to
first standardize the hydrolysis time which can vary depending upon the
tissue, fixation or type of tissue preparation (i.e. smear, imprint or
section). A typical hydrolysis curve is shown in Figure 3. A repre-
sentative Feulgen absorption curve for stained red blood cells is shown
in Figure 4.
From measures of Io and Is at both wavelengths, transmissions TI and T2
are determined and the following calculations performed.
T = IS/I0 at X, T2 = IS/IQ at X2
= L2/L1 C = 1/2 - Q In 1/Q - 1
99
-------
o
o
Two Beam Recording Microspectrophotometer used
for Validation of Analytical Procedures.
-------
The functions with Q as the parameter have been previously tabulated
in the literature by Patau (1952), and the correction factor, C, is
listed for corresponding values of Q.
M = KALjC A = r2 for relative measurements.
K = a constant (1/e) where e is the
extinction coefficient at \1 and
may be disregarded for relative
measurements.
A close approximation of the amount of absorbing molecules within a
measured area is given by the value of M, which is presented in arbitrary
units (A.U.).
A source of difficulty in the method stems from the involved series of
entries and calculations required for each object measured. This repre-
sents a potential source of human error and makes the method time
consuming and tedious (Mendelsohn, 1966). A Fortran 4 program (Figure 5)
was therefore prepared and all calculations computed by an IBM 360-67
computer. Data cards were prepared and supplied with five values in the
following order: the aperture diameter of upper viewing tube (circular
aperture diameter selected to enclose the image of the object to be
measured with a minimum amount of surrounding clear area); photometer
readings of Is at A and X: and photometer readings of IQ at A2 and X1.
The program is designed to calculate and print out values of Tl, T2,
Lj , L2, Q, C, and M. Final M values, which are the absorbing molecule
concentrations in individual nuclei are printed out in order of decreasing
magnitude such that the range of values may be easily subdivided and
presented in histogram form.
The standard plug method (Swift, 1950) was also employed for cytophoto-
metric analyses when using double staining techniques. Again, two
wavelengths must be selected, each representing the maximum extinction
for the dye complex under study. After defining a photometric field
within each of several homogeneous areas of the specimen, the two wave-
lengths are selected and extinctions determined as previously described.
Representative absorption curves for trout liver nuclei stained by the
FDNB/Sakaguchi and FDNB-azure B methods are shown in Figure 6. As is
evident, the shapes of spectral curves obtained are quite different from
those obtained using a single stain such as alkaline fast green (Figure 7)
Once the proper wavelengths are selected, the establishment of a
standard optical plug through a cellular structure is required. In an
effort to minimize possible sources of error, measurements at both pre-
determined wavelengths were made without moving the optical plug. The
background was then recorded for each wavelength. To measure the
total nuclear (lysine + tyrosine) and arginine, it is first necessary
to determine the extinction of an optical plug which passes through
the center of each nucleus. The number of dye-absorbing molecules in
the plug is equivalent to KEA, where K is a constant and may be
101
-------
Q
i
C
01
00
01
of.
10 20 30
180
.5
.4
60 90 120
Hydrolysis Time (Minutes)
(A.U. - Arbitrary Units)
Figure 3. 5 N HC1 hydroloysis curve of Feulgen stained erythrocytes
.6 r-
.3
Q
ID
D.
O
. 1
460 500 550 600
Wavelength (mu)
650
Figure -» . Absorption spectrum of Feulgen stained erythrocytes.
102
-------
FSU-WATFOR *** 5-1
1 IMPLICIT REAL *8{A-I,L.M.Q-Z), INTEGER * 4(P)
2 LOGICAL * 1 TITLE (80)
3 DIMENSION A(100Q)
4 CALL TRAPS (0,1,100,1,1)
5 K = 0
6 READ 5, TITLE
7 5 FORMAT (80A1)
8 PRINT 6, TITLE
9 6 FORMATC1',T25,80A1/'0',T6,'D',T11,'ISAMPB',T19,'ISAMPA1 ,
T27, 'ISOL1VB',T35,'ISOLVA',T45,'TRANSB',T55,'TRANSA',
T68,'LB',178,'LA1,188,'2Q',198,'C',T108,'M1)
10 9 READ(5,8, END 20) D, ISAMPB,ISAMPA,ISOLVB,ISOLVA
11 8 FORMAT (5F4.1)
12 K = K+l
13 A(K) = O.ODO
14 TRANSA - ISAMPA/ISOLVA
15 TRANSB = ISAMPB/ISOLVB
16 LA l.ODO TRANSA
17 LB = l.ODO
18 IF(LA.GT.O) GO TO 19
19 WRITE(6,69)D,ISAMPB,ISAMPA,ISOLVB,ISOLVA
20 69 FORMAT('0',5F8.1,T45.' LA IS LESS THAN OR EQUAL TO ZERO)
21 GO TO 9
22 19 Q = LB/LA
23 IF(Q.G.E.2.0DO) GO TO 11
24 IF (Q.T.I.ODD) GO TO 10
25 11 WRITE(6,12) D,ISAMPB,ISAMPA,ISOLVB,ISOLVA.Q
26 12 FORMAT('0',5F8.1,T45, 'Q INCORRECT Q ',D15.8)
27 GO TO 9
28 10 C (1.0DO/(2.0DO-Q)*DLOG(1.0DO/(Q-1.0DO)
29 M = C*LA*(D/a.ODO)**2
30 A(K) = M
31 WRITE(6,13)D,ISAMPB,ISAMPA,ISOLVB,ISOLVA,TRANSB,TRANSA,
LB,LA,Q,C,1M
32 13 FORMAT('0',5F8.1, 7F10.3)
33 GO TO 9
34 20 N = K-l
35. DO 17 J 1,N
36 PJ - J-l
37 DO 17 P = PJ,K
38 IF(A(J).GT.A(P)) GO TO 17
39 TEMP _ A(J)
40 A(J) - A(P)
41 A(P) -- TEMP
42 17 CONTINUE
43 WRITE(6,22)
44 22 FORMAT(T ,T25, 'IN DECREASING ORDER THE M VALUES ARE')
45 WRITE(6,18) (A(J), J 1,K)
46 18 FORMAT('0',10F10.3)
47 STOP
48 END
Figure 5. Fortran program for Feulgen-DNA calculations using two
wavelength method of microspectrophotometry
103
-------
.5r
.3
_
< .2
o
400 450 500 550
WAVELENGTH (nm)
600
.5
-4
-3
o
Q.
O
FDNB
-AZURE
B
J_
J_
_L
JL
_L
J
400 450 500 550 600 650 700
WAVELENGTH (nm)
Fi gure b.
Spectral absorption curves of FDNB-azure B and
FOKB-Sakaguchi complexes in trout liver nuclei.
104
-------
.7
.6
.5
-------
disregarded when making relative measurements, E is the extinction
determined at the selected wavelength, and A is the area of the plug.
Since the area of the plug is TTR^, where R is the radius of the plug,
the plug = KErrR . The cylinder obviously contains only a fraction of
the total volume of the nucleus; therefore, to obtain a value for the
total nuclear volume, the value obtained for the plug must be multi-
plied by the ratio of nuclear volume over the plug volume. In using
the plug method, particular attention should be paid to the distri-
bution of the chromophore under study, since inhomogeneous distribu-
tion may lead to serious errors in the measurements. Care should be
taken to find entire nuclei in the section, since in tissue, sections
are cut at different planes. Care must also be taken that the diameter
of the plug is small enough so that the rounded surface of the sphere
may be considered to be flat. Usually an R is used which is not more
than one-third the diameter of the nucleus. The final calculation for
the chromophore under study is:
chromophore .. = KEA
plug
„ volume nucleus
chromophore . = choromphore n X —^ =
nucleus plug volume plug
If the nucleus is a sphere, as in a tissue section, then:
3
chromophore . = KEirR X —=
nucleus R2^
= 2/3KEfrr2
where r is the radius of the nucleus.
DATA RELATING TO WATER QUALITY MEASURES
AND EXPLORATORY BIOCHEMICAL TESTS
Four tables are included as examples of water analyses run on laboratory
aquaria water and field streams (Tables 1, 2, 3 and 4). Figure 8 is a
map of Pennsylvania giving locations of stream sites where field
investigations were conducted.
Hemoglobin was extracted for 16 acute acid exposed and 16 control trout,
and five fractions separated by polyacrilamide electrophoresis. Typical
microdensitometer tracings following staining of gels with benzidine
dihydrochloride are shown in figure 9. No differences were found
between experimental and control blood samples.
Blood histones were extracted, electrophoresed, stained with 0.5 percent
amido black for 18 hrs, destained by diffusion in 7 percent acetic acid
and scanned using a Gilford densitometer. Figure 10 illustrates typical
scans from acid exposed and control trout when too high a concentration
106
-------
Table 1. Ion analysis of water used In the gravity flow diluter system.
pH
4.85
5.86
6.36
6.30
7-02
3.00
7.80
H2°d
H2°d
Tank
1
2
3
4
5
6
7
1
2
3
4
5
6
7
P
1.1
1.3
1.1
0.7
1.2
0.9
0.3
0.9
1.6
3.2
1.5
1.7
1.1
0.7
1.6
K
3.7
5.4
6.2
6.5
7.7
7.3
4.0
8.5
2.5
2.7
9.6
8.7
6.2
6.3
7.3
Ca
10.5
5.2
6.7
4.8
4.8
1.2
3.4
1-.L
7.3
5.6
7.2
6.1
5.5
0.9
3.9
Mg
(parts per D
0.2
0.2
0.2
0.2
0.2
0.1
0.1
0.2
0.1
0.1
0.2
0.2
0.2
0.0
0.2
Mn
lillion)
.00
.01
.00
.00
.00
.01
.01
.01
.00
.03
.02
.01
.01
.01
.01
Fe
.02
.05
.04
.04
.07
.10
.03
.08
.01
.12
.08
.07
.05
.06
.07
Cu
.01
.01
.01
.01
.01
.02
.00
.04
.00
.13
.02
.01
.'01
.02
.02
Values too low so absolute
probably questionable
B
.00
.01
.01'
.01
.02
.02
.00
.02
.00
.01
.02
.02
.01
.01
.02
levels
Zn
.12
.08
.07
.10
.12
.17
.12
.16
.08
.05
.10
.07
.10
.02
.27
Na
34.2
34.2
38.8
35.1
38.2
38.6
33.6
0.1
27,7
18.5
33.9
42.9
41.8
0 0
43.8
Al is too low for proper detection.
~,
is a distilled water control.
Tab] e 2. Ion analysis of streams used in acute acid exposure field studies.
K Na Ca Zn Mg Mn Fe Cu B Al
Upper Three Runs
(Acid branch)
7.00 5 7.6 1.6 5.0 5
0.06 0.07 5
Upper Three Runs
(Below acid branch)
0 1.1 6 0.03 5.3 0.98 000 0.76
Upper Three Runs
(Above acid branch)
0 0.9 2 0
0.01 0 0.01 0 0.07
Black Moshannon
(Below acid branch)
0 1.3 7 0.05 3.6 0.48 0.02 0.01 0 0.36
Black'Moshannon
(Above acid branch)
0 0.7 2 0.03 0.02 0.01 0.01 0
107
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Table 3. Water quality in Black Moshannon Creek 29 April
1970 and during trial run period-
pH
Alkalinity (ppm)
Acidity (ppm)
Sulphate (ppm)
Specific Conductance
(micromhos 13 25C)
Control
6.6*
8
3
12
39
Test
Station
5.7**
0
6
23
73
*19 May 6.4
**19 May 4.9
Table I*. Water quality in Upper Three Runs 29 April 1970
and during trial run period.
pH
Alkalinity (ppm)
Acidity (ppm)
Sulphate (pptn)
Specific Conductance
(micromhos @ 25C)
Control
6.7*
2
2
8
36
Acid
Tribu tary
3.3
0
100
320
870
Test
Station
5.0**
0
6
28
88
*18 May to 25 May b.43
**18 May 5.0; 22 May 5.5; 25 May 5.5;
27 May 4.5 (flood water)
108
-------
Figure 8. Map of Streams Used in Field Studies
A. Control Site - Hack Mo*hannon Creek
B. Test Site - Black Noshannon Creek
C. Control Site - Upper Three Runs
D. Test Site - Upper Three Runs
-------
I.Or
.50
tn
z
1.0
t-
o.
o
.50
NONBREEDIN6 CONTROL
pH 7.0 x 5d
NONBREEOING ACID
pH 4.0-3.5 x 5d
Figure 9. Hemoglobin mi crodensitometric tracings (scan speed
1 ^m/min, 5^0 mv) of polyacrylamide gels from
nonbreeding, acute acid exposed trout {pH t.0-3.5 x 5d)
110
-------
I.Or
SLR
NON8REEDING CONTROL
pH 7.0x 5d
AR - ARGININE RICH
SLR-SLIGHTLY LYSINE RICH
A -ARGININE
L-LYSINE
L1 - LYSINE
NONBREEDING ACID
pH 4.0-3.5 x 5d
Total blood histone microdens.itometric tracings (scan
speed - 1 cm/min, 660 mu) from polyacrylamide gels from
nonbreeding, acute acid exposed trout (pH 'i.O-3.5 x 5d).
Ill
-------
of extract is loaded on the gel. Only two major peaks (AR and SLR)
are resolvable. Figure 11 shows that better resolution is obtained
with a lesser concentration of histone extract. In this instance,
two SLR fractions are separated. However, if one is interested in
quantifying changes in minor fractions it is preferable to use higher
concentrations of extract.
i
The final figure 12 shows the Mount-Brungs gravity flow system used
in our studies. This was equipped with siphons, solenoids, pH meter
and related timing components for automatic monitoring of pH
following circuitry diagrams supplied by Dr. R. W. Andrews of the
Duluth Water Quality Laboratory.
i.o
.50
1.0
0.
o
.50
SLR
NONBREEDING CONTROL
pH 7.0 x 28d
SLR
SLR
NONBREEDING ACID
pH 4.9 x 28d
AR
AR - ARGININE RICH
SLR-SLI6HTLY LYSINE RICH
A - ARGININE
L- LYSINE
L1-LYSINE
Figure 11. Total blood histone microdensitometric tracings (scan
spetd - 1 cm/nun, 660 mu) of polyacrylamide /^els from
nonbreeding, acid exposed trout (pH h.9 * 26-^a).
112
-------
Figure 12. Mount-Brungs Type Gravity Flow Diluter System
iHJ.S. GOVERNMENT PRINTING OFFICE: 1972 484-482/351-3
113
-------
1
Accession /Vumber
w
5
r. Subject Field &; Croup
Organization
Pennsylvania State
SELECTED WATER RESOURCES ABSTRACTS
INPUT TRANSACTION FORM
University
Department of Biology
University Park, Pennsylvania 16802
Title
HISTOCHEMICAL AND CYTOPHOTOMETRIC ASSAY OF ACID STRESS IN FRESHWATER FISH
I Q Author(s)
Anthony, Adam
Cooper, Edwin L.
Mitchell, Robert B.
Neff, William H.
Therrien, Chester D.
16
Project Designation
FWPCA, Grant No. 1805ODXJ
21
Note
22
Cita ti on
23
Descriptors (Starred First)
*Fish Physiology, *Bioassay, *Acidic Water, *Water Pollution,
Pennsylvania, Analytical Techniques, Microscopy, Bioindicators
25
Identifiers (Starred First)
*Fish Histochemistry, * Analytical Histochemistry, *Microspectrophotometry,
Cytophotometry , Microspectroscopy
27
A bstract
The feasibility of using histochemical and histopathological changes in
brook trout, longnose dace and fathead minnows as bioindicators of acid
pollution was investigated. Laboratory studies entailed using a gravity flow
diluter system. Field studies involved using net traps in polluted streams.
Exposure durations were 4-5 days and 28-30 days. Histochemical and cytophoto-
metric analyses were made of gills, Stannius corpuscle, blood, spleen, kidney
and liver.
The primary mode of acid toxicant action is gill damage which results in
impaired respiratory, excretory and liver functions. Short term indices of
acid stress include: colloidal iron and PAS staining of gills and renal
Stannius corpuscles. A useful bioindicator of prolonged acid exposure is
decreased azure B-RNA staining of liver cells; this assesses the extent of
liver impairment and reflects a reduced tolerance of fish to other toxicants.
Sublethal levels of acidity are not cumulative. However, pH levels of about
5.0 should be considered hazardous since they prove toxic to breeding fish
having increased oxygen needs and also reduce the liver's ability to detoxify
noxious substances present in acid polluted waters.
Abstractor
Adam Anthony
Institution
Pennsylvania State University
WR:102 (REV. JULY 1969)
WRSI C
SEND, WITH COPY OF DOCUMENT, TO: WATER RESOURCES SCIENTIFIC INFORMATION CENTER
U.S. DEPARTMENT OF THE INTERIOR
WASHINGTON, D. C0 20240
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