COMPLETION REPORT
May 1, 1984
Impact of Surface Water Acidification
on Commercially and Recreatlonally Important Salmonid Fishes:
Effects on Reproductive Success and Recruitment
U.S. Environmental Protection Agency
Interagency Agreement No. AD-14F21A100
with U.S. Fish and Wildlife Service
Submitted by
Carl B. Schreck. and Hiram W. Li
Oregon Cooperative Fishery Research Unit
Oregon State University
Corvallis, Oregon 97331
Project Staff: Gary S. Weiner
with contribution by Bruce A. Barton
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EXECUTIVE SUMMARY
•Reproductive failure has been cited as an important factor in the
gradual process of fish population extinction in acidified surface
waters. Effects of low environmental pH on salmonid reproduction
were evaluated in this regard, using rainbow trout as a model.
•Adult trout were exposed to pH levels 4.5, 5.0, 5.5, and control
(range 6.5 to 7.1) during the final 6 weeks of reproductive
maturation. Plasma sex steroid levels and calcium concentrations
revealed no gross physiological abnormality in adult trout. Plasma
sodium levels tended to decrease in acid-exposed fish and fell sharply
in fish exposed to pH 4.5 for 42 days.
•Reduced survival rates in unacidified water of the progeny of acid-
exposed trout indicate that spermatogenesis and oogenesis are sensitive
to pH levels 5.5 and below. Survival rates of the progeny of
unexposed parents were reduced by exposure to pH levels 4.5, 5.0,
and 5.5.
•These results demonstrate that gametogenic performance and
subsequent survival and fitness of early life history stages of
salmonids may be influenced by environmental acidity even at pH
levels which do not elicit clinical indications of reproductive
stress in adult fish.
•Exposure of juvenile rainbow trout to acidic conditions for 5 days
altered plasma glucose, plasma sodium, and hematocrit levels, but
not plasma Cortisol, a primary stress response factor and frequently
employed indicator of stress.
•Subjection of the juvenile trout to severe handling stress after
acid exposure resulted in much greater elevations of plasma Cortisol
levels in fish exposed to pH 4.7 than in fish held at ambient pH
(6.6).
•These results indicate that acid-stressed fish are more sensitive to
additional stresses even though they may appear to be physiologically
normal.
•Planning of management strategies such as stocking fish in acidified
waters must take into account the possibility that acid-exposure of
juvenile trout may exacerbate responses to additional stress.
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ABSTRACT
Reproduction of salmonid fishes in acidic water was studied,
using the rainbow trout (Salmo gairdneri) as a model. Adult trout
were exposed to pH levels 4.5, 5.0, 5.5, and control (6.5 to 7.1)
during the final 6 weeks of reproductive maturation. Reduced
survival rates of the progeny of acid-exposed females through 7 days
of development, hatching, and yolksac absorption demonstrate that
oogenesis is sensitive to environmental acidification. Similar
reductions in the survival of the progeny of acid-exposed males
indicate the sensitivity of spermatogenesis to low ambient pH. The
progeny of unexposed adults had lower survival rates through 7 days
of development and hatching when they were reared at pH levels
4.5, 5.0, and 5.5 than did embryos reared at the control pH level.
No embryos exposed to pH 4.5 survived to the eyed stage. Plasma
estradiol-17S, androgen, and l7a-hydroxy-2O0-
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TABLE OF CONTENTS
INTRODUCTION 1
Acidic deposition and water quality alterations 2
Fish mortalities and toxicity bioassays 3
Physiological responses to ambient acidity 6
Population extinction and reproductive failure 8
METHODS 12
RESULTS 17
DISCUSSION 23
BIBLIOGRAPHY 30
APPENDIX 1 41
References 57
Table 1 63
Figures
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LIST OF TABLES
Table Page
Survival rates of the progeny of acid-exposed
and unexposed adult rainbow trout 18
2 Calcium and sodium concentrations in plasma
of acid-exposed adult rainbow trout 21
3 Estradiol-178, androgen, and
17o-hydroxy-203-dihydroprogesterone
concentrations in plasma of
acid-exposed adult rainbow trout 22
1 Appendix 63
Mean plasma Cortisol, plasma glucose,
plasma sodium, and hematocrit in
juvenile rainbow trout after
continuous exposure to reduced
environmental pH
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LIST OF FIGURES
Appendix: Page
Figure 1 Responses of plasma Cortisol in 64
juvenile rainbow trout subjected
to a 30-s handling stress after
5 days of continuous exposure to
low environmental pH
Figure 2 Responses of plasma glucose in 66
juvenile rainbow trout subjected
to a 30-s handling stress after
5 days of continuous exposure to
low environmental pH
Figure 3 Responses of plasma sodium in 68
juvenile rainbow trout subjected
to a 30-s handling stress after
5 days of continuous exposure to
low environmental pH
Figure 4 Responses of hematocrit in 70
juvenile rainbow trout subjected
to a 30-s handling stress after
5 days of continuous exposure to
low environmental pH
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PREFACE
This completion report fulfills the concerns of an Interagency
Agreement Qlo. AD-14F2A10QX entitled "Impact of Surface Water Acidifi-
cation on Commercially and Recreationally Important Salmonid Fishes:
Effects on Reproductive Success and Recruitment" between the United
States Environmental Protection Agency's Corvallis Environmental Research
Laboratory and the United States Fish and Wildlife Service's Oregon
Cooperative Fishery Research Unit. Work for this Interagency Agreement
was carried out under a Fish and Wildlife Service Research Work Order
(No. 14-16-0009-1512, WO No. 1).
In addition to the specified tasks outlined in the Interagency
Agreement, an ancillary study was conducted to optimize usage of the
experimental fish-rearing facilities established for this agreement.
This project on the "Effects of Prior Environmental Acidification on
Physiological Stress Response of Juvenile Rainbow Trout to Acute Handling
Stress" is described in the Appendix I of this report.
We wish to acknowledge the help provided to us for our studies by
the staff of the Environmental Protection Agency's Western Fish
Toxicology Station. In particular, we appreciate the help of Don Stevens
in designing and setting-up the acidification dilution and delivery
system and Gary Chapman for facilitating our work at the station. We
also appreciate the help of Robert T. Lackey of the Corvallis Environ-
mental Research Laboratory regarding the study design and the help of
numerous students and staff of the Oregon Cooperative Fishery Research
Unit, not identified in this report, with the rearing and breeding
aspects of the study, as veil as with laboratory analysis of samples.
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Although the research described in this report has been funded wholly
or in part by the Environmental Protection Agency through an Interagency
Agreement, it has not been subjected to Agency's required peer and policy
review and therefore does not necessarily reflect the views of the Agency.
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INFLUENCES OF ENVIRONMENTAL ACIDIFICATION ON SALMONID REPRODUCTION
INTRODUCTION
Surface water acidification, presumably due in part to acidic
precipitation, has been documented in northern Europe (Aimer et al.
1974; Leivestad et al. 1976; Wright et al. 1980) and eastern North
America, including the northeastern region of the United States
(Haines 1981). The occurrence of acidic precipitation has been
reported in other parts of the United States, such as the Los Angeles,
San Francisco, Seattle, and Denver areas (Lewis and Grant 1980; Powers
and Rambo 1981), and these areas contain lakes and streams that are
susceptible to acidification (Omernik and Powers 1983). Thus, current
and potential impacts of acidification on aquatic biota are receiving
widespread attention.
Fish populations have been eliminated as a result of
acidification in some systems and are endangered in others (Jensen
Snekvik 1972; Schofield 1976; Harvey 1980). Because of the commercial
and recreational Importance of salmonid fisheries in Europe and North
America, much of the concern has focused on the impacts of
acidification on salmonid populations. The literature review in this
section establishes the context for a study of the reproduction of
rainbow trout (Salmo gairdnerl) in a low pH environment, which will be
reported in the following sections.
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Acidic deposition and water quality alterations
The combustion of fossil fuels results in the emission of sulfur
dioxide and nitrogen oxides into the atmosphere, where they undergo a
complex process of transportation and transformation into sulfuric and
nitric acids (Dovland and Semb 1980). Along with other chemicals from
various natural and man-made sources, these acids may reach watersheds
and aquatic systems through wet precipitation, dry deposition,
impaction of aerosols, and adsorption of gases (Galloway and Cowling
1978).
The alterations of surface water chemistry by acidic deposition
depend upon the nature of the receiving system and the quantity and
mode of acidic input. The chemical composition of precipitation is
modified as it moves through the watershed and Interacts with the
geological substrate, soil, and vegetation (Likens et al. 1979). The
acid-neutralizing capacity of the bedrock and soils, hydrologic flow
characteristics, soil and mineral types, and age of the soil influence
the sensitivity of particular systems to acidification (Hendrey et al.
1980).
Temporal variations in acidification processes are also
important. Heavy rains and rapid snowmelt may result in sharp
episodic pH depressions and metal inputs (Gjessing et al. 1976;
Schofield and Trojnar 1980). Chronic low pH conditions develop in
waters in which the bicarbonate buffering system is gradually depleted
(Henriksen 1980).
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The characteristics of watersheds that determine water quality
responses to acidic inputs are sufficiently divergent to warrant
caution in describing "typical" conditions in impacted systems.
Gjessing et al. (1976) thoroughly reviewed this subject. They noted
increased acidity, decreased alkalinity, replacement of bicarbonate by
sulfate as the predominant anion, and increased concentrations of
calcium, magnesium, and aluminum as common effects of acidic
precipitation on the water chemistry of susceptible lakes and
streams.
Fish mortalities and toxicity bioassays
Observations of fish mortalities have been correlated with sudden
episodic acidification events. Jensen and Snekvik (1972) described
mortalities of Atlantic salmon (Salmo salar) in southern Norway
associated with snowmelt. The pH of samples taken from a river during
the fishklll ranged from 3.9 to 4.2. Similarly, an acidic pulse due
to snowmelt coincided with a massive kill of brown trout (Salmo
trutta) in the Tovdal River of Norway (Leivestad and Muniz 1976).
Schofield (1977) reported mortalities of adult, yearling, and larval
brook trout (Salvelinus fontinalis) held in tanks supplied with water
from Little Moose Lake, New York, during a spring snowmelt. The
relatively minor change in lake water pH and significantly elevated
aluminum concentrations implicated aluminum as the dominant toxic
factor rather than hydrogen ion. Grahn (1980) theorized that a kill
of ciscoe (Coregonus albula) in an acidified Swedish lake resulted
from the exposure of fish to precipitating aluminum hydroxide.
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Bioassay studies have provided information about several factors
that influence acid toxicity. Leivestad et al. (1976) pointed out
that high hydrogen ion concentrations are relatively more toxic in
waters with low concentrations of dissolved salts. Kwain (1975)
showed a positive relationship between temperature and median lethal
pH levels for rainbow trout fingerlings. Packer and Dunson (1972) and
Swartz et al. (1978) determined that sulfuric acid, hydrochloric acid,
and acid mine waste are not of equal toxicity.
As illustrated by the tabulation of acute and chronic pH exposure
bioassays prepared by Spry et al. (1981), differences in test
conditions make it difficult to generalize about the relative toxicity
of acidic water to fishes. However, some important conclusions can be
drawn. The effects of elevated hydrogen ion concentrations appear to
be species-specific. For example, Grande et al. (1978) concluded that
the relative order of tolerance to low environmental pH conditions for
some salmonids is (from least tolerant to most tolerant): rainbow
trout, Atlantic salmon, brown trout, and brook trout. Johansson
et al. (1977) demonstrated that the early development of Atlantic
salmon is more sensitive to hydrogen ion toxicity than is the early
development of brown trout or brook trout. Intraspecific differences
in tolerance have been demonstrated for brook trout (Robinson et al.
1976; Swartz et al. 1978) and brown trout (Edwards and Gjedrem 1979).
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In addition to species specificity of tolerance to ambient
acidity, the toxicity of acidic water varies among life history stages.
Bioassay results (Menendez 1976; Daye and Garside 1977; Craig and Baksi
1977; Carrick 1979) reveal as a general trend that yolksac larvae are
the least tolerant of acid stress followed by post-yolksac larvae,
embryos, juveniles, and adults, in order of increasing tolerance.
Aluminum, leached from soils by acidic waters and carried into
lakes and streams through watershed runoff, can reach high enough
concentrations to be toxic to fish (Cronan and Schofield 1979).
Schofield (1977) identified aluminum as the primary toxicant in
snowmelt runoff into Little Moose Lake, New York, and discussed the
complex interactions between aluminum and pH. Driscoll et al. (1980)
determined that inorganic aluminum forms contribute most to fish
toxicity, whereas organically complexed aluminum is of relatively low
toxicity. Baker and Schofield (1982) further evaluated the effects of
aluminum and hydrogen ions on early life history stages of white sucker
(Catostomus commersonl) and brook trout. They concluded that the
effects of aluminum vary for different life history stages; aluminum
toxicity is dependent upon pH; and brook trout and white suckers are
not equally sensitive to aluminum and hydrogen ions. Interestingly,
they showed that aluminum actually enhanced survival of eggs at pH
levels between 4.2 and 4.8. Damage and mucous clogging of gills due to
aluminum exposure have been reported frequently (Freeman and Everhart
1971; Schofield 1977; Schofield and Trojnar 1980; Muniz and Leivestad
1980; Baker and Schofield 1982).
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Physiological responses to ambient acidity
Studies of the physiological responses of fish to ambient acidity
have usually involved acute exposures to very low pH. Spry et al.
(1981) commented that these results may be of limited applicability in
assessing chronic effects of acidification. Nevertheless, they do
contribute to an understanding of some important physiological
processes, especially ionoregulation and acid-base balance.
Leivestad and Muniz (1976) correlated the losses of sodium and
chloride from the plasma of brown trout with acid stress in the Tovdal
River of southern Norway and in tank experiments. Packer and Dunson
(1970, 1972) attributed the decline in total body sodium of
acid-exposed brook trout to decreased influx and increased efflux
rates. Similar depletions in plasma sodium and chloride have been
demonstrated with rainbow trout (McDonald and Wood 1981; Booth et al.
1982). McWilliams and Potts (1978) determined that high hydrogen ion
concentrations may be associated with a shift in gill transepithelial
potential from negative to positive, resulting in increased
permeability of the gill epithelium to sodium. Furthermore, they
reported that the presence of calcium in the water reduced the
permeability of the gill epithelium to sodium. Spry et al. (1981)
related this information to a field survey by Wright and Snekvik
(1977) in which fish population status was positively correlated with
lake calcium concentration.
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McDonald et al. (1980) found that exposure of rainbow trout to
high levels of hydrogen ions in soft water resulted in severe
ionoregulatory dysfunction and minor acidosis. In contrast, exposure
to ambient acid in hard water showed little effect on plasma ions and
more serious acidosis. Lowered blood pH has been reported in several
other studies of rainbow trout (Neville 1979; McDonald and Wood 1981;
Booth et al. 1982), brook trout (Packer 1979), and carp (Cyprinus
carpio) (Ultsch et al. 1981). Neville (1979) found increases in
hemoglobin, hematocrit, and erythrocyte levels as additional effects
of acid exposure on blood characteristics.
Daye and Garside (1976) studied the histopathology of brook
trout surficial tissues in low pH waters. Their findings included
hypertrophy of mucous cells in the gills, nares, and integument; and
epithelial damage in the gills, corneae, integument, and esophagus.
Fritz (1980) speculated that such injuries to corneal and olfactory
tissues could affect salmonid imprinting, homing, and spawning.
Integumental damage in prehatching embryos and gill epithelial
alterations in alevins contributed to acid-induced mortalities of
young Atlantic salmon (Daye and Garside 1980).
Falk and Dunson (1977) observed behavioral indications of stress
in brook trout at pH 5.0 and 5.8. Johnson and Webster (1977)
determined that spawning female brook trout avoided upwellings of pH
4.0 and 4.5. They suggested that this behavior could aid in increasing
egg and larval survival in systems where such an avoidance response is
possible.
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Population extinction and reproductive failure
Occasional fish kills provide dramatic evidence of detrimental
effects of acidification, and physiological studies of acute acid and
metal toxicity add to our understanding of some relevant processes.
However, the persistence of resources in impacted systems is of
greater concern from a fisheries perspective.
In a survey of southern Norway, for example, the percentage of
lakes devoid of trout populations increased gradually from 3.8% to
60.0% as lake pH decreased from 5.5 or more to 4.0 (Jensen and Snekvik
1972). A later study of 700 lakes in southern Norway (Wright and
Snekvik 1977) revealed that about 40% of the lakes were devoid of fish
and another 40% had sparse populations. Brown trout is the principal
species of the region.
Fish population surveys of 50 Swedish lakes that ranged in pH
from 4.40 to 7.45 were reported by Aimer et al. (1974). The roach
(Leuciscus rutilus) was missing from ten of the low pH lakes and
reproductive failure was apparent below pH 5.5. Populations of perch
(Perca fluviatllis), pike (Esox lucius), minnow (Phoxlnus phoxinus),
arctic char (Salvelinus alpinus), brown trout, ciscoe, and eel
(Anguilla vulgaris) were also sparse or nonexistent.
Concern for possible acidification and metal deposition in the
La Cloche Mountain Lakes of Ontario caused by a smelter prompted
several studies of fish population status. Beamish and Harvey (1972)
chronicled the gradual reductions and losses of populations of seven
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species as the pH of Lumsden Lake dropped from 6.8 in September 1961
to 4.4 in August 1971. Beamish (1974) described the decline of
populations in Ontario Society of Artists (O.S.A.) and Muriel Lakes.
The average pH of O.S.A. Lake during the study was 4.5 and the average
pH of Muriel Lake was 4.7. Beamish et al. (1975) studied the fish
populations of George Lake, where the pH ranged from 4.8 to 5.3
between February 1972 and August 1973. They reported changes in the
average size of age classes, reduction of population sizes, and
disappearance of some species.
A survey of 217 high elevation lakes in the Adirondack Mountains
of New York (Schofield 1976) showed that 51% of the lakes had pH
values below 5.0. No fish were present in 90% of the low pH lakes.
Schofield and Trojnar (1980) determined that in 53 acidified lakes
of the Adirondacks, mean aluminum concentrations were inversely
correlated with presence of stocked brook trout. The mean
concentrations were 0.29 mg Al/liter for lakes in which trout were
absent and 0.11 mg Al/liter for lakes in which trout were present.
Reproductive failure has been cited as a major contributor to the
gradual extinction of fish populations in acidic waters (Jensen and
Snekvik 1972; Beamish et al. 1975; Beamish 1976; Ryan and Harvey 1977,
1980). Of the many factors that determine reproductive success,
investigators have specified mortality of early life history stages
(Jensen and Snekvik 1972: Leivestad et al. 1976) and physiological
disruptions during gametogenesis (Beamish et al. 1975; Lockhart and
Lutz 1977) as the causes of reproductive failure due to acidic
conditions.
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As noted earlier, bioassays have established that early life
history stages are relatively more sensitive to hydrogen ion than are
juveniles and adult fish. Other studies have demonstrated impairment
of adult reproductive physiology due to acid stress. Craig and Baksi
(1977) showed reduced egg production and egg fertility in flagfish
(Jordanella floridae) at low pH. Similarly, Lee and Gerking (1980)
exposed adult desert pupfish (Cyprlnodon nevadensis nevadensis) to
acidic conditions and observed decreased egg production, egg laying,
and egg viability. Ruby et al. (1977) determined that exposure to high
levels of hydrogen ion of adult female flagfish inhibited the
deposition of secondary yolk in the cytoplasm of oocytes. Beamish
et al. (1975) and Lockhart and Lutz (1977) concluded that acidic water
may impair plasma calcium regulation associated with vitellogenesis in
reproductively maturing female fishes.
The goal of the research described here was to increase
understanding of current and potential consequences of low
environmental pH for the reproduction of salmonid fishes. This goal
was addressed by studying final reproductive maturation and progeny
survival of rainbow trout, a relatively acid-sensitive species among
the salmonids (Grande et al. 1978), in a low pH environment. Plasma
ion and sex hormone levels were evaluated as physiological indicators
of reproductive stress in acid-exposed adult trout. Estradiol-170 is
involved in vitellogenesis and calcium regulation in maturing female
salmonids (Whitehead et al. 1978) and may serve as an indicator of
reproductive stress early in the reproductive cycle. Similarly, the
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androgens are Che primary gametogenic hormones In male salmonids and
they co-vary with estradiol-173 in females (Sower and Schreck. 1982).
The performances of the gametes from the acid-exposed fish were
compared to those from unexposed trout to demonstrate effects of high
levels of hydrogen ion on oogenesis and spermatogenesis. Specifically,
We were interested in the ontogenetic effects of low environmental pH
on reproductive success and in evaluating the potential for maternal
and paternal effects.
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METHODS
Adult rainbow trout (age 2+), averaging 57.4 ± 3.2 cm in length
and 2.2 ± 0.5 kg in weight, were obtained in June 1982 from the Roaring
River Hatchery (Oregon Department of Fish and Wildlife, Scio, Oregon)
and transported to the Western Fish Toxicology Laboratory of the United
States Environmental Protection Agency, Corvallis, Oregon. Blood
samples were obtained (prior to October 5, 1982) by caudal puncture
with a needle and ammonium-heparinized syringe, and gender of the fish
was determined by the Ouchterlony Immunodiffusion Assay Technique
through which vitellogenin is specifically detected in the plasma of
female fish (Terry Owen, Helix Biotech, Ltd., Richmond, British
Columbia, Canada). The fish were distributed on October 27, 1982 into
eight 757-liter circular fiberglass tanks, with three females and four
males in each tank. The tanks were set up outdoors to provide a
natural photoperiod.
The inflowing well water was gradually softened by reverse
osmosis (Purification Techniques, Inc., Brielle, New Jersey) from 23
mg/liter as CaC03 to 10 mg/liter over a period of five days. After an
additional 3 days of acclimation, reagent grade sulfuric acid was
introduced into the system over 5 days until pH levels of 4.5, 5.0, and
5.5 were reached in duplicate treatment tanks, while two control tanks
(pH range 6.5 to 7.1) remained on softened water.
Total water hardness, determined at least twice daily by the EDTA
titrimetric method (American Public Health Association et al. 1980),
was 9.2 ± 1.2 mg/1 as CaCOa
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softened, vigorously aerated water was acidified with sulfuric acid and
held at pH 4.0 ± 0.1 with a Beckman Model 942 pH Monitor (Beckman
Instruments, Inc., Fullerton, California). The acidified stock
solution was vigorously aerated to remove carbon dioxide that may have
been generated by the addition of acid, and pumped to the head tank of
a gravity-flow diluter. The acidic stock solution was diluted with
softened well water to attain appropriate pH levels and delivered at a
rate of 4.5 liters/minute to the tanks in the flow-through system.
Treatment pH levels were automatically monitored and recorded every
3 hours and the diluter was manually adjusted as necessary in order to
maintain exposure levels within ± 0.1 pH units of 4.5, 5.0, and 5.5.
Water temperatures during the experiment ranged from 10.9 to 14.0 C and
alkalinity, determined by Gran titration as described by Stumm and
Morgan (1981), was 252 ± 11 yeq/1 prior to addition of acid. Metal
levels were below limits of detection by atomic absorption flame
spectrophotometry.
Blood samples from each fish were drawn from the caudal artery
with a needle and ammonium-heaparinized syringe under anesthesia
(MS222, 50 mg/liter) and the plasma was stored at -20 C. Anesthetic
solutions were titrated to treatment pH levels with 0.1 M NaOH to
minimize stress. The first blood sample was taken following tank
acclimation, just prior to the addition of acid to the system. The
second sample was taken 7 days after treatment pH levels were reached,
and the final sample was drawn at the time of spawning*
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Fish were examined for reproductive maturity weekly, beginning
with the fourth week of exposure. After 42 days of exposure
(December 21, 1982), the ripe fish were anesthetized for blood
sampling, killed with a blow to the head, and manually spawned. The
gametes were collected separately from each fish and matings were
conducted according to the following scheme, employing dry
fertilization and 1-hour water hardening:
A) Unexposed females X unexposed males
Eggs from unexposed females were fertilized with sperm from
unexposed males. Water hardening and rearing took place at
each of the pH levels used in the prespawning exposure in
order to assess the performance at low pH of the progeny of
unexposed adults.
B) Acid-exposed females X unexposed males
Eggs from females exposed to each pH level were fertilized
with sperm from unexposed males. Water hardening and
rearing took place at the control pH level to determine the
effects of acidic water on oogenesis.
C) Acid-exposed males X unexposed females
Eggs from unexposed females were fertilized with sperm from
males from each pH exposure group. Water hardening and
rearing took place at the control pH level to determine the
effects of acidic water on spermatogenesis.
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D) Acid-exposed females X acid-exposed males
Eggs from females exposed to each pH level were fertilized
with sperm from males exposed to the same pH level• Water
hardening and rearing took place at the respective parental
exposure pH levels to assess reproductive performances when
both parents and the progeny were exposed to low pH.
Aliquots of embryos from each fertilization group were placed in
incubation cells within drawers of Heath incubators for rearing. The
cells consisted of 4.6 cm sections of 6.4 cm diameter PVC pipe with
plastic mesh screen attached across one end, forming a cup. Similar
cups, constructed of 1.3 cm sections of 7.6 cm diameter PVC pipe, were
inverted over each cell to confine the fish after hatching. The flow
rate through the incubators was 2 liters/minute. The incubators were
covered with black plastic sheets to exclude light. Dead eggs and
larvae were counted and removed daily except as noted subsequently. A
different incubator was used for each test pH level (4.5, 5.0, 5.5, and
ambient). Within a treatment, cells of eggs were randomly distributed
throughout the incubator.
Plasma estradiol-173, androgen (testosterone and
11-ketotestosterone) and 17a-hydroxy-2O0-dihydroprogesterone
concentrations were determined by radioimmunoassay, following the
general method of Korenman et al. (1974). Details of the procedure
are described by Sower and Schreck (1982). Estradiol-170 was
determined in 10 yl of plasma for males and 25 pi of plasma for
females. Androgens were determined in 10 yl of plasma and
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17a—hydroxy-203-dihydroprogesterone was determined in 25 yl of
plasma. Samples were extracted twice with diethyl ether and
extraction efficiency was about 90% for all assays. Antiestradiol-17g
and antitestosterone were provided by Dr. G. Niswender (Colorado State
University, Fort Collins, Colorado) and anti-17a-hydroxy-20$-
dihydroprogesterone was provided by Dr. A. P. Scott (Ministry of
Agriculture and Fisheries, Lowestoft, Suffolk, United Kingdom).
Antibody characteristics are given by Sower and Schreck (1982) and
Scott et al. (1982). Plasma calcium concentrations were determined by
atomic absorption flame emission spectrophotometry, and sodium
concentrations were measured by flame photometry.
Progeny survival rates were statistically analyzed with
contingency tables (Cochran and Cox 1957). Plasma calcium and sodium
concentrations were evaluated by analysis of variance and Duncan's new
multiple-range test for comparisons among means when appropriate (Steel
and Torrie 1980).
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RESULTS
Survival rates of the progeny of acid-exposed and unexposed trout
appear in Table 1. On the eighth day of development, some eggs became
infected with Saprolegnia fungi. Thus, mortalities through the last
day before the occurrence of infection (7 days of development) were
tallied, the infected eggs were removed, and the remaining eyed eggs
were monitored for survival through hatching. Survival through
yolksac absorption was then determined among the hatched fish.
The progeny of unexposed adults that were reared at pH 5.5 and
5.0 (groups B and C) had lower survival rates through 7 days and
slightly lower hatching success than those reared in unacidified water
(group A). No embryos reared at pH A.5 (groups D and H) survived
through 7 days.
Influences of acid-exposure on oogenesis are indicated by the
reduced hatching percentages among the progeny of females that had
been exposed to pH levels 5.5 and 4.5 (groups E and G). The progeny
of females that had been exposed to pH 5.5 (group E) had lower
survival from hatching through yolksac absorption than did controls
(group A).
Impaired spermatogenesis at low pH is evident in the performances
of the progeny of acid-exposed males (groups H, I, and J). The
progeny of males exposed to pH 4.5 prior to spawning (group J) had
lower survival through 7 days of development and through hatching than
did the progeny of unexposed males (group A). The progeny of males
exposed to pH levels 5.5 and 5.0 (groups H and I) had lower survival
rates through hatching and yolksac absorption than did controls (group A).
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Table 1. Survival rates of the progeny of acid-exposed and unexposed adult rainbow trout.
Total numbers of eggs or larvae In each case are 1n parentheses,
Parental
prespawnlng
exposure pH level
Progeny
rearing
pH level
Percent
survival
through
7 days
Percent
of eyed eggs
successfully
hatched
Percent
of larvae
surviving
to yolksac
absorption
^ Cumulative
percent
survival
Female
Male
A
control
control
control
91.3(689)
93.8(465)
86.2(436)
73.8
B
control
control
5.5
*64.2(749)
*89.2(381)
92.4(340)
52.9
C
control
control
5.0
a54.7(766)
a80.1(294)
81.5(238)
35.7
D
control
control
4.5
8 0 (761)
--
0
E
5.5
control
control
91.9(211)
*57.8(154)
*70.8(89)
37.6
F
5.0
control
control
*99,1(439)
91.1(426)
88.9(388)
80.3
G
4.5
control
control
93.1(189)
*26.4(148)
89.7(39)
22.0
H
control
5.5
control
93.8(689)
*80.8(386)
*79.5(312)
60.2
I
control
5.0
control
93.2(883)
*75.0(641)
*82.7(481)
57.8
J
control
4.5
control
*87.4(933)
*58.6(534)
86.6(313)
44.4
K
5.5
5.5
5.5
60.3(136)
^57,8(71)
b82.9(41)
28.9
L
5.0
5.0
5.0
C76.8(555)
84,0(374)
77.7(314)
50.1
M
4.5
4.5
4.5
0 (244)
—
—
0
a Different from group A at the corresponding stage of development, P < 0.05.
b Different from group B at the corresponding stage of development, P < 0.05.
c Different from group C at the corresponding stage of development, P < 0.05.
d Calculated as the product of the survival rates 1n the three preceding columns.
-------
19
When both parents and the progeny were exposed to pH 5.5
(group K), hatching success and survival through yolksac absorption
were reduced in comparison with the performances at pH 5.5 of the
progeny of unexposed adults (group B). Females exposed to pH 5.0
produced gametes of high quality as reflected in the survival of their
progeny in unacidified water and at pH 5.0 (groups F and L) through
seven days of development in comparison with the performances of the
progeny of unexposed females (groups A and C).
Calcium concentrations in the plasma of the adult trout are
presented in Table 2. No clear indications of acid stress are
apparent in the calcium levels of the females. Plasma calcium
decreased in males that were exposed to pH 4.5 for 42 days (P < 0.05).
Plasma sodium levels in the adults tended to decrease after 7 days of
exposure to each treatment pH level (Table 2). Fish exposed to pH
4.5 for 42 days had sharply reduced sodium concentrations in
comparison with unexposed fish (P < 0.05). Estradiol-170, androgen,
and 17a-hydroxy-20f5-dihydroprogesterone levels in adult trout revealed
no differences between treatments (Table 3). There was no apparent
effect of the acid exposure on rate of sexual development since the
fish all ovulated at the same time. When pressure was applied to the
abdomen, eggs flowed easily from the ovipore. No eggs appeared to be
atretic. In two of the females held at pH 4.5, approximately 10% of
the eggs in the ovaries had not ovulated and were held tightly in the
skein, although the remainder of the eggs were fully nature and
ovulated. All males were ripe, having copious quantities of sperm at
the time of spawning.
-------
20
Hatching occurred synchronously among embryos reared in
unacidified water and at pH levels 5.5 and 5.0. Three adult
mortalities occurred at pH 4.5. One adult mortality occurred in
unacidified water.
-------
21
Table 2. Calcium and sodium concentrations (mean t standard error) In plasma of acid-exposed
adult rainbow trout.
Prior to 7 days of
acidification acid exposure 42 days of
(57 days (35 days acid exposure
Sex/Exposure PH prespawnlnq) prespawnjnq) I spawn)nq)
Calcium (mill1equ1valents/11ter)
females
4.5
7.6
1
0.7
6.2 *
0.5
5.5
±
0.8
5.0
6.4
*
0.5
7.0 4
0.4
7.5
±
1.5
5.5
5.8
t
0.6
6.3 *
0.6
5.7
t
0.8
control
8.2
t
0.5
8.7 *
0.7
7.3
i
1.6
Mai es
4.5
5.5
t
0.2
5.5 ±
0.3
*4.8
±
0.1
5.0
5.4
1
0.1
5.5 t
0.2
5.3
t
0.1
5.5
5.2
t
0.1
5.2 i
0.2
5.8
±
0.3
control
5.3
1
0.2
5.8 1
0.2
5.2
±
0.1
Sodium (mil 11equ1valents/l1 ter)
Male and Female
4.5
153
1
2
145 ± 4
*129
±
4
5.0
153
t
1
146 *
3
147
±
4
5.5
152
1
3
143 ± 4
143
±
6
control
153
1
1
152 i
1
154
±
2
'Different from mean value In same exposure group prior to acidification
and mean value In control group at spawning, P < 0.05.
-------
22
Table 3. Estradiol-17ft, androgen, and 17p-hydroxy-20e-d1l«ydroprogesterone concentrations 1n plasma
of acid-exposed adult rainbow trout.
Sex/Exposure pH
females
4.5
5.0
5.5
control
Prior to
acidification
(57 days
prespawnlng)
mean
2.9
3.0
3.4
2.9
range
1.1-4.1
1.0-5.5
1.4-7.6
1.2-5.6
7 days of
acid exposure
(35 days
prespawninq)
mean
3.3
4.0
3.7
3.4
range
Estradiol - 17 e (ng/ml )
1.1-4-8
1.7-5.4
2.4-5.5
1.3-5.6
42 days of acid
exposure
(spawning)
tan range
0.7
0.7
0.3
0.6
<0.1-1.5
0.3-0.8
0.1-0.5
<0.3-1.3
Miles
4.5
5.0
5.5
control
Females
4.5
5.0
5.5
control
0.1
0.1
0.1
0.J
21
19
18
23
<0.1-0.2
<0.1-0.3
<0.1-0.2
<0.1-0.2
16-26
13-23
14-24
15-31
0.1
0.1
<0.1
<0.1
<0.1-0.2
<0.1-0.3
<0.1-0.1
<0.1-0.1
27
26
25
26
20-33
10-67
15-42
13-48
0.1
0.1
<0.1
<0.1
Androgens (ng/ml )
14
15
18
16
<0.1-0.1
<0.1-0.1
<0.1-0.1
<0.1-0.1
13-15
11-29
13-23
13-18
Males
4.5
5.0
5.5
control
26
33
24
29
18-37
16-59
21-27
17-48
31
19
31
22
19-64
17-25
17-67
13-35
33
21
27
21
14-61
10-34
15-35
13-46
17a-hydroity-206-d1hydroprogesterone (ng/ml)
Females
4.5
5.0
5.5
control
<0.1
<0.1
0.1
0.2
all <0.1
<0.1-0.3
<0.1-0.7
<0.1-0.7
1.2
0.6
0.7
0.6
0.4-5.8
<0.1-1.0
<0.1-2.4
<0.1-2.9
37
26.7
32.1
24.6
36.2-39.5
6.8-50.9
21.1-43.1
15.3-33.9
-------
23
DISCUSSION
Reproductive success of salmonids in acidic water is influenced by
gamete quality and the developmental environment of the early life
history stages. Exposure of adult rainbow trout to pH levels 5.5, 5.0,
and 4.5 during final maturation affected gamete quality as demonstrated
by the reduced survival in non-acidic water of the progeny of
acid-exposed fish. Both oogenesis and spermatogenesis were impaired.
Eggs from females that had been exposed to pH levels 5.5 and 4.5 prior
to spawning had lower survival rates than did eggs from unexposed
females, indicating detrimental effects of ambient acid during
oogenesis. In addition, two of the five females in the pH 4.5 exposure
group that survived through final maturation were incompletely
ovulated and it is possible that the eggs that were ovulated at this
level were not fully developed. In addition, two of the five females
in the pH 4.5 exposure group that survived through final maturation
were incompletely ovulated; in salmonid fishes, all eggs ovulate
synchronously, suggesting an aberrant condition in the females with two
distinct egg stages.
The high survival rates of the progeny of females exposed to
pH 5.0 prior to spawning suggest that some physiological aspects of
final reproductive maturation may actually be enhanced at this pH
level. Physiological processes are sensitive to alterations in
acid-base balance due to the pH-dependence of protein function
(Spry et al. 1981). Thus, it is possible that oxygen exchange at the
ovary or enzymatic reactions during ovulation, for example, are
-------
24
facilitated at some pH levels, but the actual effects of acid observed
at this level remain enigmatic. The response of eggs from females
exposed to pH 5.0 prior to spawning appears not to be an experimental
artifact. The proportions of survival of eggs from individual females
within and among replicate pre-spawning exposure groups were compared
and found to be similar in each case before they were pooled for
comparisons between treatments. Thus, the performance of eggs from the
females exposed to pH 5.0 cannot be explained by individual variability
in adult sensitivity to acid-exposure during gametogenesis. This
performance also cannot be accounted for by error in the acid delivery
system, since there were no unusual responses by the males that were
exposed to pH 5.0 prior to spawning, and plasma sodium levels in adults
of both genders declined at this pH level. The enhancement of
oogenesis at pH 5.0 should not be interpreted as indicating that
reproductive success of salmonids will be increased by acid exposure at
this pH level. Impaired spermatogenesis is apparent in the decreased
survival rates of the progeny of male trout held at pH 5.5 and lower
before spawning. Effects of acid exposure on gametogenesis have
previously been demonstrated histologically and through determinations
of egg production and fertility in flagfish at pH 6.0 and lower
(Craig and Baksi 1977, 1978). Similarly, Lee and Gerking (1980) showed
reductions in egg quality and quantity in desert pupfish below pH 7.0.
The present study provides evidence that salmonid reproductive
physiology is sensitive to environmental acidity as well. Examination
of the cumulative percent survival of the offspring of acid-exposed
fish suggests that oogenesis is affected more seriously than is
-------
25
spermatogenesis at pH levels 5*5 and 4.5 in rainbow trout, while the
apparent enhancement of oogenesis at pH 5.0 remains unexplained.
Direct effects of environmental acidity on early life history
stages are shown by the increased mortality at pH 5.5, 5.0, and 4.5 of
the progeny of unexposed trout. No embryos that were exposed to pH 4.5
survived to the eyed stage. Embryos that were exposed to pH levels 5.5
and 5.0 were most sensitive to the acid during the first 7 days of
development and during hatching. These sensitive periods have been
identified for toxicants in general (Rosenthal and Alderdice 1976).
Plasma calcium levels are known to increase in conjunction with
vitellogenesis in many teleosts, including salmonids (Bromage et al.
1982). Vitellogenesis is generally completed prior to the final
6 weeks of reproductive maturation and calcium levels decline gradually
during this period in trout (Whitehead et al. 1978). Plasma calcium
levels in the acid-exposed female trout tended to decrease as expected,
but variability among individuals precluded any clear indication of
reproductive stress based on calcium regulatory failure. Observations
of low female to male plasma calcium ratios among mature fish in
acid-stressed populations were ascribed to disruption of calcium
regulation associated with vitellogenesis (Beamish et al. 1975;
Lockhart and Lutz 1977). However, these observations occurred after
vitellogenesis was probably completed. Perhaps episodic exposure to
low pH earlier in the reproductive cycle or chronic exposure would
result in discernible calcium regulatory dysfunction. Indeed, plasma
calcium levels in male trout tend to be relatively invariant
-------
26
(Whitehead et al. 1978), but they were reduced after exposure to
pH 4.5 for 42 days.
The low plasma sodium levels in adult trout exposed to pH 4.5
for 6 weeks are consistent with current theory on the effects of
acidic conditions on ionoregulation. Packer and Dunson (1970) showed
that low pH conditions decrease sodium influx rates and increase efflux
rates. Ambient acidity is believed to increase the permeability of the
gill epithelium to sodium, accounting for the increased efflux rates
(McWilliams and Potts 1978). McDonald et al. (1980) determined that
ionoregulation and acid-base balance in acid-exposed rainbow trout are
influenced by water hardness. Acute acid exposure in soft water
produces severe ionoregulatory dysfunction, but only minor acidosis.
However, in hard water, ionoregulation is slightly affected and
profound acidosis may occur. Sublethal effects on sodium regulation
of adult trout at pH 4.5 may have little meaning in the reproductive
process, since no embryos survived to the eyed stage at this pH level.
Sex hormone concentrations did not reveal any gross physiological
abnormalities due to acid exposure. Hormone profiles in the
acid-exposed fish followed patterns typical for this species (Scott and
Sumpter 1983). Estradiol-170 decreased just prior to spawning in the
females and remained low throughout final maturation in the males.
Androgen levels gradually declined in both males and females.
17a-hydroxy-20f5-dihydroprogesterone concentrations were low in the
females until ovulation, when they rose dramatically. Because these
hormone levels are dynamically regulated and highly variable between
-------
27
individuals (Schreck et al. 1972; Hille 1982), they are unlikely to
provide useful warnings of reproductive impairment at sublethal pH
levels.
Hatching was synchronous among the embryos reared at pH levels 5.5
and 5.0 and in unacidified water. Delayed hatching has been noted in
Atlantic salmon and rainbow trout embryos that were exposed to low pH
after they had reached the eyed stage (Peterson et al. 1980; Nelson
1982). Atlantic salmon embryos that were continuously exposed to low
pH from fertilization through hatching, however, were not delayed
(Peterson et al. 1980). Delays that have occurred in some studies
may be due to inhibition of the hatching enzyme, chorionase, or to
reduced activity of embryos just before hatching (Peterson et al. 1980;
Peterson and Martin-Robichaud 1983).
Lethargic behavior was characteristic of rainbow trout yolksac
larvae at pH levels 5.5 and 5.0. Rombough (1982) observed similar
behavior in buttoned-up pink and chum salmon at sublethal pH levels.
In an unprotected environment, such behavior might have important
implications for the survival of young fish. For instance, salmonid
larvae that remain inactive in the gravel may be vulnerable to
predation by benthic invertebrates or other fish species, like some
sculpins which are known to prey upon salmonid eggs and fry (Scott and
Crossman 1973). This vulnerability to predation would depend, of
course, on the tolerance of the potential predators for acidic
conditions as well.
-------
28
Salmonid reproductive success in acidic surface waters is
determined by an array of water quality factors and biological
responses. The experiment reported here demonstrates that salmonid
reproduction is endangered at pH levels 5.5 and lower through effects
on gamete quality and direct exposure of developing embryos and fry to
acidic conditions. However, several other considerations are
necessary for approaching a broad understanding of impacts of low
environmental pH on salmonid reproduction.
One such consideration is the possible influence on reproductive
success of acute or chronic exposure of adult fish to acidic water
earlier in the reproductive cycle. In this study, continuous exposure
to low environmental pH for 6 weeks prior to spawning affected gamete
quality. Yet, ovarian maturation generally commences as early as
6 months before spawning in rainbow trout (Whitehead et al. 1978).
Acidic conditions throughout this period of maturation or acute
episodic exposures could exert additional influences on gamete quality.
For example, disruptions of calcium balance in maturing female fishes
as described by Beamish et al. (1975) may become important if
acid-exposure occurs at the onset or peak of vitellogenetic activity,
but were not observed as a consequence of acid-exposure during final
maturation when vitellogenesis had probably been completed.
Studies of salmonid reproduction at low pH under controlled
conditions may show physiological effects that are reflected in gamete
quality, but they do not address potential effects on spawning
behavior. Female brook trout avoided areas at pH 4.0 and 4.5 in
-------
29
selecting sites for redd construction (Johnson and Webster 1977).
Regardless of gamete quality, if female rainbow trout are unable to
locate suitable spawning habitat, reproductive failure may occur.
As yet, pH levels at which spawning behavior would be inhibited have
not been investigated for rainbow trout.
This study partitions out the direct effects of acidic water on
gamete quality and progeny survival in salmonids. As such, these data
are useful for assessing impacts on salmonid reproduction in systems
in which a high concentration of hydrogen ions is the dominant toxic
factor. However, another factor that may be important in some acidic
systems is metal toxicity. Aluminum, leached from soils by acidic
water and transported into surface waters, may reach concentrations
that are toxic to fish (Cronan and Schofield 1979). It cannot be
assumed that aluminum toxicity and hydrogen-ion toxicity will simply
be additive. Baker and Schofield (1982) found that the presence of
aluminum was antagonistic to hydrogen-ion toxicity between pH 4.2 and
4.8. Interactions between acids and metals could conceibably alter
gamete quality as well as survival of early life history stages.
Acidic waters of pH 5.5 and lower pose a threat to salmonid
reproduction and, therefore, valuable fishery resources may be
endangered. Prudent evaluations of current impacts or predictions of
future impacts of low environmental pH on fish populations must
recognize that many physiological and behavioral aspects of fish
reproduction may be affected by low pH conditions and high metal
concentrations. Caution should be taken to avoid overly simplistic
models and techniques for assessment purposes.
-------
30
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Nelson, J. A. 1982. Physiological observations on developing rainbow
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calcium ion concentrations. Journal of Fish Biology 20: 359-372.
Neville, C. M. 1979. Sublethal effects of environmental acidification
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Omernik, J. M., and C. F. Powers. 1983. Total alkalinity of surface
waters—a national map. Annals of the Association of American
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Packer, R. K. 1979. Acid-base balance and gas exchange in brook
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Journal of Experimental Biology 79: 127-134.
Packer, R. K., and W. A. Dunson. 1970. Effects of low environmental
pH on blood pH and sodium balance of brook trout. Journal of
Experimental Zoology 174: 65-72.
Packer, R. K., and W. A. Dunson. 1972. Anoxia and sodium loss
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Biochemistry and Physiology A, Comparative Biochemistry 41: 17-26.
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Peterson, R. H., P. G. Daye, and J. L. Metcalfe. 1980, Inhibition of
Atlantic salmon Salmo salar hatching at low pH. Canadian Journal
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Peterson, R. H., and D. J. Martin-Robichaud. 1983. Embryo movements
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Powers, C. F., and D. L. Rambo. 1981. The occurrence of acid
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Rombough, P. J. 1982. Effects of low pH on eyed embryos and alevins
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Rosenthal, H., and D. F. Alderdice. 1976. Sublethal effects of
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Ryan, P. M., and H. H. Harvey. 1977. Growth of rockbass, Ainbloplites
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changes in reproductive function of the rainbow trout (Salmo
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Wright, R. F., R. Harriman, A. Henriksen, B. Morrison, and L. A. Caines.
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Wright, R., and E. Snekvik. 1977. Acid precipitation: chemistry
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SNSF Project, Norway. TN37/77. 84 pp.
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41
APPENDIX 1
Effect of Prior Environmental Acidification
on Physiological Stress Responses of Juvenile Rainbow Trout
to Acute Handling Stress
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ABSTRACT
When juvenile rainbow trout, Salmo gairdneri, were exposed to
acidic conditions for 5 d, plasma glucose, plasma sodium and hematocrit
levels were significantly altered. At the lowest pH (4.7), plasma
glucose increased from 88 to 258 mg/dL and plasma sodium decreased from
154 to 130 mmol/L by day 5, and hematocrit generally remained higher
than control levels throughout the exposure. Plasma Cortisol was
affected only slightly during initial hours of exposure to test pH's of
5.7, 5.2, and 4.7. However, when the fish were subsequently subjected
to a 30-s handling stress, the post-stress plasma Cortisol level rose to
a peak of 338 ng/mL at pH 4.7 as compared with 136 ng/mL in fish held at
ambient pH (6.6). Effects on plasma glucose or sodium levels from the
handling stress were masked by the continuous response to the chronic
acid exposure. As judged by the corticosteroid response, the results
indicate that acid-stressed fish were more sensitive to additional
handling stress, even though they appeared to be physiologically normal
after 5 d. Plasma glucose and sodium appeared to be better indicators
than plasma Cortisol of chronic acid stress alone, but the greater
Cortisol response to a handling stress at the low pH suggested that this
may be a useful method of detecting increased interrenal activity during
early stages of environmental acidification.
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INTRODUCTION
The detrimental effects of acidic precipitation on fisheries
resources is a global problem of which biologists are now well aware
(see reviews by Haines 1981; Haines and Johnson 1982). In addition to
direct effects upon survival and reproductive success, the sublethal
effects of acid stress upon physiological mechanisms in fish are
becoming increasingly better understood (see reviews by Fromm 1980;
Spry et al. 1981; Wood and McDonald 1982), By comparison, relatively
little work has addressed the potentially compounding effects of chronic
acid exposure and additional stresses, such as physical disturbance or
exercise, on physiological responses in fish. Graham et al. (1982)
reported that post-exercise changes in a number of hematological and
other physiological conditions—for example, plasma ion concentrations—
were more severe in acid-exposed rainbow trout, Salmo gairdneri, than in
control fish, when held in soft water. Graham and Wood (1981) had
earlier found that severe exercise significantly increased the toxicity
of H^SO^ to rainbow trout.
The suitability of both plasma Cortisol and glucose as indicators
of certain kinds of stress in fish is now well established (see reviews
by Donaldson 1981; Schreck 1981; Wedemeyer and McLeay 1981). Recently,
Brown et al. (1984) found increases in plasma Cortisol and glucose in
rainbow trout, as well as changes in interrenal histology and plasma
thyroid hormones, during a 21-d chronic exposure to acid. To evaluate
the possibility that responses to other subsequent stresses are modified
in fish stressed by acidification, we assessed the changes in Cortisol
and glucose in rainbow trout resulting from an acute handling stress
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following a chronic exposure to low environmental pH. We also
determined plasma sodium and potassium, and hematocrit as Indicators of
possible impairment of physiological performance after exposure of fish
to acid.
METHODS AND MATERIALS
Experimental Design
Juvenile rainbow trout (Willamette R. stock) used in the experiment
weighed 51.7 ±1.9 g (mean ±SE) and had a fork length of 17.4 ±0.2 cm.
The fish were reared and maintained in ambient flow-through well water
at the Western Fish Toxicology Station, Environmental Protection Agency,
Corvallis, Oregon. Twelve days prior to the experiment (in April 1983),
the fish were transferred for acclimation to covered, outdoor
1.2-m-diameter circular tanks, each containing 350 L and receiving
4 L/min aerated, treated (soft) well water with ambient temperature of
17-19° C; pH was 6.6, alkalinity 12.6 mg/L as CaC03, total calcium 1.5
to 2.3 mg/L, and total hardness between 7.3 and 12 mg/L as CaC03.
Soft-water conditions were established and maintained by a reverse
osmosis system (Purification Techniques Inc., Brielle, New Jersey).
A total of 120 fish per tank were held at a density of 18 g/L under a
natural photoperiod and were fed daily to satiation with Oregon Moist
Pellets up to and including the day before, but not during, the
experiment.
For the experiment, aerated soft water was acidified with
concentrated reagent-grade H2SQt» and held at pH 4.0 (±0.1) with a
Beckman Model 942 pH Monitor (Beckman Instruments, Fullerton, California).
The acidified stock solution was vigorously reaerated to remove CO2,
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45
pumped to a head tank of a gravity-flow diluter, and then diluted with
softened well water to attain each experimental pH in separate mixing
chambers that flowed directly into the experimental tanks. Treatment pH
levels were automatically monitored and recorded every 3 h, and the
diluter was manually adjusted whenever necessary to maintain exposure
levels within ±0.1 pH unit.
Fish were subjected to acidic conditions by changing the inflow
supply over to the acid-blended water through a single valve at the
start of the exposure without disturbing the experimental tanks. From
the ambient pH of 6.6, final pH levels of 5.7, 5.2, and 4.7 were reached
in 20 h, and were within 0.3 pH unit of the final pH at 6 h and within
0.1-0.2 pH unit at 10 h. Temperature remained between 17 and 19° C
throughout the experiment. Duplicate tanks of fish for each pH,
including ambient, were established for sampling; an additional two
tanks with fish at ambient pH were left undisturbed as controls for the
handling phase of the experiment.
At 2, 4, 6, 10 and 20 h, and at 2, 3 and 5 d after the initial
changeover to the acidic water, five or six fish were quickly removed
with a hand net from each tank for blood sampling according to the
protocol described in the next section. After 5 d, the fish were
subjected to an acute handling stress by first capturing them with hand
nets and then holding them, as a group, out of the water in a bag seine
for 30 s and returning them to the tank; the 5-day acid exposure samples
also served as initial values for this phase of the experiment. At
1, 3, 6, 12, 24 and 48 h after handling, five or six fish were obtained
from each tank for sampling. As control, samples were simultaneously
obtained from the previously undisturbed fish in the two additional
tanks at ambient pH.
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Sampling and Analysis
Sample fish removed from the tanks were rapidly anesthetized In a
200-mg/L concentration of trlcalne methanesulfonate (MS-222); total time
for removal and complete immobilization was < 1 mln. Individual fish
were then taken serially from the anesthetic for blood sampling; total
time per tank was < 10 mln. This method has been successfully used for
Cortisol analysis in juvenile rainbow trout (Barton et al. 1980; Barton
and Peter 1982) and was verified for use under our present conditions
before this study. After severing the caudal peduncle, blood was
obtained from the caudal vasculature with an ammonium-heparinized 0.25 mL
Natelson capillary tube. Plasma was separated by centrifugation and
stored at -20 C for future assay. Additional blood was also collected
in heparinized microhematocrit tubes for immediate hematocrit determination.
Plasma Cortisol was determined by the ^-radioimmunoassay described
by Foster and Dunn (1974) and modified in our laboratory (Redding et al.
1984b) for use with salmonid plasma; inter- and intra-assay coefficients
of variation (CV) have consistently been ca. 5%. We assayed plasma
glucose by an ortho-toluidine colorometric procedure modified from
Wedemeyer and Yasutake (1977) using Sigma® pre-mixed reagent; prior to
this study, inter- and intra-assay CV were found to be 7% and 3%,
respectively. Plasma sodium (as Na+) and potassium (as K+) were
measured with a self-calibrating NOVA 1 Sodium/Potassium Analyzer
(Nova Biomedical, Newton, Massachusetts); according to manufacturer
specifications, the instrument has inter- and intra-assay CV of
<2.0% and <1*0%, respectively, with pooled serum. We determined
hematocrit as percent packed cell volume (% PCV) by direct measurement
after centrifugation, using a CRITOCAP* tube reader.
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47
Since duplicated results were in agreement, data from each pair of
tanks for each time and treatment were pooled. One-way analyses of
variance (ANOVA) were conducted both within treatments through time
and among treatments for specific times. Homogeneity of variance was
assessed using Bartlett's test (Snedecor and Cochran 1967) and, where
appropriate (i.e. for Cortisol and glucose), data were transformed to
logarithmic values for recalculation of ANOVA. Significant differences
among means were then determined using Duncan's new multiple-range test
at the 5% level (Steel and Torrie 1980). For ease of interpretation,
arithmetic means and standard errors are presented.
RESULTS
Responses to Reduced Environmental pH
Plasma Cortisol from fish held at pH 4.7 appeared to be elevated
during the course of the 5-d exposure compared with that from fish
maintained at the ambient pH of 6.6 (Table 1). Similarly, Cortisol
levels were also slightly higher in fish held at pH 5.2 and 5.7 than
in those from the ambient pH throughout this period; significant
differences from ambient were apparent during initial stages of
acidification at 2, 4, 6 and 20 h for all three test pH's (Table 1).
At 10 h, Cortisol was higher than at other times in the ambient pH
group, and was not different from the test groups. A significant
elevation in plasma glucose was evident at 2 d in the pH 4.7 group and
was ca. 300% higher, at 258 mg/dL, than in the ambient pH group at the
end of the 5-d exposure (Table 1).
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Plasma sodium was significantly depressed in fish exposed to pH
4.7 after 5 d, being 13% lower at 130 mmol/L, than the pH 6.6 group
(Table 1). Plasma potassium (data not shown) generally showed a
declining trend at all pH's from levels of around 4 to 5 mmol/L at the
beginning of the experiment to around 1 to 2 mmol/L at the end of the
experiment 7 d later. No differences from ambient pH in plasma
potassium attributable to either the chronic acidity or to the
subsequent handling stress were apparent.
Hematocrits tended to be higher in the acid-stressed fish and were
significantly so at 6 and 20 h, and at 3 and 5 d (Table 1). However, it
is not known if the differences evident at 3 and 5 d were due to the
increased environmental acidity, or to the significant decline in
hematocrit observed in the ambient pH group, possibly caused by some
other factor.
Responses to Additional Handling Stress
All groups demonstrated a significant elevation (P<0.01) in
plasma Cortisol after being subjected to the acute handling stress
(Fig. 1). However, Cortisol levels was ca. 250% higher at 1 h in fish
from pH 4.7 than it was in fish from the ambient pH, being 338 and
136 ng/mL, respectively. Plasma Cortisol also tended to remain higher
in all three acid-treated groups than in the ambient pH group for
subsequent samples, with significant differences occurring at all sample
times following handling (Fig. 1). Two fish died between 3 and 6 h
after handling at pH 4.7.
After handling, there were significant transient increases in
plasma glucose between 1 and 12 h (inclusive) in the pH 6.6 (P<0.01)
and 5.7 (P<0.05) groups (Fig. 2). However, in the pH 5.2 and 4.7
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49
groups, transient changes in plasma glucose due to the additional
handling did not differ significantly from the elevated background
levels caused by the chronic acid exposure. In the pH 4.7 group, plasma
glucose, which was already elevated at the onset of handling, continued
to increase and reached a maximum of 335 mg/dL at 48 h after handling
(Fig. 2). Plasma glucose in the pH 5.2 group also remained elevated
above that in the ambient pH group at 48 h post-handling (Fig. 2).
All groups, including ambient pH, showed transient decreases in
plasma sodium after handling (Fig. 3); temporal changes were significant
in pH groups 6.6 (PC0.05), 5.7 (P<0.01) and 4.7 (P<0.05). Plasma sodium
in fish at pH 4.7, already depressed just before handling, continued to
decline significantly to the low level of 114 mmol/L observed at 48 h
post-handling (Fig. 3).
A transient increase in hematocrit to 57% in the pH 4.7 group 3 h
after handling was significantly greater than the hematocrit of 45%
observed in the ambient pH group at the same time (Fig. 4). Although
hematocrit generally tended to be higher in lower pH groups after
handling, differences among treatments at other time intervals were not
significant. There was also a significant increase in hematocrit over
the 48-h post-handling period in the unhandled control group held at
ambient pH (Fig. 4).
DISCUSSION
Acid-stressed fish appear to be more sensitive to additional
handling stress than unstressed fish. When the rainbow trout in our
experiment were subjected to the 30—s handling stress, the effect of
prior acidification on interrenal activity was clearly apparent in the
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50
higher plasma Cortisol response to handling at the low pH. However, any
possible effects on glucose or sodium levels from handing were masked by
the continuous response to the chronic acid exposure. From the acid
exposure alone, plasma glucose, plasma sodium and hematocrit levels were
significantly altered after 5 d, particularly at pH 4.7, but plasma
Cortisol was not.
There was a slight acute corticosteroid response to the acid
conditions during the initial 20 h of exposure, which appeared to be
recovered by 2 d. Mudge et al. (1977) also reported a transient rise in
plasma Cortisol in brook trout, Salvelinus fontinalis, at 1-3 h during
exposure to pH 4.0, with titers returning to control levels within 24 h.
Our findings of apparently recovered, or resting, plasma Cortisol levels
after 2-5 d of acid exposure are consistent with those of Brown et al.
(1984) for the early phase of chronic acid exposure, as they did not
show changes in plasma Cortisol in rainbow trout during environmental
acidification until after 8 d; in their study, sampling began after Id.
Moreover, Lee et al. (1983) showed no significant change in plasma
Cortisol of rainbow trout after 2- or 3-wk exposures to pH's as low
as 4.2, although interim samples were not taken. The small transitory
increase in plasma Cortisol at 10 h in the ambient pH group may have
resulted from increased activity around the tanks or from repeated
sampling, although effects of these factors were not evident in the
handling phase of this experiment, nor in other investigations
(Barton et al. 1980; B.A. Barton anb C, B. Schreck, unpubl. data).
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When our fish were subjected to the 30-s handling stress after 5 d
of acidification, the plasma Cortisol response of the pH 4.7 group was
about double that of the other pH groups including the ambient pH. This
result supports that of Brown et al. (1984), who concluded that chronic
acid exposure stimulated increased activity of interrenal cells, as
demonstrated by interrenal hyperplasia and increased size of nuclei
after 4 d. By comparison, Mudge et al. (1977) found evidence of
decreased cell and nuclear size, suggesting that activity of interrenal
tissue decreased after acid exposure. However, it should be noted that
their histological investigation was restricted to measurements taken
after 3- to 24-h exposure periods only. On the basis of the findings of
Brown et al. (1984) of increased interrenal activity and our results
from the subsequent handling stress, we speculate that clearance rate of
Cortisol may also have increased during chronic acidification to
compensate for the increased interrenal output, as a measure directed
toward maintaining homeostasis. For example, an increase in plasma
corticosteroid clearance rate in response to a chronic environmental
change was observed by Redding et al. (1984a), who subjected freshwater-
adapted juvenile coho salmon, Oncorhynchus kisutch, to continuous
salinity. Thus, the hypothalamic-pituitary-interrenal axis may have
been stimulated in response to chronic acid stress, but the differences
in response—i.e., at pH 4.7 as compared to ambient—were not apparent
until the fish experienced an additional rapid plasma corticosteroid
increase, caused by the 30-s handling stress. The pattern of rapid
plasma Cortisol elevation, followed by recovery, in response to the
handling stress was characteristic of what has been previously shown for
juvenile salmonids (Strange and Schreck 1978; Barton et al. 1980;
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Pickering et al. 1982; B. A. Barton and C. B. Schreck 1983, unpublished
results); this subject has been reviewed extensively by Donaldson (1981)
and Schreck (1981).
Our results demonstrating an increase in plasma glucose after 2-d
exposure to pH 4.7 corroborate those of Brown et al. (1984) who found
significant rises in plasma glucose of rainbow trout after 4-d exposures
to both pH 4.7 and 5.2. Similarly, Lee et al. (1983) reported that
plasma glucose levels were higher in rainbow trout exposed for 2 wk to
pH 4.2 and pH 4.8, than in those held at higher pH's. When fish were
given the handling stress after the acid exposure, plasma glucose levels
in the ambient and pH 5.7 groups showed response-recovery patterns
typical of salmonids subjected to an acute physical disturbance (Nakano
and Tomlinson 1967; Wedemeyer 1972; Wydoski et al. 1976; Casillas and
Smith 1977; Perrier et al. 1978; Pickering et al. 1982; B.A. Barton and
C.B. Schreck 1983, unpublished results). However, at the two lower
pH's, handling responses were masked by the higher background glucose
levels that resulted from acid exposure; this was particularly evident
at pH 4.7. At 6-7 d of acidification at pH 4.7, plasma glucose was
elevated to 300-350 mg/dL, which is similar to the level Brown et al.
(1984) reported after a 21-d exposure to this pH.
We suspect that the comparatively high glucose elevation observed
at 6-7 d in response to the acid stress in our experiment was a
combination of: the relatively soft ambient water, the aggravating
effect of the additional handling at 5 d, and the relatively high
ambient temperature used in our experiment. Recent evidence with
juvenile chinook salmon, Oncorhynchus tshawytscha, (B. A. Barton and
C.B. Schreck 1984, unpublished results) has clearly shown that the
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53
glucose response to a similar 30-s handling stress was three times
greater in fish acclimated to 21° C than in those acclimated to 12 or
7° C, and was very similar to that found for the ambient pH group in
this experiment. The apparently more rapid response of plasma glucose
to the acid stress alone in our fish than in those of Brown et al.
(1984), may also have been because of the relatively soft water used in
our study. For example, Graham et al. (1982) found that physiological
responses were more severe in acid-exposed trout subjected to strenuous
exercise when held in soft water rather than hard water, and
post-exercise mortality doubled.
Leach and Taylor (1980) provided evidence to indicate that
increased endogenous Cortisol may have a functional role in sustaining
elevated glucose levels in response to stress. Possibly, increased
interrenal activity resulting from acid exposure, demonstrated
histologically by Brown et al. (1984) and in our experiment by the
secondary handling stress challenge, may be responsible for an increased
Cortisol output to maintain high levels of blood glucose as a readily
available energy source. Mobilization of energy reserves would
presumably be necessary to allow the fish to cope with the effects of
chronic acid stress, such as by increasing metabolic or ionoregulatory
activity.
The decline in plasma sodium concentration in fish exposed to pH
4.7 for 5 d was similar to the decline observed at pH 4.5 in the adult
rainbow trout as described and discussed earlier in the main body of
this report. In that investigation, plasma sodium decreased from 153 to
145 mmol/L after 7 d exposure of pH 4.5. Reduced plasma sodium as a
result of acid exposure has also been reported by others in juvenile
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54
salmonids (Lee et al. 1983; Saunders et al. 1983; see reviews by Fromm
1980; Wood and McDonald 1982). Decreased sodium Influx rates and
increased efflux rates at low pH were previously demonstrated in brook
trout by Packer and Dunson (1970). Furthermore, ionoregulatory
dysfunction in rainbow trout exposed to acidic conditions was
exacerbated in soft water (McDonald et al. 1980).
The perturbations in plasma sodium within the first 24 h after
handling in pH 6.6 and 5.7 water are consistent with those which Redding
and Schreck (1983) observed for juvenile coho salmon chronically
stressed by confinement in fresh water. These results support the
notion that under normal conditions, there is a short-lived reduction in
ionoregulatory ability when fish are subjected to physical disturbances.
The results differ from those of Graham et al. (1982), who observed an
elevation in plasma sodium of rainbow trout after 6 min strenuous
exercise at pH 7.5 in both hard and soft water. Graham et al. (1982)
attributed an increase in blood constituents to high intracellular
lactate loading, causing water to move across the osmotic gradient with
a resultant decrease in blood volume. Although there was an indication
of a slight increase in plasma sodium 1 h after handling in the ambient
and higher pH groups, we postulate that the handling stress used in our
experiment was insufficient to sustain through time the lactate response
normally associated with severe exercise. For example, we found that in
juvenile chinook salmon subjected to a 30-s handling stress similar to
that used in the present study, there was a transient rise in plasma
lactate but the fish recovered within 3 h (B. A. Barton and C. B. Schreck
1983, unpublished results). By comparison, Graham et al» (1982)
reported that lactate levels peaked at 2 h and took 8 h or more to
recover.
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55
The continuous decline in plasma sodium at pH 4.7, like that of
plasma glucose, masked any transitory changes that may have resulted
from the additional handling stress. Although less obvious, this
decreasing trend was probably responsible for the lack of significance
of the handling response at pH 5.2 as well.
The tendency towards increased hematocrit in the acid-exposed
fish is in agreement with earlier studies (Dively et al. 1977; Neville
1979; Milligan and Wood 1982; Saunders et al. 1983), and Milligan and
Wood (1982) attributed the increase to erythrocyte swelling.
Furthermore, they found a redistribution of body water, resulting in
decreased plasma volume with no change in total body water. Handling
stress has also been shown to increase hematocrit (Fletcher 1975; Soivio
and Oikari 1976) and erythrocytic swelling (Soivio et al. 1977) in fish.
In the handling phase of our experiment, only the combined stress of
handling and acidity at pH 4.7 was sufficient to cause a significant
increase in hematocrit above the pre-handling level.
As a management strategy, fish stocking will probably be used
more extensively in the future to provide short-term fisheries
in waters which no longer support natural reproduction because of
reduced pH. Since we have shown that acid-stressed fish could have an
increased sensitivity to additional stresses, particularly at pH's
below 5, fish stocked in acid waters may appear to be physiologically
normal (e.g. Cortisol titer) within a few days after planting, when in
fact they are not. The observed mortality in the pH 4.7 group, 3 to 6 h
after handling, supports that view. Thus as a management consideration,
extra attention should be given to ensure survival of fish stocked in
acidic waters, especially if the fish are likely to encounter other
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56
stressful conditions in the new environment. As physiological stress
Indicators, our results show that both plasma glucose and plasma sodium
appeared to be more suitable for chronic stress than plasma Cortisol;
this observation for glucose agrees with the conclusion of Brown et al.
(1984). However, a secondary acute stress challenge, such as the 30-s
handling stress employed in our study, may prove to be a useful method
for determining possible increases in interrenal activity at an early
stage of environmental acidification as an alternative to histological
examination.
ACKNOWLEDGMENTS
We sincerely thank Scott B. Brown, Freshwater Institute, Canada
Department of Fisheries and Oceans, and Larry R. Curtis, Department of
Fisheries and Wildlife, Oregon State University, for critically
reviewing the manuscript prior to submission. We also thank Reynaldo
Patino and Jane Linville, Oregon Cooperative Fishery Research Unit, for
their technical assistance during the sampling phase of the study, and
the staff at the Western Fish Toxicology Station, Corvallis, Oregon, for
providing and maintaining fish and facilities for the experiment.
Mention of trade names or manufacturers does not imply
U.S. Government endorsement of these commercial products.
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57
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63
Table 1. Mean plasma Cortisol (ng/mL, n«10), plasma glucose (mg/dL,
n«8), plasma sodium (mmol/L, n«8) and hematocrit (Z PCV, n=8-13)
values ±SE in juvenile rainbow trout after continuous exposure to
reduced environmental pH. Sample sizes (n) represent pooled data
from duplicate treatments. Values marked with an asterisk (*)
indicate a significant difference from the ambient pH (6.6) at that
time point (Duncan's new multiple-range test at 5%).
Time
pH 6.6
pH 5.7
- pH 5.2
pH 4.7
2 h
Cortisol
Glucose
Sodium
Hematocrit
16 ±5
86 ±7
151 ±3
46 ±1
39 ±15*
87 ±6
148 ±3
48 ±2
44 ±15*
94 ±13
154 ±2
48 ±2
27 ±9*
88 ±14
154 ±2
50 ±1
4 h
Cortisol
Glucose
Sodium
Hematocrit
6 ±3
92 ±6
149 ±4
48 ±1
49 ±15*
80 ±3
148 ±4
47 ±1
44 ±10*
95 ±11
153 ±2
49 ±1
50 ±34*
80 ±3
154 ±1
51 ±1
6 h
Cortisol
Glucose
Sodium
Hematocrit
10 ±4
99 ±11
152 ±2
47 ±1
44 ±10*
91 ±6
152 ±2
50 ±1
32 ±9*
84 ±6
152 ±2
50 ±2
33 ±10*
80 ±4
155 ±2
53 ±1 *
10 h
Cortisol
Glucose
Sodium
Hematocrit
34 ±6
93 ±10
149 ±1
49 ±2
29 ±10
90 ±9
149 ±2
50 ±1
58 ±12
99 ±14
153 ±1
50 ±2
55 ±19
92 ±5
151 ±2
51 ±1
20 h
Cortisol
Glucose
Sodium
Hematocrit
16 ±5
74 ±3
149 ±2
48 ±1
47 ±11 *
91 ±12
147 ±2
50 ±1
48 ±6 *
109 ±13
152 ±1
53 ±1 *
49 ±12*
89 ±6
143 ±2 *
53 ±2 *
2 d
Cortisol
Glucose
Sodium
Hematocrit
21 ±9
77 ±6
144 ±1
48 ±2
10 ±3
92 ±10
142 ±1
49 ±1
8 ±2
101 ±7
139 ±3
50 ±1
26 ±8
166 ±21*
138 ±2
53 ±2
3 d
Cortisol
Glucose
Sodium
Hematocrit
15 ±6
87 ±9
147 ±2
43 ±1
16 ±8
94 ±8
149 ±2
50 ±2 *
12 ±4
110 ±19
150 ±2
50 ±1 *
30 ±7
154 ±19*
143 ±4 *
52 ±2 *
5 d
Cortisol
Glucose
Sodium
Hematocrit
9 ±3
85 ±10
149 ±2
42 ±1
9 ±7
79 ±5
145 ±3
47 ±1 *
11 ±3
143 ±30
143 ±4
46 ±1 *
23 ±9
258 ±42*
130 ±3 *
49 ±1 *
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64
Figure 1* Responses of plasma Cortisol (ng/mL ±SE) in juvenile
rainbow trout subjected to a 30-s handling stress after
5 d during continuous exposure to pH's of 5.7, 5.2 or 4.7,
or ambient pH of 6.6. Sample sizes of n«10 represent
pooled data from duplicate treatments. Values marked with
an asterisk (*) indicate a significant difference from the
ambient pH at that time point (Duncan's new multiple-range
test at 5%). Open squares are values for unstressed
controls at ambient pH.
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450n
pH 6.6
-o pH 5.7
o-
pH 6.2
pH 4.7
350-
o unhandUd control* at pH 6.6
E
m
9
E
250
E
s
o
*
150
9
c
100 -
.4.7
5.7
6.6
01 3
6
48
12
24
Hours after Handling Stress
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66
Figure 2. Responses of plasma glucose (mg/dL ±SE) in juvenile
rainbow trout subjected to a 30-s handling stress after
5 d during continuous exposure to pH's of 5.7, 5.2 or 4.7,
or ambient pH of 6.6. Sample sizes of n=8 represent pooled
data from duplicate treatments. Values marked with an
asterisk (*) indicate a significant difference from the
ambient pH at that time point (Duncan's new multiple-range
test at 5%). Open squares are values for unstressed
controls at ambient pH. (See Fig. 1 for legend.)
-------
450-i
350H
4.7
E
»
co
E
250-
x
CO
o
o
X
3
o
9 150
E
5.2
5.7
100
6.6
50-^
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68
Figure 3. Responses of plasma sodium (mmol/L ±SE) in juvenile
rainbow trout subjected to a 30-8 handling stress after
5 d during continuous exposure to pH's of 5.7, 5.2 or 4.7,
or ambient pH of 6.6. Sample sizes of n*8 represent pooled
data from duplicate treatments. Values marked with an
asterisk. (*) indicate a significant difference from the
ambient pH at that time point (Duncan's new multiple-range
test at 5%). Open squares are values for unstressed
controls at ambient pH. (See Fig. 1 for legend.)
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160
(0
E 140
s»
CO
CL
5.7
5.2
-I
s
E
3
4.7
¦O 120
O
to
100
48
3
24
Hours after Handling Stress
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70
Figure 4. Responses of hematocrit (Z PCV ±SE) in juvenile rainbow
trout subjected to a 30-8 handling stress after 5 d
during continuous exposure to pH's of 5.7, 5.2 or 4.7,
or ambient pH of 6.6. Sample size6 of n«6-13 represent
pooled data from duplicate treatments. Values marked with
an asterisk (*) indicate a significant difference from the
ambient pH at that time point (Duncan's new multiple-range
test at 5%). Open squares are values for unstressed
controls at ambient pH. (See Fig. 1 for legend.)
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60
4.7
50
5.2
5.7.
40
35
30
0 1 3
6
12
24
48
Hours after Handling Stress
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