THE EFFECTS OF LOW PH ON SALMONID EMBRYOGENESIS AND
VITELLOGENESIS
David B. Parker
B.Sc.,Simon Fraser University, 1977
THESIS SUBMITTED IN PARTIAL FULFILLMENT OF
THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE
in the Department
of
Biological Sciences
@
David B. Parker 1985
SIMON FRASER UNIVERSITY
January, 1985
All rights reserved. This work may not be
reproduced in whole or in part, by photocopy
or other means, without permission of the author.
APPROVAL
NAME:
-
David B
. Parker
DEGREE :
Master of Science
TITLE OF THESIS:
T h e effects of l o w p H on s a l m o n i d e m b r y o g e n e s i s
and v i t e l l o g e n e s i s
.
EXAMINING COMMITTEE:
Chairman :
Dr.
P.C.
Oloffs
D r . B. A. M c K e o w n , S e n i o r Supervisor
-.
D r . G .H. ' G e e n
D r . T.A. W a t s o n , E n v i r o n m e n t a l S t u d i e s C o - o r d i n a t o r ,
B.C. H y d r o
I
1
"
- ,
& -
rell, P u b l i c Examiner
'i
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ABSTRACT
The effects of low pH water on embryogenesis and
vitellogenesis in kokanee and sockeye salmon ( O n c o r h y n c h u s
n e r k a ) were investigated. Eggs were exposed to low pH from
fertilization to 45 days post-median hatch or to an episodic
exposure at pH 4.0. Adult kokanee were also exposed to low pH
just prior to ovulation and spawning. Alkali-labile vitellogenin
(Vg)-bound phosphates and calcium levels were measured in the
plasma of acid exposed rainbow trout (Salrno g a i r d n e r i
).
The
effect of low pH on calcium metabolism was also investigated by
measuring gill c a 2 + - ~ ~ p a and
s e i n v i t r o gill osmotic water
uptake from rainbow trout exposed i n v i v o .
The most sensitive stages of development during chronic or
episodic exposure to low pH were early embryonic development and
newly-hatched alevins. Incubation of eggs at low pH caused a
lower median survival, delayed hatching, higher alevin mortality
and reduced the efficiency of yolk conversion to tissue of
yolk-sac alevins. Those effects were more pronounced when the
eggs were fertilized at low pH.
Exposure of sexually mature kokanee salmon to acidified
water reduced egg and alevin survival, delayed embryo hatching
and decreased the percent hatch. Those effects were more
pronounced when their eggs were incubated at low pH.
Rainbow trout exposed to pH 5.0 had significantly lower
gonadosomatic and hepatosomatic indices compared to control
fish, and did not exhibit an increase in plasma Vg-bound
iii
phosphates indicating that egg production was affected. An
increase in environmental calcium concentration significantly
affected the plasma vitellogenin levels in fish. No evidence of
bone demineralization was found in females exposed to low pH.
After 14 days exposure to different levels of pH and
environmental calcium, trout exposed to an increased calcium
concentration had a lower plasma osmolarity independent of the
environmental pH. Gill Ca2+-ATPase activity was not affected by
low pH, but was reduced in fish exposed to Ca2+ enriched water.
The increase in gill osmotic water permeability in fish exposed
to Ca2+-enriched water may have been associated with the
increase in osmolarity of the ambient media.
The effect of low environmental pH on freshwater survival
of salmonids is discussed with particular reference to
embryogenesis and vitellogenesis, and the ameliorating effects
of water hardness.
ACKNOWLEDGEMENTS
I would like to extend my sincere appreciation to my
supervisor and friend Dr. B.A. McKeown for his continuous
support and guidance throughout the duration of my studies. I
would also like to thank Dr. T.A. Watson for his help on assay
techniques used in this study, and for his advice and friendship
through some difficult moments. I wish to extend my gratitude to
Dr. G.H. Geen for his advice and helpful criticism of this
thesis.
To my parents, Jean and Bern, I thank them for their
patience and moral support. I wish to thank my wife, Kathleen,
for her love, support and encouragement.
Funds for this study were provided, in part, through a
G.R.E.A.T.
award by the Science Council of British Columbia in
collaboration with the I.P.S.F.C.
and B.C. Hydro. Special thanks
to Drs. J.A. Servizi (1.p.S.F.C)
and R.G. Ferguson (B.C. ~ y d r o )
for their involvement in this project.
I thank Jeff Johansen for his technical assistance. I
express special thanks to Bethan Chancey for her love,
encouragement and support during those intensive weeks before my
defence.
TABLE OF CONTENTS
.................................................... i i i
Acknowledgements ..............................................v
...
~ i s tof Tables .............................................vlll
List of Figures ..............................................ix
A . General Introduction ....................................... 1
Abstract
B
. Chapter
I-The effects of low pH on egg and alevin
survival of kokanee and sockeye salmon. O n c o r h y n c h u s
................................................... 4
I . Introduction .........................................5
I1 . Materials and Methods ..............;................8
Kokanee salmon experiment: ...............8
Sockeye Salmon Egg Experiments ..........13
Water quality analysis ..................14
Statistics ..............................15
I11 . Results ...........................................1 6
Kokanee salmon experiments ..............16
Sockeye salmon experiments ..............24
IV . Discussion .........................................3 6
nerka
C
. Chapter
11-Effects of pH and/or calcium-enriched
freshwater on plasma levels of vitellogenin and Ca2+.
and on bone calcium content during exogenous
vitellogenesis in rainbow trout
........................44
Introduction
I1
.
..............................49
Experimental Fish .......................49
ater rials and Methods
Acid Dilution Apparatus
................... 54
Statistical Analyses ....................55
I11 . Results ...........................................57
IV . Discussion .........................................65
Analytical Procedures
D
. Chapter
111- Effects of pH and/or calcium-enriched
freshwater on gill Ca2+-Adenosine triphosphatase
( A T P ~ S activity
~)
and osmotic water inflow in rainbow
trout
..................................................73
I . Introduction ....................................... :74
I1 . ater rials and Methods ..............................78
Experimental Fish .......................78
Dilution Apparatus ......................7 8
Experimental design .....................7 9
Gill perfusion ..........................80
Gill Preparation for C a 2 + - ~ ~ p a Assay
se
..81
c ~ ~ + - A T Assay
P ~ s ~.......................8 2
Osmotic Water Inflow ....................8 4
Statistics ..............................8 5
I11 . Results ...........................................8 7
Blood Parameters .......................8 7
ca2+-A~paseActivity ....................8 7
Osmotic Water Inflow ....................90
Plasma Calcium. Hematocrit and
Osmolarity
.......................9 4
................... 101
E . Summary ..................................................105
References ..................................................107
Osmotic Water Uptake
vii
LIST OF TABLES
TABLE
1
PAGE
Median survival time of kokanee salmon eggs
exposed to low pH
................................ 18
Days to hatching, 50% hatch and % hatch kokanee
salmon embryos exposed to low pH
.................
20
The efficiency of yolk to tissue conversion in
kokanee salmon alevins exposed to low pH
......... 2 2
Length of kokanee alevins after 118 days exposure
to low pH
........................................
23
Median survival and hatching data of sockeye
salmon eggs exposed to low pH
.................... 27
Hatching data of sockeye salmon embryos exposed to
low pH for 24 h .................................. 30
Calcium content of the caudal centra of rainbow
trout exposed to low pH and/or
calcium-enriched water
........................... 6 3
Female / male plasma Ca2+ ratio of rainbow trout
exposed to low pH and/or calcium-enriched
water
............................................ 6 4
Blood parameters of rainbow trout after 14 days
exposure to low pH and/or calcium-enriched
freshwater
....................................... 88
Ca2+-ATPase activity in rainbow trout after 14
days exposure to low pH and/or
calcium-enriched water
........................... 8 9
Protein concentrations and C a 2 + - A T P ~ Sin~ rainbow
trout after 14 days exposure to low pH and/or
calcium-enriched water
........................... 92
viii
LIST OF FIGURES
FIGURE
PAGE
Line diagram of the experimental apparatus used to
expose sexually maturing kokanee salmon to low
pH
...............................................
Line diagram of the egg incubation chambers ..........
Survival curves of kokanee salmon eggs and alevins
exposed to low pH ................................
11
12
17
Survival curves of sockeye salmon eggs and alevins
exposed to low pH
................................ 2 5
Survival curves of sockeye salmon eggs exposed to
low pH ........................................ 28
Survival curves of sockeye salmon eggs and alevins
exposed to pH 4.0 for 2 4 h ....................... 2 9
Dry tissue / dry yolk
tissue ratio of sockeye
salmon embryos exposed to low pH ................. 3 3
Dry tissue / dry yolk + tissue ratio of sockeye
salmon embryos exposed to pH 4.0 for 24 h ........ 3 4
+
Total length of sockeye salmon embryos exposed to
low pH
........................................ 3 5
Line diagram of the experimental apparatus used to
exposed rainbow trout to low pH and/or
increased C a + 2 concentrations
.................... 5 3
Plasma alkali-labile phosphorus levels in female
rainbow trout exposed to low and/or increased
Ca+2 concentrations
.............................. 5 9
Plasma calcium ion concentrations in female
rainbow trout exposed to low and/or increased
C a + 2 concentrations
.............................. 6 0
Effect of low pH and/or calcium-enriched water on
the GSI and HSI of female rainbow trout .......... 61
Effect of low pH and/or calcium-enriched water on
the i n vitro osmotic water inflow of the gills
of rainbow trout
................................. 9 3
A. General Introduction
Acid deposition is a global problem receiving a great deal
of study and public interest with respect to its impacts upon
aquatic ecosystems (Likens
et
al., 1979; Cowling, 1 9 8 2 ) . The
basis of acid deposition is an increase in emissions of mineral
acids from fossil fuel combustion and other anthropogenic
sources (Schindler, 1979; Spencer, 1 9 8 2 ) . These compounds enter
aquatic ecosystems as dry deposition or react with oxygen and
water vapour in the atmosphere to form sulphuric as well as
nitrous and nitric acids, which enter freshwater lakes and
streams by precipitation and surface runoff. quat tic systems
particularly sensitive to acid deposition are generally soft,
poorly-buffered waters low in calcium and magnesium
right
et
a l . , 1980; Henriksen, 1 9 8 2 ) .
In northwestern North America, in particular, the west
coast of British Columbia, granitic rock and thin soils low in
buffering and ion exchange capacity, occupy a large portion of
coastal areas ( ~ i e n s ,1 9 8 4 ) . Although these areas are not
presently undergoing significant acidification, bedrock geology
and water quality data suggest these areas would be sensitive to
acid input.
Acidification of lakes and streams has resulted in reduced
or complete elimination of fish populations in Scandanavian
countries and northeastern North America (~eamishand Harvey,
1972; Almer e t
Leivistad
et
al., 1974; Beamish, 1974a; Harvey, 1975;
al., 1976; Schofield, 1976; Harvey and Lee, 1982;
Schofield, 1 9 8 2 ; ) The reduction in fish populations of acidified
lakes has been attributed, in part, to decreased recruitment
from a failure of mature fish to spawn (~eamishand Harvey,
1972; Beamish e t al., 1 9 7 5 ) and increased mortality in
developing embryos (Peterson
et
al., 1 9 8 2 ) .
While numerous workers have investigated the effects of low
pH on embryo and alevin development in salmonid species
(Krishna, 1953; Kwain, 1975; Menendez, 1976; Daye and Garside,
1977;
Trojnar, 1977; Carrick, 1979; Daye and Garside, 1979;
Daye, 1980; Peterson
et
al., 1980; Brown and Lynam, 1981;
Nelson, 19821, none had observed the effects of low pH on
Pacific salmon species (Oncorhynchus ) when the present study
commenced. No studies have investigated the effects of
acidification on salmonids during the vitellogenic period.
Many British Columbia waters support salmonid species,
either resident or migratory populations, and many of these
waters are sensitive to acidification (Swain, 1 9 8 4 ) . Generally,
sockeye salmon (Oncorhynchus nerka) migrate further into
headwater streams and spend longer in freshwater before
spawning, compared with other pacific salmon species. Therefore
acidification of freshwater lakes and streams may potentially
affect gonadal maturation as well as embryonic development in
sockeye salmon. Kokanee salmon are the landlocked or freshwater
form of sockeye salmon and consequently, may be even more
suscbptible to the impacts of acidification.
To elucidate the potential impacts of acidification on
Pacific salmon species, I investigated the effects of low pH on
both the ovulation and early development of 0. nerka, and on
vitellogenesis in rainbow trout
(Salmo
gairdneri). In this
manner, the most sensitive stages of development between
exogenous vitellogenesis and the fry stage could be identified.
Since it was not possible to obtain kokanee or sockeye salmon in
the earlier stages of ovarian development, rainbow trout (S.
gairdneri) were selected for use in studies described in Chapter
1 1 . Among salmonids rainbow trout make a good subject to study
the effects of low pH on vitellogenesis, as they are considered
similar to sockeye salmon in their sensitivity to acid
ombo bough, 1982). Calcium uptake is important during the
vitellogenic period in fish and any disturbance in calcium
metabolism might inhibit vitellogenin production. Therefore, in
Chapter I 1 1 the effects of low pH on calcium ion transport and
osmotic water uptake in the gills of rainbow trout was
investigated.
B. Chapter I-The effects of low pH on egg and alevin survival of
kokanee and sockeye salmon, Oncorhynchus nerka
I. Introduction
Low pH may increase fish egg and alevin mortality, reduce
the percent hatch, inhibit hatching, and reduce alevin growth
(see review by Peterson
et
al.,
1982). Only a few studies have
demonstrated these effects on salmonid embryos fertilized at low
pH and subsequently incubated at low pH (~enendez,1976;
Trojnar, 1977; Carrick, 1979; Brown,1982b). Other studies
involving exposure of maturing adult fish, and subsequent
fertilization and embryo incubation at low pH have been
performed on non-salmonid species (Mount, 1973, fathead minnows;
Craig and Baksi, 1977, flagfish; Lee and Gerking, 1980, desert
pupfish).
The results from studies investigating the effects of low
pH on salmonid embryo development are inconsistent and are
complicated by differences in prior adult exposure to low pH.
Also these studies did not compare all possibilities, ie. eggs
fertilized at low pH and incubated at control pH levels versus
eggs fertilized at control pH incubated at low pH. Studies on
eggs not fertilized at low pH before incubation could
underestimate lethal pH levels. Episodic exposure of eggs during
fertilization or at later developmental stages, or continuous
low pH exposure from fertilization may produce differences in
embryo survival.
In acidified lakes and streams pH levels are variable, and
during periods of high precipitation and snowmelt water bodies
having non-toxic pH levels may experience large increases in
hydrogen ion concentration. These events generally occur in
spring or fall, coinciding with the reproductive events of
salmonid fish.
Nelson (1982) presented indirect evidence that reduced
embryo growth at low pH may result from an inhibition of yolk
conversion into adult tissue. The results of several other
studies have also suggested reduced yolk conversion in alevins
(~rojnar,1977; Brown and Lynam, 1981).
Menendez (1976) found that exposure of adult brook trout,
(Sal vel i nus f o n t i nal i s), to low pH af fected the survival of the
eggs and alevins. Exposure of female fish to low pH has been
shown to affect gonadal maturation, resulting in reduced egg
production and yolk quality ( ~ o u n t ,1973; Beamish, 1974a; Craig
and Baksi, 1977; Lee and Gerking, 1980). These effects are
probably a result of the inhibition of yolk production and/or
incorporation (Ruby et al. , 1977). However, low pH exposure
during final egg maturation and ovulation prior to spawning may
also affect embryo survival.
In this chapter I investigated the effects of low pH on
embryo survival, hatching, and alevin growth and mortality, in
eggs obtained from sexually mature kokanee salmon (0.nerka)
exposed to low pH during final maturation and ovulation. Sockeye
salmon (0.n e r k a ) eggs were exposed to episodic events of low pH
or continuously incubated at low pH, with or without
fertilization at low p H . Therefore, the most sensitive stages of
embryological development in 0. n e r k a exposed to low p H could be
determined, as well as the effects of prior adult exposure and
fertilization at low p H .
IS.
ater rials and Methods
Kokanee salmon experiment:
Kokanee salmon (0.nerka) (77-136 g body weight and 21-24
cm fork length) were caught in a trap net set at the mouth of
the Upper Chilliwack River, ~ritishColumbia. Fish were caught
during their upstream spawning migration ( ~ u l y28 to Aug. 4) and
were transferred to the aquarium facilities at the Sweltzer
Creek Laboratory of the International Pacific Salmon Fisheries
Commission (1.p.S.F.C.).
The fish were held in a 1500-L
fiberglass tank supplied with continuously flowing freshwater
(hardness, 80-85 mg*L- ' CaCO,; alkalinity, 60.6-68:2
mg*L- '
CaCO,; pH 7.0-7.28; dissolved oxygen, above 90% saturation;
temperature 9.0+1.0•‹C).
After 7 days, 10 female and 10 male kokanee salmon were
transferred to each of the 3 experimental tanks and pH of the
water was adjusted to experimental levels over 24 h. The fish
were exposed for 24-36 d to the following pH (mean+S.E.):
Tank 1 : pH 5.1 f 0.13
Tank 2 : pH 5.6 f 0.46
Tank 3 : pH 7.1 + 0.06
These pH levels were selected to determine the sublethal
effects of low pH exposure. During the exposure period female
fish were examined routinely to determine their time of
ovulation. Approximately 100-150 eggs were collected from mature
females in each tank and wet fertilized with sperm from males
held at the same pH. Eggs were incubated at pH 4.3+0.1,
5.1k0.08, 5.520.1 and control, 7.6k0.02 (mean
+
S.E.).
Eggs and alevins were removed for histological observation
as numbers permitted during the exposure period. Five eggs were
removed from each basket on days 3, 17, 33 and 51. Ten alevins
were sampled on day 89 and 118, anesthestized in ethyl
m-aminobenzoate (MS-222, 50 rng@~-')and their weight ( 9 ) and
body length (mm) were measured. Eggs and alevins were preserved
in either Stockard's solution (50 ml formaldehy.de, 40 ml glacial
acetic acid, 60 ml glycerin, 850 ml distilled H,O) or 5%
buffered (pH 7.0) formalin (50 ml formaldehyde, 4.0 g sodium
phosphate monobasic, 6.5 g sodium phosphate dibasic, made up to
1 L with distilled water). Yolk material was separated from the
body tissue of preserved alevins and both parts were dried at
80•‹C for 24 h to determine dry weight.
Dead eggs, identified by yolk opaqueness, were counted and
removed daily. Alevins were considered dead when their yolk sac
turned opaque and their heart stopped beating. Identification of
the kokanee egg development stages were made according to those
stages outlined by Velson (1980) for sockeye salmon. Eggs were
stained with toluene blue after removal of the egg capsule to
-
provide contrast to the embryonic structures.
Fish exp0sur.e tanks
Freshwater obtained from the hypolimnetic zone of Cultus
Lake flowed into buckets and exited through a plexiglass down
tube at approximately 500 Lob-' into the exposure tanks (1500
~)(~ig.
1).
The flow rate allowed for 90% replacement of water
in 7 h and a flow of 4.15 L of water for every kg of fish as
recommened by Sprague, (1973). The water in the tanks was
continuously aerated with compressed air. Acidic pH levels were
obtained by adding the required volume of H2S0, stock solutions
to the water in the buckets by peristaltic pump.
Egg Exposure Chambers
Incubation chambers were made of black plexiglass and
covered with a black lid ( ~ i g .2).
A
continuous flow of water
into the 'header' tanks was regulated by 'stop cock' valves. To
obtain the desired pH in the exposure tanks, H2S0, stock
solutions were added to the water in the 'header tanks' by
peristaltic pumps. Acidified water exiting from the bottom of
the 'header tank' was regulated by 'stop cock' valves to
maintain a flow rate of 200 mlomin-' into the incubation
chambers.
Fig. 1 . Line diagram of the experimental apparatus used to
expose sexually maturing kokanee salmon to various pH levels.
Water flow
f
I Peristaltic
m
Hole size cut to allow
a flow of approximately
500 L.h-1
llb
(
H2YU41
Stock
Somon
I
U
Fig. 2. Line diagram of the egg incubation chambers and
experimental apparatus used to expose kokanee and sockeye salmon
eggs and alevins to acidified water.
Sockeye Salmon Egg Experiments
Sockeye salmon eggs were obtained November 1 , 1981, at the
Weaver Creek spawning channel, British Columbia. Eggs from four
females were mixed together before dividing them in separate
groups.
Groups of eggs were wet fertilized in water of pH 7.6, 5.5,
5.0, 4.5 and 4.0, and allowed to water harden for 1 h before
transporting them to the Sweltzer Creek Laboratory. Eggs
fertilized in control water were incubated at pH 4.lf0.1,
4.620.06, 5.0+0.13, 5.720.05 or control, 7.620.02 (mean+S.~.)
and eggs fertilized in acidified water were incubated at the pH
of fertilization. Two groups of eggs were fertilized at pH 4.0
and 5.0 then subsequently incubated at pH 7.6f0.02. Other groups
of eggs were exposed for 24 h at pH 4.0 after 4, 18, 38 and 77
days incubation at control water conditions(pH 7.6). These
exposure times corresponded to early blastula development,
gastrulation, early organogenesis just after reaching the 'eyed'
stage, and 50% hatch respectively.
Each group of eggs was separated into two replicates of
approximately 250 eggs. Eggs were sampled for histological
observation at 24 h, 5 d, 10 d and every 10 d period thereafter.
-
Eggs sampled during the first 10 days of exposure were preserved
in Stockard's solution and eggs sampled 10 days after
fertilization were preserved in 5% buffered formalin. Dead eggs
and alevins .were identified as previously described and removed
daily. Developmental stages of sockeye salmon eggs were
identified following Velson (1980). Alevins were anesthetized in
MS-222 then blotted dry to determine body weight and length. Dry
weight of alevins and yolk sac material were determined as
previously described for kokanee salmon.
Dilution apparatus
To achieve the exposure pH levels, H,SO, was diluted using
a proportional diluter constructed similar to that designed by
Mount and Brungs, ( 1 9 6 7 ) . Since the exact pH levels required
could not be obtained directly from the proportional diluter
cells, freshwater regulated by 'stopcock' valves was used to
achieve the nominal pH levels. The flow rates into the
incubation chambers were maintained at 200 mlemin-'. Chambers
were placed in a water bath to maintain a constant temperature
in all incubation chambers. Incubation chambers used for the
sockeye salmon egg exposures are shown in Fig. 2.
W a t e r qua1 i t y anal y s i s
Water temperature and pH were measured daily in both
kokanee and sockeye salmon egg experiments. Dissolved oxygen,
free CO, and hardness were measured periodically during the
exposure period. Dissolved oxygen was measured according to the
Idometric method using the Azide Modification Technique and was
maintained above 90% saturation (American Public Health
Association, APHA, 1976). Free CO, was measured by the
Titrimetric method (APHA, 1976) and ranged from 4.0 mg C02@L-'
at pH 7.6 to 34.6 mg CO,@L-' at pH 4.0. Water hardness was
measured by the EDTA Titrimetric method (APHA, 1976).
Survival curves and median survival times were calculated
according to Cutler-Ederer (1958) using the BMDP (Biomedical
Computer programs) data analysis system, Life Tables and
Survival functions program (BMDP Statistical Software, 1983).
The differences in percentage hatch and alevin survival were
compared with their respective controls by testing for
differences among proportions (Zar, 1974). The efficiency of
yolk conversion to tissue was calculated by dividing the dry
tissue weight by the dry yolk plus dry tissue weight. ~fficiency
of yolk conversion to tissue and alevin length data were
analyzed by using the SPSSX (Statistical Package for the Social
sciences) system, SPSSX Manova program (SPSSX User's Guide,
1983). Efficiency group means or alevin length means were
compared by analysis of variance (~iner,l971), followed by a
Student-Newman Kuels Test to determine the difference between
.
all possible pairs of means (~50.05).
111.
Results
Kokanee salmon experiments
Adult fish exposed to pH 5.1 were more lethargic and had a
build-up of mucus covering their bodies. Thirty percent of these
fish died during the exposure period while no mortalities were
observed at pH 5.6 and pH 7.1.
Mortality was only recorded until
the time of egg taking. Two female and three ma.le fish were used
to obtain fertilized eggs from fish held at pH 5.1, four female
and six male fish from pH 5.6 and seven female and six male fish
from pH 7.1.
Since sexual-dimorphism was difficult to determine
phenotypically, far more males than females were found in the
tanks. This, in addition to the fact that some of the females
released their eggs in the tanks, accounts for the apparent low
number of females used for spawning.
All groups had a different survival curve than the control
suggesting prior adult exposure, and/or fertilization and
incubation at low pH decreases embryo survival. Eggs fertilized
and incubated at pH 5.1 and below, independent of prior adult
fish exposure, had similar survival curves ( ~ i g .3 ) . ~ortality
was high in the early stages of development (Fig. 3 ) with a 50%
survival at 2 6 - 3 3 days at pH 5.1 and 2 0 - 2 5 days at pH 4.3
1).
(Table
Fig. 3. Survival curves of kokanee salmon eggs and alevins
exposed to low pH levels from fertilization to 1 1 0 days
post-fertilization. Each curve represents one group of eggs.
~ g g sfertilized and incubated
obtained from fish exposed to
or obtained from fish exposed
or obtained from fish exposed
at
pH
to
to
pH 7 . 6 ,
7.1 (control);
pH 5 . 6 ;
pH 5 . 1 ;
Eggs fertilized and incubated
obtained from fish exposed to
or obtained from fish exposed
or obtained from fish exposed
at
pH
to
to
pH 5 . 5 ,
Eggs fertilized and incubated
obtained from fish exposed to
or obtained from fish exposed
or obtained from fish exposed
at
pH
to
to
7.1;
pH 5 . 6 ;
pH 5 . 1 ;
pH 5 . 1 ,
7.1;
pH 5 . 6 ;
pH 5 . 1 ;
Eggs fertilized and incubated at pH 4.3,
obtained from fish exposed to pH 7 . 1 ;
or obtained from fish exposed to pH 5 . 6 ;
- 0
0---0
A-
---- A
.
40
60
DAYS
80
Table
1.
Median survival time of kokanee salmon eggs
incubated at different pH levels (mean+S.E.).
*more than 50% survival at day 1 1 0
Group
Eggs fertilized and
incubated at pH 7.6 obtained
from fish exposed to:
pH 7.1 (control)
pH 5.6
pH 5.1
Eggs fertilized and
incubated at pH 5.5 obtained
from fish exposed to:
pH 7.1
pH 5.6
pH 5.1
Eggs fertilized and
incubated at pH 5.1 obtained
from fish exposed to:
pH 7.1
pH 5.6
pH 5.1
Eggs fertilized and
incubated at pH 4.3 obtained
from fish exposed to:
pH 7.1
pH 5.6
Median survival (days)
----*
----*
84.00
Eggs obtained from fish exposed to pH 5.1 and incubated at pH
5 . 5 or 7.6 had a higher mortality that resulted in a lower
median survival time when compared with eggs incubated at the
same pH levels •L•E able1 ) . Prior exposure of adult fish to pH 5 . 6
also decreased the survival of kokanee salmon eggs incubated at
pH 7.6, compared with eggs obtained from fish exposed to control
pH levels incubated at pH 7.6 (Fig. 3). This same effect was n'ot
observed in eggs incubated at pH 5.5.
Development rate of surviving kokanee salmon embryos was
not inhibited by exposure of adult fish to low pH or by
incubation of the eggs at low pH.
Only eggs fertilized and incubated at pH 5 . 5 and above
survived to hatching (Table 2). Eggs fertilized and incubated at
pH 5 . 5 irrespective of prior adult fish exposure, had a 7-12 day
delay to median hatch compared to all other groups. Exposing
parent fish to low pH had no additional effect on the day to
median hatch of eggs incubated at pH 5 . 5 . For eggs incubated at
control pH levels, prior exposure of adult fish to pH 5.6 and
5.1 resulted in a 6-day delay to first hatch but only a 3-day
delay to 50% hatch. A few embryos hatched before 84 days but
died emerging. A significantly ( ~ 5 0 . 0 5 ) lower percent hatch was
observed in all groups compared with the control (Table 2). The
data for eggs fertilized and incubated at pH 5 . 5 are variable,
however eggs from adult fish exposed to pH 5.1 showed a marked
reduction (13.3%) in the number of eggs hatching. Even by
discounting mortality to the beginning of hatching, prior
Table 2. Days to hatching, 5 0 % hatch and % hatch of kokanee
salmon embryos incubated at different pH levels.
Group
Days
to 1st
hatch
Days
to 5 0 %
hatch
Hatch*
Eggs fertilized and
incubated at pH 7 . 6
obtained from
fish exposed to:
pH 7.1 (control)
pH 5 . 6
pH 5.1
78
84
84
84
87
87
100
100
60.4""
Eggs fertilized and
incubated at pH 5 . 5
obtained from
fish exposed to:
pH 7.1
pH 5 . 6
pH 5.1
86
90
95
94
94
96
*
**
+
%
%
Hatch+
Alevin
survival
(%)
76,6
54.9**
41.4**
64.6"" . 36.9**
85.6""
62.4""
85.7**
13.3%"
95.6
91.2
87.5
74.4%"
87.7
100.0
% hatch calculated from the number of eggs alive at 1st hatch
% hatch from total number
Significantly lower than the control at p 1 0 . 0 5
adult exposure affected the hatching success of the embryos
•€ able
2).
The efficiency of yolk conversion to tissue in developing
alevins was not affected by the exposure of adult fish to low
pH, providing the eggs were incubated at control pH levels
able
3). However, the efficiency was significantly ( ~ ~ 0 . 0 5 )
reduced in alevins hatched from eggs incubated at pH 5.5
obtained from acid exposed adults, compared to alevins from
control fish. Fertilization and incubation of eggs at pH 5.5
reduced the efficiency of yolk to tissue conversion in alevins,
independent of the pH to which adults were previously exposed
(Table 3 ) .
Growth (length) was significantly reduced in alevins
incubated from fertilization, in all groups exposed to pH 5.5
(Table 4, ~10.05). Exposure of adult fish to pH 5.1 only had a
significant effect on the growth of alevins incubated at control
pH levels (~10.05).
Table 3. The efficiency (tissue/yolk + tissue) of yolk to tissue
conversion in kokanee salmon alevins after 1 1 0 days
exposure to different pH levels (mean 5 S . E . )
Group
Eggs fertilized and
incubated at pH 7 . 6
obtained from
fish exposed to:
pH 7.1 (control)
pH 5.6
pH 5.1
Eggs fertilized and
incubated at pH 5.5
obtained from
fish exposed to:
pH 7.1
pH 5.6
pH 5.1
n
Tissue/yolk+tissue
10
9
10
0.71820.017
0.74120.034
0.70120.017
a
a
Table 4. Length of kokanee salmon alevins after 110 days
exposure to different pH levels (meanfS.E.).
Group
Eggs fertilized and
incubated at pH 7.6
obtained from
fish exposed to:
pH 7.1
(control)
pH 5.6
pH 5.1
Eggs fertilized and
incubated at pH 5.5
obtained from
fish exposed to:
to pH 7.1
pH 5.6
pH 5.1
n
Length (cm)
Sockeye salmon experiments
A malfunction in the acid-dilution apparatus resulted in
variable acidity conditions in groups of eggs incubated at pH
5.0. A dose of very acidic water ( p 3.6)
~
from day 67 to 69
caused mortalities in pH 5.7 groups and the eggs incubated at pH
5.0. The results of groups incubated at pH 5.0 are not reported
and mortality curves for groups incubated at pH 5.7 were only
calculated to day 65.
In all groups, subsamples of dead eggs were examined to
determine the percent of unfertilized eggs. This percentage was
used to'determine the number of fertilized eggs that died. The
corrected values were used to calculate survival.
Development rate of the embryos was only affected at day 30
in eggs incubated at pH 5.0 and below. These eggs lagged 2
stages behind all other groups, However, prior to and after day
30, no differences in developmental rate were observed. Episodic
exposure of the eggs to pH 4.0 did not affect their
developmental rate.
The survival curves of embryos incubated at pH 4.0 had the
same distribution independent of pH during fertilization ( ~ i g .
4). At pH 4.6 survival of the embryos (eggs and alevins) had two
.
sensitive periods, a high mortality during early embryonic
development (0-20 days) and a sudden decrease in survival just
after hatching ( ~ i g .4 ) . Also, those eggs fertilized at pH 4.5
Fig. 4. Survival of sockeye salmon eggs and alevins exposed to
low pH levels from fertilization to 110 days post-fertilization.
Each curve represents one group of eggs.
.~ g g sfertilized at pH 7.6, incubated at pH 7.6 (control); e
incubated at pH 4.6;
G.....~....
incubated at pH 4.1.
A,,,,
0
A
Eggs fertilized at pH 5.0 and incubated at pH 7.6.
van- ---v
Eggs fertilized at pH 4.0 and incubated at pH 7.6.
+---+
Eggs fertilized at pH 4.5 and incubated at pH 4.6.
0-0
Eggs fertilized at pH 4.0 and incubated at pH 4.1
.
($---
0
DAYS
and subsequently raised at pH 4.6, initially had a high
mortality resulting in 50% survival by 46 days, compared with a
median survival of 109 days for eggs fertilized at control pH
levels, incubated at pH 4.6 (Table 5). The survival of sockeye
salmon eggs fertilized at pH 5.5 and incubated at pH 5.7 had the
same survival distribution as the controls (Fig. 5). However,
eggs fertilized at pH 7.6 and incubated at 5.7 had a higher
mortality with only 25-60% survival at 65 days (Fig. 5 ) . The
survival of eggs fertilized at pH 4.0 or 5.0 and incubated at pH
7.6 was similar to the control group to 50 days exposure, at
which point mortality increased resulting in a. lower (10%)
survival at day 110 (Fig. 4). Exposure of eggs to pH 4.0 for 24
h had the most significant effect on survival when the eggs were
exposed during early development (day 4) (Fig. 6). At later
stages of development, a 24-h exposure to pH 4.0 had no apparent
effect on egg survival. However, hatched embryos exposed to pH
4.0 for 24 h at 77 days (50% hatch) were more lethargic during
the acid exposure.
No eggs hatched when incubated at pH 4.1
able 5). Eggs
fertilized at pH 7.6 and incubated at pH 4.6 had a 12-day delay
to 50% hatch (Table 5). The median hatching time was delayed 16
days in eggs fertilized at pH 4.5 and incubated at pH 4.6. The
delay in hatching of embryos incubated at pH 5.7 is probably a
result of the lethal (pH 3.2) pH exposure at day 67 (see above).
The most pronounced effect of the 24 h exposures
Table 5. Median survival and hatching data of sockeye salmon
eggs exposed to different pH levels (means2S.E.).
Group
Median
survival
(days>
Time to
%
%
5 0 % hatch
Hatch
(a)
Hatch
(b)
(days)
Alevin
survival
%
Eggs fertilized
a t - p 7~. 6 and
incubated at:
pH 7 . 6
(control)
pH 4 . 6
pH 4.1"
Eggs fertilized
at pH 5 . 0 and
incubated at:
pH 7 . 6
Eggs fertilized
at pH 4 . 5 and
incubated at:
pH 4 . 6
Eggs fertilized
at p H 4 . 0 and
incubated at:
pH 7.6
pH 4.1"
* no eggs hatched
** significantly different
from the control, p 5 0 . 0 5
a % hatch calculated from those eggs surviving to 1st hatch
b % hatch calculated from the total number of eggs
Fig. 5. Survival of sockeye salmon eggs exposed to low pH
levels for 65 days post fertilization.
Eggs fertilized and incubated at pH 7.6, control.
a
-.
Eggs fertilized at pH 7.6 and incubated at pH 5.7.
A--A
Eggs ferti1ize.d at pH 5.5 and incubated at pH 5.7.
V----V
I
I
I
I
10
20
30
40
DAYS
50
60
Fig. 6. Survival of sockeye salmon eggs and alevins exposed to
pH 4.0 for 24 h, once during the 110-day incubation period at pH
7.6. Eggs were continuously exposed to pH 7.6 (control..-.)
or exposed to low pH at day 4 ( A A ) , day 18 (v------V),
day 38 (C------==---=a),
and day 77 i .---.).
60
DAYS
Table 6. Percent hatch and alevin survival of sockeye salmon
eggs exposed to water of pH 4.0 for 2 4 h (meankS.E.).
Exposure
Day
a
b
*
Time to
50% hatch
(days)
%
%
Hatch
(a)
Hatch
(b)
Alevin
survival
%
% hatch calculated from those eggs surviving to 1st hatch
% hatch calculated from the total number of eggs
significantly lower than control, p10.05
on median hatching time was observed on eggs during cleavage
(day 4)
able 6).
Eggs raised at pH 4.6 had a significant reduction in the
percentage hatch compared with the controls
a able 5).
~ertilizationof the eggs at pH 4.5 further reduced the
percentage hatch (37.9%) of eggs incubated at pH 4.6, compared
with those eggs fertilized at pH 7.6 and incubated at pH 4.6
able 5). Alevin mortality was significantly greater at pH 4.6
compared with the controls and increased by 15.8% with
fertilization of the eggs at low pH
able 5, p10.05).
pis odic
exposure of eggs did not affect the percentage hatch of those
eggs surviving to 1st hatch (Table 6). However, a significant
reduction (31.6%) in percentage hatch of the total number of
eggs was observed when eggs were exposed to pH 4.0 for 24 h on
day 4 (pS0.05). Significant decreases in the total percentage
hatch were also observed for eggs exposed to pH 4.0 for 24 h at
day 18 (5.9%) and 38 (10%)(pS0.05).
Alevin mortality was not
significantly increased as a result of a 24-h exposure to pH 4.0
at any of the developmental stages.
The efficiency of yolk to tissue conversion was inhibited
in unhatched embryos exposed to pH 4.6 at day 70 compared to
control embryos (Fig. 7). At day 110, alevins still had a
significantly lower efficiency than the controls (p10.05). The
lower efficiency of yolk to tissue conversion in alevins exposed
to pH 5.7 is probably a result of the drop in pH at day 67 (see
above). Efficiency was significantly lower in unhatched embryos
exposed to pH 4.0 for 24 h at day 4, compared with the controls,
however by day 110 no difference was observed ( ~ i g .8 ) . Alevin
growth (length) was also significantly reduced by exposure to pH
4.6, independent of the pH at fertilization (p10.05) ( ~ i g .9 ) .
Fig. 7. Dry tissue / dry yolk + tissue ratio of sockeye salmon
embry*os continuously exposed to low pH levels from
fertilization. Each point represents the mean value of 6-8 fish
at day 70, and 5 fish at days 90 and 110. Vertical bars
represent the S.E. * indicates values significantly different
from the control at p10.05.
Eggs fertilized and incubated at pH 7.6 (control).
Eggs fertilized at pH 7.6 and incubated at pH 5.7.
Eggs fertilized at pH 5.5 and incubated at pH 5.7.
Eggs fertilized at pH 7.6 and incubated at pH 4.6.
Eggs fertilized at pH 4.5 and incubated at pH 4.6.
I
I
I
I
I
70
80
90
100
110
DAYS
Fig. 8. Dry tissue / dry yolk + tissue ratio of sockeye salmon
embryos exposed to pH 4.0 for 24 h at different developmental
stages. Each point represents the mean value of 6-8 fish at day
70, and 5 fish at days 90 and 110. Vertical bars represent the.
S.E. * indicates values significantly different from the control
at ~ 5 0 . 0 5 .
Control,
*a
-
Eggs exposed to pH 4.0 on day 4,
A--
Eggs exposed to pH 4.0 on day 18,
v-----IT
Eggs exposed to pH 4 . 0 on day 38,
a=--=----=-=--0
Eggs exposed to pH 4.0 on day 77,
.
A
.
M
I
I
I
I
70
80
90
100
DAYS
I
110
Fig. 9. Total length of sockeye salmon embryos exposed to low
PH levels from fertilization. Each value represents the mean of
1 1 - 14 fish at day 70 and 10 fish at day 90 and 1 1 0 . Vertical
bar s represent the S.E. * indicates values significantly
di f ferent from the control at p S 0 . 0 5 .
Eggs fertilized and incubated at pH 7 . 6 , control.
Eggs fertilized at pH 7 . 6 and incubated at pH 5 . 7 .
A--A
Eggs fertilized at pH 5 . 5 and incubated at pH 5 . 7 .
Vg--"-V
Eggs fertilized at pH 7 . 6 and incubated at pH 4 . 6 .
0"""""""'C]
Eggs fertilized at pH 4 . 5 and incubated at pH 4 . 6 .
0
.
0
DAYS
IV. Discussion
Few studies have reported on the effects of low pH on early
life stages of Pacific salmon (Rombough, 1982; Geen
et
al., In
Press). These studies were either of short duration (10 days)
using 'eyed' eggs, alevins and fry (Rombough, 1982) or of
chronic exposure (140 days) of eggs exposed from the 'eyed'
stage (Geen
et
al., In Press). The present investigation is the
first to report the effects of low pH on Pacific salmon from
fertilization to 45 days postmedian hatch. These early stages of
embryonic development must be examined before lethal pH levels
for Pacific salmon can be determined properly (Rombough, 1982).
Decreases in pH to 5.5-and below will have a significant
effect on the survival of kokanee and sockeye salmon embryos.
The median survival time for sockeye salmon at p~ 4.5 and 4.1
was higher than for kokanee salmon at pH 5.1 and 4.3
respectively, which suggests a difference in tolerance between
stocks. Swarts e t al., (1978) found differences in resistance to
low pH between different strains of brook trout.
The most sensitive stages of development to low pH were
cleavage and newly-hatched alevins, with those stages during
gastrulation and organogenesis being more tolerant. Survival of
kokanee salmon eggs to the 'eyed1-stagewas less than 32% at pH
5.1 and below, and between 18% and 55% at pH 4.6 and below for
sockeye salmon. Exposure of sockeye salmon eggs to pH 4.0 during
late cleavage (day 4 ) reduced the percent survival to the 'eyed'
stage. However, acid exposure for 24 h during gastrulation and
organogenesis had no effect. These results are in agreement with
other studies which have reported that early embryo development
(cleavage), and newly-hatched and buttoned-up alevins are more
sensitive to low pH than other stages of development (~ohannsen
et
al., 1973; Daye and Garside, 1977; Daye and Garside, 1979;
Lee and Gerking, 1980; Peterson
et
al., 1980; Brown and Lynam,
1981; Brown, 1982b). Gjedrem (1980) exposed brown trout eggs to
low pH at different stages of development and found an increased
survival the later the transfer to low pH was made, suggesting a
more graded effect of low pH on embryo mortality.
At pH 4.0 most sockeye salmon embryos (64%) were dead
before the 'eyed' stage, independent of the pH at fertilization
and water hardening. However, at pH 4.6 only 55% survival of
acid fertilized eggs to the 'eyed' stage was observed compared
with 90% survival of eggs fertilized at pH 7.5 and raised at pH
4.6. Trojnar (1977) reported an estimated survival of 82% to the
'eyed' stage of brook trout eggs fertilized and incubated at pH
4.65. Carrick (1979) observed an increased mortality in Atlantic
salmon, sea trout and brown trout eggs fertilized at low pH.
Carrick also found salmon eggs fertilized at low pH had an
increased mortality (50% within 4 wk) at pH 4.0, in contrast to
.
the results in the present study which observed high mortalities
at pH 4.0 independent of the pH at fertilization. Brown (1982b)
found no effect of acid-fertilization at pH 4.5 on the survival
of brown trout eggs.
Exposure of adult kokanee salmon to pH 5.5 and below just
prior to ovulation and spawning reduced egg survival. This
effect was more pronounced when the eggs were taken from parents
exposed to low pH and subsequently incubated at pH 5.5 and
below, suggesting an inhibitory effect of low pH during final
egg maturation (Fig. 3). Kennedy (1980) also observed that
previous exposure of adult lake trout to low pH affected
gametogenesis and fertilization causing significant mortality in
the early stages of embryonic development.
Fertilization and incubation of kokanee salmon eggs at pH
5.5 and below increased embryo mortality. Sockeye salmon eggs
fertilized and incubated at pH 4.5 and 4.6 respectively, had a
lower median survival time than eggs fertilized at control pH
and subsequently incubated at pH 4.6. However, the effect of low
pH on fertilization and water hardening may be only temporary
unless the eggs are subsequently incubated at low pH, since the
survival of sockeye salmon eggs fertilized at pH 4.0 or 5.0 and
incubated at control pH was only reduced by 10%. These results
may also infer that a time-dependent exposure is involved. Eggs
acid-fertilized at pH 4.0 and 5.0 were transferred to control pH
levels within 3 h. Peterson and Martin-Robichaud (1982) found
100% mortality of newly fertilized ova of Atlantic salmon after
-
8 h exposure to pH 4.0.
Exposure of parent fish to low pH delayed hatching and
reduced the percent hatch of kokanee salmon eggs irrespective of
the incubation pH. Eggs exposed to low pH, independent of
parental exposure, also had a delay in hatching time. Delayed
hatching of eggs from acid exposed parents has been observed for
brook trout (~enendez,1 9 7 6 ) ~fathead minnows ( ~ o u n t ,1973) and
desert pupfish ( ~ e eand Gerking, 1980).
The delay in time to median hatch in sockeye salmon eggs
fertilized at pH 4.5 and subsequently incubated at pH 4.6 is in
contrast to other findings. Peterson
et
a1
.
(
1980) found no
delay in hatching of Atlantic salmon eggs reared at low pH from
fertilization. Whether these eggs were fertilized at low pH or
transferred to low pH water after fertilization' is not reported
by the authors. Daye and Garside (1979) observed no delay to
median hatch for Atlantic salmon eggs fertilized at control
conditions and incubated at low pH. However, Peterson
et
al.
(1980) observed delayed hatching in eggs exposed to low pH from
the 'eyed' stage. A delay in time to median hatch and a reduced
hatchability of eggs exposed to low pH from the 'eyed' stage
have been reported in other studies
Nelson, 1982; Geen
et
al
., In Press).
warts
et
Peterson
al., 1978;
et
al
.,
(1980)
and Nelson (1982) suggested that adaptation during early
development may have occurred to allow for normal hatching.
Adaptation may occur in eggs during early development, however
results in the present study suggest hatching is inhibited if
fertilization and water hardening take place at low pH.
The results of the sockeye salmon experiments show a median
lethal pH level of 4.6 for a 50% hatch of eggs fertilized in
control water and raised at low pH.
wain (1975) reported a
median lethal pH value of 4.75 at 10•‹c for a 50% hatch of
rainbow trout eggs under similar fertilization and incubation
conditions. However, in the present study, eggs raised from
fertilization at pH 4.6 had a much lower percent hatch (16%)
than eggs placed in low pH water after fertilization. Carrick
(1979) also found approximately the same percent hatch (5%) for
~tlanticsalmon eggs fertilized and raised at pH 4.5. These
results suggest that studies in which eggs are not fertilized at
low pH may underestimate the lethal pH levels.
No eggs survived to hatching at pH 4.0 irrespective of the
pH level during fertilization and water hardening. These results
are similar to those obtained by Peterson
et
al.
(1980) who
found hatching of Atlantic salmon eggs was prevented at pH 4-0.
Kwain (1975) observed no survival of rainbow trout embryos below
pH 4.49 regardless of temperature.
Exposure of parent kokanee salmon to low pH had a negative
effect on the survival of newly-hatched alevins subsequently
raised at control pH levels. The mechanism by which parental
exposure to low pH in the later stages of egg maturation affects
the survival of eggs and alevins is not known. The results in
the present study do not allow for interpretation of the
mechanisms involved. Menendez (1976) also observed an increase
in alevin mortality as a result of parental exposure at pH 5.09.
The kokanee alevin mortality data were variable, but do suggest
a pH effect on eggs raised at pH 5.5 and below. Menendez (1976)
found 56% alevin mortality at pH 5.09 in alevins hatched from
eggs obtained from control fish.
The mortality of sockeye salmon alevins was greater for
eggs that had been fertilized and raised at low pH, than eggs
fertilized at control pH levels and incubated at low pH. By 30
days post median (50%) hatch, alevin mortality was 68-83% at pH
4.6. Rombough (1983) found newly hatched sockeye salmon alevins
and buttoned-up alevins had ten-day L C s 0 (Sprague, 1973) pH
values of 4.57 and 5.02 respectively. Geen e t al. (1n press)
also observed significant mortality in newly hatched chinook
salmon alevins at pH 5.0 and below.
The efficiency of yolk conversion to tissue and the growth
(length) of kokanee and sockeye yolk-sac alevins was reduced at
pH 5.5 and 4.6, respectively (Table 3 and ~ i g .7). These results
confirm assumptions made by Nelson (1982) that reduced growth at
low pH may result from an inhibition of yolk conversion into
tissue. Nelson (1982) found a reduced concentration of
water-soluble proteins in acid exposed rainbow trout alevins,
which suggested an inhibition of the conversion of yolk-sac
material to tissue. This disruption of normal protein anabolism
could be a result of 1 ) direct deactivation of protein synthesis
by H';
2 ) an inhibition of large yolk sac proteins into low
molecular weight fragments or 3) a reduced absorption and
transport of these fragments
elso son 1982). Reduced growth and
efficiency rate in sockeye salmon alevins exposed to pH 5.7 was
a result of the drop in pH which occurred at day 67. However,
these results do show that a 2-day exposure to a lethal pH value
of 3.2 will result in reduced growth of surviving alevins raised
at pH 5.7.
Lethal pH values (pH 4.5-5.0) reported in this study are
slightly lower than pH values reported in the literature for
salmonids of similar sensitivity to low pH. In part, the
difference is attributed to the fact that eggs were fertilized
in acidified water in the present study, compared with many
other studies that fertilized eggs at control pH and then
transferred the eggs to low pH. Also, increased free CO,
concentrations probably accounted for the lower .lethal pH
values, especially in newly hatched alevins. Lloyd and Jordan
( 1 9 6 4 ) observed lower resistance times of rainbow trout at pH
values above 4.0 when dissolved C02 was above 20 ppm. However,
embryos and alevins may be exposed to increased free CO,
concentrations during incubation in redds. The concentration of
CO, in stream-bed interstitial water has been observed to
increase with increasing depth of the substrate (Williams and
Hynes, 1974). Acidification of streams and rivers may further
increase interstitial water CO, concentrations
, which may be
more important to egg survival than pH.
In summary, the results show that water of pH 5.0 or less
will significantly reduce embryo survival. Further, exposure of
adult fish to pH 5.6 prior to ovulation and spawning, results in
reduced egg survival. These results are in general agreement
with survival data for other teleost fish reported in the
literature.
Delayed hatching, inhibition of hatching, and reduced
efficiency of yolk to tissue conversion as well as other
observed effects of low pH on developing embryos, may be due to
an inhibition of enzyme systems involved in embryonic
development, and/or a reduced egg viability resulting from an
inhibition of the processes involved in egg production and
maturation in female fish exposed to low pH.
C. Chapter 11-Effects of pH and/or calcium-enriched freshwater
on plasma levels of vitellogenin and c a 2 + , and on bone calcium
content during exogenous vitellogenesis in rainbow trout
I. Introduction
Four physiological periods have been identified in the
reproductive cycle of female rainbow trout, S. gairdneri, by van
Bohemen et al., (1981). These are the previtellogenic period
arch-~pril), a period of endogenous vitellogenesis ( ~ a y - ~ u l y )
when the oocytes synthesize their own yolk, a period of
exogenous vitellogenesis (~ugust-~ecember)
when the oocytes take
up vitellogenin from the blood and a period of ovulation and
spawning. Exogenous vitellogenesis is a period where oviparous
vertebrates incorporate large amounts of yolk proteins into
developing oocytes. This involves the production of yolk
proteins in the liver which are secreted into the bloodstream,
transported to the ovary and incorporated into the oocytes by
micropinocytosis (wallace, 1978; van Bohemen et al., 1981).
Histological studies of the liver show dramatic structural
changes and an increase in cell size during exogenous
vitellogenesis (van Bohemen et al., 1981).
Vitellogenin (Pan et al., 1969) in teleosts has been
identified as a female-specific lipophosphoprotein molecule that
binds calcium ( ~ o r iet a1
., 1979; whitehead
et a1
.,
1983).
During the period of exogenous vitellogenesis blood phosphorus
and calcium ion levels, which are part of the vitellogenin
complex, rise considerably (whitehead el a1
., 1983).
The extra
calcium required for the protein complexes is taken up from the
external environment and/or internally from demineralized bone
(Fleming
et
al., 1964; Ma, 1976). Consequently, calcium
regulation in fish also becomes important during ovarian
maturation.
Loss of fish populations from acidified lakes is partially
caused by a decreased recruitment due to a failure of mature
fish to spawn ( ~ l m e re t al., 1974; Beamish, 1974a; Beamish and
Harvey, 1972; Beamish
et
al, 1975). Abnormal development of the
ovaries has been observed in lake herring and rock bass caught
in acidified lakes (Beamish, 1974a). Ovarian maturation and
spawning may be the most sensitive of all physiological
processes related to the survival of fish populations in
acidified lakes (Beamish, 1976).
Laboratory studies have shown that fish exposed to low pH
have a reduced frequency of spawning, lower number of eggs per
female and a decreased number of viable eggs (~rishna,1953;
Mount, 1973; Menendez, 1976; Craig and Baksi, 1977; Lee and
Gerking, 1980). The quality of eggs in fish exposed to low pH is
also reduced. Mount (19731, for example, found the eggs of
fathead minnows exposed to pH 5.9 lacked turgidity and had
fragile shells. Egg quality, as determined by opaqueness of the
eggs and yolk diameter which are associated with exosmosis and
protein coagulation, was reduced in desert pupfish (Cyprinodon
nevadensis n.) exposed to acidified water ( ~ e eand Gerking,
1980). A reduction in the ability of oocytes to form mature eggs
due to an inhibition of secondary yolk deposition was observed
in the ovaries of flagfish exposed to pH 6.0 and below (Ruby et
al.,
1977).
A
reduction in yolk deposition in growing oocytes
may not only be a result of an inhibition of yolk uptake at the
ovary but also an inhibition of vitellogenin production and
secretion in the liver. Although no studies have measured plasma
vitellogenin levels in sexually maturing female fish exposed to
acidified water, abnormally low serum calcium levels in female
fish during ovarian maturation (Beamish
et
1975) may
al.,
indicate inhibition of vitellogenin synthesis. Low pH exposure
may affect calcium metabolism resulting in low serum calcium
levels during ovarian maturation which consequently effect
regulator factors involved in vitellogenesis.
.
Field (Wright and Snekvik, 1978) and laboratory (McDonald
et
al, 1980; Brown, 1982b; McWilliams, 1982; Nelson, 1982)
studies have shown that environmental calcium concentration
affects the survival and physiological process in fish exposed
to acidified water. Therefore, environmental calcium
concentration in acidified waters may ameliorate the inhibitory
effects of increased H + concentration on ovarian development.
vitellogenesis in salmonids may be affected at sublethal pH
levels (pH 5.0) which could result in complete inhibition of egg
production or an increase in the production of abnormal eggs
resulting in high mortalities during embryonic development. In
the present study the effects of acidified water and different
. environmental calcium ion concentrations on the plasma
vitellogenin and calcium ion levels in salmonid fish during the
period of exogenous vitellogenesis were investigated. Bone
calcium content and the gonadosomatic and hepatosomatic indices
were determined in trout after low pH exposure during the
vitellogenic period.
11.
Materials and Methods
E x p e r i m e n t a l Fish
Three-year old autumn spawning rainbow trout, S. g a i r d n e r i ,
approximately 250-600 g body weight and 28-38 cm in fork length
were obtained from Sun Valley Trout Farm, Mission, British
Columbia. The fish were kept in a 2000-L fiberglass tank
supplied with flow through dechlorinated tap water (Ca2+
0.045-0.069
6.3-6.50)
mM, Na+ 0.024 mM, Hardness 5.2 mg*~-'CaCO,,pH
with the temperature ranging from
11"-15•‹C
over the 6
week acclimation period. The photoperiod was changed weekly to
simulate a natural photoperiod (~ug.-Nov., 50" Latitude,
~ancouver). The fish were fed dried pellets (Moore and clark,
Laconner, Washington) daily a d l i b i t u m . Fish that were not
feeding or did not appear healthy were removed from the holding
tank and were not used for experimental purposes.
After acclimation, fish were randomly selected and
anesthetized in a buffered (sodium bicarbonate, NaHCO,) solution
of ethyl m-aminobenzoate (MS-222, 50 mg*L-'1. Approximately
0.5-0.7
mL of blood was taken by caudal vascular puncture with
an ammonium heparinized (10,000 i.u.1
syringe. Body weight and
fork length were recorded at the beginning and end of the
subsequent exposure period and each fish was marked with
coloured beads attached with nylon line immediately anterior to
the dorsal fin. The fish were revived in fresh water and exposed
for 64-70 days to one of the following solutions.
a)
Group I. Dechlorinated water, pH 6.66k0.13
Ca2+=0.0520+0.006 mM, number (N) of fish=lO
b)
Group 11. Dechlorinated water, pH 6.66k0.12
~a~+=1.52+0.31mM, N=8
c)
Group 111. Dechlorinated water, pH 5.03k0.39
~a~+=0.052080.006
mM, N=8
d)
Group IV. Dechlorinated water, pH 5.1420.34,
Ca2+=1.55k0.27 mM, N=8
At two week intervals the fish were anesthetized and blood
was sampled by caudal vascular puncture. After 70 days exposure,
the fish were killed by severing the spinal cord just posterior
to the medulla oblongata, and the gonads and liver were excised
for wet weight measurements. Blood samples were analyzed for
haematocrit, alkaline-labile phosphate and Ca2+ concentrations.
Caudal centra were excised from the caudal peduncle immediately
above the insertion point of the last anal fin ray and frozen at
-20•‹C for analysis of total calcium content.
A c i d Di 1 ut i o n A p p a r a t u s
The dilution apparatus received dechlorinated water into a
230-L fiberglass holding tank ( ~ i g1 0 ) . A submersible pump
transferred the water to a 100- L plexiglass 'header' tank
equipped with an overflow pipe. The incoming water flow was
regulated by a 'rotoflow' valve and the pH was adjusted to pH
6.70 with potassium hydroxide. The water exited from the bottom
of the header tank through 1/2" tygon tubing into one of the
four treatment tanks (800 L). Water flow into the exposure tanks
was regulated by 'rotoflow' valves at 2
em in-'. The water in
the tanks was continuously aerated with compressed air filtered
through a charcoal air filter. This apparatus met the conditions
suggested by Sprague (1973) for holding fish. There was more
than a liter of water per 10 g of fish with a flow rate slightly
less than one liter per minute for each kilogram.of fish
resulting in approximately 75% replacement of water in 8 h.
Low pH and/or high calcium solutions were prepared by the
addition of sulfuric acid (H2S0,) and/or calcium nitrate
Ca(NO,),
respectively, to the water flowing from the 'header'
tank. Both H2S04 and Ca(~0,), were added to the water by a
peristaltic pump (~anostat)from stock solutions through 5/32"
tygon tubing connected to a 13-gauge needle. The needle was
inserted into the water line by a rubber serum stopper connected
to a Nalgene 'Y' junction. A float switch located in the header
tank was connected to the peristaltic pump which would shut off
the pump if the water in the header tank dropped too low. This
ensured that the fish did not receive a concentrated solution of
. H + and/or Ca2+ if the water pressure dropped.
Water temperature, pH and ca2+ concentration were monitored
daily. Dissolved oxygen and free CO, were analyzed weekly during
the exposure period. The calcium concentration of the water was
measured by the E D T A titrimetric method ( A P H A , 1 9 7 6 )
standardized with an atomic absorption spectrophotometer. Oxygen
was measured by an iodometric method using an azide modification
technique and remained above 80% saturation ( A P H A , 1 9 7 6 ) . Free
CO, was measured by the titrimetric method and never exceeded
2.0 mg CO,@L-' ( A P H A ,
1 9 7 6 ) . The
temperature ranged from 15•‹C at
the beginning of the experiment to 9 ' ~after 70 days.
Fig. 10. Line diagram of the experimental apparatus used to
expose rainbow trout to low pH and/or increased C a + 2
concentrations.
Inflow
Overflow
'Rotoflow'
1
Air
Air
1
Acid or
Calcium
A n a l yt i c a l P r o c e d u r e s
After collection of the blood, each sample was placed in a
1.5 mL polypropylene micro-centrifuge tube and the plasma was
collected by centrifugation. The plasma was removed and stored
in 400-)JL polypropylene micro-centrifuge tubes at -20•‹C.
Hematocrit was determined by centrifuging a separate sample of
the blood in 100-L heparinized hematocrit tubes. Plasma Ca2+ was
measured by atomic absorption spectrophotometry (Pye Unican, SP
191), using lanthanum chloride to reduce phosphate interference.
~emale/male plasma calcium ratios were calculated by dividing
the plasma Ca2+ concentration of each female by the mean plasma
ca2+ concentration of males from all groups. The mean plasma
ca2+ concentration of males in the experiment was used since no
significant difference in plasma Ca2+ concentration was found in
males among the groups (p>0.05).
Plasma vitellogenin content was estimated indirectly by
measuring the alkali-labile protein phosphorus concentration.
This technique, although not as accurate as many other time
consuming methods which measure vitellogenin directly (Plack e t
a2
, 1971 ; Goedmakers and Verboom, 1974; Idler
et
a1
, 1979; van
Bohemen e t al, 1 9 8 1 ) ~is a reliable indicator of the levels of
vitellogenin in the plasma of fish (Emmerson and Peterson, 1976;
Nath and Sundararaj, 1981). Alkali-labile protein was prepared
from duplicate samples according to the technique of Wallace and
Jared (1968) with minor changes in the volumes used. The amount
of inorganic phosphate in the solution was determined by the
method of Martin and Doty ( 1 9 4 9 ) ~using 1/5 the recommended
volumes. Phosphate standards were made up from reagent grade
potassium dihydrogen phosphate (KH~PO,),dried at 1 1 0 • ‹ ~in
,
double distilled water.
.
Total Ca2+ concentration of the caudal centra was
determined by the method described by Wendelaar Bonga and
Lammers (1982). Two caudal centra from each fish were cleaned in
1.0 N KOH for 2 h and subsequently rinsed 3 times in 100%
ethanol. Samples were dried at 90•‹cfor 18h to.determine dry
weight and then dissolved in 3.0 mL ION nitric acid (HNO,) for 2
h.
A
0.5 mL aliquot of the HNO, plus bone solution was diluted
with 4.5 mL of double distilled water. Total Ca2+ concentration
was measured with 50
rL duplicate samples by atomic absorption
spectrophotometry.
Glassware used to prepare chemical solutions was acid
washed and glassware used to make solutions for c a 2 + analysis
was also rinsed in 10 mM EDTA.
Statistical
Analyses
All statistical analyses were performed by using the SPSSX
data analysis system, SPSSX Manova program (SPSSX User's
Guide,l983). A multivariate analysis of variance was used to
test whether plasma Ca2+ or alkali-labile phosphate measurements
differed among the treatment groups (Winer, 1971). Homogeneity
of group variances was tested using Bartlet's test and normality
by ~olmogorov-Smirnov goodness of fit test. ~eterogeneityof the
alkali-labile phosphate group variances was reduced by applying
a ln(x +
1)
transformation. Group means of the total calcium
content of the caudal centra, were compared by an analysis of
variance
.
Gonadosomatic index (GSI) was calculated by dividing the
gonad weight by somatic weight minus the gonad weight, (body wt.
-
gonad wt.) multiplied by 100. The hepatosomatic index (HSI)
was obtained by dividing the liver weight by somatic weight
multiplied by 100. To determine the effects of low pH and/or
calcium on GSI and HSI, the data were transformed to arcsine
values and compared by an analysis of variance, followed by a
Student-Newman-Keuls test to determine the differences among the
group means ( ~ a r ,1974). Significance was accepted at the 95%
confidence level with trends being discussed at the 90%
confidence level.
111.
Results
One fish in each group, except for the control, died during
the experimental period. Acidification of the water could not be
attributed to the cause of death of these fish. Aggressive
activity between the fish may have caused some mortalities.
Exposure of fish to low pH and/or calcium-enriched
freshwater for 65-70 days had no significant effect on
hematocrit (p>0.05).
The ovulation period for the stock of fish used in the
present experiment was approximately October 15-November 30.
Plasma calcium concentrations measured in female fish one month
prior to the beginning of- the experiment, were already elevated
in some fish. Because of this asynchronous timing of gonadal
maturation, those fish with initially high plasma vitellogenin
content and elevated plasma calcium levels were not included in
the results. By excluding these early maturing fish, a
comparison could be made between more synchronized fish. One
female fish from each of group I and I 1 were excluded and two
fish from group 1 1 1 . No fish were excluded from group IV. These
fish were left in the tanks throughout the exposure period to
keep the relative number of fish per tank the same.
The range of plasma Vg-bound phosphates reported here (0.15
-
17.64,umolam~-' plasma) is close to those values (0.49
-
25.61
~ m o l a m ~ - lreported
)
for rainbow trout by other investigators
(Whitehead et a l . , 1980, 1983) and within the range for (0.35 29.81 pmol*m~-')catfish, Het e r o p n e u s t e s f o s s i l i s , ( ~ a t hand
Sundararaj, 1981).
Only trace amounts of total protein-bound phosphates were
detected in male fish. In female fish, environmental calcium had
a greater effect on Vg-bound phosphate production than pH.
Plasma Vg-bound phosphate levels in females exposed to
calcium-enriched water were not significantly different at day
0. However, significantly higher levels were observed at day 28
and 65-70 ( ~ i g .lla, pS0.05). The results of the multivariate
tests, although not significant, did suggest that low pH has a
tendency to inhibit Vg-bound phosphate production. This trend is
shown in Fig. llb, where Vg-bound phosphate levels are lower at
day 28 and 65-70. The significance (~10.05) of the univariate
tests at day 28 and 65-70 further support the observation that
low pH had some influence on plasma vitellogenin levels.
Plasma calcium ion concentrations in female fish followed a
similar pattern as the alkali-labile phosphate levels (Fig. 12a
and 12b). As expected increasing the environmental Ca2+
concentration had a greater effect on plasma c a 2 + concentration
than decreasing the pH level. Plasma Ca2+ concentrations were
significantly (~10.05)higher in fish exposed to
calcium-enriched water at day 28 and 65-70.
Exposure of females to pH 5.03 (group 111) was associated
with a significant (PS0.05) reduction in the GSI at day 65-70,
compared with the other groups (Fig. 13).
Fig. 1 1 . Plasma alkali-labile phosphorus levels in female
rainbow trout ( N = 6 ) exposed to (a) high C a 2 + ( rn , 1.52-1.55 mM)
or low C a Z + (
, 0.052 mM) concentrations independent of pH and
(b) low pH ( o , pH 5.0-5.1) or control pH levels ( rn , pH 6 . 7 ) ,
independent of the Ca2' concentration, for 65-70 days. *
indicates a significant difference between the groups at p10.05
by analysis of variance. Thin vertical lines represent the
standard error ( S . E . ) .
Fig. 12. Plasma calcium ion concentration in female rainbow
trout ( ~ = 6 ' exposed
)
to (a) high C a 2 + (
, 1.52-1.55 m ~ or
) low
c a 2 + ( 13 , 0.052 m ~ concentrations
)
independent of pH and (b)
low pH ( o , pH 5.0-5.1) or control pH levels (
, pH 6.7),
independent of the Ca2+ concentration, for 65-70 days. *
indicates a significant difference between the groups at p10.05
and t indicates a significant difference between groups at
psO.10 by analysis of variance. Thin vertical lines represent
the standard error (S.E.).
Fig. 13. Effect of low pH and/or calcium-enriched water on the
gonadosomatic aand hepatosomatic indices of sexually maturing
female rainbow trout ( ~ = 3 )after 65-70 days exposure. Thin
vertical lines represent the standard deviation (S.D.). Standard
errors not wide enough to be shown graphically are omitted. *
indicates values significantly different from the control (Group
I). I-pH 6.66, [Ca2+]=0.052 mM; II- pH 6.66, [Ca2+]=1.520mM;
III-pH 5.0, [Ca2+]=0.052 mM; IV-pH 5.1, [Ca2+]=1.550 mM.
FEMALES
FEMALES
However, calcium-enrichment completely ameliorated the effects
of low pH exposure on gonad size since no significant change in
the GSI was observed compared with the control (p>0.05). The HSI
of females exposed to acidified water was significantly lower
than the HSI of all other groups ( ~ i g .13, pS0.05). HSI and GSI
of male fish were not statistically tested due to the small
sample size ( N = 1 ) in group 111.
Bone calcium content of females from the different groups
was not significantly different
able 7, pS0.05). Male fish
exposed to calcium-enriched freshwater (groups I1 and IV
combined) showed a higher (~10.05)bone calcium content in
comparison to fish exposed to freshwater (groups I and 11,
combined). This difference may have been biased by the low
sample size in group I11 (Table 7). However, by combining males
and females, the analysis showed a significant effect of
environmental calcium on bone calcium content (Table 7, pS0.05).
Low pH caused a significant reduction in the female/male
plasma c a 2 + ratio while an increased environmental calcium
concentration resulted in a significantly higher ratio (Table 8,
p10.05). Although non-significant, fish exposed to acidified,
low calcium ion freshwater (group 111) tended to have a lower
female/male plasma calcium ratio compared with all other groups
(Table 8 ) .
-
Table 7: The effect of pH and/or environmental calcium on
the calcium content of the caudal centra in rainbow
trout (mean + S.E.).
(I)
pH 6.66, C a + 2 0.052 mM
( 1 1 ) pH 6.66, C a + 2 1.520 mM
( 1 1 1 ) pH 5.03, C a + 2 0.052 mM
( I V ) pH 5.14, C a + 2 1.550 mM
Group
Sex
n
Calcium
(mEq*100 g -
* includes all fish in the tanks
b+d>a+c, p10.05
'
bone)
Table 8: Ratio of female/male plasma ~ a concentration
+ ~
for fish exposed to the following solutions
S.E.).
for 65-70 days (mean
( I ) pH 6.66, ~ a 0.052
+ ~ mM
( 1 1 ) pH 6.66, Ca+' 1.520 mM
( 1 1 1 ) pH 5.03, Ca+' 0.052 mM
(IV) pH 5.14, C a + 2 1.550 mM
+
Group
n
~emale/male ratio
IV. Discussion
In the present study, female trout exposed to acidified
water (pH 5.03) had a significantly lower GSI than control fish,
indicating egg production was inhibited by low pH.
his agrees
with earlier studies that have shown a reduced egg production in
fathead minnows (Pimephales ~ r o r n e l a s ) exposed to pH 5.9 and
below ( ~ o u n t ,1 9 7 3 ) ~flagfish (~ordanellafloridae) exposed to
pH 6.0 and below (Craig and Baksi, 1977) and in desert pupfish
(Cyprinodon n. nevadensis) exposed to pH 7.0 and below ( ~ e eand
Gerking, 1980). Conversely, Menendez (1976) found that total egg
production was not affected when female brook trout (Salvelinus
fontinalis) were exposed to pH 5.0 although the viability of
eggs was reduced significantly.
Field observations have shown that white suckers
(Catastomus commersoni) failed to reproduce in acidified lakes
because they did not reach spawning condition (Beamish and
Harvey, 1972). Beamish (1974a) observed abnormal development of
the ovaries in lake herring from an acidified lake (pH 4.4 4.9). These fish had only a portion of their ovaries containing
developing eggs, which were abnormal in texture and size,
indicating a reduction in yolk deposition. Ruby
et
al. (1977)
observed that primary oocyte development and secondary yolk
deposition in flagfish ovaries were impaired at pH 6.0 and
below. Desert pupfish exposed to pH 5.0 and 5.5 also had fewer
developing oocytes in their ovaries than control fish, although
the difference was not significant (Lee and Gerking, 1980).
Peterson
et
a1
(1982) suggested that if low pH caused a
disturbance in ribonucleic acid (RNA) or protein synthesis
during early oocyte development, then this may have caused an
inhibition of primary and secondary yolk deposition later on.
It appears from the above information that the effects of
acid stress on physiological parameters associated with gonadal
maturation are related to an inhibition of oocyte development
and incorporation of vitellogenin into the ovary. However, yolk
deposition in the ovaries may also be inhibited.by a reduction
in vitellogenin synthesis by the liver. Rainbow trout exposed to
pH 5.0
-
5.1 (group, I11 and IV) for 65-70 days, for example,
had a lower rate of Vg-bound phosphate production (Fig. llb).
Fish held in acidified water (group 111, pH 5.0) were the only
fish not showing a rise in plasma Vg-bound phosphates during the
experimental period. In addition, the significantly lower HSI in
trout exposed to pH 5.03 (group 111) also suggests that
vitellogenin synthesis in the liver was inhibited. Van Bohemen
et
al.
(1981) found that the HSI increased during exogenous
vitellogenesis in rainbow trout.
If yolk deposition in the ovaries was impaired rather than
vitellogenin production in the liver, then plasma Vg-bound
.
phosphates might be expected to increase. Yaron e t a1 (1977)
observed exaggerated levels of calcium and protein in
ovariectomized and estradiol-injected fish. Therefore, the lower
GSI in trout exposed to pH 5.03 (Group 111) may be a result of
low Vg-bound phosphate production by the liver as well as some
inhibition of yolk deposition in the ovaries.
Environmental calcium concentration may limit vitellogenin
production since fish exposed to low environmental calcium had
significantly lower plasma Vg-bound phosphate levels. This might
be explained on the premise that a higher concentration of
environmental calcium promotes a "healthier" fish and that
CaZ+
required during vitellogenesis is obtained with less energy
expenditure from calcium rich water compared with low calcium
water and/or the loss of calcium from the body .is greater in low
calcium environments. In a survey of 700 acidified lakes, Wright
and Snekvik (1978) reported that calcium content of the water
was as important to the fish as was pH. The calcium
concentration of acidified waters influences the survival times
of fish as well as the number of species present ( ~ r o w n ,1982a).
Calcium is bound to the plasma Vg-bound phosphates in
teleosts and increases in concentration during exogenous
vitellogenesis (Oguri and Takada, 1967; Yaron
et
a l l 1977;
Whitehead e t all 1980), thus inhibition of calcium metabolism in
female fish during gonadal maturation may disrupt normal
vitellogenin production in the liver or impair yolk deposition
at the ovary. Female trout exposed to acidified water (pH 5.03,
.
group 111) only reached a female/male plasma calcium ratio of
1.15 while for females in the other groups the ratio was greater
than 1.4. Beamish e t a1 (1975) found that suckers experiencing
reproductive failure in acidified lakes had femal@/male
serum
calcium ratios below 1.4.
Abnormally low plasma calcium ratios or levels in female
fish during vitellogenesis observed in the present study and
reported from field observations by Beamish e t
a1
(1975), may be
due to an inhibition of active ca2+ transport. Ma (1976)
suggested that the gills were the site of calcium uptake since
gill Ca2+-ATPase enzyme activity increased during growth and
sexual maturation in rainbow trout. However, Ma's experiments
se
between
also showed no difference in gill C a 2 + - ~ ~ p a activity
spawning female and male rainbow trout. That was explained by
suggesting that extra calcium required by females is most likely
obtained from the external environment during growth and sexual
maturation, and stored until required for vitellogenesis. An
alternate explanation for the lack of any difference in
C a 2 + - ~ T P a s eactivity between spawning female and male rainbow
trout may be due to the time period at which the measurements
were made. Ma (1976) measured C a 2 + - ~ ~ p a enzyme
se
activity in
trout and salmon during the spawning period, and not the period
of exogenous vitellogenesis and subsequent increase in plasma
calcium levels. Vitellogenin levels in female rainbow trout peak
during late vitellogenesis and decrease during ovulation and
spawning (van Bohemen e t a l , 1981; Scott and Sumpter, 1983).
Lockhurt and Lutz, (1979) found that female white suckers did
not have an elevated serum calcium concentration in comparison
with males during early gonadal maturation and spawning.
However, these authors did find female white suckers to have
elevated serum calcium levels during ovarian development.
Therefore, even though Ma (1976) reported no difference in
Ca2+-ATPase activity between spawning female and male trout, an
increase in C a 2 + - ~ T ~ a activity
se
may occur during the preceding
period of exogenous vitellogenesis.
Further, if extra calcium taken up from the external
environment by female fish is stored internally, then
differences in bone calcium levels between sexually mature
females and males should be evident. In this study, however, no
significant difference in bone (caudal central .calcium
concentrations were observed between females during ovarian
maturation and male fish. Fraser and Harvey (1982) found that
centra calcium concentrations in white suckers did not differ
between sex or age, although the stage of sexual maturity of the
fish was not reported.
No evidence of bone demineralization was found in female
fish exposed to low pH or low pH and calcium-enriched water.
Therefore, these low calcium levels observed during
vitellogenesis in rainbow trout are probably a result of an
increased loss of calcium or an inhibition of calcium uptake.
These results are in agreement with Lockhurt and Lutz (1977) but
contrast to those reported by Fraser and Harvey (1982). Lockhurt
.
and Eutz (1977) found no indication of any loss of calcium from
rib bones, muscle, scales or skin from white suckers caught in
acidified or non-acidified lakes. Fraser and Harvey ( 1 9 8 2 ) on
~
the other hand, found the calcium concentration of backbone
8-
centra was reduced 16% in fish from acidified lakes relative to
fish from non-acidified lakes. These authors explained their
findings in comparison with Lockhurt and Lutz (1977) as the
difference in analyzing rib bones versus centra. Fraser and
Harvey (1982) found ribs and opercular bones were not
decalcified in acid stressed fish but hypurals, trunk and caudal
centra were. Rainbow trout in the present study may not have
been exposed to low pH water long enough to cause any
significant changes in bone decalcification or show any reduced
calcification during growth. Field observations. showing fish
with low bone calcium content ( ~ r a s e rand Harvey, 1982) and
skeletal deformities (~eamishe t a!,
19751, are probably a
result of an inhibition of calcium uptake and calcification
during growth since Nelson (1982) found ossificaion of skeletal
parts in developing rainbow trout alevins was inhibited by low
Rainbow trout exposed to pH 5.0 fed with less activity than
fish in non-acidified water and calcium-enriched acidified or
non-acidified water. In general, intestinal calcium uptake in
teleost fish is minimal (pang
et
ai, 1978). However, some of the
extra calcium required by female fish during ovarian maturation
may come from dietary sources. The effects of food availability
.
on gonadal maturation in fish has been reported by several
authors (see Lam, 1983). Scott, (1962) reported a delayed
maturation and reduced egg number in rainbow trout fed on a
restricted diet. Therefore, abnormal development of the ovaries
A
in acid stressed fish could be due to;
1)
reduced feeding
activity of the fish and/or, 2 ) reduced food resources caused
directly or indirectly by acidification. Beamish (1974b) found
no relationship between food availability, and reduced growth
and increased mortality of white suckers in two acidified lakes.
However, in a laboratory study, Beamish (1972) observed that
white suckers exposed to pH 5.0 and below had a reduced feeding
intensity. Yellow perch in acidified lakes had a reduced growth
rate in older age classes which may have been due to food supply
and a change in prey species ( ~ y a nand Harvey, .1981). Some
physiological parameters and biochemical changes occur in fish
during starvation such as anemia, low blood sugar and reduced
serum protein, which were not evident in fish sampled from
acidified lakes (Lockhart and Lutz, 1977). However, these data
are from surviving fish and may represent a more tolerant group
of individuals within the population.
In summary, the results of this study suggest that low pH
exposure of sexually maturing rainbow trout will inhibit
exogenous vitellogenesis. Inhibition of vitellogenin production
and/or incorporation of yolk proteins into developing oocytes,
may result in non-viable eggs and a failure of mature fish to
spawn. High embryo mortalities due to poor egg viability will
result in a decreased recruitment and a reduced rainbow trout
population.
Environmental calcium has a ameliorating infuence on the
effects of low pH exposure, since fish held in low pH, hard
water, did not show an inhibition of vitellogenin production. In
fact, increasing the environmental C a + 2 concentration
significantly enhanced plasma vitellogenin levels above those
levels in fish held in soft water.
Calcium complexes with vitellogenin and therefore an
inhibitory effect of low pH on calcium metabolism may be
partially responsible for the reduced vitellogenin levels
reported here. An inhibition of Ca2+ transport mechanisms may
impair the ability of female fish to obtain extra calcium
required during ovarian maturation as well as maintain normal
calcium homoestasis.
(
D. Chapter 1 1 1 - Effects of pH and/or calcium-enriched freshwater
on gill Ca2+-Adenosine triphosphatase ( A ~ ~ a s eactivity
)
and
osmotic water inflow in rainbow trout
I. Introduction
The gills of teleost fish are the main site of monovalent
ion and some divalent ion (Ca2+)transport, and osmotic water
movement (Potts, 1977; McDonald, 1983). These interrelated
processes are partially responsible for maintenance of ionic and
osmotic equilibria.
The effect of acidification on plasma osmolarity, ionic
balance and other physiological parameters have been reviewed by
several authors (Fromm, 1980; Spry
et
al., 1981; McDonald,
1983). The reduction in plasma Na' of acid-exposed fish is a
result of an increased Na' efflux (packer and Dunson, 1970;
McWilliams and Potts, 1978; McDonald and Wood, 1981) and an
inhibition of Na+ influx ( ~ c ~ i l l i a mand
s Potts, 1978;
McWilliams, 1980; McDonald and Wood, 1981). Increased Na' efflux
may be due to an increased gill permeability caused by the
removal of surface bound Ca2+ by H + (~cwilliams,1983).
A
decrease in Na' uptake may be a result of an inhibition of gill
Na+-K'-ATPase enzyme activity (~ohnstonee t al., 1983).
Plasma c a 2 + concentrations do not vary after exposure of
rainbow trout to acid water conditions (Leivestad
et
al, 1976;
McDonald and Wood, 1981). McDonald and Wood (1981) found that
ca2+ ion fluxes across the gills of trout exposed to low pH did
not change, although urinary Ca2+ losses increased.
'Beamish
et
a1 (1975) observed that female suckers in
acidified lakes did not develop an elevated plasma C a + 2
concentration normally associated with gonadal maturation. Also,
female rainbow trout exposed to low pH water had a female/male
plasma calcium ratio lower than fish held at control pH levels
(Chapter 11). This suggests that c a 2 + regulation in female fish
had probably been impaired.
The chloride cells in the gills of freshwater rainbow trout
have been identified as the site of calcium uptake (Payan
et
al,
1981). A Ca2+ dependent ATPase enzyme located in the gill plasma
membranes may be involved in c a 2 + transport (Ma. e t al, 1974;
Fenwick, 1979; Ho and Chan, 1980). Doneen (1981) described two
kinetic forms of C a 2 + - ~ T ~ a s one
e , with a high substrate affinity
(assayed at 100 p M Ca2+) and the other with a low substrate
affinity (assayed at 4 mM Ca2+).
Flik
et
a1 (1983) have recently re-evaluated the
characteristics of C a 2 + - ~ T ~ a activity
se
and found that
Ca2+-activated ATP hydrolysis in the plasma membranes of eel
gills partially results from non-specific alkaline phosphatase
activities. These authors also suggest that more than one type
of enzyme is involved in transepithelial Ca2+-transport and that
the high affinity C a 2 + - ~ T ~ a activity
se
may represent calcium ion
transport. The effect of low pH on active Ca2+ transport
.
mechanisms in fish gills has not been studied. Most studies
concerned with acid toxicity on fish iono regulation have dealt
with the movement of monovalent ions in fish exposed to acutely
lethal pH levels.
Since ionic problems in fish are partially an indirect
consequence of their gill permeability to water ( ~ o t t s ,19771,
the effects of low pH on ionic regulation may be partially due
to changes in osmotic permeability. Structural changes in the
gill epithelium would also result in changes in gill
permeability to water as well as ions. This study represents the
first to investigate the effects of low pH on the gill osmotic
water uptake of rainbow trout.
Calcium ions in the external media have been shown to
affect the permeability of the gills to monovalent ions (Potts
and Fleming, 1971; Isaia and Masoni, 1976; McWilliams, 1982) and
water movement ( ~ o t t sand Fleming, 1970; Ogawa, 1974; 1saia and
Masoni, 1976; Wendelaar Bonga and Van Der Meij, 1981).
Therefore, increased survival of fish in acidified water of
increased Ca2+ levels (Brown, 1981, 1982a, 1982b11983) may be
due to the ameliorating effects of Ca2+ on ion losses across the
gills.
The inability of fish exposed to low pH to regulate plasma
calcium levels during gonadal maturation, might be due to an
inhibition of active ca2+ transport processes or ion imbalances
caused by changes in gill permeability to water. The long-term
regulation of plasma calcium may be more important than the
short-term loss of ions at more lethal pH levels (Spry
1981).
et
al.,
C
Although plasma Ca2+ concentrations in fish exposed to low
pH do not change markedly, transport processes may be inhibited,
with Ca2+ levels being maintained by such mechanisms as bone
demineralization at an added energy expenditure. Changes in gill
osmctic permeability due to exposure to low pH may also result
in ionic disturbances. However, the environmental calcium
concentration may be an important factar in determining to what
extent low pH effects ionic and osmotic regulation. Therefore,
this study was designed to investigate the effects of low pH
) high and low external Ca2+ concentrations on
levels ( ~ ~ 2 5 . 0at
Ca2+-ATPase activity presumably reflective of active calcium
transport, and osmotic water uptake in the gills of rainbow
trout ( S a l m o g a i rdneri
)
.
11.
Materials and Methods
Experimental F i s h
Rainbow trout (S. gairdneri, 200-300 g) were purchased from
Sun Valley Trout Farm, Mission, British Columbia and transported
to the aquarium facilities at Simon Fraser University. The fish
were held in 2000-L fiberglass tanks supplied with continuously
flowing, aerated and dechlorinated water (~ardness5.2 mgaL- ' as
CaCO,; Ca2+ 0.045-0.069 mM; Na' 0.024 mM; dissolved oxygen, 80%
saturation; pH 6.3-6.5; 14+0.5•‹C). The fish were fed Oregon
moist pellets daily a d libitum, and kept under a simulated
natural photoperiod (~uly-~ugust,
50' Latitude, Vancouver) for
at least one month prior to experimental use. Fish were not fed
48 h prior to experimentation or throughout the duration of the
experimental period.
Di 1 ut i o n Apparat u s
The dilution apparatus used to obtain the desired Ca2+ and
pH levels is shown in Fig 10 (Chapter 111, except the final
solutions from the header tank drained into 104-L black
plexiglass exposure chambers. Chambers were subdivided into six
14-L compartments with a series of holes through each
compartment wall to allow for a complete circulation of the
water. Incoming water flowed into a 18.5-L compartment in front
of the six holding compartments then through a series of holes
in each holding compartment and out the back drain holes. The
flow rate was maintained at 5 Lemin-'. Airstones with compressed
air bubbling through them were placed in the front compartment
and maintained oxygen concentrations above 80% saturation. Water
temperature, pH and Ca2+ concentration were recorded daily.
Calcium ion concentrations were measured by the EDTA titrimetric
method (American Public Health Association, APHA, 1976). Oxygen
concentrations were measured periodically during the exposure
period according to the azide modification technique (APHA,
1976). Free CO, was measured by the titrimetric method (APHA,
1976) and never exceeded 2.0 mg*~-'.
E x p e r i m e n t a1 d e s i g n
Before the start of the experiment fish were selected at
random and anaesthetized in a buffered (NaHCO,) solution of
MS-222 (50.0 rng*~").
Weight and fork length were recorded and a
sample of blood was collected by caudal puncture with an
ammonium heparinized (Sigma) needle. An aliquot of blood was
transferred to two 100 ul heparinized hematocrit tubes and
centrifuged for 3 min to obtain hematocrit values. The remaining
blood was centrifuged for 5 min in a 1.5 mL polypropylene
micro-centrifuge tube and the plasma collected and stored in
-r1 polypropylene tubes at -20'~.
400
Following anaesthesia, fish
were placed in exposure tanks for 14 days (photoperiod 1 1 L : 13
D). Plasma Ca2+ was measured by atomic absorption
spectrophometry (Pye Unican, SP 191), using lanthanum chloride
to reduce phosphate interference. Osmolality was measured with a
vapour pressure osmometer (model 5100 Four groups of fish ( ~ = 7 )
were exposed to the following solutions:
a) Group I
Ca2+
0.052 mM, pH 6.62
b) Group I1
Ca2+
1.50 mM, pH 6.63
c) Group I11
Ca2+
0.052 mM, pH 5.00
d) Group IV
ca2+
1.50 mM, pH 5.10
.
Calcium concentrations were increased from 0.0520 mM to
1.50 mM in groups I1 and IV to represent the calcium content of
lakes of intermediate hardness (100-150 m g @ ~ - 'as CaCO,).
Gill P e r f u s i o n
After the 14-day exposure period, the fish were
anaesthetized in a 50 mg@L-' solution of MS-222 buffered ( p ~
7.0) with NaHCO, and their weight and fork length recorded.
During the gill perfusion, a continuous flow of an aerated 20
m9.L-l
solution of MS-222 was maintained over the gills of the
fish. A longtitudinal incision was made on the ventral surface
to expose the heart and bulbus arteriosus. Blood was collected
by heart puncture with an ammonium heparinized (sigma) needle.
Hematocrit values were recorded and the plasma collected and
stored as previously described. A cannula was inserted into'the
bulbous artericsus and tied off around the ventral aorta with
surgical thread immediately anterior to the branchial arteries.
To drain the blood during the perfusion, the caudal peduncle was
cut off and an incision made in the ventricle. The gills were
perfused with heparinized (0.1%) teleost saline ( ~ a C 1 ,7.25
g*L-'; KC1, 0.38 g * ~ - ' ;CaCl,, 0.162 g*L-'.; MgS04*7H20, 0.23
g*L-'; NaHC03, 1.0 g*L-'.; NaH2P0,*H,0, 0.36 g * ~ - ' ;Glucose 1.0
g*L-'; Wolf, 1963) at a rate of 5.1 mlamin-'. with a 50 cc
syringe connected to a syringe pump (model 341 A, Sage
Instruments).
Gill P r e p a r a t i o n f o r C a 2 + - A T P a s e A s s a y
Gills were excised, rinsed in ice cold saline (0.9% NaC1)
and blotted dry. The gill lamellae were trimmed from the gill
arches and placed in a preweighed 10-mL beaker containing 5.0 mL
of homogenizing media (0.25 M sucrose, 5.0 rnM EDTA, 10 mM
imidazole buffer) and weighed to determine gill filament weight.
Homogenizing medium was added to the gill filaments to provide a
10% w:v preparation. The gill filaments were homogenized with a
glass mortar and pestle (15 passes). The homogenate was
centrifuged in a Sorvall superspeed RC2-B centrifuge at 1,000g
for 10 min to sediment nuclei. The supernatant was decanted off
and centrifuged at 25,000g for 20 min to sediment the
mitochondria. The supernatant was then placed into
ultracentrifuge tubes and centrifuged at 35,000g for 1 hour at
3OC (~eckmanL2-65B) to obtain the heavy microsomal fraction.
The microsomal pellet was resuspended in 5mM Tris-HC1 buffer,
0.5 mM imidazole buffered to pH 7.8 at 25% weight by volume of
the gill filament weight. This enzyme homogenate was placed on
ice and used immediately for the C a 2 + - A T ~ a s e
assay.
C a 2 + - A T P a s e Assay
Enzyme activity was measured by the Caz+-dependent
adenosine triphosphate (ATP) hydrolysis of inorganic phosphate.
To measure the low affinity ATPase activity, 0.1 mL of enzyme
suspension was mixed with 1.8 mL 3mM CaCl,, 20 mM Tris-HC1 ( p ~
8.0) and allowed to preincubate for 10 min at 20'~. The reaction
was started by the addition of 0.1 mL vanadate-free sodium ATP
at a final concentration of 5 mM and incubated for 20 rnin at
20•‹C. The reaction was stopped by the addition of 0.5 mL cold
30% trichloroacetic acid. Reaction vials were centrifuged at 900
g for 5 min to isolate precipitated proteins.
High affinity ATPase activity was measured by using 100)(M
CaCl,, 20 mM ~ris-HC1,(pH 8.0) incubation media. Assay
procedures followed those described above for the low affinity
enzyme.
All glassware was acid washed in chromerge for 24 h and
rinsed with 10 mM ethylenediamine-tetraacetate (EDTA). Double
distilled ca2+-free water was used to make up the incubation
media and all other chemical solutions used in the assay. Blanks
contained either 1.9 mL 3 mM CaC1, or 1.9 mL 1 0 0 p M CaC1, and
0.1 mL Na,ATP. All measurements were done in triplicate for each
individual fish.
Inorganic phosphate was measured in 1.0 mL aliquots as
described by Peterson (1978) using subsequent modifications by
Doneen (1981). The concentration and quantity of sodium dodecyl
sulfate (sDS) was changed as well as the addition of
p-methylaminophenol sulfate (ELON) in place of
aminonaphtholsulfonic acid (ANSA). Briefly, the 1.0 mL sample
was added to 1.0 mL of 2.5% ammonium molybdate in 4 N HC1,
followed by the addition of 1.0 mL 8% SDS and 0.5 mL 2% ELON in
5% sodium sulfate as the reducing agent (Lebel
et
al.,
1978).
The volume was then increased to 5 mL with double distilled
water, vortexed and read on a perkin-~lmer spectrophotometer
olema man 55) at 700 nm after standing for 30 minutes at 20•‹C.
Standard phosphate solutions were prepared by diluting a stock
solution of KH,PO,. The standards and blanks were treated the
same as the sample solutions. All glassware was acid washed and
rinsed in double distilled water. This method provided good
colour stability and retention of linearity in the range of tihe
standard curve.
c a 2 + - ~ ~ p a activity
se
was expressed as pmol Piamg
protein-'ah-'. Total protein in the enzyme homogenate was
measured by a modification of the Albro (1975) technique. The
procedure involved the addition of 50 uL of enzyme homogenate to
2 mL of 0.25 N NaOH. After a 10 rnin preincubation, 1.0 mL of
alkaline copper solution (10% Na,CO,, 0.1% sodium tartrate,
0.05% cupric sulfate) was added followed by another 10 rnin
incubation. Four mL 0.111 N phenol was then added, incubated at
55•‹C for 5 rnin and then cooled rapidly in ice water for 1 min.
The samples were then read at 650 nm within 30 rnin on a
Perkin-Elmer spectrophotometer. Bovine serum albumin (Sigma) was
used as the standard protein solution over the range of 10
pg*m~' to 200 )~g.mL-
'.
Osmotic Water Inflow
Following the gill perfusion, the first two anterior gill
arches on the right side of the fish and the first anterior gill
arch on the left side were excised and rinsed in ice cold
teleost saline (0.9% ~aC1). In v i t r o osmotic water uptake by the
gills was determined by the procedure outlined by Ogawa et al.
(19731, Ogawa (19741, and Lock (1979). The isolated gills were
placed in 20 ml of oxygenated teleost saline (270 mOsm) and
incubated for 20 rnin at 2 0 " ~ .After the 20 rnin incubation period
the gills were blotted dry for 15 sec and their weight
determined. The gills were then placed in double-distilled water
( 2 0 " ~ )for 15, 30 and 45 min. They were weighed before and after
each 15 rnin time interval, After the final weighing the arches
were dried at 1 1 0 " ~for 24 h to determine dry weight.
St at i s t i c s
A11 statistical analyses were performed by using the SPSSX
data analysis system, SPSSX Manova program (SPSSX User's Guide,
1983).
Before performing statistical analyses, all variables were
checked for homogeneity of variances (Bartlett's test) and
normality. A factorial analysis of variance was used to test for
differences among the group means for each of the following
parameters, Ca2+-ATPase activity, osmolarity, plasma Ca2+ and
microsomal protein (Winer, 1971). An arcsine transformation was
performed on hematocrit data before comparing the differences
among group means by analysis of variance.
An analysis of covariance was used to test whether osmotic
water inflow rates differed among the four treatment groups
( ~ i n e r ,1971; Neter and Wasserman, 1974; SPSSX User's Guide,
1983). The effect that the initial water content of the gills
had on water retention and osmotic inflow rates was tested and
corrected for in the analysis, as a covariate. There was no
significant difference (p10.05) in dry weight of the gills
between the groups. Significance was accepted at the 95% level
(~10.05).
The dry weight of the gills was not significantly different
(p>0.05) among the groups, therefore differences in osmotic
water influx were not affected by the size of the gills. After a
20 min preincubation in a media of known osmotic value, the
osmotic water influx during the incubation periods in distilled
water should, therefore, be dependent upon the condition of the
gill membrane as well a s the osmotic gradient rather than the
mass of gill tissue.
111.
Results
B l o o d Parameters
No significant differences in the plasma Ca2+ levels of the
blood were observed between the groups (Table 9 , p>0.05).
Exposure of fish to low pH and/or calcium-enriched freshwater
had no significant effect on hematocrit.
Fish exposed to calcium-enriched freshwater
(
1.5 mM Ca2+)
exhibited a significant reduction in plasma osmolarity in
comparison with fish exposed to water of 0.052 mM ca2+ (p10.05).
Exposure of fish to pH 5..0-5.1, regardless of the environmental
calcium concentration, caused a slight but non-significant
decrease in plasma osmolarity
Ca2+-ATPase Act i v i
able 9).
t y
In fish exposed to pH 5.0 or 5.1, specific activity of the
low affinity (assayed at 3 mM c a 2 + ) C a 2 + - ~ T P a s eenzyme was not
significantly different from fish exposed to control pH levels
(p>0.05). Low-affinity ca2'-~Tpase activity was significantly
reduced in fish exposed to calcium-enriched freshwater,
regardless of pH
able 10, p10.05). There were no significant
effects of low pH and/or increased ambient calcium
Table 9: Blood parameters of rainbow trout after
14 days exposure to water of different
pH and/or calcium concentration (mean k S.E.).
( I ) pH 6.62, C a 2 + 0.052 mM;
(11) pH 6.63, C a 2 + 1.50 mM;
( 1 1 1 ) pH 5.00, C a 2 + 0.052 mM;
(IV) pH 5.10, C a 2 + 1.50 mM;
Group
Hematocrits
%
Osmolarity
(mOsm*~g-')
CaZ+
(mM)
Table 10: C a + 2 - ~ T P a s eactivity in rainbow trout
after exposure to solutions of different pH and/or
calcium concentration (mean k S . E . ) .
( I ) pH 6.62, C a 2 + 0.052 mM;
(11) pH 6.63, C a 2 + 1.50 mM;
(111) pH 5.00, C a 2 + 0.052 mM;
( I V ) pH 5.10, C a 2 + 1.50 mM;
Group
N
C a 2 + - ~ ~ p a sactivity
e
(umol Piemg-'proteinah-')
[ C a + 2 ]3mM
[ C a 2 + ] 100
yM
concentration on the specific activity of the high affinity
(assayed at 100 p M C a 2 + ) Ca2+-ATPase enzymes (Table 10).
High-affinity C a 2 + - ~ ~ p a activities
se
showed a trend similar to
the low- affinity enzyme in fish exposed to calcium-enriched
freshwater.
Although not significant, the quantity of protein recovered
in the heavy microsomal fraction of the gills showed a tendency
(P=0.077) to be higher in fish exposed to calcium-enriched water
(Table 11).
The total enzyme activity in the gills, expressed as
pmol Pioh-log-' gill filament tissue, showed no significant
difference in fish exposed to low pH and/or calcium-enriched
freshwater
able
11).
A slight increase in the gill microsomal
protein without a simultaneous increase in total enzyme activity
may suggest a synthesis of proteins other than those from
enzymes involved in ATP hydrolysis.
Osmot i c V a t e r I n f l ow
Exposure of fish to calcium-enriched freshwater
significantly enhanced the rate of osmotic water influx,
regardless of pH (Fig. 14, ~10.05). Although pH did not
significantly affect the rate of weight increase of the gills of
group I11 fish (low C a 2 + , pH 5.0) the graph showed a slightly
.
higher weight increase than the control group (Fig. 14, p>0.05).
The graph of group I11 is linear and coincides with the graphs
of the calcium-enriched freshwater groups (11 and IV) to the 30
min incubation point. After the 30 min interval the rate of
weight increase drops off and water uptake is no longer linear.
This sudden drop in the linear uptake of water might indicate a
change in the structural integrity of the gill membranes (Lock,
1979)
from exposure to low pH and low calcium.
Table 1 1 : Protein concentrations and low affinity ~ a + ~ - ~ ~ ~ a s e
(PM ~ i e g - 'gill filament tissue) in rainbow
trout after 14 days exposure to different pH and/or
calcium ion concentrations (mean + S.E.).
(I)
pH 6.62, C a 2 + 0 . 0 5 2 mM;
(11) pH 6.63, C a 2 + 1.50 mM;
(111) pH 5.00, C a 2 + 0 . 0 5 2 mM;
(IV) pH 5.10, C a 2 + 1.50 mM;
Group
N
Microsomal Protein
( ~ g e m g'- gill tissue)
CaZ+-ATPase activity
(pmol Pi@h-l@g-l gill)
Fig. 14. Effect of low pH and/or calcium-enriched water on the
i n v i t r o osmotic water inflow of the gills of rainbow trout
exposed i n v i v o . At the time intervals, each point represents
the mean gill wet weight (adjusted by the covariate, initial
water content) of all fish in each group.
TIME (min.)
IV. Discussion
Plasma Cal ci urn, Hemat ocri t and Osmol ari t y
The ability of rainbow trout to maintain normal plasma Ca2+
concentrations during exposure to acidified water (pH 5.0 or
5.1) in the present study, agrees with the findings of Leivestad
and Muniz, ( 1 9 7 6 ) and McDonald and Wood, (1981). However,
McKeown et al., ( 1 984) recently reported some .fluctuation in
plasma calcium ion levels in rainbow trout exposed to pH 4.9 and
a tendency for lower plasma calcium ion levels in suckers
(Cat ost omus macrocheil us). reared at pH 4.9 and 5.6 relative to
control fish. Other studies have also reported some tendency for
plasma calcium ion levels to fall (McDonald et al., 1980) or
fluctuate (Saunders et a1
.,
1983) in fish exposed to low pH
freshwater.
Plasma ca2+ concentrations in fish exposed to
calcium-enriched freshwater did not change relative to fish
exposed to low environmental calcium concentrations. These
results are in agreement with observations made by other authors
on fish exposed to increased external calcium concentrations
(~meharaand Oguri, 1978; McDonald et al., 1980; Wendelaar Bonga
and Van Der Meij, 1980; Shephard, 1981).
The absence of a change in hematocrit of acid ( p 5.0-5.1)
~
exposed fish suggests: 1 ) no significant hematological
disturbances occurred and/or 2)the exposure period was long
enough and/or the pH was high enough for fish to recover from
initial disturbances. Hematocrit values reported in the
literature for fish exposed to low pH are variable. These
discrepancies may have been dependent on exposure time of the
fish, species differences, field versus laboratory measurements
as well as the H + concentration of the ambient environment.
Chronic exposure of fish to water of pHS4.0 (Neville, 1979) or
acute exposure to pH<5.0 (McDonald e t al., 1980) caused an
increase in hematocrit while chronic exposure of fish to pH>4.2
had no effect on blood hematocrit (see ref. Wood and McDonald,
1982). Chronic exposure was designated by Wood and McDonald
(1982) as greater than 7 days and acute exposure 7 days or less.
Fish that survive chronic exposure to low pH water appear to
eventually compensate for hematological changes that occur
during the acute exposure period. The increase in hematocrit
associated with acid exposure is believed to be a result of at
least three processes; erythrocyte swelling, a reduction in
plasma volume, and mobilization of erythrocytes from the spleen
(Milligan and Wood, 1982). Most studies monitoring acid-base
balance, hematological disturbances, plasma ions and other
.
physiological parameters have been performed on fish exposed to
pH values below pH 4.5 for acute-exposure periods.
Plasma osmolarity decreased in fish exposed to high ambient
Ca2+ concentration relative to fish maintained in low external
calcium concentrations. This decrease may have been due to a
drop in plasma ion concentrations. McDonald
et
al., (1980) found
plasma Na+ and C1- concentrations were significantly lower in
rainbow trout acclimated to high c a 2 + , low salt concentration,
compared with fish acclimated to soft water. Their results make
a good comparison since c~(NO,), was used to increase the water
calcium concentration to approximately the same concentration
(1.35 m ~ )
as in the present study. Wendelaar Bonga and Van Der
Meij, (1980) also observed a significant decrease in plasma
osmolarity in trout exposed to calcium-enriched (20.6 mEqeL-'1
freshwater. However, these authors did not observe any
concurrent changes in plasma Na+, C1- or c a 2 + concentrations
making the reason for the drop in plasma osmolarity obscure.
Plasma N a + and C1- levels were not measured in the present
study. Therefore the reason for a decrease in plasma osmolarity
is not completely clear. However, since osmotic water inflow
increased in the gills of fish adapted to calcium-enriched
freshwater ( ~ i g .11, urine flow may have also been enhanced.
This may have increased the renal loss of Na+ and C1- (Lock,
1979) accounting for the drop in plasma osmolarity.
The reduction in plasma osmolarity may partially be a
.
result of changes in the transepithelial potential (TEP).
Changes in pH and c a 2 + concentration of the external media can
cause reversible changes in the TEP (Mc~illiamsand Potts,
1978). In their study, acidification of the external media
caused the TEP to reverse from negative(inside) to
positive(inside). Increasing the Ca2+ concentration also
produced a reversible change in the TEP in a positive direction,
since C1- diffuses out more rapidly than Na+ when the C a + 2
concentration exceeds 0.5 mM. Changes in the diffusional fluxes
of these monovalent ions due to positive shifts in the TEP
across the gills may account for loss of ions responsible for
changes in plasma osmolarity. Although TEP was not measured in
the present study the environmental calcium concentration in the
calcium enriched groups was 1.5 mM, which would have been
adequate to cause a positive shift in the TEP.
Changes in water content between different tissues (kidney,
liver, muscle) and the blood, may have accounted for the
decrease in plasma osmolarity of trout exposed calcium-enriched
freshwater. Oduleye (1975) found environmental calcium
concentration affected total body water content in brown trout.
This author also observed that body water is contained within
different tissue compartments and these various tissues exchange
water with the blood at different rates.
Plasma Ca2+ concentrations and C a + 2 - ~ ~ ~ activity
ase
were
not affected by exposure to pH 5.0 suggesting that calcium
metabolism in immature trout or trout in the previtellogenic
period is not affected. Therefore, disturbances in calcium
metabolism caused by exposure to low pH, in trout in the process
of vitellogenesis.(see Chapter II), are probably not a result of
the inhibition of calcium transport at the gills. Whether active
Ca2+ uptake in fish exposed to low pH is inhibited has not been
previously studied. However, branchial ca2+ fluxes in rainbow
trout exposed to low pH were measured by McDonald and Wood
(1981) and did not change significantly. Other ATPase ion
transport systems have been measured by Johnstone e t al., (1983)
in the gills of Atlantic salmon (Salmo s a l a r ) exposed to low pH.
These authors found no changes in ~ g ~ + - ~ T P aand
s e Na+-ATPase
enyzme activities in fish exposed to pH 4.9 relative to those in
control fish. Inhibition of Mg2+-ATPase activities (~ohnstonee t
al., 1983) and Na+ K+-ATPase activities (Johnstone e t al., 1983;
Saunders e t al., 1983) did not occur in salmon until they were
reared in freshwater acidified to pH 4.7 and below.
In the present study a decrease in C a 2 + - ~ ~ p a s
enzyme
e
activities were observed in fish exposed to calcium-enriched
freshwater. When the external ca2+ concentration is lower than
the blood ca2+ concentration a passive efflux of calcium occurs
(~ilhaude t al., 1977). If the concentration gradient is
decreased by increasing the ambient c a 2 + concentration then the
passive loss of Ca2+ should be reduced. A reduction in Ca2+
.
efflux as the external media increases in Ca2+ concentration
might reduce the requirement for active transport of C a 2 + into
the fish. These results are in agreement with some of the work
reported in the literature but at variance with a few other
studies.
Fenwick ( 1 9 7 9 ) reported a higher ca2+-activated ATPase
activity in the gills -of freshwater eels than in seawater
acclimated eels. Low affinity Ca2+-ATPase activity increased in
freshwater adapted Gillichthys in comparison to a hypersaline
group (170%) although no differences were observed between
seawater and freshwater adapted fish
oneen en,
1981). Ma ( 1 9 7 6 )
also observed no changes in C a * + - ~ ~ P a activity
se
when fish were
transferred from freshwater to seawater. Absolute C a 2 + - ~ ~ p a s e
enzyme activity in roach (Rutilus rutilus L ) did not differ
after exposure to high or low external Ca2+ concentrations for
seven weeks (Shephard, 1981). However, field studies by Shephard
( 1 9 8 1 ) showed fish living in waters of varying calcium
concentrations had enzyme activities proportional to the
external calcium concentration. Umehara and Oguri ( 1 9 7 8 ) also
found little change in the activities of Ca2+-A~Pasesin the
gills of goldfish after 3 weeks exposure to calcium enriched
freshwater. Only one study, by Burdick et al. ( 1 9 7 6 1 , reported
higher ca2+-A~paseenzyme activities in the gills of killifish
adapted to calcium-enriched freshwater in comparison with those
fish adapted to freshwater. Higher Ca2+-ATPase activites in
seawater adapted fish in comparison with freshwater adapted fish
have been reported by some authors (~urdicket al., 1976; Ho and
Chan, 1980). Differences in the literature regarding Ca2+-ATPase
activities in the gills of fish exposed to different levels of
environmental calcium, may be partially related to differences
in the renal excretion of Ca2+ in different species, time of
adaptation of the fish and differences in Ca2+ concentration of
the assay media for assaying optimum activity of the enzyme.
Shephard ( 1 9 8 1 ) suggests this enzyme may be capable of
short-term responses to a change in external calcium
concentration. Further studies of fish populations in hard and
soft water, and chronic exposure to calcium-enriched water under
laboratory controlled conditions, are required to determine the
properties of this enzyme.
Inhibition of Ca2+-ATPase enzyme activities in fish adapted
to calcium-enriched freshwater may have been due to the release
of an inhibitory factor 'hypocalcin' (pang
et
al., 1 9 7 4 ) . This
compound is secreted from the Stannius Corpuscles (~urdicke t
al., 1976; f en wick, 1976; Ma, 1976; So and Fenwick, 1 9 7 9 ) .
Ca+2-AT~aseactivity increased when calculated on the basis of
tissue weight in stanniectomized eels in comparison with sham
operated eels (Fenwick, 1 9 7 6 ) . The activity of the Stannius
Corpuscles is greater in seawater adapted killifish than those
held in Ca2+ deficient seawater (pang, 1 9 7 3 ) .
Doneen ( 1 9 8 1 ) suggested that Ca2+ transport in fish gills
may involve both low and high affinity Ca2+-ATPases operating at
different sites or by different mechanisms. Ca+2-ATPase assays
.
were measured for low ( 3 mM) affinity and high affinity ( 1 0 0
UM)
activities in the present study. This would give an indication
whether changes in environmental pH and/or Ca2+ concentrations
affect these C a 2 + - ~ T p a ~
kinetic
e
forms differently. The activity
of the low affinity ATPase decreased in fish exposed to the
calcium-enriched freshwater, independent of pH, while the high
affinity form did not change significantly. Doneen (1981)
observed similar changes in Gi 1 l i c h t h y s where the low af f inity
activity was higher in freshwater adapted fish compared with
seawater (170%) adapted fish while the high affinity activity
remained unchanged.
Osmotic Water Uptake
Fish exposed to low pH and low environmental calcium,
displayed a tendency for an increased gill osmotic water influx
(Fig.
14).
This trend is likely caused by a loss of bound ca2+
from the surface of the gill membranes and a resultant increase
in gill permeability ( ~ c ~ i l l i a m s1983).
,
Increased branchial
osmotic water uptake may be balanced by an increased renal
output as well as water entering intracellular compartments.
Exposure of rainbow trout to pH 4.0 -4.5 has shown a net loss of
Na+ and C1- across the gills resulting in electrolyte shifts
from intracellular to extracellular compartments with water
moving in the opposite direction (Wood and McDonald, 1982).
Although plasma N a + and C1- were not measured in the present
study, a reduction in plasma Na+ and C1- of fish exposed to low
.
pH has been observed in other studies ( ~ c ~ e o wetn a1 , 1984;
McDonald
&
Wood, 1981; McWilliams
&
Potts, 1978, Spry et a l . ,
1981).
The increase in osmotic permeability of the gills in fish
exposed to Ca2+-enriched freshwater at both pH levels is in
contrast to other studies that show increasing the ambient C a 2 +
concentration reduces the osmotic and ionic permeabilities of
fish gills ( ~ o t t sand Fleming, 1970, 1971; Ogawa, 1974; Isaia
and Masoni, 1976; Wendelaar Bonga and Van der Meij, 1981). This
increase is possibly a result of the higher water osmolarity of
the calcium-enriched groups. Wendelaar Bonga and Van der Meij
(1981) found that the gills of fish exposed to a hypo-osmotic
media ([Cab1=0.8 mM) showed an increase in the net osmotic
water flow rate with increasing osmolarity of the ambient media,
becoming maximal when isosmotic. Therefore, the osmotic
permeability of the gills is inversely related to the magnitude
of the osmotic gradient between the plasma and external media
when the ambient media is hypo-osmotic (Wendelaar Bonga and Van
der Meij, 1981). In media of high Ca2+ concentration (10 mM Ca2+
Wendelaar Bonga and Van der Meij (1981) found ambient osmolarity
had no effect on osmotic water inflow which is always low.
As the external media becomes more hyposmotic to the blood
of fish, the gills become less permeable to water to reduce
hydration. The increase in osmolarity between the low and high
calcium groups may have had a greater effect on increasing gill
permeability, than the increase in Ca2+ concentration to reduce
gill permeability. In a hyposmotic media, high calcium
concentrations (10 mM) have a greater effect on reducing gill
permeability than osmolarity has on increasing it. The majority
of studies cited above, showing that external calcium reduces
the osmotic permeability of fish gills, have used calcium
concentrations of 10 mM Ca2+. This may partially account for the
discrepancy between the present study and other studies.
However, Potts and Fleming ( 1 9 7 0 ) found
~
an increase in the
exchange of tritiated water in the gills of F u n d u l u s when the
ambient Ca+' concentration was reduced to 0.1 mM CaZ+ from 1.0
mM Ca2+. Whether differences in the osmolarities of the media
used by Potts and Fleming, (1970) or species differences are the
cause of the discrepancy between their study and the present
study is not clear. An alternate explanation may be the
difference in techniques, Potts and Fleming ( 1 9 7 0 ) measured the
exchange of tritiated H 2 0 in the intact animal, in comparison
with the measurment of osmotic water inflow of isolated gills
used in the present study.
McWilliams (1982) found that calcium had its greatest
influence on gill permeability to ions at concentrations between
0 and 1 mM Ca2+ and increasing the ca2+ concentration above 1 mM
produced no further effect. This suggests that the threshold
level at which the ambient c a 2 + concentration affects gill
permeability may be higher for osmotic compared with ionic
regulation, since an external calcium concentration of 1.5 mM
.
Ca2+ did not reduce osmotic water inflow. Although ionic and
osmotic regulation are often linked, Kostechi and Jones (1983)
found a higher mortality in rainbow trout due to the effects of
ionic stress in comparison to osmotic stress.
In summary, the results of this study suggest calcium
metabolism is not affected in trout exposed to intermediate
acidic pH levels (pH 5.0-5.1).
This is based on the observations
that plasma calcium levels and gill C a 2 + - ~ ~ p a activities
se
did
not change in trout after exposure to low pH. It is possible
that pH 5.0 represents a threshold pH level and that increasing
the H + concentration above this level may significantly affect
the calcium balance in trout.
E. Summary
The results of this study show that pH levels 5.5 and less
reduce the survival of kokanee and sockeye saimon eggs and
alevins. Exposure of mature fish to pH 5.6 and below prior to
ovulation and spawning also increases embryo mortality. Further,
vitellogenesis in rainbow trout exposed to pH 5.0 was inhibited
and may result in non-viable eggs or a failure of trout to
spawn. The effects of low pH on egg quality and production in
broodstock fish may be more important to the survival of fish
populations than losses due to egg and alevin .mortalities.
Studies only investigating the effects of low pH on embryo
survival and not incorporating the effects on vitellogenesis and
egg maturation are under-estimating lethal pH levels affecting
the survival of fish populations.
Reduced vitellogenin production in acid exposed fish may
have been caused by the absence of an increased plasma calcium
ion concentration. Low plasma calcium levels may be attributed
to an inhibition of calcium uptake and/or an increased loss of
calcium from the body. However, no effect of low pH exposure of
se
activity was observed.
rainbow trout on gill C a 2 + - ~ ~ p a enzyme
Although not measured, loss of calcium through the renal system
and from skin and scales may have been significant enough in
acid exposed fish to inhibit vitellogenesis, while not severely
disturbing normal plasma calcium ion levels.
Alternatively, an inhibition of the endocrine system
controlling the synthesis and release of vitellogenin may have
occurred in rainbow trout exposed to pH 5.0 accounting for both
low plasma vitellogenin and calcium ion levels.
Fish held in water enriched with calcium had higher plasma
vitellogenin levels, suggesting environmental calcium
concentration benefitted the reproductive the health of the
fish. Gill Ca2+-ATPase activity and gill osmotic water inflow
were also decreased and increased, respectively, in fish exposed
to water enriched with calcium. Although no significant
interactive effects of c a 2 + and pH were observed, the data did
suggest an ameliorating influence of calcium on the
physiological responses of salmonids to low pH exposure.
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