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This is to certify that the

thesis entitled

EFFECT OF pH ON IN_VITRO OXYGEN CONSUMPTION AND MUCUS

PRODUCTION OF GILL TISSUE OF RAINBOW TROUT (SALMO GAIDNERI)

 

presented by

Hamdan Hj. Mohd. Noor

has been accepted towards fulfillment
of the requirements for

M. S . degree in Physiology

@141 mm

Major professor

 

Date AUgUSt 26, 1982

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EFFECT OF pH ON IN_VITRO OXYGEN CONSUMPTION AND MUCUS

PRODUCTION OF GILL TISSUE OF RAINBOW TROUT (SALMO GAIRDNERI)

 

By

Hamdan Hj. Mohd. Noor

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE
Department of Physiology

1982

ABSTRACT

EFFECT OF pH ON IN_VITRO OXYGEN CONSUMPTION AND MUCUS
PRODUCTION OF GILL TISSUE OF RAINBOW TROUT (SALMO GAIRDNERI)

 

By

Hamdan Hj. Mohd. Noor

The mechanism causing fish death after exposure to low pH remains
undefined. Anoxia, as evidenced by decreased oxygen consumption, has
been suggested as 51 causative factor. The transfer capacity of fish
gills is influenced by their oxygen consumption and by the diffusion
distance across the gill. This investigation was conducted to determine
the effect of pH on both of these parameters. Trout were exposed to
acid water (pH 6.0, 5.0 and 4.0) for 2 h and oxygen consumption and
mucus content of the gill tissue were determined. Gill tissues from
exposed fish showed higher oxygen consumption and greater mucus
production than controls (pH '7.4). Histologically, exposed tissues
revealed more mucus cells and exhibited greater damage. It is suggested
that increased mucus production influences the oxygen transfer capacity
of gills three ways: (a) by increasing diffusion distance, (b) by

preventing proper ventilation and (c) by increasing gill metabolism.

DEDICATION

To my beloved parents
Hj. Mohd. Noor Hj. Daud and Hjh. Asma’ Hj. Saad

From a grateful son

11

ACKNOWLEDGEMENTS

I would like to express my sincere thanks to Dr. P.O. Fromm who has
been extraordinarily patient while teaching and guiding me through my
graduate work in the department. Without his constant support and
encouragement this thesis would not have been possible.

I would also like to express my appreciation to Dr. J.R. Hoffert
for his ever open door and ears and for his aid in the research work and
in the preparation of this thesis.

I would also like to thank Dr. W.L. Frantz for his helpful review
of this thesis.

Special thanks go to the members and friends of the fish lab: Paul
Desrochers, Bill Kreft, Ken Pratt and especially Esther Brenke for all
their help and friendship.

This research was supported by NSF Grant ENV77-123OO and by the
Michigan Agricultural Experiment Station, Project 122.

I am indebted to the Government of Malaysia for having sponsored my

education at Michigan State University.

iii

TABLE OF CONTENTS
, Page
LIST OF TABLES..............0....................................... Vi
LIST OF FIGURES......0...0.......................0.................. Vii
INTRODUCTION AND LITERATURE REVIEW.................................. 1

A. Water Pollution and Its Consequence.......................... 1

N

B. Effect of Acid Stress on Growth and Reprodution of Fish......

C. Physiological Studies on the Effect of Acid Stress on Fish...
1. Failure of Ionoregulation...............................
2. Failure of Acid Base Regulation.........................
3. Failure of Oxygen Uptake................................

(DUI-Pk»;

RESEARCH RATIONALE. . . . . . . O O . O . ..... O O O O O O O I I O O O O . . . O O . I . O . . O O O . . . . . . 1 1
MATERIALS AND METHODS. . . . . . . . . . . . . . . O . O O . O . . O O O O O O I O . . . . . . C . . . . . . . . . 14
Experimental Animals . . . . . . . . . O . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
Gill Tissue Preparation. . . . . . . . . . . . . . . . . . . . . . . . O . . . . O O . O I . . O I . . . 14
oxygen consumption Of Gill Tissue. . . . . . . . . O . . . . . . . O O O O . O O O O O . . . . 15
Sialic ACid content Of Gill Tissue. . . . . . . . . . . . . . . . . . . . . . . . . . I . . . 16
Histological Observation of Gill Tissue......................... 16
Statistical Treatment Of Data. . . . . O . . . . . . . . . . . . . . O . . . . O . . . O . . . . . 17
RESULTS.........0.0.................................................18
A. Oxygen Consumption of Gill Tissues........................... 18

1 . Experiment 1 . . . . . . . I . I O O O . . . . . . O . I . . . . . O . O O . O O O . O . . O O I . . 18

2. Experiment 2. . . . . . . . . . . O . . O O O . . . . . . . . . . . O . . . . . . . O . . . . . . . 18

b. EXPerj-ment 'j. . . . . . . O O . O . . . . . . . . . . . . O O . . . . . O . . . . O O . . . . . . . 2.5

4. Experiment 4. . . . O . . O . . O . O . I O . . O I O . O O O O O . . . . . . . . O . . . O . . O . 2'5

5 . Experiment 5. . . . . . O I O . . . O O O . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50

B. Sialic ACid content Of Gill Tissue. . . . . . . . . . . . . O . . . O . . O I O . O O . 30

C. HiStOlogical Observations..........................0...00.... j1

iv

DISCUSSIOIq. . . . . . . . . . . . . O I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
CONCLUSIONS. . . . . . . . . . O . . . O . . . O O . . O O . O O O . . . . . . . O . . . O . . . . . . . . . . . O . O . . .
APPENDIX I: comPOSitj-on Of Pe rquion Fluid . . . . . . . . . . . . . . . . . . . . . O . . . .

APPENDIX II: Composition of Citric Acid/Sodium Phosphate
Buffer at Different pH Level-8.......................

APPENDIX III: Thiobarbituric Assay for Aialic Acid -
For use in Estimating mucus...........0.............

LITERATURE CITED.........................0..........................

D6
43
44

45

47

49

LIST OF TABLES

Table Page

1. Effect of exposing the fish for 2 h at various pH
levels on sialic acid content of the gill tissue................ 30

vi

LIST OF FIGURES

Figures Page

1. Effect of pH of the incubation media on oxygen consumption
0f the gill tissue.O..0.000.0...OOOOOOOOOOOOOOCOOOOIOOOO00...... 20

2. Effect of exposing gill tissues for one hour in the
incubation media prior to measuring oxygen consumption.......... 22

3- Effect Of PO on oxygen consumption of gill tissues............. 25
2

4. Effect of exposing the fish for two hours at various pH
levels on in vitro oxygen consumption of gill tissues........... 27

5. Effect of prior exposure of fish for two hours at various pH
levels on in vitro oxygen consumption of gill tissues........... 29

6. Photomicrograph of longitudinal section of gill filaments
from rainbow trout exposed to pH 7.4 for two hours.............. 35

7. Photomicrograph of longitudinal section of gill filaments
from rainbow trout exposed to pH 6.0 for two hours.............. 35

8. Photomicrograph of longitudinal section of gill filaments
from rainbow trout exposed to pH 7.4 for two hours.............. 55

9. Photomicrograph of longitudinal section of gill filaments
from rainbow trout exposed to pH 6.0 for two hours.............. 55

10. Effect of exposing the fish for 2 h at various pH levels
on in vitro oxygen consumption of gill tissue................... 40

vii

INTRODUCTION AND LITERATURE REVIEW

A.Water Pollution and Its Consequence

 

Pollution of our surface waters has been a problem worldwide for
many years. Many fish and other aquatic animals which are directly
affected by pollution, not only serve as an important protein source for
2a lot of people, but also provide various medically and industrially
important chemicals. Therefore, anything which is a threat to the life
of these animals is undesirable. Currently acidification of fresh water
lakes and rivers is perhaps the most widespread serious problem of water
pollution.

Acidification of our environment is indirectly due to the steady
increase in our demand for energy. Acid loading of lakes arises either
from dilute acid in precipitation or from dry deposition of oxides of
sulfur or nitrogen which, on contact with water, form their respective
strong acids (Conroy 33 al., 1976; Dillon 33 al., 1978; Likens 32 al.,
1979). Galloway, Likens and Egerton (1976) concluded that sulfuric and
nitric acids are the primary sources of excess H+ in acid precipitation.
Petroleum used for power generation and in internal combustion engines
is a prime source of oxides of nitrogen whereas combustion of coal in
electric power plants, smelters and steel mills leads to production of

oxides of sulfur.

2

Some lakes are more susceptible to acidification than others because
of a lower buffering capacity of the watershed (Likens 33 al., 1979).
For example, the pH of George Lake in Ontario, a poorly buffered lake,
declined at an estimated rate of 0.13 pH units per year from 1961 to
1973 (Beamish 3311,; 1975).

Research on the Ifiological effects of water pollutants during the
past 30 to 40 years has been primarily concerned with the determination
of concentration of pollutants which kill test animals. The European
Inland Fisheries Advisory Commission (1969) pointed out that the safe
range of environmental pH for fish survival appears to be 5—9 and for
maximum productivity the pH value should be between 6.5 and 8.5. These
criteria have been generally accepted.

Freshwater fish are vulnerable to lowered environmental pH because
the buffer capacity of their body fluids is low and consequently is
easily overwhelmed if acid enters the fish or if metabolic acid is
prevented from being excreted. Since the plasma PCO2 of fish is low
(2-3 mmHg), the bicarbonate content must be cOrrespondingly low in order
to maintain the pH in the physiological range. In addition, blood
protein and hemoglobin in fish are low compared to mammals; thus the
non-bicarbonate buffer capacity is also low. Furthermore, fish do not
have the luxury of manipulating the bicarbonate/carbonic acid buffer
system by changing respiratory ventilation, viz., respiratory
ventilation is dedicated to providing adequate oxygen to the fish.

B. Effect of Acid Stress on Growth and Reproduction of Fish

Data from field studies on the effect of acid stress on growth of

fish are difficult to assess due to a variable food supply.

Environmental acidifiction is often accompanied by an increase in heavy

3

metal ion concentration and it is very difficult to separate the effects
of these ions and that of pH on growth (Beamish, 1974). There are
conflicting results from well controlled hatchery-laboratory experiments
where there was no increase in, heavy' metal ion concentration. with
increased acidification. Leivestad _e_1_:_ 31;. (1976) showed that brook
trout fed §d_libitum grew two times faster at pH 6.0 than those at pH
4.6. Jacobsen (1977) found that brown trout yearlings fed a daily
ration of 2.9% of initial body weight had similar growth rates at pH
6.26, 5.44 and 5.00 over a 48 day test period. Menendez (1976) reported
that in a 5-month experiment adult brook trout exposed to pH 4.5 and 5.0
grew slower than controls (pH 7.0) during the first 3 months but there
was no difference during the next 2 months.

Acid stress significantly affects the reproduction and development
of fish. Beamish (1976) observed that most females of a particular
species taken from acidified lakes did not release their ova prior to
death and this failure to spawn was associated with the inability of the
females ‘un maintain. normal serum calcium levels. Under' laboratory
conditions Mount (1975) reported that egg production and egg
hatchability of fathead minnows exposed to pH 5.9 and below were reduced
and that all eggs were abnormal. Most researchers agree that the no
effect level of pH for successful reproduction of fishes is around 6.5
(Craig & Baski, 1977; Menendez, 1976; Mount, 1973).

C. Physiological Studies on the Effect of Acid Stress on Fish

In addition to field studies documenting fish loss, many
physiological studies have been performed in an attempt to clinically
describe the cause of death of fish due to acid stress (reviewed by

Fromm, 1980). The gills of teleost fish are in direct contact with

4

water, and continuous exposure of fish to acid stress can cause
alteration of gill membranes and/or coagulation of gill mucus. Damage
to the gill epithelium may also cause disturbances in gill functions
such as ionoregulation (Packer' and Dunson, 1970; Lloyd and .Jordan,
1964), acid-base regulation (Reeves,1977) and oxygen uptake (Packer and
Dunson,1972). These changes may actually represent the primary cause of
death of fish exposed to severe acid stress. The actual mechanism of
these processes, however, is not fully understood.

1. Failure of Ionoregulation

 

Since freshwater fish are hyperosmotic to their environment, there
is anl Osmotic influx of water and diffusive loss of ions across the
gills. The excess water is excreted via the kidneys with a concurrent
loss of ions in the copius volume of dilute urine. The ions lost may be
recovered from the diet or absorbed directly from the water via gill
NaI/H+ or Na+/NH4+ and Cl'/HC03" exchanges or other active transport
pumps. In addition to aiding the maintainance of the NaCl balance of
the freshwater fish, gill ion exchange is important because the
excretion of HC03' provides for a net loss of metabolic C02 and
excretion of H+ provides for the net loss of metabolic acid. Excretion
of NH4+ represents a net loss of both acid and nitrogenous waste.

Total body SOdium and chloride of acid exposed fish decrease with
decreased pH of the environmental water (Packer & Dunson, 1970;
Leivestad_gt_al., 1976; McDonald & Hood, 1981). At low pH the influx of
Na+ and Cl" is knocked, but when fish are transferred to clean water,
influx returns to normal (Packer & Dunson, 1970). McWilliams and Potts

(1978) measured Na+ influx and efflux across brown trout gills at an

ambient pH ranging from 7-0 to 4-0 (H2504) and found that at pH below

5

6.0 a reduced influx and increased efflux probably contributed equally
to salt loss. Based on their measurements of gill electrical potentials
they also concluded that Na+ effluxes were due to simple diffusion and
that the permeability of the gill to Na+ and to H” increased as pH
decreased.

Water-borne calcium ion also affects the rate of sodium loss from
freshwater fish (McWilliams and Potts, 1978). If calcium is high,
diffusional salt losses are relatively slow and acid-base disturbance is
the predominant symptom noted in acid stressed fish. At low calcium,
failure of ionoregulation is the predominant symptom. The increased
gill permeability with decreasing water—borne calcium and decreasing pH
would account for the observed loss of Na+ and Cl‘ down their
electrochemical gradients.

In summary, failure of ionoregulation in acid exposed fish may be
due to:

a) H+ interfering with Na+ uptake directly by competing with Na+ for

carrier sites

b) H+ altering ion transport mechanisms

0) H+ depressing the metabolic rate of transporting cell

d) H+ displacing Ca++ in the gill thus producing an overall increase

in permeability.
Ionic imbalance due to loss of NaCl from the body may be one of the
causes of death of fish subjected to severe acid stress.

2. Failure Of Acid Base Regulation

Failure of acid-base regulation in fish as a result of exposure to
acid stress will cause variations in blOOd pH, which in turn affect

functions as diverse as blood oxygen transport (Riggs, 1970), cardiac

contractility (Poupa & Johansen, 1975) and plasma ionoregulation
(DeRenzis & NMetz, 1975). More importantly, protein function will be
affected if blood pH is changed. Reeves (1977) has pointed out that the
ultimate effect of acid-base regulation is to preserve the net charge of
protein. Severe fluctuations 511 blood 1&1 when acid-base regulation
fails may therefore cause death of fish by alterations in cellular
metabolism.

Decreases in blood pH can be due to respiratory acid (i.e., C02),
metabolic acids, e.g., lactate which could be metabolized, or "fixed"
acid which must be excreted. The latter can arise directly from the
influx of environmental H+ across the gill.

How much is arterial blood changed in response to a decrease in
environmental pH? Neville (1979b) found that there was a gradual change
in arterial blood pH from 7.92 to 7.47 over 5 days in rainbow trout
exposed to pH 4.00 (HCl). Packer (1979) showed that the arterial blood
pH of brook trout exposed to pH 5.15 - 5.50 decreased from 7.8 to 7.2.
Dively 3£_al. (1977) exposed brook trout to pH 4.2 for 5 days and found
that the arterial blood pH decreased by 1.5%, i.e., from 7.8 to 7.6.
McDonald and Wood (1980) have shown that blood acidosis seems relatively
minor during acid exposure in soft water as opposed to hard water. A
decrease in blood pH and blood total carbonate has been observed in fish
exposed to acidic hard water (Lloyd & Jordan, 1964; Neville, 1979a;
Packer, 1979; McDonald and Hood, 1980; 1981). At sublethal pH, there is

little change in blood P in salmonids (Neville, 1979a; Packer, 1979;

C02

McDonald and Wood, 1980; 1981) but there is an increase in arterial PCO
2

and a reduction in H00.)- in the white sucker, indicating a compound

metabolic and respiratory acidosis in the latter. Measurements of blood

7
lactate show that the blood metabolic acid load is not derived from
lactic acid (Neville, 1979a; McDOnald and Wood, 1980). Therefore, the
H+ responsible for the metabolic acidosis observed in acid stressed fish
almost certainly comes from the environment and is not endogenous in
origin.
Decreases in blood pH can also be caused by environmental

hypercapnia (increased PCO ). Cameron and Randall (1972) found that
2

there was a slight decrease in arterial pH when rainbow trout were
exposed to increased ambient 002, Lloyd and Jordan (1964) have
demonstrated the joint toxic action of environmental hypercapnia and low
pH by exposing rainbow trout to pH 4.5 (H01) combined with elevated
water P002, (about 19 mmHg) for one 'week. They' found that this
condition caused a much greater reduction in blood pH than when the fish
were exposed to acid alone. This observation was supported by Neville
(1979b) who found that fish exposed to hypercapnic and acid stress
experienced a significantly more rapid decrease in pHa than those
exposed 1%) acid stress alone. Hypercapnia also increased ventilation
rate, whereas acid expOsure alone did not.

An impOrtant mechanism fOr pH regulation in mammals (Pitts, 1974)
and in fish is intracellular buffering of H+. Since this is only an
internal redistribution of H+, the excess H+ must eventually be excreted
to return the animal to normal status (Wood & Caldwell, 1978). Eddy
(1976) infused approximateLy 4 mEq H+/ml Of blood and calculated that
20% Of this lOad was buffered in the blood, the rest being buffered by
interstitial fluid and intracellular fluid (ICF).

Ion losses can Occur as a result of acid-base disturbance because

the H+ entering across the gill is not completely removed by the kidney

8
(McDonald and Wood, 1981). The H+ then progressively penetrates the ICF
at the expense of other cations which must come out of the cell under
the cOnstraint of electroneutrality. This considerable ionic
disturbance could have complex physiological effects on the fish.

5. Failure of Oxygen Uptake

 

The respiratory process basically consists of 1) ventilation;
2) diffusion of oxygen from water to bloOd across the gill; 5) transport
of oxygen via the blood to the tissues; and 4) consumption of oxygen at
the tissues. Any interruption of the process can cause a reduction in
the general metabolism or oxygen consumption. Wiebe £3 _a_]_._. (1954)
showed that fish had progressively greater difficulty extracting oxygen
from test solutions down to pH 5, below which no data were reported.
Packer and Dunson (1972) noted variable but eventual decreases in oxygen
consumption to death over a period of 5 to 6 hours in brook trout
exposed to a range of pH from 2.0 to 5.25, and they suggested that
oxygen deprivation is the primary cause of death at low pH. Ultsch
(1978) has shown that there is a decrease in Oxygen consumptiOn measured
over a pH range of 7.0 to 5.5 in channel catfish, but bluegills and

goldfish were unaffected in these acute experiments.

BlOOd P02 is governed by the uptake of oxygen at the gill surface
and by removal of 02 from the plasma for tissue metabolism. In fish, as
Opposed to mammals, blood oxygen level, not blOod 002, controls
respiratOry ventilation (Wood and Jackson, 1980). Janssen and Randall
(1975) found that there was a gradual increase in the ventilation volume

when rainbow trout were exposed to pH 5.0 which they suggested might

have been due to hypOxemia arising from gradual accumulation of mucus on

9
the gills. An increase in ventilation is one of the compensatory
mechanisms for hypoxemia caused by low ambient pH. Vaala (1972)
suppOrted this observation with his report that blood P0 was decreased

2
in acid stressed fish but observations by others indicate that arterial

P0 is unaffected by acid exposure, or that there is only a transient
2
decrease in this blood parameter. Eddy (1976) and Neville (1979b) both

reported that arterial P0 was little changed in fish exposed to acid
2

stress.

Data obtained (n1 oxygen consumption and ventilation rates indicate
that the hypoxia associated with acid exposure may be the primary cause
of death only at very low ambient pH (probably pH 5 or below).

How is hypoxic stress induced by acid exposure? A number of studies
have been done to determine if this is due to decreased diffusion of
oxygen across the gill or reduced transport by the blood. The volume of
oxygen transferred across the gills (702) is inversely related to the
thickness (t) of the barrier separating water from blood as expressed in

the following equation:

-1
V = (KA’AP ).t
02 02

where:

K = Krogh permeation coefficient (ul 02 h'1mmHg‘1cm‘1)

A = area of respiratory surface (cm2)

t = thickness of barrier separating water from blood (cm)

Alterations in fish gills induced by water pollutants including H+
are usually associated with hypersecretion of mucus. The mucus may
increase the thickness of the barrier separating water from blood and

thereby decrease the diffusiOn of Oxygen acrOss the gill.

10
Ultsch and Gros (1979) found that the diffusion rate through body

mucus iSOIated from acid exposed carp was 70% of that through water.
They suggested that the major effect of mucus was to reduce or prevent
water circulation between secondary gill lamellae rather than acting as
a diffusiOn barrier. The net result wOuld be reduced oxygen flux from
water to blOod.

Wiebe at _a_i. (1934) showed that fish are less able to extract
Oxygen when the pH of the ambient water is decreased, but they did not
indicate that this was due to gill mucus coagulatiOn. Ellis (1957)
stated that impairment of gills at low pH was due initially to
precipitatiOn and cOagulation of mucus on the gills and then by
coagulation of“ gill membranes themselves. In their review of the
literature Doudoroff and Katz (1950) concluded that the death of fishes
at low pH caused by inOrganic acids was due to mucus coagulation.

Plonka and Neff (1969) found bits of cellular debris embedded in
mucus On the gills of brOok trout exposed to acutely toxic acid pH.
They suggested that mucus adhering to cellular debris from the
respiratOry epithelial cells is involved in the mechanism of coagulum
formation which decreases ventilation Of secOndary lamellae. Janssen
and Randall (1975) observed that there was a build-up of mucus on the
gills Of rainbOw trOut subjected to hypercapnic stress. They suggested
that this buildéup cOuld have interfered with oxygen transfer and that
the subsequent rise in ventilation vOlume may have been related to a
slowly develOping hypoxemia. Daye and Garside (1976) observed that the
mucus cells Of gills and the integument Of brOOk trout char exhibited
progresive degrees of hypertrOphy and excessive secretion of mucus with

increased pH stress.

RESEARCH RATIONALE

Studies on the effects of various toxicants such as H+ on the
transfer capacity of fish gills for oxygen could provide valuable
information on possible subtle effects that environmental acidification
has on a fundamental or basic process common to all freshwater aquatic
communities, namely oxygen uptake.

If death by anoxia does occur, it may be by a decrease in the rate
of transfer of oxygen across the gills resulting from an increase in the
oxygen consumption of the gill tissue and/or increased diffusion
distance (due to mucus deposition on the gill). Anoxia may also be
caused by an inhibition of the oxygen transport capacity of the blood
via a combined Root and Bohr shift, which would simultaneously lower
both the oxygen capacity and the oxygen affinity of the blood.

Isolated-perfused gill preparations .have been. employed ‘ho study
various aspects of the physiology of fish gills. Girard & Payan
(1976), Smith (1977) and Wood (1974; 1975) studied vascular resistance;
Bergman, Olson & Fromm (1974) and Haywood, Isaia & Maetz (1977) worked
on urea influx as an index of branchial surface area; Sorenson and Fromm
(1976) found that heat exchange could be used as an index of branchial
vascular geometry; Payan_et'§i. (1975) studied various ionic exchanges
and Jackson and Fromm (1980) worked on the physical and biological
factors affecting the movement of 'water’ across the gill using the

isolated-perfused gill Imeparation. However, the fundamental parameter

ll

12

isolated-perfused gill Imeparation. However, the fundamental parameter
on which the physiology of fish gills is based, viz., respiratory gas
exchange, has received no such experimental attention. Wood, McMahon &
McDonald (1978) used a totally perfused whole trout preparation to
examine the effects of variations in internal and external perfusion
flow rates, adrenergic stimulation and blood pressures on oxygen
exchange and vascular resistance in the branchial and systemic vascular
beds. They stated that the oxygen used by gill tissue i.e., oxygen
taken up from the external ventilatory flow' by the gills but not
transferred to the internal perfusate, was significant and must be
considered in calculating the amount of oxygen exchanged across the
gill.

The effect of ambient pH on the transfer of oxygen across the gill
could be studied using the isolated-perfused gill preparation. However,
before the preparation is used for this purpose a few questions
regarding the basic characteristics of the gill tissue itself need to be
answered. They are:

1. What is the oxygen consumption of the gill tissue?

The isolated perfused gill technique measures the difference in P02
of the fluid going into the gill (afferent vessel) and the fluid going
out of the gill (efferent vessel) in calculating the transfer capacity
of the gill for oxygen. Therefore, the oxygen consumption of the gill
tissue itself has to be considered in making the calculation.

2. Is the oxygen consumption of the gill tissue affected by P02 of

the bath solution and the time taken to make the measurement?

5. Is the oxygen consumption of the gill tissue affected by the pH

of the bath solution?

13

4. Is there any difference in oxygen consumption of the gill tissue
if the fish are exposed to acid water prior to measuring the
oxygen consumption of the gill tissue?

5. Mucus may interfere with diffusion across the gill and has been
reported frequently but has not been quantified. How much mucus
is produced by the gill of the fish exposed to various pH?

Experiments were done in an attempt to answer the above questions.

MATERIALS AND METHODS

Experimental Animals

 

Rainbow trout (Salmo gairdneri) weighing between 150-550 g were used

 

as experimental animals. The fish were obtained from a local hatchery
(Midwest Fish Farm Enterprises, Inc., Harrison, Michigan) and maintained
at Michigan State University in large fiberglass tanks supplied with
flowing tapwater (PH 7-4 : 0.2) from which chlorine and excess minerals
had been removed. The temperature of the room in which the fish were
kept was maintained at 10:2C. Animals were fed twice weekly and starved
at least 5 days prior to experimental use.

Fish to be exposed to various pH levels were acclimatized in small
tanks (58x55x7cm) in a 001d room (1011C) for two days prior to exposure.
In other tanks of similar size, concentrated H2804 was used to adjust
the pH to the desired level. Fish were then transferred carefully from
the acclimatization tanks to the exposure tanks. Exposure time was two
hours.

Gill Tissue Preparation

 

The fish were immobilized by severing the spinal cord immediately
posterior to the opercula and placed ventral side in a V-shaped trough.
A rubber tube inserted in the mouth directed flowing tapwater over
gills. A three-inch incision was made ventro-caudally from the jaw to

expose the heart. The ventricle was cannulated with PE 90 tubing which

14

15

was attached via a pump to a perfusion fluid reservoir. The tubing was
secured in place with ligature around the bulbus arteriosus. The gills
were pump-perfused (for jperfusion. fluid composition. see .Appendix 1)
until cleared of blood (5-10 minutes).

0f the four gill arches, the second arch of the right side was
removed and used for oxygen consumption study, the left second arch was
used for histology and the first, third and fourth arches were used for
sialic acid determination.

Oxygen Consumption of Gill Tissue

 

A sample of gill filaments (60-90 mg) was cut from the middle of the
arch, Excess fluid was blotted off and the samples were weighed and
suspended in 5 Iml of citric acid/sodium phosphate buffered incubation
medium (Appendix II) having an osmotic concentration of 270-500 mOsm/kg.
The incubation medium was aerated with room air for 10 minutes prior to
use and the oxygen content was determined using the Winkler procedure.
The oxygen consumption was then measured polarographically with a Y.S.I.
model 55 biological oxygen monitor (Yellow' Springs Instrument Co.,
Yellow Spring, 0H), and recorded with a two channel strip chart recorder
(Linear Instruments Mbdel 486, ldnear Instrments Corp., Irvine, CA).
The temperature of the samples in the incubation vials was maintained at
10:0.5C with a constant temperature water bath (Haake, Inc., Model DI,
Temperature Control Equipment Div., Saddle Brook, NJ).

The recorded tracings for each experiment were digitized at one
minute intervals. The oxygen consumption at each digitized point (time
t) was calculated based on the difference between the initial oxygen

content of the incubation medium (100% saturation) and the content at

16

time t. The formula for calculating the oxygen consumption is as

follows:
éo - (APO °a)t-1W 1
where:
902 = oxygen consumption in ul/h/mg dry wt.
APOZ = A% saturation x barometric pressure x 0.21 (mmHg)
t = time in hours
w = dry weight in mg
a = initial oxygen content (ul) at 100% saturation.

The data used to compare treatment effects were based on the oxygen
consumption during the first 20 nflnutes statistically extrapolated to
time zero utilizing an exponential curve fit routine.

Sialic Acid Content of Gill Tissue

 

The first, third and fourth gill arches of both sides were used.
The filaments were trimmed from the cartilagenous arch and blotted free
of excess fluid. The tissue was then weighed and frozen in distilled
water after it was sonified for 5 minutes using a sonifier cell
disruptor (Heat Systems Co., Melville, NY). A puccedure described by
Warren (1965) was used to determine the amount of sialic acid present.
(Appendix III).

Histological Observation of Gill Tissue

 

The second arch of the left side was removed from the fish and fixed
in Detrick fixative. The tissues were dehydrated in tetrahydrafuran and
embedded in paraffin. Tissue sections of 8 pm thickness were stained in

haematoxylin and eosin and observed for any histological changes. Gill

17

tissue sections for histochemical identification of mucous cells were
stained using Periodic Acid Schiff technique (Pearse, 1968).
Statistical Treatment of Data

Data for oxygen consumption and sialic acid content were subjected
to a one-way analysis of variance and the Student-Newman—Keul test was

used to compare treatment means. Fiducial limits were set at 0.05.

RESULTS

A. Oxygen Consumption of Gill Tissues

 

1. Experiment 1. What is the effect of pH of the incubation media

 

on the oxygen cOnsumptiOn of gill tissues?

To answer this questiOn samples of gill tissue were taken from fish
which had been exposed to a non-stressful ambient pH of 7.4. The
samples were placed in incubatiOn media having a pH of 7.4, 6.0, 5.0 and
4.0 and oxygen consumption was measured. The results, displayed in
Figure 1, shOw that as pH of the incubation media decreased there was a
decline in Oxygen cOnsumptiOn. The means for Oxygen consumption at pH
5.0 and 4.0 were bOth significantly lower than that for controls
(pH 7.4).

2. Experiment 2. What is the effect of exposing the gill tissues

 

for One hOur in the incubation media prior to measuring their oxygen
cOnsumptiOns?

Gill tissue samples ‘were obtained from fish. as .noted above and
divided into twO grOups. One group was placed in aerated (room air)
incubatiOn media having a pH Of 7.4 and the other at pH 6.0. One hour
was allOwed to elapse priOr to determining the Oxygen consumption of
these tissues. The data Obtained were compared to appropriate control
(i.e., same pH) values frOm Experiment 1 and are displayed in Figure 2.
Results show that the one hOur exposure Of gill tissue to _:_’L_r_1_ 11.32

conditiOns had no effect On Oxygen cOnsumption.

18

Figure 1.

19

Effect of pH of the incubation media on oxygen consumption of
gill tissueS- Mean values 1 S.E. are plotted and the number
in each column indicates the N values. Means (ul/h/mg dry
wt.) not underlined by the same line are significantly
different (P<0.05).

pH 7.4 pH 6.0 pH 5.0 pH 4.0

2.51io.2o(9) 1.98:0.17(6) 1.65:0.22(4) 0.76:0.1s(4)

 

 

 

'P

02 ConsumptioMpl/h/mg dry wt.)

 

20

 

 

 

 

 

 

 

 

 

9 6 J4

 

 

 

4

 

pH 7.4 pH so pH5.0

Figure 1

pH 4.0

Figure 2.

21

Effect of exposing gill tissues for one hour in the
incubation media prior to measuring oxygen consumption. Mean
values 1' S.E. are plotted and the number in each column
indicates the N value. Means (ul/h/mg dry wt.) not
underlined by the same line are significantly different

(P<0.05).

pH 7.4 pH 6.0
No exposure 1-h exposure No exposure 1-h exposure

2.51io.2o(9) 2.3410.06(4) 1.98:0.17(6) 1.8410.05(4)

 

 

g dry wt)

0 Consump1i0n(11I/h/m

3L)

9’
Q

 

OI)

 

9

 

pHZ4

 

22

D No Exposure

lhour

 

Exposure

      

 

6 as:
pH 6.0

Figure 2

23

 

5. Experiment 5. What is the effect of P02 of the incubation media
on Oxygen cOnsumption of gill tissues?

Oxygen cOnsumption data for tissues measured at pH 7.4 in Experiment
1 were used to cOmpare the effect of P02 on the oxygen consumption of
gill tissue. Since the Y.S.I. mOdel 55 biological oxygen monitor used
to make the measurements is a closed system, the P0 of the incubation
medium drOps as Oxygen is consumed. The average rate of oxygen consumed
f9? each 20 mm Hg drop in P02 was calculated for gill tissues measured
at pH 7.4. The results are shOwn in Figure 5. During the time required
to reduce the P02 from 150 mm Hg or above to 90 mm Hg (20 minutes or
less) there was a slight but statistically insignificant decline in
Oxygen consumption. AS P02 decreased further there was an accompanying
decrease in oxygen consumption and at sOme PO2 between 10 and 30 mm Hg

the decline in Oxygen cOnsumptiOn appeared to become greater.

4. Experiment 4. What is the effect of exposure of fish to various

 

pH levels prior to obtaining gill samples and measuring the oxygen
cOnsumptiOn at the pH of expOsure?

Gill tissue samples were taken from fish which had been exposed for
two hours at pH 7.4, 6.0, 5.0 and 4.0. The samples were then placed in
the incubatiOn chamber containing incubation media at a pH similar to
that of exposure. Rasults of this experiment are shown in Figure 4.
The mean oxygen cOnsumption of gill tissue showed a uniform and
statistically significant decrease as pHI Of‘ the exposure ‘water"was

decreased.

24

Figure 3- Effect Of P02 on oxygen consumption of gill tissues. Plotted
are mean 1 S.E. Means (pl/h/mg dry weight) not underlined by
the same line are significantly different (P<0.05). 11 = 6
for each mean.
P02 (mmHg)
>130 110-130 90-110 70-90 50-70 30-50 10-30 (10
+2.85 +2.54 +2.26 +2.04 +1.76 +1.57 +1.28 +0.74
-0.04 -0.30 -0.31 -0.27 -0.20 -0.15 -0.12 -0.05

 

 

 

 

25

 

o_v

 

Onao_

 

onxom

 

Ohnon

 

 

omuON

 

0:10m

 

 

On_:O:

 

 

on:

 

 

3.0-

O.

. . o
2 I
:3 be oE\;\_3 538350 No

(mmHg)

_ P02

Figure 3

Figure 4.

26

Effect of exposing the fish for two hours at various pH
levels on .in_ litre oxygen consumption of gill tissues.
Measurements of oxygen consumption were made at pH of
exposure. Plotted are mean values 1 S.E. and the numbers in
each column indicate N. Mean values (ul/h/mg dry wt.) are

as follows:

pH 7.4 pH 6.0 pH 5.0 pH 4.0

2.54io.21(s) 2.06:0.18(8) 2.ooio.17(4) 1.75:0.5o(7)

 

The oxygen consumption at pH 4.0 is significantly lower than

at pH 7.4 (P<0.05, Student t-test).

27

 

 

 

 

 

 

 

 

 

 

 

 

 

3.0-7

1.

o. o _
2 I
33 ft 9:223 coiesmeoo No

00

pH6.0 pH 5.0 pH4.0

pHZ4

Figure 4

Figure 5.

28

Effect of prior exposure of fish for two hours at various pH
levels on in yitrg oxygen consumption of gill tissue. All
measurements of oxygen consumption were made at pH 7.4.
Plotted are mean values : S.E. and the numbers in each column

indicates N. Mean values (ul/h/mg dry wt.) are as follows:

pH 7.4 pH 6.0 pH 5.0 pH 4.0

2.54:0.21(8) 2.1910.o7(3) 2.73io.34(3) 2.85:0.71(5)

Statistically (P<0.05) there were no differences between any

of the means.

O2 ConsumptioMpI/h/mg drywt.)

' 0.0

3.5-)

3.0-

2.5.

2.0-

1.5-

1.0-

0.5‘

 

 

 

 

8

29

 

 

3

 

 

3

 

 

 

 

3

 

pH7.4 pH6.0 pH5.0 pH4.0

Figure 5

 

30

5. Experiment 5. What is the effect of exposing the fish for two

 

hOurs at various pH levels prior to measuring the oxygen consumption at
pH 7.4?

The fish and gill tissue samples were treated as in Experiment 4
except that all oxygen consumption measurements were made in incubation
media at pH 7.4. Results from this experiment are shown in Figure 5.
The oxygen consumption of’ gill tissue samples from. fish. previously
exposed to different pH levels were nOt significantly different from
cOntrols and no unifOrm trend in the data was evident.

B. Sialic Acid Content Of Gill Tissues

Since sialic acid is an impOrtant component of mucus, an assay for
this acid was used to estimate the mucus content of gill tissues of
experimental animals. Intact fish were transferred from a holding tank
and acclimated fOr two days in small plastic tanks. After equilibration
they were exposed for two hours to water at pH 7.4, 6.0, 5.0 and 4.0.
Table 1. Effect of exposing the fish for 2 h at various pH levels on

sialic acid content of the gill tissue. Amount of sialic acid
is in uMoles/g wet weight. The amount of sialic acid in gill

tissues of fish exposed to pH 4.0 is significantly more than
the amont in controls. Data are presented as MeansiS.E.(N).

 

Control Exposed to
(no exposure) __pH 7.4 ipH 6.0 pH 5.0 pH 4.0

1.07:0.o4(4) 1.10:0.o9(4) 1.25:0.06(4) 1.50:0.06(4) 1.42:0.04(4)

 

The sialic acid content of gill tissue from fish exposed to pH 7.4
was compared to that of contrOls, i.e., gill tissue from fish obtained
from the holding tank, to test fOr the effect of transfer from the

holding tank to the small exposure tanks. Data for fish exposed to pH

31
6.0, 5.0 and 4.0 were compared to data fOr fish at pH 7.4. The data are

presented in Table 1. Transfer of fish from the hOlding tank tO the
small exposure tanks and 48 hOurs of acclimatiOn had no effect on gill
sialic acid content. The shOrt, twO-hour expOsure to acidic cOnditions
caused a significant increase in the sialic acid (mucus) content of gill
tissue.
C. Histological Observations

Figures 6, 7, 8 and 9 indicate that as the stress of lowered pH is
increased there is a prOgressive increase in the tissue damage,
accompanied by an increase in the number of mucOus cells. A more

detailed descriptiOn is given in the legends fOr each phOtOmicrograph.

Figure 6.

Figure 7.

32

Photomicrograph of longitudinal section. of’ gill filaments
from rainbow trout exposed to pH 7.4 for two hours. Mucus
cells which are stained dark (m) are very few in number at
the junctions of the gill lamellae (l) and gill filaments
(f), and very few in the bodies and tips of of the lamellae.
Practically' no mucus can be seen ‘between. the lamellae.
Arrows indicate the few areas of separation between the
epithelial cells (e) and the pilaster cells (p). No
separation. is seen. in the areas around the tips of the

lamellae.

Photomicrograph of longitudinal section. of' gill filaments
from rainbow trout exposed to pH 6.0 for two hours. Mucus
cells are few in number at the junctions of gill lamellae and
gill filaments and in the bodies and tips of the lamellae.
Practically no mucus can be seen between the lamellae.
Arrows indicate the areas of separation between the
epithelial cells and pilaster cells. The separation is seen
in almost all lamellae especially at their bases. No

separation is seen in areas around the tips of the lamellae.

33

 

Figure 8.

Figure 9.

34

Photomicrograph of longitudinal section. of' gill filaments
from rainbow trout exposed to pH 5.0 for two hours. More
mucus cells are seen at the junctions of the gill lamellae
and gill filaments and also at the tips of the lamellae.
Dark areas between the lamellae represent secreted mucus. The
areas of separation betwen the epithelial cells and pilaster

cells are more pronounced.

Photomicrograph of longitudinal section. of' gill filaments
from rainbow trout exposed to pH 4.0 for two hours. Mucus
cells fill the junctions of the gill lamellae and gill
filaments and more are present at the tips of the lamellae.
Mucus can be seen filling the spaces between the lamellae.
The areas of separation between the epithelial cells and

pilaster cells are very conspicuous.

 

35

 

. a ,1 -_.’ .
1"»6‘ - "AME,
FIGURE 9

DISCUSSION

The purpose of this study was to measure the rate of oxygen
consumption of fish gill tissue and to determine whether oxygen
consumption and mucus production of gill tissue are affected by lowered
pH.

In the procedure used to measure oxygen consumption three factors

(PH' P02 and time) may interactively affect the data obtained. Three
series of experiments were done to separate out the effects of these
variables. Data from Experiment 2 show that after removal of gill
tissue from a fish and storage in incubation media aerated with room air
for 1 hour, the oxygen consumption of this tissue was not different from
the oxygen consumption cu? gill tissue which was measured immediately
upon removal. After the start of the measurement period the rate of
oxygen consumption showed the same decrease with time in both of the
aforementioned cases. Therefore, in the experiments reported here, the
time period from removal of gill tissue from fish until the end of the
oxygen consumption determination can be eliminted as a factor
influencing oxygen consumption. Thus any change in oxygen consumption
must be due to other variables, viz., P02 and/or pH.

Data from Experiment 5 show that the P02 of the incubation media

does not affect oxygen consumption of the gill tissue until the PO
2

drops below 90 mm Hg. The time required to reduce the P02 to around 90

mm Hg averaged 25 minutes. Data used to‘ study the effect of' pH

36

37

(Experiment 1) were obtained during the first 20 minutes of the
measurement period. The data show that gill tissue oxygen consumption
at pH 5.0 and 4.0 are significantly lower than that for controls (pH
7.4). Therefore, any change in oxygen consumption during the first 20
minutes of the measurement period is due to pH alone.

The pH of the incubation media causes a decrease in the oxygen
consumption of the gill tissue possibly by causing physical damage to
the tissue membrane. This in turn could lead to edema, ionic imbalance
and possible intracellular pH change. This pH change could affect the
activity of the respiratory enzymes.

The next experiments were done to determine if exposure of fish to
various pH levels prior to obtaining gill samples and measurement of
oxygen consumption had any effect on the rate of oxygen consumed by the
gill tissue. Data from Experiment 4 show that there is a uniform but
not significant decrease in oxygen consumption as the pH of the exposure
water is decreased. When these data were compared to the appropriate
data in Experiment 1, the oxygen consumption of the gill tissue from the
exposed fish were consistently higher than those taken from the
unexposed fish but only those exposed to pH 4.0 showed a statistically
significant difference. The oxygen consumption of the gill tissue from
fish exposed to pH 4.0 showed a significantly higher oxygen consumption
than those taken from unexposed fish. (See Figure 10).

The above data suggest that when intact fish are exposed to acid
water for 2 hours the detrimental effect of pH on metabolism of the gill
tissue is overridden by an opposite effect (increased metabolism) which
is also caused by low ambient pH. When fish are exposd to low pH

increased rate of metabolism of gill tissue may simply reflect the

38

increased energy requirement caused by the increase in mucus production.
In order to verify the two opposite effects of pH on the metabolism of
the gill tissue, Experiment 5 was done. When the fish were exposed to
pH 7.4, 6.0, 5.0 and 4.0 for two hours in small tanks and all the oxygen
consumption of the gill samples was measured at pH 7.4, there were no
significant differences in the oxygen consumption and no uniform trend
was evident. When the data were compared to those of Experiment 1
(measured at pH 7.4 only), again there were no significant differences.
Therefore the pH of the water in which the fish had been exposed to does
not significantly affect the oxygen consumption of the gill tissue if
the oxygen consumption measurement is made at pH 7.4. However, when
the data are compared to the appropriate data in Experiment 1, the
oxygen consumption of the gill tissue of fish exposed to pH 4.0 and
measured at pH 4.0 is significantly lower than the oxygen consumption of
the gill tissue from fish exposed to pH 4.0 and measured at pH 7.4.
(See Figure 10).

In Salmonidae, the major epithelial mucin component appears to be
sialic acid (Harris , Watson and Hunt, 1975). Lemoine and Olivereau
(1971) used sialic acid assay to determine the degree of skin

mucification in Anguilla anguilla. The assay of sialic acid may

 

therefore provide evidence of the presence of an environmental stressor,
producing alterations in the secretion of gill mucus.

Miller and Mackay (1982) studied the relationship of secreted mucus
to copper and acid toxicity in rainbow trout. They found that the
amount of mucus secreted into the water by fish exposed to pH 4.0 for
eight hours was significantly greater than that secreted by fish exposed

to pH 7.5. The data cannot be used to explain a decrease in oxygen

39

Figure 10. Effect of exposing the fish for 2 h at various pH levels on
.13 32:33 oxygen consumption of gill tissue. Experiment 1:
Controls i.e., fish not exposed; O2 consumption measurements
made at pH 7.4, 6.0, 5.0, and 4.0. Experiment 4: Fish
exposed to pH 7.4, 6.0, 5.0 and 4.0, and the oxygen
consumption of the gill tissues were measured at the pH of
exposure. Experiment 5: Fish exposed as above and the
tissues measured at pH 7.4. Plotted are mean values (pl/h/mg
dry Wto):S.E. The numbers in each column indicate N values.
The oxygen consumption of gill tissue from fish exposed to pH
4.0 and mesured at either pH 4.0 or pH 7.4 is significantly

greater than that of controls.

4O

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

pH 5.0 pH 4.0

pH 6.0

 

 

 

 

 

2 I
33 be @8223 cozaszmcoo No

1.45
mm...
eem
mmm
.n.n.n
he as a»
nu. nu. nv
VA VA VA
EEE
mmm
o O. . o 1o
3 0

Figure 10

41

transfer across the gill because mucus secreted into the water does not
act as a diffusion barrier. Furthermore, mucus secreted into the water
does not indicate gill mucus alone; mucous cells are widely distributed
over the general body surface of fishes. It is believed that an assay
for the amount of mucus synthesized and/or secreted by the gill tissue
itself is the only valid method that can be used to explain the decrease
in oxygen transfer across the gill.

A series of experiments was done to determine mucus production of
the gill tissue of fish exposed to pH 7.4, 6.0, 5.0 and 4.0. The data
represent the mucus secreted on the gill tissue as well as the mucus
produced by the mucous cells but not yet extruded. The data show that
the amount of mucus steadily increased as the ambient pH was decreased.
The gill tissue taken from fish exposed to pH 4.0 had a significantly
greater amount of mucus than those taken from the controls (pH 7.4). A
histological study was done on acid stressed gill tissues to illustrate
the response of this tissue to low ambient pH. The gills exhibited two
types of reaction to pH stress. Hypertrophy' of' mucous cells was
observed at the base of the secondary lamellae with exposure to
increased hydrogen ion concentration. The accumulation of mucus on the
gill is shown in the photomicrographs and increased mucus production is
also indicated by the data on the sialic acid content. In addition, as
pH stress increased more tissue damage, especially near the tips of the
lamellae, was apparent. A more pronounced separation of epithelial
cells from the underlined cell layers is more evident with increased pH

stress.

42
Acid stress (low pH)

Tissue damage Increase in mucus production
Decrease in Q Increased diffusion Increased in Q
0 . . O
2 distance across glll 2

+

Decrease in proper
ventilation of secondary

 

 

lamellae,
V 1 V
Increase in gill Decrease in gill Decrease in gill
transfer capacity transfer capacity transfer capacity
for oxygen (V02) for oxygen (702) for oxygen (702)

The data on gill oxygen consumption and mucus production presented
above indicate that exposure of this tissue to acidic conditions may
result in multiple effects. It is postulated that pH stress, in one
case, causes tissue damage which leads to a decrease in gill oxygen
consumption. Other things being equal, this would result in an increase
in the transfer capacity of gills for oxygen. On the other hand, pH
stress stimulates mucus production which in turn could produce variable
effects. As shown in the diagram above an increase in mucus production
could increase oxygen consumption i.e., energy must be provided for the
synthetic process. Secondly, increased mucus production could result in
a decrease in the proper ventilation of secondary lamellae and also
increase the diffusion distance for oxygen across the gill from the
ventilate to the vascular space. Both of these effects should result in

a decrease in the transfer capacity of the gills for oxygen.

CONCLUSIONS

The is xiigg oxygen consumption of gill tissue from rainbow trout
not previously exposed to acid water decreased as the pH of the
incubation media was decreased.

The 13 11232 oxygen consumption of gill tissue from rainbow trout
exposed to lowered pH decreased as the pH of the incubation media
(similar to exposure pH) was decreased.

The iguyiggg oxygen consumption (measured at pH 4.0) of gill tissue
from rainbow trout exposed to pH 4.0 was higher than of none exposed
fish.

There is a progressive increase in sialic acid content as the pH of
exposure decreases. However, the only significant difference noted
is the sialic acid content at pH 7.4 and pH 4.0.

Histologically gill tissue of the more severely acid-stressed fish
had more mucous cells and exhibited a greater degree of filamental

damage.

43

APPENDICES

APPENDIX I

Composition of Perfusion Fluid

NaCl
CaCl2

KCl

NaH2P04 H2O
NaHCOU

D
MgSO4 7H20
Ms012 6H20
Glucose
Albumin

Distilled water

44

0.10
1.00

50.00

to 1

liter

APPENDIX II

Composition of Citric Acid/Sodium Phosphate Buffer

at Different pH Levels.

Procedure:
. . *
1- Prepare 0'2 M NaZHPO4 in 1% Cortland saline .
2. Prepare 0.1 M Citric Acid in 1% Cortland saline.
5. To make the buffer at different pH levels, take different volumes of
0'2 M Na2HP04 and 0.1 M Citric acid as indicated in the table below.
4. Adjust the osmolarity of the buffer to 270-500 mOsm/Kg either by

adding NaCl or distilled water.

 

.33--.. 0-2M Na2HPO4, ml 0.1M Citric Acid, ml
4.0 8.0 12.0
5.0 10.0 10.0
6.0 12.0 8.0
7.4 17.0 5.0

 

45

46

*
Composition of Cortland saline:

NaCl 7.25 g
CaCl2 2H20 0.23 8
KCl 0.38 8
NaHZPO4 H20 0.41 g
NaHCO3 1.00 g'
MeSO4 7H20 0.23 g
Glucose 1.00 g

Distilled water to 1 liter

APPENDIX III

Thiobarbituric Assay for Sialic Acid - for Use in Estimating Mucus.

Reagents:

0.1N H2504
Sodium metaperiodate (0.2M) in 9M phosphoric acid (55%)
Sodium arsenite (10%) in 0.5M sodium sulfate solution

2-Thiobarbituric acid (0.6%) in 0.5M sodium sulfate solution

Cyclonexanone

Procedure:

Trim filaments from gill cartilage and place 1 g in 5.5 ml sulfuric
acid.

Sonify for 5 minutes.

Incubate at 80 C for one hour.

Centrifuge at 10,000 rpm for 10 minutes.

Put 0.2 ml supernatant in test tube.

Add 0.1 ml sodium periodate.

Mix and allow to stand at room temperature for 20 minutes.

Add 1.0 sodium arsenite solution. Shake vigorously.

Add 5.0 ml 2-thiobarbituric acid solution. Mix by inversion.

47

48

Place in boiling water bath exactly 15 minutes.
Cool in tap water for 5 minutes.
Add 4.0 ml cyclohexanone. Shake vigorously.
Centrifuge briefly.
Transfer top,clear cyclohexanone phase to cuvette of 1 cm light path.
Determine optical densities at 552 nm and 549 nm.
Micromoles of sialic acid = (0.084 x OD549)-(0.031 x ODSBZ)
NOTE: The derivation of this equation is dependent upon the molecular
extinction coefficients at 552 nm and 549 nm of sialic acid and the

interfering material (considered 2-deoxypentose) (Warren, 1965).

LITERATURE CITED

LITERATURE CITED

Beamish, R.J. 1974. Loss of fish populations from unexploited remote
lakes in Ontario, Canada as a consequence of atmospheric fallout of
acid. Water Res., 8:85-95.

Beamish, R.J. 1976. Acidification of lakes in Canada by acid
precipitation and resulting effects on fishes. Water, Air and Soil
Poll., 6:501-514.

Beamish, R.J., W.L. Lockhart, J.C. Vanloon. and H.H. Harvey» 1975.
Long-term acidification of a .lake and the resulting leffects on
fishes. Ambio., 4:98-102.

Bergman, H.L., K.R. Olson and P.0. Fromm. 1974. The effects of
vasoactive agents on the functional surface area of
isolated-perfused gills of rainbow trout. J. Comp. Physiol.,
94:267-286.

Cameron, J.N. and D. J. Randall. 1972. The effects of increased

ambient CO on arterial C0 tension, CO2 content and pH in rainbow
trout. J.2Exp. Biol., 57:675-680.

Conroy, N., K. Haney, W. Keller and C. Lafrance. 1976. Influences of
the atmosphere on lakes in the Sudbury area. J. Great Lakes Res.,

2:146-165.

Craig, G.R. and W. F. Baksi. 1977. The effects of depressed pH on
flagfish reproduction, growth and survival. Water Res., 11:621-626.

Daye, P.G. and E.T. Garside. 1976. Histopathologic changes in
surficial tissues of brook trout, Salvelinus fontinalis (Mitchill),
exposed to acute and chronic levels of pH. Can. J. 2001.,
54:2140-2155.

DeRenzis, G. and J. Maetz. 1975. Studies on the mechanism of chloride
absorption by the goldfish gill: relation with acid-base regulation.
J. Exp. Biol., 59:559-558.

Dillon, PJ., D.S. Jeffries, W. Snyde, R. Reid, N.D. Yan, D. Evans, J.
Moss and W.A. Scheider. 1978. Acid precipitation in south-central
Ontario: recent observation. J. Fish Res. Board Can., 55:809-815.

Dively, J.L., J.E. Mudge, W. H. Neff and A. Anthony. 1977. Blood P

PCO and pH changes in brook trout exposed to sublethal levels 8f
aClgity. Comp. Biochem. Physiol., 57A:547-551.

49

50

Doudoroff, P. and M. Katz. 1950. Critical review of literature on the
toxicity of industrial wastes and their components on fish. I.
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