VOL. V, N o . 3
MARCH 1928
THE ABSORPTION OF OXYGEN BY PLAICE EGGS
BY S. T, BURFIELD, BJL, M.SC.
From the Zoological Department, Liverpool University,
{Received 10th February 1927.)
(With One Text-figure.)
1. INTRODUCTION.
IN experiments made to determine the oxygen consumption of aquatic animals
various observers have noted that if the experiments be extended over a number
of hours the rate of consumption per hour falls. Dakin (3) has lately drawn attention
to this, quoting the results of Moore with a sponge and Cancer, Branow with the
crayfish and Lipschiitz with goldfish. In the course of some work on plaice eggs
Dakin made a few experiments which showed that these also exhibited an apparent
fall in their rate of oxygen consumption when kept in a closed volume of sea water.
The consumption was always greater during the first half-hour or hour than during
the second and third. Professor Dakin informs me in a private communication
that he obviated the difficulty in his own experiments by keeping the eggs in continuous motion. If this fall in oxygen consumption be a definite and constant
phenomenon under the conditions mentioned, it may be due to one or more of
the following factors: (a) handling the specimens and so causing an abnormally
high rate of consumption at the beginning; (h) the gradual absorption of the
available oxygen as the experiment proceeds; (c) the gradual accumulation of waste
products, e.g. CO 2 , nitrogenous excreta; (d) the gradual fall from a high rate of
consumption at the beginning, the latter being due to a meal taken immediately
before the experiment.
By using plaice eggs the factors (a) and (d) may be assumed to be absent. The
factor (b) can also be eliminated by using sufficiently large volumes of sea water
for the experiments1. Thus it seemed worth while to carry out some further experiments on plaice eggs with two objects in view, viz. to confirm or otherwise
the fall in oxygen consumption, and to try and obtain an indication of the effect
of factor (c) if the fall were found to be a regular phenomenon.
See also discussion of results of Exp. 1 below.
S. T . BURFIELD
2. TECHNIQUE.
Samples of plaice eggs were obtained from the fish hatchery at the Port Erin.
Biological Station, where the work was done. For each experiment from 1500 to
2000 eggs were taken. Such a quantity could be removed in a small net, the
number being judged by eye, and immediately transferred to an experimental
bottle containing sea water obtained from the same source as that supplying the
hatching boxes. The bottle was then temporarily closed with a glass plate in which
was a hole just too small to permit an egg passing through. The whole was immersed in an empty hatching box through which -sea water was running, and the
glass plate replaced by the bottle stopper. The hole in the plate allowed any bubbles
of air to escape, and the stopper replaced the plate by sliding off the latter and
closely following up with the stopper. Thus a sample of eggs was obtained with
a minimum of manipulation in a closed known volume of sea water. The exact
number of eggs included was obtained by counting at the end of the experiment.
The experimental bottles used had well-fitting glass stoppers and were of such a
size that a large excess of available oxygen was always present for the above number
of eggs. Seven bottles were used having volumes as follows: 404-55 404-5, 420-45
397-8, 410-6, 417-2, 4077 c.c.
The oxygen content of the water at the beginning and end of an experiment
was estimated by the Winkler method, the sodium thiosulphate solution being
tested every day by standardising against a solution of potassium biiodate of known
strength. For the oxygen estimations as large a sample as possible of the water
was taken to eliminate errors. The sample was obtained
by rapidly siphoning from an experimental bottle. The
sampling bottles had well-fitting glass stoppers, and care
was taken that no air bubbles were included when inserting
the stopper. The sampling bottles had an average volume
of about 270 c.c, so that well over half the water in an
experimental bottle was used for an oxygen determination.
During all the experiments the bottles containing the
eggs were kept immersed in running sea water in the same
empty hatching box, so that the conditions of temperature
were as nearly constant as possible.
The eggs for any one experiment were all obtained
from the same hatching box, it having previously been
determined that these eggs were all in about the same
stage of development. Thus, when a number of separate
&* ^?P9f£&££££J£££££2l
samples of 1500 to 2000 were used for an experiment, ~.
Celluloid cage (only
these samples could be considered equivalent except in a portion of the perforations
exact number (determined later) without any appreciable have been drawn).
error. In addition to this, experiments were carried out on one particular sample of eggs
by changing the conditions. In order that a sample of eggs could be transferred
quickly and without loss from one experimental bottle to another, a cage was
LID
> v O O o O O O O O O O O O O OOOOOO
300 0 O O o OO OO
OOOOOQOOOOL
1OOOO O O O O O O O O O O O C O O O O O
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
00000
The Absorption of Oxygen by Plaice Eggs
179
devised to contain the eggs (Fig. 1). This was made of perforated celluloid, the
perforations being just too small to allow the eggs to pass through. The cage
was made in cylindrical form by sewing the pieces of celluloid through the perforations with thread. The lid fitted closely so that no eggs could escape, and the
cage was of such a size that it just passed through the neck of the experimental
bottles. In order to manipulate the cage and to prevent the possibility of the lid
coming off when removing from a bottle, a short piece of thread was passed through
the perforations of the box and lid, and the whole could then be picked up by
grasping the ends of this thread with a pair of forceps.
The volume of the cage was obtained by weighing dry and calculating, taking
the specific gravity of celluloid as 1*4.
The perforations were sufficiently large to allow a free circulation of the sea
water all round the eggs when immersed.
3. EXPERIMENTAL RESULTS.
The following is an account of a representative sample of the experiments
carried out. The experiments described here are_ numbered consecutively for
reference. For the sake of brevity the complete series and the detailed calculations
are not given. Two of the cages mentioned above were used, the volume of their
substance being 4-1 and 4*9 c.c. respectively. For the purposes of calculation the
volume of an egg was taken as -003479 c.c. In those experiments which required
the number of eggs to be known for comparison of oxygen absorption over various
periods, the standard taken was the amount absorbed by 2000 eggs. In the course
of the experiments eggs from three hatching boxes were used, called a, /? and y,
and the eggs in each of these were progressively younger in that order.
A. Experiments were first made to confirm or otherwise the falling-off in
oxygen consumption. In these cases samples of eggs were left for periods varying
from one to six hours. The eggs were allowed to remain with very little movement.
Exp. 1. Quantities of oxygen absorbed by 2000 eggs from box a:
In
„
„
„
„
1 hr.
3 hrs.
4 „
5 „
6 „
...
...
...
...
...
-195 rag.
-406 mg. or -zn mg. in foil, z hrs. or gen. aver, of "135 mg.
-500 „
-094 „
4th hr.
„
-125
-521 „
-021 „
5th „
„
-104
-IOO
-602 „
-081 „
6th „
„
per hr.
»
„
„
Thus there was a distinct fall in the rate of oxygen consumption, in the sixth hour
of this experiment, the quantity of oxygen absorbed being less than half that in the
first hour. These figures bear out the results obtained by Dakin ((3), p. 318). At
the end of six hours the total of dissolved oxygen in the bottle was only reduced
by about 14 per cent., and at the end of the first hour by less than 4 per cent. It
might be said that the fall in oxygen consumption was due to the relatively quiescent
condition of the eggs and water, resulting in a temporary fall in available oxygen
in the immediate neighbourhood of the eggs. Professor Dakin informs me that
he made some experiments on the effect of reducing the partial pressure of the
12-2
l8o
S . T . BURFIELD
dissolved oxygen. This was done by carefully warming some sea water to drive
off dissolved oxygen, cooling, and adding to some untreated sea water. This mixture,
which showed a reduced oxygen content when the latter was determined by the
Winkler method, was used for some samples of eggs in closed bottles. The reduction in the partial pressure of the dissolved oxygen was found to have no appreciable
effect on the rate of oxygen absorption by the eggs. From this, and from the
results of the work of several other investigators1, it would appear that the vertebrates, on the whole, show themselves largely independent of small changes in
the partial pressure of oxygen. Thus it would seem probable that it is the factor (c)
above which is important in causing the fall in oxygen consumption. Such an
accumulation of CO 2 , etc. would be accentuated by the relatively quiescent condition of the eggs.
B. The fall in oxygen consumption under the given conditions having been
confirmed, experiments were then made on samples of eggs under exactly equivalent conditions as in A5 except that the eggs were kept frequently in motion
by carefully turning over the bottles at intervals. The results of two of this series
of experiments are given.
Exp. 2. Quantities of oxygen absorbed by 2000 eggs from box a:
In
„
„
n
1 hr.
4 hrs.
5 „
7 »
...
...
•••
•••
0-231 mg.
1*028 mg. or -797 ing. in foil. 3 hrs. or gen. aver, of -257 mg. per hr.
i'384 »
-356 „
5th
far.
„
-276
i"7°4 »
*32O ,, foil. 2 hrs.
„
-243
„
The general average absorption per hour for the whole series of 17 hours is
•255 mg.
Exp. 3. Quantities of oxygen absorbed by 2000 eggs from box y:
In 1 hr.
„ 2 hrs.
>> 3 )>
55
4
>J
5 >>
>>
„ 6 „
•090 mg.
•230 mg. or -140 mg. in 2nd hr. or gen. aver, of -115 mg. per hr.
•304 „ -°74 „
3rdd
,,
•396
„
-092 „
4th
„
-099
•422
„
-026 „
5th
„
-084
•541
„
-119 „
6th
„
-090
The genera! average absorption per hour for this series of 21 hours is -094 mg. In
this experiment the total available oxygen in five hours was reduced by 10-5 per cent.
These experiments show that if the eggs be kept frequently in motion there
is no fall in the rate of oxygen absorption. It will be observed that in experiments
under these conditions there would be no serious error involved in taking the
oxygen absorption for the first hour as a general average for the sample. Small
fluctuations occur in the rate of absorption, but the average over an extended period
does not differ to any great extent from the quantity absorbed during the first hour.
C. In each of the remaining experiments the cages described above were
used, and attempts were made to discover the effect of changing the conditions on
the oxygen absorption of a sample of eggs.
Exp. 4. A batch of eggs from box a were allowed to remain with little movement, and the oxygen absorbed in three-quarters of an hour measured. The eggs
1
E.g. Roaf (6).
The Absorption of Oxygen by Plaice Eggs
181
Were
then removed to another bottle of fresh sea water and the oxygen absorbed
during a second period of three-quarters of an hour measured.
The eggs used -086 mg. of oxygen In the first period and -133 mg. during the
second period, showing that there was no fall in the amount of oxygen absorbed.
D. In a different type of experiment a batch of eggs (Q) in a cage was allowed
to absorb oxygen In a closed bottle for one hour. A second batch of similar eggs (P)
in another cage was also placed In a closed bottle and the oxygen absorption for
one hour measured. The batch Q was then removed, and the batch P placed In
the bottle of water previously occupied by Q. The oxygen absorbed by the batch P
during a second hour was then measured. During this second period the batch P
was still In the presence of a large excess of available oxygen.
Exp. 5. Eggs from box
Batch Q= 1845 eggs.
The available oxygen was reduced by batch Q In one hour by 3-5 per cent
Batch P= 2193 eggs.
In one hour batch P absorbed -148 mg. of oxygen In fresh sea water, and
•123 mg. in "used" sea water.
Thus there was a distinct fall In oxygen consumption, and this type of experimental result. In conjunction with those obtained under B and C, points to the
fact that there Is excreted Into the water some substance which exerts a considerable influence on the eggs.
E. The foregoing results Indicate that the fall In oxygen consumption is
probably due to the accumulation of either carbon dioxide or possibly nitrogenous
excretion. Experiments were now made to see what effect, If any, was produced
by adding carbon dioxide or a typical nitrogenous excretory substance, urea?. to
sea water containing eggs.
Carbon dioxide was first added to sea water by using small quantities of "soda
water." Preliminary experiments were made to find the effect of adding a small
quantity of soda water on the pH and "excess base" of the sea water. The pH
was determined by the Indlcatometric method, using phenol red, cresol red, thymol
blue and meta-cresol purple as Indicators, In conjunction with a series of standard
buffer solutions and the coloured chart given by Clark and Lubs (2), It was found
that the addition of very small quantities of soda water had a distinct effect In
lowering the rate of oxygen absorption.
Exp. 6. Number of eggs = i960 from box j8:
Soda water added in the proportion of 1 c.c. to 300 c.c. sea water.
This quantity lowered thepH from 8-65 to 8-60.
The eggs absorbed -139 mg. of oxygen In three-quarters of an hour In fresh
sea water, and ^050 mg. In the following three-quarters of an hour In CO2
sea water.
182
S . T . BURFIELD
Thus, with this very small addition of C0 2 , the rate of oxygen absorption was
considerably reduced.
Exp. y. In order to cut out any possible effect which might follow on even a
very slight change in the sea water, other than the addition of carbon dioxide, produced by adding soda water, the effect of placing eggs in sea water which had had
carbon dioxide gas bubbled through it was tried. Again preliminary experiments
were made to find out the alteration produced in the pH of the water by bubbling
CO2 through it at a given rate for a known time1. It was found that the addition
of CO2 had a very marked effect on the eggs. A batch of eggs from box y absorbed
•061 mg. of oxygen in one hour in fresh sea water.
This batch, when placed for a further hour in sea water, the pH of which had
been lowered from 8-65 to 8-45 by bubbling in CO 2 , absorbed -004 mg. of oxygen.
Thus the oxygen absorption had practically ceased, and at the ead of the second
hour there was no detectable change in the|?H from 8-45.
Exp. 8. The effect of adding urea in small quantities to the sea water appeared
to be nil.
A batch of 2031 eggs from box j8 absorbed -163 mg. of oxygen in one hour in
fresh sea water.
This batch, when transferred for a further hour to sea water to which urea had
been added (-05 gm. to 2 litres), absorbed ^252 mg. of oxygen. Thus there was
no fall in the rate of absorption, but a result similar to that obtained when the
transfer to fresh sea water was made.
4. CONCLUSIONS.
The experiments show that when left practically undisturbed young plaice
eggs, in a closed volume of water, show a marked fall in the rate of oxygen absorption, even in the presence of an abundance of available oxygen. Small changes in
the partial pressure of oxygen have no effect on the rate of absorption. If the eggs
be frequently moved so as to prevent an accumulation of excreted products no
fall in oxygen absorption is found, arid the amount absorbed during the first hour
is a fair average of the succeeding hours. Positive experiments show that while
a nitrogenous substance such as urea has practically no effect, the addition of
carbon dioxide causes a marked fall in the rate of oxygen absorption. In this
connection it is of interest to note that various workers have made experiments
showing the varying influence of carbon dioxide tension on different animals.
Roaf (6), working on barnacles and fish, has concluded that the influence of carbon
dioxide on the rates of respiratory movements is due to its effect on the hydrogen
ion concentration. Whitley<7), working on plaice eggs, states that the amount of
variation from the normal concentration of hydrogen and hydroxyl ions in sea water
which the eggs of the plaice will tolerate is very small, and that a disturbance of
the equilibrium towards the acid side is much more fatal than the opposite.
1
Control determinations showed that an ample supply of dissolved oxygen was present after
such treatment.
The Absorption of Oxygen by Plaice Eggs
183
Thus the general conclusion reached is that the fall in the rate of oxygen consumption observed in plaice eggs when relatively quiescent in a closed volume of
water is due to the accumulation of carbon dioxide in the neighbourhood of the
eggs, and the effect of this on the hydrogen ion concentration, the latter being
possibly the direct cause of the lowering of the respiration exchanges.
5. THE RESPIRATORY QUOTIENT OF PLAICE EGGS.
While obtaining data as to the quantity of oxygen absorbed by plaice eggs? and
incidentally noting the pH changes in the sea water (vide E above), several determinations of the respiratory quotient of the eggs were made. In order to do this
use was made of the useful curves given by Braced), connecting the pH of sea
water with the volume of carbon dioxide in solution per litre.
Several batches of eggs were allowed to absorb oxygen in a closed bottle for
periods of six to seven hours, the eggs being frequently moved to prevent any
fall in normal oxygen absorption. The amount of oxygen absorbed was found as
in the preceding experiments. The ^H of the water was determined carefully at
the beginning and end of the experiment, the change in pH being thus noted. Byusing the curves mentioned above, in conjunction with the known "excess base"
of the sea water used, the volume of carbon dioxide corresponding to the known
change inpH could be found. Thus the data necessary for the respiratory quotient
were obtained. Cresol red and thymol blue with Clark and Lubs* chart were used?
and also meta-cresol purple with a set of standard buffer solutions. Two determinations are given here.
Exp. 9. Batch of eggs (1330) from box a:
In 6J hours these absorbed -758 ex. of oxygen at N.T.P. The pH. of the water
during this period was lowered by -z pU (from 8-5 to 8-3).
This represents a concentration of 1*5 c.c. of carbon dioxide per litre at N.T.P.
In the bottle used in the experiment the amount of carbon dioxide produced
was -59 c.c. at N.T.P.
Thus the Respiratory Quotient = — ^ = -78.
Exp. 10. Batch of eggs from box y:
In 6J hours these absorbed -68 c.c. of oxygen at N.T.P. The ^H of the water
was lowered by -i pH (from 8-45 to 8-35).
This represents a concentration of 1-2 c.c. of carbon dioxide per litre at N.T.P.
In the bottle used in this experiment the amount of carbon dioxide produced
was -49 c.c. at N.T.P.
Respiratory Quotient = - ^ = -72.
These represent extreme values found, and the average Respiratory Quotient is
probably about 7 5 . In this connection it is of interest to note that Jolyet and
Regnard(4) give the Respiratory Quotient of the adult sole as *8i. The figure
obtained for the plaice egg is also of interest in connection with the energy-source
for the eggs at the stages used for the determinations, e.g. vide Needham(c).
S . T . BURFIELD
6. SUMMARY.
1. The rate of oxygen absorption by plaice eggs, contained in a closed volume
of sea waters falls when the eggs and water are allowed to remain relatively quiescent.
This fall takes place even when a large volume of water is used.
2. It has been shown that small changes in the partial pressure of dissolved
oxygen do not appreciably affect the rate of absorption, so that this is not the
factor concerned in the fall.
3. The rate of oxygen absorption in a closed volume of sea water does not fall
if the eggs be frequently moved.
4. The removal of eggs to a closed volume of fresh sea water at the end of a
period prevents the rate of absorption from falling during a second equal period.
5. The removal of eggs to a closed volume of "used" sea water at the end of
a period causes the rate of absorption to fall during a second equal period.
6. The addition of urea in small quantities to the sea water has no effect on
oxygen absorption.
7. The addition of carbon dioxide to the sea water has a marked effect in
lowering the rate of oxygen absorption, and the accumulation of excreted carbon
dioxide is probably the factor causing the fall in absorption mentioned under
paragraphs 1 and 5.
8. This effect is possibly directly due to the alteration in the hydrogen ion
concentration produced by the carbon dioxide.
9. The Respiratory Quotient of young plaice eggs is about -75.
I have to thank Professor Dakin for suggesting the investigation given in this
paper, and for communicating the results of some unpublished experiments.
I must acknowledge also the help given by the authorities of the Biological Station
at Port Erin in supplying material and by Mr J. R. Bruce in making several helpful
suggestions.
7. REFERENCES.
(1)
(2)
(3)
(4)
(5)
(6)
(7)
J. R. (1924). "Apii method for determining the carbon dioxide exchanges of marine,
brackish water and fresh water organisms." Brit. Journ. Exp. Biol. 2, 57.
CLARK, W. M. and LUBS (1922). The Determination of Hydrogen Ions. Baltimore.
DAKIN, W. J. and CATHERINE, M. G. (1925). " T h e oxygen requirements of certain aquatic
animals and its bearing upon the source of food supply." Brit. Journ. Exp. Biol. 2, 293.
JOLYET and REGNARD (1877). " Recherches physiol. sur la respiration des animaux aquatiques."
Arch, de Physiol. ame se'r. 4, 584.
NEEDHAM, J. (1926). "The energy sources in ontogenesis." Brit. Journ. Exp. Biol. 3, 189.
ROAF, H. E. (1912). " T h e influence of the carbon dioxide and oxygen tensions on rhythmical
movements." journ. Physiol. 43, No. 6, 449.
WHITLEY,^ E. (1905). "A note on the effect of acid, alkali and certain indicators in arresting or
otherwise influencing the development of the eggs of Pleuronectes platessa and Echinus
BRUCE,
esculentus." Proc. Roy. Soc. B ? 77, 137.