ABSTRACT
Methods have been developed for observing the effects of
DDT on the development of the larval skeleton in the purple sea
urchin (Strongylocentrotus purpuratas). These include uptake studies
with C -labelled DDT to determine the amount taken up by the dev-
eloping embryo, direct observations of skeletal growth, and obser-
vations of other factors which might indicate a general depressing
effect of DDT such as rate of cleavage, time of hatching, and rate
of protein synthesis.
-2.
INTRODUCTION
Preliminary studies with unfertilized eggs and developing
embryos of the sea urchin indicated the presence of a carbonic an¬
hydrase which was inhibited by DDT. The role of this particular
carbonic anhydrase during development is not known. Since the
skeleton of the sea urchin larva consists of calcium carbonate
(Yasamasu, 1959), and since carbonic anhydrase is involved with
calcium carbonate deposition in other forms ((Wilbur, 1964), I
postulated that this enzyme might be involved in skeleton form-
ation. If so, DDT might have a very specific effect on the rate
of growth of the skeleton, but not on other aspects of development.
Experiments were therefore initiated to observe the
effect of DDT on skeletal growth. Although methods were devel-
oped, reliable results were not obtained in the time available.
The purpose of this paper is to report these methods and to out-
line the experimental approach.
The methods of study will be discussed in three sections:
(1) uptake and accumulation of DDT, (2) measurements of skeletal
growth, and (3) measurements of other parameters of embryo activity
and development.
MATERIALS & METHODS
Shedding of gametes of S. purpuratas was induced by
intracoelomic injection of 0.5 N. KCl. Undiluted sperm were
stored at 5° C. The eggs were washed at least twice in filtered
sea water, and fertilized with a freshly diluted suspension of
sperm. Millipore-filtered sea water (0.45 micron) was used in
all experiments. The fertilized eggs were washed twice in sea
water and adjusted to a 0.1% suspension (V/V).
As great care must be taken in the handling of DDT
-3.
because of its codistillation with water, all experiments were
done with stopperred flasks, and solutions were exposed to open
air as infrequently as possible. For growing cultures, 25-50 ml.
of 0.1% egg suspension were placed in a stopperred 125 ml. flask
in a water bath at 14-15° C. Under these conditions the embryos
were in a single layer and development proceeded normally for
up to four days.
UPTAKE AND ACCUMULATTON OF DDT
Radioactivity was measured in a Nuclear Chicago Mark II
Scintillation Counter. For counting cellular material, the cells
were dissolved in Nuclear Chicago Solubilizer (NCS) and then
counted in a toluene-based scintillation fluid (4 g/1 2,5-diphenyl-
oxazole (PPO), 0.1 g/1 1,4-bis/2-(5-phenyloxazolyl)benzene (POPOP),
in toluene). Bray's scintillation fluid was used for aqueous
solutions (100 ml. ethanol, 20 ml. ethylene glycol, 4 g. PPO,
0.2 g. POPOP, 60 g. naphthalene to 1 liter in dioxane).
C-DDT, uniformly labelled in the ring (Amersham/Searle),
specific activity 19 momM was used. A stock solution of 100 ppm
was prepared in 954 ethanol. Incubation solutions were prepared
either by adding the stock solution of C -DDT to sea water, or
by adding a small amount of the CDDT stock toldilutions of non-
radioactive DDT. Because of pipetting variations, the actual
concentration of C-DDT (and hence the specific activity of the
incubation media) were calculated from the radioactivity present
in a l ml. aliquot.
Stock solutions of non-radioactive DDT (Aldrich Chemical
Company) were prepared in 95% ethanol, and appropriate dilutions
made with sea water. To avoid high concentrations of ethanol,
the DDT stock solutions were made as high as 10,000 ppm. As DDT
tends to precipitate above the solubility limit of about 1.2 ppm
in water, and since-DDT tends to stick to glassware, the sea water
dilutions were made fresh daily.
For studying kinetics of uptake in unfertilized eggs,
1 ml. of a 2.5% suspension of eggs was added to 24 ml. of incu-
bation medium in a 125 ml. Erlenmeyer flask. The stopperred
flask, kept in a water bath at 14-15° C., was swirled every 15
minutes. For each measurement, the contents of four flasks were
pooled, the eggs were packed by hand centrifugation, the supernat-
ant sea water decanted, and the eggs washed twice with sea water.
They were then dissolved in 1 ml. NCS and the radioactivity determined.
As a result of these studies, I decided to pulse dev-
eloping embryos in appropriate concentrations of CDDT for
one-half hour in order to observe the effects of DDT on develop-
ment. An accumulation of DDT about 50 times greater than that in
the incubation medium was predicted by the studies with unfertil-
ized eggs. For the studies with developing embryos, 1 ml. of
a 10% suspension of freshly fertilized eggs, or of 32 hour old
embryos, was added to 29 ml. of incubation medium in a stopperred
125 ml. flask and swirled every 10 minutes. After 30 minutes,
the supernatant sea water was removed, and the embryos washed
twice with sea water. One-fourth of them were returned to a
stopperred 125 ml. flask in a total volume of 25 ml. of sea water
for further observations of development. The remaining embryos
were dissolved in 1 ml. of NCS and the radioactivity determined.
OBSERVATTONS OF SKELETAL GROUTH
Cultures were observed in random sequence at intervals
of 2-6 hours beginning about 36 hours after fertilization. At
this time, the triradiate spicule has become apparent. The flask
-5-
was gently shaken to evenly distribute the embryos, and a drop
or two transferred to a glass slide. The sample was covered
with a glass coverslip and excess fluid drawn off with a pieceof
filter paper. Enough fluid must be removed so that the embryos
are pressed flat, and the skeleton is revealed but not crushed.
Measurements were made of the longest arm of at least 40 spicules
(20 embryos) using a 200 unit ocular micrometer, a 12.5x ocular,
and a 4Ox objective under phase contrast conditions.
MEASURENENTS OF OTHER PARANETERS
Cleavage
Examination of the cultures was begun immediately
after pulsing (about 2 hours after fertilization). Samples
were observed without a coverslip under low power in random
sequence, and at least 40 embryos from each culture were observed.
Embryos were classified as 1-, 2-, 4-, 8-, and 16-or-
more-cell stages. A number from 1 to 5 was assigned to each of the
stages respectively. Unfertilized eggs were ignored. Embryos
in the process of cleavage were scored with the later stage.
A weighted average of the cleavage stages l to 5 was made for
each time of observation, andiis referred to as the Cleavage
Index. Thus if there were 21 embryos in the 2-cell stage (2),
18 in the 4-cell stage (3), and one in the 8-cell stage (4),
the cleavage index would be (21x2 4 18x3 + 1x4)/40, or 100/40 - 2.5.
Hatching
Beginning about 16 hours after fertilization, observations
were made every hour to determine the number of embryos which
had hatched. For each sample, the culture was shaken and a few
drops applied as a streak along a glass slide and examined from
end to end with a dissecting microscope at 25x. This procedure
2
6.
made it possible to keep track of the swimming blastulae already
counted. Unfertilized eggs were ignored, and the number of
swimming or moving blastulae was compared with the number of
non-motile blastulae. With little practice it was easily possible
to recognize these various forms under the dissecting microscope.
Protein Synthesis
One-half ml. of a 10% suspension of fertilized eggs was
pulsed for one-half hour in a solution of non-radioactive DDT,
washed as usual, and placed in 50 ml. of sea water in a 125 ml.
flask. At about 7, 19, and 50 hours after fertilization (during
cleavage, hatching, and skeletal growth), the amino acid incorp-
oration of these embryos was determined.
C phenylalanine (International Chemical and Nuclear
Company), specific activity 300mcm was used, and 0.148 micro-
curies added to a 12 ml. aliquot of embryo culture in a 30 ml.
beaker. After incubation for five minutes at 14-150 C., the
sample was divided into two 5 ml. amounts in conical centrifuge
tubes. Five ml. of 10% trichloroacetic acid (TCA) was added to
Tube I (incorporated amino acid), which was then placed in ice.
Tube II (total amino acid uptake) was hand centrifuged to remove
the sea water supernatant, and the embryos washed once with sea
water. Tube I was then washed with 5% TCA, 5% TCA containing
0.01 M. phenylalanine, and again with 5% TCA. Both samples were
then dissolved in NCS (0.5 or 1.0 ml.) and the radioactivity
determined. Percent incorporation was computed from the ratio
of incorporated amino acid to total amino acid uptake.
RESULTS
These results are described in order to give an idea as
to the type of findings obtained and the further work that is
necessary. They are not intended to be final or definative.
UPTAKE AND ACCUMULATION
Results of the kinetics of uptake studies are shown in
Figures 1 and 2. It is apparent from Figure 1 that the eggs take
up and concentrate DDT immediatoly and continue to do so for
some time. Thus, by one minute, the concentration of DDT inside
the eggs is 2.5 times greater than that in the incubation medium,
and by four hours has increased to 333 times the initial concen¬
tration in the medium. Figure 2 indicates that the amount of DDT
taken up is almost linearly proportional to the concentration in
the incubation medium. This is true for both the one minute
and 30 minute incubation periods.
Uptake results from all experiments are summarized in
Table 1. Clearly more studies should be done to establish if
the difference in uptake between unfertilized or just-fertilized
eggs and later-stage embryos is significant. As the purpose of
these measurements was only to deter mine the actual concentration
of DDT in the embryos, further studies were not done.
SKELETAL GROWTH
Figure 3 indicates the rate offgrowth of the longest
skeletal arm. Extensive measurements from three control cultures
are shown, plus a single control measurement from another exper-
iment. There is close agreement among cultures 1, 2, and 4.
Culture 3 shows a much slower rate, although growth began at about
the same time. This difference may reflect the abnormal develop-
ment often observed near the end of the sea urchin breeding
8
season.
The effect of DDT applied 32 hours after fertilization
is shown in Figure 4. At 4 ppm DDT (determined in the embryos) the
rate of growth is slowed, but the final length achieved is the
same as the sea water controls. As DDT inhibits the enzymatic
hydration with carbonic anhydrase, but does not inhibit the
spontaneous reaction, such a result might be predicted. It
should be pointed out that this is a single experiment, and
proper ethanol controls were not run.
DDT pulsing with very low concentrations just after
fertilization was also done with the questionable culture (f3)
noted above. As indicated in Figure 5, ethanol alone seemed to
have an effect similar to 13 ppb DDT (determined in the embryos).
Table 2, however, indicates that in Experiment 4, at the same
concentrations of DDT, a much different response occurred. Another
factor which makes it difficult to interpret these data is that
there is a 2.5 hour delay between the measurement of the ethanol
control and the 13 ppb DDT cultures.
OTHER PARAMETERS OF DEVELOPMENT
Figure 6 indicates the rate of cleavage in sea water,
ethanol controls, and 13 ppb DDT (determined in the embryos).
There is no apparent difference, and the average is one cleavage
every 34 minutes. Subjective observations indicated abnormal or
incomplete cleavage was occurring in the higher concentrations
of DDT, but normal development followed.
Kinetics of the hatching process are shown in Figure 7.
(Notice that because of variability in hatching time, examination
of only "50% hatched" would not give an accurfate picture.) It
is apparent that the DDT had no effect on the time of hatching
3.
The relative protein synthesis at different times after
fertilization is indicated in Table 3. Two concentrations of
DDT, and one concentration of DDE (the principle breakdown product
of DDT) equivalent to the lowest DDT concentration are shown.
(The concentrations determined inside the embryos are not knoun
accurately, because only non-radioactive DDT was used. The indi-
cated concentrations are from another culture which was pulsed with
C -DDT at the same concentrations.) Pulsing with DDT, DDE,
or ethanol increased protein synthesis by as much as two-fold.
Table 4 shows the average total uptake of C-phenyl-
alanine at the different times. It is interesting that during
hatching the amount taken up is nearly twice that during cleavage
or during the growth of the skeleton.
DISCUSSTON
The results described are clearly not definative, but
do indicate possible trends and areas of further research.
The effect on rate of skeletal growth is interesting,
whether it is caused by DDT, or by the short exposure to ethanol.
It may be that ethanol modifies the cell membrane in such a way
as to disturb normal salt balance; an interesting possibility
since Davis (1961) reports that a high ionic strength inhibits
carbonic anhydrase.
Further studies of the effect on skeletal growth should
certainly be dine with complete ethanol controls, as well as a
more careful and consistent use of DDT concentrations. Considera-
tion should also be made of the effects of pulsing with DDT at
different times. It might be possible to alter the growth oftthe
skeleton after it has already begun normal growth in sea water.
In order to simplify and speed up the measuring procedure, I
30
-10-
wCuld suggest the use of a camera lucida to make rapid sketches
which can be used for later, detailed measurements. This would
also allow for determination of the width of the growing arms,
and a computation of the length-width ratio. This, as pointed
out by Okazaki (1962), is a more significant measurement of the
deposition of calcium carbonate since the length is dependent
upon the shape of the organic matrix in which the skeleton develops.
The measurements of cleavage and hatching seem to
indicate that the various experimental condidtions did not affect
these aspects of development.
The results of determinations of protein synthesis are
interesting, but without more consistent and reliable results on
effects in other aspects of development, no suggestions as to the
significance of these measurements can be made. In future studies
of this kind, a parallel measurement of C-DDT should be done
to determine more accurately the uptake of cold DDT in the exper-
imantal cultures. Double labelling could also be done using
C14-DDT and a tritium-labelled amino acid.
Other parameters of normal development could also be
measured to determine the specificity of any effect on skeletal
growth. These might include the rate of gastrulation, respiration
rates, or aspects of behavior such as ciliary beat and phototropisms.
A complete study of the phenomenon would also include
gas chromatographic determinations of the "natural" level of DDT
in sea urchin eggs. Also to correlate the results properly
with a demonstration of the role of the carbonic anhydrase, a
complete in vitro study of the enzyme should be done. This would
include quantitative determinations of the in vitro effect of DDT,
as well as determinations of the activity of the enzyme at diff-
erent stages in development.
SUMMARY
1) Methods are described for measuring:
a)the uptake of C-DDT by developing sea urchin embryos,
b)the rate of growth of the larval skeleton,
c)the rate of cleavage in early development,
d)the kinetics of the hatching process, and
e)protein synthesis during development.
2) There is some indication of an inhibition of the rate of growth
of the skeleton by either DDT or ethanol, while
3) there is no measured effect of DDT on either cleavage or hatching.
4) Both DDT and ethanol increased the rate of protein synthesis.
5) Suggestions are made for a more complete and reliable study.
ACKNOWLEDGEMENT
The author wishes to express his thanks to Dr. David Epel for
his continuing help and guidance, and to Dr. John Phillips and the
staff of Hopkins Marine Station for their helpful comments and
support. Thanks are also due for Dr. Barbara Bruner and Jeff Davidson
for their advice about the care and raising of sea urchins. This
research was supported in part by a grant from the National Science
Foundation, GY-5878.
BIBLIOGRAPHI
Davis, R. P., 1961. Carbonic anhydrase. In: The Enzymes, 5, 545-562.
ed. Boyer, Lardy, Myrbäck. Academic Press, New York & London.
Okazaki, K., 1962. Skeletal formation of sea urchin larvae. IV. Correl-
ation in shape of spicule and matrix. Embryologia, 7, 21-38.
Wilbur, K. M., 1964. Skeletal formation and regeneration. In: Physiology
of Mollusca, 1, 247. ed. Wilbur and Yonge. Academic Press, New
York & London.
Yasumasu, I., 1959. Spicules of the sea urchin larvae. Zool. Mag.,
„ 42 (in Japanese).
TABLE LEGENDS
Table 1 - Uptake of C1-DDT by different types of embryos from
different types of incubation media. All incubations are
for one-half hour at 14-15° C.
Table 2 - Effect of incubation of fertilized eggs in 4.5 ppm DDT
and 0.475% ethanol, and 0.475% ethanol. The concentration
of DDT in the embryos is 13 ppb in Experiment 3, and should
be the same in Experiment +4, although the measurement was
not made. Time is given in Hours: Minutes after fertilization.
Length is given in ocular units (1 ou. - 1.25 microns).
Table
- Relative protein synthesis at different times in devel-
opment. DDE concentration (0.040 ppm) is the same molarity
as the DDT concentration (0.045 ppm). Measurements during
cleavage were made between 7 and 8 hours, hatching between
19 and 20 hours, and skeleton formation between 49.5 and
50.5 hours.after fertilization.
Table 4 - Total C - penylalanine uptake at different times in
development. Measurements during cleavage were made between
7 and 8 hours, hatching between 19 and 20 hours, and skeleton
formation between 49.5 and 50.5 hours after fertilization.
Unfertilized eggs:
Fertilized eggs:
32-hour old
embryos:
TABLI
AKE OF CI
DDT
CONCENTRATION DDT (ppm,
INCUBATION SOLUTION
IN CELLS
5.6 X 10
2.4 x 10-2
9.5 X 10
7.2 X 10"
9.4 X 10
6.2 x 10
3.1 X 10
2.05
3.1X 10
3.66
3.(
30.0
851
APPROXIMATE INCREASE
IN CONCENTRATION
43x
76x
66x
66x
5ux
1.22x
28.4x
EXPT #3:
Sea water
Ethanol
13 ppb DDT
EXPT 44:
Sea water
Ethanol
13 ppb DDT
ACTUAL TIME
47:07
46:38
49: 04
48:38
48:28
48:17
TABLE 2
FECT ON SKELETAL GRONTH
LENGTH
£ OF SW CONTROL
35.4
100%
51%
18.1
50%
17.7
72.
100%
68.7
94%
23.7
339
VUMBER OF MEASUREMENTS
40
40
INCUBATTON MEDIUM
Sea Water
Ethanol (0.5%
DDE (.040 ppm in
.0078 EtOH)
DDT (.045 ppm in
.005% EtOH)
DDT (4.5 ppm in
O.5% EtOH)
TABLE 3
RELATIVE PROTEIN SYNTHESI!
PROTEIN SYNTHESIS RELATIVE TO S. W. CONTROL
AT TIME OF:
u
CLEAVAGE
SKELETON FORMATION
HATCHING
L.0
1.0
1.0
2.0
2.3
1.4
1.7
1.9
--
2.1
Average Total Uptake
(Moles)
Relative Uptake
TABLE 4
AMINO ACID UPTAKE
CLEAVAGE
HATCHING
5.0x10-12
9. kx1012
1.9
SKELETON FORMATTON
5. 5x10-12
1.1
FICURE LEGEND
Figure 1 - Uptake of C-DDT by unfertilized eggs from an incuba-
tion medium of 0.56 ppb DDT.
Figure 2 - Rate of uptake of C-DDT (ppm/time) from incubation
media of different concentrations. Solid circles, hour
incubation time; empty circles, 1 minute incubation time.
Numbers in parentheses indicate relative rates.
3 - Growth of the longest skeletal arm in sea water at
Tigure
14-15° C. Length is given in ocular units (1 ou. - 1.25
microns). Solid circles, Expt. 1; empty circles, Expt.
12; triangles, Expt. 13; squares, Expt. 74.
Figure 4 - Experiment f2. Effect of incubation of 32-hour-old
embryos in 3 ppm DDT and 2.95% ethanol. The concentration
of DDT in the embryos is 4 ppm. Length is given in ocular
units (1 ou. - 1.25 microns). Solid circles, sea water
control;; empty circles, DDT. Numbers in parentheses indi¬
cate the length relative to the sea water control.
- Experiment 43. Effect of incubation of fertilized eggs
Figure
in 4.5 ppm DDT and 0.475% ethanol, and in 0.475% ethanol.
The concentration of DDT in the embryos is 13 ppb. Length
is given in ocular units (1 ou. - 1.25 microns). Solid
circles, sea water control; empty circles, DDT; triangles,
ethanol.
Figure 6 - Cleavage in sea water (solid circles), and with a
concentration of 13 ppb DDT in the embryos (empty circles).
See text for definition of Cleavage Index.
Figure 7 - Kinetics of hatching. Solid circles, sea water control;
empty circles, 7 ppb DDT in the embryos; solid triangles,
62 ppb DDT in the embryos; empty triangles, 2 ppm DDT in
the embryos; squares, 16 ppm DDT in the embryos.
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