ABSTRACT
Sea urchins (Strongylocentrotus purpuratus and Strong-
ylocentrotus franciscanus) in the vicinity of the Los An¬
geles County sewer outfall at White's Point have abnormal
spination (stunted and missing primary spines). There are
very high DDT residue levels (mainly DDE) in the tissues
of these animals compared with normal appearing animals
found at Pigeon Point, San Mateo County. Calcification
rates in regenerating spines of S. purpuratus from White's
Point were one-half the rates of regenerating spines of
S. purpuratus from Pigeon Point. DDT in concentrations
above 1 ug/ml appears to supress calcification in regen-
erating spines of S. purpuratus from Pigeon Point while
no suppression of calcium uptake occurred in regenerat¬
ing spines of S. purpuratus from White's Point exposed
to DDT concentrations up to 10 pg/ml. Accumulated pesti¬
cide residues contained in the spines of the White's Point
urchins apparently suppresses calcification and may be
a major cause of their stunted, abnormal appearing spina-
tion.
35
INTRODUCTION
Pearse, et.al.(1970) noted that sea urchins (both
Strongylocentrotus purpuratus and Strongylocentrotus fran¬
ciscanus) in the White's Point area of Palos Verdes Pen-
insula, Southern California, were without many of their
major spines, suggesting an unhealthy condition. Moreover,
the proportion of unhealthy sea urchins in the area appears
to have increased between September 1970 and May 1971
(J.S. Pearse, personal communication). The cause of the
unhealthy condition is unknown, but high levels of DDT
residues in the area suggests a possible cause.
Burnett (1971) found extremely high levels of DDT
residues in the sand crab, Emerita analoga, only a few
miles from White's Point (10 ppm in the whole animal).
Swarbrick (1971) analyzed specimens of Pagurus samuelis
collected at White's Point and found DDT residue levels
4 times greater than levels found in the Monterey Bay or
San Francisco Bay areas. Marian (1971) also found DDT
residue levels in Hermissenda crassicornis that were much
higher than those found in the Monterey Bay area. Carry
and Redner (1970) reported that the Los Angeles County
sewer outfall located at White's Point (dispersion is
- 1 -
actually 1500 to 3600 meters offshore) discharged an aver¬
age of 1.44 million cubic meters (380 million gallons) of
primary treated sewage per day with an average concentra¬
tion of 34 ppb of chlorinated hydrocarbons (105 lbs. per
day) almost all of which were DDT metabolites. Therefore,
there may be a correlation between high DDT residue con¬
centrations in the area, and the abnormal spination of
sea urchins from White's Point; this is examined in the
present paper by studying calcification rates of spines
of S. purpuratus and S. franciscanus regenerating in vitro.
Heatfield (1970) studied calcification of regenerat-
ing spines of S. purpuratus using calcium-45 as a radio¬
active tracer. He examined the effects of time, tempera¬
ture, and inhibition of carbonic anhydrase on calcium up¬
take in regenerating spines. Heatfield's system was very
sensitive to calcium metabolism and therefore provides
a convenient method for examining the possible effect of
DDT on calcification.
MATERIALS AND METHODS
Specimens:
Specimens were collected at both White's Point on
16 May 1971, and Pigeon Point on 22 April 1971 (Fig. 1).
Only specimens that appeared to be distinctly unhealthy,
including both S. purpuratus and S. franciscanus, were
collected at White's Point from 1 to 3 meters of water.
Pigeon Point urchins (S. purpuratus only) were collected
- 2 -
39
from the intertidal region; all were very healthy in ap-
pearance. The urchins were maintained at Hopkins Marine
Station in running sea-water kept at 15 C I I C and fed
unlimited amounts of the brown algae, Macrocystis integ-
rifolia.
Measured Pesticide Residue Levels:
Pesticide residue levels in tissues of S. purpuratus
were measured by gas chromatography using the techniques
developed by Stanley and LeFavoure (1965) and Kadoum (1967)
except that smaller amounts of tissues were used (0.5 to
2.5 grams versus about 100 grams) and the digest medium
used for the spine extraction was 12N HCl. The gonads,
spines, lantern structure, and coelomic fluid of 3 Pigeon
Point urchins and the spines and gonads of 2 White's Point
urchins and the gonads of an additional animal were ana-
lyzed for DDT residues. Only very unhealthy appearing spec¬
imens from White's Point were analyzed, while the speci¬
mens from Pigeon Point appeared normal.
Measurement of DDT Effects on Calcification:
Heatfield (1970) found no difference in calcium up¬
take rates of spines regenerating in vitro and in vivo;
linear rates of calcium uptake were established in 4 days.
Accordingly, spines analyzed for DDT effects on calcium¬
45 uptake were allowed to regenerate in vivo for 4 days
before being exposed to test conditions in vitro. The
spines from healthy urchins (Pigeon Point) were all taken
from the 2 major rows of primary spines of each of 5 in¬
terambulacral plates to minimize effects due to size
differences. This was not possible for animals collected
at White's Point because the extremely unhealthy specimems
chosen had few spines and it was difficult to get just
the 25 spines necessary for the experiment.
All individual experiments were done with spines
from the same animal. The spines (about 40 per animal)
were fractured 3 mm above the milled rings as specified
by Heatfield (1970) and allowed to regenerate for 4 days
still attached to the animal. After 4 days the stubs were
removed intact and gently placed in 4 250 ml beakers (about
10 spines per beaker) containing 1 uci/ml calcium-45 (Amer¬
sham/Searle - 71.4 mCi/milli-mole) in 20 ml of artificial
sea-water maintained at 15 C I 1 C. Additional amounts of
ethanol (0.02, 0.1, and 0.2 ml) of different concentrations
of DDT (0.1 mg/ml and 1.0 mg/ml) were added to test sol¬
utions. After 24 hours the stubs were removed from the
solutions and assayed for calcium-45 uptake as described
by Heatfield (1970)
Ethanol Controls:
Since ethanol was being added to test solutions, it
was essential to isolate any effects due to ethanol.
Therefore, controls with varying amounts of ethanol were
analyzed for effects on calcium uptake. Concentrations of
ethanol up to 0.91% in sea-water had no effect on the rate
of calcification (Fig. 2). Salk (1969) found a similar
break at 0.95% ethanol in sea-water in his calcification
experiments with S. purpuratus larvae. Therefore, in all
calcification experiments on regenerating sea urchin spines,
the total percentage of ethanol in sea-water was less than
or equal to 0.91%.
Carbon-14 DDT Uptake:
In vivo DDT uptake was measured by adding 0.2 ml of
1 ug/mlC-DDT (Amersham/Searle - 19.1 mCi/milli-mole)
in 95% ethanol to 2 liters of filtered sea-water in which
4 specimens of S. purpuratus form Pigeon Point were placed.
These animals all weighed approximately 30 grams. At time
intervals one spine (that had not been fractured) was re¬
moved from each urchin, weighed, and placed in a plastic
scintilaation vial with 1 ml of NCS Solubilizer (Amersham/
Searle). After 5 minutes in NCS, 10 ml of scintillation
fluid (4 grams 2,5-di-phenyloxazole (PPO) and 0.1 gram
1,4-bis- 2-(5-phenyloxazole)
benzene (POPOP) per liter
toluene) was added. Counts were quantified by the channel
ratio technique with corrections made for quenching (nu¬
clear Chicago Liquid Scintillation Counter Mark II).
For in vitro uptake measurements, spines first frac-
tured as previously described and then allowed to regen¬
erate for 6 days on the urchin were then removed and
placed in 250 ml beakers containing 20 ml of filtered
sea-water with 0.1 ml of 1 ppm C-DDT in ethanol added.
(15 stubs from 3 S. purpuratus collected at Pigeon Point
were placed in 3 beakers.) The concentration of C-DDT
in the in vitro experiments (5,000 ppg/ml) was 50 times
higher than that in the in vivo experiment (100 pug/ml).
Assay for C was done in the same way as for the in vivo
experiment.
- 5
RESULTS
Measured Pesticide Residue Levels:
In the 3 specimens collected at Pigeon Point and
analyzed for pesticide residues, only the gonads showed
a trace of DDE, between 0.5 and lng/g (ppb), near the
detection limit of the gas chromatograph. No detectable
amounts of DDE, DDD, or DDT were found in the spines,
lantern structure, or coelomic fluid.
In contrast to the specimens collected at Pigeon
Point, 3 animals collected at White's Point, that were
maintained at Hopkins Marine Station for 2 weeks and then
analyzed, had measurable amounts of DDT residue (mainly
DDE) in their tissues (Fig. 3). The DDT residue levels in
the spines of 2 animals were about twice the levels found
in their gonads. The spines of the third specimen were
not analyzed but it is likely that they also had a higher
content of DDT residues than the gonads, which would
mean their content would be well over the 420 ppb found
in the gonads.
-DDT Uptake Rates:
Uptake studies showed a striking difference between
spines incubated in vivo and in vitro (Fig. 4). While
spines incubated in vitro (regenerating) took up DDT with
increasing time, spines still attached to urchins (not
regenerating) lost their initial content of DDT until they
equilibrized after 1 hour to approximately a constant
level. Spines incubated in vitro for 48 hours at 5 ng DDT/ml
C
showed a comparable DDT content to the levels found in
White's Point urchins (see Fig. 3).
DDT Effect on Calcification:
There was no effect on Ca uptake by DDT on the
spines of animals from Pigeon Point tested at concentra¬
tions of DDT up to 1 ug/ml. However, when the concentra¬
tion was raised above the saturation concentration of
1.2 ug/ml (Salk, 1969), there was a striking decrease
(32% at 10 ug DDT/ml) in calcium absorbed between spines
exposed to DDT and controls not exposed to DDT (Fig. 5).
DDT, even at concentrations of 10 jg/ml, had no ef-
fect on calcium uptake in spines of the White's Point
S. purpuratus (6A - Fig. 6). However, the amount of Ca
taken up by this specimen, even without DDT in the incu¬
bation medium, was relatively low in comparison to amounts
of Ca taken up by the spines of urchins from Pigeon
Point (see Fig. 5). Indeed, amounts of Ca taken up by
spines of the spines of the White's Point animal incubated
without DDT was most similar to that amount of Ca taken
up by spines of the Pigeon Point animal incubated in 10
ng DDT/ml (Fig. 7). The uptake of Ca45 in regenerating
spines of a 10 gram animal from White's Point was also
measured without exposure to DDT; the value of Cat ab-
sorbed was 9.6 ng f 1 ng (compared to 16.24 for the 30
gram specimen shown in Table 1 and Figs. 6A and 7).
- 7 -
DISCUSSION
C1DDT Uptake by Spines:
The results of the uptake study (see Fig. 4) show
that spines regenerating in vitro do take up DDT at con-
centrations of 5 ng DDT/ml and hence probably do so at
the higher concentrations used during the calcification
experiments. The in vivo curve of non-regenerating spines
is interesting in that it shows that DDT taken up in the
first 5 minutes, probably by adsorption, is somehow lost
until equilibrium is reached after about 1 hour. The fate
of the lost DDT is unknown. Perhaps the DDT is translocat¬
ed into the urchin until it reaches equilibrium. Another
possible explanation is mucous secretion of the spines
and subsequent loss of mucus containing high concentra¬
tions of DDT.
DDT Effect on Calcification:
The high levels of pesticide residues found in the
White's Point specimens are in accordance with the high
pesticide residue concentrations in the water and sedi¬
ments around the sewer outfall (Carry and Redner, 1970)
Moreover, because the spines are almost 99% inert calcium
carbonate by weight, the actual DDT content per weight
of organic tissue is much higher than indicated in Figs.
3 and 4. Marian (1971), Burnett (1971), and Swarbrick
(1971) also found very high DDT residue concentrations
in other animals in the White's Point area. However,
whether these high levels of DDT residues are responsi¬
- 8 -
3
ble for the unhealthy appearance of the urchins at White's
Point is not certain.
Statistical analysis (Table 1) shows that calcifica¬
tion in only 1 animal (50) from Pigeon Point was suppressed
by DDT and suppression occurred at concentrations above
jg DDT/ml. In contrast, the S. purpuratus (6A) from
White's Point showed no suppression at the same concen¬
trations of DDT in sea-water (1,5,10 ug DDT/ml). However,
as Figure 7 shows, calcium uptake in the regenerating
spines of the White's Point specimen (6A), incubated with¬
out DDT, was similar to the suppressed calcium uptake by
the spines of the Pigeon Point animal (5C). A possible
explanation is the size difference between the stunted
primary spines of the White's Point in comparison to the
normal spines of the Pigeon Point urchin. However, the
uptake of calcium by the S. franciscanus (6B) specimen
collected at White's Point, which had spines at least
twice the size of normal spines of S. purpuratus, was not
significantly different from amounts of calcium taken up
by spines of the S. purpuratus collected at White's Point
(6A). This suggests that size difference is not the major
cause of the different amounts of calcium taken up by
spines incubated without DDT from Pigeon Point and White's
Point. It seems more probable,he that the high
levels of DDT residues present in the spines of the
White's Point urchins (see Fig. 3) inhibit normal calcium
uptake and mask the effect of additional amounts of DDT
added to the water.
Heatfield (1970) found that complete inhibition of
carbonic anhydrase, an important enzyme involved in cal¬
cium metabolism, only resulted in a partial suppression
of calcium uptake in regenerating spines of S. purpuratus.
Past studies of the effect of DDT and its metabolites on
calcification, specifically on the enzyme carbonic anhy¬
drase, have been conflicting. Peakall (1970) and Bitman
et.al.(1970) noted that DDT induced inhibition of carbon-
ic anhydrase in the shell glands of birds. Keller (1952)
observed inhibition of carbonic anhydrase extracted from
bovine red blood cells and Nance (1969) found DDT inhibit¬
ed carbonic cnhydrase purified from Mytilus californianus
and Tequla funebralis as well as bovine red blood cells.
However, Anderson and March (1956) found no inhibition
by DDT of carbonic anhydrase extracted from cockroaches,
and recnetly Maren, et.al.(1971) retested the effects of
DDT on carbonic anhydrase taken from bovine red blood
cells and found no effect in concentrations less than
500 mg DDT/ml. Maren did observe a partial suppression
of calcium uptake, however, at concentrations above
500 mg DDT/ml where DDT was found to precipitate out of
solution. Precipitation of DDT from sea-water was also
noted to take place in experiments done in concentrations
above 1 ug DDT/ml (5C and 6A). The partial effect (32% in
Pigeon Point urchins and 50% between controls of speci¬
mens 50 and 6A could then possibly be due to a carbonic
anhydrase-independent element of the calcification mechanism.
- 10
2
DDT is toxic because of its tendency to accumulate
in tissues and hence cause an effect over a longer period
of time. This long range effect is probably responsible
for the lower calcification rates of regenerating spines
of the White's Point urchin. Longer time studies of the
effect of pesticide residues should be done - and ideally
on more animals than analyzed here. Trace metal effects
should also be studied. Cadmium especially has been
known to effect calcium metabolism (Nilsson, 1970) and
should be examined in the animals at White's Point. In
conclusion, it is not certain whether DDT is the major
cause of the abnormal spination of the sea urchins at
White's Point; however it probably is at least partly
responsible because there are high DDT residue concen¬
trations in the spine tissues, and high DDT concentrations
do seem to inhibit calcification rates.
SUMMARY
.
1. Sea urchins found at White's Point, Los Angeles County,
were missing many of their major spines and appeared
to be in an unhealthy condition. Very high levels of
DDT residues (mainly DDE) were found in animals collect¬
ed around the White's Point area, especially in the
spines which had more than 200 ppb. In contrast, DDT
residues were barely detectable in healthy appearing
animals from Pigeon Point, San Mateo County.
2. Uptake studies using C-DDT showed that non-regener¬
ating spines incubated in vivo tended to lose initially
absorbed levels of DDT and equilibrate at about O.5
ppb after 1 hour. Regenerating spines incubated in
vitro showed the opposite trend, concentrating DDT
with time; levels of 419 ppb were reached in 48 hours,
Calcium uptake by regenerating spines in vitro from
animals collected at Pigeon Point was not affected
significantly by DDT concentrations less than 1 g/ml
but was affected by concentrations greater than 1 ug/ml,
when the drug precipitated from the sea-water.
4. Calcium uptake rates by spines of one specimen of S.
purpuratus collected at White's Point was lower than
that in the specimens collected at Pigeon Point and,
although they were not affected by external DDT con¬
centrations up to 10 g/ml, levels already present
in the spines may have suppressed calcification.
5. It is suggested that the stunted spination of the sea
- 12 -
urchins of White's Point, may be due, in part, to high
DDT residue levels in their spine tissues.
13 -
40
ACKNON LEDGEMENTS
This work was done as undergraduate research in
Biology 175H at Hopkins Marine Station of Stanford Uni¬
versity. I would like to thank the entire staff and grad¬
uate students of Hopkins Marine Station for all the under-
standing and help they gave so generously. Especially I
would like to thank Dr. John S. Pearse for his help in
collecting specimens at White's Point, designing exper¬
ments, and for critically reading this manuscript. I
would also like to thank Dr. Vicki B. Pearse for her help
in using the Automatic Scintillation Counter, Phillip R.
Murphy for his help with preparing necessary equipment,
and Robin Burnett for his invaluable background study of
the White's Point area.
- 14 -
REFERENCES CITED
ANDERSON, A.D. AND MARCH, R.B., 1956, Inhibitors of Car¬
bonic Anhydrase in the American Cockroach, Periplaneta
americana, Canad. J. Zool., 34:68-74.
BITMAN, J., CECIL, H.C., AND FRIES, G.F., 1970, DDT-Induc¬
ed Inhibition of Avian Shell Gland Carbonic Anhydrase:
A Mechanism for Thin Eggshells, Science, 168:594-596.
BURNETT, R., 1971, DDT Residues: Their Distribution Along
Coastal California, submitted to Science.
CARRY, C.W. AND REDNER, J.A., 1970, Pesticides and Heavy
Metals, Progress Report, December 1970, County Sani¬
tation District of Los Angeles County, 51 pp.
HEATFIELD, B.M., 1970, Calcification in Echinoderms: ef¬
fects of temperature and diamox on incorporation of
calcium-45 in in vitro regenerating spines of Strong-
ylocentrotus purpuratus, Biol. Bull., 139:151-163.
KADOUM, A.M., 1967, A Rapid Micromethod of Sample Cleanup
for the Gas Chromatograph Analysis of Pesticidal Res¬
idues in Plant, Animal, Soil, Surface and Ground Water
Extracts, Bull. Environ. Contam. Toxicol., 2:264-267.
MAREN, T.H., DVORCHIK, B.H. AND ISTEN, M., 1971, Does
DDT Inhibit Carbonic Anhydrase?, Science, 172:728-729.
MARIAN, R., 1971, Assimilation and Transfer of DDT Res¬
idues in a Hydroid:Nudibranch:Tectibranch Food Chain
from the California Coast, (unpublished MS. on file
at Hopkins Marine Station Library).
NANCE, P., 1969, Inhibition of Carbonic Anhydrase by DDT
and DDE, (unpublished MS. on file at Hopkins Marine
Station Library), pp. 241-250.
NILSSON, R., 1970, Aspects on the Toxicity of Cadmium
and its Compounds, Swedish Natural Science Research
Council, Ecological Research Committee Bulletin No.
7, 48 pp.
PEAKALL, D.B., 1970, p-p'-DDT: Effect on Calcium Metabol¬
ism and Concentration of Estradiol in the Blood,
Science, 168:592-594.
PEARSE, J.S., CLARK, M.E., LEIGHTON, D.L., MITCHELL, C.T.,
AND NORTH, W.J?, 1970, Marine Waste Disposal and Sea
Urchin Ecology IN: Kelp Habitat Improvement Project,
Ann. Rept., 1 July 1969 - 30 June 1970, Appendix, pp.
1-93, Calif. Inst. Tech., Pasadena, Calif.
4
SALK, D., 1969 Methods for Assessing the Effects of DDT
on Larval Skeletal Development in the Sea Urchin,
strongylocentrotus purpuratus, (unpublished MS. on
file at Hopkins Marine Station Library), pp. 291-310.
STANLEY, R.F. AND LEFAVOURE, H.T., Rapid Digestion and
Cleanup of Animal Tissues for Pesticide residue anal¬
ysis, J. Assoc. Offic. Agr. Chem., 48:666-667.
SWARBRICK, S., 1971, DDT Residues in the Marine Intertidal
Hermit Crab Pagurus samuelis (Crustacea:Decapoda) in
California Waters, (unpublished MS. on file at Hopkins
Marine Station Library).
40
0
o
0
1+ 1+ 1+
0
0
9 8

6 6
1+
16
5

1+

i
0
O
8
11

—
10.





1 H

1
t
3
5
4
H +
82
22

3H
1
AH
0 -
c9
0
O H

9

0

1+ 1+
O A

14
LEGEND OF FIGURES
1. Map of California showing collection sites of Pigeon
Point and White's Point.
2. Effect of ethanol of Calcium-45 uptake by regenerating
spines of Strongylocentrotus purpuratus;the means and
standard deviations are shown.
3. Measured DDT residue levels found in Strongylocentrotus
purpuratus collected at White's Point. T-DDT, D-DDD.
E-DDE,
4. Carbon-14 DDT uptake rates by spines of Strongylocen¬
trotus purpuratus in vivo (not regenerating) and in
vitro (regenerating).
5. Effect of DDT on Calcium-45 uptake by in vitro regen¬
erating spines of 3 specimens of Strongylocentrotus
purpuratus (A,B,C) collected at Pigeon Point; the means
and standard deviations are shown.
6. Effect of DDT on Calcium-45 uptake of in vitro regen¬
erating spines of Strongylocentrotus purpuratus (A)
and Strongylocentrotus franciscanus (B) collected at
White's Point; the means and standard deviations are
shown.
Comparison of Calcium-45 uptake of in vitro regener¬
ating spines of Strongylocentrotus purpuratus from
White's Point and Pigeon Point exposed to the same
concentrations of DDT; the means and standard devia¬
tions are shown.
40.
25

130—



kt


CAPE
MENDOCINO

FRANCISCO
PIGEON.
POINT
POINT
/CONCEPTION



DIEGO

WHITE'S
—.——
POINT
FIGURE


1205



405
5
50
40
30
20
10
0.09
FIG. 2
91
% ETHANOL
2.25

4.5

9.1
40
5
450
300
250
200
150
100
50
FIG. 3

TDEEDT
I
TDEEDI
SPECIMEN
1DE
SPINES
GONADS
41
i
-
-
-
o-
S
2





ng DOIg
-N
50
40
20
10
(A)
0 0.1 0,5 1.0
FIG. 5
COLLECTED AT PIGEON POINT
50
(B)
401
20
104

0 0.1 0,5 1.0
ug DDTm
50
40
30
20
10
(C)
L
0 1 5 10
41
40
30
20
10
COLLECTED AT WHITES POINT
S. PURPURATUS
40
(A)
304
20
ug DOTm
FIG. 6
S. FRANCISCANUS
(B)
0.1
0.5
1.0
474
50
40
301
20
10
FIG. 7
WHITE'S POINT (4A)
PIGEON POINT (50)

ug DOT/m
415