A PRELIMINARY SURVEY OF THE EFFECTS
OF SEWAGE ON THE DEVELOPMENT OF
ECHINODERMS
Susan Marie Ott
Hopkins Marine Station
Pacific Grove, Californi.
June 4, 197
INTRODUCTIO
For many years cities around the Monterey Bay
have been dumping their sewage into the ocean, but
no one has studied the biological significance of this
In an attempt to study some effects of sewage. I used
narine embryos, since developmental processes are
sensitive to environmental factors. Dendraster
ecentricus and Strongylocentrotus purpuratus were chosen
because it is easy to obtain their gametes and they
I observed the
can be fertilized and grown in vitro.
rate of development by timing morphological changes
and measuring lengths of skeletons. The sewage didn't
seem to harm the embryos in dilutions less than 5%.
In fact, they often grew slightly better in 1% sewage
than in sea water. However, development was either
slower or abnormal in 5g and greater sewage concen-
trations. Several other factors, including the temp-
erature, type of container, concentration of embryos,
and natural variation of eggs, were also found to effect
the rate of development.
ETHOD
he adult sand dollars and sea urchins were
ollected and kept in an aquarium with sea water and
aeration. Shedding of gametes was induced by intra-
coelemic injection of O.5M KCl. Eggs were collected
y setting the animal over a beaker filled with sea
water; sperm were collected "dry" with a pipett.
he eggs were washed three times with sea water by
jecantation and resuspension. They were fertilized
addition of one or two drops of sperm suspension
one drop of sperm fluid in 10ml. sea water.) (1,3)
then transferred the embryos to small perti
dishes or flasks which contained sewage dilutions. SAt
different intervals they were examined microscopicall,
or general morphological attributes. To measure the
ate of skeleton growth, I removed a few drops from
each culture, placed them on a depression slide, and
added several drops of Clorox. After about a minute
the skeletons could be measured. I always measured th
distance from the top to the end of the postoral arm.
(Figure 1
To keep the temperature constant, I placed the
etri dishes on a tray which floated on the sea table
water. The temperature remained between 15 and 18°c.
Several times I changed the water in the petri dishes
of the older plutei. This was done by removing about
half the water with a syringe, avoiding the embryos,
and adding fresh water or dilutions of sewage. Daily
the plutei were fed one or two drops per petri dish
of algae from a culture obtained from Ralph Hine-
gardner. (These same algae are known to sustain sea
urchin embryos through netamorphosis. (4) Where
antiblotic solutions were used, streptomycin and pen-
cillen were added to a final concentration of 0.1 mg/ml
The sewage was obtained from the Honterey treat
each.
ment plant. The algae were grown in a medium which
was half the concentration described by Guillard and
Ryther. (2
ESUITS
The stages of development were defined in terms
of gross morphological changed. These are shown in
gures 2, 3, and 4. The first stages (71-14) are sim
lar to those described by Karnofsky and Simmel. (5)
The times of the first cleavages were not
effected by the sewage. No effects from sewage were
observed for about ten hours, after which retarded
development or abnormalities were seen in 5% and 10%
sewage. (Figures 5,6) The abnormal embryos were of
varying shapes and were darker than controls. They
didn't die, but remained approximately the same size
and continued to swim for several weeks
In one trial run using sand dollars I observed
that chlorinated sewage from Pacific Grove had a greater
effect than unchlorinated Nonterey sewage. Thus,
in a 10% dilution, the eggs never divided. In exper
iments with sea urchins and starfish gametes a relat-
vely low concentration of chlorine can completely block
fertilization. (7,9)
To determine the rate of development more
precisely, I measured the skeleton lengths. Embryos
grown in 14 sewage grew slightly faster than those in
sea water, but those in 5% and greater sewage dilutions
showed harmful effects. (Figure 7) The skeleton lengths
varied with each set of cultures, but the average length
was shorter in 5% sewage than in sea water. (Figures
9,10
did student "tr tests (10) on these measure.
ments to see if the groups were significantly different.
The skeletons from 14 sewage were statistically either
the same or longer than those from sea water. (In one
case they were shorter, however.) But after they had
been growing for 100 hours, the skeletons were longer
in the sea water controls. The embryos from 2% sewage
were never consistantly different from those in sea
water. (Figure 11)
My observations point to some experimental
conditions that must be kept constant in order to
observe sewage effects. First, the embryos should not
be too crowded. The type of container is significant.
The embryos died after a week in 125ml. flasks, but
hey survived in the petri dishes. Temperature and ph
must be constant. Addition of antibiotics did not
correlate with health or rate of development.
DISCUSSION
From my observations, I conclude tha concen-
trated sewage (5%) adversely effects the development
of embryos, but that lower concentrations may slightly
nhance growth, at least during the first 100 hours
The effects of temperature may also have
significance in field conditions. Thus, the temp.
erature around the outfall was found to be several degrees
higher. Also, the sewage contains organic and inorganic
nutrients which might be beneficial at low concentrations
explaining the initial slight enhancement in dilute
sewage. (8) Many factors, including various metals,
proteolytic enzymes, and dyes, are known to produce
exogastrulation, one of the abnormalities found in 5/
sewage. (5,6) The sewage may certainly contain some
of these factors. The osmotic differences in the higher
sewage concentrations may also have caused retardation.
Finally, the chlorine probably has strong effects. In
comparing development and fertilization, the latter is
a more sensitive process, particularly to chlorine.
Working with Pateria, Rotkis found that once eggs were
fertilized, they reached the blastula stage, a result
which agrees with my data. (9) However, though the
embryos look normal, are all the systems working properly'
Will embryos raised in sewage be able to undergo meta-
morphosis? These questions suggest further research.
SUMIART
The effects of sewage on sand dollar and sea
urchin development were studied. Time required to reach
different morphological stages and rate of skeleton
growth were used as parameters. Studies were also
arried out on culture conditions necessary to raise
embryos beyond the pluteus stage. The development was
adversely effected by sewage if the concentration was
greater than 5% (unchlorinated.) A slight enhancement
was often observed in 14 sewage, åt leasthduring the
first 100chours.
ACKNOWLEDGEMENT
Part of this project was fiananced by the National
Science Foundation Undergraduate Research Program Grant
Vo. GY-7288. I would like to thank Dr. Hinegardner for
giving me the algae he had cultured, and my roommate
Welanie MoCabe for bringing the culture from Santa Cruz.
am grateful to Dr. David Epel, my advisor, and Dr. Vic
Vacquier for giving me many helpful suggestions and
encouragement. The staff, professors, and other students
f Hopkins Marine Station helped me a lot, particularly
im Sutton, my T.A. Finally, I want to thank my friend
Victor Anderlini, who took all the photographs for me
FERENCES
1. Costello, D.P., M.E. Davidson, A. Eggers, M.H. Fox,
and C. Henley, 1957. Methods for Obtaining and
Handling Marine Eggs and Embryos. Marine Biolog
ical Laboratory, Woods Hole, Mass.
Guillard, R.R.L., and J.H. Ryther, 1963. Studies
on marine planktonic diatomes. I Cyclotella nana
Hustedt and Detonula confervacea (Cleve, Gran.
Can. J. Microbiol.,0: 229-239
Hinegardner, R.T., 1967. Echinoderms, pp. 139-155.
in: F.H. Wilt and N.K. Wessells, Eds., Methods
in Developmental Biology. Thomas Crowell Co.,
New York
Biological Bulletin,Vol 137
Hinegardner, 1969
No. 3, pp. 465-475
Karnofsky and Simmel, 1963. Progr. in Experimental
Tumor Research, Vol. 3254-294
Advances in Morphogenesis Vol.
Lallier, R., 1964.
pp. 147-196
7. Muchmore, Doug, 1970 Personal communication.
. Remsen, James Van, 1970. Personal communication.
. Rotkis, Tom, 1970. Personal communication.
10. Snedecor, George, 1956. Statistical Methods, lowa
State University Press.
e.
Gand dollar skeletons
postoral arm
STAGE 14
dntero lateral arm
postord arm
STAGE 15
Apest oral arm
Hanterolateral arm
pre ord arm
Losterdora
arm
STAGE 18


v 300


1 2 4504
C
A

7..


**.
o


piqment granoles



. ..
9.:

L

UNFERTILIZED EGG

SPINDLE

POUR CELS

MORULA
jella lager
DIVIDING
).:

71
4

. . ......

qur cetus
blastocoel
BLASTULA
95
-fertilization membrane
JUST HFTER FERTILIZATION
:
?
O CELS
SIXTEEN CELLS
un fertilized egg
stage!
Stage3
2008
oox
200x
serClized egg
stage 2
Stage 41
900x
200x
200x
9
25


°
HATCHING
389

Fi
69
3I.
-
—

skeleton

Formq
PRISM
erimary


mesenchyme
.*......
cells
. ....
Vt
GRSTRULATION
L

+
1
-***.*

.....
Fir
DE
mooth
441.
J.


qnus
side view
secondary
mesenchym
cells


LENVAGINATION
S
T
Side viee
postoral
am
qnos
10
stage 5
stage 7
stage 4-10
200.
aoox
100:
stage 6
Stage:
stage
100x
200x
aoox
Hmool
6
anus
PLUTEUS
S
/....
-.....

10





side vieu
.



BOTTOM VIEW




S

-moot
sand dollar.
4
37
1
mooth
moth
stage 17
stad
stage 12
200
100x
1OOX
stage 15
stage 15
stage 18
1OOX
100x
Oox
100
stage 19
oox
stage 19
125 1
I


6
8
8
3
-9TAGES


889
Jo=





5

18
96
10.
N




e
J



S
18
29 Rpcil
E
5

8ray
— 12
13 May

4 Mey
o

.
LaG IN DEVELOPRE NT N1OJ SEUAGE
10
20
25
15
/0
10
15
10
25
15
20
25
10
10
20 25
15
sea orchin
15
20
25
ter Fer
lizat
Hoors
S.W.
/o
30
30
30
30
30
35
S.00.
1o
35
35
35
545.
10
105
30.
7o
Time o
Hors
fertilizahon
da
ions
cod
n-
3 May, 12 34
7
19
10
7
qo
HMay, 100
5Om
Clasks
44
125m Sak
96.5 12
P.G.sevoge
94
10
985
12
40
19 May, 50
44
23
Gask al
35
e
43
25
Flask 1 2
70
27
petri dishe
20
12
a1 Pay. 320
25
11
u
10
1a
23 Pay, Gl.
1a
48
17
a04
11
25 Pay, 10
51
24
24
62
80
24
1144 1 20
108
Skeleton lengtis,
sand dollac
Average length of skeletons groon in sewage concentrations
sea
2%
17
n.
10%
57
ne
ne
n-
water
se skelebs
8
q
131 216
14.3 12.4
10
10.118
14.0 416
9
110115
11.5440
36.1110
10
4004
10
110112
10
477132
430165
50.0423
la
10
g
47.143
43.0113
15
8
25.914.2
164t23
9
51.1124
31.113.0
4
2.1129
342129
8
33.5105
36.212.2
373250 15
10.
335 t14
10
38
26.212.
31.5 41.8
33. 19
34671.6
26
29
381122
145:25
44.1 43.2
38.7:3
35
30
30041.8
25
28
23.712.
33.0:22
316411
25
456119
43.213.7
27.4140
145115
24
3estac
20
26
21
41.1 447
59.243.2
7
43.045.5
12
53.3:7.8
516150
17
8
34.5-4.
23
24.814.4
354 ta1 a2
33.612.5
2a
508153
C4s:60
56.42.7
16
66 946.5
7
9
33.114.6
15
27.312.0
2937 17
16
10
13
32.2144
241165
22
36.1435
34.6 :da
55,448.4
39.7158
315:43
100:2.3 24
35.0124 24
33.75 23 24
24
43.2123
48.8130
34.114.7
480 15
24
24
24
24
469129
43.913.9
540:39
44.41134 24
14.5 43.
24
24
529133
24
44.4 140
8 509437 4
52.043.8 13
48.4: 30 5
57.4453
FIGOR
2
Time of
fectilization
26 Pay, 200
pr
overerouded
26 Ma, 510
Jertilized
1a Pay, 12
INay,105
SHELETON LENGTS SAND DOLLAR, CONTINUED
Hours
Average sheleton lengtus i sewage concentrations of
old
se
5%
107
water
170
afe
ne
n-
n-
n2
n2
45
354422 24
33.9 217
370:20 24
32.212.2
24
24
44.1132 24
445.842.6
76
18.8 226
24
18.9126 24
24
51.4 : 4.6
46.0 26. 20
45.2: 4.7
50.0:40 20
37
20
20
18.3 1 3.8
34.0449
448 46.7
120
10
10
10
24.0412
20
20
24.8 : 2.8
34
20
25.8 4 2.3 20
26 215
36.511.7
44.5
20
20
20
36.3193
340 413
20
38.912.1
SKÉLETON LENGTHS, SER URCHIN
Days
Average skeleton length in sevage concentrations of
ola
sea
a7
1.7.
5%
0.5 %
n-
n:
n-
n:
Dater
13
33.143
31.8123
12
397133
34.75.7
10
22.7109
28.71 2.5
3
300:25
5
29.2711
27.823.
8
10
8
4
35 +13
33.512.3
10
8
31.1 19.2
35.01
6
30.5228
7
7
7
30.81 5.4
10
28.4:2.
10
6.5
38.0:46
37.3420
21.214.2
20.412.5
36.54 30 10
36.312.7
26.84 3
29.5 13.5
17
20
10
33
38.412.7
38.5 : 30 20
2o.143.7
10
25
275:29
20
a8.211.8
13
5F.5: 3.8 12 2734 18 13
10 14 34,022.1 15
255:24 326 255
FIGURE 7B
10
N.
5.
X
00

sss
SKELE TON LENGTH  7 SEURGE
3 Hay
normbers on ei
age
50
40
—
20
44

—10
34
10
2 4 5
Stl. 5
60
ja Play

5 50
8
a

2 40
4/


1
30

—20
flask 41
—.—
flask 12
petei dish
10
50
10
40
30
B.W.
60
50
40
30
— a0
50
4 May
20
84
0
2
107
50 ml flosk
—--—- 125 ml fask
—— PG sewage
965
10
5
al May
42
952

50
40
30
20
10
— 60
5
2 40
10
80.


6.W0.
176
14
SW
1
SKELÉTON LENGTHIS SEURGE, coatinted
25 May
23 May
204
u0

20
10
2 5

510
2
26 May
50
10
Lur
30
33
20
5W
10
2
5
23 May
10
10
10
40
35
30
15
20
3 15
5
E
10
4
Skeleton length us Percent sewage
Strengglecentretos
15%0
1odag
Tude
3dags
2.5 dags
O%o
27
17e
27.
10%
(sea Ucter)
PER CENT SEWAGE
FIGURE 9
57
purpuratus
10
1 3
3
87
kaa
NUMEER OCOLAR UNITS

Z

I
V
222


3

25
9
5

2




NUMBER OCULAR ONITS
18
Student "t" values from sewage
Age in
ertilized
hours
concentrations of 1,2,5,10% vs. 0% (S.W.)
50
10%
89
0.45
1.3000
3 May
1.86
5.
3.16
2.13
14 May
56
96.
99
.16
1.650
(Pacific
Grove sewage
0.890
W1
15.8-
2.30
19 May
12.37
8.83
flasks Hi
7.66
6.53.
2.24+
5.91
flasks 42
2.79
13.9 + 18.4
2.28
2.9
3.58-
0.71
etri dish 121
3.51
5.59
1.380
2.75
21 May
111
b.304
8.39
0.67
30
6.40+
23 Maj
6.26
1.35
4.114
5.47
0.850
4.65
25 Ma.
5.
80
3.47
4.90
590
5.74
2.404
2.37
0.79
144
08
1.840
2.08
26 Ma,
2.84
0.76
0.090
5.04 4
2.174
0.7
5.09
1. (
28 May
no significantdifference
sea water skeletons longer
sea water skeletons shorter
RE