INTRONUCTTON
Pt. Pinos is located in Pacific Grove at the
southern end of Monterey Bay. A large portion of the point
is a rocky intertidal region. The rock-filled tide pools
and abundant algal growth provides an ideal area for the
starfish Patiria miniata, (Brandt, 1835) to grow. However
inthe immediate vicinity of the Pacific Grove sewage
outfall the intertidal region has been seriously disturbed,
and there are no Patiria living there. My project was
designed to determine whether the paucity of Patiria
could be attributed in part to the effects the sewage
might have on either the adults, the gametes, or the
early developmental processes.
I found that, in the highly polluted areas near the
outfall, the sewage killed the adults and in lower con-
centrations inhibited their feeding processes. In addition
chlorinated Pacific Grove sewage reduced the success in
fertilization with full fertilization only being achieved
below a 0.1% dilution. Even very low concentrations of
sewage were strong enough to cause a decrease in the rate
of development in the embryos.
Hypochlorite, the only individual component of the
sewage which was tested for toxicity, appeared to be the
major toxic constituent. It caused a reduction in the
fertility of the eggs and a decrease in the ability of
the sperm to effect fertilization.
METHODS AND MATERIALS
For each experiment gametes were taken from a single
male and a single female. The web between two rays was
cut to the central disc, and the gonads removed with
tweezers. To prepare the ovaries, they were washed twice
with filtered sea water and placed in 25 ml of sea water
in a beaker. Two ml of 10 M 1-methyladanine was then
added to the eggs to stimulate shedding and meiosis
(Stevens, 1970). One to two hours were necessary to
complete the shedding process.
The eggs were then divided into separate aliquots
for the various experiments. A testes, removed from a
male 5 to 10 minutes before fertilization, was washed
once with filtered sea water and then placed in 50 ml of
filtered sea water. Four-tenths ml of this solution was
used for fertilization of each aliquot of eggs. Since
fertilization success is related to the sperm concen-
tration, this standard volume provided a large excess of
sperm, while it kept the amount of sperm added to each
sample constant within any one experiment. Using a
standard also minimized the variation between expeniments.
One hundred and fifty to two hundred eggs were
counted to yield the percentages for the recorded data.
The series of sewage dilutions that were used in the
experiments was 100, 50, 10, 5, 1, 0.5, and 0.1% by volume.
Filtered sea water was used to prepare these dilutions, and
the solution was made isotonic by the addition of sea salts.
In the experiments using hypochlorite, a stock solu-
tion of 40 ppm was prepared using filtered sea water and
reagent grade Naocl. To terminate the exposure of eggs to
hypochlorite, the solution was drawn off and the eggs
washed twice with filtered sea water. Rather than wash
sperm to conclude their exposure to hypochlorite, an excess
of Na,S,O, was added to neutralize the hypochlorite.
The temperature was maintained between 12° and
15 9c by placing the test vessels in a plastic tub on the
sea table.
RESULTS
Population Survey
A rough study was made of the Patiria population
on Pt. Pinos. The intertidal region was examined at low
tide on three successive days and the general location
and number of any Patiria found was noted on a map. The
study may not reflect the natural distribution because of
the difficulty in locating all of the animals, and also
because the point is accessible to the public and animals
may have been removed or transfered.
There were no Patiria found on the south side of the
point from the channel west of the outfall all of the way
through Cove C. Three adults placed in a cage in area A
about 15 meters from the outfall were killed within 24
hours.
The starfish living closest to the outfall was
found on the edge of a highly damaged area at the mouth
of Tide Pool wl. There were a few individuals found in
the rocks mainly on the seaward side of that tide pool
where they were getting fresh sea water during most of the
day. The population density continued to increase toward
the western end of the point. On the northern side there
was an even distribution all of the way down the point.
Feeding Behavior
To test the effect of sewage on feeding behavior
Patiria were starved for two weeks by keeping them in a
tank without food. They were then placed into dilutions
of both chlorinated and unchlorinated sewage and 16 hours
later a fresh Mytilus californianus was cracked open and
placed under each animal. In a 5% concentration of sewage
Patiria were less apt to eat than when they were in fresh
sea water. When the concentration was increased to 10%
they did not eat at all. The starfish kept in sewage were
then placed back in fresh sea water. After 5 hours they
were again given a mussel, and all of them began to eat
within 5 minutes.
Embryonic Development in Pt. Pinos Water
Current studies of Pt. Pinos, done by students of
Hopkins Marine Station during April, 1970, indicate that
the sewage leaving the outfall was carried toward stations
1, 2, and 3. Fertilization was drastically affected in
water samples taken from this area (fig. 1). However once
the egg was fertilized, except in water from station 3
more than 94% of the eggs were able to reach the blastula
16.
stage. No bipinnaria larvae were later found in the samples
raised in water taken from stations 1, 2, or 3 and the
larvae raised in waten taken from stations 4, 5, and 6 were
indistinguishable from the control.
Fertilization
The process of fertilization is affected by
sewage (fig. 2). Full fertilization is not attained until
there is a substantial dilution of the sewage. If it is
chlorinated, fertilization is affected over an even
greater range of dilutions (fig. 2).
When fertilized and allowed to develop for an hour
before being placed into sewage, the eggs were still sen-
sitive to sewage more concentrated than 5% (fig. 3). In
100% Monterey sewage, most of the eggs reached the 8- to
16-cell stage before stopping. Eggs placed in the
unchlorinated Pacific Grove sewage only attained the 4-
to 8-cell stage gefore being arrested. None of the eggs
in the chlorinated Pacific Grove sewage got beyond
1 cell, and most of them were cytolyzed and had lost all
internal unity (plates 1,2).
Because of the differences shown between the chlor-
inated and unchlorinated Pacific Grove sewage, further work
was done on the effects of hypochlorite (the major
product of chlorination, see Imhoff and Maskew, 1956).
In sea water containing 40 ppm hypochlorite, the
tails of the sperm became twisted and often broke. As the
solutions became more dilute, physical effects were not as
16
apparent; however no fertilization was achieved with sperm
that had been exposed to concentrations of hypochlorite
greater than 0.4 ppm. In contrast to the effects of
hypochlorite, no morphological effects of unchlorinated
sewage were observed with sperm or eggs.
The eggs are not as sensitive to the hypochlorite
as the sperm. After being exposed for 45 minutes to a
40 ppm solution there is no fertility, but full fertili-
zation is achieved by eggs when they are exposed to a 4.0
ppm concentration of the hypochlorite, washed, and then
fertilized.
The above results suggest that hypochlorite is the
major toxic ingredient in chlorinated sewage. To test
this, a hypochlorite solution in sea water was prepared
which was equivalent to the 'available chlorine' content
of a sample of chlorinated sewage. Eggs were then fer-
tilized in various dilutions of these two solutions. The
results, seen in figures 4 and 5, show that the effects on
both fertilization and development were very similar,
with the hypochlorite solution being only slightly less
toxic.
DISCUSSION
The gametes, early developmental stages, and adult
Patiria are all affected by sewage and its associated
levels of chlorination. Through its gametes, the starfish
is sensitive to concentrations of hypochlorite higher than
66
0.2 ppm. Effects on the adult ranged from an abstinence
from feeding behavior in concentrations greater than 5%
sewage to death within 24 hours when placed near the
outfall.
The most probable reason the animals did not eat in
highly polluted water is that Patiria locates food by
chemoreception (Hyman, 1955). In high concentrations of
sewage the sensors might not be able to recognize or detect
the presence of food. Monterey sewage, which was unchlor-
inated, did not inhibit the feeding behavior below a
concentration of 10% sewage. In contrast Pacific Grove
sewage affected the feeding behavior at dilutions of 5%;
perhaps the hypochlorite has an effect on chemoreception,
also.
The results showed that the fertilization process
was highly susceptible to sewage and hypochlorite. Thus,
more than twice as many embryos were able to reach the
blastula stage in 10% and 5% sewage dilutions if the eggs
were fertilized before they were placed in the solutions.
That not all embryos in 5% to 10% sewage dilutions
successfully reached the blastula stage suggests that
there are other toxins in the sewage, which interfere
with processes of division or blastulation. Alternatively
the same toxins may be present, but the embryo may be
partially isolated from the environment by the presence
of the fertilization membrane.
The comparison of chlorinated versus unchlorinated
sewage showed that chlorinated sewage was far more toxic
16.
to development. As shown, the active agent in chlorinated
sewage was hypochlorite, and the sperm were more sensitive
than the eggs. The sperm were sensitive to hypochlorite
concentrations as low as 0.2 ppm, whereas eggs could be
fertilized after an exposure to 4 ppm. Thus the sperm
was the critical gamete in regards to chlorinated sewage.
The experiment comparing chlorinated sewage to a
sea water solution containing the same amount of 'avail-
able chlorine' in the form of hypochlorite, yielded the
surprising result that the pure hypochlorite solution
was almost as toxic as the chlorinated sewage. The effects
of the hypochlorite simply over-rode most of the effects
of the sewage rather than being additive. This is some-
thing that should be more thoroughly investigated since
light, agitation, and organic constituents can cause a
breakdown in hypochlorite, and an increased rate of
hypochlorite decomposition in the sewage solution might
have made the two solutions appear more similar than they
really were. It is surprising that the oxidized and
chlorinated organics resulting from the chlorination did
not present a greater barrier to fertilization and
development.
These results are not conclusive enough to esplain
the observed distribution of adult Patiria on Pt. Pinos.
However they do indicate that the region on the south
side of the point from the channel west of the outfall
eastward through Cove C is unsuitable as a breeding site
for Patiria. Most importantly, the present results show
that fertilization is sensitive to very low levels of
hypochlorite. Therefore, down current from the outfall
in areas where the sewage has been so diluted as to
produce no obvious damage and to be hard to detect, it
may still present a significant ecological threat.
SUMMARY
The gametes, early stages of development, and
adult Patiria are all affected by sewage and its asso-
ciated levels of chlorination. Effects on the adult
ranged from an abstinence from feeding behavior in
concentrations of sewage greater than 5%, to death within
24 hours when placed near the outfall.
The fertilization process was somewhat sensitive
to unchlorinated sewage and highly sensitive to chlorinated
sewage. Thus, more than twice as many embryos were able
to reach the blastula stage in 10% and 5% sewage solutions
if the eggs were fertilized before being placed in the
sewage dilutions.
Hypochlorite was found to be the active ingredient
in chlorinated sewage. The sperm were more sensitive to
the hypochlorite than the eggs, showing a reduction in
the ability to fertilize eggs in concentrations as low as
0.2 ppm.
Because of the high sensitivity of fertilization
to both chlorinated and unchlorinated sewage, the ecological
effects of the sewage may extend far down current from
the damaged area near the outfall.
16
C
C
S
Toesdo
4
TIDEPOOL
2.

3
OUTFAL
28
V

/6.
100
80
60
40
20
a
alb alb alb
4
a
alb
16
5
—-
O
71
bab
17
81

——



S
17.
54

S
oh
17
200x 14 aee at 15 ae.ded.
oseded.
7
EIGURE LEGEND
Abscissa: station number; ordinates: percent response.
Fig. 1. Eggs fertilized and raised in water taken from
stations at Pt. Pinos. a: percent fertilized; b,
percent of the fertilized eggs that reach the blastula
stage in 14 hours.
Figs 2-5. Uchlorinated Pacific Grove sewage, OC
unchlorinated Pacific Grove sewage, AA Monterey sewage,
kAsea water with hypochlorite added.
Abscissa: percent sewage concentration; ordinates;
percent fertilized.
Fig. 2. comparative graphs for fertilization in sewage
dilutions. Chlorinated Pacific Grove sewage had 37.4 ppm
hypochlorite.
Abscissa:
percent sewage concentration: ordinates;
percent of the eggs fertilized that reached blastula
stage in 22 hours.
Fig. 3. comparative curves for development in sewage
dilutions. Chlorinated Pacific Grove sewage had 37.4 ppm
hypochlorite.
Abscissa: percent sewage or hypochlorite by volume;
ordinates: percent fertilized.
Fig. 4. comparative curves for fertilization in chlorinated
Pacific Grove sewage and sea water with hypochlorite.
The undiluted solutions both had 14.9 ppm hypochlorite.
Abscissa: percent sewage or hypochlorite by volume;
ordinates: percent of the fertilized eggs that reach the
blastula stage in 12 hours.
Eig. 5. comparative curves for development in chlorinated
Pacific Grove sewage versus sea water with hypochlorite.
The undiluted solutions both had 14.9 ppm hypochlorite.
Fig. 6. a normal fertilized Patiria egg. The fertili-
zation membrane and polar bodies may be seen.
Eig. 7. a cytolyzed Patiria egg after exposure to a
20 ppm hypochlorite solution.
ACKNONLEDGENENTS
am especially indebted to Dr. David Epel for
the encouragement and assistance he gave me on my
project. I should also like to thank the faculty and
staff of Hopkins Marine Ststion for making this quarter
the valuable experience that it was.
RERERENCES
1. Hyman, L H, The Invertebrates, McGraw-Hill Book
Company, New York (1955) Vol IV, 273.
2.
Imhoff, K and Gordon M Fair, Sewage Treatment.
John Wiley and Sons, Inc., New York (1940)
162-171.
3. Stevens, P, Explt cell res 59 (1970) 482-485.