ABSTRACT
The sensitivity of sea urchin egg fertilization was
tested in waters of various degrees of chlorinated and
unchlorinated domsetic sewage. Chlorine in sewage, especially
diluted sewage, was found to strongly inhibit sea urchin
fertilization. Removal of chlorine suggested that there
were other pollutants present in sewage effluent. The
sea urchin fertilization bioassay may be useful in indicating
pollution though it cannot act as the sole indicator of water
quality.
INTRODUCTION
In recent years, with the rising awareness of pollution
in the environment, it has become necessary to find ways of
measuring environmental quality. Much work has been done
to find effective bioassays which can indicate environmental
deterioration. For an arganism to be a good bioassay it
must show effects clearly, simply, and quantitatively.
Moreover, the organism must be sufficiently abundant year
round to be used any time. Many organisms have been tested
as bioassays for indicating pollution including diatoms,
crustaceans, bivalves, and many fish, especially stickel¬
backs, with varying success (Wilber, 1969; Warren, 1971;
Tarzwell, 1971). Some of the major difficulties have been
the hardiness of the organisms and the length of time required
to get results. The organisms that are hardy enough to
culture in the laboratory are usually too hardy to show
subtle changes in environmental quality.
Sea urchin gametes have the potential for being excellent
test material. In many regions of the world, several species
occure withe overlapping spawning seasons so gametes can
be found at any time of the year. Sea urchins can be kept
for long periods of time in the laboratory, and can be chem¬
ically induced to shed eggs and sperm. While the mature
organism is relatively hardy, the eggs and especially the
sperm seem sensitive to environmental changes. Furthermore,
the development rates of sea urchins are relatively rapid
so changes can be detected over a short period of time.
Kobayashi (1971) proposed that water quality could be
bioassayed by the ability of sea urchin eggs to be fertilized
in test waters. He tested the quality of the water in the
vicinity of Seto Marine Biological Laboratory, Japan, using
fertilization, cell division, gastrulation, polyspermy, and
exogastrulation as indicators. Using eggs and sperm of
Hemicentrotus pulcherrimus amd Anthocidaris crassispina
he tested water from the open sea side of Hatakejima Island,
the land side of Hatakejima Island, Tsunashirazu Cove, and
the running sea water system of the laboratory. He concluded,
"...any signs of biologically significant pollution have
seemingly not yet been observed in the laboratory water
and the water from the open sea side of Hatakejima Island,
but a considerable pollution is already difinable in the water
from the land side of the island and the water from Tsuna¬
shirazu Cove, especially in the latter." Using this work as
a basis for further studies, I conducted experiments with
Strongylocentrotus purpuratus on water taken from the Mon-
terey sewage outfall and the Pacific Grove sewage outfall.
MATERIALS AND METHODS
Animals
The animals were collected from Pigeon Point, San Mateo,
California, on April 21, 1971, and stored in large tanks
27
with flowing sea water. They were fed unlimited amounts
of the brown algae, Macrocystus.
Water samples
Pacific Grove, California, discharges approximately
1.26 million gallons of primary treated sewage daily with
a peak hour flow of 6.0 million gallons per day from an outfall
on Point Pinos. The end of the outfall is located off a
rocky intertidal area with a southern exposure and lies
one foot below mean lower low water in a region of heavy
surf. Samples were taken at low tide from three sites:
site 1, the outfall; site 2, five meters to the west; and site
3, five meters to the east (fig.1). The rocks at site I
were deviod of any but microscopic life. The rocks at the
other two collection points were populated almost solely
by stunted specimens of the red algae Prionitus lanceolata.
The lack of species diversity reflects the influence of
pollutants in the water. I observed a turbidity in the water
samples due to particulate matter coming from the outfall.
The water samples taken from the three sites had a consistent
grading of turbidity of 1 greater than 2 greater than 3.
Monterey, California, discharges an average of 2.67
million gallons of secondary treated sewage per day with a
peak hour flow of 8.6 million gallons per day into the
Monterey Bay through a submerged pipe approximately 10 meters
deep. Samples were taken from the surface between 9:00 and
10:00 a.m. at markers on the boil and 100 and 200 meters in
each cardinal direction, and stored at 4°C. Though there
was a slight turbidity to the samples, there was no noticable
gradation.
Water, considered to be unpolluted, was taken off Hopkins
Marine Station, filtered through a 0.45 u millipore filter
and stored at 4°C. This water was used as the unpolluted
"control"against the water from Pacific Grove and Monterey
throughout the experiment.
EXPERIMENTAL DESIGN
All experiments were conducted at 15°Ciunder artificial
light. Twenty-five ml. Erlenmeyer flasks were used for the
experiments because of their relatively large bottom surface
area. Before transfering the water to the flasks all samples
to be tested were well mixed. The water samples were tested
undiluted and the following dilutions: 1:2, 1:4, 1:8, and
1:16. Eggs and sperm were obtaineddby injecting O.5M KCI
into the sea urchins and collectinggthe shed gametes in filtered
sea water. The eggs were washed twice with filtered sea
water. Approximately 0.5 ml. of egg suspension or enough eggs
to form a single layer on the bottom of the flask, and 0.3 ml.
of sperm suspension or approximately one hundred sperm per
egg were used for each test. The sperm was allowed to sit
in the samples for ten minutes before the eggs were added.
This means these tests were actually on the effects of
different water on sperm viability. Ten minutes after the
eggs were mixed with the sperm, aliquots were removed and
observed under the microscope for elevation of the fertili¬
zation membrane. Two hundred eggs of each aliquot were counted.
294
The water from each sample was tested twice: first on the
day of collection of the water, and then on the following
day. Development was observed six hours after the eggs were
mixed with the sperm by counting the percentage of fertilized
eggs which had reached the eight cell stage.
Muchmore (1970) researching the effectsoof chlorine
on sea urchin fertilization, used sodium thiosulphate to
reduce free chlorine and neutralize its effect. Following
this I also neutralized the chlorine in some of my samples
with excess sodium thiosulphate.
RESULTS
Pacific Grove
The results from the Pacific Grove water samples show
that chlorine strongly inhibits fertilization. Inhibition
of fertilization by chlorine was especially evident after
dilution of the sewage. (figr 2,3,4185)at Foreekampletat the
outfall, inhibition of fertilization could be removed by
removal of the chlorine from dilutions of more than 1:4.
Further from the outfall, at sites 2aand 3, removal of chlorine
at dilutions above 1:4 increased the percent of fertilization.
Therefore, at high sewage concentrations, materials other
than chlorine suppressed fertilization. Allowing the samples
to sit in stoppered bottles overnight usually caused a
decrease in the effect of the inhibitor, but this was not
due only to decrease of chlorine in the samples (see, for
example 20 May, fig 3).
Monterey
The results from the Monterey experiments also show
inhibition by both chlorine and some other pollutant (fig.586).
The Monterey Health Department rated May 24, 1971 as an average
day with the chlorinator adding 2 ppm. chlorine to the sewage
effluent. However on May 3 and May 10 the chlorinator had
been malfunctioning. On May 3 the addition of chlorine had
been very low and then spurted relatively high. The coloform
counts taken at the same time as my samples were low, only
230 mpn. at the boil, also indicating that the chlorine was
high at that time. On May 10 there was less than 1.0 ppm.
chlorine being added to the effluent at the time of the sampling,
and the coloform counts were high, 4600 mpn. at the boil,
indicating a very low chlorine count. Fertilization of the
sea urchin gametes was low on May 3 and high on May 10,
suggesting that the different amount of chlorine in the
effluent directly influenced the water quality. Variations
in the effluent over a period of time are indicated in the
results from May 24 with the outer stations showing lower
values than the boil. The lower values on May 3 at the boil
and 100 meters to the north and west indicate the effluent
was dispersing from the boil in those directions.
I ran developmental tests on samples from both Monterey
and Pacific Grove (fig.7). The Monterey samples showed some
cases of slight variations in development from the filtered
sea water while the Pacific Grove samples indicated that
those eggs that were fertilized developed normally.
396
INTERPRETATION
These results show that high concentrations of sewage
can have definite effects on sea urchin fertilization, but
low concentrations have little effects. The system, there¬
fore, does not seem very sensitive for sewage pollution.
Chlorine in the sewage appears to strongly effect fertilization;
however other substances in the effluent also suppress fert¬
ilization, especially when at high concentrations.
Kobayashi (1971) found much lower inhibition of ferti-
lization in his samples than I found in mine (fig.8). Four
out of nine cases fromthealand side of Hatakejima and four
out of six cases from Tsunashirazu Cove varied more than one
standard deviation fron the mean value of the unpolluted
samples from the laboratory. Only four values varied more
than two standard deviation units. The occurance of values
below one standard deviation for development supports his
conclusions that the water on the land side of Hatakejima
and Tsunashirazu Cove werecpolluted, but in all his
experiments the development values were relatively high,
over 90%.
Muchmore (1970) with dilutions of sewage effluent from
the Pacific Grove outfall of 33%, 10%, and 5% neutralized
for chlorine, concluded, "Tests show that the effects of
unchlorinated domestic sewage on fertilization is relatively
slight, especially when compared to the effect of chlorinated
sewage." I also found at low concentrations, unchlorinated
sewage has little effect on fertilization. Other pollutants,
however, can effect gametes. Allen (1971), testing the
29
effects of oil on sea urchin fertilization, found that while
most crude and refined oils saturated in sea water did not
appreciably effect the sperm before fertilization, development
was inhibited almost completely for most oils at dilutions
down to 6.25%.
Originally the experimental design called for the use of
artificial sea water as the unpolluted control and for making
the sewage effluent-sea water dilutions. This seemed desirable
because the components of the water would be known and constant.
However the Ward's artificial sea water that I used inhibited
the fertilization from 10 to 90%.
The varying effect of different toxicants on sea urchin
fertilization and development show the system to be a poor
overall pollution indicator. While a high coloform count
at Monterey on May 10 showed high levels of sewage in the
water, the sea urchin test was not sensitive to it. Sample
waters had very little effectoon developmentin Kobayashi's
(1971) and my data, but an overwhelming effect was found
by Allen (1971) with oils. It seems unlikely that any bio¬
assay can be found that will be a good indicator for all
pollution. Rather a system could be devised that uses several
bioassays for different toxicants. Coloform counts are
now in use to determine human waste concentrations. The
sea urchin bioassay might be another useful and relatively
straightforeward indicator of water quality, especially
with respect to chlorine, heavy sewage, and petroleum pro¬
ducts. While this bioassay cannot prove the water is not
29
polluted, if there is inhibition to fertilization it is a
good indicator that the water is polluted and corrective
steps should be taken.
CONCLUSION
1. The sensitivity of fertilization of sea urchin eggs
was tested in waters of various degrees of sewage pollution.
2. Fertilization seems very sensitive to chlorine
and removal of chlorine from sewage polluted water often
restores water quality with respect to ability of fertili-
zation of sea urchin eggs.
3. However, in some cases, removal of chlorine did not
restore water quality, and fertilization was still very low.
This suggests that other pollutants which effect sea urchin
fertilization are present in sewage effluent.
4. It is suggested that the use of fertilization of
sea urchin eggs might be an easy and valuable technique
for testing water quality, although it cannot be the sole
standard for water quality.
ACKNOWLEDGEMENTS
I would like to thank Dr. John Pearse and Dr. Vicki
Pearse for their patience and help, and Mr. Tony Weaver
for help in collecting water samples.
BIBLIOGRAPHY
Allen, Heather. 1971. The effects of petroleum fractions
on the fertilization and cleavage of the sea urchin
Strongylocentrotus purpuratus. Hopkins Marine Station Final
Papers 175H.(ase Marie Pellihe Bulletin, jn press, Sept.issne)
Kobayashi, Naomasa. 1971. Fertilized sea urchin eggs as
an indicatory material for marine pollution bioassay,
Publications of the Seto Marine
preliminary experiments.
Biological Laboratory. (6)319-424
Muchmore. Douglas B. 1970. The effects of unchlorinated
and chlorinated sewage on sea urchin fertilization. Hopkins
Marine Station Final Papers 175H, pp. 430 451.
Tarzwell, C.M. 1970. Measurements of pollution effects on
living organisms. Proceedings of the Royal Society of London,
vol. 177, number 1048, pp. 279-285.
Warren, Charles E. 1971. Biology and Water Pollution,
W.B. Saunders Company, Philadelphia, pp. xvi + 434.
Wilber, Charles G, 19691 The Biological Aspects of Water
Pollution, Charles C. Thomas, Springfield, Illinois,
pp. vii + 296.
300
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PIPE
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figure 1
36
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GROVE
PACIFIC
%0 FERTILIZATION
dilutions
sea
1:4
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figure 2
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100
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MON TEREY
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S
MONTEREY
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100 m
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figure
30,
figure 8
KÖBAYASHI- aP FERTILIZATION
mean-92.24
standard deviation-
7.13
laboratory
open sea side
land side
cove
98.
94.9 90.1
5-21-70
93.1
98.4
98.5
92.0 97.8
93.5
—
—
99.6
86.5 98.2
97.6 84.2
99.8
99.7
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7-21-70
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8-18-70 80.4 82.1
91.2 82.7 83.4 90.1
77.1181.0 84.1 68.2 69.1 75.3
KOBAYASHI- SP GASTRULA STAGE
mean-98.01
s.d.-1.55
laboratory
open sea side
land side
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93.9 97.6
—
5-21-70 98.6
96.5
94.5 90.1
95.3 —
93.1
97.7
7-21-70
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97.6 98.7
97.6
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8-18-70 98.8 98.8
99.3 98.8 98.6 98.9 97.5
96.9 97.6 96.7 95.8 96.7
LISSANT-DEVELOPMENT-MONTEREY
100 m
200 m
boil
97.0 100
76.0
100
100
92.(
94.5
98.5
100
98.5
99.5
98.0
94.0
100
99.5 100
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98.5 100
99.5 99.5 100
100
98.5 100
100
NT-PACIEIC GROVE
LISSANT-DEVELOPME
1:16 1:8 1:4 1:2
fsw
1:1
-
——
——
100
100
99.5
site 1
---
100
99.0
———
—
99.(
—
100
100
100
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—
site 2
99.5 100
97.0 —
—
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site 3
100
100
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99.5 97.0
fsw- filtered sea water