THE EFFECTS OF UNCHLORINATED AND CHLORINATED
SEWAGE ON SEA URCHIN FERTILIZATION
DOUGLAS B. MUCHMORE
HOPKINS MARINE STATION
JUNE, 1970
Home Address:
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INTRODUCTION
External fertilization is widely used by marine
organisms and may be especially susceptible to environmental
pollution. In the present study the effects of primary
treated sewage from Monterey, California and Pacific
Grove, California were observed on fertilization in the
purple sea urchin, Strongylocentrotus purpuratus. Tests
were run with unchlorinated sewage from both cities and
chlorinated sewage from Pacific Grove only. (Sewage from
these two cities is almost exclusively domestic waste;
few industries contribute to the effluent.) The inhibition
of fertilization by unchlorinated sewage is minimal when
compared to the striking inhibition caused by the heavily
chlorinated Pacific Grove sewage.
The action of chlorine on fertilization proved to
be of central interest, and tests using sodium hypochlorite
showed that this was the active inhibitory agent in
chlorinated sewage. Low concentrations of hypochlorite
rapidly (within one second) inactivate sea urchin sperm,
but do not affect the egg. Concentrations as low as O.04
parts per million (ppm) available chlorine, which
corresponds to a 1000 fold dilution of heavily chlorinated
sewage, can affect fertilization. As such, chlorinated
sewage could have drastic ecological consequences.
METHODS AND MATERIALS
Gametes were obtained by intracoelomic injection of
0.5 M KCl. Eggs were collected in sea water; seminal
fluid was collected dry. Sewage was obtained fresh at
9:00 AM each day. Eggs and/or sperm were treated in
various sewage or hypochlorite dilutions prior to insemination;
treatment time was 5 minutes unless otherwise stated.
Sperm concentrations of 0.33%, 0.10% and 0.033%, all
of which are sufficient for 100% fertilization in control
suspensions, were used for most experiments. Fertilization
success was determined after 20 minutes by scoring
presence or absence of the hyaline layer; samples of
approximately 200 eggs were counted.
In studies to determine the sensitivity of the
fertilization process to hypochlorite, aliquots of a
fertilized suspension were removed at intervals and
treated two seconds with sodium hypochlorite before the
addition of excess sodium thiosulfate. Temperature was
held at 15° C.
Available chlorine was assayed in sewage and
hypochlorite solutions using an iodometric method, adding
a known amount of sodium thiosulfate to the sample and
titrating the unreacted remainder with iodine solution
using a starch indicator. A disparity between known
hypochlorite concentrations and the available chlorine assay
values was found. This could be due to decomposition
of the hypochlorite or the result of reaction of the
hypochlorite with the sea water to which it was added.
Chlorine levels cited here are available chlorine values
as assayed.
ESUTRS
As shown in Table 1, unchlorinated Monterey sewage
inhibits fertilization strongly at a concentration of 10%;
inhibition is less complete at 5% concentration. No attempt
was made to restore osmotic balance of sewage dilutions.
Unchlorinated Pacific Grove sewage is generally weaker as
a fertilization inhibitor. The composition of the sewage
is sufficiently variable to make the numbers in Table 1
typical rather than absolute; the concentration of sewage
sufficient to yield a given inhibition level may vary by
a factor of 1 from day to day.
That the effect of unchlorinated sewage is on the
sperm or the egg-sperm interaction is shown by experiments
in which eggs and sperm were separately treated. Eggs
were treated ten minutes in 10% sewage, washed and then
fertilized with untreated sperm. Fertilization success
was 100%. Attempts to wash treated sperm by centrifugation
at 3K rpm and resuspension in fresh sea water were
sporadically successful in that control sperm did not
always yield 100% fertilization. As shown in Table 1
when control sperm survived the treatment, fertilization
success for the centrifuged sperm was comparable to that
of unwashed treated sperm, further substantiating that the
effect is essentially on the sperm. Incubating sperm
for longer times resulted in corresponding lower fertilization
success.
The inactivation effect resulting from unchlorinated
sewage treatment can be partially reversed after treatment
by diluting the suspension with fresh sea water. A O.33%
sperm suspension treated 5 minutes in 5% sewage gave 7%
fertilization success. The same sperm, diluted 1:3 after
treatment gave 32% success despite dilution.
The far more drastic effect of chlorinated sewage
from Pacific Grove is shown in Table 2. As seen, even
0.2% chlorinated sewage is more detrimental to fertilization
than 10% unchlorinated sewage. That this is due to the
chlorine is supported by the additional data in Table 2;
when chlorinated sewage is treated with sodium thiosulfate
which reduces OCl to C1", the strength of the sewage as
an inhibitor falls to that of unchlorinated sewage. Chlorine
levels in the sewage vary from 15 to 45 ppm; the inhibition
effects vary accordingly.
Further evidence of chlorine as the dominant inhibitor
in chlorinated sewage is seen in Table 3. In this
experiment hypochlorite was added to sea water to a level
comparable to that found in sewage. A sodium hypochlorite
solution of 12.8 ppm available chlorine was a slightly
better inhibitor than sewage with 11.0 ppm chlorine.
That the effect of chlorination is primarily on the
sperm and not the egg is seen in Table in which treated
ggs were inseminated with untreated sperm and vice versa.
Morphological study of treated sperm (5 minutes at 0.19 ppm.
indicates an increased tendency for the flagella to fall
off and the occasional presence of an unidentified fiber
of very small diameter attached to the sperm head. Whether
or not the acrosome is caused to evert is as yet unknown.
Adding sodium thiosulfate to sperm suspensions exposed
to 0.04, 0.08 and 0.21 ppm available chlorine for 5 minutes
and then fertilizing eggs with this sperm results in
improved fertilization success as shown in Table 5. This
could indicate some reversibility effect on the thiosulfate
teated sperm; I favor the alternate hypothesis that
hypochlorite might also have a deleterious effect of the
sperm-egg interaction.
An experiment was done to determine how long it takes
hypochlorite to inactivate sperm; very short exposure to
hypochlorite was made possible through neutralization
of the unreacted hypochlorite by addition of excess
sodium thiosulfate, an agent with no observable effect
on eggs, sperm or fertilization. Exposure of sperm for
1 second to 1.9 ppm available chlorine greatly reduces
fertilization as shown in Table 6: Table 7 shows a
preliminary study in which gametes were exposed for two
seconds to hypochlorite of concentration .76 ppm at
different times after insemination. The results show
that the sensitivity of the fertilization process to
hypochlorite becomes markedly reduced by 40 seconds after
insemination; more stringent work remains to be done.
Of ecological significance is the observation that
the potency of chlorinated sewage or a hypochlorite solution
as a fertilization inhibitor drops with time as shown in
Table 8. Bubbling air through fresh sewage dilutions for
three hours did not hasten the decompostion.
DISCUSSTON
Tests show that the effect of unchlorinated domestic
sewage on fertilization is relatively slight, especially
when compared to the effect of chlorinated sewage. Sperm
are considerably more susceptible than are eggs. The
inactivation is partially reversible with unchlorinated
sewage (providing lethal levels are avoided); reversibilit
is effected by dilution with sea water. The hypochlorit
effect on sperm appears to be slightly reversible if the
sperm are treated with sodium thiosulfate before fertilization.
However, this apparent reversibility could result from
removing a chlorine effect on the egg-sperm association
at fertilization. Irregular fertilization with centrifuged
control sperm make testing this effect directly more
difficult.
The effect of a given treatment on fertilization b
different dilutions of sperm is of great interest and also
of ecological importance. Thus, exposure of sperm to O.19 ppm
available chlorine (Table 4) shows the percent fertilization
success at 0.33%, 0.19% and 0.033% sperm concentrations
to be 94, 45 and 7 respectively. As seen in Table 1,
a similar relation between inhibition level and sperm
concentration was observed at appropriate unchlorinated
sewage concentrations. This effect is far greater than
the respiratory dilution effect described by Tyler and
Tyler (1966); it is typical of results obtained using other
fertilization inhibitors as discribed by Metz (1966).
I interpret this sharp reduction in success as the requirement
for a fiven level of "inactivation factor" per sperm
before fertilization is affected; thus, any slight increase
in inhibitor concentration or decrease in sperm concentration
would affect fertilization once this critical level is
reached. The reversibility effect noted above for
unchlorinated sewage is consistent with this interpretation.
Further tests of the usefulness of hypochlorite and
thiosulfate in rate studies, such as performed by Baker and
Presley (1969) to elucidate intermediate steps in
fertilization, need to be performed. Neutralization of
hypochlorite can be effected with sodium thiosulfate.
which in turn has no effect on eggs, sperm or fertilization.
These characteristics of the hypochlorite — thiosulfate
pair suit them particularly well to rate studies of
fertilization; preliminary work is promising in the regard.
The finding that a 1000 fold dilution of heavily
chlorinated domestic sewage can significantly reduce sea
urchin fertilization is of great ecological interest.
Under such conditions of chlorination the presumably safe
dilution of sewage nutrients and noxious components of
1 to 100 is unacceptable for successful fertilization ir
sea urchins.
ABSTRACT
Unchlorinated sewage is a relatively mild fertilization
inhibitor.
Chlorinated sewage is a very strong fertilization
inhibitor, and a concentration of available chlorine as
ow as 0.04 ppm can reduce fertilization.
The primary effect of both chlorinated and unchlorinated
sewage is on the sperm.
4) The effect of unchlorinated sewage is partially reversible.
The sodium hypochlorite — sodium thiosulfate pair is
potential tool in rate study determinations of fertilization.
UNCHLORINATED
SEWAGE
CONC
SPERM
CONC
Monterey
33
Pacific
Grove
Monterey
.10%
Pacific
Grove
Monterey
.033%
Pacific
Grove
TREATED SPERN
NOT WASHED
5%
10%
94%
100%
9%
100%
48%
98%
78%
97%
20%
72%
80%
92%
TABLE 1
TREATED SPERM
CENTRIFUGED AND WASHED
5%
CONTROL
10%
98%
86%
100%
49%
99%
92%
18%
62%
95%
CHLORINATED
SEWAGE
CONC
SPER
GONC
.33
.10%
.033%
NO Na»S.0.
TREATMENT
0.5%
0.2%
59%
22%
18%
20%
1%
TABLE 2
1%
0%
SEWAGE TREATE
WITH Na,S,O.
24
100%
100%
99%
99%
5%
90%
10%
98%
384
SPER
CONC
.330
.10%
03.
HYPOCHLORITE
CONC
0.2%
0.57
100%
100%
23
100%
100%
1%
6%
14
19
CHLORINATEI
SEWAGE CONC.
0.2%
O.5%
100%
100%
100%
56%
100%
14%
09
TABLE
HYPOCHLORITE
CONC.
SPERM
ZONC.
.33%
.10%
.033%
EGGS
TREATEL
26
986
99%
986
TABLE
SPERM TREATED
0.5/
0.2%
94%
906
459
78%
40%
17
15%
HYPOCHLORITE
CONC
SPER
CONC.
33%
10%
033%
Na,S,03 ADDE
BEFORE FERTILIZATION
0.1%
0.2%
0.5%
2%
80
98%
97%
No
67%
TABLE 5
NO Na»S.0.
TREATMENT
0.2%
0.1%
97%
60%
1%
96%
16
0%o
30
0.5%
0%
SPERM
CONC
33%
106
033%
HYPOCHLORITE
CONC.
5%
10%
TABLE
146
SPERM
DNC.
33%0
4%
EXPOSURE TIME (SECONDS
20
10
15%
13%
34%
TABLE
40
70%
20%
44
SPERM
CONC
33%0
.10%
.033%
Fresh Sewage
72 Hr. Sewage
Fresh Sewage
72. Hr. Sewage
Fresh Sewage
72 Hr. Sewage
TABLE 8
SEWAGE
CONC.
0.5%
1%
1%
78%
71%
2%
0%
476
11%
1%
0%
24%
10%
44
TABLE LEGEND
Fertilization success after 5 minute
TABLE 1:
treatment of sperm in dilutions of Monterey and Pacific
Grove sewage is given in per cent values. Fertilization
with unwashed sperm was in sewage dilution; washed sperm
were used to fertilize in fresh seawater.
Drastically lower fertilization success is
TABLE 2:
seen with dilutions of chlorinated sewage (42.5 ppm
available chlorine). Treatment of the sewage with
sodium thiosulfate markedly reduced the inhibition of
fertilization.
TABLE 3:
Comparison of the inhibitory effect of the
same dilutions of chlorinated sewage (11.0 ppm available
chlorine) and hypochlorite in seawater (12.8 ppm available
chlorine) shows hypochlorite to be the major fertilization
inhibitor in chlorinated sewage.
Eggs treated in a 2% dilution of a hypochlorite
TABLE 4:
solution (38.5 ppm available chlorine) show no inhibition
after treatment and washing; sperm treated in lower
concentrations of hypochlorite solution show great
reduction of fertilization capacity
Fertilization success for sperm treated in
TABLE 5:
different dilutions of a 42.5 ppm hypochlorite solution
15
with and without thiosulfate treatment before fertilization
shows a slight apparent reversibility of the effect
which is attributed to a hypochlorite effect on the
fertilization process.
One second exposure of sperm to a 5
TABLE 6:
dilution of a 38.5 ppm available chlorine hypochlorite
solution reduces fertilization greatly; thiosulfate
was added one second after hypochlorite treatment was
initiated.
Susceptibility of fertilization to
TABLE 7:
hypochlorite (2% dilution of a 38.5 ppm solution) as
a function of time after insemination shows decreasing
sensitivity for the first minute; treatment was limited
to two seconds by addition of thiosulfate after hypochlorite.
The stability of hypochlorite in chlorinated
TABLE 8:
sewage is shown in a comparison of the inhibition effects
of fresh sewage and sewage three days in seawater dilution.
444
BIBLIOGRAPI
Baker, P. F., and R. Presley, 1969. Kinetic Evidence
for an Intermediate Stage in the Fertilization of
the Sea Urchin Egg. Nature, 221: 488-490.
Metz, C., 1961. Use of InhibitigAgents in Studies on
Fertilization Mechanisms, pp. 219-254. In: Bourne
G. H., and J. F. Danielli, Eds., International Review
tology, 11. Acad. Press, New York.
of
Physiology of Fertilization
Tyler, A., and B. Tyler, 1966.
and Early Development, pp. 683-742. In: Boolootian,
Interscience
R., Ed., Physiology of Echinodermata.
Publishers, New York.
44
ACKNOWLEDGEMENT
I wish to thank the faculty and students of
Hopkins Marine Station for assisstance in various phases
of this work. Thanks go in particular to Dr. David Epel
for aid and consultation on all aspects of the project
This work was funded in part by the National Science
Foundation Undergraduate Research Program, Grant Number
GX 7288.