-1
INTRODUCTION
Sewage which has undergone only primary treatment, i.e. removal
of grease and floatables, preliminary digestion, and treatment with
Chlorine, has been discharged into the intertidal areas at Pacific Grove
and Carmel, California until recently. Under an order from the State
Regional Water Quality Control Board to "clean up" their sewage because
of high Coliform counts, very large amounts of Chlorine were introduced
into the effluent in the winter of 1969, in order to disinfect the sewage
effluent.
In fresh water systems, chlorination has proven to be a highly
effective and relatively inexpensive method of controlling harmful
bacteria. Chlorination of public swimming pools and fresh water supplies
are common practices, and some cities are even chlorinating their storm
sewer runoffs. (Pavia, et. al,1969) Enough information about such chlorination
systems has been accumulated in order to understand both the beneficial
and deleterious effects of Chlorine and Sodium Hypochlorite on the flora
and fauna indigeneous to those fresh water systems. However, the effects
of liquid Chlorine and Sodium Hypochlorite upon the marine flora remains
largely unknown.
A comprehensive study of all the effects of liquid Cl, and Naocl on
marine algae is a prodigious undertaking and is clearly beyond the scope
of this project. Therefore, this examination has been limited to determining
the effects of Chorine residuals on the primary productivity of Prionitis
lanceolata Harvey, Ulva lobata (Kützing ) Setchel and Gardner, and
Macrocystis pyrifera (Linnaeus ) C.A. Agardh, a red, green, and brown
alga respectively.
.
Chlorine residual studies and subjective observations that I made
of the City of Carmel's sewage outfall suggested that a correlation between
algal photosynthetic rates and the chlorine residual activity of the sea
water might exist; however, a primary productivity analysis by the Winkler
method for dissolved oxygen was impossible in this case, as both Cl, and
OCl react very quickly with S,0,, the standard reagent in such analyses.
The use of a modification of the Bean et. al. (1953) C0, uptake method
circumvented this reaction, allowing the photosynthetic rates at different
chlorine residual concentrations in the selected marine algae to be measured.
MATERIALS AND METHODS
The chlorine residual analysis was carried out using the Lodometric
method with a starch endpoint described in Standard Methods for the Exam-
ination of Water and Wastewater (1965). Samples were collected in 250 ml
plastic reagent bottles and brought back to the lab within one hour of
sampling and tested immediately. It appeared from preliminary data that
in a 24- hr.period after the sampling, bacteria in the sewage capable of
living on S,0, would cause the results to be much higher that they should
have been. This was combatted by adding a drop of CCl, to each sample
bottle along with the standard S,O, reagent and by immediate testing.
The 10 sampling stations around the Carmel outfall and their relation to
the onshore currents are shown in Figure 1.
The results of the chlorine residuals analysis around the Carmel
outfall area are presented in Figure 2. This figure shows a rapid decrease
of the chlorine residuals in the sea water with increasing distance from
the point of sewage discharge.
Beyond 110 feet, the residual chlorine level
2/
had decreased to less than 0.1 ppm. Beyond 130 feet to either side of
the sewage discharge, residual chlorine in the water was not detectable
with this test. Sampling in a line parallel to the shoreline and nearly
1000 feet out to sea revealed no detectable residual chlorine; however,
others (Grey and Nakata, personal communication) found chlorine residuals
as far as 3000 feet to the south of the outfall. From this it might be
inferred that the sewage field was staying close to shore and moving south.
Figures 3,4,5, and 6 give the results of the current studies done at the
Carmel outfall area. It can be seen that the currents do indeed run just
offshore in the vicinity of the outfall and head south toward Monastery
Beach and Whaler's Cove.
After the residual chlorine levels in sea water had been established,
the 3 algae, Prionitis lanceolata, Macrocystis pyrifera, and Ulva lobata
were selected for the primary productivity tests. Prionitis (Rhodophyta)
was selected because it was the commonest of all the algae near the Pacific
Grove sewage outfall where the chlorine residuals in the water were found
to be greater than 10 ppm (Remsen, personal communication). Ulva lobata
(Chlorophyta) was selected because it was conspicuously absent form the
immediate area around the same outfall. Finally, Macrocystis,in addition
to belonging to the brown algae group, was selected because the proposed
Carmel sewage outfall extension would move the effluent boil out into the
kelp beds, and it would be of interest to know what effects Chlorine
might have upon Macrocystis before the outfall pipe was extended.
Labelling of the algae was accomplished by allowing small discs,
approximately 5 mm in diameter, cut from the algae with a +2 cork borer
to carry on photosynthesis in a sea water solution of Nahc 0, with and
without NaoCl. In an attempt to limit the natural variation in photosyn
S
Janes Schreiber
hetic rates from blade to blade, the dises were eut from the blades
the following manner. With Macrocystis, 5 discs were cut from an
area of 4 cm. near the tips off 4 blades taken from the same plant
near the center of the frond. One disc of each set of 5 discs from each
ade was used for each concentration of Ocl". In this way, even if one
blade had a photosyntheitc activity much more or less than the other blades
one disc from that blade would be in each set of the four replicates run
at each of the 5 Ocl concentrations,(0, 1, 5, 10, and 15 ppm), which woul
distribute any such effect equally among the results.
With Prionitis, all the discs were cut from different blades because
he blades were not big enough to allow more than one disc from near the
apical point of the blade. The blades were chosen all of approximately
he same length, from the same thallus, and from as close to the top
the thallus as possible.
Uva, however, was the hardest from which to get reproducible samples
The dises were cut from near the margins of the blade, as close together
as was feasible. It is recognized that this would not necessarily remove
all the natural variabliity of photosynthetic activity in the dises, bu
was assumed that such close sampling would limit it as much as was
ssible.
After the wet weights of the dises were taken, the dises, weighing
om 1.6 mg to 2.4 mg for Ulva, from 5.6 to 6.2 mg for Prionitis, and
com 5.9 to 6.3 mg for Macrocystis, were placed in 12 ml graduated centrifuge
ubes with enough freshly filtered sea water (0.45 micron millipore filter
make 5 ml of total solution when the 0.20 ml of 5.C/ml Nanc 0, solutior
and  ml of the Naocl solution were added. Naocl was chosen as the chlorine
residual to work with because it is the major component of the chlorine
residuals in sea water due to the following reaction; ((Baker, 1969
+ HC
- Hocl
C19 + H,0
The NaoCl solution was made up with distilled water and not filtered sea
water. It was felt that the OCl would be more stable in distilled water,
as it normally will very rapidly begin to decompose in the presence of an
easily oxidized material, such as would be present even in freshly filtered
sea water.
After weighing, 0.2 ml of 5C/ml NaHC 0, solution was added to each
centrifuge tube containing the algal discs. This was immediately followed
by the addition of the appropriate amount of NaoCl solution necessary to
produce the proper initial concentration of hypochlorite, which was never
more than 0.24 ml. The centrifuge tubes were then sealed with vaccine caps,
swirled to mix the solution, and placed in the incubator for i hr. This
incubator consisted of an outer plastic tub with both a water inflow and
outflow tube for cooling water, an inner clear plastic box lined with
aluminum foil and filled with fresh water, and a foil covered, wooden
tube rack that was slanted at approximately 450 to the counter top in the
hood. This slant allowed the algal discs to rest at the bottom of the
tubes in a plane parallel with the counter top and perpendicular to the
majority of the incident light. The water level in the inner plastic box
was set such that the water would cover the centrifuge tubes up to the
5 ml mark, and the rate of flow of the cooling water through the outer
plastic tub was set as necessary to keep the temperature of the inner bath
at 20° c.
The light source was a fluorescent light bank consisting of two 18
watt, 18 inch, soft white, G.E. bulbs in a aluminum foil covered hood. The
light bank was placed over the centrifuge tube rack so that it was 4"
from the algal discs. It was not possible, however, to eliminate outside
radiation completely. In order to take the outside radiation into account,
a complete set of runs was made only from 2 p.m. to 5 p.m. or from 11 p.m.
to 2 a.m.. This was necessary because it had been observed in preliminary
runs that the photosynthetic rates of those discs tested in the daylight
hours of bright sunlight were consistently about 10 % higher than those
discs tested under the incubator light source alone.
As soon as the testing period was finished, the sea water solution of
NaHC0, and NaoCl was drawn off with a 10 ml disposable syringe. The dscs
were washed vigorously with 4 5 ml portions of filtered sea water, and
the water was drawn off as before. The discs, still in the centrifuge
tubes, were covered with 0.5 ml of 2 N methanolic KOH, and the tubes placed
in an 80°C sand bath for thirty minutes. After thirty minutes, 30 %
20 was added to the solution in the tubes in 0.05 ml increments as
necessary to dissolve the disc completely. When the H,O, activity had
ceased, the contents of the centrifuge tube were diluted to 3.0 ml total
volume with distilled water. Two 1.5 ml aliquots of this solution were
transferred quantitatively from the centrifuge tube by means of a disposable
pipette with two 15 ml aliquots of Bray's scintillation fluid into 2
scintillation vials and counted for 10 minutes for C activity on a
Nuclear Chicago Scintillation Counter Model Unilux II.
RESULTS AND DISCUSSION
The comparative rates of photosynthesis for each concentration of
NaoCl were determined by dividing the total disintegrations per minute (dpm.
obtained from the dissolved algal discs by the milligrams wet weight of
7
the discs. Each disc was run for exactly 1 hr, so the above rate values
have the following dimensions of dpm/ mg wet weight/ hr. Comparison of
these experimental rates was done by taking the mean C''0, uptake rate
of the control discs (O ppm Chlorine residuals) as 100%. Dividing the
mean experimental C0, uptake rates by the mean control rate gåve the
values in per cent of the mean control CO, uptake rate, which were then
compared by plotting them against the appropriate concentration of OCl
in parts per million. The results for Macrocystis are presented in Fig. 7,
those for Prionitis and Ulva are presented in Figs. 8 and 9 respectively.
These figures show that the concentrations of 15 ppm and 10 ppn OCl cause
a definite decrease in the photosynthetic activities of each of the 3
algae tested; however, the results for the OCl concentrations of 5 ppm
and éspecially i ppm are much less certain due to a greater degree of internal
variation than was observed for the control discs and the discs in 15 and
10 ppm OCl. (See Table 1) Nonetheless, the OCl" concentration of 5 ppm
does show a decrease of 25% in the photosynthetic rate of Ulva lobata
and a 12% decrease in the photosynthetic rate of Macrocystis pyrifera.
Prionitis lanceolata, however, did not show an effect for either 5 ppm
or 1 ppm OCl", and further, the reductions in photosynthetic rates of
Prionitis for the 10 and 15 ppn concentrations were considerably less than
those found for Macrocystis and Ulva at the same OCl concentrations. This
is presented graphically in Fig. 10. It would appear that Prionitis
lanceolata (Rhodophyta) can resist the effects of OCl better than
Macrocsytis pyrifera (Phaeophyta) or Ulva lobata (Chlorophyta), at least
in the lab. However, close observation of both the Carmel subtidal sewage
outfall and the Pacific Grove intertidal sewage outfall reveals that
Prionitis lanceolata Harvey and Prionitis andersonii Eaton are the dominant
James Schreiber
species of macroscopic plant life within 5 feet of the end of the outfall
pipes. At the Pacific Grove outfall, where the chlorine residuals
sometimes reach levels greater than 10 ppm (Remsen, per. comm.), Prionitis
lanceolata is the only macroscopic plant within 5 feet of the sewage
discharge, and they seem abundant; yet there are no green algae or brown
algae within this area. This could be expected in such an area of high
chlorine residuals, judging from the results of this study
It should also be noted from Figure 10 that Ulva lobata shows the
greatest decrease in photosynthetic activity at lower concentrations of
OCl. This might be a function of the particular pigments that Ulva, a
green algae, would have;oi.e. the chlorophylls may be more susceptible
to chlorine than the pigments of the red and brown algae. It might alse
be explained by the fact that the blades of Ulva lobata are much thinner
than the blades of Prionitis lanceolata or Macrocystis pyrifera. This
might have allowed the penetration of OCl into the intracellular centers
of photosynthetic activity to be more pronounced in Ulva than in Prionitis
or Macrocystis, but the data cannot support either of these hypotheses to
the exclusion of the other.
The experimental results that have been obtained do apparently show
an adverse effect of chlorine residuals on the rates of photosynthesis
in the algae examined; however, other interpretations are possible.
Since no attempt was made to make the OCl solution isotonic with the
filtered sea water used in the experiments, it might be argued that the
gradient of salinity present in the samples caused these effects. But the
greatest decrease in the salinity was present in the 15 ppm OCl test
solution. Only 0.24 ml of 312.4 ppm OCl and 0.20 ml of Nac Ho, solutions
were added to 4.56 ml of filtered sea water in order to make up this 15 ppm
2
9 -
James Schreiber
OCl experimental solution. This represents a decrease of only 8.8 % in
the total salt concentration of the experimental media from the control
media, but this would hardly account for the 80 % decrease in the photo¬
synthetic activity of Macrocystis pyrifera at 15 ppm OCl or the 40 %
decrease of Prionitis lanceolata at 15 ppm OCl".
The variability of the results for 1 and 5 ppm OCl can be explained
using the fact that only the initial concentration of OCl in the centrifuge
tubes was known. Since OCl is a highly reactive ion that rapidly undergoes
reduction in the presence of any easily oxidized substance, the actual
residual OCl concentration could have varied due to the different levels
of oxidizable materials in each centrifuge tube. Furthermore, no additional
OCl could be added to the tubes after the run was started because this
would have changed the critical concentration of available NahcO, in the
tubes; consequently, any natural variation in the resistance of an alga¬
to the chlorine effects might have been accentuated by the variable lowering
of the actual OCl concentrations in the centrifuge tubes. If this were
the case, one would expect to see the standard deviation of the replicates
decreasing with increased OCl concentrations because the small deviations
from the initial OCl concentrations would become less important and because
the differences in the resistance to chlorine would be less at higher Ocl
concentrations. The experimental results show rather definitely that the
standard deviations do decrease with increasing OCl concentration. (See
Table 1 )
The experimental results suggest several types of experiments that
need to be done before the mechanism of this effect can be understood.
One such experiment would be to expose an entire plant to a concentration
of OCl found previously to be detrimental to that plant for several hours,
remove the plant to a fresh sea water aquarium, and then sample that plant
a
10
James Schreiber
for C0, uptake experiments at 1 hour intervals or so. In this way, it
might be determined whether the chlorine residuals were affecting the
plant reversibly or not. This experiment could then be coupled with a
concurrent analysis of the plant pigments, in order to ascertain if the
exposure to OCl had caused any damage to these crucial parts of the
photosynthetic apparatus. From the results of these experiments, it might
be possible to decide if the chlorine was primarily attacking the pigments
or other vital components of the photosynthetic process. However, until
such time as these types of experiments can be performed, it is only
possible to state that residuals Chlorine levels of 5 ppm and greater de
adversely affect the photosynthetic activities of the three algae studied,
Prionitis lanceolata, Ulva lobata, and Macrocystis pyrifera
11
James Schreiber
SUMMARY
This project involved the determination of the persistence of
residual Chlorine in sea water and the determination of any effects,
either adverse or positive, of residual Chlorine from chlorinated
sewage effluents on the photosynthetic activities of three representative
algae, Prionitis lanceolata, (Rhodophyta ), Ulva lobata (Chlorophyta),
and Macrocystis pyrifera (Phaeophyta ). By using the technique of
quantitative C0, uptake to measure the rates of photosynthesis, a
correlation between the concentrations of Sodium Hypochlorite in the
experimental media and the experimental rates of photosynthetic activity
was discovered.
The results show that a residual level of 5 ppm Chlorine in sea
water does adversely affect the photosynthetic activities of these algae.
in the case of Ulva lobata by as much as 25 %. Higher concentrations of
residual Chlorine have even greater effects,with 15 ppm residual Chlorine
giving an 80 % reduction in the photosynthetic rate of Macrocystis
pyrifera.
2
-12
LITERATURE CITED
Characteristics of Chlorine Compounds. Journal
Robert J., 1969.
Baker,
of Water Pollution Control Federation 41-3: 182 - 485.
Bean, R. C.,,E. W. Putnam, R. D. Trucco and W. Z. Hassid, 1953. Preparation
4 labbelled d-galactose and glycerol. J. Biol. Chem.,
Of
204: 411 - 425.
Pavia, Edgar H., Crawford T. Powell, Storm water disinfection at New Orleans.
Pollution Control Federation, 41 - 4: 591 - 605.
Journal of Water
des
- 13 -
ACKNOWLEDGEMENTS
My sincere thanks go to Dr. Isabella A. Abbott for her excellent
advice and timely encouragement. I would also like to thank Dr. John
H. Phillips and Phillip Murphy for their advice and technical assistance
without which this project could not have been completed. This project
was supported in part by the National Science Foundation's Undergraduate
Research Program Grant No. GY - 7288.
-14.
CAPTIONS
Figure 1:
Chlorine residuals sampling stations: Detailed map of the Carmel outfall
area showing the onshore currents and the sampling stations.
Figure 2:
Chlorine residual levels at the Carmel outfall: Curve A represents the
values found for high tide. Curve B represents the values found for low
tide.
Figure 3:
Surface currents 1000 feet offshore from the Carmel outfall: 12 drift
bottles filled with fresh water were released from a skiff and followed
from shore. Dotted lines show the path of the bottles.
Figure 1:
Surface currents near Monastery Beach: 12 bottles were released in a line
perpendicular to the beach. Dye packets were attached to drift bottles
to follow their paths. Dotted lines represent the path that the bottles
followed.
Figure 5:
Bearings to the bottles at selected times: Both currents studies were
carried out by taking a bearing to the bottle at a specified time and
plotting these on a map.
Figure 6:
Drift bottle recoveries: Location and dated of recovery are listed along
with bottle number.
Figure 7:
0, uptake rate of Macrocystis vs the concentration of OCl : Curve
represents the closest fit to the mean values. Standard deviation of
the replicates is presented as a range of values around the mean.
symbolizes the mean value.
Figure 8:
Standard
0, uptake rate of Prionitis vs the concentration of Ocl :
symbolizes
deviation presented as a range around the mean value.
the mean values.
0, uptake rate of Ulva vs the concentration of OCl : Standard deviation
Symbolizes the means
presented as a range around the mean values.
values.
Figure 10:
Mean values are plotted
Comparison of Prionitis, Ulva, and Macrocystis;
Prionitis
and the curves are fitted to these mean values.
Macrocystis.
Ulva.
Table 1
Results of the tests for each alga: Values for individual replicates are
listed along with the mean values and the relative standard and standard
deviations.
75



a(
5
—
J
ap


2


2
6
Carmel Bay
CURRENT
5

STUDY
May 11,1970
O930-1600


Mission Point

18
lee
0
50
1
e
1

V
S
Pt. Lobos

Scale
O REF.PT.
Outfall
OREF. PT. 2
6
Monastery
Beach
1:10000
N
1 2
Fie


Mission Point

Carmel Ba
Hen
STUDY
CURRENT
II
May 25,1970—1100-1300


Outfall

4

o
H
--
g-.

-

----

---+

17




a
4

REF PT 3

Scale
Pt. Lobos
N
Monastery
Beach
1:10000
A
Time
1032
1039
1102
1122
1132
1142
1232
1252
B
Time
1115
1130
1145
1215
124
Ref.
Pt.
—

2
2
2
Ref.
Pt.
200
185
197
185
210
206
205
200
188°205
178
185
182
181
34
22
15
12
20
12
8
10
CURRENT STUDY I
No.
Bottle
4
215
222
205
212
207
215
218
206
210
202
214
216
227
230
222
213
209
212
208
218
215
206
215
212
205
216
210
193
198
200
187
195
195
187
189
190
168
CURRENT STUDY II
Bottle No.
4
925
345
330
310
339
345
342
34
353
350
35/
350
358
350
4
355
0
355
3
3
7
10
244
233
236
224
229
222
214
24
233
214
215
238
2/0 °228
215
224
208° 202
200
202
195
20
195
8
10
322
318
320
335
335
337
339
331
345
354
353
353
254
230
240
224
218
210
199
183
3/6
334
336
350
358
12
260
238
248
248°
235
230
12
315
330
335
350
358
3,
O
C
Stuc
II
Bottle No.
8
12
10
12
Location
Monastery Beach
N. Pt. Lobos
Monastery Beach
Approx. 50 yds. off
north end of
Monastery Beach
Date
5/21/70
5/25/70
5/22/70
70
5/27
5/27/70
P1e
05
9.
5
u
95

00

3



60
50
40
20
100
ppm of Chlorine
F16
1 -

00


jomof Chlorine Residuals
N

2
3
.
932
2.
90
1000
90
80
2 10
60
50
5

40
S
30
20
ppin of Chlorine
Ple. 8
8
95.
9



1004
50
—
ppm of Chlorine
F16.
932
X
90
14


20

E
50
110
100
ppm
of Chlorine
Fie
10
Macrocystis
ave.
rel. 5.
Prionitis
ave,
rel. §.
Ulva
ave
re 5
dom
940 ma
1037
870
104
987.5
6t.7
6.7 %
603.4
521. 6
911. 0
620.0
66+.o
147.4
227
8100
632.6
403.6
530.
627.
718.1
7711.0
147. 3
21%
TABLE
om Chlorine
pp
15
44
54
23
158
119.8
56.
5.6%
328
442
334
443
399
10.6
11.6%
242.8
343.9
217.6
135.6
247.5
78.7
169
10
443
495
352
674
603.5
14
II.3 %
484.8
649.5
574.0
468.2
546.7
18.2
16%0
410.8
318.4
125.
—
121.2
21%
09
1293
505
173
835
274.7
28%
818.0
674.3
57.3
43.6
156.7
23%
384.0
1014.0
343.0
54.0
591.3
290
41 7
770
1066
368
/301
873.5
347.
37.3 %
418.3
640.3
868.6
(42.3
186.2
28.470
278.
119.5
07.2
357.4
621.1
180.6
25 7