ABSTRACT
California sea mussels (Mytilus californianus) of 2 to 5 cm length
were collected from intertidal areas in Pacific Grove, California, during
May. They were placed in various dilutions of primary unchlorinated
sewage and their respiration was measured. Results indicate that sewage
causes changes in respiratory activity, and that high concentrations are
extremely toxic. An attempt was made to isolate the toxic factors but
proved unsuccessful. Data for the excretion of dissolved inorganic
phosphate by the mussels are also presented.

INTRODUCTION
The city of Pacific Grove, California (population approximately
15,000) disposes household wastes in the intertidal area of Point Pinos.
The outfall discharges wastes at an average rate of 1.6 million gallons
per day. The sewage undergoes primary treatment and is chlorinated
prior to discharge, but the detectable level of free chlorine in the
water decreases rapidly within a few feet of the outfall.
One of the irregularities in the intertidal community in the vicinity
of the outfall which is in contrast to adjacent intertidal areas is the
absence of the California sea mussel, Mytilus californianus (Conrad, 1837).
Fifty feet northwest of the outfall, large M. californianus beds begin
and continue up the side of the point. These locations are relatively
unpolluted since the areas are washed by heavy surf at high tide, exposed
to the air at low tide, are in waters which test low in excess nutrients
and chemical pollutants, and have surface currents which keep the sewage
out of the area. Southeast of these beds, in a direction toward the
outfall, the intertidal rocks are barren of all life. But rocks in close
approximation to the outfall and in a southernly direction down the point
are densely covered with clusters of the barnacle Pollicipes polymerous.
In uncontaminated intertidal areas, Pollicipes and Mytilus individuals
often grow in the same area, or in adjacent clusters. Close macroscopic
examination of the Pollicipes communities on the rocks in polluted areas
showed no more than three to five Mytilus individuals per 15-20 Pollicipes
clusters (each cluster with 20 - 30 individuals). None of the
californianus were larger than 3.5 cm in length. Further south
70.
down the point the population of M. californianus increased with occasional
small clusters of 7-10 animals. Thus there seemed to be a gradient of
density of M. californianus on the coastline southeast of the outfall,
absence of any individuals at the outfall, and a large proliferation
northwest of the pipe in relatively uncontaminated waters.
The feeding habits of M. californianus suggest its susceptibility
to any effects of sewage pollution. Fox (1936) stresses the great
importance of finely divided organic debris or detritus that constitutes
a large part of the material ingested by mussels and other lammellibranchs.
He states that M, californianus normally feeds almost exclusively on
detritus and the smaller planktonic organisms, and although mollusks
have organs specialized for dealing only with small particles, selection
of food is entirely nonqualitative. Anything which is not too large
or spiny is ingested, although it may not be digested. Later, Fox (194-
again notes that although M. californianus eats dinoflagellates, diatoms
and bacteria, the largest fraction of its nutrituve material is detritus.
It is assumed that organic material is ingested in a finely divided or
partly colloidal state-not as dissoived organic matter. Thus
M. californianus is a scavenger, which feeds largely on the types of
materials which are likely to be found in sewage. If sewage has an effect
on intertidal animals, it is likely that M. californianus is extremely
susceptible.
Tests were made on the acute toxicity and on the less severe metabolic
stresses of primary unchlorinated sewage on M. californianus. Acute
toxicity is scored by the death of an individual, or by the severe
incapacitation of an individual's normal activity of byssus thread formation.
To determine less severe stresses on metabolic activity, changes in
respiration of individuals in various concentrations of sewage were measured.
Acute toxicity was demonstrated with extremely high amounts of sewage,
and respiration seems to be affected at lesser concentrations. However,
conclusions from respiratory rate data are not well substantiated.
In addition to these studies on the toxicity of sewage, two less
intensive studies were conducted: 1) changes in the level of dissolved
inorganic phosphates in the environment of the experimental animals, and
2) a preliminary investigation into the effects of the amines and amino
acids in sewage on M. californianus. The level of dissolved inorganic
phosphates in the sewage was used as a characterization of the amount
of pollutants present. The level of phosphates in the solutions was
higher after 24 hour contact with the mussels. Concerning the amines
and amino acids, Fox (1936) states that a turbid sea water suspension
of marine bacterial organisms, maintained by the addition of Witte's
peptone, proved injurious to mussels: valves were kept tightly closed
or death occurred if left immersed too long. He believes that the
nitrogenous bacterial metabolites which are characteristic of decomposing
protein matter caused the deleterious effects on the mussels. Fox cites
Harvey (1925) as showing that with the copepod Calanus, bacterial growth
supported by peptone is far more toxic than when supported by sugar.
I extracted the amines and amino acids from a dilution of sewage twice
as great as shown to be damaging, and subjected experimental animals to
them. There was no acute toxicity demonstrated-no deaths nor absence
of byssus thread formation.
64
METHODS AND MATERIALS
For the study on respiratory effects in sewage dilutions, two
experiments were conducted. The first experiment was carried out by
mixing primary unchlorinated sewage from the Monterey treatment plant
with sea water—the salinity was different in each of the separate dilutions
of sewage. The second experiment was the same, except salinity differences
were resolved by making the solutions isotonic with intertidal sea water.
This was accomplished by adding 3.35 gm of sea salts to every 100 ml
of sewage. Both experiments had the same array of M, californianus:
five individuals in each of six jars. Each jar contained 750 ml of fluid:
a control with 0% sewage, and jars with 0.5% sewage, 1% sewage, 2% sewage,
10% sewage and 50% sewage. Each jar had five Mytilus with approximately
corresponding lengths of 5 cm, 4 cm, 4-3 cm, 3 cm, and 2 cm, (Table 1).
The 30 individuals in the experiment with differing sewage concentrations
were collected at Hopkins Marine Station. The solutions in the jars
were changed daily, temperature was maintained at that of sea water,
the jars were kept in a room with dim lighting, and were aerated by
vigorously bubbling air through glass tubes at the top of the jars,
just below the water line.
Respiration tests were conducted by placing an individual in a
ground glass-stoppered bottle filled with a measured amount of fresh
sea water, and incubating the bottles in a small aquarium under the same
conditions of temperature (sea water) and light (dim) for 3 hours. At
the end of the incubation period, the amount of dissolved Oo left in
the sea water was determined according to the Winkler method as described
in Strickland and Parsons (1968). This value was then subtracted from
the amount of Op initially present in the sea water. The amount initially
present was determined by incubating several known volumes of sea water
without Mytilus in the jars, and averaging the values. The amount of
02 left in the bottle was calculated according to the following equations:
360
ml 0/liter - Va - R.
st- R 2 50 2 V5-2
where Vg - volume of thiosulfate titrated (ml)
Rb = reagent blank (ml)
Vst" standardization titration (ml)
Vb - volume of bottle (ml)
In this equation, 50 ml aliquots are used for the
titration.
m1 02 - m1 0g/liter x V, (in liters)
Respiration is expressed in ml 0/hr/gm wet weight; the mussel was removed
from the valves and weighed without its byssus threads. The experiment
with variable sewage and salinity was conducted for a period of 4 weeks.
The experiment with only variable sewage concentrations lasted five days.
Due to deaths in the 50% sewage concentration, I prepared another
jar of five Mytilus (lengths of 5.1 cm, 4.0 cm, 3.6 cm, 3.0 cm, and
2.0 cn), of 506 sea water and 506 tap water to observe byssus thread
formation and deaths.
To characterize the amount of dissolved inorganic phosphates in
the jars, 100 ml aliquots were sampled and analyzed according to the
procedure of Murphy and Riley (1962), using the Klett electrophotometer.
To test deaths and byssus thread formation in an environment of
amines and amino acids present in sewage, the following extraction
procedure was used with 800 ml of primary unchlorinated sewage from
Monterey:
Oe
raise pä to 10 with Naon
extract three times with 240 ml aliquots of diethyl ether
extract the ether fraction three times with 240 ml aliquots
of O.1 M HCI
4) evaporate the aqueous fraction to dryness
dissolve the dry residue with 800 ml sea water
The solution was characterized by titrating a 50 ml sample with 0.1M
HOl and O.1 M NaoH, measuring pH as the dependent variable. The remaining
750 ml were put into a jar with two Mytilus (lengths of 4.4 om.and
2.9 cm) and a control jar was set up with 750 ml sea water and two Mytilus
(lengths of 4.5 cm and 3.0 cm). These animals were kept under the same
conditions as all the other experimental animals. Observations were
recorded concerning byssus thread formation and death.
RESULTS
Acute toxicity. In jars with sewage and salinity dilutions, acute
toxicity occured in the 50% dilution. (Table 2). After two days in
the 50% sewage:salinity environment, none of the mussels formed byssus
threads. By the tenth day one was dead, and by the thirteenth day, all
had died. From the sixth to thirteenth day, the mussels indicated a
moribund condition by showing sluggishness in closing their valves when
the mantle edge was prodded with forceps.
In the jar with 50%-sea water: 50% tap water, the mussels did not
form byssus threads for the first two days, but by the third day three
out of five were connected to the jar. By the twelfth day, the mussels
byssus thread formation was normal. They continued to function normally
for the remaining eight days of the experiment.
In the jars with various sewage concentrations, acute toxicity was
observed in the 50% dilution. The valves remained closed for the entire
four days, and there was no byssus thread formation by any of the animals.
No deaths were observed in 50% sewage over the course of the experiment.
To determine if amines and/or amino acids in the sewage were toxic,
two mussels were placed in a jar with 750 ml of sea water containing
amines and amino acids extracted from 750 ml of primary unchlorinated
sewage. The characterization titration with 0.1 M NaOH and 0.1 M HOI
did not detect the presence of any amines and detected only one amino
acid (pk approx. 6.1) in the 50 mi sample. (Figure 1). Two control animals
were placed in 750 ml of uncontaminated sea water. These animals were
observed for 11 days, and no acute toxicity was evident. Normal byssus
thread formation was present in all four animals from the first day onward.
Since the solutions in the two jars were not changed, byssus thread
formation became erratic towards the end of the 11 day observation
period, but irregularities occured in both the control and experimental
animals.
Respiration. The respiration of 30 mussels in jars of sewage and
salinity dilutions was measured from three to five times and the values
then averaged. The 30 averages representing the respiration of the 30
individuals were graphed as ml 0/hr/gm wet weight. (Table 3 and Figure
. In the 50% dilution of sewage and salinity, in which all the animals
eventually died, respiration was lower than the control values in four
out of the five test animals. It was approximately 30% lower for the
5 cm mussel, 274 lower for the 4 cm mussel, 20% lower for the 3 cm mussel,
and 35% lower for the 2 cm individual. In the 1% dilution of sewage and
salinity, respiration increased fromethe control value in four out of
five test animals. It increased approximately 294 for the 4 cm mussel,
23% for the 3-3.5 cm mussel, 10% for the 3 cm mussel (up to 17% in the
2% dilution), and 18% for the 2 cm individual.
The respiration of the 30 mussels in the jars of sewage dilutions
with sea water salinity was measured two to three times and the values
averaged (Table 4 and Figure 3). In the 50% dilution of sewage, respiration
was higher than the control values in four of the five individuals. It
was approximately 17 higher for the 5 cm mussel, 48% higher for the
3.5-4 cm mussel, 32% for the 3 cm mussel, and 28% for the 2 cm mussel.
In the 0.5% dilution of sewage, four of the five individuals respired
less than control, but the decrease is not very large for the 5 cm and
4 cm mussels.
Phosphate levels. The changes in the phosphate levels in the six
jars of the sewage:salinity dilutions, the 50% tap water:50% sea water
jar, and a jar of 10% sewage without mussels were measured over a 24
hour period. (Table 5). The amount of dissolved inorganic phosphate
increased in all the jars except in the 50% tap water: 50% sea water.
The increases in the control, 0.5%, 1%, and 2% dilutions are roughly
equivalent; the 10% dilution increase is larger. No readings were made
on the 50% dilution. The level of phosphates also rose in a jar of 10%
sewage without any test animals in the solution.
DISCUSSION
The experiments on acute toxicity indicate that primary unchlorinated
sewage is toxic to Mytilus californianus if present at high levels in
the environment. In other toxicity studies conducted by my colleagues
with primary sewage from Pacific Grove, (chlorinated and unchlorinated),
proved to be even more injurious to intertidal test animals than primary
unchlorinated effluent from Monterey. Thus it is possible that the
paucity of M, californianus on the rocks covered with Pollicipes
polymerous southeast of the Point Pinos outfall is due to sewage pollution
of that area. Although acute toxicity was demonstrated only at 50g
concentrations in my work, observations were made for only a two-week
period, and the sewage I used was not from Foint Pinos.
The data on the respiration of the California sea mussels in deleterious
environments are not conclusive; more tests should have been performed.
However, examination of the data in Tables 3 and 4 shows that in a large
number of cases, the figures for onygen consumption of each mussel were
gery similar. Moreover, when the data is graphed, (Figures 2 and 3),
it shows variation of respiratory activity with age. This concurs with
experiments on Nytilus perna by Bayne (1967), and adds credibility to
my work.
The respiration graphs show several interesting trends. In Figure
2 where the 50% dilution proved lethal, respiration of the moribund
mussels was substantially lower than the controls. Figure 3 (variable
sewage and constant salinity) shows the opposite trend for the mussels
in the 50% dilution; this indicates a different response to an environment
where salinity is not a factor. I believe that the mussels show respiration
values higher than control values because they were making up an oxygen
debt during incubation which was acquired while in the sewage. This
hypothesis is supported by several facts. Fox (1936) has shown that
chemotactic responses are demonstrated by M. californianus for certain
substances. The mussel remains with its valves shut tight in such hostile
1
10-
environments. In a series of experiments with M, californianus in
various concentrations of sea water, Fox (1936) noted that in water of
salinity below 14.94 %/00 the mussels either remained with valves closed,
or opened only after many hours and then reclosed permanently. Both of
these observations suggest that my mussels could detect a hostile sewage
environment and responded by isolating themselves from it. The hypothesis
is further supported by two observations I made in the lab. During the
respiration test incubation, I noticed that a few of the individuals
from the 50% sewage dilution had fixed themselves to the bottles with
byssus threads. This happened only occasionally with any of the mussels,
including the control animals. Perhaps the mussel detected a favorable
change in the environment and began functioning to make up for its
decreased activity in the sewage. Also, the turbidity of the 50% sewage
dilution decreased very little in the 24 hours it was in contact with
the experimental animals. This is in contrast to my observations on
the other sewage dilutions and the sewage:salinity dilutions. It is
also inconsistent with the fact that Mytilus is known to be very effective
in lowering the turbidity of murky waters in a matter of hours (Fox, 1936).
In summary, I believe that the mussels could not survive in the 50%
sewage dilution, and when placed in a healthy environment, they attempted
to make up an oxygen debt acquired during incubation.
The changes in respiration demonstrated at 1% sewage:salinity and
1% sewage dilutions indicate that the respiratory activity of M. californianus
is extremely sensitive to small changes in the environment. The meaning
of the changes in respiration at 10 dilutions is not clear; they may
be an indication of increased feeding activity due to a solution slightly
richer in detritus than the control, or may be a reflection of osmotic
stress in a less than normal salinity.
The data on dissolved inorganic phosphate levels serve the practical
purpose of roughly characterizing the sewage from Monterey used in the
experiments. The results also suggest an hypothesis concerning phosphate
excretion in M, californianus. The values for the control animals, the
0.5%, 1%, and 24 sewage:salinity dilutions indicate that the mussels
increased the level of phosphate by about 20 Klett units in 24 hours-
approximately 0.07 ugm-at/1 (an increase of 0.053 ugm-at in these 750
ml jars). The mussels in the 10% dilution increased the environmental
level of phosphate by a measure of 10 Klett units,more, but this can
be accounted for by the fact that sewage under the same conditions but
without mussels increased in phosphate by 5-10 Klett units in the same
period.

ERENCE
Bayne, B.L. 1967. The respiratory response of lytilus perna L.
(Mollusca: Lamellibranchia) to reduced environmental oxygen.
Physiological Zoology 40 (3): 307-313.
Fox, D. L., G. W. Marks, F.0. Austin. 1936. The survival period of
adult mussels in sea water of various concentrations. In Fox,
D. L. (ed.). The Habitat and Food of the California Sea Mussel.
California University Scripps Institution of Oceanography Bulletin
4: 1-10.
Fox, D.L. 1936. Certain physical and chemical properties of the fecal
pellets and some experimental feeding studies. In Fox, D.L. (ed.).
The Habitat and Food of the California Sea Mussel. California
University Scripps Instiution of Oceanography Bulletin 4: 49-60.
Fox, D.L. 1943. Biology of the California sea mussel (Nytilus
californianus) II. Nutrition, metabolism, growth and calcium
deposition. Journal of Experimental Zoology 93: 205-249.
ACKNONLEDGEMENTS
I would like to acknowledge the help given me by Dr. John Phillips
in my amine extraction work, and for his valuable criticisms of
project.
also thank my advisor, Dr. Ellsworth Wheeler, for his patience
and encouragement throughout the quarter.
My work was supported in part by the National Science Foundation
Undergraduate Research Program, grant no. GY-7288.
TABLE LEGENDS
Table 1. Measurements of length and weight of each of the 60 mussels
used in the respiration experiments. Note that there are 5 in
each dilution of sewage:salinity, and 5 in each dilution of sewage.
Table 2. Data on acute toxicity-byssus thread formation and death
used as criteria. Empty squares indicate normal functioning, X's indicate
absence of byssus threads in all five animals, and blacked-out squares
indicate that all five are dead. 2b, 3b,4b, indicate the number of
mussels with byssus threads (short of all 5), and 10, 30 show the
number dead (short of all 5).
Table 3. Raw data from the sewage:salinity experiment, expressed in
ml 02/3 hrs, used to graph the respiration curves. in Figure 2.
Table 4. Raw data from the sewage experiment, expressed in mi 0,/3 hrs
used to graph the respiration curves in Figure 3.
Table 5. Phosphate readings measured in Klett units. Measurements
were taken in the 50% salinity jar, the jars of sewage: salinity
dilutions, and a jar of 10% sewage: salinity with no mussels in it.
SEWAGE ANL
-N
SALINTT
VARIABLE
SEWAGE
VARIABLE
5.2 cm
38 gm
4.2
1.98
5.5
1.84
3.2
0.87
2.0
D.21
5.2 cm
3.27 gm
2.05
6
1.48
2.0
0.3
5.2 cm
2.69 gm
4.2
2.00
91
3.2
cm
5.1
gm
3.37
4.0
2.21
1.06
3.0
0.84
2.2
% DILUTION
5.4 cm
5.3 cm 5.2 cm
3.50 gm 3.13 gm 2.97 gm
1.59
2.71
3.4
3.6
1.24
1.16
1.2.
3.2
3.2
0.8
O.81
2.2
0.42
0.22
4.8 cm
5.3 cm
5.0 cm
3.52 gm
3.64 gm
3.07
3.9
1.67
1.50
2.14
3.8
3.4
3.5
1.38
1.07
1.51
3.0
3.C
2.9
1.05
O.78
2.0
2.1
O.40
0.
5.3 cm
4.00 gm
2.04
1.67
1.23
2.2
0.48
4.9 cm
3.52 gm
1.97
3.6
1.00
3.1
O.8
O.31
e
155




ee
1r

—
155

e
—

15

— 15


E


e








e



125
L

LL


—
e5

able
117
cm
4 cm
-3.5
cm
3 cm
2 cm
54684
.72720
3470
70380
49900
39662
39250
.57962
.48412
47012
48919
50654
5820
.32976
.27464
.09217
.11648
.13424
% SEWAGE: SALINITY
0.5
54694
.45092
.52504
64989
.67946
.60210
54885
.71134
49036
3892
44562
51727
48874
.47588
.47038
.50841
54838
44024
43030
50504
41823
.50484
48620
.46791
50724
165
34452
.22894
36960
.20042
33513
.19662
.21788
.18850
.13624
.14770
.08976
.10010
.14391
13810
.38604
.60021
.60360
57336
62625
.63862
2552
6.
.60850
4059.
33425
143
5774
122
42714
54969
39599
2442
20683
.12402
.1476
.51038
57840
.65462
233
.6
34281
3855
34656
35464
46033
14661
34853
57843
44447
34650
.28784
28590
25116
.18915
.14742
.17991
50
.50604
.65475
.47156
46354
5762
44940
.273
.2.
.64116
49212
238
58538
.41605
16485
.27825
.08672
.27664
.10686
.16038
.08258
Le
cm
4 cm
3.5-4
cm
cm
2 cm
.70830
5832
55863
51926
34375
40378
4305
738
34645
16544
.15400
% SEWAGE
.64638
.65105
65554
.62314
437
55010
.57465
48169
34426
.50427
34761
49734
27326
30589
.23530
.27131
.12025
.10912
.0999
.09698
.74601
.71914
.28675
37802
30400
.31642
35139
34047
14616
.18244
.741
.7893
.58050
.55160
360
.43848
34541
.21138
.27562
.19810
.17976
.83510
84280
.63825
57200
.40225
.50427
49
544
46384
.47866
.19117
410
4 TAP:g SEA
0% DILUTION
O.5% DILUTION
1% DILUTION
27 DILUTION
10% DILUTION
50% DILUTION
10% DILUTION
WITH NO MUSSELS:

initial value
10,25
3, 10, 10
15, 20, 15
20, 30,
35, 35, 45.
150, 165, 160, 190
no data
150, 165
increase after 24 hrs
-7.0
15, 30, 15
15, 20, 10
5, 20, 2
55, 20, 20, 30
35, 10, 30, 25
5, 10
FIGURE LEGEND
Figure 1. The titration curve used to characterize the amine/amino acid
extraction. A 50 ml aliquot of the 800 ml total extractionwas titrated.
Figure 2. The respiration curves for the 30 mussels in sewage:salinity
dilutions. The % SEWAGE:SALINITY axis is distorted between the 10% and
The approximate
50% dilution in order to make the graph more compact.
lengths of the mussels are noted above their curves.
Figure 3. The respiration curves for the 30 mussels in sewage dilutions
with the salinity made up to equal control. The % SEWAGE axis in distorted
between the 10% and 50% dilution in order to make the graph more compact.
The approximate lengths of the mussels are noted above their curves.
p




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5
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