ABSTRACT
1. In the period 29 April to 27 May 1970 studies of the distribution,
reproduction, recruitment, and pigments of Pollicipes polymerus were
carried out on populations in both polluted and unpolluted areas.
2. Near sewage outfalls Pollicipes clusters may be abundant; however,
they lack associated biota of polychaetes, amphipods, limpets and
epizoic algae. They are loosely attached to rocks and are usually
covered with an algal slime.
3. Within a 230 foot radius of an outfall with high residual chlorine
the barnacles were not reproducing. Recruitment occurred only on the
outer fringes of the area. At a second outfall with lower residual
chlorine a few Pollicipes were reproducing and some recruitment was
occurring within four feet of the mouth of the pipe. The levels of
reproduction and recruitment here were lower than in unpolluted areas.
4. Pollicipes shows decreasing damage with increasing distance from
the outfalls.
5. Thin layer chromatogram studies show that several carotinoid
pigments found in the stalk hemolymph of normal Pollicipes are lacking
in individuals living near sewer outfalls.
6. Pollicipes appears to have some value as a quantitative indicator
of the effects of marine sewage pollution.
INTRODUCTION
The effects of sewage pollution on most marine organisms are
almost unknown, and good indicator organisms by which pollution damage
can be measured are lacking. Counts of coliform bacteria are routinely
used to indicate degree of pollution. These counts may be extremely
variable, even from hour to hour, and they give no indication of possible
pollution damage to shore organisms. A better indicator is needed; one
possibility is the common stalked barnacle Pollicipes polymerus (Sowerby,
1833).
This barnacle is abundant on wave-swept rocky shores from the Bering
Strait to the middle of Baja, California, being restricted to the upper
two-thirds of the intertidal zone (Ricketts and Calvin, 1969). It is
the only pedunculated barnacle found in the intertidal zone in North
America (Cornwall, 1951) and it does not occur subtidally (Barnes and
Reese, 1959). Pollicipes is one of the few sedentary marine animals
which is numerous not only in unpolluted areas but also occurs close
to the shoreline sewer outfalls at Pacific Grove and Carmel, California.
Hilgard (1960) determined that the animals reproduce for eight
months of the year. Preliminary observations indicated that while the
animals away from the outfall were reproducing normally those close to
it were not, suggesting that reproduction in this species might provide
a useful measure of pollution damage.
2
METHODS
Pollicipes polymerus used in this study were taken from the
following areas: (1) the Pacific Grove sewage outfall area at Point
Pinos (14 stations); (2) the Carmel sewage outfall area north of
Monastery Beach (6 stations); (3) the Pacific Grove shoreline from
Lovers Point to the Great Tide Pool (8 stations); and (4) a control
area at Mussel Point, Pacific Grove (6 stations).
All barnacles were taken in areas and at intertidal levels where
they were sizable and abundant. Only the largest individuals occurring
in close association with others of their kind (and which therefore had
opportunity for cross fertilization) were taken. When possible, about
60 barnacles were taken at each station.
After collection each Pollicipes was measured (distance from
rostrum to carina) and examined for young recently settled on the stalk.
Then it was cut open and examined for the presence of ovarian masses
in the stalk and ovigerous lamellae in the mantle cavity. Enlarged
ovarian masses indicated that the individual was a reproductively active
member of the population. The presence of ovigerous lamellae (oval,
flattened masses of developing larvae being brooded by the adult)
indicated successful fertilization. Animals with neither were considered
to be non-reproducing members of the population.
After examination, the percentage of barnacles which had enlarged
ovaries or ovigerous lamellae or both was determined and recorded as the
percentage of the sample reproducing (% reproducing). The percentage of
the sample with ovigerous lamellae was determined and recorded as
3 -
384
% w/ ovigerous lamellae. The percentage of the sample with young
recently settled on the stalk was determined and recorded as the %
w/ young
4.
RESULTS
Point Pinos
For present purposes, the region of the Pacific Grove outfall can
be divided into three areas (Fig. 1) showing obvious qualitative
differences. Areas B and C are apparently relatively normal. Pollicipes
here always grow in association with Mytilus. Amphipods are abundant in
Pollicipes clusters, and at least three species of limpets are found on
Pollicipes valves. Balanus glandula may also attach to Pollicipes
capitulum. Isopods are infrequent, and never more than two occur on an
individual barnacle. The stalks are very firmly attached to the substratum,
and frequently have coralline algae or young brown algae growing on them.
A dense mat of detached Phyllospadix blades is often intertwined around
the bases of the stalks.
Area A (Fig. 1), which receives the most direct contact with the
effluent, is conspicuously different. Here no adult Mytilus are found,
though occasionally small (§1 cm.) mussels, having a yellow, translucent
shell, are seen. Amphipods and limpets are absent from Pollicipes clusters.
Balanus glandula occurs on nearby rocks but never on the Pollicipes
capitulum. Isopods are common, and as many as eight may be found on an
individual barnacle. The Pollicipes themselves are small, few being of
minimum reproductive size (rostro-carinal distance of 17.2 mm; Hilgard,
1960). None were found measuring more than 25 mm. Maximum sizes of
30.5 mm. occurred in areas B and C, and animals measuring up to 37 mm.
were found at Mussel Point. The stalks of Pollicipes in area A are
loosely attached to the substratum, and can be removed by hand. No
coralline or brown macro algae are attached to the stalks; instead the
5
latter are often covered with an algal slime composed of brown (Feldmannia
chitinicola), green (Blidingia minima), and blue green (Oscillatoria and
Entophysalis deusta) algal species. The same slime occurs in the crevices
between capitular plates; it may become so thick that it covers the
calcareous spines on the stalk, but it does not occur inside the mantle
cavity. (Outside of area A, algal slimes occur on the stalk but never
as thickly.) The bases of the stalks do not have Phyllospadix blades
intertwined about them, but have entrapped large amounts of sand.
Figure 2 shows the results of the survey of reproduction and
recruitment in the three samples made at Point Pinos. In area A, directly
exposed to the effluent, there is tremendous damage. In a total sample
of 692 Pollicipes there are no ovigerous lamellae and essentially no
production of ova and no settlement of cyprid larvae from the plankton.
At stations 3 and 4 many isolated animals were found. These indicate
the former presence of large clusters of Pollicipes (see Barnes and
Reese, 1960). At station 4, among 27 of the largest barnacles only
eight were above minimum reproductive size. Pollicipes closest to the
outfall are dying; 11% of the samples taken 26 May 1970 from station 2
had rotted cirri and decaying stalks.
Animals in area B show some evidence of reproductive inhibition.
Not only was the onset of reproduction delayed in this population, but
the percentages of individuals brooding ovigerous lamellae and bearing
newly settled young are consistently below those of Mussel Point
(Fig. 3) and those of area C (Fig. 4). Areas B and C follow the same
trends, but area B does less well. Statistically, differences in
percentage with young are always significant (P §.05) and differences
6 -
in percentage with ovigerous lamellae are significant in the first two
sampling periods (P «.05). Area B receives more contact with effluent
than does C; other physical parameters in the two areas are roughly similar
Reproductive differences between Pollicipes populations in area C and the
controls at Mussel Point are not consistently statistically significant.
Area C does significantly better in recruitment in the latter two sampling
periods (P (.05)
Pacific Grove Shoreline
Current studies of Point Pinos carried out in July 1969 and April
1970 (using fluorescene dye packets, drift bottles and a team of observers
from the Hopkins Marine Station), indicated a current moving northward
around Point Pinos and into Monterey Bay (Fig. 1). Many drift bottles
released at the point landed on Pacific Grove beaches. It appears that
some of Pacific Grove's sewage is not moving out to sea but is being
carried back into the bay. The reproductive condition in samples of
Pollicipes from along the southern shore of Monterey Bay is shown in
Figure 5. Station 7 is about 75 feet from a 12" drain pipe which serves
not only as a storm drain but also for occasional dumping of sewage
when the treatment plant is overloaded. Animals here show the poorest
development of ovaries and a very low proportion of barnacles bear ovigerous
lamellae. The other populationson this shore showing low proportions of
ovigerous lamellae occur at stations 2 and 3, lying closest to Point Pinos.
Figure 6 compares stations 2, 3 and 7 with stations 1, 4, 5, 6, and
8 and with Pollicipes at Mussel Point. Statistically, there is no
difference between the latter set of stations and the barnacles on Mussel
Point (P «.05 for all three categories). The differences between the
first set of stations and the second are significant (P (.Ol for all
three categories), indicating that not all of the effluent is diluted
at sea but some comes back into shore at harmful levels.
36
Carmel Outfall Area
Some sampling of the Pollicipes population was carried out in
the area of the City of Carmel sewer outfall, Carmel Bay, California
(Fig. 7). While irregular mixing by waves occurs at the inshore
rocky reef, strong southerly currents move offshore water from Mission
Point toward Point Lobos (Bachelor, et al. 1970). Correlated with
this, Pollicipes show increasing levels of reproduction and recruitment
at increasing distances south of the outfall. At Point Pinos the
current system is much more complex and such an obvious gradient of damage
does not exist.
Conditions at the Carmel sewer outfall are much less harmful to
Pollicipes than those at the Pacific Grove outfall. Some successful
fertilization occurs four feet from the pipe (station 2), and young
settle out from the plankton in much larger numbers than in area A
at Point Pinos. Studies of dilution of the effluent (Bachelor, et al,
1970) indicate that it comes inshore on the beach south of station 5
and is fairly well diluted south of there. The Pollicipes data are
consistent with this finding; note the sudden jump in the proportion
of animals reproducing and in those bearing newly settled young between
stations 5 and 6.
9:
362
Pigment Analysis
Normal Pollicipes, both mature and immature, have a reddish-orange
hemolymph in the stalk which is released when the stalk is sliced open.
In Pollicipes from area A at Point Pinos, stalk fluid is colored a
dirty gray.
Holter (1969) found six carotinoid pigments occurring in the stalk
fluid of Pollicipes: B-carotine, isozeaxanthin (?), an astaxanthin ester,
lutein, zeaxanthin, and astaxanthin. Cheesman and others (1967) found
that astaxanthin-protein complexes are frequently linked to reproductive
and developmental processes. Holter hypothesizes that this lipo-protein
complex mediates the transport of large water soluble molecules across
lipid-bound membranes. This transportation could be important when
transfer of critical supplies for egg production is essential. Pollicipes
hemolymph appears to act as the store for the astaxanthin lipo-protein
complex.
The stalk fluids in Pollicipes from areas A and B on Point Pinos
and from Mussel Point were analyzed using thin layer gel chromatography
to determine qualitative differences in the pigments present.
In each area, about 125 ml. of stalk fluid was collected from
two clusters of Pollicipes (about 150 animals in each cluster), and centrifuged
to remove sand and grit. The pigments were extracted from the fluid with
acetone. Petroleum ether (b.p. 20-40° C) was added and the carotenoids
transferred to the petroleum ether phase by the addition of water. The
petroleum ether was repeatedly washed with cold tap water until free
of acetone, and then concentrated under a nitrogen stream. Schleicher
and Schuell precoated silica gel plates (^1500), 250/thick, were used
10
Se
for the chromatography. Each sample was spotted on the plate and the
spots were run in a 30% petroleum ether-acetone mixture. A standard
composed of B-carotene, astaxanthin, lutein and echino-none was also
spotted on the plate and run. The absorption spectrum from 550 nm.
to 380 nm. for each sample was taken on a Beckman DK2A Ratio Recording
Spectrophotometer.
All the pigments noted by Holter (1969) were found in the stalk
fluids of animals from area B, at Point Pinos, and from Mussel Point.
In area A however, three pigments were missing from the stalk fluid--
astaxanthin, lutein, and the astaxanthin ester. Only B-carotine,
isozeaxanthin and zeaxanthin were present.
The absorption spectra for area B and Mussel Point are identical.
Both absorptions rise steadily to a broad plateau from 476 to 450 nm.
Expanding the plateau shows two peaks, at 472 nm. and 453 nm. The
absorption spectrum for the stalk fluid from animals from area A
reflects the changed pigment composition here. The absorption rises
to a slight plateau at 472 nm., then continues to rise to the major
peak at 432 nm. It falls off slowly until 413 nm. and rapidly thereafter.
Possible quantitative differences in the amount of pigment exist
between animals in areas A and B and those at Mussel Point. Less
pigment was extracted from area B animals than from the Mussel Point
controls. Considerably less pigment was extracted from animals in
area A. If quantitative differences do exist, pigment determination
would be a valuable additional indicator of pollution damage.
11
DISCUSSION
Raw sewage in moderate concentrations could have beneficial
effects for Pollicipes. This barnacle, an opportunistic rather than
selective feeder, takes a large amount of particulate matter as food
(Howard and Scott, 1959). Near an outfall, the level of organic
particulate matter is certainly higher than normal, so that under other
favorable physical conditions such as those at Point Pinos, Pollicipes
could flourish, being helped by the sewage. Mytilus californianus,
Pollicipes' major competitor for space along the coast, is also an
efficient filter feeder, but it dies close to sewer outfalls. Pisaster
ochraceus, for which Pollicipes ranks high as a food source (Feder, 1959),
is also absent near the Point Pinos outfall. These three conditions;
increased food, absence of competition, and lack of predation could
enhance Pollicipes growth near an outfall.
In area A at Point Pinos the barnacles, although currently doing
very poorly, are the only large sedentary animals abundant on the rocks.
Their clusters cover much of the suitable substratum. According to
Barnes and Reese (1960), Pollicipes grow very slowly, not reaching sexual
maturity before the fifth year and not achieving full growth until the
twentieth. Although it is possible that some of the largest Pollicipes
in area A at Point Pinos were there before the outfall was built,
this is unlikely, as there are many small barnacles and young do occasionally
settle here. Comparison of present conditions on Point Pinos with those
described in a study by Mitter (1969) indicate that the grossest detrimental
effects of the sewage are recent ones.
12
366
Photographs taken in the spring of 1969 show that the rocks near the
pipe were covered with macroalgae at that time; now they are covered
with Hildenbrandia or a yellow, diatomacious slime. The general area
appeared much less damaged a year ago than it does now. Heavy chlorination
of the sewage was begun only about five months ago (early in 1970).
suggesting that chlorine or chlorinated compounds may be the most damaging
agent in sewage.
Both Carmel and Pacific Grove sewage treatment plants use primary
treatment and handle mostly domestic wastes. The major difference between
the two effluents is in the residual chlorine. In Pacific Grove sewage
the chlorine residuals can reach levels of up to at least 45 parts per
million (ppm) at the outfall mouth, but in Carmel sewage they are 2-5
Ppm. The fact that Pollicipes at Carmel show some reproduction and
recruitment within four feet of the mouth of the outfall pipe suggest
that chlorine or chlorinated compounds are the likliest agents causing
damage.
Pollicipes in area A are not recruiting enough larvae from the
plankton to sustain their current population level. The clusters will
probably disappear as the adults die off. In area B, recruitment is
probably sufficient to maintain the population close to current levels.
Pollicipes polymerus shows possibilities for use as an indicator
of pollution damage. Of the parameters investigated, fertilization
(as indicated by the presence of ovigerous lamellae) appears to be the
most sensitive to pollution. The proportions of the population bearing
new juveniles and the proportion reproducing are useful as well. Sperm
content represents another possible variable, (in area A at Point Pinos
13
86
no barnacle had copious sperm). These parameters can be sampled and
measured by personnel with minimal biological training. Quantitative
pigment differences, while more difficult to measure, would also be
indicative of damage. Further work needs to be done on the natural
variability of Pollicipes in an unpolluted enviornment to aid detection
of lesser degrees of damage from pollution.
14 -
ACKNOWLEDGMENTS
I wish to acknowledge the generous assistance and encouragement
given to me by Dr. Donald P. Abbott, both during the research and in
reading the manuscript. Dr. Welton Lee aided in the chromatography,
Dr. Isabella Abbott identified the algal species, and James Sutton
gave much helpful aid and advice throughout the work. This research
was supported in part by the National Science Foundation Undergraduate
Research Program Grant No. GY-7288.
36.
REFERENCES
Bachelor E., D. Grey, M. Nakata, J. Schreiber, and J. Welsh. 1970.
Unpublished
Barnes, H. and E. S. Reese. 1959. Feeding in the pedunculate cirripede
Pollicipes polymerus, J. B. Sowerby. Proc. Zool. Soc. Lond. 132:
569-585.
Barnes, H. and E. S. Reese. 1960. The behaviour of the stalked intertidal
barnacle Pollicipes polymerus J. B. Sowerby, with special reference
to its ecology and distribution. J. Anim. Ecol. 29: 169-185.
Cheesman, D. F., W. L. Lee, P. F. Zagalsky. 1967. Carotenoproteins
in invertebrates. Biol. Rev. 42: 131-160.
Cornwell, I. E. 1951. The barnacles of California (Cirripedia).
Wasmann J. Biol. 9: 311-346.
Feder, H. M. 1959. The food of the starfish, Pisaster ochraceus,
along the California coast. Ecology. 40: 721-724.
Hilgard, G. H. 1960. A study of reproduction in the intertidal barnacle,
Pollicipes polymerus, in Monterey Bay, California. Biol. Bull.
119: 169-188.
Holter, A. R. 1969. Carotenoid pigments in the stalked barnacle Pollicipes
polymerus. Comp. Biochem. Physiol. 28: 675-684.
Howard, G. K. and H. C. Scott. 1959. Predacious feeding in two common
gooseneck barnacles. Science. 129: 717-718.
Mitter, C. 1969. Unpublished.
Ricketts, E. F., J. Calvin and J. Hedgpeth. 1969. Between Pacific
Tides. (4th ed.). Stanford Univ. Press. 614 p
16
36
FIGURE CAPTIONS
Figure 1. Three areas of Point Pinos based on the relative degree of
pollution.
Figure 2. Survey of the Pacific Grove sewage outfall area at Point
Pinos showing levels of reproduction and recruitment at various times.
Outside the box: station number and N - number of Pollicipes in the
sample. Inside the box: the top number is the percentage of the sample
reproducing; the middle number is the percentage of the sample bearing
ovigerous lamellae; the bottom number is the percentage of the sample
upon which young have recently settled.
Figure 3. Survey of Mussel Point (unpolluted area) showing levels of
reproduction and recruitment at various times. N - number of Pollicipes
in the sample. Inside the box: the top number is the percentage of the
sample reproducing; the middle number is the percentage of the sample
bearing ovigerous lamellae; the bottom number is the percentage of the
sample upon which young have settled.
Figure 4. Graph contrasting levels of reproduction and recruitment
between Mussel Point and areas B and C at Point Pinos at various times.
Numbers are number of Pollicipes taken from each sampling area.
Figure 5. Survey along the south shore of Monterey Bay showing levels
of reproduction and recruitment. Outside the box: station number
and N = number of Pollicipes in the sample. Inside the box: the top
number is the percentage of the sample reproducing; the middle number
is the percentage of the sample bearing ovigerous lamellae; the bottom
number is the percentage of the sample upon which young have recently
settled.
17 -
370
Figure 6. Graph comparing levels of reproduction and recruitment along
the south shore of Monterey Bay at various areas (see Fig. 5). N -
number of Pollicipes taken from each sampling area.
Figure 7. Survey of Carmel sewage outfall area showing levels of
reproduction and recruitment south of the outfall. Outside the box:
station number and N = number of Pollicipes in the sample. Inside the
box: the top number is the percentage of the sample reproducing; the
middle number is the percentage of the sample bearing ovigerous lamellae;
the bottom number is the percentage of the sample upon which young have
recently settled.
18
a
Outfall-
p
d
N
CURRENTS AT POINT PINOS
AREA A: Most direct contact with effluent
AREA B: Some contact with effluent
AREA C: No apparant contact with effluent
L
S
-22
81.8
31.8
90.9
N-39
87.2
23.1
69.2
0
0
4.9
- 61
0
O
1.7
N-58
O
N-60
N—61
95.1
41.0
68.9
N-62
88./
29.0
72.6
N-90
86.7
3.3
60.0
OUTFALL
1.6
N-62
1.6
0
N-63
O
N-64
4.8
N- 21
O
N-21
N-64
N-63
N-65
83.1
84.4
98.4
49.0
37.0
26.2
44.4
59.4
36.9
N-56
N-79
N-71
83.9
92.4
85.9
25.0
35.4
28.2
51.9
49.3
71.4
N-54
N-42
N-40
79.6
92.9
87.5
7.1
3.7
5.0
44.4
64.3
22.5
10



26-27 MAY
14-16 MAY
1-5 MAY
6.3
O
4.8
N-63
0
8
O
5.3
19.7
N-16
N-76
N-64
98.4
53.1
70.3
N- 67
98.5
50.7
79.1
N-56
92.9
17.9
60.0
12
-73
91.8
45.2
60.3
N-70
90.0
32.9
62.9
N-54
85.2
14.8
33.3
8.1
274
N-62
5.3
17.5
N-57

26-27 MAY
14-16 MAY
1-5 MAY
N-66
89.4
39.4
71.2
N-69
94.2
31.9
56.5
-64
98.4
20.0
70.3
14
26-27 MAY
14-16 MAY
1-5 MAY
370
N-64
90.6
32.8
67.1
N-66
93.9
24.2
65.2
N-78
96.2
56.4
56.4
N-62
98.4
69.4
69.4
N-60
95.0
48.3
55.0
-61
95.1
41.0
80.3


N-63
N-66
N-64
N-68
94.1
100
88.9
90.9
54.7
25.0
28.6
25.8
21-22 MAY
49.2
54.7
52.9
78.8
N-67
N-66
N-47
100
97.0
83.0
18.2
21.3
59.7
12 MAY
27.7
56.1
78.8
N-67
N-64
5 MAY
92.2
85.1
32.8
4.5
7 MAY
68.8
50.5
N-45
29 APRIL
91.1
8.9
2 MAY
57.8
O


L
O
MUSSEL POINT

374
te
% 100


Reproducing
80
70
19o- -- - -- - - --0

Voung

60
50


40


Ovigerous lamellae
30+
Mussel Point
20
N-172 242 403
—-o---0--- Point Pinos—Area C
N-213 228 203
....c.........o Point Pinos —Area B
10
N226 268 253
2122
2627
12 14 15
MAN
38
5
N-63
N-60
N—67
N-71
N—57
N-57
100
83.6
90.1
90.0
63.5
93.0
36.7
8.5
42.1
26.3
9.5
28.4
45.1
51.7
84.2
63.2
87.3
80.6
POINT PINOS
OUTFALL
GREAT TIDE POOL
LOVERS POINT
MUSSEL
POINT
100
92.9
98.4
94.8
92.2
83.0
97.0
47.6
17.2
32.8
21.3
59.7
69.4
18.2
35.7
68.8
27.7
56.1
24.1
69.4
78.8
N-58
N-42
N-47 N-67
N-62 N-64
N-66
PACIFIC GROVE SHORELINE
7-12 MAY 1970
37
% 100
80
60
20
Ovigerous
Reproducing
lamellae
N-306
Mussel Point
N-283
Stations 1-4-5-6-8
N-192
Stations 2-3-7
Voung
4

37
2
C
N—65
86.2
200
26.2
N-63
95.2
20.6
71.4
POINT LOBOS

OUTFALL
N-62
62.9
6.5
37.2
N-69
24.6
O
N-62
75.8
17.7
24.2
N-63
63.5
6.3
41.3
CARMEL
OUTFALL
AREA
25 MAY 1970
CARMEL

MISSION
POINT
CARMEL
RIVER

STRONG

SOUTHERLY
CURRENTS
MONASTERY
BEACH



+
378