ABSTRACT
1. Qualitative studies of reproduction and quantitative
studies of total and individual carotenoid content in
the stalked barnacle Pollicipes polymerus were carried
out for animals in polluted and nonpolluted areas in the
Monterey Bay area of California.
2. Reproductive activity was retarded in areas very close
to point sources of pollution.
3. The total carotenoid content in the stalk fluid of
Pollicipes was invariably lowest in animals close to point
sources of pollution, concomitantly, the amount of carotenoid
tended to increase with increasing distance from such pollution
sources.
1. It was suggested that total carotenoid content may be
fected by proximity to sewage effluent whereas astaxanthin
content may be specifically effected by increaséd chlorine
concentrations.
5. Possible mechanisms governing the decrease in Pollicipes
carotenoid content are discussed.
96
INTRODUCTION
A growing awareness of environmental pollution has led
to recent attempts to evaluate the biological impact of local
domestic sewage discharges into Monterey Bay. One of these
studies (Holstrom, 1970) showed an effect on the stalked
barnacle Pollicipes polymerus, a natural and abundant inter-
tidal organism along the central California coast. Holstrom
found that samples of populations adjacent to sewage outfalls
had reduced reproductive capabilities. He also noted a
qualitative difference in the amounts of carotenoid pigments
in the animals, including an apparent total lack of
astaxanthin, astaxanthin ester, and lutein in animals from
highly polluted areas. This mutual effect on reproduction
and on carotenoid content seemed to be linked to levels of
chlorination rather than to any other factor.
These apparent effects on carotenoid content are most
intriguing in light of recent thoughts concerning the rela-
tionship of carotenoids to reproduction in marine invertebrates
(Cheesman et al., 1967). Some of the carotenoids in the
animal, especially astaxanthin, are found as lipoprotein
complexes (Holter, 1969). Glyco-lipoprotein complexes, which
have been noted in the blood of many invertebrates, may be
involved in the transport of carotenoids (Cheesman et al.,
1967). Likewise, a carotenoid attached to a lipoprotein could
possibly mediate the transport of large water-soluble mole-
cules across lipid-bound membranes (Holter, 1969). Such
properties would be importantat the onset of reproduction
79
when there is a transfer of nutrient materials to the egg.
The blue pigment in the ovaries and developing embryos of
Lepas fasicularis is a carotenoprotein with astaxanthin
attached to euglobulin (Ball, 1944). Ball showed that
developing nauplii change their color from blue to pink,
representing the liberation of free astaxanthin from the
protein complexes. Such phenomena are quite common in
marine invertebrates (Cheesman et al., 1967). Recently
suggestions have been made that these huge protein complexes
may be food storage sites for early developmental stages
(Zagalsky et al, 1967). The carotenoids are thought to
stabilize the glyco-lipid proteins and the organism in
question somehow frees these carotenoids, allowing the
proteins to be utilized (Cheesman et al., 1967). The eggs
of Pollicipes polymerus chang from an orange color to a
brown color as they mature. However Lee and Gilchrist (1971)
have found that a similar color chang in the sand crab,
Emerita analoga, is strictly chromataphoric.
Unextracted stalk fluid from Pollicipes polymerus in
highly polluted areas is yellow-gray, as opposed to the
bright orange color found in animals from unpolluted areas.
This yellow-gray color could be the result of interference
with either the synthesis of carotenoids, namely astaxanthin,
or the formation of a stable linkage between carotenoids and
proteins.
An investigaton of the quantitative effects of sewage
pollution on the pigments of Pollicipes polymerus is made
easier by a recent study by Holter (1969) describing the
80
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carotenoid pigments in Pollicipes. He isolated and identifiid
the following pigments: B-carotene; astaxanthin; astaxanthin
ester, lutein, zeoxanthin; iso-zeoxanthin.
The present study was designed to: (1) obtain a
quantitative estimate of the effects of domestic sewage
pollution on carotenoids; (2) compare qualitatively animals
from a polluted area with animals from a healthy area; and
(3) assess the possible relationship between carotenoids as
effected by pollution and their reproductive role.
8
C
MATERIALS AND METHODS
Animals
Barnacles were collected at six sites along the Carmel-
Monterey coastline (Fig. 1 and 2) and one site in Southern
California. The areas were as follows: (1) the Pacific
Grove sewage outfall area at Point Pinos (3 stations),
on April 28th and May 27th, 1971; (2) the Carmel sewage
outfall area north of Monastary Beach (5 offshore stations
and  inshore stations);(3) Mussel Point; (4) Hovden
cannery, north of Mussel Point, on April 29th and May 6th,
1971; (5) Sea Otter Point, approximately ½ mile south
of the mouth of Malpaso Creek along California Highway
No. 1; (6) the Highlands Inn outfall at Carmel Highlands
south of Point Lobos; (7) White's Point on the south side of
the Palos Verdes Peninsula in Southern California, directly
inshore of the Los Angeles County sewage outfall.
The number of animals collected at each of these
sitesranged from 30 to 65 (Table 1).
The areas were selected to represent highly polluted
areas: Point Pinos; Carmel; White's Point, areas polluted
by relatively small discharges: Highlands Inn, and unpolluted
areas: Hovden cannery, Mussel Point, Sea Otter Point.
The white to orange ovaries and the orange to
yellow-gray stalk fluid of the Pollicipes polymerus were
used for measuring carotenoid content as Holter (1969)
showed these two tissues to be the highest in carotenoid
content. Stalk fluid was collected by cutting whole clumps
8
of barnacles from the rocks and allowing the stalk fluid to
drain. The stalk fluid and the animals from which it was
obtained were immediately placed on ice and transported
to the laboratory as soon as possible. The animals were
then dissected and the ovaries removed. The stalk fluid
was centrifuged at 10,000 R.P.M. for 20 minutes to remove
particulate matter. Acetone was added to both the fluid and
the ovaries and they were refrigerated up to 12 hours until
needed.
Extraction of Carotenoids
The ovaries and stalk fluid were extracted in acetone
and transferred to di-ethyl ether, saponified, and re-
dissolved in di-ethyl ether according to the methods of
Lee (1966). Before saponification the extract was transferred
to petroleum ether (B.P. 20-1000) and the absorption spectra
(500-130 nm) of the extracted carotenoids determined with a
Beckman DB-G gradiant spectrophotometer. These readings
were used to calculate total carotenoid content by
applying the formula:
ug carotenoids/mg ovary
- 1 X OD at peak x volume extracted
or
original amount
ml fluid
Pigment Identification
The extracted pigments were applied to 20x20 and
5x20 Mannogram prepared silica gel-G thin layer chromatography
plates. The plates were run in a mixture of 25% acetone
in hexane. The separate bands were then scraped off the
plates into a mixture of 10% acetone in petroleum ether
83
and the absorption spectrum was again determined and the
amounts of the individual pigments were calculated as
outlined above. These individual pigments were identified
by the wavelength of maximum absorbance in petroleum ether
and by comparison with pigment standards which were also
applied to TLC plates and run under the same conditions.
The sources of these standards were identical to those
reported in Lee and Gilchrist (1971).
84
RESULTS
Point Pinos
The two Point Pinos samples taken in April and May
each contained subsamples taken from three locations (Fig.1):
area P, within 20 yards of the pipe and highly polluted;
area B, a moderately polluted area west of the pipe; and
area C, a slightly polluted area south of the pipe.
Both samples from area P consisted of undersized
individuals with rostro-carinal distances of 15-20mm(17.2mm
is minimum size for reproduction; Hilgard, 1960). The average
range encountered in all healthy populations was 25-35 mm.
These animals all had yellow-gray stalk fluid as compared
to the usual bright orange color. Few had developed ovaries
and none had ovigerous lamellae. In both the April and
May samples the amount of total pigment in the stalk fluid
(Fig.3) was well below that found in the samples taken from
areas B and C. The total pigment in the ovaries was comparable
to that found in all other Point Pinos samples. The most
abundant stalk fluid pigment in the April sample taken
from area P (Fig.5) was lutein, which amounted to 74% of
the total pigment complement of the animals.
The samples from area B appeared healthy and had
bright orange stalk fluid. Many had developed ovaries
and ovigerous lamellae. The amount of pigment in the stalk
fluid of both samples (Fig. 3) was approximately six times
the amount of pigment in area P animals and 0.75 times the
amount in area C animals. The amount of pigment in the
85
ovaries, however, was the highest recorded for any of the
Point Pinos samples and almost equivalent to the amount of
pigment found in the ovaries of animals collected from
unpolluted sites such as Sea Otter Point (Fig. 1). The
most abundant pigment in samples from area B was astaxanthin
which amounted to 66% of the total.
The two area C samples appeared healthy although there
were hardly any ovigerous lamellae seen in the individuals
collected. These animals consistently had the highest amount
of pigment in the stalk fluid as compared to the other
Point Pinos samples (Fig. 3), and, in the ovaries, more
pigment than individuals from area P but less pigment than
individuals from area B (Fig.4). Similar to the results
obtained from area B, the most abundant pigment in the
April stalk fluid samples was astaxanthin, amounting to
61% of the total carotenoid content.
In both April and May there appeared to be a
definite gradient in the amount of pigment in the stalk
fluid relative to the proximity of sewage effluent. The
pigment in the ovaries however, did not show such a gradient.
The predominant pigment in the animals that appeared healthy
(areas B and C) was astaxanthin, while lutein was clearly
dominant in the unhealthy animals (area P), almost fully
replacing the astaxanthin in relative per cent.
Carmel
Offshore: All animals collected at these sites (Fig. 2)
appeared to be healthy. The occurrence of developed ovaries
86
was highly variable in this group of samples. Station 1
animals which were collected very close to the outfall
were relatively large (30-35mm) but had no developed
ovaries and no ovigerous lamellae. Animals from stations
2,4, and 5 were smaller (25-30mm) but almost all had
developed ovaries and ovigerous lamellae were often present.
Animals from station 3 were small and half of them had
developed ovaries. There were also ovigerous lamellae
present. The stalk fluid from all these animals (stations
1-5) was bright orange. The amount of pigment in the stalk
fluid was lowest in animals from station 1 and comparable to
the level of pigment in the animals from station P at Point
Pinos (Fig. 3). The stalk fluid pigment levels in
animals from stations 2,3, and 5 were highest compared to
all the offshore samples at Carmel, and were close to the
levels found at station B at Point Pinos. Compared with
2, 3, and 5, the pigment level in station l animals was
low. The pigment in the ovaries was lowest at station 1.
The ovary pigment levels at 2 and 3 were approximately
1.5 times the level at station 1. The levels in the ovaries
at 4 and 5 were approximately 2 times this level (Fig. 1).
Astaxanthin was most abundant in animals from stations
1 and 5, amounting to 53% and 55% respectively. These
astaxanthin levels are about 10% lower than those found
at Point Pinos. B-carotene was most abundant at station
2, amounting to 59%. Lutein was most abundant at stations
3 and 4, amounting to 61% and 59% respectively.
9
Generally the amount of pigment in both the stalk fluid
and the ovaries of these offshore samples increases as the
distance south of the outfall (station 1) increases, although
the levels at station  are consistently low. Th relative
amounts of individual pigments present show no definite
trend as they appeared to in the Point Pinos samples.
Inshore: The stalk fluid from all of these animals was
bright orange although station 1 and 2 animals did not
have many developed ovaries and only station 5 animals
had ovigerous lamellae. The amounts of pigments in the
stalk fluid of station 1,2, andl animals was low (close
to the level L animals in the offshore samples). The
stalk fluid pigment level for station 5 animals was
very high, 6 times the amount at 1, 2, and  and second
only to the levels obtained for animals from Sea Otter
Point (Fig. 3). The total amount of pigment in the ovaries
was lowest for station  as was the amount of stalk fluid
pigment. The value for station 1, in the area of the pipe,
was about 6 times the value for the other inshore stations.
There was not enough pigment to obtain values for the indi-
vidual pigments.
Others: Animals from White's Point had bright orange stalk
fluid although only 3 in 60 had well developed ovaries and
no ovigerous lamellae. The amount of pigment in the stalk
fluid was low, 33.6ug/ml, close to the value obtained at the
Carmel inshore station 1. The amount of pigment in the
ovaries was close to that in animals from area C at Point
88
Pinos.
The sample from Highlands Inn appeared to be the health-
iest of all the samples collected. The fluid was bright
orange and almost every animal had a well developed ovary.
However, the values for total amount of pigment were very low
for both the stalk fluid (close to the value at White's
Point and station 1 in the inshore Carmel series) and the
ovaries (close to the value at station L in the inshore
Carmel series). Lutein was the most abundant pigment, amount-
ing to 60% of the total pigment.
Few of the animals from Mussel Point had well developed
ovaries. The stalk fluid was a dull orange. The amount of
pigment in the stalk fluid was close to station C at Point
Pinos in May (Fig. 3). The amount of pigment in the ovaries
was highest of any of the samples collected. Astaxanthin
(44%) and lutein (55%) were almost equally abundant.
The animals from Sea Otter Point were healthy with
bright orange stalk fluid,many developed ovaries, and ovi-
gerous lamellae. The amount of pigment in the stalk fluid
was higher than any of the other samples collected. The
amount of pigment in the ovaries was slightly higher than
that found at station B in May at Point Pinos.
The animals from the cannery appeared healthy, with
bright orange stalk fluid, many developed ovaries, and ovi-
gerous lamellae in the later sample. However the amount of
pigment in the stalk fluid was close to the levels taken in
May at stations B and C at Point Pinos. The amount of
89
pigment in the ovaries of the earlier sample was very high,
close to that of Mussel Point (Fig.4).
DISCUSSION
Tth results of this study coincide in part with
Holstrom's (1970) observations of decreased reproductive
capability in Pollicipes polymerus occurring in the areas of
concentrated sewage pollution.
Quantitatively, the levels of total carotenoid
pigment in the stalk fluid, and less consistently in the
ovaries, suggest that carotenoid pigments are somehow
adversely effected by the sewage effluent. The data from
Point Pinos shows the most obvious correlations. The area
where the effluent is most concentrated (sample area P)
shows evidence of decreased reproduction and a definite
drop in carotenoid pigments in the stalk fluid as compared
to the pigment in the animals from the healthier areas.
The amount of pigment in the ovaries suggest a similar trend,
although here is is not as marked or as consistent. There
is some indication that astaxanthin in the polluted areas
may decrease with a concomitant increase in lutein.
The data from Carmel does not give as clear a picture,
possibly because the currents around the outfall have
been shown to be very complicated
Chlorine residues at Carmel have been measured at
1-2 ppm whereas those at Point Pinos have been measured
as high as 10 ppm. This greater amount of chlorine input
at Point Pinos may explain the more definitive results
here as compared with the Carmel stations, although the
stalk fluid pigments at some Carmel sites seemed to be just
as effected as those at Point Pinos. The carotenoid levels
in the ovaries from Point Pinos and Carmel were generally
similar. The amount of pigment in the ovaries at inshore
station 1 seemed inordinately high. A possible explanation
may be related to local current patterns. Polluted water
from the outfall sweeping back towards shore (Fig. 2) may
miss station 1. The values for the offshore stations,
however, show a more consistent pattern with a lower
amount of pigment being found in the areas closest to
the outfall.
These observations tend to support Holstrom's contentions
that decrease in reproductive capability may be linked to
a decrease in total amount of pigment. However, the only
consistent change in the proportions of the individual
pigments takes place in the Point Pinos samples where there
is a larger concentration of chlorine in the water.
It therefore may be suggested that increased chlorination
may directly effect the oxidation of astaxanthin, whereas
unchlorinated or minimally chlorinated effluent may effect
total carotenoid content. It was found that if a sample
were left at room temperature or refrigerated for 18
hours it would turn yellow-gray and show a decreased
amount of total pigment. This seems to indicate that dena-
turing the proteins in the stalk fluid effectsthe carotenoid
level by maximizing the opportunity for oxidation. This in
turn could mean that the sewage effluent is effecting the
protein and therefore secondarily altering the pigment
90
complement of the animals.
It was hoped that a quantitative study of the indivi-
dual pigments would show some kind of relationship between
amount of sewage pollution and relative abundance of B¬
carotene, astaxanthin, and lutein. However, the data
obtained allows no such correlations to be made and appears
to be inadequate. This was brought about by the fact that
the amount of pigment in the individual Pollicipes polymerus
is very small and it is therefore difficult to obtain
a good working volume of extract. Furthermore, astaxanthin,
the major pigment in Pollicipes, is very labile and is
readily oxidized even after extreme precautions have been
taken.
The mechanisms which bring about the changes in the
amounts of total pigment are unknown. They may be due to
replacement of carotenoids and carotenoid precursors in
the diet of Pollicipes polymerus by non-nutrient containing
particulate matter from the effluent; they may be due to
some substance in the effluent which effects the biochemical
processes of pigment production; or they may be due to
denaturation of the protein structures, specifically the
stalk fluid and egg carotenoproteins, also by some substance
in the effluent.
Whatever the mechanism, sewage pollution appears
to have a definite measurable effect on total carotenoid
levels in Pollicipes polymerus.
13
REFERENCES
Ball E. G. (1911) A blue chromatoprotein found in the
eggs of the goose-barnacle. J. biol. Chem. 152,
627-631.
Cheesman D. F., Lee W. L., & Zagalsky P. F. (1967) Caroteno-
proteins in invertebrates. Biol. Rev. 12, 131-160.
Hilgard G. H. (1960) A study of reproduction in the inter-
tidal barnacle Mitella polymerus, in Monterey Bay,
California. Biol. Bull., Woods Hole 119(2),
169-188.
Holstrom M. V. (1970) Distribution, reproduction, recruit-
ment, and pigments of the stalked barnacle Pollicipes
polymerus in the vicinity of sewage outfalls at Pacific
Grove and Carmel, California. unpublished MS on file
at Hopkins Marine Station Library.
Holter A. R. (1969) Carotenoid pigments in the stalked
barnacle Pollicipes polymerus. Comp. Biochem. Physiol.
28, 675-681.
Lee W. L. (1966) Pigmentation of the marine isopod
Idothea montereyensis. Comp. Biochem. Physiol. 18,
17-36.
Lee W. L., Gilchrist B. M. (1971) Carotenoid metabolism
and reproduction in the sand crab Emerita analoga
(crustacea, anomura). in preparation.
Zagalsky P. F., Cheesman D. F., & Ceccaldi H. J. (1967)
Studies on carotenoid-containing lipoproteins isolated
from the eggs and ovaries of certain marine inverte-
brates. Comp. Biochem. Physiol. 22, 851-871.
24
O
CAPTIONS
Figure 1: Pacific Grove sewage outfall at Point Pinos,
showing current patterns and collection sites. Station
P was 20 yds from the pipe.
Figure 2: Carmel sewage outfall, located between the
Carmel River and Monastary Beach. Stations 1-5 on the
right are the inshore collection sites; those on the left
are the offshore sites.
Figure 3: Total carotenoid pigment in the stalk fluid of
Pollicipes polymerus. Carotenoid levels are given in ug/ml
for each collection site. The collection sites from left to
right are: Point Pinos, April and May,1971, P, B, and C are
the areas shown on Fig. 1; Carmel, offshore and inshore
areas as shown on Fig. 2; others include White's Point (W),
Highlands Inn (H), Hovden cannery in April and May, 1971,
(C), Mussel Point (M), and Sea Otter Point (S).
Figure 1: Total carotenoid pigment in the ovaries of
Pollicipes polymerus. Carotenoid levels are given in ug/g.
The collection sites are as in Fig. 3.
Figure 5: Individual carotenoid pigment in the stalk fluid
of Pollicipes Polymerus. Top graph shows the per cent values
for the collection points at Point Pinos (P,B,C as in Fig.1)
in April, Carmel offshore samples (Fig. 2), Highlands Inn (H),
Mussel Point (M). The bottom graph shows amounts for
the same locations as as above, in ug/ml.
Table 1: Dates of collection, number of animals collected,
and amount of total carotenoid pigment in the collected
samples. Amounts are in ug/ml and ug/g.
96
point pinos
OUTFALIO

K
6

P NEXT TO PIPE.
B SOME EFFLUENT
C NO APPARENT EFFLUENT
OUTFALL
OFFSHORE COLLECTION
CARMEL OUTFALL AREA
195


Fig 2
INSHORE COL LECTION
--
MONASTARY BCH
CARMEL R.
:::.
kikii
.
34 5
BC
BC
OFFSHORE
APRIL
MAY
POINT PINOS
CARMEL
Collection Sites

2 4 5
INSHORE
.
WHICC
OTHERS
a
.
POINT PINOS
2183315

234
FHORE
12SHO
CARME
COLLECTION SITES
WHCGM
OTHERS
kän


in

.



.
9

8

.








—

3
Fig 5
10
2
COLLECTION
SITE
POINT PINOS
CARMEL
offshore
inshore
5
OTHEI
Mussel Pt.
Hovden Onry
Highlands
White's Pt.
Hovden Cnry
Sea Otter Pt.
TABLE 1
DATE ug carot.
of
ANIMALS
govary
1/28
30
756
1/28
990
1/28
60
714
5/28
138
5/28
1067
5/28
655
30
5/28
1110
5/28
665
5/28
625
997
30
5/28
950
30
5/30
50
5/8
1690
989
5/8
30
157
5/10
30
716
5/10
30
10
5/4
1911
60
5/6
1810
5/29
30
115
5/15
768
176
30
5/29
50 5/12 1197
ug carot.
ml fluid
21
121
175
86
20
129
132
105
55
25
76
63
32
31
13
312
102
C
KNOWLEDGEMENTS
I would like to thank Dr. Welton L. Lee for
his aid and advice in the lab and with this paper.
103