C
Abstract
The effects of different concentrations of several
heavy metals on the righting behavior of Littorina
planaxis were studied. Part of the animals' shells
were removed. They were then submerged in sea water
that contained different concentrations of cadmium,
copper, lead and silver. The effects of these treat¬
ments were assayed by timing the animals' righting response.
The results obtained show that cadmium, copper and
silver have significant effects only at high concentrations.
Lead was shown to have no significant short-term effects.
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Introduction:
Much public attention has been drawn recently to
pollution of the environment by heavy metals. Many
symposia on the subject have been held to discuss problems
such as cadmium poisoning and aerosol lead fallout.
(Symposium on Metals and Ecology, 1969) Very little has
been published in this field that deals with intertidal
invertebrates and nothing has been done to assay the
relative toxicities of heavy metals and their effects on
animal behavior.
The most common animal in the intertidal splash zone
is Littorina planaxis, which ranges from Puget Sound to
southern lower California (Ricketts and Calvin, 1952)
It is found 1,5-3.1 meters above the height reached by
the highest spring tides. (Light et al., 1954 and Hyman,1964)
This is sigmificant because it means that littorines are
never submerged by the tides and are exposed to the air
at almost all times. While littorines are never completely
covered with water, they are splashed with water from the
air-water interface. The detrital matter and microscopic
algae whicharewashed up by this spray suppl the
animals' major source of food. The significance öf
these facts is that littorines are exposed to pollution
from both the air and the water. They are splashed by
and eat the detrital matter washed up by the air-water
interface, which is thought to concentrate heavy metals.
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(Baxter and Lopez, 1971). Being exposed to the air,
littorines are subject to aerosol metal fallout, notably
that of lead. As littorines are scraper feeders, they
ingest any metal fallout from the surrounding rocks when
they eat. These facts make L. planaxis an interesting
animal to study in connection with heavy metal pollution.
L. planaxis is also useful because it has a marked an
dependable behavior pattern. As it has a gill partially
adapted for air breathing, it cannot live indefinitely in
water and if forcibly submerged, it will ultimately
drown (Ricketts and Calvin, 1952). It therefore reacts
quickly to being submerged in sea water, and will immediately
right itself and start to crawl up the nearest vertical
surface. This behavior has been used in the past to assay
the toxicity of sea aenemone nematocysts (Phillips and
Abbott, 1957), and it provides an almost ideal system in
which to study the effects of almost any toxicant on
behavior.
Heavy metals such as cadmium, copper, and silver
would be expected to affect behavior in that they are
toxic substances. These metals block the activity of
many enzymes and would therefore be expected to alter
bodily functions such as respiration and digestion.
Lead affects the production of porphyrins, the respiratory
pigments present in most animals and plants. In its organic
form, lead causes severe nerve damage. Any one of these
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things would interfere with the animals' normal functions
and would therefore probably alter behavior.
The purpose of this experiment was to assay the
various toxicitiea of cadmium, copper, silver and lead and
to see how much several different concentrations of each
metal would differentially affect behavior of L. planaxis.
I would like to acknowlege the patient and generous
help of Dr. Malvern Gilmartin, Dr. D.P. Abbott, and
Dr. John Martin in preparing this project.
Materials and Methods:
a. Sampling procedures:
The snails were collected fresh for each day's experi-
ments. The animals were used immediately after capturen
and throughout the day up to 12 hours from the collecting
time. They were selected from a single location at Hopkins
Marine Station that was exposed to splash from heavy surf
at high tide. (See figure 1) All animals selected were
collected 1.8-3.1 meters above the high tide mark and were
of as uniform size as possible. They varied in size from
0.8-1.6 grams with an average size of 1.2 grams. Since
it has been shown that orientation to gravity effects the
behavioral response (Klabunde, 1964) only animals from
exposed, vertical surfaces were collected. As May falls
within the breeding season for L. planaxis, only copulating
pairs of snails were selected to insure a male-female
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ratio as close to l:l as possible, although it is recognized
that such pairs are not necessarily male-female and may
be male-male. (Gibson, 1964)
b. Assay of the effects of heavy metals
Solutions of cupric nitrate, lead nitrate, and silver
nitrate were made separately in distilled water so that each
principal metallic ionic species was in a concentration of
300,000 ug/1. Cadmium sulfate was dissolved in filtered
sea water to the same concentration. The presence of Cd'
ions already in the sea water was ignored as not being
significant. These solutions were kept in plastic bottles
and were used as the stock solutions for each day's work.
The test solutions were made up daily in sea water that had
been filtered through Whatman GF/C filter pape. The
dilutions for silver, copper and lead were 3,000 ug/1,
600 ug/1, 300 ug/1 and 30 ug/1 in sea water. The presence
of any metallic ions in the sea water was ignored as being
insignificant at the concentrations used. The concentrations
of 3,000 ug/1 for these metals actually represent saturated
solutions in sea water.
The cadmium solutions were made up in filtered sea water,
and as cadmium is much more soluble than the other metals,
it was therefore possible to meke up much more concentrated
solutions. The solutions used were: 300,000 ug/1,
30,000 ug/1, 15,000 ug/1, 6,000 ug/1, 3,000 ug/1, 300 ug/1,
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and 30 49/.
Controls for copper, silver and lead were made by
adding 1 ml of distilled water to 99 ml of filtered sea
water, with successive 5,2, and 10 fold serial dilutions.
The control for cadmium was filtered sea water. The test
solutions were made in 50 mà Erlenmeyer flasks. All
glasswear was rinsed in 4 N nitric acid before use.
c. Testing procedures
The animals were chosen randomly from the collected
samples to make up test groups of 15 snails each. The
first whorl of the animals' shells were filed off, thereby
exposing the mantle cavity. The animal was further exposed
by picking off the second whorl with a dissecting needle
while being careful not to puncture the membranes surrounding
the animals' internal organs. This procedure only uncovers
the digestive gland (Fretter and Graham, 1962) and has
no immediate effect on the amimal's behavior. However it
can only be used for short-term experimants, as it
leads to dessication.
The animals were introduced to the testsolutions
and kept forcibly submerged by wedging plastic screening
down the neck of the flask. The test solution were refrig-
erated at 12 C for 24 hours.
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After the incubation period, the test animals were
removed from the solutions and blotted on apaper towel.
They were then dropped, spire-downward, at 2 second intervals
into 15 150 mmx 16 mm round-bottom test tubes that had
been filled with fresh, normal sea water.
The time it took the animals to right themselves and
the time it took them to climb 90 mm up the test tubes
were noted to the nearest 10 seconds. For convenience,
30 minutes was chosen as the trial cut-off point. Three
trials of 15 snails each were used for each concentration of
each metal and the control.
As 8% of the controls did not right themselves in
30 minutes, these animals were considered aberrant in
behavior and were eliminated from statistical calculations.
The highest 8% of the righting times were eliminated from
all concentrations of the metals. For each concentration
of each metal, the mean, variance and standard deviations
were found. Each concentration of control was treated
separately. Student's t-test were run for each control
group against every other control group. (Simpson et al.
1960). It was shown that the controls were all the same
within the.05 probability range. Then the mean, variance
and standard deviation of all the controls, taken as a
group were used in t-test with the concentrations of metals.
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Results:
The relative toxicity of each metal at different
concentrations is shown by figure 2 which plots the
difference between the percentage of controls that
righted themselves and the percentage of treated animals
that righted themselves within a certain period of time.
The relative toxicities of all the metals at a single
concentration are graphed in figure 3. The vertical
axis represents differences in percentages between the
numbers of righted animals in the conrrol and the test
solutions. The horixontal axis represents time in minutes.
The means variances, and standard deviations with each
concentration of all the metals arelisted in fiugre 4.
To reach these figures 8% of the highest numbers were
rejected. Student"s t-tests were run to determine
uniqueness of groups. The starred groups were those found
to be different from the control at the.05 level.
The data on crawling times for the snails was rejected
as being too erratic.
Discussion:
The results obtained by these experimants show that
differentsconcentrations of cadmium, copper, and silver
definitely affect the righting response of L. planaxis.
With the exception of lead, the greater the concentration of
the metallic ion in water, the longer the righting response
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was delayed. This trend is especially evident in the cadmium
solutions where it was able to concentrate the solution
enough to reach a lethal dose. Cadmium is also interesting
in that there is a very marked threshold effect between
6,000 ug/1 and 3,000 ug/1; all the animals died at 6,000 u9/
and all lived at 3,000 ug/1 albeit they sho ed a delayed
righting response.
Silver and copper also  trends that seemed to
indicate that threshold effects would be observed at
higher concentrations. Unfortunately 3,000 ug/1
represents a saturated solution in sea water for these
metals. The only metal that had no clear cut effects
on righting response was lead,
As shown in figure 2, at high concentrations, all
other things being equal, copper and silver showed a
trend in being more toxic than either lead or cadmium.
In lower concentrations, the relative toxicities of the
different metals was not so obvious. Et lower concentrations
copper semms to be the most toxic of the tested solutions,
The data on crawling times was rejected because
t-tests revealed them to be random functions. This can
be explained by the fact that there are many more variables
involved with crawling time than with righting time.
Crawling time is influenced by the past history of the
animal, notably the surface from wheih it was collected.
It is also influenced by such physical factors such as
183
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light intensity and direction. All such factors make the
experiment difficult to control.
A problem with this experiment is that the concentrations
of metals used in these experiments do not reflect the possible
situations in nature. The actual concentrations of these
metals in sea water are: Cd: 0.1 ug/1, Pb: 0.03 ug/1.
Cu: 3 ug/1, and Ag: 0.3 ug/1. (Goldberg, 1963). Consequently,
the results observed are due to acute conditions and could
not possibly be duplicated in nature during the same length
of time. A series of experiments that would better reflect
the situation in the real world would be to dose the snails with
heavy metals in low concentrations for a longer period
of time. However, if these experiments were undertaken, the
technique would have to be changed as th animals would show
the effects of dessication after two days. A possible
technique for long-term experimentation would be
to fill the mantle cavity several times daily with treated
water. Another method would be to culture microscopic algae
so that heavy metals were concentrated in the culture which
would then be fed to the littorines.
The relative toxicities of any set substances depends
greatly on how they actually act in the body. It is surprising
t hat copper, which is contained in hemocyanin in moalusc
blood, is so toxic to them. Copper in small amounts is an
essential element for life, but in larger amounts, it is
extremely toxic. The electronegative elements such as
1+ and Ag' have an affinity for amino, imino, and sulphydryl
185
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groups which are reactive sites for many enzymes and therefore
undoubtedly block these enzyme functions. (Bowen, 1966).
Cadmium is another trace metal that blocks enzyme action. It
competes with zinc for binding sites on thiol groups; it
is bound more firmly to these groups than zinc. (Schroeder
et al., 1967). Cadmium is not an essential element for life.
Lead, on the other hand, does not necessarily block
enzyme functions, and, therefore, would not cause any immediate
acute effects. It does have several long-term effects in
that it can cause abnormalities in the synthesis of porphyrins.
and, in the organic form, can cause nerve damage. (Air
Resources Board, 1967). However, the long-term effects
of these doses are quite toxic. Lead is particularly interesting
with respect to L. planaxis in that this animal is one
of the few intertidal animas that rarely, if ever, get
sub merged by tides. Therefore they would also pick up
more lead fallout when feeding because they scrape the tops of
the rocks to gather up food and would, at the same tine.
pick up lead aerosol fallout. It would prove to be an
interesting experiment to measure lead content in L. planaxis
from different areas to see how concentrations varied
with location.
185
References
Air Resources Board. 1967. Lead in the Environment and
Its Effects on Humans. Sacramento. 81p.
Baxter, Keith and Glenn Lopez. 1971. Trace Metal
Concentrations at the Air-water Interface and
Sub-surface waters of Monterey Bay. Unpublished
manuscript on file Hopkins Marine Station.
Bowen, H.J. 1966. Trace Elements in Biochemistry. Academic
Bress. New York. 241p.
Fretter, Vera and Alastair Graham. 1962. British
Prosobranch Molluscs. Ray Society. London. 756p.
Gibson, Daniel G. 1964. Mating Behavior in Littorin
planaxis. Unpublished manuscript on file Hopkins
Marine Station.
Goldberg, E.D. 1963. The Oceans as a Chemical System. p. 3-25.
In M.N. Hill (ed.) The Sea, v. 2. Interscience. New York.
Hyman, Libbie Henrietta. 1967. The Invertebrates. v. 6.
Mollusca I. McGraw-Hill. New York. 762p.
Klabunde, Baul Jon. 1964. The Geotactic Responses of
Littorina planaxis. Unpublished manuscript on file
Hopkins Marine Station.
Light et al. 1954. Intertidal Invertebrates of the Central
California Coast. University of California Press.
Berkeley. 446p.
Phillips, John H. and Donald P. Abbott. 1957. Isolation
and Assay of Nematocyst Toxin of Metridium Senile
Fimbriatum. Biol. Bull. 113:296-301.
786
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Figure Legends
Figure 1: Map of China Point showing collection site.
Figure 2: Effects of different concentrations of
heavy metals on righting time.
Figure 3: Relative toxicities of heavy metals at
different concentrations.
Figure 4: Means, variances, and standard deviations
righting times for Littorina planaxis tested
at several concentrations of heavy metals
in sea water.
188
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6,000
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300
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600
300
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Pb 3,000 ug/1
600
300
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Ag 3,o00 ug/1
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300
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Control, dist.water
Control, sea water
Figure 4
Stan.
Variance Dey.
Mean
*
0*
8.95*
40.19
6.34
3.56
2.43
1.56
5.46
87.42
9.35
13.51*
96.70
9.83
6.91
79.74
8.93
86.89
8.17
9.32
4.76
13.00
3.61
5.39
24.27
4.93
3.65
9.61
3.09
3.42
3.06
1.75
4.29
16.11
4.01
13.964
103.80
10.19
4.60
5.72
2.39
3.18
3.37
1.84
2.76
1.92
1.39
3.71
10.76
3.28
4.01
31.16
5.52
72