Introduction.
Mytilus californianus is an abundant, suspension feeding
lamellibranch which attaches to surf-beaten intertidal rocks
on the west coast from Alaska to Mexico. Like other marine
organisms analyzed for residues of chlorinated hydrocarbon
pesticides, this mussel concentrates DDT in its tissues.
Risebrough (1966) found 19 to 84 ppb DDT residues in the Mytilus
californianus on the California coast. Mussels are eaten by
several marine invertebrates (Pisaster; Feder, 1959), birds
(Night Heron; Palmer, 1962, and Black Oyster-catcher; Vebster
1941), sea otters (Limborough, 1961) and man.
Mytilus is exposed to DDT residues in the environment.
It tolerates considerable change in salinity (Fox et al, 1936),
and can inhabit areas receiving run-off from land. It feeds
on particulate matter to which DDT adheres readily. When Mytilus
concentrates plankton and detritus in insoluble pseudofeces and
feces, particles carryimg DDT are passed to the benthos.
The physiological effects of sublethal concentration of
DT in Mytilus are not known. Possible effects of DDT here
include alteration of shell deposition, since DDT is known to
affect carbonic anhydrase (Keller, 1932), and changes in meta-
abolic rate, foot and valve movement, ciliary beat, byssal
fiber secretion, and strength of the feeding current.
Filtering rate and respiration were chosen as indices of
damage to Mytilus caused by DDT taken up directly from sea
water or ingested with food. Both respiration and filtering
are performed by the gill. The gill musculature regulates the
size of the interfilamentary gill slits which in turn affects
filtration rate. Ciliary activity on the gill filaments is
affected by acetylcholine (Bulbring et al, 1953), and by other
activating compounds, such as serotonin (Aiello, 1960; Gosselin,
1961). Wurster (1969) suggests that DDT sometimes acts as a
neurotoxin which induces repetivive firing in the nerve axon.
If this occurs in the gill, aberrant filtering and respiration
rates might result.
Test organisms used in these experiments were collected
from April to June on Mussel Point in Monterey Bay, California.
The Mytilus population in this area declined sharply during
1969. I felt this decline might be attributable to increasing
levels of DDT in the bay, caused by extensive flooding of ad¬
jacent farmlands and massive run-off in January and February,
1969.
II. Determination of the rates of filtering and respiration
in Mytilus.
Filtering rate was determined by measuring the rate of
clearing by Mytilus of colloidal graphite particles ("Aquadag'
by the Acheson Co.) from suspension in sea water. Changes in
suspension density were measured in a Klett electrophotometer.
The organisms tested were sufficiently small (0.5 to 1.5 cm.
long) to be placed directly in the Klett tubes, alleviating
the need for sampling of the suspension. Other reasons for
selecting small mussels were that they are less sensitive to
handling than larger organisms (Jorgensen, 1949), and they
are less likely to spawn. Klett tubes containing Nytilus
were kept at about 15°C since increased temperature may induce
resting (Vallengren, 1905), while at normal sea temperatures,
Mytilus pumps continuously. All animals used were taken from
the same elevation because Mytilus from the lower intertidal
pump more rapidly than those from higher up (Segal et al, 1953)
The average diameter of the particles was kept betveen three
and 7.5 microns because particle size affects rate of filtering
(Tammes & Dral, 1955). Suspensions in the Klett tubes were
stirred with a glass rod at ten minute intervals, taking care
not to disturb the mussels. The density of the suspension
was read in the Klett immediately after the tubes were stirred.
Since the rate of filtering decreases with an increase in the
concentration of the suspension (Tammes & Dral, 1955), I chose
an initial suspension density of about 150 Klett units (10'
particles per ml.). Rate of particle precipitation is negli-
gible as compared with the average rate of removal of particles
by a healthy Mytilus (fig. 1). During the experiments with
DT, the rate of removal of particles was recorded for three
ten minute intervals after the suspension was down to 150 Klett
units. The average rate of removal of particles by Mytilus
D.5 to 1.5 cm. long was 222 units per hour.
Jörgensén (1949) points out that this method does not
determine the total amount of water filtered unless the sus-
pended material is retained by the gills. As part of my
preliminary study, I drilled holes in the shells of several
Mytilus, allowed them to recover, and observed them under the
microscope. When placed in a graphite suspension, these
mussels moved the particles down the filaments and along the
food groove. Most particles were then extruded as pseudofeces;
very few carbon particles were found in gut samples. MacGinitie
(1941) observed that Mytilus in a graphite suspension tend to
stop forming a mucous sheet on the gills, though the cilia
continue to beat. Considering this effect, the removal of
graphite particles is an index of filtering but not necessarily
an index of feeding.
Respiration was determined by measuring the uptake of
oxygen by Mytilus placed individually in 125 ml. reagent bottles
filled with filtered sea water which had been bubbled with air
until saturated. The initial and final oxygen content of the
bottles were determined by the Winkler method (Strickland, 1968).
known amount of Nitzschia closterium minutissima (2 x 10 cells/ml.
was added to each bottle as this suspension increased the mussel's
measurable activity. Bottles were incubated at 15°C in dim
light for six hours.
Experimental conditions, results and discussion.
A. The effect of direct uptake of DDT from sea water.
Tour groups of five test organisms each were incubated
at 15°C in concentrations of DDT of 1.0, 0.1, 0.01, and 0.001. ppt
in filtered sea water, for periods of 12 to 132 hours. Each
group of five test organisms was placed in 200 mls. of filtered
sea water with DDT in a closed 400 ml. air tight container in
order to prevent loss of DDT by codistillation. The DDT inoculant
was dissolved in 95% ethanol. The control organisms were
given an equal concentration of ethanol in filtered sea water.
Following exposure to the DDT, the mussels were tested for any
change in filtering rate.
In all cases, no damage attributable to the DDT was observed.
Even at the highest concentration, one ppb DDT (fig. 2), there
was little difference between the mean filtering rates of the
experimental organisms and the controls. However, Mytilus in
most experiments showed an initial increase and subsequent
decrease in filtering rate. Uptake of DDT during this experiment
was not determined, but parallel experiments using C-DDT under
similar conditions, indicate levels of about 150 ppb (Davis, 1969
wet weight of the soft tissues were probably attained.
8. The effect of ingestion of DDT with food.
In order to increase the concentration of DDT in the mussels,
test organisms were fed Nitzschia closterium minutissima incubated
in sea water containing DDT. Nitzschia was selected because it
is small (thirty microns long) and stays in suspension (Galstoff,
1937). Preliminary feeding experiments and gut analyses showed
that the Nitzschia was ingested. Some diatoms passed through
the Mytilus unaffected, but skeletons of others were found in the
fecal pellets. The Nitzschia (2 x 10 cells/ml.) were incubated
in one ppb DDT in filtered sea water for six hours. 200 mls. of
the Nitzschia-DDT suspension were added to each of three 400 ml.
air tight jars each containing five mussels. The 200 ml. sus-
pensions in the jars were changed every three hours over a 36
hour period so the DDT available to the Mytilus would not be
steadily depleted. Controls contained ethanol and Nitzschia
suspension, but not DDT. At twelve hour intervals, five test
Mytilus and five control organisus were tested for changes in
rate of respiration, then for changes in rate of filtering.
Mussels were tested individually, and tests were run alternately
on experimental and control organisms. After measuring respir-
ation and filtering rates, the organisms were sacrificed and
the soft parts were extracted and weighed.
The amount of DDT taken up by the Mytilus was determined
by using C-DDT in a parallel experiment. Mytilus incubated
in radioactive DDT with Nitzschia were allowed to remain in
filtered sea water for six hours after removal from the radio-
active suspension so that radioactive food in the gut would be
eliminated in fecal pellets. The soft parts of the organism
which had taken up C-DDT were weighed, and then homogenized
in four mls. of N,N, dimethyl-formimide. Ten mls. of Toluene
scintillating fluid were added; the vials were counted in a
scintillation counter.
The rate of filtering, respiration and C14-DDT uptake
were determined at 12, 24, and 36 hours (fig. 3). The average
ppb DDT (wet weight) accumulated by the mussels during these
incubations were 150, 438, and 549 respectively. The filtering
rate decreased in both the control and test animals. Respiration
increased and then decreased in both, but experimental animals
showed greater changes in respiration than the controls. The
trend toward initial increase in respiration after exposure to
39
DDT has also been found in studies on the effect of DDT on
phytoplankton (Bailey, 1969) and zooplankton (Hunter, 1969)
his experiment was repeated and extended to 96 hours.
The only changes in procedure were that the suspensions of
Nitzschia in DDT were changed every four hours, and the res-
piration and filtering measurements were conducted at 24 hour
intervals. The parallel C-DDT uptake experiments indicated
that the average DDT uptake was 562, 880, 1253, and 1307 ppb
wet weight) at the end of four sequential 24 hour incubation
periods. The greatest DDT concentration shown by any individual
Mytilus was 2.7 ppm at the end of a 96 hour incubation.
gain in this experiment (fig. 4), there were no statis-
tically significant differences in the rates of respiration
and filtering between the test and the control organisms.
Changes in the rate of filtering and in respiration associated
with an average uptake of 1.3 ppm DDT and lesser amounts are
obscured by the variability shown by individuals exposed to
milar conditions. Some variability is expected because single
organisms can vary the pore size of their gill filters and can
selectively exude a mucous sheet which will affect filtering rate.
Variability cannot be attributed to differences in established
tidal rhythms for oxygen consumption rate (Gompel, 1937, 1938)
and rate of water propulsion (Rao, 1954), since all animals used
in any particular experiment were collected at the same time
and place. Further experiments need to be run using Mytilus
which have attained still higher tissue levels of DDT.
IV. General Summary.
Mytilus californianus maintained in solutions of filtered
sea water of 1.0, 0.1, 0.01, and 0.001 ppb DDT showed no con¬
sistent change in rate of respiration or filtering.
Mytilus fed on Nitzschia in a one ppb DDT solution increased
the level of DDT in the soft tissues to 562 (range 29 to 911)
opb in 24 hours; to 880 (range 451 to 1358) ppb in 48 hours; to
1253 (range 390 to 2056) ppb in 72 hours; and to 1307 (range 641
to 2692) ppb in 96 hours. Measurements of respiration and
filtering rates taken of subsamples of the test group at 24 hour
intervals showed no significant difference between the test
organisms exposed to DDT and the controls.
Mytilus californianus can concentrate DDT in its soft
issues up to 2.7 ppm by wet weight with no apparent reduction
in the rates of filtering or respiration.
Lelet
RATE
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60
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130
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Acknowledgements:
sincerely thank Dr. Donald P. Abbott for his advice
and inspiration. I am also very grateful for the assistance
of Sam Johnson and John Miller.
his work was supported in part by the Undergraduate
Research Purticipation Program of the National Science Foundation,
Grant + 64-5878.
References cited:
Aiello, E. L. 1960. Factors affecting ciliary activity on the
gill of the mussel Mytilus edulis. Physiol. zool. 33: 120-
35 (as cited in Jørgensen, 1966).
Bailey, Susan 1969. Inhibition of the photosynthetic process in
marine phytoplankton by DDT and its derivatives (unpublished
m.s., on file at Hopkins Marine Station).
Bulbring, E. 1953. Acetylcholine and ciliary movement in the
gill plates of Mytilus edulis. Proc. roy. soc. Lond. B. 141:
445-66 (as cited in Jørgensen, 1966).
Davis, Karen 1969. Personal communication at Hopkins Marine Station
regarding studies on the uptake of DDT by Mytilus californianus.
Feder, H.M. 1959 The food of the starfish Pisaster Ochraceus along
the California coast. Ecology 40: 721-3.
Fox, D.L., G.W. Marks, D.O. Austin. 1936 The survival period of
adult mussels in sea water of various concentrations, in:
Fox, D. L. The habitat and food of the California Sea Mussel.
Bull. Scripps Inst. Oceanogr. Tech. Ser. 4:1-64.
D. L., W.R. Coe.1943. Biology of the california sea mussel
Fox,
(Mytilus californianus) J. exp. zool. 93:205-9.
Galstoff, J.G.N., F.E. Lutz, P.L. Welch 1937. Culture methods for
invertebrate animals. Dover Pub. 590pp.
Gompel, M. 1938. Recherches sur la consommation d'oxygene de
quelques animaux aquatiques littoraux. C.R. Acad. Sci. Paris,
205: 816-818 (cited in Rao, 1954).
Gosselin, R. E. 1961. The cilio-excitatory activity of serotonin.
J. cell comp. physiol. 58:17-26 (cited in Jørgensen, 1966)
Hunter, Alice 1969. Personal communication at Hopkins Marine Station
regarding studies on the effect of DDT on the respiration rate
rate of Calanoid copepods taken from Monterey Bay, California.
Jørgensen, C.B. 1949. The rate of feeding by Mytilus in different
kinds of suspensions. J. mar. biol. assn. U.K. 28:333-43.
Jørgensen, C.B. 1966. Biology of Suspension Feeding, Pergamon Press.
Keller, H. 1952 Naturwissenschafter 39:109 (cited in H.U. Bergmeyer 1963.
Methods for enzymatic analysis. Academic Press N.Y. p.626).
Limborough, C. 1961. Observations of the California sea otter.
J. of mammology. 42:271-273.
MacGinitie, G.E. 1941. On the feeding of four Pelcypods, Biol. Bull.
80:18-25.
Palmer, R.S. 1962. Handbook of North American birds. 1 Yale
Univ. Press. 567pp.
K.P. 1954. Tidal rhythmicity of rate of water propulsion
Rao,
in Mytilus, and its modifiability by transplantation. Biol.
bull. 106:353-359.
Risebrough. R.W., Mezel, Martin, Olcott 1967. DDT residues in
Pacific sea birds; a persistent insecticie in marine food
chains. Nature. 216:
jegel, E., K.P. Rao, and T.W. James 1953. Rate of activity as
a function of intertidal height within populations of some
littoral molluscs. Nature. 172:1108-9/
Strickland, J.C. and Parsons 1968. A practical handbook of sea
water analysis, Fish. Res. Brd. of Canada, Ottowa.
Tammes, P.M.L. and A.D.G.Dral 1955. Observations on the straining
of suspensions by mussels. Arch. neer. zool. 11:87-112.
Wallengren, H. 1905 (cited in Tammes and Dral 1955).
Webster, I. 1941. Feeding habits of the Black Oyster-catcher.
Condor 43:175-180
Wurster, Charles F. lecturing at Hopkins Marine Station on May 14, 1969.
Fig. 1.
Rate of clearing of colloidal graphite from suspension by
tilus. The most uniform clearing rates occurred at densities
between 150 and 50 Klett units.
Fig. 2.
Filtering rate of Mytilus exposed to 1 ppb DDT in filtered sea
water. Bars show means, 95% confidence limits, and ranges.
Striped bars indicate experimental animals, white bars the controls.
Fig. 3.
Rates of filtering and respiration in Mytilus fed on Nitzschia
in sea water containing one ppb DDT. Bars show means, 95%
confidence limits, and ranges. Solid black bars indicate
the C14-DDT uptake (use right hand scale.) Striped bars rep
resent test organisms, white bars the controls.
ig. 4.
Rates of filtering and respiration in kytilus fed on Nitzschia
in sea water containing one ppb DDT. Bars show means, 95%
confidence limits, and ranges. Solid black bars indicate
the C14-DDT uptake (use right hand scale.) Striped bars rep-
resent test organisms, white bars the controls.