Concentrations of DDT sublethal and lethal to Calanus
helgolandicus (Claus), and effects of sublethal concen-
trations on respiratory rates
A. Decker Hunter
Hopkins Marine Station, Stanford Universityl
Running head: Effects of DDT on calanoid copepods
ABSTRACT
Marine copepods (Calanus helgolandicus) were classified as visi-
bly unaffected, visibly affected, or dead after varying periods
of incubation in varying concentrations of DDT. At 24 hours 50%
were affected (visibly affected plus dead) by a predicted 60 ppb;
at 48 hours 50% were affected by a predicted 5.8 ppb. It was
found that 1 ppb DDT increased the rate of respiration over that
of the controls.
8.
INTRODUCTION
Until recently, little was known of the effects
of DDT on marine organisms. With analyses demonstra-
ting the presence of DDT in such animals as the pere¬
grine falcon (Risebrough, 1968) and various other sea
birds (Risebrough, personal communication), blue fin
tuna, northern anchovy, English sole, Pacific jack
mackerel, and squid (Risebrough, 1967), the preva-
lence of the pesticide in the ocean was established.
Its deleterious effects have been documented in ani-
mals high in the pelagic food web; little work, how-
ever, has been done on the effects on those animals
at the base of the food web, the zooplankton.
The marine copepod, Calanus helgolandicus, was
chosen because it is representative of the zooplank-
ton, easily obtainable, and has been the subject of
many respiration studies (Marshall and Orr, 1966; Cono-
ver, 1960; Zeiss, 1963; Raymont and Gauld, 1951).
The aims of this study were to determine the
visible effects of various concentrations of DDT in
the medium and to make preliminary investigations
of the effects of 1 ppb DDT on the rate of oxygen
consumption of these animals.
The author wishes to thank the staff of the
Hopkins Marine Station, and in particular, Dr. E. H.
Wheeler, Jr., without whose advice and assistance
B
also
this project could not have been undertaken.
thank Thomas C. Malone for his helpful suggestions on
the respiration experiments. This work was supported
in part by the Undergraduate Research Participation Pro-
gram of the National Science Foundation, Grant foY-58
METHODS
Were obtained from Monterey Bay, Call
in late April and throughout Nay, 1960
With a meter net of 0.333 mm mesh aperture both ver-
tical and oblique tows were made from depths of 25 m
Immediately after capture indivi
to the surface.
duals were placed in pre-cooled sea water until s.
were
ting; adult femaleg, used in most experiments,
kept in the laboratory in membrane filtered (0.8u
During the last two weeks of May only
sea water.
thes
stage five copepodite females were available and
The ex
were used with no apparent change in results.
perimental animals were maintained in semi-darknes:
in large, uncovered glass jars at an approximate
density of 1 animal/20 ml. The sea water, kept a
The copepods were
C. was changed twice weekly.
fed with mixed phytoplankton cultures of Skeletonema
costatum, Thalassiosira sp., and Chaetoceros sp.
They were removed from feeding jars at least thre
hours prior to the beginning of any experiment.
Under these conditions the animals survived
ix weeks.
for 24 or
were carried out
Incubations in DDT
hours in closed glass liter jars. Because DDT co-
distills with water, stock and experimental jars
were opened as infrequently as possible. Since the
pesticide has a low solubility in water (1.2 ppb
fresh stock solutions were made up daily to prevent
partieles from settling out. Ten adult females were
placed in each jar at a density of 1 animal/50 ml
and appropriate concentrations of p,p' DDT dissolved
in 954 ethanol were added. For the 24 hour experi-
ments Dor concentrations ranged frou 10-500. ppb, for
the 48 hour experiments from 1-50 ppb. The values
were chosen after preliminary screenings at higher
and lower concentrations. Ethanol controls were run
with all experiments.
Atter incubation, copepods were removed indivi
dually from the jars and examined under a dissecting
microscope. They were classifled as visibly unaf-
fected, visibly affected, and dead. Visibly affected
animals were those unable to swim actively or at-
tempt to escape the presence of the pipette. They
rested without moving on the bottom of the jars or
twitched spasmodically and asymmetrically. Swimming
movements were ineffectual. If no twitching was ob-
served, heart beat and/or anal contractions identi-
fied a visibly affected animal.
The animals in the respiration experiments were
ncubated at 1 ppb in the closed jars described a-
bove for 0-144 hours. As before ethanol controls
were run with all experiments. Twenty animals were se-
lected from the closed jars with a large pipette at 24
hour intervals and placed directly in 300-ml dark
BOD bottles (1 animal/15 ml) containing 1 ppb DDT in
previously aerated sea water for eight hours. In
each set of experiments initial oxygen concentrations
were determined on duplicate bottles with correspon-
ding ethanol concentrations. Titrations were done
using the standard Winkler technique with 0.02 N thio-
sulfate. The water in the incubation jars was changed
periodically and results were not corrected for bac-
terial respiration. All phases of these experiments,
except titrations, were conducted at 12° C.
RESULTS
Figure 1 shows representative results of the 24
hour incubations in DDT. There is a clear difference
between the effect on the experimental animals and
that on the controls. From the regression line,
fifty per cent of the animals are affected at 60 ppb.
The rank difference coefficient is 0.905.
The data from the 48 hour experiments is illus-
trated in Figure 2. Because of the longer incuba-
tion period lower concentrations of DDT are effec-
tive. From the regression line, fifty per cent of the
organisms are affected at 5.8 ppb. The rank difference
coefficient is 0.95.
Two typical respiration experiments are sum-
marized in Figure 3. At each point in a particular
experiment, the oxygen consumption of the experi-
wental animals was kigher than that of the controls.
There is an obvious peak at 48 hours in both experi-
ments.
ESCUSSTON
concentrations below 50 pb do ne
Aitroeh
learly affect the copepods in 24 hours, the 18 hour
xperiments show a significant effect at as low as
This could indicate a cumulative effect
ppb DDT.
ith time until a critical level is reached, or that
with low concentrations, a longer time period is re-
quired to cause the metabolic damage which presumably
incapacitates the animals. Copepods were in-
cills or
cubated up to lu4 hours at 1 ppb before succumbing
to the pesticide; at this point the controls were
still lively. Asithe concentrations were increased,
the time required before visible effect was observed
decreased.
Concentrations as high as 10,000 ppb were tested
even at this level all animals were not killed
2b hours, although 100% were affected. Because so
much of the DDT precipitated at levels above 1000 ppb
most experiments were conducted at lower concentra-
to ensure as much accuracy and retention of the
tion:
original concentration as possible. This method was
not infallible, however, as white particles presumed
be DDf or associated with DDT were observed on the
appendages and antennae of the copepods incubate
for 48 hours at concentrations as low as 30 ppb
C
Control animals were never affected in this manner.
To clarify the data presented in Figures 1 and
2 no differentiation was made between visibly affected
and dead organisms. However, it is interesting to
note that all copepods at 5 ppb for 48 hours were
visibly affected but able to move sporadically, while
those at 10 ppb for 48 hours were all dead (33%) or
moribund (67%). At 20 ppb for 48 hours, 90% were dead
and 10% were moribund. (see Table 1.) Moribund ani-
mals are those in which only heart beat and/or anal
contractions were observed.
Since even the lowest concentrations used here
are higher than those in the natural marine environ-
ment, longer experiments at much lower concentrations
should be run. Studies should also be done on the
methods and sites of DDT action within the organism.
To determine the permanence of DDT damage, copepods
could be incubated in, and then removed from, varying
concentrations of DDT and their progress followed.
Copepods react to DDT with violent, rapid twitching;
a more complete study of these reactions should be
undertaken. The lower limit of DDT activity on cope¬
pods should be compared with levels found in both the
ocean and the phytoplankton they eat.
The respiration data given in Figure 3 has two
main characteristics: 1) the peak in respiration at
48 hours; 2) the fact that respiration of the expe-
rimental animals is always higher than that of the
controls. The difference in rates is neither cons-
tant nor consistent within any experiment, but ranges
from 0.06 to 2.07 ul 0,/animal/hour. In all experi-
ments at 1 ppb both of these characteristics appear.
After 96 hours of incubation in the storage jars,
the animals in Experiment B treated with DDT were be-
ginning to show effects of the pesticide. By 144
hours they were all moribund while the controls were
fairly lively throughout the experiment. Despite
this the respiration of the treated animals at 144
hours remained 0.356 ul 02/animal/hour higher than the
controls' respiration. Since respiration in cope-
pods is usually related to movement these results
are difficult to explain unless the amount of stress
experienced under such conditions is taken into ac-
count.
The peak found at 48 hours appears to be re-
lated to the presence of ethanol and not to that of
DDT. No such peak was found in sea water controls.
Possibly the ethanol causes a peak in activity around
48 hours which is reflected in the respiratory rate.
REFERENCES
Article: Conover, Robert J. 1960. The feeding behavior and re-
spiration of some marine planktonie Crustacea. Biolo-
gical Bulletin, 119(3): 399-415.
Artiele: Marshall, S. M., and A. P. Or. 1966. Respiration and
feeding in some small copepods. Journal of the Marine
Biological Association of the United Kingdom, U6: 513-530.
Artiele: Raymont, J. F. C., and D. T. Gauld. 1951. The respira-
tion of some planktonic copepods. Journal of the Marine
Biological Association of the United Kingdom, 28: 681-683
Article: Risebrough, R. W. 1967. DDT residues in Pacific sea
birds: a persistent insecticide in marine food chains.
Nature, 216(5115) 589-591.
1968. Polychlorinated biphenyls in the glob
Article:
ecosystem. Nature, 220(5172) : 1098-1102.
Article: Zeiss, F. Ralph, Jr. 1963. Effects of population den
sities on zooplankton respiration rates. Limmology and
Oceanography, 8: 110-1
FOOTN
Present address
DDr obtained from Aldrich Chem. Co., Milwaukee, Wisconsin.
able
Breakd
ected
3 concentrations DDT.
DDT
Affected
twitching
ibund
PDL
100
dead
LEGENDS
FICURE
in sea water. Three
DD
Per cent copepods affected
Fig.
24 hour experiments. Open circles: DDT; closed circles:
controls.
affected by DDT in sea water; three
Fig. 2. Per cent copepods
48 hour experiments. Open circles: DDT; closed circles:
controls.
Respiration rates of copepods incubated in 1 ppb DDT
Fig. 3.
Dashed line: exp. A; solid line: exp. B. Closed circles:
DDT; open circles: controls.
S
O
O
4
O
O
N
O
O
O
% Affected
D
O(
%
Affected
147
—
4.0 -
3.0
2.0
1.O
O.O
O
24 48 72
Hours
—
96 120 44
146