Concentration of C-DDT by a marine
planktonic copepod: from sea water and
from consumed phytoplankton
Mona Baumgartel
Hopkins Marine Station of Stanford California
Pacific Grove, California 93950
Abstract
The herbivorous marine planktonic copepod Calanus
helgolandicus was incubated in sea water which contained
C+4-DDT or phytoplankton labeled with C+4-DDT to study
the entrance of DDT into the marine food web at a low
trophic level. The copepods concentrated C++-DDT by a factor
of about 10,000 in 24 hours and continued to take up C+4-DDT
from low levels in the water in ten-day experiments.
C+-DDT was also found in copepods which had been fed
C+-DDT-containing phytoplankton.
32
Introduction
The damage which DDT is causing to birds at the
upper end of the marine food web is well documented
(Wurster and Wingate, 1968). Although the levels of DDT
which contaminate the world's oceans are low (of the
order of parts per trillion), the pesticide is concen-
trated in the food web and stored in lipid deposits so
that it may be over a million times more concentrated
in high level carnivores such as the Bermuda petrel and
peregrine falcon. There has been no direct evidence
of this concentration phenomenon at lower trophic levels.
I examined the entrance of DDT into the marine food web
by measuring the uptake of radioactively labeled DDT
by the planktonic copepod Calanus helgolandicus. Groups
of these animals were incubated in sea water containing
1-DDT or were fed C1-DDT-labeled phytoplankton.
Methods and Materials
Calanus helgolandicus individuals were selected from
zooplankton tows taken in Monterey Bay, California.
A one-meter diameter net (0.33 mm mesh aperture) was
towed for 5-15 minutes at various depths. In April and
early May, 1969, surface tows brought up large numbers
of adult female C. helgolandicus, but later in May, only
younger stages could be found and only at 20-60 meters.
This species was much less abundant through the summer,
and at times it was not possible to get enough individuals
because of large numbers of salps. However, one tow in
early August was nearly all Calanus helgolandicus.
Stage V copepodites were used in some experiments.
The copepods were kept in open jars at 9-14°C under
reduced illumination. Once or twice a week stocks were
transferred by pipette to fresh sea water which had been
filtered through cotton or Whatman 1 filter paper and
were fed small quantities of phytoplankton. The appear-
ance of fecal pellets was evidence that the animals were
feeding. Adult females survived very well under these
conditions. After the first few days in the artificial
environment, nearly all lived for many weeks. Stage V's
survived less well, possible because of the high mortality
during molting (Mullin and Brooks, 1967).
-4-
Incubations to determine the uptake of C-DDT from
sea water were carried out in screw-capped gallon jars
or sealed quart Mason jars to prevent loss of the pesticide
by codistillation. The jars were kept in water baths
at 9-14°0 under reduced illumination. The gallon jars,
which were used for short term experiments, contained
40 copepods at a density of 50 ml/animal in previously-
aerated, membrane-filtered sea water. The quart Mason
jars, used for ten-day experiments, each contained 10
copepods in 500 ml of sea water. In both situations,
C-DDT was added to the water in test jars before the
animals. C-DDT was either used directly from a stock
solution (100 ug of ring-labeled C-DDT from Amersham/
Searle Corporation per ml of absolute ethanol) or diluted
first in ethanol or sea water so that very low concentra¬
tions would be made conveniently. In a control run with
0.258 ethanol (the highest ethanol concentration used
in experiments), the behavior and survival of the copepods
did not appear altered by the alcohol. At various inter-
vals in the uptake experiments, copepods were removed
from the gallon jars in groups of about ten along with
enough water to maintain the original density of 50 ml/
animal. When quart jars were used, the contents of one
whole jar were removed and the remaining animals were
transferred to fresh C'"-DDT-containing sea water to
35
approximate conditions of constant flow. The copepods
were filtered onto Whatman l filter paper, washed with
distilled water, and place in liquid scintillation vials
to which a solution of the dyes PPO (4 g/1) and POPOP
(0.1 g/1) in toluene was then added. The vials were
counted on a Nuclear-Chicago Unilux II Liquid Scintilla-
tion Counter (95% confidence limits: 2n, where n
is the number of counts). It was discovered that even
if each copepod exoskeleton was punctured, the radio-
activity did not dissolve completely in the toluene
solution for 2-4 days. Hence, all the final data is
based on counts made four days after the completion of
each experiment. The specific activity of the C-DDT
was 19 mC/mM, so that one disintegration per minute
(dpm) represented 8.38 pug of C-DDT.
Since drying C"-DDT-containing copepods may drive
off radioactive DDT and contaminate equipment, average
dry weight determinations were made on large numbers of
stock animals instead of drying test organisms. Animals
were dried at 50°C for 12 hours in 10 or more groups of
10 or 20 in aluminum cups. This was done for animals from
each new tow. Computations using the student's t test
showed there was little variability among groups from
a single tow, but the range was great between the means
for different tows, probably because of varying selector
Je
-6
biases: 120-200 ug for adult females and 90-120 ug
for stage V copepodites. This means that experiments
done with animals from a single tow are comparable
because of a common factor, while data from experiments
using animals from different tows can be related only
qualitatively since the amount of radioactive DDT taken
up by the copepods is expressed as uug C-DDT per ug
average dry weight (or parts per million).
Diatom cultures were grown in 250-500 ml Ehrlen¬
meyer flasks containing 50-200 ml of an enriched sea
water medium (500 mole/1 KNO,, 50 mole/1 K,HPO), 500 mole/1
Na,SiO, plus trace amounts of Fecl,, MnSo, Znso,,
Na,Moo, Cocl,, CuSO, EDTA, thiamine, B,», and biotin).
The flasks were kept under constant illumination at 14°0
and were shaken occasionally. Fresh cultures were started
by one-milliliter inoculations into fresh medium. The
populations were mixed, but in April and May were rich
with Skeletonema costatum, along with Thalassiosira sp.,
Chaetoceros spp., Nitzschia sp., and Asterionella sp.
The cultures flourished much less consistently in the
summer months with many not growing at all. The most
successful diatom in June, July, and August was Asterionella.
Skeletonema did not appear in summer phytoplankton tows,
and the old Skeletonema cultures could not be recultured.
Rhizosolenia was common in tows but did not grow in
-7-
cultures. The earlier Skeletonema cultures were followed
closely by counting them in a Sedgwick-Rafter counting
chamber. Doubling time was about 12 hours until the cell
density reached 1 to 2 x 102 cells/ml. No successful
means was found for counting the later cultures since the
bouyant cells (mostly Asterionella and Chaetoceros) did
not sink to the bottom of the counting chamber. An attempt
to follow cell density by measuring optical density with
the Klett spectrophotometer was also unsuccessful since the
difference between no cells at all and maximum density
was only about 10 Klett units. Those summer cultures
which grew reached their peak in a week or longer.
Only cultures which were still in logarithmic phase
were used for feeding experiments. The cells were incu¬
bated in 10 ug/ml C-DDT for 15 minutes. Previous work
(Södergren, 1968) indicates that the unicellular alga
Chlorella removes 52-77 of a low concentration of C-DDT
(0.6 ug/ml) from the medium in 15 seconds. The cultures
were centrifuged briefly, washed twice in membrane-filtered
sea water, and resuspended to the original volume. Non-
radioactive cells which had undergone this treatment
recovered and grew in fresh medium. Nearly all of the
cells in predominantly Skeletonema cultures came down
in the pellet so the final resuspension had the same
density as the original culture. The washing procedure
-8-
diluted C-DDT not taken up by the cells to less than
0.01%. However, this procedure involving centrifugation
was not successful with Chaetoceros cells and those in
all the later cultures since these cells did not spin
down. This unfortunate circumstance meant that only a
few feeding experiments were successfully carried out.
Radioactivity in the phytoplankton resuspensions was
measured by counting 2.0 ml samples dissolved in 10 ml
of Bray's solution (100 ml ethanol, 20 ml ethylene glycol,
4 g PPO, 0.2 g POPOP, and 60 g naphthalene to one liter
in dioxane) in the liquid scintillation counter.
Feeding experiments were carried out under the same
conditions as uptake experiments with 30 animals per
gallon jar in indirect light to maintain the phytoplankton.
C'-DDT-labeled phytoplankton was added at a concentration
of about 10" cells per ml of copepod culture. When the
copepods were removed and filtered, they were gently
washed off the first filter and refiltered to reduce
contamination from labeled phytoplankton cells.
Results
Figures 1 and 2 show uptake of C-DDT by adult
female Calanus helgolandicus over a 32-hour period from
0.1 and 1 part per billion (ug/1) in the water. In both
cases, the DDT was about 10,000 times more concentrated
in the animals, in terms of dry weight, in 24 hours.
The copepods removed 3-4% of the C-DDT which had been
added to the culture. A similar curve was obtained for
stage V copepodites incubated in 25 part per trillion
for four and a half days (Figure 3). Over the longer
time period, the animals accumulated DDT from water of
low concentration to intra-animal concentrations nearly
as high as from water with four times more DDT.
Figure 4 shows the data for uptake of C-DDT by
adult female C. helgolandicus over a ten-day period from
50 ppt in the water in three separate experiments.
The curves are not identical, probably because of varia¬
tions in the exact amounts of C-DDT present and in
the animal dry weights (see discussion above in Methods
and Materials), but each shows that the rate of uptake of
C-DDT decreases after the initial rapid rate and suggests
that concentrations in the animals may be stabilizing.
Experiments with lower sea water DDT concentrations
(Figure 5) also show this decrease in rate, and, in fact,
20
-10-
these curves suggest after an initial period (two days),
the copepods takeup
-DDT at a constant rate which
is proportional to the amount of C-DDT present.
In all of these experiments, the amount of C-DDT was
not limiting as less than 10% of that in the water over
any period of time was removed by the animals.
Figure 6 shows that the copepods did take up
radioactive DDT from ingested phytoplankton in three
tests with stage V copepodites. That the animals were
accumulating C"-DDT as they ate the diatoms is clear,
although it is not known why in one test, the second group
of copepods removed had less C-DDT than the first.
Of the stage V's in two of these test, more died in the
test jars than in the control: 11 of 32 and 10 of 34
compared to 6 of 31 in the control. In a separate
preliminary experiment with stage V's, molting individuals
suffered a higher mortality rate in water with 1 ppb
DDT than in 10 ppt DDT or in the control. But in the
feeding experiment, two dead copepods from each of the
test jars were filtered, washed, and counted and were
found to contain less C-DDT than those which remained
alive, so that whether DDT was associated with these
deaths is unclear. In these two test situations, approxi-
mately 243 of the starting radioactivity was accounted for
in the live and dead copepods. The C-DDT concentrations
C
-11-
in the animals were of the same order of magnitude as
for uptake from 1 ppb in water although there was about
one-tenth as much radioactive DDT present. This data.
combined with a very rough determination of phytoplankton
dry weight, suggests that the copepods ate 4 ug, dry
weight, of phytoplankton tissue in 24 hours.
-12-
Discussion
The solubilities of DDT in oil and water are about
10% (Södergren,1968) and 1.2 parts per billion (Bowman,
Acree, and Corbett, 1960). In view of this high partition
coefficient, it is not surprising that the copepod Calanus
helgolandicus rapidly concentrates DDT from the surrounding
sea water into its lipid-containing tissues, and the copepod
may be compared to a small drop of lipid with a high
surface-to-volume ratio.
The experiments done in sea water C-DDT concentra¬
tions of 0.1 and 1 ppb are not ecologically significant
because, although DDT is soluble in water up to 1.2 ppb,
natural concentrations are much lower even in areas with
high local contamination. But it is significant that
uptake was detected at 5, 25, and 50 ppt, or nearer
supposed natural ambient levels. The ten-day experiments
suggest that a steady rate of uptake of DDT from low
concentrations in the water into higher concentrations
in the animals is established after a few days of rapid
uptake and that this rate is higher when DDT in the water
is more concentrated. Since the C-DDT taken up in these
experiments was in addition to that cold DDT already
taken up in the environment and since the natural levels
of DDT in C. helgolandicus populations and in Monterey
43
-13-
Bay water are not known, the environmental significance
of the experimentally observed kinetics of uptake cannot
be evaluated.
ci
DDT also concentrates in phytoplankton, and
Calanus helgolandicus, by feeding, can take up this DDT
more readily than that which is dissolved in sea water.
This is direct evidence of transfer of DDT from a primary
producer to a herbivore in the marine food web. Although
the phytoplankton were exposed to non-ecological levels
of C-DDT to load them for feeding to the copepods,
transfer has nevertheless been demonstrated. These findings
suggest that, in their natural environment, copepods may
accumulated DDT from their food, as well as, to a lesser
extent, straight from the water. It would appear that
copepods would continue to get more and more DDT as they
eat more DDT-containing diatoms.
The DDT which is accumulated and stored in Calanus
helgolandicus is further concentrated and stored in higher
trophic levels because these copepods are part of the diet
of some kinds of fish and other zooplankton predators.
Uptake of DDT directly from water by planktonic organisms
may help to maintain very low levels in the surrounding
water while functioning as an entry point for DDT into
the marine food web at its broad base.
-14-
Acknowledgements
I wish to thank Professor Ellsworth H. Wheeler,
Jr., for many kinds of help in this research. This work
was supported in part by the Undergraduate Research
Participation Program of the National Science Foundation,
Grant 164-5878.
-15-
References
Bowman, M. C., F. Acree, and M. K. Corbett. 1960.
Solubility of carbon-14 DDT in water. Agricultural
and Food Chemistry, 8:406-408.
Mullin, M. M., and E. R. Brooks. 1967. Laboratory
culture,agrowth rate, and feeding behavior of a planktonic
marine copepod. Limnology and Oceanography, 12:657-666.
Södergren, A. 1968. Uptake and accumulation of C-DDT
by Chlorella sp. (Chlorophyceae). Oikos, 19:126-138.
Wurster, C. F., and D. B. Wingate. 1968. DDT residues
and declining reproduction in the Bermuda petrel.
Science, 159:979-981.
-16.
PPO:
diphenyloxazole; POPOP:
phenyloxazolyl) benzene.
1,4 biss2-(5.
47
-17-
Figure Legends
Figure 1. Uptake of C1"-DDT by adult female Calanus
helgolandicus from sea water containing 0.1 parts per
billionC -DDT.
Figure 2. Uptake of C14-DDT by adult female Calanus
helgolandicus from sea water containing 1 ppb C -DDT.
Figure 3. Uptake of C1"-DDT by stage V copepodid Calanus
helgolandicus from sea water containing 0.025 ppb C -DDT.
Figure 4. Uptake of C"-DDT by adult female Calanus
helgolandicus from sea water containing 0.050 ppb C -DDT.
Figure 5. Uptake of C'"-DDT by adult female Calanus
helgolandicus from sea water containing 0.005 and 0.025
ppb C -DDT.
Figure 6. Uptake of C-DDT from C-DDT-labeled phyto-
plankton by stage V copepodid Calanus helgolandicus.
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