UPTAKE AND DEPOSITION OF DDT-C1+ BY THE INTERTIDAL
TELEOST CEBEDICHTHYS VIOLACEUS AYRES
by
James Edward Sutton"
Hopkins Marine Station of
Stanford University
Pacific Grove, California 93950
* Send proofs and correspondence to:
James Edward Sutton
Hopkins Marine Station
Pacific Grove, California 93950
INTRODUCTION
That DDT and other chlorinated hydrocarbons are con-
centrated by freshwater fishes is well documented by nu-
merous studies (Johnson, 1968). The uptake has been shown
to occur by concentration through the food chain (Hickey
and Anderson, 1968; Woodwell, 1967), as well as by direct
uptake from environmental waters (Holden, 1962; Anderson
and Peterson, 1969). Recent studies have indicated sig.
nificant amounts of DDT in marine organisms, particularly
marine fish and birds (Risebrough et al., 1967). Although
in birds DDT is taken up through the food chain, the mech-
anism of DDT uptake by marine fishes has not been exten-
sively studied.
In order to assess the ability of marinefishes to
concentrate DDT from the environment, a study was made
of the intertidal teleost Cebedichthys violaceus Ayres,
the monkey-faced blenny eel. Rates of uptake and sites
14
of deposition were studied through the use of DDT-C
Studies were also made of the ability of the fish to take
up DDT from extremely dilute concentrations to determine
if significant amounts could be absorbed at the levels
of DDT naturally present in sea water.
MATERIALS AND METHODS
Specimens of Cebedichthys violaceus Ayres were obtained
during periods of low tide in April and May, 1969. They
were taken from the lower intertidal region, primarily
in the harbor of Monterey, California, and also off the
Point Pinos and Hopkins Marine Station beaches. The fish
were not fed during captivity, and for the most part only
fish of 7-9 cm in length were used in the experiments.
During experiments each fish was incubated in 500 ml
of sea water in a one quart capacity screw cap jar, to
which was added 0.005-0.05 ml of DDT-C.dissolved in
ethanol (specific activity: 19 mC/mM). The jars were
placed in running sea water at a temperature of 1441°C.
Controls were run under similar conditions except that
no DDT was added. No ill effects were observed in any
of the test animals, or in controls placed in twice the
volume of ethanol in sea water as the test animals. To
prevent loss of DDT from the water by evaporation or co¬
distillation (Acree et al., 1963; Wurster,1968), the jars
were kept closed.
After incubation each fish was weighed, swabbed with
95% ethanol to remove surface DDT, and sacrificed. The
brain, gills, liver (excluding gall bladder), and a sample
of skeletal muscle from the body wall were removed, placed
in tared pieces of aluminum foil, and wet weights obtained
on a Mettler H15 or HéT balance. The tissues were then
homogenized in 2.0 ml of N,N-dimethylformamide (DMF) (Mathe-
son, Coleman and Bell) and a 1.0 ml aliquot placed in a
scintillation vial. To this was added 10.0 ml of a tol-
uene-PPO-POPOP scintillation fluid: Counts were taken for
ten minutes in a Nuclear Chicago Unilux II Liquid Scin-
tillation Counter. Background vials contained 1.0 ml DMF
and 10.0 ml scintillation fluid. Quench correction was
made by the channels-ratio method.
RESULTS
Preliminary analyses on the extent of DDT-C uptake
were made on skin, skeletal muscle, liver, intestine,
stomach, heart, gills, brain, eye, and bladder of Cebe¬
dichthys. A twelve hour incubation in one part per billion
(ppb) DDT showed that liver and brain concentrated DDT
to the greatest extent, and on that basis these tissues
were chosen for further study. Although muscle did not
concentrate DDT to very high levels, it was also selected
for study as it constitutes the bulk of the animal's weight.
Entrance of DDT into the body is through the gills,
intestine, and skin. Gills were selected for further
study because levels in this organ were nearly as high
as those of brain tissue, and because there is evidence
from studies with non-feeding freshwater fish that the
gills are the primary means of entry (Holden, 1962).
Low levels were found in the stomach, but the intestine
was much higher, with concentrations approximating those
found in the gills. This may have been due to swallowed
water carrying DDT into the intestine, or to the blood
supply of this organ. Only very small amounts of DDT
were found in the skin, suggesting that absorption through
the skin contributes little to the total amount entering
the body. As the fish secretes a rather heavy mucus coat,
the constant shedding of this mucus might have limited
the penetration of DDT. From the above considerations
liver, brain, gills, and skeletal muscle were chosen for
further analyses of uptake phenomena.
Table 1 and Figure 1 show the uptake of DDT-Ct" into
the above organs from a 1 ppb solution in sea water as a
function of time. Each point in Figure 1 is the mean of
three fish incubated for 2/3, one, two, four, and eight
hours. It is seen that in each tissue there is a very
rapid absorption during the first two hours of incubation,
followed by a decreased rate of uptake. The high amount
of DDT indicated in the brain after one hour is the result
of an extremely high reading in one fish (114.6 ppb as
compared to 47.5 and 44.7 ppb in the other two fish); this
disproportionately affected the mean uptake.
Table 2 and Figure 2 indicate the extent of uptake
by the four tissues at three different concentrations
after eight hours of incubation. Each point in Figure 2
is an average of three fish, except for the brain and gill
readings in the 0.1 ppb series, which is the average of
only two fish. In all cases the liver accumulated the
greatest amount of DDT. It is also interesting to note
that the amount of DDT in the brain and liver is much
greater than in the gills at the higher concentrations
than at the lower concentrations.
Table 3 and Figure 3 show the number of times the
DDT has been concentrated by the tissues over the concen¬
tration originally present in the sea water (i.e., the
concentration factor). Although an apparent decrease in
the concentration factor is seen for liver tissue in the
0.015 ppb series, this is actually due to an unusually
high concentration (393.9 ppb), as compared to 87.3 and
41.6 ppb in the other two fish, in one fish of the 0.1
ppb series. The concentration factor would increase from
the 0.1 ppb series to the 0.015 ppb series if this one
value were disregarded.
DISCUSSION
The results of these experiments are in general agree-
ment with those performed on other teleosts (Holden, 1962;
Bridges et al,, 1963; Anderson and Everhart, 1966). Other
workers at the Hopkins Marine Station, using similar tech-
niques, have found closely related results in studies of
the speckled sanddab, Citharichthys stigmaeus, and the
Northern anchovy, Engraulis mordax (Phillips, 1969; Kap-
lan, 1969). Liver tissue appears to concentrate DDT to
the greatest extent, but it is possible that much of the
DDT in this organ is actually present in the blood, as
is suggested by Johnson (1962). This may also be the
case in the brain. However, as Bridges et al. (1963)
found 10.1 ppm of DDT in bullhead brain tissue as opposed
to only 2.8 ppm in muscle and 2.7 ppm in the blood, ample
amounts of blood may not be sufficient to account for
the high levels in these organs. Similarly the large amounts
of lipid in brain tissue would certainly seem to be con-
ducive to absorption of significant amounts of DDT in that
organ.
Holden (1962) in his study of uptake of DDT-C by
brown trout found that 80-90% of the DDT in the incubation
tank was removed from the water by the fish within the
first ten hours of the experiment. Attempts were made
to measure the amount of DDT remaining in the water after
the completion of the present experiments, but the results
were highly inconsistent, probably due to sampling problems.
Since DDT adheres to surfaces and codistills with water,
it might be expected to have a markedly heterogeneous
distribution (Acree et al., 1963). This would not only
affect sampling, but also change the effective concen-
tration in the immediate volume of water surrounding the
fish. Thus the fish could be concentrating DDT from water
containing much less than the amount in the original
solution.
This great reduction in the amount of DDT actually
available in the water might account for the decrease in
the rate of uptake observed in all tissues after the first
two hours of incubation. Ittis also possible that the
tissues have become partially saturated and so cannot
absorb DDT as fast as before, or that a redistribution
of DDT to other tissues begins to occur, so that the amount
in the measured tissues decreases as the level in the other
tissues increases. As regards redistribution, however,
since uptake in all tissues decreased about the same time
(two hours after the start of incubation), it would not
appear that redistribution is the factor. As regards
saturation, since much higher concentrations of DDT have
39.
been found in natural populations of many other fish, it
is probable that the saturation point of the tissues of
this fish has also not been reached. For these reasons,
it would seem that the decreased rate of uptake resulted
from the decreased availability of DDT in the sea water.
The ability of Cebedichthys to greatly concentrate
DDT directly from sea water indicates several important
points. First, it shows that DDT can enter organisms,
and so the food web, directly at many levels and can con¬
tribute significantly to the overall buildup of DDT in
tissues. Thus, the amount of DDT in the animal cannot
of itself be considered a reliable indicator of trophic
level.
Second, the fact that Cebedichthys can take up sig-
nificant amounts of DDT from as low as 15 parts per trillion
(ppt) from sea water (also containing an unknown amount
of DDT dissolved in it) is ecologically significant.
Bailey and Hannum (1967) have reported concentrations of
DDT at the Golden Gate of San Francisco Bay of 20-60 ppt,
with an average of 41 ppt. The experiments I performed
show that Cebedichthys is able to concentrate DDT from
similar or more dilute solutions in a short period of time.
Thus, even the dilute concentrations found in sea water are
sufficient to permit DDT to be taken up into organisms, and
so enter the food web, where further concentration through
the food chain can occur. Since species closely related
both taxonomically and ecologically to Cebedichthys are
the prey of predatory fish and fish-eating birds, partic-
ularly the guillemot (Carl, 1964), it might be expected
that Cebedichthys may contribute to the further concen-
tration of DDT in the higher trophic levels.
Thirdly, it is apparent that continued direct uptake
of DDT from environmental waters may result in the accumul-
ation of very high levels of DDT in tissues within rela-
tively short periods of time. For example, if sea water
contained 15 ppt DDT, the liver would concentrate to a
level of 25.5 ppb per eight hour period, or 76.5pppb per
day. At the end of one year the fish would have 27.9 ppm
of DDT in its liver. This does not include the unknown
but presumably substantial amounts of DDT which would
be accumulated as a result of feeding on contaminated
food sources. There are several hypotheses to explain
why such levels are not often found in natural populations:
attainment of lethal concentrations and thus death; sat-
uration of tissues; partial metabolism to DDE or DDD
(Bridges et al., 1963); complete metabolism and/or excre-
tion; or simply that the concentration of DDT insea water
is much less than 15 ppt, although measurements of similar
or higher levels have been made (Bailey and Hannum, 1967;
Odemar et al., 1968).
Although lesser amounts of DDT are taken up from
more dilute solutions, the concentration factor was found
to increase. This significant finding indicates that the
animals are able to concentrate DDT more efficiently from
more dilute solutions, such as would be found in sea water,
than from the more concentrated and probably unnatural
solutions used in most experiments. The reasons for this
phenomenon are unknown. It may be that the dilute solu-
tionsis more homogeneous and hence that a greater percen-
tage of the DDT in the solution is available for uptake.
It may also be that the uptake from more dilute concen-
trations occurs at a greater rate.
It is apparent that numerous aspects of this problem
remain to be studied, including ambient concentrations
in natural populations, effects of DDT on metabolism and
reproductive activities, rates of uptake lethal doses,
and the accumulation of DDT through the food chain. These
must be accompanied or preceded by studies of the ecology,
behavior, reproductive, and feeding habits of Cebedichthys,
which have not been studied to date. It is difficult
to draw conclusions concerning the uptake and effects
of DDT when such parameters remain unknown.
SUMMARY
A study was performed on direct uptake and deposition
of DDT-C from sea water by the intertidal teleost Cebe-
dichthys violaceus Ayres. It was found that the liver
concentrated DDT to the greatest extent, followed by brain,
gills, and muscle. After a rapid uptake during the first
two hours of incubation, the rate was seen to decrease
to a certain extent, possibly due to removal of most of
the DDT from the water.
The ability of the fish to absorb and concentrate
DDT from concentrations comparable to those found in natural
waters was also studied, and it was found that the total
amount of DDT absorbed decreased in more dilute solutions,
but that the factor of concentration greatly increased.
The significance of direct uptake from very dilute con-
centrations is discussed.
10
ACKNOWLEDGEMENTS
I wish to thank the entire faculty and staff of the
Hopkins Marine Station for their kind assistance and advice,
and in particular Dr. David Epel for many valuable sug-
gestions and for his patient guidance throughout all phases
of this research.
I also wish to thank Mr. John Miller for his long
hours of technical assistance in the lab and with the scin-
tillation counter, and Mr. Sam Johnson for his assistance
in the lab.
This project was supported in part by the Dietz Fel-
lowship of Stanford University.
11
FOOTNOTE
1. The scintillation fluid is a mixture of 1.0 liter of
toluene, 4.0 grams of 2,5-diphenyloxazole (PPO) (Amer-
sham/Searle; Scintillation Grade) and 0.1 gram of 1,4-
bis (2-(5-phenyloxazolyl)) benzene (POPOP) (Amersham/
Searle; Scintillation Grade).
12
408
Table 1.
Table 2.
Table 3.
TABLE LEGENDS
Uptake of DDT-C by Cebedichthys violaceus, as
a function of time, from a 1.0 ppb solution of
DDT in sea water.
Uptake of DDT-C by Cebedichthys violaceus, as
a function of concentration, after an eight
hour period of incubation in 1.0, 0.1, and 0.015
ppb solutions of DDT in sea water.
Factor of concentration (the number of times
the DDT was concentrated in the tissues above
that of the original solution) by Cebedichthys
violaceus, after an eight hour period of incuba-
tion in 1.0, 0.1, and 0.015 ppb solutions of
DDT in sea water.
13
405
Figure 1.
Figure 2.
Figure 3.
FIGURE LEGENDS
Uptake of DDT-C from a 1.0 ppb solution of
DDT in sea water by tissues of Cebedichthys
violaceus, as a function of time. Each point
is an average of three fish incubated for the
indicated length of time. Key: O, brain;
E, gills; A, liver; Q, muscle.
Uptake of DDT-C from 1.0, 0.1, and 0.015 ppb
solutions of DDT in sea water by tissues of
Cebedichthys violaceus, Each point is an average
of three fisn incubated for eight hours, ex-
cept for the brain and gill tissues in the
O.1 ppb series, which are the averages of two
fish. Key: B, brain; G, gills; L,liver; M,
muscle.
Factor of concentration of DDT-C by tissues
of Cebedichthys violaceus as a function of
incubation concentration. The horizontal
scale decreases from 1.0 ppb on the left to
0.O ppb on the right. Key: O, brain; m,
gills: A, liver; o, muscle.
14
4
LITERATURE CITED
Acree, Fred, Jr., Morton Beroza, and Malcolm C. Bowman.
1963. Codistillation of DDT with water. J. Agri.
Food Chem. 11: 278-280.
Anderson R. B., and W, H. Everhart. 1966. Concentrations
of DDT in landlocked salmon (Salmo salar) at Sebago
Lake, Maine. Trans. Amer. Fish. Soc. 95: 297-309.
Anderson, John M., and Margaret R. Peterson. 1969. DDT:
sublethal effects on brook trout nervous system.
Science 64: Lho-Lli.
Bailey, Thomas E., and John R. Hannum. 1967. Distribution
of pesticides in California. Jour. Sanit. Engineer.
Div., ASCE, 93(SA5) Proc. Paper 5510: 27-13.
Bridges, W. R., B. J. Kallman, and A. K. Andrews. 1963.
Persistance of DDT and its metabolites in a farm pond.
Trans. Amer. Fish. Soc. 92: 421-127.
Carl, G. Clifford. 1964. Some common marine fishes of
British Columbia. British Columbia Provincial Museum.
Dept. of Recreation and Conservation Handbook 23: 75-76.
Hickey, Joseph J., and Daniel W. Anderson. 1968. Chlorin-
ated hydrocarbons and eggshell changes in raptorial
and fish-eating birds. Science 162: 271-273.
Holden, A. V. 1962. A study of the absorption of C labeled
DDT from water by fish. Ann. Appl. Biol. 50: 167-477.
15
19.
Johnson, Donald W. 1968. Pesticides and fishes-a review
of selected literature. Trans. Amer. Fish. Soc. 97
398-424.
Kaplan. 1969. Personal communication.
Odemar, Melvyn W., Paul W. Wild, and Kenneth C. Wilson.
1968. A survey of the marine environment from Fort
Ross, Sonoma County, to Point Lobas, Monterey County.
Calif. Dept. of Fish and Game MRO Reference No. 68-12.
Phillips, Gregory. 1969. Personal communication.
Risebrough, Robert W., Daniel B. Menzel, D. James Martin,
Jun., and Harold S.OOloott. 1967. DDT residues in
Pacific sea birds: a persistent insecticide in marine
food chains. Nature 216: 589-591.
Woodwell, G. M. 1967. Toxic substances and ecological
cycles. Scient. Amer. 216: 24-31.
Wurster, Charles F., Jr. 1968. DDT reduces photosynthesis
by marine plankton. Science 159: 1474-1475.
16
40
INCUBATION
(HOURS)
2/3
2/3
2/3
2/3
2
2
4
TISSUE
Brain
Gills
Liver
Muscle
Brain
Gills
Liver
Muscle
Brain
Gills
Liver
Muscle
Brain
Gills
Liver
Muscle
Brain
Gills
Liver
Muscle
TABLE 1
SAMPLES
MEAN
(PPB
28.0 49.6 18.6
32.1
21.0 34.6 27.7
27.8
76.3 133.6 110.8
106.9
14.9 27.0 17.6
19.8
114.6 47.5 44.7 69.0
41.0 5.7 35.7 27.5
150.4 72.7 178.3
133.8
39.9 0.0 14.0 16.0
56.0 63.9 58.2
59.4
32.4
32.7 27.0 37.6
147.9 165.0 171.0
161.3
10.9 19.7 11.6
14.1
62.6 141.5 66655
90.2
50.h 78.7 45.4
58.2
244.0
172.0 280.2 279.7
29.3 62.9 18.6
36.9
118.0 92.7 156.4 122.1
111.3 54.1 147.5
104.3
257.4 318.7 321.4 299.2
44.3
4h.1 43.0 45.7
STANDARD
DEVIATION
15.9
6.8
28.9
6.4
17.1
19.0
54.7
13.9
4.1
5.3
6.9
4.9
44.5
18.0
62.3
23.1
31.6
47.1
35.9
1.4
CONCENTRATION TISSUE
(PPB)
1.0
Brain
1.0
Gills
1.0
Liver
Muscle
1.0
Brain
O.1
O.1
Gills
0.1
Liver
Muscle
O.1
0.015
Brain
0.015
Gills
0.015
Liver
Muscle
0.015
TABLE 2
SAMPLES
MEAN
STANDARD
DEVIATION
24.7
138.1 139.8 181.7
153.2
85.2 24.4
57.3 102.7 995.5
181.5 213.0 309.8
234.7
66.9
13.4
30.3 44.6 55.
12.5
93.7 71.4 ---
82.6
15.7
90.1 38.0 ---
64.0 36.8
393.9 87.3 95.5 192.2 174.7
5.4
18.2 8.3 9.7
12.7
24.7 22.2 16.7
21.2
3.9
21.1 16.2
38.9 17.4 7.1
11.2 13.7 21.5
25.5 14.1
4.4
4.6 4.2 4.3
0.2
o
CONCENTRATION
(PPB)
1.0
1.0
1.0
1.0
O.1
O.1
0.1
0.1
0.015
0.015
0.015
0.015
TABLE
TISSUE
CONCENTRATION FACTOR
MEAN
STANDARD
DEVIATION
Brain 138.1 139.8 181.7
24.7
153.2
24.4
Gills 57.3 102.7
85.2
95.5
Liver 181.5 213.0 309.8
234.7
66.9
13.4
44.6 55.3
Muscle
12.5
30.3
714.5 ---
825.6
Brain 936.7
157.2
———
640.2
Gills 900.7
379.6
368.5
873.0 955.2
1922.5 1747.1
Liver 3939.3
Muscle 182.2
83.1 97.0
126.8
55.3
Brain 1646.3 1476.9 1111.9 1411.7
273.1
1081.6
Gills 2593.8 1159.8 474.3 1409.3
241.8
Liver 2744.0 916.3 1435.7 1698.6
13.6
Muscle 308.2 282.6 287.6
292.8
kog
o
O
O
300-
275
250
225
150
—
125
2 100
75.
50-
25-

Figure
ime
in
OUTS
250
225
200

175
9.
2 150
a
125
100
75
50
25
O
(
DDI
Figure 2
BGL
M
M
0.015
Incubation Concentration, in Parts
per Billion
M
-

20
10
Figure 3
05
O.10.015
DDT Incubation Concentration, in Parts
Billion
per