Abstract
The ambient levels of DLE (approximately 80% of total DDT
residues) were determined in the Northern Anchovy from gas
chromatographic analysis of 25 samples of 6 different tissues:
female gonad, male gonad, liver, muscle, gill, and brain. Levels
were found to range between O.11 and 0.35 ppm by wet weight.
Direct uptake from sea water by live fish was measured with the
use of C+-labeled DDT for h hour incubations in 1 ppb concentrations.
Results showed very high concentrations of the DDT in liver, gill,
and brain. In h hours liver tissue concentrated DDT an average
of 240 times over the initial level, with gill and brain tissue
averaging 65 and 45 fold concentrations, respectively, in the
same time period. Results show large variations between individual
fish in amount and site of deposition. Comparison of normal
levels and uptake show that DDT is highly mobile within the fish
and seems to move from liver to muscle tissue after original
uptake.
Introduction
The work of Risebrough (Risebrough et al, 1967) and others
with chlorinated hydrocarbons in the marine ecosystem has
generated growing concern for the concentration and effect of
these pesticides in the world's oceans. The harmful effects of
DDT and its residues on marine birds has been a topic of much
discussion (Wurster and Wingate, 1968). Since the bird is at the
top of the marine food web and because DDT is passed on and
accumulated as it moves up the food chain, it would be expected
that this animal would be among the first organisms affected by
increasing concentrations of this hydrocarbon in the environment.
In order to study the relationship between DDT and the next
lower trophic level, the fishes, an extremely important pelagic
bait fish was chosen for this work. The Northern Anchovy is
abundant in California coastal waters and is the primary source
of food for most of California's game fish and a large number
of its coastal birds. In turn, this species is a voracious
omnivore, feeding extensively on phytoplankton and zooplankton.
Thousands of tons of anchovy are taken commercially each year
and used in various forms for human consumption and for raising
hatchery fish, poultry, and livestock. This fish is easily
obtainable, can be kept alive in captivity, and is of small size.
The study analysed ambient levels of p,p'-DDT (1,1,1-2-
2bis (p-chlorophenyl) ethane) and its residues in 5 tissues of
the anchovy (gonad, liver, muscle, gill, and brain). The uptake
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and deposition of C+DDT from sea water was investigated to relate
the normal levels and uptake results.
Materials and Methods
Specimens of E. mordax were taken from Monterey Bay and kept
in a large circular cement pool with a circulating current. The
sample was relatively homogeneous, consisting of about 300 fish
ranging from 150mm to 170mm standard length. They were mature
adults, estimated at 1-5 years. Frozen brine shrimp were given as
food each evening. Water temperatures in the pool ranged from
11C to 15°0. The fish survived with only low mortality after
adjustment to these conditions.
The gas chromatographic analysis was done with a Beckman GC h
gas-liquid chromatograph using a 3% OF 1 column on 80-100 mesh
chromosorb W and as elctron capture detector. The acid extraction
method of Stanley and LeFavoure (1965) was used to isolate the
pesticide. All chemicals were checked for possible pesticide
contamination. The glassware was carefully washed and then rinsed
with petroleum ether and nannograde hexane, and the last hexane
rinse was condensed and injected into the column to check for
contamination. The analysis itself was done on each of 6 tissues
from a group of 25 fish, except for the male and female gonads.
where only 15 fish were included in the samples. The results are
thus an average of the 25 and 15 fish samples, respectively. In
addition, samples of two anchovy products, oil and meal, were
obtained from a commercial plant processing Monterey Bay anchovy
3.
15
catches. Only DDE was measured because the chromatograph was
unable to resolve DDT or DDD significantly and because DDE makes
up approximately 80% of the total DDT residues in the Northern
Anchovy (Risebrough, Menzel, Martin, and Olcott, unpublished
results).
The uptake experiments were conducted with fish chosen to
be approximately the same size (160mm). One fish was placed in
each one gallon screw-capped jar containing 2 liters of sea water
at 8 - 10 C. The sea water was aerated for thirty minutes before
each experiment. The radioactive DDT (100 ppm in ethanol) was then
added. For the bulk of these uptake experiments, 20ul of clppT
solution was added to each two liters of sea water to give a 1 ppb
concentration. The jars were kept tightly closed to prevent codistil-
lation of the radioactive DDT and were kept at 8°-10'C. It was
previously determined that the anchovies could only live from
A to 6 hours in this type of incubation, thus l hour limits were
placed on the incubations to insure that the fish would live
through the experiments. At the end of h hours, the fish were
killed and dissected. Six types of tissue were removed for
counting: the gonads, the liver, a portion of the muscle or flesh,
a portion of the skin, the gills, and the brain. Each tissue
was weighed and then homogenized in 2 ml of N,N-dimethylformamide.
One ml of this homogenate was pipetted into a scintillation vial
and 10 ml of toluene scintillation fluid (l gm PPO and O.1 gm
POPOP per liter) were added to each vial before counting on a
184
Nuclear-Chicago Unilux II two-channel scintillation counter. All
equipment which touched any tissues was rinsed twice with 952
ethanol between each tissue homogenization. Each vial was counted
for one minute and disintegrations per minute were converted into
parts per billion uptake for the h hour period. Variations in the
number of tissue samples are due to the use of other tissues at the
beginning of the study which were later standardized to the 6
listed above.
Three fish were also run at 15 ppt C+DDT under the same
conditions to simulate more closely the 15 ppt concentration found
in normal sea water as determined by the California Department of
Fish and Game (Odemar et al, 1968). One feeding experiment, with
fish, was run under the same conditions using frozen brine shrimp
which had been incubated in 10 ppb C+DDT for 1 hour and then
washed twice with sea water. This last experiment was not pursued
because results showed only very limited actual feeding due to the
excited state of the fish. Limitations on the size of containers
for the experiments were imposed by the problems of handling
volütile, radioactive materials.
Results
The results of the gas chromatography are listed in Table 1.
The results of the uptake experiments at 1 ppb for H hours are
listed in Table 2, with one standard deviation. The results of the
incubations in 15 ppt were in the same percentage proportions as
those of Table 2, with the highest values being 6.80 ppb in the
5.
liver and 8.20 ppb in the brain for the h hour study. The feeding
experiment also showed approximately the same proportions among
the tissues except that the stomach tissues averaged 21.6612.86
per cent as compared to the l.671 1.20 per cent found in A stomachs
of fish run in the normal 1 ppb uptake experiments. Ten samples of
muscle from the same fish were run to determine accuracy of the
uptake technique. Results showed an average of 30.99 with a standard
deviation of+5.30.
Discussion
The results of the gas chromatography are slightly lower than
those found by Risebrough for Monterey Bay anchovies in November.
1965 (Risebrough, Menzel, Martin, and Olcott, unpublished results).
These analyses were of whole fish and averaged O.65 ppm with a
standard deviation of 0.19. However, the lipid content of the
Northern Anchovy fluctuates greatly during the year and is much
higher in November than in May, when the results of Table 1 were
determined. This is due to reproductive cycles and is significant
because the gas chromatographic method is based on lipid extraction.
The lipid content in the spring averages 7% as compared to an average
of 15% by weight in the fall (Parish, personal communication).
The uptake experiments showed high variation between individual
fish under constant conditions, as shown by the high standard
deviations for all tissues in Table 2. Much of the data on
pesticide residues is cited for a limited number of samples, often
one or two, and the variability found in this study tends to show
that little confidence can be placed in small sample sizes where
DDT is concerned. Figure 1 shows the differences in 6 uptake
experiments.
Figure 2 shows the variations between sites of DDT concentration
in ambient levels and in uptake. This suggests that the DDT is
highly mobile within the anchovy after initial uptake. Liver tissue
was a site of heavy concentration in the uptake experiments, while
its value in ambient level is very low. The opposite is true in muscle
tissue, indicating that DDT residues seem to move from the liver
to muscle after uptake. There is also a positive indication
that the anchovy is capable of either metabolizing DDT or excreting
it directly. This conclusion comes from the extrapolation of the
15 ppt uptake results to the ambient levels in approximately 6 days.
The ambient level values for the processed oil and meal are significant
in that DDT residue concentration in the oil is above the Food and
Drug Administration's interstate trade limit of 5 ppm in products
for human consumption, and because the meal is fed directly to
animals intended for human consumption.
The results of the one feeding experiment are significant
in that the high stomach concentration shows feeding was taking
place and concentrations are again seen in liver, brain, and gill
tissue (in that order).
Further work is needed to determine the far-reaching effects
of these results. The ambient levels must be determined throughout
the year in order to understand the relationship of DDT content to
lipid cycles. Further work is also needed on the movement of
pesticide residues within the anchovy, possible metabolism and
detoxification, and excretion back into the environment. This
study has shown the Northern Anchovy to be a link in the movement
of DDT and its residues in the marine food web.
Acknowledgements
The author would like to thank those of Hopkins Marine Station
in Pacific Grove who were so helpful throughout this study.
Special thanks are given to Dr. Ellsworth Wheeler, John Miller
Phillip Murphy, and Joe Balesteri of the Hopkins staff, and to
Richard Parish of the California Department of Fish and Game.
This work was supported in part by the Undergraduate Research
Participation Program of the National Science Foundation, Grant
fGY-5878.
8.
1
References
Odemar, M.W., P.W. Wild, and K.C. Wilson. 1968 A Survey of the
Marine Environment From Fort Ross, Sonoma County, to Foint
Lobos, Monterey County. Final Report of the California
Department of Fish and Game to the San Francisco Bay-Delta
Water Quality Control Program: 140.
Risebrough, R.W., D.B. Menzel, D.J. Martin, Jr., and H.S. Olcott.
1967. DDT Residues in Pacific Sea Birds: A Persistant
Insecticide in Marine Food Chains. Nature, 216: 589-591.
Stanley, R.L., and H.T. LeFavoure. 1965. Rapid Digestion and
Cleanup of Animal Tissues for Pesticide Residue Analysis.
Journal of the Association of Official Agricultural Chemists, 48:
666-667.
Wurster, C.F., and D.B. Wingate. 1968. DDT Residues and Declining
Reproduction in the Bermuda Petrel. Science, 159: 979-981.
15
O
Toßle 1.
Tissue
Gonad
Female
Gonad
Male
Liver
Muscle
Gill
Brain
Processed
Meal
Processed
Oil
PPM
Wet Weight
0.1613
0.1065
0.1844
0.3502
0.3328
0.2750
———
6.8117
PPM
PPM
Dry Weight Lipid Lipid
0.50u1
2.20 7.3186
0.3328
2.76 3.8553
0.2526
3.33 5.5347
0.5837
1.96 17.8462
0.7396
3.35 9.9265
0.3274
11.13 2.4702
0.6248
3.43 18.2225
6.8117
1100.00 6.8117
C
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1olt 2
Tissue
Gonad (13)
Male (6)
Female (7)
Liver (11)
Muscle (13)
Skin (12)
Gill (12)
Brain (12)
Total
Average PPB
17.1518.31
18.91
15.62
240.70 +120.27
17.7517.64
19.29 112.95
64.11 +25.96
16.15 123.33
Total Average
8 in Fish
1.2672.29
18.98 +12.02
1.62 11.75
13.51*1.29
16.10 15.69
16.72 15.35
C
161
5001
400-
300-
PB
E
4DDT
200
00
90 1
Pg
SAMPLE
baute 2.
500
400

300+
P
B
ODDT
200
00
MUSCLE
GLL
BRAIN LIVER GONAD GONAD
M.
SAMPLE
C
Captions
Table 1. Ambient concentrations of DDE in Engraulis mordax Girard
as determined by gas chromatography. All tissues except male and
female gonads are averages of 25 fish samples. Gonad samples
included 15 fish.
Table 2. Results of CDDT uptake experiments with Engraulis mordax
Girard. Number of samples of each tissue in parenthesis. Incubations
were in 1 ppb concentrations for 4 hours in sea water at 8 -10 C.
Figure 1. Example of variations in uptake results at 1 ppb for
I hour incubations with Engraulis mordax Girard. Brain tissues
are open circles and liver tissues are closed circles. Both tissues
are from the same fish in each sample.
Figure 2. Comparison of ambient levels and uptake results as to
site of depostion in Engraulis mordax Girard. Uptake results (open
circles) are added to ambient levels (closed circles) to show
variations.
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Footn
1. Author's Permanent Address:
Paul F. Kaplan
3710 W. 230th Street Apt. 5
Torrance, California 90505
166