C
O
THE KINETICS OF DDT RESIDUES IN THE
NUDIBRANCH HERMISSENDA CRASSICORNIS
Ronald E. Marian
Hopkins Marine Station
of Stanford University
The accumulation of DDT residues 'in higher trophic levels
by magnification through the food chain is now well documented.
However, the mechanisms for accumulation at the species level are
not fully understood. There is presently no full account of the
kinetics of DDT residues in a marine invertebrate. This report
describes some aspects of DDT flow in the nudibranch Hermissenda
crassicornis (Escholtz). A preliminary investigation indicated
that a Hermissenda of any age will have been exposed to more
DDT residues, available through feeding and surface uptake, than
is reflected in the total body burden. This suggests a considerable
turnover of the pesticide, either by a high rate of loss or by
a physical equlibrium between tissue concentration and input dosage.
Equilibria establishing a constant DDT residue concentration have
(2,3,4)
The
been suggested in a variety of vertebrate systems.
problem presented is one of establishing the pathways for DDi
residue flow and their relative contributions in determining the
amount of DDT residues in the animal. This problem is approached
with a) the determination of field levels and the relation of
DDT residue concentration to age, b) measurement of uptake by
surface adsorption and feeding, c) measurement of losses of DDT
residues, and d) an attempt to relate this information to what
may be happening under natural conditions.
DDT Residue Levels in Hermissenda
Hermissenda for this study were collected subtidally from
the pilings of Wharf No. 2 in the harbor of Monterey, California
during the spring and summer. To determine the DDT residue levels
in various sized specimens, a number of the animals were divided
59
into weight classes. Approximately 3 gram samples of each weight
class were digested over steam by a l:l mixture of glacial acetic
and perchloric acid, and the non-polar fraction extracted with
The extracts were cleaned up using silica gel micro¬
hexane.
27 The concentrated samples were analyzed with a Beckman
columns.
(6)
GC-4 gas chromatograph equipped with electron-capture detector
Glassware for GLC analysis was combusted overnight at 400 C to
remove any DDT contamination.
The results, presented in Figure 1, show a definite increase
in the total-body DDT residue concentration with increasing size.
Since several specimens in the higher weight class samples
contained spawn, possible accounting for the higher concentration,
egg masses were collected and analyzed. Measured levels were too
low for accurate quantification- less than 2 PPB (parts per billion).
Another possibility for the higher concentration in larger animals
would be a larger percentage content of lipid. To check this,
Hermissenda of a full size range were weighed, dried and re-weighed,
digested in the acid mixture, and extracted with ethanol and
petroleum ether. The extracts were evaporated until no ether odor
was detectable, and the residue weighed. The results (Eig. 2) show a
constant percentage of ether-extractable lipids independent of
size. This does not entirely rule out the possibility of a
relatively higher number of DDT residue accumulation sites in
the larger animals, since DDT residue storage might depend on
the character and distribution of lipids as well as amount.
Though size increases with age, the relation is unlikely
to be linear. The growth curve in Hermissenda probably begins
to flatten out around 0.8 grams weight as they begin to divert
360
3.
food energy into spawn production. Eggs at the time of release
accounted for 30% of the wet weight of a 0.78 gram specimen, and
18% of a 1.0 gram specimen. If the data in Figure l is to be
considered in terms of DDT residue concentration vs. age, the
weight scale would then be compressed at the lower end, resulting
in somewhat straighter curves.
Uptake of DDT From Seawater
Measurement of surface uptake of DDT from water was accomplished
using ring-labelled C+ DDT as a tracer. Hermissenda were held
in closed one-gallon glass jars with 3 liters of unfiltered
seawater containing the CDDT. The jars were kept in dim light
at a constant 15° C. Before the addition of the animal, the
jars were allowed to stand for 24 hours in order to allow the
DDT to adsorb to equilibrium with the glass surface. To maintain
a fairly constant concentration of DDT in the water and prevent
stagnation, the water was changed daily. With animals present,
the decrease of CDDT concentration over 24 hours ranged from
20% for an initial 30 PPT (parts per trillion) to 50% for an initial
5 PPT. Consequently, in order to obtain average concentrations
of 5. 10. 20, and 30 PPT, the initial concentrations were prepared
at 7. 12, 23, and 33 PPT. Each day, before being moved to
fresh water, the Hermissenda were allowed to feed on Obelia.
Uptake with time was measured by periodic sampling of a few of
the animals cerata, dorsal extensions of the body wall containing
diverticula of the digestive gland. Comprising on the average
36% of the body dry weight, the cerata contain 95% of the animals'
total DDTüresidues. Cerata samples were digested in tha acid
mixture and extracted with hexane. Sample activities were counted
36/
in a Nuclear Chicago Unilux II liquid scintillation counter."
Curves of the accumulation of CDDT in the cerata for the
four water concentrations are shown in Figure 3. Points represent
data from several runs, and concentrations are based primarily
on wet weights, with dry weight concentrations set to the same
scale by a wet/dry conversion factor of 12.15. While the
uptake curves level off after 2-3 days for animals in water of
5 and 10 PPT CDDT, the curves for the higher concentrations
do not level off at the same time or at proportionate values.
Two possibilities accounting for this are: (i) an equilibrium
between the total DDT concentration in the cerata and the con-
centration in the water, with an increasin equilibrium constant
for higher water concentrations, and (ii) an uptake pathway
consisting of two consecutive steps or compartments, with the
first compartment establishing an equilibrium DDT concentration
when the concentration in the water is around 5-10 PPT. With
some sort of limit on the DDT capacity of the first compartment,
higher concentrations in the water result in an overflow into
the second compartment where the DDT is continuously accumulated.
An additional experiment lends support to the latter possibility:
Two Hermissenda were first exposed to water with 30 and 60 PPT
of unlabelled DDT for 60 hours, and then placed in water containing
5 and 10 PPT respectively of CDDT. The uptake curves of the
labelled DDT (Figure 4) are very similar to those in the previous
experiment, despite the initial period of uptake from a six times
higher concentration in the water. This suggests that the cerata
uptake curves resulting from DDT water concentrations of 5 and 10
PPT represent a turnover of a certain portion of the DDT in the
Soa
cerata, independent of the total amount present. Again, this
might be explained by an "equilibrium compartment" and a
"storage compartment" An earlier experiment in which the
concentration of DDT in the water was continuously measured
may also support this theory. As shown in Figure 5, as the
DDT concentration in the water drops below 10 PPT, the curve
for Hermissenda uptake (the entire animal in this case) begins
to follow fluctuations in the water concentration curve, even
DDT from the animal when the concentration
showing a loss of the
drops below 5 PPT. There is also basis for this theory in the
strucure of the cerata themselves, since they consist of an
outer layer of body wall tissue and a core of digestive gland
tissue- with not neccessarily the same DDT affinities.
DDT Uptake Through Feeding
Hermissenda crassicornis is reported as primarily a non-
specific hydroid feeder.0
Animals in this study accepted a
variety of genera including Obelia, Aglaophenia, Sertularia,
Eucopella, and Syncoryne. The hydroid appearing to be the
primary food source on the wharf pilings was Obelia dichotoma.
However, they were also observed to feed occasionally on the
bryozoan Bowerbankia, on a small species of Dendronotus abundant
on the Obelia, and readily on dead or disabled fellow Hermissenda.
Original measurement of DDT residues in Obelia gave a value
of approximately 15 PPB wet weight. However, these samples were
collected from the sides of floating piers, presumably being
exposed to higher levels of contaminants from surface fallout,
and also consisted of entire stolons with some inseparable non-
hydroid material. When the hydroid population on the submerged
36.
pilings became abundant in the early summer, a number of samples
were collected consisting of only the younger portion of the
stolons, selectively grazed by Hermissenda. Very low levels
of DDT residues were found, again precluding accurate quanti¬
fication, but appearing to be less than 1 PPB wet weight.
To determine the general distribution of ingested DDT,
14 small Hermissenda were fed the chopped-up bodies of Hermissenda
labelled with CDDT. The animals were placed in a flask of
DDT-free seawater for 48 hours and then dissected into pooled
samples of cerata, total viscera, and remainder of the body-
consisting of the muscular foot and head. Of the C recovered
from the bodies, 83.7% was in the cerata, 8.7% in the viscera,
and 5.6% in the remainder. Feces were also collected from the
flask and accounted for 3.9% of the total label recovered.
Analysis of the water in the flask during the 48 hours showed
no measurable increase, although a significant total amount in
the two liters could have been undetected in the 8 ml aliquots
sampled. Possible adsorption to the glass surface was not
measured. Thus the value of 96% for the efficiency of retention
of DDT from food is probably high.
Being rather small, few of these specimens contained spawn.
In order to check for accumulation in eggs and the accumulation
in the cerata with time, a single 0.8 gram Hermissenda was
allowed to feed continuously on Obelia labelled to a concentration
of 7 PPB CDDT. Eggs were released during the 9th and 10th
days, coinciding with a marked increase in the cerata concentration
(Figure 9). At the time of the third sampling, the total amount
of CDDT in the Hermissenda was calculated as 300 picograms, a
368
and the total recovered from the eggs was 285 picograms,
meaning that during the first week 49% of the DDT retained in
the animal accumulated in the eggs, and that once eliminated
as an accumulations site, nearly all the DDT taken in was then
accumulated in the cerata.
The important role of egg production in DDT residue flow
also became evident in a comparison of the pathways for DDT loss.
Losses of DDT Following Surface Uptake
Four Hermissenda of nearly equal size were held in seawater
containing 40 PPT C
DDT for a period of 48 hours. Each was then
placed in a stoppered 2 liter flask containing DDT-free water.
At one to two day intervals the animals were removed, sampled for
concentration in the cerata, and returned to a fresh flask of
water. DDT lost from the animal was recovered by shaking the
entire contents of the flask with three washes of petroleum
ether. On the first day feces were collected and analyzed
separately and found to account for a mean of 8% of the C“ DDT
recovered from the flasks. Feces collection was difficult due
to rapid disintegration, and thereafter feces were extracted
along with the rest of the flask contents. Large egg masses
were deposited by all four animal during the first day. Smaller
batches were released 12 to 14 days later.
The concentration of CDDT in the cerata over the two weeks
of the experiment is shown in Figure 6. There is no clear
explanation for the fluctuation excpt to note that changes
in the concentration might be due either to transfer of DDT
between the cerata and the body, or to changes in the wet weight
165
of the cerata. It may also be noted that large decrease in
concentration coincided with the release of the second batch
of eggs, though the absolute amount of C+ DDT represented by
the drop was not reflected in either the amount recovered from
the eggs or from other losses.
1 recovered from the flasks presumably included all DDT
contained in the water, feces, mucustrails, and adsorbed to
the glass. Results are shown in Figure 7 in terms of the rate
of loss from the animals. No correlation between rate of loss
and concentration in cerata was found.
The DDT contained in the eggs accounted for 28% of the
total taken up and 73% of the total lost (Figure 8). The small
amount of DDT in the second eggs indicates that though the eggs
accumulate a sizable portion of the DDT as it is taken up, they
cannot accumulate much of the DDT already stored elsewhere in
the animal.
DDT Residue Flow in Hermissenda
Under Natural Conditions
The major problem in describing the situation in the field
is in predicting the actual input dosages. Data presented here
has indicated that long-term uptake of DDT residues from water
may be very slight or very great, depending on the ambient
concentration. Estimates of DDT residue levels in inshore
waters of the Pacific coast are in the range of a few parts per
trillion.9) The harbor of Monterey acts as a catch-basin for
the southern end of Monterey Bay, and would be expected to have
higher than average levels of pesticide contaminants.
Jog
An estimate of between 5 and 15 PPT seems reasonable, and it
would thus be likely that surface uptake in Hermissenda is
either in equilibrium or accounts for a very slow accumulation.
The question of effective food input dosage is complex and
unclear. Hermissenda is a rapidly growing organism, with the
amount of food being utilized for new tissue prodction probably
far greater than that required for metabolic maintenance. If the
rate of tissue production- including DDT accumulation sites-
were to remain constant as were the feeding rate, then a constant
residue concentration might be maintained. However, in three
Opisthobranchs whose nutrition has been studied, the efficiency
of conversion of food into new tissue and spawn decreases with
age. Since the efficiency of removal of DDT residues from food
would seem independent of the efficiency of food utilization,
this suggests an increasing dosage from food in relation to
the production of new storage sites. Thus a higher concentration
of residues in older animals might reflect an equilibrium between
tissue content and increasing dosage, as well as a continual
accumulation from a constant dosage. Two other factors are
probably operating to confuse the issue: First, once Hermissenda
reach sexual maturity, a portion of the DDT residue input is
accumulated and eliminated in the spawn, thus periodically
reducing the dosage to the rest of the body. Secondly, the
dosage may vary as a result of a possible change in feeding
habits eith age. Smaller Hermissenda were almost always found
on a hydroid-bryozoan substrate, while larger ones were observed
as often on colonies of Amaroucium. The largest specimens were
consistently found on the muddy bottom among the fallout around
the bases of the pilings.
J67
10.
Summary
Results show an equilibrium for surface uptake, estab-
lishing a constant DDT concentration in the cerata with a
5 to 10 PPT concentration in the water, a level likely to
be present in the habitat. A two compartment model is pro-
posed for the uptake mechanism. Spawning is shown to be the
primary pathway for DDT output, eliminating as much as 50%
of the residues taken up during each 10 to 20 day egg pro-
duction period, but not eliminating DDT residues already
stored in the cerata. Field levels show an increasing whole-
body concentration with increasing age, probably dependent on
feeding dosage, but difficult to determine without additional
autecological knowledge of Hermissenda.
360
References and Notes
1. DDT residues include technical DDT and all it's non-polar
metabolites. In this study, only p,p'DDT 1,1,1-trichloro-
2,2-bis(p-chlorophenyl)ethane, p,p'DDD 1,1-dichloro-2,2-
bis(p-chlorophenyl)ethane, and p,p DDE 1,1-dichloro-2,2-
bis(p-chlorophenyl)ethylene were detected and measured.
2. J. Robinson, A. Richardson, A. N. Crabtree, J. C. Coulson,
and G. R. Potts, Nature 214 (5095): 1307-1311 (1967).
3.
J. J. Jeffries and C. H. Walker, Nature 212: 533-534 (1966).
J. W. Gillett, Bull. Environ. Contam. Toxicol. 4 (3) 159-
168 (1969).
5. A.M. Kadoum, Bull. Environ. Contam. Toxicol. 3, 65 (1968).
Column coating was 5% mixed bed of DC-200 and QF-1 on
Chromosorb W. All GLC parameters were those suggested in
Pesticide Analytical Manual (U.S. Dept. of Health, Education
and Welfare, Food and Drug Admin., revised 1968) vol.2.
7. Extracts were evaporated to.5 ml in scintillation vials,
and 10 mls of scintillation fluid was added consisting of
4 grams PPO and .1 gran POPOP per liter toluene.
8. L. H. Hyman, The Invertebrates,vol VI Mollusca I (McGraw Hill,
New York, 1967)
9. J. L. Cox, Ph.D. Thesis, Stanford University,1970.
10. T. H. Carefoot, Comp. Biochem. Physiol. 21, 627-652 (1967).
367
C
C
Acknowledgements
I thank Mr. Robin Burnett for his patient assistance.
This study was supported by NSF grant GY 8950
O
O
Fig. 1
Concentrations of DDT residues in Hermissenda
of varying size. Concentrations are expressed as
weight of residue per unit wet weight Hermissenda.
Points are plotted at the mean weight per animal
in the pooled samples representing each.2 gram
size class.
100
80
60
50
30
FIGURE 1
TOTAL

* *
D 8
1
0 u. .


DDT 3—
X15
Xx—
2 4 .6 .8 10 1.2 14 1.6 1.8
GRAMS WET WEIGHT
37.
O
Fig. 2 Ether-extractable lipid content, expressed as
percentage dry weight, of Hermissenda of varying
size.
O
C
Q

L
0
O
0
0

9
Gid Naod
71
O
Fig. 3
Increases in the concentration of CDDT
with time in the cerata of Hermissenda during uptake
from seawater containing 5, 10, 20, and 30 parts per
trillion of the CDDT. Data is from several runs
under the same conditions, with animals of the same
approximate size. Concentrations are based on both
wet and dry weights.
8

8
288
OeX
8
4
X
8
Noria dad siavd

40
. *
S
.00
0

90
o
O
1
37
Fig. 4
Uptake in the cerata from water containing
5 and 10 PPT C"'DDT after an initial 60 hour
exposure to water with 30 and 60 PPT unlabelled
DDT. The broken lines are the predicted uptake
curves based on data of Fig. 3.
0
0

00
60
o
O0

00
o
o
5
0
V
O
8
8
Noug ad sLävd
5

70
O

Fig. 5 Increase of total-body concentration of CDDT
in Hermissenda exposed to 20 liters of seawater
with varying concentration of CDDT.
95
•
. ../. *
* 0
11
S
9
—Q
10
8
&a
8
(alVN) Noralad sive
(YGNASSIWNSH) Nortis asa siävd
o0
O/0
O
3
00

8
0
380
O
Fig. 6 Uptake of C DDT in the cerata of a.8 gram
Hermissenda allowed to feed constantly on Obelia
labelled to 7 PPB. Spawning occured on the 9th
and 10th days.
D6

FIGURE 6
—5
EGGS DEPOSITED


—

DAYS
10
30
C
O
Concentration of CDDT in the cerata of
Fig. 7
four Hermissenda during the ten days following
a 48 hour exposere to the labelled DDT. Values
are based on wet weight only.
30
50
30
20
10

A
0
100
5
X
O
-—-
HOURS
FIGURE 7
.
200
300
38
Fig.
8
Rates of loss of CDDT from the four
Hermissenda, expressed in  grams of C DDT
lost per hour. Rates were determined by the
measurement of label recovered from the flasks
in which they were maintained.
8
90
80
70
5 60
50
40
30
20
FIGURE 8
885
8
300
400
200
HOURS
O
8
50 100
386
C
Diagram representing the relative amounts
Fig. 9
of CDDT lost by the four Hermissenda after
initial uptake from water.
387
V101 j0 Nod
9

S
a
X1
uoO



388