Abstract
Large motor aberrations were observed in the sand
crab, Emerita analoga, allowed to concentrate DDT
from seawater to 1.4 ppm. This is close to some
levels found in the field.
In recordings made from a nerve innervating the
abdominal flexor muscles, 1 ppm DDT in the bathing
medium greatly increases spontaneous neuronal
bursting, length of tactually stimulated bursts and
frequency of firing of the larger motor neurons.
These effects appear cumulative and irreversible.
Animals bathed in 100 ppb DDT and animals with
1.4 ppm DPT taken up over 12 days showed lesser but
similar neurophysiological effects. Behavioral
effects at concentrations found in the natural
environment is deemed possible.
235
Introduction
The effects of the insecticide DDT (11) on the
nerves of various animals has been the focus of many
neurophysiological studies in the past. These studies
have mainly attempted to explain the mode of action
and effect of DDT on single neurons. The changes
shown in single cells are (a) repetitive afterdischarge
and (b) prolongation of the action potential (6; 7).
These two effects appear to manifest themselves
behaviorly in hyperexcitability, ataxia, convulsion
and death. Also DDT's effect both on single cells
and on observable behavior increase with amount of
exposure to DDT (3; 6; 8). This work, however, was
all done using high concentrations of DDT far above
levels found in the natural environment.
Since DDT has effects which accumulate with
exposure, it seems reasonable that more subtle
behavioral effects may also occur before the obvious
symptoms described. I believe that electrophysiological
techniques may make a sensitive indicator of such
subtle behavioral aberrations.
This study focused on the question of whether
levels of DDT found in the natural environment and
levels that cause measurable motor changes in the
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laboratory overlap.
I. Behavioral Analysis
In order to test for gross behavioral effects 3
adult female sand crabs, Emerita analoga (Simpson),
were placed in each of six 2 liter beakers containing
1 liter of constantly areated seawater, 1 cm. layer
of sand and differing concentrations of DDT.
Concentrations (in ppb (12)) of 5000, 1000, 100, 10,
5 and 0 (control) DDT were obtained by adding 1 cc.
of acetone containing the appropriate amount of a
mixture of DDT and C+4-DDT (10,000:1) to each beaker.
Observations were made at least twice daily for
the 12 day experimental period. One animal from each
beaker was analyzed for amount of DDT taken up
after the 12 days. The C+-DDT was extracted 3 times
with 10 mls. petroleum ether from a 50% glacial
acetic:50% perchloric acid digest of the animal.
The extract was evaporated to approximately 1 ml..
mixed with 10 mls. of toluene scintillation fluid and
counted using a liquid scintillation counter (Nuclear¬
Chicago, Unilux II). The remaining animals were
saved for neurophysiological analysis.
Refer to Table I for the results of these studies.
II. Neurophysiological Analysis
Animals were pinned ventral side up with their
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abdomens extended and dissected to expose the third
and fourth abdominal ganglia. Seawater between 14
and 19° C was used as a bathing medium (10).
Electrophysiological recordings were made with
a single suction electrode (the nerve was cut distill
to the electrode). Nerve activity was displayed on
an oscilloscope and photographed. Tactile stimulation
was done by touching or brushing with a glass rod.
Electrical recordings of neural activity were
made before and after the introduction of DDT. The
DDT solutions were the same as described in section
I as was the 0.1% acetone control. The one preparation
which had taken up DDT from the 100 ppb
solution of section I (1.4 ppm whole animal concentraion)
was dissected and recordings made in seawater.
Characterization of Nerves Studied
Results of the anatomical study are shown in
figure 1. The nerve (ng) studied leaves the nerve
cord between the third and fourth abdominal ganglia
and apparently innervates the fast flexor muscles
of the fourth abdominal segment.
Six different neurons were identified by their
spike shapes (figure 24) though a seventh may be
present (figure 2B). This seventh spike, labeled
3', was usually seen next to 3 in photographs and
with its similar shape and heigth may be the same.
24
Neurons 1 and 2 seem to be inhibited (these are
normally firing all the time) while one of the
larger neurons, believed to be spike 4, is firing.
This is shown in figures 3E and 4E.
Short bursts of activity could be elicited
from the nerve, ng, by tactile stimulation of either
the head region (particularly the antennae) or the
telson region. Tactile stimulation to the telson
still gave bursting after the nerve cord was cut
anterior to the third ganglion. A cut was then made
just below the third ganglion and bursting could no
longer be elicited by telson stimulation. In
another preparation when the nerve cord was cut
just below ny, anterior to the fourth ganglion,
tactile stimulation to the head region still elicited
bursts. I concluded that the nerve ny is associated
primarily with cells in the third abdominal ganglion.
Telson stimulation, however, may not elicit all 6
spikes, leaving the possibility that some of these
motor neurons may come directly from higher centers
(i.e. more anterior). Also, all 6 neurons may not
have been recorded in these animals so a small number
of the cells could come from the fourth ganglion.
Spiking activity in the nerve, ng, usually
exhibits "size princinle" (2); the smaller spikes
(smaller motorneurons) firing much more often while
the larger spikes are harder to elicit and occur near
-5
the center of bursts when all neurons are firing.
This is shown in the fast traces in figure 2.
The Effect of DDT
One ppm DDT caused an increase in both the amount
of spontaneous bursting (figure 3) and the length of
bursts elicited tactually (figure 4). In the seven
preparations done at this concentration, spontaneous
bursting occurred 5 to 30 minutes after DDT was
applied. By 60 minutes, firing of neurons 1 thru 4
was nearly continuous; large bursts, that included
neurons 5 and 6, were as frequent as every 5 to 10
seconds. After 60 minutes, tactually elicited bursts
continued 5 to 30 seconds after stimulation.
Previously quiescent spike 6 fired frequently after
DDT treatment. Attempts to reverse the effects by
returning the animal to seawater were not sucessful.
Two control preparations were run and these
showed only isolated spiking (neurons 1-4) and
infrequent small bursts (approx. one every 20-30
minutes). The two largest neurons could not be
elicited.
A bathing liquid concentration of 100 ppb DDT
elicited spontaneous bursting only after 1 hour and
this never reached the level seen with the higher
concentration solution. The length of bursting
elicited tactually, however, increased markedly and
very large bursts containing all the neurons were
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common under these conditions.
DDT treatment in all these cases increased the
output of normal firing in ng; coordination of the
spikes, as viewed through the "size principle" and
inhibitory neuronal responses, remained as before
treatment.
In the one preparation taken from the 100 ppb DDT
solution of section I (1.4 ppm whole animal concentration)
recordings were made in seawater. Though little
spontaneous bursting occurred, tactually stimulated
bursts continued for 2 to 12 seconds after stimulation.
clearly longer than either control. Also neurons 5
and 6 could be easily tactually elicited. Again the
coordination between neurons appeared normal.
Discussion
The behavioral observations of the first section
indicate that a whole animal concentration of 1.4 ppm
is about at the threashold where large behavioral
effects can be observed. While most of the DDT found
in Emerita in the field is probably stored in lipids,
under the experimental conditions the crab might not
have been able to transfer much to the lipids,
leaving more free to be taken up by the nervous
system. Nevertheless, this level is comparable to
the 0.92 ppm DDT or 1.2 ppm DDT plus DDD concentrations
found in animals from the Los Angeles area (1). (This
ag
second concentration may actually be more meaningful
since somewhat similar effects to those of DDT have
been reported for the metabolite DDD (3).)
The neurophysiological results show that 1 ppm
DDT increases neuronal discharge in at least na.
This is consistent with the present theory on the
mechanism of DDT action which proposes that the DDT
treated membrane remains depolarized for a longer
than normal time after a spike has fired (4; 5; 6;
7; 8; 9). The effect on tactually stimulated bursts
and the ability of the larger neurons (particularly
6) to fire seem the more sensitive indications of
change; these are most clearly seen in the lower
concentration (100 ppb) and uptake (1.4 ppm whole
animal concentration) preparations.
Finally, if Davis' concept that recruitment of
larger motorneurons results in more forceful movements (2)
applies here then behavioral aberrancies might be
expected at low concentrations wherein neurons 5 and
6 fire at rates higher than controls.
Summary
The nerve of the sand crab, Emerita analoga, which
leaves the main nerve cord between the third and fourth
abdominal ganglia innervates what appears to be the
fast flexor muscles of the fourth abdominal segment.
Six motor axons have been identified, though a seventh
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may be present; spikes 1 and 2 seem to be inhibited
while neuron 4 is firing. Bursting activity
occuring during tactile stimulation of the head region
or telson seems associated with cells in the third
abdominal ganglion. "Size principle" was demonstrated
and discussed.
Gross motor effects were observed in animals
allowed to absorb DDT from seawater. Animals whose
whole body concentrations reached 1.4 ppm DDT were
seen to exhibit large motor effects yet not die within
12 days. This level is close to that reported for
some Emerita in the natural environment.
Neurophysiological recordings show that 1 ppm DDT
in the bathing medium of a dissection greatly increases
(1) amount of spontaneous bursting (2) length of
tactually stimulated bursts and (3) the frequency of
firing of the larger motorneurons. These effects
seemed to increase with exposure to DDT and could not
be reversed. Lower concentrations (100 ppb) and
animals which had taken up DDT in the behavioral
studies showed lesser but similar effects; the
continuance of tactually elicited bursts and the
frequency increase in the large neuron's firing were
the most prominent results. The increased activity
generally seemed to leave coordination between spikes
unchanged. The possibility of behavioral effects
at concentrations found in the natural environment
are discussed.
Acknowledgments
I would like to thank the faculty and students of
Hopkins Marine Station for their interest and
encouragement. In particular Dr. George Mpitsos and
Mr. Robin Burnett have been a continual help during
all parts of this study except in fucking around with
editing the paper.
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References and Notes
1. Burnett, R. Science. (submitted).
Davis, W.J. 1971. Functional significance of motorneuron
size and soma position in swimmeret system of the
Lobster. J. Neurophysiol. 34: 274.
3.
Lalonde, D.I.V., and A.W.A. Brown. 1954.
The effects of
insecticides on the action potentials of insect nerve.
Can. J. Zool. 32: 74.
4.
Matsumara, F., and R.D. O'Brien. 1966a. Absorbtion
and binding of DDT by the central nervous system of
the American cockroach. J. Agr. Food Chem. 14: 36.
Matsumara F., and R.D. O'Brien. 1966b. Interactions
of DPT with components of American cockroach nerve.
J. Agr. Food Chem. 14: 39.
6. Narahashi, T., and H.G. Haas. 1967. DDT: Interaction
with nerve membrane conductance changes. Science.
157: 1438.
Narahashi T., and T. Yamasaki. 1960a. Mechanism of
increase in negitive afterpotential by dicophanum
(DDT) in the giant axons of the cockroach. J. Physiol.,
(London). 152: 122.
8. Narahashi, T., and T. Yamasaki. 1960b. Behaviors of
membrane potential in the cockroach giant axons poisoned
by DDT. J. Celluar Comp. Physiol. 55: 131.
O'Brien, R.D., and F. Matsumara. 1964. DDT - A new
hypothesis of mode of action. Science. 146: 657.
Paul, D.H. 1970. Swimming behavior of Emerita analoga
10.
(Crustacea, Anomura): A Neurophysiological analysis.
PhD. Thesis. Stanford University.
11. DDT stand for 1,1,1-trichloro-2,2-bis(p-chlorophenyl)
ethane.
ppm stands for parts per million (ug/g
12.
ppb stands for parts per billion (ug/g).
Both are per gram wet weight when animals concentrations
are referred to.
25
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Figure
A. The undissected abdomen with the telson
extended.
B. Dissection showing the third thru fifth
abdominal ganglia. Electrophysiological
recordings were from ng.
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Figure 2.
A. Six different identified spikes from ny are shown.
B. Spikes 3 and 3' are shown. Since they differ only
slightly in size and shape and usually occur close
together they could be from the same neuron.
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953
Figure 3.
Spontaneous activity seen in the nerve n- after
application of DDT. (A) before DDT applièation;
(B) 10 minutes after application; (C) 30 minutes;
(D) 45 minutes; (E) 60 minutes. Trace E also
shows inhibition of spikes 1 and 2 during spike
4. Gain: A-D .5 mV/cm.
.2 mV/cm.
26
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Figure 4.
Activity elicited by tactile stimulation of the
head region after DDT application. (A) before DD
application; (B) 20 minutes after application;
(C) 30 minutes; (D) 45 minutes; (E) 60 minutes.
E shows the inhibition of the two small spikes
during spike 4."" indicates start of stimulation;
"" indicates cessation of stimulation.
Gain: A-D
.5 mV/cm.
.2 mV/cm.
2