United States
Environmental Protection
Agency
Health Effects
Research Laboratory
Research Triangle Park, NC 27711
Research and Development
EPA/600/S1-88/005 Jan. 1989
&EPA Project Summary
Behavioral Effects of
Microwaves: Relationship of
Total Dose and Dose Rate
Mary Ellen O'Connor and Robert Strattan
The goal of this research was to
compare the relationship of whole
body averaged specific absorption
rate (SAR) and specific absorption
(SA) to determine if dose rate or
dose was a better predictor of
biological effects. Sperm positive
Long-Evans female rats were
exposed to 2450 MHz CW microwave
radiation for 1-3 hours at
approximately 10 W/kg. The maternal
subjects were then observed for
natural delivery of their litters.
Sensitivity to thermally induced
seizures and huddling were studied
in the offspring. Analyses revealed
that there were no statistically
significant differences between
exposed and control offspring on the
behavioral indices. The behavior did
not appear to be effected by prenatal
exposure to microwave radiation at
these levels. The huddle sizes
became smaller as the pups aged
both in exposed and control
offspring.
This Project Summary was
developed by EPA's Health Effects
Research Laboratory, Research
Triangle Park, NC, to announce key
findings of the research project that
is fully documented in a separate
report of the same title (see Project
Report ordering information at back).
Introduction
The purpose of the investigation was
to define the relationship of exposure
duration and average whole body
specific absorption rate (SAR) of
continuous wave (CW) radiofrequency
radiation (RFR) using biological
endpomts The research was conducted
in two phases. In the first phase, female
Long-Evans rats were exposed at SARs
of 2, 4, 6, or 8 W/kg for durations of 1, 2,
3, 4, 5, or 6 hours. Subsequently, CF-1
mice were exposed at 2, 4, and 8 W/kg
for durations of 1,2, 3, 4, 5, or 6 hours.
Colonic temperatures were taken
immediately before and immediately
following each exposure session.
In the second phase of the research
sperm-positive Long-Evans female
rats were exposed at SARs of 10 W/kg
for durations of 1 and 3 hours. The
exposures occurred on gestational days
12 through 18. The maternal subjects
were irradiated and then observed for
natural delivery of their litters. The pups
constituted the subject pool for
subsequent behavioral studies. The
behavioral tests included sensitivity to
thermally induced seizures and huddle
size.
Experimental Procedures
All maternal exposures occurred in a
Lmdgren 4-Shield RFR anechoic
chamber The sham chamber was
located adjacent to the microwave
exposure facility and shared the same air
flow system with the anechoic chamber.
The temperature at the location of the
subject differed by no more than 1°C
between the anechoic and sham
chambers 2450-MHz CW microwave
radiation was emitted from a horn
antenna mounted on the ceiling of the
anechoic chamber. During exposure, the
animals were placed in Plexiglas
cylindrical containers that were
positioned on top of a bilayer styrofoam
platform. The pup seizures in the second
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phase were performed using a 2450-
MHz circularly polarized waveguide
placed inside the sham chamber to
provide ambient temperature and
humidity control.
The SAR was determined using twin
well calorimetry. The power settings for
the estimated range of masses were
determined from measurements of the
temperature change of equal masses of
Ringer's solution in plastic bags Animal
carcasses were then used to determine if
the selected power levels did in fact
produce equivalent SAR measurements.
In the second phase, the exposure levels
were determined using a pre-
established table for the desired SAR of
10 W/kg based upon the mass of the
maternal subject. The power level was
changed each day according to the body
mass in order to provide a constant SAR
throughout the exposure period
The subjects were female Long-
Evans rats and female CF-1 mice from
the animal colony at The University of
Tulsa. The rats used in the first phase of
the study ranged from 64 to 100 days of
age while the mice ranged from 56 to
133 days of age The rats were chosen
from the available animals based on a
body mass between 180 and 190 g while
the mice had body masses between 28
and 30 g. The rats for the second phase
were primaparous females between 90
and 180 days of age at the time of
breeding. The day on which a sperm
plug was detected was considered Day 1
of gestation.
The schedule for obtaining
measurements at durations of 1,2, 3, 4,
5, or 6 hours for each of the SAR levels
was determined randomly. Animals were
exposed singly and all of the exposures
were scheduled such that the end of the
exposure occurred at the same hour of
the day (15:00). The body mass of the
animals available for exposure was
measured between 8:00 and 9.00 h.
Colonic temperatures were taken
immediately prior to placing the animal in
the Plexiglas holder for exposure and
again at the end of the exposure period.
In the first phase, the rat exposure
schedule was repeated three times
resulting in three rats in each of the 24
conditions. The mouse exposure
schedule was repeated twice resulting in
two mice in each of the 18 exposure
conditions.
In the second phase, the same
procedures were followed for the 1 and 3
hour exposure periods. The exposures
began on Day 12 of gestation and
continued through Day 18. Each day, a 1
hour and a 3 hour exposure session was
conducted. Morning and afternoon
exposures were counterbalanced for
exposure groups. After the exposure
session on Day 18, the maternal subject
was placed in a plastic maternity cage
and monitored until the day of birth of
the litter At this time the pups were
counted, weighed, and assigned to either
the seizure or huddle study.
On day two of age the pups were
counted and all pups in a litter over
seven were chosen randomly and used
in the seizure study. The seizures were
observed by placing the pup in a beaker
inside the circularly polarized waveguide.
The pre-exposure skin temperature, the
post-exposure skin temperature, the
condition of the pup, and the ambient
temperature were all recorded.
The huddle sizes were measured at
5, 10, and 15 days of age On each of
the three days, six pups were removed
from the home cage and placed in a
plastic test cage. After a 15 minute
acclimation period, huddles were
photographed from directly overhead
using a tripod-mounted 35 mm camera.
One photograph was taken every 15
minutes until four photographs had been
taken of each litter. The developed slides
were projected onto a chalkboard and
the outer circumference of the huddles
was traced onto a sheet of paper taped
to the board. Two methods for measuring
the size of the huddle were used The
method termed perimeter measures
traced the perimeter of the huddle as if
the pups were enclosed in a rubberband
resulting in a convex polygon entirely
enclosing all pups in the litter. The other
measure is referred to as the individual
pup measure and was obtained by
tracing the circumference of the huddle
including the outline of the pup without
extended tails and limbs.
Results and Discussion
In general the average temperature
change (post-minus pre-exposure
colonic temperature) for the exposed
Long-Evans rats reflected increased
colonic temperatures. With two
exceptions, (SAR 2W'kg for 4 or 5 hours)
the colonic temperature increased during
the exposure. For a given SAR, the
temperature change did not vary
considerably as an effect of duration of
exposure. However, the sham exposed
rats showed a decrease in colonic
temperature that is more evident for
longer exposure durations. Any exposure
longer than one hour resulted in a
colonic temperature decrease in the
sham exposed rats.
The correlation between pos
exposure colonic temperature and SA i
significant (r - 0 46, p < 0.0001) as i
the correlation between post exposur
colonic temperature and SAR (r = 0.75
p < 0.0001). It is of interest to note the
SAR is a much better predictor of pos
exposure temperature in the ra
accounting for 62% of the variance
While SA is significant, it only account
for 21% of the variance. Of th
correlations between duration o
exposure and post exposure colom
temperature at each of the four SAI
levels, only the 2 W/kg condition wa
significant (r = -055, p < 001), an>
this correlation was negative.
These same comparisons wer
made for the CF-1 mice. For duration
of exposure longer than one or two noun
all of the mice had lower post exposur
colonic temperatures The averag
temperature change for the shar
exposed mice also reflected a decrease
The post exposure coloni
temperature was not related significantl
to SA and was only mildly related to SAI
(r = 0.43, p < 0 005). The correlation
between duration of exposure and poj
colonic temperature were all negative an
only the 2 W/kg condition was significar
(r = -0 57, p < 0.03) The pos
exposure temperature was lower than th
pre-exposure temperature for all but fiv
mice and the relationship between pos
exposure temperature and duration c
exposure (r = -057, p < 00001) a
well as the relationship betwee
temperature change and duration c
exposure (r = -0.35, p < 0.02) wer
significant.
The average for the whole bod
average SAR was 10.45 W/kg (± 1.0
SD) The highest SAR value was 12.
W/kg and the lowest was 8.5 W/kg. Dat
from 59 gravid dams were used in th
analyses; 18 cage control, 21 shar
exposed and 20 microwave exposed
All treatment variables associate'
with the characteristics of the maternj
subjects and their exposure as well a
the variables associated with the litte
characteristics and behavioral measure
were analyzed using a three factc
analysis of variance. Post hoc test
(Tukey HSD) were performed only whe
a significant mam effect was followed b
a significant interaction
Although the average mass of th
dams in the different treatment group
did not differ, there was a statistical!
significant difference in the mass game
from Day 1 through Day 18 (F = 5 87, ,
< 001). This difference was due to th
sham exposed groups gaming more tha
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the microwave exposed groups. The
cage control group was not different in
weight gain from either the sham
exposed or the microwave exposed
group. Time of day of exposure and
duration of exposure were not significant.
As expected, the microwave
exposed groups experienced an increase
in colonic temperature during exposure.
There was a significant difference in
temperature change associated with all
three of the major variables: exposure
condition (F = 289.98, p < 0.001),
duration of exposure (F = 11.8, p <
0.002), and time of day of exposure (F =
4.21, p < 0.05). The average
temperature change for the microwave
exposed groups was 2.04°C. The sham
exposed groups had a decrease in tem-
perature with an average of -051°C.
The one hour groups increased more
than the 3 hour groups while the morning
exposure groups increased less than the
afternoon Groups. There were no
significant interactions.
Litter sizes for the pups at birth were
not significantly different with averages of
1122, 11.76, and 1060 pups for the
cage control, sham exposed and
microwave exposed groups. Pups from
each litter were selected for the seizure
study when they were 2 days of age.
There were no differences for the mass
of the pups, the pre- and post-
treatment skin temperature, the
difference between the post- and pre-
treatment skin temperature or the
average latency to seizure.
Huddle sizes were analyzed on days
5, 10, and 15 of age. Both the perimeter
measurement (F = 3.51, p < 0 05) and
the individual pup measurement (F =
40.42, p < 0001) produced significant
differences based upon the age of the
pups. Older pups form smaller and
smaller huddles. The proportion of
variance accounted for by the two
measurement techniques was
calculatedusing the efa2 method
suggested by other investigators The
perimeter measure accounted for 8% of
the variance in huddle size across the
three age groups while the individual
measure accounted for 57%.
Conclusions and
Recommendations
Under identical exposure conditions
and identical rates of energy deposition
(SAR) the thermal response as measured
by colonic temperature is dramatically
different for the mouse and the rat. Mice
actually decrease their body temperature
while being dosed with energy at 2, 4,
and 8 W/kg even when the duration of
exposure is as long as 6 hours. The
thermal response of the microwave
exposed mice is not distinguishable from
that of sham exposed mice when
measured by colonic temperature.
The rats did not display this efficient
thermal regulation of body temperature
under these conditions of exposure.
Levels of radiation at 2, 4, 6, and 8 W/kg
raised body temperature to a given
degree and the rats maintained this
increased temperature for durations of
exposure as long as 6 hours Sham
exposed rats showed a reduction in body
temperature at all durations of exposure
greater than one hour.
The use of colonic temperature as
an indication of thermal responsivity
results in opposite results in sham
exposed and microwave exposed rats
but does not indicate differences in sham
and microwave exposed mice. Such a
dramatic species difference under
identical laboratory exposure conditions
argues for extreme caution in attempting
to generalize to human exposure
conditions. In particular, biological effects
observed in only one non-human
species should be used in establishing
human exposure conditions only when
the procedure allows for the inclusion of
considerable caveats regarding the lack
of essential data. The procedure should
also ensure that these caveats will be
included in the tables or figures used by
practitioners in attempting to abide by a
recommendation.
In phase 2 there were significant
differences in weight gain between the
microwave and sham exposed maternal
subjects. However, this difference was
not accompanied by differences in either
litter size or pup mass.
The results of the second phase
indicated that post-natal measures of
thermally induced seizure sensitivity or
huddle size in pre-natally exposed rat
pups were not significantly effected by
the microwave exposure at 10 W/kg. Two
measures were used to determine huddle
size. The age of the pups was a
significant variable and the proportion of
variance accounted for was much greater
for one of the two measures. Behavioral
research often demonstrates the
importance of operational definitions in
which variables and procedures are
defined by the methods used to measure
them. Like species differences, the
importance of operational definitions in
laboratory research and its interpretation
is a basic experimental principle learned
by most investigators in the most
elementary research methods courses.
However, bioelectromagnetics research is
replete with investigations relying on one
species and one experimental
measurement technique. Some of the
ambiguities in the data base might be
explained by these two basic
experimental principles.
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Mary Ellen O'Connor and Robert Strattan are with The University of Tulsa, Tulsa,
OK 74104.
Ezra Barman is the EPA Project Officer (see below).
The complete report, entitled "Behavioral Effects of Microwaves: Relationship of
Total Dose and Dose Rate," (Order No. PB 89-118 6401 AS; Cost:
$15.95, subject to change) will be available only from:
National Technical Information Service
5285 Port Royal Road
Springfield, VA22161
Telephone: 703-487-4650
The EPA Project Officer can be contacted at:
Health Effects Research Laboratory
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
United States
Environmental Protection
Agency
Center for Environmental Research
Information
Cincinnati OH 45268
BULK-RAT^-
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EPA
Official Business
Penalty for Private Use $300
EPA/600/S1-88/005
0000329
IL 60604
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