*
X-/EPA
Research and Development
EPA-600/S2-81-234 Feb. 1982
Project Summary
HERL Biological Exposure
Chamber Conceptual Design
Technical Note
R. M. Parks
Because of the current interest in
biotesting of potentially hazardous air
pollutants, the Health Effects
Research Laboratory (HERL) of
EPA/RTP has contracted Radian to
design biological exposure chambers
that can be used to expose test organ-
isms to the secondary aerosol effluent
of the MARC (Mobile Aerosol Reac-
tion Chamber). The purpose of this
technical note is to describe the
conceptual design of the biological
exposure chambers.
The three organisms HERL desires
to use for bioassays will be exposed in
four different ways. They are (1) Sal-
monella in Petri dishes, (2) Salmonella
in Erlenmeyer flasks (3) Drosophila in
nylon mesh cages, and (4) Tradescan-
tia cuttings in pots and/or beakers.
The physical environment within the
four exposure chambers will be con-
trolled to expose the organisms with
minimum stress and within published
tolerance limits. The four streams that
HERL would like to test for biological
activity are: (1) the diluted source
stream; (2) the filtered MARC exit
stream; (3) the unfiltered MARC exit
stream; and (4) the clean air supply.
The report describes the chamber
design and rationale behind the
design. The report also discusses the
connecting of the biochambers to the
MARC.
This Project Summary was develop-
ed 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
Radian Corporation is currently under
contract to the Environmental Protec-
tion Agency (EPA) to design, build, and
test a Mobile Aerosol Reaction Chamber
(MARC) to generate secondary pollu-
tants from advanced fossil fuel technol-
ogies.
HERL desires to expose several
organisms to the MARC effluent,
including Salmonella, Tradescantia,
and Drosophila. Radian has designed
the HERL Chamber in such a way that
the organisms can be suitably exposed
and the MARC is unaffected by the
chamber operation. The process of
designing the chamber required consid-
eration of the following:
• Insuring that the conditions within
the chamber are conducive to the
growth and survival of the organ-
isms and consistent with estab-
lished experimental protocols,
• matching the HERL Chamber to
the MARC so that the aerosols are
introduced into the chamber with
minimum impact on the MARC,
and
• designing a sampling system so
that certain specimens in the
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chambers can be treated in such a
way that their exposure to the
chamber environment is stopped
while the remainder of the speci-
mens are continuously exposed.
Sections 2 and 3 of the technical note
are concerned with the conditions with-
in the chambers. Section 4 is concerned
with connecting the biochambers to the
MARC, and Section 5 is concerned with
sampling the organisms in the
chambers.
Size, Shape and Mixing
The size and shape of the HERL
biological exposure chamber will be
determined mainly by the constraints of
the trailer presently housing the MARC.
While the chamber must be .designed to
perform the functions HERL desires, the
exposure chamber must fit into the
available space in the trailer and not
perturb the operation of the MARC.
Radian recommends a rectangular
configuration for the chamber shape.
While this shape will not be the best for
mixing the contents of the chambers, it
will make the best use of the available
trailer space. A cylindrical vessel that
would fit in the same space and give
better mixing performance would cut
the number of Petri dish samples by half
and would not offer any advantages
over the rectangular shape. Also, there
will be shelves and containers m the
chambers which will have more of an
effect on the mixing characteristics than
the vessel shape. Although Radian does
not anticipate any major mixing prob-
lems, thoroughly testing and documen-
ting of the mixing characteristics of
each chamber is recommended before
biotesting is initiated.
The three organisms HERL desires to
use for bioassays will be exposed in four
different ways. They are:
Salmonella
Petri dishes
Erlenmeyer flasks
Drosophila Nylon mesh cages
Tradescantia Pots or beakers
Since the chambers will be used to
expose three different types of organ-
isms, the internal configuration of the
chambers will vary for each organism.
When Salmonella are exposed in
Erlenmeyer flasks, appropriate fittings
can. be used to introduce the aerosols
directly from the MARC to the liquid
medium in the flasks. Small rotary
shakers will mix the contents of the
flasks. These shakers can be placed
directly in the biochambers if tempera-
ture control is a problem, or they can be
left out of the chambers if temperature
control is not a problem.
To expose the Drosophila to the
MARC effluent, the top shelf of the bio-
chambers can be removed to allow
stacking of cages containing the organ-
ism in the chambers. The number of
cages that can be located in the
chambers for a given experiment can be
predetermined by HERL when they size
cages. This allows HERL a greater
degree of flexibility in experimental
design.
Cuttings of Tradescantia can be
exposed in a chamber of configuration
similar to the ones used for Drosophila.
Cuttings can be placed in a small pot or
beaker and the beakers inserted in the
chambers to obtain multiple exposures.
This method of exposure allows for
large amounts of data collection which
would aid HERL in obtaining statistical
valid results.
For both the Drosophila and Trades-
cantia exposures, a feed stream for each
biochamber will be introduced in the
upper part of the vessel and the output
will be in the lower part of the chamber.
These requirements can be readily met
with the chambers as described above
for Salmonella.
Temperature, Lighting and
Humidity
The physical environment within
each chamber will be controlled to
expose the organisms with minimum
stress and within published tolerance
limits.
Temperature
The temperature that should be main-
tained in the biochambers while
exposing the Drosophila and Trades-
cantia can be the ambient trailer
temperature with no damage to the
organisms. For runs utilizing these
organisms, as well as experiments
exposing Salmonella at ambient condi-
tions, the heating or cooling of the
chambers will be no problem. The trailer
housing the MARC is sufficiently condi-
tioned to hold the MARC as well as the
biochambers at the desired tempera-
tures. However, when running
Salmonella at the higher temperatures,
37 ±0.5°C, auxiliary heating equipment
will be used to elevate the chamber
temperature. Also, adequate insulation
will be provided around the chambers to
reduce heat loss.
Humidity
The relative humidity in the bio-
chambers needs to be kept at 60 to 80
percent for all the organisms. When the
MARC is operated at the high relative
humidity condition, there will be little
problem maintaining the necessary
moisture in the biochambers when they
are operated at ambient temperatures.
However, for the high temperature
Salmonella runs, as well as the MARC
runs at low humidity levels, moisture
will have to be added to the bio-
chambers to raise the relative humidity
to the desired level.
Lighting
Both Drosophila and Salmonella can
be exposed to the secondary aerosols in
the dark. However, Tradescantia should
be exposed in a lighted environment for
a maximum response to the aerosols.
The amount of light will not be critical,
but should be photosynthetically active.
radiation. By exposing the Tradescantia\
system under lighted conditions, the
stamens of the organism remain open
and diffusion of the aerosols into them
will be maximized.
Interfacing the Biochambers
to the MARC
Since the biochambers are consider-
ed auxiliary testing devices for the
aerosols produced in the MARC, they
must be attached to the MARC in such a
way that they do not disturb the opera-
tion of the aerosol reaction chamber.
Although there are to be four compart-
ments in the biochamber module, only
two of these will directly utilize the
MARC effluent.
Flow Patterns for Chamber
Interface
Connecting the HERL biochambers to
the MARC will be done in such a way as
to supply the biochambers with the
desired aerosol effluent while not
causing any adverse effects on the
MARC. Two of these MARC streams can
be sampled with no perturbation to the
system. A
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1. The clean air supply.
2. The diluted source stream (the
MARC feed stream).
Since the clean air system is capable of
delivering up to 10 cubic feet per minute
(10 cfm) and the MARC will only require
1-3 cfm, there will be enough excess
clean air to feed the clean air section of
the biochamber. The flow rate of this
stream to the biochamber will be in the
range of 0.25 to 0.5 acfm. Since the
chamber volume is 6.75 ft3 the resi-
dence time (t) in the clean air chamber
will be 13.5 to 27 minutes. Provisions
will be made to humidify this chamber
since the clean air is dry (a 0% relative
humidity). The feed stream to this
chamber will be tapped from the clean
air feed just downstream of the clean air
generator. This will cause the clean air
chamber to operate at a positive pres-
sure. The exit from this chamber can be
routed to the atmosphere with no
further treatment.
The diluted source stream is the
second stream which should be in
plentiful supply and should pose no
sampling problems. This stream will be
sampled after all dilutions have
occurred and will be transported to the
appropriate biochamber by a line large
enough to keep the pressure drop
across the system to a minimum. The
stream will be tapped off the MARC feed
stream between the inlet manifold and
the inlet sampling pump. This arrange-
ment will mean that the biochamber is
operating at a pressure slightly jDelow
atmospheric. The flow rate of this
stream will be between 0.25 and 0.5
acfm to give an average residence time
(t) of 13.5 to 27 minutes for the 6.75 ft3
chamber. Also, because the stream will
be sampled before it is humidified,
provisions for humidifying this chamber
will be necessary to provide the
exposure conditions discussed in
Section 3 of the report. The exit stream
from this reactor can be routed to the
atmosphere.
Two samples remain that HERL would
like to test for biological activity.
1. A filtered MARC exit stream.
2. An unfiltered MARC exit stream.
Both of these streams will come directly
from the MARC.
Of the two samples, the filtered efflu-
ent sample can be most easily obtained.
One of these streams goes to a sampling
manifold where periodic samples are
taken for chemical characterization. The
excess from this manifold is pumped
through a final filter and exhausted
through a dry test meter to the atmos-
phere. Since the flow rate of this line
will be on the order of 1 cfm, the bio-
chamber can be inserted between the
final outlet filter and the outlet sampling
pump. The feed to the biochamber will
be the stream coming from the outlet
filter and the exit stream from the bio-
chamber would be returned to the outlet
pump. The pump exhaust could be
dumped to the atmosphere as originally
planned or filtered again for sample
collection. Since the feed to the
biochambers is taken upstream of the
pump intake, the biochamber will
operate at a negative pressure. The
pressure drop through the biochamber
is expected to be small and there should
be little disturbance to the MARC
system.
The final sample HERL would like to
bioassay is the unfiltered MARC efflu-
ent. Since this sample will contain
aerosols, it will be best not to transport
the sample any great distance. A
separate output port will be placed in
the MARC at the rear of the trailer in the
area where the biochambers are
located. While this separate output line
from the MARC will represent an ideal
feed for the biochamber, several things
have to be considered before a success-
ful interface can be achieved. At
present, the total flow rate of material
through the MARC has not been deter-
mined and since a mass balance has to
be satisfied around the MARC (Input to
MARC = Output from MARC) only the
extra aerosol beyond that needed for
chemical characterization will be
available for the biochamber feed. If
baseline chemical characterization
tests show that enough aerosols can be
collected with a high throughput of feed
materials, then there will be enough
aerosols remaining for the biochamber
feed. The final decision on this matter
will have to await the completion of the
chemical characterization of the MARC
output.
As in all the other cases, the line
taking the effluent sample to the bio-
chamber will be sized to prevent
significant pressure drop across the
system. Depending on the final outcome
of the chemical test, the flow rate to this
chamber could be 0.25 to 0.5 acfm,
giving a residence time (t) of 13.5 to 27
minutes. Also, water will have to be
added to this chamber to bring the rela-
tive humidity up to the desired level for
the high temperature runs.
The biochamber will need to have a
filter and pump downstream of the exit
and will operate at a slight negative
pressure. There should be only a slight
pressure drop associated with passing
the effluent through the biochamber
and connecting lines and should cause
no problems in the MARC operation. At
present, a restricting orifice is used to
adjust the back pressure in the chamber
by controlling the flow in the vent line.
The size of this orifice will have to be
adjusted to allow the proper pressures
and flows in the MARC allowing the two
reaction vessels to operate at almost the
same pressure.
Since three of the chambers are
operating at negative pressures, the
output of the pumps will vent to the
atmosphere and are not anticipated to
cause any more problem than venting
the associated MARC process stream
itself.
A separate flow system will be
provided to flush all the chamber with
clean, sterile air before the start of each
experiment. This stream will be
provided by filtering air from the existing
MARC compressor, or a separate
compressor may be used. Filters will
have to be installed in line to provide the
required quality of air. This arrange-
ment will insure that the experiments
are started with sterile air in the
chambers.
Sampling Systems
To determine the effect of dosage,
samples must be periodically isolated
from the exposure stream. This type of
operating procedure will enable HERL
personnel to develop dose-responses
information on the organisms exposed
to the aerosols. The dosage variable can
in this way be expressed in units of time.
To do this, the exposure chambers have
to be filled with the appropriate number
of test organisms, and certain ones
isolated at given intervals. For Salmo-
nella exposed in Petri dishes, it will be
possible to cover the Petri dish at the
appropriate time and limit exposure.
This will be the easiest and least dis-
turbing method of sample control for the
chamber. For Tradescantia and
Drosophila, the organisms also can be
isolated from the aerosol environment
of the biochambers. The provisions for
handling the samples in this way will
have to be designed into the chamber
sampling system.
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Several techniques can be employed
to isolate the organisms, Tradescantia
and Drosophila, from the reactive
aerosols. The simplest system is to
insert the organisms into an isolation
vessel contained inside the biocham-
bers. The isolation vessel can be purged
of the aerosol reactants and the biolog-
ical exposures effectively quenched.
The biochambers have been designed to
accommodate this type of isolation
procedure. The glove/glove box design
of the front panel of the biochambers
will allow easy access into all areas of
the chambers and facilitatethe isolation
of selected samples. The isolation
vessel will be a flexible-walled contain-
er that can be flushed with clean air
from the clean air system. Appropriate
converters will be used to pass the clean
air from the generating system outside
the chambers to the isolation vessel
inside the biochambers. When the
entire experiment is completed, the
front panel of the chambers can be
removed and the specimens recovered.
While each of the three types of
organisms will require their own type of
sampling protocol, the glove arrange-
ment will offer the maximum protection
and flexibility to all the samples.
R. M. Parks is with Radian Corporation. 3024 Pickett Road. Durham. NC27709
Larry Claxton is the EPA Project Officer (see below).
The complete report, entitled "HERL Biological Exposure Chamber Conceptual
Design: Technical Note," (Order No. PB 82-114 646; Cost: $7.50, 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
U S GOVERNMENT PRINTING OFFICE, 1982 — 559-017/7448
United States
Environmental Protection
Agency
Center for Environmental Research
Information
Cincinnati OH 45268
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Penalty for Private Use $300
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