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July – September 2012 1
The organophosphates and closely related chemicals
include not only important insecticides, but also potent
chemical warfare agents (CWA) that have been used by
militaries and terrorists alike. Important organophos-
phate insecticides include malathion, parathion, and chlo-
rpyrifos which are used in a broad range of insect control
programs from agriculture to public health. Among the
related CWA are the neurotoxins sarin, tabun, and VX.
Decontamination of areas containing these chemicals
may be necessary following the inappropriate use of in-
secticides or following a terrorist attack with a CWA. For
instance, in the early 1990s, several illegal applications
of methyl-parathion, a potent agricultural insecticide,
were used to control cockroach infestations in human
residences in Ohio, Mississippi, Louisiana, Illinois, and
Mississippi.1 More than 4,500 homes were affected and
2 children were killed. More recently, resurging bed bug
populations in the United States led to the illegal use of
malathion, carbaryl, and cypermethin in over 70 houses
in New Jersey, eventually requiring varying levels of
decontamination.2 Similar decontaminations have also
been required after improper termiticide applications
in military housing.3,4 The threat to public health pre-
sented by inappropriately used insecticides is obvious,
but the CWA are also of interest to people working in
homeland security and national defense. The intentional
use of sarin in the Tokyo subway system in 1995 is an
example of terrorists’ use of CWA. One event resulted
in 12 deaths and approximately 5,000 injuries, including
injuries to fi rst responders.5
Decontamination of the CWA has been defi ned as the
“process of making any person, object or area safe by ab-
sorbing, destroying, neutralizing, making harmless, or
removing chemical…agents.”6 Consistent with this defi -
nition, early attempts at CWA decontamination included
washing with soap and water, absorbing with Fuller’s
earth, and simply leaving the chemicals to weather natu-
rally over time. Actual chemical degradation of the tox-
in often relied on harsh chemicals such as calcium oxide
and chlorine dioxide. New decontaminating compounds
have been developed that are more effective or more en-
vironmentally friendly, including organophosphorous
acid anydrolase (a hydrolyzing enzyme), and decon-
taminating foam with hydrogen peroxide. Much of the
research required to quantify CWA decontamination re-
quires sophisticated instrumental analytical techniques
such as liquid or gas chromatography, which involves
expensive equipment and trained personnel.7-9 Extensive
reviews of analytic detection and monitoring techniques
are provided by Witkiewicz et al
10 and Kientz.11 Such
techniques are considered defi nitive but may provide
A Rapid and Inexpensive Bioassay to Evaluate
the Decontamination of Organophosphates
CDR (Ret) David M. Claborn, MSC, USN
Skylar A. Martin-Brown, MS
Sanjay Gupta Sagar, BS
Paul Durham, PhD
All authors state that they have no confl icts of interest involving the research reported in this manuscript and do not have invest-
ments or business with the companies mentioned. Mention of any company or product should not be seen as an endorsement by
Missouri State University or any branch of the US military. This study was approved by the Institutional Review Board of Missouri
State University.
ABSTRACT
An inexpensive and rapid bioassay using adult red fl our beetles was developed for use in assessing the decon-
tamination of environments containing organophosphates and related chemicals. A decontamination protocol
was developed which demonst rated t hat 2 to 3 applications of 5 % bleach solution we re required to obtain nearly
complete decontamination of malathion. The bioassay was also used to screen common household cleaners as
potential decontaminating agents, but only 5% bleach was effective at improving survival of insects on steel
plates treated with 25% malathion. A toxic degradation product (malaoxon) was detected using gas chromatog-
raphy/mass spectrophotometry; this toxin affected the decontamination effi cacy and resulted in continued tox-
icity to the beetles until subsequent decontaminations. The bioassay provides evidence to support the use of red
fl our beetles as a sensitive, less expensive method for determining safety levels of environments contaminated
with malathion and other toxins, and may have application in the study of chemical warfare agents.
2 http://www.cs.amedd.army.mil/amedd_journal.aspx
only indirect measures of the biological toxicity. Often
such processes document the breakdown of the target
chemical into degradation products that are also toxic,
though perhaps much less so than the original toxin.
As Munnecke12 stated, “the true change in toxicity of a
pesticide containing medium can only be measured by
conducting pertinent in vivo bioassays….” The same is
probably true for CWA.
An inexpensive and rapid bioassay would be a useful
screening tool with which to assess potential decontam-
inating agents for subsequent, more defi nitive testing by
chemical analysis. Such a screening test would also be
useful in quantifying or confi rming changes in biologi-
cal toxicity as a result of decontamination efforts. This
article describes such a bioassay using an easily main-
tained insect colony. The bioassay is primarily intended
to be a rapid screening tool. It is based on a previously
published study by the fi rst author and colleagues that
demonstrated the detoxifi cation of insecticides by cer-
tain types of paint used on ships.13 In that study, the red
fl our beetle, Tribolium castaneum (Herbst), was used as
a test organism on painted and unpainted steel plates
treated with three different pesticide formulations. The
mortality rate of beetles exposed to some of the insecti-
cides was lower on painted steel plates when compared
to unpainted plates. This study used a similar technique
as a bioassay to investigate the level of decontamination
of an organophosphate insecticide that has also been
used as a CWA simulant.
MATERIALS AND METHODS
Development of Bioassay
The red fl our beetle (RFB) was selected as a test organ-
ism with which to develop a rapid, inexpensive, and
sensitive bioassay for evaluation of organophosphate
decontamination. The RFB was used because it is easy
to rear and handle in the laboratory, and has a long his-
tory of use in insecticide tests.13 A colony of insects
was obtained from the USDA Stored Products Labora-
tory in Manhattan, Kansas, in April 2011. The insects
were raised in 946.4 ml canning jars on a diet of whole
wheat fl our and baker’s yeast (10:1 mixture). The center
of the jar lid was replaced with an unbleached coffee
fi lter and held in place with the ring. A new colony was
started every week by moving a few adults and a spoon-
ful of fl our with larvae into a new jar with the fl our:yeast
mixture. The jars were kept at room temperature (21°C
to 24°C) in a dark cabinet with an open pan of water
to provide moisture. All of the insects used for these
experiments were from colonies started on April 15 or
April 20, 2011.
Unpainted steel plates (20 cm by 20 cm) were cleaned
with ethanol and allowed to air dry. The plates were treat-
ed with a 1-ml aliquot of 25% malathion. A commercial
formulation of 50% malathion (Spectracide, Chemsico,
St. Louis, MO) was used as the stock solution and was
further diluted to 25% with xylene, which was also the
diluent in the commercial formulation. This percent-
age was used because greater concentrations produced
nonuniform dispersal on the plates as evidenced by oily
droplets after decontamination. At 25% concentration,
the malathion could be applied uniformly and it dried
enough to allow the insects to move freely on the sur-
face. The 1-ml aliquot was dripped onto the plate from
a pipette, then spread evenly with a dry, 2.54 cm wide
nylon bristle brush, covering the surface area of one side
of the steel plate. Applications were made under a hood
and the plates were allowed to dry for 48 hours. The
plates then received the fi rst decontamination.
All decontaminations were applied with an air brush
(Iwata Revolution, Iwata-Medea Inc, Portland, OR) to
prevent any unintended physical removal of the mala-
thion that might occur with a brush or other application
technique. The propellant for the air brush was 1,1-di-
fl ouroethane in a pressurized can. Decontaminant ap-
plications were all done in 1-ml aliquots. Potential de-
contaminants included:
Standard household bleach (5% sodium hypochlo-
rite), Clorox Bleach, The Clorox Company, Oak-
land, CA
95% ethanol
Lysol All-Purpose Cleaner (active ingredient 3.2%
lactic acid), Reckitt Benckiser Inc, Parsippany, NJ
Simple Green Concentrate, Sunshine Makers Inc,
Huntington Beach, CA
Pine Sol Concentrate (active ingredient 8.7% pine
oil), The Clorox Company, Oakland, CA
Bleach and ethanol were both selected because they are
commonly used for insecticide decontamination, and
bleach is a standard decontamination agent for nerve
agent weapons.9 Lysol, Simple Green, and PineSol are
common household cleaners that could be used by resi-
dents of a contaminated facility to clean an insecticide-
contaminated area. Ethanol and bleach were used as de-
contaminants in all tests, but only one of the 3 household
cleaners was used in each of 3 replications. The decon-
tamination schedule is shown in Table 1.
In each replication, the plates were decontaminated
3 times consistent with the schedule in Table 1. After
each decontamination, the plates were allowed to dry
A RAPID AND INEXPENSIVE BIOASSAY TO EVALUATE
THE DECONTAMINATION OF ORGANOPHOSPHATES
THE ARMY MEDICAL DEPARTMENT JOURNAL
July – September 2012 3
for 48 hours before the insect bioassay was performed.
An untreated, nondecontaminated control was used for
each replication as well as a control that was treated
only with 1 ml of 5% bleach and another treated with
¾ ml of the xylene diluent. In replication No. 1, Lysol
was compared to bleach and alcohol; in replication No.
2, Simple Green was substituted for Lysol, followed by
Pine Sol in replication No. 3. Three to 4 plates received
each treatment/decontamination combination in each of
the 3 replications. A separate bioassay was run to com-
pare the toxicity of each of these household cleaners to
an untreated (no malathion) control.
For the bioassay, 10 RFB adults were counted into small
disposable Petri dishes and allowed to starve overnight.
They we re obser ved the n ex t d ay to ensure that they we re
still alive, then one side of the Petri dish with the RFB
inside was inverted on the steel plates, exposing the in-
sects to the treated surfaces. After one hour, the plates
were inverted, collecting the RFB back into the Petri
dish and the insects were observed for toxic effects. All
of the insects in each dish were immediately observed
and placed into one of the following 3 categories:
Category 1: Alive (moves when prodded with a probe).
Category 2: Knockdown (moribund but showing some
movement of legs or head)
Category 3: Dead (total lack of movement even when
prodded with a probe)
The lids were replaced on the Petri dishes and the insects
were allowed to sit undisturbed on the bench top un-
til they were evaluated again for toxic effects 24-hours
postexposure. This process was performed after each of
the 3 decontaminations. At the end of each bioassay, the
tested insects were destroyed and not used for subse-
quent bioassays.
Statistical Analysis
Data were analyzed using the PROC ANOVA proce-
dure in the SAS 9.2 software (SAS Institute Inc, Cary,
NC). The models were used to describe the effects of
decontamination on survival (those classifi ed as alive)
at 1-hour postexposure and at 24-hours postexposure.
Another category of some movement combined the
categories of alive and knockdown. Means of survival
and survival with knockdown were compared using the
Tukey’s HSD (honest signifi cant difference) test.
Comparison to Standard Chemical Assay
The described steel plate assay was used to compare to
a standard analytical process using gas chromatography/
mass spectrophotometry (GC/MS). Twelve steel plates
were set up as in the assay as previously explained, and
then smaller steel plates (5.08 cm by 5.08 cm) were
placed in the center of each plate as coupons. The cou-
pons were taped onto the larger steel plates with only
a small edge of the laboratory tape extending onto the
coupon. This was done to prevent treatments from con-
taminating the underside of the coupon. The plates and
coupons were then treated with 1-ml aliquots of 25%
malathion and dried for 24 hours. Four of the coupons
were then removed, placed in glass jars, and transported
to the chemistry laboratory at the Jordan Valley Innova-
tion Center, Missouri State University, for analysis. At
that time, the remaining plates with coupons received a
single decontamination with 5% bleach identical to that
described earlier. After 24 hours, 4 more coupons were
removed for chemical analysis, and the remaining plates
with coupons received a second decontamination treat-
ment. Following another 24 hours, two of the coupons
were removed for analysis and the last two plates with
coupons received a third decontamination treatment.
After another 24 hours, the bioassay was performed on
the larger steel plates, placing the Petri dishes on treated
(malathion) and decontaminated (bleach) areas next to
the sites where the coupons had been removed.
Reagents and Materials. Malathion and malaoxon
PESTANAL analytical standards were purchased from
Sigma-Aldrich (St Louis, MO). Optima grade acetone
was purchased from Fisher Scientifi c (Fair Lawn, NJ).
Stock standards of 150 μg/ml and 100 μg/ml were
freshly prepared in acetone each week and stored at 4ºC
in opaque Nalgene bottles (Fisher Scientifi c). Ca libr a -
tion standards (0.1 μg/ml-150 μg/ml) were also pre-
pared weekly in acetone from the stock standards by
serial dilution in acetone and stored at 4ºC in opaque
Nalgene bottles.
Sample Extraction. The sample extraction procedure
was adapted from Rogers et al.14 The coupons were re-
moved from the treated steel plates and placed in 250
ml glass straight-sided jars (Fisher Scientifi c) to which
80 ml of acetone was added. The samples were then
sonicated for 30 minutes. After sonication, 1 ml was re-
moved from the jar and added to an autosampler vial for
GC/MS analysis. A single extraction cycle proved to be
suffi cient for the steel coupons.
Table 1. Application and bioassay schedule for decontam-
ination of malathion with common household cleaners.
Day 0 25% malathion applied to plates
Day 2 First decontamination
Day 4 First bioassay; second decontamination of plates
Day 6 Second bioassay; third decontamination of plates
Day 8 Third bioassay
4 http://www.cs.amedd.army.mil/amedd_journal.aspx
GC/MS Analysis. A Varian 450 GC (Varian Medical
Systems Inc, Palo Alto, CA), coupled to a Varian 320
triple quadropole mass spectrometer was used for the
analysis. The instruments were interfaced to a computer
running Varian MS workstation version 6.9.1 for instru-
ment control and data processing. Instrument conditions
were similar to those used in Rogers et al.14 The column
used for separation was a Zebon ZB-1701 with 5 mm
Guardian guard column (Phenomenex, Torrance, CA).
The column dimensions were 30 mm by 0.25 mm by
0.15 μm fi lm thickness. A 1 μl sample was injected in
splitless mode at 200ºC. The GC oven was programmed
as follows: 100ºC hold for 2 minutes, increased to 180ºC
at a rate of 10ºC/minute, increased to 220ºC at a rate of
5ºC/minute, increased to 260ºC at a rate of 20ºC/minute
and held at 260ºC for 2 minutes. Total run time was 22
minutes. Helium was the carrier gas with a fl ow rate of
0.8 ml/minute. The mass spectrometer was operated in
electron impact mode. The transfer line and ion source
temperatures were set to 280ºC and 230ºC, respectively.
Retention times were determined and parent ions were
verifi ed in full scan mode. Quantifi cation and qualifi ca-
tion ions were selected and collision energies were de-
termined experimentally by tandem mass spectrometry.
Analyte specifi c information is shown in Table 2.
Peak areas of standards were plotted using a quadratic
function with weighting scaled by the inverse of analyte
concentration. A minimum of 6 points was required for
an acceptable calibration curve. Both calibration curves
had correlation coeffi cients of r2>0.990.
Statistical Analysis. Mean concentrations were calcu-
lated with the PROC MEANS procedure in SAS 9.2 for
both malathion and malaoxon on the coupons. Results
were graphed using a Microsoft Excel 2010 spreadsheet.
RESULTS
Bioassay Development
Survival on plates that did not receive an application of
malathion or a decontaminant was not different from
plates that received only a xylene application (0.75 ml)
or 1 to 3 applications of 5% bleach (r
2=0.01, P=.65).
This fi nding indicated negligible toxicity of the diluent
(xylene) and the standard decontaminant (5% bleach).
Due to the lack of toxicity on these plates, the control
throughout the study was subsequently defi ned as plates
that did not receive a malathion application or any de-
contamination treatment, or that received only a bleach
or xylene application. Similarly, survival on plates that
were treated only with the decontamination agents of
Lysol, Pine Sol, Simple Green, and ethanol was not sig-
nifi cantly different from an untreated (no malathion)
and undecontaminated control.
Survival levels on plates treated with malathion but
which were not decontaminated was consistent through-
out the multiday study for each replication. The last
bioassay on each replication was run 8 days after the
initial application of malathion, but survival levels on
control plates on the last bioassay was not different from
that of the fi rst or second bioassays in each replication
(r2=0.002, P=.97). This fi nding provides evidence that
in the protected environment of the laboratory, malathi-
on was not degraded and it remained active throughout
the duration of the biological testing.
Table 3 displays the survival of 10 RFB confi ned for
one hour on steel plates treated with malathion, then de-
contaminated with 1, 2, or 3 treatments of 5% bleach
solution. Bleach was used as the standard decontamina-
tion treatment for this study. To monitor changes in the
level of toxicity to each treatment, survival levels were
measured one hour and 24 hours after initial exposure,
and as a combined measurement of survival and knock-
down (some movement) 24 hours after initial exposure.
A means separation test indicated that the fi rst applica-
tion of a decontaminant on Day 2 of the experiment did
not result in signifi cant detoxifi cation. This fi nding was
consistent with all 3 measures of toxicity. However, af-
ter a second application of the bleach solution on Day 4,
survival was signifi cantly increased on the plates as
measured by the bioassay on Day 6. This fi nding was
also consistent with all 3 measures of toxicity. After a
third decontamination of the plates on Day 6, the surviv-
al level as measured on Day 8 was slightly greater, but
was not signifi cantly different from that of the second
decontamination. Survival levels after the second and
third decontaminations were not signifi cantly different
from that on plates that did not receive an application
of malathion except as measured by sim-
ple survival (not knockdown) at 24 hours.
That measure indicated a difference in
survival on malathion-treated plates that
received only 2 decontaminations as com-
pared to plates that received no malathion
but did receive bleach applications. Of
the 3 measures of toxicity, the measure of
“some movement” explained the greatest
A RAPID AND INEXPENSIVE BIOASSAY TO EVALUATE
THE DECONTAMINATION OF ORGANOPHOSPHATES
Table 2. Analyte information for gas chromatography/mass spectrophotometry
analysis.
Analyte MW Parent
Ion
Retention
time (min)
Quantifi cation
Ion (m/z)
Qualifi cation
Ion (m/z)
Collision
Energy
Malathion 330.36 331 17.2 285 173 5 eV
Malaoxon 314.30 315 17.7 173 127 6 eV
MW indicates molecular weight.
m/z indicates mas s to charge ratio.
THE ARMY MEDICAL DEPARTMENT JOURNAL
July – September 2012 5
amount of variation in the model as demonstrated by an
r2 value of 0.70.
None of the household cleaners appeared to decrease
toxicity, with only bleach demonstrating a decontami-
nating effect as demonstrated by the bioassay. Table 4
compares the standard (5% bleach) to common house-
hold and laboratory cleaners in their capacities to de-
contaminate malathion. The measures of toxicity were
the same as used earlier. All treatments for each de-
contaminant were combined for comparison in Table 4.
There was decreased survival on plates treated with
Pine Sol and Simple Green alone as compared to the
nondecontaminated control, a fi nding that
is inconsistent with the control studies that
indicated no toxicity due to the decontami-
nating agents alone. Survival on bleach-
treated plates was signifi cantly greater than
on plates decontaminated with any of the
other agents.
GC/MS assay
To correlate data from the bioassay experi-
ments to the degradation of malathion, GC/
MS was used to measure the amount of mal-
athion and malaoxon, the oxidative byprod-
uct of the decontamination of malathion. Be-
cause malaoxon is toxic, the concentration
of this chemical was also determined in the
GC/MS assay. Concentrations of malathion
and malaoxon after 0 to 3 decontaminations
are graphically depicted in the Figure. The
presence of malaoxon as a toxic byproduct of the oxida-
tion of malathion continued after 1 and 2 applications of
bleach decontaminant, but was completely removed af-
ter a third application Three decontaminations resulted
in almost complete degradation of malathion.
COMMENT
The simple bioassay demonstrated in t his study provides
a quick screening mechanism that can be used to inves-
tigate factors affecting the decontamination of neuro-
toxic chemicals, particularly the organophosphates. It
allowed the identifi cation of an effective application rate
of bleach for use as a decontaminating agent. This is
perhaps the greatest utility of the bio-
assay. When the toxic agent was 25%
malathion, about twice as much 5%
bleach by volume was required to sig-
nifi cantly improve survival of RFB on
malathion treated plates. Nearly com-
plete decontamination was obtained
by 3 subsequent applications of bleach
with each application being the same
size by volume as the 25% malathion.
This rapid assessment of effi cacy can
be useful when putting together decon-
tamination protocols for toxic agents,
especially because it measures actual
biological toxicity. Further research is
necessary to determine if lower con-
centrations of the bleach decontami-
nant or smaller aliquots might be effec-
tive in repeated decontaminations. The
concentration used in this study (5%)
was very high and would not be suit-
able for use in many situations.
Table 3. Postexposure percentage of surviving Tribolium castaneaum after
confi nement for one hour on 20 cm by 20 cm steel plates which had been
treated with 1 ml of 25% malathion, then decontaminated sequentially with
1, 2, or 3 applications (1 ml) of 5% bleach solution.
Number of
decontamination
treatments n
Survival
1-hour
postexposure
% (SD)
Survival
24-hours
postexposure
% (SD)
Some movement*
24-hours
postexposure
% (SD)
0 36 59.7 (26.4)a 30.8 (22.9)a 30.8 (22.9)a
1 12 44.2 (28.1)a 12.5 (31.0)a 24.2 (35.0)a
2 12 82.0 (28.0)b 55.0 (40.5)b 85.8 (18.3)b
3 12 96.7 (4.5)b 80.0 (34.9)b,c 97.5 (4.5)b
Bleach only 27 97.7 (4.4)b 98.8 (3.3)c 98.8 (3.3)b
r20.40 0.62 0.70
n indicates the number of steel plates that received the designated treatment.
Note: Values in a column followed by the same let ter are not significantly different (Tukey
test, P=.05).
*Combined category including insects that were alive or knocked down.
Table 4. Postexposure percentage of surviving Tribolium castaneaum after con-
fi nement for one hour on 20 cm by 20 cm steel plates which had been treated
with 1 ml of 25% malathion, then decontaminated sequentially with a common
household cleaning product.
Decontaminant n
Survival
1-hour
postexposure
% (SD)
Survival
24-hours
postexposure
% (SD)
Some movement*
24-hours
postexposure
% (SD)
No decontamination 98 8.1 (20)a 9.5 (20.0)a 30.8 (22.0)b
Pine solvent 9 3.3 (5.0)a 1.1 (3.3)a 3.3 (5.0)a
Simple Green 12 8.3 (11.0)a 1.7 (3.9)a 15.0 (23.5)a,b
Ethanol 23 3.9 (6.6)a 16.0 (25.0)a 37.8 (32.0)b
Lysol 12 18.3 (25.1)a 20.0 (26.9)a 41.7 (31.6)b
Bleach (5%) 24 61.2 (38.4)b 67.0 (39.0)b 91.7 (14.3)c
No malathion/no
decontamination 37 96.6 (7.7)b 95.2 (12.0)b 98.6 (3.5)c
r20.85 0.80 0.79
n indicates the number of steel plates that received the designated treatment.
Note: Values in a column followed by the same letter are not significantly different (Tukey test,
P=.05).
*Combined category including insects that were alive or knocked down.
6 http://www.cs.amedd.army.mil/amedd_journal.aspx
Chemical analysis of the residual toxin on
decontaminated plates confi rmed that a
signifi cant amount of malathion remains
on the plates after one decontamination;
the concentration of degradation byprod-
ucts was also increased. When 25% mala-
thion was decontaminated with 5% bleach,
nearly complete decontamination of the
surface was achieved with 3 treatments, a
fi nding consistent with both the bioassay
and the GC/MS analysis. The presence
of a toxic byproduct of decontamination
(malaoxon) was demonstrated by both
the chemical analysis and suggested by
the bioassay, demonstrating the need for
validated protocols for decontamination
processes.
A rapid screening of household cleaners us-
ing the RFB bioassay failed to identify any
additional decontaminating agent other
than the common bleach solution already
known to be an effective decontaminant.
Other cleaners like Simple Green and Pine
Sol might be useful in the physical removal
of the agent, but do not demonstrate a re-
duction in toxicity of malathion as determined by the in-
sect bioassay. The results of the bioassay, however, were
not always straightforward. The increased toxicity on
malathion-treated plates decontaminated with Simple
Green and Pine Sol was unexpected. These undiluted
substances are slightly viscous. Perhaps this physical
characteristic impedes the insect’s movement or covers
the spiracles leading to asphyxiation. Alternatively, the
cleaners may break down protective characteristics of
the insect cuticle, thereby increasing the insect’s suscep-
tibility to the toxin. Insects exposed to plates that re-
ceived only Pine Sol or Simple Green applications, but
no malathion, did not elicit greater mortality than did
plates that had received no application. This suggests
that these 2 cleaners may somehow synergize the action
of the malathion, though this possibility would require
more research to confi rm. Another interesting fi nding
in this screening was the lack of effi cacy of ethanol as
a decontaminating agent against malathion. Ethanol
has been used as a decontaminant for other insecticides,
specifi cally organochlorines,4 but did not show any ef-
fi cacy against an organophosphate.
The benefi ts of this bioassay include its rapidity, very low
expense, and its actual measurement of biological toxic-
ity. The latter is important given that analytical chemis-
try-based measures can quantify the breakdown of the
target chemical, but may fail to measure the toxicity of
degradation products. No expensive equipment is re-
quired and this bioassay could actually be performed
in a fi eld situation with only minor modifi cations. This
type of bioassay provides almost immediate results and
can easily be adapted to test a variety of surfaces such as
concrete, wood, and tile. It can also be used to study the
impact of environmental variables such as temperature,
humidity, and insolation on the decontamination of toxic
chemicals. However, this bioassay does not identify the
mode of toxic action, nor does it rule out the possibility
of other forms of toxicity such as endocrine disruption
or carcinogenicity. Since this bioassay does not identify
the mechanism by which the insects are killed, it is not
a replacement for the standard analyses involving ana-
lytical chemistry. Also, the surfaces to be tested must be
dry. Wet surfaces lead to concentration of the toxins or
decontaminants and wetting of the insect cuticle, both of
which can cause inconsistent measures of toxicity. This
phenomenon was particularly observable with the Lysol
applications and may limit the utility of the bioassay for
such decontaminants.
Future research using this bioassay will include investi-
gations of decontamination effi cacy on various surface
types, extended screening of potential decontamination
agents, and evaluation of environmental factors such
as temperature and humidity on decontamination pro-
cesses. Although the current screening was done with
A RAPID AND INEXPENSIVE BIOASSAY TO EVALUATE
THE DECONTAMINATION OF ORGANOPHOSPHATES
Mean malathion and malaoxon residuals (μg /ml) on steel plates after 0, 1, 2,
or 3 applications of 5% sodium hypochlorite (bleach) solution applied with an
air brush.
THE ARMY MEDICAL DEPARTMENT JOURNAL
July – September 2012 7
decontamination of an insecticide on steel plates as a
model, this bioassay may also serve as a method to study
the decontamination of a variety of toxic environments
such as facilities that have been contaminated during
inappropriate termiticide applications, chemical war-
fare agent attacks by terrorists or national militaries, or
even houses contaminated by the illegal manufacture of
methamphetamines. It would be most useful when used
as an initial screening tool as it is not a replacement for
the more comprehensive and expensive analytical tests.
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AUTHORS
CDR (Ret) Claborn is an Assistant Professor of Public
Health and Homeland Security, Missouri State Univer-
sity, Springfi eld, Missouri. A career US Navy medi-
cal entomologist, his last assignment was the Uniformed
Services University of the Health Sciences, Bethesda,
Maryland.
Ms Martin-Brown is a chemist with the Center
for Biological and Life Sciences, Jordon Valley
Innovation Center at Missouri State University,
Springfi eld Missouri.
Mr. Sagar is a graduate assistant in the Master of
Public Health program at Missouri State University,
Springfi eld, Missouri.
Dr. Durham is a Professor of Biology and Director of
the Center for Biological and Life Sciences, Jordon
Valley Innovation Center, Missouri State University,
Springfi eld, Missouri.
