s 5
O
5 V^T77 2 EPA/600/R-04/069
May 2004
PRO^
Vapor Sampling Device for
Interface with Microtox Assay
for Screening Toxic Industrial
Chemicals
Principal Investigator
K.R. Rogers
U.S. Environmental Protection Agency
Office of Research and Development
National Exposure Research Laboratory
Human Exposure and Atmospheric Sciences Division
Exposure & Dose Research Branch
944 East Harmon Avenue
Las Vegas, NV 89119
and
USGS
Columbia Environmental Research Center
Columbia, MO
-------
-------
Notice
The information in this document has been funded by the United States Environmental Protection
Agency. It has been subjected to the Agency's peer and administrative review and has been approved for
publication as an EPA document. Mention of trade names or commercial products does not constitute
endorsement or recommendation for use.
iv
-------
V
-------
Foreword
The characterization of multi-component detection systems for chemical and biological agents is an
integral part of the EPA National Homeland Security Research Center, Safe Buildings Program. The work
described in this report was designed to meet the following guideline referred to in the EPA's Safe Buildings
Program "conduct research to adapt existing technology for the purpose of homeland security." More
specifically, each of the herein reported sampling and assay technologies has been previously described for
an application other than homeland security.
The primary goal of this project is to adapt semipermeable membrane devices (SPMDs) sampling
technology to chemical vapor monitoring, and interface this technique to commercially available bioassay
methods. This EPA report is a product of this effort and describes the characteristics and potential use of
a toxicity-based screening system for the cleanup and remediation of buildings that may have been
contaminated with toxic industrial chemicals.
vi
-------
vii
-------
Abstract
A time-integrated sampling system interfaced with a toxicity-based assay is reported for monitoring
volatile toxic industrial chemicals (TICs). Semipermeable membrane devices (SPMDs) using dimethyl
sulfoxide (DMSO) as the fill solvent accumulated each of 17 TICs from the vapor phase. Uptake kinetics
experiments for one of these compounds (acrolein) indicated that it was significantly sequestered (i.e., 10
percent of the 24 hr maximum) in as little as 10 min and was concentrated by a factor of over 200 in 24 hr
as measured using both mass and toxicity assays. The effect of each of the TICs on the Microtox bacterial
luminescence assay was determined both from a direct assay and a vapor accumulation assay using SPMDs.
Microtox EC50 values (concentration yielding 50 percent inhibition) were determined for each of the TICs
analyzed and ranged from 0.070 parts per million volume (ppmv) for diketene to 322 ppmv for 1,2-
dibromoethane. The rank order of the Microtox EC50 values for each compound measured directly from
liquid was similar but not identical to the Apparent (App) EC50 values determined from the vapor
accumulation assay. The ratios of the EC50 and the AppEC50 values were used to calculate toxicity-derived
Concentration Factors (i.e., the toxicity equivalents of compound that concentrate from vapor into the
SPMD). These Concentration Factors ranged from 17 to 5400 and primarily reflected differences in
partitioning characteristics between air and DMSO for each compound. Acrolein was chosen as a
representative compound for the vapor dilution experiment and it showed a toxicity-based detection limit of
19 mg/m3 which was less than the LD-50 by a factor of 100, but greater than the National Institute for
Occupational Safety and Health (NIOSH) 40 hr/week exposure limit also by a factor of 100. Consequently,
for acrolein, this system in its current configuration, shows potential for development as an initial screening
tool for mid to high acute vapor phase toxicity determinations.
viii
-------
IX
-------
Table of Contents
Foreword v
Abstract vii
List of Figures xi
List of Tables xiii
List of Acronyms xv
Acknowledgments xvii
Section 1 - Background 1
Section 2 - Methods 3
2.1 Reagents and Protocols 3
2.2 Calculations and Data Handling 4
Section 3 - Results and Discussion 7
3.1 EC50 Values for Test Compounds in Solution 7
3.2 AppEC50 for Test Compounds Accumulated from Vapor 8
3.3 Concentration of TICs into SPMD/DMSO 9
3.4 Effect of Microtox Assay Time on EC50 Values 9
3.5 The Effect of DMSO Concentration 10
3.6 Vapor Accumulation Kinetics 11
Section 4 - Conclusions 17
Section 5 - Future Directions 19
References 21
Appendix A - Selected Physical Parameters for TICs .23
Appendix B - Microtox Omni Test Report 25
Appendix C - SPMD Samples 27
X
-------
-------
List of Figures
Figure Page
Number Description Number
1 Experimental protocol diagram 4
2 Rank order for EC50 values 7
3 Rank order for AppEC50 values 8
4 Rank order for Concentration Factors 9
5 Time dependence of the Microtox assay 10
6 Representative chromatogram for acrolein 11
7 Accumulation of acrolein into SPMDs 12
xii
-------
xiii
-------
List of Tables
Table Page
Number Description Number
1 Summary of Microtox Data 14
2 Effect of Solvent Volume on the AppEC50 Values for Acrolein and Octanethiol 15
3 Effect of Acrolein Vapor Concentration on the AppEC50 Measured Using
the SPMD/DMSO Sampler 15
4 Comparison of Detection Limits for Acrolein Toxicity Using the
SPMD/DMSO-Microtox Assay 15
xiv
-------
XV
-------
List of Acronyms
AppEC50 Similar to the EC50 except that the assay was performed using a stock solution of
DMSO that had accumulated the test compound through an SPMD.
CWA chemical warfare agent
DMSO dimethyl sulfoxide
DoD Department of Defense
DOE Department of Energy
EC50 For the purpose of this report, the EC50 is the concentration of test compound yielding
50 percent inhibition in luminescence response (as compared to a control assay) and
performed using a DMSO stock solution of this compound.
GC/MS gas chromatography/mass spectrometry
LD-50 The concentration of a test compound that yields 50 percent mortality under specified
conditions and for a specified species.
HSARPA Homeland Security Advanced Research Project Agency
NHSRC National Homeland Security Research Center
NIOSH National Institute for Occupational Safety and Health
OSHA Occupational Safety and Health Agency
ppbv parts per billion by volume
ppmv parts per million by volume
SPMD semi permeable membrane device
TIC toxic industrial chemical
USGS-CERC United States Geological Survey-Columbia Environmental Research Center
XVI
-------
xvff
-------
Acknowledgments
The Principal Investigator (PI) would like to acknowledge the advice and technical assistance of a
number of scientists at the US Geological Survey-Columbia Environmental Research Center (USGS-
CERC) including T. Johnson, R. Gale, J. Huckins, C. Orazio and others at the USGS-CERC. The PI
would also like to acknowledge G. Robertson at the EPA Exposure and Dose Research Branch whose
advice was and assistance was invaluable for this study.
xviii
-------
XIX
-------
Section 1
Background
Monitoring of toxic industrial chemicals in support of potential building remediation applications
presents some unique analytical challenges. More specifically, remediation and re-occupation of a
chemically contaminated building typically takes place over a period of months to years. Because structural
building materials that have been contaminated with volatile or semi-volatile toxic industrial chemicals
(TICs) may slowly release these compounds, there exists a potential for human exposure throughout the
cleanup process. Consequently, continuous and time-integrated air monitoring for possible exposure to these
acutely toxic and potentially genotoxic compounds is a task of considerable importance. Although several
analytical methods are available to measure a limited number of specific compounds, air monitoring
techniques able to capture and screen for a broad range of compounds and agent simulants that are toxic, but
not expected to be detected by currently available chemical sensor technologies, may prove to be extremely
useful in the cleanup of chemically contaminated buildings.
In contrast to the analytical needs of the DoD, DOE, and Department of Homeland Security (through the
Homeland Security Advanced Research Project Agency, HSARPA) which appear to be primarily focused
on prevention and first responder monitoring needs [2,3], the analytical needs of the EPA's Safe Buildings
Program are focused on support of cleanup and remediation of contaminated buildings. In addition, the
efforts of HSARPA, as evidenced by recent calls for proposals, have been focused on 20 target compounds,
more than half of which are well known chemical warfare agents (CWAs) and focused little attention on the
dozens, if not hundreds, of potentially toxic industrial chemicals.
The research strategy reported herein focuses on two areas. The first area involves the development of
sampling technology that will provide a time-integrated concentration record for numerous locations
throughout a potentially contaminated building. The next involves the use of screening assays to detect
accumulated toxic chemicals on the basis of their potential biological / biochemical function. This report
describes the adaptation and characterization of semipermeable membrane devices (SPMDs) as time
integrated air monitors. SPMDs have been extensively used for water monitoring and are often interfaced
with toxicological and genotoxicological screening assays for exposure assessment and remediation of
environmental pollution, particularly in ecological assessment of sediments [4,5]. The adaptation of these
techniques for a screening level assessment of indoor air is expected to provide new tools in the remediation
of buildings that have been contaminated with an unknown mixture of hazardous compounds. These devices
have been integrated with the Microtox acute toxicity screening assay. Although the Microtox assay has also
been extensively used to screen for hazardous chemicals associated with water quality in waste water
treatment systems [6,7], it has not been widely used for air monitoring, nor has it been evaluated for many
of the herein reported TICs. Potential applications for this work with respect to building decontamination
will involve providing (i) a time-integrated record of chemical toxin concentrations present in the air over
a specified time period; (ii) a passive sample collection system to which acute toxicological screening assays
can be interfaced; and (iii) a demonstration of the feasibility of toxicity-based assays such as the Microtox
system for use in building decontamination applications.
1
-------
2
-------
Section 2
Methods
2.1 Reagents and Protocols
All reagents, other than the Microtox reagents, were obtained from Sigma/Aldrich (St. Louis, MO). A
complete list of the TICs evaluated in this study is found in Appendix A.
The Microtox assay is based on observed changes in luminescence due to exposure of the test organism
Vibrio fisheri to toxic chemicals. The organisms are supplied as lyophilized reagent that is reconstituted in
2 percent NaCl buffer prior to assay. The assay measures light output of the organism after exposure to the
sample as compared to the control blank. Light output is measured after 5 and 15 min exposures. A serial
dilution profile is used to determine the concentration of sample resulting in a 50 percent reduction in the
luminescence as compared to the blank under the same conditions. This value is reported by the Microtox
Omni software as the EC50. For an example of the Microtox report see Appendix B.
The Microtox 500 analyzer and acute toxicity reagent (i.e., lyophilized V. fisheri) were obtained from
SDI (Newark, DE). Assays were performed following the SDI basic protocol for pure compounds [8]. The
EC50 values for both phenol and zinc sulphate, typically used as positive controls, were similar to previously
reported values [8]. For direct measurement of the Microtox toxicity responses to the TICs, stock solutions
were prepared in dimethyl sulfoxide (DMSO). From these stocks a dilution of 1 to 100 was made into the
Microtox assay buffer. This solution was the maximum concentration in the Microtox serial dilution profile.
The "Basic Protocol for Pure Compounds" was used with 4 or 9 dilutions. EC50 values for the 5 and 15 min
assays were recorded along with 95 percent confidence limits.
The SPMDs were prepared using low density polyethylene tubing (25 mm x 88 p-m wall thickness) as
previously described [5] with the exception that DMSO rather than triolein was used as the trapping solvent.
The tubing was cut into 75 mm lengths and heat sealed on one end, followed by addition of 100 p-L DMSO
and heat sealed on the other end. These SPMDs were then placed into 40 mL vials or 4 L glass bottles
(suspended near the center of volume). For accumulation experiments, a specific volume of compound was
spiked onto the side of the vial without contacting the SPMD and the container sealed. Each of the
compounds analyzed completely vaporized in the vial head space in less than 2 min. After a specified time
(typically 24 hr), the SPMD was removed from the vial, cut open and the DMSO analyzed for toxicity
(Figure 1). Photographs of SPMDs and exposure chambers are shown in Appendix C. Apparent EC50 (App-
EC50) values were also calculated using the "Basic Protocol for Pure Compounds" from dilutions of the
DMSO in the SPMDs after exposure to vapor phase TICs.
GC/MS analyses were conducted using a Finnigan Voyager system. Separations were performed using
a 0.1 (J.L head space injection onto a capillary column maintained at 30° C until elution of the acrolein, then
the temperature was ramped to 200° C for elution of the DMSO. The 55 + 56 mass ions were used to
construct a calibration curve.
3
-------
Direct Microtox Assay
Neat
TIC
Stock
in
DMSO
V I
Dilution
in Microtox
Assay
Reagent
Vapor Accumulation Assay
SPMD
DMSO
r\
a*e
Neat Stock
TIC in
Methanol
Exposure
Chamber
Dilution
in Microtox
Assay
Reagent
EC
50
Figure 1.
AppEC50
Experimental protocol diagram.
2.2 Calculations and Data Handling
The Microtox Omni software calculates the EC50 values for 5 and 15 min assays based on the comparison
of luminescence between treated and control organisms at various times and at various dilutions [8]. For the
pure compound protocols, the primary operator-entered variable is the initial concentration of the test
compound. EC50 values for each test were determined using stock solutions of the compounds in DMSO.
The value of the initial dilution (i.e., highest concentration) used in the Microtox assay allows the software
to calculate the EC50 value for a series of serial dilutions.
In the case of the vapor accumulation experiments, the actual concentration of the test compound present
in the DMSO was not known. Although the vapor concentration of the compound added to the exposure
chamber was known, the amount of compound that actually accumulated into the DMSO in the SPMD was
not directly measured (except for in the case of acrolein). The amount of test compound introduced to the
Microtox assay from the DMSO and entered into the Omnitox software was based on an estimate of the
lowest expected concentration of test compound (i.e., no concentration of the test compound vapor into the
SPMD). Because AppEC50 values were lower than EC50 values, this observation indicated the accumulation
of test compounds from the vapor phase to solution phase in DMSO.
To determine the extent that a compound was concentrated in the DMSO in the SPMD, the following
rationale was used: if the toxicity measured using the Microtox assay for a 100 |J.L solution (DMSO) spiked
with a specific volume of pure test compound was the same as in the 100 |J.L of DMSO in the SPMD, then
the Concentration Factor (calculated as the ratio of EC50 to AppEC50) would be one.
The timescale for which the Microtox organisms are inhibited is not the same for all compounds.
Consequently, 5 and 15 min incubation protocols were used. To compare assay results for these protocols
among various test compounds, a relative change indicator was used. The percent relative change in EC50
between 5 and 15 min was calculated using the following relationship:
4
-------
EC50/5 - EC50/15
% Relative Change = -———— — x 100
Fr
50/5
where EC50/5 is the EC50 value determined for a 5 min assay time and EC5o/15 for a 15 min assay time.
Safety note: Due to the volatile and toxic nature of the compounds analyzed, all manipulations using
these compounds with the SPMDs were performed in a fume hood with appropriate personal protection
equipment.
5
-------
6
-------
Section 3
Results and Discussion
3.1 EC50 Values for Test Compounds in Solution
The toxic industrial chemicals included in this study are listed in a recent National Institute of Justice
Report. For the herein reported study, these compounds were evaluated for acute toxicity using the Microtox
assay [1], Table 1 shows data from assays using stock solutions prepared directly in DMSO from neat
compounds (listed as EC50 values) and those sampled from DMSO that had been placed in SPMDs and
exposed to compound vapors for 24 hrs (listed as AppEC50 values). The EC50 values were calculated for both
5 min and 15 min assays (i.e., corresponding to the incubation time of the sample with the Microtox reagent).
The EC50 values were determined for each of the TICs yielded values ranging from 322 parts per million
by volume (ppmv) to 0.070 ppmv (Figure 2). These EC50 values form a continuum but appear to fall into 3
groupings consisting of low (e.g., diketene - sulfuryl chloride), medium (e.g., formaldehyde - methyl-
hydrazine) and high (acetone cyanohydrin - 1,2-dibromoethane) values. It should be noted that low EC50
values indicate a more toxic response.
300-
250-
(i) a)
(- (0 Q) Q)
— ^ c
a> O
cq a)
< H 5 w
Figure 2. Rank order for EC50 values. EC50 values were
determined using TICs assayed from solution.
7
-------
3.2 AppEC50 for Test Compounds Accumulated from Vapor
AppEC50 values were determined for the accumulation of compounds in the vapor phase into DMSO
present in the SPMD. These vapor phase experiments were conducted using smaller (40 mL) or larger (4
L) incubation chambers to measure the 24 hr accumulation of test compounds into SPMD/DMSO. For the
vapor measurements, DMSO was recovered from SPMDs and diluted 100 fold prior to being assayed (see
Figure 1). Although the initial vapor phase concentrations of compounds in the vials were known, the
concentrations of the compounds in the DMSO (sampled by SPMDs) from the vapor phase (with the
exception of acrolein) were not directly determined. The Microtox assay responses (i.e., toxic activities) of
compounds sampled from the SPMD, however, were determined and reported as AppEC50 values (Figure 3).
1000-
>
CL
CL
0
LLI
-*—«
c
0)
1
03
CL
CL
<
800
600
400
200-
Q. O)
81^.1®
Figure 3. Rank order for AppEC50 values. AppEC50 values
were determined from TICs accumulated into
SPMDs from vapor.
If the molar concentration of a particular compound per unit volume was the same in the DMSO from
the SPMD as in the vapor phase in the exposure vial (i.e., no concentration effect), then the AppEC50 would
be the same as the EC50. This was not the case for any of the compounds tested, however, and the AppEC50
values ranged from 1000 parts per billion by volume (ppbv) to 0.035 ppbv (Figure 3). These values were
between 2 and 3 orders of magnitude smaller than the EC50 values which were determined from solution
(Figure 2). This result indicated that the compounds were, on average, concentrated by between 200-400
times.
Although the rank order of EC50 values (Figure 2) and AppEC50 values (Figure 3) were not identical, the
compounds on the left (e.g., diketene through sulfuryl chloride) and compounds on the right (e.g.,
methylhydrazine through 1,2 dibromoethane) of both figures remained in their low / medium / high
groupings. Small shifts in the rank order of these compounds between Figures 2 and 3 may be expected due
to differences in partition coefficients between vapor and DMSO. Because of the highly reactive nature of
many of these compounds, the relative shift in assay response (apparent toxicity) may not be entirely due to
partition coefficients alone. More specifically, some of these compounds may react with the DMSO or water
and form the breakdown products that may show higher or lower toxicity than the parent compounds.
8
-------
3.3 Concentration of TICs into SPMD/DMSO
For all compounds measured, the vapor appeared to concentrate into the DMSO. This observed
concentration effect can be measured as the ratio of EC50 to AppEC50 and is reported as a Concentration
Factor for each compound. The Concentration Factors reflected the accumulation of the TICs into the
SPMD/DMSO as measured from the 5 min Microtox assay and ranged from 17 to 5400 (Figure 4). Again,
these values formed a continuum but tended to segregate into 3 groups with most of the values falling
between 200 and 400. Because it is likely that the Concentration Factors were highly influenced by
partitioning between air/polyethylene/DMSO and kinetics of steady state accumulation, it was not expected
that the Concentration Factors would be directly related to the EC50 values. It is interesting to note that
diketene, which was particularly toxic to the Microtox organisms, as indicated by its low EC50 value, did not
appear to readily concentrate into the SPMD/DMSO and yielded a Concentration Factor of only 17 (Table 1).
Nevertheless, it remained at the left (i.e., low AppEC50) side of the chart (Figure 3). This is contrasted by
phosphorus oxychloride which was also particularly toxic but showed significant accumulation into the
SPMD/DMSO with a Concentration Factor of 5400. These two factors contributed to this compound being
the most toxic of the TICs measured using the SPMD-Microtox assay.
a> co
2 w
a> 0
Figure 4.
Rank order for Concentration Factors.
Although the Concentration Factor indicates the proportion of compound that concentrated into the
DMSO from the vapor under particular conditions, it does not account for the amount of compound that
concentrated into the polyethylene portion of the SPMD. These values were not determined for this study,
however, previously reported SPMD studies suggest that for compounds of similar molecular weight, about
1/3 of the mass of these compounds concentrates into the polyethylene and 2/3 concentrates into the triolein
which has been traditionally used in these devices [5].
9
-------
3.4 Effect of Microtox Assay Time on ECS0 Values
Because different compounds affect the light output of V. fischeri by different mechanisms and with
different kinetics, the Microtox assay protocol suggests the use of 5 and 15 min exposure times for unknown
compounds and samples. Results for the compounds measured using the 5 and 15 min protocols reflect these
differences. Figure 5 shows the relative change in EC50 values between 5 and 15 min with bars extending
to the left of center indicating compounds that were measured more effectively using the 5 min protocol;
those near the center responding similarly for the 5 and 15 min protocols; and those with bars extending to
the right indicating compounds more effectively measured after a 15 min assay. Responses for reference
compounds such as phenol (i.e., more effective at 5 min) and zinc sulfate (more effective at 15 min) were
typical of those previously reported [8]. It should be noted that the decrease in the apparent toxicity with
extended incubation for compounds such as methane sulfonyl chloride, trichloroacetyl chloride, and
phosphorus oxychloride (-1400% not included in Figure 5) may be due to the reaction/breakdown of these
compounds upon introduction to an aqueous matrix. These compounds showed visible signs of reactivity
with water and the breakdown products are less likely to be as toxic as the highly reactive parent compounds.
Whatever the mechanism for differences between 5 and 15 min assay protocols, given the range of responses,
it would appear prudent to include both protocols in any attempt to apply this assay to unknown compounds.
Trichloroacetyl chloride
Methanesulfonyl chloride
Sulfurylchloride
Hypochlorite (bleach)
More Effective
with 5 min. Assay
Phenol
Methyl chlorosilane
Methyl chloroformate
1,2-Dibromoetharie
1-Octanethiol
Stilbine
More Effective
Listen ne
with 15 min. Assay
Allyjamine
Formaldehyde
Methylhydrazine
Choroacetone
Acetone cyanohydrin
Acrolein
Diketene
Zinc Su fate
% Relative Change in EC5Q Between 5 and 15 min.
Figure 5.
Time dependence of the Microtox assay.
3.5 The Effect of DMSO Concentration
The effect of DMSO on the Microtox assay was examined for final solvent concentrations between 1 and
10 percent (Table 2) for compounds (i.e., acrolein and 1-octanethiol). For this experiment, the SPMDs
contained 500 |iL DMSO rather than the 100 |iL typically used. This allowed for various amounts of the
DMSO containing toxicant from the same SPMD to be directly compared in the Microtox assay. In the case
of the 1-octanethiol, the AppEC50 values for both 5 and 15 min assays did not appear to be greatly affected
by the DMSO over the range of 1 to 10 percent. For the acrolein, the EC50 values for DMSO concentrations
between 2.5 and 10 percent were also similar to each other. For the 15 min assay, however, the EC50 value
at 2.5% DMSO was somewhat lower than expected.
10
-------
3.6 Vapor Accumulation Kinetics
Due to its intermediate observed toxicity, acrolein was chosen to conduct uptake kinetics and mass
comparison studies. The kinetics for the accumulation of acrolein as measured by both mass and Microtox
assay response (toxicity) was compared. The mass of acrolein recovered in the SPMD/DMSO was measured
by GC/MS. A calibration curve for acrolein in DMSO was constructed by monitoring the 55 + 56 mass ions.
A representative chromatogram for acrolein is shown in Figure 6. The acrolein eluted at 5.44 min, prior to
the DMSO solvent peak which eluted at 10.69 min. A relatively low column temperature of 30°C followed
by a temperature ramp to 200°C maintained acrolein peak integrity (i.e., prevented excess tailing) and
allowed for the removal of the DMSO solvent peak, thus, regenerating the column for the next injection.
RT: 0.00
100 tl
95
90
85
80
75
70
65 ^
60 :
a 55 -
22.27
3
50
45 -
40
35
30
25
20 d
15
10 :
5 -
NL:
6.31 E5
TIC MS
FD2003091
0-001
11,00
0.75 1.31 1.87 3.41 4.2_3 4.56
'_15.55 16,97 17.94 19.3l_g>-1J~
0 I I I | | | I | . | | I I 1 | 1 1 1 1 | 1 1 1 1 [ 1 1 1 1 | | 1 1 | | | | | | | ! , | | | | | | | | | 1 | | 1 | 1 | | 1 | | 1 | 1 1 II | | | | | 1 | | | | | | I I I I | I J II | 1 I I I | I I 1 I | I II I j I II I j I
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
Time (min)
Figure 6. Representative chromatogram for acrolein.
The time course for acrolein uptake on the basis of mass and Microtox assay response is shown in
Figure 7. To compare the two uptake profiles, the relative toxicity as measured by the Microtox assay and
mass as measured by GC/MS were normalized to a 24 hr maximum value and fit to a sigmoidal curve. An
accumulation time of 2 hrs yielded about a 50 percent uptake as compared with the 24 hr maximum.
11
-------
Significant uptake of compound as measured by both mass and Microtox assay response (i.e., 10 percent)
could be measured after as little as a 10 min exposure of the SPMD/DMSO sampler to the acrolein vapor.
The determination of the mass of accumulated acrolein as a function of time also allowed a comparison of
the Concentration Factor calculated on the basis of Microtox assay response (see Table 1) to a value
calculated on the basis of mass. The Concentration Factor for acrolein based on Microtox assay response
was 246 and the mass-derived value was 210. This difference most likely arises from the toxicity
measurement protocol which required that the acrolein be diluted into an aqueous matrix and may have
resulted in the slight degradation of this compound. The amount of acrolein accumulated into the DMSO
in the SPMD accounted for 52 percent of that introduced into the vial as vapor. The amount sequestered into
the polyethylene of the SPMD was not determined.
Because the exposure chamber had a finite volume and the SPMD (in the case of acrolein) accumulated
a significant percentage of the total test compound added, it is expected that the observed accumulation rate
was lower than would have been observed if the vapor concentration were not to have been depleted during
the course of the experiment. It is also likely that the final concentration of test compounds that concentrated
into the DMSO would have been greater if the compound vapor in the exposure chamber were not depleted.
The lowest vapor concentration of a particular compound yielding a response for the SPMD-Microtox
assay would depend on several variables including: EC50 (5 or 15 min depending on the compound),
Concentration Factor, exposure time (for the SPMD to the toxic environment), and volume of DMSO
sampled in the Microtox assay as well as factors not examined in this study such as temperature. To
determine the detection limit of this assay for acrolein, a serial dilution experiment was performed using a
4 L incubation chamber with the typical 75 mm SPMD containing 100 |iL DMSO. Shown in Table 3, an
acrolein vapor concentration as low as 0.1 [J.L/L could be detected using both the 5 min and 15 min protocols,
whereas a 0.01 [iL/L concentration could be detected using the 15 min protocol. This, in fact, represents a
conservative approach because in order for the Microtox software to calculate an AppEC50 value, the highest
concentration sample in the nine tube dilution series must inhibit the assay response by 30 to 40 percent.
100-
0-
¦ % Toxicity
• % Mass
0 5 10 15 20 25
Accumulation Time (hr)
Figure 7. Accumulation of acrolein into SPMDs. Toxicity
was determined as Microtox assay response and
mass determined by GC/MS.
12
-------
Experience with this assay has indicated that inhibition values as low as 15 to 20 percent can be reproducibly
observed.
The lowest concentration of acroline for which an AppEC50 could be determined using the
SPMD/DMSO-Microtox assay is shown in Table 4. The accumulation time for the SPMD/DMSO sampler
was 24 hr and the Microtox assay time was 15 min. The limit of detection for acrolein using the
SPMD/DMSO-Microtox system was 19 mg/m3 which was about 100 times less than the LD-50 value listed
in the NIOSH database and about 100 times greater than the NIOSH occupational exposure limit.
Consequently, as evidenced for acrolein, this system in its current configuration, shows the potential for
development as a screening tool for acute toxicity determinations. Areas that require further characterization
include mass accumulation and Microtox response to chemical mixtures.
13
-------
Table 1. Summary of Microtox Data
Name
Liquid
EC50
(ppmv)a
95%
confidence
Liquid
EC50
(ppmv)b
95%
confidence
%
Change
5-15C
Vapor
AppEC50
(pprnv)"
95%
confidence
Vapor
AppEC50
(ppmv)e
95%
confidence
Cone
Factor
Acetone cyanohydrin
187
112-310
66
41-103
65
0.49
0.33-0.70
0.21
0.10-0.42
382
Acrolein
0.32
0.26-0.38
0.11
0.10-0.13
66
0.0013
0.0010-0.0016
0.0004
0.0002-0.0008
246
Allylamine
18
2-146
17
2-147
6
0.11
0.05-0.19
0.09
0.02-0.36
164
Chloroacetone
26
21-33
9
7-11
65
0.041
0.037-0.044
0.012
0.009-0.015
634
1,2-Dibromoethane
322
33-3000
346
89-1300
-7
0.68
0.65-0.72
0.75
0.67-0.83
474
Diisopropyl fluorophosphate
43
33-58
44
35-56
-2.3
0.15
0.12-0.17
0.16
0.14-0.18
287
Diketene
0.070
0.053-0.090
0.022
0.005-0.080
69
0.0041
.0036-.0045
0.0013
.0010-.0016
17
Formaldehyde
13
12-14
7
5-8
46
0.28
0.20-0.37
0.19
0.16-0.21
46
Methanesulfonyl chloride
1.8
0.8-3.6
2.5
0.8-7.0
-39
0.027
0.021-0.035
0.03
0.02-0.04
67
Methyl chloroformate
22
9-53
25
NA
-14
0.06
0.01-0.23
0.06
0.01-0.36
367
Methyl chlorsilane
29
18-45
33
16-65
-14
0.09
0.06-0.12
0.12
NA
322
Methylhydrazine
56
52-59
28
23-31
50
1
0.2-4.6
0.4
0.1-1.4
56
1-Octanethiol
3.9
3.4-4.3
41
3.6-4.7
-5
0.005
0.004-0.006
0.007
0.004-0.013
780
Phosphorus oxychloride
0.19
0.17-0.19
3.0
0.7-11
-1400
0.000035
0.000027-0.000038
0.00008
0.00007-0.00011
5400
Stilbene
2.4
2.2-2.5
2.5
2.2-2.7
-4
0.012
0.010-0.014
0.016
0.011-0.022
200
Sulfuryl chloride
4.8
2.4-10
6
NA
-25
0.015
0.005-0.025
0.02
NA
320
Trichloroacetyl chloride
1.2
0.7-1.9
1.9
1.3-2.8
-58
0.0044
0.0038-0.0050
0.0060
0.0044-0.0085
273
Listerine
0.15%
0.12-0.18
0.15%
0.12-0.18
0
NA
NA
NA
NA
NA
Phenol
18
15-21
21
18-24
-16
NA
NA
NA
NA
NA
Zinc Sulpate
37
34-41
8.4
8.1-8.8
77
NA
NA
NA
NA
NA
a Microtox 9-point protocol for pure compounds, dilution into DMSO, 5 min incubation protocol. Each EC50 or AppEC50 value was representative of at least 3 assays,
b Same as (a) with 15 min protocol
c Time-dependence of Microtox assay, data from Figure 4. Positive values indicate that the 15 min protocol is more effective and negative values indicate that the 15
min protocol is more effective.
d Microtox 9-point protocol for pure compounds, SPMD-DMSO was exposed for 24 hr to vapor then run using the 5 min incubation protocol,
e Same as (d) with 15 min protocol
f Concentration Factors were determined using the ratio of the EC50 to AppEC50 for the 5 min incubation protocols.
-------
Table 2. Effect of Solvent Volume on the AppEC50
Values for Acrolein and Octanethiola
Compound
%
DMSO
App EC50
5 min
(PPbv)
App EC50
15 min (ppbv)
Acrolein
2.5
1.6
0.26
5
1.6
0.40
10
1.2
0.44
Octanethiol
1
2.4
2.8
2.5
1.8
2.0
5
1.8
2.2
10
2.2
2.4
a Experiments were performed in 40 mL exposure
chambers using a 75-mm SPMD containing 100 )JL
DMSO.
Table 3. Effect of Acrolein Vapor Concentration
on the AppEC50 Measured Using the
SPMD/DMSO Sampler
Dilution
(|JL/L)a
AppEC50, 5 min
(PPbv)b
AppEC50,15 min
(ppbv)
10
0.60
0.17
1
0.65
0.18
0.1
0.60
0.21
0.01
NAC
0.20
a Experiment performed in a 4 L exposure chamber
using a 75-mm SPMD containing 100 mL DMSO.
b Microtox 9-point calibration with 95% confidence
limits.
c AppEC50 value could not be established using the
Microtox Omni software.
Table 4. Comparison of Detection Limits for Acrolein Toxicity Using the SPMD/DMSO-Microtox
Assay
SPMD-Microtox
(ML/L)
SPMD-Microtox
(mg/L)a
Acrolein
conversion
(mg/m3)b
LD-50 (cat)
(mg/m3/2 hr)°
NIOSH
(mg/m3/hr)d
OSHA
(ppm/40 hr)d
0.01
0.0084
19
1570
0.25
0.10
a Density determined from Aidrich Materials Safety Data Sheet (MSDS)
b Conversion Factor from reference 9
c See reference 10
d See reference 11
15
-------
16
-------
Section 4
Conclusions
Several significant goals were accomplished by the development of methods to extend the SPMD-
Microtox assay system to volatile TICs. These goals included the following: 1) The SPMD sampler was
shown to accumulate (to varying degrees) each of the assessed TICs. The use of DMSO rather than triolein,
typically used in SPMDs, allowed the Microtox assay to be run directly from the SPMD solvent without the
usual processing and cleanup procedures. 2) Microtox EC50 values were determined for each of the 17 TICs
analyzed and ranged from 0.070 ppmv for diketene to 322 ppmv for 1,2-dibromoethane. AppEC50 values
measured from vapor accumulation into the SPMD/DMSO were lower than the EC50 values measured from
liquid and ranged from 0.035 ppbv for phosphorus oxychloride to lOOOppbvformethylhydrazine. Although
the 24 hr vapor accumulation values for the SPMDs include variability due to accumulation and possible
reaction or breakdown, each of the compounds was concentrated and consequently increased in apparent
toxicity as measured by the Microtox assay.
Listed in Appendix A is a summary table for AppEC50 values for each of the TICs examined as well as
LD-50, NIOSH and OSHA occupational limits (where available). Whereas acrolein appears to be somewhat
average, it is not representative of all listed compounds. Because the values for AppEC50, LD-50 and
occupational exposure limits vary widely, and often without a clear relationship to each other, it is difficult
to predict the safety margin afforded by a negative response to the SPMD/DMSO-Microtox assay. These
results, however, suggest that the SPMD/DMSO-Microtox assay would respond to the TICs examined in this
study at concentrations below their LD-50 values.
17
-------
18
-------
Section 5
Future Directions
The Microtox assay was shown to be stable in its response to specific compounds and robust in its
application to air toxics. However, characterization of the Microtox assay for a structurally and mechan-
istically diverse group of TICs has shown a variable range of toxicity responses. It appears that the key to
the application of this assay to screening of air toxics would be the pre-concentration of the compounds
followed by elution or solvent exchange into a Microtox-compatible solvent such as DMSO or assay diluent
(essentially anNaCl solution). Preliminary experiments have suggested that carbon/Empore membranes can
accumulate several TICs to a significantly greater extent than DMSO alone. The contaminated membranes
can then be extracted with DMSO with a relatively high yield. Continued research will optimize these
protocols and correlate mass and toxicity measurements. In addition, GC/MS analyses will be used to
indicate reaction or breakdown of these compounds prior to the Microtox assay.
19
-------
20
-------
References
1. National Institute of Justice Guide for the Selection of Chemical Agent and Toxic Industrial Material
Detection Equipment for Emergency First Responders, 100-00.
2. Homeland Defense, SBCCOM ON LINE, http://hld.sbccom.armv.mil/ahout_us.htm.
3. Department of Homeland Security, http://www.dhs.gov/.
4. Johnson, BT, Long, ER (1998). Rapid toxicity assessment of sediments from estuarine ecosystems: A
new tandem in vitro testing approach. Environ. Toxicol. Chem. 17,1099-1106.
5. Petty, JD, Orazio, CE, Huckins, JN, Gale, RW, Lebo, JA, Meadows, JC, Echols, KR and Cranor, WL
(2000). Considerations involved in the use of semipermeable membrane devices for monitoring
environmental contaminants J. Chromatogr. A, 897, 83-95.
6. Cassells, NP, Lane, CS, Depala, M, Saeed, M, Craston, DA (2000). Microtox testing of
pentachlorophenol in soil extracts and quantification by capillary electrochromatography (CEC): A rapid
screening approach for contaminated land. Chemosphere 40, 609-618.
7. Amoros, I, Connon, R, Garelick, H, Alonso, JL, Carrasco, JM (2000). An assessment of the toxicity of
some pesticides and their metabolites affecting a natural aquatic environment using the Microtox system.
Water Sci. Technol. 42, 19-24.
8. AZUR Environmental, Microtox / Microtox Omni (now SDI, Newark, DE), www.sdix.com.
9. International Programme on Chemical Safety, http://www.inchem.org/documents/hsg/hsg/hsgQ67.htm.
10. National Institute for Occupational Safety and Health, http://www.cdc.gov.niosh/rtecs/as/OOSQO.html.
11. Agency for Toxic Substances and Disease Registry, http://www.atsdr.cdc.gov/tfacts 124.html.
21
-------
22
-------
Appendix A
Selected Physical Parameters for TICs
Name
FW
BP
Density
Vapor
EC-50-5
(ppbv)
Vapor
EC-50-15
(ppbv)
LD-50
LC-50
(mg/m3/hrs)
OSHA
ppm
NIOSH
mg/m3/hr
Formula
Acetone cyanohydrin
85.11
82
0.93
490
210
70-2hr
(CH3)2C(OH)CN
Acrolein
56.06
53
0.84
1.3
4.0
1570-2hr
0.1
0.25
h2c=chcho
Stilbene
180.25
82
12
16
NA
0.1
0.50
c6h5ch=chc6h6
Methyl chlorsilane
Mixed
155
1.20
90
120
100
NA
NA
NA
Allylamine
57.10
53
0.76
110
90
320
NA
NA
h2c=chch2nh2
Chloroacetone
92.53
119
1.16
41
12
262-1 hr
NA
NA
cich2coch3
Diketene
84.07
70
1.09
4.1
13
3000-1 hr
0.50
0.90
1,2-Dibromoethane
187.87
131
2.18
680
750
14,000-0.5hr
20
0.045
BrCH2CH2Br
Methyl chloroformate
94.50
71
1.22
60
60
180-2hr
NA
NA
cico2ch3
Methanesulfonyl chloride
114.55
60
1.48
27
30
620-2hr
NA
NA
ch3so2ci
Methylhydrazine
46.07
87
0.87
1000
400
270-4hr
0.2
0.35
ch3nhnh2
Phosphorus oxychloride
153.33
106
1.64
0.035
0.80
404-2hr
NA
NA
POCI3
Sulfuryl chloride
134.97
69
1.68
15
20
10 ppm-6hr
NA
NA
so2ci2
1-Octanethiol
146.30
197
0.84
5
7
NA
NA
NA
CH3(CH2)7SH
Trichloroacetyl chloride
181.83
115
1.63
4.4
6
400
NA
NA
CCI3COCI
Formaldehyde
30.03
1.09
280
190
204-4hr
0.75
0.016
HCHO
Phenol
100.07
182
1.14
NA
NA
NA
NA
NA
C6H4OH
DFP
184.15
62
1.05
150
160
NA
NA
NA
[(CH3)2CH0]2P(0)F
-------
24
-------
Appendix B
Microtox Omni Test Report
Dai*/Tinu\ 0/18.;2003 03:18 PM
Test Protocol" 90% Basic lest for Pure Compounds
Data file: Untitk'd Data File
Pkst of Gamma vs Concentration
•00;
1
10- *
E 1
a
rr\
W
0.1 a
0.01
* ix 9#
* 8#
* & 7#
' 6#
10
Concentration
5 4 15 Fit
100
Pioi of%El!ea vs Concentration
100:
75-
M 50 f-
i
V \
ss
9#
8#
25 i-
0
-25
A -"?#
20 40 60 80 100
Concentration
¦ ¦ 5 a '5
5 Wins Data
Sample
Cone
lo
it Gamma
% effect
It Gamma
% effect
Control
r, fioo
95.05
67 47-0 7098 #
80 83 •• 0.6405
4
t
0 1953
104.8?
75 44-0 0151 1
-1.536%
88.97 - 0 0279
.
-2 876%
2
0 3906
102 29
.*2 at? U 003 2 •
-0 3316%
86 08 - 0 0085
•
"0 8 539%
3
,, -rS13
100 61
"3 25 0 0250 1
-2.56?%
65.S8-0 0218
-2 233%
1,563
102 S3
72 p - v af 14 •
1 127%
)0 - 0 C977
ft
8.902%
s
3,125
100,04
70 2: 0CIf? •
1,073%
51.14- 0 2530
§
A.tSn
& ] 6 250
102,26
66.98-0 08:?' *
i -m~-
3§ 09 ¦> 0 6758
ft
.32%.
7 | 12.50
100,32
£7.75 - 0.233! #
18.90%
24.48 1525
#
81 80%
25 00
102.21
45.51 - 0.5942 H
jr 27%
12.32-4.314
#
f-.
9
»C< CO
99.1$
m 70 1 4hi #
L3"
4'55 >2B8
#
^<2 i'S*' <
# = Used in calculation
" = Invalid data
D - Deleted from calcs
-------
L' I,, < >iU,i til! •!lo<' 3 4 ilti! '! {*#i ' »i i t .? t'v 1 !• < I t1* 80V.
si*,.f* ^ v\•> ¦ r<>. f i ^-'•J
!iu» Li eiU'w1!.. i (K • J" s'*1 \ i On v> i
Coeff. of Determination CR1); 0.9993
26
-------
Appendix C
SPMD Samples
SPMD in 40 tnL VOA vial
(colored dye added for contrast)
SPMD in 4 L bottle
(colored dye added for contrast)
-------
------- |