Screening for toxic industrial chemicals using semipermeable membrane devices with rapid toxicity assays

Analytica Chimica Acta 543 (2005) 229–235

Screening for toxic industrial chemicals using semipermeable membrane devices with rapid toxicity assays夽 K.R. Rogers ∗ , S.L. Harper, G. Robertson U.S. EPA, National Research Exposure Laboratory-LV, 944 E. Harmon Avenue, Las Vegas, NV 89119, USA Received 29 October 2004; received in revised form 29 March 2005; accepted 7 April 2005 Available online 3 May 2005

Abstract A time-integrated sampling device interfaced with two toxicity-based assays is reported for monitoring volatile toxic industrial chemicals (TICs). Semipermeable membrane devices (SPMDs) using dimethylsulfoxide (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 concentrated (i.e., 10% of the 24 h maximum) in as little as 10 min and was concentrated by a factor of over 200 for a 24 h exposure time as measured using both mass and toxicity assays. The effect of each of the TICs on the Microtox bacterial luminescence assay and IQ-Tox Daphnia magna fluorescence assay was determined both from a direct assay and a vapor accumulation assay using SPMDs. Microtox EC50 values (concentrations yielding 50% inhibition) were determined for each of the TICs analyzed. The rank order of the Microtox EC50 values for each of the compounds measured by direct dilution of the TICs into assay buffer was similar but not identical to the Apparent (App) EC50 values determined from the vapor accumulation assay. The ratios of the EC50 to the AppEC50 values were used to calculate apparent toxicity-derived concentration factors (i.e., the toxicity equivalents of compound that concentrate from vapor into the SPMD). EC50 values for the IQ-Tox assay as measured using a 90 min fluorescence assay were, in most cases, similar but not identical to the Microtox EC50 values for individual compounds. © 2005 Elsevier B.V. All rights reserved. Keywords: Toxic industrial chemicals; Acute toxicity; Daphnia magna; Vibrio fischeri

1. Introduction Due to the potential risk of human exposure from an accidental or intentional release of toxic chemicals, there is a current and expanding need for field screening methods to measure these compounds that may be released into a variety of environmental settings [1,2]. Compounds of interest with respect to possible air contamination primarily include chemical warfare agents (CWAs) and toxic industrial chemicals (TICs). Although a wide range of instruments, sensors 夽 The United States Environmental Protection Agency through its Office of Research and Development has funded and managed the research described here. It has been subjected to the Agency’s administrative review and approved for publication as an EPA document. Mention of trade names or commercial products does not constitute endorsement or recommendation for use. ∗ Corresponding author. Tel.: +1 7027982298; fax: +1 7027982106. E-mail address: [email protected] (K.R. Rogers).

0003-2670/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2005.04.016

and chemical test kit methods have been reported, many of which are commercially available, most of these techniques detect a limited range of specific compounds or compound groups [1]. For the most part, the development, demonstration and marketing of these technologies has been directed toward a narrow range of compounds (i.e., the CWAs) that might be used in an intentional attack on civilian targets. For example, many well characterized screening methods have been reported for detection of CWAs from the following classes: nerve agents such as sarin, tabun, soman, VX, etc.; blood agents such as cyanide compounds; choking agents such as chlorine and phosgene; blister agents such as mustard and lewisite compounds [3]. These screening assays include color change reactions in the form of indicator papers and test kits, enzyme-based assays and immunoassays, ion mobility spectroscopy, infrared absorption spectroscopy, surface acoustic wave technology, electrochemical sensor methods, and aerosol mass spectroscopy [1].

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Although TICs are also considered to be a civilian hazard, they have not been widely addressed with respect to field analytical methods. The definition of a TIC is relatively broad and necessarily vague. There are, however, a number of organizations and agencies that have compiled lists of compounds prioritized with respect to acute toxicity, physical characteristics and potential for terrorist threats [1,4]. The consensus among these reports is that the medium to high priority rating for TICs relate to issues such as acute toxicity, volatility, volume of production and relative accessibility. Because these compounds are manufactured, stored, shipped and sold throughout the US, they perhaps constitute a more significant risk to potential human exposure through accidental or intentional release [4] than CWAs. Field screening methods that can be most readily adapted to the detection and identification of specific compounds include ion mobility spectroscopy (IMS) and mass spectroscopy (MS) [5]. Although these techniques show significant versatility in detection of a range of compounds, they are typically expensive and require a significant degree of training to operate, even for field adapted systems. Another approach to field screening for TICs and other environmental pollutants involves the use of biochemically based toxicity assays. These bioanalytical techniques are highly versatile and in some cases easily adaptable to a field screening format [6]. One of their inherent limitations as compared to chemical analysis methods is their inability to identify specific compounds. Nevertheless, one of their advantages is the ability to identify the presence of a wide range of toxic compounds [7] that may be outside the calibration set or library spectra for IMS or MS methods, respectively. Biologically based toxicity screening assays include the use of bacteria, daphnids, rotifers earthworms, plants and fish [8]. Although these assays have been shown to be sensitive to a wide range of environmental pollutants, some CWAs, and waste water pollutants, they have not been widely adapted or applied to the detection of TICs, particularly in the vapor phase [9]. In addition, the application of these toxicity-based assays to emergency response decisions associated with a toxic chemical release involving manufacturing or transport, requires that they can be operated in near-real-time and be adapted for field operation. Most toxicity screening assays are run in aqueous phase. Sampling and analysis of acutely toxic hydrophobic pollutants in environmental water samples have been widely demonstrated using semipermeable membrane devices (SPMDs) [10]. These passive sampling devices have also been used in conjunction with a wide range of biological assays [11–14]. In addition, passive sampling devices have been used to measure toxic vapors in air [15–17]. More specifically, air sampling devices that have been used to trap toxic vapors include charcoal traps and SPMDs that incorporate triolein [16] or solid phase sorbants such as polydimethyl siloxane or silicone elastomer [17]. Charcoal has been reported to trap petroleum hydrocarbons which were analyzed for toxicity using the Microtox assay [15]. In another report, urban air was sampled using SPMDs and analyzed for cy-

totoxic and genotoxic effects using the micronucleus assay, Microtox assay and Mutatox assay [16]. Although these reports both show the use of vapor trapping devices interfaced with cytotoxicity or genotoxicity assays, they are not easily adapted to real-time field analysis due to extensive extraction and solvent exchange procedures required. Our intention for this study is to demonstrate the feasibility of a near-real-time passive air sampling system (i.e., SPMDs with DMSO as a fill solvent) that can be sampled directly (i.e., no dialysis or solvent exchange) by rapid and potentially field portable prokaryotic (i.e., Microtox) and eukaryotic (i.e., IQ-Tox) toxicity screening assays. In this report we demonstrate an SPMD-based vapor sampler that does not require solvent extraction steps. We also characterize the acute toxicity effects of a number of TICs that have not been previously reported for the Microtox and the IQ-Tox assays. 2. Experimental 2.1. Reagents Chemicals were obtained from Sigma/Aldrich (St. Louis, MO). The dimethyl sulfoxide (DMSO) used for these experiments was hybridoma grade. Reagent grade DMSO inhibited the Microtox assay at concentrations less than 1%, most likely due to toxic contaminants. The toxic industrial chemicals used for this study are listed in Table 1. 2.2. SPMDs The SPMDs were prepared using low density polyethylene tubing (25 mm × 88 ␮m wall thickness from Brentwood Table 1 Sampling concentrations for toxic industrial chemicals Compound

Initial concentrations Vapor concentrations for Microtox and in exposure chamberb a IQ-Tox (␮L/L) (␮L/L)

Diketene Phosphorusoxychloridec Acrolein Trichloroacetyl chloridec Methanesulfonyl chloride Stilbene 1-Octanethiol Sulfuryl chloridec Formaldehyde Allylamine Methyl chloroformate Chloroacetone Methyl chlorsilane Diisopropylfluorophosphate Methylhydrazine Acetone cyanohydrin 1,2-Dibromoethane

1 1 1 10 10 10 20 10 100 100 100 100 100 100 100 100 100

1 1 10 10 10 10 1 100 100 100 100 100 100 100 100 100 100

a Assuming solubility in Microtox and IQ-Tox assay solutions with 1% DMSO. b Compounds vaporized from 4 ␮L drop of neat or ethanol stock solution. Residue completely disappeared within 2 min. c Compounds are reactive in aqueous solution [21].

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Plastics Inc., 8764 Manchester, St. Louis, MO) as previously described [18] 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 ␮L DMSO and heat sealed on the other end. These SPMDs were then placed into 40 ␮L vials (suspended near the center of volume). For accumulation experiments, 40 ␮L of stock solution for each compound (see Table 1 for stock concentrations) 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 h), the SPMD was removed from the vial, cut open and the DMSO analyzed for toxicity (Fig. 1). 2.3. Chemical analysis GC/MS analyses were conducted using a Finnigan Voyager system. Separations were performed using a 0.1 ␮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. 2.4. Microtox assay The Microtox assay is based on observed changes in luminescence due to exposure of the test organism Vibrio fischeri to toxic chemicals. The organisms were supplied in lyophilized form and were reconstituted in 2% NaCl buffer prior to assay. Aliquots of the bacterial cell suspension were then added to serial dilutions of the toxic compounds (about 107 cells/mL final concentration), incubated and the luminescence recorded. The assay measures light output of the organism after exposure to the sample as compared to the untreated control. Light output was measured after 5 and 15 min exposures. EC50 values determined at 5 and 15 min incubations varied in both directions (i.e., for some

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compounds the EC50 for 5 min was somewhat higher than the 15 min value and for some compounds was lower). Because we are characterizing the feasibility of this assay as a rapid screening method, we report data for the 5 min assay only. A serial dilution profile is used to determine the concentration of sample resulting in a 50% reduction in the luminescence as compared to the untreated control under the same conditions. The Microtox instrument is operated through interactive software (Microtox Omni). Once the luminescence for each sample is read, the values were automatically transferred to the software data set, compared to a negative (i.e., no toxin) control and EC50 values determined as previously reported [19]. The Microtox 500 analyzer and acute toxicity reagent (i.e., lyophilized V. fischeri) were obtained from SDI (Newark, DE). Assays were performed following the SDI basic protocol for pure compounds [19]. The EC50 values for both phenol and zinc sulfate, typically used as positive controls, were similar to previously reported values (results not shown) [19], For direct measurement of the Microtox toxicity responses to the TICs, stock solutions were prepared in DMSO. From these stocks, a dilution of 1–100 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 nine dilutions. EC50 values for the 5 and 15 min assays were recorded along with 95% confidence limits, which were less than a factor of 2 for each reported value (data not shown). 2.5. IQ-Tox assay Test organisms were obtained from Kingwood Diagnostics (469 Point Breeze Road, Flemington, NJ 08822) and cultured according to the provided directions. Briefly, the Daphnia magna were maintained using algal and mineral supplement solutions in aerated nanopure-treated water. Adult organisms were separated from offspring and 5-day old neonates were used for all exposure experiments.

Fig. 1. Experimental protocol diagram.

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The D. magna IQ-Tox assay is based on the inhibition of galactose metabolism by various toxins. Galactose metabolism is measured by cleavage of the pro-fluorescent galactose substrate to a methylumbelliferyl compound that fluoresces throughout the crustacean. The assay is conducted by exposure of six organisms to each of five serial dilutions of the toxin for 60 min followed by a 15 min incubation with substrate. The TICs were added from stock solutions in DMSO with final solvent concentrations of 1%. DMSO alone at this concentration showed no measurable effect on the IQ-Tox assay as compared to controls. Fluorescent organisms were counted and EC50 values determined using a Probit plot. 2.6. 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 [19]. For the pure compound protocols, the primary operator-entered variable is the initial concentration of the test compound. EC50 values denote concentrations expressed on the basis of the highest concentration in the assay dilution series. 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. Each test was run in duplicate using nine sample concentrations and a negative control. 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 in the case of acrolein). The amount of test compound introduced to the Microtox assay from the DMSO and entered into the Microtox Omni 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 ␮L solution (DMSO) spiked with a specific volume of pure test compound was the same as in the 100 ␮L of DMSO in the SPMD, then the Apparent Concentration Factor (calculated as the ratio of EC50 to AppEC50 ) would be 1.0. Values greater than 1.0 indicate that the TIC was concentrated into the DMSO.

SPMDs were performed in a fume hood with appropriate personal protection equipment.

3. Results and discussion 3.1. Vapor accumulation kinetics Acrolein was used to conduct uptake kinetics and mass comparison studies. The kinetics for the accumulation of acrolein as measured by both mass and Microtox assay response (i.e., toxicity) were compared. The mass of acrolein recovered in the SPMD/DMSO was measured by GC/MS (chromatogram not shown). A calibration curve for acrolein in DMSO was constructed by monitoring the 55 + 56 mass ions. The acrolein eluted at 5.44 min and 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. The time course for acrolein uptake on the basis of mass and Microtox assay response is shown in Fig. 2. 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 h maximum value and fit to a sigmoidal curve. An accumulation time of 2 h yielded about a 50% uptake as compared with the 24 h maximum. Significant uptake of compound as measured by both mass and Microtox assay response (i.e., 10%) could be measured after as little as a 10 min exposure of the SPMD/DMSO sampler to the acrolein vapor. SPMDs have been used for monitoring environmental pollution on a long-term time scale. For example, air sampling

2.7. Safety note Due to the volatile and toxic nature of the compounds analyzed, all manipulations using these compounds with the

Fig. 2. Relative accumulation profiles for acrolein into the SPMD/DMSO on the basis of toxicity (i.e., Microtox EC50 values) and mass (measured using GC/MS).

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for priority toxic compounds using SPMDs with triolein as the fill solvent indicated a sequestering of these compounds over a 21-day period [16]. Air toxics such as fluoranthrene and benzene were concentrated to levels of 4.96 and 2.47 mg per SPMD in triolein, respectively [16], By contrast, our data indicate that compounds such as acrolein begin to saturate the DMSO within 24 h. 3.2. Microtox 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 directed toward emergency first responders [1]. These compounds were ranked as medium hazard based on physical characteristics, toxicity and production quantities (i.e., in excess of 30 t/year/facility). The TICs were evaluated for acute toxicity using the Microtox assay. The EC50 values were determined for each of the TICs from stock solutions prepared in DMSO and diluted into the assay medium. These values ranged from 0.070 to 322 ␮L/L (Table 2). The EC50 values fell into three groupings consisting of low (e.g., diketene – sulfuryl chloride), medium (e.g., formaldehyde – methylhydrazine) and high (acetone cyanohydrin – 1,2-dibromoethane) values. EC50 values are inherently empirical with the lower EC50 values indicating a more toxic response. It should be noted that several of the compounds tested (i.e., phosphorus oxychloride, sulfuryl chloride and trichloroacetyl chloride) are described as highly reactive with water [21]. Consequently, it is likely that the observed toxicity for these compounds is due, Table 2 Response of V. fischeri (Microtox assay) to toxic industrial chemicals sampled from direct dilution (EC50 ) and from vapor accumulation (AppEC50 ) Compound

EC50 a (␮L/L)

AppEC50 b (␮L/L)

App CFc

Diketene Phosphorus oxychloride Acrolein Trichloroacetyl chloride Methanesulfonyl chloride Stilbene 1-Octanethiol Sulfuryl chloride Formaldehyde Allylamine Methyl chloroformate Chloroacetone Methyl chlorsilane Diisopropylfluorophosphate Methylhydrazine Acetone cyanohydrin 1,2-Dibromoethane

0.070 0.19 0.32 1.2 1.8 2.4 3.9 4.8 13 18 22 26 29 43 56 187 322

0.0041 0.000035 0.0013 0.0044 0.027 0.012 0.0050 0.015 0.28 0.11 0.060 0.041 0.090 0.15 1.0 0.49 0.68

17 5400 246 273 67 200 780 320 46 164 367 634 322 287 56 382 474

a Values were determined from 5 min assay. Nine dilutions were used to determine each EC50 value. b Apparent EC values were determined as described in Methods. Nine 50 dilutions were used to determine each AppEC50 value. c Apparent Concentration Factors were determined as the ratio of EC50 /AppEC50 .

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in part, to their aqueous breakdown products. Although this is an inherent limitation for aqueous-based toxicity screening assays, it is also an advantage given that the biological effect rather than identity of the specific toxicant is the intended indicator. 3.3. Microtox 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 40 mL incubation chambers to measure the 24 h accumulation of test compounds into SPMD/DMSO. For the vapor measurements, DMSO was recovered from SPMDs and diluted 100-fold prior to the assay. The Microtox assay responses (i.e., toxic activities) of compounds sampled from the SPMD were determined and reported as AppEC50 values. 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. If the 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 0.035 to 1000 nL/L (Table 2). These values were between two and three orders of magnitude smaller than the EC50 values which were determined from solution. This result indicated that the compounds were, on average, concentrated from the vapor in the exposure chamber into the DMSO by between 200 and 400 times. Although the rank order of EC50 values and AppEC50 values were not identical, the compounds with the lowest EC50 values (e.g., diketene through sulfuryl chloride) and compounds with the highest values (e.g., methylhydrazine through 1,2-dibromoethane) were of similar ranking (Table 2). 3.4. 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 (measured from the compound diluted into assay buffer) to AppEC50 (as measured from the accumulation of vapor into the SPMD) and is reported as an Apparent Concentration Factor for each compound. The Apparent 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 (Table 2). Most of the values were between 200 and 400. Although lipophilicity has been shown to be a significant factor in predicting toxicity of organic compounds [20], it was expected that the Apparent Concentration

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Factors would be primarily influenced by partitioning between air/polyethylene/DMSO and kinetics of steady state accumulation. 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 2). This is contrasted by phosphorus oxychloride which was particularly toxic and also showed significant accumulation into the SPMD/DMSO with a Concentration Factor of 5400. Possible explanations for these empirical observations, however, are complicated by the highly reactive nature of several of these TICs. The determination of the mass of accumulated acrolein also allowed a comparison of the Apparent Concentration Factor calculated on the basis of Microtox assay response to a value calculated on the basis of mass. The Apparent Concentration Factor for acrolein based on the Microtox assay response was 246 and the mass-derived value was 210. This difference may arise from the toxicity measurement which required that the acrolein be diluted into an aqueous matrix and may have resulted in the slight polymerization of this compound [21]. The amount of acrolein accumulated into the DMSO in the SPMD after a 24 h incubation period accounted for 52% 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 partially depleted during the course of the experiment. It is also likely that the final concentration of other test compounds that concentrated into the DMSO would have been greater. Although the Apparent 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. 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 [18]. Because of differences in partitioning constants for DMSO and triolein, these relative accumulation values may not be the same for the current study. 3.5. IQ-Tox EC50 values for test compounds in solution EC50 values for the Microtox assay and IQ-Tox assay are compared in Fig. 3. The EC50 values varied significantly between assays for the tested TICs. In some cases, such as for chloroacetone and 1,2-dibromoethane, the IQ-Tox EC50 values were more than an order of magnitude lower than for the Microtox assay. In other cases, such as for phosphorus

Fig. 3. Comparison of Microtox and IQ-Tox EC50 values.

oxychloride, acrolein, diketene, trichloroacetyl chloride and sulfuryl chloride, the Microtox EC50 values were considerably lower than those for the IQ-Tox assay. Differences in the EC50 values between the Microtox assay and D. magnabased IQ-Tox mortality assay have been previously reported for specific pesticides [22]. For some compounds such as linuron, differences are small and for other compounds such as tributyltin, differences in EC50 values are greater than two orders of magnitude [22]. Differences in EC50 values up to an order of magnitude between these assays have also been reported for compounds such as colchicine and dicrotophos [9]. Most of the compounds that were tested are listed as corrosive to biological tissues and due to their volatility would be primarily expected to damage the respiratory systems of mammals. Inhibition of both luminescence in the Microtox assay and substrate uptake and metabolism in the IQ-Tox assay, were most likely due to destruction of cellular membranes and inhibition of metabolic enzymes [23]. Differences in membrane barriers and their effect on uptake rates for various toxicants would be expected sources of variation in the observed values between prokaryotes and eukaryotes.

4. Conclusions Summary of results for this study include the following: (1) The SPMD sampler was shown to accumulate (to varying degrees) each of the TICs. The use of DMSO rather than triolein typically used in SPMDs, allowed the Microtox and IQ-Tox assays to be run directly from the SPMD solvent without the typical processing and cleanup which consists of a 48 h dialysis in cyclohexane followed by evaporation and solvent exchange into DMSO prior to toxicity testing [16].

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(2) Microtox EC50 values were determined for each of the TICs analyzed and ranged from 0.070 ␮L/L for diketene to 322 ␮L/L for 1,2-dibromoethane. AppEC50 values measured from vapor accumulation into the SPMD/DMSO were lower than the EC50 values and ranged from 0.035 nL/L for phosphorus oxychloride to 1000 nL/L for methylhydrazine. (3) Comparison of the Microtox and IQ-Tox assays showed significantly differing sensitivities with respect to various TICs suggesting that both assays should be used as biological endpoints when screening for the presence of unknown TICs. These results indicated that the SPMD/DMSO vapor sampler coupled with both the Microtox and IQ-Tox assays respond to each of the TICs examined in this study. Further studies, however, are required to determine detection limits for individual compounds or mixtures of compounds using this combination of toxicity assays.

Acknowledgments The authors 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.

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