Environ. Sci. Technol. 2004, 38, 5127-5133
Toxicity Characterization of Complex Mixtures Using Biological and Chemical Analysis in Preparation for Assessment of Mixture Similarity LESLIE CIZMAS,† THOMAS J. MCDONALD,‡ TRACIE D. PHILLIPS,† ANNIKA M. GILLESPIE,† REBECCA A. LINGENFELTER,† LEON F. KUBENA,§ TIMOTHY D. PHILLIPS,† AND K I R B Y C . D O N N E L L Y * ,†,‡ Department of Veterinary Anatomy and Public Health, Texas A&M University, 4458 TAMU, College Station, Texas 77843, Department of Environmental and Occupational Health, School of Rural Public Health, Texas A&M University System Health Science Center, Wells Fargo Plaza, 3000 Briarcrest Drive Suite 300, Bryan, Texas 77802, and United States Department of AgriculturesAgricultural Research Service, Southern Plains Agricultural Research Center, Food and Feed Safety Research Unit, 2881 F&B Road, College Station, Texas 77845
In the United States, several proposed approaches for using bioassays for the risk assessment of complex hazardous mixtures require that selected mixtures be “sufficiently similar” to each other. The goal of this research was to evaluate the utility of a protocol using in vitro bioassays and chemical analysis as a basis for assessing mixture similarity. Two wood preserving wastes (WPWs) containing polycyclic aromatic hydrocarbons and pentachlorophenol were extracted and fractionated to generate potentially similar mixtures. Chemical analysis was conducted using gas chromatography/mass spectrometry. Genotoxicity was evaluated using the Salmonella/ microsome and Escherichia coli prophage induction assays. The crude extract of one WPW was also tested in the chick embryotoxicity screening test (CHEST) assay. The CHEST assay provided the most sensitive measurement of toxicity. Overall, the biological potency of the samples was not well correlated with predicted potency based on chemical analysis. Although several mixtures appeared similar based on chemical analysis, the magnitude of the response in bioassays was often dissimilar. Fractionation was required to detect the genotoxicity of mixture components in vitro. The results confirm the need for an integrated protocol, combining chemical analysis, fractionation, and biological testing to characterize the risk associated with complex mixtures.
* Corresponding author telephone: (979)845-7956; fax: (979)8478981, e-mail:
[email protected]. † Department of Veterinary Anatomy and Public Health, Texas A&M University. ‡ Department of Environmental and Occupational Health, Texas A&M University System Health Sciences Center. § United States Department of AgriculturesAgricultural Research Service. 10.1021/es035287p CCC: $27.50 Published on Web 08/21/2004
2004 American Chemical Society
Introduction In the United States, complex mixture risk assessment has typically been conducted based on the risk associated with the most hazardous mixture components (e.g., benzo[a]pyrene [BP]) rather than on the toxicity of the whole mixture (1). However, it is recognized that a component-based approach may not adequately characterize complex mixture toxicity (2, 3). The actual toxicity of a complex mixture can differ from predicted toxicity due to factors such as unidentified toxic chemicals in the mixture (3, 4) and/or unexpected interactions between mixture components that influence mixture toxicity (2-4). To better evaluate the risk associated with whole mixtures, a number of mixture-based approaches have been developed. These include the comparative potency approach (3, 5), the sufficiently similar mixture reference dose/concentration (RfD/C) or slope factor approach (3), and the surrogate mixture approach (5). A key assumption of each of these methods is that at least two mixtures are “sufficiently similar” to each other (3, 5). To determine if two mixtures are sufficiently similar, the toxicity of each mixture must first be characterized in order to provide a basis for comparison. One or more of the following criteria may be used by the risk assessor to characterize mixtures and evaluate whether “sufficient similarity” exists (3, 5): (i) a common source of emission or formation, (ii) a common mode of toxic action, and/or (iii) the presence of components that are common across the mixtures, in similar ratios or proportions (particularly potent compounds such as BP). With the third criterion, attention should be paid to whether the proportions are different enough to change the magnitude or type of effects (3). The U.S. Environmental Protection Agency (U.S. EPA) guidelines (3), which discuss only the first two risk assessment approaches listed above, specify two additional criteria: (i) consistency in the results of short-term screening assays and (ii) similarity in chemical structure or chemical class. These guidelines also note that the risk assessor should evaluate data for all sensitive end points and should consider whether the mixture of concern will change over time in the environment (3). Although these criteria provide some guidance for determining whether mixtures are sufficiently similar, there are no quantitative guidelines for deciding whether a given criterion has been met, nor is it well-specified how many criteria must be met in order for the mixtures to be considered similar. Instead, this is left to the judgment of the risk assessor. Previous studies of a series of complex industrial wastes have found that biological activity is not well-predicted from chemical analysis alone, suggesting that both biological and chemical analysis are valuable for characterizing complex mixtures (4, 6). The objective of the present research was to evaluate whether a battery of in vitro bioassays coupled with chemical analysis could be used to adequately characterize the toxicity of complex mixtures. Polycyclic aromatic hydrocarbons (PAHs) and pentachlorophenol (PCP) have been found to co-occur at a number of hazardous waste sites across the United States (7, 8). Two mixtures containing PAHs and PCP were collected from two sites formerly used for wood preservation. They were extracted to obtain solvent-extractable organics and fractionated to isolate mixture components by chemical class. The purpose of fractionation was 2-fold: (i) to assess whether mixture toxicity was “unmasked” by fractionation, i.e., whether toxicity that remained undetected VOL. 38, NO. 19, 2004 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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with the parent mixture was detected following fractionation, and (ii) to evaluate whether fractionation could produce sufficiently similar fractions that were suitable for risk assessment using a surrogate mixture-type approach, even if the whole mixtures were not sufficiently similar. Following characterization of the whole mixtures and fractions, the biological and chemical data were used to evaluate whether similar sets of mixtures could be considered sufficiently similar based on the criteria cited above for assessing mixture similarity. The U.S. EPA guidelines (3) state that any developmental toxicity is also an important concern. An additional objective of this research was to evaluate whether developmental toxicity should be considered a sensitive end point for PAHand PCP-containing wastes and evaluated when characterizing the toxicity of these mixtures. The crude extract of one wood preserving waste (WPW) was tested in the chick embryotoxicity screening test (CHEST) assay; the mortality, changes in mean embryo mass, and other developmental effects following exposure to this mixture were assessed.
Experimental Methods WPW and Sample Collection. This study utilized WPW from two former wood preserving sites that had used creosote and PCP. The waste from the first site (WPW-1), a bottom sediment waste collected from a surface impoundment in the southern United States, was collected as described previously (9). The second waste (WPW-2) consisted of the nonaqueous phase layer (NAPL) from a WPW-contaminated aquifer in the northwestern United States. At this site, contaminated groundwater and NAPL were being pumped into an oil-water separator tank. The NAPL was collected in amber 250-mL glass I-Chem jars from this tank. The two WPWs as well as the fractions from these mixtures were considered hazardous, and as such, appropriate handling techniques and personal protective equipment were utilized. Solvent Extraction. The solvent extraction of WPW-1 was conducted as previously described (9). The solvent extraction of WPW-2 was conducted differently because WPW-2 contained no apparent particulate matter and was less viscous than WPW-1. Each aliquot of WPW-2 was solvent-extracted three times using a liquid-liquid extraction 1:1 (v/v) with dichloromethane. The organic extracts from each aliquot of WPW-2 were combined, vacuum filtered through a No. 41 Whatman filter (20-25 µm particle retention) (Whatman Inc., Clifton, NJ), and concentrated on a Buchi Rotovapor (Buchi Labortechnik AG, Switzerland). The extracts were combined and homogenized in preparation for acid/base/neutral fractionation. Fractionation of WPW-1 and WPW-2. One aliquot of WPW-1 crude extract was separated into fractions A1 and A2 using alumina column chromatography, and a second aliquot of WPW-1 crude extract was fractionated into acid, base, and neutral isolates using a liquid/liquid acid/base/neutral (A/B/N) procedure as previously discussed (9). The WPW-2 crude extract was fractionated into acid, base, and neutral isolates using the A/B/N procedure utilized for the WPW-1 (9) with the following modifications. The isolation of the acid fraction from the crude extract was scaled up 25-fold. Each 250-mL aliquot of crude extract was shaken 4 min in a separatory funnel with 500 mL of dichloromethane and 500 mL of 0.2 N NaOH. The aqueous phase (acid fraction) was removed, and the remaining organic phase was extracted at least twice more until there was no visible material partitioning into the aqueous phase. The aqueous extracts were combined and adjusted to pH < 2 using H2SO4. Each aliquot of aqueous phase was then extracted three times using a 3:1 (v/v) ratio of aqueous phase:dichloromethane. The organic extracts were combined to produce the acid fraction. Next, the base/neutral isolate was extracted three 5128
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times (300 mL of base/neutral isolate:900 mL of 1 N H2SO4). The aqueous aliquots (base fraction) were combined and extracted twice (900 mL aqueous phase:750 mL of dichloromethane) to recover neutral components. All aliquots of neutral fraction were combined. The aqueous phase (base fraction) was adjusted to pH 12 with NaOH and extracted three times with dichloromethane to extract the base fraction into the organic phase. In preparation for biological and chemical analysis, aliquots of the crude extract and acid, base, and neutral fractions were taken to dryness under nitrogen with gentle heat (
