Environ. Sci. Technol. 1994, 28, 1819-1828
Combined Filtration-Solid-Phase Extraction Method for Recovering Organic Substances from Natural Waters in Preparation for Mutagenicity Testing John L. Durant,'*t** Peter A. Monchamp,* Arthur L. Lafleur,* and Harold F. Hemondtl* Parsons Laboratory for Water Resources and Aquatic Sciences, Department of Civil and Environmental Engineering, and Center for Environmental Health Sciences, Massachusetts Institute of Technology, Cambridge, Massachusetts 02 139
Preparation of water samples for studying toxicological activity requires that methods for capture and concentration of water-borne chemicals do not chemically alter constituents or add or remove toxicants. In this paper we describe the development of an artifact-free filtrationsolid-phase extraction system that recovers particulate organic material (POM) and dissolved organic material (DOM) from >1OO-L samples of natural waters in preparation for mutagenicity determination and chemical characterization. The system consists of 0.45-hm poly(vinylidene difluoride) membrane filters connected in series to columns packed with equal amounts of CISand CN bonded-phase sorbents. Extracts of cleaned filters and bonded-phase sorbents were shown to be free of interferences that are toxic or mutagenic to human B-cells and Salmonella typhimurium. Likewise, the filtered and sorbed fractions of a 100-Lhigh-purity water sample were neither toxic nor mutagenic to human B-cells or S. typhimurium, demonstrating that the system does not generate biologically active artifacts. Recovery studies in which fluoranthene, an S. typhimurium mutagen, was added to 100-L high-purity water samples yielded >97 % recoveries and showed that the mutagenicity of the sample was conserved. The average recovery of eight organic compounds added as mixtures to 100-L high-purity water samples was 77 f 13% (mean f SD, n = 2). The POM extract of one of two water samples from the Aberjona River (eastern Massachusetts) was found to be mutagenic to human B-cells. Benzo[a]pyrene-a potent human B-cell mutagen-was present in this extract at concentrations sufficient to account for 20-25% of the observed mutagenicity. Introduction Man-made organic chemicals released into the aquatic environment may pose risks to human health. In order to better assess these risks, considerable effort has been made to detect and measure the concentration of known pollutants in natural waters and treated drinking water. However, many of the man-made organic chemicals in water have yet to be identified and/or fully characterized in terms of their toxicological properties. Likewise, the risks posed by exposure to complex mixtures of organic pollutants in water are, at best, poorly understood. Therefore, risk assessment methods that are based solely on determining the concentration of known pollutants may underestimate the actual risks or worse may fail to measure risk when hazardous chemicals are present. As an alternative or complementary risk assessment strategy,
* Corresponding author; e-mail address: JLDurant@Athena. MIT.EDU. Parsons Laboratory for Water Resources and Aquatic Sciences, Department of Civil and Environmental Engineering. 8 Center for Environmental Health Sciences. f
0013-936X/94/0928- 1819$04.50/0
0 1994 American Chemical Society
water samples suspected of containinghazardous chemicals are often tested for their ability to cause mutations in laboratory organisms. Mutagenicity testing can be used to distinguish mutagenic from nonmutagenic samples, detect compounds that may be dominant in forms of risk despite relatively low concentrations, and identify as yet unknown pollutants. Studies in which in vitro mutation assays have been used have shown that mutagens are present in natural waters (1-15), chlorinated drinking water (15-22), swimming-pool water (23), municipal wastewater treatment plant effluent (15, 24-27), and industrial effluents (28). The most widely reported mutagens have been newly-discovered chlorinated compounds in some chlorine-treated drinking waters (16,19, 22) and industrial effluents (28) and well-established genotoxic chemicals such as certain polycyclic aromatic hydrocarbons (7,211 and aromatic amines (21). The majority of organic constituents in natural waters are present at concentrations of parts per million or lower; therefore, in order to obtain sufficient mass of organic material for biological and chemicalanalysis, water samples must typically be highly concentrated prior to testing. Commonly reported enrichment techniques include (i) concentration methods such as vacuum distillation, lyophilization, freeze concentration, reverse osmosis, and ultrafiltration in which water is removed while dissolved and particulate material are retained and (ii) extraction methods such as liquid-liquid and solid-phase extraction in which constituents are removed from water as a result of equilibrium partitioning into a contacting liquid or onto a solid surface (29). While it is generally recognized that all of these methods have advantages and disadvantages and that no single method is capable of recovering all organic constituents from water (301, many investigators favor solid-phase extraction with styrene divinylbenzene copolymer-basedand methyl methacrylate polymer-based resins (Le., XAD series) (1-6,11,14-19,22-24,26-28,31). These resins can recover organic compounds from relatively large volumes of water (e.g., >lo0 L) and require only small amounts of solvents (typically 100 L) water samples.
analysis are bonded-phase silica sorbents. These sorbents are formed by reacting activated silica with different organosilane functional groups (e.g., cyanopropyl, octadecyl, and aminopropyl); thus, depending on the nature of the functional group, bonded-phase sorbents can be tailored to extract specific classes of organic chemicals. Bonded-phase sorbents (notably octadecyl) have been used to recover hydrocarbons (34-37), phenols and priority pollutants (38,39),and pesticides (39-44) from seawater (34,40),surface water (36,411,and groundwater (43).Some studies have reported >85% recoveries for organic compounds in the nanogram to microgram per liter concentration range (39, 40). Cyanopropyl bonded-phase sorbents have been shown to preserve mutagenicity in mutagenic complex mixtures during chromatographic fractionation (45, 46). Our own work (reported herein) indicates that solvent extracts from bonded-phase sorbents are relatively free of organic contamination and are neither toxic nor mutagenic in Salmonella typhimurium and human B-cell assays. These results motivated us to construct a large-volume extraction system based on bonded-phase sorbents to isolate organic material from water samples in preparation for mutagenicity determination. In this paper, we report on the development of this system. Results of materials extract analyses, recovery studies, and testing of high-purity and river water samples for mutagenicity are presented and discussed. 1820 Envlron. Sci. Technol., Vol. 28, No. 11, 1994
Materials and Methods
Apparatus. The extraction system was designed to accommodate water samples in excess of 100 L so that organic material could be recovered in sufficient quantities for mutagenicity testing and retesting (if necessary) of whole extracts, for mutagenicity-directed fractionation to isolate the most genotoxic fractions and subfractions, and for chemical analysis. The system that was developed consists of two basic components: 0.45-pmfilters mounted in high-pressure stainless steel filter holders connected in series to high-pressure liquid chromatography (HPLC) columns packed with equal amounts of octadecyl (CIS) and cyanopropyl (CN) bonded-phase sorbents (Figure 1). The filters were 90 mm diameter, 0.45-pm pore size Durapore poly(viny1idene difluoride) membrane filters made by Millipore Corp. (Bedford, MA). HPLC columns (25 cm in length and 22.5 mm i.d.) obtained from Alltech Associates Inc. (Deerfield, IL) were cut in half, packed with 20 g each of 40-pm (average particle size) CN and CIS (J. T. Baker Inc., Phillipsburg, NJ), capped a t both ends with 10-pm pore size stainless steel frits (Alltech), and fitted with stainless steel column end fittings. The filter holders and HPLC columns were connected by stainless steel 0.25-in. (outer diameter) tubing to the outlet of a Liquid Metronics Inc. (Acton, MA) piston metering pump (Model D101-97). The wetted parts of the pump were made of Teflon and stainless steel. The pump inlet was
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pg of POM extract per mL of cell culture Figure 2. Mutagenicity and toxicity induced at the tk locus in human B-cells (MCL-5) by 72-h exposure to extracts of particulate organic matter filtered from two Aberjona River water samples.
connected to a foot valve which was submerged in a 20-L glass sample reservoir. The reservoir was placed on a magnetic stirring plate, and water samples were stirred continuously to ensure uniform mixing of samples and to reduce the settling of particulate material. Water samples were pumped through the system at 70-80 mL/min at
