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Identification of Nonpolar Toxicants in Effluents Using Toxicity-Based Fractionation with Gas ChromatographylMass Spectrometry Lawrence P. Burkhard* and Elizabeth J. Durhan National Effluent Toxicity Assessment Center (NETAC),U.S. Environmental Protection Agency, 6201 Congdon Boulevard, Duluth, Minnesota 55804 Marta T. Lukasewycz National Effluent Toxicity Assessment Center, ASCI,6201 Congdon Boulevard, Duluth, Minnesota 55804
A toxlclty-based method to identify nonpolar organic toxicants In effluents has been developed. This method has low artifactual toxlclty and excellent detection llmlts, allows multlpie toxicant situations to be easlly handled, and features the use of cladocerans and flshes as test organisms and gas chromatographylmass spectrometry (GWMS) to identify the toxIcants. This method uses reverse-phase chromatography lechnlques to extract and fractlonate the nonpolar organic toxlcants from the effluent. GWMS analyses are performed on the toxlc tractlons, and lists of tentatlve compound Identlflcatlons are made by lnterpretatlon of the mass spectra and elution lnformatlon from the chromatographic separations. These Initial lists are refined by assembllng and then comparing toxlclty data of the identified chemlcals to the toxicity of the fraction. The reflned llsts of suspect chemlcais are further evaluated by pure chemical toxicity testlng, and thls process ultimately leads to toxicant ldentlflcatlon. The fractlonatlon scheme, Instrumental parameters, the toxlcant ldentlflcatlon process, an example lllustratlng the method, and discussion relevant to the method are presented.
INTRODUCTION The U.S. Environmental Protection Agency (EPA) has developed a set of methods to identify toxic pollutants in complex effluents (1-3). Effluents can contain thousands of chemicals, but usually only a few chemicals are responsible for any observed toxicity. The goal of EPA’s toxicity-based method is to separate the toxicant from the nontoxic components by using the response of an aquatic organism along with fractionation techniques. This paper describes one aspect of this method; the identification of nonpolar organic toxicants in effluents. The concept of toxicity-based (bioassay-directed) sample fractionation has been successfully applied in numerous studies concerning the separation of toxic compounds from complex mixtures of chemicals. With this approach, mutagenic compounds have been isolated from air particulates (4-9), effluents ( l o ) ,and sediments (11,12)by using bacterial test species, naturally occurring insect repellents have been isolated from plants by using leafcutter ants (13),and shellfish poisons have been isolated with mice as the test species (14, 15). In general, methods used for these toxicity-based fractionations have involved the extraction of the sample with an organic solvent and, after some preliminary clean up, fractionation of the extract using primarily normal-phase chromatography. These methods have been successfully used to identify nonpolar and moderately polar toxic organic compounds (4-17). However, these approaches have been much less successful when the test species were aquatic organisms 0003-2700/91/0363-0277$02.50/0
such as cladocerans (water fleas) and fishes (18-21) for a number of reasons. First, for most of the above fractionation schemes, large quantities of source material (e.g. sediments) with relatively high concentrations of the toxic chemicals were readily available. Consequently, large quantities of the biologically active chemicals can be readily collected, i.e. milligram or gram amounts. In contrast, in effluents, numerous chemicals exert toxic effects at nanogram or microgram per liter concentrations, making the collection of adequate quantities of toxic materials significantly more difficult since very large volumes of water may need to be processed. Second, since there are lower concentrations of the toxicants, interferences from background materials will increase as decreasing amounts of the toxicants are present. These interferences could make toxicant identification and quantitation more difficult since in many cases the toxicants will only be a minor chemical component, even in fractionated subsamples. Third, many of the manipulations used in these procedures (e.g., solvent extractions) cause so much artifactual toxicity that it is impossible to accurately track toxicity due to the actual toxicants. Fourth, the above toxicity-based fractionation schemes routinely use procedures, such as solvent exchange into dimethyl sulfoxide and evaporation to dryness, as a way of removing incompatible organic solvents before testing. However, losses of volatile toxicants can occur during these steps and thus, toxicants may be lost prior to testing. When smaller quantities of the toxicants are available, e.g. nanogram or microgram amounts, these losses can be significant. When larger amounts of the toxicants are present, e.g., milligram or gram amounts, large losses may be permissible. Our research has focused on overcoming the limitations of these current toxicity-based fractionation techniques for use with aquatic test species. With the above toxicological and chemical constraints imposed, we have developed a toxicitybased sample fractionation method to identify acutely toxic nonpolar organics in effluents. This method has low artifactual toxicity, excellent detection limits, allows multipletoxicant situations to be easily detected and resolved, and features the use of cladocerans and fishes as test organisms and gas chromatography/mass spectrometry (GC/MS) to identify the toxicants. In this report, the fractionation method, instrumental analysis parameters, the toxicant identification process, an example illustrating the method, and discussion relevant to the method are presented. APPARATUS The HPLC fractionationwas performed on a Hewlett-Packard (HP) Model 1090 liquid chromatograph equipped with a 5-pm CI8 reverse-phase column (25 cm X 4.6 mm i.d.) and a fraction collector (ISCO Foxy). An injection of 100 pL is made with a methanol/water solvent flow of 1 mL/min. An example of the methanol/water solvent set points for HPLC fractionation are as follows: 50% methanol composition at injection, 60% methanol 0 199 1 American Chemical Society
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at 1 min, 90% methanol at 13 min, 100% methanol at 20 min, and constant 100% methanol until 25 min. The solvent composition changes linearly with respect to time from one set point to the next set point. The GC/MS system used was an H P Model 5790B mass-selective detector with an H P Model 5980A gas chromatograph, HP Model 7673A automatic liquid sampler, and HP-UX series ChemStation. Injections of 1 or 2 jtL were made onto a 30 m X 0.25 mm i.d. DB-5 capillary column (J & W Scientific). The GC temperature program was 50 "C for 4 min, 50-175 "C at 10 "C/min, 175-275 "C at 5 OC/min, and 275 "C for 20 min. The splitless injector and transfer line temperatures were 250 and 280 "C, respectively. Mass spectral data were collected by using a scan range of 50-550 amu and a scan rate of approximately 1 scan/s. Library searches were performed by using HP-UX ChemStation searching algorithms with the EPA/NIH/NBS mass spectral library.
REAGENTS High-purity water was obtained from a SuperQ system of Millipore Corp. High-purity methanol (Burdick & Jackson) and high-purity water were used for all fractionations. The dilution water for all toxicity testing was 10% dilute mineral water, which consisted of one part Perrier (Vergeze, France) and nine parts high-purity water. The general characteristics of the water were a pH of 7.9 and hardness and alkalinity of 39 and 30 mg/L as CaC03, respectively. Various sizes and sources of C18solid-phase extraction (SPE) columns are readily available. The following method and discussion is predicated upon the use of two SPE column sizes produced by Baker, the 6-mL HC that handles lo00 mL of sample and the 1-mL size that handles up to 100 mL of sample. Note, the column is used as described by the manufacturer. If other sizes and/or sources of columns are used, the following elution volumes, conditions, etc. should be modified according to column size and manufacturer's specifications. METHOD The overall scheme for the isolation and identification of nonpolar organic toxicants is described below. For a more detailed discussion on methods for aquatic toxicity identification, both the analytical and toxicological aspects for nonpolar organic toxicants and other toxicants, readers are urged to consult Mount and Anderson-Camahan (1,2)and Mount (3)for further details. Whole effluent is fractionated by using solid-phase extraction technology, and the resulting SPE fractions are toxicity-tested. Only the toxic SPE fractions are then HPLC-fractionated. The resulting HPLC fractions are also toxicity-tested, and GCjMS analysis is carried out on the toxic HPLC fractions. The purpose of the SPE fractionation is to crudely isolate the toxicants from the majority of the other effluent components, followed by HPLC fractionation to more precisely isolate the toxicants from components that would interfere with the interpretation of the GC/MS data. After GC/MS analysis of the toxic HPLC fractions, a list of potential toxicants is created. The list is refined by a process of elimination using both concentration estimates and toxicological information, the ultimate goal being a list of identified toxicants. In the following subsections, detailed information for each of these steps is presented. SPE Fractionation. A 1-jtm glass fiber filter is prepared by passing two 50-mL volumes of high-purity water through the filter. Subsequently, 200 mL of dilution water (described above) is passed through the filter and, after most of the volume has passed, sample is collected for the filter toxicity blank. The effluent is then filtered. If more than one filter is required to obtain a sufficient volume of filtered effluent, additional filters should be prepared as above. A separate blank should be prepared for each additional filter. The 6-mL HC C18 SPE column is conditioned by passing 25 mL of methanol through the column with a pump set at a flow rate of 5 mL/min. Before the packing goes dry, 25 mL of highpurity distilled water is pumped through the column. As the last of the water is passing through, 25 mL of dilution water is added, and the last 10 mL of dilution water is collected for a column blank toxicity test. Elution blanks are collected from the prepared SPE column by passing a graded sequence of methanol/water solvents through
the column; i.e. 25,50,75,80,85,!30,95, and 100% methanol. For each solvent, 2 X 1.5 mL aliquots are passed through the column and are collected in a clean glass vial. The resulting eight eluates are the SPE elution blanks. The same SPE column is again conditioned with 25 mL of methanol and 25 mL of high-purity water, as described above. Before the column goes dry, 1000-mL of filtered effluent is pumped through the column at a rate of 5 mL/min, conditions recommended by the manufacture of the SPE column. Three ca. 30-mL samples of the post C18 column effluent are collected after 25,500, and 950 mL of the sample passes through the column. Eight 3-mL methanol/water fractions in glass vials are obtained from the column by using the same procedure as used for the elution blanks and are called the SPE fractions. Toxicity testing is performed on the whole effluent, the filtered effluent, all post SPE effluent samples (three per column) and the eight methanol/water fractions obtained from the SPE column for the sample. Toxicity testing is also performed on all filter blanks, all SPE column blanks, and all SPE elution blanks. Concentration of Toxic SPE Fractions. The toxic SPE fractions are concentrated for HPLC fractionation by using a back-dilution technique of Durhan et al. (22). Briefly, a 1-mL C18SPE column is conditioned with 10 mL of methanol and rinsed with 5 mL of high-purity water. The toxic SPE fraction and its corresponding SPE blank are diluted 10-fold with high-purity water. The blank fraction is drawn through the 1-mL column under a pressure of 15 in. of Hg with a vacuum manifold. The column is dried for 5 min with nitrogen a t a flow rate of 13 mL/min. The column is then eluted with three 100-pL volumes of pure methanol, and the eluate (approximately 200 pL) is collected. The corresponding toxic SPE fraction is treated similarly; this results in two methanol concentrates, one of the blank and another of the toxic SPE fraction. HPLC Fractionation. The HPLC fractionation of an SPE fraction concentrate is carried out on a C18 column with a methanol/water gradient at a flow of 1 mL/min. The gradient used is dependent on which SPE fractionts) istare) to be fractionated. An example of a gradient for the fractionation of the 75% methanol/water SPE fraction is listed in the apparatus section. The HPLC fractions are collected at 1-min intervals, into clean glass vials, and then sealed with foil-lined caps. The SPE blank concentrate is fractionated first, followed by its corresponding toxic SPE concentrate. Toxic HPLC fractions are concentrated, and their water content is eliminated for GC/MS analysis by the same procedure that is described above for the concentration of toxic SPE fractions. GC/MS Analysis. Concentration estimates were derived for all components by using the response factor for l,4-diiodobenzene (Aldrich) or a deuterated polyaromatic hydrocarbon (Cambridge Isotope Laboratory). These internal standards were added to the sample just prior to injection, and an internal standard method of quantification was used. However, if additional toxicity testing and/or fractionation was planned, the internal standards were not added to the sample prior to injection. These samples were quantified with an external standard method because of the toxicity of the internal standards. To assign tentative identifications to chromatographic components in the GC/MS data, automated library searches followed by expert interpretation are made. Automated library search procedures are instrument specific. However, when possible, both forward and reverse searching algorithms should be used and spectra should be background-corrected before searching. In addition, due to the large amount of computer processing required, a rapidly responding computer is most helpful. After the library search results are available, the search report for each chromatographic peak is examined. By this manual examination, the analyst must decide if the identification is valid and reasonable. The overall process of expert interpretation consists of examining the library search results, manually interpreting the MS data, and considering site- and fraction-specific information, all of which result in a list of tentative component identifications for a toxic fraction. Toxicant Identification. Toxicant identification is an iterative process consisting of estimating or determining toxicity and concentration values for each chemical on the tentative compound identification list. Then a comparison is made between the
ANALYTICAL CHEMISTRY, VOL. 63, NO. 3, FEBRUARY 1, 1991
concentrations of identified chemicals and their corresponding toxicity. (Acute toxicity is often expressed as the concentration of a chemical where there is 50% mortality of the test species. This concentration is referred to as the LC50.) After evaluation, chemicals which do not appear to explain the observed toxicity are eliminated from the list. With each iteration, the quality of the quantification and toxicity estimates improve and, thus, the list of suspect chemicals gradually shrinks to a few chemicals from which the toxicant can be identified. The initial toxicity estimates are obtained from one of three sources: (a) the literature, (b) quantitative structure activity relationships (QSAR), and/or (c) in-house toxicity databases derived from past work with the sample matrix. The literature and QSAR toxicity values on the initial iteration can have considerable error if toxicity data are for a different aquatic species than the one used to evaluate the effluent. Species differences in sensitivity can be as large as 1OOX and, sometimes,1OOOX. In addition, the initial concentration estimates based upon diiodobenzene or a deuterated polyaromatic hydrocarbon can have errors on 1-2 orders of magnitude. Given these sources of uncertainty, chemicals whose LC50 concentrations agree within lOOX of their LC50 toxicities are often put in the list at least through the initial evaluation. After the initial evaluation, a list of suspect chemicals is obtained. This list is refined by obtaining pure chemicals for which analytical measurements are made and acute LC50 values are experimentallydetermined. As better toxicity and analytical data are acquired, concentration differences of a smaller magnitude are used to eliminate suspect chemicals. Often, factors of 5 or less are used in the final iteration. TOXICITY TESTING Acute toxicity tests were performed as described in detail elsewhere (I). Briefly, 48-h toxicity tests using Ceriodaphnia dubia and 96-h toxicity tests using Pimephales promelas were conducted at 25 “C in 10- and 15-mL volumes, respectively. The test end point was mortality. The C. dubia used for testing were cultured at NETAC and were 148 h old at the beginning of each exposure. Larval fathead minnows (P.promelas) were obtained from our in-house culture unit and were 524 h old at test initiation. LC50 values for the effluent, fractions, and reference toxicant samples were calculated by using the trimmed Spearman-Karber method (23, 24). SPE Fraction Toxicity Testing. The whole effluent, filtered effluent, post SPE effluent samples, and the SPE column blanks are toxicity-tested by using serial dilutions with the highest concentration having no dilution. All methanol/water fractions are toxicity-tested by injecting 120 pL of each blank and sample fraction into 10 mL of dilution water. If dilutions are to be made, the initial volumes are increased to 240 pL/20 mL to provide 10 mL with which to make serial dilutions. This dilution ratio yields a 1.2% methanol concentration in the test solution and a nominal concentration factor of 4 times the whole effluent for 1 L of effluent fractionated with one SPE column. Concentrate Toxicity Testing. The methanol concentrates, of the blank and toxic SPE fractions, are toxicity-tested by injecting 8 pL into 10 mL of dilution water. This test dilution yields a 0.08% methanol concentration and a nominal concentration factor of 4 times the whole effluent. When trace amounts of the toxicants are present, effluent concentration factors of 1OX and 20X are suggested. HPLC Fraction Toxicity Testing. HPLC fractions are toxicity-tested by injecting 120 pL into 10 mL of dilution water, yielding a nominal 1.2% methanol concentration in later eluting fractions. The toxic HPLC fractions are concentrated by using the SPE concentration technique described above, and the concentrates are toxicity-tested by using the techniques described previously. DISCUSSION The toxicity-based fractionation method reported within overcomes many limitations of current procedures for toxicity testing with cladocerans and fishes by using reverse-phase rather than normal-phase chromatography for fractionation. With reverse-phase chromatography, a relatively nontoxic solvent system, water and methanol, is used. In comparison, current procedures which use normal-phase chromatography
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have solvent systems that are extremely toxic to aquatic organisms. The LC50 for methanol ranges from 2.0 to 4.0% (v/v) for numerous freshwater species ( I ) . By testing solutions with methanol concentrations of 1.2% or less, useful acute toxicity data with aquatic species can be obtained (I). This tolerance of methanol by aquatic species allows direct testing (after dilution) of the methanol/water fractions and methanol concentrates in this method. In the normal-phase methods, direct testing of the fractions and concentrates is not possible due to the high artifactual toxicity of the organic solvents, e.g., the LC50 for hexane is 0.001% (v/v). When these methods are used, solvent exchange and/or evaporation procedures for removing these solvents are required before toxicity testing can be done with cladocerans and fishes. These procedures and the uncertainties associated with them are not found in the developed method. To extract the nonpolar toxicants from the effluent, octadecyl solid-phase extraction (SPE) (25-27) rather than classical solvent extraction is used. With classical extraction techniques, unacceptable amounts of artifactual toxicity arise from the extraction solvents. With currently available SPE techniques, nonpolar chemicals have, traditionally, been eluted from the SPE column with a nonpolar solvent such as hexane, chloroform, or ethyl acetate (25-27). To avoid artifactual toxicity from these solvents, we decided to use a series of graded methanol/water solutions to elute the S P E column. With this gradient, elution of the nonpolar toxicants from the column has always occurred even though these solvents are more polar and, thus, have less elution power than the traditional nonpolar solvents. By the use of methanol/water solvents, artifactual toxicity caused by nonpolar solvents in the S P E method is avoided. This extraction procedure is simple and fast and allows extracts from an effluent to be prepared with minimal artifactual toxicity which can be toxicity-tested (after dilution) with cladocerans and fishes. A secondary benefit of the SPE technique is that this method allows discrimination between the biologically available and unavailable portions of the toxicant (28). In general, the available or “truly” dissolved portion is retained by the SPE column and unavailable or that “associated” with the dissolved organic matter passes through the column (28). Classical extraction techniques tend to measure the combined, “truly” dissolved plus “associated”, amount of the toxicant. Consequently, the SPE technique provides a better measure of the available portion of the toxicant than the classical techniques. For toxicity-based fractionation methods, being able to make this distinction is important when the toxicology of a chemical is significantly influenced by its association with the dissolved organic matter. The fractionation is performed in this method in two steps. After the effluent has passed through an SPE column, the column is eluted with eight graded methanol/water solutions. The resulting eight fractions are collected and subsequently toxicity-tested. This initial fractionation process eliminates much of the polar biogenic materials extracted by the S P E column. In general, the lower percent methanol SPE fractions contain these materials and the higher percent methanol fractions contain the nonpolar organic toxicants. Since these columns are disposable and inexpensive, messy environmental samples can be processed without destroying expensive HPLC columns. The composition and volume of the graded methanol/water elution solvents were found largely by trial and error. The second step in the fractionation process takes the toxic SPE fractions and refractionates them by using reverse-phase HPLC with methanol/water solvents to create 25 HPLC fractions. The gradient chosen for the HPLC fractionation
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Table I. C18SPE Fractionation Data sample
toxicitp LC50, %
whole effluent 35 (-)* whole effluent control NMC effluent 1st effluent 2nd effluent 3rd column blank 25% effluent 25% blank 50% effluent 50% blank 75% effluent 75% blank 80% effluent 80% blank control
sample
toxicity LC50, %
filtered effluent 39 (34-45) filter blank NDd filter control NM
Post Cis SPE
CCCI
............................................
SAMPLE
8
$60
_'
2
,
23 40
.................................................................................................
-,
...........................................................................................
v)
NM NM
20 -.. ..............................................................................................
NM ND C18 SPE Fractions NM 85% effluent NM 85% blank NM 90% effluent NM 90% blank 23 (17-32) 95% effluent NM 95% blank 35 (-) 100% effluent NM 100% blank NM
27 (20-36) NM 71 (-)
NM NM NM NM NM
80
BLANK
75-80-85-90% CIS SPE Concentrate effluent
blank control
