Identifying toxicants: NETAC's toxicity-based approach
Lawrence P. Burkhard Gerald T. Ankley U.S.Environmental Protection Agency Nm'onal Efluent Toxiciiry Assessment Center (NEUC) Duluth, MN 55804 The Federal Water Pollution Control Act of 1972, as amended by the Clean Water Act of 1977, states that ". . . it is the national policy that the discharge of toxic pollutants in toxic amounts be prohibited" (1). The major mechanism for ensuring this goal is found in Title IV of the legislation, which describes the National Pollutant Discharge Elimination System (NPDES). NPDES enables chemical and toxicity limits to be set for effluents from point-source dischargers through permit programs administered by either EPA or authorized state agencies. Dischargers who violate the limits specified in their permits can be subject to prosecution. Initially, l i t s in NPDES permits were based primarily on physical factors such as suspended solids, color, and biological oxygen demand. Later, toxic compounds of special concern were monitored through the use of chemical-specific analyses that emphasued 129 priority pollutants (I). However, by limiting priority pollutant concentrations in effluents, compliice with the Clean Water Act is still not ensured because many chemicals other than priority pollutants cause toxicity. Consequently, in 1984, EPA issued a policy statement recommending an integrated approach to NPDES permit policy that featured the use of wholeeffluent toxicity tests combined with chemical-specific analyses (2. 3). The
inclusion of toxicity lipits in effluent permits is currently the best approach for requiring and ensuring compliance with the Clean Water Act. Successful implementation of the NPDES program with toxicity limits requires routine toxicity tests for monitoring as well as protocols for performing Toxicity Reduction Evaluations (TREs). TREs are performed when dischargers are not in compliance with their permits, and are intended to determine measures needed to maintain toxicity at acceptable levels. An integral part of the TRE is the Toxicity Identification Evaluation (TIE), which actually identifies the toxicants.
1438 Environ. Sci. Technoi., Vol. 23, No. 12,lWS
Toxicant identi6cation Effluents, whether from municipal or industrial sources, contain thousands of chemicals; usually only a handful of chemicals are responsible for the observed toxicity. The goal of any TIE method is to identify quickly and cheaply those chemicals causing toxicity, However, components causing toxicity in effluents vary widely. Potential toxicants include cationic metals; polar and nonpolar organics; anionic inorganic~;and ammonia and chlorine, two common toxicants in effluents. In addition, when multiple toxicants are present, the proportion of the overall toxicity due to each toxicant often varies significantly over time. The matrix of an effluent will also change significantly over time. These changes are often cyclical, occumng daily, weekly, monthly, and seasonally, and can be spurious due to weather conditions and other unknown or unpredictable events. Many matrix parameters, including total organic carThis article not subject 10 U S copyright. Published 1989 American Chemical SociElY
bon, suspended solids, pH, hardness, and ionic strength can strongly affect the potency of toxicants. Also, in multiple toxicant situations, the importance of matrix effects can be compounded by the possibilities of synergistic and antagonistic interactions among toxicants. For a TIE method to be successful, it must be able to resolve all of the analytical and toxicological problems posed by effluents. Successful toxicant identification requires a complete understandiig of the concentration-response curve for the toxicants, including the influences of the effluent math on the toxicants and synergistic and antagonistic interactions among toxicants. Conventional (chemical-specific) apprnacb for TIES. This approach to identifying toxicants in an effluent is essentially an expansion of the priority pollutant monitoring method. Here, nontarget, chemical-specific analyses are performed that identify numerous chemicals in the effluent. Subseauentlv. an evaluation of the identified ;he& cals is performed to determine which chemicals may be responsible for the observed toxicity. However, this apuroach is fraught with difficulties. F i t , this iethod presumes that the toxicant(s) will be detected by the analytical instrumentation employed. However, there is no assurance that the analytical methods used will be sensitive enough to detect the toxicants or that the correct analytical instrumentation was used (e.g., GUMS analyses will not d e w cationic metals). Second, this method presumes that the toxicities of the identified chemicals are known. Third, this method presumes that techniques for evaluating the toxicity of complex chemical mixtures are available. However, toxicity data bases, models for predicting toxicity, and methods for evaluating the toxicity of complex chemical mixtures are limited. Consequently, evaluation of the toxicities including synergistic and antagonistic interactions and matrix effects for all identified chemicals is almost impossible. In addition, both the identitication and toxicity evaluation tasks are quite formidable when thousands of chemicals are present. In view of the above limitations, the conventional (chemical-specific) approach appears to be somewhat impractical for performing cost-effective TIEs. In situations where well-defined effluents are available, this approach may be viable. 'hicity-basedapproaeh for TIES. The goal of the toxicity-based approach to TIEs is to separate the toxicants from the nontoxic components in the effluent prior to performing instrumental analyses. To isolate the toxicants, sample fractionation techniques in combination
with toxicity tests (toxicity tracking) are used. This approach allows the physical and chemical IIaN1.e of the toxicant(s) to be determined prior to instrumental analysis. Consequently, the correct analytical technique can be selected for detecting as well as identifying the toxicant($ in the subsample. In addition, significantly fewer chemical components are in the subsamples than are in the original effluent, and thus the task of deciding which component is causing the toxicity is significantly easier. With the toxicity-based approach, detection of synergistic and antagonistic interactions as well as matrix effects for the toxicants is possible via toxicity tracking. A priori knowledge of the toxicants' behavior in the effluent is not required, as is the case with the conventional approach for TIES. One of the major shortcomings of the conventional TIE approach is that no direct relationship exists between the toxicants and the results of the analytical methods. In contrast, the toxicitybased approach enables direct relationships to be more easily established between toxicants and measured analytical data because toxicants are tracked through a l l sample manipulations using the most relevant detector available, the test organism. Establishing this relationship ultimately results in easier, quicker, and less costly TIEs. The concept of toxicit);-based sample fractionation has been applied reas~nably successfully in studies concerning the separation of mutagenic compounds from complex mixtures of chemicals, where the test species of interest is bacterial (4-6). Toxicity fractionation schemes using higher organisms and responses such as acute or chronic toxicity have been much less successful (7-10). This lack of success can be attributed to a number of factors. First, 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 caused by the actual toxicants. In other instances, fractionation steps are not specific enough to cause much of a degree of separation of compounds, thereby resulting in a still very complex mixture of possible toxicants. Finally, many of the proposed schemes routinely use procedures such as solvent exchange that can result in the loss of certain classes of toxicants (e.g., volatile compounds) before analytical procedures are initiated. These problems are largely avoided in TIE approaches recently developed and reported by EPA's National Effluent Toxicity Assessment Center (NETAC) (11-13) and other researchers (14). The following review will present the framework and some of the procedure$ Envlron. Scl. Technol., Vol. 23, No. 12, 1989 1439
-b-lb(. Toxic effluent sample
recently developed by NETAC for
TIES. These procedures were developed for performing T I E s for acutely
toxic chemicals. Investigators are urged to consult NETAC's documents (11-13) for detailed information. This approach and its procedures were developed over the last three years using municipal and industrial effluents from more than 30 sites. They have been used with a number of aquatic species, including cladocerans and fishes. These procedures have proven to be successful in identifying acutely toxic substances in mare than 90% of the samples to which they have been applied. The relationship of this TIE approach to the overall TRE is shown in Figure 1. The TIE approach of NETAC is divided into three. phases. Phase I consists of methods to identify the physical and chemical nature of the constituents causing acute toxicity. Phase I results are intended as a first step in identifying the toxicants, but the data generated also can be used to develop treatment methods to remove toxicity without identiEdon of the toxicants. Phase II describes fractionation schemes and analytical, methods to identify the toxicants, and Phase ID de1440
Environ. ScI.TochnOl., Vol. 23,No. 12, 1989
scribes procedures to confirm that the suspected toxicants are the cause of the observed toxicity,
lbxicant characterization Phase I characterizes the physical and chemical properties of effluent toxicants by altering or rendering biologically unavailable generic classes of compounds with similar properties. Toxicity tests, performed in conjunction with the manipulations, provide information on the nature of the toxicant(s). Successful completion of Phase I occurs when both the nature of the components causing toxicity as well as their consistency over time can be established. After Phase I, the toxicant(s) can be tentatively categorized as having chemical characteristics of cationic metals, nonpolar organics, volatiles, oxidants, substances whose toxicity is pH dependent, andlor substances whose toxicity is not influenced by Phase I methods (Le., possibly a polar organic andlor anionic inorganic). Phase I also provides information on the filterability, volatility, and solubility of the toxicant(s), which is useful for the development of effluent treatment processes to reduce toxicity.
Figure 2 shows an overview of tht effluent manipulations employed ir Phase I. Not shown in Figure 2, bu performed on all effluents, are routini water chemistry measurements includ ing pH, hardness, conductivity, and dis solved oxygen. These routine measurements are needed for designing effluent manipulations and interpreting test data. The manipulations shown in Figure 2 are usually sufficient to characterize toxicity caused by a single chemical. When multiple toxicants are present, various sequential combinations of the Phase I manipulations will most likely be required for toxicant characterization. Many of the manipulations in Phase I require samples that have been pH-adjusted. The adjustment of pH is a powerfd tool for detecting cationic and anionic toxicants in effluents because their behavior is strongly influenced by pH. By changing the pH of an effluent, the ratio of ionized tn un-ionized species in solution for a chemical is changed significantly. The ionized and un-ionized species have different physical and chemical properties as well as toxicities. In Phase I, pH manipulations are used to examine two different ques-
tions. First, Is the toxicity of the effluent different at various pHs? and second, Does changing the pH, performing a sample manipulation, and then readjusting the effluent to ambient pH affect toxicity? The graduated pH test examines the first question; the pH adjustment, aeration, filtration, and solid-phase extraction (SPE) manipulations examine the second. In the graduated pH test, the pH of an effluent is adjusted withim a physiologically tolerable range (Le., pHs 6.0, 7.0, and 8.0) before toxicity testing. Generally, the un-ionized form of a toxicant is able to cross biological membranes more readily than the ionized form and thus is more toxic. This test is designed primarily for ammonia, a relatively common toxicant in effluents whose toxicity is extremely pH-dependent (15). However, different pHs can strongly affect the toxicity of many w m n ionizable pesticides and may d u e n c e the bioavailabdity and toxicity of certain heavy metals (16,17). Aeration tests are designed to determine whether toxicity is attributable to volatile or oxidizable compounds. Effluents at pH, (ambient pH of the effluent), pH 3, and pH 11 are sparged with air for one hour, readjusted to pH,, and tested for toxicity. The different pHs affect the ionization state of polar toxicants, thus making them more or less volatile, and also affect the redox potential of the system. If toxicity is reduced by air sparging at any of the pHs, the presence of volatile or oxidizable compounds is suggested. To distinguish the former from the latter situation, further experiments are performed using nitrogen to sparge the sample(s) rather than air. If toxicity remains the same, oxidizable materials are implicated, if toxicity is again reduced, volatile compounds are suspected. The pH at which toxicity is reduced is also important. If nitcogen sparging decreases toxicity at pH,, neutral volatiles are present, w h e w if toxicity decreases at pH 11.0 or pH 3.0, basic or acidic volatiles, respectively, are implicated. Fitration provides information concerning the amount of toxicity associated with filterable components. Of all of the Phase I manipulations, filtration is the least useful for identifying specific classes of toxicants; however, the data generated could be helpful for defining treatment strategies for toxic ef5uents. In this test, samples at PHI, pH 3.0, and pH 11.0 are passed through 1 - p glass fiber filters, readjusted to pH,, and tested for toxicity. Reductions in toxicity due to filtration could be related to factors such as decreased physical toxicity (18) or removal of particlebound toxicants, which could be important, particularly if filter-feeding
ase I characterization results and suspect toxicant classification two effluents, from NETAC'9 Toxicity ldentlfication Evaluatlon
Phase I test
Oxidant reduction EDTA chelation Graduated pH twt pH adjustment Filtration Aeration Solid-phase extraction
EfRuent one (suspeu toxicant clsasificatim: nonpolar organk6)
NRb NR
Eftlvent two lwapsa toxkanl dsssitkation: nonpolsr agankr( catiark metal@ NR RC
NR
NR NR NR NR
NR NR NR R
R
.EPA's Natlonal Effluent Toxicity Assessment Center dNoreduction in twcity 'RBd"cl10" in t0XiCIt)r
organisms such as cladocerans are the test species. Reversephase SPE is designed to d e termine the extent of effluent toxicity due to compounds that are relatively nonpolar at pH,, pH 3.0, or pH 9.0. This test, in conjunction with asmiated Phase ll analytical procedures, is an extremely powerful TIE tool. In this procedure, filtered sample aliquots at pH,, pH 3.0, and pH 9.0 are passed through small columns packed with an omdecyl (C18)sorbent. AtpHI, the CI8 sorbent will remove neutral compounds such as certain pesticides (19).By shifting ionization equilibria at the low and high pHs, the SPE column also can be used to extract organic acids and bases (2U). During extraction, the resulting post-column effluent is collected and tested for toxicity in order to determine if the manipulation removed toxicity and if the capacity of the column was exceeded. If sample toxicity is decreased, a nonpolar toxicant is suspected. The presence of toxicity due to cationic metals is tested through additions of ethylenediaminetetraacetic acid (EDTA), a strong chelating agent that produces nontoxic complexes with many metals. As with SPE, the specificity of the EDTA test for a class of ubiquitous toxicants makes it a powerful TIE tool. Cations chelated by EDTA include certain forms of aluminum, barium, cadmium, cobalt, copper, iron, lead, manganese, nickel, strontium, and zinc (21). EDTA does not complex anionic forms of metals and only weakly chelates certain cationic metals (e.g., silver, chromium, thallium) (21). Because EDTA nonspecifically binds mono-, di-, and tri-valent metals, the appropriate range of EDTA coneentrations to use in the test is highly dependent on calcium and magnesium concentration (hardness) and salinity, as well as the sensitivity of the test organism to EDTA. The oxidant reduction test is de-
signed to determine the degree of toxicity associated with chemicals reduced by sodium thiosulfate. Chlorine is a particularly common effluent component whose toxicity is decreased by sodium thiosulfate; however, other compounds such as bromine, iodine, and manganous ions also are neutralized by this treatment. Sodium thiosulfate, l i e EDTA, is only slightly toxic to most aquatic organisms, so a relatively wide range of concentrations can be tested.
Toxicant identilieation Initial laboratory work in Phase II is focused on isolation of the toxicants using chemical fractionation techniques with toxicity tracking. The ideal isolation process would create a subsample from the effluent that contains one chemical, the toxicant. Depending on the nature of the toxicants, there can be wide differences in the techniques as well as in the amount of effort required for fractionation. After fractionation, instrumental analyses are performed on the toxic subsamples, and lists of identified chemicals are assembled for each subsample. For each chemical in a list, an LCso value (i.e., the chemical concentration resulting in acute lethality to 50% of the test population) is obtained, usually from the literature or occasionally from structure activity models (22). By comparing concentrations of the identified chemicals to their LC,o values, a list of suspect toxicants is made. This list is then refined by actually determining LCso values for the suspects using the TIE test species. If only one toxicant is present, it should be easily identified. For samples with multiple toxicants, identification be comes significantly more protracted be cause interactions among toxicants will need to be examined. If none of the suspect toxicants appear to explain the toxicity, the true toxicants were probably not detected during instrumental analysis. Usually additional separation, Environ. Sci.Technol..Vol.23, No, 12,1989
1441
combined with concentration steps, is required to increase the analytical sensitivity for toxicant identification. The information obtained in Phase I provides the analytical road map for performing the fractionation and identification tasks in Phase 11. To illustrate the relationship between Phase I data and analytical approaches employed in Phase 11, results for two typical Phase I TIE evaluations are presented in Table 1. The Phase II methods and approaches appropriate for these examples are discussed below. In the first effluent, SPE reduced toxicity. In Phase 11, the SPE column is eluted with graded, increasingly nonpolar methanoliwater solutions, and toxicity testing is performed on each fraction. Although solvents other than methanol are routinely used in analytical work with CIS columns, the low toxicity of methanol to aquatic organisms (e.g., LC50 > 1.5% for commonly tested cladocerans) makes it the solvent of choice for toxicity tracking in the fractions. If no toxicity occurs in the fractions, the toxicant(s) have been lost and further characterization (Phase I) work is required. If toxicity occurs in the fractions, Phase 11 methods feature concentration of the toxic methanoliwater fractions, high performance liquid chromatography fractionation of the concentrate (again with a CISmethanol/ water solvent system) with concurrent toxicity testing of the fractions, and ultimately, identification of suspect toxicants in the toxic fractions via GCIMS. In the second effluent, both EDTA additions and SPE reduced toxicity. The reduction of toxicity by EDTA strongly suggests the presence of toxic concentrations of cationic metals. Thus, Phase I1 procedures would include both metal analyses and the concentration, fractionation, and identification techniques described for nonpolar organics in the first example. If analyses identify specific metal(s) at concentrations high enough to cause toxicity, various mass balance procedures can be used to ascertain what portion of the sample toxicity is due to the suspect metal(s) and what portion of the toxicity is due to the suspect nonpolar compound(s). Table 1 shows only a very small subset of possible Phase I results, particularly when one considers that three of the tests (aeration, filtration, SPE) are conducted at three different pHs. A complete discussion of the types of Phase I results that may be encountered and subsequent Phase I1 strategies that could be implemented is beyond the scope of this review. Toxicant confirmation After Phase n identification proce1442
Environ. Sci. Technol., Vol. 23.No. 12, 1989
dures implicate suspect toxicants, Phase IIl is initiated to confirm that the suspects are indeed the true toxicants. Confirmation is perhaps the most critical step of the TIE process because procedures used in Phases I and II may create artifacts that could lead to erroneous conclusions about the toxicants. Furthermore, there is a possibility that substances causing toxicity change from sample to sample. Phase III enables both situations to be addressed. The tools used in Phase 111include correlation, relative species sensitivity, observation of symptoms, spiking, mass balance, and deletion techniques. In most instances, no single Phase III test is adequate to confirm suspects as the true toxicants, so it is necessary to use multiple confirmation procedures. In the Correlation approach, observed toxicity is regressed against expected toxicity due to measured concentrations of the suspect toxicant(s) in samples collected over time. For the correlation approach to succeed, temporal variation has to be wide enough to provide a range of values adequate for meaningful analyses. A significant correlation occurs when the slope and intercept of the regression are not significantly different from 1.0 and 0.0, respectively, and the square of the correlation coefficient for the regression (r2) is greater than 0.60. Figure 3 shows two sets of correlation results (13). In the first example, sample toxicity correlates very well with expected toxicity caused by the suspect toxicant; that is, slope and intercept are not significantly different from 1.0 and 0.0, and rz is greater than 0.60, thus providing support for the suspect as the true toxicant (Figure 3a). In the second example, correlation of effluent toxicity with toxicity due to the suspect toxicant was poor, suggesting that other toxic compounds may be present (Figure 3b). Further Phase I and I1 work indicated the presence of one additional suspect. When both suspects were included in the analysis, the resulting correlation was quite good (Figure 3c), indicating the importance of both suspects in determining toxicity. In order to use the correlation approach effectively when there are multiple suspect toxicants, it is obviously necessary to generate data concerning the additive, antagonistic, and synergistic effects of the toxicants in ratios similar to those found in the samples. These data also are needed for the spiking and mass balance techniques that are described below. The relative sensitivity of different test species can be used to evaluate suspect toxicants. If there are two (or more) species that exhibit markedly different sensitivities to a suspect toxicant
in pure chemical toxicity tests, and the same patterns in sensitivity are seen with the effluent sample, this provides evidence that the suspect is the true toxicant. Another Phase 111procedure is observation of symptoms in poisoned animals (e.g., time to mortality). Although this approach does not necessarily provide support for a given suspect, it can be used to provide evidence against a suspect toxicant. If the symptoms observed in a pure chemical toxicity test with a suspect toxicant are much different than those observed with the effluent sample (which contains similar concentrations of the suspect toxicant), this is strong evidence for a misidentification. Confirmatory evidence can be obtained by spiking samples with the suspect toxicants. Although not conclusive, if toxicity is increased by the same proportion that the concentration of the suspect toxicant is increased in the sample, this suggests that the suspect is correct. To get a proportional increase in toxicity from the addition of a suspect toxicant when in fact it is not the true toxicant, both the true and suspect toxicants would have to have very similar toxicity levels and would also have to be additive. Mass balance calculations can be used as confirmation steps when toxicity can be at least partially removed from the effluent and subsequently recovered. This approach can be useful in instances when SPE removes toxicity. The methanol fractions eluted from the SPE column are evaluated individually for toxicity; these toxicities are summed and then are compared to the total amount of toxicity lost from the sample. When possible, one of the best confirmation techniques is deletion of the suspect toxicant from the effluent for some period of time. This can be done with certain industrial effluents but may not be feasible with municipal effluents. Other techniques, including alteration of water quality characteristics (e.g., pH, salinity) in a manner designed to affect the toxicity of specific compounds, and analysis of body burdens of suspect toxicants in exposed animals, also can be useful confirmation steps. Final comments Although these TIE methods were developed by NETAC (11-13) using complex effluents, they can be applied to other acutely toxic aqueous samples including river and lake waters, sediment interstitial waters, and leachates from solid-waste sites. Thus they represent a significant advance in the area of
(b) Cortelatlondata for an effluent with
complex-mixture toxicology in aquatic systems. Even so,there is considerable mom for modification and improvement of existing methods. For e m ple, tecbnques to fractionate and identify toxic anionic metals and polar organic compounds are not yet well developed. More importantly, because certain TIE procedures result in marginal toxicity, the TIE approach of NETAC is appmpriate only for samples exhibiting acute toxicity and would not be useM for identifying chronidy toxic compounds. Research addressing these problems is ongoing at EPA. Aeknowledgmmt The work summarized here bas resulted from the effortsof the entire NETAC staff. Funds for this work have b e q provided by the Officeof Research and Development and the Offce of Water, Enforcement and
Permits of EPA. This article was reviewed for suitability as an ES&Tfeaiure by Jon Doi, Monsanto Campany, St. Louis, MO 63163 and Michael DeGraeve, Battelle Labs, Columbus,
OH 43201.
Reperenees (I) Kovalic, 1. M. Ihe Ckan mter Act of 1987; Wter Pollution Control Federation: Alexandria, VA, 1987. (2) Fed. Regisf. 198449. 48. (3) "Technical Support Document for Watw Quality-Based Toxics Control"; U S . Environmental Protection Agency. US. Government Rinting Office: Washingmu,DC,1985; EPA/440/4-85-032. (4) Samoiloff, M. R. el al. Envimn Sci. Technol. 1983.17. 329-34. ( 5 ) Holmbm, 8. et al. Environ. Sci. TechMI. 1984,18, 333-37. (6) West, W. R. et al. Environ. Sa'. Technol. 1988,22,224-28. (7) Reece, C. H.;Burks, S . L. In Aquatic
Toxicology and Hazard Assessment: Seventh Symposium; Carwell, R. D.; Purdy, R.; Bahner, R. e., Eds.; American Soci-
ety for Testing and Materials: Philadel-
phia, PA, 1985; ASTM STP 854; pp. 319-22. (8) Parkhurst, B. R.; Gehrs, C. W.; Rubitt, I. B. In Aquatic Toxicology; Marking, L. L.; Kimerle, R. A,, Eds.; American Society for Testing and Materials: Philadelphia, PA, 1979; ASTM STP 667; pp. 122-30. (9) Lopez-Avila, V. et al. Application of Chem'col Fmctio~tion/Aquatie Bioassay Procedure to Hazardous Waste Site Monitoring; U.S. Environmental Protection Agency. US. Government Printing Office: Las Vegas, W,1986; EPA/600/485/059. (IO) oalass, S.; Battsglia, C.; Vigano, L. Chemosphere 1988.17, 783-87. (11) Mount, D. 1.; Anderson-Canuhan, L. Methods for Aquarie %xicity I d e m M tion Evalunrions: Phase I Toxidry 8 ha; actenmion Procedures: U.S. Envimnmental Protection Agency. U.S. Government Printing Office: Duluth, MN., 1988: EPAIMOI1-R8-034. .... . (12) Mount, D. I.; Anderson-Carnahan, L. Methods for Aquaric Toicily 1denIi M rion Evaluations: P h e I1 Toxicityd n t ; Jicarion Procedures; U S . Environmental Protection Agency. US. Government Printin Office: Duluth. MN. 1988 EPA/ hMl?.f*A?+ "._"._-._-_-. (13) Mount, D. 1. Methodsfor Aquotic Taririry IdmriJicdon Evolunrionr P h e 111
Lawrence I! B d h a n i , a research chemist for EPA. obtained his B.S. degreefrom rhe Pennsylvania State Universityand his M.S. and Ph.D. degreesfrom the University of Wisconsin-Madison. His research interesfs include the fare and behavior of orgMic microconraminants in the enviranment, Molytical methodologiesfor organic microconraminants, and roxicologically based Molyrid techniques.
Enviroimenial Protection Agency. US. Government Printing Office: Duluth, MN, 1988; EPA/600/3-88-036. (14) Doi, J; Grothe, D.R. In Aquatic Toxicolom and Environmmol Fare: Eleventh fikposium; Suter 11, G.w.; Lewis, M.A., Fds.; American Society for Testing and Materials: Philadelphia, PA, 1987: ASTM STP 1007: m.128-38. (15) Ambient Wter Qunliw'&iteria for Ammonia-1984; U.S. Environmental Protection Agency. U S . Government Printine Office: Duluth. MN. 1985: Efi/440/5-8%saOl. ' (16) Campbell, p.G.C.; Stokes, p. M. Can.J. Fish. Apunt. Sci'. 1985,42,2034-49. (17) Doe, K. G. el al. Can. 3. Fish. Aqunt. Sci. 1988.45.287-93, (18) Chapman,P.M.etal. Water.AirSoilPollut. 1987,33,295-308. (19) Junk, G. A,; Richard, J. 3. Anal. Chem. 1988,W. 451-54. (20) Wells, M.I.M.; Michael, J. L. 3. Chromtog,: Sci. 1987,ZJ. 345-50. ,
G e d 'I: AnLlqK a research roxicolo isr for EPA, obtained his B.S. degree in 1$82 from Michigan Srare University and his Ph.D. degree in 1987from the University of Georgia. His research interests include toxicological assessment techniques for complex effluents and contamihfed sediments.
~
Toxicin, Confirmion Procedures: US.
(21) SNmm. W.: Morgan. J . I . Aquntie chpmts1t-y.
2nd Ed ;Wiley: New York. 1981.
1221 Uwr Monuol for OSAR Susrem: InstiNte
for Bi;lo& aid Chk;.ical Process Analysis; Montana State University: Bozeman, MT, 1986.
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