Biological Testing of Waterborne Organic Compounds - Advances in

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Biological Testing of Waterborne Organic Compounds John C. Loper Department of Microbiology and Molecular Genetics, and Department of Environmental Health, University of Cincinnati, Cincinnati, O H 45267-0524 Increased attention to the possible adverse effects of compounds in environmental waters has been stimulated not only by detection of known toxic chemicals as contaminants, but also by evidence for the presence of multiple unknown genotoxic compounds among waterborne organics. Of numerous genetic tests, bacterial mutagenicity assays have been the most revealing. Examples will be discussed to show that less than 10% of such mutagens have been chemically identified. This situation is true whether the studies involved surface or ground water, industrial wastes, or products of the chlorination of humic acids. Roles of mutagenicity testing will be discussed in relation to evaluating collection procedures, examining origin and fate of mutagens, guiding chemical fractionation of residue mixtures for compound identification, and developing criteria of water quality.

T H E C H E M I C A L C O M P L E X I T Y of residue organic mixtures in drinking water and the formidable problems in assessing the role of organic compounds in drinking water within the full range of possible toxic effects have been the focus of much research. A priority approach was put forth for compound isolation, identification, and toxicological characterization. The unknown chemicals in these mixtures were ranked for attention on the basis of molecular weight, relative hydrophobicity, and concentration in the water (la). Our laboratory and those of several others have applied a more direct biological approach. Interest in gaining a general indication of possible adverse effects of drinking water residue organics has led to analyses via various short-term tests of genotoxicity. Most of the progress has occurred through the use of mutagenicity tests, such that 0065-2393/87/0214/0595$06.00/0 ® 1987 American Chemical Society

Suffet and Malaiyandi; Organic Pollutants in Water Advances in Chemistry; American Chemical Society: Washington, DC, 1986.

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over the past few years a far better understanding has been gained of the bioactive properties of waterborne organics as mutagens. This chapter is an abridged overview presenting what biological testing has revealed of the presence of mutagens in water; their nature and origin; and approaches for their prevention, destruction, or removal. Some proposals are presented for future directions.


Background The perception of waterborne organics has changed during the past 20 years. Chlorination of an urban water supply was introduced in Jersey C i t y , N e w Jersey, in 1908 (lb). Since that time until n o w and for the foreseeable future, the overriding concern is for a reliably disinfected drinking water free f r o m hazards of waterborne infection. U n t i l recently, the other criteria for water quality were chiefly taste, odor, and color—properties the public could directly recognize. Similarly, there was a strong public rejection of nonbiodegradable detergents when they appeared as foam b u b b l i n g out of the faucet. Also, questions were raised b y the occasional presence of thousands of fish killed b y toxic chemicals released into rivers that were used as drinking water sources. The unacceptable taste and odor of drinking water f r o m a tributary of the upper O h i o River led Frank M i d d l e t o n and his collaborators in the U . S . Public Health Service to apply their granular carbon extraction-chloroform elution procedure to obtain residue samples of that water in 1962 (2). The first preliminary suggestions that such residues were carcinogenic were presented b y Hueper and Payne (3) the following year. F o r whatever reason, those observations appeared to have had little impact. By the early 1970s, however, the public perception about the origins of cancer had changed dramatically. As early as 1967, D o l l (4) had documented the geographical patterns of the incidence of different common cancers. O n the basis of that documentation and a flood of other data (5), it became understood that the large majority of cancer risk is cultural and environmental. In 1974, trihalomethanes (6, 7) and other possible carcinogenic substances (8) were discovered in water. Epidemiological studies examining cancer incidence in relation to drinking water were widely discussed. (For a recent review of this topic, see reference 9.) It became important to determine the presence and to assess the risk to human health of compounds in water that do not have offensive taste, odor, color, or foam and that do not show acute lethal effects on test organisms. It was shown that the great majority of these organic compounds are nonvolatile and require concentration for study. In the meantime, the analytical chemists continued to extend the range of analysis, and the biologists and biochemists developed an array



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of biological assays with various toxicological end points. O f the biological assays, the Salmonella mutagenesis assay and similar short-term assays proved to be successful detectors of a majority of k n o w n chemical mutagens, carcinogens, and procarcinogens. These assays were also rapid and inexpensive relative to other toxicological assays. Although many toxicological tests have been applied to drinking water, the biological data on water residue mixtures and their chemical constituents have accumulated most quickly f r o m these short-term assays of mutagenicity.

Heterogeneity of Mutagens in Water Residues Many questions remain as to the most appropriate extraction or concentration procedures. Nevertheless, residues f r o m similar water sources have n o w been obtained b y using a variety of techniques. The patterns of mutagenic activity obtained f r o m these residues are sufficiently similar so that it is clear the mutagens are generally not artifacts of concentration. Chemical assays and bioassays of these mixtures typically reveal that among the thousands of largely unknown compounds present at l o w concentrations, there is a diverse minority of mutagens. Evidence for this mutagenic diversity comes chiefly from data regarding the following properties: specificity for tester strains, including nitroreductase" strains; response to microsomal activation systems (S9); additivity of response; class separation; size; chromatography (thin-layer chromatography, reverse-phase high-performance l i q u i d chromatography [ H P L C ] ) ; U V absorption; and sensitivity to inactivation b y heat, alkali, and 4-nitrothiophenol. Examples are cited in this chapter or appear in other chapters of this book. The tests are those of Ames and co-workers (10, I I ) , who have provided procedures for their use. T o conserve sample, often only strains T A 9 8 and TA100 are used in surveys and to guide fractionation. Partially isolated fractions can be characterized further b y using strains such as TA1535 and TA1538 and the several nitroreductase-deficient strains developed b y Rosenkranz and co-workers (12, 13). Microsomal activation for bacterial assays typically has involved crude supernatant fractions (S9) f r o m livers of rats induced b y polychlorobiphenyl mixtures, usually Aroclor 1254. These S9 mixtures contain a spectrum of mixed function oxidases and other enzymes active in biotransformation. In such mixtures, a given compound might be activated to become more mutagenic, may be inactivated, or may remain unaffected. A l l three types of response have been observed with various water residues (9, 14). Fractionations b y all these methods are well-documented b y chapters in this book. O u r o w n work has emphasized fractionation b y reverse-phase H P L C . By using water-to-acetonitrile elution gradients, Suffet and Malaiyandi; Organic Pollutants in Water Advances in Chemistry; American Chemical Society: Washington, DC, 1986.


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mutagenic activity was found predominantly among the m i d - to nonpolar fractions (15). A m o n g a multitude of peaks absorbing at 254 n m , there were several across a broad range of the elution profile that showed different levels of activity for strains T A 9 8 and TA100. This population included many active fractions that expressed only l o w levels of mutgenesis (15). A feature generally true for these mixtures was that mutagenic subfractions appeared directly additive or antagonistic rather than synergistic (15, 16). Such interactive determinations sometimes are complicated b y the presence of toxicity in parent mixtures, b y technical problems of recovery and reconcentration of subfractions for bioassay, and b y l o w levels of some mutagens in relation to the background mutagenesis inherent in the assay.

Potential Mutagens of Industrial Origin Several samples of drinking water have yielded residue mixtures with relatively high mutagenic activity. F o r some of these, mutagenicity has been used as a guide to mutagen isolation. In these cases, the high activity appears to be caused b y only one or a few potent mutagens among the m y r i a d of other compounds. Such was the case with the o l d residue sample f r o m Ohio tributary drinking water, which we obtained from M i d d l e t o n . B y using our Salmonella b i o a s s a y - H P L C fractionation procedure (17), w e traced the bulk of the activity in this residue to a single potent promutagen (18). This substance was presumptively identified as 3-(2-chloroethoxy)-l,2-dichloropropene (CP), a previously undescribed compound (19). The mutagenic potency of C P for TA100, entirely dependent upon the presence of S9, is 75 net revertants/nmol (19). Although C P was the first compound isolated f r o m drinking water residue on the basis of its mutagenic properties, several other laboratories are examining potent mutagens in other residues (17, 19-22). Table I lists these studies of mutagen isolation. A l l of these involve drinking water residues, except for the study b y Maruoka and Yamanaka (22). Their samples are of urban river water taken f r o m a major tributary that contributes to the drinking water supply of more than 10 million people. Based upon the properties of tester strain specificity and dependence on, or independence of, microsomal activation, this list includes several different mutagens. The study conducted b y Heartlein et al. (20) implicated spring runoff of agricultural herbicides as the source of an activation-dependent mutagen for strain TA100 (20). The pollutant under investigation b y Zhou et al. (21) is active with strain T A 9 8 in the absence or presence of S9. The one under investigation b y Maruoka and Yamanaka (22) appears likely to be quite potent. This substance showed its highest mutagenic potency as an S9-dependent mutagen for strain TA1538.


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Table I. Isolation of Drinking Water Mutagens Source Industry


Agriculture City Industry (raw water)

Activity

Compound

Ref.

S9 dep, TA1535, TA100 S9 dep, TA100 TA98, + or -S9 S9 dep, TA1538, TA98

3- (2-chloroethoxy ) 1,2-dichloropropene herbicides? ?

17, 19 20 21 22

?

N O T E : dep denotes dependent.

Although most of these mutagens are i n drinking water residues, i n dustrial or agricultural chemical sources are implicated for their origin. It is probable that all of the stable, potent mutagens readily identifiable in drinking water residues have as their origin commercial anthropogenic chemicals or byproducts. Rappaport et a l . (23) tested organic waste water concentrates from six treatment plants for mutagenicity i n the Ames assay. These researchers concluded that all of the definitely posi tive samples were obtained f r o m plants that treated mixed domestic and industrial wastes, whereas plants that treated wastes strictly from domestic sources were always negative or marginal for mutagenic activity. Similar results involving mutagenic levels that ranged to much higher levels were reported b y H o p k e and Plewa (24) i n 1984. A more recent illustration of this pattern is f r o m a study currently directed b y Tabor for a neighboring set of facilities: one treating a mixture of domestic and industrial wastes and one treating only domestic wastes. The mutagenic activity in net revertants per liter of waste water influent to the treatment plant for strain T A 9 8 was as follows for domes tic-industrial sources: —S9, 90; +S9, 1.2 Χ 10 . F o r domestic sources only, the following data were obtained (net revertants per liter): —S9, plates showed l o w activity that was not dose-dependent; +S9, 4 Χ 10 . F o r TA100, no mutagenic activity was detected. These results were the averages of two assays (see also reference 25). Such properties of S9dependent activity specific for strain T A 9 8 are typically seen among frame shift mutagens of industrial origin. Other data i n this study indicate that part of this activity is deposited i n the sludge and part is released in the plant stream effluent (see also reference 25). 4

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Mutagenic Byproducts of Chlorination By contrast, numerous studies have shown that raw water sources not affected b y industry do not yield microsomal-activation-dependent



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mutagens active on strain T A 9 8 (9, 14, 26). Residues of finished water from these sources typically show direct-acting mutagenesis for TA100 only, or for both T A 9 8 and TA100, w h i c h is decreased b y the presence of S9 mix. Such mutagenicity arises as a result of chlorination (9, 14). The diversity and the chemical nature of this population of mutagens are described b y several of the chapters in this book. Clues as to the identity of some of these compounds may be found in results f r o m mutagenic and chemical studies of other chlorination byproducts. A m o n g these are byproducts of the pulp and paper industry caused during chlorine bleaching processes. These compounds were described in two recent reviews (27, 28). A major mutagen produced b y the bleaching of softwood kraft pulp has been isolated and identified b y H o l m b o m et al. (29, 30) as 3-chloro-4-dichloromethyl-5hydroxy-2(5/f)furanone. This newly identified mutagen is extremely potent in assays using strain TA100 —S9; reported values are 2800-10,000 net revertants/nmol. Strains TA1535, T A 9 8 , and TA1537 were much less responsive. Another c o m p o u n d considered to be a major contributor to mutagenicity in such chlorination liquors is 2-chloropropenal. Kringstad et al. (31) reported the direct-acting mutagenic activity of this compound for TA1535 to be 320 net revertants/nmol; earlier, Rosen et al. (32) reported the activity for TA100 to be about one-third of this value, 113 net revertants/nmol. Other compounds include 1,3-dichloroacetone, 1,1,2,3-tetrachloropropene, and numerous other polychlorinated propenes and propanones identified as SalmoneUa mutagens (27, 28), plus resin acids and phenolics detected as mutagenic compounds in a yeast bioassay (27). Similar products have been characterized as mutagenic following the chlorination of humic acids as a possible model for drinking water mutagens (33). These include compounds such as 3,3-dichloropropenal, dichloroacetonitrile, and l , l - d i c h l o r o - 2 - p r o p a n o n e (34). T o date, however, for humic acids, only a small fraction of the mutagenesis obtained following chlorination has been accounted for b y the identified compounds (33). Chian et al. (35) presented an analysis of the chemical products of humic acid chlorination in relation to those contained in residues of chlorinated water f r o m a surface source.

Mutagen Prevention, Destruction, and Removal The recognition that many of the numerous and unknown diverse mutagens present in drinking water are byproducts of chlorination has stimulated efforts at their prevention, destruction, or removal. Extensive


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studies on the chemistry of alternative disinfection procedures have been conducted. These have been directed at the use of other species of chlorine (C10 , chloramines) or at combination treatments that require little or no chlorine exposure. Separate studies parallel the observations made of the paper industry mutagens. Early in their work on this subject, Ander et al. (36) showed the mutagenicity of bleach process liquors was decreased in the presence of microsomal activation. In a subsequent study, this activity was shown to be alkali sensitive and was reduced when a lime decolorization precipitation step was included or when sulfite bleaching was included (37). Mutagenic residues of chlorinated drinking water were then shown to be alkali sensitive and to be susceptible to nucleophiles such as 4-nitrothiophenol. Current applications of these approaches are represented in this book.


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Removal of mutagens f r o m drinking water b y treatment with granular activated carbon ( G A C ) also has been examined b y several workers. Recent work in our laboratory confirmed observations of others (38, 39) that G A C can remove mutagens long after the level of total organic carbon ( T O C ) in the effluent has risen to a level that parallels that in the influent water (26). As w e l l as preexisting mutagens, compounds capable of yielding mutagens upon chlorination also are preferentially removed (26). However, apparently these eventually break through during prolonged use of G A C (38-40). Chemical and mutagenic analyses were conducted on residues extracted from used G A C taken f r o m the top, middle, and bottom of the G A C column; more polar compounds, including some mutagens, were detected in the residues f r o m the middle and bottom samples (40).

Risk Assessment A feature of this overview has been to highlight the progress of combined biological and chemical analysis in the characterization of waterborne organics. The major social impetus for such work has been unresolved questions on the biological side. What are the risks posed b y these organics, primarily to human health and also to the environment? The Salmonella test and similar microbial short-term tests are inadequate for a total assessment of genetic effects, not to mention additional important targets of toxicological or environmental damage. Nevertheless, these mutagenicity tests have served as practical tools both in the characterization of residue mixtures and in the isolation of their constituents. V i e w e d solely from the perspective of mutagenicity, source waters for drinking purposes can be divided into four categories:


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1. Waters contaminated with appreciable levels of stable, potent mutagens that require metabolic activation for their shortterm effects. These chemicals appear primarily, if not exclusively, to be of industrial or agricultural origin. 2. Water with relatively insignificant amounts of man-made chemicals but having appreciable T O C levels that yield mutagens as chlorination byproducts. These mutagenic mixtures generally do not require metabolic activation but rather are less active when assayed +S9. 3. Waters that contain appreciable T O C levels and that are also subject to repeated periodic contamination b y man-made mutagens. Included in this category are rivers and lakes affected b y industrial spills and discharges and b y agricultural and urban runoff. 4. Uncontaminated l o w - T O C water, such as is found in pristine wells. In setting priorities for the risk assessment of residue mixtures of waterborne organics, it seems prudent to pursue the source of these potent stable mutagens in category 1 waters. Although only a few have been examined to date, the mutagenic data of industrial municipal waste waters suggest major mutagens are being released f r o m point sources. A n individual compound isolated as a bacterial mutagen may prove nontoxic in higher organisms, and a c o m p o u n d not detected on the basis of its bacterial mutagenicity may still have significant toxicological effects. Nevertheless, the predictive value of Salmonella mutagenesis in the detection of potential carcinogens is high. Stable, microsomal-activation-dependent compounds are among those of greatest potential importance, and it appears they are amenable to isolation f r o m water residue organics for identification b y using coupled mutagenic-chemical fractionation. Considerable information of a general nature is available for uncontaminated water subject to the production of disinfection byproducts. The mutagens produced b y drinking water chlorination appear to be numerous, but they exist either at l o w levels or are of l o w potency. F o r both the unresolved mixtures and for the few mutagenic compounds thus far identified, activity is readily reduced or destroyed b y treatment with alkali or 4-nitrothiophenol and may be removed b y G A C treatment. F r o m water sources subject both to mutagen formation v i a disinfection and to periodic contamination b y toxic chemicals, experimental full-scale G A C treatment systems have provided mutagenfree water. The other remaining priority is the assessment of ground water. It has been a long time since we could complacently regard ground water as falling under category 4 in this list of water quality. Because of


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different circumstances, ground water may contain relatively high T O C levels, and in many cases is either contaminated or subject to imminent contamination b y toxic, man-made chemicals. Unfortunately, the specific circumstances vary greatly f r o m case to case so that general solutions may not be possible. Local application of chemical and bioas say analyses w i l l be important i n the evaluation and preservation of specific ground water resources.

Acknowledgments Research reported from this laboratory was supported i n part b y a grant to the University of Cincinnati b y the U . S . Environmental Protection Agency ( U S E P A ) . This chapter has not been subjected to U S E P A review and therefore does not reflect the views of that agency and no official endorsement should be inferred.

Literature Cited 1a. Neal, R. A. Environ. Sci. Technol. 1983, 17, 113A. 1b. National Research Council Drinking Water and Health; National Academy of Sciences: Washington, DC, 1977; pp 4-5. 2. Middleton, F. M.; Pettit, H. H.; Rosen, A. A. Proc. 17th Ind. Waste Conf. Purdue Univ. Ext. Serv. 1962, 122, 454. 3. Hueper, W. C.; Payne, W. W. Am. J. Clin. Pathol. 1963, 39, 475. 4. Doll, R. Proc. R. Soc. Med. 1972, 65, 49. 5. Berg, J. W. In Origins of Human Cancer; Hiatt, H. H.; Watson, J. D.; Winsten, J. Α., Eds.; Cold Spring Harbor Laboratory: Cold Spring Harbor, NY, 1977; pp 15-19. 6. Rook, J. J. Water Treat. Exam. 1974, 23, 234. 7. Bellar, Τ. Α.; Lichtenberg, J. J. J. Am. Water Works Assoc. 1974, 66, 739. 8. U.S. Environmental Protection Agency Report 906/10-74-002; U.S. Govern ment Printing Office: Washington, DC, November 1974. 9. Kool, H. J.; van Kreijl, C. F.; Zoeteman, B. C. J. CRC Crit. Rev. Environ. Control 1982, 12, 307-359. 10. Ames, Β. N.; McCann, J.; Yamasaki, E. Mutat. Res. 1975, 31, 347. 11. Maron, D. M.; Ames, Β. N. Mutat. Res. 1983, 113, 173. 12. Rosenkranz, H. S.; Speck, W. T. Biochem. Biophys. Res. Commun. 1975, 66, 520. 13. Rosenkranz, H. S.; McCoy, E. C.; Hermelstein, R.; Speck, W. T. Mutat. Res. 1981, 91, 103. 14. Loper, J. C. Mutat. Res. 1980, 76, 241, 15. Loper, J. C.; Tabor, M. W. Environ. Sci. Res. 1983, 27, 165. 16. Loper, J. C.; Tabor, M. W.; Miles, S. K. In Water Chlorination: Environmen tal Impact and Health Effects; Jolley, R. L.; Brungs, W. Α.; Cotruvo, J. Α.; Cumming, R. B.; Matrice, J. S.; Jacobs, V. Α., Eds.; Ann Arbor Science: Ann Arbor, MI, 1983; Vol. 4, pp 1199-1210. 17. Tabor, M. W.; Loper, J. C. Int. J. Environ. Anal. Chem. 1980, 8, 197. 18. Loper, J. C.; Tabor, M. W. Environ. Sci. Res. 1981, 22, 155. 19. Tabor, M. W. Environ. Sci. Technol. 1983, 17, 324.



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20. Heartlein, M. W.; DeMarini, D. M.; Kutz, A. J.; Means, J. C.; Plewa, M. J.; Broekman, H. E. Environ. Mutagen. 1981, 3, 519. 21. Zhou, S. W.; Xu, F. D.; Liu, J. L., unpublished data. 22. Maruoka, S.; Yamanaka, S. Environ. Sci. Technol. 1983, 17, 177. 23. Rappaport, S. M.; Richard, M. G.; Hollstein, M. C.; Talcott, R. E. Environ. Sci. Technol. 1979,13, 957. 24. Hopke, P. K.; Plewa, M. J.; Stapleton, P. L.; Weaver, D. L. Environ. Sci. Technol. 1984, 18, 909. 25. Tabor, M. W.; Loper, J. C. Organic Pollutants in Water: Sampling and Analysis; Suffet, I. H.; Malaiyandi, M., Eds.; Advances in Chemistry 214; American Chemical Society: Washington, DC, 1986; Chapter 33. 26. Loper, J. C.; Tabor, M. W.; Rosenblum, L.; DeMarco, J. Environ. Sci. Technol. 1985, 19, 333. 27. Douglas, G. R.; Nestmann, E. R.; McKague, A. B.; Kamra, O. P.; Lee, E. G.-H.; Ellenton, J. Α.; Bell, R.; Kowbel, D.; Liu, V.; Pooley, J. Environ. Sci. Res. 1983, 27, 431. 28. Kringstad, K. P.; Lindstrom, K. Environ. Sci. Technol. 1984,18,236A. 29. Holmbom, B. R.; Voss, R. H.; Mortimer, R. D.; Wong, A. Tappi 1981, 64(3), 172. 30. Holmbom, B. R.; Voss, R. H.; Mortimer, R. D.; Wong, A. Environ. Sci. Technol. 1984, 18, 333. 31. Kringstad, K. P.; Ljungquist, P. O.; deSousa, F.; Stromberg, L. M. Environ. Sci. Technol. 1981, 15, 562. 32. Rosen, I. D.; Segall, Y.; Casida, J. E. Mutat. Res. 1980, 78, 113. 33. Meier, J. R.; Lingg, R. D.; Bull, R. J. Mutat. Res. 1983, 118, 25. 34. Coleman, W. E.; Munch, J. W.; Kaylor, W. H.; Streicher, R. P.; Ringhand, H. P.; Meier, J. R. Environ. Sci. Technol. 1984, 18, 674. 35. Chian, E. S. K.; Giabbai, M. F.; Kim, J. S.; Reuter, J. H.; Kopfler, F. C. In Organic Pollutants in Water: Sampling and Analysis; Suffet, I. H.; Malaiyandi, M., Eds.; Advances in Chemistry 214; American Chemical Society: Washington, DC, 1986; Chapter 9. 36. Ander, P.; Eriksson, K.-E.; Kolar, M.-C.; Kringstad, K. Sven. Papperstidn. 1977, 80, 454. 37. Eriksson, K.-E.; Kolar, M.-C.; Kringstad, K. Sven. Papperstidn. 1979, 82, 95. 38. Monarca, S.; Meier, J. R.; Bull, R. J. Water Res. 1983, 17, 1015. 39. Kool, H. J.; van Kreijl, C. F. Water Res. 1984, 18, 1011. 40. Loper, J. C.; Tabor, M. W.; Rosenblum, L.; DeMarco, J. In Water Chlorina tion: Environmental Impact and Health Effects; Jolley, R. L.; Bull, R. J.; Davis, W. P.; Katz, S.; Roberts, M. H., Jr.; Jacobs, V. A. Eds.; Lewis: Chelsea, MI, 1985; pp 1329-1339. R E C E I V E D for review August 14, 1985. A C C E P T E D December 17, 1985.


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