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6 Adsorbent Accumulation of Organic Pollutants for

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Bioassays BONITA A. GLATZ Food Technology Department, Iowa State University, Ames, IA 50011 COLIN D. CHRISWELL and GREGOR A. JUNK Ames Laboratory, USDOE, Iowa State University, Ames, IA 50011

The attention given to organic compounds in drinking water has shifted from their role in causing undesirable odors and tastes to their potential effects on human health. These effects cannot be ascertained using data from traditional water quality parameters such as chemical oxygen demand (1), biological oxygen demand(2),total organic carbon content (3), fluorescence (4), ultraviolet absorbance (5) and carbon adsorbable material (6). Some help for the evaluation of health effects has been provided by the progress made during the past decade in the identificaton of individual organic components present in drinking water. The number of identified compounds has grown to over 700 according to a recent compilation (7). Bioassay results with some of these compounds have shown them to be toxic or potentially carcinogenic. However, all compounds identified in drinking water have not been tested thoroughly and some possibly detrimental substances have undoubtedly eluded even the most complete identification efforts. An alternative to identifying organic chemicals in water and then determining their biological activity is the direct bioassay of mixtures accumulated from drinking water. This approach can provide part of the data for the preliminary assessment of health risks. The data can also be used to select those water sources on which the most strenuous identification efforts should be directed. Mixtures exhibiting considerable activity can be separated into chemical classes and bioassays can then be used to identify the active fractions. Ultimately the individual culprit chemicals would be identified. In this manner, efforts are directed towards the more detrimental components. A forerunner to this approach was published in 1963 when organic chemicals accumulated from water by carbon adsorption and soxhlet extraction were found to cause tumors in laboratory animals W. However, the two-year period for completion of the animal tests of the accumulated mixture was a serious drawback. 9 _ 1 4

Several short-term bioassay procedures i J have been developed recently which are applicable to detecting mutagenic and potential carcinogenic activity of organic substances. The SaimonelJa/mammalian microsome assay or Ames Test i * 1°) has been the most frequently applied and its efficacy has been well documented. This assay has also been applied to complex mixtures (19-22) to reduce greatly the time 5 -

0-8412-0480-2/79/47-094-091$05.00/0 © 1979 American Chemical Society Schuetzle; Monitoring Toxic Substances ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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MONITORING TOXIC SUBSTANCES

and effort required for a preliminary determination of adverse health effects. The Salmonella assay is rapid and inexpensive compared to whole animal testing and a close correlation has been demonstrated between mutagenic activity indicated by this procedure and carcinogenic activity (23,24j Preliminary results with mixtures isolated from drinking water have also been described ( > 26). t

25

The organic material accumulated from water and used for bioassays should be representative of that originally present in the water. A s much unaltered material as possible should be accumulated. In this sense, the desire for effective accumulation is the same whether the organic mixture is to be separated for identification purposes or used directly for bioassays. The primary intent of this report is to describe some accumulation techniques which are applicable to bioassay requirements and to pre sent preliminary results of bioassays performed on organic mixtures accumulated from drinking water. Accumulation Techniques Accumulation as used in this report refers to the concentration of organic constit uents within the water matrix or to their removal from water and their recovery in an unaltered form either in another matrix or as a neat mixture. Some traditional tech niques for accumulating organic compounds from water include distillation, solvent extraction, freeze drying, and freeze concentration. Other more recent techniques include purge and trap ( ) and closed loop purge and trap ( ). A l l these techniques are inconvenient and/or costly when large volumes of water must be processed to accumulate sufficient quantities of material for bioassay. These large volumes of water are better accommodated using reverse osmosis or solid sorbents such as activated carbon and a variety of synthetic polymers. 27

2β

Reverse osmosis procedures concentrate over 90% of the total organic material present in water into an aqueous brine ( λ A problem has been the efficient transfer of the organic components to a solvent suitable for the bioassays (3°). Another problem is the loss of chemicals having molecular weights below 200-400. 2 9

Solid sorbents such as those identified in Table 1, have also been reported to be effective for accumulation of organic materials. The granular activated carbons have been most popular and they are normally used for removing organic impurities from drinking water and wastewater. They can also be used in analytical schemes for measuring organic contaminants. One of the first such procedures involved adsorbing contaminants on activated carbon, desorbing the organic compounds by soxhlet ex traction, evaporating the solvent to dryness, and weighing the residue More defi nitive results can be obtained by applying gas chromatography-mass spectrometry techniques for separation and identification of the components in the residue. The Environmental Protection Agency used this approach during the National Organics Reconnaissance Survey P2j. The synthetic polymer sorbents are useful alternatives to the activated carbons for accumulating organic compounds (33-36). \ 1977 it was demonstrated that Amberlite X A D - 2 resin was more efficient for accumulating many organic materials from water than was Filtrasorb 300 activated carbon (FS-300) P^J. In another study (38) greater amounts of gas chromatographable organic compounds were accumulated from water using the polystyrene-divinylbenzene copolymers, Amberlite X A D - 2 and X A D - 4 and Duolite L-863, than were accumulated using activated carbons, acrylic ester based resins, a carbonaceous resin, phenol-formaldehyde resins, and weak-base anion ex change resins. The composited plots of these data in Figure 1 illustrate clearly the n

Schuetzle; Monitoring Toxic Substances ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

GLATZ ET AL.

Adsorbent

Accumulation

of

93

Pollutants

TABLE I. Identification of Solid Sorbents Sorbent

Type

Supplier

XAD-2

Polystyrenedivinylbenzene resin

Rohm and Haas


Diamond Shamrock


Rohm and Haas

XAD-7

Acrylic ester resin

Rohm and Haas

XAD-8

Acrylic ester resin

Rohm and Haas

S-761

Phenolformaldehyde resin

Diamond Shamrock

Anion exchange resin (weak-base)

Diamond Shamrock


Diamond Shamrock


Diamond Shamrock

Granular activated carbon

ICI-USA


Calgon

Granular Activated carbon

Westvaco


Westvaco


National Carbon


National Carbon

Carbonaceous resin

Rohm and Haas

L-863 XAD-4

S-37 A-7 ES-561

DARCO FS-300 WVB WVG G-216 G-107

XE-340



0

20 4 0 6 0 8 0 J00

140

180

220

% ACCUMULATION Figure 1. Comparison of gas chromatographable material accumulated by 16 solid sorbents at four water plants (see Table I for sorbent identification). Accumulation normalized to XAD-2.


6.

GLATZ ET AL.

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Accumulation

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superior accumulation by the styrenedivinylbenzene based resins. In the absence of any accumulation procedure which is 100% effective, the superiority of these resins suggests that they are a suitable, and probably the most desirable, starting point for accumulating organic components from water for bioassay purposes. Experimental One of the more efficient solid sorbents, Amberlite X A D - 2 resin, has been used to accumulate organic materials from fourteen raw and finished waters (26J. Accumulations were performed in water utility facilities using 1/2" χ 6" columns filled with the resin. Plant streams were sampled directly without any prefiltration or other treatment. Water was passed through the resin-filled columns at a flow rate of approximately 100 m l / m i n until a total volume of 200 L had been sampled. After sampling, each resin bed was eluted with 100 ml of diethyl ether, residual water dissolved in the ether was frozen out, and the ether was decanted into a concentration flask where the volume was reduced to 1.00 ml by distillation. Aliquots of samples accumulated at monthly intervals during the winter of 1976-77 were composited for use in performing bioassays. Dimethyl sulfoxide was added to each composite and the ether removed by free evaporation. The final volume of the dimethyl sulfoxide con centrates was adjusted so that each 10-μ1 aliquot would contain organic materials accumulated from 15 L of water. These concentrates were assayed for mutagenic activity using the spot test variation of the Ames test. Each sample was tested in replicate with each of the strains, TA98, TA100, TA1535, TA1537 and TA1538. In addition, assays were performed in duplicate with the same strains with the microsonal fraction of Aroclor 1254 activated rat liver added to each test plate. Each set of assays was accompanied by positive controls (known mutagens) and negative controls (solvent and sorbent blanks). The bioassay results are given in Table II. Mutagenic activity was detected in eleven of the finished and six of the corresponding raw waters. These results indicate mutagenic agents can be accumulated from water using X A D - 2 resin. Additional investigations have been performed to compare X A D - 2 with other sorbents. Organic contaminants were accumulated from the drinking waters of four cen tral Iowa communities using sixteen different test sorbents. The accumulated materi als were assayed for mutagenic activity and these results are listed in Table III. Three of the four waters showed activity with the most positive results obtained when the polystyrene-divinylbenzenes, X A D - 2 , L-863 and X A D - 4 , were used. Fewer positive results were obtained using the acrylic ester based resins, X A D - 7 and X A D - 8 . Only marginal activity was observed when the phenol-formaldehyde polymer, S-761, was used. Use of all the other solid sorbents yielded either no or highly limited evidence of mutagenic activity. Solid sorbent procedures are currently being compared to reverse osmosis tech niques I ) for accumulating organic materials for bioassays but results are not yet available. The apparent and unexplained correlation between the accumulation of mutagenic and gas chromatographable material from water is also being investigated. 25

Conclusions Polystyrene-divinylbenzene resins are the most efficient solid sorbents tested for the accumulation of mutagenic agents from water. This greater efficiency recommends their use in bioassay programs for the preliminary assessment of health risks associ-


95


96

TABLE II. Mutagenic Activity in the Organic Material Accumulated from 14 Raw and 14 Finished Water Supplies. 1

2

Mutagenic Activity Water Supply 1 2 3 4 5 Ό

7 8

Raw

-0

0 — — 0

Without S-9 Finished

+ + + 0

+ 0 0

Raw

With S-9 Finished

-+ -—

0

+ +

-0

—

—

0

0

+

+ + +

Q

y 10 11 12 13 14

+ +

-+

+ + +

+

0

-+

+

^Sample size for each test adjusted to represent the accumulated components from 15 L of water. Salmonella typhimurium strains, TA98, TA100, TA1535, TA1537 and T A 1538 were used. Each sample was tested with and without the addition of microsomal fraction of activated rat liver designated S-9. 2

Mutagenic activity was recorded if at least one strain showed an increased reversion frequency in response to the test sample. (—) = no activity detected; (+) = at least 2x the number of colonies appeared as on a control plate situated i n a ring around the sample disc; (0) = an increased colony count was noted, but not 2x the control value, or not i n a ring.


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GLATZ ET AL.

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Accumulation

of

97

Pollutants

T A B L E III. Mutagenic Activity i n Organic Material Accumulted from 4 Water Supplies Using 16 Different Sorbents. 1

Sorbent

A

XAD-2 L-863 XAD-4

+ + + + +

XAD-7 XAD-8 S-761 S-37 A-7 ES-561 DARCO FS-300 WVB WVG G-216 G-107 XE-340

Mutagenic Activity-Site Β

+ + +

0

0 0 0

—

—

-

-

— 0

-

--

—

+

2

c

D

+ + + +

—

0 0

-

-0

—

—

-

ISample size for each test adjusted to represent the accumulated components from 10 L of finished water. Salmonella typhimurium strains, TA98, TA100, TA1535, TA1537 and TA1538, were used. 2

S e e explanation of activity code in Table II footnote.


98


ated with ingestion of various waters. These sorbent procedures are readily upgraded to the accumulation of the larger amounts of material necessary for whole animal tests when these are deemed necessary. If the health assessments are followed by the chemical characterization of the organic material then specialized, more efficient accumulation procedures could be developed. It is suspected that chlorination may play a role in the increased occurrence of activity for the finished water relative to the raw waters. This suspicion is based largely on the known production of trihalomethanes and the suspected production of other halogen containing compounds during chlorination. Any trihalomethanes accu mulated by the resin procedure are largely lost during the subsequent distillation and evaporation steps, but remaining chlorinated products may be responsible for the observed mutagenic activity. However, treatment chemicals and treatment chemical impurities may not be ruled out as the responsible agents based on current results. Furthermore, treatment may have removed some substances that inhibit the muta genic response of materials present in raw water. These explanations for the increased occurrence of mutagenic activity in finished water are clearly speculative and require more definitive studies for verification. No doubt, these studies will include the identi fication of the individual chemical(s) responsible for the mutagenic activity.

Acknowledgements Primary fundings for these investigations were provided by the American Water Works Research Foundation and U.S. Environmental Protection Agency. The work was supported by the U.S. Department of Energy, Division of Environmental and Biomedical Research. The co-operation and advice of James S. Fritz, Harry J. Svec, Ron Webb, O. Thomas Love, Michael Taras and the various representatives of the fourteen water utilities are gratefully acknowledged.

Literature Cited

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8. HUEPER, W. C. and PAYNE, W. W. "Carcinogenic Effects of Adsorbates of Raw and Finished Water Supplies." Am. J.Clin.Pathol, 39, 475 (1963). 9. CHU, Ε. H. Y. "Induction and Analysis of Gene Mutations in Mammalian Cells in Culture", in HOLLAENDER, A. (ed.), Chemical Mutagens, Principles and M ods for Their Detection, Vol. 2, p. 411, Plenum Press, New York, NY, 1971. 10. ABRAHAMSON, S. and LEWIS, Ε. B. "The Detection of Mutations in Drosophila melanogaster", ibid., p. 461. 11. UNDERBRINK, A. G., SCHAIRER, L. A. and SPARROW, A. H. "Tradescantia Stamen Hairs: A Radiobiological Test System Applicable to Chemical Mutagene sis", ibid., Vol. 3, p. 171. 12. ZIMMERMAN, F. K. "Detection of Genetically Active Chemicals Using Various Yeast Systems", ibid., p. 209. 13. MOREAU, P. and DEVORET, R. "Potential Carcinogens Tested by Inductions and Mutageneisis of Prophage λ in Escherichia coli K12", in HIATT, Η. H., WATSON, J. D. and WINSTEN, J. A. (eds.), Origins of Human Cancer, p. 1451, Cold Spri Harbor Laboratory Publ., Cold Springs Harbor, NY, 1977. 14. MISHRA, Ν. K. and DiMAYORCA, G. " In Vitro Malignant Transformation of Cells by Chemical Carcinogens." Biochim. Biophys. Acta, 355, 205 (1974). 15. AMES, Β. N., DURSTON. W. E., YAMASAKI, E. and LEE, F. D. "Carcinogens are Mutagens: A Simple Test System Combining Liver Homogenates for Activation and Bacteria for Detection." Proc. Nat. Acad. Sci. U.S.A., 70, 2281 (1973). 16. AMES, Β. N., LEE, F. D. and DURSTON, W. E. "An Improved Bacterial Test System for the Detection and Classification of Mutagens and Carcinogens." ibid., 782 (1973). 17. McCANN, J. and AMES, Β. N. "The Salmonella/Microsome Mutagenicity Test: Predictive Value for Animal Carcinogenicity", in HIATT, Η. H., WATSON, J. D. and WINSTEN, J. A. (eds.), Origins of Human Cancer, p. 1431, Cold Springs Harbor Laboratory Publ., Cold Springs Harbor, NY, 1977. 18. AMES, Β. N., McCANN, J. and YAMASAKI, E. "Methods for Detecting Carcino gens and Mutagens with the Salmonella/Mammalian-Microsome Mutagenicity Test." Mutat. Res., 31, 347 (1975). 19. EPLER, J. L., CLARK, B. R., HO, C. H., GUERIN, M. R. and RAO, T. K. "Shortterm Bioassay of Complex Organic Mixtures. Part II, Mutagenicity Testing." Pre sented at the Symposium on Application of Short-term Bioassays in the Fractiona tion and Analysis of Complex Environmental Mixtures, Williamsburg, VA, Feb., 1978. 20. COMMONER, B., VITHAYATHIL, A. J. and DOLARA, P. "Mutagenic Analysis of Complex Samples of Air Particulates, Aqueous Effluents, and Foods." ibid. 21. FISHER, G. L. and CHRISP, C. E. "Physical and Biological Studies of Coal Fly Ash." ibid. 22. PELLIZZARI, E. D. and LITTLE, L. W. "Integrating Microbiological and Chemical Testing into the Screening of Air Samples for Potential Mutagenicity." ibid.


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23. McCANN, J., CHOI, E., YAMASAKI, E. and AMES, Β. N. "Detection of Carcino gens as Mutagens in the Salmonella/Microsome Test: Assay of 300 Chem Proc. Nat. Acad. Sci. U.S.A., 72, 5135 (1975). 24. McCANN, J. and AMES, Β. N. "Detection of Carcinogens as Mutagens in the Salmonella/Microsome Test: Assay of 300 Chemicals, Discussion," ibid., 7 (1976). 25. GLATZ, Β. Α., CHRISWELL, C. D., ARGUELLO, M. D., SVEC, H. J., FRITZ, J. S., GRIMM, S. M. and THOMSON, M. A. "Examination of Drinking Water for Muta genic Activity." J. Am. Water Works Assoc., in press (1978). 26. LOPER, J. C. AND LANG, D. R. "Mutagenic, Carcinogenic and Toxic Effects of Residual Organics in Drinking Water." Presented at the Symposium on Applica tion of Short-term Bioassays in the Fractionation and Analysis of Complex Envi ronmental Mixtures, Williamsburg, VA, Feb., 1978. 27. BELLAR, Τ. Α., LICHTENBERG, J. J. and KRONER, R. C. "The Occurrence of Organohalides in Chlorinated Drinking Water." J. Am. Water Works Assoc., 66, 703 (1974). 28. GROB, K. and ZÜRCHER, F. "Stripping of Trace Organic Substances From Water: Equipment and Procedures."J.Chromatogr., 117, 285 (1976). 29. KOPFLER, F. C. Personal Communications. Jan., 1978. 30. LANG, D. R. Personal Communications. Feb., 1978. 31. Standard Methods for the Examination of Water and W American Public Health Assoc., New York, NY, 1971. 32. KOPFLER, F.C.,MELTON, R. G., LINGG, R. D. and COLEMAN, W. E. "GC-MS Determinations of Volatiles for (NORS) of Drinking Water", in KEITH, L. H. (ed.), Identification and Analysis of Organic Pollutants in W Science, Ann Arbor, MI, 1976. 33. BURNHAM, A. K., CALDER, G. V., FRITZ, J. S., JUNK, G. Α., SVEC, H. J. and WILLIS, R. "Identification and Estimation of Neutral Organic Contaminants in Potable Water." Anal. Chem., 44, 139 (1972). 34. JUNK, G. Α., RICHARD, J. J., GRIESER, M. D., WITIAK, D., WITIAK, J. L., ARGUELLO, M. D., VICK, R., SVEC, H. J., FRITZ, J. S. and CALDER, G. V. "Use of Macroreticular Resins in the Analysis of Water for Trace Organic Contami nants."J.Chromatogr., 99, 745 (1974). 35. RICHARD, J. J. and FRITZ, J. S. "Adsorption of Chlorinated Pesticides from River Water with XAD-2 Resin." Talanta, 21, 91 (1974). 36. BURNHAM, A. K., CALDER, G. V., FRITZ, J. S., JUNK, G. Α., SVEC, H. J. and VICK, R. "Trace Organics in Water: Their Isolation and Identification." J. Am. Water Works Assoc., 65, 722 (1973). 37. CHRISWELL, C. D., ERICSON, R. L., JUNK, G. Α., LEE, K. W., FRITZ, J. S. and SVEC, H. J. "Comparison of Macroreticular Resin and Activated Carbon as Sor bents." ibid., 69, 669 (1977). 38. CHRISWELL, C. D., FRITZ, J. S. and SVEC, H. J. "Evaluation of Sorbents as Organic Compound Accumulators." Presented at the American Water Works As sociation Water Quality Technology Conference, Kansas City, MO, Dec, 1977. RECEIVED November 17, 1978. Schuetzle; Monitoring Toxic Substances ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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