velopment, EPA, Research Triangle Park, N.C., EPA Report 650/2-75-014. (10) Hurn, R. W., Cox, F., Allsup, J. R., Office of Research and Development, EPA, Research Triangle Park, N.C., EPA Report 600/2-76-026. (11) Zweidinger, R. B., Tejada, S. B., Sigsby, J. E., Bradow, R. L., “Ion Chromatographic Analysis of Environmental Pollutants,” Sawicki, E., Mulik, J.,Wittgenstein, E., Eds., Ann Arbor Science Publishers, Ann Arbor. Mich., 1978. (12) Richards, W., Rockwell International Air Monitoring Center, private communication, 1977. (13) Dahlgren, G., Anal. Chem., 36,596 (1964). (14) Dunn, S. R., Simenhoff,M. L., Wesson, L. G., Jr., Anal. Chem., 4x,41 (1976). (15) Tetlow, J . A,, Wilson, A. L., Anal)st, 89,453 (1964). (16) Dietzmann, H. E., Smith, L. R., Parness, M. A,, Fanick, E. R., Environmental Sciences Research Laboratory, EPA, Research Triangle Park, N.C., EPA Report 600/2-79-017. (17) DiCorcia, A,; Samperi, R., Anal. Chem., 48,977 (1974). (18) Wartheson, J. J., Scanlan, R. A., Bills, D. P., Libbey, M., J . Agric. Food Chern., 23, 298 (1975). (19) Cadle, S.H., Chock, D. P., Monson, P. R., Heuss, J. M., J. Air Poiiut. Control Assoc., 27, 33 (1977). (20) Shapley. D., Science, 191, 268 (1976).
10) could not detect amines in the exhaust of noncatalyst cars, we feel it is unlikely that any of the standard gasoline cars on the road today are emitting significant quantities of amines.
Literature Cited (1) Fine, D. H., Rounbehler, D. P., Belcher, N. M., Epstein, S. S.,
Science, 192, 1328 (1976). (2) Pellizzari, E. D., Environmental Sciences Research Laboratory, EPA, Research Triangle Park, N.C., EPA Report 600/7-77-055. ( 3 ) Urban, C. H., Garbe, R. J.,Paper 790696 presented at the Society of Automotive Engineers Passenger Car Meeting, Dearborn, Mich., June 1979. (4) General Motors Advanced Emission Control System Development Progress, submitted to the Environmental Protection Agency, Dec 15, 1976, pp VIII-24. (5) Hare, C. T.,Banes, T. M., Paper 770719 presented at 1977 Society of Automotive Engineers, Off-Highway Vehicle Meeting, MECCA. Milwaukee, Wis., Sept 1977. (6)Hanst, P . L., Spence, J. W., Miller, M., Enciron. Sci. Techno/., 11,403 (1977). ( 7 ) Glasson, W. A,. General Motors Research Puhlication GMR-81. Sept 1977. ( 8 ) Grosjean, P., Cauwenberghe, K., Schmid, J., Pitts, J., paper presented at the 4th Joint Conference on Sensing Environmental Pollutants, New Orleans, La., Nov 1977. (9) Hurn, R. W., Allsup, J. R., Cox, F., Office of Research and De-
Receiued for reczeu April 9, 1979. Accepted March 4 , 1980
Transformation of the Mouse Clonal Cell Line R846-DP8 by Mississippi River, Raw, and Finished Water Samples from Southeastern Louisiana William Pelon’”, Thomas W. Beasley, and David E. Lesley Department of Microbiology, Louisiana State University Medical Center, 1100 Florida Avenue, New Orleans, La. 70119
Cultures of the mouse cell line R846-DP8 were exposed to 303 Mississippi River, raw, and finished water samples, collected in southeastern Louisiana between 1974 and 1976. Nineteen samples (6%)induced morphological transformation of this cell strain. Transformation was seen with 7 of 118 (6%) Mississippi River water samples, 7 of 70 (10%) raw water samples, and 5 of 115 (4%) finished water samples. The Mississippi River is the raw water source of drinking water for 1.5 million people living in southeastern Louisiana. Investigations have reported the presence of numerous organic compounds, including some carcinogens, in samples of Mississippi River and New Orleans finished water (1-3). Despite their occurrence. there was no evidence that sufficient concentrations were present to affect in any manner those consuming such water. Studies were undertaken to ascertain if unconcentrated river, raw, and finished water samples could induce genetic changes in biological test systems. We have reported that numerous water samples caused statistically significant rates of reversion to histidine independence among histidine-dependent mutant strains of Salmonella typhimurium (4, 5 ) . Many of the water samples reverted the bacterial strains only when liver enzyme fractions were present. This suggested the presence of compounds which had to be metabolically activated to be mutagenic. Most carcinogenic compounds have a similar requirement. Studies of the water samples also were conducted with a mammalian cell system. The results of this phase are presented herein. Address correspondence to: Microbiology, Louisiana State University Medical Center, 1542 Tulane Ave., New Orleans, La. 70112. 0013-936X/80/0914-0723$01.00/0
@
Material and Methods Water Samples. The collection of Mississippi River, raw, and finished water has been described ( 4 , 5 ) .The collection sites are listed in Table IV. Mississippi River water was collected subsurface using a conventional brass water sampler. Raw and finished water samples were collected simultaneously at the various water purification plants and then transported to the laboratory in wet ice. A total of 118 river water, 70 raw water, and 115 finished water samples were collected over an 18-month period. When we received the samples a t the laboratory, they were clarified (when necessary) and sterilized by positive pressure filtration using water-pumped nitrogen gas. Sterile water samples were stored frozen in glass bottles at -20 “C in the dark until assays were performed. The time between collection and assay did not exceed 4 months. Mammalian Cell Used. A NIH Swiss mouse embryo cell line designated as R846-DP8 was received through the courtesy of Dr. John S.Rhim (Microbiological Associates, Bethesda, Md.). This cell line, originating from a clone, contained the murine leukemia virus gs antigen within its genome, and has been employed in cell transformation studies (6). Cell stocks were passaged weekly, using Eagle’s minimal essential medium (MEM) containing 0.22% NaHC03, 10% agamma calf serum, arginine (126 mg/L), penicillin (100 U/ mL), streptomycin (100 pg/mL), polymyxin (100 U/mL), kanamycin (100 pg/mL), and mycostatin ( 2 5 U/mL) as the outgrowth medium. In this study, passage levels ranged from 24 to 34. Spontaneous transformation as evidenced by morphological alteration was not observed in the cell stocks. Assay Method. Trypsin-dispersed R846-DP8 cells were suspended in the growth and maintenance medium at a concentration of 75 000 cells/mL, and distributed in 4.0-mL quantities either into disposable petri dishes (Falcon 3002) or into disposable flasks (Falcon 3013).
1980 American Chemical Society
Volume 14, Number 6, June 1980
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Table 1. Composition of the Culture Medium Prepared with River, Raw, and Finished Water Samples components
mL
water samplea Earle's balanced salt solution (1OX) amino acid mixture, EBMb (1OOX) vitamin mixture, EBM (1OOX) glutamine (200 mM) arginine (1OOX)c NaHC03 (7.5%) agamma calf serum penicillin (100 000 U/mL) streptomycin (100 000 pg/mL) polymyxin (100 000 U/mL) mycostatin (10 000 U/mL) kanamycin (10000 pg/mL) a
72.45 10.0 1.o 1.o 1.o 1.o 2.9 10.0 0.1 0.1 0.1 0.1 12.64
Table II. Assay of Water Samples with R846-DP8 Cells
-1
0 2 5
6 7
9 12 13 14
63
regimen employed
R846-DP8 cells planted in petri dishes or flasks outgrowth medium replaced with 8 mL of the water sample containing medium water sample medium replaced water sample medium replaced water sample medium replaced cells dispersed, 10% of cell population subcultured in each of two flasks or Petri dishes in outgrowth medium outgrowth medium replaced outgrowth medium replaced outgrowth medium replaced one of the two cultures subdivided as on day 7; the remaining culture is fixed and stained regimen from day 7 to day 14 maintained for a total of 7 weeks
All cultures were incubated at 37 "C in a COz incubator. After 24 h, the outgrowth medium was decanted and the attached cells were rinsed with phosphate-buffered saline (PBS), pH 7.4. Eight milliliters of Eagle's basal medium (EBM) containing approximately 72% of a given water sample (Table I) was added, and the cultures were reincubated. Each culture was exposed to a total of 32 mL of a water sample containing medium over a period of 7 days; then 10% of the cell population was passaged weekly in each of two culture dishes or flasks in the presence of the outgrowth medium for an additional 7 weeks (Table 11).At 7-day intervals beginning on day 14, both cultures were examined microscopically. One culture was passaged as described (Table 11), while the remaining culture was fixed with methyl alcohol and stained with Giemsa. Negative control cultures propagated in EBM medium prepared with sterile deionized water were included with each assay. The medium for positive control cultures was prepared by the addition of 2-fluorenamine (100 pg/mL) to the negative control medium. The regimen for both controls was identical with that of the water sample exposed cultures. We recognize that the protocol described above has shortcomings. It would have been preferable if all progeny of cells originally exposed to each of the water samples, as well as those of the control cultures, had been followed over the 8week period of observation. Unfortunately, R846-DP8 cells are prolific. Because of this, only 10% of the cell population 724
Environmental Science & Technology
no. tested
no. causing cell transformation
river raw finished total
118
7 (6%) 7 (10%) 5 (4%) 19 ( 6 % )
70 115 303
Table IV. Water Samples from Various Collection Sites Causing Transformation of R846-DP8 Cells
0.25
Sterilized by positive pressure filtration. Eagle's basal medium.
water sample type
a A total of 50 negative control cultures, each propagated over 8 weeks, showed no evidence of cell transformation.
g/L.
day of incubation
Table 111. Transformation of R846-DP8 Cells by Water Samples Collected in Southeastern Louisiana (August 1974-May 1976) a
sampling site
river
St. Francisville Baton Rouge Plaquemine Lutcher Luling Jefferson New Orleans Belle Chasse Pointe a la Hache Port Sulfur
01198 NC 2/18 1/18 2/19 NC NC 1/22 1122 NC
a
sample types raw
NC NC NC NC 1/15 1/14 2/12 1/15 NC 2/14
finished
0113 0113 NC 1/12 2/15 0116 1/15 1/16 NC 0115
Number of samples causing cell transformation/numberof samples tested.
NC = not collected.
could be passaged per flask to minimize overcrowding of the subculture before the end of the 7-day incubation period. Thus, to follow all cell progeny, each flask under observation would have had to have been subcultured into a total of 10 flasks. If this ratio were maintained, all available incubator space would have soon been occupied by the descendants of cells originally exposed to a single water sample. We had 303 water samples to investigate within a limited period of time. Criteria for Cell Transformation. In their studies, Rhim et al. described transformed cells appearing as randomly oriented, spindle-shaped cells piled up like jackstraws upon a uniform monolayer of cells (7, 8). Although we observed similar phenomena, we were unable to establish with certainty whether these resulted from cell transformation, were clumps of cells which had not been trypsin dispersed, or were fragments of detached cells which had reattached and from which new cells were migrating. Accordingly, cell transformation was considered to have occurred when virtually all of the cells in one or in successive cultures exhibited an abnormal growth pattern and an abnormal cell morphology when compared with control cell cultures (Figure 1).Since such cell changes were best seen in fixed and stained cultures, karyotyping or in vivo tumor production was not attempted.
Results R846-DP8 cells were exposed to a total of 303 Mississippi River, raw, and finished water samples collected from various sites in southeastern Louisiana between August 1974 and May 1976. A total of 19 water samples (6%)caused cell transformation (Table 111).Seven of 118 (6%)Mississippi River water samples, 7 of 70 (10%) raw water, and 5 of 115 (4%)finished water samples showed cell-transforming capabilities. The distribution of the different types of water samples from various sites causing cell transformation appeared to be random (Table IV). There was no concentration of cell-transforming water samples from a particular site regardless of sample type.
Figure 1. (A) Normal R846-DP8 cells. (B) R846-DP8 cells transformed following exposure to finished water
collected at Luling, La., on June 13,
1975. Giemsa stained
While no spontaneous transformation was observed among the progeny of the negative controls, transformation was seen among subcultures of cells initially exposed to 2-fluorenamine at 5 weeks or more of incubation. Discussion Our objective was to answer the question: Do cells exposed to various water samples collected in southeastern Louisiana or the progeny of such cells become morphologically transformed, taking on the characteristics of malignant cells? When we incorporated river, raw, or finished water as a major component of growth medium for cells in culture, we assumed that substances could have been present which.could have had a deleterious effect. Often we observed no obvious ill effects upon the test cell population when compared to the-control population. In some instances, we did see effects in the form of reduced cell multiplication when cell growth was compared with that of the control cultures. We never observed the death of all cells in cultures exposed to a given water sample. Where apparent cessation or reduction in cell proliferation was seen, these effects were found to reverse when cells were exposed to the regular outgrowth medium. The mechanisms adversely affecting cell growth were not investigated. Our interests dealt with the genetic effects of substances contained in the water samples upon those cells which survived and upon their progeny, whether these represented the total or a portion of the cell population originally employed. If our investigations were of a quantitative nature where data regarding actual numbers of cells transformed were of interest, information relating to the proportion of the original cell population surviving exposure to a given water sample would have heen obtained. Such information would have been of value by providing a theoretical rate of cell transformation which would have occurred, had there heen no cell death due to exposure. Such a quantitative study was not our ohjective. Some samples of finished and raw water were capable of transforming normal mammalian cells into those having morphologic characteristics of malignant cells. Cultures of transformed cells were not observed before the fourth week of incubation. The small proportion of water samples which induced cell transformation most likely represented only a fraction of those possessing this capability. Others were probably not detected either because of the limitations imposed by the methods
employed, or because of our criteria for transformation. We believe that cell transformation by additional water samples would have heen observed if a larger proportion of the cells originally exposed to the water samples, as well as their progeny, had been passaged during the observation period. Because of physical limitations, the number of samples assayed would have had to he decreased proportional to the increase in the proportion of the cell population under ohservation. There is an accumulation of data questioning the quality of drinking water in southeastern Louisiana. These include analytical studies of concentrated extracts (1), investigation using gas chromatographic-mass spectrophotometric analyses (2,3),our findings as well as those of others using the bacterial assay system ( 4 , 5 , 9 ) , epidemiologic investigations of certain cancers which have occurred in southeastern Louisiana (10, 11 1, and finally our data regarding cell transformation presented herein. On these bases, there appears to be sufficient reason for more extensive investigations directed toward resolving what appears more and more to be an important public health prohlem. Summary Cultures of the mouse cell line R846-DP8 were exposed to 303 Mississippi River, raw, and finished water samples, collected in southeastern Louisiana between 1974 and 1976. Nineteen samples (6%) induced morphological transformation of this cell strain. Transformation was seen with 7 of 118 (6%) Mississippi River water samples, 7 of 70 (10%)raw water, and 5 of 115 (4%) finished water samples. Acknowledgments We thank Mr. L. R. Kuss and the staff of the D.iviSion of Water Pollution Control, Louisiana State Department of Wildlife and Fisheries, for their cooperation in this investigation and T. L. Bradley and W. C. Dixon for their invaluable assistance in providing the water samples. Also, appreciation is expressed to G . F. Craun, Project Officer, for his administrative assistance. Literature Cited (1) U.S. Environmental Protection Agency, “Industrial Pollution of the Lower Mississippi River in Louisiana”, Region VI, Surveillance and Analysis Division, Dallas, Tex., April 1972. (2) Dowty, B., Storer, J., Laseter, J. L., Science, 187,75 (1975). (3) Dowty, B., Laseter, J. L., Anal. Lett., 8.25 (1975).
Volume 14, Number 6, June 1980 725
(4) Pelon, W., Whitman, B. F., Beasley,T. W., Enuiron. Sci. Technol., 11,619 (1977). (5) Pelon, W., Whitman, B. F., Beasley,T. W., Lesley, D. E., Enuiron. Int., in press. (6) Rhim, J. S., Cho, H. Y., Rabstein, L., Gordon, R. J., Bryan, R. J., Gardner, M. B., Huebner, R. J., Nature (London), 239, 103 (1972). (7) Rhim, J. S., Cho, H. Y., Jaglekor, M. H., Huebner, R. J., J . Natl. Cancer Inst., 48,949 (1972). ( 8 ) Rhim, J. S., Creasy, B., Huebner, R. J., Proc. Natl. Acad. Sei. U.S.A., 68,2212 (1971). (9) Louer, J. C.. Lana, D. R., Smith. C. C.. Water Chlorination: En"iron. Impact H e d t h Eff., Proc. Conf., 2,433 (1978). (10) Page, T., Harris, R. H., Epstein, S. S., Science, 193,55 (1976).
(11) Harris, R. H., Page, T., Reiches, N. A., in "Origins of Human Cancer", Hiatt, H. H., Watson, J. D., Winsten, J. A., Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1977, p 309.
Receiued for review May 18, 1979. Accepted March 10, 1980. This project has been financed primarily with federal f u n d s from the Environmental Protection Agency under Grant N o . R800188, awarded by the Water Quality Division, Health Effects Research Laboratory, Cincinnati, Ohio. The contents d o not necessarily reflect the views and policies of the Environmental Protection Agency, nor does mention of trade names or commercial products constitute an endorsement or recommendation for use.
An Experimental Study of the Effect of Temperature upon Aerosol Charge State Hsu-Chi Yeh" and Yung-Sung Cheng Inhalation Toxicology Research Institute. Lovelace Biomedical and Environmental Research Institute, P.O.Box 5890, Albuquerque, N. Mex. 871 15 Average electrostatic charges (polarity ignored) on three sizes of monodisperse fused aluminosilicate spherical particles were measured as a function of heat treatment temperature. The range of temperatures studied was 45 to 1150 "C. Results show that there are two charge peaks at about 600 and 1000 "C, respectively. The first peak is probably due to electron emission, while the second peak is attributed to positive ion emission. It was also found that the initial charge state has minimal effect on the final charge state for temperatures higher than 600 "C for the fused aluminosilicate particles studied here. Possible implications of the thermionic emissions are also discussed. The electrostatic charge of particles is an important property of aerosols that will influence aerosol behavior ( I ) . In many aerosol studies, a knowledge of the aerosol charge state is desired or necessary. Particles may acquire electrostatic charges by various mechanisms and processes. Aerosols are usually highly charged when produced by nebulization of , direct resuspension of a dry solutions or suspensions ( 2 , 3 ) by powder ( 4 ) , or by a vibrating orifice aerosol generator ( 5 ) . Particles also may be charged by exposing the aerosol to a unipolar or bipolar ionic atmosphere ( I ) , or by the electrical induction method (6). In the case of a radioactive aerosol, particles may gain charges due to the self-charging mechanism (7,8).One other mechanism by which an aerosol can acquire electrostatic charges is by thermionic emission when it is heated to a sufficiently high temperature. This process has not received adequate attention. Since the first discovery of thermionic emission by Thomas Edison in 1883 ( 9 ) , a large volume of work has been done, primarily in the field of vacuum tubes, involving the study of the emission of electrons from a metal surface at high temperature under high vacuum and an applied electric field. Feeney et al. (IO) and Blewett and Jones ( I I ) reported techniques for producing sources of positive ions of various alkali metals from aluminosilicate, also under vacuum and the presence of an electric field. Only limited information exists concerning thermionic emissions from aerosol particles. Shuler and Weber (12)reported that electron concentration in rich hydrocarbon flames was found to be higher by one to two orders of magnitude than for a purely thermal flame ionization. They attributed this higher electron concentration to the thermionic emission of electrons from soot particles formed 726
Environmental Science & Technology
in the flame. Almost no reports exist concerning direct measurement of the aerosol charge state or changes in aerosol charge state as a function of temperature change. High-temperature heat treatments of aerosols are sometimes used in the laboratory to obtain a desired physicochemical form of the aerosol (13). Particles produced and released from power plants or internal combustion engines have also been subjected to high temperature. In this paper, experimentally observed changes in aerosol charge state with temperature are reported.
Materials and Methods Aerosol. Monodisperse fused aluminosilicate spherical particles (FAP) were chosen as the test particles for their ability to withstand temperatures up to about 1200 "C. The preparation of the monodisperse FAP involved: (1)treating an aqueous suspension of finely ground montmorillonite clay with 30% hydrogen peroxide solution to remove organic impurities, (2) packing exchange sites with sodium and removing excess sodium by running water dialysis, (3) aerosolizing using a nebulizer and passing aerosols through a heating column at 1150 O C , (4) separating into monodisperse fractions using a Lovelace Aerosol Particle Separator, LAPS ( 1 4 ) , and ( 5 ) resuspending the desired monodisperse fraction and aerosolizing using a nebulizer. Details of the production procedures of the monodisperse FAP aerosol have been reported (13, 15). Charge Measurement. The aerosol charge was measured using a parallel plate electrical mobility spectrometer previously described (7). The spectrometer consisted of two parallel metal plates between which a voltage was applied. The aerosol was introduced into the spectrometer through a narrow inlet slot nozzle as a thin ribbon and sheathed with clean air on all sides. The electrical mobility, Z,, of an aerosol particle was calculated using the equation:
2, = 2 u h 2 / V x where u (centimeterslsecond) is the mean flow velocity inside the spectrometer, h (centimeters) is the half-interplate distance of the spectrometer, x (centimeters) is the distance from the aerosol inlet nozzle at which a particle having electrical mobility 2, deposits on the plates, and V (statvolts) is the potential difference between the plates. The theoretical electrical mobility of a spherical particle is given by:
0013-936X/00/0914-0726$01 .OO/O @ 1980
American Chemical Society
