Environ. Sci. Technol. 1987, 21, 252-260
Smith, J. H.; Bomberger, D. C., Jr.; Haynes, D. L. Chemosphere 1981, 10, 281-289. Atlas, E.; Foster, R.; Giam, C. S. Environ. Sci. Technol.
1982,16,283-286. Rathbun, R. E.; Tai, D. Y. Water Res. 1981,15,243-250. Rathbun, R. E.; Tai, D. Y. Water, Air, Soil Pollut. 1982, 17, 281-293. Rathbun, R. E.; Tai, D. Y. In Gas Transfer at Water Surfaces; Brutsaert, W.; Jirka, G. H., Eds.; Reidel: Dordrecht, The Netherlands, 1984; pp 27-34. Roberts, P. V.; Dandliker, P. G. Environ. Sci. Technol. 1983, 17, 484-489. Mackay, D.; Yeun, A. T. K. Environ. Sci. Technol. 1983, 17, 211-217. Rathbun, R. E.; Tai, D. Y. J. Enuiron. Eng. Diu. (Am.SOC. Ciu. Eng.) 1982, 108, 973-989. Mackay, D., Yuen, T. K. Water Pollut. Res. J. Can. 1980, 15, 83-98. Rathbun, R. E.; Tai, D. Y. Chemosphere 1984, 13, 1009- 1023. Rathbun, R. E. J. Hydraul. Diu., Am. SOC.Civ. Eng. 1977, 103,409-424. Winter, T. C. Bibliography of U.S. Geological Survey Studies of Lakes and Reservoirs-The First 100 Years;U.S. Geological Survey Circular 895; U.S. Geological Survey. U.S. Government Printing Office: Washington DC, 1982; 35 p. Jobson, H. E.; Sturrock, A. M., Jr. Comprehensive Monitoring of Meteorology, Hydraulics, and Thermal Regime
of the Sun Diego Aqueduct, California; U S . Geological Survey Prof. Paper 1137; U.S. Geological Survey. U.S. Government Printing Office: Washington DC, 1979;29 p. (22) Rathbun, R. E.; Tai, D. Y. J . Environ. Eng. Diu. (Am. SOC. Ciu. Eng.) 1983, 109, 1111-1127. (23) Rathbun, R. E.; Tai, D. Y. Chemosphere 1984,13,715-730. (24) Rathbun, R. E.; Stephens,D. W.; Shultz,D. J.; Tai, D. Y. J. Environ. Eng. Diu. (Am. Soc. Civ. Eng.) 1978, 104, 215-229. (25) Rathbun, R. E.; Tai, D. Y. Environ. Sci. Technol. 1984,18, 133. (26) Berglund, R. L.; Conway,R. A.; Waggy, G. T.; Spiegel, M. H. Environ. Sci. Technol. 1984, 18, 133-134. (27) Danckwerts, P. V. Ind. Eng. Chem. 1951,43,1460-1467. (28) Dobbins, W. E. In Proceedings of the International Conference on Water Pollution Research; Pergamon: London, 1964; pp 61-76. (29) Mackay, D.; Shiu, W. Y.; Bobra, A.; Billington, J.; Chau, E.; Yeun, A.; Ng, C.; Szeto, F. U.S. EPA Report 600/382-019; U.S. GovernmentPrinting Office: Washington DC, 1982; NTIS PB 82-230939. (30) Reid, R. C.; Prausnitz, J. M.; Sherwood, T. K. The Properties of Gases and Liquids;McGraw-Hik New York, 1977. (31) Duda, J. L.; Vrentas, J. S. Am. Inst. Chern. Eng. J. 1968, 14, 286-294. Received for review May 20, 1986. Accepted October 9, 1986.
Effects of Chlorine and Chlorine Dioxide on Mutagenic Activity of Lake Kinnereth Water Naoml Guttman-Bass,
* p t
Mlryam BaIrey-Albuquerque,+ Shlmon UlItzur,$ Alan Chartrand,+ and Chalm Rav-Acha * g t
Environmental Health Laboratory, Hadassah Medical School and School of Public Health, Hebrew University, Jerusalem, Israel, and Department of Food Engineering and Biotechnology, Technion, Israel Institute of Technology, Haifa, Israel
w Water from Lake Kinnereth (Israel) was tested for the presence of mutagenic activity, with and without disinfection by chlorine and chlorine dioxide. The samples were assayed for activity with two Ames Salmonella typhimurium tester strains, TA 104 and TA 100, and by a luminescent genotoxic assay with a dark mutant strain of Photobacterium fischeri. The water concentrates were mutagenic in strain TA 104 and in the luminescent assay, reaching positive mutagenic activities in the equivalent of 20 mL of water. Chlorination did not greatly affect the net mutagenic activity, although C102apparently reduced it. Humic acids were isolated from lake sediment and were assayed with and without disinfection in distilled water and in lake water from which the organic components were removed. The humic acids were mutagenic in both test systems, and treatment with C12generally decreased the net activity. CIOz also tended to decrease the mutagenic activity, and cytotoxic effects were observed in some of the samples. Conversely, commercial humic acid was mutagenic only after chlorination on strain TA 100. Introduction Mutagenic activity has been detected in natural waters in many parts of the world (1-7). Possible sources of the mutagenic components are industrial and domestic wastes and agricultural runoff (8))as well as natural processes occurring in the water (9). The presence of mutagenic f
f
Hebrew University. Technion, Israel Institute of Technology.
252
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1987
activity in water that is used as a potable source is of particular concern, due to the possible health effects of drinking mutagens. Finished drinking water has been reported to contain mutagens and carcinogens (10-16). A major source of the mutagenic activity in drinking water is the treatment processes, such as disinfection, which are used to ensure the microbiological quality of the water. In addition, mutagenic activity can increase in distribution systems, following final treatment of the water (17). The specific health effects of the presence of mutagens and carcinogens in drinking water have not been defined, although a number of epidemiological studies have indicated that increased levels of specific cancer types were associated with poorer organic quality of drinking water (8). Lake Kinnereth is a large freshwater lake in Israel that supplies drinking water to about 40% of the population. It has a relatively high content of organic material and bromide, and a high pH. Until recently, the major treatment process was chlorination, but there has now been a partial move to chlorine dioxide. The major reason for this change is the relatively high concentrations of trihalomethanes (THMs), mainly bromoform, detected after chlorination of this water (18). Although CIOz does not form THMs (19) and produces very few nonvolatile chlorinated products (18))its advantage from the point of view of health is questionable, since the organic products it produces in water are largely unknown. The only available information about CIOz organic byproduds in actual water treatment plants was obtained by Stevens (20))who identified some low molecular weight aldehydes formed in Ohio River water after disinfection with C102. Addi-
0013-936X/87/0921-0252$0 1.50/0
0 1987 American Chemical Society
tional information has been obtained from laboratory experiments with CIOz and aquatic humic substances. The main products identified in these studies (21,22)were diand tricarboxylic acids, along with a few chlorinated derivatives of these compounds. Most aldehydes and carboxylic acids have not been found to be mutagenic or toxic. Recently, some ketones and quinones have been identified in Lake Kinnereth water after CIOz treatment. Their structure is under investigation, and their toxicological properties are as yet unknown (23). As the isolation and identification of each product formed during water disinfection is difficult and timeconsuming, simpler methods of ascertaining risks, such as short-term mutagenic assays, are useful alternatives. A number of groups have used such tesh to assess the effects of disinfection on the mutagenic activity present in water. A study using the Salmonella/microsome assay to assess the effects of disinfection on stored water from the Rhine and Meuse Rivers revealed a significant increase in mutagenicity after disinfection with chlorine or chlorine dioxide and a decrease in mutagenicity after ozonation (24). The mutagenicity after treatment with C12 or CIOz was similar and most pronounced without S-9 metabolic activation. Although gas chromatography/mass spectrometry (GC/MS) measurements showed the presence of increased concentrations of some known mutagenic compounds after C12 and C102 disinfection, the association between the levels of these compounds and the mutagenic activity was unclear. The nature of the mutagenicity was not identified, and the precursors of the mutagenic compounds were not determined. A potential source of the mutagenic precursors is the humic substances, which are the permanent, naturally occurring, major part of the organic content of water. A second source may be the micropollutants that are found in low and varying concentrations in water and may be loosely associated with the humic substances. Such micropollutants include pesticides and PAHs (22). On the basis of the fact that following disinfection humic substances in distilled water showed levels of mutagenicity similar to those of drinking water, Meier et al. (25) concluded that the humic substances are likely precursors for mutagen formation. The unchlorinated humic acids were not mutagenic, while a dose-related mutagenic activity was observed after chlorination, with the Salmonella/microsome assay. Again, metabolic activation reduced the mutagenic activity. Neither lyophilization nor purging significantly reduced the mutagenic activity after chlorination, and it was concluded that 80-90% of the mutagenicity was associated with nonvolatile compounds, rather than THMs or other volatile materials. This work was carried out with commercial humic acid, however, which may differ from naturally occurring aquatic humic substances. This study explored the effects of disinfection on Lake Kinnereth water and on humic substances extracted from the lake sediment, in comparison with commercial humic acid. The extent to which the humic materials contributed to mutagen formation as a result of disinfection was evaluated for C12and CIOz, both to demonstrate the source of the mutagenic precursors and to compare the potential of the two disinfectants for mutagen formation. To determine the mutagenic activity of the water and its organic fractions, a new Ames Salmonella typhimurium tester strain, T A 104, was used. It is a histidine auxotroph and was chosen for its high sensitivity to oxidative mutagens (26). Its sensitivity to mutagens in water was compared with that of TA 100, a strain that has been
Table I. Lake Kinnereth Water Quality PH turbidity, NTU alkalinity, mg/L hardness, mg/L ammonia N, mg/L chloride, mg/L bromide, mg/L permanganate value, mg/L TOC, mg of C/L DOC, mg of C/L chlorine demand, mg/L
7.7 1.2 125 226 0.23 2.11 1.7
2.9 4.5 3.5 2
commonly used in water studies (1-4,ll-14,24,25,27-40). In addition, a new assay that utilizes dark variants of luminous bacteria to detect chemicals that can restore the luminescent activity was used. Mutagenic and genotoxic agents can be detected by this system (41, 42), and its sensitivity, rapidity, and simplicity make it an attractive alternative to Ames tests. Materials and Methods Water Samples. Lake Kinnereth water was obtained near the intake for the National Water Carrier where the water is processed for drinking. The water was maintained at 4 "C in the dark until processed in the laboratory. The chemical characteristics of the water are listed in Table
I. Removal of Organic Material from Lake Water. The organic content of Lake Kinnereth water was removed by the following procedures: The water was filtered in series through a Whatman No. 1 filter and a Millipore nitrocellulose filter (0.45-pm nominal pore size). The filtrate was adjusted to pH 2.2 and passed through a column of Amberlite XAD-2 resin (43),at a filtration rate of 60 mL/min. The eluate was filtered through a Whatman No. 1 filter. The filtrate was tested for the presence of organic material by absorbance at 200-400 nm in a Varian spectrophotometer, Model 635 (35). The absorbance of the lake water was reduced to that of distilled water. Purification of Humic Materials. Humic and fulvic acids were isolated from Lake Kinnereth sediment by a modification of the methods described by De Serra and Schnitzer (44) and Mantura and Riley (43). Air-dried sediment (100 g) from Lake Kinnereth was decalcified with 0.1 N HzS04for 24 h according to the published procedure (44). The decalcified solid was shaken with 1 L of 0.1 N NaOH in an ultrasonic bath for 24 h while Nz was bubbled into the system. The humic acid was precipitated by acidification of the supernatant to pH 2 with 5 N H2S04 for 24 h at room temperature. The precipitate was separated by centrifugation and dried in a vacuum desiccator over Pz05. The dried solid was shaken for 12 h with 1 L of a dilute solution of HF-HC1, washed with distilled water, centrifuged, and dried over Pz05.The infrared spectrum of the humic acid was similar to that reported in the literature (45). Anal. for humic acid: C, 46.5; H, 6.67; N, 5.3; 0 (by differentiation), 41.5. The numberaverage molecular weight of the humic acid, as determined by analytical ultracentrifugation, was 15 000. Two isolations of the humic acid were made. In the first (method l), the HF-HC1 purification step was omitted, while in the second (method 2) it was included. In addition, the samples were obtained at different times of the year; the first sample (method 1) was obtained during the winter, while the second (method 2) was from the summer. The fdvic acid (FA) was isolated by passing the acidified supernatant obtained after removal of the humic acid Environ. Scl. Technol., Vol. 21, No. 3, 1987 253
through a 35 X 2.5 cm column of XAD-2 resin. After absorption of the FA, the column was washed with distilled water to remove excess chloride and other inorganic impurities. The FA was eluted with a 1:l mixture of methanol-NH40H. After evaporation of the eluant, the solid was dried over Pz05. Anal. for FA: C, 40.01; H, 6.45; N, 2.4; 0 (by differentiation), 51.14. Its number-average molecular weight was below 5000. Preparation of Humic Acid Solutions. Humic acid from lake sediment or commercial humic acid (Fluka, Switzerland) was dissolved in distilled water or in lake water without organic materials at a concentration of 20 mg/L at pH 12 and stirred overnight at room temperature. After the material dissolved, the samples were stored at 4 "C. Before each experiment, the pH was adjusted to 7.0. Disinfection. Each sample was divided into three equal portions that were processed in parallel: one was disinfected by chlorine, the second was disinfected by chlorine dioxide, and the third was not disinfected but was otherwise treated like the disinfected samples. Initial volumes were 4 L for the lake water and 2 L for the humic acid samples. The chlorine was prepared as a solution of NaClO (Frutarom Chemical Laboratories, Israel) in distilled water. Concentrations of free and bound chlorine were determined by the iodometric method (46). The chlorine dioxide was prepared at a concentration of 300 mg/L according to the procedure of Masschelein (47). Determination of the concentration of CIOz in the solutions was by the method of Wheeler (48). The solutions were stored in the dark at 4 "C until use. The disinfectants were added to the water samples at a ratio of 1:l CkC a t pH 7.0. The reaction proceeded for 72 h in the dark at 20 "C in Teflon-capped containers leaving no headspace. Preliminary experiments (data not shown) indicated that by 24 h most of the reaction was complete. The pH of the samples was adjusted to 7.0, and the initial volumes of 2-4 L were concentrated to 100 mL by lyophilization. Mutagenic Assays. (a) S . typhimurium Assays. Tester strains of S. typhimurium were kindly supplied by B. N. Ames and D. Maron (Department of Biochemistry, University of California, Berkeley, CA). The assays were carried out as described (49),with strains TA 98, TA 100, and TA 104 (26). Each experiment included positive controls, which were sodium azide for TA 100, methyl methanesulfonate for TA 104, and 4-nitro-0-phenylenediamine for TA 98, as well as the recommended negative controls. All of the assays were carried out in duplicate at a minimum of three sample concentrations, and the majority were repeated on separate days. Sterilization of the samples was by filtration through a 0.22-pm filter (No. FP 030/3, Schleicher & Schull, Dassel, West Germany). The preincubation modification of the plate incorporation test (49) was used routinely. The mutagenic activity of selected samples was tested both before and after filtration, and no loss of activity was detected. Samples were considered to be weakly mutagenic with mutation ratios between 1.5 and 2, and above 2 they were considered to be mutagenic. Strain-specific genetic markers that were routinely tested were histidine dependence,UV sensitivity, crystal violet sensitivity, ampicillin sensitivity, biotin dependence, spontaneous reversion rates, and response to positive controls. Although the presence of histidine was not assayed for directly, there was no growth of the bacteria in plates containing the samples but no added histidine. This indicates that endogenous histidine was probably not responsible for the elevated bacterial counts. 254
Envlron. Sci. Technol., Vol. 21, No. 3, 1987
9, c
m
300
o)
U
So)-
Ub cn=
.cW E4 E' 3
0 5 10 15 20 Water Volume (equivalent ml )
Figure 1. Mutagenicity of Lake Kinnereth water. Lake Kinnereth water was disinfected and concentrated as described under Materials and Methods and assayed for mutagenicity In TA 100 (A), TA 104 (E), and P . fischerl (C). Water volumes tested are indicated as the equivalent amount of untreated water. Closed symbols indicate points that were above MR = 2 for the Salmonella strains and above MR = 3 for P . flscheri. Symbols: (A)untreated; (0) CI, disinfected; (0)CIO, disinfected.
(b) Bioluminescent Assay (BLT). The BLT assay employed dark-variant PF13 of Photobacterium fischeri. The test was carried out as described by Ulitzur et al. (41, 42). The light was determined by liquid scintillation counting (Packard Tricarb Model 2002) repeatedly for up to 15 h at 18 OC. Samples with a mutation ratio of 3 or more were considered mutagenic. Results Mutagenicity of Lake Kinnereth Water. Lake Kinnereth water was obtained from a location close to the intake site of the Israeli National Water Carrier. It was divided into three portions, one of which disinfected with chlorine, the second disinfected with chlorine dioxide, and the third left untreated. Following treatment, the samples were concentrated by lyophilization and tested for mutagenic activity in two bacterial test systems. The systems were the mutagenicity assay developed by B. N. Ames (49) using S. typhimurium strains TA 100 and TA 104 and the luminescent genotoxic test using P. fischeri developed by S. Ulitzur (41,42). Metabolic activation was not used in these studies, since, to date, direct-acting mutagens have been reported to form a major part of the mutagenic activity in water (1,11,31, 32, 40). With TA 100, which detects primarily base-pair substitutions at a G-C site (49),no mutagenic activity was detected in the water concentrates, in up to 20 mL (equivalent volume) of water (Figure 1A). This represents 80 pg of organic material, on the basis of an average TOC value of 4 pg/mL typically found in the water (Table I). It should be noted, however, that in the samples without disinfection and those with chlorination there was an increase in the mutation ratio (MR) at the higher concentrations (Table 11). Although in these experiments the MR did not exceed 1.5 for either sample, it is possible that at even higher concentrations the material might show a clearer dose-response relationship. However, the small amount of material available limited the number of experiments that could be performed. The activity of the sample disinfected with CIOz remained slightly below the
Table 11. Mutagenicity of Lake Water with and without Disinfection' sample no disinfection
Clz disinfection
CIOz disinfection
vol, mLb 4 8 16 20 4 8 16 20 4 8 16 20
TA 100 net rev/plate
MR
6f8 13 f 10 20 f 18 36 f 13 -4 f 22 -4 f 4 8 f 24 35 f 15 -6 f 17 -6 f 41 -20 f 8 -12 f 6
TA 104 net rev/plate 292 f 133 310 f 211 353 f 214 434 f 132 133 h 71 362 f 88 376 f 208 398 f 179 191 f 49 77 f 90 119 k 7 99 i 83
1.04 1.10 1.15 1.28 0.99 0.97 1.09 1.30 0.97 0.99 0.85 0.92
MR 1.69 1.71 1.82 2.05 1.36 1.88 1.88 1.94 1.50 1.22 1.32 1.28
"Lake Kinnereth water was tested for mutagenic activity in strains TA 100 and TA 104. In parallel, samples disinfected with C1, or CIOz were assayed. The results are expressed as average net revertants (total revertants minus spontaneous revertants) per plate f SD and are the average of two experiments, each carried out in duplicate. The MR (mutation ratio) is total revertants/spontaneous revertants, where the average spontaneous reversion rates were 124 i 25 for TA 100 and 411 f 104 for TA 104. bVolume before concentration.
spontaneous reversion rate for all concentrations tested (Table 11). With TA 104, which detects base-substitution mutagens at an A-T site (26),mutagenic effects were noted with the nondisinfected and non-chlorinated samples (Figure 1B). The untreated water sample reached an MR = 2 at the highest equivalent volume tested, and even the lowest amount tested (4 mL) had an MR greater than 1.5 (Table 11). The C12-treated sample had an increasing MR, which reached a plateau of just under 2.0 at the higher concentrations (Table 11). The sample disinfected with CIOz did not show a linear response, although all of the results were above a MR of 1 (Table 11). The results obtained for the untreated Lake Kinnereth water were analyzed statistically for significance by unpaired t tests and Wilcoxon rank-sum tests, and the results of the two tests were in agreement. The samples containing the water concentrates were significantly different (p < 0.05) from the control without lake water for TA 104, with the exception of the sample containing an equivalent of 8 mL of water. None of the samples tested on TA 100 were significantly different from the control. The second assay for mutagenic activity is a recently developed system using dark variants of a strain of luminous bacteria, which revert to luminescent after exposure to toxicants or mutagens (41,42). Genotoxic activity is detected as'an increase in luminescence, while toxic agents result in a prompt depression of the background luminescence of the dim mutant (42). In all of the samples tested with the BLT, the effects were noted after several hours of exposure, indicating that the activity was mutagenic rather than toxic (data not shown). The results with the water samples resemble those obtained with T A 104 (Figure IC). That is, the untreated and C1,-treated samples were mutagenic, reaching MR = 7.25 and MR = 6.49, respectively, after exposure to the equivalent of 20 mL of water. The ClO,-treated sample did not demonstrate a mutagenic effect. In summary, concentrates of Lake Kinnereth water contained mutagenic activity that was detected in TA 104 and the BLT. In addition, although T A 100 was less sensitive to the activity, a clear trend to increasing induced mutations with increasing sample concentrations was observed. Similar results were obtained with the chlorinated samples, and the net mutagenic activity was not greatly influenced by this treatment. It is possible, however, that the chlorination procedure reduced the endogenous mutagenic activity and generated new active fractions. Finally, the chlorine dioxide treatment resulted in a mixed
Table 111. Mutagenicity of Lake Water without Organic Material" vol, p L b 0 100 200 500
TA 100 av rev/plate MR 112 f 1 110 f 6 109 f 34 104 f 28
1.00 0.98 0.97 0.93
T A 104 av rev/plate MR 512 f 94 527 f 48 545 f 3 593 f 110
1.00 1.03 1.06 1.16
"Organic material was removed from Lake Kinnereth water as described under Materials and Methods. The mutagenic activity was tested on strains TA 100 and TA 104 and is expressed as average revertants per plate f SD for duplicate plates. MR is the mutation ratio. *For each sample the volume of water was made up to 500 pL with appropriate additions of distilled water.
picture, including nonlinear response (TA 104), slight cytotoxicity (TA loo), and no apparent effect (BLT). Removal of Organic Content of Lake Kinnereth Water. Characterization of the mutagenic activity detected in Lake Kinnereth water required fractionation of the components. The organic and inorganic fractions of the water were separated by passage over a column of XAD-2 resin to remove the organic material, as described under Materials and Methods. The unadsorbed eluate was tested directly for mutagenic activity in strains TA 100 and TA 104 (Table 111). A maximum of 0.5 mL per plate could be assayed in unconcentrated form. No mutagenic activity was found for either tester strain. This water was used as solvent for the organic fractions tested in the experiments described below, in order to determine whether the background of dissolved inorganic chemicals in the water would influence the mutagenicity of the organic materials. Mutagenicity of Humic Acid from Lake Kinnereth Sediment. In order to determine whether the mutagenic activity was associated with the humic acid fraction, humic acid was isolated (method 1)from Lake Kinnereth sediment taken from the same region as the water sample. The humic acid was dissolved either in distilled water or in Lake Kinnereth water from which the organic material had been removed. Samples were subjected to disinfection by C12or CIOz or were left untreated (Figure 2). The material was concentrated and tested for mutagenic activity by the three assays. The untreated sample containing humic acid was mutagenic for T A 100 in both types of water (Figure 2A,B), reaching mutation ratios greater than 2 with 160-200 Fg of organic material (Table IV). The chlorinated samples did not exceed MR = 2, although a mutation ratio greater Environ. Sci. Technol., Vol. 21, No. 3, 1987 255
Table IV. Mutagenicity of Humic Acid Extracted from Lake Sedimenta sample
water
no disinfection
distilled
Lake Kinnereth C1, disinfection
distilled
Lake Kinnereth CIOz disinfection
distilled
Lake Kinnereth
humic acid, mg 0.02 0.04 0.08 0.16 0.20 0.40 0.04 0.08 0.20 0.02 0.04 0.08 0.16 0.20 0.40 0.04 0.08 0.20 0.02 0.04 0.08 0.16 0.20 0.40 0.04 0.08 0.20
TA 100 net rev/plate
50 83 53 121 138 f 2 150 34 f 3 83 f 0 163 f 36 72 75 9 36 61 f 41 57 24 f 27 50 f 3 56 f 27 50 115 ND 66 104 f 43 88 -7 f 1 17 f 71
MR
TA 104 net rev/plate
MR
1.60 1.94 1.50 2.13 2.45 2.40 1.26 1.64 2.24 1.82 1.85 1.10 1.34 1.66 1.50 1.20 1.40 1.43 1.60 2.31 ND 1.61 2.12 1.82 0.95 1.12
35 160 f 81 234 297 426 & 20 465 355 f 119 358 f 50 411 27 273 f 90 384 450 452 f 8 467 294 f 78 292 f 28 306 f 22 259 107 f 53 127 10 208 f 83 -50 160 f 102 -199 f 55
1.08 1.33 1.50 1.60 1.91 1.92 1.82 1.84 2.03 1.06 1.60 1.80 1.89 1.95 1.92 1.69 1.69 1.73 1.60 1.23 1.25 1.02 1.45 0.90 1.37 0.54
-
-
-
"Humic acid was extracted from Lake Kinnereth sediment by method 1, as described under Materials and Methods. The humic acid was resuspended in distilled water or Lake Kinnereth water from which the organic material had been removed. Disinfected and undisinfected samples were concentrated and assayed for mutagenic activity, and the results are expressed as described in Table 11. Experiments were carried out twice, although not every concentration was tested in both experiments. ND = no data; (-) = cytotoxic effect (sparse lawn). The average spontaneous reversion rates were 98 f 13 for TA 100 in distilled water and 131 f 4 in Lake Kinnereth water and 481 f 33 for TA 104 in distilled water and 427 40 in Lake Kinnereth water.
*
than 1.5 was noted at several concentrations of the distilled water sample (Table IV). The C102-treatedsamples were cytotoxic in the lake water and had a nonlinear response in the distilled water, while at some concentrations the MR exceeded 2. The mutagenic activity of the humic acid on TA 104 was consistent with that found for the unfractionated Lake Kinnereth water (Figure 2C,D). The untreated humic acid reached mutation ratios above 1.5 by 80 pg in distilled water and 40 lug in the lake water without organic material (Table IV). The mutation ratios approached or exceeded MR = 2 by 200 pg of humic acid. The chlorinated sample in distilled water generally had slightly higher and the samples in lake water had slightly lower mutation ratios than the untreated humic acid. However, in both cases the qualitative trends were similar, and mutation ratios approaching 2 were achieved. In contrast, the C102-treatedsamples were cytotoxic, and no conclusions about their mutagenicity can as yet be drawn. The BLT indicated the presence of genotoxic activity in the untreated humic acid in both types of water (Figure 2E,F). In the Lake Kinnereth water without organic material, the mutation ratio reached 7.67 and was greater than 3 by 40 yg of humic acid. In the sample containing humic acid in distilled water, maximal genotoxic activity was observed by 40 pg. The chlorinated samples indicated weak genotoxic activity at low concentrations of humic acid, which decreased at higher concentrations. The samples treated with CIOz did not demonstrate mutagenic activity. In summary, the humic acid from Lake Kinnereth sediment was mutagenic in both test systems. The chlorinated samples exhibited mutagenic activity, particularly in TA 104. The C102-treated humic acid generally resulted in 256
Environ. Sci. Technol., Vol. 21, No. 3, 1987
0
100
200 400 100 Humic acid ( p g )
201
Flgure 2. Mutagenicky of humic acid from lake sediment. Humic acld isolated from Lake Kinnereth sediment (method 1) was resuspended in distllled water (A, C, and E) or lake water from which organic materlal was removed (8, D, and F). The samples were disinfected, concentrated, and assayed for mutagenlcky in TA 100 (A and B), TA 104 (C and D), and P. fischeri (E and F). Symbols are as described in the legend to Flgure 1.
lower mutagenic activity and in some cases was clearly cytotoxic. The two types of water used as solvents in the experiments generally resulted in similar levels of mutagenic activities, with some exceptions noted above. These exceptions were more quantitative than qualitative. One
Table V. Mutagenicity of Humic and Fulvic Acids from Lake Sediment' tester strain TA 100
concn, mg/plate 0
0.05 0.20 0.50
TA 104
0
0.02 0.05 0.20
TA 98
0
0.02 0.05 0.20
av rev f SD
humic acid net rev
MR
av rev f SD
82 f 19 112 f 19 165 f 43 199 f 4 372 t 92 536 f 34 691 f 13 797 15.5 f 0.7 18.5 f 3.5 24 f 4 32 i 6
0 30 83 117 0 164 319 425 0 3 8.5 16.5
1.00 1.37 2.01 2.43 1.00 1.44 1.86 2.14 1.00 1.19 1.55 2.06
82 f 19 123 f 18 148 f 14 160 f 6 372 f 92 601 f 18 612 f 25 732 36 15.5 f 0.7 20.5 A 5 19.5 f 2 21 f 7
fulvic acid net rev 0
41 66 78 0 229 240 360
*
0
5 4 5.5
MR 1.00 1.50 1.80 1.95 1.00 1.62 1.64 1.97 1.00 1.32 1.25 1.35
'Humic and fulvic acids isolated from Lake Kinnereth sediment (method 2) were assayed for mutagenic activity at the indicated concentrations on three Ames tester strains. The results are expressed as average revertants f SD, calculated from duplicate plates. Net revertants and mutation ratios were calculated as described in Table 11. trend that was apparent for the Ames strains was that the C102-treatedsamples in distilled water were less cytotoxic than those in lake water without organic materials. However, even in the distilled water a variable response was obtained with increasing amounts of humic acid. It may be noted that the background of inorganic materials, which were concentrated from the water after treatment, did not contribute greatly to the mutagenic activity, since in most cases the mutagenic activities were similar to the corresponding samples in distilled water. Mutagenicity of Humic and Fulvic Acid from Lake Kinnereth Sediment. Fulvic and humic acids isolated from Lake Kinnereth sediment by a slightly different method (method 2) were assayed for mutagenic activity in the two tester strains used previously and in TA 98 to detect frame-shift mutagens (Table V). The humic materials were tested only in distilled water and were not subjected to disinfection as in the previous series of experiments. The humic acid was mutagenic for all three tester strains, exceeding MR = 2 by 200 pg of humic acid. The fulvic acid was slightly less mutagenic for TA 100 and T A 104 than the humic acid at the same concentration. For TA 98, a mutation ratio greater than 1.5 was not achieved at any of the concentrations tested. However, the limited amount of material available did not allow for retesting, and definitive conclusions about the relative mutagenicity of the different organic fractions cannot as yet be drawn. The mutagenic activities of the humic acid samples isolated by different methods and at different times of the year were in agreement. This indicates a degree of consistency in the mutagenic activity associated with the humic acid, as opposed to substantial seasonal variations found in experimenh described in the literature (35). Mutagenicity of Commercial Humic Acid. The use of commercially available humic acid has been proposed as a model system to study the byproducts of the disinfection of organic materials in water (25,35). In order to compare our results with those reported in the literature for a standard source of humic acid and to study the sensitivity of TA 104 to a commonly used humic acid, the following experiments were performed. Humic acid from a commercial source (Fluka) was dissolved in either distilled water or Lake Kinnereth water from which the organic content was removed, and samples were disinfected as described above. The concentrates were assayed for mutagenic activity in the tester strains TA 100 and TA 104 (Figure 3). The untreated and C102-treatedsamples did not show strong mutagenic activities on TA 100, for either type of
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*
/.
/ /'
r
D
water (Figure 3A,B). In contrast, the chlorinated sample was mutagenic (Table VI), although mutation ratios greater than 2 were reached at lower concentrations in the lake water (90pg) than in the distilled water (200 pg). The lack of mutagenic activity for TA 100 and the generation of activity by chlorination are in agreement with results reported by others using this humic acid (25, 35). The treatment with C102did not result in a significantly altered mutagenic profile when compared with the untreated humic acid. The mutagenic response of TA 104 to the commercial humic acid was pronounced in all samples (Figure 3C,D). The chlorinated samples had the greatest mutagenic activity, with weakly mutagenic levels at all concentrations tested in both types of water (Table VI). Mutation ratios greater than 2 were reached by 40-80 pg. The untreated humic acid was weakly mutagenic at concentrations of 40 Fg and above for both water types, and in distilled water the mutation ratio increased to above MR = 2 at the highest concentration tested. Finally, the samples treated with C102 were weakly mutagenic at all concentrations, with a mutagenic pattern similar to that obtained with the untreated humic acid. T A 104 was apparently more senEnvlron. Sci. Technol., Vol. 21, No. 3, 1987
257
Table VI. Mutagenicity of Commercial Humic Acid" sample no disinfection
water distilled
Lake Kinnereth C12 disinfection
distilled
Lake Kinnereth C102disinfection
distilled
Lake Kianereth
humic acid, mg 0.02 0.04 0.08 0.16 0.20 0.04 0.08 0.20 0.02 0.04 0.08 0.16 0.20 0.04 0.08 0.20 0.02 0.04 0.08 0.16 0.20 0.04 0.08 0.20
TA 100 net rev/plate ND -17 f 35 -7 f 8 2 22 f 11 11 f 9 81 f 74 27 f 45 ND 53 f 33 94 f 56 120 147 f 31 134 f 43 171 f 91 277 f 58 ND 16 f 20 39 f 1 39 56 f 8 34 48 f 5 10 f 6
MR
ND 0.88 0.95 1.01 1.20 1.05 1.61 1.20 ND 1.40 1.60 1.99 2.08 2.03 2.32 3.12 ND 1.11
1.30 1.30 1.41 1.27 1.37 1.08
TA 104 net rev/plate
131 358 ND ND 526 276 i 4 276 f 50 272 f 46 302 637 ND ND 1063 359 i 60 453 f 173 568 f 146 268 365 ND ND 425 355 f 119 314 f 65 361
MR 1.29 1.80 ND ND 2.16 1.66 1.66 1.64 1.67 2.40 ND ND 3.35 1.85 2.08 2.35 1.60 1.80 ND ND 1.94 1.82 1.73 1.90
"Commercial humic acid was tested for mutagenic activity, as described in Table 11. The spontaneous reversion rates were 139 f 7 for TA 100 in distilled water and 131 f 4 in Lake Kinnereth water and 452 f 54 for TA 104 in distilled water and 427 f 40 in Lake Kinnereth water.
sitive than TA 100 to the mutagenic activity present in the humic acid and might thus prove a useful additon to the standard strains used for water testing. The commercial humic acid was tested in the BLT in a background of lake water from which the organic content had been removed (data not shown). Although there was a slightly elevated response, the MR did not exceed 3 at a concentration range of 5-80 pg, The chlorinated sample reached MR = 3.44 with 80 pg of humic acid, which was the largest amount tested. The C102-treated sample had a nonlinear dose-response curve and at one concentration of humic acid (40 pg) reached a peak value of MR = 3.84. Thus, the mutagenic profile generated by this system was somewhat different than that found with the Ames strains. It is possible that the luminescent system is less sensitive to the toxic effects of the CIOz treatment and was thus more sensitive to its mutagenic effects. Discussion Lake Kinnereth water was found to be mutagenic in two test systems, and isolated humic substances from the lake sediment had a similar mutagenic pattern. Although natural waters have been reported to contain mutagenic activity (1-7), mutagenic activity has not generally been found in association with purified humic materials. Humic acids isolated from Black Lake in North Carolina was not active in T A 100 and TA 98, with or without metabolic activation (35),and commercial humic acid (Fluka) had a similar lack of activity. It has been proposed that Fluka humic acid may serve as a model for studies on the interaction between disinfectants and natural humic materials (25, 35). Although our results were similar when Fluka humic acid was tested, the natural humic acid from Lake Kinnereth sediment gave different results. In fact, the humic acid from the sediment provided a mutagenic spectrum similar to that of the water itself and may therefore be a more appropriate model for studying the effects of various disinfectants on the mutagenic activity than the commercial humic acid. 258
Environ. Scl. Technol., Vol. 21, No. 3, 1987
Of interest was the finding that the mutagenic activity of the isolated humic materials in distilled water was similar to that obtained for the same humic material in lake water from which the organic materials had been removed. This indicated that the inorganic content had little, if any, effect on the total mutagenic activity, with the exception of the increased toxicity found with the C102-treatedhumic acid from the lake sediment. Overall, the major contribution to the mutagenic activity may be attributed to the organic fraction of the water. The mutagenic activities of humic and fulvic acids from lake sediment were compared. It might be expected that fulvic acid would be more active on a per weight basis, since it has a much smaller average molecular weight and contains more oxygen-containing functional groups than the humic acid. The latter may be particularly important for TA 104, which is sensitive to oxidative mutagens with a relatively high oxygen content, such as peroxides, aldehydes, and ketones (50). We found, however, that the fulvic acid was not more mutagenic than the humic acid and in fact appeared to be slightly less active. One possibility is that the humic acid has a higher chelating power and may contain mutagenic materials that are not an integral part of the humic acid parent structure. This has not yet been investigated. Fulvic acid did, however, contain some mutagenic activity, and it may thus be an important contributor to the total mutagenic activity of the water, since it is usually found in water in higher concentrations than humic acid (51). It has been shown by a number of laboratories that disinfection of water with C12 leads to the formation of chlorinated organic compounds and increases the mutagenic activity of the water (13,15,25,29,35).Our results with commercial humic acid were in agreement with those of other investigators (25,35),with an increase in mutagenicity detected in T A 100 after chlorination, and we noted the same effect in the new tester strain TA 104. In addition, TA 104 detected mutagenicity in the untreated sample. The natural humic acids that were tested here
did not show a substantial increase in mutagenicity after chlorination, nor did the water itself. It may be noted that the lake water contains relatively high levels of bromide, which in the presence of chlorine resulta in the formation of brominated organic compounds. Many of these compounds are known to be more mutagenic than their chlorinated counterparts in TA 100 (28). However, we found that the inorganic content of the water, which contains relatively high levels of bromide, had little influence on the mutagenicity of either the natural or commercial humic acids. In contrast with Cla, which produces primarily chlorinated byproducts, C102reacts more specifically as an oxidant rather than a chlorinating agent (23). By use of high-performanceliquid chromatographyand MS analysis, it was recently found that typical organic products found in Lake Kinnereth water after treatment with C102 are mainly ketones and quinones (52). Similarly, using MS/GC, Stevens (20) detected primarily low molecular weight aldehydes and ketones in Ohio River water disinfected with Clop Therefore, we expected that the new Ames tester strain, TA 104, would be particularly useful for the detection of mutagenicity in water treated with CIOz, since it has been reported that this strain is particularly sensitive to oxidative compounds (26) and, specifically, to aldehydes (50). Treatment with C102 tended to decrease the mutation ratios of the humic materials, in some cases below the spontaneous reversion rates. This may be due to the destruction of the mutagenic materials, to the formation of toxic products as a result of the treatment, or to a combination of the two. In addition, these effects may mask the presence of newly formed mutagenic materials, which possibly were not detected due to the cytotoxicity. It has been reported that C102oxidizes various known mutagens to inactive products (53). In addition, toxic compounds have been detected after treatment of organic materials with CIOz. For example, treatment of water with C102 increases the phenolic content of water (21))and these phenols are subsequently oxidized by C102 to quinones, some of which have been found to be cytotoxic and to interact with native DNA (54). In some of our samples the toxic effects were very apparent, and in other cases the mutagenic response was nonlinear. Fractionation and assay of the organic products, which might separate mutagenic from toxic elements, might allow separation of these effects. In addition, methods that decrease the toxic effects in the bacteria, such as the addition of a glutathione chase, might allow the detection of mutagenic activity if present (50). If such an approach were effective, it might enable the routine use of the highly sensitive TA 104 strain for water monitoring.
Acknowledgments We thank D. Alkaslassy, who performed the isolation of the humic substances as part of her Ph.D. research work. Registry No. CIOz, 10049-04-4.
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OzonelChlorine Dioxide Oxidation Products of Organic Materials; Rice, R. G.; Cotruvo, J. A., Eds.; Ozone: Cleveland, OH, 1978; pp 189-200. (22) Colchough, C. A.; Johnson, J. D.; Christman, R. F.; Millington, D. s.In Water Chlorination: Enuironmental Impact and Health Effects; Jolley, R. L.; Brungs, W. A.; Cotruvo, J. A.; Cumming, R. B.; Mattice, J. S.; Jacobs, V. A., Eds.; Ann Arbor Science: Ann Arbor, MI, 1983; Vol. 4, pp 219-229. (23) Rav-Acha, Ch.; Blits, R. Water Res. 1985,19, 1273-1281. (24) Zoeteman, B. C. J.; Hrubec, J.; de Greef, E.; Kool, H. J. Enuiron. Health Perspect. 1982, 46, 197-205. (25) Meier, J. R.; Lingg, R. D.; Bull, R. J. Mutat. Res. 1983,118, 25-41. (26) Levin, D. E.; Hollstein, M.; Christman, M. F.; Schwiers, E. A.; Ames, B. N. Proc. Natl. Acad. Sci. U.S.A. 1982, 79, 7445-7449. (27) Loper, J. C.; Lang, D. R.; Smith, C. C. In Water Chlorination, Environmental Impact and Health Effects; Jolley, R. L.; Gorchev, H.; Hamilton, D. H., Jr., Eds.; Ann Arbor Science: Ann Arbor, MI, 1978; Vol. 2;pp 433-450. (28) Simmon, V. F.; Tardiff, R. G. In Water Chlorination, Enuironmental Impact and Health Effects; Jolley, R. L.; Gorchev, H.; Hamilton, D. H., Jr., Eds.; Ann Arbor Science: Ann Arbor, MI, 1978; Vol. 2, pp 417-431. (29) Cheh, A. M.; Skochdopole, J.; Koski, P.; Cole, L. Science (Washington, D.C.) 1980,207, 90-93. (30) Coleman, W. E.; Melton, R. G.; Kopfler, F. C.; Barone, K. A.; Aurand, T. A.; Jellison, M. G. Environ. Sci. Technol. 1980,14,576-588. (31) Denkhaus, R.; Grabow, W. 0. K.; Prozesky, 0. W. Prog. Water Technol. 1980,12,571-589. (32) Loper, J. C. Mutat. Res. 1980, 76, 241-268. (33) Maruoka, S.; Yamanaka, S. Mutat. Res. 1980,79,381-386. (34) Neeman, I.; Kroll, R.; Mahler, A,; Rubin, R. J. Bull. Enuiron. Contam. Toxicol. 1980,24, 168-175. (35) Bull, R. J.; Robinson, M.; Meier, J. R.; Stober, J. Environ. Health Perspect. 1982,46, 215-227. (36) Kfir, R.; Grabow, W. 0. K.; Hilner, C. A. Bull. Enuiron. Contam. Toxicol. 1982,28, 641-646. (37) van Rossum, P. G.; Willemse, J. M.; Hilner, C.; Alexander, L. Water Sci. Technol. 1982,14, 163-173. (38) DeLuca, S. J.; Chao, A. C.; Smallwood, C., Jr. J. Enuiron. Eng. Diu. (Am. SOC.Ciu. Eng.) 1983, 109, 1159-1167. Environ. Scl. Technol., Vol. 21, No. 3, 1987 259
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(47) Masschelein, W. J. In Chlorine Dioxide; Ann Arbor Science: Ann Arbor, MI, 1979; p 129. (48) Wheeler, G. L.; Lott, P. F.; Yau, F. W. Microchem. J. 1978, 23, 160-164. (49) Maron, D. M.; Ames, B. N. Mutat. Res. 1983,113,173-215. (50) Marnett, L. J.; Hurd, H. K.; Hollstein, M. C.; Levin, D. E.; Esterbauer, E.; Ames, B. N. Mutat. Res. 1985,148,25-34. (51) Visser, S. A. Freshwater Biol. 1984, 14, 79-87. (52) Rav-Acha,Ch.; Blits, R., EnvironmentalHealth Laboratory, Hebrew University, Jerusalem, unpublished results, 1985. (53) Reichert, J. K. Arch. Hyg. Bakteriol. 1968, 152, 265-276. (54) Trevors, J. T.; Bursuraba, J. Bull. Environ. Contam. Toxicol. 1980, 25, 672-675.
Received for review February 4,1986. Revised manuscript received September 9,1986. Accepted October 21,1986. M.B.-A. was partially supported by a grant from the Israel Interior Ministry.
Atmospheric Concentrations and Chemistry of Alkyllead Compounds and Environmental Alkylation of Lead C. Nicholas Hewltt
Department of Environmental Science, University of Lancaster, Lancaster LA 1 4YQ, U.K. Roy M. Harrison"
Department of Chemistry, University of Essex, Coichester C04 3SQ, U.K. The atmospheric chemistry of alkyllead compounds was investigated by determining the ratio of alkyllead to total lead in a variety of different air masses. Maritime air was found to contain significantly more alkyllead relative to total lead than continental or urban air. The presence of gas-phase trialkyllead in the rural atmosphere was confirmed, with trialkyllead becoming progressively more important relative to other lead species with increasing distance from urban source areas. On the basis of estimates of the atmospheric lifethnes of the various lead species, it is concluded that these enhanced alkyllead ratios are explicable in terms of the atmospheric chemistry of the various species, with pollutant tetraalkyllead decomposing in the atmosphere to the relatively stable trialkyllead derivatives. It is therefore probably not necessary to invoke the hypothesis of the natural alkylation of lead to explain these enhanced ratios. Notwithstanding this, evidence is also presented from experiments using intertidal sediments both with and without the addition of a labeled lead tracer that indicates that lead(I1) nitrate can be inefficiently alkylated by a sediment system. Introduction Evidence for the environmental formation of alkyllead compounds from inorganic lead is, a t the present time, available from several sources, but despite extensive research is mainly circumstantial in nature. Experiments with environmental media (1, 2) and experiments with chemical systems (3) and environmental monitoring (4) all provide evidence that has been used to support the hypothesis that the alkylation of lead takes place in the environment. We present here the results of a series of experiments and atmospheric monitoring that indicate that the atmospheric chemistry of alkyllead is considerably more complex than was previously believed, with species other than tetraalkyllead (TAL) being present in the gas phase in both urban and rural air. Smog-chamber studies in260
Environ. Sci. Technoi., Vol. 21, No. 3, 1987
dicate that these other alkyllead compounds, the ionic trialkyl and dialkyl species (TriAL and DiAL), are formed from TAL by reaction with hydroxyl (HO). They are more stable in the atmosphere than TAL, which casts doubts on the validity of the use of alkyllead-in-air data as an indicator of environmental alkylation. Notwithstanding this, we have also obtained experimental evidence using intertidal sediments both with and without the addition of a labeled lead tracer that indicates that lead(I1) nitrate can be inefficiently alkylated by a sediment system. Prior to this study little was known of the atmospheric chemistry of alkyllead apart from the concentrations of TAL in the urban and rural atmosphere (5)and the rates of the main TAL decomposition reactions (6). No information was available concerning the nature or atmospheric concentrations of the products of these reactions, and it was assumed that these intermediate compounds (formed in the inevitable decay of TAL to inorganic Pb) were not significant in the atmosphere compared with TAL. Consequently, it was anticipated that measuring the ratios of alkyllead to total P b in different air masses, both in continental air close to urban areas and in maritime air remote from anthropogenic lead sources, would be a suitable method for determining whether or not the natural alkylation of lead takes place in the environment (4).This method assumes that both the gas-phase organic lead and the inorganic lead aerosol have similar atmospheric lifetimes, of the order of a few days. However, we show here that this is not the case. The concentration of particulate lead found in the at mospheric aerosol in rural and maritime regions is now well established, with several recent papers containing extensive data sets of such measurements (7-10).Unfortunately, however, the same cannot be said of the organic lead species for which only a very few data are available for rural air (11-15), and none, to date, have been reported for true maritime air. We present here data on organic lead concentrations obtained at several rural sites in N.W.
0013-936X/87/0921-0260$01.50/0
0 1987 American Chemical Society
