Envlron. Sci. Technol, 1903, 17, 272-277
of PEG 600 that we obtained in the separate experiment using the culture adapted to LPAE (the same as in the third experiment) supports the above explanation. During the biodegradation experiments of tert-octylphenol ethoxylates, the highest biodegradation degree that could be ascribed to gradual culture adaptation was not observed. Conclusions (1) The biodegradation test of nonionic surfactant is significantly simplified by introducing direct electrochemical determination of surfactants in synthetic sewage and in effluents. (2) The proposed electrochemical method is very sensitive and selective to surfactants contained in detergents and can be used for their direct determination in the presence of organic matter in synthetic sewage and in effluents. (3) Unlike many other methods, the electrochemical method is applicable to the determination of different types of nonionic surfactants regardless of the number of ethylene oxide groups including polyethylene glycols and biodegradation intermediates with a low degree of ethoxylation. (4) The biodegradation degree of nonionic surfactants obtained by the electrochemical method is generally lower than by the modified Wickbold method. Registry No. Polyethylene glycol, 25322-68-3;polyethylene glycol tert-octylphenyl ether, 9036-19-5; polyethylene glycol tert-nonylphenyl ether, 37281-58-6; polyethylene glycol p-tertoctylphenyl ether, 9002-93-1.
(5) Linhart, K. Tenside Deterg. 1972, 9, 241. (6) Kozarac, Z.; ZutiE, V.; CosoviE, B. Tenside Deterg. 1976, 13, 260. (7) Kozarac, Z.; ZvonariE, T.; iutiE, V.; CosoviE, B. Thalassia Jugosl. 1977, 13, 109. (8) CosoviE, B.; ZutiE, V.; Kozarac, A. Croat. Chem. Acta 1977, 50, 229. (9) Kozarac, A.; &aoviE, B.; Branica, M. J. Electroanal. Chem. 4976, 68, 75. (10) CosoviE, B.; VojvodiE, V. Limnol. Oceanogr. 1982,27,361. (11) CosoviE, B.; Hrlak, D. Tenside Deterg. 1979, 16, 262. (12) Rosen, M. J.; Hua, X.; Bratin, P.; Cohen,A. W. Anal. Chem. 1981, 53, 232, (13) ZvonariE, T.; ZutiE, V.; Branica, M. Thalassia Jugosl. 1973, 9, 65. (14) Kozarac, A.; iutib, V.; CosoviE, B. Fourth Yugoslav Symposium on Surface Active Substances, Dubrovnik 1977,
Yugoslavia.
Literature Cited (1) Berth, P.; Heidrich, J.; Jakobi, G. Tenside Deterg. 1980,
(15) Pollution by detergents; Determination of the biodegradability of anionic surface active agents; Publications de l’OCDE, Paris 1972. (16) Hrlak, D.; Bolnjak, M.; Johanides,V. Tenside Deterg. 1981, 18, 137. (17) Wickbold, R. Tenside Deterg. 1973, 10, 179. (18) Longman, G. F. ”The Analysis of Detergents and Detergent Products”;Wiley: New York, 1975; p 513. (19) CosoviE, B.; Branica, M. J. Electroanal. Chem. 1973,46, 63. (20) Radej, J.; RuiiE, I; Konrad, D.; Branica, M. J. Electroanal. Chem. 1973,46,261. (21) CosoviE, B.; Batina, N.; Kozarac, Z. J. Electroanal. Chem. 1980, 113, 239. (22) Gerike, P.; Schmid. R. Tenside Deterg. 1973, 10, 186. (23) Schoberl, P.; Kunkel, E.; Espeter, K. Tenside Deterg. 1981, 2, 64. (24) Swisher,R. D. “SurfactantBiodegradation”;Marcel Dekker: New York, 1970.
17, 228. (2) Schoberl, P.; Bock, K. J. Tenside Deterg. 1980, 17, 262. (3) Kinkel, E. Tenside Deterg. 1980, 17, 247. (4) Jehring, H. “Electrosorptionsanalyse mit der Wechselstrompolarographie”; Akademie-Verlag: Berlin, 1974.
Received for review May 6, 1982. Accepted December 13,1982. This work was supported by the Self-management Authority for the Scientific Research in SR Croatia and by Grant NBSI 6-261 from the National Bureau of Standards, Washington, D.C.
Mutagenicity of Municipal Sewage Sludges of American Cities John G. Babish,*t Brian E. Johnson,? and Donald J. Llskt Department of Preventive Medicine, NYS College of Veterinary Medicine, Cornell University, Ithaca, New York 14853, and Toxic Chemicals Laboratory, NYS College of Agriculture and Life Sciences, Cornell University, Ithaca, New York 14853 ~_____
Dichloromethane extracts of sludge samples from 34 cities were tested for mutagenicity by using the Salmonella/mammalian microsome assay. Of the 34 samples, 33 demonstrated a dose-related increase in revertants in at least 1 of the 5 tester strains; of these 33 samples, 12 were positive with 2 or more strains. Seventy-six percent of the positive samples required metabolic activation to demonstrate mutagenicity, while 18% were mutagenic with and without the S-9 fraction, and 6% were mutagenic only in the absence of any metabolic activation. No association existed between the independent variables percent industrialization, wastewater treatment scheme, and chemical additives when tested individually against mutagenicity. Introduction Approximately 8 X loe gallons of municipal waste containing some 17 000 dry tons of sediments (sludges) are t Department of
Preventative Medicine.
t Toxic Chemical Laboratory. 272
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produced daily in the United States (1). These sludges are generated from residential, commercial, and industrial sources, and may contain human excreta, pathogenic bacteria (2),viruses (3))and a myriad of chemicals ( 4 , 5 ) . Disposal methods have included incineration, fresh water dilution, ocean dumping, disposal in landfills, and limited use on lawns, ornamentals, forests, and agricultural land (6, 7). Estimating the potential hazards from these waters requires that their toxic properties be determined. Once these characteristics are known, environmentally sound methods of disposal may be developed and employed. As part of our studies on ambient exposures to carcinogenic and mutagenic compounds, we evaluated the mutagenicity of organic extracts of sewage sludges from 34 American cities. Experimental Section In 1980, a letter and questionnaire describing our proposed study was sent to 65 cities. They were requested to participate by returning a representative sample of their
0013-936X/83/0917-0272$01.50/0
0 1983 American Chemical Society
sludge to us with the completed questionnaire describing the respective sewage treatment process. The cities that responded and the data they provided are presented in Table I. Upon arrival the sludge samples were spread on plastic sheets and allowed to air dry at ambient temperature. After grinding in a hammer mill, each sample was thoroughly mixed in a rotating mixer. Thirty-gram subsamples were taken for mutagenicity assays. The subsamples were placed in a 45 X 123 mm cellulose extraction thimble, which had been washed three times with 100 mL of redistilled methylene chloride, covered with a glass wool plug, and placed in a Soxhlet extraction apparatus containing 250 mL of redistilled methylene chloride. After 12 h of refluxing in the Soxhlet apparatus, the methylene chloride was removed under a vacuum at 30 "C. This procedure extracted approximately 10% of the starting material. The residue was dissolved in a minimal amount of dimethyl sulfoxide (Me2SO)for testing in the bacterial mutagen assay. Individual sample concentrations in Me2S0 were used for dose-response calculations. Five strains of histidine-requiring (his-) auxotrophs of the bacterium Salmonella typhimurium were used. These strains were a gift from Dr. B. N. Ames, University of California, Berkeley. Strains TA1535 and TAlOO have been used to detect base-pair substitution mutagens; TA1538, TA1537, and TA98 are strains that are susceptible to frameshift mutagens. Strains TAlOO and TA98 have an ampicillin-resistant R factor plasmid which codes for an error-prone DNA repair enzyme (8),thereby amplifying the mutagenic response of these two organisms. All strains were stored as permanent cultures at -80 "C in nutrient broth with Me2S0. The master plates made from these frozen permanent cultures and stored at 4 "C were used as the source of inoculum for overnight cultures used in the mutagenesis assays. The overnight cultures were grown in Oxoid nutrient broth no. 2 at 37 OC for 16 h with agitation in a water bath shaker. Cultures contained (1-2) X log viable cells/mL. For each assay, cultures were checked for crystal violet sensitivity and ampicillin resistence. In addition, the responses to the following mutagens were monitored with each assay: 2-aminoanthracene(all strains), 2-nitrofluorene (TA1538 and TA98), sodium azide (TA1535 and TAlOO), and 9-aminoacridine (TA1537). The procedure reported by Ames et al. (9) for preparation of Aroclor 1254 induced S-9 was used with no modifications. Each mL of S-9 mix contained 100 pL of S-9. Sterility of S-9 was also monitored with each test. In order to maximize the sensitivity and decrease the within and among day variation in response, we incorporated previously reported (10) modifications of the procedure of Ames et al. (9). Biotin (17 pg/plate) and Lhistidine.HCl(1OO pg/plate) were placed in the bottom (15 mL), rather than the top (2 mL) agar phase. The amount of glucose was maintained at 2% in the bottom agar phase. In the top agar 0.5% NaCl solution was replaced by Vogel-Bonner medium (11). The agar concentration in the top agar was 0.75%. Depending on the tester strain and the compound tested, the sensitivity of the assay was increased by these modifications. Furthermore, since the modified procedure reduced within group variability to usually less than lo%, dose-related significant mutagenic activity could be determined reproducibly at relatively low levels. The modified procedure has also been shown (10) to be less sensitive to exogenous histidine than the original procedure of Ames et al. (9). A minimum of five geometrically spaced doses of extract were used. All samples were initially screened to toxic
levels by using strains TA1538, TA98, and TAlOO without S-9. The two extreme dose ranges in the study were 5-20 pg per plate and 700 pg to 3.7 mg per plate. Triplicate determinations with and without S-9 were made at each dose level. Plates were incubated for 72 h at 37 OC. Colony counts were made by using an automatic colony counter. A positive response was defined as a dose-related increase in the number of colonies per plate. A Student's t test for slope = 0 was used to determine a positive dose-response. Regression analysis was performed on the square root of the response variable vs. log dose. Appropriate spontaneous control values were subtracted before transformation. The square root transformation was made on the data in order to stabilize the variance more effectively since enumeration data such as bacterial counts tend to be distributed in a Poisson fashion (12,13). A minimum of four test levels were used for computing the parameters of slope (revertants/log dose), potency (revertants/mg of extract), and lowest significant dose (LSD). The 95% confidence intervals were calculated for both the slope and potency. The LSD for each sample waas determined from the intersection of the lower limit of the 95% confidence interval with the x axis in the plot of the square root of excess revertants vs. log pg of extract. This value represents the lowest concentration of sludge extract that would produce a statistically significant (p C 0.05) increase in revertants in this test. The more commonly reported measure of potency (revertants/unit weight) is confounded by the toxicity of the extracts to the bacterial tester strains. Since the LSD reflects extremely low levels of extract, it is less affected by toxicity and can be used to quantitate the mutagenic potency of complex environmental mixtures. Simple correlations (Pearson's correlation coefficient, r) were computed to assess the relationship between several characteristics of sludges and the parameters of mutagenicity. All statistical procedures were performed according to Snedecor and Cochran (14). The probability of rejecting the null hypothesis when true was set at the nominal 5% level.
Results and Discussion Table I relates the information obtained from the questionnaires describing the samples. Percent of wastewater volume from industrial sources ranged from a high of 65% for Toledo to a low of only 1%for Lexington. Individual wastewater treatment, sludge handling, chemicals added, and the ultimate sludge disposal methods are also listed in Table I. As seen in Table 11, only one sample, Dallas Central, failed to demonstrate a dose-related increase in revertants in any of the five tester strains with or without metabolic activation (S-9). The other 33 sludge samples all exhibited a positive mutagenic response with at least 1strain; 12 of these 33 samples were positive with 2 or more strains. Seventy-six percent (25/33) of the positive samples required metabolic activation to demonstrate mutagenicity, while 18% (6/33) were mutagenic with and without the S-9 fraction, and 6% (2/33) were mutagenic only in the absence of any metabolic activation. The extraction blank run with these samples was negative (data not shown). Of the five tester strains, TAlOO was most responsive (30/33) and TA1535 least responsive (0/33). This is interesting since TAlOO was derived from TA1535 and differs only in the presence of the R factor, pKM 101 (9). As previously mentioned, this plasmid codes for an errorprone DNA repair enzyme (B), which increases the sensitivity of TAlOO to mutagens. I t also results in strain Envlron. Sci. Technol., Vol. 17, No. 5, 1983
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TAlOO responding to both base-pair substitution and frameshift mutagens (8). Therefore, in the absence of any response of strain TA1535, it must be inferred that the mutagens contained in these samples induced primarily frameshift mutations. Strains TA98 and TA1538 are also identical except for the pKM 101 plasmid in strain TA98. Although it is generally recognized that the sensitivity to most mutagens is increased by the plasmid, strain TA1538 responded positively to 8 of the 34 samples while TA98 was mutated by only 5 of the 34 samples. Concordant positive responses of the two strains occured only twice. Therefore, for general screening of environmental samples, the exclusion of TA1538 seems unadvisable. The requirement for metabolic activation of 76% of the positive responses indicates the presence of relatively inert, nonpolar compounds illiciting the mutagenic response. Although such compounds can be degraded by soil microorganisms, many factors such as soil pH, 02,and sunlight influence the rate of metabolism of these compounds and hence the hazard associated with the application of these compounds to open lands (15). Additionally, the components of sludge itself can alter the capacity of soil microorganisms to degrade these refractory organic compounds. Metals commonly found in sludges (Zn, Cd, Cr, Pb) have been shown to inhibit soil bacterial and fungal activity (16, 17). Other studies have demonstrated that addition of municipal sludges to field soil in vitro inhibited microbial carbon transformations (18-20). The distributions for all parameters of mutagenicity (revertants/mg of extract, slope of dose-response curve, and LSD) were computed. Over all strains and samples, the median response of revertants/mg of extract was 36. The cutoff values for the upper and lower quantile were 65 and 20 revertants/mg, respectively. The lowest response was 2 revertants/mg (Kalamazoo, TA1537 with activation), while the highest response was 264 revertants/mg (Philadelphia, Northeast, TAlOO with activation). These results are in good agreement with those of Weaver et al. (21) and Hopke et al. (22). In these studies, crude acetone extracts of sewage sludge obtained from five Illinois municipal sanitary districts induced between 27 and 58 revertants/mg of extract; approximately 155 pL of neat sludge resulted in a doubling of the number of revertant colonies with TA98 after activation with rat S-9. The median slope for the dose-response curves for all ' sludge extracts was 19.8 revertants/(log dose), with values for the upper and lower quantile of 33.1 and 12.2, respectively. Extracts of Galviston airport sludge produced the shallowest slope (1.9 revertants/(log dose), TAlOO with activation) and Buffalo sludge had the steepest slope (119.3 ' revertants/ (log dose), TAlOO with activation). Although the cities with the highest and lowest mutagenicity on a revertants/mg of extract basis were not the same cities with the steepest and shallowest slopes, there was a significant, positive association between revertants/mg of extract and slope of the dose-response curve (p < 0.01, r = 0.489, n = 54). This indicates the frequency with which the initial portion of the dose-response curve was described, since a high response in terms of revertants/mg of extract and a shallow slope are only possible when the initial portion of the dose-response curve is omitted. LSD values were in the microgram range; the median value over all strains and samples was 2.6 pg of extract. This indicates that only 2.6 pg of extract would be necessary to produce a statistically significant (p < 0.05) increase in revertants. The upper and lower quantiles were 16.9 and 1.0 hg, respectively. The lowest LSD was 0.02 Environ. Sci. Technol., Vol. 17, No. 5, 1983
275
Table 11. Mutagenicity of Methylene Chloride Extracts of Municipal Sewage Sludge
sample Baltimore, Back River Boston, Deer Island Boston, Nut Island Buffalo
Cincinnati Mill Creek Denver Northside Detroit Duluth, Regional Galveston, Airport Galveston, Main Groton, NY Houston Kalamazoo Knoxville, Kuwahee Knoxville, Fourth Creek Lexington, Town Branch Lexington, West Hickman Los Angeles, Hyperion Memphis, North Memphis, T.E. Maxson Milwaukee, South Shore
Milwaukee, Jones Island Philadelphia, Northeast Philadelphia, Sputhwest Portland Phoenix Salt Lake City San Diego Point Loma San Francisco, Richmond Sunset San Francisco, North Point Seattle Syracuse Toledo
a
tester strain TAlOO TA1537 TAlOO TA1538 TAlOO TA15 38 TA98 TAlOO TAlOO TA1538 TAlOO TAlOO TA98 TA1538 TAlOO TAlOO TAlOO TAlOO TA1537 TA1538 TA1538 100 TA1537 TAlOO TA1537 TAlOO TAlOO TA98 TAlOO TAlOO TA1538 TA 100 TAlOO TAlOO TAlOO TA98 TAlOO TAlOO TAlOO TAlOO TAlOO TAlOO TAlOO TAlOO TA1538 TAlOO TAlOO TAlOO TAlOO TAlOO TAlOO TA1538 TA98 TAlOO
s-9 activation revertants/mg (-/+) of extract + 138 (129-146) + 106 (95-118) + 86 (64-107) 37 (14-60) + 21 (19-22) + 24 (23-25) +90 (87-94) 104 (98-110) + 172 (165-178) + 147 (145-149) + 172 (171-173) + 35 (34-36)
-
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-
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7.5 (7.1-8.0) 8.6 (8.1-9.0) 35.5 (34.8-36.1) 78.6 (71.8-85.5) 3.6 (3.3-3.9) 12.2 (11.8-12.6) 61.6 (60.5-62.7) 30.6 (29.6-31.5) 119.3 (118.2-120.4) 29.7 (29.5-29.9) 12.2 (12.1-12.3) 12.0 (11.9-12.1)
1 9 (18-20) 1 6 (8-23) 29 (28-30) 44 (43-45) 1 5 (14-16) 39 (38-40) 28 (26-29) 59 (54-64) 1 3 (12-14) 1 9 (18-20) 2 (2-2) 5 (4-6) 4 (3-5) 35 (34-36) 65'( 62-68) 5 (3-7) 41 (39-44) 31 (30-32) 18 (14-22) 46 (44-49) 20 (18-23) 21 (17-24) 31 (29-33) 9 (4-13) 1 5 (14-17) 33 (31-35) 24 (23-26) 264 (255-273) 7 1 (57-85) 144 (133-155) 50 (46-54) 26 (26-26) 6 (5-7) 42 (41-43) 27 (25-29)
38.4 (37.9-38.9) 32.8 (29.0-36.7) 21.1 (21.0-21.3) 10.3 (10.1-10.4) 1.2 (1.1-1.3) 18.5 (18.3-18.6) 1 4 . 1 (13.9-14.4) 40.9 (40.2-41.6) 30.1 (29.8-30.3) 13.7 (13.5-14.0) 16.3 (15.8-16.9) 13.0 (11.8-14.2) 3.8 (3.5-4.1) 29.6 (29.6-29.6) 12.4 (12.1-12.6) 7.2 (5.9-8.61 31.2 (30.4-32.0) 9.7 (9.4-9.9) 29.2 (26.7-31.8) 23.2 (22.2-24.2) 11.3 (10.7-11.9) 8.1 7.5 -8.6) 33.1 32.4-33.8) 18.0 15.7-20.2) 17.8 17.1-1 8.4) 16.0 15.4-1 6.5) 13.1 12.5-1 3.6) 63.1 62.0-64.3) 14.0 12.5-15.6) 29.6 28.4-30.8) 21.0 20.3-21.7) 6.0 6.0-6.0) 4.5 3.9-5.1) 31.2 30.5-31.9) 5.2 5.0-5.5)
44 (41-47) 46 (44-48) 40 (34-45) 7 1 (70-72) 45 (33-57) 57 (53-60) 172 (169-175)
24.4 42.7 40.0 35.6 56.4 17.9 61.1
23.8-25.1) 42.2-43.2) 38.4-41.6) 38.9-36.3) 52.2-60.3) 16.8-1 9.0) 60.5 -6 1.6)
LSD, HLp 0.1 0.9 0.9 137.4 0.2 4.4 8.8 0.5 5.8 3.8 0.1 0.2 84.4 143.1 5.4 0.8 0.1 1.0
19.5 34.7 60.2 1.6 230.1 14.1 22.6 2.6 0.4 86.6 7.9 0.3 78.0 2.3 2.1 2.0 16.9 113.3 19.3 1.1
1.5 1.5 2.6 1.6 1.3
