Environ. Sci. Technol. 1984, 18, 909-916
Comparison of the Mutagenicity of Sewage Sludges Phlllp K. Hopke," Mlchael J. Plewa, Patrlcla L. Stapleton, and Danny L. Weaver
Institute for Environmental Studies, University of Illinois, Urbana, Illinois 6 180 1 Samples of five municipal sewage sludges from Illinois cities have been subjected to a multiorganism testing program to determine the presence or absence of mutagenic activity. Chicago sludge has been the most extensively tested by using the Salmonella/microsome reverse mutation assay, the micronucleus test in Tradescantia, the wx locus assay in Zea mays, and sister chromatid exchange induction in human lymphocytes. Mutagenic activity has been observed in the sludges from Chicago and Sauget, IL. Sludges from primarily domestic sewage do not apparently contain significant mutagenic activity. Introduction For about a decade there has been increasing interest in land application of sewage sludges as a disposal method, as a supplemental treatment method, and as a means to increase the organic content of disturbed soils such as strip-mined lands. One of the major problems that has been identified with land application of sludges is the heavy metal contamination, particularly cadmium, often found in municipal sewage sludge. The concern over cadmium incorporation in the human food chain has led to regulations ( I ) limiting the annual cadmium loading in order to avoid excessive cadmium in food-chain crops. Recently, the presence of organic compounds in sludges, particularly compounds that have genetic activity, have been reported in Chicago sewage sludge (21,a sludge used extensively in reclaiming strip-mined land through land application. Babish et al. (3) have reported a survey of 34 sludges that were tested for mutagenicity. In their study they air-dried the samples and then extracted them 12 h with methylene chloride in a Soxhlet apparatus. The extract was vacuum distilled to dryness and the residue taken up in dimethyl sulfoxide (Me,SO) for mutagen testing with several of the Salmonella bacterial strains. The median slope of the dose-response curve for all of their extracts was 19.8 revertants/ (log dose) with values of 33.1 and 12.2 for the upper and lower quartile, respectively.. Their observation of mutagenicity in all of the samples analyzed might be anticipated on the basis of reports of the mutagenicity of human fecal material (4-6). It has also been reported (7) that the mutagenicity of feces increases with anaerobic digestion. Thus, sewage sludge from municipal wastewater treatment might be expected to have some mutagenicity. In order to ascertain how common it is to find substantial mutagenicity in municipal sewage sludge, and to determine the effects of their mutagenicity in a variety of genetic tester organisms, samples were obtained from five different locations in Illinois: Champaign, Chicago, Hinsdale, Kankakee, and Sauget. The treatment plants for Champaign, Hinsdale, and Kankakee primarily or exclusively treat household wastes. The Sauget plant treats the effluent from a large, synthetic organic chemical manufacturing plant in addition to domestic sewage. Additional information regarding these sludge samples is provided in Table I. The plants at Champaign, Chicago, Hinsdale, and Kankakee are all activated sludge secondary treatment plants. Champaign and Hinsdale aerobically 0013-936X/84/0918-0909$01.50/0
digest their sludge for greater than 30 days. The West Southwest Plant of the Chicago Metropolitan Sanitary District digests the sludge anaerobically at 35 f 2 "C for 14-15 days. This sludge sample used in these studies is the same as described by Hopke et al. (2). The Kankakee sludge is anaerobically digested in a conventional manner for 30-40 days. The Sauget plant uses a simple physical/chemical primary treatment. It adds lime to the sludge to improve coagulation, vacuum filters it through a cloth filter, and landfills the sludge. These samples were subjected to a variety of assays with organisms ranging from bacteria to whole plants to assess a wide range of possible genetic damage from components within the sludge as well as to determine if the mutagens can be translocated into plants. Experimental Procedures Sludge Fractionation. The sludge samples were centrifuged at 12 500 rpm in a Sorvall GSA rotor (25400g) for 60 min. The supernatant aqueous phase was discarded, and the samples of sludge solids were subjected to an extraction/fractionation scheme outlined in Figures 1and 2. In our earlier work (2),acetone and hexane had been used as simple sequential extraction agents with no further subsequent separation. The extraction protocol was altered here in order to begin to define the chemical classes to which any observed mutagenic activity may belong. Chloroform/methanol (C/M) was chosen because it is considerably less polar than acetone although more polar than hexane. This modification has advantages in that it permits further chemical fractionation of the crude extracts and it reduces the amount of water sequestered during the extraction of the wet solids. The sequestered water is eliminated from these extracts by extracting lyophilized solids rather than the wet solids originally employed. The yields of residue obtained from the C/M extraction of these five sludge samples were 9.1% while the acetone yields for Champaign and Chicago had averaged 7.5%. These mass yields are similar to those obtained by the acetone extraction (2). The extraction yields given by Babish et al. (3)were 10% so these results are quite comparable. These crude extracts were then subjected to a further fractionation as outlined in Figure 2. These additional separation steps are useful to determine the presence or absence of mutagenic activity and to minimize synergistic and antagonistic effects of other compounds potentially present in these complex mixtures. This procedure separates the extracts into four general chemical classes: strongly acidic, weakly acidic, basic, and neutral compounds. The yields for each of these fractions are given in Table 11. These yields are needed to permit the calculation of the level of mutagenic activity per unit mass of material being tested. The Kankakee sludge did not separate well as it formed emulsions that could not be broken. Therefore, the testing of individual fractions of the Kankakee sludge was not possible. The data in Table I1 have been placed in the order of the highest to the lowest yields of extracted mass. The majority of the mass in each case was in the neutral fraction (63-92%). The other classes show the following ranges: basic (1.67-25 % ),
0 1984 American Chemical Society
Envlron. Sci. Technol., Vol.
18, No. 12, 1984 909
Table I. General Information on the Municipal Sewage Sludge Samples To Be Examined for Mutagenicity municipality sampled
sampling date
source typen
treatment process
nature of sample
% dry solids, w/w
Chicago Champaign Kankakee Hinsdale Sauget
10179 11/80 4/81 4/81 7/81
ind/resid residential indlresid ?/resid ind/resid
anaerobic aerobic aerobic/anaerobic aerobic aerobic
slurry slurry slurry solid slurry
3.5 4.0 3.5 13.9
Twes: ind, industrial; resid, residential, Table 11. Yields of the Various Chemical Fractions Obtained from the Chloroform/Methanol Extraction of Municipal Sewage Sludges
3 6 Dry Weight of S1ud;e S o l i d s E x t r a c t w i t h 3 0 0 4 of Chloroform-Uothanol ( 4 / 1 v o l / v o l )
w i t h 100 ml 1:l Ch1oroform:Methanol
sample
t o 150 m1 of Crude Extract
Chemical F r a c t i o n a t i o n (Figure 2)
I
Ch1oroform:Uethanol
Crude E x t r a c t ( 3 0 1 )
I
I
Figure 1. Outline of the original extraction protocol.
Z O O l l crud* Extract Extraot 3 1 ' 1 with 1 0 0 4 oP 0.1N NaCB Pool Extract
Adjnst to pE 9 , EItract 3 x 1 8 . 1 0 0 4 Diethylether, Pool !?ztr.et
Extrict 3 x + s , 0.1N RC1,
100-1
Pool Extr1ot
Acidic Compounds
Figure 2. Schematic outline of the modified fractionation protocol.
weakly acidic (3.5-12%), and strongly acidic (2.9-6.3%). These various extracts and fractions were then examined for mutagenic activity by several test systems described in subsequent sections of this report. Materials and Methods. (1) Microbial Assay. The Salmonella/microsome reversion assay employing Ames tester strains TA98 and TAlOO was conducted as described in our previous report (2) where the top agar plate incorporation procedure described by Ames et al. (8) is employed by incorporating the recommendations of deSerres and Shelby (9). The materials were tested for mutagenic 910
Environ. Sci. Technol., Vol. 18, No. 12, 1984
fraction
CHMP neutral basic weak acid strong acid CHI neutral basic weak acid strong acid HNSD neutral basic weak acid strong acid SAUG neutral basic weak acid strong acid
total vol of fraction, mL
total amount of residue, mg
% of total
199 79.5 38.2
300 300 300 300
59.8 23.8 11.5 164.8
23.0 9.2 4.4 63.4
294 53.2 26.4 25.0
300 300 300 300
88.3 15.9 7.9 7.5
73.8 13.3 6.6 22.5
1020 17.9 38.2 31.6
300 300 300 300
305 5.4 11.5 9.6
92 1.6 3.5 2.9
525 19.4 51.8 33.6
300 300 300 300
158 5.8 15.5 10.1
83.4 3.1 8.2 5.3
concn, MlmL
activity both with and without the addition of the hepatic S-9 fraction prepared from male Sprague-Dawley rats induced with either Aroclor 1254 or phenobarbital as described by Ames et al. (8). (2) Tradescantia Micronucleus Test. The Tradescantia paludosa micronucleus test measures chromosome damage in tetrads following meiosis of the pollen grain mother cells ( 1 0 , I I ) . A micronucleus may result from the induction of a multipolar nuclear division or by chromosome aberrations that result in acentric fragments. The fragment of broken chromosome can be observed as a separate micronucleus within the tetrad. A figure illustrating a tetrad containing micronuclei was presented in our earlier report (2). Inflorescencesof Tradescantia clone 03 plants were used throughout this study. The use of a single clone ensured that all of the inflorescences were isogenic. The procedures for exposing the inflorescences and scoring the micronuclei was presented by Hopke et al. (2). The advantage of this assay is that the whole sludge can be directly tested for activity. Dilutions can be made with Hoagland's solution for the more active sludges. Concentration of the less active sludges can be obtained with vacuum distillation so that a range of doses can be administered. (3) Maize wx Locus Assay. The wx locus assay using inbred strains of Zea mays was discussed in detail in our earlier report (2). The forward mutation test was conducted to detect a broader range of mutational events and to allow the administration of high concentrations of sludge. Inbred Early-Early Synthetic was chosen because its convenient growth characteristics permit its use in plant
Table 111. Mutagenicity of Crude Chloroform/Methanol Extracts in the Absence and Presence of 5-9
sample
vol, pL
CHMP
100 100
CHI
HNSD
KANK
SAUG
100 100
100 100
100 100
concn factor 0.1 1.0 10.0 10.0 30.0 0.1 1.0 10.0 10.0 30.0 0.1 1.0 10.0 10.0 30.0
0.1 1.0 10.0 10.0 30.0
100
100
0.1 1.0 10.0 10.0 30.0
equiv vol, pL concn, pg/mL 100 10 100 1000 1500 3000 100 10 100 1000 1500 3000 100 10 100 1000 1500 3000 100 10 100 1000 1500 3000 100 10 100 1000 1500 3000
0 650
0 770
0 470
0 1250
0 1390
mass per plate, pg 0.0 6.5 65.0 650.0 975.0 1900.0 0.0 7.7 77.0 770.0 1155.0 2300.0 0.0 4.7 47.0 470.0 705.0 1400.0 0.0 12.5 125.0 1250.0 1875.0 3750.0 0.0 13.9 139.0 1390.0 2086.0 4170.0
TA98,” revertants per plate -s-9 +S-9‘ +S-9d 25 27 26 36
59 55 57 43
32 40 48 31
Ke 20 21 18 26
K 57 64 58 35
34 40 32 39 62
K 28 22 36 45f
28 75 70 71 76
42 40 47 55 57
4lf 28 24 28 18
58 63 44 37 54
63
K 28 29 608 3778
K 63 79 214 7418
11368
33638
TA100,b revertants per plate -s-9 +S-9‘ 80
119
88 95 86 66 58
107 113 129 84 93
59 45 31 31 99
71 63 98 50 152
97 74 85 55 100
132 151 157 95 131
98 39 30 K 85
105 43 31 K 165
94 115 116 82
199r 5318 6858 7088
Results from duplicate plates per treatment. *Results from six plates per treatment. Aroclor-inducedS-9. Phenobarbital-induced S-9. K absence or partial absence of bacterial lawn due to killing. fsignificantly greater than control, p I 0.05. #Significantly greater than control, p 5 0.01. (I
e
growth chambers where well-controlled conditions can be assured. The genetic end point of this assay is the forward mutation at the wx locus in pollen grains. The plants used in this test were homozygous for the dominant Wx allele. Mutation is to the recessive wx allele, and the presence of tan-staining pollen grains after treatment with iodine is the visual end point. Inbred Early-Early Synthetic 2. mays kernels were obtained from sibling crosses and are homozygous for the dominant Wx allele (12). Five plants were grown as a control group in individual 10-cm diameter plastic pots filled with a standard soil mixture and watered with deionized water. Standard potting soil consists of four parts of loam, two parts of peat moss, and one part of sand. Once a week 50 mL of a 20/20/20 nitrogen/phosphorus/potash fertilizer solution was added to each pot. Three treatment groups of five plants each were prepared with differing amounts of sludge added to the initial soil mixture. Only Chicago and Champaign sludges were tested with this assay. For the highest treatment group, one part sludge was added to two parts of standard soil. These plants were watered with a 113 dilution of sludge in deionized water. For the next treatment group, one part of sludge was added to five parts of soil. The plants were watered with a 116 dilution of sludge. In the lowest treatment group, 11 parts of soil were combined with one part of sludge, and these plants were watered with a 1/12 sludge/deionized water solution. The treatment groups were grown in a separate chamber from the control plants. The chambers were set for a 17-h photoperiod of 300 PEinsteins m-2 s-l with day and night temperatures of 25 and 20 OC, respectively. The plants in the treatment
groups were watered with the appropriately diluted sludge until tassel emergence and then watered with deionized water only. A t early anthesis the tassels were harvested, labeled, and stored in 70% ethanol for subsequent analysis. The pollen grains were then analyzed in the same manner as described previously (2).
Results and Discussion Microbial Assays. The crude extracts from all five samples, Champaign (CHMP), Chicago (CHI), Hinsdale (HNSD), Kankakee (KANK), and Sauget (SAUG), were examined for mutation induction. The data are presented in Table 111. The table shows the method of preparing the plates. In these experiments the volume of a MezSO solution of a relative concentration shown was pipetted onto the plate giving an equivalent volume of original extract added to the plate. From the mass concentration of the extract, the mass of materials added to each plate was calculated. In the case of the TA98, duplicate plates were prepared and counted while six plates at each treatment level were employed in the TAlOO assays. The results for each treatment level were then compared with the corresponding concurrent controls to determine if the mean values were different at a given level of probability of random occurrence (0.05 or 0.01). The values that are signficantly different are so marked. The Champaign, Chicago, Hinsdale, and Kankakee crude extracts did not induce either a doubling over the control or a dose-dependent increase in the number of revertants per plate. Only for the Hinsdale extract in the absence of S-9 was the number of TA98 revertants statistically greater than the controls at the 0.05 probability Environ. Scl. Technol., Vol. 18, No. 12, 1984
911
Table IV. Mutagenicity of Champaign Municipal Sewage Sludge Fractions with and without S-9
sample neutral
bases
weak acids
strong acids
equiv vol, p L
mass per plate, pg
0 50 1000 3000 6000 0 50 1000 3000 6000 0 50 1000 3000 6000 0 50 1000 3000 6000
0 10.0 199.0 597.0 1194.0 0 4.0 79.5 238.5 477.0 0 1.9 38.2 114.6 229.2 0 27.5 550.0 1650.0 3300.0
TA98,” revertants per plate -s-9 +s-9c +S-9d
TA100,* revertants per plate -s-9 +s-9c
24 29 26 27 19 24 25 34 64O 97e 24 27 40 50 60 24 32 34 71 116
141 144 123 145 104 153 143 367f 45d 574f 142 144 159 515’ 289f 155 143 483f 78d 833f
53 34 52 48 39 53 47 54 51 99 53 58 54 88 88 53 44 58 109 117e
32 36 32 39 41 32 36 59 50 69 32 40 42 67f 7If 32 32 41 72f
loaf
175 148 123 189 200 177 162 151 216e 216e 167 182 126 228 227 167 187 220e 198 288O
Results from duplicate plates per treatment. Results from six plates per treatment. Aroclor-induced S-9. Phenobarbital-inducedS-9. OSignificantly greater than control, p I0.05. fsignificantly greater than control, p I0.01. (I
Table V. Mutagenicity of Chicago Municipal Sewage Slud
sample neutral
bases
weak acids
strong acids
equiv vol, p L
mass per plate, pg
0 50 1000 3000 6000 0 50 1000 3000 6000 0 50 1000 3000 6000
0 14.7 294.0 882.0 1764.0 0 2.7 53.2 160.0 319.0 0 1.3 26.4 79.2 158.4 0 1.3 25.0 75.0 150.0
0
50 1000 3000 6000
Fractions with and without S-9 TA98,” revertants per plate -s-9 +S-9‘ +S-9d
TA1OO,brevertants per plate -s-9 +S-9‘
16 19 17 26 14 16 23 26 66O 58e 16 22 15 52O 38 16 20 25 49 53f
90 113 103 109 84 136 148 175O 267f 533f 104 101 59 287f 21d 151 143 238f 374f 599f
49 57 64 60 53 49 63 59 70 76 49 65 65 87 66 49 64 67 94e 99e
40 39 45 37 36 40 22 34 40 40 40 36 34 41 41 40 31 37 36 63
131 122 122 245e 238e 152 124 114 138 160 114 127 79 158 143 131 119 130 203f 204e
Results from duplicate plates per treatment. *Results from six plates per treatment. Aroclor-induced S-9. Phenobarbital-inducedS-9. OSignificantly greater than control, p I0.05. f Significantly greater than control, p I0.01.
level. The Kankakee extract was quite toxic to both strains of bacteria, and therefore, mutagenic effects could not be determined. Therefore, these crude extracts were not strongly mutagenic. Similarly, these extracts did not demonstrate a signficant response with the addition of either type of S-9 in either bacterial strain. The Sauget crude extract was very mutagenic in TA98 both with and without S-9 mix. However, it was only mutagenic to TAlOO with the addition of the S-9 mix. At least a doubling over the control in the number of TA98 revertants per plate was induced by 139 vg of Sauget crude extract residue per plate in the absence and presence of Aroclor-induced S-9. For TA100, only with S-9 was a significant increase in the revertants per plate observed. The slopes of the responses indicate that the Sauget crude extract residue induced 270 TA98 revertants/mg without S-9, 780 TA98 revertants/mg with Aroclor-induced S-9, 912
Environ. Sci. Technol., Vol. 18, No. 12, 1984
and 137 TAlOO revertants/mg with the Aroclor-induced S-9. The presence of Aroclor-induced S-9 enhanced the yield of revertants per milligram of residue. These values are among the higher values reported by Babish et al. (3). The highest direct-acting response they observed was 104 revertants of TA100/mg of extract of sludge from Buffalo, NY. The highest activated response was 264 revertants of TA100/mg of extract of sludge from Philadelphia, P A (Northeast). The TA98 response for the Sauget crude extract was much larger, and the S-9 activated response was almost 3 times greater than the highest reported TAlOO response on a revertant per milligram basis. Thus, the Sauget sludge is one of the most mutagenic sludges yet reported for both direct-acting and activatable mutagenicity. The results of the mutation assays using TA98 and TAlOO with each separated subfraction are presented in
Table VI. Mutagenicity of Hinsdale Municipal Sewage Sludge Fractions with and without S-9 TA100,b revertants per plate
TA98,’ revertants per plate
sample neutral
bases
weak acids
strong acids
equiv vol, rL 0 50 1000 3000 6000 0 50 1000 3000 6000 0 50 1000 3000 6000
0 50 1000 3000 6000
e
mass per plate, pg
-s-9
+s-9c
+S-9d
-s-9
+s-9c
0 51.0 1020.0 3060.0 6120.0 0 0.9 17.9 53.7 107.4 0 1.9 38.2 114.6 229.2 0 1.6 31.6 94.8 189.6
32 30 43 32 39 32 31 31 58e 58e 32 24 36 82e
85 93 100 116 106 85 95 95 93 103 85 84 90 105 113 85 83 72 97 112
40 47 36 43 60 40 32 35 43 65 40 39 39 66e 67f 40 41 49 67
143 152 144 170 146 136 143 205f 271f 471f 143 160 1971 545f 711f 143 164 409’ 471f 653’
183 163 191 211 225 183 169 181 187 150 178 188 191 225 242 178 191 210 242f 240‘
79’ 32 32 32 69 94
a Results from duplicate plates per treatment. Results from six plates per treatment. Aroclor-inducedS-9. dPhenobarbital-induced S-9. Significantly greater than control, p I 0.05. f Significantly greater than control, p I 0.01.
Tables IV-VII. The equivalent volumes and mass per plate were calculated as in Table 111. Similarly, significant levels were also calculated and reported. The Kankakee crude extract formed an emulsion that could not be separated, and thus results are not available for individual fractions. The Sauget fractions were not tested with phenobarbitol-induced S-9. For the Champaign, Chicago, and Hinsdale extracts, only a few of the doses used elicited a statistically significant response in TA98. It should be noted that there are several instances of apparent doublings of the control frequency where that value was still not statistically significant because of the rarge variance present within a treatment level. For all three of these sludges, even the statistically significant increases only indicated weak activity mutagenic to TA98 present in these sludges. A substantially higher response is observed in TAlOO particularly for direct-acting mutagens. It appears that for the Champaign, Chicago, and Hinsdale fractions, S-9 deactivates the mutagenicity in these samples. The basic and acidic fractions all induce statistically significant mutagenicity that is much greater than the apparent activity of the crude extract. There would seem to be antagonistic or toxic effects that reduce the observable effect of the crude extract. Separation into fractions permits the activity to be expressed. All of the Sauget fractions induced a mutagenic response in TA98 and TAlOO (Table VII). Two types of dose-dependent responses induced by the Sauget fractions in the absence or prescence of Aroclor-induced S-9 are shown in Figures 3 and 4. In Figure 3, the neutral fraction results show the increased level of mutagenic activity in the presence of the S-9 mix. In contrast, Figure 4 shows that, for the weakly acidic fraction, the addition of S-9 substantially deactivates the mutagens present. A similar deactivation pattern clearly holds for the strongly acidic fraction while the basic fraction gives a pattern indicative of toxicity. The Sauget fractions show a quite different pattern of mutagenicity than does the crude extract. The crude extract showed substantial activation for both TA98 and TA100. For the fractions, only the neutral fraction shows this pattern of activatable mutagenicity. The other frac-
1 SAUGET ----
+
NEUTRAL
0
/
s-9 s-9
, 800 a,
a cn + C
/
/
/
/
/
1
L
a,
’ a,
[y.
0
/
/
/ /
1 0
z
I
I
1000
2000
-
3000
Residue P e r Plate (pg) Figure 3. Dose-response curves for the Sauget neutral fraction in the absence and presence of S-9 activation in strain TA100. 700
a
0
1 0
100
200
300
Residue P e r P l a t e (pug) Figure 4. Dose-response curves for the Sauget weak acid fraction in the absence and presence of S-9 activation in strain TA100.
tions show deactivation particularly in the TAlOO strain. However, since the neutral fraction has the vast majority of the mass, it apparently controls the properties of the crude extract. On a revertant per milligram of extract basis, the basic fraction was the most mutagenic to TA98 with a value of 1910 revertants/mg without S-9 and 1650 revertants/mg with the S-9 mix. The weak and strong acid fractions were Environ. Sci. Technol., Vol. 18, No. 12, 1984
913
Table VII. Mutagenicity of Sauget Municipal Sewage Sludge Fractions with and without S-9 TA98," revertants
per plate sample
equiv vol, p L
neutral
mass per plate, pg
-S-9
+S-gC
equiv vol, p L
mass per plate, pg
0 26.3 52.5 262.5 525.0 0 1.0 2.0 9.9 23.6 0 2.6 5.2 25.9 62.2 0 1.7 3.4 16.8 40.3
19 21 45' 64' 98' 19 20 29 43d 65' 19 24 26 33 62e 19 20 22 31 55d
50 18d 96' 197' 282' 50 59 56 64' 93d 50 7 5d 52 6gd 77' 50 44 52 56 68O
0 50 1000 3000 6000 0 50 1000 3000 6000 0 50 1000 3000 6000 0 50 1000 3000 6000
0 26.3 525.0 1575.0 3150.0 0 1.0 19.7 59.1 118.2 0 2.6 51.8 155.4 310.8 0 1.7 33.6 100.8 201.6
0
bases
weak acids
50 100 500 1000 0 50 100 500 1200 0 50 100 500 1200
strong acids
0 50 100 500 1200
148 138 161 199' 234e 129 152 98 283' 155 147 155 21ge 436' 65ge 151 119 259O 317O 457'
Results are from triplicate plates per treatment group. Results are from six plates per treatment group. Significantly greater than control, p 5 0.05. e Significantly greater than control, p 5 0.01. (I
Table VIII. Results of Different Whole Sludge Samples from Five Cities with the Tradescantia Micronucleus Test sample control +control (50 mM MH)c Chicago sludge Kankakee sludge Hinsdale sludge Champaign sludge sample 1 sample 2 Sauget sludge"
no. of MCN
MCN/100 tetrads
6174 8887 8071 7048 6333
180 972 515 347 314
2.92 f 0.55 10.94 f 1.42 6.38 f 1.61 4.92 f 0.80 4.48 f 0.74
control +control (50 mM MH)" Kankakee sludge
8225 6460 382b
342 282 114
4.16 f 0.57 4.37 f 0.57 29.8
control +control (50 mM MH) Hinsdale sludge
most active in TAlOO with values of 1680 and 1540 revertantslmg without s-9,respectively. With the s-9 present, the activity drops to 260 and 160 revertantslmg, respectively. Again these values must be contrasted with the much lower apparent activity of the crude extract. This is particularly true for TAlOO where the crude extract without S-9 shows no mutagenicity while the basic and acid fractions show strong direct mutagenic activity. It appears that caution may be needed in interpreting low apparent activity of unfractionated extracts as indicated by the lack of mutagenicity in the sample. Tradescantia Assay. It was previously reported (2) that whole Chicago sludge showed a significant increase in the frequency of induced micronuclei in Tradescantia for both whole sludge and a 1/1 dilution with Hoagland's solution. To provide a comparison with the other sludges, the result for the Chicago samples is presented in Table VIII. The value of micronuclei/lOOtetrads is the average over a number of slides. Each slide is prepared from a single inflorescence and typically is counted for 500-1000 tetrads. The number of slides per sample is typically six to eight from which the mean and standard errors are calculated. For the Sauget sample, only one slide was used for reasons discussed below. 914
Environ. Sci. Technol., Vol. 18, No. 12, 1984
174 194d 445e 690' 988' 156 150 132 186 124 167 162 182 232' 2380 167 177 184 190 206
Aroclor-induced S-9.
Table IX. Results of Concentrated Sludge Samples from Several Cities with the Tradescantia Micronucleus Test
no. of tetrads
Sludge was reconstituted to contain 76% of the solids found in the original whole sludge. bMeiosis stopped in 9 of 10 inflorescences, limiting the number of observable tetrads. Maleic hydrazide.
TA1OO,b revertants per plate -S-9 +5-9c
sample
6.7X 13.3X 20.0x
6.7X 13.3X 20.0x
Champaign sludge 2x 4x
control Sauget sludge 0.25X 0.50X 0.76X
no. of tetrads
no. of MCN
MCN/100 tetrads
5297 7989
200 445
3.8 f 0.60 5.6 f 1.35
5486 6408 3027
348 404 298
6.3 f 0.88 6.3 i 0.76 9.8 f 2.71
10449 15032
539 1263
5.2 f 0.63 8.4 f 1.57
10030 6271 7202 14135 10263 10051
610 319 447 1059 460 533
6.1 f 0.86 5.1 f 1.00 6.2 f 1.10 6.6 f 0.78 4.5 f 0.57 5.3 1.03
3833
104
2.7 f 0.68
1607 2289 382
126 309 114
7.8 f 0.73 13.5 f 2.24 29.8
*
Maleic hydrazide.
Two samples of whole Champaign sludge were assayed for their ability to induce micronuclei. Neither sample induced a significant increase in cytogenetic damage (Table VIII). Whole sludge was concentrated by lyophilization. The solid sludge was then mixed with Hoagland's solution at rates of 0.3 g14.7 mL and 0.6 gl4.7 mL which corresponded to approximately 2X and 4X concentrations, respectively. The results of these experiments are presented in Table IX, and none of the Champaign sludge samples induced a significant increase in the frequency of micronuclei compared to the negative control for the Champaign and Hinsdale samples that were analyzed simultaneously. Whole sludge samples were assayed, and a negative response was observed (Table VIII). The Hinsdale sludge was concentrated by lyophilization. The sludge solids were mixed with Hoagland's solution at rates of 1g/5 mL, 2 g/5
mL, and 3 g/5 mL which corresponded to 6.7X, 13.3X, and 20.0X concentrations, respectively. The data for the concentrated Hinsdale sludge are presented in Table IX. The concentrated Hinsdale sludge samples did not significantly increase the induction of micronuclei. These data agree with the results obtained with the unconcentrated samples of Hinsdale sludge. Kankakee whole sludge samples were assayed and found to be negative (Table VIII). Whole sludge was concentrated by lyophilization, and 6.7X, 13.3X, and 20.0X concentrations in Hoagland’s solution were prepared and assayed. The results are presented in Table IX, and only the 20X sludge concentration induced a positive response. Thus, it appears that a low concentration of mutageds) is present in Kankakee sludge that can induce chromosome damage in meiotic cells of Tradescantia. The Sauget sludge was difficult to score for micronuclei because it was so toxic, it inhibited meiosis. The sample was lyophilized and the dry sludge was reconstituted with distilled water and 0.3 mL of dimethyl sulfoxide. Samples of 3.53, 7.07, and 10.6% dry weight solids were prepared. The original sludge contained 13.9% dry weight solids. The 10.6% dry weight samples were so toxic that meiosis was totally inhibited in 9 of the 10 inflorescences and tetrads were scored in only 1 of the inflorescences. Consequently, there were very few tetrads available for scoring. Of those that were found, a very high value of the frequency of micronuclei was found (Table VIII), once again indicating that this sludge is much more mutagenic than any of the other samples including Chicago (Table 1x1. Two other dilutions of the sludge also gave substantially enhanced frequencies of micronuclei induction. wx Locus Assay. The results of the induction of forward mutation at the wx locus in Early-Early Synthetic maize for the Chicago sludge sample were presented previously (2) and clearly showed that the administration of Chicago sludge increased the frequency of mutation by 2 orders of magnitude. The frequencies of mutant pollen grains decreased with increasing amounts of added sludge, indicating that even the lowest concentration of applied Chicago sludge was sufficient to reach the toxic region of the mutational dose-response curve. The percentage of pollen abortions was higher at all of the sludge concentrations compared to the control group. The control frequency was 13.3% while it was 19.9% in the highest treatment group. There was, however, no dose dependence to the abortion data. There was a clear indication that Chicago sludge induced forward mutation in the pollen grains with a substantial increase in frequency over the control group value. The increased abortion rate indicated the presence of toxic as well as mutagenic agents. Within the statistical limits of the experiment, the Champaign sludge sample did not induce an increased frequency of forward mutations in this assay. These results are in agreement with the Tradescantia assay that showed that whole Champaign sludge did not contain activity that can induce micronuclei. The microbial assays demonstrated weak mutagenicity in the basic and acidic fractions that do not seem to be able to be translocated into the plante and cause mutations.
Conclusions This study has determined that mutagenic activity is present in some municipal sewage sludges. Sludges from Champaign, Chicago, Hinsdale, Kankakee, and Sauget, IL, were studied. The Chicago and Sauget municipal sewage sludge samples clearly have components that induce a variety of mutagenic responses. Their sludge or sludge extracts induced a response in all of the species employed.
The Champaign, Hinsdale, and Kankakee sludge samples showed direct-acting mutagenicity and deactivation following S-9 activation in the TAlOO Ames Salmonella assay with samples obtained by the chemical fractionation of the chloroform/methanol extracts. Much lower activity was observed with strain TA98. The Sauget sample demonstrated substantial direct-acting mutagenic activity in both strains and strongly activatable mutagenicity in the neutral fraction. The other three Sauget fractions were deactivated by the S-9 enzymes. The Tradescantia assays clearly indicate that whole or diluted Chicago and Sauget sludges induce chromosome aberrations in meiotic cells. When whole sludge samples from each municipality were evaluated, a pattern of response emerged. A ranking in order of decreasing mutagenic response is that of Sauget, Chicago, Kankakee, Hinsdale, and Champaign. When concentrated, whole sludge samples are evaluated, the same ranked order is maintained. However, the Sauget sludge is much more potent than any of the other sludges, and Chicago sludge is more mutagenic than the Kankakee sludge samples. A 1/2 dilution of whole Chicago sludge induced a mean value of 5.19 micronuclei (MCN)/100 tetrads as compared to a value of 6.30 MCN/100 tetrads for the 13.3X concentration of Kankakee whole sludge. Neither the Hinsdale nor Champaign sludge samples induced a signficant frequency of micronuclei in Tradescantia tetrads. It appears that plants that process only domestic wastewater have lower potency sludges than those plants with industrial influents. Laboratory studies using the wx locus in 2.mays show substantial uptake of mutagens from Chicago sludgeamended soils and no uptake of mutagens from the Champaign sludge. These studies indicate that mutagens present in sludge-amended soil are transported into a crop plant and can induce genetic damage in germ cells. These studies do not provide any indication of mutagenicity in the kernels grown on sludge-amended soil or transfer of the mutagens from the sludge to groundwater or surface water. However, these results do suggest the need for further study of the possible adverse effects of land application of sludge from industrialized cities. The separation of extracts showed that there may be effects that mask the mutagenic activity in the sample. Chemical fractionation removes some of these effects and can allow observation of much higher mutagenicity than was obtained with the whole extract. It also makes clear that some sludges from municipal sewage treatment plants like Sauget are quite genotoxic and their disposal should be made with considerable care.
Acknowledgments We would like to express our appreciation to Luther Landon of the Illinois Environmental Protection Agency for providing us with the samples from Hinsdale, Kankakee, and Sauget, IL, and to the Champaign-Urbana Sanitary District for the sample from the Champign plant. We thank Thomas Hinesly for providing us with the Chicago sludge.
Literature Cited (1) Fed. Reg. 1979,44. (2) Hopke, P. K.; Plewa, M. J.; Johnston, J. B.; Weaver, D.; Wood, S. G.; Larson, R. A.; Hinesly, T. Enuiron. Sci. Technol. 1982,16,140. (3) Babish, J. G.; Johnson, B. E.; Lisk, D. J . Enuiron. Sci. Technol. 1983,17,272-277. (4) Dion, P. W.; Bright-See, E. B.; Smith, C. C.; Bruce, W. R. Mutat. Res. 1982,102,27-37. (5) Kuhlein, H. V.; Kuhnlein, U. Nutr. Cancer 1980,2,119-124. Envlron. Scl. Technol., Vol. 18, No. 12, 1984 915
Environ. Sci. Technol. 1904, 18, 916-922
Dion, P. W.; Bruce, W. R. Mutat. Res. 1983,119,151-160. Lederman, M.; Van Tassell, R.; West, S. E. H.; Ehrich, M. F.; Wilkins, T. D. Mutat. Res. 1980,79,115-124. Ames, B. N.;McCann, J.; Yamasaki, E. Mutat. Res. 1975, 31. 347.
deserres, F.J.; Shelby, M. D. Science (Washington, D.C.) 1979.203.564.
Ma, T.Hi;Sparrow, A. H.; Schairer, L. A.; Naumann, A.
F.Mutat. Res. 1978,58,251. Ma, T.H.Mutat. Res. 1980,64, 307.
(12) Plewa, M. J.; Wagner, E. D. Environ. Health Perspect. 1981, 37,61-73.
Received for review August 18,1983.Revised manuscript received April 11,1984.Accepted May 29,1984.This work was supported by U.S. Environmental Protection Agency Grant R807009 and by Grant S-095-ILLfrom the Water Resources Center. Although supported by the Agency, this report has not been reviewed by it, and no official endorsement of this report should be inferred.
Impliclt-Adsorbate Model for Apparent Anomalies with Organic Adsorption on Natural Adsorbents Rane L. Curl’ and Gregory A. Keolelan Department of Chemical Engineering, The University of Michigan, Ann Arbor, Michigan 48109 Adsorption-desorption hysteresis, the adsorbent-concentration effect, and other apparent anomalies that have been observed in studies of adsorption of organic chemicals on natural adsorbents can be explained by an implicitadsorbate model. The model is based on the competitive adsorption, between an adsorbate under study, A, and an implicit adsorbate, B, initially on the adsorbent. During batch adsorption-desorption experiments B desorbs and uncovers sites for binding A, increasing the apparent partition coefficient. The model agrees well with several sets of experimental data from the literature, but ranges of model parameters such as the true adsorption partition coefficient for A give equivalently good agreement. The true adsorption parameters are not determinable uniquely from presently available data, in the presence of strong hysteresis or adsorbent-concentration effects. They are determinable if the implicit adsorbate could be identified and characterized.
Introduction The interaction between organic pollutants and soils and other natural adsorbents is important in determining the fate and distribution of organic pollutants in the environment. Numerous adsorption-desorption experiments have been performed and reported to characterize this interaction. In many of these experiments an aqueous solution of the organic chemical has been equilibrated with the soil or sediment and the equilibrium concentration of each phase determined. The distribution of the adsorbate between the adsorbed phase and the aqueous phase for linear partitioning is defined by the apparent partition coefficient as CA! KA* = (1) CA where CAtand CA are the adsorbed and aqueous equilibrium concentrations of A, respectively. In studies conducted to measure the apparent partition coefficients and characterize adsorption-desorption behavior, several apparent anomalies have been discovered, which have not yet been adequately explained. The three most significant anomalies are the adsorbent-concentration effect, adsorption-desorption hysteresis, and endothermicity of the adsorption process. Adsorption studies conducted by Grover and Hance ( I ) , O’Connor and Connolly (2),Di Tor0 et al. (31, and Voice et al. ( 4 ) have demonstrated the adsorbent-concentration 916
Envlron. Sci. Technol., Vol.
18, No. 12, 1984
effect. They observed linear adsorption isotherms but a decrease in the apparent partition coefficient with increasing adsorbent concentration. Figure 1 shows this dependence for typical data from O’Connor and Connolly (2) and Huang and Liao (5). The figure shows that the dependence of the apparent partition coefficient on the adsorbent concentration follows a variety of patterns. The maximum effect appears to be a near reciprocal dependence of KA* on m / V . O’Connor and Connolly (2) correlated the data with an empirical relationship but did not explain the effect. Grover and Hance (1)suggested that the adsorbentconcentration effect can be attributed to the variation in the degree of soil aggregation with soil concentration. The first quantitative model of the adsorbent-concentration effect was recently proposed by Voice et al. (4). This will be contrasted with the model developed here under Discussion. A variety of organic chemical-soil systems have been shown to display adsorption-desorption hysteresis or “nonsingularity”. Rao and Davidson (6) and Di Toro and Horzempa (7) describe a number of experimental studies demonstrating this phenomenon. In these studies batch adsorption experiments were conducted, followed by either single or consecutive desorption experiments. After initial adsorption and centrifugation steps, a portion of the supernatant was removed and replaced with adsorbate-free solution. The equilibrium concentrations of both phases were then determined. This procedure was repeated for consecutive desorption experiments. The hysteresis effect is exhibited when the equilibrium desorption points are not coincident with the apparent adsorption isotherm. Figure 2, from Swanson and Dutt (8),shows typical adsorption-desorption hysteresis, here for atrazine on Mohave soil. In the consecutive desorptions shown, each starting with a different initial atrazine concentration, more atrazine is retained by the soil than is predicted by the initial adsorption isotherm. Three suggestions for causes of hysteresis presented by Rao and Davidson (6)are (1)artifacts due to some aspects of the method, (2) nonattainment of equilibrium during adsorption, and (3) chemical and/or microbial transformation of the adsorbate during the experiment. Di Toro et al. (9) modified adsorption and desorption experiments to test these factors and concluded that they were not the cause of hysteresis. Many investigators have suggested that hysteresis is produced by irreversible adsorption (10-13, 7). Di Toro and Horzempa (7) modeled the ad-
0013-936X184/0918-0916$01.50/0
0 1984 American Chemical Society
