PB84-232560
Use of Short-Term Bioassays to Evaluate Environmental Impact of Land
Treatment of Hazardous Industrial Waste
Texas A&M University
College Station, Texas
Aug 84
U.S. DEPARTMENT OF COMMERCE
National Technical Information Service
KITS
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EPA-600/2-84-135
August 1984
USE OF SHORT-TERM BIOASSAYS TO EVALUATE
ENVIRONMENTAL IMPACT OF LAND TREATMENT OF
HAZARDOUS INDUSTRIAL WASTE
by
K. W. Brown, K. C. Donnelly and J. C. Thomas
Texas Agricultural Experiment Station
Texas A&M University
College Station, Texas 77843
Grant No. CR-807701-01
Project Officer
John Matthews
Robert S. Kerr Environmental Research Laboratory
P. 0. Box 1198
Ada, Oklahoma 74820
ROBERT S. KERR ENVIRONMENTAL RESEARCH LABORATORY
U.S. ENVIRONMENTAL.PROTECTION AGENCY
ADA, OKLAHOMA 74820
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPORT NO.
EPA-600/2-84-135
3. RECIPIENT'S ACCESSION-NO.
PB8U-232560
4. TITLE AND SUBTITLE
Use of Short-term Bioassays to Evaluate Environmental
Impact of Land Treatment of Hazardous Industrial Waste
5. REPORT DATE
August 1984
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
K. W. Brown, K. C. Donnelly, and J. C. Thomas
8. PERFORMING ORGANIZATION REPORT NO,
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Texas Agricultural Experiment Station
Texas A&M University
College Station, TX 77843
10. PROGRAM ELEMENT NO.
CBRD1A
Coop. Agree
CR807701
12. SPONSORING AGENCY NAME AND ADDRESS
R. S. Kerr Environmental Research Laboratory
P. 0. Box 1198
Ada, OK 74820
13. TYPE OF REPORT AND PERIOD COVERED
Final 9/1/80 - 11/30/83
14. SPONSORING AGENCY CODE
EPA/600/15
15. SUPPLEMENTARY NOTES
A four phase study was conducted to evaluate utility of short-term bioassays
in monitoring environmental impact of land treatment of hazardous waste. During phase
one, three microbial bioassays were conducted to define chronic toxic potential of
each waste selected for study. Acid, base, and neutral fractions of each of three
wastes studied induced genetic damage in at least two of the three bioassays.
Phase two was conducted to evaluate efficiencies of blender and soxhlet extraction
procedures, as well as potential interactions between known mutagens and soil compo-
nents. Results indicate that there was no appreciable difference in mutagenicity of
the extract using either procedure. Using the blender procedure extraction efficiency
for pure compounds added to soil averaged greater than 85%, as measured by High Pressur
iquid Chromotography.
Phase three consisted of a greenhouse study in which each of three wastes was
applied to two soils. Results from chemical analyses indicate that waste constituents
*ere degraded in soil during a 360 or 340 day interval. Increased mutagenic activity
*as exhibited in some soil and water extracts during this same interval. When compared
an an equivalent volume basis, however, mutagenic potential of waste-amended soils was
reduced over time and, in some cases, was reduced to a non-mutagenic level.
Wood-preserving bottom sediment was applied to barrel-sized lysimeters in the fina'
>roject phase to compare results of soil-core and soil-pore liquid monitoring. Differed
:vpes of compounds TJPT-O ijofoq^o^ jn EOJl-Corc 3nd Tm'1 nnrj 1i~i..i.l l"
~~ _ _ *p^^^^^T^^^^ u \J .i. ,fc, 1^"^^^W
7.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.lDENTIFIERS/OPEN ENDED TERMS C. COS AT I Field/Group
Industrial Wastes
Bioassays
Waste'Treatment
Land
Land Treatment
Operational Monitoring
Mutagenicity
Microbial Tests
68C
8. DISTRIBUTION STATEMENT
Release to public
19. SECURITY CLASS (ThisReport/
Unclassified
21. NO. OF PAGES
386
20. SECURITY CLASS (Thispage)
Unclassified
22. PRICE
EPA Form 2220-1 (9-73)
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DISCLAIMER
Although the research described in this article has been funded wholly
or in part by the U.S. Environmental Protection Agency under assistance agree-
ment CR807701 to Texas A&M University, it has not been subjected to the Agency's
peer and administrative review and therefore may not necessarily reflect the
views of the Agency, and no official endorsement should be inferred.
ii
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FOREWORD
EPA is charged by Congress to protect the Nation's land, air and water
systems. Under a mandate of national environmental laws focused on air and
water quality, solid waste management and the control of toxic substances,
pesticides, noise, and radiation, the Agency strives to formulate and imple-
ment actions which lead to a compatible balance between human activities and
the ability of natural systems to support and nurture life.
The Robert S. Kerr Environmental Research Laboratory is the Agency's
center of expertise for investigation of the soil and subsurface environment.
Personnel at the Laboratory are responsible for management of research pro-
grams to: (a) determine the fate, transport and transformation rates of
pollutants in the soil, the unsaturated zone and the saturated zones of the
subsurface environment; (b) define the processes to be used in characterizing
the soil and subsurface environment as a receptor of pollutants; (c) develop
techniques for predicting the effect of pollutants on ground water, soil and
indigenous organisms; and (d) define and demonstrate the applicability and
limitations of using natural processes, indigenous to the soil and subsurface
environment, for the protection of this resource.
This project was initiated to determine the utility of short-term
bioassays in monitoring the operation of land treatment systems. Results
indicate that selected organic hazardous wastes can be treated in land
treatment units and that bioassays can be used to monitor detoxification
of organic hazardous waste constituents. This information should prove
useful to those responsible for regulating, designing, and operating
hazardous waste land treatment systems.
Clinton W. Hall
Director
Robert S. Kerr Environmental
Research Laboratory
m
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ABSTRACT
A four phase study was conducted to evaluate the utility of
short-term bioassays in monitoring the environmental impact of the land
treatment of hazardous industrial wastes. In the waste characterization
phase, the acute toxicity of ten wastes was evaluated in bioassays using
Bacillus subtilis, Salmonella typhimurium, and haploid and
diploid forms of Aspergillus nidulans. Based on the results of the
acute toxicity study and a chemical characterization, three wastes were
selected for an evaluation of chronic toxicity. The acid, base, and
neutral fractions of each of the three wastes induced genetic damage in
at least two of the three bioassays.
In the second phase, the additive effect of 2-nitrofluorene or
benzo[a]pyrene to the soil was evaluated, using both the prokaryotic
point mutation assay and high performance liquid chromatography. The
results from this portion of the study have been used to evaluate the
efficiency of the blender and soxhlet extraction procedures, as well as
the potential interactions between known mutagens and soil components.
The results indicate that while greater quantities of hydrocarbons were
extracted using the Soxhlet method, there was no appreciable difference
in the mutagenicity of the extract using either procedure. In addition,
when pure compounds were added to the soil, the extraction efficiency
averaged greater than 85%, as measured by HPLC; while there was no
statistical difference in the mutagenicity of the pure compound or the
extract of the soil plus the compound.
Phase three consisted of a greenhouse study in which each of three
wastes was applied to two soils. Soil, plant, and runoff samples were
collected at various times over a 360 or 540 day interval. The results
from chemical analysis indicate that waste constituents were degraded in
soil; however, the bioassay results indicate that the degradative
process may have increased the mutagenic potential of soil and water
extracts, as well as converting indirect acting mutagens to direct
acting compounds.
Barrel-sized lysimeters were used in the final phase of the project
to compare soil-core and soil-pore liquid monitoring. A wood-preserving
bottom sediment waste was applied to three lysimeters. Three additional
lysimeters to which no waste was applied served as a control. Leachate
and soil samples were collected prior to, 30, and 90 days after waste
application. The results from this portion of the study indicate that if
a land treatment facility is not properly managed, mutagenic
iv
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constituents from land applied waste may migrate through the soil. The
results from this research have demonstrated that hazardous industrial
wastes may contain significant quantities of mutagenic materials. Soil
incorporation of three wastes resulted in the transformation or
degradation of mutagenic materials, although these reactions did not
always reduce the mutagenic activity of soil extracts. In addition, for
the waste, soil, and loading rate evaluated in the lysimeter study,
attenuation of waste constituents was not sufficient to prevent
mutagenic compounds from migrating to a depth of 90 cm. This research
has demonstrated that short-term bioassays can be used to trace the
environmental fate of mutagenic constituents in land applied hazardous
industrial wastes.
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CONTENTS
Abstract
Figures vi
Tables xviii
Acknowledgements xxvi
1. Introduction 1
2. Conclusions 5
3. Recommendations 7
4. Biological Analysis 8
Introduction 8
Differential DNA Repair 9
Test System Description 9
Test System Protocol 9
Test System Results 11
Point Mutations: Prokaryote 15
Test System Description 15
Test System Protocol 18
Test System Results 21
Point Mutations and Chromosome Damage: Eukaryote 36
Test System Description 36
Test System Protocol 37
Test System Results 44
Evaluation of the Battery of Bioassays 48
5. Waste Characterization 50
Introduction 50
Materials and Methods 51
Wastes 51
Extraction 55
Chemical analysis 57
Biological analysis 57
Results and Discussion 61
6. Soil Characterization 135
Introduction 135
Materials and Methods 136
Soil 136
Biological analysis 137
Results and Discussion 138
7. Quantification of Soil Extraction 155
Introduction 155
Materials and Methods 156
Selection of Mutagenic Compounds and Soils 157
Soil Preparation and Chemical Addition 157
vi
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Extraction Procedure and Sample Preparation 159
Bioassay 159
High Performance Liquid Chromatography 160
Results and Discussion 160
8. Mutagenic Activity of Runoff Water From Hazardous
Waste Amended Soil 176
Introduction 176
Materials and Methods 176
Waste 176
Soil 177
Greenhouse boxes 177
Extraction procedures 180
Chemical analysis 180
Biological analysis 181
Results and Discussion 182
9. Affect of Degradation on the Mutagenic Activity of
Waste Amended soil 217
Introduction 217
Materials and Methods 218
Waste 218
Soil 218
Extraction Procedures 218
Chemical analysis 218
Soil Sample Collection 218
Biological analysis 219
Results and Discussion 220
10. Soil Mobility and Degradation of Mutagenic Constituents
from a Wood-Preserving Bottom Sediment 321
Introduction 321
Materials and Methods 321
Soil 321
Waste 323
Lysimeters 323
Leachate Sample Collection 323
Soil Sample Collection 325
Biological analysis 325
Results and Discussion 325
11. Literature Cited 340
Appendices (on file at Ada, Oklahoma).
A. Quality Assurance Program
B. Standard Operating Procedures
1. Biological
2. Chemical
3. Agricultural (Greenhouse)
C. Raw Data
1. Waste characterization
2. Greenhouse study
3. Lysimeter study
4. Extraction study
5. Aspergillus
D. Computer Programs
vii
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FIGURES
Number Page
1 Potential effects of environmental mutagens on human
cells 3
2 Lethal effect of increasing doses of benzo(a)pyrene on
repair proficient (168 wt) and deficient strains of
B.subtilis with metabolic activation 16
3 Lethal effect of increasing doses of 2-aminoanthracene
on repair proficient (168 wt) and deficient strains of
B.subtilis with metabolic activation 17
4 Variability of mutagenic activity of TA100 induced by MMS. . . 27
5 Variability of mutagenic activity of TA100 induced by MNNG. . . 28
6 Variability of mutagenic activity of TA98 induced by 2NF. ... 30
7 Variability of mutagenic activity of TA100 induced by B(a)P. . .31
8 Variability of mutagenic activity of TA100 induced by 2AA. . . .32
9 Variability of mutagenic activity of TA98 induced by B(a)P. . . 33
10 Variability of mutagenic activity of TA98 induced by 2AA. ... 34
11 Genotype of Aspergillus nidulans diploid 20 43
12 Fractionation scheme used for waste and waste-amended soils . . 56
13 Percent survival of JB. subtilis strain 168, j>.
typhimurium strain TA100, and A. nidulans diploid 109,
after exposure to (a) wood-preserving bottom sediments waste,
(b) acetonitrile purification column waste, (c) wood
preserving liquid waste, (d) slop-oil emulsion solids waste . . 62
14 Percent survival of IJ. subtilis strain 168, j>.
typhimurium strain TA100, and A. nidulans diploid 109,
after exposure to (a) dissolved air floatation float waste,
(b) methyl ethyl ketone waste,(c) storm-water runoff impound-
ment waste, (d) combined API separator/waste-water treatment
sludge waste 63
viii
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FIGURES (continued)
Number page
15 Mutagenicity, as measured with j>. typhimurium
strain TA98, of fractions of acetonitrile waste 65
16 Mutation frequency induced by neutral fraction of
methyl ethyl ketone (MEK) waste in A. nidulans
methionine system 69
17 Growth inhibition induced by fractions of methyl ethyl
ketone waste in DNA repair proficient (168 wt) and
deficient strains of IJ. subtilis 71
18 Mutagenic activity, as measured in j>. typhimurium
strain TA98, of fractions of methyl ethyl ketone waste
(MEK) 72
19 Mutagenic activity, with metabolic activation, of crude
(A), acid (B), base (C), and neutral (D) fraction of
PENT S (wood preserving bottom sediment) from two blender
extractions and one soxhlet extraction 74
20 Mutagenic activity of fractions of the PENT S (wood
preserving bottom sediment) waste 78
21 Mutagenic activity, as measured in S_. typhimurium
strain TA100, of fractions of PENTS (wood preserving
bottom sediments) waste 79
22 Mutagenic activity of acid fraction of PENT S waste as
measured with high, medium, and low levels of S9 in the
S9 mix 82
23 Mutagenic activity of base fraction of PENT S waste as
measured with high, medium, and low levels of S9 in the
S9 mix 83
24 Mutagenic activity of neutral fraction of PENT S waste as
measured with high, medium, and low levels of S9 in the
S9 mix 84
25 Growth inhibition induced by fractions of the wood
preserving bottom sediment in repair proficient (168 wt)
and deficient strains of B. subtilis 87
~~^^^^^^~ *
26 Induced mutation frequency and fractional survival in A.
nidulans following exposure to acid fraction of PENT S
waste 89
ix
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FIGURES (continued)
Number Page
27 Induced mutation frequency and fractional survival in
A. nidulans following exposure to base fraction of
PENT S waste 90
28 Induced mutation frequency and fractional survival in
A. nidulans following exposure to neutral fraction of
PENT S waste 91
29 GC/MS chromatograph of acid fraction of PENT S waste 100
30 GC/MS chromatograph of base fraction of PENT S waste 101
31 GC/MS chromatograph of neutral fraction of PENT S waste. . . . 102
32 Mutagenic activity of fractions of SWRI waste 106
33 Mutagenic activity of subfractions of neutral fraction
of SWRI waste 107
34 Induced mutation frequency and fractional survival in A.
nidulans following exposure to acid fraction of SWRI
waste 112
35 Induced mutation frequency and fractional survival in A.
nidulans following exposure to base fraction of SWRI
waste 113
36 Induced mutation frequency and fractional survival in A.
nidulans following exposure to neutral fraction of SWRI
waste 114
37 Mutagenic activity of fractions of COMBO waste 117
38 Mutagenic activity of subfractions of neutral fraction of
COMBO waste 119
39 Mutagenic response of base fraction of COMBO waste using
metabolic activation from Aroclor 1254 or phenobarbitol
induced rat liver 120
40 Induced mutation frequency and fractional survival in A.
nidulans following exposure to acid fraction of COMBO
waste 124
41 Induced mutation frequency and fractional survival in A.
nidulans following exposure to base fraction of COMBO
waste 125
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FIGURES (continued)
Number Page
42 Induced mutation frequency and fractional survival
in A. nidulans following exposure to neutral
fraction of COMBO waste 126
43 GC/MS chromatograph of acid fraction of COMBO waste 128
44 GC/MS chromatograph of base fraction of COMBO waste 129
45 GC/MS chromatograph of neutral fraction of COMBO waste. . . . 130
46 Mutagenic activity of organic extract of three agricul-
tural soils as measured with J3. typhimurium, strain
TA98, with metabolic activation 140
47 Mutagenic activity of organic extract of three agricul-
tural soils as measured with £5. typhimurium, strain
TA100, with metabolic activation 141
48 Mutagenic activity of one gram of soil as compared to
cigarette smoke condensate 142
49 Mutagenic activity of organic extract of three agricul-
tural soils as measured with j>. typhimurium, strain
TA98, without metabolic activation 144
50 Induced mutation frequency and fractional survival in
A. nidulans following exposure to organic extract of
Bastrop soil 146
51 Induced mutation frequency and fractional survival in
A. nidulans following exposure to organic extract of
Norwood soil 147
52 GC/MS chromatograph of organic extract of Norwood soil .... 150
53 GC/MS chromatograph of organic extract of Bastrop soil .... 151
54 Extraction efficiency, as measured with j>. typhimurium
strain TA98, of 2-nitroflourene from Norwood soil 162
55 Extraction efficiency, as measured with J5. typhimurium
strain TA98, of 2-nitroflourene from Bastrop soil 163
56 Extraction efficiency, as measured with J3. typhimurium
strain TA98, of benzo(a)pyrene from Norwood soil 166
xi
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FIGURES (continued)
Number Pag£
57 Extraction efficiency, as measured with j>. typhimurium
strain TA98, of benzo(a)pyrene from Bastrop soil 167
58 HPLC chromatograph from (A) dimethylsulfoxide and (B)
Norwood soil 169
59 HPLC chromatograph from (A) dimethylsulfoxide and (B)
Bastrop soil 170
60 HPLC chromatograph from (A) 5.0 mg/ml 2-nitroflourene,
and (B) Norwood soil +5.0 mg/ml 2-nitrof lourene 171
61 HPLC chromatograph from (A) 5.0 gm/ml 2-nitroflourene,
and (B) Bastrop soil + 5.0 mg/ml 2-nitrofluorene 172
62 HPLC chromatograph from (A) 5.0 gm/ml benzo(a)pyrene, and
(B) Norwood soil + 5.0 mg/ml benzo(a)pyrene 173
63 HPLC chromatograph from (A) 5.0 gm/ml benzo(a)pyrene, and
(B) Bastrop soil + 5.0 mg/ml benzo(a)pyrene 174
64 Schematic diagram of greenhouse boxes used in degradation
study 179
65 Mutagenic activity with metabolic activation of runoff
water from PENT S amended Norwood soil 183
66 Mutagenic activity without metabolic activation of runoff
water from PENT S amended Norwood soil 186
67 Mutagenic activity with metabolic activation of runoff
water from PENT S amended Bastrop soil 187
68 GC/MS chromatograph of organic extract of runoff water
from unamended Bastrop soil collected on day 360 190
69 GC/MS chromatograph of organic extract of runoff water
from PENT S amended Bastrop soil collected on day 0 191
70 GC/MS chromatograph of organic extract of runoff water
from PENT S amended Bastrop soil collected on day 360 192
71 Mutagenic activity with metabolic activation of runoff
water from SWRI amended Norwood soil 198
72 Mutagenic activity with metabolic activation of runoff
water from SWRI amended Bastrop soil 199
xii
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FIGURES (continued)
Number Page
73 Mutagenic activity with metabolic activation of runoff
water from COMBO amended Norwood soil.
200
74 Mutagenic activity with metabolic activation of runoff
water from COMBO amended Bastrop soil 204
75 Extractable hydrocarbons (mg/1) and mutagenic activity
(revertants/10 ml) in runoff water from PENT S amended
Norwood soil 207
76 Total mutation frequency per 10 survivors in A.
nidulans induced by the extractable hydrocarbons in
runoff water from waste amended soils 208
77 Extractable hydrocarbons (mg/1) and mutagenic activity
(revertants/10 ml) in runoff water from control and
PENT S amended Bastrop soils 209
78 Extractable hydrocarbons (mg/1) and mutagenic activity
(revertants/50 ml) in runoff water from control and SWRI
amended Norwood soils 212
79 Extractable hydrocarbons (mg/1) and mutagenic activity
(revertants/50 ml) in runoff water from control and SWRI
amended Bastrop soils 213
80 Extractable hydrocarbons (mg/1) and mutagenic activity
(revertants/50 ml) in runoff water from control and COMBO
amended Norwood soils 214
81 Extractable hydrocarbons (mg/1) and mutagenic activity
(revertants/50 ml) in runoff water from control and COMBO
amended Bastrop soils 215
82 Degradation rate of total extractable hydrocarbons in
Norwood and Bastrop soils amended with PENT S (PS), SWRI
(SI), and COMBO (CO) waste 225
83 Mutagenic activity of acid fraction of PENT S amended
Norwood soil as measured with jj typhimurium strain
TA98 with and without metabolic activation 228
84 Mutagenic activity of base fraction of PENT S amended
Norwood soil as measured with J3 typhimurium strain
TA98 with and without metabolic activation 229
xiii
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FIGURES (continued)
M v Page
Number
85 Mutagenic activity of neutral fraction of PENT S amended
Norwood soil as measured with j> typhimurium strain
TA98 with and without metabolic activation 230
86 Mutagenic activity of acid fraction of PENT S amended
Norwood soil as measured with ji typhimurium strain
TA100 with and without metabolic activation 231
87 Mutagenic activity of base fraction of PENT S amended
Norwood soil as measured with J5 typhimurium strain
TA100 with and without metabolic activation 232
88 Mutagenic activity of neutral fraction of PENT S
amended Norwood soil as measured with J5 typhimurium
strain TA100 with and without metabolic activation 233
89 Mutagenic activity of acid fraction of PENT S amended
Bastrop soil as measured with j> typhimurium strain
TA98 with and without metabolic activation 240
90 Mutagenic activity of base fraction of PENT S amended
Bastrop soil as measured with J5 typhimurium strain
TA98 with and without metabolic activation 241
91 Mutagenic activity of neutral fraction of PENT S amended
Bastrop soil as measured with j» typhimurium strain
TA98 with and without metabolic activation 242
92 Mutagenic activity of acid fraction of PENT S amended
Bastrop soil as measured with j> typhimurium strain
TA100 with and without metabolic activation 246
93 Mutagenic activity of base fraction of PENT S amended
Bastrop soil as measured with j> typhimurium strain
TA100 with and without metabolic activation 247
94 Mutagenic activity of neutral fraction of PENT S amended
Bastrop soil as measured with j> typhimurium strain
TA100 with and without metabolic activation 248
95 Mutagenic activity of acid fraction of SWRI amended
Norwood soil as measured with j>. typhimurium strain
TA98 with and without metabolic activation 252
96 Mutagenic activity of base fraction of SWRI amended
Norwood soil as measured with j>. typhimurium strain
TA98 with and without metabolic activation 256
xiv
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FIGURES (continued)
Number page
97 Mutagenic activity of neutral fraction of SWRI amended
Norwood soil as measured with j^. typhimurium strain
TA98 with and without metabolic activation 257
98 Mutagenic activity of acid fraction of SWRI amended
Bastrop soil as measured with J3. typhimurium strain
TA98 with and without metabolic activation 262
99 Mutagenic activity of base fraction of SWRI amended
Bastrop soil as measured with £. typhimurium strain
TA98 with and without metabolic activation 263
100 Mutagenic activity of neutral fraction of SWRI amended
Bastrop soil as measured with J5. typhimurium strain
TA98 with and without metabolic activation 264
101 Mutagenic activity of acid fraction of COMBO amended
Norwood soil as measured with j>. typhimurium strain
TA98 with and without metabolic activation 268
102 Mutagenic activity of base fraction of COMBO amended
Norwood soil as measured with j>. typhimurium strain
TA98 with and without metabolic activation 269
103 Mutagenic activity of neutral fraction of COMBO amended
Norwood soil as measured with Si. typhimurium strain
TA98 with and without metabolic activation 270
104 Mutagenic activity of acid fraction of COMBO amended
Bastrop soil as measured with j>. typhimurium strain
TA98 with and without metabolic activation 273
105 Mutagenic activity of base fraction of COMBO amended
Bastrop soil as measured with J5. typhimurium strain
TA98 with and without metabolic activation 275
106 Mutagenic activity of neutral fraction of COMBO amended
Bastrop soil as measured with £. typhimurium strain
TA98 with and without metabolic activation 277
107 GC/MS chromatograph of base fraction of PENT S amended
Norwood soil collected on day 0 283
108 GC/MS chromatograph of neutral fraction of PENT S amended
Norwood soil collected on day 0 284
xv
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FIGURES (continued)
Number Page
109 GC/MS chromatograph of acid fraction of PENT S amended
Norwood soil collected on day 360 285
110 GC/MS chromatograph of base fraction of PENT S amended
Norwood soil collected on day 360 286
111 GC/MS chromatograph of neutral fraction of PENT S amended
Norwood soil collected on day 360 287
112 GC/MS chromatograph of acid fraction of PENT S amended
Bastrop soil collected on day 0 288
113 GC/MS chromatograph of base fraction of PENT S amended
Bastrop soil collected on day 0 289
114 GC/MS chromatograph of neutral fraction of PENT S amended
Bastrop soil collected on day 0 290
115 GC/MS chromatograph of acid fraction of PENT S amended
Bastrop soil collected on day 360 291
116 GC/MS chromatograph of base fraction of PENT S amended
Bastrop soil collected on day 360 292
117 GC/MS chromatograph of neutral fraction of PENT S amended
Bastrop soil collected on day 360 293
118 GC/MS chromatograph of acid fraction of SWRI amended
Bastrop soil collected on day 360 296
119 GC/MS chromatograph of base fraction of SWRI amended
Bastrop soil collected on day 360 297
120 GC/MS chromatograph of neutral fraction of SWRI amended
Bastrop soil collected on day 360 298
121 GC/MS chromatograph of acid fraction of COMBO amended
Norwood soil collected on day 0 301
122 GC/MS chromatograph of base fraction of COMBO amended
Norwood soil collected on day 0 302
123 GC/MS chromatograph of neutral fraction of COMBO amended
Norwood soil collected on day 0 303
124 GC/MS chromatograph of acid fraction of COMBO amended
Norwood soil collected on day 360 304
xvi
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FIGURES (continued)
Number Page
125 GC/MS chromatograph of base fraction of COMBO amended
Norwood soil collected on day 360 305
126 GC/MS chromatograph of neutral fraction of COMBO amended
Norwood soil collected on day 360 306
127 GC/MS chromatograph of acid fraction of COMBO amended
Bastrop soil collected on day 0 307
128 GC/MS chromatograph of base fraction of COMBO amended
Bastrop soil collected on day 0 308
129 GC/MS chromatograph of neutral fraction of COMBO amended
Bastrop soil collected on day 0 309
130 GC/MS chromatograph of acid fraction of COMBO amended
Bastrop soil collected on day 360 310
131 GC/MS chromatograph of neutral fraction of COMBO amended
Bastrop soil collected on day 360 311
132 Total extractable hydrocarbons and mutagenic potential
of equivalent volumes of PENT S amended Norwood (NW)
and Bastrop (BA) soils as measured with ^. typhimurium
strain TA98 with and without metabolic activation.
Dashed line ( ) is equal to 2.5 times solvent control. . . . 314
133 Total extractable hydrocarbons and mutagenic potential
of equivalent volumes of SWRI amended Norwood (NW) and
Bastrop (BA) soils as measured with J3. typhimurium
strain TA98 with and without metabolic activation.
Dashed line ( ) is equal to 2.5 times solvent control. . . . 315
134 Total extractable hydrocarbons and mutagenic potential
of equivalent volumes of COMBO amended Norwood (NW) and
Bastrop (BA) soils as measured with J3. typhimurium
strain TA98 with and without metabolic activation.
Dashed line ( ) is equal to 2.5 times solvent control. . . . 316
135 Total induced mutation frequency of equivalent volumes
of PENT S amended Norwood (NW) and Bastrop (BA) soils as
measured in A. nidulans methionine system with and
without metabolic activation. Dashed line (~7~) is equal
to total induced mutation frequency of 5.0/10 survivors . . 317
xvii
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FIGURES (continued)
Number
136 Total induced mutation frequency of equivalent volumes
of SWRI amended Norwood (NW) and Bastrop (BA) soils as
measured in A. nidulans methionine system with and
without metabolic activation. Dashed line (~g~) is equal
to total induced mutation frequency of 5.0/10 survivors . . 318
137 Total induced mutation frequency of equivalent volumes
of COMBO amended Norwood (NW) and Bastrop (BA) soils as
measured in A. nidulans methionine system with and
without metabolic activation. Dashed line (~T~) is equal
to total induced mutation frequency of 5.0/10 survivors . . 319
138 Schematic diagram of leachate collection system used in
the lysimeter study 324
139 Extractable hydrocarbons and mutagenic activity from soil
core samples collected at various depths on day 90 327
140 GC/MS chromatograph of crude extract of soil core sample
collected on day 90 at a depth of 0 to 15 cm from unamended
Norwood lysimeter 331
141 GC/MS chromatograph of acid fraction of soil core sample
collected on day 90 at a depth of 0 to 15 cm from PENT S
amended Norwood lysimeter 332
142 GC/MS chromatograph of base fraction of soil core sample
collected on day 90 at a depth of 0 to 15 cm from PENT S
amended Norwood lysimeter 333
143 GC/MS chromatograph of neutral fraction of soil core
sample collected on day 90 at a depth of 0 to 15 cm
from PENT S amended Norwood lysimeter 334
144 Mutagenic activity of leachate water from control and
PENT S waste amended lysimeters 335
145 GC/MS chromatograph of soil pore liquid sample collected
from unamended Norwood lysimeter on day 90 337
146 GC/MS chromatograph of soil pore liquid sample collected
from PENT S amended lysimeter on day 90 339
xviii
-------
TABLES
Number Page
1 Strains of Bacillus subtilis used for mutagen
testing.
10
2 Comparison of lethal effect of various samples on DNA
repair deficient and proficient strains of B.
subtilis 12
3 Survival of wild-type and repair-deficient strains
of %._ subtilis exposed to varying concentrations
of DNA damaging chemicals 13
4 Survival of wild-type and repair-deficient strains
of B_._ subtilis exposed to varying concentrations
of 2-aminoanthracene and benzo(a)pyrene with and
without metabolic activation 14
5 Characteristics of Salmonella strains used for
mutagenesis testing 19
6 Effect of a 1:10 MeCl_:DMSO solution on the
spontaneous reversion and induced mutation frequency
in S_._ typhimrium 23
7 Response obtained in prokaryotic mutagenesis assays
using S_._ typhimurium or B_^ subtilis with
negative and positive controls 24
8 Variability of S^_ typhimurium strains TA98 and
TA100 with negative and positive controls for each year
of the project 25
9 The effect of storage at 4 C on the mutagenic activity
of two runoff samples (samples were received 5/13/82) 35
10 Surviving fraction and induced mutants per survivor
in haploid A. nidulans following treatment with
solvent and positive controls 45
11 Summary of genotoxic effects induced by controls in
segregant colonies of diploid A._ nidulans 46
xix
-------
TABLES (continued)
Number Pa§e
12 Summary of genotoxic effects induced by controls in
abnormal colonies of diploid A^ nidulans 47
13 Gross characteristics of hazardous wastes collected
for study 52
14 Biological systems used to detect genotoxic compounds
in environmental samples 59
15 Mutagenic activity of liquid stream from acetonitrile
purification column as measured with S. typhimurium
strain TA98 and TA100 with and without metabolic
activation 66
16 Comparison of lethal effects of acetonitrile (ACN)
waste fractions on DNA repair deficient and proficient
strains of B^_ subtilis 67
17 The effect of the neutral fraction of a methyl ethyl
ketone waste on the frequency of induced mutations in
Aspergillus nidulans 70
18 Mutagenic activity of methyl ethyl ketone waste , . . 73
19 Distribution of mutagenic activity in fractions of
wood-preserving bottom sediment (PENT S) waste
extracted using blender or soxhlet technique 75
20 Mutagenic activity of fractions of wood-preserving
bottom sediment 76
21 Mutagenic activity of PENTS waste in four plasmid
containing strains of S^_ typhimurium 80
22 Mutagenic activity of PENTS waste fractions as measured
with j>. typhimurium, strain TA98, with high (0.5 ml
S9/ml: S9/mix), medium (0.3 ml S9/ml S9/mix), and low
(0.1 ml S9/ml S9 mix) concentrations of aroclor 1254
induced rat liver in 59 mix 81
23 Capacity of fractions of wood-preserving waste to
induce increased lethal damage in DNA repair deficient
strains of ]$._ subtilis 86
24 Fractional survival of repair proficient and deficient
strains of B. subtilis 88
xx
-------
TABLES (continued)
Number Page
25 Surviving fraction and induced mutation frequency of
A._ nidulans following exposure to acid fraction
of PENT S waste 92
26 Surviving fraction and induced mutation frequency of
A.^ nidulans following exposure to base fraction of
PENT S waste 93
27 Surviving fraction and induced mutation frequency
of A^ nidulans following exposure to neutral
fraction of PENT S waste 94
28 Summary of genotoxic effects observed in segregant
colonies of diploid A^ nidulans following exposure
to controls and waste fractions 95
29 Summary of genotoxic effects observed in abnormal
colonies of diploid A. nidulans following exposure
to controls and waste fractions 97
30 Selected properties of compounds identified in the
fractions of the wood-preserving bottom sediment (PENT S). . 103
31 Mutagenic activity of fractions of storm-water runoff
impoundment 105
32 Fractional survival of repair proficient (168 wt) and
deficient strains of B^ subtilis exposed to
subfractions of combined API-seperator/slop-oil emulsion
solid (COMBO) or storm-water runoff impoundment (SWRI).
Survival was measured in the presence of metabolic
activation 108
33 Surviving fraction and induced mutation frequency of A._
nidulans following exposure to acid fraction of SWRI
waste 109
34 Surviving fraction and induced mutation frequency of A._
nidulans following exposure to base fraction of SWRI
waste 110
35 Surviving fraction and induced mutation frequency of A._
nidulans following exposure to neutral fraction of SWRI
waste Ill
xxi
-------
TABLES (continued)
Number Page
36 Mutagenic activity of fractions of combined API separator/
slop oil emulsion waste H8
37 Surviving fraction and induced mutation frequency of A._
nidulans following exposure to acid fraction of COMBO
waste 121
38 Surviving fraction and induced mutation frequency of A.
nidulans following exposure to base fraction of COMBO
waste 122
39 Surviving fraction and induced mutation frequency of A.
nidulans following exposure to neutral fraction of COMBO
waste 123
40 Selected properties of compounds identified in the
fractions of the combined API-seperator/slop-oil
emulsion solids COMBO waste 131
41 Summary of results obtained from testing waste
fractions in biological test systems 133
42 Physical properties of the three soils 137
43 Mutagenicity of organic extracts of three soils as
measured with and without metabolic activation 139
44 Effect of increasing doses of organic extract of
Bastrop and Norwood soils on survival and induced
mutation frequency in Aspergillus nidulans with
and without metabolic activation 145
45 List of compounds identified in Norwood and Bastrop
soils 148
46 Diagnostic mutagens used to evaluate the efficiency of
extraction procedures 158
47 Mutagenic activity, as measured with S. typhimurium
TA98, of 2-nitrofluorene, and the solvent extract of the
Norwood and Bastrop soils amended with various levels of
2-nitrofluorene 161
48 Mutagenic activity, as measured with j>. typhimurium
TA98, with metabolic activation, of benzo(a)pyrene, and
the solvent extract of the Norwood and Bastrop soils
amended with various levels of benzo(a)pyrene 165
xxii
-------
TABLES (continued)
Number
49 Extraction efficiency, as measured with HPLC, of the
Bastrop and Norwood soils amended with mutagenic
compounds. 168
50 Distribution of mutagenic activity in fractions of
hazardous waste and the two soils used in the greenhouse
study 178
51 Mutagenic activity, as measured with S^_ typhimurium
strain TA98 of runoff water from PENT S amended Norwood
soil 184
52 Mutagenic activity, as measured with S^ typhimurium
strain TA100 of runoff water from PENT S amended Norwood
soil 185
53 Mutagenic activity, as measured with S. typhimurium
strain TA98 of runoff water from PENT S amended Bastrop
soil 188
54 Mutagenic activity, as measured with S. typhimurium
strain TA100 of runoff water from PENT S amended Bastrop
soil 189
55 Mutagenic activity, as measured with S^_ typhimurium
strain TA98 of runoff water from Norwood soils amended
with SWRI waste 194
56 Mutagenic activity, as measured with S. typhimurium
strain TA100 of runoff water from Norwood soils amended
with SWRI waste 195
57 Mutagenic activity, as measured with J5._ typhimurium
strain TA98 of runoff water from Bastrop soils amended
with SWRI waste 196
58 Mutagenic activity, as measured with S. typhimurium
strain TA100 of runoff water from Bastrop soils amended
with SWRI waste 197
59 Mutagenic activity, as measured with j>^_ typhimurium
strain TA98 of runoff water from Norwood soils amended
with COMBO waste 201
xxiii
-------
TABLES (continued)
Number
60 Mutagenic activity, as measured with js._ typhimurium
strain TA100 of runoff water from Norwood soils amended
with COMBO waste 202
61 Mutagenic activity, as measured with S. typhimurium
strain TA98 of runoff water from Bastrop soils amended
with COMBO waste 203
62 Mutagenic activity, as measured with ^-_ typhimurium
strain TA100 of runoff water from Bastrop soils amended
with COMBO waste 206
63 Total hydrocarbons extracted from runoff water using
combined XAD2 and XAD7 resins 211
64 Total hydrocarbons extracted from soil amended with
wood-preserving bottom sediment 221
65 Total hydrocarbons extracted from soil amended with
storm-water runoff impoundment 222
66 Total hydrocarbons extracted from soil amended with
combined API separator/slop-oil emulsion solid 224
67 Mutagenic activity of acid fraction of PENT S waste
amended Norwood soil as measured with S^. typhimurium
strain TA98 with and without metabolic activation 234
68 Mutagenic activity of base fraction of PENT S waste
amended Norwood soil as measured with S.typhimurium
strain TA98 with and without metabolic activation 235
69 Mutagenic activity of neutral fraction of PENT S waste
amended Norwood soil as measured with S_. typhimurium
strain TA98 with and without metabolic activation 236
70 Mutagenic activity of acid fraction of PENT S waste
amended Norwood soil as measured with j>. typhimurium
strain TA100 with and without metabolic activation 237
71 Mutagenic activity of base fraction of PENT S waste
amended Norwood soil as measured with J5. typhimurium
strain TA100 with and without metabolic activation 238
72 Mutagenic activity of neutral fraction of PENT S waste
amended Norwood soil as measured with j>. typhimurium
strain TA100 with and without metabolic activation 239
xxiv
-------
TABLES (continued)
Number ^SS£
73 Mutagenic activity of acid fraction of PENT S waste
amended Bastrop soil as measured with J5. typhimurium
strain TA98 with and without metabolic activation 243
74 Mutagenic activity of base fraction of PENT S waste
amended Bastrop soil as measured with j>. typhimurium
strain TA98 with and without metabolic activation 244
75 Mutagenic activity of neutral fraction of PENT S waste
amended Bastrop soil as measured with js. typhimurium
strain TA98 with and without metabolic activation 245
76 Mutagenic activity of acid fraction of PENT S waste
amended Bastrop soil as measured with j>. typhimurium
strain TA100 with and without metabolic activation 249
77 Mutagenic activity of base fraction of PENT S waste
amended Bastrop soil as measured with j>. typhimurium
strain TA100 with and without metabolic activation 250
78 Mutagenic activity of neutral fraction of PENT S waste
amended Bastrop soil as measured with J3. typhimurium
strain TA100 with and without metabolic activation 251
79 Mutagenic activity of acid fraction of SWRI waste
amended Norwood soil as measured with j>. typhimurium
strain TA98 with and without metabolic activation 253
80 Mutagenic activity of base fraction of SWRI waste
amended Norwood soil as measured with j>. typhimurium
strain TA98 with and without metabolic activation 255
81 Mutagenic activity of neutral fraction of SWRI waste
amended Norwood soil as measured with J3. typhimurium
strain TA98 with and without metabolic activation 258
82 Mutagenic activity of acid fraction of SWRI waste
amended Bastrop soil as measured with j^. typhimurium
strain TA98 with and without metabolic activation 259
83 Mutagenic activity of base fraction of SWRI waste amended
Bastrop soil as measured with S^. typhimurium strain
TA98 with and without metabolic activation 260
84 Mutagenic activity of neutral fraction of SWRI waste
amended Bastrop soil as measured with £!. typhimurium
strain TA98 with and without metabolic activation 261
xxv
-------
Number
TABLES (continued)
Page
85 Mutagenic activity of acid fraction of COMBO waste
amended Norwood soil as measured with j^. typhimurium
strain TA98 with and without metabolic activation 265
86 Mutagenic activity of base fraction of COMBO waste
amended Norwood soil as measured with J5. typhimurium
strain TA98 with and without metabolic activation 266
87 Mutagenic activity of neutral fraction of COMBO waste
amended Norwood soil as measured with S^ typhimurium
strain TA98 with and without metabolic activation 267
88 Mutagenic activity of acid fraction of COMBO waste
amended Bastrop soil as measured with j>. typhimurium
strain TA98 with and without metabolic activation 272
89 Mutagenic activity of base fraction of COMBO waste
amended Bastrop soil as measured with J3. typhimurium
strain TA98 with and without metabolic activation 274
90 Mutagenic activity of neutral fraction of COMBO waste
amended Bastrop soil as measured with J3. typhimurium
strain TA98 with and without metabolic activation 276
91 List of compounds detected in PENT S waste amended soil . . . 280
92 List of compounds detected in COMBO waste amended soil. . . . 299
93 Chemical properties of the Norwood soil series and
physical properties of the Norwood soil series 322
94 List of compounds detected in soil core sample collected
on day 90 from control Norwood soil at 0-15 cm depth .... 328
95 List of compounds detected in soil core samples
collected on day 90 from PENT S waste amended Norwood
soils at various depths 329
96 List of compounds detected in soil pore samples
collected on day 90 from PENT S waste ameded Norwood
soil 336
XXVI
-------
ACKNOWLEDGEMENTS
Valuable advice and assistance was obtained from Carlton Wiles, EPA
Project Officer for years 1 and 2, and from John Matthews, the EPA
Project Officer for the third year. Dr. D. Kampbell of the USEPA's R. S.
Kerr Environmental Research Laboratory at Ada, Oklahoma performed
analytical work on waste and soil extracts.
A great deal of technical support was provided in all phases of the
project by Phebe Davol. Rosella Saltarelli assisted in maintenance of
microbial strains and Lea Maggard conducted the study on extraction
efficiency. All biological analyses using A. nidulans were
conducted by Dr. B. R. Scott with Phoenix Co., Smithville, Texas. Mr.
D. Anderson assisted in the location and collection of the wastes. A
special acknowledgement is also given to Nora Sai and Pamela Antilley
for patient typing and editing of the text, respectively.
xxvn
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SECTION 1
INTRODUCTION
Industrial activities in the United States have undergone a rapid
expansion over the past three decades concurrent with the development of
numerous new chemicals. These activities have been responsible in part
for a dramatic improvement in the quality of life while affecting a
reduction in the frequency of infectious disease. An unavoidable
by-product of our industrial growth has been the generation of large
volumes of complex industrial wastes. Since 1950, the world production
of organic chemicals has risen from 7 million tons to a projected 150
million tons by 1985 (U.S. Tarriff Commission, 1974). Presently,
approximately 700 new chemicals are introduced into commerce each year,
in addition to the more than one-half million chemicals already in use
(National Cancer Institute, 1976). For the majority of these chemicals,
there is little information available pertaining to their toxicological
and environmental aspects (Fishbein, 1979).
This lack of sufficient information and the fact that the majority
of chemical carcinogens are considered to be products of our increasing
agricultural and technological sophistication (Chemical and Engineering
News, 1975) dictate the need to develop techniques for monitoring the
disposal of hazardous chemicals. The EPA (1983) estimated that 150
million metric tons of hazardous waste are generated annually in the
United States. Past disposal practices have generally included deep sea
dumping, incineration, and landfilling. All of these methods have
environmental or economical drawbacks which limit their utility. The
alternative disposal method of land treatment, i.e., the incorporation
of waste into the surface layer of soil resulting in the degradation or
attenuation of hazardous waste constituents, is being used more
frequently for the disposal of selected industrial wastes. EPA (1980)
regulations state that a waste cannot be land applied unless the waste
is rendered less or non-hazardous by chemical or biological reactions in
the soil. In order for land treatment to be a viable method of
disposal, techniques are needed for the monitoring of hazardous waste
constituents and their metabolites. The use of a combined biological and
chemical testing protocol may provide the most practical means of
efficiently monitoring hazardous waste disposal. The use of chemical
analysis alone fails to account for the interactions of the components
of a complex mixture, the production of mutagenic metabolites via
degradative pathways, and chemical reactions between non-toxic
precursors that may result in the formation of mutagenic compounds. An
-------
appropriately selected bioassay should be capable of integrating these
effects. The use of biological analysis alone, however, could fail to
account for artifacts generated in the collection or extraction
process. A combined testing protocol utilizes microbial bioassays to
measure primary DNA damage and a chemical analysis to identify causitive
agents in genotoxic samples. Although not all chemical mutagens" are
established carcinogens, most chemical carcinogens have been determined
to be mutagenic in sub-mammalian systems (Stolz et_ al. , 1974; Miller
and Miller, 1974; Committee 17, 1974). The utility of microbial
mutagenesis assays is further enhanced by recent evidence that a point
mutation in a human cell can give the cell malignant properties (Santos
ejt al., 1984). Thus, microbial bioassays may be utilized to evaluate
the mutagenic activity of waste and environmental samples as an
indication of their potential for inducing mutagenic damage in the
human population. Mutagenic compounds in the environment affecting
somatic cells may induce cell death, cancer, aging, and heart disease
(Ames, 1979); while mutations in germ cells may result in birth defects,
sterility, and abortions (Brusick, 1981) (Figure 1). The human genome
is our most precious heritage (de Serres, 1979), and analytical
techniques must be developed to reduce the transmission of mutagenic
defects to future generations. A need exists to determine if mutagenic
constituents of land treated industrial waste will migrate from the site
of application to locations where the human population could be exposed
to them.
The objectives of this research were to characterize the genotoxic
constituents of three hazardous wastes, to monitor waste degradation in
soil, and to determine the environmental fate of mutagenic waste
constituents following land application. In order to meet these
objectives and to develop a set of test protocols which can be used to
monitor environmental contamination, this project was divided into four
main phases. The waste characterization phase included an acute
toxicity evaluation of ten wastes, a complete characterization of the
mutagenic potential of seven subfractions of three selected wastes, and
a chemical characterization of major organic constituents. In the
second phase, direct and indirect acting mutagens were added to the soil
in order to quantify extraction procedures and to determine the affect
of soil components on the activity of mutagenic compounds.
Phase three consisted of a greenhouse study in which three wastes
were applied at one loading rate to two soil types packed in boxes.
Simulated rainfall was applied and runoff and soil samples were
collected at various time intervals during a 360 or 540 day period. The
results from this phase of the project were used to evaluate the affect
of degradation on the mutagenic activity of waste amended soil and the
potential for the removal of mutagens in runoff water. In the final
phase of the study, one waste was applied to an undisturbed soil
enclosed in lysimeters in order to monitor the movement of mutagens
through soil. The information generated by this research should provide
a sound basis for determining the utility of short-term bioassays to
-------
HUMANS
GERM CELLS
(REPRODUCTIVE)
DOMINANT
MUTATIONS
ALTERATION
IN DNA
OR
CYTOPLASMIC
MATERIAL
MUTATION
RECESSIVE
MUTATIONS
SOMATIC CELLS
(NON-REPRODUCTIVE)
BIRTH DEFECTS
GENETIC DISEASE
ABORTIONS
STERILITY
EXPRESSED AS GENETIC
DISEASE IN FUTURE
GENERATIONS
CELL DEATH.
CANCER
AGING
HEART DISEASE
OTHER ILLNESS
Figure 1'. Potential effects of environmental mutagens on human cells.
-------
evaluate the environmental impact of the land treatment of hazardous
industrial wastes.
-------
SECTION 2
CONCLUSIONS
1. The battery of biological test systems employed in this research
provided detection capabilities for a range of genotoxic damage and
a variety of genotoxic compounds.
2. The acid, base, and neutral fractions of each of three hazardous
wastes contained compounds capable of inducing DNA damage, point
mutations, or chromosome damage.
3. An agricultural soil with no previous history of waste application
will have an inherent level of mutagenic activity. The magnitude
of this activity will be directly related to the past history of
agricultural practices, including biocide applications,
fertilization, and cultivation.
4. Through use of the soxhlet technique, significantly greater
quantities of hydrocarbons were extracted from the waste than were
extracted using the blender technique. However, the composition of
the extracted hydrocarbons appeared to be similar using either
technique, as there was no appreciable difference in the
mutagenicity of the residues.
5. The extraction efficiency for 2-nitrofluorene and benzo(a)pyrene
from soil using the blender technique and measured by HPLC averaged
greater than 85%, while the mutagenic activities of the pure
compound and the pure compound extracted from soil were within one
standard deviation of each other.
6. The mutagenic potential of runoff water from soil amended with two
of the three wastes was reduced 360 days after waste application.
For the third waste, the mutagenic potential was increased,
although the amount of extractable hydrocarbons in the runoff had
decreased.
7. Degradation increased the mutagenic potential of the extractable
hydrocarbons present in the acid and base fractions of all three
waste amended soils. However, the mutagenic potential of
equivalent volumes of waste amended soils was reduced and, in some
cases, was reduced to a nonmutagenic level.
-------
8. The wood-preserving waste collected for this study contained a
limited number of highly mobile and highly mutagenic constituents
that were identified by chemical and biological analysis.
Different types of compounds were detected in soil core and soil
pore liquid samples.
9. Chemical assisted biological analysis provided more accurate
information for monitoring complex mixtures in the environment than
either method alone.
-------
SECTION 3
RECOMMENDATIONS
1. A battery of bioassays, capable of detecting point mutations, DNA
repair damage, and chromosome damage should be used to define the
genetic toxicity of a hazardous industrial waste.
2. The bioassay(s) used for environmental monitoring need only consist
of the least complex test system(s) found to be sensitive to the
type of genetic damage and the types of genotoxic compounds in the
waste.
3. Both chemical and biological analysis should be used to monitor
hazardous waste land treatment.
4. A field study would provide a more accurate representation of the
potential of the three wastes used in the present study to be
rendered less hazardous by soil incorporation.
5. A biological testing protocol used to monitor hazardous waste land
treatment should include both chronic and acute toxicity bioassays.
6. Additional areas of research not evaluated in the present study
which merit future investigation include the mutagenic potential of
different wastes and the affect on detoxification of loading rate
and fertilizer additions.
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SECTION 4
BIOLOGICAL ANALYSIS
INTRODUCTION
Bioassay techniques measure the toxic effects of a substance by
comparing its effect on a living organism to that of standard substances
with known toxicological effects. A battery of short-term bioassays was
used in this research to evaluate the genotoxic potential of various
samples associated with land treatment of hazardous industrial waste.
Through the use of a battery of bioassays to detect a range of genetic
damage, environmental monitoring can be made both more economical and
environmentally sound.
Genotoxic compounds in a hazardous waste should be monitored in
order to control accidental exposure to mutagenic, carcinogenic, or
teratogenic agents and to prevent the transmission of related genetic
defects to future generations. Because no single test system has been
identified which can detect all types of genetic damage and all
genotoxic compounds, genetic toxicity can best be determined using a
battery of biological test systems. The battery of test systems should
be capable of detecting inhibition of DNA repair, gene mutations, and
various types of chromosome damage.
The most common biological test systems employ microorganisms to
detect point mutations in a haploid genome. Since the composition of DNA
is essentially the same in all organisms, an agent which induces
mutagenesis in a microorganism is also assumed to be capable of mutating
mammalian DNA. While chemical mutagenesis is a relatively simple
process involving the induction of heritable change in the genome,
mutagenic events may constitute only the initial step of a multi-stage
process in chemical carcinogenesis (Brusick, 1981). The eventual
outcome of chemical carcinogenesis will be greatly influenced by enzyme
activation and detoxification systems (Miller and Miller, 1974), as well
as cocarcinogenic and promotional factors (Brusick, 1980). Therefore,
not all microbial mutagens will also be carcinogens; although in several
validation studies, 80 to 90% of carcinogens were detected as mutagens
(McCann e^ aj^., 1980).
The most efficient battery for evaluating the genotoxic potential
of environmental samples should be capable of detecting both a range of
genetic damage and a range of genotoxic compounds. Each test system
-------
should include provisions for solvent and positive controls that will
demonstrate the sensitivity of the test systems, the functioning of the
metabolic activation system, and act as an internal control for the
biological system. The following discussion concerns the standard
protocols for the bioassays utilized in this research, as well as a
comparison of the variability over time of each system, the types of
damage and compounds detected, and the limitations of each system.
A. Differential DNA Repair
1. Test System Description
Repair assays are used to detect primary DNA damage and evaluate
differential growth inhibition of DNA repair proficient and deficient
pairs of bacteria. Increased growth inhibition in a DNA repair deficient
strain of bacteria implies that the agent being tested has reacted with
cellular DNA to produce a repairable DNA lesion. Agents which produce
DNA damage are likely to be mutagenic, carcinogenic, as well as
effectors of chromosome aberrations (Kada, 1978). Numerous test systems
are available which employ pairs of IS. coli (Slater et^ al. ,
1971), B. subtilis (Kada et^ al., 1977; Tanooka, 1977; Felkner
et_ al. , 1979), J?. mirabilis (Adler et^ al. , 1976), and J5.
typhimurium (Ames e_t^ al. , 1975). These test systems offer the
advantage of being rapid, simple, and inexpensive. In addition, since
these tests are capable of detecting DNA damage at any locus and by any
mechanism, they are capable of detecting many compounds not detected by
point mutation assays (Kada, 1980; Rosenkranz and Poirier, 1979).
Limitations of DNA repair assays in bacteria include the minimal
response from many compounds that are poorly soluble in water, the lack
of response from large molecules due to the impermeability of the cell
wall, and an inability to interpret borderline responses from the spot
test (Leifer et al., 1981). The following modifications to the
standard protocol were used to enhance the sensitivity of the DNA repair
assay.
2. Test system protocol: DNA repair
To test for lethal DNA-damage, five different strains of IJ.
subtilis deficient in different recombination (Rec ) and/or excision
(Exc ) repair were employed. _ These included the Rec _ strains
recAS, recE4, and mc-1; Exc strain her-9; and Rec , Exc
strain fh2006-7. These strains are all isogenic with ]}. subtilis
strain 168 which has all repair intact (Table 1). The DNA-repair assay
was performed using the procedure of Felkner (1977) on strains Dr. I. C.
Felkner kindly supplied. Metabolic activation was incorporated into the
spot test by combining 0.5 ml of the test chemical with 0.5 ml of S-9
mix. The S-9 mix is composed of enzymes obtained from the supernatant of
homogenized rat liver which was centrifuged at 9,000 x g, and a NADPH
generating system. This mix, when incorporated into the bioassay,
simulates the in vivo biotransformation of the test material (Muller
-------
TABLE 1. STRAINS OF BACILLUS SUBTILIS USED FOR MUTAGEN TESTING
Strain
Genotype
Source
168 wt
rec E4
rec A8
rec B2
hcr-9
fh2006.;
mc-1
TKJ5211
TKJ6321
Wild-type
trp, thr, Rec_
trp, thr, Rec
trp, thr,. Rec
trp, thr, Exc_
trp, thyz Rec , Exc
trp, Rec
his, meth, Exc .
his, meth, polAlSl, spp-1, Exc
Dr. I. C. Felkner
1. Auxotrophic mutations: trp (tryptophane), thr (threonine), thy
(thymine), his (histidine), meth (methionine).
Repair Deletions: Rec (recombination), Exc (excision), polAlSl
(polymerase).
Additional Mutations: spp-1 (spore-repair deficiency marker).
10
-------
et_ al., 1980). Aroclor 1254 induced rat liver was obtained from
Litton Bionetics (Charleston, S.C.) and prepared according to the
procedure of Ames £t al. (1975). The chemical and S-9 mixture were
incubated with shaking for 30 min at 37 C. Inocula from overnight
cultures were streaked radially on a nutrient agar plate to either a
centered sensitivity disk containing 100 pi of the test sample or a
well in the agar plate containing 200 pi of the chemical S-9 mixture.
The plates were incubated overnight at 37 C, and the distance of
growth inhibition was measured from the periphery of the disk or well.
Dimethyl sulfoxide (DMSO) was used as a negative control. Mitomycin C
(Sigma), methylmethane sulphonate (Aldrich), and sensitivity to
ultraviolet light were used as appropriate positive controls.
Fractional survival (N/N ) was evaluated for the most sensitive
strains and for strain 168 using the procedure of Felkner et al.
(1979). Cells were inoculated into Difco brain-heart infusion broth
(BHI) and incubated for approximately 16 hours until an O.D.-,,. of 0.3
was reached. The cells were then diluted with fresh BHI to an O.D.,.,-
of 0.1 (approximately 1.2 x 10 cells/ml) and serially diluted witn
Spizizen's Minimal Salts (Spizizen, 1958). To determine fractional
survival, 100 ul of the bacterial suspension at a minimum of three cell
concentrations was added to 2.5 ml of nutrient top agar in a dry bath at
47°C. To this mixture, either 100 pi of the test sample or 200 pi
of the sample S-9 mixture was added at 37 C and plated onto nutrient
agar in duplicate. Plates were incubated for 24 hours at 37 C, and
the surviving fraction was determined by dividing the colony count on
the treated plate (N) by the colony count for the solvent control
(N ). The results shown are the average of two independent
experiments with duplicate platings at each of three cell
concentrations.
3. Test system results
The results presented in Table 2 indicate that water soluble
mutagens such as mitomycin C (MitC) are readily detected using the spot
test, while compounds such as benzo(a)pyrene [B(a)p] or 2-nitrofluorene
(2-NF) which require metabolic activation or are sparingly soluble in
water give no response. An evaluation of fractional survival in two
repair-deficient strains recE4 and fh2006-7 (Table 3) indicates that
2-NF does produce increased lethal damage in the repair-deficient
strains. The use of a plate-incorporation assay to evaluate survival
apparently provided a greater opportunity for cellular exposure to 2-NF,
and thus the positive response was obtained. Both MitC and MMS
continued to induce a positive response in the plate incorporation
assay, and results from this assay were more easily transformed into a
dose-response curve than for the spot test (Table 3).
For the indirect-acting mutagens B(a)p and 2-AA, no response was
obtained in the spot test with or without activation, or in the survival
assay without activation (Table 1 and 4). However, when metabolic
11
-------
TABLE 2. COMPARISON OF LETHAL EFFECT OF VARIOUS SAMPLES ON DNA REPAIR DEFICIENT
AND PROFICIENT STRAINS OF B. SUBTILIS
Inhibition Radius
Sample
Mit C
MMS
2NF
B(a)p
2AA
DMSO
S-9 Dose/Plate
(pg)
20
2
500
+ 50
50
+ 100
100
100 ill
+ 100 pi
168 wt
RP
4.3
6.8
0
0
0
0
0
0
0
recE4
12.4
13.0
0
0
0
0
0
0
0
fh2006.7
7.2
15.9
0
0
0
0
0
0
0
(mm)
recAS
repair
11.5
15.6
0
0
0
0
0
0
0
mc-1
deficier
10.2
17.8
0
0
0
0
0
0
0
n
her. 9 Response'"
ll_
15.1
12.1
0
0
0
0
0
0
0
+++
H-+
-
-
-
-
-
-
1- Repair Proficient
2- Response: (zone of inhibition in repair deficient strain-zone of inhibition in repair
proficient strain) <3 = -; 3,4 = +; 5-7 = +»; >7 = +++.
-------
TABLE 3. SURVIVAL OF WILD-TYPE AND REPAIR-DEFICIENT STRAINS OF
IJ. SUBTILIS EXPOSED TO VARYING CONCENTRATIONS OF
DNA DAMAGING CHEMICALS
u>
Sample
Methylme thane
sulfonate
Mitomycin C
2-nitrof luorene
Dose/Plate
(pg)
20
2
0.2
0.02
2
1
0.1
0.01
1,000
500
250
25
2.5
Fractional
168 wt
0.27
0.83
0.92
1.04
0.72
0.91
0.97
0.91
0.95
0.82
1.04
0.99
1.00
Survival
recE4
0.0007
0.60
0.43
0.69
0.03
0.13
0.62
0.77
0.47
0.60
0.53
0.51
0.90
(N/N )
o
fh2006.7
0.0005
0.26
0.70
0.44
0.20
9.14
0.62
0.57
0.34
0.52
0.42
0.39
0.45
-------
TABLE 4. SURVIVAL OF WILD-TYPE AND REPAIR-DEFICIENT STRAINS OF B. SUBTILIS EXPOSED
TO VARYING CONCENTRATIONS OF 2-AMINOANTHRACENE AND BENZO(A)PYRENE WITH AND
WITHOUT METABOLIC ACTIVATION
Fractional Survival N/N
Sample
Benzo(a)pyrene
2-aminoanthracene
Dose/Plate
(ug)
10
5
0.5
0.05
0.005
10
1
0.1
0.01
0.001
168 wt
+ S-9
0.70
0.91
0.86
0.81
0.85
0.52
0.47
0.57
0.86
0.102
- S-9
_
0.71
0.60
0.65
0.82
0.73
0.81
0.85
0.83
0.94
recE4
+ S-9
0.24
0.39
0.33
0.29
0.65
0.24
0.35
0.49
0.42
0.76
- S.9
_
0.46
0.73
0.63
0.69
0.54
0.86
0.61
0.76
0.94
fh2006.
+ S-9
0.38
0.43
0.48
0.57
0.47
0.37
0.38
0.49
0.67
0.71
7
- S.9
_
0.75
0.83
0.93
0.76
0.43
0.51
0.47
0.61
0.55
-------
activation was incorporated into the survival assay by pre-incubation of
the chemical S-9, both B(a)p and 2-AA induced increased lethal damage in
DNA repair deficient strains (Table 4, Figures 2 and 3). After exposure
to the 2M:S-9 mixture at the highest dose level, the survival ratio of
recE4:168 was 0.46. This would be considered a positive response, and
atlower dose levels this effect was reduced. The survival ratio for
recE4:168 at 5 and 0.5 ug/plate exposure to B(a)p:S-9 mixture was
0.43 and 0.33, respectively. These results indicated that while both
2-AA and B(a)p induced increased lethal damage in DNA-repair deficient
bacteria after metabolic activation, the DNA-damaging activity of B(a)p
appeared to be greater.
This protocol increased the utility of a plate-incorporation assay
that employed several different DNA repair-deficient strains of bacteria
with metabolic activation. In the Salmonella/microsome assay, the
frequency of induced mutations per nanomole was found to be four times
greater for 2AA than for B(a)p (McCann et al. , 1975). This may have
been due in part to increased lethal damage induced by B(a)p in the
excision repair deficient strains used for mutagenesis testing. The
addition of a DNA-repair assay to a battery of test systems for
detecting potential carcinogens should enhance the sensitivity of the
battery towards compounds such as MitC which cause DNA damage at low
dose levels. By utilizing a plate incorporation assay with metabolic
activation, the sensitivity of the DNA repair assay can be enhanced
toward compounds which are indirect-acting or sparingly soluble in
water. This slight modification to the standard procedure for measuring
fractional survival in 15. subtilis produces an increased sensitivity
towards certain genotoxic compounds and should increase its utility as
part of a battery of biological test systems. The DNA repair assay using
13. subtilis offers the additional advantages of increased
permeability of the gram positive organisms and the use of multiple
strains of repair deficient organisms which can detect inhibition of
different types of DNA repair (Tanooka, 1977).
B. Point Mutations: Prokaryote
1. Test system description
Microbial mutagenesis assays measure primary DNA damage by
detecting small changes in the nucleotide sequence of DNA. The more
common reverse mutation assays detect changes (point mutations) by
measuring bacterial growth in a media free of a specific substrate which
the bacteria require for growth. Increased colony formation (or growth)
following exposure to a specific agent implies that the agent has
induced reverse mutations. Damage to DNA by environmental mutagens may
contribute to human cancer, genetic defects, aging, and heart disease
(Ames, 1979). However, since reverse mutations are highly specific in
the type of required DNA interaction, a compound that does not increase
the frequency of reverse mutations may still induce other types of
genetic effects (Anderson and Longstaff, 1981). For this reason, a more
15
-------
1.0-
0.9-
0.7-
05-
I
I
tr
u.
0.3-
168 wt
rec E4
fh2006-7
0.1-
3 4 5 10
DOSE/PLATE (>rg-B(a)p)
Figure 2. Lethal effect of increasing doses of benzo(a)pyrene
on repair proficient (168 wt) and deficient strains
of B.subtilis with metabolic activation.
16
-------
STRAIN
168 wt
recE4
fh2006-7
e
OJ-
1.0
2345 10
DOSE/PLATE (*g-2AA)
Figure 3, Lethal effect of increasing doses of 2-amino-
anthracene on repair proficient (168.wt) and
deficient strains of B.subtilis with metab.olic
activation-.
17
-------
accurate risk assessment is obtained when a battery of bioassays are
used to detect a range of genetic damage.
The most widely validated microbial mutagenesis assay is the
Salmonella/microsome assay developed by Ames et al. (1973). Using
the Salmonella assay, test data has been published on more than 5,000
chemicals (Environmental Mutagen Information Center Index, 1982) and
several validation studies have been conducted (McCann et_ al., 1975;
Purchase et^ al., 1976; Sugimura ej^ al., 1976; Bartsch et
al., 1980; De Flora, 1981). The correlation between carcinogenicity
and mutagenicity using this test is currently estimated to be about 83%
(McCann and Ames, 1981). Categories of carcinogens which are poorly
detected by the Salmonella assay include azonapthols, carbamyls, and
thiocarbamyls; phenyls; benzodioxoles; polychlorinated aromatics,
cyclics, and aliphatics; steroids; antimetabolities; and symetrical
hydrazines (Rinkus and Legator, 1979).
In the present study, two bacterial assays have been utilized to
evaluate the capacity of various samples to induce point mutations. The
IJ. subtilis reverse mutation assay, as described by Tanooka (1977),
was used with selected waste fractions to supplement the Salmonella
assay. The IJ. subtilis assay has been found to be sensitive to many
compounds poorly detected by the Salmonella assay (Felkner, 1981) and
offers the advantage of being a gram positive organism that is more
permeable to certain compounds (Tanooka, 1977). The following discussion
will include the protocols used for the two prokaryotic mutagenesis
assays and the results obtained using various diagnostic mutagens.
2. Test system protocol: prokaryote mutagenesis
The Salmonella/microsome assay as described by Ames et al.
(1975) was used to measure the ability of a sample to revert strains of
bacteria to histidine prototrophy. A description of the J3.
typhimurium strains that were provided by Dr. B. N. Ames (University
of California, Berkeley, CA) is given in Table 5. The procedures used
were essentially the same as Ames et^ al. (1975). Overnight cultures
were grown from individual colonies picked from nutrient agar master
plates and inoculated into Oxoid Nutrient Broth No.2 (KC Biological,
Lenexa, KS). Cultures were incubated for 16 hr at 37 C. All strains
were checked quarterly or when new master plates were made for
nutritional markers for the following characteristics: the deep rough
character, the presence of the R factor, and sensitivity to ultraviolet
light. Top agar and petri plates were prepared as described by Ames
e_t al. (1975). Samples were tested with strains TA98 and TA100 in
two independent experiments at five dose levels on duplicate plates with
and without metabolic activation (0.3 ml rat liver/1.0 ml S9 mixture;
0.5 ml S9 mixture/plate). Aroclor 1254-induced rat liver was obtained
from Litton Bionetics (Charleston, S.C.). Positive controls, as well as
solvent and sterility controls, were run on each test date. The
positive control used to verify functioning of the metabolic activation
18
-------
TABLE 5. CHARACTERISTICS OF SALMONELLA STRAINS USED FOR MUTAGENESIS TESTING
Histidine
Strain
TA1535
TA100
TA1538
TA98
TA1537
Mutation
his
his
his
his
his
G46
G46
D3052
D3052
C3076
Additional
Mutations
LPS;
LPS;
LPS;
LPS;
LPS;
Exc
Exc .R
Exc |
Exc R
Exc
2
SMF
39
120
16
26
9
»
i.
IMF IMF + S9"
2774
2282
2158
2421
160
440
1833
870
1594
371
1- LPS - lipopolysaccharide cell wall.
Exc - excision repair deficient.
R - plasmid pKMlOl
2- Historical spontaneous mutation frequency for lab with DMSO, without S9.
3- Induced mutation frequency; TA1535, TA100-MNNG: TA98, TA1538-2NF; TA1537-9AA
4- Induced mutation frequency 2-acetylaminofluorene with activation.
-------
system for all strains was 10 ug 2-aminoanthracene (Sigma, St. Louis,
MO); while direct acting positive controls for TA98 and TA1538 were 25
ug 2-nitrofluorene, for TA100 and TA1535 2 ug
N-methyl-N'-nitro-N-nitrosoguanidine, for .. TA97 and TA1537 10 ug
9-aminoacridine, and 1 ug mitomycin C for TA102. Dimethyl sulfoxide
(DMSO) was used as a negative control.
A limited number of samples were also analyzed using the Bacillus
subtilis reverse mutation assay (Tanooka, 1977; Felkner git al. ,
1979). The two IJ. subtilis mutants were supplied by Dr. I. C.
Felkner (Clements Assoc., Washington, D.C.) and are described in Table
1. These strains may be used to detect either reverse mutation £o
His or Met or a forward mutation to prototrophy as His , Met .
In the present study, only the reverse mutation assay was utilized.
The methods employed were basically the same as described by
Tanooka (1977). Cells of TKJ5211 or TKJ6321 were grown overnight at
37°C with shaking in Oxoid Nutrient Broth No. 2. The cells were then
harvested, resuspended in an equal volume of glucose minimal medium, and
starved for 1 h at 37 C with shaking. To detect the His mutation,
0.2 ml of culture was plated with 2.5 ml soft agar on a suppplemented
minimal media (Tanooka, 1977). Plates were incubated for 72 hrs at
37°C.
Statistical AnalysisMutagenicity in the Salmonella/microsome
assay was determined using the modified two-fold rule (Chu e_t al.,
1981). To determine the mutagenicity of a sample, the mutagenic
activity ratio (MAR) is first calculated (Commoner, 1976) at the four
highest non-toxic dose levels. The mutagenic activity ratio is defined
as (E-C)/C. , where E is the experimental number of revertant
colonies (average of four plates); C is the number of revertant colonies
on the control plates (average of eight plates) obtained on the same day
with the same strain and microsome preparation; and C. is the
historical average of revertant colonies on control plates obtained the
year the test was run with the same strain and microsome preparation. A
sample was considered mutagenic if the MAR was greater than 2.0 for at
least two consecutive dose levels or in the last non-toxic dose level.
In addition, at least two of these consecutive doses must possess a
dose-response relationship showing an increase in the number of mutant
colonies at increasing dose levels. Chu et^ al. (1981) calculated
that when using the modified two-fold rule to determine the mutagenic
potential of a sample, false positives and false negatives will occur at
a rate of 4.1% and 1.8%, respectively. In an evaluation of seven
different methods for statistical analysis of data from microbial
mutagenicity assays, Chu et^ al. (1981) determined that the modified
two-fold rule was one of three analytical methods that gave an
acceptable rate of false positives and negatives. All raw data from the
Salmonella assay was first entered into a TRS-80 computer using a
program that calculated the means and standard deviations. This
information is provided in Appendix C. Selected data was then entered
20
-------
into a second TRS-80 program to calculate the mutagenic activity ratios.
A printout of the programs utilized to process data is provided in
Appendix D.
3. Test system results: prokaryotic mutagenesis
Prokaryotic mutagenesis assays using both J3. typhimurium and
B. subtilis were employed in this research. However, the vast
majority of the work was conducted using the Salmonella/microsome
assay of Ames e_t al. (1975). Recent modifications to the standard
protocol (Maron and Ames, 1983) and the addition of two new tester
strains (Levin ££ al., 1982a; Levin e£ al., 1982b) have enhanced
the sensitivity of this bioassay. Brusick (1983) reviewed the
sensitivity (mutagenic carcinogens/total carcinogens), specificity
(nonmutagenic noncarcinogens/total noncarcinogens), and predictive value
(mutagenic carcinogens/total mutagens) of the Salmonella assay as
reported in the literature. Based on studies which reviewed at least
100 chemicals, Brusick (1983) reports that the sensitivity ranges from
54 to 93%, the specificity ranges from 77 to 100%, and the predictive
value ranges from 87 to 100%. Overall, Brusick (1983) reports that the
true correlation coefficient is about 80% when compared to tumor
responses in mice and rats. Groups of chemicals which are poorly
detected in bacterial mutagenesis assays include antimetabolites, azo
compounds, carbamyls and thiocarbamyls, halogenated compounds, steroids,
cross-linking agents, inorganic compounds, and promoters (Brusick,
1983). Most of these groups can be detected by using microbial assays
which employ DNA repair deficient bacteria (Shiau et al., 1980;
Kanematsu et al., 1980) or eukaryotic organisms (Scott et^ al.,
1982; Kafer et^ al., 1982). Thus, when employed as part of a battery
of biological test systems, the Salmonella/microsome assay can provide
a highly efficient tool for monitoring genotoxic chemicals in the
environment.
The final interpretation of the results from the Salmonella/
microsome assay may be influenced by a variety of factors including the
selection of solvents, the size of the inoculum, the presence or absence
of metabolic activation, and the presence of histidine containing
substances. Organic solvents are used in biological testing in order to
dissolve compounds which are sparingly soluble in water and may affect
the results of the bioassay due to the solvents' toxicity or
interractions with cellular membranes and proteins (Abbondandolo et
al., 1980). Organic solvents which are compatible with the
Salmonella test include dimethyl sulfoxide, glycerol formal, dimethyl
formami'de, formamide, acetonitrile, 95% ethanol, acetone, ethylene
glycol dimethyl ether, l-methyl-2-pyrrolidinone, p-dioxane,
tetrahydrofurfuryl alcohol, and tetrahydrofuran (Maron e£ al. ,
1981). Abbondandolo et_ al. (1980) observed that ethylene glycol,
2-methoxyethanol, and methanol were also compatible with mutagenesis
test systems. While these 15 solvents provide a useful array of
characteristics, certain fractions of a hazardous waste may contain
21
-------
substances which are insoluble in any of these solvents. The data
provided in Table 6 indicate that a 1:10 mixture of dichloromethane in
dimethyl sulfoxide may be used to increase solubility without
significantly affecting the results. The use of such a combined solvent
system can, however, affect the results of the Bacillus and
Aspergillus assays (data not shown), and its use should be restricted
to preliminary testing in Salmonella.
The standard procedures for the Salmonella assay (Ames et
al., 1975) recommend the addition of 0.1 ml of an overnight (16 h)
culture, or approximately 1 x 10 bacteria to 2.0 ml of top agar. In
the revised methods, Maron and Ames (1983) suggest reducing the
incubation period to 10 h in order to increase the viability of the
cells plated. Matney (1981) and Green and Muriel (1976) state that the
number of spontaneous revertants per plate is completely independent of
the initial inoculum size within the range of 10 to 10 cells per
plate. As for the effect of inoculum size on mutagen yield, both Belser
et^ al. (1981) and Salmeen and Durison (1981) found no appreciable
difference in the yield of revertants induced by 2-nitro.f luorene at
plating densities ranging from 1 x 10 to 1 x 10 , although
increasing the cell density to 1 x 10 did produce a 2-fold increase
in revertant yield (Salmeen and Durison, 1981). MacPhee and Pallister
(1983) predicted that inoculum size would have a significant affect on
the results when testing a mixture of toxic and mutagenic compounds.
They observed that the sensitivity of the assay fiwas increased by
decreasing the inoculum size to approximately 1 x 10 cells per plate.
Barber et^ al. (1983) also observed a relationship between growth
and reversion frequency in the Salmonella assay. They found that the
final reversion frequency will be dependent on the L-histidine
concentration in the top agar, and that no reversion takes place during
the first four hours after the bacteria are plated (Barber et^ al.,
1983). The fact that no reversion takes place during the first four
hours after plating may explain the limited sensitivity of the plate
incorporation assay to volatile compounds (Barber £_t al., 1983).
Additional factors may also affect the reversion frequency in the
Salmonella assay. Peak £t al. (1983) demonstrated that the
liver-microsome S9 enzyme produces a highly significant increase in the
spontaneous reversion frequency. This data is consistent with that of
the present study (Table 7 and 8) which indicate that the rat-liver S9
extract alone increases the number of revertant colonies on control
plates.
Trace quantities of histidine which may be present in biological
material can also affect the results of the Salmonella/microsome assay
(Aeschbacher e£ al., 1983). This affect may be especially important
in the analysis of soil extracts. Aeschbacher et^ al. (1983)
recommend using a solvent or sorbent extraction to eliminate
interference from histidine. In the present study, XAD resins and
dichloromethane were used to extract water and soil samples,
22
-------
fO
OJ
TABLE 6. EFFECT OF A 1:10 MeCl -DMSO* SOLUTION ON THE
SPONTANEOUS REVERSION AND INDUCED MUTATION
FREQUENCY IN J5. TYPHIMURIUM
Revertants /Plate
Strain
TA98
TA100
TA1535
TA1538
TA1537
Sample
2-nitrof luorene
(25 pg)
2-aminoanthracine
(10 pg)
-
-
-
*~
DMSO
21
3008
2007
96
19
13
10
MeCl2:DMSO
24
2485
1949
91
21
11
9
* Dichloromethane:dimethyIsulfoxide.
-------
POSITIVE CONTROLS
Total Revertanta Mean » SD
Chemical S-9 Dose/Plate TA1535
None
ONSO
2-NP
MNNC
MMS
B(a)p
2AA
0
t 0
100 |il
» 100 Ml
250
25
2.5
4
2
0.2
20
2
0.2
50
t 500
50
5
.5
10
+ 100
10
1
36 » 9
22 7 4
39 t 10
22 7 6
NT
NT
NT
2,774 * 136
2,547 t 124
36 » 10
494 t 457
268 7 392
222 t 212
31 * 4
49 * 5
54 t 6
50 » 10
32 + 4
54 » 8
440 » 188
346 7 94
44 * 13
TAI538
18
16
35
1,765
2,158
850
22
188
156
124
52
33
870
t 8
NT
* a
* 9
* 572
7 749
+ 253
NT
NT
NT
NT
NT
NT
11
15
30
22
9
t 8
NT
* 343
~ NT
TA98
30
40
26
37
2.421
2.197
632
28
507
524
609
86
37
1,122
1.548
581
+ 8
7 15
* 6
7 10
t 423
+ 445
7 205
NT
NT
NT
NT
NT *
NT
« 7
7 99
7 137
7 152
± *'
* 13
7 469
« 444
+ 319
TA100
127
126
120
122
1,743
2,282
452
1.296
1.826
555
131
778
889
707
197
146
868
1,833
671
* 24
7 36
+ 38
7 27
NT
NT
NT
t 927
7 404
7 455
* 118
7 415
* 294
* 21
+ 247
7 178
7 130
t 31
+ 37
7 133
+ 554
7 446
TKJ5211
25 »
NT
31 *
21 7
417 t
93 7
NT
2,292"
39**
26"
393 «
54 7
29 *_
21 *
329 7
380 *
149 +
26 +
60"
341"
774"
196"
9
8
6
227
49
281
6.4
11
5
96
33
130
0.7
TKJ6321
103 * 14
NT
131 * 40
112 » 45
438 * 205
203 7 78
NT
1.519"
118"
109"
261"
133"
108"
124"
996 t 175
371 7 34
91 7 26
"NT
131 * 3
608 t 81
718 « 274
253 7 46
Negative controls: none-no additions to top agar; DHSO-100 |il Dimethylsulfoxide; Postive controls: 2-NF-2-Nitrotluoreoe;
MNNC-N-methyl-N'-nitro-N-nitroaoguanidine; MMS-methyl methane sulfonate; B(a)P- benzo(a)pyrene; 2AA-2-aminoanthracene;
NT " not tested.
** Represent one experiment with duplicate plates, standard deviation not provided.
-------
TABLE 8. VARIABILITY OF S. TYPH1HUR1UH STRAINS TA98 AND TA100 WITH NEGATIVE AND POSITIVE CONTROLS FOR EACH YEAR OF THE
ro
Ol
PROJECT
Strain Sample 3-9 C
TA 98 None
+
DHSO
+
HeCl
2NF
B(a)P
+
2AA
+
TAIOO None
+
DMSO
f
MeCl
MNNG
MMS
B(a)P
+
2AA
+
Year2 1981
lose/Plate
:
100 pi
10 pi
25 pg
50 pg
10 pg
-
100 pi
10 pi
2 Pg
2 pg
50 pg
10 pg
(N)
(NT)
(NT)
(72)
(105)
(NT)
(NT)
(34)
(NT)
(NT)
07)
(42)
(NT)
(NT)
(56)
(91)
(NT)
(NT)
(28)
(NT)
(NT)
(NT)
(22)
(35)
Hean + SD (N)
(246)
(94)
20 + 4 (529)
28 T 10 (449)
(80)
(60)
1.760+424 (112)
(8)
(10)
24 * 8 (123)
2,142 + 15 (126)
(144)
(88)
87 + 11 (416)
112 + 27 (374)
(83)
(55)
2,374 + 602 (106)
(8)
(6)
(6)
109 + 24 (113)
2,054 + 667 (110)
1982
Hean * SD
29+8
38 + 10
25+6
34 + 10
33 + 12
37 + 12
2.257 + 594
27 + 10
501 + 116
37 + 16
1.878 + 431
119 + 22
123 + 27
113 + 26
114 + 28
116 + 29
114 ^ 27
2.321 + 528
1,854 + 254
124 + 24
603 T 75
142 + 39
1,799 + 580
1983
(N)
(217)
(123)
(440)
(448)
(NT)
(NT)
(93)
(6)
(6)
(96)
(88)
(197)
(135)
(386)
(373)
(NT)
(NT)
(78)
(NT)
(8)
(8)
(91)
(85)
Hean + SD
30 + 8
42 +; 15
29 + 7
42 «; 10
-
1.943 + 315
28 + 9
731 + 88
41 + 12
1.751 + 392
132 + 25
128 + 42
132 + 55
132 + 26
-
2,197 + 549
-
137 + 19
1.104 *. 256
160 + 38
1,787 T 473
I. Negative controls: none - 3.0 ml top agar only; DHSO - dimethylsulfoxide; HeCl. - dichloromethane, 1:10 in DHSO;
Positive controls: 2NF - 2-nitrofluorene; B(a)P BerizoU)pyrene; 2AA 2-aminoanthracene|
MHNC » N-methyt-N'-nitro-N-nitrosoguanidine; HMS - methyl methaneaulfonate.
2. Year: 1981 - includes data from 6/1/81 through 12/31/81; 1982 Include* data from 1/1/82 through 12/31/82 and 1983
includes data from 1/1/83 through 9/31/83; (N) number of observation*; NT - not tested.
-------
respectively. It is anticipated that these procedures should eliminate
any histidine in water and soil samples. Additional factors which may
affect the results of mutagenicity tests include the method of media
preparation and amount of medium per . plate (Friederich et al.,
1982), the inducer used for the liver enzymes (Venitt, 1980), and the
presence or absence of visible light (MacPhee and Imray, 1974). Belser
et al. (1981) reported that the greatest source of variability is
nonuniformity of the top agar thickness. Because of these numerous
sources of variation, it is essential that microbial mutagenesis assays
be conducted under a Quality Assurance/Quality Control Program with
standardized protocols and that each experiment include sufficient
positive and negative controls to accurately assess the variability.
The extent of inter- and intralaboratory variation will be an
important factor in determining the utility of short-term bioassays as a
monitoring tool. The results presented in Table 7 demonstrate the
interlaboratory variation with four Salmonella strains and two
Bacillus strains for negative controls and diagnostic mutagens for the
entire project. For the negative controls, the variability ranged from
14% for TKJ6321 with no additions to 50% for TA1538 with DMSO (Table 7).
Cheli et^ al. (1980) observed that the plasmid containing strains
(TA98 and TA100) were generally less variable than the corresponding
non-plasmid strains (TA1538 and TA1535), although no such trend was
found in this laboratory. While a greater degree of variability was
observed with the diagnostic mutagens than with the negative controls,
none of the variations were sufficient to influence the final
interpretation of the results. That is to say, in all cases, the
no-effect dose level and the dose level at which a positive response was
obtained were constant. The overall variability of both prokaryotic
mutagenesis assays were comparable; however, for the compounds tested, a
greater mutagenic response was obtained in strains TA98 and TA100 than
in either of the TKJ strains.
The majority of the biological testing performed in this research
was conducted with strains TA98 and TA100. The variability of these two
strains for each of the three test years is provided in Table 8. Both
strain TA98 and TA100 exhibited a broad range of variability, although
in general the mean for each year was within one standard deviation of
the mean for the other two years. In all three years, the mean
spontaneous reversion frequency falls within the range considered
acceptable in a survey of eight laboratories conducted by the
Environmental Mutagen Information Center (1978).
Periodically during this research, titrations were conducted with
diagnostic mutagens in order to evaluate the sensitivity of the tester
strains. The results from testing the direct-acting mutagens MMS and
MNNG in strain TA100 are provided in Figures 4 and 5. For both
compounds on all three test dates, the results are comparable, although
a broad range of variation was observed at all dose levels. The results
from testing the direct-acting mutagen 2-nitrofluorene with strain TA98
26
-------
3000
tn
"c
a
> '500
2
IE
O
O
600-
300i
MMS
x 9/24/8!
O 9/2/83
9/5/83
5 10
DOSE/PLATE (ul)
15
20
Figure 4. Variability of mutagenic activity of TA100 induced
by MMS
27
-------
3000-
2100
1500
(ft
£
O
O
600-
300-
.25 .5
MNNG
X 9/24/81
7/23/82
9/13/82
2 3
DOSE/PLATE (ug)
Figure 5. Variability of mutagenic activity of TA100 induced
by MNNG.
28
-------
is provided in Figure 6. While there was a broad range of variability
for the 18 month monitoring period, the variability was comparable to
that observed by Cheli £t al. (1980) over an 11 month period.
The addition of metabolic activation is another parameter which can
significantly affect the variability of the Salmonella assay. Cheli
et al. (1980) observed that the addition of S9 and 2-aminoanthracene
resulted in a broad range of mutagenic responses. The responses
obtained with benzo(a)pyrene (B(a)P) and 2-aminoanthracene (2AA) in
strain TA100 are given in Figures 7 and 8. The variability of the
response from activated B(a)P is smallest at a dose level of 50 ug per
plate and increases at higher and lower dose levels. With 2AA, the
variability of the responses are greater than for B(a)P, although the
optimum dose level was consistently 10 ug per plate. Similar results
were obtained with activated B(a)P and 2AA in strain TA98 (Figures 9 and
10). The response induced by B(a)P was less variable than that induced
by 2AA; however, the overall mutagenic response induced by 2AA was also
greater. For both strains TA98 and TA100, the optimum dose level for
B(a)P and 2AA with activation remained constant on all test dates.
An additional comparison was conducted to evaluate the effects of
storage on the variability of the mutagenic activity of water extracts
over time. The results from two different water extracts tested in
three independent experiments at intervals of 2, 5, and 14 months after
collection indicate that there was no significant change in the
mutagenic activity at all dose levels in either sample (Table 9). There
was a large increase in the total induced mutation frequency for both
samples with activation on the last test date. However, these results
did not deviate significantly from the mean and may have been influenced
by the spontaneous reversion frequency that was almost double the
spontaneous reversion frequency from the initial test date. Thus,
microbial mutagenesis testing has indicated that storage of extracted
water samples at 4 C will not appreciably alter their mutagenic
potential over a 14 month interval. While retesting did clarify the data
interpretation, there was no difference in the final interpretation of
the data when compared to the results from each individual testing date.
These results, and those discussed previously, emphasize the need for
adequate positive and negative controls as well as duplicate experiments
in microbial mutagenicity testing. In addition, the Salmonella assay
exhibited a wide range of variability over time, although the optimum,
toxic, and no-effect dose level remained fairly constant.
The Salmonella/microsome assay is a rapid and simple test
procedure that can be used to evaluate the mutagenic and potential
carcinogenic activity of test sample. As part of a battery of
biological test systems, the Salmonella/microsome assay is considered
to be the most widely validated and most sensitive test system (Griem
e_t al., 1980). In addition, this is one of a limited number of
bioassays for which a standard protocol has been developed and which has
been subjected to numerous intralaboratory evaluations (Dunkel, 1979;
29
-------
3000
2 I 00-
-------
3000
2100
-------
300CH
2AA+S9
x 9/24/81
o 6/18/82
* 7/23/82
n 11/12/82
9/3/83
A 9/5/83
10
20 ' 100 300
DOSE /PLATE (ug)
500
Figure 8. Variability of mutagenic activity of TA100 induced
by 2AA.
32
-------
30001
2100^
09
*>»
C
ta
1 1500
00
05
600-
300-
B(a)p+S9
x 4/2/82
o 9/13/82
A 9/24/82
al1/12/82
5/31/82
A 9/2/83
9/5/83
-o
10
20
100
300
500
DOSE/PLATE Cug)
Figure 9. Variability of mutagenic activity of TA98 induced
by B(a)P.
33
-------
3000
2100
£ 1500
i_
>
4.
!E
oo
l-
600-
300-
2AA+S9
x 6/18/82
o 7/23/82
a 9/13/82
a 9/24/82
11/12/82
A 9/2/83
9/5/83
10 20 100
DOSE/PLATE(ug)
150
Figure 10. Variability of mutagenic activity of TA98
induced by 2AA.
34
-------
TABLE 9. THE EFFECT OF STORAGE AT 4 C ON THE MUTAGENIC ACTIVITY OF TWO RUNOFF
SAMPLES (SAMPLES WERE RECEIVED 5/13/1982)
to
Ul
t
Dose/plate
Sample
0582006
0582007
(rag)
0
.01
.05
.1
.5
1.0
0
.01
.05
.1
.5
1.0
7/9/82
17
33
34
42
91
Toxic
17
34
28
37
48
51
-S9
10/12/82
31
18
23
28
70
Toxic
31
18
18
15
28
26
TA98 his
+ *
Revertants
+ S9
7/18/83
34
17
24
42
60
Toxic
34
22
23
24
29
25
Mean
27
23
27
37
74
Toxic
27
25
23
25
35
34
7/9/82
20
54
32
66
129
120
20
22
65
79
145
115
10/12/82
39
28
48
65
102
107
39
32
55
73
87
70
7/18/83
39
56
58
91
201
275
39
46
56
76
138
164
Mean
33
46
46
74
144
167
33
33
59
76
123
116
* Average of two plates.
-------
de Serres and Ashby, 1981; Grafe et^ al., 1981). Because the plate
incorporation assay does not provide data as to the viability of exposed
cells, quantitation of this assay is difficult if not impossible
(Matney, 1981). As a result, risk assessment from bioassay data is also
impossible (Clive, 1980; Griem et^ al., 1981). Additional
limitations of the Salmonella/ microsome assay include its inability
to detect volatile compounds, inorganic compounds, and chlorinated
hydrocarbons (Brown et^ al., 1982). However, the Salmonella assay
is generally accepted as the primary test in a battery of bioassays
because it has a high sensitivity and specificity, and because it is
rapid and inexpensive to run (Brookes and de Serres, 1981).
C. Point Mutations and Chromosome Damage: Eukaryotic Organism
1. Test System Description
Eukaryotic assays for measuring genetic damage are capable of
detecting point mutations and small deletions as well as changes in
mitotic segregation resulting from induced nuclear damage.
Investigations of chemical mutagenesis in haploid Aspergillus
nidulans using the methionine assay has been shown to detect base-pair
changes and small deletions (Alderson and Hartley, 1969; Hartley, 1969),
while investigations using the diploid strains can detect changes in
mitotic segregation resulting from crossing-over, non-disjunction,
breakdown of mitosis, or chromosome aberrations (Kafer e_t al.,
1982).
The Aspergillus methionine assay detects forward suppressor
mutations by measuring colony formation on a media lacking methionine.
Restoration of the ability to synthesize methionine in Aspergillus has
been shown to arise from a forward mutation in any of six genes (Scott
and Alderson, 1971; Lilly, 1965). For the detection of mitotic
segregation or chromosome aberrations, diploid conidia heterozygous for
recessive color markers, non-utilization nutritional markers, and
resistant markers were utilized. Agents inducing mitotic segregation
are initially detected by visual observation of phenotypic changes using
diploid organisms grown on a complete media that contains the chemicals
to be tested. An increased frequency of phenotypic changes, e.g.,
changes in conidial color or colony morphology, implies that the agent
being tested has induced some form of genetic damage.
Although Aspergillus has only been tested with 150 compounds, the
same response was obtained in 26 of the 27 compounds for which data from
both animal carcinogenesis bioassays and Aspergillus were available.
In addition, five of the compounds giving a positive response in the
Aspergillus system and carcinogenesis bioassays were negative in the
Salmonella assay (Scott ^t al., 1982). The following is a
discussion of the procedures utilized for the haploid and diploid
bioassay, and the results obtained with solvent and positive controls.
36
-------
2. Test System Protocol
a. Methionine SystemThe bioassay using the haploid form of
Aspergillus nidulans has been used to assess the mutagenic potential
of various samples by evaluating the induction of mutations at the
methionine suppressor loci. The parent strain of Aspergillus was
obtained from Dr. T. Alderson, MRC Radiobiology Laboratory, Harwell,
England. The parent strain was stored in silica gel at 4 C. Stock
cultures were obtained by growing Aspergillus crystals on complete
medium. Working cultures were grown from organisms picked from the
stock culture. The requirements for biotin and methionine were checked
individually on media lacking the appropriate amino acid, and the
untreated experimental controls indicated the presence of an excessive
number of methionine suppressor mutants in the conidial suspension
before exposure to the test chemical. An acceptable overall spontaneous
mutation frequency should be within the range of 2.5 mutants per million
conidia assayed. Slightly higher spontaneous mutation frequencies can
be expected when conidia are exposed to 37 C or metabolic activation.
Conidia from four to five single colonies of the meth Gl; biAl
(requiring methionine and biotin) Glasgow strain of Aspergillus
nidulans grown for 5 to 6 days on a complete medium (CM) at 37 C
were used for each experiment. The conidia were suspended in dilution
fluid, i.e., water containing Tween 80, vortexed for 15 min to break up
conidial chains, and filtered through sterile cotton to remove mycelial
debris. The conidia were then concentrated on a 0.45 um millipore
filter (Millipore-Flouropore filters, Millipore Corp., Detroit, MI),
washed with at least 20 ml of dilution fluid, and resuspended in
phosphate buffer (pH 7.0) containing Tween 80.
Removal of germination inhibitory substances from the conidia of
Aspergillus is essential to achieve consistent levels of survival
after mutagenic treatments (Scott et al., 1972). Inhibitor-depleted
conidia were prepared by the addition of a 1% (v/v) solution of diethyl
ether to the conidial suspension. The flask containing the 50 ml of
conidial suspension with diethyl ether was placed on a shaker for 1 hour
at 37 C. The conidia were then collected on a 0.45 um millipore
filter, washed with at least 20 ml of dilution fluid, and resuspended at
a density of 240 million conidia/ml in Tween 80 and phosphate buffer (pH
7.0). This method for removal of the germination inhibitor is described
in Bertoldi e_t al_. (1980).
Test compounds were dissolved in DMSO and added to
inhibitor-depleted conidia to yield the appropriate final concentrations
of the test chemical. All manipulations and exposures were completed
under yellow light. Throughout the exposure, the conidial suspensions
were enclosed in a teflon sealed pyrex glass test tube and incubated
with mild agitation at a temperature of 37 C for the desired period.
The treatment was stopped by the addition of 9 ml of ice cold dilution
fluid to 1 ml of the reaction mixture. For the non-activated system,
37
-------
1 ml of approximately 24 million conidia in Tween 80 phosphate buffer
was added to the exposure tube. At the appropriate time, 10 pi of the
appropriate concentration of the chemical in DMSO was added to the
conidial suspension.
For the activated system, 0.1 ml of approximately 240 million
conidia in Tween 80 phosphate buffer was added to the exposure tube
containing 0.9 ml of the S9 mix. Thus, the exposure concentration of the
conidia was 24 million conidia per ml. At the proper time, 10 ul of
the appropriate concentration of the chemical in DMSO was added to the
conidial suspension, and the previously described procedure was then
followed.
Immediately before and after treatment, the viability and
mutability of a conidia population from the biAl, methGl strain of
Aspergillus was assayed by platting on the appropriate minimal media.
To estimate the viability, minimal medium plates containing methionine,
biotin, and sodium deoxycholate were used. Appropriately diluted
suspensions were spread over the surface of five plates and incubated
for at least two days at 37 C before the viable colonies were manually
counted. Media of a similar composition, although lacking methionine
and sodium deoxycholate, were used to detect all the methionine
suppressors. The methionine-free minimal media was melted in a water
bath at 45 C to prevent gelling. Mutants were assayed by pipetting
samples of an appropriately diluted conidial suspension into 100 ml of
melted minimal media. The resultant suspension was then poured onto 12
petri dishes. The volume added was regulated by the potency of the
agent. These plates were incubated for five to six days at 37 C. At
this time, the mutants were classified as A (large green colonies), B
(brown heavily pigmented colonies), and C (small green colonies with a
white hyaline edge) in accordance with the classification system
described by Lilly (1965).
For meaningful quantitative data with this system, not only is it
important to ensure complete removal of any "germination inhibitory"
substance but also to restrict the average total number of methionine
mutants per 100 mm diameter petri dish to less than 20 (Scott et
aj^., 1973).
For the haploid mutation system, 8-methoxypsoralen (8-MOP) and near
ultraviolet light were used for the positive control. In the absence of
near UV, 8-MOP can also act as a negative control. The advantage of
using this chemical is twofold. First, this particular photosensitizing
reaction randomly induces mutation at all loci; therefore, it
quantitatively and qualitatively checks the response of the multilocus
system, in addition to acting as a positive control. Second, the
compound alone exhibits no mutagenic properties with the biAl;
methGl strains in the absence of light (Alderson and Scott, 1970;
Scott and Alderson, 1971). DMSO exposures under non-growth conditions
yield a negative response in this system. This control and the
38
-------
activation mixture control were included in the experimental procedure
to ensure that no undetected substances were inadvertently included in
the experiment which could lead to a false positive response.
Benzo(a)pyrene requires activation in this assay system and was included
in the procedure to insure the proper functioning of the activation
mixture.
Each piece of raw data was recorded following good laboratory
procedures. Raw data was not averaged, transformed, or corrected before
recording. Data was recorded in tabular form to indicate numbers of
identified mutants and classification of mutants e.g., morphological
types. Sufficient detail was recorded so that verification of survival
and mutation frequencies could be undertaken if necessary.
The data generated from this study was interpreted in the following
four ways. First, data was analyzed by a 3 x N contingency chi square.
Second, mutation frequency vs concentration was compared to the
spontaneous background mutation frequency. Third, mutation frequency vs
exposure time at a fixed sample concentration was compared to background
mutation frequency. If the mutation frequency of the latter two analyses
exceeded twice the spontaneous level, the test was considered positive.
The fourth method of analysis employed an adapted version of the
mathematical model of Munson and Goodhead (1977). The points were
plotted according to the equation:
M-M = -m In (S/S )
o o
where:
S = population of viable cells at time 0,
M = mutation frequency/viable cell at time 0,
S = number of viable cells after exposure to a dose D
of the waste fraction in question,
M = mutation frequency/viable cell after exposure to a
dose D of the compound in question.
Data were plotted such that the y-axis represents induced mutation
frequency (M-Mo) and the x-axis denotes lethal hits (ln(S/S ). Linear
regression analysis was used to determine the best fit of an unweighted
straight line through the data points. If the homogenity as measured by
chi square was not significant, the slope of the regression line was
obtained. When the slope is less than or equal to 2.5 per million
survivors for all suppressor mutants, the compound is regarded as
non-mutagenic. If the slope has a value of greater than 5 per million
survivors, it is regarded as mutagenic. The values for a negative
response of the individual classes of mutants are less than 1.25 per
million surviving conidia for A or B and 0.25 for C. Values greater
than 2.5 (Class A and B) or 0.5 (Class C) per million surviving conidia
are regarded as mutagenic.
39
-------
b. Diploid AssayTreatment of heterozygous diploid conidia with
mutagens and various chemicals can either increase one of the processes
of spontaneous segregation or it can induce mutations that include
chromosome aberrations and may lead to apparent increases in
segregation. Basically, the four following types of effects can be
identified by the relative frequencies and types of stable and unstable
segregants: mitotic crossing-over, mitotic non-disjunction, breakdown of
mitosis, and mutations with deleterious dominant effects.
The type of mitotic crossing over detected in heterozygous diploids
occurs between chromatids at the four strand stage of mitosis. These
reciprocal exchanges between markers involving two homologous chromatids
can produce complementary diploid recombinants that are homozygous for
all markers distal to the exchange, i.e., segregants homozygous for all
markers of one chromosome arm. No change in ploidy and viability occurs
in this exchange, and the selection is not operative. This type of
exchange is usually detected in colonies growing on solid CM by visual
identification of segregant sectors that express recessive mutants
present in the heterozygous diploid as markers. The most conclusive
evidence for mitotic crossing over are twin spots or twin sectors, i.e.,
recovery of both products of a single reciprocal exchange as paired
sectors.
Mitotic non-disjunction of typically single chromosomes produces
2n+l types in the first instance; in Aspergillus, the reciprocal 2n-l
product is inviable. The trisomic 2n+l types show no segregation of
recessive markers but have a somewhat reduced growth rate compared to
the original diploid. Although typically not distinguished from other
genetic damage in the original growing conidia, they can be identified
through a secondary plating of the original abnormal head. This
secondary plating probably increases the rates of secondary
non-disjunction or chromosomal loss, as is common for trisomics of many
species. The 2n+l types, therefore, may produce diploid 2nd-order
segregants with relatively high frequencies. The random loss of one of
the three homologues of a 2n+l can easily be seen when trisomic conidia
are plated at low density. Absence of competition leads to fairly good
but not normal growth of the 2n+l type in the center of colonies. At
various points in the colony, faster growing diploid sectors are
observed which are due to the loss of the extra chromosome. One third of
these diploid improved sectors are showing segregation of a chromosomal
type, i.e., all recessive markers of 1 chromosome show up simultaneously
in the non-disjunctional diploid sectors. The detailed growth pattern,
especially size and conidiation of the trisomic center, are different
and characteristic for each of the 8 2n+l, trisomics for any one of the
8 linkage groups or chromosomes. Each can be identified on standard
media if no chromosomal aberrations are present. For spontaneous
mutation frequencies, non-disjunction is the main process producing
spontaneous haploids, which are rarer than non-disjunctional 2n
segregants; these, in turn, are 5 to 10 times less frequent than 2n
mitotic crossing over. For induced mutation frequencies, these ratios
40
-------
are altered.
Misdistribution of chromosomes leading to haploidization rarely
occurs but is a frequent effect of treatments with spindle poisons. If
the products of single treated nuclei are analyzed carefully, a large
number of somewhat-related segregants are found, all of them showing
chromosomal type segregation. These include any aneuploid types that
are viable, i.e., 2n+l up to 3 and n+1 up to 3. Specific aneuploid
types are recognizable only if conidia from the most abnormal-looking
areas are replated at low density. The stable types, however, are
mainly haploids, in addition to some "non-disjunctional" diploids, i.e.,
diploid segregants homozygous for all markers of 1 or several
chromosomes. Haploids are the most viable and best growing types if
mitotic inhibitor is present during growth of the mycelium. Such agents
are, therefore, used for haploidization of diploids in order to verify
genotypes or detect translocations and recessive lethals.
Induced mutation with dominant effects on viability, semi-dominant
lethals, deletions, and other unbalanced aberrations, lead to formation
of abnormal colonies. The original unbalanced nuclei are at a
disadvantage, resulting in poor growth and the formation of few conidia
even if plated at low density. However, as is the case of aneuploids,
the original unbalanced nuclei spontaneously produce well-growing
2nd-order segregants, either by mitotic crossing over, non-disjunction,
chromosome loss, or any combination of these. In competition with the
growing mycelia of normal nuclei, only the euploid final segregants will
be recovered from the original unbalanced nuclei. These induced
mutations are of the same type as produced by the three preceeding
mechanisms and vary primarily in relative frequencies. Increase of
haploid and diploid crossing over is usually diagnostic for chromosomal
aberrations, since none of the preceeding three types of effects produce
both types of segregants simultaneously.
Mutations in diploids that result in recessive lethals or balanced
translocations will predominantly produce no obvious change in
phenotype. In other words, there will be no change in morphology or
growth, nor any detectable segregation of markers. Such mutations can,
however, influence the recovery of haploid types when these are selected
from heterozygous diploids. Any recessive lethal on a specific
homologue prevents recovery of the marker located on this chromosome.
Balanced translocations generally cause complete linkage of markers that
are located in the two involved chromosomes.
Diploid strains of Aspergillus are not completely stable; test
diploids must be synthesized fresh from heterokaryons between 2 suitable
haploid components. This has the advantage that strains are easily
tested for all markers, except for certain cases of epistatic types,
using standard procedures.
41
-------
c. Diploid strains for analysis of induced segregationThere are
two main considerations in the construction or choice of test diploid
that work in the opposite directions. First, a minimum of testing
should be needed for the identification of small increases in the
frequencies of the three main types of stable segregants so that large
numbers can be handled for preliminary screening. Secondly, a diploid
should contain enough markers for detailed analysis of the type of
induced genetic damage. Ideally, the diploid should have the following
features:
a. Markers to distinguish all haploids from diploids by simple
tests;
b. Markers on both arms of one or more chromosomes to
distinguish diploid crossing over from diploid
"non-disjunctional" types;
c. Two recessive color markers on the same chromosome arm,
heterozygous in repulsion, to identify twin sectors; these
may also permit visual identification of certain haploids
from diploid segregants;
d. One marker in each linkage group to check segregation of the
8 different chromosomes in haploids;
e. Markers on each homologue, or semi-dominant markers in each
group to follow all chromosomal segregation in diploid, as
well as haploid segregants;
f. Many markers on both arms of at least one linkage group to
follow segregation patterns after induction of chromosome
aberrations or semi-dominant lethals.
New diploids are constantly being constructed to meet the above
requirements. The diploid used in this investigation was Diploid 20, a
cross of haploids F475 x F513 (Figure 11). Haploid strains were obtained
from Fungal Genetic Stock Center (FGSC, Arcadia, California).
Data from the diploid assay is expressed as the induced segregation
index with respect to the negative control or the solvent control. The
induced segregation index is calculated by dividing the frequency of
mutations induced by a specific sample by the spontaneous mutation
frequency (negative control). A response is considered positive if the
induced segregation index is statistically greater than 1.0. Following
treatment of heterozygous diploid conidia, the abnormal or segregating
colonies are replated to obtain secondary segregation or pure primary
segregation, respectively. These isolates are further characterized by
replicate plating onto various nutritional and resistant media.
42
-------
CO
fpaB37 galD5 suAadE20 -I- rlboA anA pabaA yA2 adE20 blA
, '
-t- + + sulA *-+*- + adE20 +
sD85 fwA2 4-
AcrA ActA pyroA4 facA303 lacA sB3 choA -f -f chaA
H nr EZ
Figure 11. Genotype of Aspergllus nidulans diploid 20.
-------
The nature of the genetic damage can then be determined by the
response on the various nutritional and resistant media. The mutation
frequency for each class and overall genetic damage is then determined
by dividing the number of colonies in each class by the total number of
colonies examined for each exposure.
3. Test System Results (Eukaryote)
Two groups of compounds were used as solvent and positive controls
in the haploid and diploid assays using A. nidulans. In the haploid
methionine system, DMSO and 8-MOP served as negative controls, where as
8-MOP+NUV and benzo(a)pyrene (B(a)P) were positive controls. Following
exposure to 10 ul of the solvent DMSO for 60 min, the frequency of
induced mutations per million surviving conidia for colony types A, B,
C, and the total were 0.47, 0.21, 0.041, and 0.72, respectively (Table
10). For the 8-MOP in the absence of near ultraviolet (NUV) light, the
total induced mutation frequency was 1.4/10 , and the induced mutation
frequencies for class A,B, and C were 0.69, 0.48, and 0.24/10 ,
respectively (Table 10). Mutations were randomly induced at all loci
when the 8-MOP was photoactivated with near UV light. The total induced
mutation frequency was 250 per million surviving conidia. In addition,
activated 8-MOP induced 89, 88, and 70 mutations/10 surviving conidia
in classes A,B, and C, respectively (Table 10). Benzo(a)pyrene in the
presence of a rat liver activation system induced 16 type A and B, 8
type C, and 40 total mutations per million survivors (Table 10). The
standard deviation of the induced mutation frequencies for these
controls observed over the three years of testing of DMSO, 8-MOP,
8-MOP+NUV, and B(a)P were 94, 69, 18, and 50%, respectively. In most
cases, the greatest deviation was observed in class C colonies. In no
case, however, was the deviation sufficient to alter the interpretation
of the results.
The negative control used in the diploid assay was the top agar
alone, while DMSO served as a solvent control, and benomyl was the
positive control. The results from the negative and positive controls
for the segregant and abnormal colonies recovered from the diploid A.
nidulans are summarized in Tables 11 and 12. The induced segregation
index for the segregant colonies with respect to DMSO was 0.04 for the
negative control and 3.7 for benomyl. While the induced segregation
index for DMSO and benomyl with respect to the negative control was 1.4
and 5.2, respectively. Benomyl induced genetic damage at all loci in
both segregant and abnormal colonies. The overall mutation frequency
observed in the abnormal colonies induced by benomyl was .157, which is
equivalent to an induced segregation index that is approximately four
times greater than both the solvent and negative controls. Significant
increases were observed in all classes of abnormal colonies induced by
benomyl, except the hyperhaploid class.
The two bioassays using Aspergillus nidulans as an indicator
organism to detect primary genetic damage provide information not
44
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TABLE 10. SURVIVING FRACTION AND INDUCED MUTANTS PER SURVIVOR IN HAPLOID A. NIDULANS
FOLLOWING TREATMENT WITH SOLVENT AND POSITIVE CONTROLS
Chemical
DMSO
8-MOP
8-MOP+NUV
B(a)p
Dose/pi Exposure Number of Surviving
(ug) Time (min) Observations Fraction
(Mean + SD)
10 pi 60 14 .98 + .02
50 90 14 .99 + .05
50 5 14 .47 + .13
11.6 20 14 .52 +_ .03
Colony
Type
A
B
C
T
A
B
C
T
A
B
C
T
A
B
C
T
Induced Mutations/
Survivor X 10
(Mean _+ SD)
.47 + .33
.21 + .37
.041+ .057
.72 + .68
.69 + .59
.48 + .46
.24 + .24
1.40 + .97
87.0 + 14.0
88.0 + 17.0
70.0 + 16.0
245.0 _+ 45.0
16.0 + 6.9
16.0 + 8.5
8.0 + 4.7
40.0 + 20.0
-------
TABLE 11. SUMMARY OF GENOTOXIC EFFECTS INDUCED BY CONTROLS IN SEGREGANT COLONIES OF DIPLOID A. HIDULANS
Sample
Control
DMSO
Benomy 1
1
Total1
619
140
299
Overall
1.9
4.0
12.7
Yellow
CO ND
0.3 0.5
0.7 0.7
1 3.5
Phenotype '
Fawn CHAT DC CR
M 2n n 2n n 2n n 2n n
0 0.3 0 0.3 0 0.3 0 0.2 0
00 0.7 2.0 0 0 0 0 0
0.3 1.4 0.3 2.3 0.7 1.0 1.3 1.0 0
ISI*
CT DO
.04
1.4
5.2 3.7
1. Total " number of segregant colonies examined; overall « total number of induced aegreganta.
2. Units - total frequency of segreganta per colonies examined.
3. Phenotype: Yellow-includes only colonies that were completely yellow, all other listed in other sections;
CO crossing-over, ND -non-disjunction, M nutations, Fawn tan sectors; CHAT * chatreuae; DG - dark green
G " green; 2n - diploid; n - haploid.
4. Induced segregation index - Total induced aegregants divided by frequency of control aegreganta. CT = evaluated
with respect to controla; DO " evaluated with respect to solvent control (DHSO). If ISI is significantly greater
than 1.0, response is considered positive.
-------
TABLE 12. SUMMARY OF GENOTOXIC EFFECTS OBSERVED IN ABNORMAL COLONIES OF DIPLOID
A. NIDULANS FOLLOWING EXPOSURE TO CONTROLS
Genetic Damage Observed
Sample
CONTROL
DMSO
BENOMYL
Total2
COL=619
MF
ISI(S)
ISI(D)
COL=140
MF
ISI(S)
ISI(D)
COL=299
MF
ISI(S)
ISI(D)
0V
22
.036
-
-
6
.043
1.19
-
47
.157
4.36
3.65
HD
14
.023
-
-
3
.021
.91
-
19
.050
2.17
2.38
NH
. 3
.005
-
- '
2
.014
2.8
-
3
.010
2.0
.71
HH BK
2 1
.003 .002
-
-
0 0
-
- -
- ' -
1 19
.003 .064
1.0 32
_ »
OT
2
.003
-
-
1
.007
2.3
-
5
.017
5.67
2.43
1. Genetic dauage observed: 0V = overall summary of genetic damage observed in
all categories; HD = hyperdiploid, results from non-disjunction of chromosomes;
NH = near hyperdiploid, probably breakdown products including a mixture of haploid
and diploid colonies; HH = hyperhaploid, results from major deletions or lethals
in chromosomes; BK = breakdown, results from mitotic spindle poisons; OT ° others,
results from mitotic recombination which has occurred prior to aneuploidy.
2. Total: col - total number of colonies examined, and total number of abnormala in
each class; MF = total mutation frequency = 0V COL; ISI(S) ° induced segregation
index with respect to control = MF (sample) MF (control); ISI(D) = induced
segregation index with respect to solvent control - MF(sample) MF(DMSO).
-------
available from the bioassays using IJ. subtilis or J3.
typhimurium. The methionine system employs a eukaryotic organism to
detect forward mutations, and the diploid assay provides additional
information on chromosome damaging agents.. While the variability of the
negative and solvent control was quite high, the spontaneous mutation
frequency is extremely low and the frequency of mutations induced by the
positive controls is extremely high. The low background mutation
frequency observed in Aspergillus bioassays does not overwhelm the
response as would occur in a test system with a high background. A
compound inducing a weak response might go undetected in a test system
with a higher background frequency. Aspergillus, because it can be
used in a haploid or diploid form, can detect a broad range of genetic
damage and, as a result, detects a range of genotoxic compounds.
The primary limitation of the Aspergillus bioassays is the
complexity of the test protocols. The time and expenses incurred in
conducting a complete Aspergillus testing protocol will restrict its
utility as part of a routine monitoring program. However, Aspergillus
is a critically important segment of the initial waste screening
protocol because it is one of the least complex and most widely
validated test systems used to detect chromosome damage.
D. Evaluation of the Battery of Test Systems
Four different biological test systems have been evaluated in this
research to determine their utility to monitor the environmental impact
of land treatment of hazardous industrial waste. The DNA repair spot
test is severely limited by problems with compounds that are insoluble
in water or require metabolic activation to reach their ultimate
mutagenic form. The modified plate incorporation assay (Donnelly et
al., 1983) enhances the sensitivity of the DNA repair assay but also
increases the cost per sample. However, since DNA repair assays are
capable of detecting mutagenic compounds not detected by the
Salmonella assay (Shiau et al., 1980), they do provide a useful
compliment as part of a battery of bioassays. Thus, the DNA repair plate
incorporation assay would be useful in a waste characterization
protocol, while the utility of the spot test would be limited to a
monitoring program provided its sensitivity to the anticipated mixtures
has been previously demonstrated.
The Salmonella/microsome assay is perhaps the least complex and
most widely validated of all the microbial mutagenesis test systems.
This bioassay is known to have a high degree of sensitivity and
specificity (Bridges et^ al., 1981), while it is insensitive to a
number of compounds that might be anticipated in a hazardous waste
(Rinkus and Legator, 1978). Although a negative result in a bacterial
mutagenesis assay is not a definitive indicator of non-carcinogenicity
due to the existence of non-mutagenic carcinogens, this test can serve
as an extremely effective screening test for those compounds which are
carcinogenic by causing damage to DNA (Bridges et^ al., 1981). The
48
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bacterial mutagenesis assay using 15. subtilis did not appear to
enhance the sensitivity of the battery of bioassays, although it was
only used to test a limited number of samples. The Bacillus
mutagenesis assay has been found to be sensitive to compounds not
detected in , the Salmonella assay (Shiau e_t al. , 1980) and may be
applicable to certain types of waste not evaluated in the present study.
This research and an earlier evaluation of a battery of bioassays by
Ashby (1981) confirmed that the Salmonella/microsome assay is
best-suited as the primary test system.
Ashby (1981) also recommended that the bioassay used to compliment
the Salmonella test be based on a eukaryotic system. The two
eukaryotic bioassays utilizing A. nidulans appear to offer the
maximum specificity and sensitivity of all the test systems evaluated in
the present research (Scott et al., 1982). Brookes and de Serres
(1981) suggest that there are two critical requirements for a test to
compliment the Salmonella assay. These include a high specificity in
order to prevent increasing the incidence of false positives and the
ability to detect those carcinogens missed by Salmonella. As a
compliment to the Salmonella assay, the Aspergillus bioassay meets
both these requirements. The primary utility of the Aspergillus
bioassay lies in its ability to detect compounds and types of genetic
damage not detected in the prokaryotic systems. Because of the
complexity of the Aspergillus test protocols, the utility of this
system is limited if a mutagenic sample is also detected in a
prokaryotic assay.
The battery of bioassays that is employed to monitor hazardous
waste disposal should be selected through the evaluation of several
factors. The test protocol used to characterize a hazardous waste
should include test systems capable of detecting DNA damage, point
mutations, and chromosome damage. The composition of the battery of
biological test systems used to characterize a waste should be
determined according to the number, structural type, and anticipated
environmental fate of the hazardous waste constituents. Brookes and de
Serres (1981) suggest that by considering these factors when selecting a
battery of bioassays, sensitivity (mutagenic carcinogens/total
carcinogens) can be as high as 100%, while specificity (nonmutagenic
noncarcinogens/total noncarcinogens) would be no less than 95%. As the
waste characteristics are defined and the fate of waste metabolites is
better understood, the intensity of the testing protocol can be reduced.
Ultimately, once the lab and field evaluations have been completed, the
monitoring of a hazardous waste land treatment facility should be
feasible using only one of the more simple spot tests.
49
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SECTION 5
WASTE CHARACTERIZATION
INTRODUCTION
Industrial wastes, defined by regulations as hazardous, may contain
chemicals that are ignitable, reactive, corrosive, or toxic (CFR, 1980).
The present study has employed short-term bioassays to detect mutagens,
as well as other potentially genotoxic agents present in waste
fractions. Genotoxic compounds constitute the relatively small group of
agents that are highly specific for nucleic acids and produce
deleterious effects in genetic elements at subtoxic concentrations
(Brusick, 1980).
All current waste disposal techniques, e.g., incineration,
landfilling, deep well injection, and land treatment, could potentially
result in the release of genotoxic compounds into the environment.
Estimates as to the environmental origin of cancer range from 60 to 90%
(Epstein, 1974). While much of the environmental cancer may result from
individual exposures, e.g., smoking, drugs, alcohol, etc., the disposal
of hazardous waste should be carefully monitored, in order to minimize
any additions to this burden.
The first step in monitoring any method of waste disposal is to
accurately assess the hazardous characteristics of the, waste. The
analytical methodology that is currently being utilized to characterize
hazardous wastes employs only a chemical analysis. Chemical analysis
alone is insufficient to evaluate the hazardous characteristics of a
waste. Component identification of a complex mixture cannot account
for potential interactions that may occur between components of the
mixture or the waste and soil components. Interactions between waste
and/or soil components may be synergistic, additive, or antagonistic.
Biological analysis can be used to evaluate the toxic potential of the
waste as a whole and the subfractions of a waste or a waste-soil
mixture. Chemical analysis can be used to supplement the results of
biological testing in order to define the nature of toxic constituents
and verify the absence of artifacts generated in the collection or
extraction process. Bioassay directed chemical analysis provides a
management tool which can be used to make semi-quantitative risk
assessments when the results are compared to reference compounds, such
as cigarette smoke condensate (Barnes and Klekowski, 1978).
50
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This research has been conducted to evaluate the mutagenic
potential of three hazardous industrial wastes. Microbial bioassays
capable of detecting point mutations, lethal damage to DNA, and
chromosome damage have been conducted on the subfractions of the three
wastes. Since the composition of DNA in microorganisms is the same as
that in humans, these test systems are anticipated to provide an
indication of the potential of waste constituents to cause genetic
damage in the human population.
MATERIALS AND METHODS
Wastes
A group of thirteen wastes were initially collected for use in the
project. These included two wood-preserving wastes, four refinery
wastes, four petrochemical wastes, a pulp and paper waste, an alum
sludge, and a paint sludge. A listing of these wastes is given in Table
13. Ten wastes were selected for acute toxicity analysis, and three of
the ten were selected for use in the waste characterization and
greenhouse studies. Waste selection for the characterization and
greenhouse studies was based on the results of a chemical
characterization and the results from the acute toxicity study. Each
waste was collected on site by study personnel. A permit was obtained
from the Texas Department of Water Resources to allow for the
collection, transport, and storage of hazardous waste.
Prior to departure for collection of the waste, materials were
assembled for waste collection, personnel protection, and waste storage.
Although most wastes were collected in-line at a refinery or
petrochemical plant, shovels, buckets, and a sludge pump were
transported to each site in order to facilitate waste collection.
Protective devices included respirators, gloves, goggles, and disposable
clothing. The collected waste was placed in a 55 gallon barrel. Each
drum was washed with soap and rinsed with water before use. Two barrels
containing approximately 50 gal of each waste were obtained. Upon
returning to the laboratory, a 4 1 reserve sample of each waste was
collected and stored at 0°C.
The wood-preserving bottom sediment, PENT S, waste was collected
from a wood-preserving plant utilizing both pentachlorophenol and
creosote as preservative agents. The effluent from the treatment
process was pumped from a concrete storage basin to a large lagoon where
the solids were allowed to settle-out while the liquids evaporated. At
the time the waste was collected, the lagoon had been in use for several
years, and the bottom sediment was approximately .9 meters in depth. The
ultimate planned disposal technique for this waste was to dredge the
lagoon and remove the sediment to a landfill. There are, however, six
existing facilities in the U.S. utilizing land treatment for disposal of
wood-preserving waste (Brown and Assoc., 1981). Hazardous constituents
anticipated in the PENT S waste include phenols, polycyclic aromatic
51
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TABLE 13. GROSS CHARACTERISTICS OF HAZARDOUS WASTES COLLECTED FOR STUDY
Waste
1 2
EPA No. Extractable Physical Use in
Hydrocarbons Form Study
Ln
S3
Wood-Preserving Bottom Sediment K001
(PENT S)
Wood-Preserving Wasteuater -
Slop-Oil Emulsion Solids K049
Combined API Separator ,
Waste Treatment Sludge (COMBO) K051
Storm-Water Runoff Impoundment (SWRI)
Dissolved Air Flotation K048
Acetonitrile K013
Methyl Ethyl Ketone
.
Phenol production -
Agricultural Chemicals -
Biosolids Waste
Primary Clarifier Pulp and Papermill
Alum Sludge
Paint Sludge
27
NT
86
41
21
5
2
97
0.2
0.2
NT
NT
NT
Sludge
Liquid
Liquid
Sludge
Sludge
Sludge
Liquid
Liquid
Liquid
Liquid
Solid
Liquid
Sludge
A.W.G.L
A
A,W
A.W.G
A.W.G
A
A,W
A,W
A
A
N
N
N
1- Percent by weight, extracted with dichloromethane; NT = not tested.
2- Physical form estimated from visual observation.
3- N=Not used; A=acute toxicity; W=waste characterization; G=greenhouse; L=lysimeter.
-------
hydrocarbons, pentachlorophenol, other chlorinated hydrocarbons, and
dioxins. Although the volume of wood preserving sludge produced by the
entire industry each year is less than 2,000 dry metric tons, these
sludges contain compounds that are toxic . even at extremely low dose
levels. This waste was utilized in the acute toxicity, waste
characterization, greenhouse, and lysimeter studies.
A second wood-preserving waste was collected from a facility using
both creosote and pentachlorophenol as preservative agents. This waste
was collected on site by pumping the effluent from a lagoon used for
spray irrigation. Being a wastewater and not a sludge, this waste was
only used in the acute toxicity study.
The slop-oil emulsion solids waste was collected from an API
oil-water separator at a petroleum refinery. The waste is generated
from skimming the emulsified oil-water layer from an API separator; it
was collected by pumping the waste from the pit into a tank truck and
discharging from the tank truck into collection barrels. More than
31,000 metric tons of hydrocarbons are generated in the U.S. from this
waste stream each year (Abrams et^ al., 1976). Slop-oil emulsion
solids typically contain 40% water, 43% oil, and 12% solids. This waste
is listed as hazardous because significant quantities of lead and
chromium may be encountered in the waste. The sample collected for the
current study was a liquid waste with a low solids content and composed
of 86% extractable hydrocarbons. Considering the separation process
from which this waste was generated, the slop-oil emulsion solids should
be composed of predominantly low molecular weight hydrocarbons. A waste
that is composed of predominantly saturated or other low molecular
weight hydrocarbons should degrade quite rapidly following land
application. The slop-oil emulsion solids waste was only used in the
acute toxicity study.
The second refinery waste consisted of the combined waste streams
from the API-separator sludge and slop-oil emulsion solids. The sludge
was being stored in a two acre lagoon. The waste was collected from the
lagoon with a backhoe and placed in the barrels for transport. The API
separator sludge and slop-oil emulsion solids account for 13.9 and 18.6%
of the total hydrocarbons in refinery solid waste, respectively (Abrams
et al., 1976). Waste streams of this nature are currently being
treated by land application at a large number of facilities in the U.S.
(Brown and Assoc., 1981). Anticipated mutagenic constituents in this
waste include polynuclear aromatic hydrocarbons and heterocyclic
nitrogen containing compounds. The combined API-separator and slop-oil
emulsion solids (COMBO) waste was used in the acute toxicity, waste
characterization, and greenhouse studies.
The third waste common to petroleum refineries that was collected
and used in the study was a storm-water runoff impoundment (SWRI) waste.
This waste is generated from plant housekeeping and rainfall runoff from
the plant site. All runoff water from the refinery from which the
53
-------
sample was collected was channeled into a large basin. Two 189.3 1
samples were collected on-site from the settling basin from which the
sludge was periodically removed for land disposal. The sludge was
collected at a location on the side of the settling basin where the
water had evaporated and was shoveled into the collection barrels.
Storm-water runoff silts account for more than 12,000 metric tons of
hydrocarbons in refinery solid wastes each year (Brown and Assoc.,
1980). The collected sample contained 21% extractable hydrocarbons. As
this waste was generated from runoff and wash water, the composition of
hydrocarbons in the SWRI waste was be more varied than the other
refinery wastes. In addition, because this waste was contaminated with
soil particles from the refinery, it may have had an indigenous
microbial population that could enhance waste degradation. The SWRI
waste was used in the acute toxicity, waste characterization, and
greenhouse studies.
The fourth refinery waste collected for the present study was a
dissolved air flotation float (DAF) waste. The DAF waste was obtained
from a truck load of waste at a land treatment facility. This waste
contained the finely divided oil, clay, and silt particles that were not
settled out in the API-separator. The DAF waste is listed as hazardous
because of its lead and chromium content. A typical DAF waste consists
of 82% water, 12.5% oil, and 5.5% solids. The collected sample contains
5% extractable hydrocarbons. The DAF waste was only used in the acute
toxicity study. Each of the four refinery sludges collected for the
present study are amenable to land treatment because of their relatively
low concentration of toxic constituents. These wastes are produced in
large volumes and are currently being treated by land application at
existing facilities. Thus, two of the refinery wastes, SWRI and COMBO,
have been used in the waste characterization and greenhouse studies.
Four wastes were collected from petrochemical plants. The bottom
stream from the acetonitrile purification column (ACN) is a very low
solids, low organic liquid waste from the production of organic
chemicals. This waste was collected in-line from the acetonitrile
purification column using equipment supplied by plant personnel. An
on-site GC analysis of the collected sample indicated a composition of
3.5% acetonitrile, 0.1% phenols, 0.9% acetamide, 0.5% heavy ends, and
approximately 95% water. The extractable hydrocarbon content of this
waste was found to be 2%. Anticipated toxic constituents include
hydrocyanic acid and polynuclear aromatic hydrocarbons (Lowenback et
al., 1978). Because the physical state of this waste would limit land
treatment, the ACN waste was used exclusively for the acute toxicity
study and characterized using the Salmonella/microsome assay.
The second waste from the production of organic chemicals was the
methyl ethyl ketone (MEK) waste. The U.S. production of methyl ethyl
ketone in 1978 was 300 million kilograms (Beck, 1979). This waste is a
very viscous liquid containing 97% extractable hydrocarbons. Since the
MEK waste may contain as much as 40% sulfuric acid, land application
54
-------
must be precluded by a pH adjustment. The amenability of this waste to
land application will be dependent on the composition of organic waste
constituents' and the concentration of sulfuric acid. The MEK waste was
used in the acute toxicity study, and only a limited characterization in
the bioassays was performed.
The bio-solids waste from the production of phenol should contain
acetophenone, phenol, cumyl phenol, and tars (EPA, 1980). The U.S.
production of phenol in 1978 was greater than one billion kilograms.
The phenol waste was collected in-line at a petrochemical plant using
equipment provided by plant personnel. This waste is a liquid waste
containing only 0.2% extractable hydrocarbons. Because of the low level
of organic waste constituents and the physical state, this waste was
used exclusively in the acute toxicity study.
The fourth petrochemical waste collected for the present study was
the bio-solids from an agricultural chemical plant. The bio-solids
waste was collected from a concrete wastewater treatment pit using
equipment supplied by plant personnel. This waste contains the combined
waste streams from a large agricultural chemical plant and may contain a
variety of hazardous constituents. The bio-solids waste from the
agricultural chemical plant was only used in the acute toxicity study
because it was a liquid waste with a 0.2% extractable hydrocarbon
content.
Three additional wastes were collected but were not used in the
current study. These include the primary clarifier from a pulp and
paper mill, an alum sludge, and a latex paint sludge. None of these
wastes contained sufficient extractable hydrocarbons to merit any
further evaluation.
Extraction
Two procedures were used for1 the extraction of hydrocarbons from
wastes and waste-amended soils. The majority of samples were extracted
using the blender technique, and comparisons were made with a limited
number of samples using a Soxhlet extractor.
Dichloromethane was selected from a group of agents to extract the
organic fractions of the wastes and the soil; dichloromethane
consistently provided the greatest extraction efficiency for the type of
materials anticipated (McGill and Rowell, 1980). Six volumes of
dichloromethane were added to approximately 25 g of the waste or
waste-soil mixture and mixed in a Waring blender for thirty seconds.
This extraction was repeated twice or until the extracting solvent
remained colorless. Solvent extractions were then combined and taken to
dryness on a Brinkman Bucci Rotary Evaporator. The residue from this
extraction was partitioned into acid, base, and neutral fractions
following the scheme outlined in Figure 12. The neutral fraction of
each waste was further separated into four subtractions using sequential
55
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CRUDE SAMPLE IN MeClg
Ul
EXTRACT WITH
IN NaOH
ORGANIC
BASE AND NEUTRALS]
2N
EXTRAC
T WITH IN HCI
1
NaOH AQUEOUS
1
BASE|
ORGANIC
EXTRACT WITH MeCI2
1
3RG
ANIC
BASES
AQUI
:ous
WATER SOLUBLE
AQUEOUS 2N HCI
I ACIDl
EXTRACT WITH MeCI2
ORGANIC AQUEOUS
iNEUTRALSl |ACIDS| |WATER SOLUBLE|
SILICA GEL
1 1 1 1
HEXANE
1 PET ETHER: MeCU METHANOL
4 MtClg *
Figure 12. Fractionation scheme used for waste and waste-amended soils.
-------
solvent extraction on a silica gel column. This extraction
approximately separated the neutral fraction into saturate, aromatic,
and condensed ring fractions according to the procedures of Warner et_
a_l. (1976).
A limited number of waste and waste-soil samples were also
extracted using a Soxhlet apparatus to compare the efficiency of the two
procedures. Fifteen to twenty grams of the homogenized waste or
waste-soil mixture were weighed into a cellulose extraction thimble and
mixed with a glass rod. The thimble and glass rod were placed in a 20
ml capacity Soxhlet extractor. Condensers and boiling flasks containing
200 ml of dichloromethane were assembled with the extractor and placed
on the heater apparatus. Refrigerated coolant was circulated through
the condensers. A 30 ml volume of solvent was introduced into the
extractor from the top to facilitate flushing of the extractor chamber.
Heat was applied to the boiling flask to initiate a 6 hour solvent
extraction of the organic components; the temperature was adjusted to
give six flushings per hour. The solvent extracts were then combined,
taken to dryness, and partitioned according to the same procedures as
used for the blender extracts.
Chemical Analysis
A chemical analysis of waste and soil-waste extracts was conducted
by the USEPA's RS Kerr Environmental Research Laboratory. The compounds
were identified using a Finnigan OWA Automated GC/MS. The GC capillary
column used was a J&W Scientific DB-5-30W. One pi aliquots were used
with a helium carrier gas flow near 36 cm/sec. The GC oven temperature
program was 60 C for 1 minute and then increased at 6 /min to
260 C with a hold time of 12 minutes. The OWA unit had a splitless
mode injector. The software used for analysis had a mass spectra
library of 31,331 organic compounds.
Biological Analysis
Acute toxicity was determined for the crude extract of all ten
wastes using at least one strain in each of the microbial bioassays.
Overnight cultures of the appropriate strain were grown to a cell
density of approximately 1 x 10 cells per ml and serially diluted
from 10 through 10 To 2.0 ml of top agar, 0.1 ml of the
microbial culture and 0.1 ml of the crude extract of each waste were
added, mixed on a vortex mixer, and plated on a complete medium. Cells
were exposed to a minimum of four dose levels of the crude extract. The
plates were incubated for 24 hours at 37°C, and fractional survival
(N/N ) was determined by comparing cell counts on exposed plates to
the cell count on plates exposed to the solvent DMSO without the waste
extract. Three of the 10 wastes were then selected for characterization
of the chronic toxic effects and for use in the greenhouse and lysimeter
studies.
57
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The ability of samples to induce genetic damage was measured in
three microbial systems (Table 14). The eukaryotic bioassay which
employs Aspergillus nidulans (a fungus) can be used to detect point
mutations and small deletions induced in a haploid genome or can be used
as a diploid organism to detect chromosome aberrations, mitotic
recombination, gene mutation, non-disjunction, recombinogenic events,
recessive lethals, and spindle poisons. These systems are capable of
detecting changes in the genetic entity that are of relevance to the
human species and are sensitive to compounds not detected in the
Salmonella assay (Lilly, 1965; Scott e£ aU , 1978; Scott et^
al., 1982). In the first phase of this study, the Aspergillus
bToassay will be used to assess the mutagenic potential of the acid,
base, and neutral fractions of hazardous industrial wastes by evaluating
the induction of mutations at the methionine suppressor loci. Conidia
from 4 to 5 single colonies of the methGl biAl (requiring methionine
and biotin) Glasgow strain of Aspergillus nidulans were grown for 5
to 6 days on a complete medium at 37 C and were used for each
experiment. Samples were tested at a minimum of three dose levels and
four exposure times. The procedures used were the same as Scott et
al. (1978). Mutant colonies were assayed by spreading exposed cells on
a methionine free medium. Mutant colonies were scored after incubation
for 5 days at 37 C. Colonies were divided by colony morphology into
three classes, i.e., A, B, C, and the total number of mutant colonies.
Each of these three classes is believed to involve two genes. The
morphology of Class A colonies appear green, Class B brown, and Class C
green with a white hyaline edge. The frequency of mutations induced by
a sample was determined by subtracting the frequency of spontaneous
mutations which occurred in Classes A, B, C, or the total from the total
mutation frequency in Classes A, B, C and the total. A sample was
considered mutagenic if there was a positive slope on the mutation
induction curve, or the induced mutation .frequency for at least two
exposure times was more than twice the spontaneous mutation frequency.
Positive controls included 8-methoxypsorlen (Sigma, St. Louis, MO),
8-methoxypsorlen plus near UV light without activation, and
benzo(a)pyrene (Aldrich, Milwaukee, WI) with metabolic activation. The
three waste fractions were also tested in a diploid bioassay. Diploid
20 (Kafer £t al., 1982) was used to test the ability of waste
fractions to induce mitotic crossing over, mitotic non-disjunction,
breakdown of mitosis, and mutations with deleterious dominant effects.
The procedures used were essentially the same as were used with the
methionine system and are described in Section 4.
A microbial DNA repair assay was used to measure the capacity of a
sample to produce increased lethal damage in DNA repair deficient
strains. Six strains of B subtilis deficient in different
recombination (Rec ) and/or excision (Exc ) repair were used to test
for lethal DNA damage. These included the Rec strains rec A8^
rec B2, rec E4, mc-1; Exc strain hcr-9; and Rec /Exc
fh 2006-7. All of these strains are isogenic with !$._ subtilis
strain 168 which has all repair intact. These strains were kindly
58
-------
TABLE 14. BIOLOGICAL SYSTEMS USED TO DETECT GENOTOXIC COMPOUNDS IN ENVIRONMENTAL SAMPLES
Ui
Organism
PROKARYOTES
Bacillus
subtilis
Salmonella
typhimurium
Genetic Event
Detected
Increased lethal
damage in DNA
Point mutation
Advantages
Sensitive to bacteriocides,
inorganics; can evaluate
several DNA repair mechanisms;
Well-validated; well defined;
Disadvantages
Insensitive to volatiles;
difficult to quantify.
Insensitive to volatiles;
References
Felkner et al., (1979).
Kada et al., (1974).
Tanooka et_ aU ,(1978).
Ames et al.,(1975).
end-point.
toxic mutagens, certain
chlorinated hydrocarbons.
Skopek et^ a^. ,(1978).
Haroun and Ames, (1981).
EUKAHYOTES
Aspergillus
nidulans
Point mutation;
chromosome damage.
Detects range of genetic
damage including terratogens;
good correlation in compounds
Limited data base;
insufficient number of
trained personnel.
Scott et. a\_., (1982).
Scott et al., (1978).
Bignami et. a_l. , (1891).
-------
supplied by Dr. I. C. Felkner of Clements Assoc., Washington, D.C.
Overnight cultures were grown in brain-heart infusion broth (Difco,
Detroit, MI) incubated at 37°C. Each strain was streaked radially on
a nutrient agar plate to a centrally placed sensitivity disc containing
100 ul of the test chemical. After incubation at 37 C for 18 hours,
the distance of growth inhibition from the disc was measured in
millimeters (Kada et al. , 1974). A response was considered positive
if the distance of growth inhibition was more than 2.5 mm greater in one
of the repair deficient strains than in the repair proficient strain
168. Mitomycin C (Sigma), methylmethane sulphonate (Aldrich), and
sensitivity to ultraviolet light were used as appropriate positive
controls. Quadruplicate plates were run at each dose level for all
samples.
Fractional survival (N/N ) was determined for those strains
showing the greatest sensitivity (inhibition) to the test chemical using
the procedures of Donnelly et_ al. (1983). Brain heart infusion
broth was inoculated with the appropriate strain and incubated at
37°C for approximately- 16 h until an ODcAQ of °'3 was reached
(approximately 1.2 x 10 cells per ml). The cells were then diluted
with -brain heart infusion media to an OD,-,0 of 0.16 (approximately 1.2
x 10 cells per ml), Aerially diluted with Spizizen's Minimal Salts
from 10 through 10 , and plated onto nutrient agar (Felkner et_
al., 1979).
The Salmonella/microsome assay of Ames et al. (1975) was used
to evaluate the mutagenic activity of waste fraction samples. The
Salmonella strains were kindly supplied by Dr. Bruce N. Ames -
(University of California, Berkeley, CA). The procedural methods were
the same as Ames et al. (1975) except that overnight cultures were
prepared by inoculation into 10 ml of Oxoid Nutrient Broth #2 (KC
Biological, Inc., Lenexa, KS) and incubated with shaking for 16 hours at
37 C. Waste fractions were tested on duplicate plates in two
independent experiments in the standard plate incorporation assay at a
minimum of 5 dose levels of the sample with and without enzyme
activation (0.3 ml rat liver/ml S-9 mix) using strains TA98 and TA100.
Positive controls included 2ug/plate N-methyl-N'-nitro-N-nitroso-
guanidine (Sigma) for TA100, 25 ug/plate 2-nitrofluorene (Aldrich) for
TA98, and 10 ug/plate 2-aminoanthracene (Sigma) which was used with all
strains to verify the functioning of the metabolic activation system.
All reagents and extracts were tested for sterility; DMSO was used as a
negative control.
The sensitivity of in vitro bioassays can be greatly enhanced by
incorporating mammalian metabolism into the testing protocol (Ames et
al. , 1973). This metabolic activation system serves to convert
promutagens into their ultimate mutagenic form. Metabolic activation
can be incorporated into the testing protocol by the addition of an S9
mix which consists of a 9,000 x G supernatent from homogenized rat liver
and an NADPH generating system. Rat liver was utilized as the source of
60
-------
enzymes because studies conducted to evaluate the activity of S9 liver
fractions from various species concluded that rat liver provides
activation for the broadest range of chemicals (Baker et al., 1980;
Muller £t al., 1980). A 50 ml batch of S9 mix consists of 85 mg
glucose-^phosphate (Sigma), 160 mg NADP (Sigma), 10 ml of 0.5 m
phosphate buffer, 25 ml cation solution, and 15 ml of S9 (Matney et
al., 1979). The majority of testing was conducted using Aroclor 1254
induced rat liver which is considered to activate the broadest range of
chemicals (Maron and Ames, 1983). In addition, a limited number of
fractions were also tested using phenobarbitol induced S9 which is
considered to work more efficiently with the basic fraction (Epler et
al., 1978). Both Aroclor 1254 and Phenobarbitol induced rat liver
were obtained from Litton Bionetics (Charleston, SC). The concentration
of rat liver in the S9 mix is critical for obtaining the optimum
response and will be variable from one compound to another (Maron and
Ames, 1983). A limited number of samples were tested at three
concentrations of S9/plate to determine the optimum concentrations for
the types of mixtures being studied. The standard test was run at 0.3
ml rat liver/ml S9 mix with additional testing conducted at 0.5 and 0.1
ml rat liver/ml S9 mix.
RESULTS AND DISCUSSION
The acute toxicity of the crude extract of ten hazardous wastes was
determined in bioassays using A. nidulans, JJ. subtilis, and J3.
typhimurium. The results for eight of the ten wastes are provided in
Figure 13a-d and Figure 14a-d. These studies were conducted in order to
establish maximum dose levels for the chronic toxicity study and to
determine the potential short-term effects of waste application on
bacteria and fungi. The acute toxicity of the bio-solids wastes from the
production of phenol and from the agricultural chemical plant are not
included as these wastes had very low organic content and almost no
acute toxic effects in the bioassays at the levels tested. For most of
the wastes studied, the acute toxic effects were greatest in prokaryotic
Salmonella and Bacillus, and least in eukaryotic diploid
Aspergillus. Only the crude extract of the methyl ethyl ketone waste
(Figure 14b) was more toxic in the eukaryotic system than in the systems
using prokaryotic organisms. For the Aspergillus system, only the
wood-preserving liquid waste (Figure 13c) exhibited a definite increase
in toxicity with increasing dose of the waste, while the slop-oil
emulsion solids exhibited a slight increase in toxicity with increasing
dose. For the Salmonella and Bacillus systems, all waste extracts
studied produced an increase in toxicity with increasing dose. At least
one dose level was determined for each waste at which less than 10% of
the exposed prokaryotic cells survived. This dose will be used as the
maximum dose level for all subsequent testing with the waste as well as
with soil or water extract.
The mutagenic potential of the fractions of five hazardous wastes
was evaluated in the battery of bioassays. The results of the
61
-------
a irr»~«u-fv» IKMUIO n
. mnm ina^tta *« i
.« * 4 4 I
Figure 13a. Percent survival of B.
subtilis strain 168, S_.
typhimurium strain TA100,
and _A. nidulans Diploid
109. The cells were
plated on a complete medium
after exposure to various
concentrations of wood-
preserving bottom sediments
waste. The percent survival
(N/No x 100Z) was then
determined.
JL Figure 13b.
Percent survival of JJ.
sub tills strain 168, .S.
typhimurium strain TA100,
and A>. nidulans Oiploid
109. The cells were
plated on a complete medium
acetonitrile purification
column waste. The percent
survival (N/No x 100%) was
then determined.
DOSE/PLATE
Figure 13c. Percent survival of ]J.
subtilis strain 168, £.
typhimurium strain TA100,
and A. nidulans Diploid
109. The cells were
plated on a complete mediua
after exposure to various
concentration of wood-
preserving liquid waste.
The percent survival (N/No
x 100%) was Chen determined.
Figure 13d. Percent survival of £.
subtilis strain 168, S_.
typhimurium strain TA100,
and A. nidulans Diploid
109. The cells were
plated on a complete medium
after exposure to various
concentrations of slop-oil
emulsion solids waste. The
percent survival (N/No x
100%) was then determined.
62
-------
i.rr»-i«u«iuM(Htnaa TUMI
t. rj«TT>.n i tango i««»t i
inn nm Tim
.
OOSE/ PLATE
-------
Salmonella/microsome mutagenicity assay of the acetonitrile waste are
presented in Figure 15 and Table 15. These results indicate that
mutagenic activity could be detected in all three waste fractions and
that primarily indirect acting mutagens were detected. The
dose-response curves for all fractions were non-linear, indicating that
the fractions are composed of constituents with non-equivalent kinetics
of mutation induction. The base and neutral fractions induced a
two-fold increase in revertant colonies in both strain TA98 and TA100,
while the acid fraction only induced a significant increase in strain
TA98. The basic fraction of the waste induced the greatest number of
revertant colonies in both strains. At an exposure level equivalent to
almost 11 g of the original waste material (5.0 mg/plate of extract),
the basic fraction induced 328 and 366 revertant colonies in strain TA98
and TA100, respectively (Table 15). In the absence of metabolic
activation, a doubling of revertant colonies was induced by all three
fractions in TA98 at dose levels greater than 5 mg/plate. The addition
of metabolic activation to the assay system resulted in an increase of
at least twice the number of revertant colonies than was obtained
without metabolic activation.
One of the known constituents of this waste, acetamide, induced
less than 14 revertant colonies/mg at a dose level of 5 mg (McCann et
al. , 1975). At the same dose level, the acid, base, and neutral
fractions induced 31, 71, and 21 net revertants/mg, respectively.
Although the acetonitrile waste contained the experimental carcinogens
acetamide (Sax, 1979), it also contained small concentrations of other
mutagenic agents or agents that promoted the activity of acetamide in
the various waste fractions.
An evaluation of the acid, base, and neutral fractions of the
acetonitrile waste using the IJ. subtilis DNA repair assay (Table 16)
indicates that none of the waste fractions induced increased lethal
damage in the repair deficient strains. Two limitations of the DNA
repair spot test are limited sensitivity to compounds which are indirect
acting and limited sensitivity to compounds which are insoluble in
water. The negative response in the DNA repair assay may have been due
to the absence of metabolic activation in this test, since metabolic
activation was required to obtain the maximum response in the
Salmonella assay.
In conclusion, the determination of the mutagenic potential of the
fractions of acetonitrile waste indicates that the fractions contain
primarily indirect acting mutagens, and the base fraction was the most
active. The base fraction induced 306 net revertants in the
Salmonella assay at a dose level equivalent to approximately 11 g of
waste. By comparison, the basic N-heterocyclic compound
10-azobenzo[a]pyrene induced 130,000 revertants/mg in an evaluation by
Ho et al. (1981). Thus, the mutaganic potential of the components of
the base fraction appears to be much lower than that of the substituted
polycyclic aromatic hydrocarbon.
64
-------
300-
U
5
a.
N»
(O
200-
OC
10
X
00
loo-
ACN WASTE
-S-9 +S-9
AA AGIO AA
OO BASE ---
DD NEUTRAL
200
tOO 600 1000
DOSE/PLATE (mg.eq.)
5000
10000
Figure 15. Mutagenicity, as measured with S^. typhimurium strain TA98, of fractions of
acetonitrile waste. .
-------
TABLE 15. MUTAGENIC ACTIVITY OF LIQUID STREAM FROM ACETONITRILE PURIFICATION COLUMN AS
MEASURED WITH S. TYPHIMURIUM STRAIN TA98 AND TA100 WITH AND WITHOUT
METABOLIC ACTIVATION
ON
Sample
Acid
Base
Neutral
DMSO
Dose Level
(mg/plate)
5.0
2.5
1.0
0.5
0.1
0.01
5.0
1.0
0.5
0.1
0.01
10.0
5.0
1.0
0.1
0.01
100 pi
Strain
TA98
+ S-9
Total His+
162 + 31
122 + 2
45 + 13
30+5
30 + 14
20+4
328 + 5
97 + 32
62 + 1
35 + 15
20 _+ 2
126 + 10
106 + 21
55+5
36 + 19
21+3
22 +_ 5
- S-9
TA100
+ S-9
- S-9
revertants/plate - Mean + S.D.
69 + 3
36 + 5
32+5
36 + 7
15+4
17+4
77 + 12
36+4
28 + 2
20 + 1
17 _+ 0
67 + 1
55 + 2
27+8
13 + 1
15 + 3
19 + 3
197 + 31
NT*
145 + 14
155 + 3
140 + 18
140 + 24
366 + 76
173 + 4
143 + 15
120 + 18
140 +_ 8
326 + 47
289 + 15
87+34
97 + 39
79 + 25
117 + + 21
188 + 58
149 + 1
85 + 11
149 + 6
80+9
80+5
109 + 5
112 + 17
182 + 12
92 + 14
87 + 15
131 + 21
NT
102 + 35
93 + 20
85+4
103 + 164
* NT = Not tested.
-------
TABLE 16. COMPARISON OF LETHAL EFFECTS OF ACN WASTE FRACTIONS ON DNA REPAIR DEFICIENT
AND PROFICIENT STRAINS OF B. SUBTILIS
Inhibition Radius (mm)
Sample
Acid
Base
Neutral
MMS2
Mit.C3
DMSO4
Dose/pt
1 mg
1 mg
10 mg
2 pi
10 pg
100 pi
168,wt
RP1
0
0
0
14
6
0
fh2006.7
1
1
0
26
11
0
recEA
0
1
0
25
11
0
mc-1
repair
i
0
1
20
12
0
her. 9
deficient
0
2
0
21
13
0
recAS
0
1
0
21
13
0
recB2
0
1
0
18
13
0
1- Repair Proficient
2- Methyl methane sulfonate
3- Mitomycin C
4- Dimethylsulfoxide
-------
The methyl ethyl ketone waste was found to be genotoxic in only one
of the three bioassays. In the Aspergillus methionine system, a
positive response was obtained with the neutral fraction (Figure 16 and
Table 17). These results indicate that for colony A, B, C and the total,
there was a significant increase in mutant colonies. The results from
the Aspergillus bioassay are in contrast with those from the
Bacillus and Salmonella assays (Figures 17 and 18 and Table 18). The
DNA repair deficient strain mc-1 gave a weak positive response when
exposed to 2.5 rag of the acid fraction, while no response was obtained
in any of the other strains with the base and neutral fractions. The
acid and neutral fractions induced a doubling of revertant colonies in
£. typhimurium strain TA98 with metabolic activation only at the
highest tested dose level. Using the modified 2-fold rule described by
Chu et^ al. (1981), this would not be considered a positive response.
Biological analysis of the methyl ethyl ketone waste disclosed the
presence of marginal genetic toxicity. The neutral fraction was
positive in Aspergillus and negative in both Salmonella and
Bacillus; however, the acid and base fractions were negative in all
three bioassays.
The most intensive analysis was conducted on the wood-preserving
bottom sediment (PENT S). Samples of the PENT S waste were collected on
two separate occasions and returned to the laboratory for fractionation
and characterization. A subsample of the second sample collected was
also extracted using the Soxhlet apparatus and evaluated using
Salmonella strain TA98. The first waste sample collected was used in
the greenhouse study, while the second sample was used in the lysimeter
study. The results presented in Table 19 indicate that although greater
amounts of extractable hydrocarbons were recovered using the Soxhlet
apparatus, there was no appreciable difference in the specific activity
of the fractions. The similarity of the mutagenic activity obtained
from the three extractions is most evident when the dose response curves
for the crude, acid, base, and neutral fractions are compared (Figures
19a-d). At lower dose levels, the mutagenic responses obtained from
each of the three extractions were within 10% of each other. At the two
highest dose levels tested, the mutagenic activity of the crude, base,
and neutral fractions of the Soxhlet extraction was lower than the other
two extractions by 20 to 50%. These results indicate that greater
quantities of hydrocarbons can be extracted from the soil using a
Soxhlet apparatus than with the Blender procedure. Both procedures did,
however, give approximately equivalent responses when tested in the
Salmonella/microsome assay, indicating that the composition of
mutagenic compounds in the extracts was similar.
The results from biological analysis of the PENT S fractions using
four strains of J3. typhimurium are provided in Table 20. A positive
response was obtained from the crude, acid, base, and neutral fractions
of the PENT S waste with strains TA98, TA100, and TA1538 in the presence
of metabolic activation. None of the waste fractions induced a positive
response in the absence of metabolic activation, and none induced a
68
-------
cr
§
a
\
o
z
UJ
tr
u_
o
s«>
O O
'O
MEK: NEUTRAL- 200>ig
NOV. 81= T 0 T DEC. 81
A A A A
B D B
C * o C
i
20
MINUTES
I i
40 60
EXPOSURE TIME
80
Eigure 16.
Mutation frequency induced by neutral fraction of
methyl ethyl ketone (MEK) waste in A. nidulans
methionine system.
69
-------
TABLE 17. THE EFFECT OF THE NEUTRAL FRACTION OF A METHYL ETHYL KETONE WASTE ON THE
FREQUENCY OF INDUCED MUTATIONS IN ASPERGILLUS NIDULANS
Exposure
Dose
(rag/pi)
0.2
0.2
0.2
0.2
0.2
0.2
Time
(min)
0
15
20
30
45
80
Number of
Cells Plated
2.4 x 10^
7
2.8 x 10'
7
1.4 x 10_
1.7 x 10,
/
1.3 x 10'
0.9 x 10
t.
Surviving Mutation Frequency Per 10 Survivors
Fraction
1.0
0.89
0.83
0.55
0.72
0.52
A
4.27
6.19
14.4
10.6
17.1
B
2.27
9.67
4.80
8.9
11.1
C
.076
.222
.409
.393
2.4
Total
6.61
16.1
19.6
19.6
30.6
-------
METHYL ETHYL KEYTONE WASTE
B . SUBTILIS DMA REPAIR
168 wt
rec E4
rec A 8
her-9
f h 2006-7
MC-I
ACID 125 mg
(POSITIVE
IRESPCNS
y^S^XXXX^
\\\\\\\
\\\\\\\\\
1
i
v\\\\\\\N
1
l
X\\\\\\\1
i
\\\\\\\\H\V
i
rr
.
BASE 10 mg
!._. -
i *
i
x£>Q<
\\»
i
\\NvN
\\
0
Xs
i
NEUTRAL lOmg
$£
\\
X-
\\
i
i
\\v
i
i
i
l
l
1
1
\\\\
1
N\
\
IGBwf
rec E4
rec A8
her- 9
fh200&7
MC-1
2468 024024
GROWTH INHIBITION (mm)
Figure 17. Growth inhibition induced by fractions of methyl ethyl ketone
waste in DNA repair proficient (168 wt) and deficient strains
of B. subtilis.
-------
ro
-------
TABLE 18. MUTAGENIC ACTIVITY OF METHYL ETHYL KETONE WASTE
Sample Dose Level
(mg/plate)
Strain
TA98
+ S-9
Revertant
Acid
Base
Neutral
2
1
0
0
5
2
1
0
0
0
5
1
0
0
.5
.0
.5
.1
.0
.5
.0
.5
.1
.01
.0
.0
.5
.1
43 +
22 +
29
14 +
33 +
18 +
24 +
28 +
29 +
22 +
49 +
25 +
24 +
24 +
3*
2
1
2
1
1
1
1
2
6*
8
1
8
- S-9
Colonies per
30
22
27
40
36
21
18
17
17
20
21
18
25
+ 9
+ 7
+ 9
+ 23
+ 4
NT
+ 6
+ 1
+ 2
± 4
+ 6
+ 7
+ 2
+ 4
TA100
+ S-9
plate + S.D.
NT**
NT
NT
NT
65+3
NT
78 + 1
NT
70+2
NT
100 + 14
79 + 5
NT
NT
- S-9
77
70
80
74
94
84
75
81
80
110
67
73
86
+
+
+
+
+
+
+
+
+
+
+
+
+
9
1
2
0
5
5
5
1
2
16
2
4
2
* - significant increase.
**- NT = Not tested.
-------
I
000.
inumucTio*
tl< CmUCTIOM
01W.IT CXTMCT
PCHT S-ACIO
iitonucnm
TUCT
0 OJ M OU Qa 1.0
I I
omc/ftATi »
0 (XI 0.4
itt cxnucnon
M«T t-MUTHM.
i iMCxnucnon
k IM tmueno"
> M tXT««CT10"
O.t 0.4 0.« 0.1 1.0
00«I/>
O.I 0.4 0.1 O.I 1.0
DOU/FLATI <«)
Figure 19. Mutagenic activity, with metabolic activation, of crude
(A), acid (B), base (C), and neutral '(D) fraction of
PENT S (wood preserving bottom sediment) from two
blender extractions and one soxhlet extraction.
74
-------
TABLE 19. DISTRIBUTION OF MUTAGENIC ACTIVITY1 IN FRACTIONS OF PENT S WASTE
EXTRACTED USING BLENDER OR SOXHLET TECHNIQUE
Sample
Blender (1)
Crude
Acid
Base
Neutral
Blender (2)
Crude
Acid
Base
Neutral
Soxhlet
Crude
Acid
Base
Neutral
1. Mutagenic
2. Revertants
Extractable
Hydrocarbon
(mg/g)
270
23
24
223
271
9
3
259
590
21
8
561
Specific
Activity
(rev/mg)
1,282
771
1,204
860
1,154
828
1,657
1,186
1,162
904
1,209
1,036
Weighted,
Activity
(rev/g)
346
18
29
192
313
7
5
307
686
19
10
581
activity as measured with strain TA98 with microsomal activatii
/mg = slope of mutation induction curve calculated using three
highest non-toxic dose levels.
Revertants/gram eq = revertants/gram material extracted; calculated by
multiplying revertants/mg x % extractable hydrocarbons.
-------
TABLE 20. HUTAGENIC ACTIVITY OF FRACTIONS OF WOOD-PRESERVING BOTTOM SEDIMENT
Total hit revertanti
Sample
Crude
Acid
Base
,
Neutral
Dose/Plate
(lag)
0
1
.5
.1
.05
.01
.005
0
1
.5
.1
.05
.01
.005
0
1
.5
.1
.05
.01
0
1
.5
.1
.05
.01
TA98
25
17
31
30
27
21
23
27
Tox
26
24
25
26
24
26
26
25
24
27
27
21
28
23
23
28
21
42
172
183
132
108
61
44
41
Tox
111
77
66
48
37
41
167
222
131
93
48
33
123
140
86
72
34
TA100
+
144
48
111
145
149
147
124
133
Tox
Tox
108
112
122
109
116
72
88
121
103
104
121
101
91
103
%
98
133
243
521
466
377
187
155
139
Tox
403
331
247
159
149
139
415
563
543
408
175
133
328
391
527
209
176
TA153S
NT
25
Tox
Tox
11
24
18
15
25
19
18
15
15
14
25
18
IS
14
16
20
NT
17
Tox
13
IS
IS
16
17
17
15
IS
16
17
17
17
15
16
10
15
IS
TA1538
NT
13
Tax
Tox
10
8
13
NT
11
16
15
10
15
8
13
19
19
20
19
15
NT
17
Tox
Tox
58
36
19
15
16
60
38
37
32
32
17
47
17
33
24
23
NT - not tested.
-------
positive response in strain TA1535 with or without metabolic activation.
The maximum response was obtained with the base fraction using strain
TA98 with metabolic activation (Figure 20). At the optimum dose level
of 500 ug/plate, the base fraction induced 222 revertant colonies, or
greater than five times the background for strain TA98 (Table 20). At
the same dose level, the base fraction induced 563 revertant colonies in
strain TA100, or slightly greater than four times the background level
(Figure 21). The mutagenic response in the two plasmid containing
strains (TA98 and TA100) were comparable; however, the absence of a
positive response in strain TA1535 indicates that constituents in the
PENT S waste may be selective for frameshift mutants such as TA1538. A
review of the literature by Wassom e_t al. (1977) observed that
tetrachlorodibenzodioxin selectively induces frameshift mutations.
The sensitivity of the Salmonella/microsome assay has been
further enhanced by the addition of two new plasmid carrying strains,
TA97 and TA102 (Levin et^ al., 1982a; Levin et al_. , 1982b).
Strain TA97 detects frameshift mutants and is intended to replace TA1537
(Levin jjt al., 1982a), while strain TA102 detects base-pair
mutations and has been found to be sensitive to oxidative mutagens
(Levin et al., 1982b). The results obtained from a biological
analysis of the fractions of the PENT S waste using strain TA97 and
TA102 were comparable to those obtained using strains TA98 and TA100
(Table 21).
Additional testing of the PENT S waste fractions was conducted with
strain TA98 with high (0.5 ml S9/ml S9 mix), medium (0.3 ml S9/ml S9
mix), and low (0.1 ml S9/ml S9 mix) concentrations of liver microsomes.
Maron and Ames (1983) recommend that these titrations be conducted as
the concentration of S9 per plate is critical for optimum mutagenesis
and can be variable from compound to compound. Rao e± al. (1978)
also observed that the concentration of microsomal enzymes can
significantly affect the results of the biological testing of complex
mixtures. The results presented in Table 22 and Figures 22, 23, and 24
indicate that maximum mutagenicity. was detected with the high
concentration of S9. There was, however, no appreciable difference
between the level of mutagenic activity in the acid and base fractions
with the high and medium concentrations of S9 (Figures 22 and 23). In
the neutral fraction, the mutagenicity with the high concentration of S9
was significantly greater than the medium and low concentrations (Figure
24). However, for all three waste fractions, the optimum, toxic, and
no-effect dose levels were the same with all concentrations of S9 mix.
These results indicate that while the medium level of S9 in the S9 mix
provides an adequate source of metabolic activation of most waste
fractions, slightly greater levels of mutagenic activity may be observed
in the presence of higher levels of S9.
A bioassay using 15. subtilis was utilized to evaluate the
capacity of wood-preserving bottom sediment fractions to produce
increased lethal damage in DNA repair deficient bacteria. The acid
77
-------
600
400-
e
o>
200-
PENTS-WASTE
XX CRUDE B B
&& ACID A A
OO BASE
0O NEUTRAL *
-S9
+S9
I 00
300
DOSE /PLATE(uj)
500 I 000
Figure 20. Mutagenic activity of fractions of the PENT S (wood
preserving bottom sediment) waste.
78
-------
600
CRUDESH
ACID
0-0 BASE
D-Q NEUTRAL
too
300
500
I 000
DOSE/PLATE Cug)
Figure 21. Mutagenic activity, as measured in _§.. typhimurium
strain TA100, of fractions of PENT S (wood preserving
bottom sediments) waste.
79
-------
oo
o
TABLE 21. MUTAGENIC ACTIVITY OF PENT S WASTE IN FOUR PLASMID CONTAINING STRAINS OF
S TYPHIMURIUM
Strain (+ Metabolic Activation)
Fraction TA98 TA100 TA97 TA102
- + - +- + - +
Acid LB1 333 LB 1556
Base LB 463 16 1721
Neutral 25 278 LB 1550
70 1419 LB 2300
15 229 LB 7
122 1975 LB 1186
1 - LB = Less than background.
-------
TABLE 22. MUTAGENIC ACTIVITY OF PENT S WASTE FRACTIONS AS MEASURED WITH S.
TYPHIMURIUM STRAIN TA98 WITH HIGH (0.5 ML S9/ML S9 MIX), MEDIUM
00
(0.3 ML
S9/ML S9 MIX) AND LOW
(0.1 ML S9/ML S9 MIX)
CONCENTRATIONS
OF AROCLOR 1254 INDUCED RAT LIVER IN S9 MIX
Fraction
Acid
Base
Neutral
Dose/plate
(rag)
0
1
.5
.1
.05
.01
0
1
.5
.1
.05
.01
0
1
.5
.1
.05
.01
High
37 + 7
NT*
126 + 10
85 + 14
53 + 10
53 + 5
37 + 7
222 + 51
226 + 24
113 + 17
95 + 11
61 + 23
37 + 7
201 + 17
210 + 32
102 + 12
88 + 7
52 + 8
Metabolic Activation
Medium
A -»- rt .- 1 1-1 »- lO^/i
Arocior i/ jQ
41+3
Tox
111 + 17
77+2
66 + 13
48 + 11
41+3
167 + 11
222 + 18
131 + 16
93 + 17
48 + 14
33 + 7
123 + 36
140 + 29
86 + 24
72 + 15
34 + 16
Low
37 + 6
NT
79 + 29
83 + 3
53 + 10
50 j+ 10
37+6
143 + 62
162 + 53
115 + 19
79 + 20
47 + 9
37 + 6
108 + 46
135 + 74
108 + 12
104 + 9
50+6
* NT = Not tested.
-------
500n
400'
I 300
«0
200-
100-
ACID FRACTION
HIGH S9
MED S9
LOW S9
1.0
DOSE /PLATE Cmg)
Figure 22. Mutagenic activity of acid fraction of PENT S waste
as measured with high, medium, and low levels of S9
in the S9 mix.
82
-------
500i
400
300
0
**
e
200-
co
O)
100-
BASE FRACTION
HIGH S9
A MED S9
LOW S9
DOSE /PLATE Cmg)
Figure 23, Mutagenic activity of base fraction of PENT S waste
as measured with high, medium, and low levels of S9
in the S9 mix.
83
-------
500
400
2 300
c
ta
05 200
0>
IOOH
NEUTRAL FRACTION
HIGH S9
MED S9
LOW S9
0.2
0.4 0.6 0.8
DOSE /PLATE (mg)
1.0
Figure 24. Mutagenic activity of neutral fraction of PENT S
waste as measured with high, medium and low levels
of S9 in the S9 mix.
84
-------
fraction was the only fraction to induce a significant inhibition in the
repair deficient strain when evaluated in the spot test (Table 23 and
Figure 25). When tested in the modified plate incorporation assay, the
base fraction also produced increased lethal damage in the repair
deficient strains, while the response obtained from the neutral fraction
was less conclusive (Table 24). The different responses obtained using
the two procedures may have been a result of the lack of diffusion of
waste fractions in the spot test. The modified plate incorporation
assay did, however, conclusively demonstrate that the acid and base
fractions of the PENT S waste contained constituents that are capable of
producing increased lethal damage in repair deficient bacteria.
The capacity of PENT S fractions to induce genetic damage in a
eukaryotic system was measured using both the Aspergillus methionine
system and a diploid bioassay. All three fractions induced a doubling
of revertant colonies both with and without metabolic activation
(Figures 26, 27, and 28). The frequency of induced mutations per
survivor for the acid and base fractions at the highest exposure level
with metabolic: activation was 100 and three times, respectively, the
response obtained in the absence of metabolic activation (Tables 25 and
26). For the neutral fraction at the highest dose level, the induced
mutation frequencies were almost the same with or without metabolic
activation (Table 27). The results in Figures 27 and 28 show that in
the absence of metabolic activation, the surviving fraction and induced
mutation frequencies for the base and neutral fractions are almost
identical at all exposure levels. The maximum mutagenic response was
obtained with the acid fraction at the greatest exposure time with
metabolic activation (Figure 26). Thus, the acid, base, and neutral
fractions of the PENT S waste induced a significant increase in point
mutations at the methionine suppressor loci both with and without
metabolic activation. While the addition of metabolic activitation to
the assay system -did increase the frequency of induced mutations,
significant increases were also obtained in the absence of metabolic
activation.
Additional tests were conducted on the fractions of the
wood-preserving bottom sediment using A. nidulans diploid 20. The
diploid assay, while more costly and time consuming than the prokaryotic
assays, offers the advantage of detecting a range of genetic damage and
genotoxic compounds comparable to that detected using in vitro
mammalian cell culture bioassays (Kafer e£ al., 1982). The summary
of the genotoxic effects observed in segregant colonies and induced by
the fractions of the PENT S waste (Table 28) indicates that each waste
fraction preferentially acted on a different group of genes in the
diploid Aspergillus. The genotoxic constituents present in the acid
fraction induced predominantly fawn and dark green segregants, whereas
the base and neutral fraction induced yellow, fawn, and chartreuse
85
-------
oo
TABLE 23. CAPACITY OF FRACTIONS OF WOOD-PRESERVING WASTE TO INDUCE INCREASED LETHAL
DAMAGE IN DNA REPAIR DEFICIENT STRAINS OF B. SUBTILIS
Waste Metabolic
Fraction Activation
Acid +
10 mg/pt
Base +
10 mg/pt
Neutral +
10 mg/pt
Strain: 168
13.9
12.0
0
0.38
0.43
2.3
Growth
wt recE4
17.6*
14.6*
0
0.35
0.37
2.2
Inhibition
recA8
14.6
11.7
1.2
0.68
0.20
3.0
(mm)
hcr-9
15.3
12.9
0
0
0
1.3
fh 2006-7
15.0
13.4
1.1
0.2
1.0
2.3
mc-1
14.2
12.9
1.5
0
0
2.1
* Significantly Inhibited.
-------
PENTA- S WASTE
_B. SUBTILIS DMA REPAIR (+METABOLIC ACTIVATION)
oo
168 wt
rec E4
rec A8
her- 9
fh 2006-7
me- I
ACID lOmg
BASE 10 mg
NEUTRAL 10 mg
I68wt
rec E4
rec A8
her-9
fh 2006-7
me-1
8 12 16
GROWTH INHIBITION (mm)
Figure .25. Growth inhibition induced by fractions of the wood-preserving
bottom sediment in repair proficient (168 wt) and deficient
strains of B. subtilis.
-------
TABLE 24. FRACTIONAL SURVIVAL OF REPAIR PROFICIENT AND DEFICIENT STRAINS OF
B. SUBTILIS EXPOSED TO SUBFRACTIONS OF WOOD-PRESERVING
BOTTOM SEDIMENT. SURVIVAL WAS MEASURED IN THE PRESENCE
OF METABOLIC ACTIVATION
00
oo
Fractional Survival (N/N ) %
o
Sample
Acid
Base
Neutral
Dose/Plate 168 wt
(pg) RP
100
50
10
1000
100
10
1000
100
10
35 + 28
88 + 28
92 j+ 12
20+3
43 + 28
MOO
5+5
17+6
.66 + 44
fh2006.7
19 + 13
34+22
50 + 17
3+2
13 + 11
63 +_ 15
6+5
15+6
43 + 12
recE4
repair deficien
6+4
26 + 28
41**
2 + 0.5
4 + 0.5
9 + 1.5
2**
2**
36**
mc-1
NT*
NT
NT
4+1.
33 + 12.
16 + 2.
76**
64**
58**
5
0
0
* - NT = not tested
**- Standard deviation not provided because only one test conducted.
-------
i65j
i
LJ
UJ
.6^
PENT- S- ACID
-S-9 !25*Q/pt. +S-9
A -A A A
!0TJ
O
LJ
O
O
100 _
15
10
o
fc
(9
CO
10
30
50
70
90
MINUTES EXPOSURE TIME
Figure 26. Induced mutation frequency and fractional survival
in A. nidulans^ following exposure to acid fraction
of PENT S waste. Data based on results from two
independent experiments.
89
-------
o
i*
PENT- S- BASE
-S-9
o o
*S-9
I tf
e
rioo^
10
o
i
CO
10 30 50 70 90
MINUTES EXPOSURE TIME
Figure 27. Induced mutation frequency and fractional survival
in _A. nidulans following exposure to base fraction
of PENT S waste. Data based on results from two
independent experiments.
90
-------
.64
D-O--..
' -.
PENT -S- NEUTRAL
-S-9 I25itg/pt. +S-9
a a 1
o
|
a
1
--a
100 --.
10 £
o
a:
(O
10 30 50 70 90
MINUTES EXPOSURE TIME
Figure 28. Induced mutation frequency and fractional
survival in A. nidulans following exposure
to neutral fraction of PENT S waste. Data
based on results from two independent
experiments.
91
-------
TABLE 25. SURVIVING FRACTION AND INDUCED MUTATION FREQUENCY OF A. NIDULANS
to
FOLLOWING EXPOSURE TO ACID
Dose/Plate
(ug)
Exposure
Time (min)
Surviving
Fraction
FRACTION OF
Mutation
A
PENT S WASTE
Frequency Per
B
io6
c
Survivors
Total
Without Metabolic Activation
41.7
62.5
125.0
250.0
With Metabolic
41.7
62.5
125.0
250.0
40
40
40
40
Activation
20
20
20
20
.75
.75
.54
.31
.75
.82
.56
.26
.84
1.4
2.4
5.4
1.9
2.2
3.8
7.1
1.4
1.8
3.6
6.8
3.2
4.2
8.7
17.6
.51
.40
.90
2.5
.51
.93
1.3
1.4
2.7
3.6
6.9
14.9
5.5
7.3
13.9
26.3
-------
TABLE 26. SURVIVING FRACTION AND INDUCED MUTATION FREQUENCY OF A. NIDULANS
VO
CO
Dose/Plate
(pg)
FOLLOWING
Exposure
Time (min)
EXPOSURE TO BASIC
Surviving
Fraction
FRACTION OF
Mutation
A
PENT S WASTE
Frequency Per
B
106
c
Survivors
Total
Without Metabolic Activation
41.7
62.5
125.0
250.0
With Metabolic
41.7
62.5
125.0
250.0
40
40
40
40
Activation
20
20
20
20
.76
.94
.64
.40
.82
.91
.64
.40
.98
1.6
3.1
2.4
1.6
2.8
6.7
10.4
1.7
6.1
4.2
6.9
2.7
9.5
10.6
17.6
.50
.88
.98
1.4
.46
1.3
3.2
3.9
3.2
8.6
8.3
10.7
4.9
13.6
20.6
31.9
-------
TABLE 27. SURVIVING FRACTION AND INDUCED MUTATION FREQUENCY OF A. NIDULANS
FOLLOWING EXPOSURE TO NEUTRAL FRACTION OF PENT S WASTE
Dose/Plate
Exposure
Time (min)
Surviving
Fraction
Mutation Frequency Per 10 Survivors
A
B
C
Total
Without Metabolic Activation
41.7
62.5
125.0
250.0
With Metabolic
41.7
62.5
125.0
250.0
40
40
40
40
Activation
20
20
20
20
.77
.83
.57
.44
.70
.78
.40
.34
.63
1.3
.96
4.1
3.3
.74
3.5
5.4
1.8
5.3
3.4
12.8
6.0
3.1
9.3
12.4
.16
.17
.22
.50
.21
.37
.91
1.1
2.6
6.8
4.6
17.4
9.5
4.2
13.7
18.9
-------
TABLE 28.
SUMMARY OF CENOTOXIC EFFECTS OBSERVED IN SEGBEGANT COLONIES OF DIPLOID A. NIDULAHS FOLLOWING
EXPOSURE TO WASTE CONTROLS AND FRACTIONS
VO
Ln
2,3
Phenotype
Sample
Control
DMSO
Benomyl
Pent S - Acid
Base
Neutral
SWRI - Acid
Base
Neutral
COMBO - Acid
Base
Neutral
Total'
619
140
299
248
306
318
369
348
332
285
285
261
Yellow
Overall
1.9
4.0
12.7
4.4
10.0
6.9
11.1
10.9
9.6
14.7
11.2
11. S
CO
0.3
0.7
1
0
2
0.6
1.6
1.4
2.1
3.9
1.8
1.1
NO M
0.5 0
0.7 0
3.5 0.
0 0
1.0 0
1.9 0
2.7 0
1.0 0
2.7 0
3.2 0
3.5 0
2.7 0
2n
0.3
0
3 1.4
0.8
1.6
1.3
1.4
2
1.5
2.1
2.5
3.1
fawn
n
0
0.7
0.3
0.8
1.3
0.3
0
0.6
0.3
0.4
0
1.5
1. Total « number of segregant colonies examined; overall total number of
2. Unit - Total frequency of segregants per colony examined.
3. Phenotype: Yellow-includes only colonies that were completely yellow, all
CHAT
2n
0.3
2.0
2.3
0.4
2.6
1.9
2.4
2.0
2.4
1.4
1.1
1.1
induced
other
DG
n 2n
0 0.3
0 0
0.7 1.0
0 0.8
0.3 0
0 0
0 0.3
0 0.9
0 0
0 0
0 0
0.4 0
GR
n 2n
0 0.2
0 0
1.3 1.0
0.8 0.4
0.3 1.0
0 0.9
1.4 1.4
1.4 0.6
0 0.6
2.5 1.4
1.8 0.7
0.4 0.8
n
0
0
0
0.4
0
0
0
0.3
0.0
0
0
0.4
ISI*
CT DO
.04
1.4
5.2 3.7
4.2 2.9
1.6 .1
2.6 .8
2.7 .9
2.1 .5
2.6 .8
1.6 .1
1.9 .3
2.3 .6
segregants.
listed in other sections;
CO ° crossing-over, ND "non-disjunction, H mutations. Fawn » tan sectors; Chat - chatreuae; DG - dark green
G " green; 2n diploid; n " haploid.
Induced segregation index - Total induced aegregants divided by frequency of control segregants. CT " evaluated
with respect to controls; DO evaluated with respect to solvent control (DMSO). If ISI is significantly greater
than 1.0, response is considered positive.
-------
segregants (Table 28). Both crossing over and non-disjunction were
observed in the yellow segregants induced by the base fraction, while
predominantly non-disjunction was observed in yellow segregants from the
neutral fraction. No yellow segregants were induced by the acid
fraction. When the induced segregation indexes are compared with
respect to the spontaneous control, all three waste fractions are
positive with the genotoxic potential of the acid fraction slightly less
than benomyl, the positive control. When the responses are compared
with respect to the solvent control, the acid and neutral fractions are
positive; while the induced segregation index of the base fraction,
which is slightly greater than one, would be considered a borderline
positive response.
In abnormal colonies, the acid, base, and neutral fractions induced
31, 15 and 25 abnormal colonies, respectively (Table 29). For all three
fractions, the maximum mutation frequency was observed in the
hyperdiploid class. The acid fraction induced significant increases in
hyperdiploid colonies, breakdown of the mitotic spindle apparatus, and
mitotic recombination; the neutral fraction induced significant
increases in the hyperdiploid class and mitotic recombination. Thus,
the acid fraction induced the maximum amount of genetic damage observed
in abnormal colonies, while the neutral fraction also induced a
significant positive response, and the base fraction induced a
borderline positive response.
The results from biological analysis of the wood-preserving waste
indicate that the base fraction induced the maximum response in both
prokaryotic point mutation assays, the DNA repair plate incorporation
assay, and the eukaryotic point mutation assay with metabolic
activation. In the prokaryotic DNA repair spot test and the eukaryotic
point assay, the acid fraction induced the maximum genotoxic response.
Although the genotoxicity of the acid fraction was greatest in the
presence of metabolic activation, a positive response was obtained in
the absence of metabolic activation in both bioassays. The acid
fraction, however, was toxic at higher dose levels in the Salmonella
and Bacillus assays, possibly a result of the presence of
pentachlorophenol. The acid fraction induced the maximum response in
j>. typhimurium strain 102. The wood-preserving bottom sediment
contained a diverse range of constituents including compounds capable of
causing induction, promotion, and inhibition, as well as mutagenic and
carcinogenic effects (Figure 29, 30, 31, and Table 30). Biological
analysis of the fractions of this waste detect compounds capable of
causing point mutations, lethal damage to DNA, and various types of
chromosome damage. While the results of biological testing do not rule
out the PENT S waste as candidate for land treatment, the presence of
pentachlorophenol and other compounds that are resistant to degradation
or mobile in the soil indicate that land treatment should probably be
conducted at a lower loading rate or be precluded by a pretreatment
method such as composting or anaerobic digestion.
96
-------
TABLE 29. SUMMARY OF GENOTOXIC EFFECTS OBSERVED IN ABNORMAL COLONIES OF DIPLOID
A. NIDULANS FOLLOWING EXPOSURE TO CONTROLS AND WASTE FRACTIONS
Sample
CONTROL
DMSO
BENOMYL
PENT S
(Acid)
PENT S
(Base)
Total2
COL=61 9
MF
ISI(S)
ISI(D)
COL=140
MF
ISI(S)
ISI(D)
COL=299
MF
ISI(S)
ISI(D)
COL=248
MF
ISI(S)
ISI(D)
COL=306
MF
ISI(S)
ISI(D)
0V
22
.036
-
6
.043
1.19
-
47
.157
4.36
3.65
31
.125
3.47
2.91
15
.049
1.36
1.14
Genetic
HD
14
.023
-
-
3
.021
.91
-
19
.050
2.17
2.38
21
.085
3.70
4.05
10
.033
1.43
1.57
Damage Observed
NH
3
.005
-
2
.014
2.8
-
3
.010
2.0
.71
3
.012
2.4
0.86
1
.003
.06
.21
HH BK
2 1
.003 .002
-
- -
0 0
- -
-
-
1 19
.003 .064
1.0 32
-
0 2
.008
4.0
-
1 1
.003 .003
1 1.5
- -
OT
2
.003
-
-
1
.007
2.3
-
5
.017
5.67
2.43
5
.020
6.67
2.87
2
.006
2.0
0.0
-------
TABLE 29 CONTINUED
VO
oo
Sample
PENT S
(Neutral)
SWRI
(Acid)
SWRI
(Base)
SWRI
(Neutral)
COMBO
(Acid)
Total2
COL=318
MF
ISI(S)
ISI(D)
COL=369
MF
ISI(S)
ISI(D)
COL=348
MF
ISI(S)
ISI(D)
COL=332
MF
ISI(S)
ISI(D)
COL=285
MF
ISI(S)
ISI(D)
QV
25
.079
2.19
1.84
28
.076
2.11
1.77
27
.078
2.17
1.81
21
.063
1.75
1.47
14
.049
1.36
1.14
Genetic
HD
15
.047
2.04
2.24
16
.043
1.87
2.05
19
.055
2.39
2.05
10
.030
1.30
1.43
7
.056
1.09
1.19
Damage Observed
NH
5
.016
3.2
1.14
4
.011
2.2
.78
2
.006
1.2
.36
7
.021
4.2
1.5
2
.007
1.4
.5
HH BK
0 1
.003
1.5
- -
1 2
.003 .006
1 3
- -
0 0
-
-
- -
1 2
.003 .006
1 3
-
0 4
.014
7
- -
OT
5
.013
4.3
1.86
5
.014
4.67
2.00
6
.017
5.67
2.43
5
.003
1
.42
1
.004
1.3
1.3
-------
TABLE 29 CONTINUED
VO
Sample
COMBO
(Base)
COMBO
(Neutral)
Total2
COL=285
MF
ISI(S)
1SI(D)
COL=261
MF
ISI(S)
ISI(D)
0V
20
.70
1.%
1.63
15
.057
1.58
1.33
Genetic Damage Observed
HD NH HH
16 1 0
.056 .004
2.43 .8
2.67 .29
300
Oil
.48
.52
BK OT
0 3
"""
3.
1.
1 11
.004
2 14
6
Oil
66
57
042
1. Genetic damage observed: 0V = overall summary of genetic damage observed in
all categories; HD = hyperdiploid, results from non-disjunction of chromosomes;
NH = near hyperdiploid, probably breakdown products including a mixture of haploid
and diploid colonies; HH = hyperhaploid, results from major deletions or lethals
in chromosomes; BK = breakdown, results from mitotic spindle poisons; OT = others,
results from mitotic recombination which has occurred prior to aneuploidy. Units =
Total frequency of abnormal segregants per colony examined.
2. Total: col = total number of colonies examined, and total number of abnormals in
each class; MF = total mutation frequency = 0V COL; ISI(S) = induced segregation
index with respect to control = MF (sample) MF (control); ISI(D) = induced
segregation index with respect to solvent control = MF(sample) MF(DMSO).
-------
PENT-3-ACID
o
o
RIC
vO CO
o
CM
CM
CM
CM
200
300
400
500
600
RT
Figure 29. GC/MS chromatograph of acid fraction of PENT S waste.
-------
co
ro
-------
a\
n
o
to
RIG
in
to
CM
CM
CO
r--
m
o\
CM
CO
oo
CM
VO
CO
CM
f-
CO
PENT 8-NEUTRAL
200
300
400
500
RT
Figure 31. OC/MS chromatograph of neutral, fraction of PENT S waste.
-------
TABLE 30. SELECTED PROPERTIES OF COMPOUNDS IDENTIFIED IN THE FRACTIONS OF THE
WOOD-PRESERVING WASTE
o
LO
Sample
Acid Fraction
Basic Fraction
Neutral Fraction
Peak
Number
287
322
331
335
342
363
434
483
537
553
239
322
336
363
185
237
288
322
335
342
363
536
553
Compound
Dimethyl napthalene
1-2 Dihydro-acenapthylene
Methylethyl napthalene
Dibenzofuran
Trimethyl napthalene
Phenalene
Pentachlorophenol
Methyl phenanthrene
Fluoranthene
Pyrene
Methyl napthalene
1-2 Dihydro-acenapthylene
Dibenzofuran
Phenalene
Napthalene
Methyl napthalene
Dimethyl napthalene
1-2 Dihydro-acenapthylene
Dibenzofuran
Trimethyl napthalene
Phenalene
Fluoranthene
Pyrene
CAS f
573-98-8
83-32-9
132-64-9
879-129
203-80-5
87-86-5
832-69-9
206-44-0
121-00-0
90-12-0
83-32-9
132-64-9
203-80-5
91-20-3
90-12-0
573-98-8
83-32-9
132-64-9
879-129
203-80-5
206-44-0
121-00-0
Formula
C12H12
P H
12 10
P H
13 14
C12V
C13H14
P H
cjictjo
C15H12
P H
16 10
P H
C16H10
C11H10
C12H10
C.-H-O
C13H10
C10H8
P H
11 10
P H
12 12
P H
12 10
C. HQ0
C13H14
C H
r13H10
16 10
P H
C16H10
Genetic
Activity
MO
Ml
X
X
MO
MO; Cl
Ml
Ml ; CO ; CC
Ml ; CO ; CC
Ml; CO
Ml; CO
Ml
X
X
MO; CO
Ml
MO
Ml
X
MO
X
M1,CO;CC
Ml ; CO ; CC
1. Potential genetic activity: P = promoter; I = inhibitor; CC = cocarcinogenic;
Ml = mutagenic; MO = nonmutagenic; Cl = carcinogenic; CO = noncarcinogenic;
X = unknown; references included in text.
-------
The storm-water runoff impoundment (SWRI) waste was one of two
refinery sludges evaluated in the complete battery of bioassays. The
distribution of mutagenic activity in the fractions of the SWRI waste
are provided in Table 31 and Figure 32. ; These results indicate that
although the maximum response was obtained from the acid fraction, the
specific activity of all three fractions was approximately equal. In
addition, when the neutral fraction was separated on a silica gel column
into subfractions, the specific activity of neutral fraction three was
approximately three times the specific activity of the unfractionated
neutral fraction (Figure 33). The dose-response curves for the
extractable hydrocarbons of the SWRI waste fractions (Figure 32) also
indicate that the overall maximum response was obtained from the acid
fraction, although the maximum mutagenic response at a single dose level
was obtained with the neutral fraction at a dose of 1.0 mg/plate. With
the exception of the neutral fraction at the highest dose, there was no
mutagenic activity detected in the absence of metabolic activation. In
the three subfractions of the neutral fraction (Figure 33), higher
levels of mutagenic activity were detected at the lower doses than were
present in the composite neutral fraction. The maximum amount of
mutagenic activity was detected in fraction three which represents the
condensed ring fraction. The results from biological analysis of the
SWRI waste indicate that predominantly indirect acting mutagens were
detected in the waste fractions, and there was no appreciable difference
in the mutagenic potential of each of the three primary fractions. The
analysis of fractions of the storm-water runoff impoundment waste in the
spot test or plate incorporation assay using DNA repair proficient and
deficient strains of ]3. subtilis did not provide any conclusive
results (Table 32). Thus, the storm-water runoff impoundment waste
contained constituent(s) that were capable of inducing point mutations
but not lethal damage to DNA.
The fractions of the SWRI waste were also evaluated in the
Aspergillus methionine and diploid bioassays. When the results from
testing the three waste fractions in the haploid methionine assay are
compared, the maximum mutagenic response was obtained from the acid
fraction in the presence of metabolic activation (Tables 33, 34 and 35;
and Figures 34, 35, and 36). While a positive response was also
obtained from the base and neutral fractions, the induced mutation
frequencies were consistently lower than were obtained with the acid
fraction. In contrast to the results of the prokaryotic bioassay,
mutagenic responses were observed for all waste fractions in
Aspergillus in the absence of metabolic activation.
Each of the three subfractions of the storm-water runoff
impoundment waste induced a positive response in the diploid bioassay.
The results presented in Table 28 indicate that in segregant colonies
the maximum genotoxic response was obtained with the acid fraction,
although the genotoxic potential of the neutral fraction was not
significantly lower than the acid fraction. Yellow and chartreuse
segregants were predominantly induced by the acid and neutral fraction,
104
-------
TABU 31. MUTAGENIC ACTIVITY Of RUCTIONS OF STOHM-UATER RUNOFF IMPOUNDMENT
O
Ul
Fraction
TA
98
TA 100
TA1535
TA1538
TAJ 537
Doaa/plata 89- + - » - * - » - *
<«>
Total taia ravertaota
Acid
Baa a
Neutral
0
1
.5
.1
.05
.01
0
1
.5
.1
.OS
.01
0
1
.5
.1
.05
.01
26 * 2
S3 7 18
32 7 S
28 7 7
24 7 7
23 7 6
26 » 2
47 7 18
34 7 3
31 7 6
28 7 6
26 7 3
25 2
79 44
49 21
28 13
25 9
36 10
29 » 6
222 7 23
158 740
88 7 23
68 7 8
34 7 10
45 » 1
1S1 7 30
144 7 23
82 7 12
68 7 9
44 7 9
44 » 6
235 7 111
134 7 25
74 7 10
66 7 24
50 7 7
US * 13
114 7 19
116 + 11
114 7 25
102 7 11
105 7 24
130 * 5
126 7 9
137 7 7
109 + 11
112 7 17
113 * 10
125 « 9
147 7 11
123 7 16
115 7 9
120 7 6
130 7 IS
109 »
249 7
25S 7
245 7
198 7
133 7
124 +
349 7
337 7
230 7
185 7
132 7
109 *
290 7
283 7
227 7
166 7
119 7
7
27
36
31
10
12
6
49
120
43
12
21
7
26
14
21
54
19
(Maaa » SO)
28 * 8
36 7 10
47 7 16
53 7 15
49 7 27
36 7 17
28 * 8
52 7 17
52 7 16
41 7 12
38 7 12
37 7 6
28 * 8
21 7 6
23 7 7
26 7 1
20 7 6
21 7 7
21 »
28 7
38 7
25 7
25 7
24 7
20 »
28 7
28 7
20 7
22 7
21 »
19 »
25 7
22 7
24 7
19 7
23 »
4
S
3
S
4
S
4
2
7
4
3
4
6
12 +
67 7
52 *
18 7
19 7
14 7
12 +
34 7
23 »
17 7
16 7
18 7
12 *
26 7
20 7
16 7
14 7
14 7
2
8
14
5
7
2
2
6
6
5
3
5
2
5
3
1
2
2
21 » 4
75 7 11
78 7 8
61 7 9
42 7 17
17 7 3
25 » 2
111 7 51
90 7 30
46 7 14
25 7 18
19 7 5
25 * 4
66 7 11
52 7 2
20 7 5
32 7 7
17 7 3
9 *
42 7
38 7
10 7
15 7
13 *_
10 *
25 7
16 7
13 7
H 7
16 7
10 *
20 7
14 7
12 7
10 7
8 7
2
4
5
2
4
3
2
6
5
2
S
2
2
12
3
4
3
3
14 * 2
42 7 14
44 7 8
33 7 11
26 7 14
25 7 3
24 » 5
27 7 8
22 7 8
20 7 5
21 7 6
13 7 1
21 * 6
42 7 10
43 * 17
39 » 16
28 '7 10
25 * 7
-------
300'
x
a
*«
.c
CO
SWRI WASTE
ACID £r-4
00 BASE Oo
DO NEUTRALOa
100-
0.2
0.8
1.0
0.4 0.6
DOSE/PLATE Cmg)
Figure 32. Mutagenic activity of fractions of SWRI waste.
106
-------
600
400
CD
0)
200-
I 00
SWRI NEUTRAL
NETJTRAL. '
FRACTION 1-8
ALL+S9
£-
3 A
300
500 1000
DOSE/PLATECug)
Figure 33. Mutagenic activity of subfractions of neutral fraction
. of SWRI waste.
107
-------
TABLE 32. FRACTIONAL SURVIVAL OF REPAIR PROFICIENT (168 WT) AND
DEFICIENT STRAINS OF IJ. SUBTILIS EXPOSED TO SUB-
FRACTIONS OF COMBINED API-SEPARATOR/SLOP-OIL EMULSION
SOLID (COMBO) OR STORM-WATER RUNOFF IMPOUNDMENT (SWRI).
SURVIVAL WAS MEASURED IN THE PRESENCE OF METABOLIC
ACTIVATION
Sample
COMBO
Acid
Base
Dose/Plate
(ug)
500
100
50
10
1000
500
100
50
Fractional
168 wt
RP
72 + 0.6
97+8
MOO + 24
96+4
31**
43 + 27
87 + 14
MOO
Survival (N/N ) %
hcr-9 ° recAS
repair deficient -
27+9 NT*
54+5
55 + 17
MOO**
27+13 NT*
64 + 10
60+8
50 + 21
SWRI
Acid
2500
1000
500
100
50
8**
52 + 33
64 + 14
100 + 0
96+8
21**
53
NT
MOO +
MOO +
0
0
32**
66
MOO
MOO
* - NT = not tested
**- Standard deviation not provided because only one test conducted.
108
-------
TABLE 33. SURVIVING FRACTION AND INDUCED MUTATION FREQUENCY OF A. NIDULANS
o
vo
FOLLOWING EXPOSURE TO ACID
Dose/Plate
(ug)
Exposure
Time (min)
Surviving
Fraction
FRACTION OF
Mutation
A
SWRI WASTE
Frequency Per
B
io6
c
Survivors
Total
Without Metabolic Activation
99
199
248
497
With Metabolic
124
248
331
497
40
40
40
40
Activation
20
20
20
20
.54
.24
.23
.03
.44
.17
.29
.09
7.7
30.0
30.7
124.0
119.0
44.4
36.2
87.1
7.2
19.0
20.3
88.6
67.4
37.2
31.1
61.0
1.0
2.3
3.9
8.9
35.4
10.5
1.4
8.7
16.0
51.0
54.9
221.5
221.9
92.1
68.6
156.9
-------
TABLE 34. SURVIVING FRACTION AND INDUCED MUTATION FREQUENCY OF A. NIDULANS
FOLLOWING EXPOSURE TO BASE FRACTION OF SWRI WASTE
Dose
(ug)
Exposure
Time (min)
Surviving
Fraction
Mutation Frequency
A
B
Per
10 Survivors
C
Total
Without Metabolic Activation
42
84
168
226
336
With Metabolic
42
84
128
168
226
40
40
40
40
40
Activation
20
20
20
20
20
.78
.46
.21
.12
.04
.76
.61
.39
.23
.21
0
1
7
9
91
0
7
8
24
8
.9
.8
.9
.0
.0
.4
.6
1
0
19
26
242
3
1
2
14
25
.7
.7
.4
.3
.7
0
0
0
0
15
0
0.8
1.6
5.8
0
1
2
26
35
348
3
9
12
44
33
.7
.6
.8
.8
.4
.1
.7
.0
.6
-------
TABLE 35. SURVIVING FRACTION AND INDUCED MUTATION FREQUENCY OF A. NIDULANS
FOLLOWING EXPOSURE TO NEUTRAL
Dose
Without
93
185
463
719
925
Exposure
Time (rain)
Metabolic Activation
40
40
40
40
40
Surviving
Fraction
.94
.82
.51
.35
.41
FRACTION OF
SWRI WASTE
Mutation Frequency Per
A
1.2
3.6
6.2
9.8
13.0
B
1.1
1.0
1.3
2.1
2.7
10 Survivors
C
0
0
0
0
0.5
Total
2.3
4.6
7.5
11.9
16.2
With Metabolic Activation
185
370
555
719
925
20
20
20
20
20
.74
.67
.49
.45
.40
9.5
4.9
13
14
11
2.4
0
1.9
2.9
1.4
0
0.6
1.3
0
1.2
11.9
5.5
16.2
16.9
13.6
-------
tr
o
(E
O
-4
UJ
o
UJ
cc -
u- 10
o
UJ
o
0
-6
10
SWRI ACID
-S 9 248ug/pt + S 9
-&
100
10
o
<
cr
u.
o
(C
CO
10 30 50 70 90
EXPOSURE TIME Cmin.)
Figure 34. Induced mutation frequency and fractional survival
in A. nidulans following exposure to acid fraction
of SWRI waste. Data based on results from two
independent experiments.
112
-------
-------
en
o
>
(E
D
CO
X
>-
o
z
UJ
3
O
UJ
(T
U.
-4
10
-5
10
-6
10
SWRI NEUTRAL
-S9 925ug/plate -»-S9
OO
O
UJ
O
13
a
rioo *
o
10
o
<
cr
LL.
CD
I 0
30
50
70
90
MINUTES EXPOSURE TIME
Figure 36. Induced mutation frequency and fractional survival in
A. nidulans following exposure to neutral fraction of
SWRI waste. Data based on results from two
independent experiments.
114
-------
while the base fraction induced a range of segregant colonies including
all possible phenotypes. The majority of yellow segregants induced by
the acid and neutral fractions were a result of non-disjunctional
events, while yellow segregants induced by the base fraction resulted
predominantly from crossing over. In addition, a relatively high
frequency of diploid colonies were recovered from chartreuse segregants
induced by each of the three fractions (Table 28). The results from the
diploid assay demonstrate that constituent(s) of the fractions of the
SWRI waste are capable of inducing a broad range of genotoxic damage in
segregant colonies.
In abnormal colonies, the observed induced segregation index with
respect to DMSO for the acid, base, and neutral fractions of the SWRI
waste was 1.77, 1.81 and 1.47, respectively (Table 29). The acid and
base fractions induced primarily hyperdiploid colonies and mitotic
recombination, while the neutral fraction induced hyperdiploid and near
hyperdiploid colonies. All three fractions of the SWRI waste induced a
positive response in the abnormal colonies. In addition, the maximum
genotoxic response was induced by the base fraction, although the
induced segregation index of the acid fraction was not significantly
lower.
The results from the chemical analysis of the fractions of the
storm-water runoff impoundment waste were far less conclusive. The
components of the acid, base, and neutral fraction of the SWRI waste
were present at levels that were below the detection limits of the
procedures used. Thus, the results from the chemical analysis of the
waste fractions are, to a limited extent, in contrast to the results
from biological testing.
Biological analysis detected genotoxic constituent(s) in all three
fractions of the storm-water runoff impoundment waste, and three of the
four bioassays detected the maximum genotoxic response in the acid
fraction with metabolic activation. Genotoxic constituent(s) in the
SWRI waste were capable of inducing point mutations and chromosome
damage; however, the concentrations of the genotoxic compounds in the
waste were below the detection limits of the analytical instruments.
The potential of the SWRI waste to be treated by land application should
be enhanced by the fact that genotoxic compounds are present in such low
concentrations. Since adsorption and degradation are frequently
concentration dependent, the SWRI waste can possibly be treated by land
application, in spite of the presence of genotoxic constituents.
However, because of the inability of chemical analysis to identify
compounds present at genotoxic levels in the waste, short-term bioassays
appear to provide the only analytical method capable of efficiently
monitoring land application of this waste.
The second refinery waste evaluated in this research was a combined
API separator slop-oil emulsion solids (COMBO) waste. The dose-response
115
-------
curves for the acid, base, and neutral fractions, as measured in the
Salmonella/microsome assay are provided in Figure 37 and Table 36.
Theseresults indicate that the maximum mutagenic response was obtained
from the neutral fraction in the presence of metabolic activation and
that significant quantities of mutagenic activity were also detected in
the neutral fraction in the absence of metabolic activation. In
addition, none of the subfractions of the neutral fraction induced a
greater mutagenic response than the whole fraction (Figure 38). Tests
were also conducted with the base fraction using phenobarbitol induced
rat liver. Epler et_ al. (1978) found that Aroclor-induced rat liver
reacted best with the neutral fraction, while phenobarbitol-induced rat
liver reacted best with the base fraction of synthetic fuel extracts.
However, the results in Figure 39 indicate that for both strains TA98
and TA100, the maximum frequency of induced mutations was observed in
the presence of Aroclor 1254 induced S9. Thus, all three waste
fractions induced a mutagenic response in the Salmonella assay.
The analysis of fractions of the combined API-separator/slop oil
emulsion waste in the spot test using DNA repair deficient and
proficient bacteria did not provide any conclusive results (data not
shown). The use of the modified plate incorporation assay (Donnelly
e_t al., 1983) did, however, provide more conclusive results. The
acid fraction induced the maximum response in the modified DNA repair
assay (Table 32). The survival ratio of the repair proficient strain
(168) compared to the repair deficient strain (hcr-9) was 0.37 for the
acid fraction, while the base fraction induced a survival ratio of 0.69.
There was no appreciable difference in the survival of the repair
proficient and deficient strains following exposure to the neutral
fraction.
Mutagenic activity was also detected in the fractions of the COMBO
using the Aspergillus methionine assay (Tables 37, 38 and 39 and
Figures 40, 41 and 42). The maximum mutagenic response was obtained
from the neutral fraction with metabolic activation, and mutagenic
activity was also detected in the absence of metabolic activation
(Figure 42). The neutral fraction induced 2575 mutations/10
survivors. This was the maximum mutagenic response induced in the
Aspergillus methionine system by any of the waste fractions tested in
the present study. Mutagenic activity was also detected in the acid
fraction both with and without metabolic activation; whereas, the base
fraction induced a positive response only in the presence of metabolic
activation (Figures 40 and 41). In the diploid system, the neutral
fraction of the COMBO waste induced the maximum genotoxic response.
Significant increases in the induced segregation index were also
observed in the base fraction; whereas, the induced segregation index in
the acid fraction, with respect to DMSO, would be considered a
borderline positive response. The acid and base fractions induced
predominantly yellow segregants, while constituent(s) of the neutral
fraction appeared to preferentially induce fawn segregants (Table 28).
The overall mutation frequencies in abnormal colonies induced by the
116
-------
400
300-
>
>
£200-
oo
o>
100-
DOSE/PLATE (mg)
Figure 37. Mutagenlc activity of fractions of COMBO waste.
117
-------
TABLE 36. HUTAGENIC ACTIVITY OF FRACTIONS OF COMBINED API SEPARATOR/SLOP OIL EMULSION WASTE
Fraction
Dose/plate
(mg)
S9 -
TA 98
TA 100
TA1535
TA1538
00
Total hi* revertaats (Mean + SD)
Acid
Base
Neutral
0
1
.5
.1
.0$
.01
0
1
.5
.1
.05
.01
0
1
.5
.1
.05
.01
24+2
45 + 26
26 + 12
22+9
24+4
22+3
17 + 1
24+6
21+4
20 + 10
22+6
17 + 1
27+4
179 + 81
100 + 39
50 + 13
43 + 13
33+9
27+6
217 + 37
118 + 13
65 + 10
51 + 15
31 * 13
25+7
224 + 13
139 + 70
53 + 10
52+3
33 + 12
32+7
269 + 97
161 + 28
90 + 26
54+8
34+17
99 + 6
98 + 15
102 + 9
86 + 16
86 + 19
78 + 10
85 + 11
100 + 41
108 + 19
85 + 16
95+9
80 + 15
117 + 6
147 + 12
123 + 16
114 + 3
120 + 6
130 + 15
99 + 16
118 + 7
128 + 24
: 144 + 29
121 + 31
103 + 23
114 + 13
NT*
207 + 35
138 + 29
112 + 19
106 +_ 35
112 + 10
248 + 40
206 + 18
172 + 18
144 + 29
113 + 12
22+3
28+6
27 + 10
34 + 13
30+9
27 + 12
23+4
22 + 13
34+9
39 + 11
39+9
24+6
21+2
26+5
33+7
37 + 12
39+3
33 + 13
18 +
24 +
29 +
29 +
20 +
20 j+
23 +
28 7
35 +
27 +
18 +
22 ±
26 +
27 +
23 +
22 +
24 +
23 +
1
4
7
6
4
3
4
3
8
6
5
7
4
13
7
4
5
5
13 +
17 +
19 +
13 +
16 +
26 +_
13 +
14 +
17 +
13 +
12 +
14 +
17 +
16 +
16 +
13 +
17 +
19 +
3
2
3
3
4
17
3
2
7
4
3
2
2
5
4
5
5
13
24+4
20 + 1
49 + 12
25 + 17
32+5
26 jf. 3
25+4
290 + 86
185 + 56
58+8
34+4
28 +_ 4
21+5
65 + 11
60+4
40+6
32+8
21 + 1
* - NT - not tested.
-------
400^
0
o
200-
COMBO NEUTRAL CALL+S9)
NEUTRALCwholc) 0 O
IOO
300
500
I 000
DOSE/PLATECug)
Figure 38. Mutagenic activity of sub fractions of neutral fraction
of COMBO waste.
119
-------
600-
400-
ui
20QJ
I 00
COMBO-BASE
TA98-Aroclor S9 OO
TA98-Phenobarbitol S9
TAI 00-Aroclop S9
TA I 00-Phenobarbitol S9
300
DOSE/PLATE(ug)
500
I 000
Figure 39.
Mutagenic response of base fraction of COMBO waste
using metabolic activation from Aroclor 1254 or
phenobarbitol induced rat liver.
120
-------
TABLE 37. SURVIVING FRACTION AND INDUCED MUTATION FREQUENCY OF A. NIDULANS
Dose/Plate
(pg)
FOLLOWING
Exposure
Time (min)
EXPOSURE TO ACID
Surviving
Fraction
FRACTION OF
Mutation
A
COMBO WASTE
Frequency Per
B
106
C
Survivors
Total
Without Metabolic Activation
131.5
436.5
657.5
873
1315
With Metabolic
164
436.5
655
873
1315
40
40
40
40
40
Activation
20
20
20
20
20
.62
.28
.11
.09
.08
.59
.34
.15
.04
.30
2.2
25.4
8.6
69.6
10.0
4.0
36.8
12.9
176.4
9.4
.39
0
2.0
0
0
3.7
68.2
24.9
367.6
20.0
0
0
0
0
0
1.0
15.8
5.9
73.5
3.6
2.6
25.4
10.6
69.6
10.0
8.7
120.8
43.7
617.4
33.1
-------
TABLE 38. SURVIVING FRACTION AND INDUCED MUTATION FREQUENCY OF A. NIDULANS
ro
Ni
FOLLOWING EXPOSURE TO BASIC
Dose/Plate Exposure
(pg)
Without
130
221
275
441
550
Time (min)
Metabolic Activation
40
40
40
40
40
Surviving
Fraction
.75
.69
.49
.37
.34
FRACTION OF
COMBO WASTE
Mutation Frequency Per 10 Survivors
A
3.2
6.0
6.0
12.6
9.0
B
.29
.89
1.8
1.6
3.1
C
0
0
0
0
0
Total
3.5
6.9
7.8
14.2
12.0
With Metabolic Activation
110
221
330
441
550
20
20
20
20
20
.83
.66
.48
.43
.39
4.7
7.0
8.3
22.7
7.9
1.7
6.7
5.8
19.1
8.8
.66
3.7
1.4
8.6
1.8
7.1
17.4
15.5
50.4
18.5
-------
TABLE 39. SURVIVING FRACTION AND INDUCED MUTATION FREQUENCY OF A. NIDULANS
KJ
CO
Dose/Plate
(ug)
FOLLOWING
Exposure
Time (min)
EXPOSURE TO NEUTRAL
Surviving
Fraction
FRACTION
Mutation
A
OF COMBO
Frequency
B
WASTE
Per 106
C
Survivors
Total
Without Metabolic Activation
85
171
214
320
428
550
With Metabolic
106.9
213.8
221.0
427.5
441
40
40
40
40
10
40
Activation
20
20
20
20
20
.87
.69
.46
.43
.82
.30
.75
.38
.34
.19
.04
3.9
7.7
8.6
15.0
1.6
17.1
2.0
5.4
36.7
34.7
176.3
3.4
6.9
8.2
13.7
2.4
15.7
.76
2.8
68.2
39.0
367.6
0
0
0
0
0
0
.68
1.4
15.8
13.0
73.5
7.3
15.4
16.8
28.7
4.0
32.8
3.4
9.6
120.7
86.7
617.4
-------
-------
cr
o
>
o:
=>
(a
o
LJ
O
tr.
u.
z
o
-
<
o
Ul
o
-4
10
-5
10
-
10
COMBO BASE
-S3 550ug/pt
100 *
O
10
o
<
(E
u.
o
I 0
30
SO
70
90
MINUTES EXPOSURE TIME
Figure 41. Induced mutation frequency and fractional
survival in A^. nidulans following exposure
to base fraction of COMBO waste. Data
based on results from two independent
experiments.
125
-------
2575
tr
o
>
ce
3
CO
o
z
LU
o
Ul
ec
<
*-
O
Ul
o
-4
10
-5
10
-
10
COMBO NEUTRAL
-S9 488ug/pt + S9
oa
100 *
o
-I 0
O
I 0
30
50
70
90
MINUTES EXPOSURE TIME
Figure 42. Induced mutation frequency and fractional
survival in ^. nidulans following exposure to
neutral fraction of COMBO waste. Data based
on results from two independent experiments.
126
-------
acid, base and neutral fractions of the COMBO waste were .049, .70, and
.057, respectively (Table 29). The induced segregation index with
respect to DMSO was 1.14 for the acid, 1.63 for the base, and 1.33 for
the neutral fraction (Table 29). The acid and base fractions induced
the maximum mutation frequency in the hyperdiploid class, while the
neutral fraction induced the maximum mutation frequency in the class
representing mitotic recombination. In addition, the acid fraction
induced a significant increase in the breakdown of the mitotic spindle
apparatus. These results indicate that in abnormal colonies the maximum
genotoxic response was induced by the base fraction, with the neutral
and acid fractions inducing slightly lower responses. Thus, in both the
haploid and diploid Aspergillus assays, the maximum overall genotoxic
response was obtained with the neutral fraction, with an intermediate
response in the acid fraction, and a borderline response in the base
fraction.
Chemical analysis identified a total of eleven, seven, and eight
compounds in the acid, base, and neutral fractions, respectively, of the
COMBO waste (Figures 43, 44, 45, and Table 40). In all three fractions,
at least half of the compounds identified were substituted napthalenes.
Only one compound, dimethyl phenanthrene,identified in the COMBO waste
fractions would be considered a potential source of mutagenic activity,
and it was identified in the neutral fraction. A study by 'LaVoie et
al. (1983) determined that several substituted phenanthrenes were
mutagetiic in Salmonella with metabolic activation.
Bioassay directed chemical analysis of the fractions of the
combined API separator/slop-oil emulsion waste detected compound(s)
capable of inducing point mutations, lethal damage to DNA, and various
types of chromosome damage. Toxic effects were not observed in the
prokaryotic point mutation assay even at the highest dose levels. The
maximum genotoxic response in the Salmonella, Aspergillus haploid,
and Aspergillus diploid bioassays was induced by the neutral fraction;
while the acid fraction induced the maximum rsponse in the Bacillus
DNA repair assay. The neutral fraction induced a significant mutagenic
response both with and without metabolic activation. This fraction also
contained dimethyl phenanthrene, the only potential mutagen identified
in the COMBO waste. Substituted phenanthrenes, however, are known to
require metabolic activation in order to reach their ultimate mutagenic
form (LaVoie £t al., 1983). Thus, these results indicate that
additional compound(s), not identified by chemical analysis and present
in the COMBO waste, are likely to be responsible for the direct-acting
mutagenic activity detected in the neutral fraction and for the
indirect-acting mutagenic activity of the other waste fractions. Both
the chemical and the biological analysis of the COMBO waste indicated
that the neutral fraction contained the maximum mutagenic potential.
The constituent most likely to be responsible for the mutagenic activity
of the neutral fraction, dimethyl phenanthrene, was detected by a
chemical analysis. However, land application of the COMBO waste should
be monitored using a combined testing protocol because biological
127
-------
RIG
to
ZOO
COMBO ACID
12
ZOO
400
5OO
RT
Figure 43. GC/MS chroraatograph of acid fraction of COMBO waste.
-------
VO
RIG
COMBO BASE
100
i
200
300
400
500
RT
Figure 44. GC/MS chromatograph of base fraction of COMBO waste.
-------
0>
n
RIG
COMBO NEUTRAL
20O
300
400
500
RT
Figure 45. GC/MS chromatograph of neutral fraction of COMBO waste.
-------
TABLE 40. SELECTED PROPERTIES OF COMPOUNDS IDENTIFIED IN THE FRACTIONS OF THE
COMBINED API-SEPARATOR/SLOP-OIL EMULSION SOLIDS WASTE
Sample
Acid Fraction
287,
Basic Fraction
Neutral Fraction
287,
Peak
Number
229
237
277
294
315
331
342
392
414
230
237
288
315
331
342
277
294
315
331
342
392
528
Compound
Trimethyl octane
Methyl napthalene
Ethyl-methyl octane
Dimethyl napthalene
Trimethyl decane
Methyl ethyl napthalene
Trimethyl napthalene
Dimethyl undecane
Hexyl tridecane
Trimethyl octane
Methyl napthalene
Dimethyl napthalene
Trimethyl decane
Methyl ethyl napthalene
Trimethyl napthalene
Ethyl-methyl octane
Dimethyl napthalene
Trimethyl octane
Methyl ethyl napthalene
Trimethyl napthalene
Dimethyl undecane
Dimethyl phenanthrene
CAS #
-
90-12-0
573-98-8
-
879-129
-
-
90-12-0
573-98-8
-
879-129
-
573-98-8
-
879-129
Formula
C11H24
C11H10
C11H24
r H
12 12
r H
13 28
C13H14
C3H4
X J A*T
C11H24
r H
11 10
C12H12
r it
13 28
C13H14
C13H149
C11H24
r it
12 12
r H
11 24
C13H14
r it
13 14
C13H28
r H
C16H14
Genetic
Activity
X
Ml; CO
X
MO
X
X
MO
X
X
X
Ml; CO
MO
X
X
X
X
Ml; CO
X
X
MO
X
M1;C1
1. Potential genetic activity: P = promoter; I = inhibitor; CC = cocarcinogenic;
Ml = mutagenic; MO = nonmutagenic; Cl = carcinogenic; CO = noncarcinogenic;
X = unknown; references included in text.
-------
analysis detected trace quantities of additional genotoxic compounds not
identified by a chemical analysis.
Biological and chemical analysis have been employed in the present
study to evaluate the mutagenic potential of the acid, base, and neutral
fractions of three hazardous industrial wastes. A summary of the
results obtained in the different biological test systems is provided in
Table 41. The base fraction of the wood-preserving waste induced the
maximum mutagenic response in the 1J. subtilis DNA repair plate
incorporation assay, the Salmonella/microsome assay (strains TA98,
TA100 and TA1538) and the Aspergillus methionine assay. In the DNA
repair spot test and Aspergillus diploid assay, the acid fraction of
the wood-preserving waste induced the maximum response. One component
of the acid fraction which may be responsible for its genetic toxicity
is pentachlorophenol (Fishbein, 1979). Pentachlorophenol does not
induce a mutagenic response in the Salmonella assay (Anderson et
al., 1972). However, in the recently developed S>. typhimurium
strain TA102, the maximum response was induced by the acid fraction. The
results of biological analysis indicate that all three fractions of the
wood-preserving waste contain genotoxic compounds, while chemical
analysis identified compounds that may be mobile and resistant to
degradation. Thus, land treatment of a wood-preserving waste should be
carefully monitored and preferrably conducted at low application rates.
Although chemical analysis failed to identify any of the constituents of
the storm-water runoff impoundment, biological analysis detected
mutagenic activity in all three waste fractions. The maximum mutagenic
response observed in three of four bioassays was induced by the acid
fraction of the SWRI waste. Biological analysis detected genotoxic
compounds in the SWRI waste; however, these compounds were present in
quantities that were below the detection limits of the chemical
analysis. Thus, the storm-water runoff impoundment may be a good
candidate for land treatment because of its relatively low concentration
of mutagenic compounds.
Chemical and biological analysis agreed that the neutral fraction
of the combined API separator/slop-oil emulsion solids possessed the
maximum mutagenic potential. In the COMBO waste, biological and chemcial
analysis identified the fraction with the greatest mutagenic potential,
although chemical analysis was unable to identify all of the mutagenic
constituents. These results indicate that for both refinery wastes,
biological analysis may provide the most efficient tool for monitoring a
land treatment facility.
This research has demonstrated the utility of a combined testing
protocol using biological analysis to measure the genotoxic potential of
waste fractions and a chemical analysis to define the type and quantity
of genotoxic compounds. These results have also demonstrated the
inability of chemical analysis to provide a comprehensive evaluation of
the genotoxic potential of a hazardous industrial waste. While it is
possible that a more intensive chemical analysis could have identified
132
-------
TABLE 41. SUMMARY OF RESULTS OBTAINED FROM TESTING WASTE FRACTIONS IN BIOLOGICAL
TEST SYSTEMS
u>
u>
Sample
Bioassay '
DNA
SALM
BAG PM
S9"
ASPMT
ASPDP
PENT S
SWRI
COMBO
Acid + +
Base - ++
Neutral
Acid - -
Base
Neutral
Acid - +
Base
Neutral
+
- ++ -
+
++ 0
+ 0
++ o
- ++ 0
++ 0
+ ++ 0
+ +
+ +
+
0 +
0 +
0 +
0 +
0 +
0 +
H+ + 0
>+ + Q
H + 0
t-+ + 0
M- + 0
H + 0
^ + 0
i- + 0
n- + 0
1. DNA = _B. subtilis DNA repair assay; SALM = £>. typhimurium reverse
mutation assay; BAG PM = E. subtilis reverse mutation assay; ASPMT = A.
nidulans methionine assay; ASPDP = A. nidulans diploid assay.
2. Response: 0 = not tested; - = <2 times background; _+ = >2 <2.5 times background;
+ = >2.5 <5 times background; + + = >5 times background.
3. S9 = 9000 x g supernatant from Aroclor 1254 induced rats.
-------
genotoxic compounds present in trace concentrations, information would
still be lacking as to the interactions of the waste constituents.
However, the results also indicate that chemical analysis is a necessary
component of a hazardous waste analytical protocol. Chemical analysis
is the only procedure that can be used to identify waste constituents
and to verify the absence of artifacts generated in the collection or
extraction process.
134
-------
SECTION 6
SOIL CHARACTERIZATION
INTRODUCTION
As the products of our industrialized society become more complex,
the need correspondingly increases to develop the technology that can
provide for the safe disposal of the waste. The use of soil to receive
and degrade waste materials has gained general acceptance while other
methods of waste disposal have become more restricted for economical or
environmental reasons. In order to prevent further loss of an already
diminishing natural resource, land disposal of waste should be managed
in order to provide the most efficient use of the land and monitored in
order to evaluate the progress of waste degradation. Land treatment is a
method of waste disposal that is designed to utilize the diverse
microbial population of a fertile soil for the degradation of waste
constituents, while the adsorptive capacity of the soil prevents
environmental deterioration. EPA (1982) regulations require that land
treatment be restricted to wastes that are rendered less or
non-hazardous through chemical and biological reactions in the soil. In
order to provide information that can demonstrate the effect of soil
incorporation on the hazardous characteristics of a waste, techniques
need to be developed for monitoring the mutagenic potential of
environmental samples before and after waste application.
Short-term bioassays have been proposed as a tool for monitoring
land treatment because biological analysis can often be used to detect
the synergistic, antagonistic, or additive interactions of the
components of a complex mixture. The first step in establishing a land
treatment program is to demonstrate that the waste is rendered less or
non-hazardous by soil incorporation. This treatment demonstration
should be preceded by an evaluation of the mutagenic potential of both
the waste and the receiving soil. This study reports on the results of a
mutagenic potential evaluation of the organic extract of three native
soils used solely for agricultural purposes. These procedures may also
prove useful for evaluating the point of sufficient reclamation for a
soil contaminated by a chemical spill.
135
-------
MATERIALS AND METHODS
Soil
Three soils were selected to represent a range in soil texture and
management practices. Physical characteristics of these soils are given
in Table 42. A Norwood sandy clay (Typic Udifluvent) was obtained from
the Texas A&M University Research Farm. This soil had been fallow for
four to five years prior to being cropped to a sorghum-sudan hybrid. The
Norwood soil has a Class I land use capability which indicates this
series has very few limitations and can be used for intensive
cultivation practices. Potential sources of mutagenic contaminants in
the Norwood soil include exhaust from the tractors and direct
application or drift of biocides from sprayed crops. The Bastrop clay
(Udic Paleustalf) was collected from a range area being used for cattle
grazing at the Texas A&M University Research Annex. The soil series at
this site consisted of a well-drained fine sandy loam surface soil
approximately 45 cm thick and underlain by a massive clay loam subsoil.
Because of the limited permeability of the subsoil, the Bastrop series
is primarily used as rangeland and is rarely cultivated. The range area
had been sprayed with a herbicide mixture of 2,4-D and 2,4,5-T in July
and August preceeding the October collection of soil. Dr. W. D. Surge
supplied a sample of Sassafrass sandy loam (Mesic Typic Hapludult) from
the Beltsville Agricultural Research Center. This soil had been fallow
for about five years until being cropped to corn (Zea mays) and
fertilized during the three years prior to soil collection. Herbicide
treatments that had been applied at acceptable rates to the Sassafras
soil included atrazine, lasso, simazine, paraquat, carbofuran, and
toxaphene. Hydrocarbons were extracted from the soil using
dichloromethane following the methods of Brown and Donnelly (1983).
Solvent extractions were taken to dryness on a Brinkman Bucci Rotary
Evaporator, and the residue was dissolved in dimethyl-sulfoxide (Grade
1, Sigma Chemical Co., St. Louis, MO) for testing in the biological
systems.
Biological Analysis
The ability of the organic extract of soil samples to induce
genetic damage was measured in a prokaryotic and eukaryotic system
capable of detecting compounds that induce point mutations. The
Salmone1la/microsome assay of Ames e£ al. (1975) utilizes a
prokaryotic organism to evaluate the capacity of a sample for inducement
of reverse mutations to histidine prototrophy. The two Salmonella
strains TA98 (a frameshift mutant) and TA100 (a base-pair substitution
mutant) were supplied by Dr. B. N. Ames (University of California,
Berkeley, CA). The methods used were the same as Ames e£ al. (1975)
except that overnight cultures were prepared by inoculation into 10 ml
of Oxoid Nutrient Broth No. 2 (K C Biological, Lenexa, KS). Soil
extracts were tested on duplicate plates in two independent experiments
in the standard plate incorporation assay at a minimum of four dose
136
-------
TABLE 42. PHYSICAL PROPERTIES OF THE THREE SOILS
Soil
Bastrop
Norwood
Sassafrass
Sand
60.3
48.2
57.27
Silt
10.0
15.2
34.86
Clay
29.7
36.6
7.87
Texture
SCL
SC
SL
PH
6.9
7.7
5.6
Extractable
Hydrocarbon
.229
.057
.025
-------
levels with and without enzyme activation (0.3 ml rat liver/ml S.9 mix).
Aroclor 1254 induced rat liver was obtained from Litton Bionetics
(Charleston, SC). Positive controls included 2 ug/plate
N-methyl-N -nitro-N-nitrosoguanidine (Sigma) for TA100, 25 ug/plate
2-nitrofluorene, (Aldrich Chemical Co., Milwaukee, WI) for TA98, and 10
ug/plate 2-aminoanthracene (Sigma) which was used to verify the
functioning of the metabolic activation system. All reagents and
extracts were tested for sterility; dimethylsulfoxide was used as a
negative control.
The eukaryotic test employed Aspergillus nidulans (a fungus) to
assess the mutagenic potential of soil extracts by evaluating the
induction of foreward mutations at the methionine suppressor loci.
Conidia from four to five single colonies of the methGl biAl
(requiring methionine and biotin) Glasgow strain of Q Aspergillus
nidulans grown for 5-6 days on a complete medium at 37 C were used
for each experiment. Samples were tested at a minimum of five dose
levels and four exposure times with and without metabolic activation.
The procedures used were the same as Scott et al. (1978). Mutant
colonies were assayed by spreading exposed cells on a methionine-free
medium. Mutant colonies were scored after incubation for 5 days at
37 C. Colonies were divided by colony morphology into three Classes,
A, B, C, and the total number of mutant;, colonies. Each of the three
classes is believed to involve two genes (Scott and Alderson, 1970).
The morphology of Class A colonies appear green, Class B brown, and
Class C green with a white hyaline edge. The frequency of mutations
induced by the soil extract was determined by subtracting the frequency
of spontaneous mutations which occurred in Class A, B, C or the total
from the total mutation frequency in Classes A, B, C and the total. A
sample was considered positive if a positive slope occurred on the
mutation induction curve, or the induced mutation frequency for at least
two exposure times was more than twice the spontaneous mutation
frequency. Positive controls included 8-methoxypsoralen (Sigma),
8-methoxypsoralen plus near UV-light without activation, and
benzo(a)pyrene (Aldrich) with metabolic activation. Only the Bastrop and
Norwood soil extracts were tested in the Aspergillus assay.
RESULTS AND DISCUSSION
The results from testing each of the three soils in the
Salmonella/microsome assay (Table 43, Figures 46 and 47) indicate that
all soil extracts contained mutagenic activity, with the greatest amount
of activity being detected in the Bastrop soil. In strain TA98 with
metabolic activation, 1,000 ug of the extract of the Norwood,
Sassafrass, and Bastrop soils induced 67, 119, and 469 revertant
colonies, respectively. The mutagenic activity of the organic extract
from one gram of the Bastrop soil was greater than the Norwood or
Sassafrass soils by almost two orders of magnitude and less than the
condensate from the smoke of one cigarette by one order of magnitude
(Figure 48).
138
-------
TABLE 43. MUTAGENICITY OF ORGANIC EXTRACTS OF THREE SOILS AS MEASURED WITH
S. TYPHIMURIUM STRAINS TA98 AND TA100 WITH AND WITHOUT METABOLIC
VO
ACTIVATION
Total his Revertants (Mean + SD)
TA98
Soil
Norwood
Sassafrass
Bastrop
Dose/plate
(pg)
0
10
50
100
500
1000
5000
0
10
50
100
500
1000
2500
0
10
50
100
500
1000
5000
-S9
26 + 6.9
23 + 3.3
NT*
27 + 6.9
NT
32 + 8.3
51 + 23
19 + 3.1
25 + 5.0
37 + 6.2
31 + 21
59 + 10
69 -i- 14
112 _+ 20
23 + 5.8
28 + 0.8
47 + 8.0
183 + 81
256 + 22
423 + 34
515 +256
+S9
.-
32 + 7.7
38 + 9.2
NT
33 + 11
NT
67 + 16
122 + 45
21 + 5.3
30 + 7.6
42 + 4.9
36 + 6.7
61 + 5.6
199 + 35
179 + 16
35+6.7
48 + 6.6
62 + 3.2
95 + 29
264 + 90
469 + 96
716 + 108
TA100
-S9
88 + 14
76+9.7
NT
87 + 15
NT
116 + 19
88 + 14
106 + 16
115 + 11
172 + 43
185 + 80
251 + 78
265 + 115
276 +_ 18
124 + 22
121 + 23
143 + 56
142 + 50
306 + 33
416 + 13
605 + 72
+S9
130 + 27
122 + 49
NT
107 + 35
NT
140 +8.1
188 +_ 52
117 + 17
146 + 20
147 + 25
117 + 18
186 + 28
288 + 65
NT
94+12
101 + 15
111 + 27
142 + 52
381 + 119
429 + 74
NT
*NT - Not tested.
-------
500
6300
100
UNAMMENDED SOILS
TA98 +S-9
O BASTROP
SASSAFRASS
o NORWOOD
2X-BKG
0.5 1.0
DOSE/PLATE (mg)
5jO
Figure 46.
Mutagenic activity of organic extract of three agricultural
soils as measured with £. typhimurium, strain TA98, with
metabolic activation.
140
-------
500-
300
o
o
5
UNAMMENDED SOILS
TA-IOO +S-9
Q BASTROP
SASSAFRASS
o NORWOOD
0.5 1.0
DOSE/PLATE (mg)
Figure 47. Mutagenic activity of organic extract of three
agricultural soils as measured with S_. typhimurium,
strain TA100, with metabolic activation.
141
-------
MUTAGENIC ACTIVITY OF I g. eq. SOIL EXTRACTS AS
COMPARED TO CIGARETTE SMOKE CONDENSATE
S9
-S9
S3
-ONE CIGARETTE
(from Kler et. al., 1974)
-BASTROP SOIL
-SASSAFRASS SOIL
-NORWOOD SOIL
10 100 1,000 10,000
TA98 hl8*revertant8
Figure 48. Mutagenic activity of one gram of soil as compared to cigarette
smoke condensate.
-------
The dose-response curves for all three soils in strain TA98 with
and without metabolic activation (Figures 46 and 49) indicate that while
direct acting mutagens were detected in the Sassafrass and Bastrop
soils, the addition of metabolic activation to the assay system
increased the number of revertant colonies. The addition of metabolic
activation produced almost twice the number of revertant colonies
obtained in the absence of metabolic activation in both the Sassafrass
and Norwood soils in strain TA98 at the highest dose levels. For the
Bastrop soil, there was only a slight increase in the total number of
revertant colonies when metabolic activation was added to the assay
system (Table 43).
The organic extracts of the Bastrop and Norwood soil were also
tested in an eukaryotic bioassay using A. nidulans. This bioassay
has been found to detect 95% of carcinogens as mutagens and is sensitive
to some compounds not detected in the Salmonella assay (Scott et
al. , 1982). The results presented in Table 44 and Figures 50 and 51
indicate that the organic extract of the Bastrop and Norwood soils
induced increased mutation frequencies with increasing exposure times in
the absence of metabolic activation. The frequencies of mutations
induced by the two soils were comparable, although the Norwood soil was
tested at a dose level ten times greater than the Bastrop soil. For
both soils, survival of Aspergillus in the absence of metabolic
activation was decreased as the exposure time or dose level was
increased. The effect of increasing dose level on the frequency of
induced mutations was less conclusive. In the Norwood soil, an
increased mutation frequency was observed as the dose level was
increased, while this effect in the Bastrop soil was not observed.
When metabolic activation was added to the system, the response
obtained in the Aspergillus assay was much less conclusive (Table 44
and Figures 50 and 51). Survival of Aspergillus was not appreciably
altered at increased dose levels or exposure times. In addition, the
frequency of mutations induced by the extract of either soil was not
significantly affected by increasing the dose level or exposure time in
the presence of metabolic activation. Thus, while mutagenic activity
was detected in the organic extract of the two soils in the absence of
metabolic activation, the addition of metabolic activation reduced or
eliminated this effect.
An analysis of the extracts of the two soils using gas
chromatographic and mass spectrometric techniques detected the presence
of several low molecular weight chemicals. A total of thirteen compounds
were identified in the Norwood soil and eight compounds in the Bastrop
soil (Table 45). The chromatographs of the Norwood and Bastrop soils
are provided in Figures 52 and 53, respectively. All thirteen compounds
identified in the Norwood soil are saturated alkanes ranging from C,,
(tetradecane) to C (docosane). Eight compounds were identified in
the Bastrop soil including eight alkanes, four of which were saturated
n-alkanes. The three additional compounds identified in the Bastrop
143
-------
500
o
r 300
o
3
100
UNAMMENDED SOILS
TA98 -S-9
a BASTROP
A SASSAFRASS
o NORWOOD
as to
DOSE/PLATE (mg)
2.0
Figure 49.
Mutagenic activity of organic extract of three
agricultural soils as measured with S_. typhimurium.
strain TA98, without metabolic activation.
144
-------
Ln
TABLE 44. EFFECT OF INCREASING DOSES OF ORGANIC EXTRACT OF BASTROP AND NORWOOD SOILS
ON SURVIVAL AND INDUCED MUTATION FREQUENCY IN ASPERGILLUS NIDULANS
WITH AND WITHOUT METABOLIC ACTIVATION
Dose/Plate
(pg)
-S9 (Exposure Time - 40 min
Surviving Induced Mutation
Fraction Frequency (x 10 )
+S9 (Exposure Time - 20 min)
Surviving Induced Mutation^
Fraction Frequency (x 10
Bastrop Soil
10
18.75
25
37.5
Norwood Soil
0.90
0.80
0.71
0.61
144
288
566
755
1133
0.94
0.75
0.69
0.52
0.41
0.55
1.2
0.29
1.3
<0
0.53
0.74
2.2
2.8
1.6
1.1
0.99
0.95
0.97
0.92
0.99
1.00
0.97
<0
<0
<0
0.33
<0
0.37
0.40
<0
<0
-------
i64
lO"5
g io
a
UJ
S io7
o
UJ
8
p.-
BASTROP SOIL
50 *g/pt
+S-9 -S-9
o o
81 B X K
X
100^
10
(O
10 20 30 SO 80
MINUTES EHX5SURE TIME
Figure 50. Induced mutation frequency and fractional survival in A.
nidulans following exposure to organic extract of Bastrop
soil. Data based on results from two independent
experiments.
146
-------
I04
10
i67J
O
UJ
o .,
NORWOOD SOIL
566 icg/pt
+S-9 -5-9
B H H
~""~~X--«. x-
^^ "X1" -»
,00-
o
I
K) £
10 20 30 40 60 80
MINUTES EXPOSURE TIME
Figure 51. Induced mutation frequency and fractional survival in
A. nidulans following exposure to organic extract of
Norwood soil. Data based on results from two independent
experiments.
147
-------
TABLE 45. LIST OF COMPOUNDS IDENTIFIED IN NORWOOD AND BASTROP SOILS
00
Soi 1 Compound
Norwood Tetadecane
Pentadecane
Methyl hexadecane
Hexadecane
Dimethyl hexadecane
Heptadecane
Trimethyl hexadecane
No octadecane
Tetramethyl hexadecane
Nonadecane
Eicosane
Heneicosane
Docosane
Formula
CH3(CH2)12CH3
CH3(CH2)13CH3
C17H36
CH3(CH2)14CH3
C18H38
CH3(CH2)15CH3
CH3(CH2)16CH3
C20H42
CH3(CH2)lgCH3
CH3(CH2)19CH3
CH,(CH0)onCH,
Genetic ,
Activity
P;CC
X
X
I
X
X
CC
X
X
P;CC
X
X
-------
TABLE 45 CONTINUED.
Soil
Bastrop
Compound
Dodecanol
Ethyl methyl pentanol
Dimethyl undecane
Trimethyl decane
Octadecane
Trimethyl dodecane
Nonadecane
Benzenedicarboxylic acid
Eicosane
Heneicosane
Docosane
Fo'rmula
C12H26°
C8H18°
C13H28
C18H38
C19H40
C8H16°4
C20H42
C22H46
Genetic
Activity
P;CC
X
X
CC
X
MO; CO
P;CC
C
1. Probable genetic activity: P = promotor; I = inhibitor; CC = cocarcinogenic
X = unknown; References listed in test.
-------
Ol
o
pic
NORWOOD SOIL
400 5OO 600 700 800 900 1000
RT
Figure 52. GC/MS chromatograph of organic extract of Norwood soil.
-------
Ln
RIG
BASTROP SOIL
200 300 4OO 500 600 700 800
RT
Figure 53. GC/MS chromatograph of organic extract of Bastrop soil.
-------
soil include two alcohols, dodecanol and ethyl methyl pentanol, and
benzene dicarboxylic acid. The conclusive determination of the source
of the compounds identified in the soil extracts is difficult given the
limited amount of available information. A chemical characterization of
the resin- and asphalt-free components from a mineral soil by Morrison
and Bick (1967) identified a number of n-alkanes, n-alkan-2-ones, and
primary n-alkanols. The n-alkanes comprised approximately 6.4% of the
wax fraction of the soil and consisted of a mixture of n-alkanes with
carbon numbers ranging from 16 to 33 (Morrison and Bick, 1967). In
addition, Ogner and Schnitzer (1970a) identified normal plus
branched-cyclic alkanes in the water-soluble soil fulvic acid. An
alternative source of the compounds identified in the Norwood and
Bastrop soil is the particulate matter from diesel tractors used to
cultivate the soil. One of the major organic constituents identified in
diesel particulate matter is n-tetradecane (Yergey e£ al., 1982).
Although this and other alkanes have been identified in particulate
matter, the absence of the more complex polycyclic aromatic hydrocarbons
indicates that the source of the alkanes in the soil extracts is
probably not diesel particulate.
The Bastrop soil also contained several alkanes in addition to two
alcohols and benzene dicarboxylic acid. The most probable source of the
alkanes is the waxy fraction of the soil (Morrison and Bick, 1967),
whereas the alcohols may be hydroxylated metabolites of soil alkanes.
The initial oxidative reaction in alkane decomposition generally
involves one terminal methyl group and results in the formation of the
corresponding alcohol, aldehyde, and fatty acid (Rowell, 1977). Esters
of benzene dicarboxylic acid have been identified in soil organic matter
(Cifrulak, 1969) and in the fulvic acid fraction of soil (Ogner and
Schnitzer, 1970). Ogner and Schnitzer (1970) state that the origin of
these compounds is uncertain although they may have been produced
biosynthetically. One potential source of the benzenedicarboxylic acid
in the Bastrop soil is the degradation of 2,4-dichlorophenoxyacetic acid
(2,4-D). The primary pathway for 2,4-D biodegradation is through
dichlorocatechol with complete dehalogenation usually following cleavage
of the benzene ring (Bourquin and Gibson, 1978). It is possible,
however, that dehalogenation of the 2,4-D in the Bastrop soil preceded
ring cleavage, resulting in the formation of benzenedicarboxylic acid.
Benzenedicarboxylic acid may also have reached the Bastrop soil as a
result of its use as a plasticizer, pesticide carrier (Fishbein, 1979),
or as a trace contaminant of the pesticides (EPA, 1980).
The results from the biological analysis of the organic extract of
three soils indicate that mutagenic activity may be associated with the
waxy fraction (alkanes). Mutagenic activity was detected in all three
soils, with the greatest amount of activity detected in the Bastrop
soil. The extract of the Bastrop and Norwood soils induced increased
mutation frequencies in both the Salmonella and Aspergillus assays,
although the mutagenic activity in Aspergillus was significantly
reduced in the presence of metabolic activation. The discrepancies
152
-------
between the results of the bioassays may have resulted from several
factors. While the sensitivity of the Aspergillus bioassay is reported
to be 96% (Scott £t al., 1982), there are some carcinogens which
give a positive response in Salmonella and no response in
Aspergillus (Bignami e£ al., 1980). It is also possible that
Aspergillus, which is more metabolically competent than Salmonella
(Dunkel, 1981), was able to detoxify mutagenic compounds with the aid of
microsomal activation.
Using chemical analysis, only saturated n-alkanes were detected in
the extract of the Norwood soil. Of the identified compounds
n-tetradecane has been found to have promoting activity (Lankas et.
al., 1978) and cocarcinogenic activity with benzo(a)pyrene
(Goldschmidt, 1981); whereas, octadecane and eicosane were also found to
have cocarcinogenic activity, and hexadecane acted as an inhibitor of
benzo(a)pyrene (Goldschmidt, 1981). The mutagenic activity in the
Bastrop soil may have been produced by degradation products from
previously applied herbicides. Benzene dicarboxylic acid, one of the
constituents of the Bastrop soil, is non-mutagenic in the Salmonella
assay (Omori, 1976). In addition, while 2,4-D, 2,4,5-T, and mixtures of
these compounds gave negative results in the Salmonella assay
(Anderson e_t al., 1972), a review of the literature by Grant (1979)
indicates that component(s) of 2,4,5-T can cause various types of
chromosome damage. Interactions of the metabolites of biodegradation
represent another possible source of the mutagenic activity in soil
extracts. Catechol, a frequent metabolite from the oxidation of aromatic
hydrocarbons in soil (Gibson, 1971), has been found to enhance the
mutagenicity of polycyclic aromatic hydrocarbons (Yoshida and Fukuhara,
1983). Other soil metabolites including dodecanol, octadecanol, and
eicosaine have been found to have cocarcinogenic activity (Goldschmidt,
1981).
The biological analysis of the organic extract of three soils used
solely for agricultural purposes has demonstrated the presence of
mutagens and potential carcinogens. These results are in agreement with
those of previous reseachers who have demonstrated that agricultural
soil (Goggelman and Spitzauer, 1982; Withrow, 1982) and municipal water
obtained from an agricultural area (Heartlein zt_ al. , 1981) may
contain significant quantities of mutagenic activity. In addition,
epidemiologic studies have identified a higher incidence of certain
types of cancer in the residents of rural communities (Lilienfeld e_t^
al., 1972; Higginson, 1980) which may be a result of the use of
agricultural chemicals. A chemical analysis of two of the three soils
used in this research was unable to conclusively identify the mutagenic
contaminants. However, the past history of these soils indicates that
the most probable source of mutagenic activity is trace quantities of
the partially oxidized residues from previous biocide applications. It
also appears that the mutagenic activity of these trace contaminants may
have been enhanced by the presence of promotors and cocarcinogens.
153
-------
This research has defined the level of background contamination in
three soils. While chemical analysis was unable to identify any
mutagenic compounds, biological analysis indicated that the soil that
had received the most recent applications of herbicide yielded the
greatest amount of mutagenic activity. Natural sources cannot be ruled
out as being responsible for the elevated mutagenic activity of the
Bastrop soil; however, the presence of direct-acting mutagens in the
soil indicates an exogenous source, since natural mutagens such as
mycotoxins and plant toxins are indirect acting (Garner e_t_ al.,
1982; Brown and Dietrich, 1979). The presence of organic mutagens in the
soil does not present a human health risk provided these materials are
retained in the soil. Indeed, organic mutagens in soil may enhance the
capacity of soil microorganisms to degrade complex molecules (Poglazova
et al., 1967). These results have also demonstrated the utility of
a combined testing protocol. Biological analysis defined the level of
mutagenic activity in each soil, while chemical analysis indicated that
the activity was not produced by an artifact, e.g., histidine or solvent
residues, from the collection or extraction process.
154
-------
SECTION 7
QUANTIFYING SOIL EXTRACTION PROCEDURES
INTRODUCTION
Industrial activities generate large quantities of hazardous waste
that must be disposed of in an economical and environmentally sound
manner. These wastes have been shown to contain constituents that may
be mutagenic, carcinogenic, or teratogenic. Chronic exposure of large
populations to genotoxic compounds in hazardous wastes represents a
serous threat to public health.
Genotoxic compounds constitute a relatively small group of agents
which are highly specific for nucleic acids and produce deleterious
effects in genetic elements at subtoxic concentrations (Brusick, 1980).
An environmental mutagen is an agent that is released into the
environment and can alter the genome or the proper functioning of a
genome (Plewa, 1981). Environmental mutagens have the potential of
changing human genetic material by inducing mutations. Mutations
affecting somatic cells may induce cancer, heart disease, aging, or
other illnesses (Ames, 1979), whereas mutations affecting germ cells may
induce birth defects, sterility, or other teratogenic effects (Brusick,
1980). A carcinogen is an agent that significantly increases the yield
of malignant neoplasms in a population (Clayson, 1962). A teratogen is
an agent that acts during pregnancy to produce physical or functional
defects in the embryo, fetus, or offspring (Meyers and Beyler, 1981).
Evidence that most carcinogens are mutagens lends validity to the theory
that cancer can be caused by somatic muations or damage to DNA (Brusick,
1980).
Hazardous waste must be disposed of properly in order to prevent
environmental contamination and the transmission of genotoxic effects to
future generations. The usual methods for disposal have been
incineration, landfilling, and deep well injection. Because these
techniques of waste disposal are not economical and/or do not provide
for the desired destruction of genotoxic constituents, their utility is
limited. Using non-destructive methods of waste disposal on the land
results in sites that must be permanently removed from productivity.
However, land treatment of a waste can result in land that may be
reclaimed and may provide for the destruction of the genotoxic organic
waste constituents (Donnelly and Brown, 1981). When properly designed
and managed, a land treatment facility provides for the recycling of
155
-------
nutrients and water, while toxic organic constituents are retained and
degraded in the upper layer of soil or soil treatment zone (Brown e_t
al., 1982a). In order to prevent contamination of the environment
with mutagenic compounds, land application of hazardous waste is
restricted by Environmental Protection Agency (1982) regulations to
include only those wastes in which the hazardous constituents can be
completely degraded, transformed, or immobilized in the soil treatment
zone.
The present research is an integral part of a larger project to
evaluate land treatment as an environmentally sound alternative in
hazardous waste disposal. This research was designed to evaluate the
efficiency of extraction procedures for recovering mutagenic activity
from soil using the Salmonella/microsome assay (Ames e± al., 1975)
and high performance liquid chromatography (HPLC) analysis. Microbial
test systems such as the Salmonella/microsome assay can be used to
evaluate the potential of hazardous waste constituents for causing
genetic damage and, also, to accurately predict the fate and mobility of
mutagenic constituents when hazardous wastes are land applied (Donnelly
and Brown, 1981).
Hazardous industrial wastes contain a complex mixture of chemicals
which may have additive, synergistic, or antagonistic interactions with
regard to their toxic or genotoxic effects. These interactions are
further complicated when wastes are mixed with the soil. Chemical
analysis alone may fail to account for the transformations in the soil
or various interactions which occur between chemicals in a complex
mixture (Donnelly and Brown, 1981). The Salmonella/microsome assay
provides a rapid and inexpensive means of measuring the mutagenic
potential of chemicals in a soil extract.
This segment of the research evaluated two extraction procedures
and determined which method most efficiently extracted the mutagenic
activity from the soil. An efficient extraction procedure is a critical
element for monitoring a land treatment facility. Levels of mutagenic
activity at a chosen site must be monitored in order to evaluate the
degradation rate of toxic organic chemicals.
MATERIALS AND METHODS
The procedures for this study involved the selection and treatment
of the soils and the organic compounds. A comparison of soil extraction
procedures and their reproducibility on treated soils was evaluated
using the Salmonella/microsome assay and HPLC analysis. Statistical
analysis was by the ANOVA procedure and the Duncan Multiple Range test
(Steel and Torrie, 1976; Freund, 1982).
156
-------
Selection of Mutagenic Compounds and Soils
Most of the experiments involved the use of either 2-nitrofluorene
(Aldrich, Milwaukee, Wis) or benzo[a]pyrene (Sigma, St. Louis, Mo.). In
addition, a complex waste mixture of known mutagenic activity was also
used for comparison. The Salmonella/microsome assay is sensitive to
2-nitrofluorene (2NF), benzo[a]pyrene (B[a]P) (McCann and Ames, 1977),
and the complex waste mixture, i.e., a wood-preserving bottom sediment
(Donnelly et_ al., 1982). B[a]P and 2NF were selected because they
are nonpolar and polar compounds, respectively; therefore, they may have
different binding affinities to the soil. This may effect the
efficiency of the extraction procedures to recover the cmpounds.
Two soils including a Norwood sandy clay (Typic Udifluvent) and a
Bastrop sandy clay loam (Udic Paleustalf) were used. The soil
characteristics are given in Table 42.
Analysis of specific organic compounds in the soils that may
influence the mutagenicity of chemical additions to the soil were
carried out by the U.S. Environmental Protection Agency Robert S. Kerr
Environmental Research Laboratory. Research on the mutagenic activity
of untreated soil is necessary because information in the literature on
this topic is almost nonexistent. Chemical constituents found in the
soil may enhance or reduce mutagenic activity or may effect the
efficiency of extraction. A comparison was made between the mutagenic
response of the known mutagens, the soil extract, and the known mutagens
or waste after soil incorporation to determine the effect of soil
incorporation on the mutagenicity of these mixtures.
Soil Preparation and Chemical Addition
Each soil was sieved through a 4 mm sieve and oven dried at 100 C
for 24 hours. To prevent biological degradation from influencing the
test results, the soils were autoclaved before chemical addition. The
soil was measured into glass jars and heat sterilized for 90 min at
121 C and 15 psi for three consecutive days. The soils were brought
to field capacity of 18 and 22% for the Norwood and Bastrop soils,
respectively, with sterile deionized water. All soils were checked for
sterility by plating on nutrient agar plates before chemical addition.
The chemicals that were used were of the highest purity available.
A description of the chemicals used in this segment of the research is
provided in Table 46. Treatment levels were selected to fall within the
detection limits of the Salmonella/microsome test. Soils were treated
with three different dose levels of the chemical; the dose levels were
selected from the optimum response in the Salmonella/microsome assay.
The dose at which the maximum mutational response occured was the medium
dose level. The high and low dose levels were an order of magnitude
higher and lower than the optimum dose level for B[a]p (5 ug/plate).
For 2NF, the high dose level was half an order of magnitude higher than
157
-------
TABLE 46. DIAGNOSTIC MUTAGENS USED TO EVALUATE THE EFFICIENCY OF EXTRACTION
PROCEDURES .
Compound
Structure
Mutagenicity
Polarity Source
Ul
oo
2-Nitrofluorene
Direct Acting Polar
Aldrich,
Milwaukee, WI
Benzo(a)pyrene
Indirect Acting Nonpolar Sigma,
St. Louis, MO
-------
the optimum dose, and the low dose level was an order of magnitude lower
than the optimum dose (25 pg/plate). Soils were spiked with the
chemicals or wood-preserving waste and incubated in a closed environment
at room temperature for different time intervals to be studied prior to
extraction.
Extraction Procedure and Sample Preparation
Hydrocarbons were extracted from the soil using a Waring laboratory
blender following procedures of Brown and Donnelly (1983). A Soxhlet
blender technique was used on the wood-preserving bottom sediment to
compare the efficiency of both extraction techniques (Brown e_t al.,
1982b). The extracting solvent, dichloromethane, was removed by
evaporating the sample to near dryness on a Brinkman-Bucci Rotary
Evaporator. Dichloromethane was used to transfer the organic residue to
a glass culture tube. The sample was taken to dryness under a stream of
nitrogen. The dried extract was dissolved in 5 ml dimethyl sulfoxide
(Sigma, St. Louis, Mo.) and brought to its original concentration with
reference to the low, medium, or high dose level. The samples were
filtered through a 0.45 pm millipore filter (Millipore Corp., Bedford,
Mass.) to remove soil particles. The sample was serially diluted in
dimethyl sulfoxide to a 10 dose level so that four concentrations of
each soil dose level were tested in the mutagenicity assay.
Bioassay
The Salmonella/microsome mutagenicity test was used to measure
the mutagenic potential of the extracted samples. Dilutions of each
extract were tested with Salmonella typhimurium strain TA98 supplied
by Dr. B. N. Ames, University of California at Berkeley, CA.
Salmonella TA98 is a frameshift mutant which contains a histidine
D3052 mutation and a deletion which invokes a requirement for biotin.
Frameshift mutations occur by shifted pairing in repetitive sequences of
DNA. The mutation is in a reverse direction to independence from
histidine auxotrophy. This bacterial strain is capable of being
reverted from a histidine requirement back to prototrophy by a wide
variety of mutagens.
Overnight bacterial cultures were grown in Oxoid Nutrient Broth No.
2 (KG Biological, Lenexa, Kansas) with shaking at 37 C for 16 hours.
Liver homogenates (S9 from rats induced with a polychlorinated biphenyl
mixture [Aroclor 1254]) (Litton Bionetics, Charleston, S.C.) were
incorporated into the test system to allow indirect acting mutagens to
reach their ultimate mutagenic form. This incorporates an important
aspect of mammalian metabolism into the test, thereby allowing those
carcinogens requiring metabolic activation to be as easily detected as
mutagens. The procedures used for the preparation of the S9 mix are
described by Ames e_t al. (1975). Positive, solvent, and sterility
controls were included in each test to act as an internal control for
the bioassay, to demonstrate the sensitivity of the test system, and to
159
-------
demonstrate the functioning of the metabolic activation system.
High Performance Liquid Chromatography
Samples were assayed using HPLC to quantify the recovery of the
chemicals from soil. Techniques used for HPLC are as described by
Schram (1980) and Yost et_ al. (1980). The analysis of polynuclear
aromatic hydrocarbons utilizes octadecylsilane bonded stationary phases
and acetonitrile/water as the mobile phase (Belinki, 1980). To optimize
the mobile phase composition, an isocratic composition of acetonitrile/
water was used. This compound is favored as it permits faster
separations than other solvent systems (Donahue e_t al. , 1978).
Acetonitrile has a much lower viscosity than does methanol; therefore, a
faster flow rate can be used without exceeding the pressure limit of the
column. Also, acetonitrile has a greater ability to dissolve many
polycyclic hydrocarbons than other solvents, thus greater amounts can be
loaded into the system per injection (Donahue et_ a_l., 1978).
RESULTS AND DISCUSSION
In order to determine the affect of storage on the extraction of
mutagenic activity from soil, the polar compound 2-nitrofluorene or the
nonpolar benzo(a)pyrene was added to the two soils and extracted
following 24 hours or 7 days of storage at 0 C. The results from
biological analysis of the extract of the Norwood or Bastrop soils
amended with the two compounds indicated that there was no statistical
difference (p<.01) between samples stored for 24 hours or 7 days.
These results demonstrate that binding of chemicals to soil particles
will not affect the mutagenic activity of sterile soil stored at 0 C
for at least one week.
The results from the biological analysis of the extract of the two
soils amended with 2-nitrofluorene are provided in Table 47 and Figures
54 and 55. At the optimum dose level (25 ug), 2-nitrof luorene induced
85 revertants/ug, while the extract from 500 mg of the Norwood and
Bastrop soils induced .13 and .11 revertants/mg, respectively. At the
highest treatment level (25 mg 2NF/50 g soil), the 2-nitrofluorene and
2-NF amended Norwood and Bastrop soils induced 5.4, 6.1, and 5.2
revertants/mg, respectively. All of these values fall within their
respective standard deviations. Both the 10 and 10 dilutions at
the high treatment level for the two soils fell slightly below the
anticipated level for 2-nitrofluorene. At the medium and low treatment
levels, the mutagenic activity of the extract of the 2-NF amended
Bastrop and Norwood soil was within the standard deviation for the pure
chemical at all dose levels. These results indicate that a blender
extraction of soil amended with the direct-acting polar compound
2-nitrofluorene will provide complete recovery of the mutagenic activity
in the soil.
160
-------
TABLE 47. MUTAGENIC ACTIVITY, AS MEASURED WITH S. TYPHIMURIUM TA98, OF
2-NITROFLUORENE , AND THE SOLVENT EXTRACT OF THE NORWOOD AND
BASTROP SOILS AMENDED WITH VARIOUS LEVELS OF 2-NITROFLUORENE
Sample
None
2-nitrof luorene
(high)
(med)
(low)
-
Dose/Plate
(mg)
0
-1
-2
-3
0(0.5)
-1(0.05)
-2(0.005)
-3(0.0005)
0(0.25
-1(0.025)
-2(0.0025)
-3(0.00025)
0(0.025)
-1(0.0025)
-2(0.00025)
-3(0.000025)
2-Nitrof luorene Bastrop
Soil
-
-
2711 + 478
- '
-
-
2560 + 409
2122 + 465
682 -i- 173
123 _+ 30
2122 + 465
682 + 173
123 + 30
30 + 7
55 + 18
39 + 17
34+10
32 +_ 11
3074 +509
2632 + 373
1145 + 251
239 + 55
2490 + 271
1870 + 177
490 + 80
93 + 24
2056 + 183
549 + 47
117 + 16
31 + 4
Norwood
Soil
64+31
31 + 12
26 + 10
25 + 10
2600 + 202
2484 + 226
966 + 113
152 + 20
2574 + 518
2231 + 289
593 + 66
103 _+ 12
1930 + 535
825 + 331
153 + 46
36 + 13
-------
NJ
.009
cost ((/tutu
Figure 54. Extraction efficiency, as measured with J3. typhimurium strain TA98,
of 2-nitroflourene from Norwood soil.
-------
Figure 55. Extraction efficiency, as measured with j^. Cyphimurium strain TA98,
of 2-nitrofluorene trom Bastrop soil.
-------
Additional tests were conducted using the nonpolar indirect-acting
benzo(a)pyrene. The results from biological analysis of the extracts of
the two soils amended with benzo(a)pyrene.are provided in Table 48 and
Figures 56 and 57. For both soils at all treatment levels, these was no
appreciable difference in the mutagenic activity of the B(a)p and the
soil amended with B(a)p. At the high and medium treatment levels, the
extract of the Norwood soil plus B(a)p was consistently less mutagenic
than the pure compound. However, the soil extract did fall within the
standard deviation of the pure compound at the same dose levels. Thus,
there was no apparent interraction between the benzo(a)pyrene and the
soil components which significantly affected the mutagenic acitivity of
the soil extract.
The soil extracts were also analyzed using high performance liquid
chromatography to determine the efficiency of the blender extraction
procedure. The HPLC chromatograms from the solvent control, the two
soils, and the soil plus the chemicals are provided in Figures 58
through 63. For the Norwood soil, the average extraction efficiency for
both compounds at all three treatment levels was 91% (Table 49), while
the average efficiency for the Bastrop soil was 89% (Table 49). The
average extraction efficiency for benzo(a)pyrene and 2-nitrofluorene in
the Norwood soil was 91 and 99%, respectively. For the Bastrop soil,
the average recovery was 84% for benzo(a)pyrene and 93% for
2-nitrofluorene. These results indicate that there was no appreciable
difference in the extraction efficiency from the Norwood or Bastrop
soils. Treatment level did not appear to influence the recovery of
benzo(a)pyrene, whereas the recovery of 2-nitrofluorene at the low
treatment level was 15% less than the high and medium levels.
When the extraction efficiencies are compared for each of the pure
compounds, the recovery of the polar compound appears to be slightly
greater. The extraction efficiency from both soils at all three
treatment levels was 90% for the nonpolar benzo(a)pyrene and 96% for the
polar 2-nitrofluorene. The lower efficiency observed in the
benzo(a)pyrene amended soils was influenced by the very low recovery
efficiency in the Norwood soil at the medium treatment level. The cause
of the low recovery efficiency for benz(a)pyrene in the Norwood soil is
at this point unknown.
Biological and chemical analysis of two soils amended with either
2-nitrofluorene or benzo(a)pyrene have indicated that the blender
extraction procedure does provide adequate recovery of mutagenic
compounds. The results from the Salmonella/microsome assay indicate
that there was no appreciable difference in the mutagenic activity of
the pure compound or the extract of the soil amended with the pure
compound at equivalent dose levels. In addition, the extraction
efficiency as measured using high performance liquid chromatography
averaged greater than 85% for both chemicals at all treatment levels on
both soils. This analytical approach, using combined chemical and
biological analysis, has demonstrated that for the compounds, levels,
164
-------
TABLE 48. MUTAGENIC ACTIVITY, AS MEASURED WITH j>. TYPHIMURIUM TA98, WITH
Ln
METABOLIC ACTIVATION, OF BENZO(a)PYRENE , AND THE SOLVENT EXTRACT OF
THE NORWOOD AND BASTROP SOILS AMENDED WITH VARIOUS LEVELS OF
BENZO(a)PYRENE
Sample
None
B[a]P
(high)
(raed)
(low)
Dose/Plate
(rag)
0
-1
-2
-3
0(0.5)
-1(0.05)
-2(0.005)
-3(0.0005)
0(0.05
-1(0.005)
-2(0.0005)
-3(0.00005)
0(0.005)
-1(0.0005)
-2(0.00005)
-3(0.000005)
B[a]P
-
500 + 139
514 + 123
505 + 163
74 _+ 33
514 + 123
505 + 163
74 + 33
35 + 10
505 + 163
74 + 33
35 + 10
Bastrop
Soil
64 + 5
47 + 8
44 + 9
45 + 14
543 + 79
612 + 122
553 + 124
90 + 17
355 + 61
293 + 195
54 + 16
49 + 9
618 + 82
119 + 18
60 + 11
48 + 7
Norwood
Soil
56 + 23
42 + 22
39 + 19
40 + 21
361 + 50
400 + 68
390 + 185
52 + 11
437 + 944
459 + 118
89 + 41
58 + 36
475 + 163
118 + 71
46 + 15
43 + 16
-------
300
IOO
900
'5
I
300
too
OOOOS .0009 .COS .05
008E
t BA8TROP B(«>f»0«H>»89
i"
.00003 .0005
.005
DOSE <»|/pl«U>
.03
.3
Figure 56. Extraction efficiency, as measured with j>. typhimurium strain TA98,
of benzo(a)pyrene from Norwood soil.
-------
900
2
CD
IOO
-f f
--I
900
300
100
.OOO03 .OOO3
.009 .OS
DOSE (|/»UU>
SOO
CO
300
100
.0009 .009
.09
B(.)f*89
k BASTROP BU>r
900
300
B<«)»«89
BA8TROP BU>»»B9
.9 .OOOO9 .OOO9
-OO3
008E (
Figure 57. Extraction efficiency, as measured with S^ typhimurium strain TA98,
of benzo(a)pyrene from Bastrop soil.
-------
TABLE 49. EXTRACTION EFFICIENCY, AS MEASURED WITH HPLC, OF THE BASTROP AND NORWOOD
SOILS AMENDED WITH MUTAGENIC COMPOUNDS
oo
Compound
2-Nitrof luorene
Benzo(a)pyrene
Application Rate Bastrop
25 mg 2NF/50g soil
12.5 mg/50g
1.25 mg/50g
Average
25 mg B(a)p/50g soil
2.5 mg/50g
.25 mg/50g
Average
Soil
104
91
84
93
89
89
73
84
Norwood Soil
104
108
85
99
97
52
99
83
Average
104
100
85
96
93
71
86
84
Average both compounds 89 91 90
-------
Dimethyl Sulfoxide
5.0mg/ml
Norwood Soil + Dimethyl Sulfoxide
5.0mg/ml
Figure 58. HPLC chromatograph from (A) dimethylsulfoxide and
(B) Norwood soil.
169
-------
Dimethyl Sulfoxide
5.0mg/ml
Bastrop Soil > Dimethyl Sulfoxide
5.0mg/ml
Figure 59. HPLC chromatograph from (A) dimethylsulfoxide and
(B) Bastrop soil.
170
-------
2-Nitrofluorene
5.0mg/ml
Norwood Soil + 2-Nitrofluorene
5.0mg/ml
Figure 60. HPLC chromatograph from (A) 5.0 mg/ml 2-nitroflourene,
and (B) Norwood soil +5.0 mg/ml 2-nitroflourene.
171
-------
2-Nitrofluor«M
5.0mg/ml
Bastrop Soil + 2-Nltrofluorene
1 S.Omg/ml
Figure 61. HPLC chromatograph from (A) 5.0 mg /ml 2-nitroflourene,
and (B) Bastrop soil +5.0 mg/ml 2-nitrofluorene.
172
-------
Benzo(a)pyrene
5.0mg/ml
Norwood Soil + Benzo(a)pyrene
5.0mg/ml
Figure 62. HPLC chromatograph from (A) 5.0mg./ml benzo(a)pyrene,
and (B) Norwood soil +5.0 mg/ml benzo(a)pyrene.
173
-------
Benzo(a)pyrene
5.0mg/ml
Bastrop Soil + Benzo(a)pyrene
5.0mg/ml
Figure 63. HPLC chromatograph from (A) 5.0 mg/ml benzo(a)pyrene,
and (B) Bastrop soil +5.0 mg/ml benzo(a)pyrene.
174
-------
and soils evaluated, there are no interractions with soil compounds, and
the blender procedure does provide efficient extraction of mutagenic
compounds from soil.
175
-------
SECTION 8
MUTAGENIC ACTIVITY OF RUNOFF WATER FROM HAZARDOUS WASTE AMENDED SOIL
INTRODUCTION
Land treatment of hazardous waste consists of a program of
controlled applications of waste to the surface layer of soil
accompanied by a carefully planned management and monitoring program.
This technique is designed to protect the environment by using the
surface layer of soil to transform, degrade, or immobilize hazardous
waste constituents. Many of the more complex organic waste constituents,
particularly those which are toxic or genotoxic, may persist in the soil
for an extended period of time (Alexander, 1981). Scant information is
available concerning the ultimate fate of resistant organic chemicals in
the environment. These chemicals may volatilize from the soil, be
translocated by plants, be removed by runoff water, or be leached into
the groundwater. A variety of analytical techniques could be used for
characterizing samples of the various streams leaving the soil.
Detailed chemical analysis would provide information on the presence or
probable presence of specific compounds. A chemical analysis would not,
however, be capable of predicting the synergistic, additive, or
antagonistic interactions of a mixture of chemicals or their effect on a
biological system. Biological analysis provides the only analytical
technique that can be used to define the toxic or genotoxic potential of
an environmental sample.
This segment of the present study was conducted to evaluate the
potential for the removal of mutagenic waste constituents by rainfall.
The EPA (1980) regulations require that the runoff from hazardous waste
land treatment facility be retained. However, monitoring techniques must
be developed to determine when runoff water can be safely discharged
following closure of the facility. In addition, these results will
provide information on the presence of water soluble mutagens in soil
and the effect of degradation on their mutagenic activity.
MATERIALS AND METHODS
Waste
Three petroleum based sludges were utilized in the greenhouse
study. A general description of their characteristics is provided in
176
-------
Table 50, while a more detailed description is included in Section 5.
The calculated amount of waste applied to the soi-io ifl each box was
based on the bulkdensity of each soil (1.44 g cm for the Norwood
soil and 1.49 g, cm" for the Bastrop soil), the volume of soil in each
box (36,225 cm ), and the percent extractable hydrocarbons in each
waste (27% for PENT S, 21% for SWRI, and 41% for COMBO). The weight of
waste applied to each box was be determined by multiplying the weight of
soil times the desired level of extractable hydrocarbons in each box and
dividing this figure by the amount of extractable hydrocarbons in each
waste.
Based on these calculations, the wood-preserving bottom sediment
(PENT S) was applied to the greenhouse boxes at a rate of 5.5 kg/box to
the Norwood soil and 6.5 kg/box to the Bastrop soil, or a hydrocarbon
loading rate of 3.1%. The application rate for the storm-water runoff
impoundment waste (SWRI) was 11.2 kg/box to the Norwood soil and 11.6
kg/box to the Bastrop soil, or a hydrocarbon loading rate of 4.5%. The
combined API-separator/slop oil emulsion sludge (COMBO) was also applied
at a hyrocarbon loading rate of 4.5%. This was equivalent to a sludge
application rate of 5.7 kg/box to the Norwood soil and 5.9 kg/box to the
Bastrop soil.
Soil
The physical and chemical properties of the soils used in the
greenhouse study are provided in Table 42. A more detailed discussion
of the Bastrop and Norwood soils is provided in Section 6. Approximately
two fifty-five gallon barrels of each soil were collected from an
uncontaminated site for use in the greenhouse study.
Greenhouse Boxes
At the time of collection, the soils used in the greenhouse study
were slightly moist to wet. The two soils were spread separately on
sheets of brown paper on greenhouse benches to air dry. Large clods were
passed through an ore crusher to reduce their size. Air dried soil was
passed through a 4 mm sieve and stored until needed.
Sifted soil was packed in wooden boxes (45 cm x 57 cm x 20 cm) in 5
cm lifts to a depth of 17 cm. The surface of the soil was smoothed to
level with the runoff collection trough (Figure 64). Waste
incorporation into the soil was accomplished by removing approximately
one-half of the soil from the box and applying the waste to the
remaining soil in the box. The removed soil was added to the box and
mixed with a small trowel until the mixture was homogenous. Twelve boxes
of each soil were prepared for the greenhouse study. These included
three boxes of each soil to serve as controls and three boxes of each
soil to receive the PENT S, SWRI, or COMBO waste. Soil samples were
collected from greenhouse boxes before, immediately after, and 45, 90,
180, 360, and 540 days following waste application.
177
-------
00
TABLE 50. DISTRIBUTION OF MUTAGENIC ACTIVITY IN FRACTIONS OF
HAZARDOUS WASTE AND THE TWO SOILS USED IN THE GREENHOUSE
STUDY
Fraction
PENT S
SWRI
COMBO
Norwood
Bastrop
Crude
Acid
Base
Neutral
Acid
Base
Neutral
Acid
Base
Neutral
Crude
Crude
Extractable
Hydrocarbon
(mg/g)
270
23
24
223
8
5
346
6
3
137
0.057
0.229
Specific
Activity -
(rev./mg)
1,282
771
1,204
860
475
418
420
368
344
497
21
297
Weighted
Activity _
(rev./mg)
346
18
29
192
4
20
145
2
1
68
0.0012
0.068
1- Mutagenic activity as measured with strain TA98 with microsomal
activation.
2. Revertants/mg = slope of mutation induction curve calculated using
three highest non-toxic dose levels.
3. Revertants/mg eq. = Revertants/milligram material extracted;
calculated by multiplying revertants/mg x % extractable
hydrocarbons.
-------
SHEET METAL RUNOFF
COLLECTION TROUGH
3/4" PLYWOOD
LABEL-BOX
OUTLET
PIPE
TYGON TUBIN
(to amber glass bottle)
20cm
57.5cm
Figure. 64. Schematic diagram of greenhouse boxes used in degradation study.
-------
Runoff samples were also collected before, immediately after, and
45, 90, 180, 360, and 540 days following waste application. A rainfall
simulator that held four boxes per simulation was used to generate
runoff samples. The boxes were placed in the chamber on a 10 slope,
and a 3.5 in/hour intensity rain was applied with a rotating disk
rainfall simulator as described by Morin et al. (1967). The water
used in the simulated rainfall was rain water collected from a trough on
the sides of a fiberglass greenhouse. Collected rain water was pumped
to a 600 gallon capacity storage system where it was stored until
needed.
Extraction Procedures
Runoff samples were extracted using the procedures of Brown and
Donnelly (1982). The samples were passed through a jmixed bed of 4.0 g
of XAD-2 and 6.3 g XAD-7, or approximately 20 cm of each resin as
suggested by Rappaport e_t al. (1979). The resins were washed prior
to use by swirling and decanting three times with ten volumes each of
acetone, methanol, and distilled water. Washed resins were stored at
4 C ttrior to use. Glass econo-columns (Bio-Rad, Richmond, CA) 1.5 x
50 cm were packed with 20 cm of XAD-2 resin followed by 20 cm
of XAD-7 resin. Dressier (1979) found that the combined XAD-2 and XAD-7
column provided the most efficient recovery for a broad range of organic
compounds, while Grabow et^ al. (1981) observed that the XAD
technique may be more efficient than a liquid-liquid extraction for the
recovery of low concentrations of mutagens. A glass wool plug was placed
above the resin in order to trap soil particles. The columns were
flushed with 1,200 ml of distilled water before loading the water
sample. Leachate or runoff water was placed in a reservoir and allowed
to pass through the column by gravity flow at about 50 ml per minute.
After loading the water sample, dry nitrogen was introduced into the
column to remove the residual aqueous phase, and the column was washed
with 120 ml of distilled water to remove residual histidine. The
adsorbed organic compounds were eluted with 160 ml of acetone. The
acetone extract was filtered through about 30 g of anhydrous Na_SO,
and Whatman No. 42 filter paper into a flat bottom flask. Extract were
reduced to less than ten milliliters on a Brinkman-Bucci Model R
roto-evaporator. The sample was added to a glass funnel filled with
sodium sulfate to remove residual water and washed with acetone. The
sample was then pipetted off into a small screw-capped glass culture
tube and taken to dryness under a stream of nitrogen. Dimethyl
sulfoxide (Sigma) was added to the dried extract, and the resultant
solution was passed through a 0.2 urn average pore diameter Teflon
filter (Millipore-Fluoropore Bedford, Mass.). Samples were stored at
4 C prior to use.
Chemical Analysis
The compounds were identified using a Finnigan OWA Automated GC/MS.
The GC capillary column used in the procedure was a J&W Scientific
180
-------
DB-5-30W (Orangeville, CA). One ul aliquots were used with a helium
carrier gas flow near 36 cm/sec. The GC oven Qtemperature program was
60°C for 1 minute and then increased ..at 6 /min to 260 C with a
hold time of 12 minutes. The OWA unit has a splitless mode injector.
The software used has a mass spectra library of 31,331 organic
compounds.
Biological Analysis
The potential of the organic extracts of runoff water to induce
genetic damage was measured with two microbial systems capable of
detecting compounds which produce point mutations.
The Salmonella/microsome assay of Ames et_ al. (1975) was used
to monitor the mutagenic activity of concentrated water samples. The
Salmonella strains were 'kindly supplied by Dr. Bruce N. Ames
(University of California, Berkeley, CA). The methods were the same as
Ames e_t al. (1975) except that overnight cultures were prepared by
inoculation into 10 ml of Oxoid Nutrient Broth #2 (KG Biological, Inc.,
Lenexa, KS) and incubated with shaking for 16 hours at 37 C. Water
extracts were tested on duplicate plates in two independent experiments
in the standard plate incorporation assay at a minimum of 4 dose levels
of the sample with and without enzyme activation (0.3 ml rat liver/ml
S-9 mix) using strains TA98 and TA100. Aroclor 1254 induced rat liver
was obtained from Litton Bionetics (Charleston, SC). Positive controls
included 2 ug/plate N-methyl-N'-nitro-N-nitrosoguanidine (Sigma) for
TA100, 25 jig/plate 2-nitrofluorene (Aldrich) for TA98, and 10 ug/plate
2-aminoanthracene (Sigma) which was used with all strains to verify the
functioning of the metabolic activation system. All reagents and
extracts were tested for sterility; DMSO was used as a negative control.
The bioassay using Aspergillus nidulans was used to detect
point mutations and small deletions induced in a haploid genome using
the methionine system. Conidia from four to five single colonies of the
methGl biAl (requiring methionine and biotin) Glasgow strain of
Aspergillus nidulans grown for 5 to 6 days on a complete medium at
37 C were used for each experiment. Samples were tested at one dose
level, usually the extract equivalent to 250 ml of water, and one
exposure time selected to yield approximately 50% of survival on five
plates in two independent experiments with and without metabolic
activation. The procedures used were the same as Scott e_£ al.
(1978) except that the cells were exposed in a 13 x 100 mm screw capped
culture tube. The exposed cells were plated on a complete medium to
determine survival and a methionine-free medium to determine induced
mutation frequency. Mutant colonies were scored after incubation for 5
days at 37 C. Colonies were divided by colony morphology into three
classes, A, B, C, as well as the total mutation frequency represented by
the sum of induced mutants from Class A, B, and C. Each of these three
classes is believed to involve two genes. The morphology of Class A
colonies is green, Class B brown, and Class C green with a white hyaline
181
-------
edge. The frequency of mutations induced by a sample was determined by
subtracting the frequency of spontaneous mutations from the total
mutation frequency. A sample was considered mutagenic if the
induced-mutation frequency was greater than 5.0. Positive controls
included 8-methoxypsoralen (Sigma, St. Louis, Mo), 8-methoxypsoralen
plus near UV light without activation, and benzo(a)pyrene (Aldrich,
Milwaukee, WI) with metabolic activation.
RESULTS AND DISCUSSION
Runoff water was collected from the Norwood or Bastrop soil amended
with PENT S waste over a 540 day period. The mutagenic activity of the
residual hydrocarbons in the runoff water from the Norwood soil
increased with each subsequent sampling date (Figure 65). When compared
to the runoff sample from day 0, the mutagenic potential of the sample
collected on day 540 was approximately eight and three times greater in
strain TA98 and TA100, respectively (Tables 51 and 52). In addition,
strain TA98 detected mutagenic activity without the S9 mix in the runoff
sample from day 540, whereas no mutagenic activity was detected in the
absence of metabolic activation in runoff samples collected prior to
this date (Figure 66).
While similar results were obtained with the runoff water from the
Bastrop soil, the increase in mutagenic activity over time was much less
dramatic than the increase observed in the runoff water from the Norwood
soil. The mutagenic potential of the residual hydrocarbons in the
runoff water from the PENT S amended Bastrop soil increased from day 0
through day 360 but remained constant or decreased slightly from day 360
to day 540 (Figure 67, Tables 53 and 54). As with the Norwood soil,
direct-acting mutagens were detected on day 540 (Table 53). The results
from a chemical analysis of runoff water from control and PENT S waste
amended Bastrop soil from day 0 and 360 are provided in Figures 68, 69
and 70. There were no compounds present in detectable levels in the
runoff water from the unamended Bastrop soil (Figure 68). Ten compounds
were detected in the runoff water collected on day 0 from the waste
amended Bastrop soil, including dimethylnapthalene (DMN),
dihydro-acenaphthylene (DHN), dibenzofuran (DBF), fluorene (FL),
methylfluoranthene (MF), pentachlorophenol (PCP), methyldibenzothiophene
(MDT), methyl- phenanthrene (MP), fluoranthene (FA), and pyrene (PY).
(Figure 69). Of these compounds, napthalene, dibenzofuran,
methydibenzothiophene, pentachlorophenol, and fluorene are not mutagenic
in Salmonella (Anderson et_ al., 1971; McCann et al., 1975;
Pelroy et al., 1983), while fluoranthene and methylphenanthrene are
strongly mutagenic (Kaden e£ al., 1979; LaVoie et^ al., 1983).
Only three compounds remained in detectable levels in the runoff sample
collected on day 360. These included pentachlorophenol, fluoranthene,
and pyrene (Figure 70). In the runoff water, the impact of reducing the
concentration of organic compounds on the mutagenic potential of the
mixture is difficult to predict because mixtures of polycyclic aromatic
hydrocarbons have been shown to cause synergistic, additive, and
182
-------
500
>
i
300
00
o>
100-
NORWOOD SOIL
PENT-S RUNOFF -t-S-9
o CONTROL
x DAY 0
DAY 180
a DAY 360
DAY 540
0.2
0.4 0.6
DOSE/PLATE (mg)
0.6
LO
Figure 65. Mutagenic activity with metabolic activation
of runoff water from PENT S amended Norwood
soil.
183
-------
TABLE 51. MUTAGENIC ACTIVITY, AS MEASURED WITH S. TYPHIMURIUM STRAIN TA98 OF RUNOFF WATER
FROM PENT S AMENDED NORWOOD SOIL
Day
BKG
0
180
360
540
S9
20 +
+ 29 +
21 +
+ 30 +
26 +
+ 36 +
25 +
+ 42 +
31 +
+ 39 +
0
1.9
8.0
8.3
4.3
4.6
7.9
3.3
5.4
4.3
3.7
Dose/Plate (rag)
1 .5
NT*
NT
Toxic
65 + 17.1
Toxic
20 +_ 35.2
47 + 11.5
153 + 47.3
166 + 92.6
514 +404.2
17 +
33 +
1.7
3.9
Toxic
87 +_ 10.1
22 +
98 +
68 +
175 +
193 +
373 +
20.0
12.1
13.3
65.5
116.1
232.0
21 +
24 +
18 +
76 +
31 +
60 +
34 +
99 +
82 +
108 +
,1
2.6
8.1
1.3
10.6
2.0
15.0
6.1
38.4
49.0
35.6
11 +
31 +
17 +
58 +
32 +
65 +
36 +
77 +
55 +
72 +
05
11.8
5.2
2.1
7.6
3.5
16.7
7.6
22.6
22.8
15.3
i
16
45
26
46
27
50
27
50
,01
NT
NT
+ 3.6
+ 17.0
+ 5.3
+ 13.9
+ 1.5
+ 2.9
+ 6.4
+ 2.3
* NT = not tested.
-------
TABLE 52. MUTAGENIC ACTIVITY, AS MEASURED WITH j>. TYPHIMURIUM STRAIN TA100 OF RUNOFF
oo
Day
BKG
0
180
360
540
S9
104
+ 81
87
+ 101
119
+ 111
167
+ 147
121
+ 120
0
+ 16.7
+_ 35.4
+ 34.4
+ 10.2
+ 8.8
+ 16.4
+ 17.0
+ 7.6
+ 9.5
+ 7.0
127
27
55
25
283
92
306
WATER
1
NT*
NT
Toxic
_+ 27.5
+ 47.9
+ 95.8
+ 42.7
+ 106.5
+ 25.5
+ 82.1
FROM PENT S AMENDED NORWOOD SOIL
Dose/Plate (mg)
.5 .1
51
82
261
63
310
126
501
124
486
+ 60.0
+ 24.7
Toxic
+ 48.0
+ 54.9
+_ 47.7
+ 35.2
+ 202.6
+ 49.2
+ 115.0
56 + 65.3
76 + 17.4
82 + 8.7
288 + 29.5
106 + 18.3
198 + 60.7
158 + 6.0
378 +238.2
112 + 2.1
273 + 79.2
52 +
73 +
81 +
208 +
108 +
239 _+
155 +
313 _+
106 +
198 +
05
58.8
25.7
10.0
27.5
16.6
49.7
10.8
177.1
13.7
28.6
f
79
97
115
143
149
179
100
130
.01
NT
NT
+ 7.2
+ 28.1
+ 10.4
+ 21.1
+ 14.1
+ 20 . 6
+ 9.5
+ 5.7
* NT = not tested.
-------
500-,
300-
fc
5
NORWOOD SOIL
PENT-S RUNOFF -S-9
o CONTROL
XDAY 0
A DAY 180
O DAY 360
DAY 540
oo
O)
100-
0.2
2X-BKG
0.4 0.6 0.8
DOSE/PLATE (mg)
Figure 66. Mutagenic activity without metabolic activation of
runoff water from PENT S amended Norwood soil.
186
-------
500
300
CD
100-
BASTROP SOIL
PENT-5 RUNOFF +S-9
o CONTROL
x DAY 0
A DAY 180
ODAY 360
DAY 540
x 2X-BKG
02
0.4 0.6
DOSE/PLATE (mg)
0.8
1.0
Figure 67. Mutagenic activity with metabolic activation of
runoff water from PENT S amended Bastrop soil.
187
-------
TABLE 53. MUTAGENIC ACTIVITY, AS MEASURED WITH S. TYPHIMURIUM STRAIN TA98 OF RUNOFF
co
oo
WATER FROM PENT S AMENDED BASTROP SOIL
Day
BKG
0
180
360
540
S9
20
+ 29
23
+ 28
24
+ 32
34
+ 47
27
+ 39
+
+
+
+
+
+
+
+
+
+
0
1.9
8.0
2.3
4.7
4.1
6.1
7.8
6.2
4.1
3.7
24
31
68
30
103
50
122
71
151
1
+ 2.6
j± 9-3
Toxic
+ 27.1
+ 16.7
+ 21.7
+ 7.1
+ 5.9
+ 33.9
+ 18.4
Dose/Plate (tng)
.5
23
27
15
92
46
112
42
114
52
102
+ 4.7
+ 28.7
+ 2.2
+ 8.4
+ 30.3
+ 4.2
+ 7.5
+ 4.9
+ 18.4
+ 2.8
20
27
18
69
27
72
35
70
31
68
.1
+ 4.8
+ 3.5
+ 5.4
+ 19.4
+ 8.0
± 2-1
+ 3.0
+ 6.1
+ 5.7
+ 7.8
22 +
30 +
20 +
57 +
22 +
49 +
31 +
62 +
26 +
59 _+
05
5.0
8.2
1.0
15.6
5.9
8.3
3.2
5.0
6.4
5.7
22
34
22
42
31
55
26
53
.01
NT*
NT
+ 6.2
+_ 5.8
+ 2.6
+ 3.5
+ 3.5
± 3-5
+ 1.4
± 3-5
* NT = not tested.
-------
TABLE 54. MUTAGENIC ACTIVITY, AS MEASURED WITH S. TYPH1HURIUH STRAIN TA100 OF RUNOFF MATER FROM PENT S
00
v£>
AMENDED BASTROP SOIL
Day
BKG
0
180
360
540
* NT -
S9
104
* 88
88
+ 104
107
+ 99
126
+ 130
136
t 142
not tested.
0
« 17.1
+ 19.5
* 10.6
+ 9.0
* 18.5
* 16.7
+ 10.7
+ 13.1
+ 12.6
+ 15.4
1
NT*
83 i 17.3
Toxic
253 + 20.6
Toxic
86 + 81.0
75 + 6.8
323 + 13.2
99 + 31.8
384 + 74.2
Dose/Plate (tag)
.5
56 *
79 +
63.0
41.5
Toxic
206 i 27.1
120 +
304 *
121 «
394 *,
141 +
318 +
16.5
60.12
8.3
51.0
24.0
62.9
50
86
84
239
96
268
122
209
141
209
.1
+ 56.9
+ 34.9
+ 9.5
i 32.5
+ 13.6
+ 11.8
+ 8.4
+ 6.1
+ 11.3
+ 22.6
51 +
86 +
73 +
202 +
105 +
183 j*
112 4
166 +
132 +
163 +
.05
57.9
19.9
12.6
27.0
21.6
25.5
9.5
14.6
16.3
2.8
77
112
101
103
113
139
127
144
.01
NT
NT
* 3.9
+ 20.7
+ 11.3
*_ 8.6
+ 9.2
± 7-5
+ 4.9
+ 30.4
-------
BASTROP CONTROL DAY 360
vQ
O
RIC
200
800
Figure 68. GC/MS chromatograph of organic extract of runoff water from
unamended Bastrop soil collected on day 360.
-------
RIG
BASTROP PS DAY 0
100
200
300
600
700
800
400 500
RT
Figure 69. GC/MS chromatograph of organic extract of runoff water from PENT S
amended Bastrop soil collected on day 0.
-------
PCP
BASTROP PS DAY 360
RIG '
VD
300
40O
500
RT
600
700
Figure 70.
GC/MS chromatograph of organic extract of runoff water from PENT S
amended Bastrop soil collected on day 360.
-------
antagonistic responses (Hass et^ al^. , 1981; Haugen and Peak, 1983). A
comparison of the runoff samples collected on day 540 indicates that
greater amounts of mutagenic activity were detected in the runoff water
from the Norwood soil than in the runoff water from the Bastrop soil
(Tables 51 and 53). The results from chemical and biological analysis of
runoff water from the PENT S amended soils indicate that the mutagenic
activity of the compounds residual on day 540 was greater than the level
of mutagenicity present in the more complex samples collected prior to
day 540.
The results from the biological analysis of the runoff water from
SWRI waste amended soils are provided in Figures 71 and 72, and Tables
55 through 58. For strain TA98, the mutagenic activity of the residual
hydrocarbons in the runoff water from the Norwood soil increased from
day 0 through day 360 (Table 55); whereas, for strain TA100 the
mutagenicity increased from day 0 to 180 and decreased slightly on day
360 (Table 56).
The mutagenic potential of the runoff water from the Bastrop soils
amended with the SWRI waste showed a similar trend. The net TA98 his
revertants with metabolic activation at a dose level of 500 ug/plate on
day 0, 180 and 360 was 86^, 88, and 206, respectively. Without
activation, the net TA98 his revertants at the same dose level were
21, 25, and 196 on days 0+ 180, and 360, respectively (Table 57). In
strain TA100, the net his revertants at 500 ug/plate with metabolic
activation increased from day 0 to 180 and decreased from day 180 to 360
(Table 58). The runoff water collected on day 0, 180, and 360 from both
soils yielded consistently similar dose-response curves when the total
mutation frequencies in strain TA98 with metabolic activation are
compared (Figures 71 and 72). In both soils, the mutagenic potential of
the residual hydrocarbons in the runoff water increased slightly from
day 0 to 180 and almost doubled from day 180 to 360. In addition,
direct-acting mutagens were detected in the samples collected on day
360, whereas only promutagens were detected on days 0 and 180.
The mutagenic potential of the runoff water from the combined API
separator/slop-oil emulsion solids (COMBO) waste amended soil did not
follow a consistent trend in either soil. In the Norwood soil, the
mutagenic activity of the residual hydrocarbons decreased from day 0 to
180, and then increased from day 180 to 360 (Figure 73). The values for
the net TA98 his revertants with metabolic activation at a dose level
of 500 ug/plate were 95, 40, and 144 on day 0, 180, and 360,
repectively (Table 59). In strain TA100, none of the runoff samples from
the COMBO waste amended Norwood soil induced a positive response (Table
60). The runoff water collected from the COMBO waste amended Bastrop
soil on day 0 induced a positive response only at the highest dose level
in strain TA98 with metabolic activation (Table 61 and Figure 74). Using
the modified two-fold rule (Chu et al. , 1981), this sample would be
considered non-mutagenic. However, the mutagenic activity induced by the
residual hydrocarbons in the runoff water from the COMBO waste amended
193
-------
TABLE 55. MUTAGENIC ACTIVITY, AS MEASURED WITH J3. TYPHIMURIUM STRAIN TA98 OF RUNOFF WATER
FROM NORWOOD SOILS AMENDED WITH SWRI WASTE
Day S9
0
180
360
0
23
28
30
50
24
39
+
+
+
+
+
2.8
7.4
4.1
4.6
1.6
4.2
37
87
65
64
197
507
Dose/Plate (mg)
1 .5
+ 8.5
+ 9.2
+ 1.0
4- 12.3
+ 106.0
+ 235.1
35 +
102 +_
52 +
148 _+_
123 +
271 +
4.0
39.4
1.0
15.0
48.0
152.8
30 +
82 +_
38 +
71 +
53 +
90 +
,1
8.3
10.2
3.2
14.5
12.0
48.5
28
67
32
57
37
57
.05
-i- 5
± 6
+ 0
± 4
+ 7
+ 14
.01
.3
.8
.6
.0
.0
.3
29 +
38 +
30 +
53 +
26 +
43 ^
2.2
15.0
1.7
8.0
2.1
h 3.0
-------
TABLE 56. MUTAGENIC ACTIVITY, AS MEASURED WITH J5. TYPHIMURIUM STRAIN TA100 OF RUNOFF
VO
WATER FROM NORWOOD
Day
0
180
360
S9
114 +
+ 97 +
128. +
+ 158 +
126 +
+ 123 +
0
9.2
11.9
15.0
11.0
14.3
8.1
1
Toxic
Toxic
167 + 12.2
342 + 21.8
256 + 120.0
328 + 119.4
SOILS
AMENDED WITH SWRI WASTE
Dose/Plate (mg)
.5 .1
122
203
159
301
201
220
+ 32.8
+ 31.4
+ 17.6
+ 10.5
+ 62.9
+ 66.4
101 +
189 +
145 +
207 +
158 +
144 +
8.5
26.8
19.1
36.7
17.9
10.6
91
156
157
172
145
128
.05
+ 17.4
+ 10.0
+ 3.5
+ 9.2
+ 12.7
+ 16.4
.01
112
109
149
162
134
125
+
+
+
+
+
+
26.0
4.2
2.5
6.0
5.1
17.2
-------
TABLE 57. MUTAGENIC ACTIVITY, AS MEASURED WITH S. TYPHIMURIUM STRAIN TA98 OF RUNOFF
vo
Day
0
180
360
S9
23 +
+ 25 +
31 +
+ 45 +
32 +
+ 43 +
WATER FROM BASTROP
SOILS AMENDED WITH SWRI WASTE
Dose/Plate (mg)
0 1 .5 .
5 . 2 NT*
10.2 NT
5.6 65 + 1.7
3.5 64 +_ 12.3
4.6 306 + 184.5
3.5 475 + 218.6
44 +
111 +
56 +
133 +
228 +
249 _+
4.4
28.7
9.8
25.1
118.1
131.1
22
60
40
62
75
76
+
+
+
+
+
+
1
6.3
12.9
1.2
1.2
37.2
15.0
.05
22 +
46 +
37 +
57 +
56 +
51 +
4.6
3.5
5.6
1.5
23.2
5.7
.01
24 +
37 +
30 +
47 +
33 +
46 _+
6.2
14.2
2.6
8.5
8.9
0.6
* NT = not tested.
-------
TABLE 58. MUTAGENIC ACTIVITY, AS MEASURED WITH S. TYPHIMURIUM STRAIN TA100 OF RUNOFF
WATER FROM BASTROP SOILS AMENDED WITH SWRI WASTE
Day
0
180
360
S9
114
+ 97
128
+ 158
126
+ 140
0 1
+_ 9.3 Toxic
_+ 12.0 Toxic
+ 15.0 169 + 12.3
_+ 11.0 304 + 7.6
+ 14.0 180*
+_ 16.0 254
Dose/Plate (mg)
.5
104 + 10.7
90 + 103.3
163 + 4.6
276 + 17.6
172
216
.1
102 + 10.5
55 + 62.0
141 + 11.4
196 + 41.6
143
166
.05
118 + 15.5
41 + 46.1
147 + 9.0
163 +14.2
137
174
116
46
147
168
126
157
.01
+ 12.4
+ 52.1
+ 3.2
+ 6.1
* Mean represents average of only two values; standard deviation not provided.
-------
600
400'
CD
CD
200
NORWOOD SWRI RUNOFF
O CONTROL
X DAY 0
DAY 180
2X-BKG
0.2
1.0
0.4 0.6 0.8
DOSE/PLATE Cmg)
Figure 71. Mutagenic activity with metabolic activation of
runoff water from SWRI amended soil.
198
-------
600
400
«
t-
4
00
0)
200'
BASTROP SWRI RUNOFF
CONTROL
DAY 0
DAY 180
DAY 360
_ -. 2X-6KG
I .0
DOSE/PLATE Cmg)
Figure 72. Mutagenic activity with metabolic activation of
runoff water from SWRI amended Bastrop soil.
199
-------
600i
400-
cd
+*
«.
>
to
O>
200-
NORWOOD COMBO RUNOFF
- 2X-BKG
0.2
0.4
0.6
0.8
1.0
DOSE/PLATE Cmg)
Figure 73. Mutagenic activity with metabolic activation of
runoff water from COMBO amended Norwood soil.
200
-------
to
o
TABLE 59. MUTAGENIC ACTIVITY, AS MEASURED WITH JJ. TYPHIMURIUM STRAIN TA98 OF RUNOFF
WATER FROM NORWOOD
SOILS AMENDED WITH COMBO WASTE
Dose/Plate (mg)
Day
0
180
360
S9
22
+ 41
35
+ 44
25
+ 42
0
+ 5.4
+ 5.8
+ 2.8
i 2.3
+ 5.4
± l-9
79
54
124
440
430
1
NT*
+ 95.
+ 11.
+ 30.
« 50.
+ 4 .
7
1
3
9
9
61
136
43
84
272
186
<
+
+
+
+
+
,5
22.9
16.6
7.5
22.2
43.1
44.5
30 +
65 +
33 +
62 +
80 +
63 +
1
11.1
45.5
4.6
11.4
16.3
1.4
33 +
61 +
30 +
50 _+
64 +
62 _+
05
16.9
11.9
2.5
1.7
11.3
9.2
34
43
30
47
33
61
.01
+ 13.2
+ 2.9
+ 1.5
+ 2.9
+ 4.9
+ 8.5
* NT = not tested.
-------
TABLE 60. MUTAGENIC ACTIVITY, AS MEASURED WITH S. TYPHIMURIUM STRAIN TA 100 OF RUNOFF
WATER FROM NORWOOD SOILS AMENDED WITH COMBO WASTE
Dose/Plate (rag)
Day
0
180
360
to
o
KJ
S9
96
+ 114
114
+ 112
145
+ 136
0
+ 6.0
+ 12.7
+ 18.0
_+ 11.6
+ 12.8
_+ 11.1
1
NT*
144 + 35.4
96 + 35.6
212 + 15.4
151 + 62.9
176 + 68.6
119
122
92
155
112
148
.5
+ 19.1
+ 24.7
+ 30.6
+ 36.8
+ 53.7
+ 56.6
114 +
136 +
94 +
123 +
108 +
109 _+
.1
17.5
25.0
35.9
12.6
50.2
63.6
102 +
111 +
87 +
117 +
100 +
108 +
05
16.6
22.2
31.8
4.9
38.2
50.2
109
83
88
108
100
96
.01
+ 9.3
+ 21.5
+ 34.9
+ 6.1
+ 38.2
+ 48.1
*NT = not tested.
-------
TABLE 61. MUTAGENIC ACTIVITY, AS MEASURED WITH S. TYPHIMURIUM STRAIN TA98 OF RUNOFF
NJ
o
Day
0
180
360
S9
27
+ 45
28
+ 46
25
+ 42
WATER
0
+ 7.2
+ 9.0
+ 4.9
± 4-2
+ 4.9
+ 1.7
FROM BASTROP SOILS AMENDED
39
86
112
175
85
160
1
+ 8.2
+ 19.3
+ 52.0
± 9-7
+ 14.6
+ 37.2
Dose/Plate
.5
39
60
81
123
56
101
+ 1.
+_ 10.
+ 28.
± 14-
+ 8.
+ 16.
WITH COMBO WASTE
(mg)
7
9
7
6
5
1
23 +
50 +
43 +
58 +
39 +
60 +
1
9.0
1.9
8.7
4.6
2.1
5.0
35
37
38
54
29
58
.05
+ 16.9
+ 6.7
+ 2.3
+ 3.6
+ 1.2
+ 4.6
30
38
31
40
22
51
.01
+ 10.8
+ 5.9
+ 2.0
+ 2.5
+ 2.5
+ 1.7
-------
600-
400'
>
>
co
0)
200-
BASTROP COMBO RUNOFF
CONTROL
DAY 0
DAY I 80
DAY 360
0.2 0.4 0.6 0.8
DOSE/PLATE Gng)
- 2X-BKG
1.0
Figure 74. Mutagenic activity with metabolic activation of
runoff water from COMBO amended Bastrop soil.
204
-------
Bastrop soil in strain TA98 with metabolic activation increased from 15
to 77 to 59 net revertants on day 0, 180, and 360, respectively (Table
61). As with the Norwood soil, in strain TA100, none of the runoff
samples from the COMBO waste amended Bastrop soil induced a positive
response (Table 62). Thus, the mutagenic potential of the residual
hydrocarbons in the runoff water from both soils was greater on day 360
than on day 0. In addition, direct-acting mutagens were detected on day
360 but not on day 0.
Degradation, infiltration, and removal will influence the quality
and quantity of hazardous organic compounds in runoff water from a land
treatment facility. In order to evaluate the influence of these factors
on the mutagenic potential of runoff water, the results from the waste
amended and control soils were compared on the basis of equivalent
volumes. Because of the limited amount of sample available, the extracts
of runoff water samples were tested in the Aspergillus assay at
volumes approximately one-fourth that tested in the Salmonella assay.
While it is unfortunate that equal volumes were not tested in the two
bioassays, some general conclusions can be drawn from the results in the
different bioassays. A comparison of the results from testing the
equivalent of 10 ml of runoff water from the PENT S amended Norwood soil
in the Salmonella assay indicates that the mutagenic potential
increases consistently from day 0 to 180 to 360 and from day 360 to 540
(Figure 75). Contrasting results were observed in the Aspergillus
assay (Figure 76). In the PENT S amended Norwood soil, the mutagenic
activity of the runoff water collected on day 360 decreased to a level
approximately 16% of that detected on day 0 (Figure 76). The surviving
fraction in Aspergillus increased significantly from day 0 to 360. If
this effect also occurred in the Salmonella assay,'the induced mutants
per survivor in Salmonella would be greater on day 0 and approximately
the same on day 360; thus, the results from the two bioassays would then
be comparable. The observed difference in the results from the two
bioassays is probably a reflection of the increased metabolic and DNA
repair capabilities of the eukaryotic system (Dunkel, 1981).
In the Bastrop soil, the mutagenic response in both bioassays
decreased from day 0 to 180 and from day 180 to 360 (Figures 76 and 77).
In both bioassays, the response induced by the sample collected on day
360 was at a level that was only slightly greater than twice background.
However, on day 540 there was an appreciable increase in both the amount
of extractable hydrocarbon and the mutagenic potential of the runoff
water from the PENT S amended Bastrop soil in the Salmonella assay.
The results obtained from the runoff water from either soil amended with
the PENT S waste as measured with the Salmonella and Aspergillus
assays indicate that significant levels of mutagenic activity are
detectable 360 days after waste application. However, the overall
results indicate that the mutagenic potential of the runoff water
collected on day 360 is less than on day 0.
The runoff water from soils amended with the SWRI and COMBO waste
205
-------
TABLE 62. MUTAGENIC ACTIVITY, AS MEASURED WITH S. TYPHIMURIUM STRAIN TA100 OF RUNOFF
WATER FROM BASTROP SOILS
Day
0
180
360
S9
96
+ 114
109
+ 105
150
+ 139
0
+ 6.
± 12-
+ 13.
+ 16.
+ 10.
0
7
9
5
3
9.0
1
100 + 19.4
173 +_ 10.8
NT*
NT
150 + 17.7
209 +11.1
Dose/Plate
.5
102
172
143
172
+ 16.
± 3-
NT
NT
+ 9.
± 5.
(mg)
5
6
5
5
AMENDED WITH
.1
95 + 13.6
140 _+ 20.7
NT
NT
145 + 4.5
145 + 9.1
COMBO WASTE
.05
105 + 12.9
131 + 14.9
NT
NT
138 + 7.5
148 + 4.2
.01
95 + 18.
99 + 10.
NT
NT
135 + 4.
130 + 8.
7
8
0
0
*NT = not tested.
-------
K)
O
W
o
01
IT
O 60
o
tc
a
Il4O-
wX,
ffl5
§ 2
S
X
60
~ 40
CD
0) 2O
z a
a-
+ 69
89
C TOX
c TOX
r^-PTTI
i
NORWOOD RENTS
RUNOFF WATER
C-UNAMENDED
P-WASTE AMENDED
180 360
TIME (D«y»)
540
Figure 75. Extractable hydrocarbons (mg/1) and mutagenic activity
(revertants/10 ml) in runoff water from PENT S amended
Norwood soil.
-------
O
CO
40i
CO
u.
2
20-
0
40-1
+ S9 ^
-S9 D
CONTROL
I Oral
40i
CO
20
NW
20-
OlNOT TESTED
N
L_ N
NW BA
J&A
awRi
I Orel
-I
I
20
-180-
TIME (Otyi)
-360
Q
bJ
ss,
^
i
BA
RENTS
2.50ml
NW BA
BA
COMBO
I Oml
BA
BA
I
180-
o
UJ
H »-
o OT i BA
z uj
i- I
-360
TIME (Day*)
Figure 76.. Total mutation frequency per 10 survivors ±n A. nidulans Induced by the
extractable hydrocarbons In runoff water from waste amended soils.
-------
N)
o
W
z
o
m
cr
<
o
o
oc
0>
O
~ 40-
oo
o
x
UJ
o
CO
0>
<
eo
20
C TOX
fZ^
BASTROP PENT-S
RUNOFF WATER
C-UNAMENDED
P-WASTE AMENDED
I
ISO
360
TIME (Dayt)
+ S9
-S9
i
540
Figure 77. Extr actable hydrocarbons (mg/1) and mutagenic activity
(revertants/10 ml) in runoff water from control and
PENT S amended Bastrop soils.
-------
contained lower levels of hydrocarbons than did the water from the PENT
S waste (Table 63). Therefore, the quality of runoff water from soils
amended with the SWRI or COMBO waste were compared on the basis of a 50
ml volume. The amount of extractable hydrocarbons in runoff water from
SWRI waste amended Norwood and Bastrop soils was less than that in
runoff water from unamended soils on day 0. On day 180, the level of
extractable hydrocarbons in the runoff water increased approximately
ten-fold and then decreased by approximately 50% on day 360. The
mutagenic potential of 50 ml of runoff water from SWRI waste amended
Norwood and Bastrop soils was non-mutagenic on day 0, increased to 3 to
4 times background on day 180, and increased still further on day 360
(Figures 78 and 79). In addition, while greater amounts of mutagenic
activity were detected with metabolic activation, the mutagenic activity
in the absence of metabolic activation also increased significantly from
day 0 to 180 and from day 180 to 360. The results from the Aspergillus
assay followed the same general trend. With metabolic activation, the
mutagenic potential of the sample collected on day 180 was approximately
three times that of the sample from day 0 (Figure 76). In addition, the
mutagenic potential of the sample from day 180 was essentially the same
as that of the same from day 360. Thus, the mutagenic activity of the
runoff water from both the Norwood and Bastrop soils amended with the
SWRI waste followed the same general trend increasing from day 0 to 180
and from day 180 to 360. In addition, slightly greater levels of
mutagenic activity were detected in the runoff water from the Norwood
soil than from the Bastrop soil.
The level of extractable hydrocarbons in the runoff water from the
Norwood soil amended with the COMBO waste decreased by approximately 25%
from day 0 to 180 and by approximately 33% from day 180 to 360 (Table
63). The level of mutagenic activity in 50 ml of runoff water decreased
by approximately 50% from day 0 to 180 but increased appreciably from
day 180 to 360 (Figure 80). The runoff sample collected on day 180 from
the COMBO waste amended Norwood soil induced 10 net revertants/50 ml
without and 50 net revertants/50 ml with metabolic activation; while,
the sample collected on day 360 induced 220 net revertant colonies/50 ml
without and 130 net revertants/50 ml with metabolic activation.
Similarly, the response obtained in the Aspergillus assay with
activation decreased by approximately 40% from day 0 to 180, while the
sample from day 360 was not tested (Figure 76). In the COMBO waste
amended Bastrop soil, none of the runoff samples induced a significant
increase in revertant colonies in the absence of metabolic activation.
In the presence of metabolic activation, the mutagenic activity in
Salmonella of the sample collected on day 0 was less than the control,
while the mutagenic activity of the sample collected on day 180 was
slightly greater than two times background; the sample collected on day
360 induced a response that was greater than 2.5 times background
(Figure 81). However, in the Aspergillus assay with activation, the
mutagenic potential of the runoff water from the Bastrop soil was
approximately equal on days 0 and 180 and significantly lower on day 360
(Figure 76). Thus, the results from the Salmonella assay indicate that
210
-------
TABLE 63. TOTAL HYDROCARBONS EXTRACTED FROM RUNOFF WATER USING COMBINED XAD2 RESINS
ISJ
Extractable Hydrocarbons (mg/1)
Sample
Wood-Preserving Waste
Norwood soil
Bastrop soil
Storm-Water Runoff Impoundment
Norwood soil
Bastrop soil
Combined API-Separator/
Slop-Oil Emulsion Solid
Norwood soil
Bastrop soil
0
60
78
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Days After Appplication
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*No sample collected.
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Figure 78.
Extractable hydrocarbons (mg/1) and rautagenic activity
(revertants /50ml) in runoff water from control and SWRI
amended Norwood soils.
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Figure 79. Extractable hydrocarbons (mg/1) and mutagenic activity
(revertants/50ml) in runoff water from control and SWRI
amended Bastrop soils.
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(revertants/50ml) in runoff water from control and COMBO
amended Bastrop soils.
-------
for COMBO waste amended soils, significantly greater quantities of
mutagenic activity were detected in the runoff water from the Norwood
soil than in the runoff water from the Bastrop soil; and in both soils,
the maximum amount of mutagenic activity was observed in the sample
collected 360 days after waste application.
The results from this segment of the research indicate that the
mutagenic potential of runoff water from hazardous waste amended soils
should eventually return to background levels. The amount of time
required for the mutagenic potential of runoff water to return to
background levels will be regulated by the quantity and degradation rate
of mutagenic compounds in the waste as well as the waste loading rate.
These results have also demonstrated that there will be dramatic
differences in the degradation rates between wastes and between soils.
As a general rule, the mutagenic potential of the runoff water from day
0 was greatest from the PENT S amended soils and least from the COMBO
waste amended soils; while the mutagenic potential of the sample from
the SWRI waste amended soil was greatest on day 180 and 360. In
addition, the mutagenic potential of runoff water from waste amended
Norwood soil was greater than that of the runoff water from the Bastrop
soil. Thus, since different soils will have substantially different
capacities to retain and degrade organic compounds, bioassays provide
the most effective analytical tool for evaluating when runoff water may
be safely released following closure of a hazardous waste land treatment
facility.
216
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SECTION 9
AFFECT OF DEGRADATION ON THE MUTAGENIC ACTIVITY
OF HAZARDOUS WASTE AMENDED SOIL
INTRODUCTION
The application of hazardous waste to soil is restricted by
regulations to include only those wastes which will be rendered less or
non-hazardous by chemical or biological reactions in the soil (EPA,
1982). These regulations assume that degradation, immobilization, and
transformation will serve to reduce the hazardous characteristics of
soil applied waste constituents. Degradation in soil is generally
assumed to be biological, and biological degradation normally accounts
for approximately 80% of soil degradation (Hamaker, 1971). However, in
a soil contaminated by the toxic and resistant compounds frequently
encountered in a hazardous waste, chemical and photochemical degradation
will also play an important role. Soil degradation may not result in
the complete mineralization of a hazardous waste, but soil degradation
can certainly render waste constituents less or non-hazardous by
removing substituted groups, by breaking an aromatic ring, or by
substitutions that produce a less reactive product. Soil degradation
may not, however, always result in detoxification of a hazardous waste.
Thus, techniques are needed to evaluate the affect of degradation on the
mutagenic activity of hazardous waste amended soil.
The technique which is currently being used to monitor land
treatment employs a chemical analysis to define the products of the
various reactions that may occur in the soil. Chemical analysis alone
may fail to account for the interactions between soil and waste
components and the affect of degradation on these interactions. In
addition, the results from a chemical analysis must be extrapolated to
estimate the toxicological endpoint in a biological system. The use of
bioassay directed chemical analysis as an alternative monitoring
technique provides more accurate information from which to obtain a risk
assessment. This technique employs a battery of short-term bioassays to
define the acute and chronic toxic potential of a sample with a chemical
analysis to enhance the result from the biological systems.
217
-------
MATERIALS AND METHODS
Waste
Three petroleum based sludges were used in the greenhouse study. A
general description of sludge characteristics is provided in Table 50,
while a more detailed description is included in Section 5. The
wood-preserving bottom sediment (PENT S), storm-water runoff impoundment
(SWRI), and combined API-separator/slop-oil emulsion solids (COMBO)
wastes were applied to the soil at hydrocarbon loading rates of 3.1,
4.5, and 4.5%, respectively. Application rates were calculated using
the formula discussed in Section 8.
Soil
The physical and chemical properties of the soils used in the
greenhouse study are provided in Table 42, while a more detailed
discussion of the Bastrop and Norwood soils is included in Section 6.
Extraction Procedures
Soil and waste samples in the greenhouse study were extracted with
dichloromethane. Dichloromethane was selected from a group of solvents
to extract the organic fractions of the soil as it consistently provided
the greatest extraction efficiency for the types of compounds present in
the waste (McGill and Rowell, 1980; Donnelly and Brown, 1981).
Twenty-five grams of the waste or waste-soil mixture were blended with
six volumes of dichloromethane in a Waring Laboratory blender for 30
sec. This extraction was repeated twice or until the extracting solvent
remained colorless. Solvent extractions were then combined and taken to
dryness on a Brinkman-Bucci rotary evaporator. The residue from this
extraction was partitioned into acid, base, and neutral fractions
following the scheme outlined in Figure 12.
Chemical Analysis
A chemical analysis was conducted on selected samples by the
USEPA's RS Kerr Environmental Research Laboratory. The procedures used
were the same as those discussed in Section 8.
Soil Sample Collection
A composite sample representing approximately 500 g of soil was
collected from each box 24 to 48 hours following rainfall events, i.e.,
before, immediately after, and 0, 45, 90, 180, 360, and 540 days after
waste application. Soil samples were composited from 6 to 10 randomly
selected plugs, each representing the surface to 17 cm soil. Each sample
was stored at 0 C in a labeled ziplock bag prior to extraction. In
addition, reserve samples from each box and each collection date will be
stored for five years following completion of the project.
218
-------
Biological Analysis
Two different microbial mutagenicity assays were employed to
evaluate the capacity of soil extracts to induce genetic damage. The
Salmonella/mi crosome assay of Ames et al. (1975) was used to
monitor the mutagenic activity of concentrated water samples. The
Salmonella strains were supplied by Dr. Bruce N. Ames (University of
California, Berkeley, CA). The procedural methods were the same as Ames
et al. (1975), except that overnight cultures were prepared by
inoculation into 10 ml of Nutrient Broth #2 (KG Biological, Inc.,
Lenexa, KS) and incubated with shaking for 16 h at 37 C. Water
extracts were tested on duplicate plates in two independent experiments
in the standard plate incorporation assay at a minimum of 4 dose levels
of the sample with and without enzyme activation (0.3 ml rat liver/ml S9
mix) using strains TA98 and TA100. Aroclor 1254 induced rat liver was
obtained from Litton Bionetics (Charleston, SC). Positive controls
included 2 ug/plate N-methyl-N'nitro-N-nitrosoguanidine (Sigma) for
TA100, 25 ug/plate 2-nitrofluorene (Aldrich) for TA98, and 10 ug/plate
2-aminoanthracene (Sigma) which was used with all strains to verify the
functioning of the metabolic activation system. All reagents and
extracts were tested for sterility; DMSO was used as a negative control.
Soil samples were also tested in the eukaryotic bioassay using A.
nidulans. The Aspergillus system was used to assess the mutagenic
potential of soil extracts by evaluating the induction of mutations at
the methionine suppressor loci. Conidia from four to five single
colonies of the methGl biAl (requiring methionine and biotin)
Glasgow strain of Aspergillus nidulans grown for 5 to 6 days on a
complete medium at 37 C were used for each experiment. Samples were
tested at one dose level, usually the extract equivalent to one gram of
soil and one exposure time selected to yield approximately 50% survival
on five plates in two independent experiments with and without metabolic
activation. The procedures were the same as Scott e± al. (1978)
except that the cells were exposed in a 13 x 100 mm screw-capped culture
tube. The exposed cells were plated on a complete medium to determine
survival and on a methionine-free medium to determine the induced
mutation frequency. Mutant colonies were scored after incubation for
five days at 37 C. Colonies were divided by colony morphology into
three classes, A, B, C, as well as the total mutation frequency
represented by the sum of induced mutants from Class A, B, and C. Each
of these three classes is believed to involve two genes. The morphology
of Class A colonies is green, Class B brown, and Class C green with a
white hyaline edge. For the acid, base and neutral fractions of the soil
extract, only the total induced mutation frequency was calculated. The
frequency of mutations induced by each sample was determined by
subtracting the frequency of spontaneous mutations from the total
mutation frequency. A sample was considered positive if the
induced-mutation frequency was more than twice the spontaneous mutation
frequency. Positive controls included 8-methoxypsoralen,
8-methoxypsoralen plus near UV light without activation, and
219
-------
benzo(a)pyrene with metabolic activation.
RESULTS AND DISCUSSION
The soils collected in the greenhouse study were analyzed using
both chemical and biological techniques. Residual hydrocarbon analysis
was used as a gross measurement of degradation, while GC/MS analysis was
used in an attempt to identify the resistant and/or mutagenic waste
constituents. Biological analysis was utilized to define the affect of
degradation on the mutagenic activity of residual hydrocarbons in the
soil and to determine if the wastes were rendered less or non-hazardous
by soil incorporation.
The amount of residual hydrocarbons in the soil at various time
intervals following waste application provides a preliminary indication
of the progress of waste degradation. The total and extractable
hydrocarbons in the acid, base, and neutral fractions were determined
for each waste/soil mixture on days 0, 90, 180, and 360, and for the
PENT S waste/soil mixtures on day 540. The results in Table 64 indicate
that the overall degradation rate of the PENT S waste after 540 days in
the Norwood and Bastrop soils were 83 and 66%, respectively. The most
rapid rate of degradation occurred in the base fraction. In the Norwood
soil, only 5% of the base fraction was residual after 540 days, while
10% of the base fraction was residual in the Bastrop soil. The neutral
fraction, which represented approximately 90% of the extractable
hydrocarbons in the waste, degraded rapidly from day 0 to 180 but slowed
appreciably from day 180 to 540. The degradation rate for the neutral
fraction over the first 180 days was 66% in the Norwood soil and 51% in
the Bastrop soil; whereas, the rate from 180 to 540 was only 50% in the
Norwood soil and 25% in the Bastrop soil. These results indicate that
significant quantities of the wood-preserving bottom sediment were
degraded in both soils. In addition, while less than 50% of the applied
hydrocarbons were residual 180 days after waste application, those
compounds residual after day 180 had a much slower rate of degradation
than the components of the initial waste/soil mixture.
For soils amended with the storm-water runoff impoundment, the most
rapid rate of degradation was observed in the Bastrop clay (Table 65).
The overall degradation rate of the SWRI waste was 53% in the Norwood
soil and 61% in the Bastrop soil. In the Norwood soil, the most rapid
rate of degradation after 360 days was 60% in the acid and base
fractions. In the Bastrop soil, the most rapid rate of degradation
after 360 days was 62%, which occurred in the neutral fraction. While
the degradation rates for the first 180 days were quite low in both
soils, less than 50% of the applied hydrocarbon remained in the soil
after day 360.
The combined API separator/slop-oil emulsion solids exhibited the
most rapid rate of degradation of the three wastes studied. The overall
degradation rate in the Norwood soil was 74% after 360 days, and 83% in
220
-------
TABLE 64. TOTAL HYDROCARBONS EXTRACTED FROM SOIL AMENDED WITH WOOD-PRESERVING BOTTOM SEDIMENT
to
Extractable Hydrocarbons (mg/g)
Sample
Norwood Soil
Acid
Base
Neutral
Total
Bastrop Soil
Acid
Base
Neutral
Total
0
7.0
3.8
122.2
135.0
11.0
4.0
124.0
139.0
Days After Appplication
90 180
4.8 + 2.1
2.7 + 1.0
42.1 + 3.8
49.7 + 6.4
6.6 + 2.6 5.3 + 2.5
5.7 + 0.2 3.1 * 2.3
82.9 + 43.4 60.6 + 8.7
95.3 + 45.8 69.0 + 4.4
Z Degraded
360
1.5 + 0.5
0.8 + 0.15
34.3 + 8.4
36.7 + 8.9
4.7 + 1.1
1.1 + 0.3
55.2 * 5.7
61.0 + 6.6
540
1.6 + 0.6
0.3 + 0.2
20.9 + 3.0
23.4 + 4.5
3.1 + 0.8
0.4 + 0.1
44.3 * 9.0
47.8 + 9.0
Day 360
79
86
72
73
57
72
55
56
Day 540
77
95
83
83
72
90
64
66
-------
TABLE 65. TOTAL HYDROCARBONS EXTRACTED FROM SOIL AMENDED WITH STORM-WATER RUNOFF
IMPOUNDMENT
NS '
t-0
to
Extractable Hydrocarbons (mg/g)
Sample
Norwood Soil
Acid
Base
Neutral
Total
Bastrop Soil
Acid
Base
Neutral
Total
0
1
0.5
39.5
41.0
0.5
0.4
52.1
53.0
Days After Appplication
90 180
1.5 +
1.0 +
32.1 +
34.7 +
0.7 +
0.3 +
33.3 +
34.3 +
0.9
1.0
15.5
13.7
0.3
0.05
7.9
8.1
0.5 +
0.4 +
31. 4 +
32.3^
0.7 +
0.4 +
37.6 +
38.7 +
0.3
0.15
6.9
6.8
0.1
0.2
11.0
10.7
%
360
0.4
0.2
18.6
19.2
0.4
0.2
19.9
20.5
+ 0.2
+ 0.1
+ 3.5
± 3-7
+ 0.2
+ 0.03
+ 7.3
+ 7.0
Degraded
Day 360
60
60
53
53
20
50
62
61
-------
the Bastrop soil (Table 66). The degradation rates of the fractions of
the COMBO waste were 78, 75, and 74% for the acid, base, and neutral
fractions in the Norwood soil. In the Bastrop soil, the degradation
rate for the acid, base, and neutral fractions were 94, 0, and 82%,
respectively. Thus, for the COMBO waste, the most rapid rate of
degradation occurred in the acid fraction and the least rapid rate in
the base fraction. In addition, the average total degradation rate for
the COMBO waste in both soils was 49% from day 0 to 180 and 60% from day
180 to 360.
A comparison of the average degradation rates of the three wastes
indicates that at day 360, the COMBO waste was 79% degraded, while the
PENT S and SWRI wastes were degraded 65 and 57%, respectively. The
average total rates of degradation for day 0 to 180 and day 180 to 360
were 57 and 18% for the PENT S waste, 24 and 44% for the SWRI waste, and
49 and 60% for the COMBO waste, respectively. For the COMBO and SWRI
wastes, the most rapid rate of degradation occurred in the Bastrop soil;
whereas, the PENT S waste degraded most rapidly in the Norwood soil
(Figure 82). Previous studies with two API-separator sludges found the
rate of degradation in the Norwood soil to be from 30 to 60% greater
than the rate of degradation in the Bastrop soil (Brown and Donnelly,
1983). The half-life of all three wastes, as measured by residual
hydrocarbon content, was less than 360 days. Thus, these preliminary
results indicate that all three wastes were transformed and degraded by
soil incorporation.
The evaluation of residual hydrocarbon data does not, however,
provide an indication of the affect of degradation on the mutagenic
activi-ty of waste constituents. Only through the use of bioassays can
these parameters be measured. One of the major limitations of using
these bioassays is the absence of a standard procedure for the
statistical evaluation of environmental data. While there have been
several procedures suggested for the analysis of bioassay data
(Commoner, 1976; Munson and Goodhead, 1979; Horn et al. , 1983; Chu
e± al., 1981; Katz, 1979; Bernstein et al., 1982; and Moore and
Felton, 1983), none of these procedures include adjustments for
background levels of mutagenic activity in environmental samples. The
bioassay data from the greenhouse study were evaluated using two
procedures. Comparisons that were made on a weight basis reviewed the
total mutation frequency at five equally spaced dose levels. Data was
accumulated on each sampling date for all three fractions of each waste
from three boxes of each soil. Thus, each value in Tables 67 to 90 and
Figures 83 through 106 represents the mean of 12 plates from at least
two independent experiments. This data is an expression of the mutagenic
potential of the residual hydrocarbons in the soil and does not account
for losses due to degradation. However, an indication of the effect of
degradation on the mutagenic potential of the residual hydrocarbons in
the soil is provided by the data. Comparisons that were made on a
volume basis reviewed the mutagenic activity ratio of a constant volume
of soil and can be used to determine the reduction of the hazardous
223
-------
TABLE 66. TOTAL HYDROCARBONS EXTRACTED FROM SOIL AMENDED WITH COMBINED API SEPARATOR
SLOP-OIL EMULSION SOLID
Ni
N>
Extractable Hydrocarbons (rag/g)
Sample
Norwood Soil
Acid
Base
Neutral
Total
Bastrop Soil
Acid
Base
Neutral
Total
0
1.5
0.4
21.1
23.0
3.6
0.7
29.7
34.0
Days After Appplication
90 180
1.3 +
0.4 +
17.7 +
19.3 +
0.8 +
0.2 +
8.7 +
9.7 +
0.1
0.1
2.3
2.3
0.2
0.1
0.9
0.6
360
0.33
0.1 +
5.5 + 0
6.0 _+ 0
0.2 +
1.0 +
5.3 + 1
5.7 + 1
.09
.4
.45
.05
.02
.2
.3
% Degraded
Day 360
78
75
74
74
94
0
82
83
-------
to
N>
Ln
I OO-i
a
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O
60-
20-
PS
Cl
1
SI
1
I
I
180
PS
i
1
i
SI
I
1
CO
I
360
WASTEt
PS-PENTS
SI-SWRI
S CO-COMBO
SOIL:
NORWOOD f~]
BASTROP
i
i
540-I
TIME (Dtyi)
Figure 82. Degradation rate of total extractable hydrocarbons in Norwood
and Bastrop soils amended with PENT S (PS), SVRI (SI), and
Combo (CO) waste.
-------
waste characteristics by land application. Comparisons made on a volume
basis account for changes in the hydrocarbon content of the soil over
time, as well as variations in the biological test system.
To determine mutagenicity on a volume basis, the mutagenic activity
ratio (Commoner, 1976) is first calculated for the four highest
non-toxic dose levels. The mutagenic activity ratio is calculated using
the equation:
C - C
MAR= n °
CA
where C is the total number of revertant colonies on experimental
plate at dose n (average of four plates); C is the number of
revertant colonies on solvent control plates the same day the test was
run using the same strain and tissue preparation (average of eight
plates); and C. is the historical average of solvent control plates
for the year the test was run. Once the MAR's were calculated for each
set of data, the mutagenic activity ratio for a specific volume of soil
was calculated using the equation:
R =[ V 3[MAR]
v W
T
where V is the volume of soil (gram equivalents); WT is the volume of
soil equivalent to dose of residue = DOSE (mg/plate) - extractable
hydrocarbons (mg/g); and MAR is the mutagenic activity ratio. The
mutagenic activity ratios which are used calculate the Ry were
calculated from the two non-toxic dose levels closest to the selected
volume of soil which fell on. the linear portion of the dose-response
curve. The selected volume of soil was that volume of soil which fell
between two dose levels which were used in the bioassays and was
comparable to the optimum dose for the volume of soil extracted on Day
0. The extracts of the thre'e fractions from each soil-waste mixture
were tested at five equally spaced dose levels ranging from .01 to 1.0
mg/plate. For the acid and base fractions, these dose levels are
equivalent to the extract from approximately .05 to 5.0 g of soil. The
neutral fraction, which accounted for approximately 90% of the residual
hydrocarbons in the soil-waste mixtures, was tested at the same dose
levels on the basis of residual hydrocarbons that were equivalent to the
extract from approximately 0.2 to 20 milligrams of soil. Thus, in order
to calculate R^ using MAR's derived from the linear portion of the
dose-response curve, the volume of soil used for comparing the neutral
fraction will be one tenth the volume used for the acid and base
fractions. By employing these calculations, the mutagenic potential of
equal volumes or masses of soil can be compared. In addition, these
calculations will account for the variability of the bioassay and the
reduction in the amount of extractable hydrocarbon in the soil over
time. The data from the Sa Imone 1 la assay was evaluated on both a
226
-------
weight and volume basis, while the data from the Aspergillus assay was
only evaluated on a volume basis.
The dose-response curves with strains TA98 and TA100 for the acid,
base, and neutral fractions of the PENT S waste amended Norwood soil are
provided in Figures 83 through 88 and Tables 67 through 72. In the acid
fraction, the maximum response in strain TA98 was induced by the sample
collected on day 180 (Figure 83 and Table 67); while, in strain TA100
the maximum response was induced by the sample collected on day 360
(Figure 86 and Table 70). In both strains, the response induced by the
sample collected on day 540 was appreciably lower than the maximum
response induced by samples collected prior to day 540. A similar trend
was observed in the base fraction from PENT S waste amended Norwood
soil. For strain TA98, the maximum response observed in the base
fraction was induced by the sample collected on day 360 (Figure 84 and
Table 68), while in strain TA100 the sample collected on day 540 induced
the maximum response (Figure 87 and Table 71). In addition, significant
levels of direct-acting mutagens were detected on day 540, whereas no
direct-acting mutagens were detected in the waste or soil samples
collected prior to this date. The maximum response induced by the
neutral fraction was observed in the sample collected on day 180 in
strain TA98 (Figure 85 and Table 69) and in the sample collected on day
360 in TA100 (Figure 88 and Table 72). These results indicate that for
the PENT S waste amended Norwood soil, the factors affecting the
mutagenic potential of the residual hydrocarbons initially increased but
eventually decreased the mutagenicity of the acid and neutral fractions.
While in the basic fraction, the mutagenicity was increased over the
entire 540 day period with an apparent increase in direct-acting
mutagenicity also occurring.
In the PENT S waste amended Bastrop soil, the overall rate of
hydrocarbon degradation and the rate of detoxification appeared to be
lower than the rate in the Norwood soil. The maximum mutagenic response
induced by the acid, base, and neutral fractions was observed in the
sample collected on day 540 in strain TA98 (Figures 89, 90, and 91, and
Tables 73, 74, and 75) and on day 360 in strain TA100 (Figures 92, 93,
and 94 and Tables 76, 77, and 78). As occurred in the Norwood soil,
direct-acting mutagens were detected in the base fraction collected on
day 540 in both strains (Figures 90 and 93 and Tables 74 and 77). Thus,
the mutagenic activity of all three fractions from the PENT S waste
amended Bastrop soil appeared to reach a maximum on day 360 as measured
with strain TA100, while the maximum response in strain TA98 was induced
by the sample collected on day 540.
The dose-response curves for the hydrocarbons extracted from the
SWRI waste amended Norwood soil are given in Figures 95, 96, and 97.
The mutagenic activity of the acid fraction increased from day 0 to 180
and from day 180 to 360 (Figure 95 and Table 79). While the mutagenic
potential of the acid fraction of the soil-waste mixture from days 0 and
180 were less than the mutagenic potential of the acid fraction of the
227
-------
600-
400-
co
o>
NORWOOD-RENTS-ACID
WASTE
DAY 0
B B 180
360
T V 540
+ S9
-S9
200-
I 00
300
DOSE/PLATECufl)
500
I 000
Figure 83. Mutagenic activity of acid fraction of PENT S
amended Norwood soil as measured with £. typhimurium
strain TA98 with and without metabolic activation.
228
-------
600
400
200
NORWOODRENTS BASE
00
300
500
000
DOSE /PLATE
-------
600
400
CO
o>
200
NORWOOD PENTS NEUTRAL
I 00
300
500 I 000
DOSE/PLATE Cuj)
Figure 85. Mutagenic activity of neutral fraction of PENT S
amended Norwood soil as measured with S_ typhimurium
strain TA98 with and without metabolic activation.
230
-------
600-
NORWOOD- PENTS'ACID
' DAYO O
H H 180 X
. 36O n
v n i iir R
-------
833
69 I
600
400
o
O
ZOO-
NORWOOD -RENTS- BASE
WASTE
DAY 0
B B 180
360
540
100
300
500 1000
DOSE/PLATE Cug)
Figure 87. Mutagenic activity of base fraction of PENT S amended
Norwood soil as measured with ^ typhimurium strain TA100
with and without metabolic activation.
232
-------
600i
NORWOOD-RENTS-NEUTRAL
WASTE £&
DAY 0 OO
B S | 80 XX
360 D O
540
400-
O
O
200-
100
300
500 I 000
DOSE/PLATECug)
Figure 88. Mutagenic activity of neutral fraction of PENT S
amended Norwood soil as measured with £. typhimurium
strain TA100 with and without metabolic activation.
233
-------
TABLE 67. MUTAGENIC ACTIVITY OF ACID FRACTION OF PENT S WASTE AMENDED NORWOOD SOIL AS MEASURED WITH
S TYPHIHURIUM STRAIN TA98 WITH AND WITHOUT METABOLIC ACTIVATION
Total TA98 his * Revertants (Mean + S.D.)
Day S9 0
j Day 0 - 21 +
+ 22 *
Jj Day 180 - 27 *
*- + 36 i
Day 360 - 28 +
* 37 ±
Day 540 - 28 4
+ 38 +
2.5
4.4
2.6
2.9
0.2
0.2
0.8
3.3
1
Toxic
Toxic
Toxic
Toxic
Toxic
85 + 35.0
21 * 8.4
44 + 19.8
Dose/Plate (rag)
.5
20
126
34
144
37
157
37
110
+
+
+
+
7
+
+
8.4
11.3
18.8
60.6
5.3
18.9
6.4
12.9
21
49
40
120
42
133
32
111
.1
+ 5.0
± 5-9
+ 5.7
+ 25.3
+ 21.8
* 6.9
+ 7.3
+ 27.9
21
42
37
100
41
112
35
72
.05
+ 5.6
* 17.7
+ 6.2
+ 37.2
+ 25.5
+ 10.5
+ 10.7
+ 23.9
17 +
17 +
33 +
59 +
42 +
68 +
23 +
43 +
01
2.6
6.2
4.4
11.3
27.3
18.9
1.1
5.3
-------
N>
CO
U1
TABLE 68. MUTAGENIC ACTIVITY OF BASE FRACTION OF PENT 8 WASTE AMENDED NORWOOD SOIL AS MEASURED WITH
S. TYPHIHURIUM STRAIN TA98 WITH AND WITHOUT METABOLIC ACTIVATION
Total TA98 his Revertants
Day S9
Day 0
+
Day 180 -
Day 360 -
+
Day 540 -
+
21 +
22 +
27 +
36 +_
29 +
39 +
27 +
40 t
0
2.5
4.4
2.6
2.9
0.9
5.0
0.8
3.3
1
24 + 6.3
112 +_ 12.4
60 + 32.9
284 ±137.8
111 + 15.4
540 + 23.7
59 + 11.0
334 ±158.0
(Mean +
Dose/Plate (nig)
.5
21 +
98 +
51 *
293 +
106 «
417 +
74 +
381 +
4.2
31.3
34.8
115.0
16.7
22.4
40.0
133.0
24 +
62 +
38 +
215 ±
62 +
170 T
52 +
274 +
S.D. )
1
3.6
20.4
9.7
53.0
22.8
14.4
28.0
147.0
20 +
37 +
35 +
157 +
55 +
151 ±
34 +
163 T
.05
7.8
7.8
16.5
26.8
34.6
2.8
29.0
19 +
22 +
31 +
72 ±
44 +
75 *_
28 +
68 +
01
3.3
7.0
6.7
25.8
28.5
11.5
.71
13.0
-------
NJ
Co
TABLE 69. MUTAGENIC ACTIVITY OF NEUTRAL FRACTION OF PENT S WASTE AMENDED NORWOOD SOIL AS MEASURED WITH
S. TYPHIMURIUM STRAIN TA98 WITH AND WITHOUT METABOLIC ACTIVATION
Total TA98 hia *
Day S9
Day 0
Day 180 -
Day 360 -
Day 540 -
21 +
21 i
26 +
36 +
28 +
36 +
27 +
39 +
0
3.6
1.8
2.4
2.9
1.0
1.0
0.8
2.9
1
22 +
86 +
204 +
281 7
50 +
184 7
50 +
144 +
Revertanta
(Mean + S.D.)
Dose/Plate (mg)
.5
6.7
7.6
154.5
78.3
5.5
24.1
11.5
44.7
19 *
96 7
126 +
220 +
36 +
160 +
41 *
136 +
5.1
28.2
68.3
40.7
1.4
16.9
5.9
22.6
24
62
38
215
62
144
31
128
.1
+ 3.6
+ 20.4
* 9.7
+ 53.0
+ 22.8
+ 20.5
+ 4.1
+ 7.7
20 +
37 *
35 +
157 *
55 +
119 +
27 +
105 +
.05
7.8
7.5
7.8
16.5
26.8
10.7
1.8
10.3
19 +
22 +
31 +
72 *
44 *
64 T
24 +
57 +
01
3.3
7.0
6.7
25.8
28.5
9.2
1.1
9.8
-------
ro
u>
TABLE 70. MUTAGENIC ACTIVITY OF ACID FRACTION OF PENT S WASTE AMENDED NORWOOD SOIL AS MEASURED WITH
S. TYPHIHURIUM STRAIN TA100 WITH AND WITHOUT METABOLIC ACTIVATION
Total TA100 his * Revertants (Mean + S.D.)
Day S9
Day 0
Day 180 -
f
Day 360 -
Day 540 -
93 +
40 J_
105 +
107 +
156 +
135 j*
115 +
126 +
0
18.9
11.9
5.1
17.8
6.0
2.2
7.5
0.7
1
Toxic
Toxic
Toxic
Toxic
Toxic
Toxic
Toxic
113 + 25.8
Dose/Plate (rag)
.5
Toxic
226 + 17.3
Toxic
307*
Toxic
287 + 157.4
72 + 11.0
235 + 31.4
84
202
94
362
147
557
113
376
.1
* 7.4
+ 16.7
+ 22.4
+116.9
+ 12.3
+ 88.9
+ 5.6
* 28.1
87
162
105
275
147
419
110
288
+
+
+
+
+
+
.05
25.4
35.4
13.7
61.6
8.8
65.8
4.7
36.6
85 +
118 +
101 +
165 i
139 +
193 +
116 +
166 +
01
11.0
29.2
14.9
5.7
8.3
9.6
14.0
17.7
* Mean represents average of only tvo samples; standard deviation not provided.
-------
Co
oo
TABLE 71. MUTAGENIC ACTIVITY OF BASE FRACTION OF PENT S WASTE AHENDED NORWOOD SOIL AS MEASURED WITH
S. TYPHIMURIUH STRAIN TA100 WITH AND WITHOUT METABOLIC ACTIVATION
Total TA100 his
Day S9
Day 0
+
Day 180 -
+
Day 360 -
+
Day 540 -
+
120
134
US
118
138
120
115
122
0
+ 21.9
± 10.6
+ 13.1
1 5-3
+ 7.7
± 1-1
+ 9.6
+ 3.4
298
56
129
154
486
691
528
1
Toxic
+ 22.0
+ 31.0
i 52.7
+ 10.5
* 98.6
* 90.9
* 53.04
Revertants (Mean -f S.D.)
Dose/Plate (mg)
.5
92 +
395 +
83 4
277 ^
148 +
621 *
433 +
884 +
11.3
86.8
15.0
74.1
22.5
108.8
153.4
63.9
.1
80 + 18.6
317 + 37.1
113 + 18.9
456 ±106.0
118 + 29.1
618 + 76.0
248 + 68.8
652 t 105.5
86 +
181 *
131 +
398 +
114 +
545 +
187 *
536 *
.05
8.8
78.8
25.6
80.8
29.4
65.4
53.6
23.0
95 +
119 +
132 +
193 +
111 +
259 +
121 +
259 *
01
15.6
27.03
55.2
51.3
30.7
82.5
6.8
65.4
-------
NJ
LO
VO
TABLE 72. MUTAGEHIC ACTIVITY OF NEUTRAL FRACTION OF PENT S WASTE AMENDED NORWOOD SOIL AS MEASURED WITH
S. TYPHIMURIUM STRAIN TA100 WITH AND WITHOUT METABOLIC ACTIVATION
Total TA100 bia
Day S9
Day 0
Day 180 -
Day 360 -
Day 540 -
0
120 + 21.9
122 ±40.1
111 + 12.7
118 ± 5.3
130 * 14.0
120 ± 1.7
120 + 4.4
117 + 8.1
1
94 +
139 *_
136 +
346 +
132 +
375 *
139 +
280 +
L
13.0
44.7
39.1
152.0
28.9
22.9
9.0
51.2
Bevertanta (Mean
Dose/Plate (mg)
.5
105 +
352 *
125 +
364 +
126 +
455 +
122 +
328 +
22.2
45.6
22.1
124.0
22.0
23.2
11.4
40.8
84 +
244 +
136 +
372 +
125 +
571 +
108 +
434 +
+ S.D.)
1
17.0
136.0
29.5
99.0
23.5
30.7
9.4
31.0
77
296
148
315
112
474
111
355
.05
+ 14.1
+ 41.1
+ 41.2
* 59.2
+ 21.6
+ 13.3
+ 15.7
±14.6
43 +
133 +
139 +
156 +
118 +
211 +
115 +
201 +
.01
47.9
5.7
38.6
9.5
14.8
22.8
3.6
39.1
-------
600i
400-
co
0)
200-
BASTROP RENTS ACID
WASTE
DAYO
S 3 I 80
360
540
500 I 000
DOSE/PLATE Cug)
Figure 89. Mutagenic activity of acid fraction of PENT S amended
Bastrop soil as measured with £ typhimurium strain TA98
with and without metabolic activation.
240
-------
60 O
400
CD
0>
200-
1006
»-
BASTROP. PENTS'BASE
A A WASTE & &
B B 1 80 * *
+S9 -S9
100
300
500 " ' I 000
DOSE/PLATE
Figure 90. Mutagenic activity of base fraction of PENT S amended
Bastrop soil as measured with £ typhimurium strain
TA98 with and without metabolic activation.
241
-------
600
400
0
o>
200-
BASTROP-RENTSNEUTRAL
,4 WASTE
- DAY 0 O
8 - B 180 X
- 360 D
T - T 540 V
K
+S9
-S9
I 00
300
DOSE/PLATECug)
500
I 000
Figure 91. Mutagenic activity of neutral fraction of PENT S
amended Bastrop soil as measured with £. typhimurium
strain TA98 with and without metabolic activation.
242
-------
TABLE 73. HUTAGENIC ACTIVITY OF ACID FRACTION OF PENT S WASTE AMENDED BASTROP SOIL AS MEASURED WITH
S. TYPHIHURIUM STRAIN TA98 WITH AMD WITHOUT METABOLIC ACTIVATION
Total TA98 hi* * Revertanta
ro
P-
U)
Day S9
Day 0
+
Day 180 -
f
Day 360 -
+
Day 540 -
+
21 +
25 +
28 +
35 +
26 +
43 +
31 *
39 +
0
4.9
4.3
2.0
3.7
2.3
3.3
2.7
5.7
1
Toxic
87 + 37.7
Toxic
Toxic
Toxic
Toxic
Toxic
513 + 390.8
(Mean + S.D.)
Doae/Plate (mg)
.5 .1
77
117
128
110
364
184
647
Toxic
+. 10.5
* 45.8
+ 41.2
+ 140.5
» 432.0
+ 123.8
+ 421.5
17 + 5.1
53 + 5.0
S3 + 3.8
138 ± 26.6
53 + 42.0
139 + 57.5
123 + 8.0
304 + 219.3
16 +
43 T
39 +
86 +
37 +
100 +
60 +
107 +
.05
2.4
4.6
2.6
9.0
6.4
23.4
29.3
29.9
16 +
27 _»
27 +
58 +_
32 +
56 +
34 +
59 +
01
1.8
7.1
3.0
2.2
5.1
10.6
8.3
13.4
-------
TABLE 74. MUTAGENIC ACTIVITY OF BASE FRACTION OF PENT S WASTE AMENDED BASTROP SOIL AS MEASURED WITH
S. TYPHIMURIUM STRAIN TA98 WITH AND WITHOUT METABOLIC ACTIVATION
Total TA98 his * Revertanta (Mean + S.D.)
Day S9
Day 0
Day 180 -
Day 360 -
Day 540 -
20 *
25 +
28 +
35 *
24 +
43 +
31 *
39 7
0
1.9
4.3
2.0
3.7
3.2
2.9
2.7
5.7
1
17 +
102 *
42 +
213 *
167 +
699 7
1394 +
1718 7
Dose/Plate (ing)
.5
6.3
24.4
6.13
48.0
93.0
372.7
1242.0
151.5
24 *
109 +.
33 +
208 7
137 *
452 +
1195 «
1600 7
4.7
14.7
3.5
17.9
70.0
169.0
1140.0
410.4
15
69
31
164
60
184
624
1006
.1
+ 7.3
7 25.25
+ 2.4
+ 14.0
+ 17.0
1 *'5
+ 827.6
7 906.0
.05
18 + 2.5
60 7 8.7
26 * 4.9
116 ± 26.0
47 + 11.0
132 > 13.0
409 * 536.0
621 + 671.0
19 t
26 7
23 +
5' 1
30 +
69 +
99 +
119 +
01
4.4
2.4
4.7
10.3
6.1
11.0
101.4
59.3
-------
Ul
TABLE 75. MUTAGENIC ACTIVITY OF NEUTRAL FRACTION OF PENT 8 WASTE AMENDED BASTROP SOIL AS MEASURED WITH
S. TYPHIHURIUM STRAIN TA98 WITH AND WITHOUT METABOLIC ACTIVATION
Total TA98 hia Hevertants
Day S9
Day 0
+
Day 180 -
Day 360 -
Day 540 -
20 +
25 +
27 +
35 i
23 +
44 +
31 +
49 +
0
1.9
4.3
0.7
3.7
1.5
3.6
2.7
5.7
1
23 +
122 +
56 +
189 +
43 +
137 +
52 +
226 +
7.7
16.8
21.7
8.2
6.4
45.0
12.0
113.0
Doae/Plate (mg)
.5
NT*
96 + 14.4
50 + 18.5
183 + 7.0
34+5.3
146 + 17.0
38 + 11.0
161 +47.0
(Mean +
18 +
56 +
34 +
149 +
34 +
132 +
35 +
121 +
S.D.)
1
4.3
13.6
5.0
15.0
4.9
28.0
.71
13.0
17 +
56 +
33 +
118 +
27 +
113 +
30 +
97 +
.05
2.0
20.6
5.9
30.5
4.9
41.0
2.8
10.6
IS +
27.3+
31 +
57 +
25 +
69 +
29 +
66 +
01
3.2
5.6
4.0
5.1
2.5
13.0
1.4
10.3
** Not tested due to limited amount of sample available.
-------
600
400
O
o
200
BASTROP- PENIS-ACID
500 1000
DOSE/PLATE (ug)
Figure 92. Mutagenic activity of acid fraction of PENT S amended
Bastrop soil measured with S_ typhimurium strain TA100
with and without metabolic activation.
246
-------
600i
400-
o
o
200-
BASTROP-PENTSBASE
A WASTE
DAY 0
ra B I 80 x X
00
3DO
500 I 000
DOSE/PLATECuj)
Figure 93. Mutagenic activity of base fraction of PENT S amended
Bastrop soil as measured with S_ typhimurium strain
TA100 with and without metabolic activation.
247
-------
600
400
o
o
200-
BASTROPRENTS'NEUTRAL
WASTE
DAY 0
180
360
540
I 00
300
500 I 000
DOSE/PLATE Cug)
Figure 94. Mutagenic activity of neutral fractionof PENT S amended
Bastrop soil as measured with £. typhimurium strain
TA100 with and without metabolic activation.
248
-------
TABLE 76. MUTAGENIC ACTIVITY OF ACID FRACTION OF PENT S WASTE AMENDED BASTROP SOIL AS MEASURED WITH
S. TYPHIMURIUM STRAIN TA100 WITH AND WITHOUT METABOLIC ACTIVATION
Total TA100 his * Revertants (Mean + S.D.)
ro
.p-
VO
Day S9
Day 0
Day 180 -
Day 360 -
t
Day 540 -
120
134
115
118
127
121
121
122
0
t 22.0
* 11.0
+ 13.0
+ 5.0
+ 9.0
± *>
+ 5.1
+ 0.7
1
Toxic
Toxic
Toxic
Toxic
Toxic
Toxic
Toxic
121 + 18.0
Doae/Plate (mg)
.5
107
61
382
90
258
Toxic
Toxic
Toxic
+ 9.2
+ 21.0
i 121.9
+ 8.0
+ 74.6
.1
82 + 11.0
278 + 64.0
98 + 2.2
358 + 19.0
93 + 21.0
448 + 56.0
119 * 5.8
369 * 14.6
97
154
127
225
97
367
121
305
.05
+ 24.0
+ 35.7
+ 21.0
T 34.0
+ 16.0
+ 46.0
+ 5.2
+ 57.0
88 +
100 +
137 +
137 +
103 +
169 +
125 +
158 +
,01
11.0
21.0
31.0
24.0
29.0
18.0
4.3
17.0
-------
N)
Ul
O
TABLE 77. MUTAGEHIC ACTIVITY OF BASE FRACTION OF PENT S WASTE AMENDED BASTROP SOIL AS MEASURED WITH
S. TYPHIMURIUM STRAIN TA100 WITH AND WITHOUT METABOLIC ACTIVATION
Total TA100 hi*
Day S9
Day 0
+
Day 180 -
4-
Day 360 -
+
Day 540 -
+
0
85 + 12.0
135 + 12.2
106 + 5.1
107 + 17.8
139 + 18.0
123 + 17.0
115 + 7.5
127 i 7.7
1
256 +
73 +
246 +
149 +
419 +
345*
433*
Revertants (Mean
Dose/Plate (mg)
.5
NT**
37.8
7.6
39.3
40.0
146.0
77 +
321 +
73 +
436 +
149 +
588 +
263*
643*
11.6
78.5
7.6
45.6
2.1
252.0
789 +
322 *
103 +
428 +
142 +
635 ±
150*
539*
+ S.D.)
1
19.0
30.7
10.0
138.0
18.0
245.0
66 +
226 +
107 +
450 +
143 +
591 +_
130*
427*
.05
7.1
19.3
7.7
121.0
15.0
260.0
84 *
140 i
115 +
184 +
139 +
272 +
121*
212*
01
4.6
28.6
15.2
10.2
16.0
118.0
* Mean represents average of only two samples; standard deviation not provided.
** Not tested due to limited amount of sample available.
-------
ro
TABLE 78. MUTAGENIC ACTIVITY OF NEUTRAL FRACTION OF PENT S WASTE AMENDED BASTROP SOIL AS MEASURED WITH
S. TYPU1MURIUH STRAIN TA100 WITH AND WITHOUT METABOLIC ACTIVATION
Total TA100 his * Revertanta (Mean
Day S9
Day 0
+
Day 180 -
+
Day 360 -
»
Day 540 -
+
0
93 + 11.4
140 + 12.9
105 + 5.3
107 j+ 17.8
137 + 14.0
124 + 17.0
115 + 0.0
126 + 0.8
92
310
230
140
368
178
488
1
+ 16.3
+ 30.0
Toxic
+ 95.3
+ 19.0
+ 121.0
+ 75.0
+ 325.0
Dose/Plate (rag)
.5
333
384
141
485
155
426
NT*
+ 39.1
Toxic
* 19.6
+ 15.0
+ 139.0
+ 49.6
+ 128.7
67 +
299 *
100 +
456 +
137 +
620 +
128 +
408 ±
+ S.D.)
1
7.8
24.7
12.6
102.0
16.0
191.0
27.6
73.9
168
93
352
132
572
119
380
.05
NT
* 46.4
+ 18.5
* 79.5
+ 15.0
+152.0
+ 16.9
+ 62.0
99 +
142 +
100 +
158 +
137 +
278 +
114 +
222 t
.01
13.1 .
21.2
8.4
22.1
18.0
93.0
4.8
23.2
* Not tested due to limited amount of sample available.
-------
600-
400-
NORWOOD-SWR!-ACID
CO
en
200-
WASTE
DAY 0 00
aa I e o xx
360 oa
too
300
500 I 000
DOSE /PLATE
-------
S3
Ul
CO
TABLE 79. MUTAGENIC ACTIVITY OF ACID FRACTION OF SWRI WASTE AMENDED NORWOOD SOIL AS MEASURED WITH
S. TYPHIMURIUM STRAIN TA98 WITH AND WITHOUT METABOLIC ACTIVATION
Total TA98 hia Revertante
Day S9
Day 0
f
Day 180 -
Day 360 -
24
30
25
44
27
34
0
* 4.5
*. 5.0
+ 3.0
+ 8.6
+ 2.9
+ 6.5
39
138
55
139
127
650
1
+ 19.4
+ 36.9
+ 2.6
+ 8.4
+ 123.0
+ 678.8
Dose/Plate (mg)
.5
40 + 7.5
102 + 9.0
37 + 1.6
137 * 4.4
92 + 71.2
369 + 336.2
(Mean * S.D.)
32
77
27
86
43
113
.1
+ 7.2
+ 13.8
+ 3.1
± '*
* 30.6
+ 29.3
26 +
44 +
21 +
69 +
34 +
76 +
.05
7.5
22.4
0.8
13.3
17.5
26.5
22
34
24
46
27
58
+
+
f
+
i
,01
7.3
10.6
0.8
7.4
9.6
0.2
-------
waste, the hydrocarbons in the acid fraction collected on day 360
induced a mutagenic response at higher dose levels that was two to three
times greater than the response induced by the waste fraction. Similar
results were observed in the base fraction with the maximum response
induced by the sample collected on day 360. The base fraction from day
360 also induced a mutagenic response in the absence- of metabolic
activation, whereas neither the waste nor the base fraction of the
samples collected prior to day 360 induced a mutagenic response without
metabolic activation (Table 80 and Figure 96). In the neutral fraction
of the SWRI waste amended Norwood soil, the maximum response observed in
the study samples was induced by the hydrocarbons extracted from the
waste (Figure 97). In addition, the mutagenic activity of the neutral
fraction from day 360 was appreciably lower than both the waste and the
day 0 sample (Table 81 and Figure 97). Since the neutral fraction
accounts for greater than 90% of the total extractable hydrocarbon in
the SWRI waste, the reduction in mutagenic activity observed in the
neutral fraction after 360 days should compensate for increases observed
in the acid and base fractions. Thus, the mutagenic potential of the
total extractable hydrocarbons should be reduced 360 days following
application of the SWRI waste to the Norwood soil.
For the Bastrop soil amended with the SWRI waste, the same general
trend over time was observed (Tables 80, 81, and 82 and Figures 98, 99,
and 100). The maximum response observed in both the acid and base
fractions was induced by the sample collected on day 360 (Tables 82 and
83 and Figures 98 and 99). In the neutral fraction, the mutagenic
activity of the residual hydrocarbons was virtually unchanged from day 0
through 360. At the highest dose level, the mutagenic activity of the
residual hydrocarbons in the neutral fraction of the SWRI waste amended
Bastrop soil from day 0, 180, and 360 was approximately one-half the
level of activity induced by the hydrocarbons present in the neutral
fraction of the waste (Table 84 and Figure 100). These results indicate
that the mutagenic activity of total residual hydrocarbons in both the
Norwood and the Bastrop soils collected 360 days after waste application
were significantly less than the mutagenic activity of the hydrocarbons
present in the waste.
The results from the biological analysis of the COMBO waste amended
Norwood soil are presented in Tables 85, 86, and 87 and Figures 101,
102, and 103. The sample collected on day 360 induced the maximum
response observed in the acid fraction from COMBO waste amended Norwood
soil (Figure 101 and Table 85). In addition, direct-acting mutagens
were detected in the acid fraction from day 360, whereas there were no
direct-acting mutagens detected in the acid fraction of samples
collected prior to day 360. In the base fraction, there was no
appreciable difference in the mutagenic potential of the extract from
the waste or the soil samples collected on day 0 and 180; however, the
mutagenic potential of the base fraction from day 360 was approximately
two times greater than the response induced by samples from previous
dates. Without metabolic activation, the base fraction from day 360
254
-------
TABLE 80. MUTAGENIC ACTIVITY OF BASE FRACTION OF SURI WASTE AMENDED NORWOOD SOIL AS MEASURED WITH
S. TYPHIHURIUH STRAIN TA98 WITH AND WITHOUT METABOLIC ACTIVATION
Total TA98 bis * Revertanta (Mean + S.D.)
(S3
Ln
Ui
Day S9
Day 0
+
Day 180 -
*
Day 360 -
+
24
30
25
45
28
33
0
+ 4.5
+ 5.0
+ 3.3
1 7-l
+ 2.6
+ 7.7
1
50 +
100 +
59 +
206 +
171 +
901 +
12.8
33.6
19.4
23.9
69.7
409.4
Dose/Plate (mg)
.5
52 + 5.9
103 + 15.5
"
46 + 16.5
149 + 6.6
138 + 49.9
400 + 159.9
31 +
65 +
43 +
78 +.
54 +
138 +
,1
5.7
6.3
22.4
9.4
17.6
30.8
24 +
61 T
"
43 +
66 *
39 +
80 +
.05
7.8
8.8
35.5
4.9
14.9
4.1
.01
19 + 2.4
44 + 10.2
~~"
36 + 21.2
46 + 4.5
25 + 5.4
46 + 8.6
-------
90
600
400
a
O)
ZOO-
NORWOOD-SWRI BASE
I 00
300
500
000
DOSE/PLATE Cug)
Figure 96. Mutagenic activity of base fraction of SWRI amended
Norwood soil as measured with £. typhimurium strain
TA98 with and without metabolic activation.
256
-------
600i
400-
NORWOOD-SWRI -NEUTRAL
+S9
-S9
CD
ff>
200-
I 00
300
500 I 000
DOSE /PLATE Cuj)
Figure 97. Mutagenic activity of neutral fraction of SWRI amended
Norwood soil as measured with S_. typhimurium strain
TA98 with and without metabolic activation.
257
-------
TABLE 81. MUTAGENIC ACTIVITY OF NEUTRAL FRACTION OF SWRI WASTE AMENDED NORWOOD SOIL AS MEASURED WITH
S. TYPHIHURIUM STRAIN TA98 WITH AND WITHOUT METABOLIC ACTIVATION
Ln
oo
Total TA98 his Revertanta
Day S9
Day 0
Day 180 -
Day 360 -
23
31
25
44
26
29
0
± 2-*
+ 5.5
+ 2.9
+ 1.2
+ 1.3
+ 2.0
58
125
44
127
39
92
1
+ 12.8
+ 19.0
+ 5.7
+ 24.9
+ 8.4
+ 19.4
Dose/Plate (mg)
.5
42 + 8.5
138 + 7.8
30 + 0.8
122 + 16.6
31 + 5.7
81 + 4.9
(Mean + S.D.)
25 +
90 +
27 +
95 +
24 +
66 +
1
6.5
9.8
3.2
11.2
3.1
12.5
30 +
79 +
23 +
71 +
24 +
52 +
.05
9.3
13.3
1.8
11.9
0.8
11.4
27
50
27
53
21
38
.01
+ 2.2
+ 18.7
+ 2.3
+ 2.6
+ 1.9
+ 7.8
-------
TABLE 82. MUTAGENIC ACTIVITY OF ACID FRACTION OF SHRI WASTE AMENDED BASTROP SOIL AS MEASURED WITH
S. TYPHIMURIUM STRAIN TA98 WITH AND WITHOUT METABOLIC ACTIVATION
1
Total TA98 hii * Revert ant*
Day S9
Day 0
l-o +
Ui
Day ISO -
t
Day 160 -
t
22
32
29
43
31
40
0
+ 3.4
+ 5.8
+ 8.3
* 4.7
+ 1.8
1
156 +
263 *
52 +
137 »
86 *
341 *
33.4
23.7
4.3
30.1
8.5
151.0
Doae/PUte (ing)
.5
129 + 26.7
188 ± 10.8
45 + 9.2
139 7 19.6
69 « 8.3
198 + 65.0
(Mean + S.D.)
39
88
32
90
40
94
.1
+ 7.0
^ 20.3
4 6.8
+ 14.2
4 12.0
+ 9.0
31
63
32
76
37
42
.05
+ 7.3
± 10.2
+ 2.3
± 6.9
* 4.2
+ 7.1
26
56
29
61
30
52
.01
4 4.8
+ 11.9
+ 2.7
t 8.0
+ 4.9
+ 5.7
-------
TABLE 83. HUTAGENIC ACTIVITY OF BASE FRACTION OF SWRI WASTE AMENDED BASTROP SOIL AS MEASURED WITH
S. TYPHIMURIUM STRAIN TA98 WITH AND WITHOUT METABOLIC ACTIVATION
NJ
C^
O
Total TA98 hia * Revertants (Mean ± S.D.)
Doae/Plate (mg)
Day S9
Day 0
+
Day 180 -
*
Day 360 -
f
0
22 + 3.4
32 + 5.8
29 + 8.3
43 * 3.0
31 +4.7
40 + 1.8
134
60
243
150
494
1
NT*
+ 30.3
+ 32.9
+ 131.9
+ 101.3
+ 291.0
70 +
120 +
50 +
192 +
84 +
319 +
,5
13.5
40.9
25.8
68.7
11.8
158.8
28 +
71 +
27 +
93 j»
38 +
89 +
1
6.4
15.5
4.1
14.1
10.3
16.0
26 +
44 +;
21 +
69 _+
28 +
73 +
.05
7.5
22.4
0.8
13.3
1.75
6.4
22 +
39 *
27 +
55 +
26 +
45 +
01
5.3
4.2
3.9
5.0
4.2
6.5
* Not tested due to limited amount of sample available.
-------
TABLE 84. MUTACENIC ACTIVITY OF NEUTRAL FRACTION OF SWRI WASTE AMENDED BASTROP SOIL AS MEASURED WITH
S. TYPHIHURIUH STRAIN TA98 WITH AND WITHOUT METABOLIC ACTIVATION
Total TA98 his Revertants (Mean + S.D.)
to
Day S9
Day 0
+
Day 180 -
+
Day 360 -
+
0
23 + 2.8
28+9.2
29 + 8.3
43 + 3.1
31 +4.4
41 + 1.8
1
35 +
117 jf
56 +
142 +_
34 +
117 +
5.6
26.4
35.7
7.8
10.2
8.7
Doae/Plate (mg)
.5
36 +
107 +.
45 +
130 +
27 +
115 +
4.0
7.0
19.0
15.6
2.6
8.7
23 +
63 +
32 +
87 +
24 +
86 +
1
4.9
9.9
4.9
7.5
2.1
10.3
28 +
41 +
28 +
69 +
23 +
73 +
.05
3.3
9.5
4.1
8.0
4.2
0.9
.01
22 +
30 +
24 +
55 +
27 +
52 +
4.9
8.1
7.1
1.6
5.1
4.0
-------
600
» 400
a
O
200-
BASTROP-SWRI-ACID
00
300
500
I 000
DOSE/PLATECug)
Figure 98. Mutagenic activity of acid fraction of SWRI amended
Bastrop soil as measured with _S_. typhimurium strain
TA98 with and without metabolic activation.
262
-------
600-
BASTROP«SWRI'BASE
400-
0
0)
200;
00
300
500
I 000
DOSE /PLATE Cug)
Figure 99. Mutagenic activity of base fraction of SWRI amended
Bastrop soil as measured with £. typhimurium strain
TA98 with and without metabolic activation.
263
-------
600-
400-
200-
BASTROP'SWRI-NEUTRAL
I 00
300
500
I 000
DOSE/PLATECuj)
Figure 100. Mutagenic activity of neutral fraction of SWRI amended
Bastrop soil measured with S^. typhimurium strain TA98
with and without metabolic activation.
264
-------
TABLE 85. HUTAGENIC ACTIVITY OF ACID FRACTION OF COMBO WASTE AMENDED NORWOOD SOIL AS MEASURED WITH
S. TYPHIMURIUM STRAIN TA98 WITH AND WITHOUT METABOLIC ACTIVATION
Total TA98 hie * Revertants (Mean + S.D.)
S3
ON
Ui
Day S9
Day 0
+
Day 180 -
f
Day 360 -
+
0
33 + 4.2
39 + 6.8
35 +0.7
45 I 2.1
24 +0.7
40 + 5.8
64
151
49
133
105
272
1
+ 5.6
+ 52.7
+ 11.2
+ 20.4
+ 39.3
+144.5
Dose/Plate (rag)
.5
57 +
114 +
42 +
114 +
78 +
148 +
11.0
15.0
5.4
15.4
34.7
49.4
32 +
83 +
30 +
66 +
42 +
66 +
1
6.9
5.9
4.7
4.2
18.0
17.4
30 +
39 _+
25 +
59 +
35 +
51 +
.05
3.6
13.7
15.2
3.5
11.3
3.6
25 +
23 +
27 +
56 +
25 +
44 +
01
2.0
8.5
1.5
7.2
2.2
5.6
-------
TABLE 86. HUTAGENIC ACTIVITY OF BASE FRACTION OF COMBO WASTE AMENDED NORWOOD SOIL AS MEASURED WITH
S. TYFHIHURIUM STRAIN TA98 WITH AND WITHOUT METABOLIC ACTIVATION
Total TA98 his * Revertanta (Mean *_ S.D.)
Dose/Plate (mg)
Day S9
Day 0
*
Day 180 -
f
Day 360 -
f
0
33 + 4.2
39 + 6.8
35 + 0.7
45 * 2.1
24 + 0.7
37 + 2.3
37
206
69
205
747
1
+ 14.2
+ 8.5
+ 17.9
+ 48.4
NT*
+ 180.0
34 +
140 +
55 +
158 ^
227 +
341 _*
,5
5.3
33.2
17.0
38.0
49.0
13.0
31 +
87 +
34 +
76 +
91 +
69 +
1
10.3
13.9
2.2
. 2.0
4.2
18.0
26 +
58 *
30 +
67 _*
63 +
59 +
.05
7.2
13.1
1.7
5.3
4.2
1.4
30
352
25
55
31
44
.01
+ 5.1
+ 12.9
+ 1.2
+ 7.0
+ 7.8
+ 4.9
* Not tested due to United amount of sample available.
-------
N>
TABLE 87. HUTAGENIC ACTIVITY OF NEUTRAL FRACTION OF COMBO WASTE AMENDED NORWOOD SOIL AS MEASURED WITH
S. TYPHIHURIUM STRAIN TA98 WITH AND WITHOUT METABOLIC ACTIVATION
Total TA98 hi» * Revertanti
Day S9
Day 0
Day 180 -
Day 360 -
33
39
35
45
24
36
0
4 4.2
_4 6.8
4 0.7
4 0.9
4 0.7
4 2.3
I
29 4
97 4
37 4
118 4
50 *
148 4
6.2
8.7
0.9
13.5
28.5
56.8
Dose/Plate (nig)
.5
26 4 4.7
85 4 10.8
34 4 2.1
108 4 1.6
40 4 15.8
101 4 27.0
(Mean 4 S.D.)
25 4
56 4
26 4
69 4
23 4
58 4
1
3.2
23.5
2.1
4.1
7.5
9.4
22
45
28
61
26
48
.05
4 6.1
4 9.2
4 3.5
4 3.9
4 4.8
4 6.4
23
39
26
49
23
40
.01
4 4.1
4 8.5
4 0.3
4 2.8
4 1.0
4 4.1
-------
600-
400-
CD
o>
200-
NORWOOD COMBO ACID
WASTE
DAY 0 Oo
H B I 80 X X
360
-S9
too
300
DOSE /PLATECug)
500
I OOO
Figure 101. Mutagenic activity of acid fraction of COMBO amended
Norwood soil as measured with S_. typhimurium strain
TA98 with and without metabolic activation.
268
-------
I 133
600'
400
NORWOOD COMBO 'BASE
* A WASTE
DAY 0
O D ISO
360
+ S9
A *
0 0
X X
0 D
-S9
CO
o>
200-
I 00
300
500 I 000
DOSE/PLATECug)
Figure 102. Mutagenic activity of base fraction of COMBO amended
Norwood soil as measured -with £[. typhimuri'im strain
TA98 with and without metabolic activation.
269
-------
600
400
0
at
ZOO-
NORWOOD -COMBO-NEUTRAL
100
300
500 1000
DOSE/PLATECug)
Figure 103. Mutagenic activity of neutral fraction of COMBO amended
Norwood soil as measured with J3. typhimurium strain
TA98 with and without metabolic activation.
270
-------
induced a response that was approximately nine times background, whereas
none of the samples from earlier dates induced a response that was
greater than twice background without activation. The neutral fraction
of the COMBO waste amended Norwood soil induced a consistently higher
response at the 1.0 mg/plate dose level from day 0 to 180 to 360.
However, this increase was relatively small, and on day 360 the
mutagenic potential of the extractable hydrocarbons in the neutral
fractions of the COMBO waste amended Norwood soil was approximately
one-half that of the neutral fraction of the waste. After 360 days of
incubation in the Norwood soil, the acid and neutral fraction displayed
a small but significant increase in mutagenic activity, while the base
fraction displayed a dramatic increase both with and without metabolic
activation.
The overall rate of hydrocarbon degradation in the COMBO waste
amended Bastrop soil was much greater on day 180 and slightly greater
than the rate of degradation in the Norwood soil on day 360 (Figure 82).
The different rates of degradation observed in the two soils appears to
have been reflected in the results from mutagenicity testing of the
extracts of the waste amended Bastrop soil. In the acid fraction, the
mutagenic potential of the samples collected on day 0 and day 180 were
.less than or equal to the mutagenic potential of the acid fraction of
the waste, while the mutagenic potential of the acid fraction from the
sample collected on day 360 was dramatically increased both with and
without metabolic activation (Table 88 and Figure 104). In the base
fraction, the mutagenic potential of the samples collected on days 180
and 360 induced at least twice the mutagenic response that was obtained
from the base fraction of the waste or the day 0 sample both with and
without metabolic activation (Table 89 and Figure 105). The mutagenic
potential of the base fraction from day 180 without activation was equal
to that of the waste sample with activation. The increase in mutagenic
activity that was observed in the base fraction from day 180 may have
resulted from the increased rate of degradation that occurred in the
Bastrop soil. However, in the neutral fraction, there was no
appreciable difference in the mutagenic potential of the waste fraction
and the soil extract from day 360 (Table 90 and Figure 106). Before
soil application, the neutral fraction of the COMBO waste induced a
positive response in the absence of metabolic activation. In the waste
amended Bastrop soil, this effect appears to have been inhibited until
day 360 when direct acting mutagens were again detected (Table 90).
Penalva e£ al. (1983) also observed that a mixture of aromatic
hydrocarbons could inhibit direct acting mutagens. Therefore, as the
neutral fraction accounts for 90% of the total extractable hydrocarbons
in the soil, the mutagenic activity of the total residual hydrocarbons
in the COMBO waste amended soils appears to be slightly increased after
360 days of soil incubation.
The alterations that were observed over time in the mutagenic
potential of the three fractions of the soil-waste extracts follows
strikingly similar trends. In most cases, the hydrocarbons present in
271
-------
TABLE 88. HUTAGENIC ACTIVITY OF ACID FRACTION OF COMBO WASTE AMENDED BASTROP SOIL AS MEASURED WITH
S. TYPH1HURIUH STRAIN TA98 WITH AND WITHOUT METABOLIC ACTIVATION
to
Total TA98 his * Revertanta (Mean «
Day S9
Day 0
*
Day 180 -
f
Day 360 -
33
39
32
44
30
41
0
+ 4.2
+ 6.8
+ 3.6
+ 1.4
+ 3.5
+ 7.5
1
55 _*
94 +
39 +
133 +
375 +
1293 +
Dose/Plate (ng)
.5
21.1
19.3
9.0
28.3
30.0
179.0
41 +
102 +
35 +
112 +
283 +
455 +
8.5
37.8
4.2
14.0
16.0
33.0
24 +
69 *
28 +
64 +
110 +
133 +
_ S.D.)
1
9.0
8.9
4.2
5.1
7.8
141.0
26
45
25
52
73
65
.05
+ 9.0
+ 6.5
+ 3.6
+ 4.9
» 49.5
+ 9.4
20 +
30 ^
26 +
53 +
43 +
48 +
01
6.3
17.1
1.2
5.0
7.1
9.2
-------
400'
o
at
200
0
BASTROP- COMBO -ACID
WASTE
- DAY 0
B E) 180
360 O
100
300
DOSE/PLATE(ug)
I 195 *
500
I 000
Figure 104. Mutagenic activity of acid fraction of COMBO amended
Bastrop soil as measured with JL typhimurium strain
TA98 with and without metabolic activation.
273
-------
NJ
TABLE 89. HUTAGENIC ACTIVITY OF BASE FRACTION OF COMBO HASTE AMENDED BASTROP SOIL AS MEASURED WITH
S. TYPHIHURIUM STRAIN TA98 WITH AND WITHOUT METABOLIC ACTIVATION
Total TA98 his Revertants (Mean ± S.D.)
Day S9
Day 0
+
Day 180 -
*
Day 360 -
+
33
39
29
48
30
41
0
+ 4
± 6
+ 6
* 7
+ 3
* 7
.2
.8
.3
.4
.5
.4
1
28 + 21.3
147 + 13.6
221*
614*
530*
734 _* 144.9
Dose/Plate (mg)
.5
39 + 37.1
149 + 16.2
146*
291*
334 + 188.0
376 + 187.0
.1
27 + 4.7
76 + 4.9
57*
102*
97 + 27.0
75 ^47.0
28 +
50 +
45*
70*
69 +
58 +
.05
8.7
12.0
12.4
21.4
27 +
46 +
26*
54*
61 +
41 +
01
5
7
33
7
.8
.0
.7
.9
* Mean represents average of only two samples; standard deviation not provided.
-------
733^
600i
400-
0
O>
200-
BASTROP-COMBO-BASE
WASTE £a
DAY 0 00
HB I 8O Xx
360 DO
1=8
I 00
300
DOSE/PLATECug)
500
I 000
Figure 105. Mutagenic activity of base fraction of COMBO amended
Bastrop soil as measured with S^. typhimurium strain
TA98 with and without metabolic activation.
275
-------
to
TABLE 90. HUTAGENIC ACTIVITY OF NEUTRAL FRACTION OF COMBO WASTE AMENDED BASTROP SOIL AS MEASURED WITH
S. TYPHIHURIUM STRAIN TA98 WITH AND WITHOUT METABOLIC ACTIVATION
Total TA98 hit + Revertants (Mean + S.D.)
Dose/Plate (og)
Day S9
Day 0
+
Day 180 -
f
Day 360 -
+
0
33 + 4.2
39 + 6.8
32 + 3.6
44 + 1.7
30 + 3.5
41 + 7.5
1
29 +
101 +
35 «
148 +
105 +
302 +
1
13.5
11.7
7.9
15.0
105.7
279.0
27 +
105 +
31 +
102 +
64 +
145 +
.5
10.2
18.7
2.9
7.5
40.0
76.0
23 +
60 i
24 +
67 +
37 +
71 +
1
2.6
7.9
1.7
9.5
13.0
19.0
24 +
57 +
27 +
64 +
29 +
53 +
.05
3.9
7.3
3.0
7.4
1.6
5.9
29 +
31 +
28 +
55 +
27 +
45 +
01
5.4
10.9
1.5
1.5
3.6
11.4
-------
600-
- 400-
BASTROP- COMBO-NEUTRAL
H Q
-59
0
09
200-
too
300
500 1000
DOSE/PLATE (ug)
Figure 106. Mutagenic activity of neutral fraction of COMBO amended
. Bastrop soil as measured with j^. typhimurium strain
TA98 with and without metabolic activation.
277
-------
the soil served to inhibit the mutagenic activity of the waste
constituents to the extent that the activity of the soil-waste extracts
from day 0 were less than the activity of the waste extracts alone. The
nature of the mutagenic materials also appreared to be affected by
degradation. The base fraction of all six waste-soil mixtures induced
direct-acting mutagenicity at some point in time following waste
application; whereas, none of the base fractions from the waste or day 0
extracts induced direct-acting mutagenicity. For the majority of the
acid and base fractions, the maximum level of mutagenic activity was
observed in the final sample collected. However, the mutagenic
potential of the neutral fraction did not exhibit a similar increase
over time and in some cases was less on day 360 than on day 0.
These results indicate that soil degradation can have a significant
impact on the mutagenic potential of hazardous waste constituents.
Alexander (1981) states that biodegradation may increase the toxicity of
some hydrocarbons. The increases in the mutagenic activity of soil-waste
extracts observed in the present study may have been a result of
hydrocarbon oxidation or a result of the reduction in the number of
different residual waste constituents in the soil. The initial
reactions involved in the oxidation of aromatic hydrocarbons are
believed to result in the formation of dioxetanes and epoxides in
microbial and mammalian systems, respectively, with subsequent reactions
producing dihydrodiols in both systems (Gibson, 1972). Although the
epoxide and not the dihydrodiol form is mutagenic (Huberman et^ al.,
1971), it appears from the results of the present study that
biodegradation can increase the mutagenic potential of hydrocarbons in
the soil. In addition, mixed function oxygenases are the enzymes
believed to account for numerous mammalian activation reactions (Miller
and Miller, 1974) and have also been found in plants (Higashi et_
al., 1981) and bacteria (McKenna, 1972; Sokatch, 1969). However, Sims
and Overcash (1981) observed that the products of soil incubated
benzo(a)pyrene (B[a]P) are much less mutagenic than the parent compound;
and in the the present study, the degradation products of dimethyl
phenanthrene appeared to be less mutagenic than the original components
of the neutral fraction of the COMBO waste. Thus, the major impact of
degradation on the mutagenicity of a complex mixture may be its affect
on the composition of the mixture.
The components of a complex mixture may have synergistic,
antagonistic, or additive effects on the mutagenic potential of the
mixture as a whole. Hass et al. (1981) suggest that even a very
slight shift in the concentration of one of the nonmutagenic chemical
species can drastically alter the response of the mutagenic species.
Hermann (1981) observed that several nonmutagenic hydrocarbons enhanced
the mutagenic activity of B(a)P, while most mutagenic polycyclic
aromatic hydrocarbons (PAH) produced a large decrease in the
mutagenicity of benzo(a)pyrene. When Penalva (1983) examined the
interactions of the vapors and aerosols emitted by road coating tar,
they found enhancement at low levels and inhibition at high levels for
278
-------
indirect-acting mutagens. However, only the inhibition effect appeared
for direct acting mutagens (Penalva, 1983). This may explain the
inhibition of direct-acting mutagenicity that was observed in the
initial extracts from the COMBO waste amended soil.
The data of Shahin and Fournier (1978) indicate that the PAH
fraction of Athabasca tar-sand suppressed the mutagenicity of
2-aminoanthracene. In a study by Kaden e_t al. (1979) the mutagenic
activity of a kerosene soot extract was 10 to 20 times higher than could
be accounted for by the amount of B(a)P present. By evaluating the
mutagenic activity of the 70 PAH identified in the soot, Kaden et
al. (1979) were able to account for the mutagencity of the whole PAH
fraction in terms of the additive mutagenic activity of the individual
components. Haugen and Peak (1983) observed that a complex mixture of
aromatic hydrocarbons isolated from a coal-derived oil suppressed the
mutagenic activity of indirect mutagens but had no effect on the
activity of direct-acting mutagens. Haugen and Peak (1983) also
demonstrated that this suppression was produced by inhibition of the
microsomal monooxygenase system. Environmental factors may have also
affected the mutagenic activity of the residual hydrocarbons (McCoy et
al., 1979; Claxton and Barnes, 1982). The information in the
literature indicates that the increased mutagenicity observed in some of
the soil-waste extracts from the present study was probably a result of
degradation reducing the number of compounds in the complex mixtures.
This reduction effectively reduced competition for activating enzymes
with a resultant increase in the mutagenicity of the residual compounds.
While this reduction in compounds appears to be the dominant factor
affecting the mutagenic activity of hazardous waste amended soils, the
increased presence of direct-acting mutagens with time indicates
microbial oxidations may also have played an important role.
The results of the present study and those in the literature
indicate that the composition of a complex mixture will have a
significant influence on the mutagenic potential of the mixture. The
composition of the acid, base, and neutral fractions from the PENT S
waste amended Norwood and Bastrop soils are given in Table 91 and
Figures 107 through 117. Apparently from the variety of constituents
identified in the extracts, the prediction of the genotoxic effects of
these samples from chemical analysis alone would be difficult if not
impossible. However, from the variety of identified initiators,
promoters, mutagens, and carcinogens, chemical analysis evidently
provides valuable information pertinent to the interpretation of the
results from the biological testing. A number of alkanes were
identified in the base and neutral fractions of the soil extracts.
These included the promoting agents dodecane, tetradecane, and
octadecane; the cocarcinogens octadecane and eicosane; and hexadecane
which is an inhibitor (Lankas £t al., 1978; Goldschmidt, 1981). The
majority of the alkanes detected in the PENT S soil extracts were also
present in the extract of the unamended soil.
279
-------
TABLE 91. LIST OF COMPOUNDS DETECTED IN PENT S HASTE AMENDED SOIL
Sample
Acid
Compound (Peak Number)*
Fraction
Base
Compound (Peak Number)*
Neutral
Compound (Peak Number)*
Norwood
Day 0
Not Determined
t-o
oo
o
Norwood
Day 360
Unknown (267)
Dimethyl nonane (303)
Unknown (377)
Methyl propyl pentanol (358)
Unknown (377)
Ethyl hexanol (399)
Pentachlorophenol (419)*HO; Cl
Dimethyl undecane (437)
Trimethyl octane (469)
Benzene dicarboxylic acid (492)*MO
Fluoranthene (S22)*H1; CO; CC
Pyrene (S38)*M1; CO; CC
Unknown (562)
Unknown (591)
Unknown (620)
Unknown (639)
Unknown (657)
Methyl napthalene (249)*M1; CO
Dimethyl napthalene (289)*HO
Dihydro acenaphthylene (314)*M1
Hexene (332)
Dimethyl butane (358)
Unknown (377)
Epoxy-methyl-pentane (397)
Unknown (435)
Unknown (461)
Trifluoro methane (472)*M1
Unknown (496)
Unknown (523)
Unknown (541)
Unknown (570)
Unknown (590)
Unknown (616)
Unknown (646)
Unknown (676)
Unknown (277)
Unknown (303)
Ethyl heptane (318)
Unknown (339)
Dimethyl nonane (359)
Unknown (378)
Dimethyl hexane (399)
Trimethyl heptane (469)
Propyl aziridine (492)
Fluoranthene (523)*H1; CO; CC
Pyrene (539)*M1; CO; CC
Unknown (586)
Unknown (619)
Unknown (650)
Unknown (681)
Unknown (238)
Methyl napthalene (251) (257)*M1; CO
Biphenyl (281)*MO; Cl
Dimethyl napthalene (291) (295)*MO
Dihydro acenaphthylene (321)*M1
Dibenzofuran (334)
Fluorene (359)*MO; CO
Unknown (384)
Unknown (406)
Dibenzothiophene (424)*MO
Phenanthrene (432)*MO; CO
Unknown (462)
Unknown (478)
Unknown (503)
Fluoranthene (531)*M); CO; CC
Pyrene (547)*M1; CO; CC
Unknown (589)
Unknown (615)
Unknown (637)
Unknown (237)
Unknown (271)
Unknown (281)
Alkane (308)
Dihydro acenaphthylene (324)*M1
Unknown (334)
Trimethyl naphthalene (347)*MO
Unknown (375)
Dimethyl octane (384)
Dimethyl biphenyl (407)
Unknown (421)
Methyl propyl pentanol (445)
Unknown (462)
Trimethyl octane (476)
Cyclopenta phenanthrene (481)*MO
Unknown (499)
Dimethyl undecane (508)
Fluoranthene (529)*M1; CO; CC
Pyrene (544)*M1; CO; CC
Unknown (568)
-------
TABLE 91 CONTINUED
Fraction
Sample
Acid
Compound (Peak Number)*
Base
Compound (Peak Number)*
Neutral
Compound (Peak Number)*
Methyl napthalene (240)*M1; CO
Dimethyl napthalene (285) (299)*MO
Dihydro acenaphthylene (317)*M1
Methyl ethyl napthalene (326)
Dibenzofuran (331)
Unknown (343)
Phenalene (352)*M1
Trimethyl napthalene (363)*MO
Unknown (365)
Alkane (405)
Dibenzothiophene (424)*MO
Methyl dibenzothiophene (461)*M1
Methyl phenanthrene (478)*M1
Phenyl napthalene (504)
Fluoranthene (533)
Pyrene (550)
Unknown (627)
Unknown (679)
Unknown (730)
Unknown (775)
Unknown (822)
Unknown (876)
Dimethyl hexane (277)
Unknown (303)
Dihydro acenaphthylene (321)*M1
Unknown (343)
Unknown (361)
Trimethyl octane (397)
Unknown (456)
Cyclopenta phenanthrene (476)*MO
Unknown (502)
to
00
Bastrop Methyl napthalene (239)*M1; CO
Day 0 Unknown (246)
Dimethyl napthalene (282) (292)*MO
Dihydro acenaphthylene O19)*M1
Alkane (326) (367) (407)
Dibenzofuran (332)
Trimethyl napthalene (346)*MO
Phenalene (353)*M1
Pentachlorophenol (429)*MO; Cl
Unknown (481)
Unknown (515)
Fluoranthene (535)*M1; CO: CC
Pyrene (553)*M1; CO: CC
Unknown (635)
Unknown (691)
Unknown (728)
Unknown (770)
Unknown (807)
Unknown (846)
Unknown (884)
Baatrop Unknown (267)
Day 360 Ethyl methyl pentanol (277)
Unknown (303)
Dihydro acenaphthylene (323)*M1
Trimethyl napthalene (345)*HO
Dodecane (358)*P; CC
Dimethyl octane (376)*P
Dimethyl undecane (398)
Pentachlorophenol (422)*C1
Unknown (220)
Methyl naphthalene (244) (250)*M1; CO
Unknown (277)
Dimethyl naphthalene (287) (293)*MO
Dihydro acenaphthylene (319)*M1
Alkane (323)
Dibenzofuran (332)
Trimethyl naphthalene (337) (343)*MO
Phenalene (358)
Alkane (364)
Unknown (378)
Unknown (392)
Methyl fluorene (405)
Dibenzothiophene (422)*MO
Unknown (438)
Methyl dibenzothiophene (467)*MO
Methyl phenanthrene (477)*MO
Cyclopenta phenanthrene (482)*MO
Phenyl napthalene (501)
Unknown (511)
Fluoranthene (531)*M1; CO
Pyrene (548)*M1; CO
Unknown (561)
Unknown 578)
Unknown (276)
TetradeCane (287)*P
Unknown (314)
Dihydro acenaphthylene (33S)*M1
Trimethyl naphthalene (359)*MO
Unknown (385)
Hexadecane (400)*A
Trimethyl octane (417) (447)
Heptadecane (429)
-------
TABLE 91 CONTINUED
Fraction
Sample Acid Base Neutral
Compound (Peak Number)* Compound (Peak Number)* Compound (Peak Number)*
Methyl propyl nonane (432) (467) (498) Dimethyl phenanthrene (514)*H1
Phenyl naphthalene (452) Fluoranthene (52l)*Hl; CO; CC Octadecane (450)*P; CC
Cyclopenta phenanthrene (473)*MO Pyrene (537)*M1; CO; CC Unknown (467)
Nonadecane (479)
Unknown (510) Cyclopentaphenanthrene (487)*MO
Fluoranthene (519)*M1; CO; CC Eicosane (510) *CC
Pyrene (534)*H1; CO; CC Fluorantbene (531)*M1; CO; CC
^j Unknown (558) Heneicosane (541)
00 Pyrene (547)*M1; CO; CC
^ Docoaane (571)
Unknown (601) (630)
* Potential genetic toxicity: I - initiator; P - promoter; A - antagonist; CC = cocarcinogen(aynergiat); HO -
nonmutagenic; HI * mutagenic; CO non-carcinogen; Cl " carcinogen; all others no information. The references
used in determining these factors include HcCann e£ al (1975); Kaden e£ al_ (1979); Goldschmidt (1981) and
DHEW (1969).
-------
1-0
00
LO
RIG
NORWOOD
RENTS BASE
DAY 0
2<
DO
i
300
400
500
600
700
RT
Figure 107. GC/MS chromatograph of base fraction of PENT S amended Norwood
soil collected on day 0.
-------
NORWOOD
RENTS NEUTRAL
DAY 0
00
RIC
200
300
400
500
600
RT
Figure 108. GC/MS chromatograph of neutral fraction of PENT S amended
Norwood soil collected on day 0.
-------
ro
oo
Ln
RIG
NORWOOD
RENTS ACID
DAY 360
200
300
400
500
600
700
RT
Figure 109. GC/MS chromatograph of acid fraction of PENT S amended Norwood
soil collected on day 360.
-------
NORWOOD
RENTS BASE
DAY 360
IsJ
cn
200
600
700
RT
Figure 110. GC/MS chromatograph of base fraction of PENT S amended Norwood
soil collected on day 360.
-------
NORWOOD
RENTS NEUTRAL
DAY 360
(o
oo
RIG
200
i
300
400
RT
500
600
Figure 111. GC/MS chromatograph of neutral fraction of PENT S amended
Norwood soil collected on day 360.
-------
oo
00
RIC
BASTROP
RENTS ACID
DAY 0
r^
-------
N>
OO
VO
RIG
BA3TROP
PENTS BASE
DAY 0
r. I- oo oo
ZOO
400
600
800
RT
Figure 113. GC/MS chromatograph of base fraction of PENT S amended BasCrop
soil collected on day 0.
-------
BASTROP
RENTS NEUTRAL
DAY 0
to
vo
o
RIC
200
300
400
RT
500
600
Figure 114. GC/MS chrotnatograph of neutral fraction of PENT S amended Bastrop
soil collected on day 0.
-------
BASTROP
PENT3 ACID
DAY 360
hO
VO
RIG
200
300
400
RT
500
600
Figure 115. GC/MS chromatograph of acid fraction of PENT S amended Bastrop soil
collected on day 360.
-------
BASTROP
PENTS BASE
DAY 360
tv>
VO
1x3
200
600
700
RT
Figure 116. GC/MS chromatograph of base fraction of PENT S amended Bastrop soil
collected on day 360.
-------
BASTROP
RENTS NEUTRAL
DAY 360
N>
VO
OJ
RIG
250
350
450
RT
550
i
650
Figure 117. GC/MS chromatograph of neutral fraction of PENT S amended Bastrop
soil collected on day 360.
-------
Several polycyclic aromatic hydrocarbons (PAH) were also identified
in the extracts of the PENT S amended soils. These included
methylnapthalene, dimethyl napthalene, trimethyl napthalene, and dihydro
acenapthylene. Of the methylated napthalenes, only methylnapthalene has
been found to be mutagenic (Kaden et_ a_l. , 1979). Although dihydro
acenapthylene was not mutagenic in the standard reverse mutation assay
(Gatehouse, 1980), it was positive when tested in the forward mutation
assay (Kaden e£ al. , 1979). Non-mutagenic PAH with three or more
aromatic rings were identified in the soil-waste extracts and included
anthracene, phenanthrene, and cyclopentaphenanthrene (McCann et al.,
1975; Kaden et^ al., 1979). Of these compounds, only anthracene has
been tested in a whole animal bioassay and was found to be
non-carcinogenic (DHEW, 1969). Identified mutagenic PAH included
methylphenanthrene, dimethyl phenanthrene (La Voie et al., 1983),
and pyrene and fluoranthene (Kaden et al., 1979) which are not
carcinogens (DHEW, 1969). Pyrene and fluoranthene have, however,
displayed cocarcinogenic activity (Hoffman et al. , 1982). A study by
La Voie e£ al. (1981) found that methylphenanthrene and
dimethylphenanthrene were mutagenic toward Salmonella and that only
dimethyIphenanthrene acted as a tumor initiator on mouse skin. Two
polycyclic aromatic sulfur heterocycles were identified in the
soil-waste extracts. Both of these compounds have been tested in the
Salmonella assay, and dibenzothiophene and methyldibenzothiophene were
not mutagenic (Pelroy et al. , 1983). The only chlorinated
hydrocarbon identified in the waste-soil extracts was pentachlorophenol.
Pentachlorophenol is not mutagenic in the Salmonella assay (Anderson
e_t al. , 1972) but has been found to induce mitotic gene conversion
in J5. cerevisae (Fahrig, 1974). In addition, Schmid ejt a_l (1983)
observed an increased incidence of chromosome damage in workers from a
pentachlorophenol plant. Thus, chemical analysis of the PENT S amended
soils identified more than fourteen polycyclic aromatic hydrocarbons,
including four that were mutagenic and two animal carcinogens.
Although a large number of compounds were identified in the PENT S
soil extracts, there were many additional compounds that were detected
but not identified. These included a large number of higher molecular
weight compounds that were possibly three to five ring PAH. The source
of the waste and some of the potential soil metabolites can provide
information as to the identity of these minor constituents. Technical
grade pentachlorophenol is known to contain a number of impurities
including tetrachlorophenol, dibenzo-p-dioxins, and dibenzofurans
(Fishbein, 1979). Of these compounds, only dibenzofuran was detected in
the waste-soil extracts from the greenhouse study. Creosote oil is
composed of a variety of aromatic hydrocarbons and has been shown to be
an indirect-acting mutagen (Bos e£ al. , 1983). Since this oil was
used in the process that generated the PENT S, the aromatic
hydrocarbons which compose creosote oil would be anticipated in the
soil-waste extracts. The biodegradation of some of the organic
compounds present in waste fractions would be expected to produce
metabolites such as hydroxylated napthalenes (Higgins and Gilbert, 1978)
294
-------
and catechol (Gibson, 1972). Catechol is not mutagenic for Salmonella
but is a co-mutagen (Yoshida and Fukuhara, 1983) and a co-carcinogen
(Van Duuren and Goldschmidt, 1976). Thus, the extracts of the PENT S
amended soil contain a complex mixture of chemicals, including mutagens,
carcinogens, teratogens, promoters, initiators, inhibitors, and
co-carcinogens.
A comparison of the GC/MS chromatograms of the PENT S soil extracts
from day 0 and 360 does not indicate any significant loss of organic
compounds nor any appreciable gain in metabolic products from soil
degradation (Figures 107 to 117). Methylnapthalene was the only
mutagenic compound present in day 0 samples that was not also identified
in the soil from day 360. Potential biodegradation products that were
identified in the soil extracts from day 360 include methyl propyl
pentanol, ethyl methyl pentanol, and ethyl hexanol. The enzymes
responsible for the formation of these compounds are mixed function
oxygenases (McKenna, 1972). If these compounds are products of soil
degradation, these results indicate that the enzymes responsible for
activating promutagens may be present in the soil. Thus, the increased
activity observed in some of the incubated soil samples was possibly a
result of soil activation or a result of decreased competition for the
activating enzymes used in the mutagenesis bioassays.
The GC/MS analysis was unable to conclusively identify any of the
components of the acid, base, and neutral fractions of the SWRI waste or
the day 0 extracts. Additional concentration of the day 360 soil
samples allowed the identification of seven compounds and the detection
of 25 additional unknown compounds. The compounds identified in the day
360 extract from the SWRI waste amended Bastrop soil included
heptadecane, nonadecane, dihydro acenapthylene, dimethyl octane, ethyl
propyl hexanol, trimethyl decane, and pyrene (Figures 118, 119 and 120).
Only two of these compounds, dihydro acenapthylene in the base and
pyrene in the neutral fraction, are potential sources of mutagenic
activity (Kaden et_ al. , 1979). Kaden ejt a_l. (1979) indicate that
the mutagenic potential of acenapthylene and pyrene are approximately
equal. However, biological analysis demonstrated that the mutagenic
potential of the base fraction was three to four times that of the
neutral fraction. An evaluation of the combined results from the
chemical and biological analysis of the day 360 samples from the SWRI
waste amended Bastrop soil indicate that the decreased mutagenic
activity observed in the neutral fraction may have resulted from
inhibition produced by the non-mutagenic components of the neutral
fraction.
A list of the compounds detected in the COMBO waste amended soils
is provided in Table 92, and the GC/MS chromatograms are given in
Figures 121 through to 131. Unlike the PENT S waste, a review of the
GC/MS chromatograms from the COMBO waste amended soil does indicate that
significant quantities of organic compounds are degraded in the soil.
The GC/MS chromatograms of the acid fraction of the Norwood soil
295
-------
BASTROP
SWRI ACID
DAY 360
to
200
300
400
RT
Figure 118. GC/MS chromatograph of acid fraction of SWRI amended Bastrop
soil collected on day 360.
-------
BASTROP
SWRI BASE
DAY 360
RIG
NJ
VO
-J
200
300
400
RT
500
600
Figure 119. GC/MS chroraatograph of base fraction of SWRI amended Bastrop
soil collected on day 360.
-------
10
10
RIG
BASTROP
SWRI NEUTRAL
DAY 360
250
350
450
RT
550
650
Figure 120. GC/MS chromatograph of neutral fraction of SWRI amended
Bastrop soil collected on day 360.
-------
TABLE 92. LIST OF COMPOUNDS DETECTED IN COMBO WASTE AMENDED SOIL
Sample
Fraction
Acid
Compound (Peak Number)*
Base
Compound (Peak Number)*
Neutral
Compound (Peak Number)*
t-o
<£>
10
Norwood Unknown (226)
Day 0 Trimethyl octane (233)
Unknown (245)
Phenyl cyclopentanol (257)
Methyl napthalene (263)*M1; CO
Trimethyl dodecane (280)
Unknown (295)
Dimethyl napthalene (300)(305)(313)*MO
Propyl heptanol (318)
Methyl ethyl napthalene (339)
Trimethyl napthalene (350)(356)(364)*HO
Methyl propyl napthalene (377)
Unknown (386)
Undecane (396)
Methyl (Meth) napthalene (405)
Ethyl tridecane (418)
Dimethyl undecane (457)
Unknown (477) (492)
Norwood Unknown (335)
Day 360 Unknown (357)
Unknown (376)
Methyl decane (397)
Unknown (468)
Unknown (492)
Unknown (527)
Unknown (548)
Unknown (576)
Dimethyl decane (272) Unknown (227)
Dimethyl undecane (300) Methyl napthalene (251)*H1; CO
Unknown (347) Unknown (258)
Unknown (385) Trimethyl octane (273)
Unknown (406) Dimethyl napthalene (288) (299)*MO
Unknown (437) Unknown (303)
Unknown (470) Unknown (322)
Unknown (517) Methylethyl napthalene (329)
Unknown (549) Trimethyl napthalene (339) (347) (352)*MO
Unknown (583) Propylmethyl napthalene (367)
Unknown (609) Unknown (375)
Unknown (643) Methyl ethyl decane (386)
Unknown (675) Unknown (395)
Unknown (708) Methyl fluorene (408)
Unknown (446)
Methyl phenanthrene (480)*M1
Unknown (505)
Dimethyl phenanthrene (519)*M1; Cl
Tridecane (236)
Unknown (258)
Tetradecane (279)*P
Dimethyl napthalene (295)*HO
Unknown (308)
Pentadecane (319)
Unknown (331)
Fluorene (355)*MO; CO
Hexadecane (360)*A
Dimethyl undecane (379)
Heptadecane (399)
Phenanthrene (428)*MO; CO
Octadecane (436)*P; CC
Unknown (456)
Nonadecane (471)
Benzene dicarboxylic acid (493)*MO; CO
Eicoaane (505)*CC
Fluoranthene (525)*Ml; CO
Unknown (330)
Unknown (353)
Unknown (353)
Unknown Branched Alkane (399)
Dimethyl nonane (415)
Unknown (429)
Unknown (468)
Dimethyl decane (479)
Unknown (489)
Unknown (510)
Unknown (533)
Unknown (572)
-------
TABLE 92 CONTINUED
Fraction
(-0
O
O
Sample
Norwood
Day 360
Bastrap
Day 0
Bastrop
Day 360
Acid
Compound (Peak Number)*
Base
Compound (Peak Number)*
Neutral
Compound (Peak Number)*
Unknown (224)
Hethylnapthalene (249)(255)*M1; CO
Alkane (272)
Dimethyl napthalene (291)(297)(305)*MO
Dihydro acenaphthylene (323)*Ml
Methyl ethyl napthalene (331)
Trimethyl napthalene (341)(349)(355)*MO
Propenyl napthalene (369)
Unknown (377) (388) (396)
Methyl fluorene (409
Dimethyl fluorene (433)
Methyl phenanthrene (481)*M1
Unknown (507) (527) (606) (641) (683)
Unknown (327)
Dimethyl napthalene (341)*MO
Dihydro acenapthylene (356)*M1
Phenalene (380)"Ml
Unknown (395)
Dimethyl undecane (407)
Unknown (457)
Trimethyl decane (467)(498)
Unknown (476) (520) (536) (558) (587)
Heneicosane (536)
Pyrene (541)*M1; CO
Docaaane (566)
Unknown (577)(596)
Methyl napthalene (253)*M1
Dimethyl napthalene (295)*MO
Ethyl napthalene (303)
Alkane (309)
Methyl ethyl napthalene (328)
Trimethyl napthalene (338)(344)
(351)*HO
Methyl propyl napthalene (364)
Unknown (405) (479) (513) (555)
(592) (657) (697) (755)
(795) (838) (877)
Not determined
Methyl napthalene (245)*M1
Alkane (271)
Dimethyl napthalene (285)(291)*MO
Propyl heptanol (303
Methyl ethyl napthalene (327)
Trimethyl napthalene (338H346)(352)*MO
Propenyl napthalene (367)
Unknown (387)
Dimethyl biphenyl (408)
Methyl phenanthrene (481)*M1
Unknown (526) (600) (689) (736) (801)
(847)
Unknown (289)
Benzene dicarboxyllc acid (484)*MO;CO
Unknown (543)
* Potential genetic toxicity: I * initiator; P « promoter; A " antagonist; CC » cocarcinogendynergiat); MO -
nonmutagenic; Ml » mutagenic; CO - non-carcinogen; Cl carcinogen; all others no information. The references
used in determining these factors McCann et_ a_l (1975); Kaden et_ a± (1979); Goldachmidt (1981) and DHEU
(1969).
-------
CO
o
RIG
NORWOOD
COMBO ACID
DAY 0
200
Figure 121.
300
400
500
RT
GC/MS chromatograph of acid fraction of COMBO amended Norwood
soil collected on day 0.
-------
OJ
o
S3
RIG
NORWOOD
COMBO BASE
DAYO
O* CO
Is* » 00 O\ **> tf\ tO
»H tt\ tfl O * I-* O
t/\ vo \o »o r*»
100
Figure 122.
200
300
400
500
600
700
RT
GC/MS chromatograph of base fraction of COMBO amended Norwood
soil collected on day 0.
-------
NORWOOD
COMBO NEUTRAL
DAY 0
LO
o
U)
RIG
200
300
400
500
RT
Figure 123.
GC/MS chromatograph of neutral fraction of COMBO amended Norwood
soil collected on day 0.
-------
NORWOOD
COMBO ACID
DAY 360
RIC
u>
o
.p-
200
300
500
600
RT
Figure 124. GC/MS chromatograph of acid fraction of COMBO amended Norwood soil
collected on day 360.
-------
RIC
U)
o
Cn
NORWOOD
COMBO BASE
DAY 360
200
300
40O
RT
500
600
Figure 125.
GC/MS chromatograph of base fraction of COMBO amended
Norwood soil collected on day 360.
-------
RIG
LO
O
NORWOOD
COMBO NEUTRAL
DAY 360
300
400
500
600
RT
Figure 126. GC/MS chromatograph of neutral fraction of COMBO amended Norwood
soil collected on day 360.
-------
BA8TROP
COMBO ACID
DAYO
300
400
500
600
700
RT
Figure 127. GC/MS chromatograph of acid fraction of COMBO amended Bastrop soil
collected on day 0.
-------
BASTROP
COMBO BASE
DAY 0
Lo
O
oo
RIG
o> n
-------
LO
o
V0
RIG
BASTROP
COMBO NEUTRAL
DAYO
o\
CO
-------
u>
I
o
RIG
BASTROP
COMBO ACID
DAY 360
2(
\ f^ .H »o o m o l\ to*of^ * in in
X^^ M » ir> eo o< «» 11 «»«
^"""^'^'Vs. ^L ^*-. JU r-^"^ ~
I 1 1
)0 300 400 500
RT
600
Figure 130. GC/MS chromatograph of acid fraction of COMBO amended Bastrop
soil collected on day 360.
-------
RIG
BASTROP
COMBO NEUTRAL
DAY 360
200
300
400
500
600
700
RT
Figure 131. GC/MS chromatograph of neutral fraction of COMBO amended Bastrop
soil collected on day 360.
-------
indicates that on day 0 there were twelve compounds identified with an
additional six unknown compounds detected (Figure 118). Of the twelve
compounds identified, only methylnapthalene is mutagenic (Kaden, 1979).
The acid fraction from day 360 contained only one identifiable and eight
unknown detectable compounds (Figure 118). However, the bioassay of the
acid fractions from day 0 and 360 did not reflect a reduced level of
mutagenic activity (Table 85). Similar results were obtained from the
neutral fraction from day 0 with ten compounds identified plus eight
unknown detected (Figure 119). While three of the ten compounds
identified in the day 0 sample were mutagenic, none of the mutagens were
also detected in the extract from day 360 (Table 90). The reverse trend
seemed to occur in the base fraction with the GC/MS chromatograph from
day 360 (Figure 125) appearing more complex than the sample from day 0
(Figure 122). The base fraction from COMBO waste amended soils also
showed a corresponding increase in mutagenic activity on day 360.
(Figures 101 and 104). The results from chemical and biological analysis
of the acid fraction of the COMBO waste amended soils appear to
contradict each other, while the chemical analysis of the base and
neutral fractions provides confirmation of the respective increased and
decreased mutagenic activity that was observed in the bioassays.
Neither chemical analysis of the soil extracts nor biological
analysis of residual hydrocarbons have been capable of clearly
demonstrating treatment of soil applied waste. The results already
presented in Figures 83 through 106 describe the mutagenicity of the
residual hydrocarbons in waste amended soil. The results presented in
Figures 107 through 131 identify the major organic constituents residual
in the soil. However, in order to determine if a waste is rendered less
or non-hazardous by soil incorporation, it is necessary to compare the
mutagenic potential of equal volumes of waste-amended soil. In the
Salmonella assay, the mutagenic potential (Figures 132 to 134) was
determined by calculating the mutagenic activity ratio (Commoner, 1976)
of two non-toxic dose levels from a five member dose-response curve
(Table 73 through 90 and Figures 83 through 106) and adjusting this
ratio by the rate- of hydrocarbon degradation. While in the
Aspergillus methionine assay (Figures 135, 136 and 137) equivalent
volumes of soil were compared by evaluating the total mutation frequency
induced by the soil-waste extract. The concentration of extract tested
in each assay was adjusted according to the rate of degradation. Thus,
the data presented in Figures 132 through 137 is not an accurate
representation of the mutagenic activity of soil-waste extracts. The
utility of these data is their ability to define the hazardous
characteristics of equal volumes of waste-amended soil. The volume
tested from the acid and base fractions of the SWRI and COMBO
waste-amended soils was the equivalent of 1 gram. However, the neutral
fraction from the SWRI and COMBO soils, and all three fractions from the
PENT S soils were tested in the Aspergillus assay at a level twice
that tested in the Salmonella assay. While it is unfortunate that
equal volumes were not tested in all assays, these results do allow some
general comparisons to be made. By comparing the mutagenic potential of
312
-------
equivalent volumes of waste amended soil over time, it is possible to
determine if a waste is rendered less or non-hazardous by soil
incorporation.
The results from evaluating the affect of soil degradation on the
mutagenic potential of PENT S amended soils are presented in Figures 132
and 135. In the Norwood soil, the mutagenic potential of the base and
neutral fractions decreased to below the significant level (two times
solvent control), in both bioassays. In the acid fraction, the
mutagenic potential with metabolic activation was reduced to below a
level at which the sample would be considered mutagenic in both
bioassays, although the response without activation in Aspergillus was
increased by more than 50% from day 0 to 360. The mutagenic potential
of the neutral fraction from PENT S amended Bastrop soil was also
decreased to below significant levels in both bioassays. In the acid
and base fractions from PENT S amended Bastrop soil, the mutagenic
potential was increased on day 180. The responses in Aspergillus from
both fractions were reduced by day 360 to less than that observed on day
0; and in Salmonella the mutagenic potential of the base fraction was
reduced by day 360 while the response from the acid fraction did not
decrease until day 540. Thus, both bioassays detected constituent(s) of
the acid fraction of the PENT S waste amended soil that were resistant
to degradation and highly mutagenic. The bioassays also indicated that
the bulk of the total extractable hydrocarbons were rendered
non-hazardous by land treatment.
The mutagenic potentials of the extracts of SWRI amended soils are
presented in Figures 133 and 136. In the Norwood soil, the mutagenic
potential of the acid and base fractions was not reduced to below the
significant level in either of the bioassays 360 days after waste
application. Although there was an appreciable reduction of the
mutagenic potential of these two fractions in the Aspergillus assay,
the mutagenic potential of the acid and base fractions on day 360, as
measured with J5. typhimurium, was approximately twice the mutagenic
potential of an equivalent volume of soil from day 0. In both
bioassays, the mutagenic potential of the neutral fraction from day 360
was approximately 25% that of the sample from day 0. The acid fraction
of the SWRI waste amended Bastrop soils induced a lower response on day
360 than on day 0; however, in both bioassays the response induced by
the sample collected on day 360 would be considered mutagenic. The
response induced by the base fraction from SWRI waste amended Bastrop
soils increased from day 180 to 360 in both the Salmonella and
Aspergillus assays. Although the response induced by the sample from
day 360 was below a level at which a sample would be considered
mutagenic in Salmonella, in Aspergillus the response from the base
fraction collected on day 360 was slightly greater than this level. The
response induced by the neutral fraction extracted from the SWRI waste
amended Bastrop soil collected on day 360 was less than the significant
level in both bioassays. A comparison of the results from the two
bioassays indicates that degradation did not render the acid and base
313
-------
Ift
LE HYDROCARBOh
«/«)
»
IAL
BA
N
1-89 0
201 -89 O
K 10
M£
NW
BA
BA
ss,
ACID IOO»J
BA
i
%
BA
20n
10
BASE I00n|
BA
1 80 - ) U - 360
TIME(Oiyi)
20
10
NEUTRAL lOng
ISO J( V 360-
TIUECOtyi)
Figure 132. Total extractable hydrocarbons and mutagenic potential of equivalent
volumes of PENT S amended Norwood (NW) and^pastrop (BA) soils as
measured with S. typhi'murium strain TA98 with and without metabolic
activation. Dashed line ( ) is equal to 2.5 times solvent control.
-------
s
o
m
a:
4
O
O
<£
a
u
a:
x
80
BA
10
3
8WRI EH
N-NEUTRAL
B-BASE
A-ACID
NOT TESTED
10
4-89
a
ACIDCI
NW
U)
I'
(Jl
20-|
1 0
BASE(I|)
TIME(D*yi>
20
10
NEUTRAL (SOmg)
. TESTED
180 > 360"
TIUE(D*yi)
540
Figure 133.
Total extractable hydrocarbons and mutagenic potential of equivalent
volumes of SWRI amended Norwood (NW) and Bastrop (BA) soils as measured
with j>. typhimurium strain TA98 with and without metabolic activation.
Dashed line ( ) is equal to 2.5 times solvent control.
-------
Co
HYDROCARBONS
/9>
* CD
O O
UJ
_l e
0 ~ in
4 10
1-
< 0
ft_ *
r^i
COUBO-EH
20,
N-NEUTRAL
B-BA8E
A-ACIO fc
* 10
BA
NW Q^
1 1 1 1 I N.OT TE8TEO I
NW
%
^
4-8
-8
B
B
9
"V y
^
^
1
H ACID(I|>
BA
NW BA NW p
T_yfy(_jffl( (^^^ NOT TESTED
X
bl
10
BASE(I|)
20
10
NEUTRAL(SOag)
TIME
TIME (Out)
Figure 134. Total extractable hydrocarbons and mutagenic potential of equivalent
volumes of COMBO amended Norwood (NW) and Bastrop (BA) soils as measured
with S^ typhimurium strain TA98 with and without metabolic activation.
Dashed line ( ) is equal to 2.5 times solvent control.
-------
RENTS
X
u_
2
+ S9
-S9
ACID CZOOmg)
SO-.
NEUTRAL C20mg)
TIME (Days)
Figure 135. Total induced mutation frequency of equivalent
volumes of PENT S amended Norwood (NW) and
Bastrop (BA) soils as measured in A. nidulans
methionine system with and without metabolic
activation. Dashed line ( ) is equal to total
induced mutation frequency of 5.0/10^ survivors.
317
-------
SWRI
50-
I 0-
BA
NW
NW
+ S9 £2
-S9 D
ACID (10)
BA BA
NW
^
i
50i
NEUTRAL C.lg)
TIME (Days)
Figure 136. Total induced mutation frequency of equivalent
volumes of SWRI amended Norwood (NW) and
Bastrop (BA) soils as measured in A. nidulans
methionine system with and without metabolic
activation. Dashed line ( ) is equal to total
induced mutation frequency of 5.0/10^ survivors.
318
-------
COMBO
CO
V
a.
2
NEUTRAL C.lg)
TIME (Days)
Figure 137. Total induced mutation frequency of equivalent
volume of COMBO amended Norwood (NW) and Bastrop
(BA) soils as measured in A. nidulans methionine
system with and without metabolic activation.
Dashed line ( ) is equal to total induced
mutation frequency of 5.0/10^ survivors.
319
-------
fractions less or non-hazardous. However, the results also indicate
that the neutral fraction, which represents greater than 90% of the
total extractable hydrocarbons, was rendered non-hazardous by
degradation in the soil.
The average rate of degradation of the COMBO waste in the Bastrop
and Norwood soils was the maximum rate observed in the three wastes
studied. This degradation rate was also reflected in the mutagenic
potential of the soil extracts as measured in the biological systems.
The response induced by the acid, base, and neutral fractions extracted
from the COMBO waste amended Norwood soil collected on day 360 was less
than a level at which the sample would be considered mutagenic in both
the Salmonella and Aspergillus assays (Figures 134 and 137).
Similarly, in the waste amended Bastrop soil the response induced by the
fractions of the samples collected on day 360 was less than that induced
by the samples collected on day 0. In addition, except for the acid
fraction from the Bastrop soil in the Salmonella assay, the mutagenic
potential of the three waste fractions from day 360 were less than the
significant level in both bioassays. Thus, if the response in the
eukaryotic assay is assumed to provide a more accurate prediction of the
mutagenic response in a mammalian system, all three fractions of the
COMBO waste were rendered non-hazardous by land treatment.
The major factors influencing the mutagenic potential of the
hydrocarbons residual at a land treatment facility include the number of
different compounds present, the concentrations of those compounds, and
the toxic effects and interactions of those compounds. Biodegradation
will influence the mutagenic potential as it reduces the concentration
of certain compounds and alters the reactivity of others. Oxidation or
substitution at specific sites have been shown to increase the
reactivity of PAH (LaVoie et_ al., 1981; Hubermann £it al. , 1978;
Ho et al., 1981). The rate of biodegradation may increase as soil
bacteria adjust to the types of hydrocarbons in the soil. Poglazova
et al. (1967), Shabad e£ al,. (1971) and Khesina et^ al..
(1969) observed that the degradation of PAH by soil microorganisms was
significantly increased in a soil previously exposed to these compounds.
It is also anticipated that bacteria capable of degrading the more
complex PAH will occur in nature, since compounds such as napthalene,
phenanthrene, fluoranthene, dibenzothiophene, carbazole, and
dibenzofuran are components of coal (Sims and Overcash, 1983). Soil
microorganisms possess a broad range of enzymatic capabilities and
should be capable of degrading an assortment of hazardous compounds
(Clark et_ al., 1979; Patil et al. 1970; Chacko et_ al,.,
1966; Bixby e± al., 1971). The results of this research
demonstrate that land treatment can reduce the hazardous characteristics
of a waste by transformation or degradation. Thus, land treatment can
render a waste less or non-hazardous if it can also be demonstrated that
hazardous constituents are immobilized in the soil.
320
-------
SECTION 10
SOIL MOBILITY AND DEGRADATION OF MUTAGENIC CONSTITUENTS
FROM A WOOD-PRESERVING BOTTOM SEDIMENT
INTRODUCTION
The interaction of environmental parameters with soil and waste
components will greatly influence the rate at which hazardous organic
constituents are transformed, degraded, or immobilized at a land
treatment facility. While the majority of hazardous organic
constituents will apparently be retained and degraded in the surface
layer of soil, there is concern about the environmental fate of those
constituents which may be mobile in soil. Mobile compounds may be
soluble in water and move with the wetting front or they may be
hydrophobic and move ahead of the wetting front. In addition, the
mobility of a compound may be modified by chemical and biological
reactions occurring in the soil.
In order for land treatment to be an environmentally sound
alternative for hazardous waste disposal, there is a need to develop
procedures for sampling and techniques for monitoring mobile compounds
in soil. Two procedures have been recommended for sampling mobile
compounds at a hazardous waste land treatment facility (EPA, 1982).
Soil core samples are intended to provide information on the movement of
"slower moving" constituents, whereas soil-pore samples are intended to
provide information on the movement of fast-moving hazardous
constituents that may be missed by a soil-core sample. This research
project was designed to provide information as to the utility of the two
sampling procedures and to monitor the mobility of the components of a
complex mixture in soil using both biological and chemical analysis.
MATERIALS AND METHODS
Soil
Undisturbed soil monoliths of the Norwood silt loam were used in
this study. The properties of this soil are discussed in detail in
Section 6, and the chemical and physical properties with depth are
provided in Table 93. The soil monolith was obtained from a virgin area
that had not received waste applications of any kind.
321
-------
TABLE 93a. CHEMICAL PROPERTIES OF THE NORWOOD SOIL SERIES
Exchangeable Cations
Depth
(cm)
0.0-15.2
15.2-30.5
30.5-61.0
61.0-91.5
91.5-119.0
119.0-122.0
122.0-152.0
pH CEC*
(meq/100 g)
7.69
7.73
7.74
7.86
7.75
7.75
7.95
19.6
21.1
22.9
17.8
16.2
30.0
13.2
Ca++
20
20
20
20
20
20
20
++
Mg
I/ 100 g -
4.0
4.0
4.0
4.0
4.0
4.0
4.0
K*
0.8
0.8
0.8
0.8
0.8
0.8
0.8
*CEC = cation exchange capacity.
TABLE 93b. PHYSICAL APROPERTIES OF THE NORWOOD SOIL SERIES
Particle Distribution
Depth
(cm)
0.0-15.2
15.2-30.5
30.5-61.0
61.0-91.5
91.5-119.0
119.0-122.0
122.0-152.0
* sc = sandy clay,
Texture
(USDA)
sc*
scl
c
cl
cl
c
scl
scl = sandy
Sand
48^2
49.6
36.0
40.5
42.0
23.1
49.1
clay loam,
Silt
/yv
\h)
15.2
15.1
18.4
22.3
25.0
20.5
15.8
c = clay, cl =
Clay
36.6
35.3
45.6
37.2
33.0
55.6
35.1
clay loam
322
-------
Waste
The waste used in this segment of the research was a wood-
preserving bottom sediment. A detailed description of this waste is
given in Section 5 and the characteristics summarized in Table 50.
Lysimeters
Six undisturbed soil monolith lysimeters were used in this portion
of the study. A detailed description of the procedures for the
collection and installation of lysimeters is given by Brown e_t al.
(1974). Briefly, the technique is as follows: Casings (28 cm x 90 cm)
were obtained from the Sharney Container Corp. (Houston, Texas). These
55 gal straight-walled barrels were cleaned and coated with an epoxy
based heavy metal free paint (Shertar, Sherwin-Williams Co). Using an
iron frame for support, the casings were forced into the ground with
pressure applied by a backhoe. As the casing was pushed into the
ground, the soil was removed around the outside to relieve the pressure.
The backhoe was used to lift the soil monolith and to roll it upside
down. Sufficient soil was removed from the bottom of the profile to
install three porous ceramic suction cups at the bottom of the profile.
The nylon tubes that were used to conduct the leachate to the surface
were threaded through a 1.27 cm diameter PVC tube installed along the
inside of one wall. A barrel gasket was sealed in place, and the bottom
was clamped on the barrel. The lysimeters were then turned upright and
installed in a location to facilitate leachate collection.
Leachate 'Sample Collection
A schematic diagram of the leachate collection system is shown in
Figure 138. Leachate from the bottom of each lysimeter was collected
through 3 porous cups (Coors Type #70001-P-6-C). Each_?had a_.bubbling
pressure of 0.5 bars, a conductivity of 1.2 ml cm min bar ,
and a surface area of 38.8 cm .
Plexiglass caps were sealed to the open end of the cups with a
water proof two part epoxy (Armstrong #34). Nylon tubes .159 cm in
diameter were cemented into the plexiglass caps with another two part
epoxy (Armstrong #6) that adhered well to nylon. The three tubes passed
through the 1.27 cm PVC pipe to a plexiglass collection manifold
approximately 7.5 cm above the soil surface. Nylon tubes of 0.32 cm
diameter were used to convey the leachate to amber glass bottles in a
refrigerator at 4 C. A continuous vacuum was maintained on all six
bottles. Each bottle was equipped with an outflow tube connected to a
trap bottle. A glass tube 0.64 cm in diameter was placed in the center
of a rubber stopper in the trap bottle and connected to a vacuum pump by
rubber tubing. Leachate samples were collected as necessary by
replacing the amber glass bottles. Leachate samples from each lysimeter
were composited for the thirty days prior to waste application, day 0 to
30 after application, and day 30 to 90 after application. All containers
323
-------
U)
to
p-
LYSIMETER
CAMBER GLASS
COLLECTION
BOTTLE
PLEXIGLASS MANIFOLD
SOIL SURFACE POROUS CUPS ^^-1/16" TUBES
Figure 138. Schematic diagram of leachate collected system used in the lysimeter
study.
-------
were cleaned with soap and water and sequentially rinsed with tap water,
acetone, and distilled water to remove all organics prior to sampling.
All samples were stored at 4 C after arrival at the laboratory.
Soil Sample Collection
Surface soil samples, 0 to 15 cm, were collected from each
lysimeter prior to and immediately after waste application. Ninety days
following waste application, the lysimeters were dissected using a
circular saw equipped with an abrasive blade. Four of the six
lysimeters were sacrificed in order to collect the amount of sample
needed. Sufficient soil was collected to conduct a biological and
chemical analysis on the soil extract, as well as to provide a reserve
sample for future use. Soil samples were collected from the center of
each lysimeter at depths of 0 to 15 cm, 15 to 45 cm, and 45 to 90 cm.
Precautions were taken to prevent cross-contamination of cores. These
included collecting the soil-cores from the center of the lysimeter to
avoid any influence due to side-channel flow, cleaning the sampling
device between corings, collecting samples from control before
waste-amended lysimeters, and collecting multiple composited samples
from each lysimeter at each depth.
Biological Analysis
The ability of the organic extract of soil and water samples to
induce genetic damage was measured in a prokaryotic system capable of
detecting compounds which induce point mutations. The
Salmonella/microsome assay* of Ames et^ al. (1975) utilizes a
prokaryotic organism to evaluate the capacity of a sample to induce
reverse mutations to histidine prototrophy. Salmonella strain TA98 (a
frameshift mutant) was supplied by Dr. B. N. Ames (University of
California, Berkeley, CA). The procedural methods were the same as Ames
e£ al. (1975), except that overnight cultures were prepared by
inoculation into 10 ml of Nutrient Broth #2 (KC Biological, Inc.,
Lenexa, KS). Soil extracts were tested on duplicate plates in two
independent experiments in the standard plate incorporation assay at a
minimum of 4 dose levels of the sample with and without enzyme
activation (0.3 ml rat liver/ml S-9 mix). Aroclor 1254 induced rat
liver was obtained from Litton Bionetics (Charleston, SC). Positive
controls included 25 ug/plate 2-nitrofluorene (Aldrich Chemical Co.,
Milwaukee, WI), and 10 jag/plate 2-aminoanthracene (Sigma) which was
used to verify the functioning of the metabolic activation system. All
reagents and extracts were tested for sterility; DMSO was used as a
negative control.
RESULTS AND DISCUSSION
The chemical and biological analysis of soil and leachate water
from control and waste amended lysimeters indicate that certain
constituents of the wood-preserving bottom sediment are capable of
325
-------
migrating through the soil. The analysis of soil-core samples from
control and waste-amended lysimeters over various depths indicated that
greater quantities of residual hydrocarbons and mutagenic activity were
present in waste amended samples up to a depth of 45 cm (Figure 139).
There was no appreciable difference in the mutagenic potential of the
soil from control and waste amended lysimeters at the 45 to 90 cm depth,
although greater quantities of residual hydrocarbons were recovered from
the waste amended soil at this depth. The mutagenic potential of the
soil-core sample from a depth of 0 to 15 cm was greatest in the presence
of metabolic activation; whereas, the soil at 15 to 45 cm gave
approximately the same response with or without metabolic activation
(Figure 139).
The results from the chemical analysis of soil-core samples from
control and waste amended lysimeters are provided in Tables 94 and 95
and Figures 140, 141, 142, and 143. The compounds identified in the
soil-core sample from the unamended lysimeter (Table 94 and Figure 140)
were predominantly the same as those identified in the Norwood soil
(Table 45 and Figure 52). Two compounds of an unknown origin were
identified including 1,2-dichlorobenzene and trimethyl-hexadione. A
larger number of compounds were identified in the waste amended soil at
all depths than were identified in the unamended soil (Table 95 and
Figures 141, 142, and 143). These included several high molecular
weight polycyclic aromatic hydrocarbons. Mutagenic compounds identified
in the surface soil sample (Figures 135, 136, and 137) included
methylnapthalene, dihydro acenapthylene, fluoranthene, pyrene, and
benzanthracene (Kaden et^ al., 1979; McCann et^ al., 1975). Other
compounds with a. potential influence on the genetic activity of the soil
extract included pentachlorophenol, several alkane promoters and
cocarcinogens, and hexadecane which is an inhibitor. Only one compound,
dihydro acenapthylene, was consistently identified in the waste and soil
core samples at all depths (Figure 94).
When compared to the soil-core samples, the analysis of soil-pore
^samples provides a slightly different perspective on the capacity of
PENT S waste samples to migrate through the soil. Biological analysis
indicated that there was no mutagenic activity, as measured with J3.
typhimurium strain TA98, detected in the leachate water collected from
control and waste amended lysimeters prior to waste application. The
mutagenic potential of the soil-pore sample collected on day 30 was
approximately seven times greater than the control, whereas the sample
collected on day 90 had a mutagenic potential approximately ten times
the control (Figure 144). Thus, the bioassays detected significantly
greater quantities of mutagenic activities in the soil-pore samples from
the PENT S lysimeters both on day 30 and on day 90.
Nine compounds were detected in the soil-pore liquid sample from
the unamended lysimeters (Table 96 and Figure 145) including
benzene-dicarboxylic acid and chloro-octane. Neither of these compounds
is known to have any genotoxic activity. There were ten compounds
326
-------
1200-
1000
60
40
10
f
-m
P
/
>
s
^
il
^
NX
^
s$
^
^
<
ru
P
9
1
I
UT51METER SOIL
a CONTROU-C ss
a TCNTS -PS ss
-S9 0»Y 3O -89
C
10,171
t
t
A
^ °
iSv
^^T
i
i
i
?w
i
1
1
^
0-1S
r
a
5
so
1.0-
P3
0-)*
4S-9O
eerrHOa)
IS »3
49-30
WASTE
Figure 139. Extractable hydrocarbons and mutagenic
activity from soil core samples collected
at various depths on day 90.
327
-------
TABLE 94. LIST OF COMPOUNDS DETECTED IN SOIL CORE
SAMPLE COLLECTED ON DAY 90 FROM CONTROL
NORWOOD SOIL AT 0-15 CM DEPTH
Compound (Peak Number)* Genetic Toxicity
Pentadecane (324)X
Unknown (351) X
Hexadecane (367) A
Unknown (388) X
Heptadecane (411) X
Octadecane (443) P; CC
Unknown (463) X
Nonadecane (475) X
Unknown (496) X
Eicosane (507) CC
Heneicosane (538) X
Unknown (550) °X
1,2-dichlorobenzene (564) Ml
Trimethyl-hexanedione (599) X
Unknown (614) X
Unknown (663) X
Unknown (684) X
* Potential genetic toxicity: I = initiator;
P = promoter; A = antagonist; CC = cocarcinogen
(synergist); MO = nonmutagenic; Ml = mutagenic;
CO = non-carcinogen; Cl = carcinogen; all others
= no information. The references used in determining
these factors are listed in the text.
328
-------
TABLE 95.
LIST OF COMPOUNDS DETECTED IN SOIL CORE SAMPLES COLLECTED ON DAY 90 FROM PENT S WASTE AMENDED NORWOOD SOIL AT
VARIOUS DEPTHS
Fraction
Sample
Acid
Compound (Peak Number)*
Base
Compound (Peak Number)*
Neutral
Compound (Peak Number)*
0-15 cm Unknown (222)
Tridecane (236)
Methyl napthalene (243)*M1; CO
Unknown (262)
Tetradecaiie (2B3)*P
Dimethyl napthalene (290)*MO
Dihydro acenaphthalene O17)*M1
Pentadecane (330)
Fhenalene (369)*Ml
llexadecane (379)*A
Unknown (396)
Heptadecane (409)
Pentachlorophenol (437)*HO; Cl
Methyl dibenzothiophene (461)*MO
Nonadecane (472)
Eicosane (503)*CC
Fluoranthene (525)*M1; CO; CC
Pyrene (540)*M1; CO; CC
Docosane (563)
Unknown (592)
Unknown (620)
Unknown (642)
Unknown (671)
Tridecane (249)
Dimethyl napthalene (296)*HO
Tetradecane (3!l)*P
Dihydro acenaphthalene (323)*M1
Unknown (337)
Pentadecane (353)
Phenalene (366)*t(l
Hexadecane (384)*A
Carbazole (402)*MO; CO
Heptadecane (414)
Octadecane (445)*P; CC
Unknown (457)
Nonadecane (477)
Cyclopentaphenanthrene (485)*MO
Eicoaane (509)*CC
Fluoranthene (530)*M1; CO; CC
Heneicosane (540)
Pyrene (545)*Ml; CO; CC
Docosane (569)
Unknown (599)
Unknown (628)
Unknown (242)
Unknown (304)
Pentadecane (338)
Dimethyl napthalene (3S1)*MO
Dihydro acenaphthylene (366)*Ml
Dibenzofuran (375)
Trimethyl naphthalene (385)*MO
Heptadecane (401)
Methyl dibenzofuran (416)
Octadecane (437)*P; CC
Dibenzothiophene (455)*MO
Nonadecane (472)
Eicosane (506)*CC
Ethyl undecane (520)
Methyl hexadecane (540)
Docoaane (569)
Branched alkane (595)
Tricosane (620)
Tetracosane (639)
Benzophenanthrene (659)
Benzanthracene (680)*M1; Cl
-------
TABLE 95 CONTINUED
15-45 cm (crude)
Compound (Peak Number)*
45-90 cm (crude)
Compound (Peak Number)*
Unknown (213)
Tridecane (234)
Unknown (267)
Tetradecane (277)*P
Unknown (303)
Dihydro acenaphthylene (311)*M1
Pentadecane (319)
Unknown (338)
Fluorene (351)*MO; CO
Hexadecane (358)*A
Trimethyl octane (378)
Heptadecane (398)
Octadecane (435)*CC
Unknown (450)
Nonadecane (470)
Benzene dicarboxylic acid (492)*MO
Eicosane (502)*CC
Unknown (514)(523)(554)(592)
Unknown (230)
Unknown (257)
Dihydro acenaphthylene (307)*Ml
Dimethyl nonane (314)
Unknown (339)
Trimethyl octane (354)
Unknown (373)
Tetrazolamine (393)
Unknown (475)
Benzenedicarboxylic acid (487)*MO
Methyl hexene (521)
Unknown (563) (589)
Ethyl methyl pentanol (604)
Ethyl methyl heptane (610)
Unknown (636)
Methyl heptanol (651)
Unknown (685) (716)
Methyl propyl pentanol (738)
Unknown (755) (780)
Potential genetic toxicity: I = initiator; P = promotor; A = antagonist;
CC = cocarcinogen(synergist); MO = nonmutagenic; Ml = mutagenic;
CO = non-carcinogen; Cl = carcinogen; all others = no information.
The references used in determining these factors are listed in the text.
-------
o
o
m
RIG
LYS. CONTROL
SOIL - CRUDE
DAY 90
oo
u>
n
200
300
400
RT
500
600
111
Figure 140. GC/MS chromatograph of crude extract of soil core sample
collected on day 90 at a depth of 0 to 15 cm from unamended
Norwood lysimeter.
-------
OJ
CO
ho
RIG
LYS. 0-6"
PENTS ACID
DAY 90
o
r4
vO
200
300
400
5dO
600
RT
Figure 141. GC/MS chromatograph of acid fraction of soil core sample collected on
day 90 at a depth of 0 to 15 cm from PENT S amended Norwood lysimeter.
-------
LYS. 0-6"
RENTS BASE
DAY 90
CO
RIG
200
300
400
500
600
RT
Figure 142.
GC/MS chromatograph of base fraction of soil core sample collected
on day 90 at a depth of 0 to 15 cm from PENT S amended Norwood
lysimeter.
-------
LYS.0-6"
RENTS NEUTRAL
DAY 90
RIG
OJ
CO
200
300
400
500
600
700
RT
Figure 143. GC/MS chromatograph of neutral fraction of soil core sample
collected on day 90 at a depth of 0 to 15cm from PENT S
amended Norwood lysimeter.
-------
U)
U)
600
1 400
O
to
00
CD
<
zoo
I C i i PS i
LYSIMETER LEACHATE
{"I CONTROL C
O PENTS
-89
PS
+89
C i i PS
1 V//A
BACKGROUND
DAY 30
C i ' P
i
DAY 90
Figure 144. Mutagenic activity of leachate water from control and
PENT S waste amended lysimeters.
-------
TABLE 96. LIST OF COMPOUNDS DETECTED IN SOIL PORE SAMPLES COLLECTED ON DAY
90 FROM PENT S WASTE AMENDED NORWOOD SOIL
Compound (Peak Number)* Compound (Peak Number)*
PENT SUnknown (164)
Hydroxyl methyl pentanone (185) CONTROL:Unknown (298)
Unknown (212) Unknown (391)
Unknown (226) Unknown (417)
Unknown (251) Benzene dicarboxylic
acid(491)*MO
Unknown (278) Unknown (542)
Octanedione (287) Unknown (572)
Unknown (308) Unknown (602)
Unknown (325) Chloro-octane (632)
Tetrachlorophenol (338)#MO Unknown (661)
Phenyl butanone (362)
Phenalene (376)*M1
Anthracene (402)*MO
Pentachlorophenol (415) (429)*MO; Cl
Benzene dicarboxylic acid (491)*MO
* Potential genetic toxicity: I = initiator; P = promoter; A = antagonist;
CC = cocarcinogen(synergist); MO = nonmutagenic; Ml = mutagenic;
CO = non-carcinogen; Cl = carcinogen; all others = no information.
The references used in determining these factors are listed in the text.
-------
u>
RIG
LEACHATE
CONTROL
DAY 90
« IN
r- o
VO
t>
200
400
600
RT
Figure 145.
GC/MS chromatograph of soil pore liquid sample collected from
unamended Norwood lysimeter on day 90.
-------
identified in the soil-pore samples from the PENT S lysimeters,
including anthracene and pentachloraphenol which were also detected in
the 0 to 15 cm soil-core sample from day 90 (Table 96 and Figure 146).
Anthracene, a non-mutagenic hydrocarbon, has been found to produce
significant enhancement when in mixture with other polycyclic aromatic
hydrocarbons (Hermann, 1981); at high concentrations, anthracene caused
significant enhancement of 2-aminoanthracene (Kawalek and Andrews,
1981). Phenalene, another compound identified in a leachate sample, was
also present in the waste but was not detected in any of the soil-core
samples. Tetrachlorophenol, a degradation product of pentachlorophenol
(Fishbein, 1977), was also detected in the 90 day leachate sample. This
compound was not present in waste or soil-pore samples. As a result,
the tetrachlorophenol may have been transformed from pentachlorophenol
at the soil water interface and was subsequently leached into the
soil-pore water 90 cm below the soil surface. The absence of
tetrachlorophenol in the 0 to 15 cm soil sample makes it unlikely that
the compound reached the soil-pore sample as a result of side channel
flow.
This analytical protocol, using both biological and chemical
analysis, has demonstrated that soil-core and soil-pore liquid
monitoring can be used to detect different types of compounds.
Mutagenic activity was detected in both soil-core and soil-pore samples,
although no activity was detected in the 45 to 90 cm soil-core sample.
Saturated alkanes, branched alkanes, and alkanols were identified in
soil-core samples, while the more complex compounds such as tetra- and
pentachlorophenol, phenalene, and anthracene were identified in the
soil-pore samples. In addition, the results from utilizing bioassay
directed chemical analysis indicated that compound(s) such as anthracene
may have served to enhance the activity of mutagenic compounds present
in the leachate in trace quantities. This combined testing protocol has
demonstrated that both soil-core and soil-pore samples are necessary to
provide an accurate evaluation of the potential for hazardous waste
constituents to migrate below the zone of incorporation at a land
treatment facility. The analysis of samples from lysimeters amended
with a wood-preserving bottom sediment indicated that mutagens and
potential carcinogens are capable of migrating to a depth of 75 cm below
the zone of incorporation within 90 days following waste application.
These results indicate that land treatment of a wood-preserving waste
should proceed with caution, and that pretreatment options or in-plant
process controls might be used to reduce the concentration of mobile
constituents in the waste prior to land application.
338
-------
P3 LEACHATE
DAY90
RIC
150
Figure 146.
550
GC/MS chromatograph of soil pore liquid sample collected from
PENT S amended lysimeter on day 90.
-------
SECTION 11
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